Naturally Occurring Pest Bioregulators 9780841218970, 9780841213050, 0-8412-1897-8

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NATURALLY OCCURRING PEST BIOREGULATORS

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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ACS SYMPOSIUM SERIES 449

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Naturally Occurring Pest Bioregulators Paul A. Hedin,

EDITOR

U. S. Department of Agriculture

Developed from symposia sponsored by the Division of Agrochemicals at the 197th and 198th National Meetings of the American Chemical Society and the International Chemical Congress of Pacific Basin Societies at the 1989 International Chemical Congress of Pacific Basin Societies

American Chemical Society, Washington, DC 1991

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Library of Congress Cataloging-in-Publication Data Naturally occurring pest bioregulators Paul A. Hedin, editor p.

cm.—(ACS Symposium Series, 0097-6156; 449)

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"Developed from a symposium sponsored by the Division of Agrochemicals and the 1989 International Chemical Congress of Pacific Basin Societies at the 197th and 198th national meetings of the American Chemical Society, and the 1989 International Chemical Congress of Pacific Basin Societies, Honolulu, Hawaii, December 17-22, 1989." Includes bibliographical references and index. ISBN 0-8412-1897-8 1. Natural pesticides—Congresses. 2. Bioactive compounds— Congresses. I. Hedin, Paul A. (Paul Arthur), 1926. II. American Chemical Society. Division of Agrochemicals. III. International Chemical Congress of Pacific Basin Societies 1989: Honolulu, Hawaii) IV. Series SB951.145.N37N38 632'.95—dc20

1991

90-22914 CIP

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48-1984. Copyright © 1991 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center, Inc., 27 Congress Street, Salem, MA 01970, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of thefirstpage of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

ACS Symposium Series M . Joan Comstock, Series Editor

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1991 ACS Books Advisory Board V . Dean Adams Tennessee Technological University

Paul S. Anderson Merck Sharp & Dohme Research Laboratories

Alexis T. Bell

Bonnie Lawlor Institute for Scientific Information

John L . Massingill Dow Chemical Company

Robert McGorrin Kraft General Foods

University of California—Berkeley

Julius J. Menn

Malcolm H . Chisholm

Plant Sciences Institute, U.S. Department of Agriculture

Indiana University

Natalie Foster

Marshall Phillips

Lehigh University

Office of Agricultural Biotechnology, U.S. Department of Agriculture

Dennis W. Hess

Daniel M. Quinn

University of California—Berkeley

University of Iowa

Mary A . Kaiser

A . Truman Schwartz

Ε. I. du Pont de Nemours and Company

Macalaster College

Gretchen S. Kohl

Conoco Inc.

Dow-Corning Corporation

Michael R. Ladisch

Stephen A . Szabo Robert A . Weiss University of Connecticut

Purdue University

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Foreword

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IHE A C S SYMPOSIUM SERIES was founded in 1974 to provide

a medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that, in order to save time, the papers are not typeset, but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the editors with the assistance of the Advisory Board and are selected to maintain the integrity of the symposia. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation. However, verbatim reproductions of previously published papers are not accepted.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Preface WHILE KNOWLEDGE ABOUT NATURAL PRODUCT STRUCTURES with biological activities toward pests has increased rapidly in recent years, agricultural scientists have generally depended upon their traditional methods for controlling plant and animal pests. Even though highly active compounds have been identified, only in a few situations have they been used for control or, as in the case of crop plants, for selecting resistant varieties. However, agents such as pheromones, antifeedants, and insect- and plant-growth regulators have found some commercial application. Also, the use of traditional pesticides is being circumscribed by environmental concerns. Growing resistance of pests to present pesticides has given greater urgency to the search for better, safer compounds and better, safer delivery systems. The need to treat more precisely has also provided additional opportunities for the use of natural products. These biologically active natural products can be considered to be bioregulators, the name modeled in part on the plant-growth hormones that were shown to regulate various plant growth and development processes. They are found to be active at ever decreasing concentrations. This has, in turn, contributed to the development of increasingly more sensitive analytical procedures to measure and identify the compounds. In fact, the elucidation of a vast array of chemical structures with diverse biological activities has been achieved, and is continuing to occur, with ever increasing frequency. This book, based on presentations at three American Chemical Society symposia, has been organized into five divisions dealing with the control of insects and other animals, diseases, and weeds. The compounds, their activities, their biosynthesis, and their mechanisms of action are explored.

The first section, Bioregulation of Insect Behavior and Development,

includes chapters on arthropod and insect repellents, the identification of a beetle pheromone, nonparalyzing factors from hymenoptera, endogenous regulation of pheromone biosynthesis and mating, and systems for controlled release of pheromones.

Mechanisms of Plant Resistance to Insects, the second section, focuses

on the role of trichomes in resistance to potatoes and tomatoes, enzymatic antinutritive defenses of tomatoes, and the roles of phytoalexins in insect control.

xi In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

The

next section, Allelochemicals for Control of Insects and Other

Animals, discusses insect resistance factors in petunias, geraniums, corn, centipede grass, sunflowers, and neem, as well as insecticidal activity of monoterpenoids and fish toxins from mangrove plants.

Phytoalexins and Phototoxins in Plant Pest Control presents a model

approach to the binding of a phytoalexin elicator to D N A , studies of phytoalexins in cotton and peanuts, chapters on phototoxic metabolites of tropical plants and on photosensitizing porphyrins as herbicides.

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The final section, Allelochemicals as Plant Disease Control Agents, looks

at black shank fungus in tobacco, suppression of Fusarium Wilt and other fungi by microorganisms, antifungal and antibacterial compounds in Peruvian plants, and the description of a countercurrent chromatographic separation of complex alkaloids in tall fescue. I hope that this book will contribute to the understanding and the subsequent adoption of additional criteria and research strategies for the control of pests. The compounds should be effective at low concentrations, selective in activity against specific pests, of limited toxicity to nontarget organisms, and environmentally nonpersistent. We also need a better understanding about their mechanisms of action. I am grateful to all of the participants for their contributions to the book. Finally, I thank the Agricultural Research Service of the U.S. Department of Agriculture for providing me with the opportunity and the support to organize the symposia and compile the book. PAUL A . HEDIN

U.S. Department of Agriculture Mississippi State, M S 39762 September 5, 1990

xii In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 1

Use

of Natural Products in Pest Control Developing Research Trends Paul A. Hedin

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Crop Science Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Mississippi State, MS 39762

The rapid growth of knowledge of natural products with biological activities toward pests now provides an option for treatment, a clearer understanding of biochemical mechanisms, and a basis for biorational approaches to the design of pest control agents. Compounds that modify insect behavior are also valuable for pest control because they are normally not toxic to the target insect or to the environment. Natural products are often a key to understanding ecological systems. When the compounds are identified, progress can be made in understanding the metabolic cycles, the enzymes that lead to their biosynthesis, and the underlying genetic controls. An understanding of the interactions should make it possible to devise minimum changes that yield maximum benefits. Some developing research trends in the utilization of natural products for pest control are reviewed. P a r t i c u l a r l y over the p a s t 25 y e a r s , t h e r e has been much a c t i v i t y d i r e c t e d t o c h e m i c a l work on the i s o l a t i o n and i d e n t i f i c a t i o n o f a wide a r r a y o f b i o l o g i c a l l y a c t i v e n a t u r a l p r o d u c t s t h a t i n some way a f f e c t the b e h a v i o r , development and/or r e p r o d u c t i o n o f p e s t s such as i n s e c t s , d i s e a s e s and the growth o f weeds. However w i t h r e g a r d t o crop p l a n t s , a g r o n o m i s t s , e n t o m o l o g i s t s , and o t h e r a g r i c u l t u r a l s c i e n t i s t s have g e n e r a l l y depended on t r a d i t i o n a l methods f o r s e l e c t i n g r e s i s t a n t v a r i e t i e s w i t h adequate y i e l d p r o p e r t i e s . Even though h i g h l y a c t i v e a l l e l o c h e m i c a l s have been i d e n t i f i e d , o n l y i n a l i m i t e d percentage of s i t u a t i o n s has c h e m i c a l guidance been the l e a d i n g f a c t o r i n s c r e e n i n g f o r the p r o p e r t i e s . On the o t h e r hand, agents such as pheromones, a n t i f e e d a n t s , i n s e c t and p l a n t growth r e g u l a t o r s , t o name a few, have found some commercial a p p l i c a t i o n . A l s o , s y n t h e t i c analogues based on the a c t i v i t y o f some n a t u r a l p r o d u c t s have found an even wider market. This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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NATURALLY OCCURRING PEST BIOREGUIATORS However, perhaps t h e day o f t h e b i o l o g i c a l l y a c t i v e n a t u r a l p r o d u c t has come. E n v i r o n m e n t a l concerns a r e c i r c u m s c r i b i n g t h e use o f t r a d i t i o n a l p e s t i c i d e s . Growing r e s i s t a n c e o f p e s t s t o p r e s e n t p e s t i c i d e s has g i v e n added urgency t o t h e s e a r c h f o r b e t t e r , s a f e r compounds, and b e t t e r , s a f e r d e l i v e r y systems. The need t o t r e a t more p r e c i s e l y has a l s o p r o v i d e d a d d i t i o n a l o p p o r t u n i t i e s f o r t h e use o f n a t u r a l p r o d u c t s . I t should be s t a t e d , however, t h a t r e s i s t a n c e can be expected t o develop t o n a t u r a l p r o d u c t s t h a t have a s i n g l e mode o f a c t i o n . B i o l o g i c a l l y a c t i v e n a t u r a l p r o d u c t s can be c o n s i d e r e d t o be b i o r e g u l a t o r s , t h e name modeled i n p a r t on t h e p l a n t growth hormones t h a t were shown t o r e g u l a t e v a r i o u s p l a n t growth and development p r o c e s s e s . Today, t h e l e v e l s r e q u i r e d f o r a c t i v i t y a r e ever s m a l l e r and s m a l l e r . F o r example, t h e pheromones have been found t o be a c t i v e a t picogram c o n c e n t r a t i o n s and below. Now, t h e neurohomones have been found t o be j u s t as a c t i v e , and t h e s e a r e j u s t two examples. The e l u c i d a t i o n o f compounds o f e x q u i s i t e l y unique s t r u c t u r e s becomes more and more common as access t o p o w e r f u l i n s t r u m e n t a l techniques r a p i d l y increases. As one surveys t h e avalanche o f new, b r i l l i a n t e l u c i d a t i o n s , i t i s t h r i l l i n g t o r e a l i z e t h a t so much power and c a p a b i l i t y has been d e v e l o p e d , p a r t i c u l a r l y i n t h e p a s t decade. The advent o f new c a p a b i l i t i e s i n b i o t e c h n o l o g y f o r e c a s t s a n o t h e r quantum l e a p as t h e c a p a b i l i t y grows t o i n s e r t genes i n t o c r o p p l a n t s . The p r e s e n t prominence o f B a c i l l u s t h u r i n g i e n s i s (BT) might suggest t h a t t h e n a t u r a l p r o d u c t s c h e m i s t can be d i s p e n s e d w i t h , b u t i t can be a n t i c i p a t e d t h a t p e s t s w i l l d e v e l o p r e s i s t a n c e t o BT sooner o r l a t e r . T h e r e f o r e , t h e b i o t e c h n o l o g i s t a p p a r e n t l y w i l l have t o r e l y on t h e c h e m i s t t o i d e n t i f y o t h e r systems t o e x p r e s s . I t thus appears t h a t t h e r e i s a f u t u r e f o r the n a t u r a l p r o d u c t s c h e m i s t . P e s t s can most b r o a d l y be c l a s s i f i e d as those a t t a c k i n g a n i m a l s o r p l a n t s . Those p e s t s t h a t a t t a c k a n i m a l s a r e o f t e n a r t h r o p o d s , u s u a l l y i n s e c t s . Other p e s t s o f a n i m a l s a r e microbes and nematodes. T h e i r t r e a t m e n t i s g e n e r a l l y c o n s i d e r e d a v e t e r i n a r y a c t i v i t y , although n a t u r a l products i n c l u d i n g a n t i m i c r o b i a l s a r e o f t e n used. Thus, a n i m a l h e a l t h c o n c e r n s , u n l e s s they a r e i n s e c t r e l a t e d , a r e g e n e r a l l y c o n s i d e r e d s e p a r a t e l y , and w i l l n o t be emphasized i n t h i s s u r v e y . The p r o t e c t i o n o f p l a n t s from p e s t s has been d i v i d e d i n t o t h r e e c a t e g o r i e s ; c o n t r o l o f (1) i n s e c t s , (2) d i s e a s e s , and (3) weeds. While p e s t c o n t r o l has t r a d i t i o n a l l y been accomplished over t h e p a s t 40-50 y e a r s w i t h p e s t i c i d e s o f s y n t h e t i c o r i g i n , t h i s was n o t the case p r i o r t o t h e development o f s y n t h e t i c p e s t i c i d e s such as DDT. I n r e c e n t y e a r s , a number o f n a t u r a l p r o d u c t s i n c l u d i n g t h o s e from f e r m e n t a t i o n have been f i n d i n g a n i c h e i n t h e market p l a c e , because o f t h e i n c r e a s i n g concerns about c l a s s i c a l p e s t i c i d e s , and more s t r i n g e n t c o n s t r a i n t s about t h e i r u s e . A c c o r d i n g l y , an e f f o r t has been made t o i d e n t i f y t h o s e n a t u r a l p r o d u c t s t h a t have been found u s e f u l f o r t h e c o n t r o l o f a d i v e r s i t y of pests. I t was r e c o g n i z e d t h a t t h e v a l u e o f t h i s r e v i e w would be i n c r e a s e d by t h e i n c l u s i o n o f as many l i t e r a t u r e c i t a t i o n s as

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

1.

HEDIN

Use of Natural Products in Pest Control

p o s s i b l e . Many of the c i t a t i o n s are of r e c e n t meeting a b s t r a c t s , p a r t i c u l a r l y ACS meetings. These should p r o v i d e an e n t r a n c e i n t o the l i t e r a t u r e . An e f f o r t has been made t o a c h i e v e a r e l a t i v e l y e q u a l emphasis f o r a l l a r e a s t h a t have been i d e n t i f i e d , but t h i s i s intended as a survey t o show impact and not t o be an e x h a u s t i v e review.

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C o n t r o l of

Insects

Sex, A s s e m b l i n g , and Other Pheromones. The r e c e n t l i t e r a t u r e ( 1 4) c o n t a i n s hundreds of c i t a t i o n s a t t e s t i n g t o the s p e c i f i c i t y , u n i q u e n e s s , and e f f i c a c y o f t h e s e compounds, a few o f which are l i s t e d i n Chapters 2 and 3 of t h i s volume. Two of the major drawbacks t o u t i l i z a t i o n o f pheromones are t h a t m a r k e t i n g o p p o r t u n i t i e s are l i m i t e d and t h a t the c o n t r o l a c h i e v e d i s u s u a l l y l e s s than complete. Pheromones may o c c a s i o n a l l y be s u i t a b l e f o r c o n t r o l , but may be most a p p l i c a b l e i n survey a p p l i c a t i o n s o r as p a r t of i n t e g r a t e d pest management programs. Pheromones have r e p e a t e d l y been used t o demonstrate the presence o f i n s e c t s where s c o u t i n g and o t h e r p h y s i c a l procedures are i n e f f e c t i v e . I t has been demonstrated t h a t pheromones can be used both f o r a t t r a c t i o n and f o r c o n f u s i o n . For c o n f u s i o n , e i t h e r l a r g e q u a n t i t i e s of the pheromone o r isomers can be used. I t has been f u r t h e r suggested t h a t where s e v e r a l i n s e c t s a t t a c k a c r o p , a l l of the pheromones may be f o r m u l a t e d t o g e t h e r . Where s e v e r a l components are r e q u i r e d f o r an i n s e c t r e s p o n s e , c o n f u s i o n may be a c h i e v e d by a l t e r i n g the r a t i o o r w i t h h o l d i n g one o r more components. In a d d i t i o n t o c o n t i n u i n g work t o i d e n t i f y pheromones, c o n s i d e r a b l e work on pheromone b i o s y n t h e s i s i s i n p r o g r e s s . Two r e c e n t symposia each i n c l u d e m u l t i p l e papers d e s c r i b i n g v a r i o u s a s p e c t s (4, 5 ) . Chapters 4-6 i n t h i s volume d e s c r i b e r e g u l a t o r y f a c t o r s of pheromone b i o s y n t h e s i s t h a t when more c o m p l e t e l y understood may f i n d f i e l d a p p l i c a t i o n s i m i l a r t o t h a t of i n s e c t growth r e g u l a t o r s . A l l e l o c h e m i c a l s . Food a t t r a c t a n t s , r e p e l l e n t s , f e e d i n g stimulants, feeding deterrents (antifeedants), o v i p o s i t i o n s t i m u l a n t s and d e t e r r e n t s , t o x i c a n t s , and n u t r i t i o n a l f a c t o r s are examples of a l l e l o c h e m i c a l s . They are u s u a l l y secondary p l a n t c o n s t i t u e n t s ( n o n - n u t r i t i o n a l chemicals a f f e c t i n g i n s e c t behavior and development). A l l e l o c h e m i c a l s g i v i n g the h o s t p l a n t an a d a p t i v e advantage are c a l l e d a l l o m o n e s , and f a c t o r s g i v i n g the i n s e c t an a d a p t i v e advantage are c a l l e d kairomones. A number of books and symposia have d e s c r i b e d work on t h e i r i s o l a t i o n , i d e n t i f i c a t i o n , and a p p l i c a t i o n (6-13). For many y e a r s , the U.S. Department of A g r i c u l t u r e conducted a r e s e a r c h program t o i d e n t i f y a t t r a c t a n t s and r e p e l l e n t s by b i o a s s a y i n g p l a n t e x t r a c t s and s y n t h e t i c compounds, many r e l a t e d t o known p l a n t compounds. A t t r a c t a n t s f o r the M e d i t e r r a n e a n f r u i t f l y , o r i e n t a l f r u i t f l y , and Japanese b e e t l e and many o t h e r s were s e l e c t e d as the r e s u l t of t h e s e programs and w i d e l y used. However, i n r e c e n t y e a r s , food a t t r a c t a n t s (kairomones) have found o n l y i n f r e q u e n t a p p l i c a t i o n s . On the o t h e r hand, a n t i f e e d a n t compounds w i t h very h i g h a c t i v i t y , n o t a b l y a z a d i r a c h t i n from neem

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NATURALLY OCCURRING PEST BIOREGULATORS ( 1 3 ) , have r e c e n t l y become the s u b j e c t o f renewed i n t e r e s t , and s e v e r a l have now been r e g i s t e r e d f o r commercial use. The i n t e r e s t s of s e v e r a l i n d u s t r i a l companies have been i n s t r u m e n t a l i n t h i s new t r e n d . The a n t i f e e d a n t s t e s t e d t o date are sometimes s p e c i f i c f o r c e r t a i n i n s e c t s . These v a r i a b l e a c t i v i t i e s may serve as the b a s i s both f o r s t r u c t u r e - a c t i v i t y s t u d i e s and m e c h a n i s t i c s t u d i e s a t the r e c e p t o r l e v e l . The use of a n t i f e e d a n t s has an added advantage o v e r t h a t of t o x i c a n t s i n t h a t the i n s e c t consumes l i t t l e i f any of the p l a n t , t h u s l i m i t i n g damage. Pest c o n t r o l m a t e r i a l s which are s p e c i f i c f o r i n s e c t s and which a f f e c t m e t a b o l i c pathways o r a n a t o m i c a l s t r u c t u r e s ( i n c l u d i n g c e l l u l a r ) which are unique t o i n s e c t s may p o s s e s s l e s s t o x i c i t y f o r mammals, and thus may be s u p e r i o r as p e s t i c i d e s . I t has a l s o been s p e c u l a t e d t h a t h i g h l y s p e c i f i c l i p o l y t i c and p r o t e o l y t i c enzymes w i l l be found t o c o n t r o l s p e c i f i c p r o c e s s e s i n i n s e c t s , as they do i n mammals; thus t h e y w i l l be i m p o r t a n t t a r g e t s f o r r e s e a r c h . Chapters 2 and 9-20 i n t h i s volume d i s c u s s the c o n t r o l of i n s e c t s w i t h a l l e l o c h e m i c a l s , and the mechanisms by which p l a n t s b i o s y n t h e s i z e t h e s e compounds and use them t o become r e s i s t a n t t o t h e i r pests. Toxicants. The d i s c u s s i o n o f t h i s s u b j e c t has been separated from t h a t of a l l e l o c h e m i c a l s t o emphasize t h a t n o v e l compounds p o s s e s s i n g s t r o n g t o x i c a c t i v i t y , and o f t e n accompanying a n t i f e e d a n t a c t i o n , have been i s o l a t e d from p l a n t s t h a t are o f t e n t r o p i c a l o r d e s e r t i n o r i g i n (12-15). One of the apparent e x p l a n a t i o n s f o r the l a r g e number o f t r o p i c a l and d e s e r t p l a n t s p o s s e s s i n g h i g h l y t o x i c compounds i s t h a t they must c o e x i s t w i t h h i g h e r i n s e c t and d i s e a s e i n f e s t a t i o n s t h a n p l a n t s grown i n temperate c l i m a t e s . In e v o l u t i o n a r y t i m e , p l a n t s t h a t are most a b l e t o c o e x i s t ( i n p a r t because of t h e i r b i o s y n t h e s i s of t o x i c a n t s ) have been s e l e c t e d . An i n c r e a s e i n the a c c e s s i b i l i t y o f t h e s e p l a n t m a t e r i a l s w i l l depend l a r g e l y on the a c t i v i t y of b o t a n i c a l and p h a r m a c e u t i c a l c o l l e c t i o n programs. Chapters 21 and 28 i n t h i s volume are p e r t i n e n t examples of d i r e c t e d e f f o r t s t o i d e n t i f y n a t u r a l t o x i n s from t r o p i c a l p l a n t s o u r c e s . A prominent example of a s y n t h e t i c p e s t i c i d e whose s t r u c t u r e was based on a n a t u r a l p r o d u c t i s the f a m i l y of p y r e t h r o i d s ( 1 6 ) . A f t e r the e l u c i d a t i o n of the n a t u r a l p r o d u c t s t r u c t u r e , a s e a r c h f o r compounds w i t h optimum p r o p e r t i e s has c o n t i n u e d f o r many y e a r s and has r e s u l t e d i n wide c o m m e r c i a l i z a t i o n . N a t u r a l l y O c c u r r i n g Hormonal Agents from I n s e c t s and P l a n t s . The j u v e n i l e , m o l t i n g , b r a i n , and diapause hormones, antihormones such as the p r e c o c e n e s , and the p r o s t a g l a n d i n s are examples of t h e s e a g e n t s . S e v e r a l books and symposia ( 5 , 10, 12, 17) p r o v i d e a summary o f the s t a t u s of t h e s e c l a s s e s of compounds. I t i s p r o b a b l e t h a t a d d i t i o n a l hormonal and a n t i h o r m o n a l agents w i t h h i g h a c t i v i t y and s p e c i f i c i t y w i l l be i s o l a t e d from p l a n t s o r i n s e c t s . Some w i l l a c t i n a d i r e c t manner w h i l e o t h e r s may promote b i o s y n t h e s i s of a secondary compound(s) w i t h t o x i c i t y o r o t h e r a c t i v i t y toward the i n s e c t . The h i g h a c t i v i t y and g r e a t s p e c i f i c i t y of t h e s e compounds make i t p r o b a b l e t h a t f i e l d

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HEDIN

Use of Natural Products in Pest Control

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a p p l i c a t i o n s can be c a r r i e d out under o p t i m a l c o n d i t i o n s , o f t e n as a p a r t of p e s t management systems. D u r i n g the past few y e a r s , a number of p e p t i d e s have been i s o l a t e d from i n s e c t s and p l a n t s w i t h v e r y p o w e r f u l hormonal o r t o x i c e f f e c t s (18, 19). Chapter 7 i n t h i s volume d e s c r i b e s work on the e f f e c t s of enzymes on c u t i c l e t a n n i n g t h a t are hormonal l i k e i n a c t i o n s and have a c t i v i t y a t low c o n c e n t r a t i o n s . M i c r o b i a l and V i r a l Agents f o r the C o n t r o l of I n s e c t P e s t s . M i c r o b i a l agents t r a d i t i o n a l l y have been v i s u a l i z e d as c o n t r i b u t i n g t o b i o l o g i c a l c o n t r o l of p e s t s . Many o f these m i c r o b i a l agents can now be d e f i n e d c h e m i c a l l y . Work i s a l s o p r o g r e s s i n g on f l y c o n t r o l i n a n i m a l p r o d u c t i o n and a n t i g e n s f o r nematodes and i n s e c t s ( 1 9 ) . The development of BT p r e p a r a t i o n s f o r c o n t r o l of i n s e c t p e s t s has a c c e l e r a t e d g r e a t l y i n the past 3-5 y e a r s , p a r t i c u l a r l y s i n c e f o r m u l a t i o n s have been developed t o extend f i e l d l i f e . Other m i c r o b i a l and v i r a l agents are a l s o b e i n g developed and show v a r y i n g degrees of promise f o r f i e l d a p p l i c a t i o n s (18-21). N a t u r a l Products as Inducers of I n s e c t R e s i s t a n c e . P l a n t growth r e g u l a t o r s have been shown t o i n c r e a s e the b i o s y n t h e s i s of c e r t a i n secondary p l a n t c o n s t i t u e n t s t h a t i n t u r n decrease p l a n t a t t a c k by i n s e c t s . $ - N a p h t h a l e n e a c e t i c a c i d , f o r example, e l i c i t s i n c r e a s e d t e r p e n e b i o s y n t h e s i s i n c i t r u s , thus d e c r e a s i n g a t t a c k by f r u i t flies. The approach of u s i n g both n a t u r a l and s y n t h e t i c p l a n t growth r e g u l a t o r s may c o n t i n u e t o f i n d a p p l i c a t i o n s i n i n s e c t control. A g e n e r a l term f o r compounds whose b i o s y n t h e s i s i s e l i c i t e d o r i n d u c e d i n p l a n t s as the r e s u l t of a t t a c k by a pest i s " p h y t o a l e x i n " . These h i g h e r p l a n t m e t a b o l i t e s are a n t i b i o t i c t o c e r t a i n p o t e n t i a l p l a n t pathogens and a l s o on o c c a s i o n t o i n s e c t s (Chapter 13 of t h i s volume). The p h y t o a l e x i n s t y p i c a l l y are b i o s y n t h e s i z e d i n g r e a t e r c o n c e n t r a t i o n s when the p l a n t i s s u b j e c t e d t o s t r e s s . T h e r e f o r e , the a t t a c k i n g agent ( f u n g i , b a c t e r i a , o r v i r u s e s i n most work t o date) e l i c i t s the i n i t i a t i o n o r i n c r e a s e d s y n t h e s i s o f p h y t o a l e x i n s ( a n t i b i o t i c compounds). I t has r e c e n t l y been shown t h a t a t t a c k by i n s e c t s and nematodes a l s o can e l i c i t the f o r m a t i o n o f p h y t o a l e x i n s . A number of c h e m i c a l e l i c i t o r s o f p h y t o a l e x i n s have a l s o been i d e n t i f i e d (11, 12, 22, 23). Induced A u t o i n t o x i c a t i o n . The g o a l of t h i s approach i s t o a p p l y p r e c u r s o r s t h a t develop a c t i v i t y o n l y i n the t a r g e t s p e c i e s . A l t h o u g h p h o t o s e n s i t i z a t i o n i s not always r e q u i r e d t o develop a c t i v i t y , t h i s type of s e n s i t i z a t i o n was r e p o r t e d a number of y e a r s ago as o c c u r r i n g when h o u s e f l i e s were f e d v a r i o u s dyes (24). A c e t y l e n e s and furanocoumarins have been shown t o a f f e c t the i n s e c t m e l a n i n s and they are p h o t o t o x i c ( 2 5 ) . The advantage of t h i s approach f o r p r a c t i c a l a p p l i c a t i o n i s t h a t i t may p e r m i t f o l i a r a p p l i c a t i o n o f compounds w i t h low t o x i c i t y and perhaps h i g h s p e c i f i c i t y f o r the t a r g e t i n s e c t . Continued s t u d i e s of the mechanisms of a c t i v a t i o n can be expected t o generate more e f f i c i e n t p r e c u r s o r s . Much of the r e c e n t work has been summarized

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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NATURALLY OCCURRING PEST BIOREGULATORS i n two symposia and a subsequent book (26, 2 7 ) , and i n Chapters 25 and 26 o f t h i s volume.

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C o n t r o l o f Weeds Weed c o n t r o l i s d e f i n e d as t h e s e l e c t i v e a p p l i c a t i o n o f s t r e s s agents t h a t can be c h e m i c a l ( s y n t h e t i c o r n a t u r a l ) , t i l l a g e , f e r t i l i z e r , c u l t u r a l p r a c t i c e , or other. I t has been suggested and i s g e n e r a l l y r e c o g n i z e d t h a t c o n t r o l o f weeds must f o l l o w t h e approach o f i n t e g r a t e d p e s t management. Some o f t h e d e s i r e d r e s u l t s a r e t h a t t h e system be energy e f f i c i e n t , c o n s e r v a t i v e t o s o i l , e n v i r o n m e n t a l l y s a f e , i n e x p e n s i v e , e f f e c t i v e , broad spectrum where d e s i r a b l e , and s e l e c t i v e . The c o n t r o l o f weeds has g e n e r a l l y been c o n s i d e r e d t o be t h e domain o f s y n t h e t i c c h e m i c a l s , but n a t u r a l p r o d u c t s a r e b e g i n n i n g t o have an impact (28, 2 9 ) . N a t u r a l P r o d u c t s t h a t R e g u l a t e P l a n t Growth. N a t u r a l p r o d u c t s t h a t possess growth r e g u l a t i n g a c t i v i t y can be b r o a d l y c a t e g o r i z e d i n t o two groups: (1) growth substances such as a u x i n s , g i b b e r e l l i n s , c y t o k i n i n s , a b s c i s i c a c i d , e t h y l e n e , and t h e i r s y n t h e t i c analogues o r mimics, and (2) t h e s o - c a l l e d secondary p l a n t growth substances such as t h e p h e n o l s , a l i p h a t i c and a r o m a t i c c a r b o x y l i c a c i d s and t h e i r d e r i v a t i v e s , s t e r o i d s , a l k a l o i d s , t e r p e n o i d s , amino a c i d s , and l i p i d s . Of t h e secondary p l a n t growth s u b s t a n c e s , some o f t h e u n s a t u r a t e d l a c t o n e s , t e r p e n o i d s , s t e r o i d s , and a l k a l o i d s tend t o have l i m i t e d s p e c i e s d i s t r i b u t i o n , a r e produced i n s m a l l q u a n t i t i e s , b u t may possess some s p e c i f i c a c t i v i t y . N e a r l y a l l o f t h e s o - c a l l e d secondary p l a n t growth substances can be viewed as o r i g i n a t i n g from t h e a c e t a t e and s h i k i m i c a c i d pathways. Mandava (30) has l i s t e d a number o f t h e s e compounds w i t h t h e i r s p e c i f i c a c t i v i t i e s . Duke (Chapter 26 i n t h i s volume) r e p o r t e d on s e v e r a l t e t r a p y r r o l e i n t e r m e d i a t e s o f heme b i o s y n t h e s i s t h a t generate h i g h l e v e l s o f s i n g l e t oxygen. Many compounds t h a t e f f e c t heme and/or c h l o r o p h y l l pathways a r e s t r o n g l y h e r b i c i d a l due t o a c c u m u l a t i o n of p h y t o t o x i c l e v e l s o f these t e t r a p y r r o l e s . I n v e s t i g a t i o n s of the r o l e that these n a t u r a l products play i n t h e metabolism o f t h e p l a n t s where t h e y a r e produced and t h e e x t e n t t o which they c o n t r o l growth and developmental p r o c e s s e s appear o f i n t e r e s t . A l s o , t h e n a t u r e o f t h e c o n t r o l mechanisms and t h e i r e f f i c a c y f o r h e r b i c i d a l a c t i v i t y may warrant i n v e s t i g a t i o n . The r e c e n t i n t e r e s t i n b r a s s i n o s t e r o i d s as p l a n t growth r e g u l a t o r s may a l s o l e a d t o an a p p l i c a t i o n f o r weed c o n t r o l (31). A l l e l o p a t h i c Agents. Weeds (and o t h e r p l a n t s ) s e c r e t e c h e m i c a l s from t h e r o o t s and t o p s t h a t i n h i b i t seed g e r m i n a t i o n and growth of proximate p l a n t s . These c h e m i c a l s can be shown t o be v e r y e f f e c t i v e i n t h e l a b o r a t o r y and t h e i r f i e l d e f f e c t s i n - s i t u a r e e v i d e n t , but a d a p t a t i o n f o r commercial use has been slow t o f o l l o w . Nevertheless, there i s a continuing e f f o r t t o i d e n t i f y a l l e l o p a t h i c a g e n t s . Two comprehensive books by Thompson (28) and W a l l e r (29) summarize much o f t h e r e c e n t l i t e r a t u r e .

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Use of Natural Products in Pest Control Control

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D i s e a s e s i n p l a n t s may be c a t e g o r i z e d as r e s u l t i n g c h i e f l y from v i r u s e s , b a c t e r i a and f u n g i . I t has been s t a t e d t h a t modern o r g a n i c f u n g i c i d e s have i n e f f e c t b i o r a t i o n a l l y e v o l v e d from the p a s t use of s u l f u r , copper s u l f a t e , and copper s u l f a t e h y d r a t e d l i m e . Over the p a s t 30-40 y e a r s , a s u c c e s s i o n of s y n t h e t i c f u n g i c i d e s and o t h e r d i s e a s e c o n t r o l agents have been developed. They have c a p t u r e d most of the market and c o n t i n u e t o h o l d i t . N e v e r t h e l e s s , l a r g e numbers of n a t u r a l p r o d u c t s have been screened f o r v a r i o u s d i s e a s e c o n t r o l a c t i v i t i e s (12, 30, 32). F u n g i c i d e s from T r o p i c a l and S u b t r o p i c a l P l a n t s . As d i s c u s s e d e a r l i e r , p l a n t s from t r o p i c a l r e g i o n s of the w o r l d are s u b j e c t e d t o severe d i s e a s e p r e s s u r e s , p a r t i c a l l y because of the heat and h u m i d i t y ( o t h e r d i s e a s e organisms r e q u i r e c o o l , humid c o n d i t i o n s ) . These p l a n t s may adapt t o p r e s s u r e of d i s e a s e s i n e v o l u t i o n a r y time by d e v e l o p i n g defense systems. One mechanism of defense i s the b i o s y n t h e s i s of h i g h l y a c t i v e a n t i f e e d a n t s f o r i n s e c t s . A number have been i d e n t i f i e d (14, 32) which suggests t h a t o t h e r t y p e s of b i o l o g i c a l a c t i v i t y such as a n t i f u n g a l a c t i v i t y a l s o may be found i n these p l a n t s . The a v a i l a b i l i t y of p l a n t m a t e r i a l from the t r o p i c s has i n c r e a s e d because of i n d u s t r i a l and p u b l i c s e c t o r groups s e c u r i n g them f o r e x a m i n a t i o n o f t h e i r p h a r m a c e u t i c a l and a n t i c a n c e r p r o p e r t i e s . The work o f M i l e s e t . a l . (Chapter 28 i n t h i s volume), who screened P e r u v i a n p l a n t s f o r s e v e r a l a n t i m i c r o b i a l a c t i v i t i e s , i s an example of t h i s approach. Snook and Chortyk (Chapter 27 of t h i s volume) found s e v e r a l tobacco r o o t p h e n o l i c s t o be a c t i v e a g a i n s t the b l a c k shank fungus. Hasegawa e t a l (Chapter 29 of t h i s volume) i s o l a t e d a n t a g o n i s t i c microorganisms from the r h i z o s p h e r e s o i l of Adzuke-bean r o o t t h a t c o n t r o l l e d Fusarium w i l t and i s o l a t e d s e v e r a l a n t i b i o t i c c o n s t i t u e n t s . A l s o , a number of a n t i f u n g a l c o n s t i t u e n t s were r e p o r t e d i n the symposium book o f C u t l e r ( 1 2 ) . A n t i b i o t i c s . The s u c c e s s f u l use of a n t i b i o t i c s a g a i n s t b a c t e r i a l d i s e a s e s of humans has l e d t o l a r g e - s c a l e s c r e e n i n g of a n t i b i o t i c s f o r p l a n t d i s e a s e c o n t r o l . They are n o r m a l l y produced c o m m e r c i a l l y f o r use by m i c r o b i o l o g i c a l p r o c e s s e s . Some o f the a n t i f u n g a l a n t i b i o t i c s are c y c l o h e x i m i d e , g r i s e o f u l v i n , b l a s t i c i d i n S, kasugamycin, p o l y o x i n , ezomycin, and v a l i d a m y c i n A. The a n t i b a c t e r i a l a n t i b i o t i c s i n c l u d e s t r e p t o m y c i n , c e l l o c i d i n , c h l o r a m p h e n i c o l , and n o v o b i o c i n . In another a r e a , S t r o b e l and Myers (33) i s o l a t e d a b a c t e r i u m n o r m a l l y found on l e a v e s of wheat, b a r l e y , and o a t s t h a t can d e f e a t the fungus r e s p o n s i b l e f o r Dutch elm d i s e a s e . The b a c t e r i u m i s a pseudomonad t h a t produces f u n g u s - k i l l i n g a n t i b i o t i c s . A l t e r n a t i v e l y , the b a c t e r i a c o u l d be d i r e c t l y used o r the b a c t e r i a l a n t i b i o t i c c o u l d be m i c r o b i o l o g i c a l l y produced and used f o r c o n t r o l . The success w i t h Dutch elm d i s e a s e fungus suggests t h a t s u i t a b l e s c r e e n i n g programs may i d e n t i f y s t i l l o t h e r d i s e a s e - k i l l i n g agents. E l i c i t a t i o n of P h y t o a l e x i n s . P h y t o a l e x i n s ( p r e v i o u s l y d i s c u s s e d b r i e f l y w i t h r e g a r d t o a c t i v i t i e s a g a i n s t i n s e c t s ) are g e n e r a l l y

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NATURALLY OCCURRING PEST BIOREGULATORS d e f i n e d as low m o l e c u l a r weight p r o d u c t s o f p l a n t b i o s y n t h e s i s t h a t have a n t i b i o t i c p r o p e r t i e s t o one o r s e v e r a l groups o f m i c r o o r g a n i s m s . The preformed l e v e l s o f p h y t o a l e x i n s a r e g e n e r a l l y low o r n o n - d e t e c t a b l e i n h e a l t h y p l a n t t i s s u e , b u t they accumulate t o h i g h l e v e l s a t t h e s i t e o f a t t a c k o f t h e p l a n t by an i n v a d i n g m i c r o o r g a n i s m . B e l l e t a l . ( 3 4 ) d e f i n e s t h o s e components o f r e s i s t a n c e t h a t a r e p r e s e n t i n h e a l t h y p l a n t s as p a r t o f a " c o n s t i t u t i v e d e f e n s e " . Chemicals t h a t e f f e c t a c t i v e defense responses a r e r e f e r r e d t o as e l i c i t o r s , and may be e i t h e r b i o t i c o r a b i o t i c i n o r i g i n . B e l l e t a l . (34) have i d e n t i f i e d a number of a b i o t i c e l i c i t o r s o f t e r p e n o i d and t a n n i n s y n t h e s i s i n c o t t o n such as c h i l l i n g i n j u r y , UV i r r a d i a t i o n , c u p r i c i o n s , polymers from m i c r o b i a l c e l l w a l l s such as p o l y s a c c h a r i d e s , and p e s t i c i d e s . I n a sense, many o f those b i o r e g u l a t o r s , n a t u r a l and s y n t h e t i c , t h a t i n c r e a s e p l a n t r e s i s t a n c e t o p e s t s c a n be viewed as e l i c i t o r s o f p h y t o a l e x i n s . Of s p e c i a l i n t e r e s t a r e t h e p o l y s a c c h a r i d e s t h a t a c t as e l i c i t o r s . V a r i o u s heterogeneous polymers o b t a i n e d from f u n g a l and b a c t e r i a l c e l l w a l l s a l s o a c t as e l i c i t o r s . These i n c l u d e e x t r a c e l l u l a r p o l y s a c c h a r i d e s , l i p o p r o t e i n p o l y s a c c h a r i d e s , and glycoproteins. S t u d i e s w i t h o t h e r p l a n t - p a t h o g e n systems have shown t h a t o l i g o m e r s from c h i t o s a n and 3-1,3-glucans can be potent e l i c i t o r s ( 3 5 ) . Both c h i t i n and t h e g l u c a n s a r e common c o n s t i t u e n t s o f f u n g a l c e l l w a l l s and a r e c l e a v e d by c h i t i n a s e s and 3-1,3-glucanases t h a t o c c u r i n p l a n t s ; c o n c e n t r a t i o n s o f t h e s e enzymes i n c r e a s e i n many p l a n t s soon a f t e r i n f e c t i o n . A s p e c i f i c o l i g o m e r s i z e ( u s u a l l y 5-8 s u b u n i t s ) o f c h i t o s a n o r 3-1,3-glucans i s required for appreciable a c t i v i t y . I s i s p o s s i b l e t h a t such s i z e s a r e c l e a v e d from l i p o p r o t e i n p o l y s a c c h a r i d e s , e x t r a c e l l u l a r p o l y s a c c h a r i d e s , and dead c e l l s o f f u n g a l and b a c t e r i a l pathogens t o a c t i v a t e defense responses i n c o t t o n . A 3-Na c e t y l g l u c o s a m i n i d a s e has been demonstrated i n c o t t o n t i s s u e ( 3 4 ) . P e c t a t e o l i g o m e r s a l s o may a c t as e l i c i t o r s and can be formed by t h e a c t i o n o f f u n g a l o r h o s t p e c t i n a s e enzymes on p e c t i n (33). Summary. N a t u r a l l y o c c u r r i n g pest b i o r e g u l a t o r s have been i s o l a t e d from a wide d i v e r s i t y o f s o u r c e s , o f t e n from g e o g r a p h i c a l l y s t r e s s e d r e g i o n s which f a v o r r a p i d growth o f p e s t populations. I n o r d e r f o r p l a n t s ( o r a n i m a l s ) t o s u r v i v e , they must develop mechanisms t o a v o i d o r cope w i t h t h e s e p e s t s . These mechanisms o f t e n i n v o l v e c h e m i c a l s t h a t d e t e r f e e d i n g , t h a t a r e t o x i c , o r t h a t slow growth and m a t u r a t i o n o f t h e p e s t . N a t u r a l p r o d u c t s a r e a l s o a key t o u n d e r s t a n d i n g e c o l o g i c a l systems. With t h e i d e n t i f i c a t i o n o f t h e c o n s t i t u e n t s , p r o g r e s s has been made i n u n d e r s t a n d i n g t h e m e t a b o l i c c y c l e s , t h e enzymes t h a t l e a d t o t h e i r b i o s y n t h e s i s , and t h e u n d e r l y i n g g e n e t i c c o n t r o l s . F o r a p p l i c a t i o n o f these n a t u r a l p r o d u c t s t o p e s t c o n t r o l , an u n d e r s t a n d i n g o f t h e p h y s i o l o g y and b e h a v i o r a r e e s s e n t i a l t o e l u c i d a t e t h e u n d e r l y i n g c h e m i c a l l y mediated i n t e r a c t i o n s between h o s t and p e s t . N a t u r a l p r o d u c t s have p r o v i d e d l e a d s t o new p e s t i c i d e s . Knowledge o f t h e b i o s y n t h e t i c p r o c e s s e s o f n a t u r a l p r o d u c t s and

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Use of Naturd Products in Pest Control

r e l a t e d i n f o r m a t i o n has o f t e n l e d t o an u n d e r s t a n d i n g o f t h e mode o f a c t i o n . A knowledge o f t h e r e q u i r e d f u n c t i o n a l groups has l e d t o t h e d e s i g n o f s y n t h e t i c p e s t i c i d e s w i t h h i g h a c t i v i t y and specificity. N a t u r a l p r o d u c t s may a l s o f i n d t h e i r p l a c e i n t h e market p l a c e . E c o n o m i c a l l y f e a s i b l e b i o r e g u l a t o r s may be produced by m i c r o b i o l o g i c a l p r o c e s s e s such as f e r m e n t a t i o n . I n o t h e r i n s t a n c e s , p r o c e s s i n g o f p l a n t m a t e r i a l may p r o v i d e c o m m e r c i a l l y adequate y i e l d s . F o r o t h e r a p p l i c a t i o n s such as t h e p r o d u c t i o n o f pheromones, hormones, o r p e p t i d e s , s y n t h e s i s o f t h e n a t u r a l p r o d u c t s may be t h e b e s t s t r a t e g y . Another a p p l i c a t i o n o f n a t u r a l p r o d u c t s t o p e s t c o n t r o l may be t h r o u g h t h e i n s e r t i o n o f genes i n t o p l a n t s e x p r e s s i n g t h e compound. T h i s approach i s j u s t now r e c e i v i n g i n c r e a s e d emphasis. When a b i o l o g i c a l l y a c t i v e n a t u r a l p r o d u c t i s i d e n t i f i e d , o f t e n more i s g a i n e d than knowledge about t h e s p e c i f i c s t r u c t u r e . An u n d e r s t a n d i n g o f how t h e n a t i o n a l p r o d u c t i s b i o s y n t h e s i z e d and by which mechanism(s) i t e x p r e s s e s i t s a c t i v i t y s h o u l d make i t p o s s i b l e t o d e v i s e a d d i t i o n a l minimal changes t h a t y i e l d maximum b e n e f i t s . Thus, those changes t h a t b e n e f i t people may be s e l e c t e d which do minimum o r no damage t o t h e ecosystem. Literature Cited 1.

Beroza, M. Pest Management with Insect Sex Attractants; ACS Symposium Series No. 23; American Chemical Society: Washington, DC, 1976; 192 pp. 2. Inscoe, M. N. In Insect Suppression with Controlled Release Pheromone Systems; Kydonieus, A. E., Beroza, M., Eds.; CRC Press: New York, Vol. I I , p. 201-295. 3. Leonhardt, Β. Α.; Beroza, M. Insect Pheromone Technology: Chemistry and Application; ACS Symposium Series No. 190, American Chemical Society: Washington, DC, 1982; 260 pp. 4. Hedin, P. Α.; Menn, J . J . ; Editors, Insect Chemical Communication: Unifying Concepts; Special Issue, J . Chemical Ecology 1988, 14, 1979-2145. 5. Carlson, D. A. Symposium on Biosynthesis and Catabolism of Insect Pheromones and Hormones; Abstracts of Papers, 199th National Meeting of the American Chemical Society: Boston, MA, A p r i l 1990, AGRO 36-40, 55-60, 74-79. 6. Wallace, J . W.; Mansell, R. L. Biochemical Interactions Between Plants and Insects; Recent Advances i n Phytochemistry, Vol. 10, Plenum Press, Ν. Υ., 1976; 425 pp. 7. Hedin, P. A. Host Plant Resistance to Pests; ACS Symposium Series No. 62; American Chemical Society: Washington, DC, 1977; 286 pp. 8. Rosenthal, G. Α., Janzen, D. H. Herbivores: Their Interaction with Secondary Plant Metabolites; Academic Press: New York, 1979; 718 pp. 9. Hedin, P. A. Plant Resistance to Insects; ACS Symposium Series No. 208; American Chemical Society: Washington, DC, 1983; 375 pp. 10. Hedin, P. A. Bioregulators f o r Pest Control; ACS Symposium Series No. 276; American Chemical Society: Washington, DC, 1985; 540 pp.

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NATURALLY OCCURRING PEST BIOREGULATORS 11. Green, M. B.; Hedin, P. A. Natural Resistance to Pest: Roles of Allelochemics; ACS Symposium Series No. 296; American Chemical Society: Washington, DC, 1986; 243 pp. 12. Cutler, H. G. B i o l o g i c a l l y Active Natural Products: Potential Uses i n Agriculture; ACS Symposium Series No. 380; American Chemical Society: Washington, DC, 1988; 483 pp. 13. Morgan, E. D.; Mandava, Ν. B. Handbook of Natural Pesticides: Vol. VI, Insect Attractants and Repellents; CRC Press: Boca Raton, FL.; 249 pp. 14. Kubo, I.; Nakanishi, K. In Host Plant Resistance to Pests; Hedin, P. Α., Ed.; ACS Symposium Series No. 62; American Chemical Society: Washington, DC, 1977; pp 165-178. 15. Mabry, T. J . ; Burnett, W. C.; Jones, S. B.; Gill, J . E. In Host Plant Resistance to Pests; Hedin, P. Α., Ed., ACS Symposium Series No. 62; American Chemical Society: Washington, DC, 1977; pp. 179-184. 16. Elliott, M. Synthetic Pyrethroids; ACS Symposium Series No. 42; American Chemical Society: Washington, DC, 1977; 229 pp. 17. G i l b e r t , L. I. The Juvenile Hormones; Plenum Press: New York, 1976; 572 pp. 18. Osburn, Β. I. Symposium on Natural Antimicrobial Peptides and t h e i r Application i n Agriculture; Abstracts of Papers, 197th National Meeting, American Chemical Society: Dallas, TX, A p r i l 1989, AGRO 13-17, 30-33. 19. Menn, J.J.; Kelly, T. J . ; Masler, E. P.; Edwards, J . V.; Cherry, J . P; Eto, M., Hammock, B. D.; Okai, H. Symposium on B i o l o g i c a l l y Active Peptides i n Insects, Plants, and Food; Abstracts of Papers, 197th International Chemical Congress of P a c i f i c Basin Societies: Honolulu, HI, December 1989, AGRO 43-47, 82-88, 105-110, 133-138, 140-145, 382-390, 398-404. 20. Cross, B.; Cody, S. Symposium on Formulation of Proteins f o r A g r i c u l t u r a l Applications; Abstracts of Papers, 200th National Meeting, American Chemical Society: Washington, DC, August 1990, AGRO 5-8, 14-18, 40-44. 21. Hedin, P. Α.; Menn, J . J . ; Hollingworth, R. M. Biotechnology for Crop Protection; ACS Symposium Series No. 379; American Chemical Society: Washington, DC, 1988, 471 pp. 22. Keen, N. T.; Bruegger, B. In "Host Plant Resistance to Pests"; Hedin, P. Α., Ed.; ACS Symposium Series No. 62; American Chemical Society: Washington, DC, 1977; pp 1-26. 23. Hedin, P. Α.; Jenkins, J . N.,; Thompson, A. C.; McCarty, J . C.; Smith, D. H.; Parrott, W. L.; Shepherd, R. L. J . Agric. Food Chem. 1988, 36, 1055-1061. 24. Carpenter, T. L; Heitz, J . R. "Abstracts of Papers", 178th National Meeting, American Chemical Society: Washington, DC, Sept. 1979; PEST 34. 25. Berenbaum, M. Science (Washington, DC): 1978, 201, 532-533. 26. Heitz, J . R.; Downum, K. R. Light Activated Pesticides; ACS Symposium Series No. 339; American Chemical Society; Washington, DC, 1987; 355 pp. 27. Towers, G. Η. N.; Kubo, I.; Tang, C. S.; Mizutani, J . Symposium on Chemical Aspects of Plant-Microogaranism, PlantInsect, and Plant-Plant Interactions; Abstracts of Papers,

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28.

29.

30.

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31.

32.

33. 34.

35.

Use of Natural Products in Pest Control

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1989 International Chemical Congress of P a c i f i c Basin Societies: Honolulu, HI, December 1989, AGRO 164-171, 195-202. Thompson, A. C. The Chemistry of Allelopathy: Biochemical Interactions Among Plants; ACS Symposium Series No. 268; American Chemical Society: Washington, DC, 1985; 471 pp. Waller, G. R. Allelochemicals: Role i n Agriculture and Forestry; ACS Symposium Series No. 330; American Chemical Society: Washington, DC, 606 pp. Mandava, Ν. B. In "Plant Growth Substances"; Mandava, Ν. B., Ed.; ACS Symposium Series No. 111; American Chemical Society: Washington, DC, 1979; pp 135-213. Cutler, H. G. Symposium on Brassinosteroids; Abstracts of Papers, 200th National Meeeting, American Chemical Society: Washington, DC, August 1990, AGRO 76-79, 100-102, 119-122, 131-133. Jacobson, M. In Host Plant Resistance to Pests; Hedin, P. Α., Ed.; ACS Symposium Series No. 62; American Chemical Society: Washington, DC, 1977; pp 153-164. Strobel, G.; Myers, D. F. Science News (Washington, DC): 1980, 117, 362. B e l l , Α. Α.; Mace, M. E.; Stipanovic, R. D. In Natural Resistance of Plants to Pests: Roles of Allelochemicals; Green, M. B, Hedin, P. Α., Eds.; ACS Symposium Series No. 296; American Chemical Society; Washington, DC, pp 36-54. Keen, N. T.; Yoshikawa, M.; Want, M. C. Plant Physiol. 1983, 71, 466-471.

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17, 1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 2

Arthropod Natural Products as Insect Repellents Murray S. Blum , Daniel M. Everett , Tappey H. Jones , and Henry M. Fales 1

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Department of Entomology and Department of Computer Science, University of Georgia, Athens, GA 30602 Laboratory of Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 1

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The defensive allomones of arthropods have been evolved to blunt the attacks of a variety of predatory species. These natural products function primarily by repelling adversaries and thus usually constitute the first line of defense. An examination of the deterrent efficacies of compounds produced by honey bees, thrips, and ants demonstrates that these exocrine products are highly effective repellents against a diversity of ant species. A potpourri of natural products including aliphatic and aromatic ketones, esters, fatty acids, and alkaloids has been determined to possess well-developed repellent properties at physiological concentrations. These results emphasize the great potential of insect-derived compounds as an outstanding source of repellents in the never-ending battle with species of pest arthropods.

Among a n i m a l s , a r t h r o p o d s a r e d i s t i n g u i s h e d by t h e i r u t t e r dominance i n terms o f b o t h numbers o f s p e c i e s and i n d i v i d u a l s . The v i r t u a l u b i q u i t y o f t h e s e populous organisms guarantees t h a t t h e y w i l l be s u b j e c t t o g r e a t p r e d a t o r y p r e s s u r e from t h e i n v e r t e b r a t e s and v e r t e b r a t e s w i t h which t h e y s h a r e t h e i r f r a g i l e w o r l d . N o t s u r p r i s i n g l y , a r t h r o p o d s themselves c o n s t i t u t e a major group o f p r e d a t o r s , and among t h e s e , i t i s n o t u n l i k e l y t h a t a n t s a r e dominant. Indeed, a n t s a r e p r o b a b l y t h e major p r e d a t o r y a n i m a l s i n t h e w o r l d , w i t h most o f t h e i r 10,000-15,000 s p e c i e s (1) e x h i b i t i n g a c a r n i v o r o u s p r o p e n s i t y combined w i t h an e f f i c i e n t system o f p r e y a c q u i s i t i o n ( 2 ) . F o r most a r t h r o p o d s , a n t s p r o b a b l y r e p r e s e n t t h e most f r e q u e n t l y encountered p r e d a t o r s w i t h which t h e y must contend. Defense a g a i n s t a n t s and t h e l e g i o n s o f o t h e r p r e d a t o r y a n i m a l s thus becomes a s i n e qua non f o r t h e s u r v i v a l o f a r t h r o p o d o u s species i n a v a r i e t y of ecological contexts. 0097-6156/91/0449-0014$06.00/0 © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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B L U M ET AL.

Arthropod Natural Products as Insect Repellents

A r t h r o p o d s s y n t h e s i z e an i n c r e d i b l e d i v e r s i t y o f n a t u r a l p r o d u c t s i n t h e i r e x o c r i n e g l a n d s (3) w h i c h a r e u t i l i z e d w i t h g r e a t e f f e c t i v e n e s s t o b l u n t t h e a t t a c k s of t h e i r a d v e r s a r i e s . The d e f e n s i v e s e c r e t i o n s o f t h e s e i n v e r t e b r a t e s can o f t e n be d e l i v e r e d w i t h great accuracy, thus ensuring t h a t aggressive p r e d a t o r s a r e s u b j e c t e d t o t h e f u l l impact o f t h e s e s e c r e t o r y e f f r o n t e r i e s ( 4 ) . S i g n i f i c a n t l y , t h e s e exudates u s u a l l y m a n i f e s t t h e i r d e f e n s i v e e f f i c a c y as e f f e c t i v e r e p e l l e n t s and t h u s c o n s t i t u t e the f i r s t l i n e o f defense o f t h e p r e y s p e c i e s . The d e t e r r e n t v a l u e o f t h e n a t u r a l p r o d u c t s i n t h e s e s e c r e t i o n s i s i n e s t i m a b l e , s i n c e i t can p e r m i t t h e i r p r o d u c e r s t o escape predators without i n j u r i o u s p h y s i c a l confrontations. This i s e s p e c i a l l y important i n encounters w i t h ants, s i n c e these s o c i a l i n s e c t s can q u i c k l y l a u n c h d e v a s t a t i n g en masse a t t a c k s . A v a r i e t y o f s t u d i e s has demonstrated t h a t a n t s may be r a p i d l y d e t e r r e d by t h e d e f e n s i v e s e c r e t i o n s of d i v e r s e a r t h r o p o d s on w h i c h t h e y attempt t o p r e y ( 5 , 6 ) . For example, many o f t h e s m a l l and d e l i c a t e s p e c i e s o f t h r i p s ( T h y s a n o p t e r a ) produce a n a l s e c r e t i o n s t h a t a r e d i r e c t e d a g a i n s t t h e a n t s w i t h w h i c h t h e y f r e q u e n t l y have e n c o u n t e r s (7). Although the d e f e n s i v e s e c r e t i o n s o f v e r y few t h r i p s s p e c i e s have been a n a l y z e d , e v a l u a t i o n o f t h e compounds p r e s e n t i n a few o f t h e s e exudates i n d i c a t e s t h a t t h e y a r e e f f e c t i v e d e t e r r e n t s f o r a n t s ( 8 , 9 ) . These r e s u l t s f u r t h e r suggest t h a t r e p e l l e n t s f o r a n t s may be commonly e n c o u n t e r e d i n t h e d e f e n s i v e s e c r e t i o n s o f a v a r i e t y o f a r t h r o p o d o u s s p e c i e s . F u r t h e r m o r e , s i n c e a n t s have frequent a n t a g o n i s t i c i n t e r a c t i o n s w i t h other species of ants, i t c o u l d be a n t i c i p a t e d a p r i o r i t h a t t h e s e f o r m i c i d s would have e v o l v e d p o w e r f u l ant d e t e r r e n t s t h e m s e l v e s . B o t h f i e l d and l a b o r a t o r y s t u d i e s have documented t h e d e t e r r e n t e f f i c a c i e s t o a n t s o f the venomous s e c r e t i o n s o f t h e s e i n s e c t s (10, Γ1, _12). For example, i t has been demonstrated t h a t t h e r a i d i n g modus v i v e n d i of one ant s p e c i e s was made p o s s i b l e by the u t i l i z a t i o n of a venom-derived a l k a l o i d t h a t i s a p o w e r f u l r e p e l l e n t f o r workers o f r a i d e d ant s p e c i e s ( 1 3 ) . A l t h o u g h a n t s s y n t h e s i z e an i n c r e d i b l e d i v e r s i t y o f venomous a l k a l o i d s ( 1 4 ) , t h e i r a c t i v i t i e s as r e p e l l e n t s f o r t h e s e f o r m i c i d s have o n l y been d e s c r i b e d p r e l i m i n a r i l y ( 1 5 ) . S i g n i f i c a n t l y , a n t s appear t o be t y p i c a l o f s o c i a l i n s e c t s i n g e n e r a t i n g d e t e r r e n t compounds i n t h e i r e x o c r i n e g l a n d s , and examples of o t h e r hymenopterans p r o d u c i n g r e p e l l e n t s , o f g r e a t s o c i a l s i g n i f i c a n c e , have r e c e n t l y been r e p o r t e d ( 1 6 ) . Honey bee queens produce a r e c t a l s e c r e t i o n w h i c h r e p e l s a g g r e s s i v e workers i n t h e c o l o n y as an example o f an i n t r a s p e c i f i c r e p e l l e n t ( 1 6 ) . I f such pheromonal r e p e l l e n t s a r e commonly produced by s o c i a l i n s e c t s , a c o r n u c o p i a o f d e t e r r e n t n a t u r a l p r o d u c t s a w a i t s i d e n t i f i c a t i o n i n the exocrine s e c r e t i o n s of these arthropods. I n the p r e s e n t r e p o r t , we p r e s e n t r e s u l t s o f v e r y r e c e n t i n v e s t i g a t i o n s on t h e c h e m i s t r y and d e t e r r e n t a c t i v i t i e s t o a n t s o f the d i v e r s e compounds i d e n t i f i e d as e x o c r i n e compounds o f t h r i p s . I n a d d i t i o n , we e v a l u a t e the r e p e l l e n c y o f some honey bee n a t u r a l p r o d u c t s t o honey bee workers as an example o f how pheromones themselves can be c a n d i d a t e s as d e t e r r e n t s

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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f o r t h e s p e c i e s t h a t produce them. F i n a l l y , d e t a i l e d a n a l y s e s o f the r e p e l l e n c i e s of a host of n i t r o g e n h e t e r o c y c l e s i d e n t i f i e d i n t h e venoms o f a n t s a r e d e s c r i b e d .

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C h e m i s t r y and R e p e l l e n c y o f T h r i p s N a t u r a l

Products

The f i r s t compound i d e n t i f i e d as a n a t u r a l p r o d u c t o f t h r i p s was V - d e c a l a c t o n e ( I ) , a p r o d u c t o f B a g n a l l i e l l a yuccae ( 8 ) . The a n a l e x u d a t e , w h i c h i s d i s c h a r g e d when t h e t h r i p s a r e d i s t u r b e d , does not c o n t a i n any o t h e r d e t e c t a b l e v o l a t i l e s . B o t h l a b o r a t o r y and f i e l d s t u d i e s demonstrated t h a t t h e s e c r e t i o n e f f e c t i v e l y d e t e r r e d a n t w o r k e r s e i t h e r as a t o p i c a l i r r i t a n t o r by r e p e l l e n c y p e r s e . Workers o f Monomorium minimum q u i c k l y withdrew a f t e r c o n t a c t w i t h t h e a n a l f l u i d and a v o i d e d s i t e s a t w h i c h t h e exudate had been r e l e a s e d ( 8 ) . The r e p e l l e n c y o f t h e a n a l exudate was c l e a r l y i d e n t i f i e d w i t h T - d e c a l a c t o n e , two t h r i p s e q u i v a l e n t o f t h i s compound e v o k i n g 50% r e p e l l e n c y f o r minimum w o r k e r s . S i m i l a r r e s u l t s were o b t a i n e d w i t h p h a r a o n i s and h u m i l i s , two o t h e r s p e c i e s o f p r e d a t o r y a n t s . The r e s u l t s o f f i e l d s t u d i e s corroborated the laboratory findings ( 8 ) .

7-Decalactone

The c h e m i s t r y and r e p e l l e n t e f f i c a c y o f t h e a n a l exudate o f t h e Cuban l a u r e l t h r i p s , G y n a i k o t h r i p s f i c o r u m , a g a l l - i n h a b i t i n g s p e c i e s , have a l s o been examined ( 9 ) . The exudate i s dominated by a 1:1 r a t i o o f h e x a d e c y l a c e t a t e and pentadecane; t e t r a d e c y l a c e t a t e , t r i d e c a n e , t e t r a d e c a n e , and heptadecane c o n s t i t u t e minor concomitants. The a n a l s e c r e t i o n o f Gj_ f i c o r u m i s b o t h an e f f e c t i v e c o n t a c t d e t e r r e n t and r e p e l l e n t f o r a g g r e s s i v e a n t s . Workers o f Wasmannia a u r o p u n c t a t a a r e r a p i d l y r e p e l l e d by an a n a l d r o p l e t from a t h r i p s t h a t i s w i t h i n a m i l l i m e t e r o f t h e a n t s (9). T o p i c a l t r e a t m e n t o f a n t s w i t h a n a l exudate c o r r e s p o n d i n g t o 0.25 t h r i p s e q u i v a l e n t s r e s u l t e d i n 100% o f t h e workers d r a g g i n g t h e m s e l v e s away from t h e scene o f t h e encounter i n much t h e same manner as o c c u r s when t h e s e a n t s c o n t a c t t h e t h r i p s under f i e l d c o n d i t i o n s . B o t h h e x a d e c y l a c e t a t e and pentadecane caused dragging behavior a f t e r being a p p l i e d t o ant workers, which responded i n a dose-dependent manner. S i g n i f i c a n t l y , a l t h o u g h h e x a d e c y l a c e t a t e i s more a c t i v e than pentadecane, a c o m b i n a t i o n o f t h e two compounds i s c o n s i d e r a b l y more e f f e c t i v e t h a n t h e e q u i v a l e n t amounts o f e i t h e r o f t h e two compounds a l o n e . T h e r e f o r e , i t i s e v i d e n t t h a t t h e e s t e r and h y d r o c a r b o n i n t e r a c t

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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2. B L U M ET A L

Arthropod Natural Products as Insect Repellents

s y n e r g i s t i c a l l y t o augment t h e d e t e r r e n t e f f i c a c y o f t h e exudate (2). R e c e n t l y , t h e c h e m i s t r y o f t o t a l e x t r a c t s o f another s p e c i e s o f G y n a i k o t h r i p s was examined ( Γ7 ). G. u z e l i i s r e p o r t e d t o produce t h e same h y d r o c a r b o n s and a c e t a t e s as G. f i c o r u m , b u t i n a d d i t i o n s y n t h e s i z e s a n o v e l monoterpene, β-acaridial, a compound r e c e n t l y i d e n t i f i e d as a n a t u r a l p r o d u c t o f a m i t e (18). βA c a r i d i a l has a l s o been i d e n t i f i e d as an i m p o r t a n t compound produced by two o t h e r s p e c i e s o f t h r i p s , V a r s h n e y i a p a s a n i i and L i o t h r i p s kuwanai (Γ7). The f u n c t i o n o f β-acaridial, w h i c h i s v e r y u n s t a b l e , i s n o t known. Another monoterpene, p e r i l l e n e , has been i d e n t i f i e d as a p r o d u c t o f t h r i p s i n s e v e r a l genera. T h i s furanomonoterpene accompanies β-acaridial i n e x t r a c t s o f V. p a s a n i i , L. kuwanai, and L. p i p e r i n u s (19, 2 0 ) . P e r i l l e n e has a l s o been d e t e c t e d as a major p r o d u c t i n e x t r a c t s o f T e u c h o t h r i p s l o n g u s , A r r h e n o t h r i p s ramakrishnae, and S c h e d o t h r i p s s p . ( 2 1 ) . Vapors o f t h i s compound produced a dose-dependent r e p e l l e n c y i n workers o f two a n t s p e c i e s , Monomorium c a r b o n a r i u m and Iridomyrmex h u m i l i s , under l a b o r a t o r y c o n d i t i o n s . Thus, p e r i l l e n e f u n c t i o n s as a r e p e l l e n t and i t s p r e s e n c e i n t h e a n a l exudates o f d i v e r s e t h r i p s s p e c i e s c l e a r l y augments t h e d e t e r r e n t e f f i c a c i e s o f t h e s e d i s c h a r g e s . On t h e o t h e r hand, i t has been suggested t h a t h i g h dosages o f p e r i l l e n e (20-200 Mg) can f u n c t i o n as an a l a r m pheromone ( 1 7 ) . A t h i r d monoterpene, r o s e f u r a n , has been i d e n t i f i e d as t h e major c o n s t i t u e n t produced by t h e t h r i p s A r r h e n o t h r i p s r a m a k r i s h n a e (21^). Rose f u r a n i s accompanied by p e r i l l e n e and i n a d d i t i o n , hexadecyl acetate i s q u a n t i t a t i v e l y s i g n i f i c a n t . Two a r o m a t i c compounds, p h e n o l and p h e n y l a c e t a l d e h y d e , a r e minor c o n c o m i t a n t s i n t h e a n a l exudate.

CHO

Rose Furan

0-Acaridial

Perillene

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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F a t t y acids are a l s o c h a r a c t e r i s t i c n a t u r a l products of s p e c i e s o f t h r i p s i n a d i v e r s i t y o f genera. A t o t a l e x t r a c t o f H o p l o t h r i p s j a p o n i c u s was dominated by ( E ) - 3 - d o d e c e n o i c a c i d w h i c h i s c o n s i d e r e d t o be an a l a r m - a g g r e g a t i o n pheromone; ( Z ) - 5 d o d e c e n o i c a c i d i s a minor c o n c o m i t a n t ( 2 2 ) . 2-Methylbutyric a c i d , a d e f e n s i v e allomone o f s w a l l o w t a i l l a r v a e ( 2 3 ) , i s the o n l y f r e e f a t t y a c i d d e t e c t e d i n the p r e v i o u s l y d e s c r i b e d a n a l exudate of Varshneyia p a s a n i i (20). I t i s b e l i e v e d t h a t the a n a l d i s c h a r g e o f t h i s s p e c i e s , t h o r o u g h l y dominated by a l k a n e s , a c e t a t e s , and oxygenated monoterpenes, e x h i b i t s an i n c r e a s e d r e p e l l e n t "punch" because o f t h e p r e s e n c e o f the C5 a c i d . The a n a l f l u i d o f a D i n o t h r i p s sp. c o n t a i n s o n l y i s o v a l e r i c and d e c a n o i c a c i d s i n e q u a l q u a n t i t i e s ( 2 4 ) . A c o m b i n a t i o n o f t h e a c i d s i s h i g h l y r e p e l l e n t t o workers of t h e f i r e ant S o l e n o p s i s i n v i c t a . Decanoic a c i d i s t h e major allomone p r e s e n t i n the a n a l f l u i d o f E u r y a p l o t h r i p s c r a s s u s and i s accompanied by dodecanoic a c i d as a q u a n t i t a t i v e l y i m p o r t a n t p r o d u c t ( 2 1 ) . The exudate o f E. c r a s s u s a l s o c o n t a i n s 2 - p h e n y l a c e t a l d e h y d e , p h e n o l , and 4o c t a d e c - 9 - e n o l i d e as minor c o n s t i t u e n t s . Dodecanoic a c i d has a l s o been i d e n t i f i e d as a major compound i n t h e a n a l exudate o f Elaphrοthrips t u b e r c u l a t u s , and i t i s accompanied by two o t h e r a c i d s , ( Z ) - 5 - t e t r a d e c e n o i c a c i d and 5,8t e t r a d e c a d i e n o i c a c i d ( 2 5 ) . On t h e o t h e r hand, a n o v e l a n i m a l n a t u r a l p r o d u c t , j u g l o n e , p r o v i d e s t h i s exudate w i t h a p a r t i c u l a r l y d i s t i n c t i v e exocrine chemistry. Juglone, which i s s y n t h e s i z e d de novo by E. t u b e r c u l a t u s , i s an o u t s t a n d i n g r e p e l l e n t f o r ant s p e c i e s i n s e v e r a l genera. The a l l e l o p a t h i c e f f e c t s of j u g l o n e , a product of black walnut (Juglans n i g r a ) , are w e l l e s t a b l i s h e d ( 2 6 ) , and i t i s r e a l l y not s u r p r i s i n g t h a t t h i s h i g h l y p h y t o t o x i c and r e a c t i v e quinone s h o u l d e x h i b i t c o n s i d e r a b l e r e p e l l e n t a c t i v i t y f o r a n t s as a consequence of a s s a u l t i n g t h e i r chemoreceptors.

ο

ο

HO

Juglone

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

2.

BLUM E T A L

Arthropod Natural Products as Insect Repellents

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Honey Bee Pheromones as R e p e l l e n t s Some pheromones o f s o c i a l i n s e c t s w h i c h f u n c t i o n as e x c i t a n t s and a t t r a c t a n t s a t low ( = p h y s i o l o g i c a l ) c o n c e n t r a t i o n s a r e p o w e r f u l r e p e l l e n t s when p r e s e n t a t super t h r e s h o l d c o n c e n t r a t i o n s ( 2 7 ) . The a b i l i t y o f t h e s e i n s e c t s t o be d e t e r r e d by h i g h l e v e l s o f t h e i r own pheromones can p r o v i d e a means o f d e t e c t i n g new r e p e l l e n t s f o r e u s o c i a l a r t h r o p o d s . Beyond t h i s c o n s i d e r a t i o n , i t i s p o s s i b l e t h a t t h e s e "pheromonal r e p e l l e n t s " might s e r v e as r e p e l l e n t s f o r a wide range o f i n s e c t s . Honey bee workers u t i l i z e a mandibular g l a n d pheromone, 2heptanone, as a low l e v e l a l a r m pheromone a t t h e h i v e e n t r a n c e ( 2 8 ) . T h i s compound, w h i c h i s a r e p e l l e n t f o r f o r a g i n g worker bees ( 2 9 ) , produces abnormal b e h a v i o r a l r e a c t i o n s when workers a r e exposed t o h i g h c o n c e n t r a t i o n s o f i t ( 3 0 ) , i n much t h e same way as i s observed w i t h 2-heptanone-producing a n t s ( 3 1 ) . S i g n i f i c a n t l y , when t h i s methyl ketone i s a p p l i e d t o t h e hands as a 0.5, 1.0, o r 2% a e r o s o l s o l u t i o n , i t i s p o s s i b l e t o m a n i p u l a t e t h e bees i n a h i v e w i t h o u t them r e a c t i n g a g g r e s s i v e l y . Workers a r e r e p e l l e d by t h e k e t o n i c super t h r e s h o l d c o n c e n t r a t i o n and r e t r e a t from i t s s o u r c e i n a n o n a g i t a t e d s t a t e . The h i g h c o n c e n t r a t i o n o f t h i s pheromone thus e f f e c t i v e l y disarms t h e workers and p r o v i d e s a r e p e l l e n t " s h i e l d " f o r t h e pheromone e m i s s i o n s o u r c e . S i n c e some cockroaches u t i l i z e 2-heptanone as a d e f e n s i v e allomone ( 3 2 ) , i t i s p o s s i b l e t h a t t h i s compound may be r e p e l l e n t t o a broad spectrum o f i n s e c t s p e c i e s . Young honey bee queens produce a r e p e l l e n t pheromone t h a t e f f e c t i v e l y t r a n q u i l i z e s workers t h a t may i n t e r a c t a n t a g o n i s t i c a l l y w i t h t h e s e v i r g i n females ( 1 6 ) . The a c t i v e compound, o-aminoacetophenone, i s a minor component i n t h e a n a l exudate t h a t i s d i s c h a r g e d by t h e molested queens ( 3 3 ) . T h i s compound i s a l s o a d e f e n s i v e allomone o f an a n t s p e c i e s ( 3 4 ) , r a i s i n g t h e p o s s i b i l i t y t h a t i t may possess g e n e r a l d e t e r r e n t a c t i v i t y f o r arthropods.

o-Aminoacetophenone

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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NATURALLY OCCURRING PEST BIOREGULATORS

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The R e p e l l e n c y of Venomous A l k a l o i d s o f A n t s A n t s i n t h e myrmicine genera S o l e n o p s i s and Monomorium a r e d i s t i n c t i v e i n p r o d u c i n g venoms t h a t a r e dominated by a l k a l o i d s r a t h e r than p r o t e i n s ( 3 5 ) . F i r e a n t s ( S o l e n o p s i s spp.) i n t h e subgenus S o l e n o p s i s c h a r a c t e r i s t i c a l l y s y n t h e s i z e p o i s o n g l a n d s e c r e t i o n s t h a t a r e dominated by t h e c i s - and t r a n s - i s o m e r s o f 6a l k y l - o r 6 - a l k y l i d e n e - 2 - m e t h y l p i p e r i d i n e s ( 3 6 ) . On t h e o t h e r hand, s p e c i e s i n t h e subgenus D i p l o r h o p t r u m f r e q u e n t l y produce 2 , 5 - d i a l k y l p y r r o l i d i n e s as venomous c o n s t i t u e n t s ( 3 7 ) , as w e l l as 3 , 5 - d i a l k y l p y r r o l i z i d i n e s ( 3 8 ) , and 3 , 5 - d i a l k y l i n d o l i z i d i n e s ( 3 9 ) . The venoms o f Monomorium s p e c i e s t y p i c a l l y c o n t a i n 2,5d i a l k y l p y r r o l i d i n e s t h a t a r e f r e q u e n t l y accompanied by 3,5d i a l k y l p y r r o l i z i d i n e s and 3 , 5 - d i a l k y l i n d o l i z i d i n e s (AO, 41, 4 2 ) . With the exception of the d i a l k y l p i p e r i d i n e s (reviewed i n 14), v i r t u a l l y n o t h i n g i s known about t h e b i o l o g i c a l a c t i v i t i e s o f t h e o t h e r c l a s s e s o f venomous n i t r o g e n h e t e r o c y c l e s . In view of the r e p o r t e d d e t e r r e n t a c t i v i t i e s of Solenopsis and Monomorium venoms under f i e l d c o n d i t i o n s (10, 11, 1 2 ) , i t seemed w o r t h w h i l e t o e v a l u a t e t h e comparative r e p e l l e n c i e s t o a n t s o f some o f t h e s e venom-derived a l k a l o i d s . I n t h i s i n v e s t i g a t i o n , t h e d e t e r r e n c y of t h e s e n i t r o g e n h e t e r o c y c l e s t o a v a r i e t y o f ant s p e c i e s was determined by u s i n g a f e e d i n g b i o a s s a y i n w h i c h t h e r e a c t i o n s o f hungry ant w o r k e r s t o a l k a l o i d - t r e a t e d f o o d were q u a n t i f i e d ( 4 3 ) . S e l e c t i o n o f a v a r i e t y o f a g g r e s s i v e ant s p e c i e s i n c o m b i n a t i o n w i t h a d i v e r s i t y of c a n d i d a t e compounds, b e l o n g i n g t o t h e main c l a s s e s o f venomous a l k a l o i d s , p r e s e n t e d an o p p o r t u n i t y t o examine t h e s e f o r m i c i d n a t u r a l p r o d u c t s i n terms o f t h e i r a c t i v i t i e s as r e p e l l e n t s . Queenright ( q u e e n - c o n t a i n i n g ) c o l o n i e s of 10 s p e c i e s of a n t s , b e l o n g i n g t o two major s u b f a m i l i e s , were u t i l i z e d f o r r e p e l l e n c y s t u d i e s . Members o f t h e s u b f a m i l y M y r m i c i n a e i n c l u d e d S o l e n o p s i s i n v i c t a , Crematogaster ashmeadi, P h e i d o l e d e n t a t a , Monomorium minimum, M. v i r i d u m and M^ p h a r a o n i s . The s u b f a m i l y D o l i c h o d e r i n a e was r e p r e s e n t e d by Iridomyrmex p r u i n o s u s , I . h u m i l i s , Tapinoma s e s s i l e , and T^ melanocephalum. Whereas S. i n v i c t a and t h e Monomorium s p e c i e s have been demonstrated t o s y n t h e s i z e a l k a l o i d - r i c h venoms (36, 40, 41_), t h e s e n i t r o g e n h e t e r o c y c l e s have n o t been d e t e c t e d as p o i s o n g l a n d p r o d u c t s o f any o f t h e o t h e r myrmicine genera ( 4 4 ) . S i n c e we have f r e q u e n t l y o b s e r v e d workers s e c r e t i n g venom d u r i n g c o m p e t i t i v e i n t e r a c t i o n s between M^ p h a r a o n i s , M. v i r i d u m , and T^_ melanocephalum i n s o u t h e r n F l o r i d a , and t h e o t h e r seven s p e c i e s i n n o r t h e r n G e o r g i a , t h e s e s p e c i e s seemed p a r t i c u l a r l y a p p r o p r i a t e f o r e v a l u a t i n g t h e p o t e n c i e s o f t h e s e a l k a l o i d s as ant r e p e l l e n t s . B o t h f o r a g i n g and f e e d i n g were s t i m u l a t e d by not p r o v i d i n g t h e ant c o l o n i e s w i t h f o o d f o r 48 hours. Food was then o f f e r e d t o t h e a n t s as d r o p l e t s o f honey t o w h i c h were added 1 o r 2 pg o f a l k a l o i d s i n 2 μΐ o f a b s o l u t e e t h a n o l . I n one s t u d y , b o t h t r e a t e d d r o p l e t s and c o l o n i e s were randomized f o r each r e p l i c a t e w h i c h compared t h e r e p e l l e n c i e s o f f o u r a l k a l o i d s t h a t i n c l u d e d a S o l e n o p s i s a l k a l o i d , t r a n s - 6 - u n d e c y l - 2 - m e t h y l p i p e r i d i n e ( 3 6 ) , two Monomorium d i a l k y l p y r r o l i d i n e s ( 4 1 ) , and a S o l e n o p s i s d i a l k y l p y r r o l i z i d i n e ( 3 8 ) . I n t h i s i n v e s t i g a t i o n , a l l s p e c i e s of

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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2.

BLUM ET A L

Arthropod Natural Products as Insect Repellents

a n t s were u t i l i z e d except p h a r a o n i s , T. melanocephalum, and I» h u m i l i s . A second r e p l i c a t e o f t h e measurements was performed 24 hours l a t e r , w i t h t h e same c o l o n i e s and b a i t s randomized i n a different pattern. I n a second s t u d y , t h e comparative d e t e r r e n c i e s o f e i g h t S o l e n o p s i s d i a l k y l p i p e r i d i n e s (36) and two S o l e n o p s i s d i a l k y l i n d o l i z i d i n e s (39) were compared. T e s t s p e c i e s i n c l u d e d two a l k a l o i d p r o d u c e r s , p h a r a o n i s and S^ i n v i c t a , and two s p e c i e s t h a t do not s y n t h e s i z e a l k a l o i d a l p o i s o n g l a n d s e c r e t i o n s , I . h u m i l i s and T^ melanocephalum (45, 4 6 ) . T h i s s t u d y , w h i c h was implemented t h e same way as d e s c r i b e d f o r t h e p r e v i o u s t e s t , was a l s o d e s i g n e d t o compare t h e d e t e r r e n t a c t i v i t i e s o f t h e c i s - and t r a n s - 2 , 6 - d i a l k y l p i p e r i d i n e s ( r e l a t i o n o f s u b s t i t u e n t s a t C-2 and C-6). I n c l u d e d were compounds i n w h i c h t h e 6 - a l k y l group was normal Cg, C^3, and C ^ ; t h e 6 - a l k y l i d e n e group was e i t h e r Z-4t r i d e c e n y l o r Z-6-pentadecenyl. Compounds a r e r e f e r r e d t o as c i s o r t r a n s i n c o m b i n a t i o n w i t h an a b b r e v i a t i o n f o r t h e c h a i n l e n g t h ( p l u s u n s a t u r a t i o n ) o f t h e group a t t a c h e d t o C-6. Thus, c i s - 6 n o n y l - 2 - m e t h y l p i p e r i d i n e ( I ) i s d e s i g n a t e d as c i s - C g and 2 - t r a n s 6-(4-tridecenyl)-2-methylpiperidine ( I I ) i s designated trans-C-^.^ ( F i g u r e 1 ) . Two d i a l k y l i n d o l i z i d i n e s , ( 5 Z , 9 Z ) - 3 - h e x y l - 5 m e t h y l i n d o l i z i d i n e (Hex Ind) and ( 5 Z , 9 Z ) - 3 - e t h y l - 5 m e t h y l i n d o l i z i d i n e ( E t I n d ) , were i n c l u d e d f o r comparison. Three r e p l i c a t e s o f t h e measurements were performed i n t h e s t u d y , u s i n g d i f f e r e n t random c o m b i n a t i o n s o f c o l o n i e s and t r e a t m e n t s , a t 7-10 day i n t e r v a l s .

CH

3

ι

H

trans-C

1 3 ; 1

(I)

F i g u r e 1. F o r t h e most p a r t , each o f t h e seven ant s p e c i e s e x h i b i t e d c h a r a c t e r i s t i c responses t o t h e f o u r a l k a l o i d s e v a l u a t e d i n t h e f i r s t s t u d y . For a l l s p e c i e s , a 2 pg dosage o f an a l k a l o i d was s i g n i f i c a n t l y more r e p e l l e n t t h a n 1 pg. However, workers o f f o u r o f t h e s p e c i e s , C^ ashmeadi, I . p r u i n o s u s , M. minimum, and M. v i r i d u m , were r e p e l l e d by each o f t h e a l k a l o i d s o f b o t h c o n c e n t r a t i o n s . Two of t h e compounds, 2 - ( l - h e x - 5 - e n y l ) - 5 n o n y l p y r r o l i d i n e ( I I I ) and 2 - ( l - h e x - 5 - e n y l ) - 5 - ( l - n o n - 8 e n y l ) p y r r o l i d i n e ( I V ) , were s i g n i f i c a n t l y more r e p e l l e n t than

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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( 5 Z , 8 E ) - 3 - h e p t y l - 5 - m e t h y l p y r r o l i z i d i n e (V) f o r a l l s p e c i e s ( F i g u r e 2 ) . The trans-C-Q p i p e r i d i n e ( V I ) was i n t e r m e d i a t e i n r e p e l l e n c y between the p y r r o l i d i n e s I I I and IV, and the p y r r o l i z i d i n e ( V ) , but was not s i g n i f i c a n t l y d i f f e r e n t from e i t h e r c l a s s of a l k a l o i d s (43).

The s p e c i e s t h a t do not produce a l k a l o i d - r i c h venoms--C. ashmeadi, T, s e s s i l e , I . p r u i n o s u s , and dentata--were s i g n i f i c a n t l y more r e p e l l e d by t h e s e n i t r o g e n h e t e r o c y c l e s than t h e t h r e e a l k a l o i d - s y n t h e s i z i n g s p e c i e s ( T a b l e 1 ) . These r e s u l t s suggest t h a t s p e c i e s t h a t g e n e r a t e venoms dominated by a l k a l o i d s may have a c o m p e t i t i v e advantage a g a i n s t s p e c i e s t h a t do not produce p o i s o n g l a n d s e c r e t i o n s c o n t a i n i n g t h e s e v o l a t i l e - - a n d r e p e l l e n t - - n a t u r a l products. Recent f i e l d s t u d i e s (47) s u p p o r t t h i s conclusion. I n t h e second s t u d y , w h i c h u t i l i z e d two s p e c i e s not known t o produce a l k a l o i d a l venoms--I. h u m i l i s and Ί\_ melanocephalum--and two a l k a l o i d - p r o d u c i n g s p e c i e s , M. p h a r a o n i s and S^ i n v i c t a , dosedependent r e s p o n s e s were o n l y o b s e r v e d f o r two o f the s p e c i e s w i t h s e l e c t e d d i a l k y l p i p e r i d i n e s ( 4 3 ) . The responses o f workers o f M. p h a r a o n i s t o the c i s - C p p i p e r i d i n e and the c i s - C ^ p i p e r i d i n e were c l e a r l y dose dependent, as was the response of workers of T. melanocephalum t o the c i s - C - ^ p i p e r i d i n e .

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991. 3

Pyrrolidine^ 46.1 ± 5.3 15.2 ± 3.3 27.6 ± 3.4

c

2-(1-hex-5-enyl)-5-nonylpyrrolidine. ^2-(l-hex-5-enyl)-5-(l-non-8-enyl)pyrrolidine. ( 5 Z , 8E ) - 3-hepty1-5-methylpyrrο1i ζ i d i n e . ^trans-6-undecy1-2-methylp i p e r i d i n e .

a

Pyrrolidine A l k a l o i d producers 46.8 ± 6.6 N o n a l k a l o i d p r o d u c e r s 15.5 ± 3.4 A l l species 28.0 ± 3.8

c

Pyrrolizidine Piperidine^ 60.5 ± 8.0 53.2 ± 8.0 36.4 ± 1.6 28.9 ± 5.3 46.0 ± 5.1 37.4 ± 4.7

Alkaloid

Table 1. Mean number of ant workers (as percent of controls) of alkaloid- and nonalkaloidproducing species feeding on baits treated with four ant-derived alkaloids.

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Workers o f b o t h melanocephalum and 1^ h u m i l i s , s p e c i e s t h a t do n o t produce a l k a l o i d a l venoms, were e s p e c i a l l y s e n s i t i v e t o t h e d e t e r r e n t e f f e c t s o f d i f f e r e n t a l k a l o i d s ; S^ i n v i c t a w o r k e r s were n o t r e p e l l e d by any o f t h e a l k a l o i d s ( T a b l e 2 ) . Workers o f p h a r a o n i s were n o t r e p e l l e d as e f f e c t i v e l y as t h o s e o f t h e two n o n a l k a l o i d - p r o d u c i n g s p e c i e s ( 4 4 ) . The r e p e l l e n c i e s o f t h e a l k a l o i d s f o r t h e s e n s i t i v e s p e c i e s were: c i s - C | 3 ι > c i s - C | 3 > cis-Cp > trans-C^3 > c i s - C i 5 . ^ > t r a n s - C j ^ . ^ > Hex I n d > t r a n s - C j 5 > c i s - C > E t I n d > EtOH ( s i g n i f i c a n t a t 5% level). These r a n k i n g s were o b t a i n e d u s i n g Duncan's M u l t i p l e Range T e s t on t h e numbers o f a n t s f e e d i n g a t each b a i t , e x p r e s s e d as p e r c e n t a g e s o f t h e a v e r a g e number o f a n t s from t h e i r c o l o n y f e e d i n g a t c o n t r o l b a i t s (see Table 1 ) . P a i r w i s e comparison o f t h e r e p e l l e n c i e s o f t h e c i s and t r a n s - i s o m e r s o f t h e 0 ^ 3 , C^5 and C^.^ d i a l k y l p i p e r i d i n e s demonstrated t h a t t h e c i s i s o m e r s o f t h e C^3 and C^.^ a l k a l o i d s a r e more r e p e l l e n t t h a n t h e i r t r a n s c o u n t e r p a r t s . On t h e o t h e r hand, no s i g n i f i c a n t d i f f e r e n c e s i n t h e d e t e r r e n c i e s o f t h e c i s and t r a n s - C ^ 5 compounds were o b s e r v e d . ;

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1 5

Table 2. Mean number of ant workers (as percent of controls [EtOH]) of alkaloidand nonalkaloid-producing species feeding on baits treated with 10 ant-derived alkaloids.

Alkaloid

3

A l k a l o i d Producer

cis-C^3 cis-Cp cis-Cj^.ι trans-C-^ 3 c_is-C].5 EtOH trans-C^5 2 trans-CjL5 EtOH Hex I n d CIS-C13.2 Et Ind :

a

61.1 69.9 68.8 73.1 86.7 99.5 92.0 86.0 100.5 84.3 57.2 86.7

+ 8.2 ± 12.3 ± 5.8 ± 10.4 ± 7.1 ± 7.1 ± 8.0 ± 9.0 ± 9.0 ± 6.8 ± 8.9 ± 5.8

N o n a l k a l o i d producer 7.5 3.8 25.1 14.8 49.9 103.8 30.4 41.1 96.2 40.8 0.5 94.4

± ± ± ± ± ± ± ± ± ± ± ±

2.1 2.2 3.8 3.6 6.0 6.0 5.2 5.0 7.7 6.3 0.2 7.6

S e e t e x t f o r f u l l names o f a l k a l o i d s . Conclusions R e p e l l e n t compounds appear t o commonly f o r t i f y t h e e x o c r i n e secretions of a d i v e r s i t y of arthropods. As i l l u s t r a t e d by t h e d e t e r r e n t s s y n t h e s i z e d by t h r i p s , bees, and a n t s , c o n s i d e r a b l e s t r u c t u r a l e c l e c t i c i s m c h a r a c t e r i z e s t h e s e n a t u r a l p r o d u c t s . The g r e a t v a r i e t y o f d e f e n s i v e a l l o m o n e s w h i c h has been e v o l v e d t o blunt the a s s a u l t s o f m u l t i f a r i o u s predators a t t e s t s t o both the b i o s y n t h e t i c v i r t u o s i t y o f i n s e c t s and t h e w e a l t h o f p o t e n t i a l candidate r e p e l l e n t s . I t would be no e x a g g e r a t i o n t o s t a t e t h a t t h e i n c r e d i b l e s u c c e s s o f i n s e c t s i s i n no s m a l l way due t o t h e i r a b i l i t y t o e f f e c t i v e l y counter, w i t h chemical a r s e n a l s , the predatory

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

2. BLUM ET AL.

Arthropod Natural Products as Insect Repellents

e f f r o n t e r i e s o f t h e i r enemies. These a l l o m o n a l d e t e r r e n t s have been " t r i e d and t e s t e d " i n t h e f u l l n e s s o f e v o l u t i o n a r y t i m e , and o f f e r humankind a t r e a s u r e t r o v e o f proven r e p e l l e n t s w i t h w h i c h to challenge pest species. I t i s time t o g i v e these arthropods c r e d i t f o r h a v i n g p e r f e c t e d c h e m i c a l w a r f a r e , and t o e x p l o i t t h e defensive products o f t h e i r success f o r s o c i e t y ' s b e n e f i t . L i t e r a t u r e Cited

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1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17. 18. 19. 20. 21. 22. 23. 24.

Brown, W. L.; Taylor, R. W. In "The Insects of A u s t r a l i a . " Univ. Melbourne Press, Melbourne, 1970; pp. 951-959. Wilson, E. O. The Insect Societies; Harvard Univ. Press: Cambridge, 1971. Blum, M. S. Chemical Defenses of Arthropods; Academic Press: New York, 1981. Eisner, T. In "Chemical Ecology"; Sondheimer, E.; Simeone, J. B., Eds. Academic Press: New York, 1970. Nutting, W. L.; Blum, M. S.; Fales, H. M. Psyche 1974, 81, 167. Wallace, J . B.; Blum, M. S. Ann. Ent. Soc. Am. 1969, 62, 509. Lewis, T. Thrips; Academic Press: London, 1973. Howard, D. F.; Blum, M. S.; Fales, H. M. Science 1983, 220, 335. Howard, D. F.; Blum, M. S.; Jones, T. H.; Fales, H. M.; Tomalski, M. D. Phytophaga 1987, 1, 163. Hölldobler, B. Oecologica. 1973, 11, 1971. Bhatkar, Α.; Whitcomb, W. H.; Buren, W. R.; Callahan, P. S.; Carlyse, T. Environ. Ent. 1972, 1, 274. Baroni-Urbani, C.; Kannowski, P. B. Environ. Ent. 1974, 3, 755. Blum, M. S.; Jones, T. H.; Hölldobler, Β.; Fales, Η. M.; Jaouni, T. Naturwissenschaften 1980, 67, 144. Jones, T. H.; Blum, M. S.; Fales, H. M. Tetrahedron 1982, 38, 1949. Blum, M. S. In " B i o l o g i c a l l y Active Natural Products"; Cutler, H.G., Ed.; ACS Symp. Ser. No. 380, 1988, pp. 438449. Post, D. C.; Page, R. E.; Erickson, Ε. H. J. Chem. Ecol. 1987, 13, 583. Suzuki, T.; Haga, K.; Leal, W. S.; Kodama, S.; Kuwahara, Y. Appl. Ent. Zool. 1989, 24, 222. Leal, W. S.; Kuwahara, Y.; Suzuki, T. Agric. B i o l . Chem. 1989, 53, 875. Suzuki, T.; Haga, K.; Kuwahara, Y. Appl. Ent. Zool. 1986, 21, 461. Suzuki, T.; Haga, K.; Kodama, S.; Watanabe, K.; Kuwahara, Y. Appl. Ent. Zool. 1988, 23, 291. Blum, M. S.; Fales, H. M. Ananthakrishnan, T. Unpublished r e s u l t s , 1988. Haga, K.; Suzuki, T.; Kodama, S.; Kuwahara, Y. Appl. Ent. Zool. 1989, 24, 242. Eisner, T.; Meinwald, Y. C. Science 1965, 150, 1733. Blum, M. S.; Whitman, D. W.; Fales, H. M. Unpublished r e s u l t s , 1989.

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25.

Blum, M. S.; Crespi, B.; Jones, T. H.; Howard, D. F.; Howard, R. W. Unpublished r e s u l t s , 1988. 26. Rietveld, W. J . ; Schlessinger, R. C.; Kessler, K. J . J . Chem. Ecol. 1983, 9, 1119. 27. Blum, M. S.; Warter, S. L. Ann. Ent. Soc. Am. 1966, 59, 774. 28. Shearer, D. Α.; Boch, R. Nature 1965, 206, 530. 29. Simpson, J . Nature 1966, 209, 531. 30. Boch, R.; Shearer, D. Α.; Petrasovits, A. J . Insect Physiol. 1970, 16, 17. 31. Blum, M. S.; Warter, S. L.; Monroe, R. S.; Chidester, J . C. J. Insect Physiol. 1963, 9, 881. 32. Moore, B. P.; Brown, W. V. J . Aust. Ent. Soc. 1979, 18, 123. 33. Page, R. E.; Blum, M. S.; Fales, Η. M. Experientia 1988, 44, 270. 34. Blum, M. S.; Brand, J . M.; Amante, Ε. Experientia 1981, 37, 816. 35. Jones, T. H.; Blum, M. S. In "Alkaloids: Chemical and B i o l o g i c a l Perspectives"; P e l l e t i e r , S. W. Ed.; John Wiley and Sons, Inc., 1983; V o l . 1, pp. 33-84. 36. MacConnell, J . G.; Blum, M. S.; Fales, H. M. Tetrahedron 1971, 26, 1129. 37. Pedder, D. J . ; Fales, H. M.; Jaouni, T.; Blum, M. S.; MacConnell, J . G.; Crewe, R. M. Tetrahedron 1976, 32, 2275. 38. Jones, T. H.; Blum, M. S.; Fales, H. M.; Thompson, C. R. J. Org. Chem. 1980, 45, 4778. 39. Jones, T. M.; Highet, R. J . ; Blum, M. S.; Fales, H. M. J. Chem. Ecol. 1984, 10, 1233. 40. R i t t e r , F. J . ; Rotgans, I. E. M.; Talman, E.; Verwiel, E.; Stein, F. Experientia 1973, 29, 530. 41. Jones, T. H.; Stahly, S. M.; Don, A. W.; Blum, M. S. J . Chem. Ecol. 1988, 14, 2197. 42. Jones, T. H.; Highet, R. J . ; Don, A. W.; Blum, M. S. J . Org. Chem. 1986, 51, 2712. 43. Blum, M. S.; Everett, D. M.; Tomalski, M. D.; Jones, T. H. Unpublished r e s u l t s , 1989. 44. Blum, M. S. In "Insect Poisons, A l l e r g i n s , and Other Invertebrate Venoms"; Tu, A. T. Ed.; Maurice Dekker, Inc., 1984; pp. 225-242. 45. C a v i l l , G. K. W.; Houghton, E. Aust. J . Chem. 1974, 27, 879. 46. Tomalski, M. D.; Blum, M. S.; Jones, T. H.; Fales, H. M.; Howard, D. F.; Passera, L. J . Chem. Ecol. 1987, 13, 253. 47. Andersen, Α.; Blum, M. S.; Jones, T. H. Unpublished r e s u l t s , 1989. R E C E I V E D May

16,

1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 3 Aggregation Pheromone of Carpophilus lugubris New Pest Management Tools for the Nitidulid Beetles Robert J. Bartelt, Patrick F. Dowd, and Ronald D. Plattner

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Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604

The existence of a male-produced aggregation pheromone in the nitidulid beetle, Carpophilus lugubris Murray, was demonstrated using a wind tunnel (flight) bioassay. The pheromone attracts both sexes. It was obtained by trapping volatiles from feeding beetles. The key purification step was HPLC on a silver-nitrate coated s i l i c a column. Based on the mass and ultraviolet spectra of the pheromone, the mass spectra of the hydrogenated derivatives, and knowledge of the pheromone of a related nitidulid, four synthetic compounds were prepared as pheromone candidates. One of these, (2E,4E,6E,8E)-7-ethyl-3,5-dimethyl-2,4,6,8-undecatetraene matched the pheromone by a l l spectral and chromato­ graphic criteria. The synthetic compound was active in the wind tunnel and also in the field. A possible biosynthetic route is discussed. The dusky sap b e e t l e , Carpophilus lugubris Murray ( C o l e o p t e r a : N i t i d u l i d a e ) , i s a s m a l l ( c a . 4 mm), d a r k c o l o r e d s p e c i e s t h a t o c c u r s t h r o u g h o u t t h e temperate and t r o p i c a l p o r t i o n s o f the Western hemisphere ( 1 ) . L i k e most n i t i d u l i d s p e c i e s , C. lugubris frequents s i t e s where p l a n t m a t e r i a l s a r e f e r m e n t i n g o r decomposing, b u t t h e s e b e e t l e s a l s o have t h e u n f o r t u n a t e tendency o f i n f e s t i n g e a r s o f c o r n i n t h e f i e l d ( 1 ) . I n the U n i t e d S t a t e s , C. lugubris i s responsible f o r t h e r e j e c t i o n o f l a r g e amounts o f sweet c o r n a t c a n n e r i e s ( 2 ) . Furthermore, b e e t l e s o f t h i s f a m i l y are able t o t r a n s m i t f u n g i i n t o c o r n w h i c h c a n cause t h e subsequent b u i l d u p o f m y c o t o x i n s Q ) . C. lugubris i s a l s o commonly found i n oak woods and i s c a p a b l e o f v e c t o r i n g t h e fungus w h i c h causes t h e oak w i l t d i s e a s e ( 4 ) . Pheromones a r e now r o u t i n e l y used as t o o l s i n t h e m o n i t o r i n g and c o n t r o l o f a v a r i e t y o f i n s e c t p e s t s , b u t u n t i l l a t e l y , n o t h i n g was known about the pheromones o f n i t i d u l i d b e e t l e s . An a g g r e g a t i o n pheromone was r e c e n t l y d i s c o v e r e d i n C. hemipterus (5), a close r e l a t i v e o f C. lugubris, and i t i n c l u d e s two t e t r a e n e h y d r o c a r b o n s w h i c h were p r e v i o u s l y unknown t o s c i e n c e : (2£,4Ε,6E,SE)-3,5,7trimethyl-2,4,6,8-decatetraene (1) and (2£,4£,6£,SE)-3,5,7-trimethy12,4,6,8-undecatetraene ( 2 ) . The pheromone i s produced by male This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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1

2

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b e e t l e s , and i t a t t r a c t s b o t h sexes. I t appears t o work p r i m a r i l y as a s y n e r g i s t o f f o o d source v o l a t i l e s . I n t h e f i e l d , we b e l i e v e i t f a c i l i t a t e s t h e assembly o f groups o f b e e t l e s a t s u i t a b l e f e e d i n g s i t e s so t h a t m a t i n g and o v i p o s i t i o n can take p l a c e . The r e s e a r c h r e p o r t e d h e r e i s a c o n t i n u a t i o n o f t h e s t u d y o f n i t i d u l i d pheromones. The p r e v i o u s knowledge o f t h e pheromone o f C. hemipterus a l l o w e d i d e n t i f i c a t i o n o f t h e C. lugubris pheromone from a v e r y s m a l l n a t u r a l sample. Beetles. Bioassays.

and Pheromone C o l l e c t i o n

The C. lugubris b e e t l e s f o r these s t u d i e s were f i e l d c o l l e c t e d d u r i n g the summer o f 1988 i n oak woods and c o r n f i e l d s near B a t h , I l l i n o i s . They were a t t r a c t e d t o t r a p s b a i t e d w i t h whole-wheat b r e a d dough i n o c u l a t e d w i t h b a k e r ' s y e a s t . The c o l l e c t e d b e e t l e s were t h e n m a i n t a i n e d i n t h e l a b o r a t o r y on an a r t i f i c i a l d i e t as d e s c r i b e d p r e v i o u s l y f o r C. hemipterus ( 6 ) . A d u l t b e e t l e s l i v e d as l o n g as 6 months under these c o n d i t i o n s . The a v a i l a b i l i t y o f f i e l d - c o l l e c t e d a d u l t s o b v i a t e d the development o f an e g g - t o - a d u l t r e a r i n g p r o c e d u r e . The l a b o r a t o r y pheromone b i o a s s a y s were c o n d u c t e d i n a wind t u n n e l as d e s c r i b e d p r e v i o u s l y f o r C. hemipterus ( 5 ) . B r i e f l y , ca. 200-400 a d u l t b e e t l e s were p l a c e d , w i t h o u t f o o d , i n t o t h e w i n d t u n n e l about 16 h r p r i o r t o b e g i n n i n g t h e b i o a s s a y s . As w i t h C. hemipterus, the b e e t l e s f l e w r e a d i l y o n l y a f t e r b e i n g s t a r v e d f o r a number o f h o u r s . A b i o a s s a y was conducted by a p p l y i n g a t e s t s o l u t i o n t o a 7-cm d i s k o f f i l t e r paper (Whatman 541), w h i c h was f o l d e d i n t o q u a r t e r s and hung, a l o n g w i t h a c o n t r o l f i l t e r paper, i n t h e upwind end o f t h e w i n d t u n n e l , the two b a i t s b e i n g s e p a r a t e d by 30 cm. The number o f b e e t l e s a l i g h t i n g on each b a i t d u r i n g a t h r e e - m i n t e s t p e r i o d was r e c o r d e d . U s u a l l y , 20-30 t h r e e - m i n b i o a s s a y t e s t s c o u l d be c o n d u c t e d d u r i n g a day w i t h one group o f b e e t l e s . The t e s t s were s e p a r a t e d i n time by 2-5 min. An a c t i v e p r e p a r a t i o n was always r u n e v e r y t h i r d o r f o u r t h t e s t t o ensure t h a t t h e b e e t l e s remained i n a r e s p o n s i v e c o n d i t i o n . The age o f b i o a s s a y b e e t l e s was n o t con­ t r o l l e d , b u t s a t i s f a c t o r y r e s u l t s were o b t a i n e d from the time the b e e t l e s were c o l l e c t e d i n the f i e l d u n t i l they were c a . 6 months o l d . The b i o a s s a y counts were s u b j e c t e d t o a n a l y s i s o f v a r i a n c e a f t e r t r a n s f o r m a t i o n o f t h e d a t a t o t h e l o g ( X f l ) s c a l e . I n the t a b l e s , s i g n i f i c a n t d i f f e r e n c e s from t h e c o n t r o l s a t t h e Ρ - 0.05, 0.01, and 0.001 l e v e l s a r e denoted by *, **, and ***, r e s p e c t i v e l y . Pheromone c o l l e c t i o n s were a l s o made as d e s c r i b e d f o r C. hemipterus ( 5 ) . Tenax porous polymer was used t o t r a p the v o l a t i l e s e m i t t e d from groups o f b e e t l e s f e e d i n g on p i n t o - b e a n d i e t . Parallel samples were d e r i v e d from males, from females, and a l s o from the d i e t medium a l o n e , f o r l a t e r c h r o m a t o g r a p h i c and b i o a s s a y comparisons. Counts o f b e e t l e s were k e p t so t h a t the pheromone p r o d u c t i o n c o u l d be q u a n t i f i e d i n terms o f b e e t l e - d a y s . (A b e e t l e - d a y i s t h e average amount o f pheromone c o l l e c t e d from one b e e t l e i n one d a y ) . B e e t l e s were s e p a r a t e d by sex and s e t up f o r pheromone c o l l e c t i o n w i t h i n a

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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BARTELT ET A L

Aggregation Pheromone of Carpophilus lugubris

29

week o f t r a p p i n g them i n the f i e l d . The f o l l o w i n g r e s u l t s were based on a c o l l e c t i o n o f c a . 5000 b e e t l e - d a y s .

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I n i t i a l Bioassays

and

Chromatography

B e e t l e - d e r i v e d samples were always b i o a s s a y e d a t a dose o f 5 b e e t l e days. I n p r e l i m i n a r y b i o a s s a y s , the Tenax e x t r a c t d e r i v e d from males was c l e a r l y more a c t i v e t h a n t h a t from f e m a l e s , w h i c h p a r a l l e l e d the p r e v i o u s r e s u l t w i t h C. hemipterus. I n seven 3-min p a i r e d comparisons, the c o l l e c t i o n from males a t t r a c t e d an average o f 14.1 b e e t l e s w h i l e t h a t from females a t t r a c t e d o n l y 1.4. B o t h sexes responded. The e x t r a c t from males was f r a c t i o n a t e d on s i l i c a g e l t o s e p a r a t e components by p o l a r i t y , as d e s c r i b e d p r e v i o u s l y ( 5 ) . The s o l v e n t s and b i o a s s a y a c t i v i t i e s o f the f r a c t i o n s a r e p r e s e n t e d i n T a b l e I . The hexane f r a c t i o n was the o n l y one t h a t showed c l e a r a c t i v i t y , s u g g e s t i n g t h a t the pheromone o f C. lugubris was a h y d r o c a r b o n . The s l i g h t a c t i v i t y o f the most p o l a r f r a c t i o n appears t o have been due t o the a r t i f i c i a l d i e t on which the i n s e c t s were f e d d u r i n g pheromone c o l l e c t i o n . Table I.

Wind Tunnel B i o a s s a y s w i t h Chromatographic D e r i v e d from C. lugubris males

A.

S i l i c a g e l f r a c t i o n s from Tenax c o l l e c t i o n

B.

Mean b i o a s s a y count Fraction Fraction Hexane 28.,4 *** 5% Ether-hexane 0,.0 10% Ether-hexane 0 .2 50% Ether-hexane 1,.2 10% MeOH-methylene c h l o r i d e 2 ,7 * AgNO^-HPLC f r a c t i o n s from hexane f r a c t i o n (above) F r a c t i o n (ml after injection) 3.0-4.5 4.5-5.0 5.0-5.5 5.5-6.0 6.0-6.5 6.5-7.0 7.0-7.5

Mean b i o a s s a y count Fraction 0.0 0.0 0.2 35.8 *** 8.5 *** 2.3 ** 0J)

Fractions

(n-4) Control 0.,2 0.,6 0.,2 0,,2 0..0

(n-4) Control 0.2 0.0 0.4 0.0 0.2 0.0 0.4

The a c t i v e hexane f r a c t i o n was s u b j e c t e d t o HPLC on a s i l v e r n i t r a t e c o a t e d s i l i c a column (2) as d e s c r i b e d f o r C. hemipterus (5); 25% toluene-hexane was used as the s o l v e n t . F r a c t i o n s 0.5 ml i n volume were c o l l e c t e d and b i o a s s a y e d (Table I ) . The a c t i v i t y e l u t e d p r i m a r i l y i n a f r a c t i o n 5.5-6.0 ml a f t e r i n j e c t i o n . T h i s e l u t i o n volume was w e l l beyond the s o l v e n t f r o n t (3.0 m l ) ; t h u s , t h e r e was e v i d e n c e f o r u n s a t u r a t i o n . However, the a c t i v i t y e l u t e d s l i g h t l y b e f o r e the t e t r a e n e s (1 and 2) p r e v i o u s l y i s o l a t e d from C. hemipterus, w h i c h o c c u r r e d i n f r a c t i o n s 6.0-7.5 ml a f t e r i n j e c t i o n .

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NATURALLY OCCURRING PEST BIOREGULATORS

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Pheromone I s o l a t i o n and

Spectra

The AgNO^ HPLC f r a c t i o n s from male and female C. lugubris were compared by c a p i l l a r y GC. The h i g h l y a c t i v e 5.5-6.0 ml f r a c t i o n from males produced a GC peak a t 7.87 min (15 m X 0.25 mm ID DB-1 column, 1.0 /im f i l m t h i c k n e s s , 100-200°C a t 10°C/min) w h i c h was c o m p l e t e l y l a c k i n g from the female sample. T h i s peak had a r e t e n t i o n i n d e x o f 15.15, r e l a t i v e t o n-alkanes ( 8 ) , and t h i s peak r e p r e s e n t e d 5% o f the t o t a l GC peak a r e a i n the m a l e - d e r i v e d f r a c t i o n ; the o t h e r peaks c o r r e s p o n d e d t o m a t e r i a l s t h a t were d i e t d e r i v e d . The m a l e - s p e c i f i c compound was p r e s e n t i n the f r a c t i o n a t c a . 100 pg p e r b e e t i e - d a y . The 6.0-6.5 ml f r a c t i o n had a s m a l l e r amount o f t h i s compound and was s t i l l f a i r l y a c t i v e i n the b i o a s s a y . The 5.5-6.0 ml AgNO^ f r a c t i o n was rechromatographed on a s i z e e x c l u s i o n HPLC column (PLGEL 50A, 30 cm X 7.5 mm ID, 10 μπι p a r t i c l e s i z e , hexane as s o l v e n t ) . The m a l e - s p e c i f i c compound e l u t e d i n a f r a c t i o n 11.0-11.7 ml a f t e r i n j e c t i o n , and by GC, appeared uncontami n a t e d by o t h e r compounds. A t o t a l o f c a . 700 ng was o b t a i n e d . Q u a l i t a t i v e l y , the b e e t l e s responded i n s t a n t l y t o 1 ng o f t h i s p r e p a r a t i o n i n the b i o a s s a y . The e l e c t r o n impact mass spectrum ( F i g u r e 1 ) , o b t a i n e d on a F i n n i g a n 4535 i n s t r u m e n t , i n d i c a t e d a m o l e c u l a r w e i g h t o f 204, w h i c h c o r r e s p o n d e d t o the m o l e c u l a r f o r m u l a , C 1 5 H 2 4 . This formula i m p l i e s f o u r degrees o f u n s a t u r a t i o n . I n g e n e r a l appearance, the spectrum was s i m i l a r t o those o f the t e t r a e n e s 1 and 2 o f C. hemipterus ( 5 ) . An u l t r a v i o l e t spectrum was o b t a i n e d i n hexane, and a b r o a d peak w i t h a maximum a t 286 nm was observed. The e x t i n c t i o n c o e f f i ­ c i e n t was a p p r o x i m a t e l y 20,000, based on GC i n t e g r a t i o n r e l a t i v e t o an e x t e r n a l q u a n t i t a t i v e s t a n d a r d . A g a i n , the UV spectrum was s i m i l a r t o those o f 1 and 2 from C. hemipterus (5). The s p e c t r a l d a t a suggested t h a t the C. lugubris pheromone was a c o n j u g a t e d t e t r a e n e r e l a t e d t o 1 and 2 b u t w i t h 15 i n s t e a d o f 13 o r 14 carbons. Hydrogénation The p u r i f i e d compound was hydrogenated (9) and a n a l y z e d by mass s p e c t r o m e t r y t o c o n f i r m the number o f double bonds and t o g a i n i n f o r m a t i o n about the carbon s k e l e t o n . The r e a c t i o n was r u n on c a . 100 ng o f pheromone i n 25 μΐ o f C ^ C ^ ; P t ^ ^ s the c a t a l y s t . The sample was i n t r o d u c e d i n t o the mass s p e c t r o m e t e r through a c a p i l l a r y GC column (DB-1, programed a t 10°C p e r m i n ) , and s p e c t r a were t a k e n a t the r a t e o f 1 per sec. A complex m i x t u r e o f p r o d u c t s r e s u l t e d ( F i g u r e 1, t o t a l i o n GC t r a c e ) . (A s i m i l a r m i x t u r e was always o b s e r v e d when 1 or 2 was hydrogenated (5) and was due t o the c r e a t i o n o f asymmetric c e n t e r s d u r i n g hydrogénation as w e l l as t o a c y c l i z i n g r e a c t i o n w h i c h competed w i t h complete hydrogénation). The h i g h e s t m o l e c u l a r weight i n the p r o d u c t s from C. lugubris was 212, w h i c h corresponded to the f o r m u l a , C ^ H ^ . * r e s u l t e d from the uptake o f 8 hydrogen atoms. T h i s i m p l i e d the o r i g i n a l compound was i n d e e d a t e t r a e n e and had no r i n g s . A l t h o u g h the m o l e c u l a r i o n (m/z 212) was o f low i n t e n s i t y f o r these p r o d u c t s , most o f t h e i r major a l k y l fragment i o n s (m/z 57, 71, ...) were e a s i l y d e t e c t e d . The s i n g l e mass chromatograms f o r these fragments ( e . g . , m/z 183, F i g u r e 1) i n d i c a t e d t h a t t h e r e were f o u r d i s t i n c t GC peaks due t o a c y c l i c , w

a n c

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

BARTELT ET A L

Aggregation Pheromone of Carpophilus lugubris

PHEROMONE BEFORE HYDROGENATION 133

100.0 n 55

175

119

91 105

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147 491 ι I 40 60

M/Z

100

120

140

204 189 161 T^ • t. . -rprrn "T 160 180 200 220 160 180

1

PHEROMONE AFTER HYDROGENATION Downloaded by COLUMBIA UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch003

TOTAL ION GC TRACE

SINGLE ION GC TRACES 57

A A /

,

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71 85 99 SINGLE ION GC TRACES

113 127

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141

M/V

155

212

183 460

440

480

460

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HYDROGENATED PHEROMONE: ALKYL FRAGMENTS 57

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ρ

143

5.000X 99

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PENTADECANE: ALKYL FRAGMENTS 57

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F i g u r e 1. A n a l y s i s o f pheromone b y mass s p e c t r o m e t r y , b e f o r e and a f t e r hydrogénation.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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32

NATURALLY OCCURRING PEST BIOREGULATORS

s a t u r a t e d d e r i v a t i v e s . The c y c l i c d e r i v a t i v e s ( m o l e c u l a r i o n m/z 210) o v e r l a p p e d w i t h t h e s e , by GC, b u t d i d n o t i n t e r f e r e w i t h mass s p e c t r a l i n t e r p r e t a t i o n f o r the a c y c l i c d e r i v a t i v e s . The g e n e r a t i o n o f f o u r GC peaks from a s i n g l e pure compound s u g g e s t e d the c r e a t i o n o f t h r e e asymmetric c e n t e r s d u r i n g hydrogénation. W i t h t h r e e asymmetric c e n t e r s , t h e r e would be e i g h t p o s s i b l e o p t i c a l i s o m e r s , w h i c h would produce, a t most, f o u r GC peaks on an a c h i r a l column. As w i t h the pheromone components o f C. hemipterus, the double bonds i n the o r i g i n a l compound p r o b a b l y i n v o l v e d t h r e e branches i n the c a r b o n c h a i n , b u t even more branches were p o s s i b l e i f the s a t u r a t e d d e r i v a t i v e s d i d n o t s e p a r a t e c o m p l e t e l y by GC o r i f the branches d i d n o t i n v o l v e asymmetric c e n t e r s . The GC r e t e n t i o n t i m e s o f the s a t u r a t e d d e r i v a t i v e s a l s o i n d i c a t e d a h i g h degree o f b r a n c h i n g . These 15-carbon a l k a n e s had r e t e n t i o n i n d i c e s o f 13.32-13.43. The i n t e n s i t i e s o f a l k y l fragment i o n s , 2 n + l ' provide i n f o r m a t i o n about l o c a t i o n s o f branches i n a l k a n e s ( 1 0 ) . The s p e c t r a o f a l l f o u r a c y c l i c d e r i v a t i v e s were n e a r l y i d e n t i c a l when o n l y t h e s e fragments were c o n s i d e r e d , and the key f e a t u r e was the r e l a t i v e l y i n t e n s e peak a t m/z 183, compared w i t h the 15-carbon n - a l k a n e ( F i g u r e 1 ) . T h i s c o r r e s p o n d e d t o the ready l o s s o f an e t h y l r a d i c a l , a f e a t u r e w h i c h was a l s o e v i d e n t i n the mass spectrum o f the u n s a t u r a t e d p a r e n t compound (m/z 175). S e v e r a l o t h e r fragments were r e l a t i v e l y s u p p r e s s e d (m/z 71, 85, 169), b u t u n f o r t u n a t e l y , the l o c a t i o n s o f branches were n o t as o b v i o u s from the f r a g m e n t a t i o n p a t t e r n o f the h y d r o g e n a t e d d e r i v a t i v e s as they had been f o r 1 and 2 (5). c

H

+

n

S y n t h e s i s o f Model T e t r a e n e s Four model compounds were s y n t h e s i z e d ( F i g u r e 2) t o a i d the i n t e r p r e t a t i o n o f s p e c t r a l and c h r o m a t o g r a p h i c f e a t u r e s o f the unknown compound, p a r t i c u l a r l y the l o s s o f an e t h y l group from the c a r b o n s k e l e t o n . Because o f the c l o s e p h y l o g e n e t i c r e l a t i o n s h i p between C. lugubris and C. hemipterus, model compounds were chosen w h i c h were s t r u c t u r a l l y s i m i l a r t o 1 and 2. Compound 3 was s i m p l y the 15-carbon homolog o f the C. hemipterus compounds. T e t r a m e t h y l t e t r a e n e 4 was chosen because i t s c a r b o n s k e l e t o n c o u l d l o s e an e t h y l group from e i t h e r end a f t e r hydrogénation, perhaps r e s u l t i n g i n an enhanced m/z 183 fragment. Compounds 5 and 6 had e t h y l groups a t the 5 and 7 p o s i t i o n s , r e s p e c t i v e l y , i n s t e a d o f m e t h y l groups, so t h a t the l o s s o f an " i n t e r n a l " e t h y l group c o u l d be s t u d i e d . No 3 - e t h y l - 5 , 7 - d i m e t h y l u n d e c a t e t r a e n e was s y n t h e s i z e d because the c a r b o n s k e l e t o n would have o n l y two asymmetric c e n t e r s ( t h e 5 and 7 c a r b o n s ) and c o u l d n o t , t h e r e f o r e , g e n e r a t e f o u r GC peaks on an a c h i r a l column. The s y n t h e s e s o f the compounds f o l l o w e d the g e n e r a l p r o c e d u r e s o u t l i n e d e a r l i e r (5) w i t h one major m o d i f i c a t i o n : e t h y l groups i n s t e a d o f m e t h y l groups c o u l d be i n c o r p o r a t e d i n t o the c a r b o n c h a i n s by u s i n g t r i e t h y l 2-phosphonobutyrate (TEPB) i n the W i t t i g - H o r n e r r e a c t i o n i n s t e a d o f t r i e t h y l 2-phosphonopropionate (TEPP). A l l r e a c t i o n s were m o n i t o r e d by GC and mass s p e c t r o m e t r y . The W i t t i g Horner r e a c t i o n s produced p r e d o m i n a n t l y Ε i s o m e r s , w h i l e b o t h Ε and Ζ isomers were o b t a i n e d from the W i t t i g r e a c t i o n s . From p r e v i o u s e x p e r i e n c e ( 5 ) , the isomers r e t a i n e d the l o n g e s t by GC were

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

3.

BARTELT ET AL.

Aggregation Pheromone of Carpophilus lugubris

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Downloaded by COLUMBIA UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch003

TEPP COOEt

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F i g u r e 2. S y n t h e t i c scheme f o r f o u r t e t r a e n e s u s e d as model compounds i n t h e s t r u c t u r a l a n a l y s i s o f t h e C. lugubris pheromone.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

33

34

NATURALLY OCCURRING PEST BIOREGULATORS

a s s i g n e d t h e Ε,Ε,Ε,Ε c o n f i g u r a t i o n ; t h e s e r e p r e s e n t e d 50-90% o f t h e s y n t h e t i c t e t r a e n e p r o d u c t s , depending on s y n t h e t i c r o u t e and t h e s t r u c t u r e . The s y n t h e t i c p r o d u c t s were p u r i f i e d f i r s t on s i l i c a g e l t h e n by HPLC on t h e AgNO^ HPLC column. The NMR s p e c t r a o f t h e p u r i f i e d s y n t h e t i c t e t r a e n e s always s u p p o r t e d t h e e x p e c t e d b r a n c h i n g p a t t e r n . By analogy t o 1 and 2 ( 5 ) , t h e Ε,Ε,Ε,Ε c o n f i g u r a t i o n was s u p p o r t e d as w e l l . By c a p i l l a r y GC (DB-1), t h e t e t r a e n e s were a t l e a s t 90% pure a f t e r AgNO^ chromatography, and t h e i m p u r i t i e s were m o s t l y t r a c e s o f o t h e r g e o m e t r i c a l i s o m e r s . Samples o f t h e p u r i f i e d t e t r a e n e s were d i l u t e d t o 1 ng p e r 10 μΐ w i t h hexane f o r b i o a s s a y s .

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Comparison o f Chemical P r o p e r t i e s between Pheromone and S y n t h e t i c Tetraenes The GC r e t e n t i o n s and mass s p e c t r a l f r a g m e n t a t i o n p a t t e r n s o f t h e hydrogenated d e r i v a t i v e s o f these s t a n d a r d s a r e summarized i n T a b l e II. I n t e n s i t i e s o f mass s p e c t r a l peaks a r e r e l a t i v e t o the base peak (m/z 57 i n a l l c a s e s ) and a r e rounded t o t h e n e a r e s t whole p e r c e n t ; "-" i n d i c a t e s t h e fragment was n o t d e t e c t e d , and Ο u ol οο ΓΗο ω ω ω c 3 ω

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

g

e

^

0

a

En

Ε

*

-

+

En

Ε

•

+ + +

+

-

-

En En En

En

L L L

L

This i s a p a r t i a l l i s t i n g . +, r e g u l a t o r y f a c t o r i s p r e s e n t i n s p e c i e s ; -, r e g u l a t o r y f a c t o r has not been r e p o r t e d i n s p e c i e s ; *, h o s t r e g u l a t i o n has been r e p o r t e d , but a n a l y s i s o f a l l r e g u l a t o r y f a c t o r ( s ) has n o t been completed. D e s i g n a t i o n s f o r the h o s t growth s t a g e s . L = l a r v a l s t a g e , L-P = p a r a s i t i s m o f t h e l a r v a l stage and emergence from the p u p a l s t a g e o f t h e h o s t , E-L = p a r a s i t i s m o f the egg and emergence from t h e l a r v a l s t a g e o f t h e h o s t , Ε = egg s t a g e . B. l o n g i c a u d a t u s c o n t a i n s a pox v i r u s . C. m a r g i n i v e n t r i s c o n t a i n s a v i r u s t h a t i s m o r p h o l o g i c a l l y d i s t i n c t from t h e p o l y d n a v i r u s e s . A l l other species contain polydnaviridae v i r u s e s . See t h e t e x t f o r r e f e r e n c e s t o m a t e r i a l i n p r e s s . P i e k and S p a n j e r (23) have c o m p i l e d an e x t e n s i v e l i s t i n g o f s p e c i e s w i t h p a r a l y z i n g venoms. I n c l u d e s t h e f o l l o w i n g s p e c i e s B. b r e v i c o r n i s . B. g e l e c h i a e . B. h e b e t o r , and B. m e l l i t o r . T. A. Coudron, USDA, ARS, u n p u b l i s h e d d a t a .

H v p o s o t e r exiguae Hvposoter f u g i t i v u s V e n t u r i a canescens Scelionidae Telenomus h e l i o t h i d i s Trichogrammatidae Trichogramma p r e t i o s u m

Ichneumonidae Camooletis sonorensis

*

+

+

*

+

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77

77

47, 58, 88, 96, 100 101, 107, 109 34, 35, 100, 102 99 126, 127

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46

NATURALLY OCCURRING PEST BIOREGULATORS

Relatively l i t t l e i s known of the physiology and metabolism of insect parasitoids. Though i t i s generally assumed that the maturing p a r a s i t i c hymenopteran eggs take up host hemolymph components and ions through i t s t h i n wall (17, 25) i t has been shown that some parasitoids rely on t h e i r protein synthetic machinery f o r growth (25, 26). Much of t h i s synthesis occurs a f t e r the parasitoid i s associated with the host (27). Parasitism has resulted i n changes i n the s p e c i f i c gravity, freezing point depression and dry weight of the hemolymph of the host. This correlates with a l t e r a t i o n s i n the concentration and composition i n the host hemolymph (for review see 18). However, the change i n host protein concentration or composition i s not l i k e l y to be due to excretion of waste material by the parasitoid. P a r a s i t i c hymenopteran larvae have an imperforate gut, with a midgut that i s not joined to the hindgut. Much of the waste generated by the parasitoid i s stored i n t e r n a l l y u n t i l pupation when the waste i s released as meconium outside the host. The developing parasitoid may secrete material into the host that a f f e c t s the composition of the host t i s s u e . Such secretions are l i k e l y to originate from the s a l i v a r y glands of the parasitoid. Phenoloxidase a c t i v i t y i s secreted from the salivary gland of Exeristes roborator and i s thought to help i n the preservation of the host tissue (28). Cotesia (= Apanteles) congregata secreted proteins i n v i t r o that arose from de novo synthesis by the wasp (29). This i s not unique to hymenopteran parasitoids. The dipteran Blepharipa s e r i c a r i a e , parasitoid of the silkworm Philosamia synthia, secretes a small peptide into i t s host that i n h i b i t s l i p i d transport by lipophorin (30). A possible b i o l o g i c a l significance of t h i s peptide may be to divert l i p i d consumption by the host during diapause. This type of secretion may be directed at providing nutrients f o r the parasitoid. Parasitism also causes a l t e r a t i o n s i n the constitutive hemolymph proteins of the host. Hemolymph storage proteins i n the cabbage b u t t e r f l y , P i e r i s rapae, decreased i n concentration following parasitism by Cotesia glomerata (31). This decrease may have been due to an observed concurrent uptake of the same proteins by the parasitoid (32). A reduced synthesis of arylphorin by the tobacco hornworm, Manduca sexta, parasitized by Ç. congregata was hypothesized to be due to i n h i b i t o r y e f f e c t s of parasitism on host fat body, food consumption and growth (33). Arylphorin concentrations increased precociously i n larvae of the cabbage looper, Trichoplusia n i , parasitized by Chelonus sp. (Kunkel, J . G.; Grossniklaus, C ; Karpells, S. T.; Lanzrein, C. Arch. Insect Biochem. Physiol., i n press). Concentrations of several hemolymph proteins decreased i n parasitized T. n i , during development of the parasitoid Hyposoter exiguae (34, 35). Several high molecular weight host proteins were detected 40 hours e a r l i e r i n hemolymph from larvae of the f a l l arrayworm, Spodoptera frugiperda, parasitized by Cotesia marginiventris than i n hemolymph of control larvae (36). Parasitism has also been reported to cause the synthesis of proteins that were not present i n unparasitized hosts (18, 37, 38). Two predominant and one minor proteins were found i n the hemolymph of M. sexta larvae parasitized by l a r v a l parasitoid, C, congregata. These proteins were apparently produced by the host during the f i n a l

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

4.

COUDRON

Host-Regulating Factors and Parasitic Hymenoptera

47

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stages of the parasitoid*s development i n d i c a t i n g their synthesis was activated i n response to the development of the parasitoid (33). In comparison, one minor high molecular weight protein was found i n the hemolymph of T. n i parasitized by the egg-larval parasitoid, Chelonus near curvimaculatus (39). I t was not determined whether the protein was encoded by the host, the parasitoid, or a factor derived from the parasitoid. However, the protein was only found i n hosts containing a developing p a r a s i t o i d . The cause of these changes i n host proteins i s unclear and i t i s uncertain what, i f any, regulatory role these changes play. Host Response to the Developing P a r a s i t o i d . Sometimes a l t e r a t i o n s i n the composition of the host tissues are part of the response of the host defense reaction. An i n i t i a l hemolymph response to foreign material i s through the hemocytes (plasmatocytes and granulocytes), produced i n hemopoietic organs near the dorsal diaphragm (40). Part of the humoral defense response i s the synthesis of several enzymic, l e c t i n , and b a c t e r i c i d a l proteins (41-43). I t i s also probable that some parasitoids e l i c i t unique humoral proteins, other than the b a c t e r i c i d a l proteins (29). Some p a r a s i t i c Hymenoptera appear equipped to protect themselves against the host defense responses. Early studies by Salt (44, 45) suggested d i f f e r e n t methods existed for parasitoid resistance to the host immune system. More recent studies indicate i n some cases the a b i l i t y of the parasitoid to overcome the host defenses i s associated with factors (e.g., teratocytes, viruses and venoms) that originate from the adult female parasitoid. These factors w i l l be discussed i n the subsequent sections of t h i s chapter. The texture and composition of the outer surface of the parasitoid egg can be a passive means of protection against the host defense reactions. Fibrous layers on the surface of Cardiochiles nigriceps (46), Campoletis sonorensis (47) and C. glomerata (48) eggs have been implicated i n the protection of the egg from encapsulation by hemocytes of their hosts. Histochemical studies showed the fibrous material was composed of neutral glycoproteins and neutral and a c i d i c raucoproteins. The mechanism by which these complex proteins prevent haemocyte adhesion i s unclear. This type of passive defense i s proposed to delay encapsulation u n t i l more permanent means of host immunosuppression ( i . e . , teratocytes, parasitoid-derived viruses or venoms) i s established (46). I t i s unlikety that the parasitoid egg secretes haemocyte-repelling substances, since i n some cases dormant and dead eggs evade encapsulation. I t should be noted that protection from host immune responses i s an important issue i n l a r v a l parasitism. However, protection may not be important i n true egg parasitoids since insect eggs apparently lack the a b i l i t y to encapsulate foreign objects (44). E f f e c t on the Host Endocrine System. Several publications document an endocrine basis of several parasitoid-host interactions (18, 20, 49, 50). However, most of the mechanisms involving the endocrine interactions remain to be c l a r i f i e d . At best, we can describe the q u a l i t a t i v e e f f e c t s but not the regulatory factors responsible for the interactions. U n t i l the characterization of

American Chemical Society Library 1155 16th St.,

N.W.

In Naturally Occurring Pest Bioregulators; Hedin, P.; Washington. D.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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48

NATURALLY OCCURRING PEST BIOREGULATORS

regulatory factors responsible f o r these e f f e c t s , i t i s perhaps most f i t t i n g to discuss these e f f e c t s i n t h i s section. Several systems have been studied and although each i s unique, some common features e x i s t . Chelonus sp. stimulate precocious spinning of a cocoon i n their lepidopteran hosts (51). Presumably t h i s i s a r e s u l t of a premature decline i n the juvenile hormone (JH) l e v e l i n the host. In contrast, other genera (e.g., gregarious Cotesia sp. and Copidosoma sp.) cause a delay or suppression of metamorphosis of the host, sometimes causing supernumerary or intercalated molts into immature stages. Presumably t h i s i s a result of a decline i n the JH s p e c i f i c esterase a c t i v i t y ( i . e . , causing a higher than normal l e v e l of JH) (52), and of a disruption of the ecdysteroid l e v e l s i n the host (33). B. longicaudatus superparasitism of the larvae of A^. suspensa causes an increase i n host JH l e v e l s and a decrease i n JH esterase a c t i v i t y that apparently i s the cause i n delayed host larval-pupal metamorphosis (53). These r e s u l t s could be due to the presence of the parasitoid but also could be a result of the extra-embryonic serosa o r i g i n a t i n g from t h i s p a r a s i t o i d . This i s addressed i n a subsequent section. There i s increasing evidence that the braconid parasitoids, Apanteles k a r i y a i (54) and M i c r o p l i t i s croceipes (55), cause a decrease i n the l e v e l s of ecdysone, v i a a mechanism other than a d i r e c t e f f e c t on the prothoracic gland, the s i t e of synthesis of ecdysone. The braconid endoparasitoids complete t h e i r development without extensive destruction of host t i s s u e s . However, redirection of metabolism i n the f a t body tissue within the host could account for an ultimate reduction i n ecdysone (55, 56). In contrast, ichneumonid parasitoids may regulate the host ecdysteroid l e v e l s by a f f e c t i n g the prothoracic gland tissue (57-59). This regulation i s accomplished through the symbiotic viruses that are the subject of a subsequent section. Indirect evidence implies that some parasitoids produce and release hormones into the host that act as regulatory factors that change host events under endocrine c o n t r o l . The homologue JH III was found i n larvae of M. sexta parasitized by C. congregata (52), though only JH I and I I are present i n unparasitized M. sexta (60). In larvae of the large white cabbage b u t t e r f l y , P i e r i s brassicae, parasitized by C. glomerata, the JH t i t e r increased a f t e r neck-ligation of the host. Both cases suggest a possible source of the hormone was the parasitoid (61). This hypothesis i s further supported by recent findings. Larvae of T. ni, parasitized by Chelonus sp. showed reduced l e v e l s of JH I I , but contained high l e v e l s of JH I I I , compared to unparasitized larvae (Jones, G.; Hanzlik, T.; Hammock, B. D.; Schooley, D. Α.; M i l l e r , C. Α.; T s a i , L. W.; Baker, F. C. J . Insect Physiol., i n press). The presence of JH I I I , together with JH I and I I , was detected i n parasitized eggs, but only JH I and I I were present i n unparasitized eggs (Grossniklaus-Burgin, C ; Lanzrein, B. Arch. Insect Biochem. Physiol., i n press). These findings suggest that JH I I I originated from the p a r a s i t o i d . In addition, a comparison revealed that JH t i t e r fluctuations i n the parasitoid were independent of changes i n the JH t i t e r i n the host, which supports the idea that the parasitoid can produce i t s own JH (Grossniklaus-Burgin, C ; Lanzrein, B. Arch. Insect Bioch. Physiol., i n press).

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

4. COUDRON

Host-Regulating Factors and Parasitic Hymenoptera

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Levels of JH I I I i n suspense superparasitized by B. longicaudatus were considerably higher than i n unparasitized A. suspensa (53). JH I I I was also found i n f i r s t instars of the parasitoid. These results suggested that the elevated JH I I I l e v e l s i n the superparasitized host may r e s u l t from a decrease i n JH esterase a c t i v i t y with a continued or elevated synthesis of the hormone i n the host or from a secretion of JH I I I by the parasitoids. Parasitoid Response to the Host. Simultaneous with the e f f e c t s of parasitism on the host, are the e f f e c t s of the host on the parasitoid. The response of the parasitoid to host parameters influences the way the parasitoid interacts with the host. This creates a continuum between the "conformers" and the "regulators." Endo- and ectoparasitoids feed i n part on the hemolymph of the host and many endoparasitoids develop within the haemocoel of the host. Both ingestion and cross-cuticular transport of host humoral material by the parasitoid are l i k e l y to occur. Apparently, parasitoids are p a r t i c u l a r l y sensitive to the endocrine milieu i n the host hemolymph (_3, 18, 20) and capable of taking up hormones from the host (Grossniklaus-Burgin, C.; Lanzrein, B. Arch. Insect Biochem. Physiol., i n press). The "hormonal hypothesis" proposed that the growth of many parasitoids was affected and possibly controlled by the hormones of t h e i r hosts (62, 63). M e l l i n i thought t h i s hypothesis was more applicable to dipteran parasitoids. However, several studies have confirmed the application of the hypothesis to p a r a s i t i c Hymenoptera as well. Many parasitoids are sensitive to disturbances of the host endocrine milieu caused by application of insect growth regulators (64-71), and by administering exogenous hormone to the host (72-74). The f i r s t l a r v a l molt of Opius concolor i s regulated by endogenous release of ecdysteroids by i t s host, the Mediterranean f r u i t f l y , C e r a t i t i s capitata, during puparium formation (73, 74). The l a r v a l ecdysis of C. congregata at emergence from the tobacco hornworra, M. sexta, i s regulated by host hemolymph ecdysteroid l e v e l s (52) and affected by topical application of JH or the JH analogue, raethoprene (69). The larval-pupal parasitoid B. longicaudatus deposits an egg i n the f i r s t , second or t h i r d instar larvae of A^. suspensa. The p a r a s i t o i d s f i r s t instar remains inactive (conformer), and l a t e r coordinates i t s molt with host pupation (75). This obligatory synchrony of the early l a r v a l molt of the parasitoid with the host's metamorphosis i s i n response to the ecdysteroid levels i n the host hemolymph (76). In addition to these cases where a d i r e c t association or response has been shown between the host hormonal milieu and the development of the parasitoid, there are other cases where the developmental synchrony of both the parasitoid and i t s host constitutes i n d i r e c t evidence of t h i s relationship (for a review see 20). 1

Teratocytes and Serosa. Teratocytes, which originate from the disintegration of an embryonic membrane or serosa (=* trophamnion) of some p a r a s i t i c Hymenoptera, appear to have a s i g n i f i c a n t role i n parasitoid-host interactions (8, 77-80, Dahlman, D. L. Arch. Insect Biochem. Physiol., i n press). A discussion of teratocytes i s

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included i n t h i s section since these c e l l s are derived from the developing parasitoid embryo and are released into the host when the embryo hatches. However, teratocytes can cause c e r t a i n e f f e c t s on the host, independent of the developing parasitoid. Though teratocytes may increase i n s i z e when they are released into the host, they do not multiply within the host (18). Secretory organelles have been observed i n teratocytes that may be important to the function of these c e l l s (80, 81). I t has been proposed that these c e l l s serve n u t r i t i o n a l and gaseous exchange functions that benefit the parasitoid (80, 82, 83). In theory nutrients could be sequestered from the host hemolymph or produced by de novo synthesis within the c e l l s . Several studies have investigated the production and secretion of regulator molecules by teratocytes. The presence of rough endoplasmic r e t i c u l a and other organelles i n the teratocytes suggests these c e l l s are a c t i v e l y metabolizing and synthesizing material (8). Substances released by teratocytes have been shown to a f f e c t phenoloxidase a c t i v i t y (9_, Dahlman, D. L. Arch. Insect Biochem. Physiol., i n press). An endocrine r o l e has also been postulated i n some species (84). Injection of teratocytes from the braconid parasitoid M. croceipes into larvae of the tobacco budworm, H e l i o t h i s virescens, caused an elevation of the ecdysteroids, an increase i n JH l e v e l s and a decrease i n the JH esterase a c t i v i t y i n the host (85, Dahlman, D. L. Arch. Insect Biochem. Physiol., i n press). Results from recent studies show that the presence of teratocytes causes a disruption of the c e l l u l a r defense system of the host (Tanaka, T.; Wago, H. Arch. Insect Biochem. Physiol., i n press, Kitano, H.; Wago, H.; Arakawa, T. Arch. Insect Biochem. Physiol. i n press). This supports e a r l i e r speculation that encapsulation was suppressed by the teratocytes (80) or substances produced by the teratocytes that i n h i b i t the host immune response, (86-88). In most p a r a s i t i c Hymenoptera studied the serosa degenerates a f t e r hatching. However, the serosa of IS. longicaudatus remains intact (Lawrence, P. 0. Arch. Insect Biochem. Physiol., i n press). Though there are no other reports of serosas that remain i n t a c t , i t i s possible that some species not recorded as having teratocytes (see Table I) may maintain an i n t a c t serosa. C e l l s of the serosa of B. longicaudatus are secretory and appear to use large coated v e s i c l e s to transport molecules from the host, and m i c r o v i l l i to release materials into the host (Lawrence, P. 0. Arch. Insect Biochem. Physiol., i n press). A polypeptide, approximately 24 Kd, was found i n the hemolymph of the host and apparently was produced i n the serosa c e l l s . Injection of the protein into healthy _A. suspensa prepupae i n h i b i t s metamorphosis (Lawrence, P. 0., University of F l o r i d a , personal communication, 1990.). The e f f e c t of 13. longicaudatus on _A. suspensa i s s i m i l a r to the e f f e c t of teratocytes of M. croceipes injected into larvae of H. virescens reviewed above (Lawrence, P. 0. Arch. Insect Biochem. Physiol., i n press). The sequestering of host material through coated v e s i c l e s could support a nutrient role of the serosa. However, d e f i n i t i v e evidence i s s t i l l lacking.

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The proximity of the serosa and teratocytes with the developing parasitoid, and their simultaneous presence i n the host, make i t d i f f i c u l t to determine which i s the source of the parasitoid regulatory f a c t o r s . I t i s also possible that regulatory factors are synthesized i n one tissue or location and released to the host at another location v i a a d i f f e r e n t tissue. These points remain to be determined i n most cases.

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Factors Associated with the Adult Female Paxasitoid Parasitoid-Derived Viruses. Symbiotic viruses found i n some members of the Ichneumonidae and Braconidae families are assembled i n the nuclei of the c e l l s of the calyx epithelium (89). C e l l s that produce the viruses may form a layer around the calyx lumen near the ovariole end of the calyx (90). The viruses are secreted into the lumina of the l a t e r a l oviduct and injected into the host with the egg at the time of o v i p o s i t i o n . Once i n the host the virus material i s expressed (91, 92). At present there i s no unequivocal evidence of r e p l i c a t i o n of these viruses i n the host (93), though t h i s continues to be an active area of i n v e s t i g a t i o n . V i r a l genomes consist of separate, multiple heterologous, double-stranded, c i r c u l a r DNA of various lengths. Viruses isolated from ichneumonids have a fusiform nucleocapsid surrounded by two unit-membrane envelopes. This i s c h a r a c t e r i s t i c of the genus Polydnavirus (94). Viruses isolated from braconids have c y l i n d r i c a l nucleocapsids of variable lengths surrounded by a single unit membrane, and morphologically have some resemblance to baculoviruses (89). These symbiotic viruses from both the ichneumonids and the braconids, have been assigned to the new virus family, polydnaviridae (95). Reports of the secretory nature of the calyx region date back over twenty years. Salt (44) recognized that secretions of the calyx of the ichneumonid Venturia (= Nemeritis) canescens affected the host immune system. Studies on the ichneumonid C. sonorensis revealed the presence of nuclear secretory p a r t i c l e s associated with the calyx c e l l s (96). Those studies were followed by the confirmation of DNA i n virus p a r t i c l e s associated with the oocytes of the braconid C. nigriceps (97). Several reports now corroborate the role of the symbiotic viruses i n inactivating defense mechanisms of the host. Washed eggs of the ichneumonid parasitoids C. sonorensis (98) and Hyposoter f u g i t i v u s (99) are encapsulated and do not develop when introduced a r t i f i c i a l l y into their hosts JH. virescens and the forest tent c a t e r p i l l a r , Malacosoma d i s s t r i a , respectively. However, when virus isolated from the calyx of the parasitoid accompanies the egg, viable parasitoid progeny develop, suggesting a role for the virus i n protecting the parasitoid egg from the immune response system of the host. In a cross-protection experiment, washed eggs of C. sonorensis and H. exiguae developed i n H. virescens larvae when heterologous combinations of eggs and symbiotic virus were used (100). These results suggest that the viruses from the two ichneumonids act i n a similar manner to promote successful parasitism. The mechanism by which the virus of Ç, sonorensis suppresses the host's a b i l i t y to encapsulate the parasitoid egg remains

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unknown. Davies et a l . (101) demonstrated that within 8 hours of i n j e c t i n g calyx f l u i d , 75% of the c i r c u l a t i n g capsule-forming hemocytes (plasmatocytes) were removed from the host hemolymph. Such an a l t e r a t i o n i n the host plasmatocytes could account for the suppression of encapsulation, though the fate of the l o s t plasmatocytes remains unknown. The a b i l i t y of the virus from the ichneumonid, H. exiguae to i n h i b i t phenoloxidase a c t i v i t y i n the host T. n i , may provide another mechanism of suppression of encapsulation (102). A similar decline i n the monophenoloxidase a c t i v i t y i n the hemolymph of M. sexta occurred after i n j e c t i o n of virus from the braconid C. congregata (Beckage, N. E.; Metcalf, J . S.; Nesbit, D. J . ; S c h l e i f e r , K. W.; Zetlan, S. R.; de Buron, I. Insect Biochem., i n press). The phenoloxidase-tyrosine enzyme system i s also thought to be part of the host immune system (103, 104). Suppression of the host immune system, observed following parasitism by two braconid parasitoids, has also been attributed, i n part, to the presence of a symbiotic v i r u s . Long v i r u s - l i k e filaments from the calyx region of the reproductive t r a c t of the braconid parasitoid M i c r o p l i t i s mediator were found attached to the surface of the chorion of the oviposited egg (105). These filaments appeared to prevent encapsulation i n half the eggs tested i n the host, the common armyworm, Pseudaletia separata. There was also evidence that the f i l o p o d i a l elongation of the host hemocytes was strongly suppressed (106). Also, half of the sephadex p a r t i c l e s , injected into P. separata together with calyx f l u i d from the braconid endoparasitoid A. k a r i y a i , were not encapsulated (15). It should be noted that a mixture of calyx f l u i d and venom material for both braconids, was more e f f e c t i v e i n suppressing encapsulation than the calyx f l u i d alone. Apparently, calyx f l u i d (containing symbiotic virus) and venom act s y n e r g i s t i c a l l y and both were essential for complete evasion of the host defense reactions. There was some evidence suggesting the calyx material affected the capsule-forming c e l l s of the host, while the venom material provided a non-antigenic protective covering over the egg (106). A similar e f f e c t was observed with a mixture of calyx f l u i d and venom material for the braconid C. nigriceps (14). Another explanation for the synergism observed between symbiotic viruses and venomous material may involve tissue i n f e c t i o n and expression of the v i r u s . Venom promoted uncoating of a braconid virus i n v i t r o and, also promoted persistence i n vivo of DNA from the virus (16). Injection of virus material has also been associated with host synthesis of new proteins. Parasitism by C. sonorensis resulted i n the synthesis of a new glycoprotein i n several of i t s habitual hosts (107). The appearance of the glycoprotein was duplicated by the i n j e c t i o n of either calyx f l u i d or p u r i f i e d v i r u s . The glycoprotein has been correlated with suppression of encapsulation by the host. It i s interesting that parasitism by C. sonorensis of two non-permissive hosts, the velvetbean c a t e r p i l l a r , Anticarsia gemmatalis, and the bertha armyworm, Mamestra configurata, did not stimulate the production of the glycoprotein by those hosts (107). D e f i n i t i v e indication of t r a n s c r i p t i o n of parasitoid-derived viruses i n the host has thus f a r eluded researchers. Parasitism by C. congregata induced synthesis of new hemolymph proteins i n the

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host M. sexta (108). Synthesis of one of the major induced proteins occurred within a few hours of parasitism. Induction was also caused by the i n j e c t i o n of ovarian calyx f l u i d from the parasitoid. Exposure of the calyx f l u i d to psoralen and UV l i g h t destroyed i t s capacity to induce synthesis of the major new protein. This suggested that the synthesis may be mediated by v i r a l nucleic acid, though i t was not determined i f i t represented a v i r a l gene product or a host protein induced by v i r u s - s p e c i f i c a c t i v i t y i n the host. Another type of i n t e r a c t i o n of the parasitoid-derived viruses with the host i s based on the endocrine regulation of development. Injections of the calyx f l u i d or i s o l a t e d virus material of C. sonorensis arrested development of 40% of the ÏÏ. virescens larvae tested (58, 109). Injections into i s o l a t e d thoraces were most e f f e c t i v e . Ecdysteroid production by the prothoracic gland of the host ceased f o r ca. 10 days following the i n j e c t i o n . Arrested development was reversed by i n j e c t i o n s of ecdysone and 20-hydroxyecdysone. The prothoracic glands of injected host larvae were p a r t i a l l y degenerated. In contrast other tissues associated with the endocrine system of the host appeared normal (59). These observations suggested that the virus material induced tissue degeneration that was s p e c i f i c to the prothoracic gland. S i m i l a r l y , parasitism of P. separata, by A. k a r i y a i or i n j e c t i o n of the calyx f l u i d or virus material from the parasitoid, caused a decline i n the host ecdysteroid t i t e r and arrested metamorphosis of the host (54). Again, i n j e c t i o n of exogenous 20-hydroxyecdysone reversed the developmental a r r e s t . In t h i s case, administration of prothoracicotropic hormone caused a reactivation of the prothoracic glands i n the treated host. These r e s u l t s showed that the virus material i n h i b i t e d the synthesis or secretion of prothoracicotropic hormone and also lowered the ecdysteroid l e v e l i n the host. Also i n t h i s case, a mixture of both venom and calyx f l u i d were needed to obtain the f u l l prolongation of the l a r v a l stage i n the host (110). Other developmental e f f e c t s caused by a symbiotic virus from a parasitoid may involve the n u t r i t i o n a l physiology of the host. An increase i n trehalose l e v e l s i n the hemolymph of Η· virescens parasitized by M. croceipes could be duplicated by the i n j e c t i o n of the calyx f l u i d from the parasitoid (111). Though there i s no known explanation f o r the increase i n trehalose, i t i s proposed to r e s u l t from the release of glucose that i s catabolized to trehalose i n the host (13). An increase i n the host hemolymph trehalose l e v e l may have a n u t r i t i o n a l benefit f o r M. croceipes which i s a hemolymph feeder. From t h i s review we see that symbiotic viruses from the braconids and the ichneumonids have some differences i n their physical structures and their apparent interaction with the host. Some variations noted i n the action of these viruses may eventually be correlated with differences i n v i r a l structures and the interaction of the viruses with venom components. Certain host tissues may be susceptible to one virus-type and r e f r a c t i v e to the other virus-type. I t i s possible that ichneumonids, which are commonly known as tissue feeders, contain symbiotic viruses that have a more prominent e f f e c t on the endocrine system of the host, thereby preserving the host t i s s u e . In comparison, braconids, which

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are commonly known as hemolymph feeders, may contain symbiotic viruses that contribute more to a l t e r a t i o n s i n the composition of the host hemolymph that would benefit the parasitoid. Other types of virus material have been recorded from p a r a s i t i c hymenopteran species. V i r a l p a r t i c l e s of parasitoid o r i g i n were reported i n tissues of the /L. suspensa superparasitized by the s o l i t a r y endoparasitoid B. longicaudatus (112). Rhabdoviruses were found i n vesicles near the basement membrane of the host epidermal c e l l s and pox viruses were found i n hemocytes adjacent to the epidermis (Lawrence, P. 0.; Akin, D. Canad. J . Zool., i n press). The rhabdoviruses were proposed to a f f e c t migration of vesicles to the c e l l apices and the a c t i v a t i o n of the molting f l u i d . The function of the pox virus i s not yet known but could relate to the changes i n the hemolymph l e v e l s of ecdysteroids, JH, and JH esterases (53). A long nonoccluded filamentous virus, morphologically d i s t i n c t from the polydnaviruses, was characterized from the reproductive t r a c t of the parasitoid C. marginiventris. Though the role of the filamentous virus i n the parasitoid-host interaction i s unknown, the virus was reported to r e p l i c a t e i n hypodermal and tracheal matrix c e l l s of the host larvae (113). Secretions from Glands and Other Specialized Tissues. In general, p a r a s i t i c Hymenoptera are known to i n j e c t secretions from glands and specialized tissues into their hosts. Those secretions, except f o r the symbiotic viruses and virus material or calyx f l u i d , are c o l l e c t i v e l y presented i n the next two sections as venomous substances. Paralyzing Venomous Substances. The best characterized regulatory factors at present are a group of substances isolated from the venom of the wasps of the p a r a s i t i c genus Bracon (= Haborbracon, = Microbracon), and the predatory sphecids Philanthus trangulum and Sceliphron caemantarium (114), and s c o l i i d Megascolia f l a v i f r o n s (115). These species cause paralysis i n their hosts or prey that varies from complete and permanent paralysis, which causes immediate cessation of feeding, to a transient or an incomplete paralysis. Though these venoms act on the nervous system of the host or prey, the active ingredients vary considerably. The chemical nature of the active ingredients range from large highly l a b i l e proteins with molecular weights of 43 kd to 56 kd found i n the braconid species, and short chain peptides (kinins) found i n the s c o l i i d species, to low molecular weight substances l i k e the polyamines found i n the sphecid species (116). Piek and Spanjer (114) l i s t members of the families Braconidae and Ichneumonidae having paralyzing venoms. Current a r t i c l e s are available on t h i s area (12, 115, 117, 118) and no further d e t a i l w i l l be given i n t h i s report. Nonparalyzing Venomous Substances. Often the i n j e c t i o n of the virus material and nonparalyzing venomous substances occur simultaneously so that the e f f e c t s are not e a s i l y separated. Reference i s made i n t h i s section to reports where venomous substances were shown to perform a function independent of symbiotic

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viruses, or no reference was made i n the report to the presence of virus material. A d i v e r s i t y of functions and a variety of developmental abnormalities have been associated with nonparalyzing venomous substances. Of the four sources of regulatory factors reviewed i n Table I, venomous substances are common to species i n a l l the families represented, by ecto- and endoparasitoids a l i k e , and the only source of regulatory substances i n ectoparasitoids known to a f f e c t the development of the host. Growth of H. virescens was arrested following parasitism by C. sonorensis (88). The mechanism for the i n h i b i t i o n i n host weight gain was unknown, but the e f f e c t could be reproduced by i n j e c t i o n s of aqueous extracts of the l a t e r a l oviducts of the female parasitoid. These contained protein but no RNA or DNA material and was inactivated by heat and exposure to organic solutions or protease action. This evidence strongly suggests the arrest of weight gain i n the host was i n response to the i n j e c t i o n of a proteinacious substance secreted by the l a t e r a l oviducts. Vinson (88) proposed the primary purpose f o r arrested weight gain was to reverse the flow of nutrients from organs of the host to the hemolymph that a i d the n u t r i t i o n of the developing p a r a s i t o i d . The scelionid egg parasitoid Telenomus h e l i o t h i d i s interrupts egg development i n i t s host R. virescens. A regulatory substance, apparently produced and secreted by the exocrine c e l l s of the common oviducts of the parasitoid, was responsible f o r the rapid arrestment of host embryogensis (77). Necrosis of host tissue i s also associated with parasitism. However, i f oviposition was interrupted prior to egg deposition, the r e s u l t i n g pseudoparasitized host exhibited arrestment of embryogenesis but no tissue necrosis. Apparently i n t h i s case the secreted substance was not associated with restructuring of host tissue f o r the n u t r i t i o n of the parasitoid. Also, the mode-of-action and the nature of t h i s regulatory substance i s unknown. Results from an early study by Shaw (119) indicated that stinging by the s o l i t a r y endoparasitic braconid Clinocentrus g r a c i l i p e s of i t s host, Anthophila f a b r i c i a n a was separable from oviposition. Stinging alone caused temporary paralysis followed by reduced feeding, precocious molting, incomplete pupal formation and death of the host. The e f f e c t s were attributed to the presence of a nonparalyzing venom from the p a r a s i t i c wasp. Proteins from the venom gland of the egg-larval parasitoid, C. near curvimaculatus are injected into the host within the f i r s t eight seconds of the oviposition period (120). These proteins remain intact i n the host f o r two days at which time they are rapidly degraded prior to hatch of the parasitoid egg. The exact role of these proteins remains unknown. Interesting i s the finding that these proteins did not cause the precocious metamorphosis followed by arrested development observed i n parasitized T. n i (120, 121). Another regulatory substance, injected a f t e r the venom proteins but before the parasitoid egg, caused the developmental redirection of host t i s s u e . The source and nature of t h i s substance i s unknown but i t i s proposed to be d i f f e r e n t from calyx f l u i d and virus material, also associated with t h i s parasitoid, and injected

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at a time d i s t i n c t from the i n j e c t i o n of other regulatory substances. In some parasitoid-host systems certain material, which would come under the heading of venomous substances as used i n t h i s chapter, may play a role i n suppression of the immunological response of the host against the parasitoid by acting as a nonreactive egg coating. Examples of these are the mucinous accessory gland secretions of the ichneumonid, Pimpla t u r i o n e l l a (122-124), and material from the venom gland of C. glomerata. This material protects the parasitoid egg without i n h i b i t i n g the encapsulation a b i l i t y of the host (125). The parthenogenetic ichneumonid V^. canescens secretes a particulate material onto the surface of the egg as i t passes through the calyx. The material, which contained antigenic determinants similar to a protein component of the host Ephestia kuehniella, formed a physical covering for the egg (126, 127). The observed s i m i l a r i t y between proteins of the secreted material and a host protein suggested the secreted protein could simply mask the egg surface and prevent the egg from being recognized as a foreign body. In t h i s way the parasitoid protein provided passive protection from the host immune system similar to the fibrous layers found on eggs of Ç. nigriceps. Though the protein occurred i n a particulate structure that gave i t the appearance of the virus material i t was found to contain glycoprotein but no detectable amount of nucleic acids. In other parasitoid-host systems certain material apparently interferes with the a c t i v i t y of the host hemocytes. Examples of these are the accessory genital gland secretions of P. t u r i o n e l l a , which i n h i b i t s capsule-forming hemocytes (128), and accessory gland secretions of the cynipid Leptopilina heterotoma, which causes deterioration of the hemocytes (129). Careful documenting of the i n h i b i t i o n of molting ( i . e . , arrest of apolysis and ecdysis) i n parasitized lepidopteran hosts i s an increasingly important area of study (114, 119, 130-133). Members of the Eulophidae family that are i n the genus Euplectrus and some Eulophus species produce a venom that causes t h i s s p e c i f i c type of response i n their hosts. In contrast, most a l l other hymenopteran ectoparasitoids (e.g., Braconidae, Ichneumonidae, Eulophidae, and Bethylidae) of lepidopteran and coleopteran larvae paralyze t h e i r hosts (134-136). The development of Orthosia s t a b i l i s was arrested after stinging by the gregarious ectoparasitoid Eulophus larvarum. During the period of arrested development, the host was recorded as mobile and continued to feed for a period of time but f a i l e d to produce a new c u t i c l e or i n i t i a t e apolysis (119). This e f f e c t was attributed to a venomous substance, presumably transferred from the parasitoid to the host at the time of stinging, and was shown to be independent of oviposition or the developing parasitoid. Euplectrus sp. are naturally gregarious ectoparasitoids that develop on noctuid and geometrid larvae (137-139). Euplectrus p u t t l e r i and Euplectrus kuwanae are host s p e c i f i c (140, 141). In contrast, Euplectrus plathypenae has several lepidopteran host species (142, 143). A l l three species arrest the development of t h e i r hosts. Recent studies have shown that Euplectrus comstockii also arrests the development of i t ' s hosts (Coudron, Τ. Α., USDA,

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ARS, unpublished data). Euplectrus sp. i n h i b i t host ecdysis once stinging ( i . e . , puncture with the ovipositor), oviposition ( i . e . , egg deposition), or parasitism ( i . e . , stinging and oviposition) has occurred (132). Molt i n h i b i t i o n usually occurs without host paralysis and prevents shedding of the c u t i c l e , which i s the s i t e of attachment of the parasitoid*s eggs. Contact with the host by parasitoid eggs or developing parasitoid larvae i s not necessary f o r the expression of the molt arrest e f f e c t . Instead, Euplectrus sp. transfer an arrestment substance to the host at the time of stinging that acts as a potent bioregulator (132, 133). The recording of superparasitism by the s o l i t a r y endoparasitoid 15. longicaudatus i n puparia of A^. suspensa (112, 144) i s the only other instance of arrestment of apolysis and ecdysis of host larvae following parasitism by an entomophagous insect i n a family other than Eulophidae. The presence of viable parasitoids was required f o r t h i s effect to be manifested and the added stress of the additional parasitoid load ( i . e . , superparasitism) was thought to cause the observed arrestment and not a venom from the parasitoid alone. Venom from E. plathypenae arrested molting i n 44 species of Lepidoptera, plus 19 species i n s i x other orders. The arrestment was expressed i n a l l natural hosts and i n most, but not a l l , insects outside the natural host range of the parasitoid (132). The active substance has been located i n hemolymph of parasitized hosts, i n a gland/reservoir complex isolated from the female ectoparasitoid, and found associated with the f l u i d and c r y s t a l l i n e structures within the gland/reservoir complex (133, 145). The developmental arrest could result from disrupting events normally under endocrine control (130, 131, 146, Kelly, T. J . ; Coudron, T. A. J . Insect Physiol., i n press). However, treatment of parasitized larvae with exogenous JH, 20-hydroxyecdysone, and several hormone analogs did not restore the normal molting process i n the parasitized host (133, 145). Molting was arrested even when parasitism occurred after the release of endogenous molting hormone (133). Clearly, t h i s system offers a new and i n t r i g u i n g area f o r research on the regulation of insect development and parasitoid-host interactions. Future Exploitation C l a s s i c a l b i o l o g i c a l control involves the action of b e n e f i c i a l organisms i n the suppression of pest insects. A wider view of b i o l o g i c a l control has evolved to embrace attempts to develop improved strains of b e n e f i c i a l organisms through controlled selection and genetic manipulation of species. Improved strains are targeted at increasing the suppression phenomenon, widening the host range, and reducing the lapse of time between parasitism, or i n f e c t i o n , and cessation of host feeding. This has enhanced our e f f o r t s to understand desirable attributes of bénéficiais and has brought on a search to i d e n t i f y the b i o l o g i c a l substances ( i . e . , bioregulators) that are responsible f o r the regulation of molecular and c e l l u l a r processes involved i n growth, development, and behavior of beneficial and harmful insects. Understanding the mechanisms used by p a r a s i t i c Hymenoptera i n regulating their host physiology may provide valuable information i n t h i s direction (147).

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The f i r s t step i s the determination of desired t r a i t s controlled by bioregulators. Second i s the chemical characterization and elucidation of the mode-of-action of the natural substances that regulate the development of pest insects. Thirdly i s the refinement of techniques to modify and regulate the expression of the substance at the c e l l u l a r l e v e l . Over the past decade genetic engineering has developed from the advances made i n molecular biology and biotechnology areas. This technology has been applied to the production of transgenic plants that are r e f r a c t i v e to extensive insect damage, and to transgenic microbials that are designed to produce desirable b i o l o g i c a l materials. These powerful tools of the molecular g e n e t i c i s t s could make substantial and rapid contributions to b i o l o g i c a l pest c o n t r o l . Applications of such contributions may include the transfer of desirable bioregulators to other b e n e f i c i a l organisms to enhance effectiveness or improve desirable q u a l i t i e s ( i . e . , speed of k i l l ) and may lead to the production of new substances that influence unique insect biochemical processes. Such contributions may take the form of insect transformations through the use of P-elements (148) and plasmids (149), or the incorporation of bioregulators into the genome of plants consumed by phytophagous insects. Refer to Kuhlemeier et a l . (150), McCabe et a l . (151) and Benfey and Chua (152) for recent reviews on the incorporation and regulation of foreign genes i n plant tissues. I t may be possible to use vectors to produce large quantities of desirable substances that can then be formulated for d i r e c t use as naturally derived pesticides. Refer to Luckow and Summers (153) and Maeda (154) for recent reviews on the use of insect baculoviruses as vectors for expression of foreign DNA. Presently, concerns over insect resistance to synthetic i n s e c t i c i d e s (155) has grown to include concerns over resistance to transgenic organisms that are designed s p e c i f i c a l l y to replace the use of synthetic chemicals (156). One potential solution for avoiding resistance to transgenic organisms i s the use of many gene t r a i t s , each of which encodes for a natural bioregulator. The r a t e - l i m i t i n g step to t h i s solution i s the determination of characters that are desirable for manipulation and the i d e n t i f i c a t i o n of the genes that express for those substances with desirable t r a i t s . The regulatory factors produced by p a r a s i t i c Hymenoptera have promising applications i n t h i s area. Though p a r t i a l descriptions can be given for the e f f e c t s of these regulatory factors on the host insect, i t i s apparent that much of t h e i r function remains unclear. Chemical characterization i s incomplete for most of these factors and a thorough understanding of their e f f e c t s , i n d i v i d u a l l y and i n combination, has yet to be detailed. However, the protein nature and independent action of several venomous substances make them amenable to approaches that involve genetic engineering technology. The synthesis and expression of genes encoding i n s e c t i c i d a l proteins derived from scorpion venoms have been reported (157, 158). Enriched genetic material (RNA and DNA) coding for these substances can be i s o l a t e d from s p e c i f i c tissues known to produce the active substances or cDNA can be constructed that encodes for the active substance. The genomic material can be transferred into microbials

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(both symbiotic or pathogenic bacteria and viruses) which can be used as vectors to transport and express the substances i n pest insects. Transgenic plants also could be constructed to produce the active substances d i r e c t l y within the s p e c i f i c plant tissue that would be consumed by the pest insect. The potential f o r constructing a r t i f i c i a l pathogens to act as delivery systems of these active substances i s also under consideration. These approaches create a basis f o r the construction of an i n s e c t i c i d a l method f o r the use of naturally occurring substances with desirable a t t r i b u t e s . Genes encoding f o r fast-acting venoms could greatly diminish the lapse of time between parasitism by a b i o l o g i c a l control agent and the r e s u l t i n g cessation of host feeding. Gene products should be selected that have minimal mammalian t o x i c i t y . Certain paralyzing toxins from the venoms of the s c o l i i d wasps are now known to act as blockers of the synthesis of acetylcholine by preventing the access of choline to the presynaptic terminals (115, 116, 118). Unfortunately i t i s generally accepted that t h i s system i s s i m i l a r i n insects and vertebrates. In contrast, an i n s e c t i c i d a l neurotoxin, found i n the venom of the scorpion Androctonus a u s t r a l i s , i s s e l e c t i v e l y toxic to several species of insects but harmless to crustaceans, arachnids and mammals (159). The venom from the Eulophid species i s also fast-acting (133) and i t appears to function on a physiological process ( i . e . , ecdysis) that i s more unique to arthropods. These q u a l i t i e s may make the active toxin i n the Eulophid venom an a t t r a c t i v e alternative to the paralyzing toxins. The delicate interplay between parasitoid and host development o f f e r s a wealth of opportunities f o r future e x p l o i t a t i o n . Avenues for exploitation w i l l expand as we continue to characterize the biochemical parameters contributing to the parasitoid-host relationships. A strategy of examining and exploiting these s p e c i f i c interactions could lead to s i g n i f i c a n t new advances i n p r a c t i c a l b i o l o g i c a l control measures. Such advances may include new pest control measures targeted at physiological and metabolic pathways s p e c i f i c to insects. An understanding of these interactions also may a i d i n the structuring of more e f f e c t i v e compounds that mimic or antagonize the e f f e c t s of the b i o l o g i c a l compounds produced by b e n e f i c i a l agents and a i d i n the production of pharmacologically active agents that are environmentally acceptable for use to control pest insects. Acknowledgments I sincerely thank Mr. Benjamin Puttier f o r h i s encouragement and many helpful comments, and Drs. N. Beckage, D. Dahlman, B. Lanzrein, P. Lawrence and M. Strand f o r providing material i n press and their discussions on t h i s subject. The author g r a t e f u l l y recognizes Drs. B. Hammock, D. Jones, P. Lawrence and D. S t o l t z f o r t h e i r review of the text, Karen Ridgwell f o r her assistance i n preparing the manuscript, and USDA, ARS f o r supporting the preparation of t h i s document.

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60 Literature Cited 1. 2. 3. 4.

5.

Downloaded by COLUMBIA UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch004

6. 7. 8. 9. 10. 11.

12.

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Askew, R. R. In P a r a s i t i c Insects; American Elsevier Press: New York, 1971; pp 316. Townes, H. In The Genera of Ichneumonidae; Mem. Ann. Entomol. Inst. 1969; Part I, No. 11. Lawrence, P. O. J . Insect Physiol. 1986, 32, 295-8. Vinson, S. B. In Evolutionary Strategies of Parasitoids and Their Hosts; Price, P., Ed.; Plenum Press: New York, 1975; pp 14-48. Beckage, Ν. E. In Molecular and C e l l u l a r Biology; Baker, R.; Dunn, P., Eds., UCLA Symposium New Series Vol. 112; Alan R. L i s s : New York, 1989; pp 497-515. Vinson, S. B. ISI Atlas Science, Animal and Plant Science 1988, 1, 25-32. Jones, D.; Jones, G.; Van Steenwyk, R. Α.; Hammock, B. D. Ann. Ent. Soc. Am. 1982, 75, 7-11. Strand, M. R.; Vinson, S. B.; Nettles, J r . , W. C.; Xie, Ζ. -N. Entomol. Exp. Appl. 1988, 46, 71-78. Strand, M. R.; Dover, B. A.; Johnson, J . A. Arch. Insect Biochem. Physiol. 1990, 113, 41-51. S t o l t z , D. B. J . Insect Physiol. 1986, 32, 347-50. van Marle, J . ; Piek, T. In Venoms of the Hymenoptera: Biochemical, Pharmacological and Behavioral Aspects; Piek, T., Ed.; Academic: New York, 1986; Chapter 2. Piek, T. In Venoms of the Hymenoptera: Biochemical, Pharmacological and Behavioral Aspects; Academic Press: New York, 1986; Chapter 1. Vinson, S. B.; Dahlman, D. L. Southwest Entomol. 1989, Suppl. 12, 17-37. G u i l l o t , F. S.; Vinson, S. B. J . Insect Physiol. 1972, 18, 1315-21. Tanaka, T. J . Insect Physiol. 1987, 33, 413-20. S t o l t z , D. B.; Guzo, E. R.; Belland, C. J . ; L u c a r o t t i , C. J . ; MacKinnon, E. J . Gen. V i r o l . 1988, 69, 903-7. Fisher, R. C. B i o l . Rev. 1971, 46, 243-78. Vinson, S. B.; Iwantsch, G. F. Q. Rev. B i o l . 1980, 55, 143-65. Thompson, S. N. Comp. Biochem. Physiol. 1983, 74B, 183-211. Beckage, Ν. E. Ann. Rev. Entomol. 1985, 30, 371-413. Salt, G. Proc. Royal Soc. London 1973, 183, 337-50. Lynn, D. C.; Vinson, S. B. J . Invertebr. Pathol. 1977, 29, 50-5. Thompson, S. N. Ann. Rev. Entomol. 1986, 31, 197-219. Thompson, S. N. J . Insect Physiol. 1986, 32, 421-3. Ferkovich, S. M.; D i l l a r d , C. R. Insect Biochem. 1986, 16, 337-45. Jones, D.; Barlow, J . S.; Thompson, S. N. Experimental Parasitology 1982, 54, 340-51. Tilden, R. L.; Ferkovich, S. M. Insect Biochem. 1987, 17, 783-92. Thompson, S. N. J . Insect Physiol. 1980, 26, 505-9. Beckage, N. E.; Nesbit, D. J . ; Nielson, B. D.; Spence, K. D.; Barman, Μ. A. E. Arch. Insect Biochem. Physiol. 1989, 10, 29-45.

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30. 31.

61

Hayakawa, Y. Fed. European Biochem. Soc. 1986, 195, 122-4. Smilowitz, Z.; Smith, C. L. Ann. Entomol. Soc. Amer. 1977, 70, 447-54. 32. Kawai, T.; Maeda, S.; Ono, H.; Kai, H. J . Fac. Agric. T o t t o r i Univ. 1983, 18, 18-26. 33. Beckage, N. E.; Templeton, T. J . J . Insect Physiol. 1986, 32, 299-314. 34. Smilowitz, Z. Ann. Entomol. Soc. Amer. 1973, 66, 93-9. 35. Thompson, S. N. Parasitology 1982, 84, 491-510. 36. Ferkovich, S. M.; Greany, P. D.; D i l l a r d , C. R. J . Insect Physiol. 1983, 29, 933-43. 37. Fisher, R. C.; Ganesalingam, V. K. Nature, 1970, 227, 191-2. 38. King, P. E.; Rafai, J . J . Exp. B i o l . 1970, 53, 245-54. 39. Jones, D. Insect Biochem. 1989, 19, 445-55. 40. Gupta, A. P. Insect Hemocytes; Cambridge Univ. Press: New York, 1979; p. 614. 41. Boman, H. G.; Hultmark, D. Trends Biochem. S c i . 1981, 6, 306-9. 42. Dunn, P. E. Ann. Rev. Entomol. 1986, 31, 321-9. 43. Boman, H. G., Hultmark, D. Ann. Rev. Micro. 1987, 41, 103. 44. Salt, G. B i o l . Rev. 1968, 43, 200-32. 45. Salt, G. Proc. Royal Soc. London 1980, 207, 351-3. 46. Davis, D. H.; Vinson, S. B. J . Insect Physiol. 1987, 32, 1003-10. 47. Norton, W. M.; Vinson, S. B. J . Invertebr. Pathol. 1977, 30, 55-67. 48. Kitano, H. XVII Int. Congress Entomol. 1984, Abstr. S4.9, 19. 49. Grossniklaus-Burgin, C.; Lanz, F.; Lanzrein, B. In Endocrinological Frontiers i n Physiological Insect Ecology; Seknal, F.; Zabza, Α.; Denlinger, D. L., Eds.; Wroclaw Tech. Univ. Press: Wroclaw, 1988; pp 437-441. 50 . Lawrence, P. O. In Endrocrinological Frontiers i n Physiological Insect Ecology; Sehnal, F.; Zabza, Α.; Denlinger, D. L., Eds.; Wroclaw Tech. Univ. Press: Wroclaw, 1988; pp 432-435. 51. Jones, D. Ann. Ent. Soc. Amer. 1985, 78, 141-8. 52. Beckage, N. E.; Riddiford, L. M. Gen. Comp. Endocr. 1982, 47, 308-22. 53. Lawrence, P. O.; Baker, F. C.; T s a i , L. W.; M i l l e r , C. Α.; Schooley, D. Α.; Geddes, L. G. Arch. Insect Biochem. Physiol. 1990, 13, 53-62. 54. Tanaka, T.; Agui, N.; Hiruma, K. Gen. Comp. Endocrin. 1987, 67, 364-74. 55. Dahlman, D. L.; Coar, D. L.; K o l l e r , C. N.; Neary, T. J . Arch. Insect Biochem. Physiol. 1990, 13, 29-39. 56. Racioppi, J . V.; Dahlman, D. L. Comp. Biochem. Physiol. 1980, 67C, 35-9. 57. Dover, Β. Α.; Davies, D. H.; Vinson, S. B. J . Invertebr. Pathol. 1988, 51, 80-91. 58. Dover, Β. Α.; Davies, D. H.; Vinson, S. B. Arch. Insect Biochem. Physiol. 1988, 8, 113-26. 59. Dover, Β. Α.; Strand, M. R.; Davies, D. H.; Vinson, S. B. I n t . J . Insect Morphol. & Embryol. 1989, 18, 47-57.

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62 60.

61. 62. 63. 64. 65. 66.

Downloaded by COLUMBIA UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch004

67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83.

84. 85. 86. 87. 88. 89. 90. 91. 92. 93.

NATURALLY OCCURRING PEST BIOREGULATORS Schooley, D. Α.; Judy, K. J . ; Bergot, J . ; H a l l , M. S.; Jennings, R. C. In The Juvenile Hormones; G i l b e r t , L. I., Ed.; Plenum Press: New York, 1976; p 101-17. Schopf, A. Entomologia Exp. Appl. 1984, 36, 265-72. M e l l i n i , E. Mem. Soc. Ent. I t a l . 1969, 48, 324-50. M e l l i n i , E. B o l l . I s t . Ent. Univ. Bologna 1975, 31, 165-203. Divakar, B. J . Experentia 1980, 36, 1332-3. McNeil, J . Science 1975, 189, 640-2. Ascerno, M. E.; Smilowitz, Z.; Hower, A. A. Environ. Entomol. 1980, 9, 262-4. Outram, I. Environ. Entomol. 1974, 3, 361-3. Vinson, S. B. J . Econ. Entomol. 1974, 67, 335-7. Beckage, Ν. E.; Riddiford, L. M. J . Insect Physiol. 1982, 28, 329-34. Smilowitz, Z.; Martinka, C. Α.; Jowyk, E. A. Environ. Entomol. 1976, 5, 1178-82. Fashing, N. J . ; Sagan, H. Ann. Entomol. Soc. Amer. 1979, 8, 816-8. Webb, Β. Α.; Dahlman, D. L. J . Insect Physiol. 1986, 32, 339-45. C a l s - U s c i a t i , J . C. R. Acad. S c i . (Paris). Ser. D. 1969, 269, 342-4. C a l s - U s c i a t i , J . C. R. Acad. S c i . (Paris). Ser. D. 1975, 281, 275-8. Lawrence, P. O. Expl. Parasit. 1982, 53, 48-57. Lawrence, P. O. In Biology of the P a r a s i t i c Hymenoptera; Gupta, V.; Brill, E. J . , Eds.; Amsterdam, 1988; pp 351-366. Strand, M. R.; Meola, S. M.; Vinson, S. B. J . Insect Physiol. 1986, 32, 389-402. Vinson, S. B.; Lewis, W. J . J . Invertebr. Pathol. 1973, 22, 351-5. Hashimoto, K.; Kitano, H. Zool. Mag. 1971, 80, 323. Vinson, S. B. J . Invertebr. Pathol. 1970, 16, 93-101. Strand, M. R.; Quarles, J . M.; Meola, S. M.; Vinson, S. B. In Vitro C e l l and Dev. B i o l . 1985, 21, 361-7. Hagen, K. S. In B i o l o g i c a l Control of Insect Pests and Weeds; DeBach, P., Ed.; Reinhold: New York, 1964; Chapter 7. Ivanova-Kasas, O. M. In Developmental Systems: Insects; Counce, S. J . and Waddington, C. H., Eds.; Academic Press: New York, 1972; pp 243-271. Joiner, R. L.; Vinson, S. B.; Benskin, J . B. Nature 1973, 245, 120-1. Zhang, D.; Dahlman, D. L. Arch. Insect Biochem. Physiol. 1989, 12, 51-61. Kitano, H. B u l l . Tokyo Gakuge Univ. 1969, 21, 95-136. Kitano, H. J . Insect Physiol. 1974, 20, 315-27. Vinson, S. B. J . Insect Physiol. 1972, 18, 1509-14. S t o l t z , D. B.; Vinson, S. B. Adv. Virus Res. 1979, 24, 125-71. K r e l l , P. J . Can. J . Microbial. 1987, 33, 176-83. Fleming, J . G. W.; B l i s s a r d , G. W.; Summers, M. D.; Vinson, S. B. J . V i r o l . 1983, 48, 74-8. Blissard, C. W.; Fleming, J . G. W.; Vinson, S. B.; Summers, M. D. J . Insect Physiol. 1986, 32, 351-9. Theilman, D. Α.; Summers, M. J . Gen. V i r o l . 1986, 67, 1961-9.

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94. 95.

Federici, B. A. Proc. Natl. Acad. S c i . USA 1983, 80, 7664-8. S t o l t z , D. B.; K r e l l , P.; Summers, M. D.; Vinson, S. B. Intervirology 1984, 21, 1-4. 96. Norton, W. N.; Vinson, S. B.; Thurston, E. L. Proc. Electron Microscopy Soc. Am. 1974, 1984, 140-1. 97. Vinson, S. B.; Scott, J . R. J . Invertebr. Pathol. 1975, 25, 375-8. 98. Edson, Κ. M.; Vinson, S. B.; S t o l t z , D. B.; Summers, M. D. Science 1981, 211, 582-3. 99. S t o l t z , D. B.; Guzo, D. J . Insect Physiol. 1986, 32, 377-88. 100. Vinson, S. B.; S t o l t z , D. B. Ann. Entomol. Soc. Am. 1986, 79, 216-8. 101. Davies, D. H.; Strand, M. R.; Vinson, S. B. J . Insect Physiol. 1987, 33, 143-53. 102. S t o l t z , D. B.; Cook, D. I. Experientia 1983, 39, 1002-4. 103. Soderhall, K. Dev. Comp. Immunol. 1982, 6, 601-11. 104. R a t c l i f f e , Ν. Α.; Leonard, C.; Fowley, A. F. Science 1984, 226, 557-9. 105. Tanaka, T. Entomophaga 1987, 32, 9-17. 106. Tanaka, T. Developmental and Comparative Immunology 1987, 11, 57-67. 107. Cook, D. I.; S t o l t z , D. B.; Vinson, S. B. Insect Biochem. 1984, 14, 45-50. 108. Beckage, Ν. E.; Templeton, T. J . ; Nielsen, B. D.; Cook, D. I.; S t o l t z , D. B. Insect Biochem. 1987, 17, 439-55. 109. Dover, Β. Α.; Davies, D. H.; Strand, M. R.; Gray, R. S.; Keeley, L. L.; Vinson, S. B. J . Insect Physiol. 1987, 33, 333-8. 110. Tanaka, T. Ann. Entomol. Soc. Amer. 1987, 80, 520-3. 111. Dahlman, D. L.; Vinson, S. B. J . Invertebr. Pathol. 1977, 29, 227-9. 112. Lawrence, P. O. Ann. Ent. Soc. Am. 1988, 81, 233-9. 113. Styer, E. L.; Hamm, J . J . ; Nordlund, D. A. J . Invertebr. Pathol. 1987, 50, 302-9. 114. Piek, T.; Spanjer, W. In Venoms of the Hymenoptera: Biochemical, Pharmacological and Behavioral Aspects; Piek, T., Ed.; Academic: New York, 1986; Chapter 5. 115. Hue, B.; Piek, T. Comp. Biochem. Physiol. 1989, 93C, 87-9. 116. Piek, T. In Venoms of the Hymenoptera: Biochemical, Pharmacological and Behavioral Aspects; Academic Press: New York, 1986; p 570. 117. Schmidt, J . O. In Ann. Rev. Entomol.; M i t t l e r , T. E., Ed.; Annual Reviews, Inc.: Palo Alto, CA, 1982; V o l . 27, pp 339-68. 118. Piek, T.; Hue, B.; Mony, L.; Nakajima, T.; Pelhate, M.; Yasuhara, T. Comp. Biochem. Physiol. 1987, 87C, 287-95. 119. Shaw, M. R. J . Invertebr. Pathol. 1981, 37, 215-21. 120. Leluk, J . ; Jones, D. Arch. Insect Biochem. Physiol. 1989, 10, 1-12. 121. Jones, D. J . Insect Physiol. 1987, 33, 129-34. 122. Fuhrer, E. Naturwissenschaften 1972, 52, 167-8. 123. Fuhrer, Ε. Z. Parasitenkd 1973, 41, 207-13. 124. Osman, S. Ε.; Fuhrer, Ε. Int. J . Invertebr. Reprod. 1979, 1, 323-32.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

64 125. 126. 127. 128. 129. 130.

131.

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132. 133. 134. 135. 136. 137. 138. 139. 140. 141. 142. 143.

144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155.

156.

NATURALLY OCCURRING PEST BIOREGULATORS Kitano, H. J . Invertebr. Pathol. 1982, 40, 61-7. Feddersen, L.; Sander, K.; Schmidt, O. Experientia 1986, 42, 1278-81. Berg, R.; Schuchmann-Feddersen, I.; Schmidt, O. J . Insect Physiol. 1988, 34, 473-80. Osman, S. Ε. Z. Parasitenkd 1978, 57, 89-100. R i z k i , R. M.; R i z k i , T. M. Proc. Natl. Acad. S c i . U.S.A. 1984, 81, 6154-8. Coudron, T. Α.; Kelly, T. J . In New Concepts and Trends i n Pesticide Chemistry; Hedin, P. Α., Ed.; ACS Symposium Series No. 276, 1985, 319. Uematsu, H.; Sakanoshita, A. Appl. Ent. Zool. 1987, 22, 139-44. Coudron, T. Α.; Puttler, B. Ann. Entomol. Soc. Am. 1988, 81, 931-7. Coudron, Τ. Α.; Kelly, T. J . ; Puttler, B. Arch. Insect Biochem. Physiol. 1990, 13, 83-94. Beard, R. L. Conn. Agric. Exp. Stn. New Haven B u l l . 1952, 562. Puttler, B. Ann. Entomol. Soc. Am. 1963, 56, 857-9. Gordh, G. USDA Technical B u l l . 1976, 1594. Noble, N. S. Science B u l l e t i n , Dept. Agric. New South Wales 1938, 63, 5-27. Neser, S. In Entomology Memoir; Dept. Agric. Tech. Ser.: Republic of South A f r i c a , 1973; p 32. Gerling, D.; Limon, S. Entomophaga 1976, 21, 179-87. Puttler, B.; Gordh, G.; Long, S. H. Ann. Entomol. Soc. Am. 1980, 73, 28-35. Uematsu, H. Appl. Entomol. Zool. 1980, 16, 57-9. Wall, R.; Berberet, R. C. Environ. Entomol. 1974, 3, 744-6. Krombein, Κ. V.; Hurd J r . , P. D.; Smith, D. R.; Burks, B. D. In Catalogue of Hymenoptera i n America North of Mexico; Smithsonian I n s t i t u t e Press: Washington, DC, 1979; v o l . 1. 1420. Lawrence, P. O. J . Insect Physiol. 1988, 34, 603-8. Coudron, T. A. J . C e l l Biochem. 1989, Suppl. 13A, 166. Uematsu, H. Jpn. J . Appl. Ent. Zool. 1986, 30, 55-7. Jones, D. Entomophaga 1986, 31, 153-7. Cooley, L.; K e l l y , R.; Spradling, A. Science 1988, 239, 1121-8. M i l l e r , L. H.; Sakai, R. K.; Romans, P.; Guadz, R. W.; Kantoff, P.; Coon, H. G. Science 1987, 237, 779-81. Kuhlemeier, C.; Green, P. J . ; Chua, N. -H. Ann. Rev. Plant Physiol. 1987, 38, 221-57. McCabe, D. E.; Swain, W. F.; M a r t i n e l l , B. J . ; Christou, P. Bio/Technology 1988, 6, 923-6. Benfey, P. N.; Chua, N. -H. Science 1989, 244, 174-81. Luckow, V. Α.; Summers, M. D. Bio/Technology 1988, 6, 47-55. Maeda, S. Ann. Rev. Entomol. 1989, 34, 351-72. Sparks, T. C.; Hammock, B. D. In Pest Resistance to Pesticides: Challenges and Prospects; Georghiou, G. P.; Saito, T., Eds.; Plenum Press: New York, 1983; pp 615-68. Muller, K. Agrichemical Age 1989, A p r i l , 14-21.

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COUDRON

157. 158. 159.

Host-Regulating Factors and Parasitic Hymenoptera

Carbonell, L. F.; Hodge, M. R.; Tomalski, M. D.; M i l l e r , L. M. Gene 1988, 73, 409-418. Dee, Α.; Belagaje, R. M.; Ward, K.; Chio, E.; L a i , M.-H. T. Bio/technology 1990, 8, 339-342. Zlotkin, E. Insect Biochem. 1983, 13, 219-236. July 2, 1990

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RECEIVED

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 5

Sex

Pheromone Production in Moths

Endogenous Regulation of Initiation and Inhibition Peter E. A. Teal and James H. Tumlinson

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Agricultural Research Service, U.S. Department of Agriculture, Gainesville, FL 32604

Recent studies on a number of moth species have indicated that pheromone production is under endogenous regulation. For many species the diel periodic production period is regulated by a neuropeptide, termed "pheromone biosynthesis activating neuropeptide", which is produced in the brain-subesophageal ganglion complex. This peptide is present in the brain-subesophageal ganglion at a l l times but is released only during specific periods of the photoperiod. This establishes the diel periodicity of pheromone production. Females of some moth species also inhibit the production of sex pheromone by endogenous means. Inhibition usually follows mating and is either the direct effect of a substance passed from the male to the female during mating or an indirect response to mating, in that the female then produces an inhibitory factor. Virgin females of Heliothis zea also produce a pheromone biosynthesis suppression factor as they senesce. The evolutionary significance of this suppression factor may be to ensure that males do not waste reproductive effort on females that will die prior to laying fertile eggs. Over the p a s t t h r e e decades c o n s i d e r a b l e e f f o r t has been d i r e c t e d towards the e l u c i d a t i o n o f the s t r u c t u r e s and b i o l o g i c a l e f f e c t s o f sex pheromones produced by moths. These s t u d i e s have been v e r y f r u i t f u l , as i s e v i d e n c e d by the g r e a t number o f s p e c i e s f o r w h i c h pheromones have been i d e n t i f i e d (1) and by the s i g n i f i c a n t advances i n knowledge about the semiochemical-mediated b i o l o g y o f these i n s e c t s ( 2 ) . As a consequence, moth pheromones a r e now commonplace i n p o p u l a t i o n m o n i t o r i n g programs and a r e b e i n g i n c r e a s i n g l y u s e d as d i r e c t c o n t r o l agents ( 3 ) , w h i c h a r e a p p l i e d p r i n c i p a l l y f o r m a t i n g d i s r u p t i o n . N o n e t h e l e s s , w h i l e so much i s known about the c h e m i s t r y and b e h a v i o r o f moth communication systems, much l e s s i s known about the endogenous mechanisms t h a t r e g u l a t e pheromone p r o d u c t i o n . These mechanisms c o n t r o l when pheromones a r e produced This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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5.

TEAL AND TUMLINSON

Sex Pheromone Production in Moths

and are t h e r e f o r e key components f o r r e p r o d u c t i v e s u c c e s s i n moth species. Females o f many moth s p e c i e s s i g n a l t h e i r r e a d i n e s s t o mate by p r o d u c i n g and r e l e a s i n g sex pheromones d u r i n g s p e c i e s s p e c i f i c times o f the p h o t o p e r i o d ( 4 ) . A t o t h e r times l i t t l e o r no pheromone i s p r e s e n t i n the pheromone g l a n d and none o f the b e h a v i o r s a s s o c i a t e d w i t h pheromone r e l e a s e are e x h i b i t e d . Studies on a number o f moth s p e c i e s have shown t h a t the i n d u c t i o n o f pheromone p r o d u c t i o n i s under endogenous r e g u l a t i o n ( 5 , 6 , 7 ) . The f a c t o r responsible f o r i n d u c t i o n of b i o s y n t h e s i s i s a neuropeptide produced i n the b r a i n - s u b e s o p h a g e a l g a n g l i o n complex. A l t h o u g h p r e s e n t i n t h i s complex a t a l l t i m e s , i t s r e l e a s e i s a p p a r e n t l y under d i e l p e r i o d i c c o n t r o l ( 8 , 9 ) . The s t r u c t u r e o f t h i s p e p t i d e , termed "pheromone b i o s y n t h e s i s a c t i v a t i n g n e u r o p e p t i d e " (PBAN), has r e c e n t l y been e l u c i d a t e d f o r H e l i o t h i s zea (10) and f o r Bombvx m o r i ( 1 1 ) . A l t h o u g h the amino a c i d sequences o f these two forms o f PBAN are somewhat d i f f e r e n t , t h e i r a c t i v i t i e s are the same inasmuch as they b o t h induce the p r o d u c t i o n o f pheromone. The e x a c t mechanism by w h i c h PBAN s t i m u l a t e s the p r o d u c t i o n o f pheromone i s unknown i n these i n s e c t s . Furthermore, r e c e n t s t u d i e s (12) have i n d i c a t e d t h a t a l t h o u g h some moth s p e c i e s , f o r example the cabbage l o o p e r moth, do produce PBAN, t h i s p e p t i d e does not appear t o s t i m u l a t e the i n s e c t s t o produce pheromone. Consequently, the f u n c t i o n o f PBAN i n these s p e c i e s i s unknown. S i m i l a r l y , the r e a s o n f o r the presence o f PBAN i n the b r a i n - s u b e s o p h a g e a l g a n g l i a complexes o f males o f H. v i r e s c e n s i s u n c l e a r because they m a i n t a i n e q u a l l y h i g h t i t e r s o f pheromone d u r i n g b o t h the scotophase and photophase ( 1 3 ) . I n a d d i t i o n to r e g u l a t i o n o f the i n d u c t i o n o f pheromone p r o d u c t i o n , moths and o t h e r i n s e c t groups (14) i n h i b i t the p r o d u c t i o n o f pheromone d u r i n g p e r i o d s when m a t i n g i s not a p p r o p r i a t e . A d u l t females o f some s p e c i e s d e l a y r e p r o d u c t i v e m a t u r i t y a f t e r a d u l t emergence f o r v a r i o u s reasons and do not engage i n r e p r o d u c t i v e a c t i v i t y u n t i l gametes are mature. For example, the s p r i n g and f a l l g e n e r a t i o n s o f P s e u d a l e t i a u n i p u n c t a are m i g r a t o r y and n e i t h e r produce nor r e l e a s e pheromone u n t i l eggs are mature ( 6 ) . Cusson and M c N e i l (6) have shown t h a t these i n s e c t s mediate pheromone p r o d u c t i o n and r e l e a s e v i a j u v e n i l e hormone (JH). Thus, low l e v e l s o f JH suppress s e x u a l b e h a v i o r w h i l e e l e v a t e d t i t e r s s t i m u l a t e b o t h pheromone p r o d u c t i o n and r e l e a s e b e h a v i o r ( 6 ) . V i r g i n f e m a l e s , a t the peak o f t h e i r r e p r o d u c t i v e p o t e n t i a l , p r o b a b l y r e g u l a t e the i n d u c t i o n and t e r m i n a t i o n o f pheromone p r o d u c t i o n based s o l e l y on the d i e l p e r i o d i c r e l e a s e o f PBAN. However, mated females o f many moth s p e c i e s , i n c l u d i n g the omnivorous l e a f r o l l e r moth, the c o r n earworm moth, tobacco hornworm moth, and the c e c r o p i a moth, s w i t c h from a " v i r g i n " c o n d i t i o n i n which the i n s e c t s r e l e a s e pheromone, t o a "mated" c o n d i t i o n i n d i c a t e d by o v i p o s i t i o n , c e s s a t i o n o f c a l l i n g , and s i g n i f i c a n t r e d u c t i o n i n the amount o f pheromone produced (15,16,17,18,19,20). S t u d i e s on the omnivorous l e a f r o l l e r have i n d i c a t e d t h a t a t o p i c a l l y a p p l i e d j u v e n i l e hormone analogue caused v i r g i n females t o o v i p o s i t a t a h i g h r a t e and a l s o i n h i b i t e d pheromone p r o d u c t i o n (17). I n the c o r n earworm moth the t i t e r o f pheromone i s reduced r a p i d l y a f t e r mating and lower t i t e r s o f pheromone are produced, w i t h r e s p e c t t o v i r g i n s , f o r a t l e a s t two n i g h t s a f t e r m a t i n g ( 4 ) . The immediate r e d u c t i o n i n t i t e r o f

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pheromone a f t e r mating i s p r o b a b l y due t o the t r a n s f e r o f a s u b s t a n c e from the male a c c e s s o r y glands d u r i n g m a t i n g ( 1 8 ) . A somewhat d i f f e r e n t s i t u a t i o n o c c u r s i n the c e c r o p i a moth. Females o f t h i s i n s e c t r e l e a s e a substance, produced by the b u r s a c o p u l a t r i x a f t e r f e r t i l e m a t i n g , which causes a s h i f t from the " v i r g i n " t o the "mated" c o n d i t i o n (16,19). The b e h a v i o r a l s w i t c h by c e c r o p i a moth females i s mediated t h r o u g h the a c t i o n o f a f a c t o r t r a n s f e r r e d from the male t o the female d u r i n g mating. S t r e t c h i n g o f the b u r s a c o p u l a t r i x appears t o t r i g g e r the p h y s i o l o g i c a l change and i t has been e s t a b l i s h e d t h a t c o n t i n u e d n e u r a l communication between the b u r s a and c o r p o r a a l l a t a i s r e q u i r e d f o r maintenance o f the "mated" c o n d i t i o n . A l l o f the above s t u d i e s i n d i c a t e t h a t i n s e c t s r e g u l a t e the p r o d u c t i o n o f pheromone and mating v e r y p r e c i s e l y by u s i n g endogenous f a c t o r s . A l t h o u g h the many f a c e t s o f pheromone m e d i a t e d biology i n c l u d i n g i n i t i a t i o n of production, termination of p r o d u c t i o n and a c t u a l r e l e a s e o f components are t i g h t l y c o o r d i n a t e d , i t i s o b v i o u s t h a t no s i n g l e n e u r a l , n e u r o e n d o c r i n e , o r e n d o c r i n e f a c t o r r e g u l a t e s the complete pheromone p r o d u c t i o n system. F u r t h e r , w h i l e the above s t u d i e s have l a i d the f o u n d a t i o n f o r u n d e r s t a n d i n g how pheromone p r o d u c t i o n i s c o n t r o l l e d , they a l s o r a i s e new q u e s t i o n s w h i c h must be addressed. I n d u c t i o n o f Pheromone P r o d u c t i o n The p i o n e e r i n g s t u d i e s conducted by R a i n a and co-workers (5,8,10) t h a t l e d t o the d i s c o v e r y o f PBAN i n H. zea have p r o v i d e d a u s e f u l t o o l f o r s t u d y i n g the p h y s i o l o g i c a l and b i o c h e m i c a l e v e n t s t h a t r e g u l a t e pheromone p r o d u c t i o n as w e l l as f o r c l a s s i c a l pheromone i d e n t i f i c a t i o n s t u d i e s . C y c l i c a l d a i l y periods during which pheromone i s produced by females o f many moth s p e c i e s i n c l u d i n g H e l i o t h i s c o r r e s p o n d w i t h b o t h the c a l l i n g p e r i o d , as e v i d e n c e d by r e l e a s e o f v o l a t i l e pheromone components ( 2 1 ) , and the p e r i o d o f response o f males ( 2 2 ) . D u r i n g o t h e r times l i t t l e o r no pheromone i s p r e s e n t i n the g l a n d . I n these females, p e r i o d s o f p r o d u c t i o n are a p p a r e n t l y r e g u l a t e d by the d i e l p e r i o d i c r e l e a s e o f PBAN. E v i d e n c e f o r t h i s comes from the f a c t t h a t e i t h e r neck l i g a t i o n o r d e c a p i t a t i o n d u r i n g the photophase i n h i b i t s the p r o d u c t i o n o f pheromone d u r i n g subsequent n i g h t s ( 4 ) , b u t pheromone p r o d u c t i o n can be s t i m u l a t e d i n these i n s e c t s by i n j e c t i o n o f PBAN d u r i n g the photophase ( 7 , 9 ) . We have found t h a t females o f the t h r e e s p e c i e s o f H e l i o t h i s c u r r e n t l y under study i n our l a b o r a t o r y can be i n d u c e d t o produce pheromone by i n j e c t i o n o f PBAN a t times when l i t t l e o r no pheromone i s p r e s e n t i n the g l a n d , f o r example, the mid-photophase ( F i g . 1 ) . T h i s i s o f s i g n i f i c a n c e because i t a l l o w s us t o conduct our s t u d i e s d u r i n g the photophase, p r e c i s e l y t i m i n g each experiment a f t e r the i n j e c t i o n o f PBAN. Our s t u d i e s on H. zea. H. v i r e s c e n s and H. s u b f l e x a have shown t h a t the amount o f pheromone p r e s e n t i n the g l a n d , as i n d i c a t e d by the t i t e r o f the major component, ( Z ) - 1 1 - h e x a d e c e n a l , i n c r e a s e s i n a l i n e a r f a s h i o n f o r a t l e a s t the f i r s t 90 min a f t e r i n j e c t i o n o f PBAN ( F i g . 2 ) . Furthermore, as i n d i c a t e d i n F i g u r e 3, the r a t i o o f components p r e s e n t i n g l a n d e x t r a c t s o b t a i n e d a t 15 min. i n t e r v a l s a f t e r i n j e c t i o n o f PBAN remains c o n s t a n t and i s not d i f f e r e n t from the n a t u r a l l y produced r a t i o . Other s t u d i e s

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TEAL AND TUMLINSON

69

Sex Pheromone Production in Moths

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T I M E (min.) F i g u r e 1. Gas chromatographic a n a l y s i s (Column - 30m χ 0.25mm i d Supelcowax 10) o f the e x t r a c t o f the pheromone g l a n d o f a 3-day-old v i r g i n female o f H e l i o t h i s v i r e s c e n s o b t a i n e d 1 h a f t e r i n j e c t i o n o f 0.5 pmoles o f s y n t h e t i c PBAN i n 10 μΐ o f water d u r i n g t h e photophase ( u p p e r ) , compared t o the e x t r a c t o f a g l a n d o b t a i n e d 1 h a f t e r i n j e c t i o n o f 10 μΐ o f water d u r i n g the t h i r d photophase. Pheromone components a r e : t e t r a d e c a n a l (14:AL, ( Z ) - 9 - t e t r a d e c e n a l (Z9-14:AL), hexadecanal (16:AL), (Z)-7-hexadecenal and ( Z ) - 9 h e x a d e c e n a l (Z7/Z9-16:AL) (no r e s o l u t i o n ) , (Z)-11-hexadecenal ( Z l l 16:AL) and ( Z ) - 1 1 - h e x a d e c e n - l - o l (Z11-16:0H).

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

70

NATURALLY OCCURRING PEST BIOREGULATORS

zea

Κ

virescens

*

κ

subflexa

1 (ng)

80 70 -




I n i t i a l l y Cg - 0 and w i l l i n c r e a s e w i t h time. As a r e s u l t , the e v a p o r a t i o n r a t e w i l l not be p r o p o r t i o n a l t o C and the degree o f d i s p r o p o r t i o n a l i t y w i l l i n c r e a s e w i t h t i m e , i . e . , the l o n g e r the e x p e r i m e n t a l d e t e r m i n a t i o n o f t ^ i s r u n , the l o n g e r w i l l be the c a l c u l a t e d t ^ . T h e r e f o r e , t h i s s i t u a t i o n i s s i m i l a r t o t h a t i n an e n c l o s e d c o n t a i n e r w i t h o u t a i r f l o w even though t h e r e i s some a i r f l o w . For d e t e r m i n a t i o n s o f t ^ t o a d e q u a t e l y r e p r e s e n t h a l f - l i v e s o u t s i d e the g l a s s c o n t a i n e r , a i r f l o w must be h i g h enough t o maintain C at n e g l i g i b l e levels. r

ο

L i m i t a t i o n s on Uses o f N a t u r a l Rubber Septa I f h a l f - l i v e s a r e v e r y l o n g , the dose needed t o produce d e s i r e d e v a p o r a t i o n r a t e s i n m o n i t o r i n g t r a p s may exceed the p r a c t i c a l a b s o r p t i o n l i m i t o f the s e p t a o f about 50-70 mg. A l s o , even i f the a b s o r p t i o n l i m i t i s not exceeded, 50-70 mg i s w a s t e f u l o f an e x p e n s i v e pheromone component. I f h a l f - l i v e s a r e v e r y s h o r t , the e v a p o r a t i o n r a t e w i l l be c h a n g i n g so r a p i d l y t h a t the d e s i r e d range o f e v a p o r a t i o n r a t e s w i l l be too s h o r t l i v e d . Thus, the r u b b e r

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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septum i s n o t an i d e a l CRS f o r 18 carbon a c e t a t e s ( t ^ a t 20° 8,000-15,000 d) n o r f o r 10 carbon a c e t a t e s and a l c o h o l s ( t ^ a t 20°, c a . 5 d and 2 d, r e s p e c t i v e l y ) . Rubber s e p t a g e n e r a l l y p r o v i d e v e r y d e s i r a b l e r e l e a s e r a t e s f o r compounds w i t h v o l a t i l i t i e s i n t h e ranges 12:Ac-16:Ac, 12:0H-16:0H, 1 4 : A l - 1 8 : A l , and 16:H-20:H. Heath e t a l . (10) suggested t h a t some c r y s t a l l i n e compounds may g i v e anomolous r e l e a s e r a t e s . Subsequently, my coworkers and I found t h a t 16:OH c r y s t a l l i z e s from s e p t a l e a v i n g a v i s i b l e s u r f a c e d e p o s i t and the r e l e a s e r a t e was f a s t e r than would be e x p e c t e d from the r e g r e s s i o n l i n e o f E q u a t i o n 4 based on the lower members o f t h i s s e r i e s ( 1 8 ) . T e t r a d e c a n o l d i d n o t show t h i s e f f e c t . S i n c e many Ε isomers o f h e x a d e c e n - l - o l a r e c r y s t a l l i n e , these compounds may a l s o show t h i s e f f e c t . F o r t u n a t e l y most L e p i d o p t e r a n sex pheromones o f 16 c a r b o n a l c o h o l s have t h e Ζ c o n f i g u r a t i o n and do n o t c r y s t a l l i z e from s e p t a . S a t u r a t e d and monoene a l c o h o l s and a c e t a t e s a r e q u i t e s t a b l e i n r u b b e r s e p t a . I n g e n e r a l i n t r a p s i n f i e l d a p p l i c a t i o n s , they n e i t h e r h y d r o l y s e n o r o x i d i z e ( 5 , 2 5 ) . However, s e v e r a l s t u d i e s showed t h a t c o n j u g a t e d d i e n e s i s o m e r i z e i n rubber t o u l t i m a t e l y form an e q u i l i b r i u m m i x t u r e (25-28). A t e q u i l i b r i u m t h e c o n t e n t i s 65-70% EE, 2-5% ZZ and t h e p e r c e n t a g e s o f EZ and ZE a r e comparable to each o t h e r (27, 2 8 ) . The e x a c t v a l u e s depend on t h e p o s i t i o n o f the double bonds. Because non-pheromone isomers may decrease o r p r e v e n t t r a p c a t c h , the u s e f u l l i f e o f a l u r e may be d e t e r m i n e d by t h i s f a c t o r . Complete e q u i l i b r a t i o n o n l y r e q u i r e s about a month (27. 2 8 ) . The r e a s o n f o r the r a p i d i s o m e r i z a t i o n appears t o be a c o m b i n a t i o n o f s u n l i g h t and c a t a l y s i s by the s u l f u r used t o c u r e the r u b b e r (26-28). An a l t e r n a t e s y n t h e t i c - e l a s t o m e r i c septum i s a v a i l a b l e w h i c h reduces the r a t e o f i s o m e r i z a t i o n by about 8 - f o l d (28). Aldehydes a r e n o t e n t i r e l y s t a b l e i n rubber s e p t a . Although c h e m i c a l breakdown o f aldehydes i n rubber s e p t a has n o t been r e p o r t e d , l o s s o f aldehydes from s e p t a as determined by t h e r e s i d u e method i s f a s t e r than l o s s as determined by t h e r a t e o f e v a p o r a t i o n , t h e r e b y i n d i c a t i n g some form o f c h e m i c a l d e g r a d a t i v e l o s s ( 5 ) . The known tendency o f aldehydes t o t r i m e r i z e has been documented i n p o l y e t h y l e n e v i a l s ( 2 9 ) . A l s o aldehydes o x i d i z e i n a i r t o c a r b o x y l i c a c i d s and o t h e r d e g r a d a t i o n p r o d u c t s . C e r t a i n a l d e h y d i c pheromone components have been shown t o form c a r b o x y l i c a c i d s and o t h e r o x i d a t i o n p r o d u c t s i n hexane under f l u o r e s c e n t l i g h t ( 3 0 ) . Aldehydes have been r e p o r t e d t o p e r s i s t f o r as l o n g as 10 weeks i n r u b b e r s e p t a i n t r a p s based on i n s e c t response ( 3 1 ) , b u t s i g n i f i c a n t d e g r a d a t i v e l o s s had p r o b a b l y o c c u r r e d . Aldehydes r e a c t w i t h amines and a t l e a s t one manufacturer o f rubber s e p t a adds amines as s t a b i l i z e r s o f the rubber. The l o s s o f l u r e e f f e c t i v e n e s s from t h i s f a c t o r i s w e l l documented (32., 33) . The amines c a n be removed by p r e - e x t r a c t i n g the s e p t a w i t h an o r g a n i c s o l v e n t ( 3 2 ) . A p p l i c a t i o n s o f Rubber

Septa

For some i n s e c t s , rubber s e p t a have p o t e n t i a l use f o r p o p u l a t i o n c o n t r o l by p e r m e a t i n g t h e a i r w i t h pheromone. F o r many c r o p s i t i s e c o n o m i c a l l y p r a c t i c a l t o p l a c e the CRS on the f o l i a g e by hand l a b o r . Rubber s e p t a c o u l d be used f o r such a p p l i c a t i o n s i f t h e h a l f - l i v e s o f t h e pheromone components were s u i t a b l e f o r t h e

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

NATURALLY OCCURRING PEST BIOREGULATORS

122

i n t e n d e d use. An e q u a t i o n r e l a t i n g the c o n c e n t r a t i o n o f pheromone ( i . e . , the amount a p p l i e d per u n i t a r e a ) , C, needed f o r c o n t r o l as a f u n c t i o n o f the f i r s t o r d e r r a t e c o n s t a n t , k, has been r e p o r t e d ( 3 4 ) . When the minimum e v a p o r a t i o n r a t e per u n i t a r e a r e q u i r e d f o r m a t i n g d i s r u p t i o n i s E , and the r e q u i r e d p e r i o d o f c o n t r o l from one a p p l i c a t i o n i s t , the e q u a t i o n i s : m

Ε exp(kt)

1

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l n 9

or s i n c e k - ~ — , \

Ε t, e x p i t t ' ^ ) C :~ ln2

I n E q u a t i o n 13, the v a l u e o f C as a f u n c t i o n o f t ^ has A t the minimum: t, - t l n 2 ~

(13)

a minimum.

0.7t

C - e E t ~ 2 . 7 E t m m where e i s the base o f n a t u r a l l o g a r i t h m s . Thus, C i s m i n i m i z e d when the h a l f - l i f e i s 0.7t. For CRS p r o v i d i n g h a l f - l i v e s s h o r t e r or l o n g e r t h a n 0.7t more pheromone i s r e q u i r e d , but the i n c r e a s e d requirement i s greater f o r s l i g h t l y s h o r t e r than s l i g h t l y longer half-lives. For t v a l u e s o f 30-100 days, t ^ v a l u e s o f 21-70 days would m i n i m i z e pheromone useage and somewhat l o n g e r t ^ v a l u e s would a l s o be p r a c t i c a l . I t i s o b v i o u s from T a b l e s I , I I , and I I I t h a t many pheromone components i n r u b b e r s e p t a , even a t the t e m p e r a t u r e s e x p e c t e d from d i r e c t exposure t o s u n l i g h t , c o u l d be e f f i c i e n t l y used i n mating d i s r u p t i o n programs. The p r i n c i p a l p r a c t i c a l a p p l i c a t i o n o f r u b b e r s e p t a as a CRS f o r sex pheromones has been i n t r a p s used to m o n i t o r f l i g h t a c t i v i t y o f insect pests. Rubber s e p t a have a l s o been used as a r e s e a r c h t o o l t o s t u d y b e h a v i o r a l responses o f i n s e c t s t o pheromones (35, 36)· W i t h the i n f o r m a t i o n now a v a i l a b l e , i t i s p o s s i b l e t o s p e c i f y b l e n d s o f pheromone components w h i c h w i l l produce d e s i r e d e v a p o r a t i o n r a t e s and v a p o r r a t i o s , and w i t h known changes o f t h e s e r a t e s and r a t i o s w i t h time, temperature and a i r speed f o r a l a r g e number o f i n s e c t s . Thus, more e f f e c t i v e use o f s e p t a i n d e v e l o p i n g m o n i t o r i n g l u r e s and i n b e h a v i o r a l s t u d i e s i s p o s s i b l e . A l s o , i n the f u t u r e , i t i s t o be e x p e c t e d t h a t a p p l i c a t i o n s o f s e p t a f o r m a t i n g d i s r u p t i o n w i l l be found. Literature Cited 1.

2. 3.

Charmillot, P. J. In P r a c t i c a l Use of Insect Pheromones and Other Attractants: Ridgway, R.; S i l v e r s t e i n , R. M.; Inscoe, Μ., Ed.; Marcel Dekker, In press, Chapter 11. Audemard, P. H.; Leblon, C.; Neumann, U.; Marboutie, G.; J . Appl. Entomol. 1989, 108, 191-207. Maitlen, J. C.; McDonough, L. M.; M o f f i t t , H. R.; George, D. Α.; Environ. Entomol. 1976, 5, 199-202.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

8. McDONOUGH 4. 5. 6. 7. 8.

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9. 10. 11. 12. 13. 14. 15.

16.

17. 18. 19.

20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Controlled Release of Insect Sex Pheromones

Golub, M.; Weatherston, J . ; Benn, M. A. J . Chem. Ecol. 1983, 9, 323-33. Butler, L. I.; McDonough, L. M. Unpublished. Butler, L. I.; McDonough, L. M. J . Chem. Ecol. 1979, 5, 825-37. Butler, L. I.; McDonough, L. M. J . Chem. Ecol. 1981, 7, 627-33. Leonhardt, Β. Α.; Moreno, D. S. In Insect Pheromone Technology: Chemistry and Applications; Leonhardt, B. A.; Beroza, M., Ed.; ACS Symposium Series No. 190: Washington D.C., 1982; Chapter 8. Heath, R. R.; Tumlinson, J . H. J . Chem. Ecol. 1986, 12, 2081-88. Heath, R. R.; Teal, P. Ε. Α.; Tumlinson, J . H.; Mengelkoch, L. J . J . Chem. Ecol. 1986, 12, 2133-43. Greenway, A. R.; Davis, S. Α.; Smith, M. C. J . Chem. Ecol. 1981, 7, 1049-56. Baker, T. C.; Carde, R. T.; M i l l e r , J . R. J . Chem. Ecol. 1980, 6, 749-58. McDonough, L. M.; Butler, L. I. J . Chem. Ecol. 1983, 9, 1491-1502. McDonough, L. M.; Brown, D. F.; A l l e r , W. C. J . Chem. Ecol. 1989, 15, 779-90. Z e o l i , L. T.; Kydonieus, A. F.; Quisumbing, A. R. In Insect Suppression with Controlled Release Pheromone Systems: Kydonieus, A. F.; Beroza, M., Ed.; CRC Press: Boca Raton, F l o r i d a , 1982; Chapter 3. Roseman, T. J . ; C a r d a r e l l i , N. F. In Controlled Release Technologies: Methods, Theory, and Applications; A. F. Kydonieus, Ed.; CRC Press: Boca Raton, F l o r i d a , 1980; Chapter 2. Daterman, G. E. USDA Forest Service Research Paper 1974, PNW-180. McDonough, L. M.; Brown, D. F.; A l l e r , W. C. Unpublished. Dal Nogare, S.; Juvet, J r . , R. S. Gas-Liquid Chromatography: Theory and Practice; Interscience Publishers: New York, New York, 1962; Chapter 3. McReynolds, W. O. Gas Chromatographic Retention Data; Preston Technical Abstracts: Evanston, I l l i n o i s , 1966. Olsson, Α.; Jonsson, J . Α.; Thelin, B.; L i l j e f o r s , T. J . Chem. Ecol. 1983, 9, 375-85. Davis, H. G.; Brown, D. F.; McDonough, L. M. Unpublished. Teal, P. Ε. Α.; Tumlinson, J . H.; Heath, R. R. J . Chem. Ecol. 1986, 12, 107-26. Landolt, P. J . ; Heath, R. R. J . Chem. Ecol. 1987, 13, 1005-18. Shani, Α.; Klug, J . T. J . Chem. Ecol. 1980, 6, 875-81. Fugiwara, H.; Sato, Y.; Nishi, K. J . Takeda Res. Lab. 1976, 35, 52-59. Davis, H. G.; McDonough, L. M.; Burditt J r . , A. K.; Bierl-Leonhardt, B. A. J . Chem. Ecol. 1984, 10, 53-61. Brown, D. F.; McDonough, L. M. J . Econ. Entomol. 1986, 79, 922-7. Dunkeblum, E.; Kehat, M.; Klug, J . T.; Shani, A. J . Chem. Ecol. 1984, 10, 421-8.

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30. Shaver, T. N.; Ivie, G. W. J . Agric. Food Chem. 1982, 30, 367-71. 31. F l i n t , H. M.; Butler, L. I.; McDonough, L. M.; Smith, R. L.; Forey, D. E. Environ. Entomol. 1978. 7, 57-61. 32. Steck, W. F.; Bailey, B. K.; Chisholm, M. D.; Underhill, E. W. Environ. Entomol. 1979, 8, 732-3. 33. Kamm, J . Α.; McDonough, L. M. Environ. Entomol. 1979, 8, 773-5. 34. McDonough, L. M. U.S. Dept. of Agriculture/Science and Education Administration/Agricultural Research Results 1978, ARR-W-1/May. 35. Grant, A. L.; Mankin, R. W.; Mayer, M. S. Chem. Senses. 1989, 14, 449-62. 36. Baker, T. C.; Hansson, B. S.; Lofstedt, C.; Lofquist, J . Chem. Senses. 1989, 14, 439-48. R E C E I V E D May

16, 1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 9

Potato Glandular Trichomes Defensive Activity Against Insect Attack

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Ward M. Tingey Department of Entomology, Cornell University, Ithaca, NY 14853-0999

Insect-resistant cultivars offer a realistic and practical foundation upon which to build an economical and environmentally sound crop protection system. Insect resistance of a wild Bolivian potato species is conditioned by the presence of glandular trichomes on its foliage. Resistance is expressed both mechanically and behaviorally. Mechanical resistance involves release of a viscous exudate from the trichome gland upon contact by an insect. The trichome secretions accumulate initially on tarsi, first impeding movement and later entrapping the insect. In addition to immobilization, entrapment and resultant mortality, glandular trichomes dramatically alter normal insect behaviors, particularly those involving host acceptance and feeding. Pest species adversely affected by glandular trichomes include the Colorado potato beetle, green peach aphid, potato leafhopper, potato flea beetle, and spider mites. The mechanical and behavioral defensive properties of glandular trichomes are conditioned by the presence of specialized chemistry in trichome exudate. The Cornell University potato breeding program has introgressed the genetic information for expression of glandular trichomes into germplasm of the cultivated potato. Commercially acceptable levels of resistance have been maintained in 8 successive breeding cycles under development for horticultural adaptation. Elite hybrids can experience greater than 85% reduction in populations of insect pests, compared to those on susceptible commercial cultivars. Insecticide use on resistant hybrids can be reduced by at least 40% of that presently needed on susceptible cultivars. To-date, over 180 wild, tuber-bearing species of Solanum are known (JJ and while many of these have been screened for insect resistance over the past ten years, the first large-scale systematic efforts to examine wild Solanum germplasm for insect resistance were initiated by Ε. B. Radcliffe at the University of Minnesota and reported in a series of papers first appearing in 1968 (2). These studies led to the identification of several excellent sources of resistance to aphids and leafhoppers. Prior to this time, very few species were known to resist attack by 0097-6156/91/0449-0126$06.00/0 © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

9. TINGEY

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major potato insects and in most cases, the underlying plant phenomena conferring resistance were unknown although the presence of steroid glycosides in the foliage of some species had been associated with resistance as early as 1950 by European workers (3-7). One of the wild potato species Radcliffe reported resistant to the potato leafhopper had been described in 1944 from a collecting expedition in Bolivia. J. G . Hawkes, the British potato systematist, described this species, Solanum berthaultii, as follows (8): "Very glandular species with pale violet-blue pentagonal sub-stellate corolla. Almost certainly formed as a natural hybrid between S. tarijense (Commersoniana) and a blue-flowered mountain species, possibly S. sparsipilum. Distribution: Bolivia, eastern slopes of Andes in rather dry valleys amongst bushes and in waste places from 2,100 - 2,700 m." In 1971, R. W. Gibson at the Rothamsted Experimental Station reported that S. berthaultii was resistant to aphids and furthermore that resistance was associated with the presence of glandular trichomes (9). Subsequent work in Britain, the U.S., and Peru has expanded the list of pest species against which S. berthaultii is defended. Currently, this species is known to resist herbivory by at least 10 major groups or species of pests including leaf miner flies, the potato tubermoth complex, aphids, leafhoppers, fleabeetles, mites , and the Colorado potato beetle (10-13). In 1977, research was initiated at Cornell University to systematically explore the nature of insect resistance in this wild potato species and to examine its potential value in management of insect pests. Glandular Trichomes of Solanum berthaultii Resistance of S. berthaultii to insects and mites is associated with the presence of two types of glandular trichomes on foliage, fruiting organs, and stolons. Type A trichomes are about 120 to 210 mu in length and bear a membrane-bound tetralobulate gland about 50 to 70 mu in diameter at their apices (14). Release of secretory material occurs upon mechanical breakage of the gland at its junction with the stalk. The tetralobulate gland is not renewed following rupture. Type Β trichomes, in contrast, are longer (ca. 600 to 950 mu in length) and bear an ovoid droplet of exudate (20 to 60 mu in diameter) at their tips (14). The naked droplets of exudate are extremely adhesive and transfer readily upon contact by an insect. Following mechanical or solvent removal, droplets of type Β exudate are renewed to their original dimensions within 8 days (15). Defensive Biology of Solanum berthaultii Many species of small-bodied pests experience classical responses to host resistance phenomena during an encounter with foliage of S. berthaultii. These include host avoidance and restlessness, reduced feeding, delayed development, reduced adult weight and reproductive performance, increased mortality, and diminished longevity. In several cases, these responses are associated with entrapment or immobilization by trichome exudate. Entrapment/Immobilization. Aphids and leafhoppers experience significant mortality by entrapment in trichome exudate. Initial contact with the foliage leads to an encounter with the tall Type Β hairs which confer a high degree of adhesiveness to the tarsi (Fig. 1). Tarsi coated with this viscous material are very effective in

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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NATURALLY OCCURRING PEST BIOREGULATORS

Contact with Type Β Droplet

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•/ -ι \ Adhesive Properties of Type Β Exudate

Surfactant Properties of CASE

Rupture of Type A Trichome ||pL g

Oxidative Polymerization of Type A Exudate

Exudate-Encased Tarsi & Mouthparts Obstruction of Tarsal & Labial Sensory Receptors Loss in Coordination

10, 11

Cessation of Feeding 12

DEATH

Figure. 1. Nature of glandular trichome-mediated aphid resistance in S. berthaultii. Key: (1) Carboxylic acid sucrose esters (CASE), (2) Viscous type Β trichome exudate, (3) Increased aphid movement and attempts to escape, (4) Adhesive aphid tarsi, (5) Enhanced rupture of type A trichome membrane?, (6) Polyphenoloxidase + 02 + substrate, (7) Aphid alarm pheromone, E (B)=farnesene, (8) Encasement of tarsi by trichome exudate, (9) Greater effective tarsal size, (10) Decreased aphid mobility, (11) Occlusion of mouthparts by trichome exudate, (12) Starvation and death. Adapted from (38).

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breaking the junction between the head and stalk of a Type A trichome thus resulting in release of Type A exudate onto the insect's body (16). Within several hours, the exudate darkens and hardens by virtue of its phenolic oxidation chemistry, immobilizing these insects or severely restricting their movements. The dispersal rate of aphids with exudate-encased tarsi on S. berthaultii and on hybrids is only 1/3 that of aphids with unencased tarsi on nonglandular plants ( I D Trichome exudate also accumulates on the mouthparts and may totally occlude the stylets of insects with sucking mouthparts, thus preventing feeding (18). The defensive activity of S. berthaultii foliage increases along with the densities of both types of trichomes and with increased volume of Type A trichome glands. Young aphids and leafhoppers experience greater mortality from an encounter with glandular trichomes than do adults, probably because of a more limited ability to escape the viscous and adhesive trichome barrier (16). Behavioral and Sensory Disturbance. In addition to entrapment and immobilization, the glandular trichomes of S. berthaultii interfere with host acceptance and condition an avoidance for oviposition and feeding. Agitation and avoidance by aphids is conferred by a complex of sesquiterpenes located primarily in Type A trichomes and released when the gland is broken (19. 20). The aphidimmobilizing potential of trichome exudate is magnified by the presence of these compounds because the resulting behavioral excitation and increased locomotion promote a greater frequency of encounters with undisturbed Type A trichomes. Interestingly, Type A trichomes of some commercial potato cultivars contain high levels of the aphid alarm pheromone, Ε-β-farnesene (20). However, aphid acceptance and feeding behavior on these plants are not adversely affected because the tetralobulate glands on foliage of the cultivated potato do not readily rupture upon contact. Type Β trichome exudate of S. berthaultii also conditions abnormal behaviors in aphids and leafhoppers, particularly delay in host acceptance as measured by an increase in time to the first probe and a decrease in feeding time (Table I) (21. 22). The avoidance/deterrance responses of aphids are conditioned by the presence of sucrose esters of short-chain branched carboxylic acids in the type Β exudate (Table II) (23. 24). Sensory receptors on the tarsi and/or antennae of aphids are the likely target sites of sucrose esters in type Β exudate (24). Table I. Effect of type Β trichome removal on feeding behavior of fourth-instar potato leafhopper nymphs on resistant and susceptible potato clones. Adapted from (22) Clone

Type Β trichomes

Preprobe time (min)

S. tuberosum X S. berthaultii (F3) Intact 14.2 a Removed 11.2 b S. tuberosum Intact 1.2 c 0.6 c Removed a

Total feeding time (min)

% of nymphs feeding

5.7 5.3

b b

25 95

b a

21.0 16.9

a a

100 100

a a

A glass microscope slide was gently pressed to the leaf surface to simulate the conditions used to remove type Β exudate from the F3 hybrid. a

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Coupled with the consequences of entrapment by Type A exudate, (i.e. high mortality, limited population development, and the disabling influence of trichome exudates on locomotion), the avoidance behaviors conditioned by sesquiterpenes in type A trichomes and by sucrose esters in type Β trichomes contribute to the reduced vector efficiency of the green peach aphid for potato virus Y as measured by impaired acquisition and transmission ability (Table ΠΙ) (25. 26). This interplay of several trichome defensive phenomena adds further value to the plant breeding attributes of S. berthaultii because it provides an additional barrier of disease protection for hybrid germplasm carrying genetic resistance to the potato virus Y pathogen. Table II. Deterrence of green peach aphid feeding by sucrose esters from type Β glandular trichomes of S. berthaultii (PI 473331) applied to diet membranes. Adapted from (24) Concentration μg/cm 2

100 33 10 3 1

Λ

No. feeding sheaths/mm Treated Untreated 0.21 0.38 1.31 1.39 1.66

/>a 0.0001 0.0001 0.06 0.33 0.15

2.24 3.36 1.77 1.17 1.30

Probability that sucrose esters have no effect on the distribution of feeding sheaths by G-test, goodness of fit.

a

Table III. Final potato virus Y (PVY) incidence as a percent of exposed target plants for four source plant/target plant combinations of S. tuberosum and S. berthaultii (PI 310927). Target plants exposed to green peach aphids for 4 weeks. Adapted from (26) Source plant/target plant Tuberosum/Tuberosum TuberosumlBerthaultii Berthaultii/Tuberosum Berthaultii/Berthaultii

% PVY incidence 39 30 17 6

S.E.

Ν

3.67 9.15 4.16 2.78

3 2 2 3

Nature of Resistance to the Colorado Potato Beetle. Over the past two decades, the Colorado potato beetle, Leptinotarsa decemlineata (Say), has become the major limiting factor in potato production in the northeastern and mid-Atlantic regions of the U.S. (27) and is an increasingly serious constraint in Europe. The magnitude of the problem has stimulated considerable interest in S. berthaultii as a source of resistance to this pest. When confined on 5. berthaultii, both adults and larvae accumulate type A and Β exudate on their tarsal pads and claws but they do not become entrapped nor does their mobility appear affected (28. 29). Rather, the expression of resistance against the Colorado potato beetle is more subtle and characterized initially by abnormal behaviors. Females display a marked reluctance to accept, feed, and oviposit on S. berthaultii. Numbers of eggs per egg mass

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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and, indeed, total fecundity on S. berthaultii are typically less than 1/2 that on commercial cultivars of potato (13. 30-33). Neonates experience significant mortality within 72 h of confinement on S. berthaultii because of starvation (29). This reluctance to accept S. berthaultii foliage for food can be largely eliminated by mechanical and/or solvent removal of glandular trichomes (Table IV). Our most recent findings indicate that the presence of type A trichomes is a fundamental requirement for expression of S. berthaultii resistance to neonate L. decemlineata. Type Β droplets containing sucrose esters increase the expression of resistance in the presence of defensively-active type A trichomes (29). However, we have been unable to demonstrate a density-dependent relationship between the two types of glandular trichomes and levels of resistance. Table IV. Effect of removing type A (wipe) and type Β (methanol dip) glandular trichomes of S. berthaultii (PI 310927) on feeding, growth, and mortality of neonate Colorado potato beetle. Adaptedfrom(29) Weight (mg)

Treatment

Ν

% Feeding

% Mortality

None

65

40 a

63 a

1.6

a

Wipe

50

60 ab

48 ab

1.9

a

MeOHdip

57

72 ab

32 be

1.2

a

MeOH dip and wipe

39

90 cd

15 cd

1.4

a

abed. Values followed by the same letter are not significantly different (p > 0.05) by R X C test of independence using the G statistic for feeding and mortality and analysis of variance, LSD for weight. Such subtle effects of the trichome barrier might explain our recent finding that feeding is a much smaller portion of the activity budget of Colorado potato beetle larvae on S. berthaultii than on non-glandular susceptible cultivars (Table V) (34) and thus provide an explanation for several of the major impacts of resistance on this pest, i.e. decreased food consumption leads to the reduced growth rates, retarded development, and their cumulative suppressive effects on survival, fecundity, and population dynamics. Genetic Manipulation of S. berthaultii and Variety Development The breeding program at Cornell University to introgress genetic information for insect resistance of S. berthaultii was initiated in 1977 by crossing selected clones of S. berthaultii (as males) with several tetraploid (2n=48) clones of S. tuberosum (35-37). The successful production of tetraploid hybrids between these two species results from unreduced male gamete production in diploid (2n=24) S. berthaultii. A l l subsequent breeding has been with tetraploids. The F2 generation was produced by random intermating of the F l generation. The F3 and F4 generations were produced by intercrossing progenies selected for presence of type Β droplets and for horticultural adaptation. The F3 generation also included outcrossing to selected F2's of S. andigena χ S. berthaultii to introduce genes for resistance to potato virus Y . In subsequent generations, the breeding method has involved a

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system in which back-crossing to S. tuberosum has been alternated with intercrossing within the hybrid population. Parents used in these latter generations had high densities of both types of trichomes, large Type Β droplets and the presence of sucrose esters in type Β exudate, high levels of trichome phenolic oxidation activity, insect, disease and nematode resistance, and horticultural adaptation. We are currently 8 generations advanced beyond the F l . Table V . Activity of neonate Colorado potato beetle larvae on excised leaflets of either S. tuberosum, S. berthaultii PI 310927, or PI 310927 from which most of the glandular trichome exudate had been removed by wiping between tissue papers. Adapted from (24)

Host plant

η

S. tuberosum 16 S. berthaultii PI 310927 (wiped) 16 PI 310927 16

% of larvae in contact with leaflet

No. of larvae feeding as % of: Total No. larvae on no. larvae leaflet

a

12.5

a

13.1 a

79 b 74 b

8.5 1.0

b c

11.1 a 1.7 b

96

Means within the same column followed by the same letter not significantly different in multiple paired t tests at α = 0.017 (experimentwise α = 0.05). The inheritance of glandular trichomes in S. berthaultii and in crosses with S. tuberosum has been studied by several groups (11. 35-37). Type Β trichome density, droplet size and presumably the presence of sucrose esters appear to be controlled by relatively few recessive genes. Heritability estimates ranged from 2030% for density of Type A and Β trichomes and 60% for Type Β droplet size. The presence of polyphenoloxidase in type A trichomes is controlled by a single dominant gene (38). The inheritance of sesquiterpenes in type A trichomes has not been studied. Recent studies at Cornell indicate that with the exception of Type Β trichomes, none of the trichome insect-resistance traits being selected in our breeding program has any deleterious associations with horticultural adaptation. In the case of type Β trichomes, their presence in hybrid clones is associated with reduced yielding ability, late maturity, and other characteristics unique to the wild parent (39). Alternate breeding schemes and somaclonal variation are being explored to determine whether gene linkage is responsible for this undesirable association (39. 40). Potential Pest Management Applications Clones selected from these hybrid populations have demonstrated excellent levels of resistance to aphids, leafhoppers, and the Colorado potato beetle in field and laboratory studies. We have demonstrated season-long reduction in aphid populations on hybrid clones of up to 60%, compared with populations on commercial cultivars (17.41). We have also demonstrated population reductions of leafhopper adults and nymphs of over 80% on hybrid clones, compared to those on commercial susceptible potato cultivars (22). This level of resistance eliminates the

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need for insecticides in management of leafhoppers. As for the Colorado potato beetle, we have documented the following negative impacts by resistant clones compared to commercial cultivars: 30% delay in time to 1st egg laying, 80% reduction in total egg production, 20% increase in larval development time, and 25% reduction in adult weight (Table VI). Under field conditions, these impacts translate into a nearly 90% reduction in densities of second generation larvae with accompanying protection from defoliation. Insecticide use on resistant hybrids can be reduced by at least 40% of that presendy needed on susceptible cultivars ($80160 per acre per growing season) (32). Although glandular trichomes can interfere with predators and parasitoids of aphids and the Colorado potato beetle, the inhibitory effect is largely associated with entrapment by type Β exudate and is minimized on plants bearing moderate densities of these trichomes (42.43). Table VI. Vital Statistics of Colorado Potato Beetle on S. berthaultii and S. tuberosum. Adaptedfrom(3D

Age at 1st Egg Laying (days) Total Eggs per Female Larval Development Time (days) Female Pupal Weight (mg) Female Adult Weight (mg)

S. berthaultii 46 351 14.3 104 88

S. tuberosum 31 2063 11.2 138 105

Future Outlook Our experience with S. berthaultii indicates that it is a useful source of insect resistance for genetic improvement of the potato. Major attributes of this resistance include its broad-spectrum nature and the diversity of its mechano-chemical impact across a range of pest species. However, insects as a group have demonstrated a remarkable ability to adapt to stress. And, in fact, the durability of some types of host resistance is not appreciably greater than that of insecticides. We believe, however, that adaptation by pests to the multiplicity of negative impacts conditioned by glandular trichomes is likely to be a lengthy process requiring substantial genetic changes involving behavior and morphology. For this reason, glandular trichomemediated host resistance to such pest species is likely to be more stable than that conditioned by the presence of a single toxic or deterrent allelochemical. Ackno wled gments I am indebted to the many present and former Cornell University academic personnel contributing to the research efforts described above and with whom I've been fortunate to interact: Dick A . Ave, Pierre Y. Bouthyette, Michael B. Dimock, Felix H . França, Joseph J. Goffreda, Peter Gregory, Robert Hoopes, Julio C. Kalazich, Stanley P. Kowalski, Stephen L . Lapointe, Zaida Lentini-Gil, Shawn A . Mehlenbacher, Jonathan J. Neal, John J. Obrycki, Robert L . Plaisted, John R. Ruberson, James D. Ryan, John C. Steffens, Maurice J. & Catherine A . Tauber, Robert G . Wright, and G . Craig Yencho. This research was supported by the International Potato Center and the United States Department of Agriculture under grant number 7800454 and subsequent grants from the Competitive Research Grants Office.

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Literature Cited 1. Ochoa,C.;Schmiediche, P. In Research for the Potato in the Year 2000 Hooker, W.J., Ed.; Int. Potato Ctr., Lima, Peru. 1983, 199 p. 2. Radcliffe, E. B.; Lauer, F. I. Minn. Agric. Exp. Stn. Tech. Bull. 1968, 259. 3. Buhr, H.; Toball, R.; Schrieber, K. Entomol. Exp. Appl. 1958, 1, 209-224. 4. Kuhn, R.; Löw, I. In Origins of Resistance to Toxic Agents Sevag, M.G.; Reid, R.D.; Reynolds, D. E., Eds. Academic Press: New York, 1955; p. 122-132. 5. Schreiber, K. Züchter 1957, 27, 289-299. 6. Schreiber, K. Entomol Exp. & Appl. 1958, 1, 28-37. 7. Tingey, W. M. Am. Potato J. 1984, 61, 157-167. 8. Hawkes, J. G. Bull. Imp. Bur. Plant Breed and Genet. Cambridge, 1944; pp 142. 9. Gibson, R. W. Ann. Appl. Biol. 1971,68,113-119. 10. Gibson, R. W.; Turner, R.H. PANS 1977, 23, 272-277. 11. Gibson, R. W. Potato Res. 1979, 22, 223-236. 12. Tingey, W. M.; Sinden, S.L. Am. Potato J. 1982, 59, 95-106. 13. Casagrande, R. J. Econ. Entomol. 1982, 75, 368-372. 14. Tingey, W. M. In The Leafhoppers and Planthoppers Nault, L.R.; Rodriguez, J.G., Eds. 1985; pp. 217-234. 15. Lapointe, S. L.; Tingey, W. M. J. Econ. Entomol. 1986, 79, 1264-1268. 16. Tingey, W. M.; Laubengayer, J. E. J. Econ. Entomol. 1981, 74, 721-725. 17. Tingey, W. M.; Plaisted, R. L.; Laubengayer, J. E.; Mehlenbacher, S. A. Am. Potato J. 1982, 59, 241-251. 18. Tingey, W. M.; Gibson, R. W. J. Econ. Entomol. 1978, 71, 856-858. 19. Gibson, R. W.; Pickett, J. A. Nature 1983, 302, 608-609. 20. Avé, D. Α.; Gregory; P.; Tingey, W. M. Entomol. Exp. Appl. 1987, 44, 131-138 21. Lapointe, S. L.; Tingey, W. M. J. Econ. Entomol. 1984, 77, 386-389. 22. Tingey, W. M.; Laubengayer, J. E. J. Econ. Entomol. 1986, 79, 1230-1234. 23. King, R. R.; Pelletier, Y.; Singh, R. P.; Calhoun, L. A. Chem. Comm. 1986, 14, 1078-1079. 24. Neal, J. J.; Tingey, W. M.; Steffens, J. C. J. Chem. Ecol. 1990, 16, 487497. 25. Gunenc, Y.; Gibson, R. W. Potato Res. 1980, 23, 345-351. 26. Lapointe, S. L.; Tingey, W. M.; Zitter, T. A. Phytopathol. 1987,77,819822. 27. Radcliffe, Ε. B. Ann. Rev. Entomol. 1982, 27, 173-204. 28. Dimock, M. B.; Tingey, W. M. Am. Potato J. 1987, 64, 507-515. 29. Neal, J. J.; Steffens, J.C.;Tingey, W. M. Entomol. Exp. Appl. 1989, 51, 133-140. 30. Dimock, M. B. Ph. D. dissertation. Cornell University, 1985. 31. Dimock, M. B.; Tingey, W. M. 1985. In: Proc. Symp. Colorado potato beetle, XVIIth Intern. Congress Entomol. Ferro. D. N; Voss, R. H., Eds. Mass. Agric. Exp. Stn. Bull. 704, 1-144. 32. Wright, R. J.; Dimock, M. B.; Tingey, W. M.; Plaisted, R. L. J. Econ. Entomol. 1985, 78, 576-582. 33. Groden, E.; Casagrande, R. A. J. Econ. Entomol. 1986, 79, 91-97. 34. Dimock, M. B.; Tingey, W. M. Physiol. Entomol. 1988, 13, 399-406. 35. Mehlenbacher, S. Α.; Plaisted, R. L. Proc. Int. Congress: Research for the Potato in the Year 2000., 1983, pp. 1228-130.

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36. Mehlenbacher, S. Α.; Plaisted, R. L . ; Tingey, W . M . A m . Potato J. 1983. 60, 699-708. 37. Mehlenbacher, S. Α.; Plaisted, R. L . ; Tingey, W. M . Crop Sci. 1984, 224, 320-322. 38. Kowalski, S. P. Ph.D. dissertation. Cornell University, Ithaca, N Y 1989. 39. Kalazich, J. C. Ph.D. dissertation. Cornell University, Ithaca, N Y 1989. 40. Lentini-Gil, Z. Ph.D. dissertation. Cornell University, Ithaca, N Y 1989. 41. Xia, J.; Tingey, W. M . J. Econ. Entomol. 1986, 79, 71-75. 42. Obrycki, J. J.; Tauber, M . J.; Tingey, W. M . J. Econ. Entomol. 1983, 76, 456-462. 43. Ruberson, J. R.; Tauber, M . J.; Tauber, C . Α.; Tingey, W. M . Can. Ent. 1989, 121, 841-851. R E C E I V E D May

16, 1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 10

Biochemical Aspects of Glandular Trichome-Mediated Insect Resistance

in the Solanaceae

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John C. Steffens and Donald S. Walters Department of Plant Breeding and Biometry, Cornell University, Ithaca, NY 14853

Glandular trichomes of w i l d Solanaceae produce an array of b i o c h e m i c a l defenses against insects. This chapter reviews our knowledge of two of these mechanisms i n the w i l d potato, Solanum berthaultii, and the w i l d tomato, Lycopersicon pennellii. Ruptured trichomes of S. berthaultii r e l e a s e a h i g h l y expressed, epidermis-specific polyphenol oxidase that polymerizes trichome exudate and entraps insects. Glandular trichomes of L. pennellii continuously secrete a viscous mixture of glucose e s t e r s which a c t as i n s e c t f e e d i n g deterrents. Glucose e s t e r a c y l c o n s t i t u e n t s are composed of branched short and medium c h a i n length f a t t y a c i d s . B i o s y n t h e t i c i n v e s t i g a t i o n s reported here show that the branched amino a c i d b i o s y n t h e t i c pathway i s used t o form 4 and 5 carbon branched acyl groups which are subsequently elongated by acetate t o form the medium chain a c y l groups. I t appears that, i n both s p e c i e s , primary metabolic enzymes have been r e c r u i t e d and m o d i f i e d f o r trichome­ - s p e c i f i c i n s e c t r e s i s t a n c e mechanisms.

Development o f p l a n t s f o r use as food crops has gradually stripped these species of their natural r e s i s t a n c e t o i n s e c t s and pathogens. C o n s e q u e n t l y , most modern c u l t i v a r s rely upon inputs of pesticides to produce an a c c e p t a b l e y i e l d . I n s e c t i c i d e r e s i s t a n c e and the i n c r e a s i n g s o c i a l and economic costs o f p e s t i c i d e application have prompted efforts t o reduce the requirement of crop plants f o r these inputs. Breeding f o r h o s t p l a n t r e s i s t a n c e i s one a p p r o a c h t o r e d u c i n g 0097-6156/91Λ)449-0136$06.00Α) © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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10.

STEFFENS AND WALTERS

Glandular Trichome-Mediated Resistance

insecticide use. Wild relatives of crop plants f r e q u e n t l y possess u s e f u l insect or pathogen r e s i s t a n c e t r a i t s , and are o f t e n c r o s s a b l e w i t h c r o p p l a n t s . E f f o r t s to t r a n s f e r these resistance t r a i t s to crop p l a n t s have thus f a r proceeded by c o n v e n t i o n a l p l a n t breeding approaches. The prospect of producing transgenic crop p l a n t s possessing resistance traits d e r i v e d f r o m w i l d s p e c i e s p r o v i d e s a new avenue f o r u t i l i z i n g r e s i s t a n c e t r a i t s o f w i l d s p e c i e s and removes the barrier to their u t i l i z a t i o n imposed by i n c o m p a t i b i l i t y with crop species. Knowledge of the basic biochemical processes that contribute to resistance is critical to the success of molecular b i o l o g i c a l approaches s i n c e i t i s , at p r e s e n t , necessary to c h a r a c t e r i z e s p e c i f i c p r o d u c t s of r e s i s t a n c e genes i n o r d e r t o i d e n t i f y and t r a n s f e r t h e s e genes t o other species. A l t h o u g h knowledge of r e s i s t a n c e mechanisms may n o t be n e c e s s a r y t o e m p l o y t r a d i t i o n a l breeding m e t h o d s , i n f o r m a t i o n on t h e b i o c h e m i c a l m e c h a n i s m o f r e s i s t a n c e may p e r m i t d i r e c t s c r e e n i n g f o r t h e m e c h a n i s m i n t h e a b s e n c e o f p e s t p o p u l a t i o n s , and c a n be f a r l e s s time consuming than t r a d i t i o n a l screens f o r i n s e c t or pathogen s u s c e p t i b i l i t y . We h a v e b e e n i n v o l v e d i n m e c h a n i s t i c s t u d i e s a i m e d at understanding the b a s i s of g l a n d u l a r trichome-based insect resistance in wild Solanum (potato) and Lycopersicon (tomato) s p e c i e s . Much e f f o r t has f o c u s e d on i d e n t i f i c a t i o n o f w i l d members o f t h e Solanaceae with potentially useful resistance traits for i n t r o g r e s s i o n i n t o Solanum tuberosum and Lycopersicon esculentum. I n many c a s e s r e s i s t a n c e h a s b e e n s h o w n t o be c o n f e r r e d by g l a n d u l a r t r i c h o m e s , m o d i f i e d e p i d e r m a l cells (JJ which f u n c t i o n as p h y s i c a l and/or chemical barriers against insect attack (2-10. Tingey, this volume). The w i l d t o m a t o , Lycopersicon pennellii, and the w i l d p o t a t o , Solanum b e r t h a u l t i i , a r e two s p e c i e s w h i c h exhibit insect resistance c o n f e r r e d by glandular trichomes. S. b e r t h a u l t i i a n d L. p e n n e l l i i h a v e b e e n the focus of e f f o r t s at C o r n e l l U n i v e r s i t y t o t r a n s f e r trichome-based insect resistance t r a i t s . This chapter reviews our knowledge of the b i o c h e m i s t r y of g l a n d u l a r trichome-based insect resistance i n these species.

Type h (VI) T r i c h o m e s Insect Entrapment

and

Polyphenol

Oxidase-Mediated

T h e r e a r e p r i m a r i l y two c l a s s e s of f o l i a r glandular trichomes that c o n t r i b u t e to the insect r e s i s t a n c e of S. b e r t h a u l t i i a n d L. p e n n e l l i i . Type A t r i c h o m e s o f S. berthaultii ( r e f e r r e d t o as Type VI i n t h e genus Lycopersicon (UJ ) condition resistance to small-bodied insect pests such as aphids and l e a f h o p p e r s by an

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a d h e s i v e entrapment mechanism. The t y p e A t r i c h o m e i s s t a l k e d w i t h a t e t r a l o b u l a t e head, a p p r o x i m a t e l y 50-65 m i c r o n s i n d i a m e t e r , and f o u n d a t h i g h d e n s i t i e s on t h e l e a f s u r f a c e o f many s o l a n a c e o u s s p e c i e s . Contact with t h i s t r i c h o m e by an i n s e c t c a u s e s t h e m e m b r a n e - e n c l o s e d head o f t h e t r i c h o m e t o r u p t u r e , c o a t i n g t h e l e g s and mouthparts of the insect with the trichome contents. Immediately t h i s c o a t i n g b e g i n s t o brown and harden; as the insect ruptures additional trichomes, the dark masses on t h e l e g s and m o u t h p a r t s i n c r e a s e d r a m a t i c a l l y . These a c c r e t i o n s d i s r u p t i n s e c t f e e d i n g by restricting movement, by o c c l u d i n g t h e m o u t h p a r t s , o r by e n t r a p m e n t o f t h e i n s e c t on t h e l e a f (12). B r o w n i n g and h a r d e n i n g o f e x u d a t e on t h e i n s e c t ' s m o u t h p a r t s a n d t a r s i i s due t o t h e e n z y m a t i c a c t i v i t y o f an 0 r e q u i r i n g o x i d a s e (13.) , a n d h a s b e e n a s s o c i a t e d w i t h b o t h p e r o x i d a s e and p o l y p h e n o l o x i d a s e activities (£, 14_f 1_5.) . We r e c e n t l y showed t h a t t h e s e enzymatic a c t i v i t i e s a r e d u e t o a 59 kD p o l y p h e n o l o x i d a s e (PPO) which i s present i n the type A trichomes of S . berthaultii at approximately 1 ng/trichome head (a concentration of approximately 0.3 mM) (1£) . The s p e c i a l i z a t i o n of the glandular trichome i s r e f l e c t e d i n t h e f a c t t h a t PPO c o m p r i s e s a p p r o x i m a t e l y 60% o f t h e t o t a l s o l u b l e p r o t e i n of the organ. T r i c h o m e PPO appears t o be an example o f an e v o l u t i o n a r y m o d i f i c a t i o n o f an e x i s t i n g p l a n t enzyme f o r use as an i n s e c t d e f e n s e mechanism. PPO e n z y m e s , o f m o l e c u l a r w e i g h t 45,000, a r e l o c a l i z e d i n t h e t h y l a k o i d a n d a r e n e a r l y u b i q u i t o u s i n t i s s u e s o f p l a n t s {12., 18.) . U n l i k e o t h e r known n u c l e a r e n c o d e d c h l o r o p l a s t p r o t e i n s , t h e 45 kD t h y l a k o i d PPO, whose f u n c t i o n i s unknown, i s t r a n s l a t e d a t i t s m a t u r e s i z e o f 45 kD, a n d d o e s n o t p o s s e s s a t r a n s i t p e p t i d e s e q u e n c e (1_&) . In contrast, the 59 kD trichome PPO is translated as a 67 kD p r e c u r s o r and l o c a l i z e d i n t h e l e u c o p l a s t s o f t r i c h o m e and o u t e r e p i d e r m a l c e l l s . Immunological and primary s e q u e n c e s i m i l a r i t i e s b e t w e e n t h e 59 kD t r i c h o m e PPO and the 45 kD thylakoid PPO underscore the close e v o l u t i o n a r y r e l a t i o n s h i p b e t w e e n t h e s e two proteins. The a p p a r e n t a d v a n t a g e o f t h e v e r y h i g h c o n c e n t r a t i o n o f PPO i n the trichome i s the high initial rate of catalysis which results upon trichome rupture, f a c i l i t a t i n g the entrapment of mobile i n s e c t s . We h a v e c l o n e d t h e 5 9 kD t r i c h o m e PPO f r o m a cDNA l i b r a r y c o n s t r u c t e d f r o m e p i d e r m a l mRNA (H. Y u a n d J . S t e f f e n s , u n p u b l i s h e d data) and a r e c u r r e n t l y u s i n g t h i s c l o n e a s a p r o b e t o i s o l a t e t h e g e n o m i c DNA e n c o d i n g t h e S. b e r t h a u l t i i t r i c h o m e PPO ( S . Newman a n d J . S t e f f e n s , unpublished data). T h e s e s t u d i e s may a l l o w us to increase the insect entrapping a b i l i t i e s of c u l t i v a t e d p o t a t o , w h i c h has r e t a i n e d low Type A t r i c h o m e d e n s i t i e s and no longer possesses the biochemical machinery 2

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

10.

STEFFENS AND WALTERS

Glandular Trichome-Mediated Resistance

necessary for insect entrapment. Study of the regulatory sequences c o n t r o l l i n g expression of this e n z y m e may p r o v i d e a n u n d e r s t a n d i n g o f how glandular trichomes are able to synthesize the high l e v e l s of s p e c i f i c products which f r e q u e n t l y c h a r a c t e r i z e s these organs in plants. Alternatively, the regulatory s e q u e n c e s o f g l a n d u l a r t r i c h o m e g e n e s may b e exploited to express other i n s e c t i c i d a l p r o t e i n s i n the trichome and e p i d e r m i s as a f i r s t l i n e o f defense a g a i n s t i n s e c t pests.

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Secretion

o f Sugar E s t e r s by

Glandular

Trichomes

A s i m i l a r degree of b i o c h e m i c a l s p e c i a l i z a t i o n e x i s t s i n g l a n d u l a r trichomes which s e c r e t e sugar e s t e r s . Glucose and s u c r o s e e s t e r s a r e p r o d u c e d by s e v e r a l g e n e r a w i t h i n the Solanaceae (20-25) . In c o n t r a s t to the PPOc o n t a i n i n g trichome, these sugar e s t e r s are c o n t i n u o u s l y secreted from the apex of a t a l l (750μπι) , t a p e r e d t r i c h o m e d e s i g n a t e d as t h e Type Β o r Type IV t r i c h o m e i n Solanum a n d Lycopersicon, r e s p e c t i v e l y (H) . Described i n i t i a l l y a s e p i c u t i c u l a r l i p i d s b y F o b e s e t a l . (2ϋ) , the glucose and sucrose esters of Solanum and Lycopers icon s p e c i e s have b e e n shown t o physically e n t r a p s m a l l p e s t s s u c h as s p i d e r m i t e s and a p h i d s (272_£; T i n g e y , t h i s volume) , and t o a c t as a p h i d f e e d i n g deterrents. As l i t t l e as 33 μg sucrose ester/cm s i g n i f i c a n t l y d e t e r s f e e d i n g by g r e e n p e a c h and p o t a t o aphids i n a r t i f i c i a l f e e d i n g c h a m b e r b i o a s s a y s (£.,HQ.) . L e v e l s o f s u c r o s e e s t e r s p r e s e n t on leaflets of S. b e r t h a u l t i i r a n g e a b o v e 100 mg/ cm ( u n p u b l i s h e d ) , and i n L. p e n n e l l i i a r e f r e q u e n t l y a b o v e 3 0 0 m g / c m (Z3.) . In a d d i t i o n t o d i r e c t l y reducing aphid f e e d i n g , sugar esters indirectly contribute to reduction in virus t r a n s m i s s i o n b y a p h i d s (3_1) . O t h e r r e s u l t s s u g g e s t t h a t sugar e s t e r s may also inhibit bacterial and fungal g r o w t h (1,21) . The s u g a r e s t e r s p r o d u c e d b y s o l a n a c e o u s trichomes a r e p r i m a r i l y composed o f s h o r t t o medium c h a i n l e n g t h f a t t y a c i d s (C4-C12) e s t e r i f i e d t o g l u c o s e o r s u c r o s e . Both branched and s t r a i g h t c h a i n a c y l components are present i n these esters. Each species produces a c h a r a c t e r i s t i c combination of f a t t y acids e s t e r i f i e d at 2 to 4 sugar hydroxyls ( T a b l e I) . Among t h e known species producing sucrose esters, a c y l a t i o n of every p o s s i b l e h y d r o x y l has been d e s c r i b e d e x c e p t t h e l , 4 , and 6 p o s i t i o n s (20-25). The d i v e r s i t y o f f a t t y a c i d c o m p o s i t i o n as w e l l as t h e acylation positions are p u z z l i n g phenomena. I n s t r u c t u r e / a c t i v i t y s t u d i e s we are unable to detect differences i n aphid feeding d e t e r r e n c e when c o m p a r i n g s u c r o s e and g l u c o s e esters. Esters bearing d i f f e r e n t chain lengths of f a t t y acid a l s o do n o t e x h i b i t significant activity differences (H) · The o r i g i n o f s u g a r e s t e r s t r u c t u r a l diversity 2

2

2

1

1

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f

139

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Group

+

Major a c y l groups,

10-methylundecanoate

dodecanoate

9-methyldecanoate_

undecanoate

*

sucrose

2,3,3',4

glucose

2,3,4

Solanum Solanum neocardensii aethiopsicum

Minor a c y l groups (0.05). Means f o r f o l i a g e w i t h and w i t h o u t trichomes are s i g n i f i c a n t l y d i f f e r e n t by F - t e s t (P 0.001, η - 75) w i t h the a b i l i t y o f H. zea t o grow on f o l i a g e . A l s o , f o l i a r PPO a c t i v i t y i n c r e a s e s w i t h age, w h i c h i s a l s o s t r o n g l y n e g a t i v e l y c o r r e l a t e d w i t h a reduced a b i l i t y o f l a r v a l H. zea t o grow (79; u n p u b l . d a t a ) . I n a d d i t i o n , i t has been shown t h a t p l a n t PPO a c t i v i t y p e r s i s t s i n t h e i n s e c t ' s g u t hours after i n g e s t i o n , and i n f a c t t h e i n s e c t ' s d i g e s t i v e p r o c e s s e s actually a c t i v a t e PPO b y 50% w i t h i n minutes a f t e r i n g e s t i o n ( 7 9 ) . Because up t o 50% o f i n g e s t e d c h l o r o g e n i c a c i d i s c o v a l e n t l y bound t o p l a n t p r o t e i n (the cause o f a s i g n i f i c a n t l o s s o f amino a c i d s ; e.g., up t o 38% o f l y s i n e and c y s t e i n e , and 10-20% o f h i s t i d i n e ) when t h e i n s e c t feeds on f o l i a g e , i t i s c o n c l u d e d t h a t PPO i s o p e r a t i n g as a s t r o n g a n t i n u t r i t i v e defense a g a i n s t these i n s e c t s . A l t h o u g h PPO i s t h e major p h e n o l a s e o f t h e tomato p l a n t , p e r o x i d a s e (POD) a l s o i s p r e s e n t ( 7 9 ) . A major l i m i t a t i o n o f t h e use o f POD as a defense i s t h a t , u n l i k e PPO, i t r e q u i r e s a continuous source o f H2O2 i n o r d e r t o o x i d i z e p h e n o l i c s . One p o s s i b l e advantage o f POD i s t h a t i t i s a b l e t o o x i d i z e a g r e a t e r v a r i e t y o f p h e n o l i c s . F o l i a r PPO h a s s i g n i f i c a n t a c t i v i t y w i t h c a f f e i c a c i d and c h l o r o g e n i c a c i d ; whereas, POD can o x i d i z e a much g r e a t e r v a r i e t y o f s u b s t r a t e s (e.g., c h l o r o g e n i c a c i d , c a f f e i c a c i d , r u t i n , e s c u l e t i n , f e r u l i c a c i d , t y r o s i n e , and coumaric a c i d ) . Moreover, POD c a n a t t a c k p r o t e i n b y o x i d i z i n g t y r o s i n y l and s u l p h y d r y l m o i e t i e s , as w e l l as d e a m i n a t i n g and d e c a r b o x y l a t i n g amino a c i d s such as l y s i n e . As p o i n t e d out b e f o r e , these r e a c t i o n s are v e r y d e t r i m e n t a l t o p r o t e i n i n t e g r i t y . Thus, t h i s greater spectrum o f a c t i v i t y may make POD a s t r o n g o r i n d i s p e n s i b l e component o f enzyme-based defense. To t h i s p o i n t our work w i t h POD i s l i m i t e d . A d d i t i o n o f POD w i t h c h l o r o g e n i c a c i d o r r u t i n t o a r t i f i c i a l d i e t s t r o n g l y reduces the n u t r i t i v e v a l u e o f d i e t a r y c a s e i n f o r l a r v a l H. zea (Table I V ) . This again i s i n part the r e s u l t o f covalent binding o f the o x i d i z e d p h e n o l i c t o the p r o t e i n . Our major e v i d e n c e f o r the a n t i n u t r i t i v e a c t i o n o f POD comes from f e e d i n g e x p e r i m e n t s w i t h f o l i a g e i n w h i c h c a t a l a s e was added t o macerated f o l i a g e . C a t a l a s e c o n v e r t s H2O2 (needed b y POD) t o w a t e r ; hence, t h e presence o f h i g h c a t a l a s e a c t i v i t y i n f o l i a g e reduced the impact o f POD on f o o d q u a l i t y . The a d d i t i o n o f c a t a l a s e t o f o l i a g e n o t o n l y reduced the l e v e l o f POD a c t i v i t y i n f r e s h l y c r u s h e d t i s s u e 3 6 - f o l d b u t c o r r e s p o n d i n g l y improved t h e r e l a t i v e growth o f l a r v a l H. zea by 0.042 mg/day/mg l a r v a (Table V ) . T h i s e v i d e n c e i m p l i c a t e s POD as a p o t e n t i a l l y i m p o r t a n t factor i n r e d u c i n g t h e growth o f H. zea. However, because t h e i n s e c t ' s g u t c o n t e n t c o n t a i n s h i g h l e v e l s o f c a t a l a s e , the a n t i b i o t i c a c t i o n o f POD may be l i m i t e d . I n c o n t r a s t , PPO does n o t r e q u i r e H 0 and remains h i g h l y a c t i v e i n the i n s e c t ' s g u t d u r i n g the d i g e s t i o n o f f o o d and even remains a c t i v e i n the f a e c e s . 2

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12.

DUFFEY AND FELTON

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T a b l e IV.

Enzymatic Antinutritive Defenses of Tomato

E f f e c t o f O x i d a t i v e Enzyme A c t i v i t y on t h e Growth o f l a r v a l H e l i o t h i s zea

Treatment % R e l a t i v e Growth P r o t e i n alone 100a (tomato f o l i a r p r o t e i n a t 0.5%) Protein + 3.5 mM l i n o l e i c a c i d 100a Protein + 3.5 mM c h l o r o g e n i c a c i d 102a Protein + lOmM H 0 105a Protein + lipoxygenase + 3.5 mM l i n o l e i c a c i d 48.1b Protein + p e r o x i d a s e + H 0 (lOmM) + 7.0 mM c h l o r o g e n i c a c i d 27.1c Protein + p e r o x i d a s e + H 0 (lOmM) + 7.0 mM r u t i n 78.Od Protein + polyphenol oxidase + 7.0 mM c h l o r o g e n i c a c i d 32 .0c R e l a t i v e growth - mg/day/mg o f l a r v a : l i p o x y g e n a s e , p e r o x i d a s e , and p o l y p h e n o l o x i d a s e were added t o d i e t a t a c t i v i t i e s c o r r e s p o n d i n g t o t h a t found i n tomato f o l i a g e : r u t i n and c h l o r o g e n i c a c i d a t 3.5 mM/kg d i e t wwt. S i g n i f i c a n t d i f f e r e n c e s between means w i t h i n a column, based 95% c o n f i d e n c e i n t e r v a l s from ANOVA, a r e shown by d i f f e r e n t l e t t e r s . PPO - 0.100 O.D./min/gm d i e t wwt.; POD - 27.0 O.D./min/gm d i e t wwt. 2

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2

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NATURALLY OCCURRING PEST BIOREGULATORS

178 T a b l e V.

E f f e c t o f Exogenous C a t a l a s e on L a r v a l Growth and F o l i a r Oxidative A c t i v i t i e s

Treatment

CAT-L

C a t a l a s e added

3570a

No c a t a l a s e added

lib

P0D

2

RGR

3

1.85a

0.296

65.20b

0.254

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1

CAT - c a t a l a s e a c t i v i t y i n units/min/gm f o l i a g e 2 POD - p e r o x i d a s e a c t i v i t y i n OD470/min/gm f o l i a g e 3 R G R - r e l a t i v e l a r v a l growth r a t e (mg/day/mg l a r v a ) . S i g n i f i c a n t d i f f e r e n c e s between means w i t h i n a column, b a s e d 95% c o n f i d e n c e i n t e r v a l s from ANOVA, a r e shown by d i f f e r e n t l e t t e r s .

We have t h e l e a s t i n f o r m a t i o n about t h e a n t i n u t r i t i v e e f f e c t s of lipoxygenase (LOX). Experiments (Table I V ) show t h a t t h e n u t r i t i v e v a l u e o f p r o t e i n t o H. z e a i s reduced by t r e a t m e n t w i t h LOX and l i n o l e i c a c i d ( 5 2 % r e d u c t i o n i n growth). Tomato f o l i a g e c o n t a i n s s i g n i f i c a n t q u a n t i t i e s o f LOX a c t i v i t y and l i n o l e i c a c i d has been shown t o be c o v a l e n t l y bound t o p r o t e i n b o t h i n v i t r o and i n p l a n t a (unpubl. d a t a ) . S t u d i e s a r e underway t o determine w h i c h amino a c i d s a r e p r e f e r e n t i a l l y d e s t r o y e d . A l s o , p r e l i m i n a r y s t u d i e s w i t h f o l i a g e demonstrate t h a t c o p i o u s q u a n t i t i e s o f m a l o n d i a l d e h y d e are g e n e r a t e d i n c r u s h e d f o l i a g e ; t h e a n t i n u t r i t i o n a l e f f e c t s o f t h i s S c h i f f base former a r e as y e t undetermined. C u r r e n t l y we a r e d e t e r m i n i n g i f t h e j o i n t a n t i n u t r i t i o n a l e f f e c t s o f POP, POD, and LOX a c t i v i t y a r e a d d i t i v e o r s y n e r g i s t i c . I f these a c t i v i t i e s a r e found t o d i f f e r e n t i a l l y d e s t r o y amino a c i d s , t h e n t h e i r b a t t e r y o f e f f e c t s may be s y n e r g i s t i c . I f t h e i r e f f e c t s a r e e q u a l (same amino a c i d s d e s t r o y e d ) , t h e n perhaps o n l y one enzyme, s a y PPO (with high phenolic levels) requires a m p l i f i c a t i o n t o prove a s u f f i c i e n t a n t i n u t r i t i v e d e f e n s e . We a r e a l s o determining i f v a r i o u s o x i d i z e d phenolics (e.g., c a f f e i c a c i d , c h l o r o g e n i c a c i d , coumaric a c i d , and r u t i n ) a r e e q u i v a l e n t i n t h e i r a b i l i t y t o a l k y l a t e and i m p a i r p r o t e i n q u a l i t y . A b r e e d i n g program t o enhance r e s i s t a n c e would b e n e f i t from the knowledge o f w h i c h enzymes and/or s u b s t r a t e s t o enhance. Our s u r v e y s o f w i l d s p e c i e s o f h v c o p e r s i c o n show t h a t c e r t a i n genotypes ( p a r t i c u l a r l y L. h i r s u t u m f . glabratum) n o t o n l y have h i g h e r constitutive levels of catecholic phenolics t h a n found i n L. e s c u l e n t u m b u t a l s o h i g h e r l e v e l s o f PPO, POD, and LOX (99; u n p u b l . data). I n d u c t i o n o f Enzymes by Feeding-Damage. A l t h o u g h t h e tomato p l a n t has c o n s t i t u t i v e l e v e l s o f PPO, POD, and LOX, t h e d e f e n s i v e a b i l i t y o f t h e p l a n t may be enhanced i f these enzymes were i n d u c e d by i n s e c t f e e d i n g damage. The i n d u c i b l e n a t u r e o f PPO, POD, and p h e n o l i c s as a r e s u l t o f i n f e c t i o n by m i c r o o r g a n i s m s has a l r e a d y been d i s c u s s e d . S i m i l a r l y , t h e i n d u c t i o n o f P i ' s by n o c t u i d l a r v a e has been p o i n t e d out. Indeed, f e e d i n g damage by l a r v a l H. z e a i s a b l e t o s y s t e m i c a l l y induce v e r y h i g h l e v e l s o f PPO a c t i v i t y i n

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

12.

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Enzymatic Antinutritive Defenses of Tomato 179

tomato f o l i a g e 24 hours a f t e r damage ( T a b l e V I ) . I n d u c t i o n o f POD and LOX by H. zea was n o t observed. I n c o n t r a s t , the Tomato R u s s e t m i t e A c u l o p s l y c o p e r s i c i d r a m a t i c a l l y i n d u c e d LOX and POD levels ( T a b l e V I ) . We have n o t y e t r e l a t e d the i n d u c t i o n o f t h e s e enzymes t o the l e v e l s o f p h e n o l s , p r o t e i n , and/or i n s e c t performance on i n d u c e d v e r s u s uninduced f o l i a g e .

Table VI.

I n d u c t i o n o f Tomato P l a n t F o l i a r O x i d a t i v e Enzymes by A r t h r o p o d s

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%Relative A c t i v i t v

Russet M i t e

Enzyme

H.

Lipoxygenase

105

2367

Peroxidase

100

264

980

96

Polyphenol

Oxidase

zea

2

1

3

undamaged p l a n t s r e p r e s e n t 100% a c t i v i t y ; represents systemic i n d u c t i o n (other leaves); represents l o c a l i z e d induction (within a l e a f ) .

Impact o f O x i d a t i v e Enzymes on A p h i d s . Tomato p l a n t s a r e a l s o a t t a c k e d by a v a r i e t y o f o t h e r a r t h r o p o d s such as w h i t e f l i e s , m i t e s , and a p h i d s (126,127). Trichomes have been s t u d i e d as a b a s i s o f r e s i s t a n c e a g a i n s t many o f these a r t h r o p o d s ( 9 9 ) . A c o n s i s t e n t feature o f r e s i s t a n c e has been the a l l u s i o n t o the sticky e n t r a p p i n g p r o p e r t i e s o f t r i c h o m e s ( t y p e V I ) . We have shown t h a t these entrapping properties result from the presence of c o m p a r t m e n t a l i z e d c a t e c h o l i c p h e n o l i c s and PPO/POD i n the t i p s o f type V I t r i c h o m e s (99: u n p u b l . d a t a ) , w h i c h upon breakage, f o r example by a p h i d s , l e a d t o quinone mediated p o l y m e r i z a t i o n o f t r i c h o m a l p r o t e i n . T h i s p o l y m e r i z e d p r o t e i n forms the c l a s s i c a l b l a c k e n e d b o o t s on a p h i d ' s f e e t . U t i l i z i n g a s e r i e s o f c r o s s e s between PI 134417 and W a l t e r [the p a r e n t s i n Kennedy's s t u d i e s -of 2-tridecanone resistance ( 1 2 8 ) ] , we have shown a significant n e g a t i v e c o r r e l a t i o n between the number o f a p h i d s , Macrosiphon e u p h o r b i a e . per l e a f l e t and the d e n s i t y o f type V I t r i c h o m e s and t h e i r phenolase a c t i v i t y . These f i n d i n g s p a r a l l e l the s t u d i e s o f T i n g e y ' s group on r e s i s t a n c e i n p o t a t o (see c h a p t e r i n t h i s volume). As mentioned above, c e r t a i n a c c e s s i o n s o f L. h i r s u t u m f . g l a b r a t u m a r e s o u r c e s o f h i g h l e v e l s o f f o l i a r p h e n o l i c s and o x i d a t i v e enzymes. Some o f the same a c c e s s i o n s o f L. h i r s u t u m f . g l a b r a t u m have been shown t o be h i g h e s t i n type V I t r i c h o m a l d e n s i t i e s w i t h commensurately h i g h t r i c h o m a l PPO/POD a c t i v i t y . Hence, i t s h o u l d be p o s s i b l e , by employing o x i d a t i v e enzymes, t o b r e e d f o r s i m u l t a n e o u s r e s i s t a n c e a g a i n s t p e s t s such as a p h i d s , H. zea and S. e x i g u a .

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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P o t e n t i a l I n c o m p a t i b i l i t y with Proteinase I n h i b i t o r s I t was suggested e a r l i e r t h a t P i ' s and PPO might s e r v e as complementary defenses because o f the demand they p l a c e upon the i n s e c t f o r s u l p h u r amino a c i d s . U n f o r t u n a t e l y , the two types o f d e f e n s e s may be i n c o m p a t i b l e . P i ' s a r e p o l y p e p t i d e s (8,000 - 40,000 mw) w h i c h have a v a r i e t y o f a l k y l a t a b l e amino a c i d s (e.g., c y s t e i n e - c y s t e i n e and l y s i n e ) . P i ' s o f t e n have l y s i n e n e a r the a c t i v e s i t e , and many P i ' s have m u l t i p l e d i s u l p h i d e bonds w h i c h are i n t e g r a l f o r a c t i v i t y (129-131). D e r i v a t i z a t i o n o f these amino a c i d s by quinones s h o u l d render P i ' s l e s s a c t i v e . Indeed, t h i s has been shown t o o c c u r b o t h i n v i t r o and i n p l a n t a (125). Treatment o f soybean t r y p s i n i n h i b i t o r ( I I ) , tomato PI ( I and I I ) , and l i m a bean i n h i b i t o r w i t h PPO and c h l o r o g e n i c a c i d i n v i t r o caused a l o s s o f ability to inhibit bovine trypsin: this loss of activity c o r r e s p o n d e d t o a l o s s o f up t o 30% o f d e t e c t a b l e amino a c i d s such as l y s i n e and c y s t e i n e as a r e s u l t o f a l k y l a t i o n . Furthermore, i t was shown the a c t i o n o f PPO p l u s c h l o r o g e n i c a c i d a g a i n s t P i ' s I and I I i n tomato f o l i a g e r e s u l t e d i n up t o 70% l o s s i n t h e i r d e t e c t a b i l i t y by immunological a s s a y w i t h a c o r r e s p o n d i n g 50% l o s s i n the a b i l i t y o f f o l i a g e t o i n h i b i t the growth o f S. e x i g u a . The l o s s o f PI i d e n t i t y and b i o l o g i c a l a c t i v i t y was magnified by wounding (124,125). Crop p l a n t s c o n t a i n a v a r i e t y o f p o t e n t i a l a l k y l a t i n g o r S c h i f f base f o r m i n g agents (e.g., g o s s y p o l , i s o t h i o c y a n a t e from mustard o i l s , "CN from c y a n o g e n s i s , DIMBOA, e p o x i d e s , sesquiterpene l a c t o n e s , o x i d i z e d t a n n i n s , and a l d e h y d e s ) w h i c h are i m p l i c a t e d as d e f e n s e s a g a i n s t i n s e c t s and pathogens. These a l k y l a t i n g agents a l s o have the a b i l i t y t o s i g n i f i c a n t l y reduce ( g e n e r a l l y 30-70%) the i n h i b i t o r y p r o p e r t i e s o f a v a r i e t y o f P i ' s ( e . g . , K u n i t z and Bowman-Birk i n h i b i t o r s , tomato PI I and I I , p o t a t o i n h i b i t o r I and I I ) . G e n e r a l l y , PPO and POD w i t h a v a r i e t y o f s u b s t r a t e s (e.g., c a f f e i c a c i d , c h l o r o g e n i c a c i d , coumaric a c i d , and e s c u l e t i n ) were the most e f f e c t i v e a t i n a c t i v a t i n g P i ' s compared t o e p o x i d e s and S c h i f f base formers. Some s e l e c t e d r e s u l t s are shown i n T a b l e V I I . Tomato PI I I was more r e s i s t a n t t o i n a c t i v a t i o n t h a n s e v e r a l o t h e r P i ' s (unpubl. d a t a ) , w h i c h s u g g e s t s t h a t c h e m i c a l i n c o m p a t i b i l i t i e s i n t r a n s g e n i c p l a n t s may be p a r t i a l l y a v o i d e d by the c o r r e c t c h o i c e o f P I . I n v i e w o f the r e c e n t emphasis on t r a n s g e n i c a l t e r a t i o n o f crop p l a n t s w i t h PI genes (119-122,132), our r e s u l t s suggest t h a t u n l e s s the c h e m i c a l m i l i e u o f the r e c e i v i n g p l a n t i s p r o p e r l y a c c o u n t e d f o r the e f f i c a c y o f P i ' s as a b a s i s o f r e s i s t a n c e may be compromised.

P o t e n t i a l Incompatibilités w i t h B i o l o g i c a l C o n t r o l Agents A n o t h e r p o t e n t i a l c o n s t r a i n t upon the use o f o x i d a t i v e enzymes as bases of host plant resistance may be their potential incompatibility with b i o l o g i c a l c o n t r o l agents. Tomato plant p h e n o l i c s ( r u t i n and c h l o r o g e n i c a c i d ) have been shown t o be

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

12.

DUFFEY AND FELTON Table V I I .

Enzymatic Antinutritive Defenses of Tomato

Impact o f S e l e c t e d P l a n t N a t u r a l P r o d u c t s upon P r o t e i n a s e I n h i b i t o r A c t i v i t y a g a i n s t Bovine Trypsin 1

C h e m i c a l Treatment Allylisothiocyanate

Inhibitor % Inactivation 32 STI 0 Tomato PI I I DIMBOA 49 STI Gossypol 5 STI 64 Tomato PI I Tannic a c i d 50 STI 74 T a n n i c a c i d + H o 0 + POD STI L i p o x y g e n a s e + l i n o l e i c a c i d STI 33 47 Tomato PI I 20 Tomato PI I I POD + H 0 + coumaric a c i d 53 STI 75 Tomato PI I 12 Tomato PI I I Data d e r i v e d from Workman, F e l t o n , and D u f f e y , u n p u b l . d a t a : % I n a c t i v a t i o n i s d e t e r m i n e d by comparing a b i l i t y o f u n t r e a t e d PI versus p r e t r e a t e d proteinase i n h i b i t o r to i n h i b i t bovine t r y p s i n h y d r o l y s i s o f TAME i n v i t r o : f o r treatment a l l c h e m i c a l s were used a t 3.5 mM: STI - soy t r y p s i n i n h i b i t o r I ; POD - p e r o x i d a s e :

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2

2

2

1

m o d e r a t e l y s a f e compared t o t o m a t i n e i n t h e i r d e t r i m e n t a l e f f e c t s upon the ichneumonid p a r a s i t o i d Hyposoter e x i g u a e (32,97). The e f f e c t s o f p h e n o l i c s i n c o n j u n c t i o n w i t h PPO and POD upon t h i s p a r a s i t o i d are unknown. T h e i r a c t i o n may be i n c o m p a t i b l e i f the growth o f the h o s t l a r v a e were s u f f i c i e n t l y r e s t r i c t e d so as t o i m p a i r the growth o f the p a r a s i t o i d ( 9 7 ) . The use o f h i g h t r i c h o m a l d e n s i t y i n c o n j u n c t i o n w i t h h i g h t r i c h o m a l PPO a c t i v i t y t o c o n t r o l a p h i d s may compromise the e f f i c a c y o f such p a r a s i t o i d s . I t i s not known i f h i g h t r i c h o m a l d e n s i t y / h i g h trichome PPO activity is c l o s e l y g e n e t i c a l l y l i n k e d w i t h the p r o d u c t i o n o f 2 - t r i d e c a n o n e i n L y c o p e r s i c o n h i r s u t u m f . glabratum. Kennedy's group has found t h a t the p r e s e n c e o f 2 - t r i d e c a n o n e i n trichomes t o be i n c o m p a t i b l e w i t h the a c t i o n o f the p a r a s i t o i d Campoletis s o n o r e n s i s a g a i n s t H. zea (98). I n the absence o f PPO, r u t i n and c h l o r o g e n i c a c i d i n h i b i t the r e p l i c a t i o n o f a n u c l e a r p o l y h e d r o s i s AcMNPV i n i n s e c t t i s s u e c u l t u r e ( T r i c o p l u s i a n i ) . These c h e m i c a l s i n a r t i f i c i a l d i e t a l s o s t r o n g l y reduced the i n f e c t i v i t y o f HzSNPV i n H. zea (133). I n the p r e s e n c e o f PPO and c h l o r o g e n i c a c i d , the s o l u b i l i t y o f the o c c l u s i o n body o f HzSNPV i s markedly reduced, and c o r r e s p o n d i n g l y , i n f e c t i v i t y i n l a r v a l H. zea i s reduced up to 90% (124,134). Such a n e g a t i v e impact upon the v i r u s a r i s e s from a l k y l a t i o n o f p o l y h e d r o n p r o t e i n s by c h l o r o g e n o q u i n o n e . Hence, r e s i s t a n c e b a s e d on PPO and POD may s e v e r e l y compromise the e f f i c a c y o f v i r a l c o n t r o l agents i n

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181

NATURALLY OCCURRING PEST BIOREGULATORS

182

the f i e l d . Furthermore, i f i n s e c t v i r u s e s a r e t o be used as v e c t o r s o f a l i e n genes ( e . g . , n e u r o p e p t i d e s o r enzymes; 132,135-137), c o n s i d e r a t i o n must be g i v e n t o t h e p o t e n t i a l o f such o x i d a t i v e enzymes t o d e t r a c t from c o n t r o l . S u p r i z i n g l y , l i p i d h y d r o p e r o x i d e s produced from t h e a c t i o n o f LOX on l i n o l e i c a c i d enhanced t h e i n f e c t i v i t y o f HzSNPV i n zea (unpubl. data; Table V I I I ) . I n a d d i t i o n , i t has been found t h a t t h e t o x i c i t y o f p u r i f i e d t o x i n from B a c i l l u s t h u r i n g i e n s i s k u r s t a k i i s enhanced up t o 50% by a l k y l a t i o n with c h l o r o g e n o q u i n o n e (Ludlum, F e l t o n , and D u f f e y , u n p u b l . d a t a ) ( T a b l e . V I I I ) . Other c h e m i c a l s such as P I from soybean and t o m a t i n e a l s o enhanced t h e a c t i v i t y o f b o t h NPV and BTk a g a i n s t H. z e a ( T a b l e V I I I ) .

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Table V I I I .

E f f e c t o f P h y t o c h e m i c a l s on the I n f e c t i v i t y o f NPV and BTK i n H e l i o t h i s z e a

Mortality 2

Ratio 3

Treatment

NPV

STI

1..34a

1.,21a

CHA

0. ,74a

1.,68b 2. 76c

CHA

+ PPO

0. ,50b

LOX

+ linolenic acid

2.,60c

Rutin

0. ,53b

Tomatine

1.,61d

BTK

1

1.,22a

STI - soy t r y p s i n i n h i b i t o r I i n d i e t a t 0.18% wwt; CHA c h l o r o g e n i c a c i d i n d i e t a t 3.5 mM/kg d i e t wwt; r u t i n a t 3.5 mM/kg d i e t wwt; t o m a t i n e a t 0.9 mM/kg d i e t wwt.: NPN - HzSnPV; LOX - l i p o x y g e n a s e ; PPO - p o l y p h e n o l o x i d a s e : m o r t a l i t y r a t i o - % m o r t a l i t y i n treatment d i e t / m o r t a l i t y i n control diet. R a t i o 1.0 shows enhancement o f i n f e c t i v i t y : , F i x e d dose o f pathogen such t h a t l a r v a e i n g e s t i n g c o n t r o l d i e t s u f f e r e d 40-50% m o r t a l i t y . S i g n i f i c a n t d i f f e r e n c e s between means w i t h i n a column, based on 95% c o n f i d e n c e i n t e r v a l s from ANOVA, a r e shown b y d i f f e r e n t l e t t e r s . 1

2

3

C r i t i c a l C o m p l i c a t i o n s i n the Use o f O x i d a t i v e Resistance a g a i n s t Noctuid Larvae

Enzymes as Bases o f

Our knowledge o f how t o most e f f e c t i v e l y u t i l i z e PPO, POD, and LOX as a n t i n u t r i t i v e bases o f r e s i s t a n c e a g a i n s t n o c t u i d larvae i s i n s u f f i c i e n t . A number o f o t h e r c r i t i c a l enzymatic and c h e m i c a l

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

12.

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Enzymatic Antinutritive Defenses ofTomato

r e a c t i o n s o c c u r i n b o t h wounded p l a n t t i s s u e and t h e i n s e c t s ' d i g e s t i v e f l u i d s t h a t might s t r o n g l y modulate t h e o v e r a l l e f f e c t . PPO has h i g h a c t i v i t y over a b r o a d pH range (5.5 - 10.0) and e f f i c i e n t l y oxidizes a variety of caffeic acid derivatives to quinones w i t h o u t r e q u i r i n g a c o f a c t o r . PPO i s o p e r a t i o n a l t h r o u g h a l l phases o f d i g e s t i o n . I n comparison t o POD, t h e use o f PPO f o r r e s i s t a n c e i s r e l a t i v e l y s t r a i g h t f o r w a r d . A l t h o u g h POD a l s o has h i g h a c t i v i t y over a b r o a d pH range, i t s f u l l a c t i v i t y i s compromised by t h e f a c t t h a t i t r e q u i r e s H 0 as a c o f a c t o r . A l t h o u g h p l a n t s a r e known t o produce a l o c a l i z e d b u r s t o f H 0 a t s i t e s o f damage ( 3 6 ) , i t i s n o t known i n t h e tomato system i f t h i s burst provides sufficient H 0 to f a c i l i t a t e degradation of p r o t e i n q u a l i t y d u r i n g t h e e a r l y s t a g e s o f f e e d i n g . Both f o l i a g e , i n s e c t r e g u r g i t a t e , midgut t i s s u e s and lumen c o n t e n t s a l l c o n t a i n catalase. In fact, t h e r e g u r g i t a t e a l o n e o f l a r v a l H. z e a s i g n i f i c a n t l y impedes p l a n t POD a c t i v i t y because o f t h e p r e s e n c e o f h i g h c a t a l a s e a c t i v i t y ( F i g u r e 3 ) . We have a l r e a d y provided e v i d e n c e t h a t a d d i t i o n o f c a t a l a s e t o c r u s h e d f o l i a g e enhances t h e a b i l i t y o f H. z e a t o grow on t h a t f o l i a g e (Table I V ) , presumably through r e d u c t i o n o f endogenous l e v e l s o f p l a n t H 0 with consequent d i m i n u t i o n o f POD a c t i v i t y . However, i n o r d e r t o f o r m a l l y e s t a b l i s h the defensive r o l e o f p l a n t POD, one must u n d e r s t a n d i t s r e l a t i o n s h i p n o t o n l y w i t h p l a n t and i n s e c t d e r i v e d c a t a l a s e , b u t a l s o w i t h o t h e r c h e m i c a l and e n z y m a t i c systems t h a t degrade o r g e n e r a t e H 0 . For example, b o t h f o l i a g e and t h e i n s e c t ' s g u t f l u i d c o n t a i n s u p e r o x i d e dismutase (SOD), an enzyme w h i c h c o n v e r t s s u p e r o x i d e i o n s (Of) t o H 0 ( F i g u r e 3) . The f o r m a t i o n o f s u p e r o x i d e i o n i s f a v o r e d i n b a s i c c o n d i t i o n s (52,138,139), and i s a l s o a b y - p r o d u c t o f semiquinone a c t i o n on 0 (73) and o f a u t o x i d a t i v e r e a c t i o n s (e.g., catecholic phenolics, t h i o l s , and l e u k o f l a v a n s ) (139). A l s o , c e r t a i n enzymes produce O^F or K 0 as end p r o d u c t s (Table I X ) . Hence, t h e c o u n t e r - b a l a n c i n g a c t i v i t i e s o f c a t a l a s e and SOD i n t h e g e n e r a t i o n o f H 0 i n t h e p l a n t and t h e i n s e c t must a l s o be a c c o u n t e d f o r i n o r d e r t o a s s e s s t h e e f f i c a c y o f POD as a p l a n t defense. H 0 c a n a l s o be g e n e r a t e d n o n - e n z y m a t i c a l l y from coo x i d a t i v e p r o c e s s e s ( F i g u r e 3) such as v i a t h e o x i d a t i o n o f a c a t e c h o l i c p h e n o l i c by an o-quinone (139-143). Quinone f o r m a t i o n can be t h e r e s u l t o f PPO a c t i v i t y , b u t a l s o c a n a r i s e from spontaneous o x i d a t i o n i n b a s i c media (142,143). A l t h o u g h we have e s t a b l i s h e d i n v i t r o t h a t c h l o r o g e n i c a c i d and c a f f e i c a c i d c a n g e n e r a t e s i g n i f i c a n t q u a n t i t i e s o f H 0 , we have n o t e s t a b l i s h e d whether t h i s p r o c e s s o c c u r s i n c r u s h e d f o l i a g e and/or t h e i n s e c t ' s gut and s i m u l t a n e o u s l y f u r n i s h e s s u f f i c i e n t H 0 t o p e r m i t POD t o o p e r a t e d u r i n g t h e d i g e s t i o n o f food. I t a l s o remains t o be d e t e r m i n e d whether c e r t a i n p h e n o l i c s (e.g., r u t i n , v s c h l o r o g e n i c acid, vs. c a f f e i c acid) a r e more e f f i c i e n t than others a t g e n e r a t i n g H 0 . A l s o , c e r t a i n enzymes p r e s e n t i n p l a n t f o l i a g e ( e . g . , c a t a l a s e , and a s c o r b i c a c i d p e r o x i d a s e ; 144-146) c o u n t e r a c t the p r o d u c t i o n o f H 0 by r e d u c i n g i t t o water ( F i g u r e 3 ) . I t may be p o s s i b l e t o b r e e d f o r t h e a p p r o p r i a t e e n z y m a t i c and c h e m i c a l m i l i e u w h i c h w i l l f a v o r a h i g h e r p r e - i n j u r y l e v e l o f H 0 production, a more r a p i d post-damage b u r s t , and/or i t s maintenance d u r i n g f e e d i n g by t h e i n s e c t , t h e r e b y p e r m i t t i n g t h e 2

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2H0

2Η Ο

Figure 3. Interrelationship of Oxidative and Reductive Processes Linked t o t h e P r o d u c t i o n o f o-Quinones. POD — p e r o x i d a s e , PPO = p o l y p h e n o l o x i d a s e , GSG - g l u t a t h i o n e , GSSG oxidized glutathione.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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j o i n t a n t i n u t r i t i v e a c t i o n o f PPO and POD. The s i m u l t a n e o u s use o f LOX may be c o m p l i c a t e d by the f a c t t h a t c a t e c h o l s such as r u t i n are known t o i n h i b i t LOX a c t i v i t y (147) as w e l l as scavenge f r e e r a d i c a l s g e n e r a t e d d u r i n g t h e s e o x i d a t i v e p r o c e s s e s (148). Other i n t e r a c t i v e f a c t o r s w h i c h w i l l d e t e r m i n e the a b i l i t y of oxidized phenolics (quinones and semiquinones) t o damage p r o t e i n are the l e v e l s o f a s c o r b i c a c i d , g l u t a t h i o n e , and o t h e r r e d u c t a n t s i n f o l i a g e and the i n s e c t ' s d i g e s t i v e system. The maintenance o f h i g h l e v e l s o f Η θ 2 , t o run POD, may be compromised by h i g h l e v e l s o f a s c o r b i c a c i d w h i c h can reduce Η θ 2 t o w a t e r ( 1 4 4 - 1 4 6 ) ( F i g u r e 3 ) . Furthermore, h i g h l e v e l s o f a s c o r b i c a c i d are a l s o c a p a b l e o f r e d u c i n g quinones t o p h e n o l i c s , w h i c h t h e n can counteract the antinutritive effects of quinone production ( F i g u r e s 1,2, & 3 ) . I n v i t r o . the p r e s e n c e o f a s c o r b i c acid i m p a i r s the p r o d u c t i o n o f c h l o r o g e n o q u i n o n e by PPO. However, t h i s c o u n t e r a c t i v e e f f e c t may be overcome by the enzyme a s c o r b i c a c i d o x i d a s e (AOX). w h i c h o x i d i z e s a s c o r b i c a c i d t o d e h y d r o a s c o r b i c a c i d . A d d i t i o n o f AOX to a r t i f i c i a l d i e t s c o n t a i n i n g ascorbic a c i d , c h l o r o g e n i c a c i d , and PPO causes a g r e a t e r r e d u c t i o n i n l a r v a l growth (unpubl. d a t a ) . Tomato f o l i a g e c o n t a i n s b o t h h i g h l e v e l s o f a s c o r b i c a c i d and AOX (60; unpubl. d a t a ) . Thus, i f one were to b r e e d f o r h i g h l e v e l s o f AOX, quinone p r o d u c t i o n would occur more rapidly because of lowered levels of the reductant/nutrient ascorbic a c i d (Figures 3 & 4). 2

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T h i s multi-enzyme approach has s e v e r a l p o t e n t i a l advantages for c o n t r o l l i n g noctuid larvae. First, ascorbic acid is a n u t r i t i o n a l r e q u i r e m e n t f o r such l e p i d o p t e r a n s (27,30,149); i t s o x i d a t i o n t o d e h y d r o a s c o r b i c a c i d demands t h a t the i n s e c t use r e d u c i n g power i n the form o f g l u t a t h i o n e and NAD(P)H t o r e c l a i m a s c o r b i c a c i d . The s i m u l t a n e o u s p r o d u c t i o n o f quinones may also p l a c e a d r a i n on r e d u c i n g power because g l u t a t h i o n e i s r e a d i l y a l k y l a t e d by o-quinones (140,141; unpubl d a t a ) r e n d e r i n g it u n r e c l a i m a b l e . G l u t a t h i o n e may a l s o reduce o-quinones d i r e c t l y or w i t h an i n t e r v e n i n g s t e p i n v o l v i n g a s c o r b i c a c i d , t h e r e b y p l a c i n g a f u r t h e r d r a i n on reducing power ( F i g u r e 4). I f H2O2 i s d e t o x i f i e d by g l u t a t h i o n e p e r o x i d a s e a f u r t h e r d r a i n i s p l a c e d on r e d u c i n g power. G l u t a t h i o n e p e r o x i d a s e and g l u t a t h i o n e may a l s o be i n v o l v e d i n d e t o x i c a t i o n o f p r o d u c t s from l i p i d peroxidation r e s u l t i n g from LOX a c t i v i t y ( F i g u r e 3 ) . Reduced s u l p h u r amino a c i d i n t a k e as a r e s u l t o f the a c t i o n o f P i ' s o r quinones may may further exacerbate the requirement for reducing power and g l u t a t h i o n e . Hence, t h i s m u l t i p l e d r a i n may be o f s i g n i f i c a n c e i n c o n t r o l l i n g the i n s e c t i f h i g h l e v e l s o f r e d u c i n g power are r e q u i r e d to simulataneously d e t o x i f y o t h e r i n g e s t e d t o x i n s such i n s e c t i c i d e s or other n a t u r a l products. S i n c e P i ' s , PPO, POD, and LOX have the p o t e n t i a l t o i m p a i r s u l p h u r amino a c i d i n t a k e and u t i l i z a t i o n by t h e s e i n s e c t s , and such i n t a k e i s i m p o r t a n t f o r d e t o x i c a t i o n (e.g., g l u t a t h i o n e ) , i t may be p o s s i b l e t o c o n t r o l H. zea and S. e x i g u a not j u s t t h r o u g h a n t i n u t r i t i v e e f f e c t s but a l s o t h r o u g h impairment o f d e t o x i c a t i v e abilities. However, a deeper knowledge of these insects' d e t o x i c a t i v e a b i l i t i e s i s e s s e n t i a l i f t h e s e enzymes are t o be used s u c c e s s f u l l y . F u t h e r c o m p l i c a t i o n s arise, for like the f o l i a g e t h e y i n g e s t (146,148,150-153), t h e i r g u t s a l s o contain i n h e r e n t c a t a l a s e , s u p e r o x i d e dismutase, g l u t a t h i o n e p e r o x i d a s e ,

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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F i g u r e 4. Some I n t e r r e l a t i o n s h i p s Between Quinones, A s c o r b i c a c i d , G l u t a t h i o n e , and Reducing Power. The c y c l e o f redox events can o c c u r from l e f t t o r i g h t w i t h o u t i n t e r v e n t i o n o f enzymes. C e r t a i n enzymes c a n f a c i l i t a t e these r e a c t i o n s : 01 - p o l y p h e n o l o x i d a s e , 02 - p e r o x i d a s e , R l - quinone r e d u c t a s e without a s c o r b i c as a s u b s t r a t e , 03 — a s c o r b i c a c i d o x i d a s e , R2 — a s c o r b i c a c i d f r e e r a d i c a l r e d u c t a s e , R3 - d e h y d r o a s c o r b i c a c i d reductase, R4 - g l u t a t h i o n e r e d u c t a s e , R5 - g l u t a t h i o n e p e r o x i d a s e , LOOH - l i p i d h y d r o p e r o x i d e , and GS - c o v a l e n t l y bound g l u t a t h i o n e .

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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peroxidase, glutathione reductase, and other enzymes and r e d u c t a n t s (147,154-157). The consequences o f t h e b a l a n c e between the p l a n t d r i v e n c h e m i c a l and/or enzymatic r e a c t i o n s and those o f the i n s e c t a r e p o o r l y understood. A v a r i e t y o f p l a n t enzymes ( T a b l e I X ) generate t o x i c by-products. I t c e r t a i n l y may be p o s s i b l e t o simultaneously challenge the i n s e c t ' s a c q u i s i t i o n o f n u t r i e n t s , i t s b a l a n c e o f d e t o x i c a t i v e r e d u c i n g power, and i t s a b i l i t y t o h a n d l e t h e d i e t a r y and b o d i l y g e n e r a t i o n o f s u p e r o x i d e ions, hydroxyl ions, free radicals and H2O2 (52,139,147,154,155,158). The degree t o w h i c h t h e s e p l a n t f a c t o r s can be g e n e t i c a l l y m a n i p u l a t e d without i m p a i r i n g the p l a n t ' s p r o d u c t i v i t y , i s undetermined.

T a b l e IX.

Some O x i d a n t s and R e d u c t a n t s / A n t i o x i d a n t s i n P l a n t s

O x i d a n t s and By-products Aldehyde o x i d a s e ( s u p e r o x i d e i o n ) Glucose o x i d a s e ( s u p e r o x i d e i o n ) Xanthine oxidase (superoxide i o n ) Lipoxygenase ( h y d r o p e r o x i d e s , e p o x i d e s , free radicals) P o l y p h e n o l o x i d a s e (quinones)

Peroxidase

Re duc t a n t s/Ant i ox i d a n t s glutathione phenolics polyamines carotene s u p e r o x i d e dismutase glutathione p e r o x i d a s e and reductase catalase

(quinones, semiquinones free radicals) c h l o r o p h y l l ( i n dark) Chlorophyll ( i n light) vitamin Ε F l a v a n dehydrogenase ( s u p e r o x i d e i o n ) ascorbic acid Galactose oxidase (superoxide ion) 2°2 generators Superoxide dismutase Amine o x i d a s e Polyamine o x i d a s e G l y c o l a t e oxidase U r i c oxidase A u t o o x i d a t i o n and c o o x i d a t i o n of/by phenolics See r e f e r e n c e s 52,139,146,148,151,152,159.

H

M u l t i p l e Onslaughts

against A c q u i s i t i o n of Nutrients

We have d e s c r i b e d an approach t o h o s t p l a n t r e s i s t a n c e t h a t i n v o l v e s t h e use o f p l a n t o x i d a t i v e enzymes t o i r r e v o c a b l y d e p r i v e the i n s e c t o f n u t r i e n t s . We have emphasized t h a t t h e c h e m i c a l r e a c t i o n s c a t a l y z e d by POD and PPO have t h e p o t e n t i a l t o d e s t r o y a v a r i e t y o f e s s e n t i a l o r l i m i t i n g amino a c i d s (Table X ) . I n p a r t i c u l a r , these r e a c t i o n s a r e adept a t d e s t r o y i n g l y s i n e and cysteine. Integral lysine i s necessary f o r proper enzymatic h y d r o l y s i s o f p r o t e i n . C y s t e i n e and m e t h i o n i n e , amongst o t h e r u s e s , a r e r e q u i r e d t o s y n t h e s i z e t r y p s i n . The a c t i o n o f PPO and POD i n c o n j u n c t i o n w i t h P i ' s a r e p r o p o s e d t o p l a c e a s e v e r e s t r a i n on t h e i n s e c t f o r h i g h s u l p h u r amino a c i d i n t a k e . T h i s s t r a i n may be f u r t h e r e x a c e r b a t e d by t h e complementary a c t i o n o f quinones

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d e p l e t i n g a v a i l a b l e glutathione v i a formation o f conjugates. Furthermore, s i m u l t a n e o u s a c t i o n o f AOX may l i m i t t h e q u a n t i t y o f e s s e n t i a l a s c o r b i c a c i d and p l a c e a f u r t h e r s t r a i n on c h e m i c a l and enzymatic r e d u c i n g power. These o x i d a t i v e c o n d i t i o n s a l s o have t h e p o t e n t i a l t o d e s t r o y e s s e n t i a l n u t r i e n t s such as t o c o p h e r o l (151) and t h i a m i n e (160).

T a b l e X.

Some N u t r i e n t s D e s t r o y e d o r Rendered L e s s A v a i l a b l e by P l a n t Chemical o r Enzymatic I n t e r a c t i o n s

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Agent Ascorbic a c i d oxidase Lipoxygenase

Peroxidase (lysine,cysteine, histidine, tryptophan),

P h e n y l a l a n i n e ammonia l y a s e Polyphenol oxidase cysteine

Nutrient ascorbic acid l i n o l e n i c and l i n o l e i c a c i d s , β-carotene amino a c i d s ( l y s i n e , cysteine, histidine) protein methionine, tyrosine, above f r e e amino acids, ascorbic acid, thiamine phenylalanine protein (lysine, methionine,

histidine,

Proteinase i n h i b i t o r s utilization

t y r o s i n e ) , above f r e e amino a c i d s , a s c o r b i c a c i d , thiamine d i g e s t i o n and of p r o t e i n ( s u l p h u r amino a c i d s )

Tomatine

T y r o s i n e ammonia l y a s e

cholesterol, sitosterol and r e l a t e d phytosterols tyrosine

LOX destroys linoleic and l i n o l e n i c acids v i a their o x i d a t i o n t o l i p i d h y d r o p e r o x i d e s . These l i p i d hydroperoxides s u b s e q u e n t l y form h y d r o p e r o x i d e s , h y d r o p e r o x i d e free radicals, epoxides and malondialdehyde which can impair the n u t r i t i v e q u a l i t y o f p r o t e i n v i a mechanisms s i m i l a r t o those mediated by POD and PPO. These u n s a t u r a t e d f a t t y a c i d s a r e e s s e n t i a l f o r normal l a r v a l growth and m a t u r a t i o n . We have p r e s e n t e d e v i d e n c e (124) t h a t p h e n y l a l a n i n e and t y r o s i n e ammonia l y a s e s , two enzymes i n d u c e d d u r i n g wounding, have the p o t e n t i a l t o l i m i t t h e i n s e c t s ' i n t a k e o f f r e e p h e n y l a l a n i n e

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and t y r o s i n e because t h e y are c o n v e r t e d t o n u t r i t i o n a l l y i n e r t cinnamic a c i d d e r i v a t i v e s . I n s e c t s a l s o r e q u i r e an exogenous s o u r c e o f p h y t o s t e r o l s ( 2 7 ) . The g l y c o a l k a l o i d tomatine i s an e f f e c t i v e p r e c i p i t a t o r o f c e r t a i n p h y t o s t e r o l s such as s i t o s t e r o l and c h o l e s t e r o l , and as such may p r o v i d e a means o f r e d u c i n g s t e r o l i n t a k e f o r n o c t u i d l a r v a e (97). The f e a s i b i l i t y o f u s i n g t h e s e a n t i n u t r i t i v e p l a n t systems as m u l t i p l e - f a c t o r / m u l t i p l e - m e c h a n i s m resistance against noctuid l a r v a e remains t o be determined. I t i s p o s s i b l e t h a t such a m u l t i p l e o n s l a u g h t a g a i n s t n u t r i e n t a c q u i s i t i o n i s redundant. I n o t h e r words, perhaps m e r e l y the use o f PPO and c h l o r o g e n i c a c i d i s s u f f i c i e n t . I t a l s o remains t o be d e t e r m i n e d whether t h i s p r o p o s e d multiple-factor/multiple-mechanism of resistance renders the i n s e c t s ' d e t o x i c a t i v e systems more s u s c e p t i b l e t o traditional c o n t r o l t a c t i c s , and whether the e v o l u t i o n o f r e s i s t a n c e t o such multiple antinutritive factors i s more difficult than to insecticides. Advantages o f the Use

o f Enzymatic Defenses

Our e v i d e n c e s u p p o r t s the c o n t e n t i o n t h a t p l a n t o x i d a t i v e enzymes can be used as c o n s t i t u t i v e and/or i n d u c i b l e a n t i n u t r i t i v e bases o f r e s i s t a n c e a g a i n s t i n s e c t s . T h i s r e s i s t a n c e i s b a s e d on the irreversible chemical degradation of multiple essential or l i m i t i n g n u t r i e n t s , w h i c h may be more d i f f i c u l t f o r the i n s e c t species to evolve biochemical r e s i s t a n c e against than against c l a s s i c a l "toxins". Other advantages a l s o a c c r u e from t h e i r use. Because t h e y are enzymes, o n l y c a t a l y t i c amounts are r e q u i r e d t o d r i v e the reactions, provided substrates are not limiting. I f one is c o n c e r n e d about the c o s t o f defense r e n d e r i n g the p l a n t l e s s agronomically efficient (15), when employing a battery of secondary gene p r o d u c t s as the bases o f resistance (e.g., t o m a t i n e , p h e n o l i c s , and 2 - t r i d e c a n o n e ) , perhaps the u t i l i z a t i o n o f PPO and POD i n c o n j u n c t i o n w i t h p h e n o l i c s i s more e f f i c i e n t . The s y n t h e s i s o f c a t a l y t i c amounts o f enzyme s h o u l d p l a c e l e s s m e t a b o l i c d r a i n on the p l a n t t h a n s y n t h e s i z i n g one o r s e v e r a l secondary p r o d u c t s t h a t u s u a l l y r e q u i r e l e v e l s o f 0.1% and above t o be a c t i v e . I n terms o f b r e e d i n g programs, i t s h o u l d be e a s i e r to manipulate the expression of primary gene p r o d u c t s through c l a s s i c a l (e.g., i n t r o d u c i n g e x o t i c genes from r e l a t e d s p e c i e s ) o r modern biotechnological procedures (e.g., amplifying gene e x p r e s s i o n ) t h a n t o m a n i p u l a t e the e x p r e s s i o n o f secondary gene products. C o n s i d e r i n g the i n c r e a s i n g l y s e v e r e c o n s t r a i n t s upon r e g i s t e r i n g g e n e t i c a l l y m o d i f i e d organisms f o r commercial use, i t might be e a s i e r t o r e g i s t e r p l a n t s t h a t c o n t a i n o n l y c a t a l y t i c q u a n t i t i e s o f enzymes, d e r i v e d from r e l a t e d s p e c i e s , t h a n t o r e g i s t e r p l a n t s t h a t must e x p r e s s h i g h l e v e l s o f t r a n s g e n i c gene p r o d u c t s ( e . g . , BT t o x i n , l e c t i n s , and P i ' s ) . Other advantages b e a r r e p e a t i n g . These enzymes have been i m p l i c a t e d i n r e s i s t a n c e t o pathogens, and hence, t h e i r d i r e c t e d utilization against insects may complement resistance to pathogens. Three enzymes (PPO, POD, and LOX) have b r o a d pH

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p r o f i l e s which permit t h e i r operation i n the a c i d i c c o n d i t i o n s o f i m m e d i a t e l y c r u s h e d f o l i a g e (pH - 5.5) and i n t h e b a s i c c o n d i t i o n s o f t h e i n s e c t ' s g u t (pH 8.5). Furthermore, PPO and POD a r e a l m o s t completely r e s i s t a n t t o d i g e s t i o n by t r y p t i c and chymotryptic enzymes and thus remain a c t i v e i n t h e i n s e c t ' s g u t d u r i n g d i g e s t i o n o f food. PPO i s a c t u a l l y a c t i v a t e d by t h e g u t p r o t e a s e s . These p l a n t enzymes a r e i n d u c i b l e by v a r i o u s k i n d s o f f e e d i n g damage, w h i c h s h o u l d e x a c e r b a t e t h e i r e f f e c t s . And, f i n a l l y , t h e phenology o f t h e tomato, l i k e most p l a n t s (151,161-164), f a v o r s the a c t i o n o f t h e s e enzymes. As tomato p l a n t s age t h e y become more o x i d a t i v e by h a v i n g l e v e l s o f PPO, POD, and LOX i n c r e a s e , w h i l e l e v e l s o f p l a n t n i t r o g e n and c a t a l a s e f a l l (79; u n p u b l . d a t a ) . Even i n t h e green f r u i t s t a g e , tomato p l a n t s a r e o x i d a t i v e . I t may be p o s s i b l e t o a c c e n t u a t e t h e o x i d a t i v e s t a t e o f t h e p l a n t t h r o u g h o u t i t s l i f e t o f a c i l i t a t e r e s i s t a n c e . However, t h i s e f f o r t might be a t odds w i t h o t h e r s ' attempts t o b r e e d l e s s " o x i d a t i v e " p l a n t s i n o r d e r t o decrease s u s c e p t i b i l i t y t o h e r b i c i d e s (165). T h i s form o f r e s i s t a n c e i s t a r g e t e d a g a i n s t i n s e c t s w i t h b a s i c g u t s ( i . e . , l e p i d o p t e r a n l a r v a e ) . The b a s i c g u t environment f a v o u r s o x i d a t i v e c o n d i t i o n s and t h e t y p e s o f r e a c t i o n s p r o p o s e d (e.g., S c h i f f base formation, alkylation, co-oxidation, and a u t o o x i d a t i o n ) . However, w i t h i n s e c t s (e.g., t h e C o l o r a d o P o t a t o b e e t l e L e p t i n o t a r s a d e c e m l i n e a t a ) h a v i n g a c i d i c g u t f l u i d (pH 6.0 - 6.5), t h e p r o p o s e d mechanisms may be o f l i t t l e v a l u e . I n a c i d i c c o n d i t i o n s , S c h i f f base f o r m a t i o n and a l k y l a t i o n o f n u c l e o p h i l i e s are n o t f a v o r e d because t h e n u c l e o p h i l i c groups a r e p r o t o n a t e d . We have shown i n l a r v a l L. d e c e m l i n e a t a t h a t p l a n t p r o t e i n i s n o t s i g n i f i c a n t l y a l k y l a t e d by PPO and c h l o r o g e n i c acid (unpubl. d a t a ) . A l t e r n a t e t a c t i c s might be n e c e s s a r y f o r s i m u l t a n e o u s c o n t r o l o f l a r v a l b e e t l e s and n o c t u i d s . Conclusion We have p r o p o s e d t h e use o f s e v e r a l p l a n t enzymes as a p o l y g e n i c b a s i s o f r e s i s t a n c e against n o c t u i d l a r v a e through a c t i v a t i o n o f b o t h " t o x i n s " and " n u t r i e n t s " t o forms t h a t c h e m i c a l l y reduce t h e n u t r i t i o n a l v a l u e o f t h e tomato p l a n t . A l t h o u g h t h i s approach may be u s e f u l i n d e v e l o p i n g r e s i s t a n c e t h a t i s d u r a b l e , t h e use o f such enymes i s c o m p l i c a t e d by t h e i r mutual i n t e r a c t i o n s and t h e c h e m i c a l c o n t e x t o f t h e p l a n t , by t h e d e t o x i c a t i v e a b i l i t i e s o f the i n s e c t , and by t h e i r u n p r e d i c t a b l e e f f e c t s upon b i o l o g i c a l c o n t r o l a g e n t s . Such an approach may be w a r r a n t e d i n v i e w o f p u b l i c d i s d a i n f o r p e s t i c i d e s ; such r e s i s t a n c e may o f f e r n o t o n l y s i m u l t a n e o u s r e s i s t a n c e a g a i n s t pathogens and i n s e c t s , b u t a l s o o f f e r resistance that i s environmentally safe, thus, lessening r e l i a n c e on c o n v e n t i o n a l c o n t r o l t a c t i c s . On a l e s s o p t i m i s t i c n o t e , t h e f o r t h r i g h t u t i l i z a t i o n o f t h e above a n t i n u t r i t i v e defenses may be p r a g m a t i c a l l y d i f f i c u l t from the s t a n d p o i n t o f breeding. The e x p r e s s i o n o f p h e n o l i c s , and l i k e l y many o f t h e o t h e r characters, a r e under q u a n t i t a t i v e genetic c o n t r o l . Hence, d e r i v i n g p r e d i c t a b l e r e s i s t a n c e , as prescribed above, w i t h o u t the h i g h l y modulating e f f e c t s o f environment and p o t e n t i a l gene i n t e r a c t i o n s may p r e s e n t i t s own s e t o f problems. Furthermore, many o f t h e c a n d i d a t e enzymes a r e m u t u a l l y and i n t i m a t e l y i n v o l v e d i n t h e p l a n t ' s defense a g a i n s t

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m i c r o b e s , i n d e t o x i c a t i o n and g e n e r a l m e t a b o l i s m , and i n p r o c e s s e s o f m a t u r a t i o n and senescence. The e f f e c t s o f m a n i p u l a t i n g the e x p r e s s i o n o f s u c h enzymes on the g e n e r a l agronomic v a l u e o f the p l a n t i s unknown. Acknowledgments T h i s work was s u p p o r t e d by the USDA C o m p e t i t i v e G r a n t s Program t h r o u g h g r a n t s USDA 89-37250-4639 t o S.S.D and G W.F., and USDA 87-CRCR-1-2371 t o S.S.D. Literature Cited

Downloaded by CORNELL UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch012

1.

2. 3. 4. 5.

6.

7. 8. 9. 10.

11.

12.

13.

14. 15. 16.

17. 18.

Hare, J . D. In Variable Plants and Herbivores i n Natural and Managed Systems; Denno, R. F.; McClure, M. S., Eds; Academic Press: New York, 1983; Chapter 18. Mattson, W. J . Ann. Rev. Ecol. Syst. 1980, 11, 119-61. Mattson, W. J . ; Haack, R. A. BioScience 1987, 37, 110-18. Price, P. W. Insect Ecology; John Wiley & Sons: New York, 1984; p 606. Scriber, J . M. In Chemical Ecology of Insects; B e l l , W. J . ; Carde, R. T., Eds.; Sinauer Associates Inc.: Sunderland, Massachusetts, 1984b; pp 159-202. Strong, D. R.; Lawton, J . H.; Southwood, S i r R. Insects On Plants. Community Patterns and Mechanisms; Harvard University Press: Cambridge, Massachusetts; 1984. White, T. C. R. Oecologia 1984, 63, 90-105. Barbosa, P.; Schultz, J . C., Eds. Insect Outbreaks; Academic Press: New York, 1987; p 578. Coley, P. D.; Bryant, J . P.; Chapin, F. S. Science 1985, 230, 895-99. Denno, R. F.; McClure, M. S., Eds. Variable Plants and Herbivores i n Natural and Managed Systems; Academic Press: New York; 1983, p 717. Mattson, W. J . ; Levieux, J . ; Bernard-Dagan, C. Eds. Mechanisms of Woody Plant Defenses Against Insects. Search for Pattern; Springer-Verlag: B e r l i n , 1988; p 416. McClure, M. S. In Variable Plants and Herbivores i n Natural and Managed Systems: Denno, R.F.; McClure, M. S., Eds.; Acadmeic Press: New York, 1983, pp 135-54. Rhoades, D. F. In Variable Plants and Herbivores i n Natural and Managed Systems; Denno, R.F.; McClure, M. S., Eds.; Acadmeic Press: New York, 1983, pp 155-220. Rhoades, D. Amer. Nat. 1985, 125, 205-38. Kogan, M. In IPM and Ecological Practise; Kogan, M., Ed.; John Wiley and Sons: New York, 1986; pp 83-134. Maxwell, F. G.; Jennings, P. R., Eds. Breeding Plants Resistant to Insects; Wiley Interscience: New York, 1980; p 683. Panda, N. Principles of Host-Plant Resistance to Insect Pests; Allanheld/Universe: New York, 1979; p 386. Smith, C. M. Plant Resistance to Insects. A Fundamental Approach; John Wiley and Sons: New York, 1989; p 286.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

191

192

NATURALLY OCCURRING PEST BIOREGULATORS 19.

20. 21.

22. 23. 24.

Downloaded by CORNELL UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch012

25. 26. 27. 28.

29. 30. 31.

32. 33. 34. 35. 36. 37.

38.

39.

40.

41.

42.

Feeny, P. P. Coevolution of Animals and Plants; G i l b e r t , L. E.; Raven, P. Η., Eds.; University of Texas Press: Austin, 1975, pp 1-19. Moore, R. F. J . Econ. Entomol. 1983, 76, 697-99. Rhoades, D. F. In Herbivores: Their Interaction with Secondary Plant Metabolites; Rosenthal, G. Α.; Janzen, D. Η., Eds.; Academic Press: New York, 1979; pp 1-54. Bernays, E. A. Ecol. Entomol. 1981, 6, 353-60. Martin, J . S.; Martin, M. M. Oecologia 1982, 54, 205-311. Martin, J . S.; Martin, M. M.; Bernays, E. A. J . Chem. Ecol. 1987, 13, 605-21. Reese, J . C.; Chan, B. G.; Waiss, A. C., J r . J . Chem. Ecol. 1982, 8, 1429-36. Zucker, W. V. Amer. Natur. 1983, 121, 335-65. Dadd, R. H. Ann. Rev. Entomol. 1973, 18, 381-419. Brodbeck, B.; Strong, D. In Insect Outbreaks; Barbosa, P.; Schultz, J . C., Eds.; Academic Press: New York, 1987; pp 347-64. van Emden, H. F. In Phytochemical Ecology; Harborne, J.,Ed.; Academic Press: London, 1972; pp 25-43. Penny, L. H.; Scott, G. E.; Guthrie, W. D. Crop S c i . 1967, 7, 407-9. Birk, Y.; Peri, I. In Toxic Constituents of Plant Foodstuffs; Liener, I. Ε., Ed.; Academic Press: New York, 1980; pp 161-82. Bloem, Κ. Α.; Kelley, K. C.; Duffey, S. S. J . Chem. Ecol. 1989, 15, 387-98. Silano, V. Cereal Chem. 1978, 55, 722-31. Silano, V.; Zahnley, J . C. Biochim. Biophys. Acta. 1978, 533, 181-5. Shukle, R. H.; Murdock, L. L. Environ. Entomol. 1983, 12, 787-91. Apostol, I.; Heinstein, P. F.; Low, P. S. Plant Physiol. 1989, 90, 109-16. Boiler, T. In C e l l u l a r and Molecular Biology of Plant Stress; Key, L.; Kosuge,T., Eds; Alan R. L i s s , Inc.: New York, 1985; pp 247-62. Boller, T. In Plant-Microbe Interactions. Molecular and Genetic Perspective; Kosuge, T.; Nester, E. W., Eds.; MacMillan Publ. Company: New York, 1987; pp 385-413. Davies, E. The Biochemistry of Plants. A Comprehensive Treatise. Physiology of Metabolism; Davies, Ε., Ed.; Academic Press: New York, 1987; Vol. 12, pp 243-64. Esquerre-Tuagaye, M. T.; Mazau, D.; P e l i s s i e r , B.; Roby, D.; Runeau, D.; Toppan, A. In C e l l u l a r and Molecular Biology of Plant Stress; Key, J . L.; Kosuge, T., Eds.; Alan R. L i s s Inc.: New York, 1985; pp 459-73. Gianinazzi, S. Plant-Microbe Interactions. Molecular and Genetic Perspective; Kosuge, T.; Nester, E. W., Eds.; MacMillan Publ. Company: New York, 1984; pp 321-42. Hahlbrock, K.; Scheel, D. In Innovative Approaches to Plant Disease Control; Chet, I.,Ed; Wiley and Sons: New York, 1987; pp 228-54.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

DUFFEY AND FELTON 43.

44.

45.

46.

Downloaded by CORNELL UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch012

47.

48. 49. 50. 51. 52.

53. 54. 55. 56. 57.

58. 59. 60.

61. 62. 63. 64. 65.

66.

Enzymatic Antinutritive Defenses of Tomato

Kahl, G. In The New Frontiers i n Plant Biochemistry; Akazawa, T.; Asahia, T; Imaseki, Η., Eds; Martinus Nijhoff/Dr. W. Junk Publishers: The Hague, 1983; pp 193-216. Kuc, J.; Lisker, Ν. In Biochemistry of Wounded Plant Tissues. Kahl, G., Ed.; Walter de Gruyter: B e r l i n , 1987; pp 203-42. Rhodes, J . M.; Wooltorton, L. S. C. In Biochemistry of Wounded Plant Tissues; Kahl, G., Ed.; Walter de Gruyter: B e r l i n , 1978; pp 243-86. Ryan, C. Α.; Bishop, P. D.; Graham, J . S.; Meyer-Broadway R.; Duffey, S. S. J . Chem. Ecol. 1985, 12, 1025-36. Butt, V. S. In The Biochemistry of Plants. A Comprehensive Treatise. Metabolism and Respiration; Stumpf, P. K.; Conn,.E. Ε., Eds; Academic Press: New York, 1981; V o l . 2, pp 81-123. Fry, S. C. Ann. Rev. Plant. Physiol 1986, 37, 165-86. G a l l i a r d , T. In Biochemistry of Wounded Plant Tissues; Kahl, G., Ed.; Walter de Gruyter: B e r l i n , 1978; pp 155-201. Gardner, H. W. J . Agric. Food Chem. 1979, 27, 220-29. Ampomah, Υ. Α.; Friend, J . Phytochemistrv 1988, 27, 253341. Elstner, E. F. In The Biochemstrv of Plants. A Comprehensive Treatise. Biochemistry and Metabolism; Davies, D. D., Ed.; Academic Press: New York, 1980; V o l . 11, pp 253-315. Mayer, A. M. Phytochemistrv 1987, 26, 11-20. Mayer, A. M.; Harel, E. Phytochemistry 1979, 18, 193-215. Mohan, R.; Kolattukudy, P. Plant Physiol. 1990, 921, 27680. Pierpoint, W. S.; Ireland, R. J . ; Carpenter, J . M. Phytochemistry 1977, 16, 29-34. Stahmann, M. A. In The New Frontiers i n Plant Biochemistry; Akazawa, T.; Asahia, T.; Imaseki, Η., Eds; Martinus Nijhoff/Dr. W. Junk Publishers: The Hague, 1983; pp 237-50. G a l l i a r d , T.; Matthew, J . A. Phytochemistrv 1977, 16, 33943. Gentile, I. Α.; F e r r a r i s , L.; Matta, A. J . Phytopathol. 1988, 122, 45-53. Hobson, G. E.; Davies, J . N. In The Biochemistry of F r u i t s and Their Products.; Hulme, A. C., Ed.; Academic Press: New York, 1971; V o l . 2, pp 437-82. Raman. K.; Sanjayan, K. P. Proc. Indian Acad. S c i . (Animal Sci.), 1984, 93(6), 543-7. Reuveni, R.; Ferreira, J . F. Phytopath. Z. 1985, 112, 1937. H u r r e l l , R. F.; Finot, P. Α.; Cuq, J . L. J . N u t r i t i o n 1982, 42, 191-211. Vaughn, K. C.; Duke, S. O. Physiol. Plant 1984, 60, 106-12. H u r r e l l , R. F.; Finot, P. A. In D i g e s t i b i l i t y and Amino Acid A v a i l a b i l i t y i n Cereals and Oilseeds; Finley, J . W.; Hopkins, D. T., Eds.; American Association of Cereal Chemists, Inc.: St. Paul, 1982; pp 233-46. Davies, A. M. C.; Newby, V. K.; Synge, R. L. M. J. Sci. Food Agric. 1978, 29, 33-41.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

193

194 67. 68. 69.

70. 71.

Downloaded by CORNELL UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch012

72. 73.

74.

75.

76. 77.

78.

79. 80. 81. 82. 83. 84.

85. 86. 87. 88. 89. 90. 91.

NATURALLY OCCURRING PEST BIOREGULATORS Leatham, G. F.; King, V.; Stahmann, M. A. Phytopathology 1980, 70, 1134-40. Matheis, G.; Whitaker, J . R. J . Food Biochem. 1987, 11, 309-27. Pierpoint, W. S. In Leaf Protein Concentrates; Telek, L.; Graham, H. D., Eds.; A v i Publ. Comp., Inc.: Wesport, Connecticut, 1983; pp 235-67. Motoda, S. J . Ferment. Technol. 1979, 57, 395-9. Stahmann, Μ. Α.; Spencer, A. K.; Honold, G. R. Biopolymers 1977, 16, 1307-18. Whitmore, F. W. Plant S c i . Letters 1978, 13, 241-5. Kanner, J . ; German, J . B.; K i n s e l l a , J . E. In C r i t i c a l Reviews i n Food Science and N u t r i t i o n ; Furia, T. Ε., Ed.; CRC Press: Boca Raton, 1987; pp 317-65. Vick, Β. Α.; Zimmerman, D. C. In The Biochemistry of Plants. A Comprehensive Treatise. L i p i d s : Structure and Function; Stumpf, P. Κ., Ed.; Academic Press: New York, 1987; V o l . 9, pp 53-90. B e l i t z , H.-D.; Grosch, W., Eds. Food Chemistry. (translated from German by D. Hadziyev); Springer Verlag: B e r l i n , 1987; p 774. Matsushita, S. J . Agric. Food Chem. 1975, 23, 150-5. Ory, R. L.; St. Angelo, A. J . In Food Protein Deterioration. Mechanisms and Functionality: Cherry, J . P., Ed.; American Chemical Society: Washington, DC, 1982; pp 5566. W i l l s , E. D. In Biochemical Toxicology, A P r a c t i c a l Approach; Snell, K.; Mullock, Β., Eds.; IRL Press: Washington, DC, 1985; pp 127-52. Felton, G. W.; Donato, K.; Del Vecchio, R. J . ; Duffey, S. S. J. Chem. Ecol. 1989, 15, 2667-94. Dryden, M. J . ; Saterlee, L. D. J . Food Science 1978, 43, 650-1. Eklund, A. Nutr. Metab. 1975, 18, 258-264. Friedman, M. Protein N u t r i t i o n a l Quality of Foods and Feeds; Marcel Dekker, Inc.: New York, 1975. Horigome, T.; Kandatsu, T. Agric. B i o l . Chem. 1968, 32, 1093-1102. H u r r e l l , R. F.; Finot, P. In N u t r i t i o n a l and Toxicological Aspects of Food Safety: Friedman, M., Ed.; Plenum Press: New York, 1984; pp 423-36. Jung, H.-J. G.; Fahey, G. C., J r . J . Agric. Food Chem. 1981, 29, 817-20. Lahiry, N. L.; Satterlee, H. W.; Wallace, G. W. J . Food Science 1977, 42, 83-85. Lee, H. S.; Louriminia, S. S.; C l i f f o r d , A. J . ; Whitaker, J . R.; Feeny, R. A. J . N u t r i t i o n 1978, 108, 687-97. Barbeau, W. E.; K i n s e l l a , J . E. J . Agric. Food. Chem. 1983, 31, 993-98. Barbeau, W. E. ; K i n s e l l a , J . E. J . Food S c i . 1985, 50, 1083-1100. Finot, P. A. Qual. Plant Foods Hum. Nutr. 1983, 32, 439-53. Anderson, P. A. In D i g e s t i b i l i t y and Amino Acid Availability i n Cereals and Oilseeds: Finley, J . W.;

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

12.

DUFFEY AND FELTON

92. 93. 94. 95. 96.

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97.

98. 99.

100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117.

118. 119.

Enzymatic Antinutritive Defenses of Tomato

Hopkins, D. T., Eds.; Amer. Assoc. Cereal Chemists, Inc.: St. Paul, 1985; pp 31-44. Igarashi, K.; Tsunekuni, T.; Yasui, T. J. Nutrition Sci. Vitaminol. 1983, 29, 227-32. Rock, G. C.; Hogdson, E. J . Insect Physiol. 1971, 17, 108797. Broadway, R. M.; Duffey, S. S. J . Insect Physiol. 1986, 32, 673-80. Broadway, R. M.; Duffey, S. S. J . Insect Physiol. 1986, 32, 827-33. Broadway, R. M.; Duffey, S. S. J . Insect Physiol. 1988, 34, 1111-17. Duffey, S. S.; Bloem, K. A. In Ecological Theory and IPM Practice; Kogan, M., Ed.; John Wiley & Sons: London, 1986; pp 135-83. Kauffman, W. C.; Kennedy, G. G. J . Chem. Ecol. 1989, 15, 2051-60. Duffey, S. S. In Insects and Plant Surfaces; Southwood, S i r R.; Juniper, Β., Eds.; Edward Arnold: London, 1986; pp 15172. H a l l , C. B.; Knapp, F. W.; S t a l l , R. E. Phytopathology 1969, 59, 267-8. Rick, C. M.; Fobes, J . F. Proc. Nat. Acad. S c i . 1976, 73, 900-4. Duffey, S. S.; Isman, M. B. Experientia 1981, 37, 574-6. E l l i g e r , C. Α.; Wong, Y.; Chan, B. G.; Waiss, A. C., J r . J. Chem. Ecol. 1981, 7, 753-8. Strack, D.; Gross, W. Plant Physiol. 1990, 92, 41-7. Ryan, J . D.; Gregory, P.; Tingey, W. M. Phytochem. 1982, 8, 1185-7. Carrasco, Α.; Boudet, A. M.; Marigo, S. Physiol. Plant Pathol. 1978, 12, 225-32. Chadha, K. C.; Brown, S. A. Can. J . Bot. 1974, 52, 2041-7. Matta, Α.; Gentile, I.; G i a i , I. Phytopathol. 1969, 59, 512-13. Mendez, J . ; Brown, S. A. Can. J . Bot. 1971b, 49, 2101-05. Pollock, C. J . ; Drysdale, R. B. Phytopath. Z. 1976, 86, 5666. Buttery, R. G.; Teranishi, R. ; Ling, L. C. J . Agric. Food Chem. 1987, 35,540-546. Buttery, R. G.; Ling, L. C.; Light, D. M. J . Agric. Food Chem. 1987, 35, 1039-42. Zamora, R.; Olias, J . M.; Mesias, J . L. Phytochemistry 1987, 26, 345-7. Hildebrand, D. F. Physiol. Plant. 1989, 76, 249-53. Hildebrand, D. F.; Rodriquez, J . G.; Brown, G. C.; Luu, K. T.; Volden, C. S. J . Econ. Entomol. 1986, 79, 1459-65. Neese, P. A. Poster STD0017; Entomol. Soc. Amer. Natl. Conf. Exh., Dec., 1988, L o u i s v i l l e , Kentucky. Ryan, C. A. In Advances i n Plant Gene Research: Verma, O. S. P.; Hohns, T., Eds.; Springer-Verlag: B e r l i n , 1984; pp 321-32. Weiel, J . ; Hapner, K. D. Phytochemistry 1976, 15, 1885-7. Thornburg, R. W.; Kernan, Α.; Molin, L. Plant Physiol. 1990, 92, 500-5.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

195

196 120.

121. 122. 123. 124.

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126. 127. 128. 129.

130. 131. 132. 133. 134. 135. 136.

137. 138. 139. 140. 141. 142. 143. 144. 145. 146.

NATURALLY OCCURRING PEST BIOREGULATORS Foard, D. E.; Murdock, L. L.; Dunn, P. E. In Plant Molecular Biology; Alan R. L i s s , Inc.: New York, 1983; pp 223-33. Hilder, V. Α.; Angharad, A. M. R.; Gatehouse, Sheerman S. E.; Barker, R. E.; Boulter, D. Nature 1987, 300, 160-3. Thornburg, R. W.; Cleveland, A. G.; Johnson, R.; Ryan, C. A. Proc. Natl. Acad. S c i . 1987, 84, 744-8. Broadway, R.; Duffey, S. S.; Pearce, G.; Ryan, C. A. Entomol. Exp. Appl. 1986, 41, 33-8. Duffey, S. S.; Felton, G. W. In Biocatalysis i n A g r i c u l t u r a l Biotechnology; Amer Chem. Soc. Symp. Ser. 389, 1989; pp 289-313. Felton, G. W.; Broadway, R. M.; Duffey, S. S. J . Insect Physiol. 1989, 35, 981-90. Lange, W. H.; Bronson, L. Ann. Rev. Entomol. 1981, 26, 34571. Kennedy, G. G. Entomol. Soc. Amer. B u l l . 1978, 24, 375-84. Dimock, Μ .Α.; Kennedy, G. G. Ent. Exp. Appl. 1983, 33, 263-8. Lee, J . S.; Brown, W. E.; Graham, J . S.; Pearce, G.; Fox, E. Α.; Dreher, T. W.; Ahren, K. G.; Pearson, G. D.; Ryan, C. A. Proc. Natl. Acad. S c i . 1986, 83, 7277-81. Liener, I. E. Ed. Toxic Constituents of Plant Foodstuffs; Academic Press: New York, 1980; p 500. Plunkett, G.; Senear, D. F.; Zuroske, G.; Ryan, C. A. Arch. Biohem. Biophys. 1982, 213, 463-72. Meeusen, R. L.; Warren, G. Ann. Rev. Entomol. 1989, 34, 373-81. Felton, G. W.; Duffey, S. S.; V a i l , P. V.; Kaya, Η. K.; Manning, J . J . Chem. Ecol. 1987, 13, 947-57. Felton, G. W.; Duffey, S. S. J . Chem. Ecol. 1989, 36. Barton, Κ. Α.; Whiteley, H. R.; Yang, N.-S. Plant Physiol. 1987, 85, 1103-09. Hammock, B. D.; Harshman, L. G.; Philpott, M. L.; Szekacs, Α.; Ottea, J . Α.; Newitt, R. Α.; Wroblewski, V. J . ; Halarnakar, P. P.; Hanzlik, T. N. In Biomechanisms Regulating Growth and Development; Steffens, G. L.; Rumsey, T. S., Eds; Kluwer Academic Publishers: The Netherlands, 1988; pp 137-73. Maeda, S. Ann. Rev. Entomol. 1989, 34, 351-72. Cadenas, Ε. Ann. Rev. Biochem. 1989, 58, 79-110. Fridovich, I. Science 1978, 201, 875-80. Cheynier, V. F.; Van Hulst, M. W. J . J . Agric. Food Chem. 1988, 36, 10-14. Cheynier, V.; Osse, C.; Rigaud, J . J . Agric. Food Science 1988, 53, 1729-33. Cohen, G.; Heikkila, R. E. J . B i o l . Chem. 1974, 249, 244752. Hanham, A. F.; Dunn, B. P.; Stich, H. F. Mutation Research 1983, 116, 333-9. Dalton, D. Α.; Russell, S. Α.; Hanus, F. J . ; Pascoe, G. Α.; Evans, H. J . Proc. Natl. Acda. S c i . 1986, 83, 3811-15. Dalton, D. Α.; Hanus, F. J . ; Russell, S. Α.; Evans, H. J . Plant Physiol. 1987, 83, 789-94. Rennenberg, H. Phytochemistry 1982, 21, 2771-81.

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148. 149. 150.

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151. 152. 153. 154.

155.

156.

157. 158. 159. 160. 161. 162. 163. 164.

165.

Enzymatic Antinutritive Defenses of Tomato

Pritsos, C. Α.; Ahmad, S.; Bowen, S. M.; E l l i o t , A. J . ; Blomquist, G. J . ; Pardini, R. S. Arch. Insect Biochem. Physiol. 1988, 8, 101-12. Torel, J . ; C i l l a r d , J . ; C i l l a r d , P. Phytochemistry 1986, 25, 383-5. Kramer, K. J . ; Hendricks, L. H.; Liang, Y. T.; Seib, P. A. J. Agric. Food Chem. 1978, 26, 874-8. Barraccino, G.; Dipierro, S.; Arrigoni, O. Phytochemistry 1989, 28, 715-17. Kunert, K. J . ; Edcere, M. Physiol. Planta 1985, 65, 85-8. Larson, R. A. Phytochemistry 1988, 27, 969-78. Meister, A. Science 1982, 220, 472-8 Ahmad, S.; Pritsos, C. Α.; Bowen, S. M.; Kirkland, Κ. E.; Blomquist, G. J . ; Pardini, R. S. Arch. Insect Biochem. Physiol. 1987, 6, 85-96. Ahmad, S.; Pritsos, C. Α.; Bowen, S. M.; Heisler, C. R.; Blomquist, G. J . ; Pardini, R. S. Free Rad. Res. Comms. 1988, 4, 403-8. Ahmad, S.; Pritsos, C. Α.; Bowen, S. M.; Heisler, C. R.; Blomquist, G. J . ; Pardini, R. S. Arch. Insect Biochem. Physiol. 1988, 7, 173-86. Pritsos, C. Α.; Ahmad, S.; Bowen, S. M.; Blomquist, G. J . ; Pardini, R. S. Comp. Biochem. Physiol. 1988, 90C, 423-7. Fridovich, I. J . B i o l . Chem. 1989, 264, 7761-4. Khan, V. Phytochemistry 1983, 22, 2155-9. Panijpan, B.; Ratanaubolchai, K. Internatl. J . V i t . Nutr. Res. 1980, 50, 247-53. Choudhuri, M. A. Plant. Physiol. Biochem. 1988, 15, 18-29. Dhindsa, R. S.; Plumb-Dhindsa, P.; Thorpe, T. A. J . Exp. Botany 1981, 32, 93-101. Stewert, R. R. C.; Bewley, J . D. Plant Physiol. 1980, 65, 245-8. Thompson, J . E. In Senescence and Aging i n Plants;Nooden, L. D.; Leopold, A. C., Eds.; Academic Press: New York, 1988; pp 51-83. Schmidt, Α.; Kunert, K. J . Molecular Strategies f o r Crop Protection;rntzen, C. J . ; Ryan, C.; Eds.; Alan R. L i s s : New York, 1987; p 443.

RECEIVED

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Chapter 13

Phytoalexins and Their Potential Role in Control of Insect Pests Jack D. Paxton

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Department of Plant Pathology, University of Illinois, 1102 S. Goodwin, Urbana, IL 61801

Plant phytoalexins [natural plant a n t i b i o t i c s ] [1] have the p o t e n t i a l of becoming a new c l a s s of u s e f u l compounds i n the c o n t r o l of insect p e s t s . Some phytoalexins have been demonstrated as d e t e r r e n t s to i n s e c t f e e d i n g . C o n s i d e r a b l e p r o g r e s s has been made to c h a r a c t e r i z e them c h e m i c a l l y and t o extend the study of t h e i r function in plant disease resistance, but e x p l o r a t i o n of t h e i r r o l e i n the c o n t r o l of i n s e c t pests i s just beginning.

Plant

Defenses

Against

Stresses

Plants have developed many responses a n d ways o f defending themselves against attacks by pathogens and i n s e c t s . Some o f t h e s e d e f e n s e s a r e p r e f o r m e d s u c h a s t h e plant cuticle, which provides a barrier against desiccation a n d which possibly even creates an i n h o s p i t a b l e environment f o rvarious pathogens. Another preformed defense against s t r e s s i s l i g n i f i c a t i o n o f c e l l walls. This creates a physical barrier t o mechanical p e n e t r a t i o n b y f u n g i o r i n s e c t mouthparts, however, t h e m o s t common p r e f o r m e d d e f e n s e t h a t h a s b e e n s t u d i e d t o date i s t h e accumulation o f chemical deterrents (allomones) [ 2 ] . In preformed defenses t h e p l a n t i n v e s t s considerable energy a n dother metabolic resources i na generalized d e f e n s e a g a i n s t some s t r e s s e s t h a t may n e v e r m a t e r i a l i z e . 0097-6156/91Λ)449-0198$06.00Λ) © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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199

T h e s e d e f e n s e s h a v e t h e a d v a n t a g e o f b e i n g i n p l a c e when the insect arrives at the plant thereby preventing a p p r e c i a b l e damage t o t h e p l a n t . A p o s s i b l e disadvantage i s t h a t a c o n s i d e r a b l e amount o f t h e p l a n t ' s resources a r e d e v o t e d t o a d e f e n s e t h a t may n o t b e n e c e s s a r y and which might u l t i m a t e l y reduce i t ' s a b i l i t y t o reproduce and compete f o r an e c o l o g i c a l n i c h e and r e s o u r c e s . Between preformed ( c o n s t i t u t i v e ) and inducible defenses exists a wide range o f compounds t h a t a r e e s s e n t i a l l y p r e f o r m e d , b u t s t o r e d i n an i n a c t i v e f o r m u n t i l damage o c c u r s t o t h e c e l l . An e x a m p l e o f t h i s i s juglone, a 5-hydroxy naphthoquinone t h a t i s accumulated as i t s 4 - g l u c o s i d e i n m e m b e r s o f t h e J u g l a n d a c e a e [2.] . Upon i n j u r y of the c e l l , b-glucosidases i n the cell release the aglucone hydrojuglone, which i s then r a p i d l y oxidized t o juglone by a i r and p l a n t cell oxidases. Juglone, f o r example, i s a potent a n t i f e e d a n t t o Scolytus m u l t i s t r i a t u s [£_] . H y d r o j u g l o n e i s r e l a t i v e l y non-toxic c o m p a r e d t o j u g l o n e [£]. A n o t h e r compound b o r d e r i n g between preformed and i n d u c e d d e f e n s e s i s g o s s y p o l . T h i s compound a c c u m u l a t e s within epidermal g l a n d s on t h e s u r f a c e o f c o t t o n p l a n t s a n d i s v e r y t o x i c t o some i n s e c t s [£.] . G o s s y p o l , and a series of structurally r e l a t e d c o m p o u n d s o n t h e same biosynthetic pathway, accumulate to substantially i n c r e a s e d l e v e l s i n p l a n t s t h a t have been c h a l l e n g e d by potential pathogens. For this reason they have been considered phytoalexins by some plant pathologists. Phytoalexins are inducible antibiotics and w i l l be discussed later. I n d u c i b l e d e f e n s e s have t h e advantage o f d i v e r t i n g p l a n t m e t a b o l i c r e s o u r c e s t o d e f e n s e o n l y when a n i n s e c t o r p a t h o g e n a t t a c k a c t u a l l y o c c u r s . W h i l e some t i s s u e may be damaged a n d l o s t , a r e s p o n s e t o t h e i n s e c t o r p a t h o g e n can prevent significant damage t o t h e p l a n t , without d i v e r t i n g t h e same a m o u n t o f r e s o u r c e s t h a t w o u l d be r e q u i r e d f o r p r e f o r m e d d e f e n s e s . F u r t h e r m o r e , some o f t h e p r e f o r m e d compounds t h a t seem t o p l a y a s i g n i f i c a n t r o l e i n d e f e n s e a g a i n s t f e e d i n g o f some i n s e c t s , a c t u a l l y a r e used as 'keys' t o f e e d i n g o n t h e same p l a n t b y other i n s e c t s t h a t a t t a c k t h a t p a r t i c u l a r p l a n t [2] . Plant

Defenses Against

Insects

Plants have evolved a number of different defenses a g a i n s t i n s e c t s . One p r o m i n e n t d e f e n s e i s h a i r s o r o t h e r s t r u c t u r e s which cover t h e surface o f t h e p l a n t and deter insect feeding. Another i s accumulation of allomones (compounds t h a t s e r v e t o d e t e r f e e d i n g o r o v i p o s i t i o n o r are otherwise toxic to the insect), especially i n c r i t i c a l p l a n t p a r t s such as t h e r e p r o d u c t i v e s t r u c t u r e s .

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Immature seeds a n d t h e i r seed c o a t s a r e o f t e n a good source o f such compounds. As an example, w a l n u t trees c o n t a i n t h e r i c h e s t source o f hydrojuglone glucoside i n young growing p a r t s and i n t h e p e r i c a r p s u r r o u n d i n g t h e s e e d [2.] ·

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Preformed

Compounds

A w i d e r a n g e o f c o m p o u n d s a r e now k n o w n t o a c c u m u l a t e i n plants and serve as allomones t o some insects and kairomones (feeding attractants o r excitants) t o other i n s e c t s [2J . A n e x a m p l e o f s u c h a c o m p o u n d i s g l a u c o l i d e A, a g e r m a c r a n o l i d e type sesquiterpene lactone found i n Vernonia spp. G l a u c o l i d e A h a s a n t i f e e d a n t a c t i o n a g a i n s t some L e p i d o p t e r a . But other Lepidoptera, such as t h e cabbage looper [Trîchoplusia ni] and t h e yellow woollybear [Spilosoma virginica] actually prefer Vernonia spp. w i t h t h i s compound [ 2 ] .

I n d u c i b l e Compounds

W h i l e i n d u c i b l e compounds t h a t d e t e r i n s e c t f e e d i n g have not been w i d e l y s t u d i e d , some i n t e r e s t i n g e x a m p l e s o f these compounds a r e known. A proteinase inhibitor i n d u c i n g f a c t o r ( P I I F ) , d i s c o v e r e d b y Ryan i n wounded tomatoes, has been extensively studied i n insect resistance [£_] . P I I F a c t i v i t y has been i s o l a t e d from t o m a t o l e a v e s a s a s i n g l e , b r o a d S e p h a d e x G-50 p e a k w i t h a Mr r a n g e o f 5,000-10,000 a n d i s p r i m a r i l y c a r b o h y d r a t e . PIIF can e l i c i t the accumulation o f two proteinase i n h i b i t o r s i n wounded tomato l e a v e s a n d t h e s e inhibitors i n t e r f e r e with p r o t e i n d i g e s t i o n by i n s e c t s . I n h i b i t o r I has a m o l e c u l a r r a t i o o f 41,000 a n d i s a p e n t a m e r . E a c h subunit has an a c t i v e s i t e , specific f o r inhibiting chymotrypsin, w i t h a K i o f a b o u t 10~^M. I n h i b i t o r I I i s a dimer with a molecular ratio o f 23,000 a n d s t r o n g l y i n h i b i t s both t r y p s i n and chymotrypsin with K i values o f a b o u t 10"~ a n d 10"" M r e s p e c t i v e l y [jl] . F u r t h e r m o r e , p l a n t and fungal c e l l w a l l fragments a c t i v a t e expression o f p r o t e i n a s e i n h i b i t o r genes f o rp l a n t defense j u s t like they a c t i v a t e phytoalexin accumulation . C a r r o l l a n d Hoffman [ H J presented evidence that a feeding stimulant f o r Acalymma vittata i s rapidly m o b i l i z e d i n t o d a m a g e d C u c u r j b i t a moschata l e a v e s . These same l e a v e s r a p i d l y accumulate a feeding inhibitor of Epilachna tredecimnotata. This plant response i s circumvented b y Epilachna tredecimnotata by trenching around t h e leaf area t o be eaten, prior t o feeding. 8

7

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Unfortunately the active compounds have not been chemically characterized. A n o t h e r g r o u p o f i n d u c i b l e compounds t h a t probably have an important role i n plant insect resistance i s phytoalexins.

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Phytoalexins

Phytoalexins a r e low molecular weight, a n t i m i c r o b i a l compounds t h a t a r e b o t h s y n t h e s i z e d b y a n d a c c u m u l a t e d i n plants after exposure t o microorganisms [1_] . S e v e r a l l i n e s o f e v i d e n c e s u g g e s t t h a t t h e s e compounds have an important r o l e i n p l a n t disease and pest r e s i s t a n c e [12J. Phytoalexins f r o m many d i f f e r e n t p l a n t s h a v e b e e n chemically characterized. The phytoalexins include isoflavanoid-derived pterocarpan compounds c h a r a c t e r i s t i c of the Leguminosae, sesquiterpenoid compounds c h a r a c t e r i s t i c o f t h e S o l a n a c e a e , p h e n a n t h r e n e compounds characteristic of the Orchidaceae and acetylenic compounds c h a r a c t e r i s t i c o f t h e C o m p o s i t a e . G l y c e o l l i n , a p h y t o a l e x i n o f soybeans, accumulates as a s e r i e s of isomers first identified by Lyne e t al. [lu]; t h e t h r e e m o s t common i s o m e r s a r e s h o w n i n F i g u r e 1. P l a n t s f r e q u e n t l y p r o d u c e a s e r i e s o f a c t i v e isomers of related phytoalexins, and soybeans a r e a u s e f u l e x a m p l e [JLA] . Phytoalexins

as Insect

Feeding

Deterrents

In t h e f i r s t work t o i m p l i c a t e p h y t o a l e x i n s i n f e e d i n g deterrent a c t i v i t y o f p l a n t s , R u s s e l l et a l . [ l u ] found t h a t 3 R - ( - ) - v e s t i t o l a n d s a t i v a n f r o m Lotus pendunculatus leaf e x t r a c t s were major deterrents f o r Costelytra zealandica larvae. Furthermore pastures c o n t a i n i n g as l i t t l e a s 2 0 % Lotus pendunculatus were r e l a t i v e l y f r e e o f t h i s insect pest. V e s t i t o l h a d a n ED50 w i t h Costelytra zealandica larvae o f about 8.5 μg/g o f medium a n d f e e d i n g was completely i n h i b i t e d a t 100 μg/g o f m e d i u m . Vestitol however could not account f o r a l lof the feeding deterrency o f t h e Lotus e x t r a c t s , and s a t i v a n was i m p l i c a t e d i n t h i s a d d i t i o n a l a c t i v i t y . The d u a l f u n c t i o n of v e s t i t o l as a p h y t o a l e x i n and i n s e c t f e e d i n g d e t e r r e n t i s o f e c o l o g i c a l i n t e r e s t . I t must s t i l l be d e t e r m i n e d whether v e s t i t o l i s induced by i n s e c t f e e d i n g or i s present i n uninfected f i e l d plants. S u b s e q u e n t r e s e a r c h f o u n d t h a t Costelytra zealandica a n d Heteronychus arator l a r v a e a r e s e n s i t i v e t o seven phytoalexins including pisatin, maackiain and medicarpin Γ161 . R e l a t e d compounds n a r i n g e n i n , apigenin, morin,

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

NATURALLY OCCURRING PEST BIOREGULATORS

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202

F i g u r e 1.

Major soybean

phytoalexins.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

13.

PAXTON

Phytoalexins and Control of Insect Pests

coumestrol, formononetin, b i o c h a n i n A and g e n i s t e i n d i d not s i g n i f i c a n t l y affect feeding a c t i v i t y . Phaseollin, v e s t i t o l , p h a s e o l l i n i s o f l a v a n , and 2 -methoxyphaseollinisoflavan were t h e most a c t i v e , showing significant f e e d i n g r e d u c t i o n a t l ( ^ g / m l o r l e s s . Heteronychus arator s e e m s m o r e s e n s i t i v e t h a n Costelytra zealandica t o these c o m p o u n d s , e x c e p t f o r p i s a t i n . P h y t o a l e x i n s may t h e r e f o r e s e r v e t w o d i f f e r e n t a n d p e r h a p s indépendant r o l e s i n t h e plant. Research by Hart e t a l . [11] on t h e e f f e c t of s o y b e a n p h y t o a l e x i n s on M e x i c a n bean b e e t l e Epilachna v a r i v e s t i s a n d t h e s o y b e a n l o o p e r Pseudoplusia includens showed t h a t t i s s u e s i n w h i c h t h e s e isomers had been elicited [by e x p o s u r e to ultraviolet irradiation] s t r o n g l y d e t e r r e d t h e Mexican bean b e e t l e but not t h e soybean looper. At the induced, physiological c o n c e n t r a t i o n o f 1% o f d r y w e i g h t , g l y c e o l l i n h a d some e f f e c t on s u r v i v a l o f 1 - d a y - o l d s o y b e a n l o o p e r l a r v a e b u t not on s u b s e q u e n t s u r v i v a l and d e v e l o p m e n t . S i n c e this i n s e c t r e a d i l y f e e d s o n s o y b e a n s , a s i t s name i m p l i e s , t h i s a b i l i t y t o t o l e r a t e g l y c e o l l i n i s not s u r p r i s i n g . In a d d i t i o n , t e s t i n g s i n g l e compounds i n an a r t i f i c i a l diet ignores the possible synergistic effects of the multitude o f p h e n o l i c compounds i n g e s t e d f r o m t h e p l a n t during n o r m a l h e r b i v o r y . A d d i t i o n a l l y a compound d i s t r i b u t e d i n a diet rather uniformly may not approximate the l o c a l i z e d , and sometimes h i g h e r c o n c e n t r a t i o n s i n p l a n t s . On t h e o t h e r h a n d t h e o l i g o p h a g o u s Mexican bean beetle clearly was deterred from feeding on t h e p h y t o a l e x i n - r i c h t i s s u e . Since t h i s insect i s not e a s i l y reared on a r t i f i c i a l diets, the direct toxicity of glyceollin t o t h e Mexican bean b e e t l e could n o t be determined i n these experiments. This i s a common experimental p r o b l e m when d e t e r m i n i n g the effect of a g i v e n compound i n v i v o . Some o f t h e s e problems have been addressed by Fischer, D.C.; Kogan, M.; Paxton, J.D., (Environ . E n t o m o l . s u b m i t t e d ) . They p u r i f i e d g l y c e o l l i n , a p p l i e d i t to common b e a n (Phaseolus vulgaris) leaves at rates of 0.22, 1.1, 2.2, 11.0 o r 22.0 μg/mg d r y w e i g h t o f l e a f tissue. These t i s s u e s were then subjected to feeding preference tests i n p e t r i plates with the southern corn rootworm {Diabrotica undecimpunctata howardi), the M e x i c a n b e a n b e e t l e (Epilachna v a r i v e s t i s ) and t h e bean leaf beetle (Cerotoma trifurcata) . Glyceollin treated l e a v e s were consumed l e s s by t h e s o u t h e r n corn rootworm and t h e M e x i c a n bean b e e t l e , even a t l e v e l s below t h a t f o u n d i n e l i c i t e d p l a n t s . On t h e o t h e r h a n d t h e b e a n l e a f b e e t l e f e d f r e e l y , e v e n a t v e r y h i g h [22μg/mg] d o s e s o f g l y c e o l l i n . T h i s s u g g e s t s t h a t p h y t o a l e x i n s may r e p r e s e n t a convergence o f defenses a g a i n s t both microorganisms and insect herbivores. 1

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203

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

204

NATURALLY OCCURRING PEST BIOREGULATORS

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Elicitation

of Phytoalexins

by

Stresses

Many e n v i r o n m e n t a l f a c t o r s s u c h a s h e a t , d r o u g h t , a n d f r o s t may a f f e c t h e r b i v o r e / p l a n t i n t e r a c t i o n s b y increasing or decreasing the level of resistance ofthe plant t o the herbivore [ϋ] . This suggests that t h e plant i s a c t i v e l y responding t o t h e f e e d i n g by t h e h e r b i v o r e a n d t h a t p h y t o a l e x i n s m i g h t t h e r e f o r e b e i n v o l v e d [20.] · This also suggests that s e l e c t i v e e l i c i t a t i o n o f p h y t o a l e x i n s i n p l a n t s might be a d e s i r a b l e s t r a t e g y f o r p r o t e c t i n g p l a n t s a g a i n s t i n s e c t s [21]. A number o f d i f f e r e n t s t r e s s e s c a n e l i c i t i n d u c i b l e compounds i m p o r t a n t i n i n s e c t i n t e r a c t i o n s w i t h p l a n t s . Some o f t h e s e c o m p o u n d s a p p e a r t o a t t r a c t i n s e c t s t o a p l a n t ' i n d i s t r e s s ' , such as t h e southern pine b e e t l e , Dendroctonus f r o n t a l i s , t o Pinus s p p . O t h e r compounds a p p e a r t o p r e v e n t a t t a c k [22]. O z o n e d e c r e a s e s s o y b e a n r e s i s t a n c e t o i n s e c t herbivory and o v e r r i d e s induced r e s i s t a n c e [ L i n , H.C.; K o g a n , M.; E n d r e s s , A.G., E n v i r o n . E n t o . 1990, i n p r e s s . ] . T h i s m i g h t be due t o a d i v e r s i o n o f p h e n o l i c s i n t o c o u m e s t r o l i n s t e a d o f more t o x i c compounds s u c h a s g l y c e o l l i n . Pathogens E l i c i t

Phytoalexins

The m o s t s t u d i e d a s p e c t o f p h y t o a l e x i n e l i c i t a t i o n i s t h a t generated by fungal pathogens o f p l a n t s . Karban e t al.[22] f o u n d i n d u c e d r e s i s t a n c e a n d i n t e r s p e c i f i c c o m p e t i t i o n between s p i d e r mites and a v a s c u l a r w i l t f u n g u s . M c l n t y r e e t a l . [2A] d e m o n s t r a t e d t h a t 7 d a y s a f t e r t o b a c c o p l a n t s were i n f e c t e d w i t h t o b a c c o m o s a i c virus, reproduction o f t h e green peach aphid, Myzus persicae, was s i g n i f i c a n t l y r e d u c e d o n t h e s e i n f e c t e d p l a n t s . Inoculation o f plants with tobacco mosaic v i r u s i n d u c e d r e s i s t a n c e t o s e v e r a l pathogens, however t h e m e c h a n i s m f o r i n d u c e d r e s i s t a n c e was n o t c h a r a c t e r i z e d . This suggests that a d i f f e r e n t type o f p r o t e c t i o n o f p l a n t s a g a i n s t i n s e c t a t t a c k i s p o s s i b l e . Few c a s e s e x i s t where such p r o t e c t i v e i n t e r a c t i o n s between pathogens a n d i n s e c t s on p l a n t s have been s t u d i e d . I b e l i e v e s u c h i n t e r a c t i o n s a r e common a n d d e s e r v e m o r e a t t e n t i o n t h a n received t o date. Insect

Feeding E l i c i t s

Plant

Responses

D e c r e a s e d f e e d i n g on soybean c a n be i n d u c e d b y p r e v i o u s i n s e c t h e r b i v o r y as w e l l as by mechanical i n j u r y .L i n Γ251 found mechanical i n j u r y and p r i o r herbivory induced r e s i s t a n c e i n s o y b e a n c v W i l l i a m s 82 t o t h e s o y b e a n

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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13.

PAXTON

Phytoalexins and Control of Insert

205

l o o p e r a n d t h e M e x i c a n b e a n b e e t l e . The l e v e l o f induced r e s i s t a n c e d e p e n d e d on t h e number o f i n j u r e d h o s t c e l l s i n c o n t a c t w i t h h e a l t h y c e l l s , and n o t on t h e amount o f l e a f a r e a l o s t . The r e s i s t a n c e i n d u c e d b y t h e s o y b e a n looper r e s u l t e d from a combination of mechanical i n j u r y a n d c o m p o u n d s i n t h e l a r v a l r e g u r g i t a n t . No c o m m u n i c a t i o n o f t h e i n d u c t i o n s i g n a l s between p l a n t s , and no s u b s e q u e n t p r o t e c t i o n , was f o u n d . I n d u c e d r e s i s t a n c e h a d a s i g n i f i c a n t r e t a r d a n t e f f e c t on d e v e l o p m e n t a n d g r o w t h o f b o t h i n s e c t s , b u t i t h a d no s i g n i f i c a n t e f f e c t o n t o t a l f o o d consumed by e i t h e r i n s e c t . L i n [25.] s e p a r a t e d , b y h i g h p r e s s u r e liquid chromatography, from soybean l e a f e x t r a c t s , twelve peaks r e p r e s e n t i n g an unknown number o f compounds. F e e d i n g by the Mexican bean b e e t l e i n c r e a s e d the areas of t h r e e p e a k s and f e e d i n g by t h e soybean l o o p e r i n c r e a s e d t h e a r e a s o f two p e a k s . These compounds were s i g n i f i c a n t l y c o r r e l a t e d w i t h a n t i f e e d i n g a c t i v i t y of the l e a f samples. K a r b a n [2£] found that cotton r e s i s t a n c e to beet a r m y w o r m s , Spodoptera exigua, c o u l d be i n d u c e d by p r i o r e x p o s u r e t o s p i d e r m i t e s , Tetranychus turkestani. In t h e s e c a s e s t h e compounds r e s p o n s i b l e f o r r e s i s t a n c e were not identified.

Use

of Phytoalexins

and

Their

Elicitors

B a s e d on t h e p r e v i o u s e x a m p l e s , t h e e l i c i t a t i o n o f p h y t o a l e x i n s and o t h e r i n d u c i b l e compounds i n p l a n t s by v a r i o u s e l i c i t o r s m i g h t be a s u c c e s s f u l t a c t i c f o r c o n t r o l l i n g i n s e c t p e s t s on i m p o r t a n t c r o p s . T h i s e l i c i t a t i o n w o u l d n e e d t o o c c u r o n l y when t h e i n s e c t f e e d s , t o p r e v e n t u n n e c e s s a r y damage t o t h e p l a n t b y a c c u m u l a t i o n o f compounds t o x i c t o p l a n t c e l l s as w e l l . B a s e d on p r e s e n t l y known c a r b o h y d r a t e e l i c i t o r s , i t seems p l a u s i b l e t h a t p l a n t s u r f a c e s c o u l d be t r e a t e d i n s u c h a way t h a t t h e e l i c i t o r i s c a r r i e d i n t o t h e p l a n t u p o n i n s e c t f e e d i n g [21]. These carbohydrate elicitors should b e s t a b l e a n d s h o u l d p o s e no e n v i r o n m e n t a l threat since t h e y a r e most l i k e l y n o n - t o x i c t h e m s e l v e s . I s u g g e s t t h a t p h y t o a l e x i n s p l a y an i m p o r t a n t role i n i n s e c t r e s i s t a n c e by p l a n t s . T h i s r o l e o f p h y t o a l e x i n s a n d p h y t o a l e x i n e l i c i t a t i o n now s h o u l d b e s t u d i e d f u r t h e r and t e s t e d under f i e l d c o n d i t i o n s .

Acknowledgments C e r t a i n aspects of the were s u p p o r t e d i n p a r t

research presented i n t h i s chapter by t h e I l l i n o i s A g r i c u l t u r a l

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

206

NATURALLY OCCURRING PEST BIOREGULATORS

Experiment Station, U.S.D.A. Competitive

The American Soybean Grants Program.

Association,

and

Literature Cited 1. 2. 3. 4.

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5. 6.

7.

8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18.

Paxton, J.D. Phytopath. Zeit. 1981, 101, 106-109. Norris, D.M., In Chemistry of Plant Protection; Haug, G.; Hoffmann, H. Eds.; Springer-Verlag: Berlin, 1986; pp 99-146. Daglish, C. Biochem. J. 1950, 47, 452-462. Gilbert, B.L.; Norris, D.M. Insect Physiol. 1968, 14, 1063-1068. Paxton, J.D.; Wilson, E.E. Phytopathology 1965, 55, 21-26. Stipanovic, R.D.; Bell, Α.A.; Lukefahr, M.J. In Host Plant Resistance to Pests; Hedin, P.Α., Ed.; ACS Symposium Series No.62; American Chemical Society: Washington, DC, 1977; Chapter 14. Mabry, T.J.; Gill, J.E.; Burnett, W.C.; Jones, S.B. In Host Plant Resistance to Pests; Hedin, P.Α., Ed.; ACS Symposium Series No.62; American Chemical Society: Washington, DC, 1977; Chapter 12. Ryan, C . A . Trends Biochem. Sci. 1978, 3, 148-150. Nelson, C.E.; Walker-Simmons, M.; Makus, D.; Zuroske, G.; Graham, J.; Ryan, C.A. In Plant Resistance to Insects; Hedin, P.Α., Ed.; ACS Symposium Series No.208; American Chemical Society: Washington, DC, 1983; Chapter 6. Ryan, C.A.; Bishop, P.D.; Graham, J.S.; Droadway, R.M.; Duffey, S.S. J. Chem. Ecol. 1986, 3, 10251036. Carroll, C.R.; Hoffman, C.A. Science 1980, 209, 414416. Hedin, P.Α., Ed. Host Plant Resistance to Pests: ACS Symposium Series No.62; American Chemical Society: Washington, DC, 1977; pp 286. Lyne, R.L.; Mulheirn, L.J.; Leworthy, D.P. JCS Chem.Comm. 1976, 1976, 497-498. Ingham, J.L.; Keen, N.T.; Mulheirn, L.J.; Lyne, R.L. Phytochem. 1981, 20, 795-8. Russell, G.B.; Sutherland, O.W.R.; Hutchins, R.F.N.; Christmas, P.E. J.Chem.Ecol. 1978, 4, 571-579. Sutherland, O.W.R.; Russell, G.B.; Biggs, D.R.; Lane, G.A. Biochem. Syst. Ecol. 1980, 8, 73-75. Hart, S.V.; Kogan, M.; Paxton, J. J. Chem. Ecol. 1983, 9, 657-72. Fukami, H.; Nakajima, M. In Naturally Occurring Insecticides; Jacobsen, M.; Crosby, D.G., Eds.; Marcel Dekker, New York, 1971; pp 71-97.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

13.

PAXTON

19.

20.

21.

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22.

23. 24. 25. 26.

Phytoalexins and Control of Insect Pests

207

Kogan, M., In The R o l e o f C h e m i c a l F a c t o r s i n I n s e c t / P l a n t R e l a t i o n s h i p s ; P r o c . XV I n t . Congr. E n t o m o l . , Washington, D.C. 1977; pp 211-227. Kogan, M.; P a x t o n , J.D. In P l a n t R e s i s t a n c e t o I n s e c t s; H e d i n , P.Α., Ed.; ACS Symposium S e r i e s No.208; A m e r i c a n C h e m i c a l S o c i e t y : Washington, DC, 1983; C h a p t e r 9. P a x t o n , J.D. In Biologically Active Natural Products, Potential Use i n Agriculture; Cutler, H.G., Ed.; ACS Symposium S e r i e s No.380; A m e r i c a n C h e m i c a l S o c i e t y : Washington, DC, 1988; C h a p t e r 8. Schoonhoven, L.M. In S e m i o c h e m i c a l s : T h e i r R o l e i n P e s t C o n t r o l ; N o r d l u n d , D.A. Ed.; John W i l e y and Sons, New York, 1981, pp 31-50. Karban, R.; Adamchak, R.; S c h n a t h o r s t , W.C., Science 1987, 235, 678-680. M c I n t y r e , J . L . ; Kodds, J.Α.; Hare, J.D. P h y t o p a t h o l o g y 1981, 71, 297-301. L i n , H. Ph. D. T h e s i s , University of Illinois, Urbana-Champaign, Illinois, 1989. Karban, R., Am. Mid. Nat. 1988, 119, 77-82.

RECEIVED May

16,

1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 14

Insect Resistance Factors in Petunia Structure and Activity

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Carl A. Elliger and Anthony C. Waiss, Jr. Western Regional Research Center, U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710

Petunia species contain an array of more than three dozen steroidal materials, formally related to ergostane, that are involved in the res­ istance of the plant toward attack by certain lepidopteran larvae. Among these compounds are Α-ring dienones and 1-acetoxy-4-en-3-ones. Epoxy functionalities may be found on the side chain in certain cases and on the A- and D-rings. Hydroxy and acetoxy substituents may be located at various positions on the steroid nucleus and the side chain. In a large number of examples, the side chain possesses a bicyclic orthoester moiety which, in certain cases, may also have a thiolester functionality attached. Alteration of ring A from the usual steroid pattern has given rise to a set of compounds which have a spirolactone at position-5. The biological activity of these substances is dependent upon various structural features, most notably the orthoester, which is essential for toxicity of the compounds toward insects. Foliage of petunia plants is poisonous to various insects. Early reports by Fraenkel, et al (1-3) indicated that although tobacco horn worm (Manduca sexta Johannson) larvae found Petunia hybrida Vilm. plant material to be highly acceptable (ie attractive, or at least non-repellent) as food, growth was not supported and premature death occurred. It was also stated that petunia was toxic to the Colorado potato beetle, Leptinotarsa decemlineata Say, and that these insects found the plant to be attractive as well. Sub­ sequently, other investigators (4,5) confirmed that several species of Petunia were extremely toxic to first and second instar M. sexta although the third and fourth instar larvae were killed less rapidly. Even so, after feeding on P. inflata Fries and P. violacea Lindl., the larger larvae exhibited convulsive symptoms within four hours and stopped eating. All larvae were dead two days later. These workers found that wash­ ing leaves of P. axillaris Lam., P. inflata and P. violacea with water, ethanol-water mixtures, or 95% ethanol did not reduce the toxicity of the leaves toward M. sexta. From this they concluded that exudates from foliar trichomes, which they considered to probably contain alkaloids responsible for activity, were not removed by this treatment. No evidence for the presence of alkaloids, other than convulsions of the larvae during toxicosis, was given. The toxic effect of P. hybrida has been used to good account by Dethier (6,7) in conducting an interesting series of experiments on food-aversion learning in certain

This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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caterpillars. Larvae of two species of woolly bear caterpillars, Spilosoma (Diacrisia) virginica Fabr. and Estigmene congrua Walk, as well as M. sexta were allowed to feed on petunia leaves until acute symptoms were observed. Most animals could recover from this effect within twelve hours after they were removed from the leaves under the conditions of the experiment. They were then subjected to preference tests involving Petunia and two other plants, one more acceptable, and one less acceptable than Petunia to control larvae. The results of these experiments suggested that aversive learning was possible for the woolly bears but not for the hornworm. Dethier mentioned that the symptoms of insect intoxication on Petunia included convulsions, partial paralysis, and excessive regurgitation, but that S. virginica and E. congrua were less affected than was M. sexta. Another wooly bear, Pyrrharctia (Isia) Isabella Smith, was also subject to poisoning when fed P. violacea (8). From the results shown above and from similar toxicity data obtained in our laboratory using the corn earworm and fall army worm, we believed that P. hybrida and various species of Petunia contained a substance or subst­ ances injurious to insects, and that these plants might provide a valuable genetic source of host plant resistance factors. Isolation of Biologically Active Materials from Petunia Initial experiments were conducted onfreeze-driedleaf material of P. hybrida by serial extraction with increasingly more polar solvents (9). Fractions obtained were assayed by incorporation into artificial diet (10) upon which newly hatched larvae of the corn earworm (Heliothis zea Boddie) were placed. This insect had previously been shown to be adversely affected by petunia foliage, and inhibition of larval growth on these artificial diets (ten-day weights) indicated which fractions contained active material. Insect-inhibitory substances of Petunia are relatively nonpolar and some of these are extracted from plant material even with hexane, but we have found that it is more con­ venient to effect complete extraction of all of the compounds with chloroform followed by chromatographic separation. After evaporation of chloroform from the initial ext­ ract, the mixture of lipids thus obtained was stirred with boiling acetonitrile and allowed to cool. Waxy materials, which are not very soluble in acetonitrile, were removed by filtration, after which the acetonitrile solution was passed through a column of prep­ arative C-18 packing to yield an insect-inhibitory, enriched mixture of steroidal mater­ ials in the eluate, free from very nonpolar lipids and chlorophyll. Further fractionation was carried out by preparative H P L C on silica, C-18, polar amino-cyano (PAC) and cyanopropyl columns as required by the properties of individual compounds. In many cases, detection of components by U V at 254 nm was satisfactory; however, a variable wavelength detector was used when increased sensitivity for a specific compound was desired (ie 232nm for certain α,β-olefinic ketones), and a refractive index detector was used when substances lacked sufficiently intense U V absorption at any wavelength. Chromatographic profiles of crude extracts could be obtained either on C-18 or silica analytical HPLC columns, but not all components were resolved on even the most effic­ ient analytical columns. However, it was possible to follow the progress of preliminary chromatographic workups by analytical H P L C and also to examine extracts of various plant parts conveniently by this means. Petunia leaves are typically coated with a sticky exudate, and it was of interest to determine if this exudate contained any of the insect-inhibitory steroids. We subjected fresh leaves to an initial five minute soak in water followed by a one minute dip in chloroform with gentle agitation. This chloroform extract contained nonpolar surface lipids and substances tentatively identified as carbohydrate esters (ca 1 % of fresh wt.), but no biologically active materials were shown to be present by H P L C analysis. Fur­ ther workup of the solvent-washed leaves by grinding with additional chloroform then yielded the usual quantities of active materials, showing that the insect-inhibitory com­ ponents were definitely neither part of the exudate nor of the leaf surface coating.

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Structures of the Petunia Steroids Petuniasterones. At the present time, thirty seven steroidal ketones, all of which are based on the ergostane skeleton, have been isolated from several species of Petunia and from P. hybrida (11-16). The structures of these petuniasterones vary considerably, depending upon the plant source, and their biological activity toward insects is structure dependent (vide infra). Many of these structures have been described in an earlier

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R

I R = CH COSMe II R = Me 2

Figure 1. Petuniasterones A (I) and D (II) showing two different orthoester types. symposium (9); consequently only the most significant types will be mentioned here. Petuniasterone A (I) shows many typical structural features (Figure 1). A n A-ring dienone is present in about two thirds of the compounds. More unusual is the pecular bicyclic orthoester system of the side chain, which in the case of I (PS-Α) is derived from the uncommon [(methylthio)carbonyl]acetate or from acetate for PS-D (II). About half of the petuniasterones have orthoesters of this sort, approximately evenly divided between the two types above. Two derivatives of PS-A bear hydroxy groups a- to the thiolester group, and a few orthopropionates (only from P. integrifolia Hook) have been found. All but one of the petuniasterones have either a 7 α-hydroxy (or acetoxy) group, and further hydroxylation or acetoxylation can also occur at positions -11,-12 and -17 of the steroid nucleus. Petuniasterones with side chains bearing functionalities other than orthoesters also occur. The important PS-B and PS-C series (Figure 2) have epoxy groups at the 24,25-position and hydroxy or acyloxy groups at position-22. We have shown (12) that 22-acyloxy epoxides easily rearrange to the bicyclic orthoesters, and also that a 22-hydroxy epoxide yields the isomeric 5-membered cyclic ether as well as a mixture of side chain triols under mild acid treatment. Epoxy groups are also found at positions in rings A , Β and D (Figure 3). The Α-ring epoxy petuniasterones, I (III) and J (IV) are hydroxylated at position -17 and acetoxylated at position -12 respectively, a pattern that is also observed for ring-A dienone 7-acetates derived from I and II. Petuniasterones Κ (V) and L (VI), bearing epoxy functionality at position -16β,17β, retain the Α-ring dienone system of I and II. The substitution pattern of petuniasterone Ο (Vu), with its keto group at position-1 and 5α-hydroxy group is not typical of the usual petuniasterone structures, more nearly resembling the substitution pattern found in many withanolides (17) which also may have 6a,7 α-epoxides. However, the characteristic orthoacetate side chain reveals the affinity of VII with petuniasterones already discussed.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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ELLIGER AND WAISS

Insect Resistance Factors in Petunia

R = CH COSMe, Me 2

Figure 2. Rearrangement of epoxy side chain petuniasterones.

V VI

R = Me R = CH COSMe

VII

9

Figure 3. Ring epoxides.

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In two examples of petuniasterones, oxidative cleavage of ring D has occurred (Figure 4). Petuniasterone Q (VIII) has a keto group on an extended side chain which still possesses the orthoacetate moiety. The remainder of this seco-steroid bears the familiar Α-ring dienone and the 7oc-acetoxy group. Petuniasterone Ν (IX), which has undergone additional oxidative processes, still retains the original number of carbons of the ergostane system. Reclosure of ring D by addition of the 13-hydroxyl group to a ketone at position-16 has given a structure for the steroid nucleus similar in many respects to that of more usual steroids, but the C,D-ring junction is now cis. Again, the orthoester functionality has been preserved. Compound DC was always obtained as

Figure 4. Ring D seco-petuniasterones. a mixture of isomers that could be separated by H P L C . Reversion to the original epimeric proportions took place in deuteromethanol much faster than did incorporation of deuterium into position-21, thereby indicating that enolization of the C-20 keto group did not occur rapidly, and that epimerization of methyl-22 was not involved in the process. Isomerization under these conditions must occur by very facile ring opening and reclosure at C-16 to give a mixture of epimers at that position. Recently, several new petuniasterones derived from the epoxides V and VI have been identified (Elliger, et al. unpublished). We found that mild acid treatment (Figure 5) of these epoxides gave 16-ketopetuniasterone D (X) and 16-ketopetuniasterone A (XI) along with a set of side chain esters (XII-XV) having the 17,22-oxido- function­ ality. Acid catalyzed opening of the oxirane ring of V and VI can occur with carbocation formation at C-17. Interception of this cation by the oxygen attached to position -22 followed by ring opening of the bicyclic orthoester system between this oxygen and the orthoester center (with addition of water) gives the new oxetane system. The

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monocyclic species remaining on the side chain, with oxygens linked to carbons -24 and -25, may open in either of two directions, to yield the mixture of 24,25-hydroxy esters XII, ΧΠΙ and XIV, X V . The individual hydroxy esters can be isolated, but they revert to an approximately equal mixture of 24,25-isomers on storage, presumably through the same cyclic intermediate. Ketone formation can occur by loss of a proton at C-16 from the same cationic intermediate mentioned above to yield the enol forms which then may equilibrate to the 16-ketones ( X and XI). It is known that the β-configuration of the side chain at C-17 is preferred for 16-ketosteroids under conditions that effect enolization (18). Although

V R = Me VI R = C H C O S M e 2

XII XIII XIV XV

R R R Rj

Ac,R =H = H , R = Ac = COCH COSMe, R = H = H , R = COCH COSMe

1 =

{

x

2

2

2

2

2

2

Figure 5. Rearrangement of D-ring epoxides. all of these substances have been isolated from chromatographic fractions of P. parodii Steere, it is likely that they are artifactual, and result from rearrangement during workup of the D-ring epoxides originally present in the plant. The rearrangement products were always found in those fractions that contained their epoxy precursors even though the chromatographic mobilities of the individual products were typically quite different. If their formation had occurred naturally then the products should have appeared in differ­ ent chromatographic zones from the plant extracts. Petuniolides. In certain species of Petunia there is considerably more insect toxicity than can be accounted for by the presence of known petuniasterones. From these var­ ieties we have isolated a number of related steroidal lactones, some of which are much more toxic than are the petuniasterones, and have named these petuniolides (19) in analogy to the well known withanolides (20). The petuniolides are based on a modified ergostane system in which ring-Α has undergone rearrangement with loss of a carbon and formation of a spirolactone (Figure 6). All petuniolides isolated have a 6α,7αepoxy group on ring-B and either orthoacetate or orthopropionate side chains. No thiolester derivatives analogous to those of the petuniasterone orthoesters have been found at the present time. Petuniolides A - E (XVI-XX) have a 9,11-double bond and differ from each other at position-12 in having ally lie acetates, ally lie keto groups or a methylene group at that position. Petuniolides A (XVI), Β (XVII), and D (XIX) were

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found in P. integrifolia whereas petuniolide C is the major petuniolide of P. parodii. Three other petuniolides have been found to occur in P. parodii in smaller concentrat­ ions (Elliger, et al., unpublished) and include petuniolides Ε (XX), F (XXI) and G (XXII). Both X X I and XXII have cis-stereochemistry at the B,C-ring junction, with petuniolide G being notable in possessing not only a 9P,19-cyclopropane ring but also an 11-hydroxy-12-ketone in ring C. X-ray crystal structure determination of XXII showed that considerable steric interaction occurs between the 11-hydroxy and the endo hydrogen of methylene-19 as well as between HA9-exo and one of the methylene hydrogens at position-4.

XVI XVII XVIII XIX XX

XXI

R = Me, R Rj = Et, R R Me,R R =Et,R Rj = Me, R x

1 =

1

2

= H, a-OAc = H, a-OAc = 0 = 0 = H

2

2

2

2

2

ΧΧΠ

Figure 6. Structures of petuniolides from P. integrifolia and P. parodii.

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Biogenesis and Interconversions of the Petunia Steroids Inasmuch as no direct evidence for specific biosynthetic pathways in the formation of these steroids is available at the present time, any proposals on this subject are necessar­ ily rather speculative. Still, speculations on certain of these pathways are nonetheless valuable in suggesting experimental approaches for their elucidation. The acid catalyzed conversion of the 22-acyloxy-24,25-epoxides into side chain orthoesters indicated pre­ viously (Figure 2) is undoubtably analogous to the process occurring within the plant. It is significant that only a few of these side chain epoxides have been found, and that they are of only two petuniasterone types, namely the l,4-dien-3-ones and 1-acetoxy4-en-3-ones (11). Both of these types have only the 7a-hydroxy group as an additional nuclear substituent. From this, it may be concluded that the more highly substituted petuniasterones arise after formation of the orthoester group. The relationship between the l-acetoxy-4-ene-3-ones and the dienones of the Α-ring is not completely clear since either type of compound may arise from the other. Elimination of acetate at position-1 from the former type can give the dienone, and addition of acetate, Michael-wise, at this position of the dienone yields the acetoxy compound. However, only one of these 1-acetoxy derivatives that also bears an orthoester on the side chain ( PS- E) has ever been isolated (12). This suggests that the dieneones are also formed at a very early stage by the elimination of acetate. The numerous hydroxy and acetoxy derivatives as well as the ring epoxides are probably elaborated after dienone formation. As mention­ ed earlier, the D-ring epoxides (Figure 5) are probably not transformed biogenetically into the D-ring ketones (X & XI) and oxetanes (XII-XV); however, it is possible to perceive a role for these epoxides in the formation of the seco-steroids VIII and IX. Petuniasterone Ο (VII) is particularly interesting from a biogenetic standpoint because its 5a-hydroxy and 6a,7oc-epoxy groups suggest that it occupies a position midway between the usual petuniasterones and the petuniolides. If we assume that VII could eventually be functionalized to a A -12-ketone such as XXIII (Figure 7) then 9,11

xxrv ο xvm Figure 7. Proposed biosynthetic scheme for conversion of petuniasterone Ο into petuniolide C.

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oxidative cleavage of ring A between carbons-1 and -2 could give the diacid X X I V , which in turn, would undergo decarboxylation at C-10 (facilitated by the vinylogous keto group at position-12). Cyclization of the pendant carboxy-bearing chain to form petuniolide C (XVIII) can occur easily. We have found that reclosure of a similar open chain derivative of petuniolide C having an additional 7,8-double bond (obtained by base treatment of petuniolide C) is very rapid. The implication of the pathway proposed above is that Δ » -12-ketones are precursors of the remaining petuniolide types. This is not inconsistent with the structures of the known petuniolides. 9

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Biological Activity of the Petunia Steroids Growth reduction of Heliothis zea. In our bioassays we have tested the Petunia steroids against the corn earworm (//. zed) as well as the fall army worm (Spodoptera frugiperda Smith) and the pink bollworm (Pectinophora gossypiella Saunders). These bioassays were carried out using pure substances deposited on cellulose powder and incorporated into artificial diet (10) at several levels. Ten neonate larvae were used for each concentration. A dose-response curve was generated in this way for each com­ pound, relating insect weight gain (compared to controls) to the dietary concentration of added material. The dose of additive required to reduce the growth of insect larvae to fifty percent of control values is termed E D . For the earworm and armyworm, response curves obtained under these conditions of chronic toxicity were smoothly sigmoidal, and even at doses above the E D all larvae survived. However, the pink bollworm gave essentially an all or nothing response with mortality occurring without much weight curtailment of the survivors. Most of our results are from studies with the corn earworm; however, the armyworm was qualitatively similar, but somewhat less sensitive for the compounds selected. We have found, for the compounds tested, that only those substances having the bicyclic orthoester moiety on the side chain are significantly active in reducing larval growth of H. zea. Table I presents bioassay results for some of these petuniasterones. 5 0

5 0

Table I. Growth Inhibition of Heliothis zea by Petuniasterones Compound PS-Α (I) PS-Α 7-acetate Πβ-Hydroxy PS-A Πβ-Hydroxy PS-Α 7-acetate 30-Hydroxy PS-A 16-Keto PS-A 7-acetate PS-D (II) 12-acetoxy PS-D 7-acetate 11-hydroxy-12-acetoxy PS-D 7-acetate PS-I (III) PS-J (IV) PS-K (V) PS-N (IX) PS-0 (VII) PS-Q (VIII)

ED

5 Q

fPPM)

130(150)* 144 >400 >800 >400 700 130 (325)* 115 93 125 >1000 >800 75 165 185

* Value obtained for S. frugiperda. It may be seen that activity is not dependent upon whether a (methylthio)carbonyl substituent is present on the orthoacetate grouping. We have examined two examples

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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of orthopropionates (not shown in Table I) which are analogous to PS-D (II) and 12-acetoxy PS-D and found them to be somewhat more active than are the orthoacetates. Placement of a hydroxy a- to the thiolester group in 30-hydroxy PS-A was significant in reducing activity since larvae fed on diets containing 400 ppm of that compound attained over 90 percent of normal growth. Generally, nuclear substitution in rings A , Β and C does not greatly affect activity. The notable exception, PS-J (IV), is very insoluble, and its true dietary concentration may be considerably less than the amount incorporated into the diet. Substitution in ring D does decrease inhibitory activity as is shown by Πβ-hydroxy PS-A, 16-keto PS-A acetate and by PS-K (V). Nevertheless, the D-ring can undergo considerable modification with retention of activity. The 17-oxa-steroid, PS-N (IX), is actually slightly more inhibitory than PS-A even though the side chain has been displaced from its usual position in respect to the steroid nucleus. Preliminary results on the entirely open analog, PS-Q (VIII) indicate that much less insect growth inhibition occurs in this case (EDc 185 ppm). At the other end of the molecule, complete alteration of ring A as in PS-0 (Vu), which has an entirely different substitution pattern than most petuniasterones, has little effect in re­ ducing activity. That the Α-ring in the Petunia steroids may be extensively modified without loss of biological activity is convincingly demonstrated by the petuniolides (Table II). All but one of these lactones is impressively active against H. zea, and the least effective com­ pound is still comparable to most of the usual petuniasterones in this respect. 0

Table Π. Growth Inhibition of Heliothis zea by Petuniolides Petuniolide A Β C D Ε F G

(XVI) (XVII) (XVIII) (XIX) (XX) (XXI) (XXII)

ED

5 Q

(PPM)

13 10 3 (69)* 2 21 170 12

* Value obtained for S. frugiperda. Other insects and a crustacean. The fall armyworm (S. frugiperda) is much more resist­ ant toward the effect of petuniolide C than is H. zea (Table II), but the pink bollworm (P. gossypiella) is more susceptible. We observe, however, that even though 70% kill took place with this compound at a dietary level of 0.25 ppm, surviving P. gossypiella larvae grew to 74% of control weights. This effect is typical for substances that are toxic to early instar larvae, but which do not adversely influence older animals. In all our studies, older lepidopteran larvae were found to be more resistant toward plant allelochemicals than were younger individuals. The pink bollworm is a highly special­ ized feeder, and its extreme susceptibility toward petuniolide C probably is a conseq­ uence of adaptation to a few plants at the expense of tolerance for allelochemicals produced by plants other than its usual hosts. Petuniolide C (XVIII) was available in reasonable amounts, and we submitted samples of the compound elsewhere for tests upon other insects. The tobacco hornworm (M. sexta) was susceptible to this compound in artificial diets (as might have been expected from early observations of larvae on the plant), with only 50% survival of animals to the prepupal stage at a dosage of 20 ppm (J. Oliver, personal commun­ ication). Another caterpillar, the variegated cutworm (Peridroma saucia Hiibner), was

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insensitive to petuniolide C. However, it was learned that this insect is a natural pest of Petunia and may be evolutionary adapted to compounds of this sort (M. Isman, personal communication). The Bertha armyworm (Mamestra configurata Walker) is about as susceptible as is the corn earworm toward artificial diets containing XVIII (Isman). Feeding experiments were also carried out using neonate larvae of the Color­ ado potato beetle (L. decimlineata). A methanolic solution of XVIII was sprayed on to leaves of susceptible tomato plants, and larvae were applied after solvent evaporation was complete. After four days, the plants were examined for surviving larvae, and their developmental stage was determined. Mortality was 70-90%, and no larvae developed beyond first instar. Controls on untreated leaves exhibited 100% survival and had reached second instar (W. Cantelo, personal communication). We also examined the effect of petuniolide C upon the brine shrimp, Anemia salina, in order to test the response of another class of arthropod toward this chemical. This crustacean has been suggested as a general experimental subject in toxicity determin­ ation of chemical substances toward invertebrates, and for the indication of cytotoxicity (21, 22). Solutions for the test were prepared by adding stock solutions of XVIII in ethanol to containers of artificial salt water (1% EtOH final concentration) with sonic agitation. Even with added ethanol, the solubility limit of the steroid was about 1.0 ppm. Newly hatched brine shrimp were added to these solutions, and controls were run using salt water and salt water containing 1% ethanol. After 24 hr., no mortality was observed for the EtOH controls, and no toxic effect was seen for petuniolide C at 1.0 ppm. Acute effects. When the concentrations of active substances in artificial diets are increased to levels well above the E D , acute effects are observed on test subjects. Heliothis zea showed immediate distress after ingesting only a few mouthfuls of diet containing 1000 ppm of PS-A, and comparable results were seen at levels of 50 ppm for petuniolide C. Larvae thus affected stop feeding and exhibit violent convulsive movements. This is accompanied by regurgitation and copious diuresis leading to dehydration and loss of body turgor. Although the animals become moribund fairly soon, death is not rapid, and some larger larvae have been observed to remain alive for several days after the onset of symptoms. At lower test concentrations, third to fifth instar larvae generally survive treatment if they are transferred to control diets. Heliothis zea larvae were subjected to topical applications of petuniolide C to determine if contact with diet treated at higher levels of this compound could produce symptoms even though no ingestion of the diet occurred. Solutions of petuniolide C in acetone were applied in 1.0 μΐ aliquots to larvae having an average weight of 10 mg; the amounts were 0.5 μg and 1.0 μg (50 and 100 mg/kg of body weight). Application was to the middle of the larva well back from the head to minimize accidental ingestion, and controls received an equivalent amount of acetone. No immediate effect was noted. After 48 hr., all larvae were alive, but the 100 mg/kg subjects were slightly shrunken in appearance. It was possible to observe a greater toxic effect at an application level of 330 mg/kg. In this case also, larvae remained alive 24 hr. after application; however, 80% were dead at 48 hr. Clearly, topical absorption of petuniolide C is not an part­ icularly important mode of entry of this compound into the insect. 5 0

Occurrence of the Petunia Steroids in the Plant We have observed that the concentrations of the Petunia steroids varies considerably between horticultural varities of P. hybrida and between various species of this plant (9). Table III shows the approximate content of a number of insect-inhibitory petuniasterones in leaves of certain Petunias that we have examined. In the examples selected, the individual compounds were isolated by H P L C from extracts of anhydrous leaves and their respective amounts represent a very conservative minimum quantity. Weights were converted to concentrations on the fresh basis assuming a water content

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of 80% in leaves of the plant. We had previously noted that the effect of active petuniasterones is additive in nature, and it is clear that the aggregate concentration of these compounds present in the plant is sufficient to account for substantial toxicity. Not shown in the table is the significant observation that leaves of P. violacea did not contain petuniasterones, and that these leaves were not toxic to H. zea larvae (9). A degree of uncertainty exists in the classification of this species, and it is considered by some that P. violacea is synonymous with P. integrifolia (23). However, using petuniasterone content as a taxonomic indicator, it would appear that these species are separate entities.

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Table ΙΠ. Petuniasterone Content (mg/kg)* of Fresh Petunia Leaves Compound

Roval Cascade

PS-A (I) Πβ-hydroxy PS-A PS-D (II) 12-acetoxy PS-D 7-acetate PS-N (IX)

P. axillaris

P. parodii

120 10 30

85 **

40 **

10

40

100 40

50 **

55 150

P.

integrifolia 90 **

320

*80% water assumed for fresh leaf. **Value not obtained. Although the petuniasterones are important resistance factors, we feel that the petuniolide content of the plant represents a better measure of insect resistance because of the much greater toxicity of the latter compounds (except for petuniolide F). We isolated (dry basis) about 300 mg/kg of petuniolide C (XVIII) from consolidated batches of P. parodii leaves and have obtained petuniolides A (XVI), Β (XVII) and D (XIX) from P. integrifolia in quantities of 100, 430 and 200 mg/kg, respectively. Petuniolides Ε (XX), F (XXI) and G (XXII) were isolated from P. parodii in respect­ ive amounts of 45, 240 and 190 mg/kg. If it is assumed that the effect of these sub­ stances is additive, then their content in fresh leaves of P. parodii would be at least twenty-times E D , and is easily enough to account for the acute symptoms which are observed for larvae eating this plant. Of the petuniolides, petuniolide C is the most important in P. parodii because of its high content and low E D value. We desired to survey its concentration in this species since the isolated quantity (above) varied from batch to batch, and it was suspected that this might represent seasonal variation in the plant. We examined samples of freezedried leaf material obtained at two week intervals from plants grown in outside beds over a five-month period. Analysis was by H P L C on a microsorb 5μ C-18 column with an acetonitrile-water gradient optimized for this compound, and detection was by U V absorption at 232 nm. The concentration of petuniolide C (dry basis) in P. parodii ranged from 240 to 920 mg/kg (average 415), but no seasonal trend could be found. Greenhouse grown plants of this species had an average content of 505 mg/kg, whereas plants of P. axillaris had a content of 175 mg/kg under this condition. 5 0

5 0

Content of petuniolide C (XVIID in plant organs. During the course of our field study, a natural infestation of Heliothis virescens Fabr. (tobacco budworm) occurred on sur­ rounding plants (mainly tomato and Physalis species). We found that whereas no leaf feeding took place on Petunia, considerable damage to the flowers did occur. Also, we noticed that seed capsules of this plant were entered by the larvae, and immature seeds were consumed. Inasmuch as it appeared likely that this selective feeding was a con­ sequence of differing Petuniolide C content in different parts of the plant, we performed

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NATURALLY OCCURRING PEST BIOREGULATORS

analyses for the compound in various plant organs. Neither the flowers nor the immature seed contained any detectable amount of XVIII. Additionally, it was determined that the wall of the capsule and its inner structure were also free of the compound. Older seed from dry opened capsules did not contain any XVIII, but seedling plants of 50 to 75 mm height had 330 mg/kg. The content of XVIII in mature plant stems was below 15 mg/kg, and that of the root was 510 mg/kg. The nil content of petuniolide C in the plant parts that are consumed by the larvae is consistent with the observation that aversive learning by insect larvae is possible, and that larvae migrating from adjoining plants are able to detect the compound without suffering fatal toxicosis.

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Implications for Improvement of Crop Plants Classically, plant breeders selecting for insect resistance in crop plants have utilized crosses between varieties within the same species or between plants of closely related species. Higher levels of chemical resistance factors can indeed be built up in this way. However, insects can potentially become adapted to these substances as a consequence of previous exposure at lower concentrations, thus being preadapted to detoxify or otherwise tolerate these chemicals. We desire to introduce completely exotic chemicals, i. e. substances to which specific pests have never been exposed. This could be done by crossing plants that are only distantly related to the crop plant and would include species from other genera, possibly from other families. The purpose of the present work was to develop a basis for the transfer of resistance factors from Petunia into plants of economic significance. Toward this end it was important to establish the identity of the allelochemical agents, to determine the efficacy of these substances and to develop means for their analysis. We have shown that petuniolides are very significant agents of resistance, and that petuniolide C is the most toxic of these substances from P. parodii. The introduction into other plants of the biochemical processes involved in formation of the petuniolides is thus of great interest. A number of gene transfers in plants using the Ti plasmid of Agrobacterium tumefaciens have been carried out for one gene. However, it is obvious that a large number of enzymatic steps are necessary for the transformation of steroidal precursors into the eventual products, and that a large number of genes would therefore have to be transferred. At the present time, we are entirely ignorant of the detailed enzymology involved within the petuniasterone/petuniolide pathway, and even if the required knowledge were available, it would still be an impractical undertaking to introduce individually the genes controlling each enzymatic step. We are currently exploring methods for the insertion of rather large quantities of Petunia genetic material into the genome of tomato and potato. Protoplast fusion is being examined; but this method, which essentially combines the entire contents of two cells, may indeed be excessive in its mixing of genetic information. Even if viable plants were to be obtained after regeneration of hybrid protoplasts, it is unlikely that these plants would have economically satisfactory properties, or that they would be easily propagated. A procedure that offers more promise is that of microinjection of chromosomes or chromosomal fragments from P. parodii into individual protoplasts of a recipient species. Less disruption of cellular physiology would take place than in the case of protoplast fusion, and it should be possible to maintain the original chromosome number. In this way a large number of genes can be transferred in one step. In an interspecific hybridization, Griesbach (24) successfully transformed P. hybrida protoplasts by microinjection of chromosomes from P. alpicola Juss. This changed the level of production of various flavonoids and phenolic acids, reflecting simultaneous changes in the activity of several enzymes. It may be possible in a similar manner to introduce from Petunia the complement of enzymes required for alteration of the usual steroid biosynthesis in a recipient plant such as tomato and potato, and obtain production of the petuniolides. We feel that intergeneric (or interfamilial) hybridizations of this sort applied to crop plants will provide needed host plant resistance while still maintaining the desirable economic characteristics of these plants.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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WAISS

Insect Resistance Factors in Petunia

223

Acknowled gments We thank M. Benson for the determination and interpretation of NMR spectra, R. Y. Wong for X-ray cristallographie structure analysis and W. H . Haddon for mass spectral determinations. Ing. Agr. Hugo A. Cordo kindly furnished samples and seed of P. integrifolia. Seeds of Petunia species were also furnished by the National Seed Storage Laboratory, Fort Collins Co.

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Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24.

Fraenkel, G . F. Science 1959, 129, 1466-70. Fraenkel, G . ; Nayar, J.; Nalbandov, O.; Yamamoto, R. T. Symp. Insect Chem. 1960, 3, 122-26. Fraenkel, G . Tagungsber. Deut. Akad. Landswirtshaftswiss. Berlin 1961, 27, 297-307. Parr, J. C.; Thurston, R. J. Econ. Entomol. 1968, 61, 1525-31. Thurston, R. J. Econ. Entomol. 1970, 63, 272-74. Dethier, V . G . ; Yost, M . T. Physiol. Entomol. 1979, 4, 125-30. Dethier, V . G . Physiol. Entomol. 1980, 5, 321-25. Shapiro, A . M . Ann. Entomol. Soc. Am. 1968, 61, 1221-24. Elliger, C. Α.; Waiss, A . C., Jr. In Insecticides of Plant Origin; Arnason, J. T.; Philogène, Β. J. R.; Morand, P., Eds.; ACS Symposium Series No. 387; American Chemical Society; Washington, DC, 1989; pp 188-205. Chan, B. G . ; Waiss, A. C., Jr.; Stanley, W. L.; Goodban, A . E. J. Econ. Entomol. 1978, 71, 366-68. Elliger, C. Α.; Benson, M . E.; Haddon, W. F.; Lundin, R. E.; Waiss, A. C. Jr.; Wong, R . Y . J. Chem. Soc. Perkin Trans 1 1988, 711-17. Elliger, C. Α.; Benson, M.; Lundin, R. E.; Waiss, A. C., Jr. Phytochemistry 1988, 27, 3597-3603. Elliger, C. Α.; Benson, M.; Haddon, W. F.; Lundin, R. E.; Waiss, A. C., Jr.; Wong, R. Y . J. Chem. Soc. Perkin Trans. 1 1989, 143-49. Elliger, C. Α.; Haddon, W. F.; Waiss, A. C., Jr.; Benson, M . J. Nat. Prod. 1989, 52, 576-80. Elliger, C. Α.; Waiss, A . C., Jr.; Wong, R. Y.; Benson, M . Phytochemistry 1989, 28, 3443-52. Elliger, C. Α.; Wong, R. Y.; Benson, M.; Waiss, A. C., Jr. J. Nat. Prod. 1989, 52, 1345-49. Kirson, I.; Glotter, E. J. Nat. Prod. 1981, 44, 633-47. Ronchetti, F.; Russo, G . ; Longhi, R.; Sportolini, G. J. Lab. Comp. & Radiopharm. 1978, 14, 687-96. Elliger, C. Α.; Wong, R. Y.; Waiss, A. C., Jr.; Benson, M . J. Chem. Soc. Perkin Trans. 1 (in press). Glotter, E.; Kirson, I.; Lavie, D.; Abraham, A. In Bioorganic Chemistry; Van Tameln, Ε. E., Ed.; Academic Press: New York, 1978; Vol. II pp. 57-95. Meyer, Β . N.; Ferrigni, N. R.; Putnam, J. E.; Jacobsen, L. B.; Nichols, D. E.; McLaughlin, J. L . Planta Medica 1982, 45, 31-34. Alkofahi, Α.; Rupprecht, J. K.; Anderson, J. E.; McLaughlin, J. L.; Mikolajczak, K. L.; Scott, B. A . In Insecticides of Plant Origin; Arnason, J. T.; Philogène, Β . J. R.; Morand, P., Eds.; ACS Symposium Series No. 387; American Chemical Society; Washington, D C , 1989; pp 25-43. Sink, K. C. in Petunia; Sink, K. C., Ed.; Springer: Berlin 1984; pp. 3-9. Griesbach, R. J. Plant Science 1987, 50, 69-77.

R E C E I V E D May 16,

1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 15

Arthropod-Resistant and -Susceptible Geraniums Comparison of Chemistry D. Hesk , L. Collins , R. Craig , and R. O. Mumma Downloaded by NORTH CAROLINA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch015

1

2

3

1

Department of Entomology, Department of Chemistry, and Department of Horticulture, Pesticide Research Laboratory and Graduate Study Center, Pennsylvania State University, University Park, PA 16802

1

2

3

Glandular trichome exudate from insect resistant and susceptible geraniums (Pelargonium xhortorum) was chemically analyzed to d e t e r m i n e differences between the two plant lines. Hplc analysis of t h e exudate from resistant plants s h o w e d that it w a s p r e d o m i n a n t l y made up of u n s a t u r a t e d a n a c a r d i c acids, with the C ω5 and C ω5 anacardic acids contributing nearly 8 0 % of the total. By contrast, the e x u d a t e from t h e s u s c e p t i b l e plants w a s chiefly saturated, with the C and C saturated anacardic acids contributing nearly 5 0 % of the total. The C ω5 and C ω 5 anacardic acids were only present in trace a m o u n t s . In addition a number of other significant peaks o b s e r v e d in the c h r o m a t o g r a m s of extracts f r o m s u s c e p t i b l e plants, a n d also seen in small amounts in the resistant profile, were isolated a n d characterized by m a s s spectrometry a n d nmr spectroscopy. Two unsaturated anacardic acids, which contributed nearly 3 0 % of the total exudate in the susceptible plant, and only seen in trace amounts in t h e resistant plant, were identified as C ω6,9 and C ω9 anacardic acids. A number of o d d chain length and branched chain anacardic acids, were also isolated a n d identified. 22

24

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0097-6156/91/0449-0224$07.75/0 © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Arthropod-Resistant and -Susceptible Geraniums 225

The common garden geranium (Pelargonium xhortorum) has been shown to possess a potent chemical defense consisting of glandular trichomes which secrete a viscous sticky exudate.(Iz5) T h i s s t r a t e g y , w h i c h is p r i m a r i l y a p h y s i c a l entrapment mechanism, rather than a toxic o n e , is effective against small a r t h r o p o d species, a n d may also provide resistance against larger herbivores. The exudate from the trichomes w a s found to be comprised of a mixture of anacardic acids, which in the mite resistant plants, w a s s h o w n to be mainly C22 a n d C24 ω 5 unsaturated anacardic acids. Small amounts of t h e saturated a n a l o g u e s w e r e also f o u n d in t h e e x u d a t e f r o m resistant p l a n t s ( l j i 4 - ) . A n a l y s i s of e x u d a t e f r o m mite susceptible geraniums, showed that it contained the same four compounds, but with the saturated materials predominating (5. 6 ) . A n a l y s e s w e r e performed on a number of resistant a n d susceptible plants in order to determine whether the chemical composition w a s constant within each plant line. While it w a s found that the chemical profile of the resistant plants w a s fairly constant, the exudate from susceptible plants exhibited v a r i a b i l i t y . F u r t h e r m o r e , on c l o s e e x a m i n a t i o n of t h e g a s chromatographic trace of the exudate from susceptible plants, it was determined that in addition to the four previously identified c o m p o u n d s , there were a number of other significant peaks, w h i c h were unidentified. In addition, as all t h e peaks eluted close together, it w a s possible that the apparent variability w a s due to unresolved peaks (5. 6). Hence, t h e objectives of this study w e r e to d e v e l o p an improved separation of the anacardic acids, to characterize the unknown compounds in order to fully compare the resistant and s u s c e p t i b l e plants a n d to d e t e r m i n e w h e t h e r t h e e x u d a t e composition w a s indeed constant within both t h e resistant and susceptible lines.

Experimental Plant S o u r c e . Geraniums were maintained in a g r e e n h o u s e environment using standard cultural practices. The plant line 7 1 17-7 and its corresponding self-pollinated progeny, family 8 7 - 5 , had been previously determined to be mite a n d aphid resistant, a n d plant line 71-10-1 a n d its c o r r e s p o n d i n g self-pollinated progeny, families 85-26, 87-13 and 87-14, had previously been determined to be mite and aphid susceptible (Z). Newly opened

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inflorescences, from 12 plants, 6 resistant and 6 susceptible, w e r e s e l e c t e d from r a n d o m locations in the g r e e n h o u s e for chemical analysis. Isolation and purification of exudate. The exudate from both the resistant and susceptible plants was collected by immersing the inflorescences (minus the petals) in methylene chloride for one hour. The extract was then dried over anhydrous sodium sulfate, filtered and evaporated to leave the crude anacardic acid residue. This p r o c e d u r e had p r e v i o u s l y been s h o w n to p r o d u c e a representative extract of anacardic acids (5. 6 ) . The carboxyl group in the anacardic acids w a s methylated ( d i a z o m e t h a n e - d i e t h y l ether, 5 minute t r e a t m e n t ) before the acids were separated from the other extracted material by thin layer c h r o m a t o g r a p h y (tic). Merck 2 0 x 2 0 c m silica gel F254 plates with a pre-concentration zone were used. The plates were developed in a two step solvent system consisting of benzene: diethyl ether: ethanol: acetic acid (50: 4 0 : 1: 0.5, by volume) followed by hexane: diethyl ether: acetic acid (84: 15: 1, by volume). Development was allowed to proceed to about half the length of the plate in the first step and the entire length of the plate in the second. The monomethylated anacardic acid band was located by viewing at 254nm and comparing the Rf value with that of a monomethyl anacardic acid standard, before it w a s s c r a p e d from the plate a n d eluted with m e t h y l e n e c h l o r i d e (3x2mL). The methylene chloride extract was filtered through a pasteur pipette containing a glass wool plug, and then evaporated to leave the purified anacardic acids. Finally the phenolic group on the anacardic acids was methylated (diazomethane-diethyl ether plus a few drops of methanol, 30 minute treatment (3.)) to give the dimethylated anacardic acids. Bulk Extraction of Anacardic Acids for NMR. Separate batches (2.5Kg each) of resistant and susceptible inflorescences w e r e s o a k e d in m e t h y l e n e chloride for one hour. The m e t h y l e n e chloride extracts were dried, filtered and evaporated as d e s c r i b e d a b o v e to leave c r u d e resistant a n d s u s c e p t i b l e exudates. A preliminary clean up of each was performed using c o l u m n c h r o m a t o g r a p h y . Each crude extract w a s dissolved in hexane (15mL) and 5mL of that w a s applied to a silica gel chromatography column (18cm χ 2cm) and eluted successively with (i) hexane (100mL), (ii) 4 % diethyl ether, 1 % acetic acid in

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hexane (200mL), (iii) 10% diethyl ether, 1 % acetic acid in hexane ( 2 0 0 m L ) , (iv) 5 0 % diethyl ether, 1 % acetic acid in hexane (200mL) and (v) 2 5 % methanol in diethyl ether (100mL). The c o l u m n eluent w a s collected in 10mL t u b e s and each w a s analyzed by tic in order to determine the elution point of the anacardic acids. The tubes containing the anacardic acids were combined and subsequently pooled with the acids collected from the other two column runs. T h e p a r t i a l l y p u r i f i e d a n a c a r d i c a c i d s , f r o m b o t h the resistant and susceptible plants were further cleaned up prior to hplc separation using Merck 20x20cm, 2mm thickness preparative tic plates. The compounds were applied to several plates as the dimethylated compounds (diazomethane, 30 minute t r e a t m e n t (fi.)), and run in the same two step solvent system described above. The plates were v i e w e d at 2 5 4 n m and the anacardic acid bands were cut and eluted from the silica using methylene chloride (3x30mL). The purified anacardic acids were then ready for separation by preparative hplc. HPLC Analysis of Anacardic Acids. The anacardic acids collected from both the resistant and susceptible plants were analyzed by high performance liquid chromatography (hplc) using a 25cm χ 4 . 6 m m Supelcosil 5μ LC8 DB column. The solvent system used consisted of isopropanol, acetonitrile, 0.01 M acetic acid (49: 14: 37, by volume) and was run isocratically at 1.2mL/min. Detection was achieved using a Waters 490 uv detector at 212nm, with the trace being recorded on a Shimadzu 3A recorder-integrator. In preparative chromatography of the the bulk extract, a 2 5 c m χ 10mm Supelcosil 5μ LC8 DB semi-preparative column was used. In this case the mobile phase was modified to isopropanol, acetonitrile, 0.01 M acetic acid (49: 15: 30, by volume) and run i s o c r a t i c a l l y at 3 m L / m i n . T h e e l u t i n g s o l v e n t f r o m each a b s o r p t i o n peak w a s collected into s e p a r a t e vials, a n d the solvent was removed by rotary evaporation. Each anacardic acid was further purified by hplc, to achieve a purity of greater than 95%. GC-MS Analysis. The d i m e t h y l a t e d a n a c a r d i c acids w e r e analyzed by gas chromatography-mass spectrometry (gc-ms). All analyses were performed in the El mode, using a Kratos model MS-25 mass spectrometer, with a 30m χ 0.53mm RTX-5 (Restek Corp., cross bonded 9 5 % d i m e t h y l - 5 % diphenyl polysiloxane) capillary column with a temperature program of 2 5 0 - 2 8 0 ° C at 5°C/minute.

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NMR Analysis. The dimethylated anacardic acids were analyzed by proton nmr s p e c t r o s c o p y . All s a m p l e s w e r e d i s s o l v e d in deuterochloroform and were run at 360Mz on a Bruker AM 360. D i m e t h y l Disulfide D e r i v a t i z a t i o n . Dimethyl disulfide (DMDS) derivatization was performed on the unsaturated anacardic acids in order to locate the position of the double bond (2.)· A solution of the anacardic acid (0.05-0.1 mg) w a s d i s s o l v e d in hexane (1.75mL). DMDS (2.5mL) and iodine solution (0.25mL of a 60mg I2/1T1L ether) were added to the reaction vial, and the reaction w a s allowed to run overnight at 40°C. After the reaction w a s complete, 5mL of a 5% (w/v) solution of sodium thiosulfate was added to the reaction mixture to remove the excess iodine. The organic layer was removed, and evaporated to leave the crude D M D S d e r i v a t i v e , which w a s purified by hplc, before being submitted for mass spectrometric analysis.

Results. HPLC Analysis. Hplc analysis of the resistant exudate (Figure 1) showed that there are two major compounds which are known to be the C22 ω5 (compound B) and C24 ω5 (compound I) unsaturated a n a c a r d i c acids from earlier work, and a n u m b e r of minor compounds of which only the C22 saturated (compound G) and C24 saturated (compound M) anacardic acids have previously been identified ( 2 - 6 ) . Of the uncharacterized compounds, Ε and J are of particular interest, as they are present in similar quantities to the C22 and C24 saturated anacardic acids. This analysis is in good agreement with the capillary gc method used by Walters, who identified the four major anacardic acids and found them in similar quantities to those seen in the hplc trace. Hplc analysis of the susceptible exudate by contrast revealed some differences in the composition to that reported by Walters (5. 6). A very complicated profile w a s seen, with at least ten peaks each contributing over 1 % to the total exudate composition (Figure 2). Of those, only the C22 saturated (compound G) and C24 saturated (compound M) anacardic acids were known and hence the majority of the anacardic acids making up the exudate were u n k n o w n s t r u c t u r e s . T h u s t h e r e w a s c l e a r l y a n e e d to characterize the unknown c o m p o u n d s to obtain a full chemical profile of the susceptible exudate. Of particular interest was the almost complete lack of the C22 ω 5 (compound B) and C24 ω 5

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Arthropod-Resistant and -Susceptible Geraniums

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In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Figure 2: HPLC Profile of Glandular Trichome Exudate From Susceptible Geraniums

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Arthropod-Resistant and -Susceptible Geraniums 231

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(compound I) anacardic acids, contrasting with the analysis by Walters, who observed that they were present in amounts varying from 3 0 % to 6 0 % of the total susceptible exudate composition ( i L fi). In o r d e r to resolve this a m b i g u i t y , the major p u r i f i e d anacardic acids from the hplc were analyzed under the same gc conditions used by Walters (Table I).

Table I: HPLC and GC Retention Times of Anacardic Acids Code A Β C D Ε F G H I J Κ L M N 0

Name C20 sat C22 ω5 C21 anteiso C24 ω6,9 C23 ω6 C22 iso C22 sat. C24 ω9 C24 co5 C23 anteiso C23 s a t C24 iso C24 sat C25 anteiso C25 sat

HPLC Rt(min) 19.0 20.5 21.9 22.8 25.2

GC Rt(Min) nd 7.74

27.8 29.0 30.7

nd 7.02 9.50

31.5

9.82

33.6 36.1 43.2 45.2 52.5 56.7

7.81 8.11 8.73 9.29 9.94 nd

nd 10.11 nd

It was found that compound J, a major peak in the susceptible co-chromatographed with the C22 ω5 (compound B) peak on gc and c o m p o u n d s Η and Ν closely chromatographed with the C24 ω 5 (compound I) peak. Thus it appears that the difference in results can be accounted for by the improved separation achieved on the hplc over the capillary gc method, and hence what were believed to be ω 5 anacardic acids in the susceptible exudate were really other compounds which happened to co-chromatograph with them. Such a problem is not encountered in the resistant exudate, as the co5 anacardic acids are by far the major components.

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Mass Spectral and NMR Analysis.

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The mass spectral and nmr d a t a from each of the unknown anacardic acids are given in Tables II and III respectively, and the results for each compound are discussed in detail below. The spectra were also obtained for the previously known anacardic acids (Compounds B, G, I, and M) and are listed in Tables II and III for comparative purposes. However because their structures are already known (5. 6). they are not discussed below. C o m p o u n d A. The mass spectrum of dimethylated A showed a molecular ion at m/z 348 and a significant peak at m/z 3 1 7 , indicating a loss of - O C H 3 (Table II). The region from 100 to 200 m/z is practically identical in all the d i m e t h y l a t e d a n a c a r d i c acids under investigation, because as can be seen from Figure 3, none of the ions in this region contain the side c h a i n , which differentiates each anacardic acid. The base peak ion occurs at m/z 161 produced by the loss of water from the ion produced by α-cleavage of the side chain from the aromatic ring (m/z 1 7 9 ) . T h e r e are also substantial peaks at m/z 148 and m/z 121, indicating losses of - O C H 3 and - ( 0 = C ) - O C H 3 , respectively from the m/z 179 fragment. The principal resonances seen in the nmr spectrum of A, together with all the other anacardic acids isolated, are shown in T a b l e III. For the p u r p o s e s of clarity, the two s i g n a l s c o r r e s p o n d i n g to m e t h y l a t e d carboxyl ( 8 3 . 8 8 p p m singlet) and phenol (53.79ppm singlet) groups have been omitted, as have the aromatic signals. In all cases the aromatic region consisted of two pairs of doublets at 66.73 and 56.79ppm, and a double doublet at 57.23ppm, fully consistent with an ABC type system. T h e nmr s p e c t r u m of A s h o w s a triplet at 5 2 . 5 1 p p m corresponding to the methylene group adjacent to the ring (Table III). The methylene group beta to the ring can also be seen as a multiplet at 8 1 . 5 4 p p m , with remainder of the methylene groups present in an intense broad resonance at 61.23ppm. No resonances in the vinylic region of the spectrum are seen, thus confirming the result from mass s p e c t r o m e t r y that A is s a t u r a t e d . The remaining signal in the spectrum is the terminal methyl group at 5 0 . 8 7 p p m , which in this case is a simple triplet, integrating for three protons. Thus as there is only one methyl group present it can be concluded that the side chain in A is unbranched, and hence is identified as the C o straight chain saturated anacardic acid. 2

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

N 0

L M m

J Κ k

I

F G H

anteiso

C25 sat.

C25

C26 ω5

C24 sat.

C24 iso

C2sœ6

C24 ω5 C23 anteiso C23 sat.

C24 ω9

C22 iso C22 sat.

2

43 41 34 41

388

418 418

390 390 416 404 404 430

402

39 61

31 36 19

40 51 31

20

73 29

362 400

376 376 402

M 46 20

Mwt 348 374

18 29

16 20 19

16 22 30 31

24 23 31

27

44 62

52 59

51 49 32

50 55 41

51 55 98 37

91

52 64

100 100

100 100 100

100 100 100 100

100 100 100

100

100 100

INTENSITY (% BASE PEAK ION) M-31(2) M/Z 161 M/Z 180 31 100 51 15. 47 100

15 14

33 15 14 20 14 56 21

15 14 30

33

15 35

M/Z 148 15 79

22 19

23 17 31 22 24 34

32

23 15 45

36

18 38

M/Z 1 19 34

Mass Spectrometric Analysis of Anacardic Acids in Glandular Trichome Exudate

Name Code A C20 sat. Β C22 ω5 C C21 anteiso D C 4 ω6,9 Ε C23 co6

Table II:

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In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

0.87f

(Straight)

0.86f

(Anteiso)

0.83m

(Straight)

0.90Γ

(Straight)

0.86f

(Iso)

0.844

(Straight)

0.89f

(Straight)

0.89f

(Anteiso)

0.83m

(Straight)

0.87f

(Straight)

0.85f

(Straight)

0.86f

(Iso)

0.83d

(Straight)

0.86f

(Straight)

0.89f

(Anteiso)

0.85m

(Straight)

1.23m

1.23m

1.29m

1.23m

1.22m

1.29m

1.29m

1.22m

1.23m

1.26m

1.23m

1.24m

1.27m

1.30m

1.24m

1.24m

1.23m

2

CH C/fCH 2

1.53m

1.55m

1.56m

1.55m

1.52m

1.57m

1.56m

1.54m

1.55m

1.55m

1.55m

1.54m

1.53m

1.57m

1.54m

1.55m

1.54m

2

CH -CH-Ar

* m=*multiplet, Mriplet, ^ d o u b l e t , ctef-double doublet

0

N

m

M

L

k

Κ

J

I

H

G

F

Ε

D

C

Β

0.87r

(Straight)

A

3

CH

Compound

2.03m

2.01m

2.00m

1.98m

1.99m

2.7Qdd

2.04m

1.99m

2

CH -CH«

2.51 f

2.52f

2.54f

2.51 f

2.51 f

2.54f

2.54r

2.51 f

2.51 f

2.51 f

2.51 f

2.51/

2.50f

2.54f

2.52f

2.51 f

2.51/

2

CH -Ar

Table III: N M R Analysis of Anacardic Acids in Glandular Trichome Exudate*

5.0Hz

3.8Hz

5.36m J-4.5, 4.7Hz

5.36m J-4.5, 4.6Hz

5.33m J-4.5, 4.6Hz

J-3.6,

5.32m

J~5.2,

5.32m

5.38m

4.7Hz

5.32m

CH-CH

J-4.6,

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15.

HESK ET AL.

Arthropod-Resistant and -Susceptible Geraniums 235

OCH

3

COOCH3

M (-OCH or HOCH3)

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v

(-R)

3

\ M-31.M-32

(-H2O)

(-OCH )>y 3

m/z148

m/z149

m/z 121

Figure 3: Mass Spectral Fragmentation of Dimethylated Anacardic Acids C o m p o u n d C. The mass spectrum again s h o w e d the c o m m o n anacardic acid ions, and a molecular ion of m/z 362, which suggested that Compound C was a C21 saturated anacardic acid. The nmr spectrum also indicated that C was saturated, and the t e r m i n a l methyl signal at 5 0 . 8 5 p p m was complexed and represented 6 protons. Hence C was identified as a C21 branched chain anacardic acid, and by comparison with the nmr spectra of iso and anteiso branched chain fatty acid standards, the terminal methyl signal was identical to the terminal methyl resonance of an anteiso group, thus C is in all probability the C21 anteiso saturated anacardic acid.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

236

NATURALLY OCCURRING PEST BIOREGULATORS

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It should be noted t h o u g h , that although the nmr spectrum will eliminate the iso and straight chain isomers as possible structures for C, it is theoretically possible for the branch to be located in a position other than the anteiso. Such c o m p o u n d s would also give an nmr spectrum very similar to the anteiso, but as such c o m p o u n d s are relatively rare in plants, C can be classified as the C21 anteiso saturated anacardic acid, with a high degree of confidence. C o m p o u n d D. The mass spectrum of D gave a molecular ion of m/z 4 0 0 , and the expected ion at m/z 368 from the loss of C H 3 O H . Compound D was therefore identified as a C24 unsaturated anacardic acid, but containing two double bonds. Nmr a n a l y s i s of D c o n f i r m e d that the c o m p o u n d w a s u n b r a n c h e d (60.89ppm, triplet, 3 protons) and unsaturated, and furthermore indicated the presence of more than one double bond, due to the extra signal at 52.78ppm, and a highly complex vinyl signal at 55.38ppm, in addition to the resonance at 5 2 . 0 4 p p m . As the latter is due to a methylene group adjacent to a vinyl group, it was postulated that the resonance at 62.78ppm could be due to a methylene group located between two double bonds. However in order to establish the position of unsaturation, it is necessary to prepare the dimethyl disulfide (DMDS) derivative. DMDS simply adds across a double bond to leave a C H 3 S - group attached both carbons. The presence of the methyl thioether g r o u p s on adjacent c a r b o n s renders the bond between t h e m susceptible to cleavage during mass spectrometry, and hence it is possible to determine the position of the original double bond (a).

DMDS has previously been used to establish the location of double bonds in mono-unsaturated c o m p o u n d s (SL); however it appeared to work very well in the case of compound D, giving one major peak on the hplc. The mass spectrum turned out to be much more complicated than expected, because of partial decomposition of the derivative under the conditions employed. From Figure 4, the ion at m/z 2 9 1 , which is derived from the loss of C H 3 O H from m/z 323 places one double bond 9 carbons in from the terminal methyl group. The position of the second double bond was localized from the ion m/z 347, which arises from the loss of a C H S C H u n i t from the 9,10 carbon bond to leave a thioepoxide group, a break across the 6,7 carbon bond and a loss of C H S H . The ion at m/z 315 arises from the loss of C H O H from m/z 347. Hence the second double bond is located 6 carbons in from the terminal methyl group and therefore c o m p o u n d D is 3

3

3

3

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

m/2 478

\

•MeSH

m/2 399

- MeOH

m/2 431

^SMe

m/2 291 (B)

m/2 323

Figure 4: Mass Spectral Fragmentation of the DMDS Derivative of D

m/2 315

• MeSH

12

• C6H SMe

MeSMe

m/2 588

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SMe

238

NATURALLY OCCURRING PEST BIOREGULATORS

identified as being a C24 ω 6 , 9 unsaturated anacardic acid. The other major ions in the spectrum can be accounted for by loss of C H 3 S C H 3 from the molecular ion (not seen) to leave m/z 526. That ion itself loses CH3SH to give m/z 478, and further losses of S C H and CH3OH from m/z 478 account for the ions at m/z 431 and m/z 399. A small ion at m/z 464 is formed from the loss of two CH3SCH3 groups to leave the double thio-epoxide.

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3

C o m p o u n d Ε. Ε was found to have a molecular ion at m/z 388 i n d i c a t i n g t h a t the s t r u c t u r e w a s c o n s i s t e n t w i t h a C 2 3 unsaturated anacardic acid. The mass spectrum of the DMDS derivative of Ε showed the expected molecular ion at m/z 482, and two significant fragments at m/z 319, and m/z 1 3 1 . The ion at m/z 131 is the fragment expected from the cleavage of the bond between adjacent methyl thioether groups, when the bond occurs six c a r b o n s from the methyl end of the side c h a i n . However, as anacardic acids containing branched chains had a l r e a d y b e e n i d e n t i f i e d , m/z 131 c o u l d also represent a fragment, with an iso or anteiso structure and hence the double bond would then occur 5 carbon atoms from the terminal methyl group. This ambiguity was easily solved by the nmr spectrum, which showed that the terminal methyl signal (80.86ppm) was a simple 3 proton triplet, which hence proved that the double bond was located at the ω 6 position. In addition coupling constants of 5.2 and 5.0Hz indicated that the the double bond was cis. Hence Ε is identified as the cis C23CÛ6 unsaturated anacardic acid. C o m p o u n d F. A molecular ion at m/z 376 and the knowledge that the retention time of F was slightly earlier than G, which was already known as the C22 straight chain saturated anacardic acid, strongly suggested that Compound F was a C22 branched chain saturated anacardic acid. The nmr of the terminal methyl signal (80.83ppm) showed it contained 6 protons, and was split into a simple doublet, consistent with an '/so type' end chain. Hence F is the C22 iso saturated anacardic acid. C o m p o u n d H . The mass spectrum shown in Figure 5 has a molecular ion at m/z 402 and the usual ions at m/z 180, 1 6 1 , 148 and 121 indicating that H is a C24 u n s a t u r a t e d a n a c a r d i c acid. The ion at m/z 370 is formed from the loss of C H O H from the molecular ion, The DMDS derivative gave the spectrum shown in Figure 6. The expected molecular ion at m/z 496 was seen and the fragment at m/z 173 located the position of unsaturation as 3

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Arthropod-Resistant and -Susceptible Geraniums 239

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15. HESK ET A L

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991. 350

ur* M/Z

4

0

0

450

Figure 6: El Mass Spectrum of the Dimethyl Disulphide Derivative of Dimethylated Compound H from Geranium

300

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500

15.

HESKETAL.

Arthropod-Resistant and Susceptible Geraniums

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being 9 carbon atoms from the end of the terminal methyl group. The fragment at m/z 2 9 1 , formed from the loss of CH3OH from m/z 323, supported this conclusion. In addition the nmr spectrum showed that the side chain was unbranched (50.85ppm, triplet), and also that the geometry of the double bond was c/s, due to J values of only 3.6 and 3.8Hz. Hence compound H is identified as the c/s C24 ω9 unsaturated anacardic acid. C o m p o u n d J . J was found to have a molecular ion at m/z 390, together with the characteristic ions in the 100 to 200 m/z r e g i o n , s u g g e s t i n g that the structure w a s a C23 saturated anacardic acid. As can be seen from Figure 7 a nmr analysis confirmed that J was saturated, and examination of the terminal methyl group signal (50.83ppm multiplet), s h o w e d it integrated for 6 protons and contained a splitting pattern consistent with anteiso branching. Hence J is more than likely the C23 anteiso saturated anacardic acid. C o m p o u n d K. The mass spectrum w a s practically identical to that of compound J , with the same molecular ion at m/z 390, but with a slightly later hplc retention t i m e , a n d hence it w a s thought likely that Κ was a C23 s t r a i g h t chained saturated anacardic acid. Confirmation of this was provided from the nmr spectrum which showed a simple three proton terminal methyl triplet at 50.89ppm, as well as the absence of any signals in the vinyl region. Hence Κ is the C23 straight chain saturated anacardic acid. C o m p o u n d k. This minor peak in resistant exudate, and not seen in the susceptible exudate, gave a molecular ion at m/z 416 suggesting that the structure was a C25 u n s a t u r a t e d a n a c a r d i c a c i d . The mass spectrum of the D M D S derivative g a v e the expected molecular ion at m/z 510, a fragment at m/z 131 and an ion at m/z 347, indicating that a 6 carbon fragment had been lost. However as in the case of Compound E, it was also possible that this 6 carbon fragment was branched, thus meaning that the double bond would be effectively located in the ω 5 position. This ambiguity w a s easily cleared up by the nmr s p e c t r u m which s h o w e d a s i m p l e triplet which i n t e g r a t e d for 3 protons at 50.89ppm, and also because of the small J values (4.5, 4.6Hz), the geometry of the double bond was shown to be cis. Hence k is the cis C25 ω6 unsaturated anacardic acid.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

241

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242

NATURALLY OCCURRING PEST BIOREGULATORS

C o m p o u n d L. The mass spectrum gave a molecular ion at m/z 4 0 4 t o g e t h e r with the other e x p e c t e d a n a c a r d i c acid ions. Furthermore as compound L had an earlier retention time on the hplc than compound M, the known C24 saturated straight chained anacardic acid, it was thought likely that L was a C24 b r a n c h e d chain saturated anacardic acid. The exact structure was confirmed by nmr spectroscopy (Figure 7b), which showed the terminal methyl group (50.84ppm) to be a 6 proton doublet, a splitting pattern consistent with iso branching. Thus L is the C24 iso saturated anacardic acid. C o m p o u n d m. This was another c o m p o u n d only seen in the resistant exudate, and from a molecular ion of m/z 430 it was identified as a C26 unsaturated anacardic acid. The spectrum of the DMDS derivative gave a molecular ion of m/z 524 and the fragment at m/z 117 placed the double bond 5 carbons in from the t e r m i n a l methyl g r o u p The ion at m/z 3 7 5 , w a s also c o n s i s t e n t with a 5 c a r b o n f r a g m e n t being lost. The nmr spectrum showed a simple triplet, integrating for 3 protons, at the t e r m i n a l methyl p o s i t i o n , s h o w i n g that the c h a i n w a s unbranched, and thus confirming the ω5 double bond position. In addition the small J values of 4.5 and 4.7 Hz placed the double bond geometry as c/s, and hence m is identified as the cis C26 ω5 unsaturated anacardic acid. Compound N. The spectrum showed the compound had a molecular weight of 418, consistent with a C25 saturated anacardic acid. Nmr analysis s h o w e d that the splitting of the terminal methyl group (80.83ppm multiplet, 6 protons) was complex and identical to the terminal methyl group splitting in the nmr spectrum of the anteiso standard. Hence Ν is in all probability the C25 anteiso saturated anacardic acid. C o m p o u n d O. This gave a practically identical spectrum to N, with the same molecular weight, and because of the slightly later retention time on the hplc it was strongly suspected that Ο was the C25 straight chain isomer. This was readily confirmed by nmr as the terminal methyl group at 50.86ppm was a simple 3 proton triplet, meaning that Ο is the C25 straight chain saturated anacardic acid.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Arthropod-Resistant and -Susceptible Geraniums 243

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15. HESK ET A L

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

NATURALLY OCCURRING PEST BIOREGULATORS

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244

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

15.

HESK ET AL.

Arthropod-Resistant and -Susceptible Geraniums

245

DigcUSSion. Having identified the majority of the anacardic acids in both resistant and susceptible exudate, it is possible to more fully highlight the differences between the two, than w a s previously the case.

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Table IV: Percentage Composition of Anacardic Acids in Geranium Glandular Trichome Exudate. Code A Β C D Ε F G H I J Κ k L M m N 0

Name C20 sat C22 ω5 C21 anteiso C24 ω6,9 C23 (û6 C22 iso C22 sat. C24 dicaffeoylquinic acids, of which the most potent were sesquiterpene lactone angelates including argophyllin A and 3-methoxyniveusin A, the diterpenoic acids grandifloric acid and its 15-angelate, and theflavonoidsnevadensin and quercetin ß-7-O-glucoside. Similarly, kaempferol was identified as a weak antifeedant from Canadian goldenrod. Two of the electrophilic germacranolide angelates with 4,5-unsaturation, when injected into rootworm adults, gave neurotoxic symptoms (hyperexcitability, enhanced egg expulsion, tarsal tetany) similar to picrotoxinin, a sesquiterpene lactone epoxide known to act on theγ-aminobutyricacid-gated chloride channel. These neurotoxic antifeedants may explain both the seven-fold decreased tolerance of western com rootworm to aldrin and its decreased longevity when fed onfloraltissues of sunflower in comparison to com. Relevance of these results to other herbivore-phytochemical associations, particularly those with chrysomelids, will be discussed.

Phytochemicals produced from secondary metabolic pathways are major mechanisms by which plants are protected from excessive herbivory. The role of foliar chemicals in retarding or preventing consumption of leaves, the primary photosynthetic organs of plants, has been clearly established. However, few studies have addressed the negative effects of floral chemistry on insect herbivory. Reproductive structures should, expectedly, be well-defended to assure adequate propagation of plant genes (1,2). Yet the attributes of flowers that attract pollinators (i.e. visual or volatile cues, nectar and pollen quality) have dominated study in the chemical basis for insect-floral relationships. The considerable amounts of flavonoids, carotenoids and steroids in pollen (2,4), alkaloids and phenolics in nectars (5), UV-quenching flavonoid nectar guides (£,2), and floral fragrances (e.g. fi) are most often associated with attraction and rewarding of essential pollinators and not with defense against floral

O097-6156/91/0449-O278$06.00/0 © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

18.

MULLIN E T AL.

Corn Rootworm Feeding on Compositae

279

consumption. This general phytochemical enhancement of pollination is consistent with animals functioning as pollinators for the majority of angiosperm species, but tends to neglect the defense of plant propagules from florivores particularly where self-pollination is evident. In the ensuing discussion, we will emphasize terpenoid and phenolic factors that protect Compositae (Asteraceae) flowers from excessive consumption by insects.

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Terpenoids as Regulators of Herbivory - Associations with Chrvsomelidae Plant sesquiterpenes and other terpenoids are major determinants of insect-plant interactions (3z 16V Many insecticidal and antifeedant terpenoids are epoxides including monoterpene (12, 18). sesquiterpene QQ, 19-23). diterpene (11, 21) and triterpene derivatives (25-2D typified by the potent antifeedant azadirachtin (28-30). Most biological effects have been determined with Lepidoptera and non-chrysomelid Coleoptera. Occasionally, the same compound, while normally inhibitory to herbivores, may for adapted insect species or at low concentrations have a stimulatory effect (13). Insects, in turn, synthesize their own defensive (31. 32) and pheromonal (22) terpenoids. Plants may utilize insect pheromones such as the sesquiterpene alarm pheromone, rran5-8-farnesene, in their own defense (34. 35). Inhibitory cyclic sesquiterpenes (Table I) and diterpenes (Table II) for insect herbivores have been identified from at least 28 genera of the terpenoid-rich Compositae. These studies were largely confined to extrafloral tissues. Floral chemistry is increasingly being associated with antiherbivore actions, particularly among terpenoids. In Gossypium, phenolic sesquiterpenoid-derived heliocides and the dimeric gossypol are anti-lepidopteran factors found in high concentration in flower parts (2, 2 i ) . The monoterpene-derived iridoid glycosides protect nectar of Catalpa from consumption by non-pollinators (22). Floral concentrations of terpenoids higher than that of leaves and externally compartmentalized into trichomes have been noted (26.2&)- In the Compositae, a plant family characterized by many self-pollinated species, both monoterpene derivatives such as the insecticidal pyrethrins from Chrysanthemum spp. (22, 40) and toxic sesquiterpene lactones and diterpenoic acids concentrated in the floret achenes of wild Helianthus spp. (41-44) are clearly protecting flowers from excessive herbivory. Both niveusin A from H. niveus and 88-sarracinoyloxycumambranolide from H. maximiliani deter feeding of the sunflower moth, Homoeosoma electellum (42). It is thought that sesquiterpene lactones in glandular trichomes of the anther prevent pollen-feeding by this sunflower pest; foliar sesquiterpenes including the epoxide, argophyllin A , I, from H. argophyllus (44), and diterpenoic acids (45) may explain antibiosis in sunflower for this pest and others (Tables I and II) including the chrysomelid Zygogramma exclamationis (F.) (41). Among the most noted of chrysomelid-terpenoid investigations have been Diabrotica spp. feeding associations with squash cucurbitacins, triterpenoidal-derived electrophiles that serve as potent feeding stimulants for corn rootworms (4£, 42). Cucurbitacin contents are particularly high within the anther and filament of Cucurbita maxima, a much preferred squash species for Diabrotica spp. as a pollen-source of food (48). Interestingly, for other chrysomelids such as the Cruciferae leaf beetles (42) and Colorado potato beetle, Leptinotarsa decemlineata (50), these compounds are strong feeding deterrents. Work with other squashfeeding Diabroticine chrysomelids has identified a number of potent antifeedants including the neem tetranortriterpenoids (Meliaceae) for striped cucumber beetle, Acatymma vittatum (F.) and southern corn rootworm, D. undecimpunctata howardi Barber (51), and the sesquiterpenoid celangulin from Chinese bittersweet (Celastraceae) for Aulacophora femoralis chinensis (52). Neem (22,20,52) as well as citrus (26) limonoids generally deter feeding of chrysomelid species. While large amounts of dietary sesquiterpene lactones from Encelia farinosa (Table I) deter the growth of the specialist herbivore, Trirhabda geminata, natural resistance by this composite species to this chysomelid appears more associated with elevated chromene levels (54). Antifeedant and toxic sesquiterpenes for the Colorado potato beetle have been identified from the wild tomato Lycopersicum hirsutum (55), the sagebrush Artemisia tridentata (50). and from other Compositae and some Apiaceae species (56 and refs therein). Antifeedant diterpenoids for this chrysomelid are also known (57). The goldenrod diterpenoids, in turn, are antifeedant to the Solidago specialist, Trirhabda canadensis (24, 5&). Various monoterpenes and cardenolides are also important as stimulators or inhibitors of chrysomelid herbivory (52), and some compounds from these terpenoid classes as well as the cucurbitacins are utilized in beetle defense against natural enemies (42.60).

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Table I.

Cyclic Sesquiterpenes from Compositae that Deter Insect Herbivores

Plant Genera Sesquiterpene

Insect species

Inhibitor of

References

Achillea Caryophyllene

Locusta migratoria

Feeding

61

Feeding Growth

50, 62-66

63,67

Artemisia Absinthin, Achillin Heliothis zea, Hypochlora Caryophyllene alba, Leptinotarsa decemlineata, ar-Curcumene Melanoplus sanguinipes, Pieris Desacetoxymatricarin rapae, Spodoptera littoralis, Dehydroleucodin Sitophilus granarius, Tribolium a-Santonin confusum, Trogoderma granarium Centaurea Cnicin Salonitenolide

Sitophilus granarius, Tribolium confusum, Trogoderma granarium

Feeding Longevity

Chrysanthemum Artecanin Canin

Sitophilus granarius, Tribolium confusum, Trogoderma granarium

Feeding

Schistocerca gregaria

Feeding

Trirhabda geminata

Growth ?

Cichorium 8-Deoxylactucin Lactucopicrin Encelia Farinosin

Eupatorium 1 -Desoxy-8-epi-ivangustin Ana cephalotes, seco-Eudesmanolide Drosophila melanogaster, Eupatoriopicrin Philasomia ricini, Euponin Sitophilus granarius, Tribolium Cadinene type confusum, Trogoderma granarium Grossheimia Grossheimin Helenium Helenalin Linifolin A Tenulin

Sitophilus granarius, Tribolium confusum, Trogoderma granarium

Homogyne Bakkenolide A

68

54

Feeding Growth Longevity Oviposition

56,67 69-73

Feeding Longevity

63, 67

Epilachna varivestis, L. decemlineata, Feeding Melanoplus sanguinipes, Ostrinia nubilalis, Growth Peridroma saucia, Sitophilus granarius, Longevity T. confusum, Trogoderma granarium Oviposition

Helianthus Argophyllins A & B Budlein A , Eupatolide Cumambranolide ester Desacetyleupasserin Niveusin A

63

63, 74-77

Feeding Growth Longevity

42-44, 78

Feeding Leptinotarsa decemlineata, Peridroma saucia, Sitophilus granarius, Tribolium Growth Longevity confusum, Trogoderma granarium

56, 71, 77

Homoeosoma electellum, Melanoplus sanguinipes, Spodoptera eridania, S. litura

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Table I (cont'd). Cyclic Sesquiterpenes from Compositae that Deter Insect Herbivores Plant Genera Sesquiterpene Inula Alantolactone Isoalantolactone

Insect species

Inhibitor of

References

Sitophilus granarius, confusum, Trogoderma

Tribolium granarium

Feeding Longevity

56, 79, 80

Sitophilus granarius, confusum, Trogoderma

Tribolium granarium

Feeding Longevity

63,75

Sitophilus granarius, confusum, Trogoderma

Tribolium granarium

Feeding Longevity

63,67

Feeding

81

Feeding Growth Longevity

82, 83

Feeding

63

Feeding Growth Heartbeat Longevity

75, 84-88

Feeding

56

Feeding

89

Feeding

90

Feeding

63

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Coronopilin Jurinea Alatolide Lasianthaea Lasidiol angelate

Atta cephalotes

Melampodium Caryophyllene oxide Atta cephalotes, Guaianol, Melampodin A Spodoptera frugiperda Melampodinin A , Spathulenol Onopordon Onopordopicrin

Sitophilus granarius, confusum, Trogoderma

Tribolium granarium

Parthenium Conchosins A & B Heliothis zea Confertin, Coronopilin Melanoplus sanguinipes Isochiapin B , Ligulatin C Spodoptera exigua Parthenin and derivatives Tribolium confusum Tetraneurins A , B & E Other species Petasites Petasitolide A Schkuhria Schkurins I & II

Sitophilus

E. varivestis, Spodoptera

Tithonia Tagitinin C Venidium Hirsutolide

granarius exempta

Philasomia ricini Sitophilus granarius, confusum, Trogoderma

Tribolium granarium

Vernonia Feeding Glaucolide A Diacrisia virginica, Sibine stimulea, 11,13-Dihydro- Spodoptera eridania, exempta, frugiperda Growth vernodalin S. ornithogalli, Trichoplusia ni Oviposition Xanthium 8-Epi-xanthatin, Xanthumin Xantholide A (= Ziniolide) Xeranthemum Xerantholide

20, 91, 92

Drosophila melanogaster

Growth

93, 94

Sitophilus granarius, Tribolium confusum, Trogoderma granarium

Feeding

63

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Table II.

Diterpenes from Compositae that Deter Insect Herbivores

Plant Genera Diterpene

Insect species

Chrysothamnus 18-Hydroxygrindelic acid Leptinotarsa decemlineata 18-Succinyloxygrindelic acid

Inhibitor of

References

Feeding

57

Feeding

95

Growth Longevity

44, 45, 96

Feeding

97

Feeding

97

Feeding

24,58

Grindelia

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6 a & B-Hydroxygrindelic acids Schizaphis

graminum

Helianthus Angeloylgrandifloric acid Heliothis virescens, H. zea, Ciliaric acid, cw-Ozic acid Homoeosoma electellum, Kaur-16-en-19-oic acid Pectinophora gossypiella 16-Hydroxykaurane 16-Hydroxykaur-l 1-en-19-oic acid Trachylobanoic acid Lasianthaea Kaur-16-en-19-oic acid Atta cephalotes Melampodium Kolavenol

Atta cephalotes

Solidago 16-Hydroxykaurane Trirhabda canadensis 15-Hydroxykaur-l 6-en-19-oic acid 17-Hydroxykaur-15-en-19-oic acid

Phenolic - Insect Associations: Relevance to Chrysomelidae Many polyhydroxylated flavonoids and related phenolics have been shown to limit insect herbivory (98-100. Hesk et al., this volume). Inhibitory actions.by phenolics often require both the high concentrations naturally present in plants and chemical structures bearing adjacent (ortho) hydroxyl groups (cf 101). although exceptions to both these trends occur with aphids (102.103). A t lower dosages or with phenolic specialists, stimulatory rather than inhibitory effects on feeding may result (cf 9JL 104). While this tendency is also found among chrysomelids, other structural features such as the type of sugar and its position of attachment may be more important in influencing activity. Flavonoid glycosides are known that both stimulate and inhibit chrysomelid feeding (105.106). Simple phenolics such as chlorogenic acid have been shown to deter a Salicaceae-feeding leaf beetle (107). Strongly UV-absorbing flavonoids (108) and other phenolic derivatives (109) with pro- or anti-insect activities are increasingly being found within floral tissues, suggesting that their adaptive roles extend beyond the visual orientation of pollinators (6,110. 111). Compositae-Corn Rootworm Interactions Adult northern com rootworm, D. barberi Smith & Lawrence (NCR), readily feed on flowers of the Compositae (Asteraceae) that are barely acceptable to western com rootworm, Diabrotica virgifera virgifera LeConte (WCR). Rearing adult W C R continuously on inflorescences of cultivated sunflower Helianthus annuus L . var. Giant Gray Stripe, or Canadian goldenrod Solidago canadensis L . var canadensis, reduces its longevity by 40% and 70%, respectively, to that on com ears. B y contrast, NCR's longevity is not significantly affected by host shifts from com to Compositae (112). Also, an antifeedant action of this food was observed for W C R in the short term (< 24 hr). A two-dimensional thin-layer chromatographic (tic) method

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developed by us to analyze flavonoid and phenolic acid aglycones within small amounts (< 30 mg) of plant or insect tissue (103) gave a lavender fluorescing compound (366 nm) at R f coordinates (0.21,0.25) that accumulates in W C R after long-term feeding on sunflower petals (Figure 1). The free acid has been identified (confirmed by U V , EIMS and ^H-NMR) as transcaffeic acid both within the plant and insect after preparative isolation on silica gel and cochromatography in four solvent systems (P. R. Urzua, W. R. Wenerick and C. A . Mullin, unpubl.). A gold fluorescing compound at R f coordinates (0.23, 0.29), that is sequestered from sunflower petals by rootworm, is the aglycone form of a quercetin flavonoid (see below). Isolation and characterization of feeding deterrents. A more systematic fractionation of floral principles responsible for the feeding deterrent and toxic effects of sunflower was then conducted, guided by a squash disk bioassay where relative consumption after 5,24 and 48 hr by adult W C R of solvent- or compound-treated flower disks was measured. This bioassay was designed to detect only highly active antifeedants that counteract the potent feeding stimulatory effect of cucurbitacins. In 1988 studies, residues from one-week extracts of petals and florets using ice-cold 95% ethanol were dissolved in water and partitioned in order by chloroform, ethyl acetate and n-butanol. Most of the original antifeedant and longevity-reducing activities concentrated into the ethyl acetate fraction, and were isolated by Toyopearl T S K HW-40F using methanol-water (75:25) to give three major phenolic components and a number of unbound polar terpenoids (dashed sunflower profile, Figure 2). Two of the phenolics had U V spectra resembling caffeoyl esters, and the other exhibited U V characteristics resembling a glycoside of quercetin with a free 3-hydroxy group. Through use of H - and ^ C - N M R in d6-DMSO, the flavonol was identified as quercetin 6-7-O-glucoside, II, and the phenolic acids as 3,5dicaffeoyl-, H I , and 3,4-dicaffeoylquinic acids, IV. Only the former was antifeedant for W C R 1

Western Corn Rootworm on Sunflower Petal

Sunflower Petal

Figure 1. Two-dimensional tic of phenolic acids and flavonoids in W C R that had fed for one month on sunflower petals. Degree of shading indicates quench at 254 nm; x = apparent cochromatography between insect and plant. See ref. 103 for details on development solvents.

HO HO

Quercetin

B-7-O-glucoside

II

3,5-Dicaffeoylquinic acid 3,4-lsomer I V

III

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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0

100

200

300

400

500

600

700

ELUTION VOLUME (ml) Figure 2. Composite chromatogram of floral ethyl acetate residues on Toyopearl HW-40F. in a squash disk bioassay (Table III). Interestingly, a mixture of 3,5- and 3,4-dicaffeoylquinic acids, the major phenolics within the floral tissues of sunflower, was stimulatory to rootworm feeding at a low dose (32 u.^disk), but not at a higher dose (129 jig/disk) more representative of the intact flower. Quercetin 7-O-glucoside has previously been isolated from the flowers Q , 113) and pollen (114) of H. annuus, and 3,5-dicaffeoylquinic acid has been identified in sunflower seeds (115). By similar procedures we isolated and identified free kaempferol from the ethyl acetate residue of an ethanolic extract of goldenrod inflorescences (solid triangle profile, Figure 2) as a weak rootworm deterrent. This flavonol was previously reported from S. canadensis (116). In order of richness (amounts per weight tissue) in floral phenolics, goldenrod was greater than sunflower which was richer than corn tassel (Figure 2). The unbound terpenoids from sunflower (Figure 2) were further purified by silica gel

Table HI. Effect of Sunflower Floral Chemicals on Rootworm Consumption of Blue Hubbard Squash D i s k s a

Compound

Dose Relative Consumption (treated/control) (^ig/disk) 5 hr 24 hr 48hr

Argophyllin A (+ some VI)

40

0.32*

0.23*

0.24*

3-Methoxyniveusin A (+ some VI)

40

0.39*

0.26*

0.26*

Quercetin B-7-O-glucoside

114

0.81

0.86*

0.74*

3,5+3,4-Dicaffeoylquinic acids

32

1.25

1.65*

2.83*

a

Dual choice tests with 8 uJ of solvent or compound per 1.5 cm flower disk * = significantly different from methanol control at p < 0.05 based on area consumed.

D

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0

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chromatography and identified as the sesquiterpene lactones argophyllin A , I, and 3-methoxyniveusin A , V , each contaminated with the lesser antifeedant l-memoxy-4,5-dihydroniveusin A , V I . These sesquiterpene lactone angelates are greater than five times more potent than quercetin 7-8-O-glucoside as antifeedants for WCR (Table III). In 1989, whole sunflower heads were extracted by solvent immersion for only two minutes to optimize the isolation of labile cuticular terpenoids probably occurring as trichome exudates. Sequential two-minute surface extraction of 84 inflorescences by petroleum ether, methylene chloride-methanol (3:1 v/v) and methanol followed by partitioning of residues (22 g) from the combined polar extracts between water and ethyl acetate, and then the ethyl acetate residues (16 g) between 90% aqueous methanol and petroleum ether yielded methanolic solubles (8 g) rich in antifeedant activity. Column, flash and thin-layer silica gel chromatography of these solubles gave more than 65 compounds, which in decreasing order of abundance were primarily diterpenoic acids, sesquiterpene lactone angelates, and methoxylated flavonoids. Compounds were identified through use of 360 M H z ^ H - N M R (including N O E , spin-decoupling), 126 M H z C - N M R (including GASPE), M S and U V / V i s spectroscopy as necessary. Purity was assessed by tic and by reversed-phase high performance liquid chromatography (hplc) on C8 and CI 8 columns using acetonitrile-water gradients. Indeed, the flower of this annual species of Helianthus was quite chemically complex. Thirty-four of these compounds were bioassayed for deterrency to W C R as above, with the order of potency; sesquiterpenes (7 compounds) » diterpenes (6) > methoxylated flavonoids (4). Bioassay data (Table IV) and structures are included here for the more potent 1 3

Table I V . Floral Feeding Deterrents for Adult Western Corn Rootworm A m o n g Ethyl Acetate Soluble Chemicals from the Sunflower^ Relative Consumption after 24 hr (treated/control)b

CHEMICAL CLASS Structure

40 ^ig/disk

compound name SESQUITERPENES I argophyllin A V 3-methoxyniveusin A VI 1 -methoxy-4,5-dihydroniveusin A VII 15-hydroxy-3-dehydrodesoxytifruticin VIII 3-oxo- derivative of VI IX 10-methoxy-3-oxo-derivative of VI DITERPENES X kaur-16-en-19-oic acid XI grandifloric acid XII grandifloric acid angelate XIII trachylobane XIV 15-hydroxytrachyloban-19-oic acid XV 7-oxo-trachyloban-15,19-diol FLAVONOIDS XVI nevadensin X V I I 5-hydroxy-4,6,4 -trimethoxyaurone c

,

a

0.23 0.75 0.97 0.97

± ± ± ±

0.04 0.08 0.09 0.16

— 0.80 ± 0.05 1.0510.21 0.91 ± 0 . 0 5 0.92 ± 0.04

— — 1.00 ± 0 . 0 1 ....

—

80 ^ig/disk

— d

d

0.30 0.71 0.75 0.72 0.64

0.85 ± 0.13d 0.61 ± 0.14 0.70 ± 0 . 1 3 1.00±0.01 0.96 ± 0 . 0 3 0.98 ± 0 . 0 1 d

d

d

0.93 ± 0 . 0 6 1.01 ± 0 . 0 1

D u a l choice tests with the Blue Hubbard squash disk bioassay using 8 ul of solvent or compound solution per 1.5 cm flower disk. Mean ± S E M for 4 replicates per dose; consumption based on area. Dosages were 50 and 100 u,g/disk, respectively. Although inactive at 24 hr, substantial feeding deterrency observed at 5 hr.

D

c

d

± 0.09 ±0.02 ± 0.08 ± 0.08 ± 0.02

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d

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O

I Argophyllin A

X Kaur-16-en-19-oic H X I Grandifloric acid OH XII Grandifloric angelate O A n g

V 3-Methoxyniveusin A CH3 H V I l-Methoxy-4,5-dihydro- H C H 3 A X K niveusin A X I H Trachylobane CH3 H H X I V 15-HydroxytrachyloO ban-19-oic COOH H OH XV 7-Oxo-trachyloban15,19-diol C H 2 0 H =0 O H

\TI

15-Hydroxy-3-dehydrodesoxytifruticin

X V I H 3-Oxo- derivative of V I H I X 10-Methoxy-3-oxo-VI CH3

OCH

X\TI

3

,

5-Hydroxy-4,6,4 -trimethoxyaurone

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antifeedants. Included among these are a sesquiterpene, I X , a diterpene, X V , and an aurone, X V I I , new to science. The most potent feeding deterrents included sesquiterpene lactone angelates of the germacranolide type with epoxidation or 4,5-unsaturatioa For the diterpenoic acids, the 16-kaurene system appeared more active than the trachylobane type, and 15hydroxylation in both types improved feeding deterrence. In general, the diterpenoic acids and the methoxylated flavonoids had less residual activity (i.e. good bioactivity up to 48 hr) than the sesquiterpenes. Also, the polar phenolics isolated in 1988 from the more extensive extraction of floral tissues were much reduced in the 1989 surface extracts, indicating the expected intracellular localization of the flavonol glucosides and caffeoylquinic acids. Natural versus Artifactual Sesquiterpenoids from Helianthus. Many of the sesquiterpenes were isolated as methoxylated derivatives that may have resulted from the interaction of methanol with precursor epoxides under acid conditions. Since the more abundant diterpenoic acids, probably present with the sesquiterpenes in the same trichomes (44,117.118). could provide the requisite low pH for these additions/rearrangements, we hypothesized that isolating the terpenoids under buffered conditions in the absence of alcoholic solvents may result in chemistry more representative of the intact plant. Thus, 139 sunflower inflorescences (with many opened disk florets) were extracted for 30 seconds with 4 by 4 L of a heterogeneous solvent (75% ethyl acetate: 25% 50 m M potassium phosphate pH 8 buffer) at the Rock Springs Field Lab immediately after cutting. Extracts were transported on ice to the lab, and the ethyl acetate residue fractionated by silica gel chromatography as before. This residue proved to be enriched with at least ten different sesquiterpene lactones. While characterization remains incomplete, it is clear that less methoxy-substituted sesquiterpene angelates occur if methanol is absent indicating that these compounds, some of which are reported by others from sunflower (11&), may be artefacts of the isolation method. Nevertheless, 3-methoxyniveusin A , V , is present, and appears to be synthesized de novo in the plant. Recent work in two other laboratories has led to the identification of eight germacranolides in extrafloral aerial tissue (primarily leaves) of H. annuus. Melek et al. (117) have identified argophyllin A , I, as the major and another epoxide, argophyllin B, and niveusin B as minor components, whereas Spring et al. (118) have identified 15-hydroxy-3dehydrodesoxytifruticin, V I I , and its hemiketal as major and argophyllin B , niveusin C, 1methoxy-4,5-dihydroniveusin A , V I , and its anhydrido analog as the minor components. Part of the discrepancy between these labs might be due to cultivar differences since a wild variety was used in the former and var giganteus was used in the latter study. However, cyclic sesquiterpene epoxides similar to the argophyllins (119.120) are sensitive to both acid and base catalyzed rearrangements that form tetrahydrofurans and ultimately conjugated systems such as VII. Sufficient acidity for these reactions may result from co-occurring diterpenoic acids on the plant surface. Thus, the argophyllins or more labile epoxides may be the actual or, at least, predominant forms in which these C-6 lactonized germacranolide angelates are present in sunflower. Interactions between antifeedant sesquiterpenes and other plant allelochemicals. Binary combinations of one of the more potent W C R feeding deterrents with another at a dose that gives weak feeding deterrence were explored with eight combinations of chemicals in the squash disk bioassay. No synergistic or antagonistic interactions for combinations of deterrents within or between the sesquiterpene ( V - V I I , I X ) , diterpene ( X I , X I I ) and flavonoid (XVII) classes were noted. This indicates that the suite of antifeedants present in sunflower inflorescences act jointly in an additive fashion. Neurotoxicity of antifeedant sesquiterpenes. The sesquiterpene, V , and the conjugated lactone, V I I , when injected as D M S O solutions (200 nl) into W C R adults at dosages of 2.5 u.g or greater per insect (avg. live wt of 17 mg), gave neurotoxic symptoms at 24 hr (excitability, hyperextension of ovipositor, egg expulsion, tarsal tetany) similar to that of picrotoxinin, a known y-aminobutyric acid ( G A B A ) antagonist, but not like that of avermectin (sluggish movements, paralysis), a G A B A agonist (121.122). The acute toxicity of these sesquiterpenes (LD50S > 50O ug/g insect), although low compared to avermectin and picrotoxinin (Table V), was substantial considering that the most active compound, I, and coadministration of synergists was not tested. Picrotoxinin, a sesquiterpene epoxide lactone from Anamirta cocculus L . , has some structural similarities to that of Helianthus germacranolides. The latter

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sesquiterpene lactones, as is the case for other antifeedants (123, 124), have electrophilic centers including allylic hemiketal, conjugated ketone, and epoxide sites in addition to the conjugate lactone which may interact with critical nucleophiles such as thiol (125) and amino groups (126) of sensory receptors. Based on the structure-activity data presented above, the lactone site is not solely responsible for feeding deterrency or probably neurotoxicity. It remains to be determined i f electrophilicity is associated with the G A B A - l i k e effects on the central nervous system. Other terpenoids are known to inhibit acetylcholinesterase (127). This is the first report of a putative G A B A antagonist (i.e. convulsant) for an insect within its native food plant.

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Deductions: Dietary Phytochemicals. Insecticide Resistance and Com Rootworm Control Pioneer populations of W C R in central Pennsylvania, depending on their food, are 90 to 1200 times more resistant to aldrin than an endemic population of NCR. Susceptibility of W C R to aldrin increased at least seven times when the adults consumed inflorescences of sunflower or other Compositae species rather than com, whereas the northern species was equally susceptible to aldrin on sunflower or goldenrod (129). The more frequent consumption of Compositae pollen and floral tissues by the northern over the more corn-specializing western species could, over many generations, have led to the loss of aldrin resistance in NOR, which had similarly high resistance as WCR prior to cancellation of the cyclodienes for rootworm control (130). The terpenoid-rich flowers of the Compositae may provide the responsible chemistry that results in increased susceptibility to the chlorinated cyclodienes. These insecticides are believed to act via their epoxides at the same GABA-regulated chloride ionophore site as picrotoxinin (121. 122). Our studies with W C R indicate a low crossresistance between this plant neuroexcitant and the cyclodienes, but the resistance ratio between species for picrotoxinin (4 times) is two orders of magnitude less than that observed for aldrin (Table V ) and does not argue solely for an insensitive G A B A site in mediating cyclodiene resistance. These rootworm populations are equally susceptible to the acetylcholinesterase inhibitors (Table V). Neurotoxic antifeedants from Compositae should provide important leads into strategies that ameliorate the control of the Diabrotica complex. Phytochemicals with combined effects that result in loss of insecticide resistance, reduced feeding, decreased life span, and neurotoxicity in rootworms may be a practical avenue to low chemical input strategies for com production. Also, phytochemical antagonism of cyclodiene resistance may have important consequences to future control of com rootworm by insecticides such as avermectins and pyrethroids (e.g. tefluthrin) which, certainly in the former case (121) and at least secondarily in the latter case (122), act on the G A B A gated-chloride ionophore complex.

Table V . Susceptibility of Adult C o m Rootworms in Central P A to Neurotoxicants Topical L D 5 0 (^g/g insect) Rootworm species

G A B A - A chloride channel ligands Aldrin

Western Northern

1980 6.0

Acetylcholinesterase inhibitors

0

Picrotoxinin Avermectinb Carbofuran

111 26.2

a

Terbufos

Isofenphos

58

1.16

2.91

3.39

24

1.05

2.78

4.58

a

5 0 % mortality determinations at 24 hr by probit analysis. bEstimated by injection; for picrotoxinin (128). a 2 hr prior topical treatment with 5 jig/insect of the cytochrome P450 inhibitor piperonyl butoxide was used.

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289

Acknowledgments This work was made possible through the support of the U.S.D.A.(CRGO 89-37263-4567 and NEPIAP 88-34050-3361) and the Pennsylvania Agricultural Experiment Statioa Literature Cited 1. 2.

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3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

McKey, D. In Herbivores, Their Interaction with Secondary Plant Metabolites; Rosenthal, G. A.; Janzen, D. H., Eds.; Academic: New York, 1979; pp 55-133. Bazzaz, F. A.; Chiariello, N. R.; Coley, P. D.; Pitelka, L. F. BioScience 1987, 37, 58-67. Barbier, M. Prog. Phytochem. 1971, 2, 1-34. Stanley, R. G.; Linskens, H. F. Pollen:Biology,Biochemistry,Management; Springer: New York, 1974. Baker, H.G.; Baker, I. In The Biology ofNectaries;Bentley, B.; Elias, T., Eds.; Columbia University Press: New York, 1983; pp 126-152. Thompson, W. R.; Meinwald, J.; Aneshansley, D.; Eisner, T. Science 1972, 177, 528-530 Harborne, J. B.; Smith, D. M. Biochem. Syst. Ecol. 1978, 6, 287-291. Whitten, W. M.; Williams, N. H.; Armbruster, W. S.; Battiste, M. A.; Strekowski, L; Lindquist, N. Syst. Bot. 1986, 11, 222-228. Stipanovic, R. D.; Bell, A. A.; Lukefahr, M. In Host Plant Resistance toPests;Hedin, P.A., Ed.; ACS Symposium Series No. 62; American Chemical Society: Washington, DC, 1977; pp 197-214. Mabry, T. J.; Gill, J. E. InHerbivores,Their Interaction with Secondary Plant Metabolites; Rosenthal, G. A.; Janzen, D. H., Eds.; Academic: New York, 1979; pp 501-537. Kubo, I.; Nakanishi, K. In Advances in PesticideScience,Part 2; Geissbuhler, H.; Brooks, G. T.; Kearney, P. C., Eds.; Pergamon: New York, 1979, pp 284-294. Koul, O. IndianRev.Life Sci. 1982, 2, 97-125. Brattsten, L. B. In Plant Resistance toInsects;Hedin, P.A., Ed.; ACS Symposium Series No. 208; American Chemical Society: Washington, DC, 1983; pp 173-195. Rodriguez, E. In Bioregulators for PestControl;Hedin, P.A., Ed.; ACS Symposium Series No. 276; American Chemical Society: Washington, DC, 1985; pp 447-453. van Beek, T. A.; de Groot, A. Recl. Trav. Chim. Pays-Bas 1986,105,513-527. Reese, J.C.;Holyoke, C. W., Jr. In Handbook of NaturalPesticides,Vol. 3B; Morgan, E. D.; Mandava, N. B., Eds.; CRC Press: Boca Raton, 1987; pp 21-66. Grundy, D. L; Still, C.C. Pestic. Biochem. Physiol. 1985, 23, 383-388. Bowers, M. D. In Chemical Mediation ofCoevolution;Spencer, K.C.,Ed.; Academic: New York, 1988; pp 133-165. Rodriguez, E.; Towers, G. H. N.; Mitchell, J.C. Phytochemistry 1976, 15, 15731580. Burnett, W.C.,Jr.; Jones, S. B., Jr.; Mabry, T. J. In Biochemical Aspects of Plant and Animal Coevolution; Harborne, J.B., Ed.; Academic: London, 1978, pp 233257. Doskotch, R. W.; Odell, T. M.; Girard, L. In TheGypsyMoth: Research Toward Integrated PestManagement;Doane, C.C.;McManus, M. L., Eds.; U.S. Dept. Agric. Tech. Bull. No. 1584; Washington, DC, 1981; pp 657-666. Ivie, G. W.; Witzel, D. A. In Handbook of Natural Toxins, Vol. 1; Keeler, R. F.; Tu, A. T., Eds.; Marcel: New York, 1982; pp 543-584. Picman, A. K. Biochem. Syst. Ecol. 1986,14,255-281. Cooper-Driver, G. A.; Le Quesne, P. W. In Allelochemicals: Role in Agriculture and Forestry; Waller, G. R., Ed.; ACS Symposium Series No. 330; American Chemical Society: Washington, DC, 1987; pp 534-550. Dreyer, D. L. In Chemistry and Chemical Taxonomy of theRutales;Waterman, P. G.; Grundon, M. F., Eds.; Academic: New York, 1983; pp 215-245. Bentley, M. D.; Rajab, M. S.; Alford, A. R.; Mendel, M. J.; Hassanali, A. Entomol. Exp. Appl. 1988, 49, 189-193.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

290

NATURALLY OCCURRING PEST BIOREGULATORS

27.

Elliger, C. A.; Waiss, A. C., Jr. In Insecticides of Plant Origin; Amason, J. T.; Philogene, B. J. R.; Morand, P., Eds.; ACS Symposium Series No. 387; American Chemical Society: Washington, DC, 1989; pp 188-205. Yamasaki, R. B.; Klocke, J. A. J. Agric. Food Chem. 1987, 35, 467-471. Jacobson, M., Ed. Focus on PhytochemicalPesticides,Vol. 1. The Neen Tree; CRC: Boca Raton, 1989; 178pp. Saxena, R. C. In Insecticides of Plant Origin; Amason, J. T.; Philogene, B. J. R.; Morand, P., Eds.; ACS Symposium Series No. 387; American Chemical Society: Washington, DC, 1989; pp 110-135. Eisner, T.; Kluge, A. F.; Ikeda, M. I.; Meinwald, Y. C.; Meinwald, J. J. Insect Physiol. 1971, 17, 245-250. Prestwich, G. D. Annu. Rev. Entomol. 1984, 29, 201-232. Bowers, W. S. InBiotechnology,Biological Pesticides and Novel Plant-Pest Resistance for Insect PestManagement;Roberts, D. W.; Granados, R. R., Eds.; Cornell University: Ithaca, 1988; pp 123-131. Gibson, R. W.; Pickett, J. A. Nature 1983, 302, 608-609. Ave, D. A.; Gregory, P., Tingey, W. M. Entomol. Exp. Appl. 1987, 44, 131-138. Stipanovic, R. D. In Plant Resistance toInsects;Hedin, P.A., Ed.; ACS Symposium Series No. 208; American Chemical Society: Washington, DC, 1983; pp 69-100. Stephenson, A. G. J. Chem. Ecol. 1982, 8, 1025-1034. Kelsey, R. G. In Biology and Chemistry of Plant Trichomes; Rodriguez, E.; Healey, P. L.; Mehta, I., Eds.; Plenum: New York, 1984; pp 187-241. Head, S. W. InPyrethrum,The NaturalInsecticide;Casida, J. E., Ed.; Academic: New York, 1973;. pp 25-53. Casida, J.E. Environ. Health Persp. 1980, 34, 189-202. Rogers, C. E. In Biology and Breeding for Resistance to Arthropods and Pathogens in AgriculturalPlants;Harris, M. K., Ed.; Texas Agric. Expt. Stn. Misc. Publ. 1451, 1980; pp 359-389. Gershenzon, J.; Rossiter, M.; Mabry, T. J.; Rogers, C. E.; Blust, M. H.; Hopkins, T. L. In Bioregulators for PestControl;Hedin, P.A., Ed.; ACS Symposium Series No. 276; American Chemical Society: Washington, DC, 1985; pp 433-446. Rossiter, M.; Gershenzon, J.; Mabry, T. J. J. Chem. Ecol. 1986, 12, 1505-1521. Rogers, C. E.; Gershenzon, J.; Ohno, N.; Mabry, T. J.; Stipanovic, R. D.; Kreitner, G. L. Environ. Entomol. 1987, 16, 586-592. Elliger, C. A.; Zinkel, D. F.; Chan, B. G.; Waiss, A. C., Jr. Experientia 1976, 32; 1364-1366. Metcalf, R. L.; Metcalf, R. A.; Rhodes, A. M. Proc. Natl. Acad. Sci., U.S.A. 1980, 77, 3769-3772. Metcalf. R. L. J. Chem. Ecol. 1986, 12, 1109-1124. Andersen, J. F.; Metcalf, R. L. J. Chem. Ecol. 1987, 13, 681-699. Nielsen, J. K. Entomol. Exp. Appl. 1978, 24, 41-54. Jermy, T.; Butt, B. A.; McDonough, L.; Dreyer, D. L.; Rose, A. F. Insect Sci. Appl. 1981, 1, 237-242. Reed, D. K.; Warthen, J. D., Jr.; Uebel, E. C.; Reed, G. L. J. Econ. Entomol. 1982, 75, 1109-1113. Wakabayashi, N.; Wu, W. J.; Waters, R. M.; Redfern, R. E.; Mills, G. D., Jr.; DeMilo, A. B.; Lusby, W. R.; Andrzejewski, D. J. Nat. Prod. 1988, 51, 537-542. Yamasaki, R. B.; Klocke, J. A. J. Agric. Food Chem. 1989, 37, 1118-1124. Wisdom, C. S.; Rodriguez, E. Biochem. Syst. Ecol. 1983, 11, 345-352. Carter, C. D.; Gianfagna, T. J.; Sacalis, J. N. J. Agric. Food Chem. 1989, 37, 1425-1428. Nawrot J.; Harmatha.J.;Novotny,L. Biochem. Syst. Ecol. 1984, 12, 99-101. Rose, A. F. Phytochemistry 1980, 19, 2689-2693. Le Quesne, P. W.; Cooper-Driver, G. A.; Villani, M.; Do, M. N. In New Trends in Natural Products Chemistry 1986; Rahman, A-U.; Le Quesne, P. W., Eds.; Elsevier: Amsterdam, 1986; pp 271-282. Mitchell, B. K. J. Insect Phvsiol. 1988, 34, 213-225. Pasteels, J. M.; Rowell-Rahier, M.; Braekman, J.-C.; Daloze, D. Biochem. Syst. Ecol. 1984, 12, 395-406.

28. 29. 30. 31.

Downloaded by PENNSYLVANIA STATE UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch018

32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

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18.

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61. 62. 63.

291

Bernays, E. A.; Chapman, R. F. Ecol. Entomol. 1977, 2, 1-18. Wada, K.; Munakata, K. Agric. Biol. Chem. 1971, 35, 115-118. Nawrot, J.; Bloszyk, E.; Grabarczyk, H.; Drozdz, B. Prace Nauk. Inst. Ochr. Ros. 1982, 24, 27-36. 64. Wisdom, C. S.; Smiley, J. T.; Rodriguez, E. J. Econ. Entomol. 1983, 76, 993-998. 65. Yano, K. J. Agric. Food Chem. 1987, 35, 889-891. 66. Blust, M. H.; Hopkins, T. L. Entomol. Exp. Appl. 1987,45,37-46. 67. Nawrot, J.; Bloszyk, E.; Harmatha, J.; Novotny, L. Z. Ang. Ent. 1984, 98, 394-398. 68. Rees, S. B.; Harborne, J. B. Phytochemistry 1985, 24, 2225-2231. 69. Nakajima, S.; Kawazu, K. Heterocycles 1978, 10, 117-121. 70. Nakajima, S.; Kawazu, K. Agric. Biol. Chem. 1980, 44, 2893-2899. 71. Harmatha, J.; Nawrot, J. Biochem. Syst. Ecol. 1984, 12, 95-98. 72. Okunade, A. L.; Wiemer, D. F. Phytochemistry 1985, 24, 1199-1201. 73. Bordoloi, M. J.; Shukla, V. S.; Sharma, R. P. Tetrahedron Lett. 1985, 26, 509-510. 74. McGovran, E. R.; Mayer, E. L. U. S. Dept. Agric. Bur. Entomol. Plant Quarantine, Entomol. Tech. 1942, E-572. 75. Picman, A. K.;Picman,J. Biochem. Syst. Ecol. 1984, 12, 89-93. 76. Arnason, J. T.; Isman, M. B.; Philogene, B. J. R.; Waddell, T. G. J. Nat. Prod. 1987, 50, 690-695. 77. Isman, M. B.; Brard, N. L.; Nawrot, J.; Harmatha, J. J. Appl. Entomol. 1989, 107, 524-529. 78. Watanabe, K.; Ohno, N.; Yoshioka, H.; Gershenzon, J.; Mabry, T. J. Phytochemistry 1982, 21, 709-713. 79. Picman, A. K.; Elliott, R. H.; Towers, G. H. N. Biochem. Syst. Ecol. 1978, 6, 333-335. 80. Streibl,M.;Nawrot, J.; Herout, V. Biochem. Syst. Ecol. 1983, 11, 381-382. 81. Wiemer, D. F.; Ales, D. C. J. Org. Chem. 1981, 46, 5449-5450. 82. Smith, C. M.; Kester, K. M.; Fischer, N. H. Biochem. Syst. Ecol. 1983, 11, 377380. 83. Hubert, T. D.; Wiemer, D. F. Phytochemistry 1985, 24, 1197-1198. 84. Sharma, R. N.; Joshi, V. N. Biovigyanam 1977, 3, 225. 85. Picman, A.K.;Elliott, R.H.;Towers, G. H. N. Can. J. Zool. 1981, 59, 285-292. 86. Isman, M. B.; Rodriguez, E. Phytochemistry 1983, 22, 2709-2713. 87. Isman, M. B.; Rodriguez, E. Environ. Entomol. 1984, 13, 539-542. 88. Isman, M. B. Pestic. Biochem. Physiol. 1985, 24, 348-354. 89. Pettei, M. J.; Miura, I.; Kubo, I.; Nakanishi, K. Heterocycles 1978, 11, 471-480. 90. Sarma, D. N.; Barua, N.C.;Sharma, R. P. Chem. Ind. 1985, 167-168. 91. Burnett, W. C., Jr.; Jones, S. B., Jr.; Mabry, T. J.; Padolina, W. G. Biochem. Syst. Ecol. 1974, 2, 25-29. 92. Ganjian, I.; Kubo, I.; Fludzinski, P. Phytochemistry 1983, 22, 2525-2526. 93. Kawazu, K.; Nakajima, S.; Ariwa, M. Experientia 1979, 35, 1294-1295. 94. Tahara, T.; Sakuda, Y.; Kodama, M.; Fukazawa, Y.; Ito, S.; Kawazu, K.; Nakajima, S. Tetrahedron Lett. 1980, 21, 1861-1862. 95. Rose, A. F.; Jones, K.C.;Haddon, W. F.; Dreyer, D. L. Phytochemistry 1981, 20, 2249-2253. 96. Herz, W.; Kulanthaivel, P.; Watanabe, K. Phytochemistry 1983, 22, 2021-2025. 97. Wiemer, D. F. Rev. Latinoamer. Ouim. 1985, 16, 98-102. 98. Hedin, P. A.; Waage, S. K In Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and Structure-ActivityRelationships;Cody, V.; Middleton, E., Jr.; Harborne, J. B., Eds.; Alan Liss: New York, 1986; pp 87-100. 99. Duffey, S. S. In Insects and the Plant Surface; Juniper, B.; Southwood, Sir R., Eds.; Edward Arnold: London, 1986; pp 151-172. 100. Harborne, J. B. In Plant Flavonoids in Biology and Medicine II: Biochemical, Cellular, and MedicinalProperties;Cody, V.; Middleton, E., Jr.; Harborne, J. B.; Beretz, A., Eds.; Alan R. Liss: New York, 1988; pp 17-27. 101. Elliger, C. A.; Chan, B.C.;Waiss, A.C.,Jr. Naturwiss. 1980, 67, 358-360. 102. Dreyer, D. L.; Jones, K. C. Phytochemistry 1981, 20, 2489-2493. 103. Mullin, C. A. In Molecular Aspects of Insect-PlantAssociations;Brattsten. L. B.; Ahmad, S., Eds.; Plenum: New York, 1986; pp 175-209.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

292 104. 105. 106. 107. 108.

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109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130.

NATURALLY OCCURRING PEST BIOREGULATORS

Schultz, J. C. In Chemistry and Significance of CondensedTannins;Hemingway, R. W.; Karchesy, J. J., Eds.; Plenum: New York, 1989; pp 417-433. Nielson, J. K. In Biology ofChrysomelidae;Jolivet, P.; Petitpierre, E.; Hsiao, T. H., Eds.; Kluwer Academic: Dordrecht, 1988; pp 25-40. Matsuda, K. In Biology ofChrysomelidae;Jolivet, P.; Petitpierre, E.; Hsiao, T. H., Eds.; Kluwer Academic: Dordrecht, 1988; pp 41-56. Matsuda, K.; Senbo, S. Appl. Ent. Zool. 1986, 21, 411-416. Hedin, P. A.; Miles, L. R.; Thompson, A.C.;Minyard, J. P. J. Agric. Food Chem. 1968, 16, 505-513. Nitao, J. K.; Zangerl, A. R. Ecology 1987, 68, 521-529. Rieseberg, L. H.; Schilling, E. E. Amer. J. Bot. 1985, 72, 999-1004. Harborne, J. B. In Introduction to EcologicalBiochemistry;3rd Ed. Academic: London, 1988; pp 42-81. Siegfried, B. D.; Mullin, C. A. Environ. Entomol. 1990, 19, 474-480. Sando, C. E. J. Biol. Chem. 1925, 64, 71-74. Ohmoto, T.; Udagawa, M.; Yamaguchi, K.ShoyakugakuZasshi 1986, 40, 172-176. Mikolajczak, K. L.; Smith, C. R., Jr.; Wolff, I. A. J. Agric. Food Chem. 1970, 18, 27-32. Batyuk, V. S.; Kovaleva, S. N. Khim. Prir. Soedin. 1985, 566-567. Melek, F. R.; Gage, D. A.; Gershenzon, J.; Mabry, T. J. Phytochemistry 1985, 24, 1537-1539. Spring, O.; Benz, T.; Ilg, M. Phytochemistry 1989, 28, 745-749. Geissman, T. A. Rec. Adv. Phytochem. 1973, 6, 65-95. Herz, W. Israel J. Chem. 1977, 16, 32-44. Matsumura, F.; Tanaka,K.;Ozoe, Y. In Sites of Action for Neurotoxic Pesticides ; Hollingworth, R. M.; Green, M. B., Eds.; ACS Symposium Series No. 356; American Chemical Society: Washington, DC, 1987; pp 44-70. Eldefrawi, M. E.; Abalis, I. M.; Sherby, S. M.; Eldefrawi, A. T. In Neuropharmacology and PesticideAction;Ford, M. G.; Lunt, G. G.; Reay, R. C.; Usherwood, P. N. R., Eds.; Ellis Horwood: Chichester, 1986; pp 154-173. Ma, W.-C. Physiol. Entomol. 1977, 2, 199-207. Schoonhoven, L. M.; Fu-Shun, Y. J. Insect Physiol. 1989, 35, 725-728. Norris, D. M. In Sterol Biosynthesis Inhibitors and Anti-Feeding Compounds; Haug, G.; Hoffmann, H., Eds.; Springer: Berlin, 1986; pp 97-146. Lam, P. Y.-S.; Frazier, J. L. Tetrahedron Lett. 1987, 28, 5477-5480. Ryan, M. F.; Byrne, O. J. Chem. Ecol. 1988, 14, 1965-1975. Siegfried, B. D.; Mullin, C. A. Pestic. Biochem. Physiol. 1990, 36, 135-146. Siegfried, B. D.; Mullin, C. A. Pestic. Biochem. Physiol. 1989, 35, 155-164. Metcalf, R. L. In Pest Resistance toPesticides;Georghiou, G. P.; Saito, T., Eds; Plenum: New York, 1983; pp 703-733.

RECEIVED July 18, 1990

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Chapter 19

Insecticidal Constituents of Azadirachta indica and Melia azedarach (Meliaceae) 1

2

3

S. Mark Lee , James A. Klocke , Mark A. Barnby , R. Bryan and Manuel F. Balandrin

4

Yamasaki ,

5

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National Product Sciences, Inc., University of Utah Research Park, 417 Wakara Way, Salt Lake City, U T 84108

The insecticidal constituents of the Indian neem tree (Azadirachta indica) and the related chinaberry tree (Melia azedarach) were investigated using bioassay-guided fractionation and isolation techniques. Azadirachtin was isolated as the principal insecticidal and antifeedant constituent of A. indica seeds. In addition, 25 volatile compounds were identified as constituents of crushed neem seeds. The major volatile constituent identified, di-n­ -propyl disulfide, was shown to be larvicidal to three species of insects. Furthermore, two new insecticidal compounds,1-cinnamoylmelianoloneand 1-cinnamoyl-3,11-dihydroxymeliacarpin, were isolated from the fruit of M. azedarach. The insecticidal activities of these new compounds, compared with those of azadirachtin and several of its derivatives, suggest structure­ -activity relationships and a mode of action that may be useful in the design of synthetic analogs.

The neem tree. Azadirachta indica A. Juss., is a tropical and subtropical species indigenous to India and Southeast Asia (1) which is now widely distributed in many tropical and subtropical regions of both the Old and New Worlds (2-4). The chinaberry tree, Melia azedarach L , is a native of tropical Asia (§), but is now also widely distributed in drier regions of the southern and western United States (§) (e.g., Texas, Arizona, southeastern

1

Current address: Chemistry Laboratory Services, California Department of Food and Agriculture, 3292 Meadowview Road, Sacramento, C A 95832

2

Current address: Division of Entomology and Parasitology, College of Natural Resources, University of California, Berkeley, C A 94720

3

Current address: Department of Entomology, University of Georgia, Athens, GA 30602

4

Current address: SERES Laboratories, Inc., 3331 Industrial Drive, Santa Rosa, C A 95403

5

Current address: Natural Product Sciences, Inc., University of Utah Research Park, 420 Chipeta Way, Salt Lake City, U T 84108 0097-6156/91Α)449-0293$06.00Λ) © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Nevada, and southwestern Utah). The two species are closely related taxonomically, with both belonging to the same subfamily (MeliokJeae) and tribe (Melieae) in the family Meliaceae (mahogany family) (7-9). Recent phytochemical studies have also illustrated the close similarities which exist between their secondary metabolites, especially with regard to their insecticidal tetranortriterpenoid limonoid constituents (meliacins) (7-11). Both plant species have long been recognized for their medicinal (12-16) and insecticidal properties (11.15.17.18). Indeed, these two species have long been recognized as possessing among the most outstanding antifeedant and insecticidal activities among members of the Meliaceae (17-20). In this chapter, our recent studies on the insecticidal constituents from these two plant species will be presented, concentrating first on the volatile organosulfur compounds recently isolated from neem seeds (21), and then on the azadirachtin-type tetranortriterpenoid limonoids present in both species (22-26). Our recent studies on the insecticidal meliacins of the azadirachtin type suggest structure-activity relationships and a mode of action which may be useful in the design of synthetic analogs. Volatile Organosulfur Compounds of Crushed A. indica Seeds Crushed and/or ripening neem tree fruits and expressed neem seed oil give off a strong alliaceous (garlic-like) odor. This characteristic alliaceous odor has also been reported to be present in the leaves, inner bark, timber, and heartwood of this tree species (1.14.21). Some of the reputed medicinal properties of neem seed oil have been attributed to the sulfurous compounds that it contains. Although the presence of sulfur-containing compounds in neem oil had previously been reported (12.14). no detailed chemical studies of neem volatile organosulfur compounds had been reported prior to our recent study (21). The volatiles from crushed neem seeds were purged with nitrogen, trapped onto Amberlite XAD-4 resin traps at room temperature, recovered and concentrated into diethyl ether using a Kuderna-Danish evaporative concentrator at 30°C, and analyzed by means of capillary gas chromatography/mass spectrometry (GC/MS). For comparative purposes, similar volatile concentrates were prepared from freshly chopped onion bulbs and garlic cloves, and blank controls were simultaneously prepared for each of the three test samples. A total of 25 compounds were identified by GC/MS analysis as constituents of the crushed neem seed volatile concentrate, 22 of which were organosulfur compounds. Most of the sulfur-containing compounds were either aliphatic disulfides (eight in all) or higher polysulfides (nine trisulfides and three tetrasulfides). A number of the di- and polysulfides present in the neem seed volatile concentrate were mixed unsymmetrical aliphatic alky! (methyl, propyl, and butyl) and alkenyl (propenyl) containing derivatives. However, no monosulfides or dimethyl polysulfides (often observed in onion and garlic extracts) were found among the neem seed volatiles, and onion-type lachrymatory factors were also absent. Nevertheless, apart from these exceptions, the neem seed volatile constituents were found to generally closely resemble those of onions (21). Most of the neem seed volatiles identified (19 out of 25) were present in concentrations of less than 1% of the total area detected in the GC/MS total ion chromatogram. Many of these minor components (e.g., isomeric dimethylthiophenes and various polysulfides (i.e., tri- and tetrasulfides)) are probably heat-generated artifacts produced upon GC/MS analysis of thermolabile precursors. The major volatile constituents of crushed neem seeds were identified by means of G C / M S as di-n-propyl disulfide (75.74% of the total area detected), n-propyl-trans-1 -propenyl disulfide (9.67%), and n-propyl^js-1-propenyl disulfide (2.76%) (Figure 1). Together, these three closely eluting compounds accounted for 88.17% of the total area detected (all of the 25 compounds identified accounted together for 98.74% of the total area detected). The corresponding trisulfides (2.19, 4.22, and 2.20%, respectively) were the next most abundant group of compounds detected. However, as previously alluded to above, they probably

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Insecticidal Constituents of A. indica & M . azedarach

n-Propyl-cis-l-propenyl

disulfide

Figure 1. Chemical structures of the principal volatile organosulfur compounds isolated from crushed neem (Azadirachta indica) seeds.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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NATURALLY OCCURRING PEST BIOREGULATORS

represent heat-generated artifacts. Furthermore, the principal neem seed volatiles (i.e., the disulfides) identified by GC/MS are probably artifacts generated from nonvolatile precursor substrates by enzymatic activity when the seeds are crushed, as is the case with onion and garlic tissues (21.27). The neem seed volatiles may be responsible for the reputed insect repellent properties attributed to neem seeds. For example, neem seeds have been reported to repel (generally at a distance, without contact) various types of insects, including book mites, locusts, planthoppers, white ants, cockroaches, moths, mosquitoes, cattle flies, post-harvest insects, and stored-products pests (1.12.14.21). We found that the major volatile component of crushed neem seeds, i.e., di-n-propyl disulfide, exhibited larvicidal properties in bioassays against the larvae of three species of insects, i.e., the yellow fever mosquito (Aedes aegvoti; 5 0 Oethal concentration necessary to kill 50% of the treated insects), 66 ppm), the tobacco budworm (Heliothis virescens: L C ^ , 1000 ppm), and the corn earworm (H. zga; L C ^ , 980 ppm) (21). The most probable chemical-ecological explanation for the presence of biologically active volatile organosulfur compounds in neem seeds is that these compounds, like many other plant secondary metabolites, may play a role in chemical defense mechanisms against herbivorous insects, higher herbivorous predators, and invading microorganisms. Because of their extreme volatility and pungency, these compounds may serve as repellents to attacking insects (27) before they can cause significant injury to neem trees, their leaves, or their seeds. In addition, the neem seed volatiles (and/or their nonvolatile precursors) may be responsible, at least in part, for some of the reputed medicinal properties attributed to neem seeds (21).

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LC

Insecticidal Tetranortrfterpenoids of A. indica and M. azedarach The potent tetranortriterpenoid limonoids of A. indica (neem) and M. azedarach (chinaberry) (i.e., meliacins) are considered to be among the most promising and interesting of the plantderived insecticidal compounds yet discovered (17). Azadirachtin (Figure 2) is considered to be the prototype compound for this class of feeding deterrent (antifeedant) and insecticidal substances (17.18.28-31). Azadirachtin and Derivatives. The potent and specific effects of azadirachtin against a wide variety of insects, including a number of economically important species of insect pests, have warranted its evaluation as both a source of and a model for new commercial insect control agents. The potential of azadirachtin lies in its several advantages, which include its potency, which is comparable to that of the most potent conventional synthetic insecticides. Furthermore, azadirachtin has a high degree of specificity (affecting behavioral, developmental, and biochemical processes peculiar to insects), it is non-mutagenic (in the Ames test) (32.33). it is biodegradable, and it has systemic activity in plants, being translocated throughout the plant following absorption through the leaves Q4) and/or root system (2QJ. Azadirachtin has several effects on susceptible insects, including potent feeding deterrency, growth inhibition, and ecdysis inhibition (disruption of the molting process) (17.34). Several analogs of azadirachtin (both natural and semi-synthetic) have been shown to exhibit activities comparable to those of the parent compound. For example, two semi­ synthetic derivatives of azadirachtin, i.e., 22,23-dihydroazadirachtin and 2\3*,22,23tetrahydroazadirachtin, were prepared and tested as growth inhibitors and toxicants against i t virescens larvae (22.26). and as antifeedants against Spodoptera frugiperda (fall armyworm) Q§). The two hydrogenated derivatives proved to be as active as azadirachtin itself in all of these bioassays (22.26.35). Furthermore, 22,23-dihydroazadirachtin also had activity comparable to that of azadirachtin against Epilachna varivestis (Mexican bean beetle) (20. In addition, other related derivatives, such as the semi-synthetic 3-deacetylazadirachtin,

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

19.

L E E ET A L

Insecticidal Constituents of A. indica & M . azedarach

l-OH

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R 0 3

OH

CH, '

Azadirachtin

Ac

C0 CH

l-Cinnamoyl-3,11-dihydroxymeliacarpin

H

CH

R,0

0

2

3

3

H

Tig Cinn

1-Cinnamoylmelianolone

H

H

Cinn

3-Acetyl-l-cinnamoylmelianolone 3,19-Diacetyl-l-cinnamoylmelianolone 1-Decinnamoylmelianolone

Ac

H

Cinn

Ac

Ac

Cinn

H

H

Cinn C

Tig C II

Figure 2. Stereostructures of some natural and derivatized insecticidal tetranortriterpenoids isolated from Azadirachta indica and Melia azedarach.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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NATURALLY OCCURRING PEST BIOREGULATORS

also have activity comparable to that of azadirachtin as growth inhibitors and toxicants against the larvae of H. virescens (22) and E. varivestis (36). In a recent study (2§), it was found that radioactively labelled (tritiated) 22,23dihydroazadirachtin administered orally to fifth (last) instar larvae of H. virescens was rapidly absorbed through the midgut and into the hemocoel, and that excretion of the compound was slow. Data obtained by thin-layer chromatographic (TLC) analysis indicated that metabolism of the dihydroazadirachtin to a more polar form(s) probably occurred following absorption through the midgut (25). Azadirachtin disrupts the normal growth and development of a variety of insect pests, possibly by functioning as a molting hormone (ecdysteroid) analog (17.25.34.36-38). In several species of insects treated with azadirachtin, ecdysis was prevented possibly by the disruption of the molting hormone titre (17.20.36). Azadirachtin was found to be tightly bound to binding proteins in the insect body, and its action is irreversible, one dose (1 JJQ) being sufficient to disrupt subsequent developmental and/or reproductive stages (25.36). For example, development to the pupal stage was disrupted by such doses (25). Insecticidal Constituents of M. azedarach. Azadirachtin has previously been reported to be a constituent of both A. indica and M. azedarach (9.17.18.39.40). However, recent, more detailed investigations suggest that azadirachtin has never been isolated from or identified in authentic M. azedarach. and that it has so far been shown to occur only in A. indica (7.8.10.11.28.31). We found that methanolic extracts of chinaberry (M. azedarach) fruits from southwestern Utah possessed insecticidal potency comparable or equivalent to that of A indica seed extracts and/or to that of azadirachtin (19.23). However, after a thorough search by means of TLC and high-pressure liquid chromatography (HPLC), we were unable to detect the presence of azadirachtin in these chinaberry extracts. Instead, in an effort to isolate and identify the principal active constituents of M. azedarach. we isolated and characterized two new potent insecticidal tetranortriterpenoid limonoids. The first of these was a novel meliacin termed 1 -cinnamoylmelianolone. It possesses a novel structure (skeletal arrangement) not previously observed among the azadirachtin-like meliacins (23). The second compound was a new derivative of meliacarpin (11.31). i.e., 1-cinnamoyl-3,11dihydroxymeliacarpin (Figure 2). The insecticidal activities of these two new compounds, plus two acetylated derivatives of 1-cinnamoylmelianolone (Figure 2) were compared to those of azadirachtin and several of its derivatives using two species of lepidopterous insects. H. virescens (Table I) and S. frugiperda (Table II). Green chinaberry (M. azedarach) fruits were collected during late September of 1986 from trees growing in Hurricane, Utah. (We have observed that chinaberries allowed to ripen and become yellow tend to ferment, giving off malodorous products) (§). Methanolic extracts of the fresh chinaberry fruits (5.7 kg fresh weight) were prepared, concentrated, and filtered. The aqueous methanolic filtrate was partitioned successively, first with hexane and then with methylene chloride. The methylene chloride partitionfraction was concentrated in vacuo at 40°C yielding an extract weighing 6.6 g which was then subjected to flash column chromatography on silica gel using a step-gradient elution involving n-hexane, ethyl acetate, isopropanol, and methanol. Effluents from this column were monitored by means of thin-layer chromatography (TLC). The fraction containing the compounds of interest (2.5 g) was rechromatographed on a low-pressure column using 60% aqueous methanol as eluting solvent system. Monitoring of collected fractions by TLC revealed the fractions containing the two compounds (meliacins) of interest (yield, 1.38 g). Further purification of this fraction was accomplished by means of droplet counter-current chromatography (DCCC) using a solvent system (mixture) incorporating chloroform-toluene-methanol-water (41), finally affording c&. 200 mg of 1cinnamoylmelianolone and £a. 300 mg of 1-cinnamoyl-3,11-dihydroxymeliacarpin. Final purification of the compounds was accomplished by means of normal- and reversed-phase

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

19.

L E E ET A L

Insecticidal Constituents of A. indica & M . azedarach

Table I. Growth-Inhibitory and LarvickJal Effects of Natural and Derlvatized Tetranortriterpenoids from Azadirachta Indica and Melia azedarach Fed in Artificial Diet to First-lnstar Larvae of Heliothis virescens.

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Test Compound

c,d

Azadirachtin 22,23-Dihydroazadirachtin 2',3',22,23-Tetrahydroazadirachtin 3-Deacetylazadirachtin 1 -Detigloyl-22,23-dihydroazadirachtin Deacetylazadirachtinol d

d

d

d

0

6

1 -Cinnamoylmelianolone 3-Acety1-1 -cinnamoyl­ melianolone 3,19-Diacetyl-1 -cinnamoyl­ melianolone 1 -Cinnamoyl-3,11 -dihydroxymeliacarpin 6

a

(ppm)

(ppm)

0.07

0.80

0.08

0.47

0.08 0.09

0.30 0.37

0.59 0.17

2.40 0.80

0.12

1.50

0.15

1.50

0.12

1.50

0.18

3.50

EC5Q is the effective concentration in ppm of additive necessary to reduce larval growth to 50% of the control values.

b

LC5Q is the lethal concentration in ppm of additive necessary to kill (usually by inhibiting ecdysis, the final stage of the molting process) 50% of the treated insects.

c

Naturally occurring compound isolated from Azadirachta indica.

d

Similar preliminary results were obtained against the corn earworm ( K zea) and the fall armyworm (S. frugiperda) (see Table II).

e

Naturally occurring compound isolated from Melia azedarach.

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NATURALLY OCCURRING PEST BIOREGULATORS

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Table II.

Growth-Inhibitory and LarvlckJal Effects of Natural and Derivatlzed Tetranortriterpenolds from Azadirachta Indica and Melia azedarach Fed in Artificial Diet to First-lnstar Larvae of Spodoptera frugiperda.

Test Compound

Azadirachtin

b

0

d

1 -Cinnamoy1melianolone 3-Aceryl-1 -cinnamoyl­ melianolone 3,19-Dlacetyl-1 -cinnamoyl­ melianolone 1 -Cinnamoyl-3,11 -dihydroxymeliacarpin d

a

(ppm)

LC5o (ppm)

0.08

1.0

0.04

1.3

0.06

2.4

0.04

2.5

0.04

1.6

EC5o is the effective concentration in ppm of additive necessary to reduce larval growth to 50% of the control values.

b

LC5o is the lethal concentration in ppm of additive necessary to kill (usually by inhibiting ecdysis, the final stage of the molting process) 50% of the treated insects.

c

Naturally occurring compound isolated from Azadirachta indica.

d

Naturally occurring compound isolated from Melia azedarach.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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LEEETAL.

Insecticidal Constituents of A. indica & M . azedarach

301

HPLC methods previously described (22.24.26.42). The cinnamoyl ester-bearing compounds were detected by ultraviolet (UV) monitoring at 280 nm. Structure elucidation of the purified compounds was carried out by means of infrared (IR) and UV spectrophotometry, proton and carbon-13 nuclear magnetic resonance (NMR) spectroscopy, and electron impact (EI-) and fast-atom bombardment mass spectrometry (FAB-MS). The structures of the two new isolates and three of their derivatives were established on the basis of spectroscopic data and spectral evidence obtained on comparison with azadirachtin (23). The two new meliacins showed general TLC chromatographic properties similar to those of azadirachtin, including giving characteristic colors typical of azadirachtin after spraying with a vanillin-H S0 -etnanol solution followed by heating. However, unlike azadirachtin, the two new compounds showed the strong UV absorptions (in methanol) at 280 nm typical of an ^-unsaturated, unsubstituted aromatic group, i.e., a trans-cinnamovl group. This identification was corroborated by IR, El- and FAB-MS, and proton and carbon13 NMR spectra (11.23.31). Downloaded by CORNELL UNIV on August 1, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch019

2

4

Structure-Activity Relationships of Azadirachtin and Its Analogs and Derivatives The insecticidal activities of the two new meliacins, two acetylated derivatives of 1 -cinnamoyl­ melianolone, and azadirachtin and several of its analogs and derivatives tested against _K virescens and S. frugiperda are shown in Tables I and II, respectively. These data, together with other findings on the insecticidal activities of closely related compounds (20.22.25.26.35.36.43). suggest that specific ester groups are not required at positions 1 and 3 in the azadirachtin nucleus in order to maintain a high level of insecticidal activity. However, the presence of certain ester groups (e.g., tigloyl, cinnamoyl) at these positions may provide a favorable hydrophilic/lipophilic balance necessary for optimum transport across various membranes and physiological compartments as these molecules make their way to their target sites or receptors. It seems probable that the ester groups at positions 1 and 3 in ring A are hydrolyzed off enzymatically in vivo (e.g., by esterases) to afford the more polar deesterrfied metabolites which are the true ultimate molecular species involved in interaction with the appropriate receptor(s) at the molecular level. A number of metabolic and physiological studies (17.25.34.36) strongly suggest that azadirachtin and related compounds act at hormonal (physiological) concentrations rather than at the more usual pharmacological or toxicological concentration levels (36). Furthermore, it has previously been observed that the effects of azadirachtin on susceptible insects (such as the disturbance of hormonal systems giving rise to ecdysis (molting) inhibition) are similar to those produced by the administration of certain plant-derived ecdysteroid analogs (phytoecdysones) such as ponasterone A (Figure 3) (34.44.45). In addition, recent studies with synthetic ecdysone agonists have produced hormonal disturbances and molting cycle (ecdysis) failures (46.47) similar to those observed upon administration of azadirachtin and/or the phytoecdysones (such as ponasterone A). These observations suggest that azadirachtin and related compounds may be acting as ecdysteroid (molting hormone) analogs, thus interfering with insect development by causing profound hormonal disturbances. In this regard, azadirachtin and related compounds may be acting either as ecdysteroid agonists or antagonists (or as a combination of both at various different receptors), either directly or indirectly (e.g., by negative feedback inhibition of hormone biosynthesis or metabolism, or related mechanisms). A comparison of the chemical structure of the deesterrfied derivative of azadirachtin with that of the insect molting hormone, ecdysterone (Figure 3), shows several structural similarities, especially with regard to the spatial disposition of the hydroxy! groups at C-3 in the A rings and in the oxygenation pattern in the B and D rings and adjacent moieties in both molecules. It is noteworthy that although the A / B ring junction is trans in azadirachtin and related compounds (with the 3-OH in the axial position) and.cjs. in the ecdysteroids (with the 3-OH in the equatorial position) (2§), the hydroxyl groups of both molecules at position

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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NATURALLY OCCURRING PEST BIOREGULATORS

Azadirachtin skeleton, deesterified (1,3-dideacylated)

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Insecticidal Constituents of A. indica & M . azedarach

303

C-3 occupy the same relative spatial relationship with regard to the rest of the structure (skeletal carbon framework) in both types of molecules (Figure 3). Thus, there may be two alternative carbon frameworks which can hold the C-3 hydroxyl oxygens in space in such a way that appropriate receptor interactions can occur. Also noteworthy is the equatorial (or pseudoequatorial) disposition of the oxygen functionalities at C-6 in the B rings and the axial (or pseudoaxial) disposition of the oxygen functionalities at C-14 in the D rings of both molecules. These observations, both with regard to biological (insecticidal) activities and chemical structures, suggest that considerable portions of the azadirachtin structure may be required in order to elicit the hormonal disturbances previously observed. Rembold has suggested a minimum structure required to elicit the desired biological activities in this class of compounds (insecticidal tetranortriterpenoids) (2S). The proposed ecdysteroid-analog mode of action and structure-activity relationships may be useful and important considerations in the design of synthetic analogs of these natural and biorational insecticides. Acknowledgments This work was supported in part by a grant awarded to J.A.K. by the U.S. National Science Foundation (PCM-8314500). We thank Ms. Deborah Camomile for typing this manuscript. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15.

16. 17.

18.

Kunkel, G. Flowering Trees in Subtropical Gardens; Dr. W. Junk: The Hague-BostonLondon, 1978; p 258. Ahmed, S.; Grainge, M. Econ. Bot. 1986,40,201-209. Lewis, W.H.; Elvin-Lewis, M.P.F. Econ. Bot.1983,37,69-70. Ahmed, S.;Bamofleh,S.; Munshi, M. Econ. Bot. 1989,43,35-38. Munz, P.A.; Keck, D.D. A California Flora and Supplement; University of California: Berkeley, 1973; p 999. Duffield, M.R.; Jones, W.D. Plants for DryClimates;H.P. Books: Tucson, 1981; p 118. Taylor, D.A.H. In Chemistry and Chemical Taxonomy of the Rutales; Waterman, P.G.; Grundon, M.F., Eds.; Academic: New York, 1983; pp 353-375. Silva, M.F. das G.F. da; Gottlieb, O.R.; Dreyer,D.L.Biochem. Syst. Ecol.1984,12, 299-310. Silva, M.F. das G.F. da; Gottlieb, O.R. Biochem. Syst. Ecol. 1987,15,85-103. Taylor, D.A.H. Prog. Chem. Org. Nat. Prod. 1984,45,1-102. Kraus, W. In New Trends in Natural Products Chemistry 1986; Atta-ur-Rahman; Le Quesne, P.W., Eds.; Studies in Organic Chemistry, Vol. 26; Elsevier Science: Amsterdam, 1986; pp 237-256. Nadkarni, K.M.; Nadkarni, A.K. Indian Materia Medica, 3rd ed.; Popular Book Depot: Bombay, India, 1954; Vol. 1, pp 776-784. Chopra, R.N.; Nayar, S.L.; Chopra, I.C. Glossary of Indian Medicinal Plants; Council of Scientific and Industrial Research: New Delhi, 1956; pp 31-32, 163-164. Dey, K.L.; Mair, W. The Indigenous Drugs of India, 2nd ed.; Pama Primlane, Chronica Botanica: New Delhi, 1973; pp 186-188. Perry, L.M.; Metzger, J. Medicinal Plants of East and Southeast Asia: Attributed Properties and Uses; Massachusetts Institute of Technology: Cambridge, Massachusetts, 1980; pp 260-262. Kapoor, L.D. Handbook of Ayurvedic Medicinal Plants; CRC: Boca Raton, Florida, 1990; pp 59-60, 225-226. Schmutterer, H. In CRC Handbook of Natural Pesticides, Vol. III, Insect Growth Regulators, Part B; Morgan, E.D.; Mandava, N.B., Eds.; CRC: Boca Raton, Florida, 1987; pp 119-170. Warthen, J.D., Jr.; Morgan, E.D. In CRC Handbook of Natural Pesticides, Vol. VI, Insect Attractants and Repellents; Morgan, E.D.; Mandava, N.B., Eds.; CRC: Boca Raton, Florida, 1990; pp 23-134.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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304 19.

20.

21. 22. 23. 24.

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25. 26. 27. 28. 29. 30. 31. 32.

33. 34. 35.

36.

37. 38. 39. 40.

41. 42. 1986, 43. 44.

45.

46. 47.

Lee, S.M.; Stone, G.A.; Klocke, J.A. 190th American Chemical Society National Meeting, Chicago, Illinois, September 8-13, 1985; American Chemical Society: Washington, DC, 1985; Agrochemicals Division Abstract No. 76. Klocke, J.A. InAllelochemicals:Role in Agriculture and Forestry; Waller, G.R., Ed.; ACS Symposium Series No. 330; American Chemical Society: Washington, DC, 1987; pp 396-415. Balandrin, M.F.; Lee, S.M.; Klocke, J.A. J.Agric.Food Chem. 1988, 36, 1048-1054. Yamasaki, R.B.; Klocke, J.A. J. Agric. Food Chem.1987,35,467-471. Lee, S.M.; Klocke,J.A.;Balandrin, M.F. Tetrahedron Lett. 1987, 28, 3543-3546. Klocke, J.A.; Barnby, M.A.; Yamasaki, R.B.; Lee, S.M.; Balandrin, M.F. Proc. 31st Internat. Congr. Pure Appl. Chem., Section 4, Chemistry and Biotechnology of Biologically Active Substances of Medical and VeterinaryImportance,1987, pp 150169. Barnby, M.A.; Klocke, J.A.; Darlington, M.V.; Yamasaki, R.B. Entomol. Exp. Appl. 1989, 52, 1-6. Barnby, M.A.; Yamasaki, R.B.; Klocke, J.A. J. Econ. Entomol. 1989, 82, 58-63. Block, E. Scient. Am. 1985,252(3),114-119. Taylor, D.A.H. Tetrahedron 1987, 43, 2779-2787. Turner, C.J.; Tempesta, M.S.; Taylor, R.B.; Zagorski, M.G.; Termini, J.S.; Schroeder, D.R.; Nakanishi, K. Tetrahedron1987,43,2789-2803. Bilton, J.N.; Broughton, H.B.; Jones, P.S.; Ley, S.V.; Lidert, Z.; Morgan, E.D.; Rzepa, H.S.; Sheppard, R.N.; Slawin, A.M.Z.; Williams, D.J. Tetrahedron 1987, 43, 2805-2815. Kraus, W.; Bokel, M.; Bruhn, A.; Cramer, R.; Klaiber, I.; Klenk,A.;Nagl, G.; Pöhnl, H.; Sadlo, H.; Vogler, B. Tetrahedron 1987, 43 2817-2830. Jacobson, M. In Natural Pesticides from the Neem Tree (Azadirachta indica A. Juss.); Schmutterer, H.; Ascher, K.R.S.; Rembold, H., Eds.; German Agency for Technical Cooperation: Eschborn, Germany, 1980; pp 33-42. Jongen, W.M.F.; Koeman, J.H. Environ. Mutagen.1983,5,687-694. Kubo, I.; Klocke, J.A. Agric. Biol. Chem. 1982, 46, 1951-1953. Klocke, J.A.; Balandrin, M.F.; Barnby, M.A.; Yamasaki, R.B. In Insecticides of Plant Origin; Arnason, J.T.; Philogène, B.J.R.; Morand, P., Eds.; ACS Symposium Series No. 387; American Chemical Society: Washington, DC, 1989; pp 136-149. Rembold, H. In Insecticides of Plant Origin; Arnason, J.T.; Philogène, B.J.R.; Morand, P., Eds.; ACS Symposium Series No. 387; American Chemical Society: Washington, DC, 1989; pp 150-163. Ruscoe, C.N.E. Nature New Biol. 1972, 236, 159-160. Leuschner, K. Naturwissenschaften1972,59,217-218. Morgan, E.D.; Thornton, M.D.Phytochemistry1973, 12, 391-392. Morgan, E.D.; Wilson, I.D. In CRC Handbook of Natural Pesticides:Methods,Vol. II, Isolation and Identification; Mandava, N.B., Ed.; CRC: Boca Raton, Florida, 1985; pp 3-81. Lee, S.M.; Klocke, J.A. J. Liq.Chromatogr.1987,10,1151-1163. Yamasaki, R.B.; Klocke, J.A.; Lee, S.M.; Stone, G.A.; Darlington, M.V. J. Chromatogr. 356, 220-226. Kubo, I.; Matsumoto, A.; Matsumoto, T.; Klocke, J.A. Tetrahedron 1986, 42 489-496. Kubo, I.; Klocke, J.A. In Plant Resistance to Insects; Hedin, P . A ., Ed.; ACS Symposium Series No 208; American Chemical Society: Washington, DC, 1983; pp 329-346. Kubo, I.; Klocke, J.A. In Natural Resistance of Plants to Pests: Roles of Allelochemicals; Green, M.B.; Hedin, P.A., Eds.; ACS Symposium Series No. 296; American Chemical Society: Washington, DC, 1986; pp 206-218. Wing, K.D. Science 1988, 241, 467-469. Wing, K.D.; Slawecki, R.A.; Carlson, G.R. Science 1988, 241, 470-472.

RECEIVED September 10, 1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 20

Toxicity and Neurotoxic Effects of Monoterpenoids In Insects and Earthworms 1

Joel R. Coats, Laura L . Karr , and Charles D . Drewes Department of Entomology and Department of Zoology, Iowa State

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University, Ames, IA

50011

The i n s e c t i c i d a l a c t i v i t y of several monoterpenoids from e s s e n t i a l o i l s was evaluated against insect pests. T o x i c i t y t e s t s i l l u s t r a t e d the b i o a c t i v i t y of d-limonene, α-terpineol, ß-myrcene, l i n a l o o l , and pulegone against i n s e c t s , including the house f l y , the German cockroach, the r i c e weevil, and the western corn rootworm. Bioassays were conducted to assess t h e i r t o x i c i t y v i a t o p i c a l a p p l i c a t i o n , fumigation, ingestion, and o v i c i d a l exposures. Growth, reproduction and repellency were also evaluated i n the German cockroach. Non-invasive e l e c t r o p h y s i o l o g i c a l recordings were used with an earthworm to investigate neurotoxic e f f e c t s of the monoterpenoids. Relevant monoterpenoid bioassay r e s u l t s i n the l i t e r a t u r e are also discussed.

Many e s s e n t i a l o i l s from p l a n t s p o s s e s s b i o l o g i c a l a c t i v i t y a g a i n s t p e s t s t h a t c o u l d be h a r m f u l t o t h e p l a n t . Some e x h i b i t a c u t e t o x i c i t y , w h i l e o t h e r s demonstrate r e p e l l e n t , a n t i f e e d a n t , o r a n t i o v i p o s i t i o n e f f e c t s o r i n h i b i t i o n o f growth, development o r r e p r o d u c t i o n ( 1 ) . Many o f t h e f r a g r a n t v o l a t i l e o i l s c o n t a i n t e n carbon hydrocarbons, o r t h e i r r e l a t e d a l c o h o l s , ketones, aldehydes, c a r b o x y l i c a c i d s , and o x i d e s , and a r e termed m o n o t e r p e n o i d s . Most are c o n s i d e r e d secondary p l a n t chemicals, w i t h l i t t l e d i r e c t m e t a b o l i c importance, b u t w i t h c o n s i d e r a b l e c o e v o l u t i o n a r y significance ( 2 ) . P l a n t - i n s e c t i n t e r a c t i o n s have been s t u d i e d f o r many y e a r s , b u t a b e t t e r u n d e r s t a n d i n g o f t h e s e complex c o a d a p t i v e r e l a t i o n s h i p s could provide a basis for using plant-derived c h e m i c a l s i n b i o r a t i o n a l approaches f o r b e t t e r management o f p e s t organisms ( 3 ) . B o t a n i c a l i n s e c t i c i d e s such a s p y r e t h r i n s and r o t e n o n e have p r o v e n t o be b o t h s a f e and e f f e c t i v e i n c o n t r o l l i n g insect pests. An improved knowledge o f t h e monoterpenoids (as w e l l as s e s q u i t e r p e n e s , d i t e r p e n e s , t r i t e r p e n e s , t e t r a t e r p e n e s ) and t h e i r e f f e c t s on i n s e c t s c o n t r i b u t e s t o u n r a v e l i n g t h e i n t r i c a t e i n t e r a c t i o n s t h a t have shaped t h e c o e v o l u t i o n o f i n s e c t s and plants. I t a l s o provides leads f o r p o s s i b l e u t i l i t y o f these safe, d e g r a d a b l e compounds i n modern p e s t c o n t r o l , and, a s more advanced g e n e t i c engineering c a p a b i l i t i e s develop, t h e p o t e n t i a l f o r

Current address: DowElanco, Walnut Creek, C A 94598 O097-6156/91A)449-O305$06.00/0 © 1991 American Chemical Society

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

NATURALLY OCCURRING PEST

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e x p l o i t i n g t h e i r e f f i c a c y by t r a n s f e r r i n g genes t o d i f f e r e n t c r o p s o r by s e l e c t i n g f o r t h e p r o t e c t i v e c h e m i c a l s i n b r e e d i n g programs. F i g u r e 1 shows t h e s t r u c t u r e s o f 2 c y c l i c and 2 a c y c l i c monoterpenoids. Knowledge o f t h e spectrum o f i n s e c t i c i d a l a c t i v i t y i s l i m i t e d f o r most o f t h e t e r p e n o i d s . The r e s u l t s o f d-limonene t r i a l s a g a i n s t a wide range o f i n s e c t groups i n d i c a t e t h a t t h i s i m p o r t a n t c o n s t i t u e n t o f c i t r u s o i l i s t o x i c t o some l i f e s t a g e s o f some s p e c i e s v i a some r o u t e s o f e x p o s u r e (4,5,6,7). I t s u t i l i t y as a b r o a d - s p e c t r u m i n s e c t i c i d e , however, does not seem f e a s i b l e . Spectrum o f r e p e l l e n t a c t i v i t y has been e v a l u a t e d f o r s e v e r a l t y p e s o f t e r p e n e s , and r e p r o d u c t i v e e f f e c t s have been d e s c r i b e d f o r some chemicals (1,8). Mechanisms o f a c u t e t o x i c i t y have not been e l u c i d a t e d f o r t h e m o n o t e r p e n o i d s , but t h e o n s e t o f symptoms i s u s u a l l y r a p i d , m a n i f e s t e d as a g i t a t i o n , h y p e r a c t i v i t y , and q u i c k knockdown (4,5.) • Our i n v e s t i g a t i o n s have a l s o i n c l u d e d e l e c t r o p h y s i o l o g i c a l s t u d i e s o f t h e t o x i c e f f e c t s o f m o n o t e r p e n o i d s on n e r v e s . Methods T o p i c a l a p p l i c a t i o n s t o house f l i e s (Musca domeatica) and German c o c k r o a c h e s (Blattella germanica), f u m i g a t i o n o f German c o c k r o a c h e s and r i c e w e e v i l s {Sitophilus oryzae) and r e p e l l e n c y t o German c o c k r o a c h e s were c o n d u c t e d by methods d e s c r i b e d p r e v i o u s l y ( 4 ) . The t o x i c i t y by i n g e s t i o n and e f f e c t s on growth and r e p r o d u c t i o n were e v a l u a t e d by i n c o r p o r a t i o n o f t h e c h e m i c a l s i n t o t h e ground c a t chow d i e t o f t h e German c o c k r o a c h e s ( 9 ) . L a v i c i d a l and o v i c i d a l a c t i v i t y a g a i n s t t h e w e s t e r n c o r n rootworm (Diabrotica vergifera vergifera) were t e s t e d i n p e t r i d i s h e s o f s o i l and on moist b l o t t e r paper, r e s p e c t i v e l y (4). R e p e l l e n c y o f t e r p e n o i d s was e v a l u a t e d w i t h German c o c k r o a c h e s i n c h o i c e t e s t s , u s i n g p a i r s o f p l a s t i c boxes (9 X 8.5 X 2cm) c o n n e c t e d by p l a s t i c t u b i n g . T r e a t e d f i l t e r p a p e r was p l a c e d i n one box, and a c o n t r o l (acetone t r e a t e d ) f i l t e r p a p e r was put i n t h e o t h e r box ( 4 ) . Hedgeapple, bay l e a v e s , and s p e a r m i n t chewing gum were t e s t e d u s i n g t h e weight o f p r o d u c t s ( i n ug) p e r u n i t o f volume o f t h e box ( i n cnr) f o r d e t e r m i n a t i o n o f e x p o s u r e c o n c e n t r a t i o n s (1 /jg/cnr = 1 ppm). N e u r o t o x i c i t y was a s s e s s e d i n t h e earthworm Eisenia fetida using non-invasive e l e c t r o p h y s i o l o g i c a l techniques described p r e v i o u s l y (10). The earthworms were exposed i n a v a p o r / c o n t a c t method i n which a s m a l l volume o f t e r p e n o i d was d e l i v e r e d o n t o a m o i s t f i l t e r p a p e r i n a v i a l which was t h e n c l o s e d t i g h t l y ( 1 1 ) . P e r i o d i c a l l y , worms were p l a c e d on an e t c h e d c i r c u i t b o a r d r e c o r d i n g g r i d f o r e l e c t r o p h y s i o l o g i c a l t e s t i n g , and g i a n t f i b e r a c t i v i t y a s s o c i a t e d w i t h t h e e s c a p e r e s p o n s e was m o n i t o r e d (12.). The method has been u s e d p r e v i o u s l y t o examine s u b l e t h a l e f f e c t s o f p e s t i c i d e s on earthworm n e u r a l a c t i v i t y ( 1 3 ) . B i o a s s a y r e s p o n s e s were c a l c u l a t e d as L D ' s , E D ' s o r E T ' s u s i n g t h e trimmed Spearman-Karber method ( 1 4 ) . Duncan's m u l t i p l e r a n g e t e s t and a n a l y s i s o f v a r i a n c e were used f o r t h e r e p e l l e n c y t r i a l s , and t h e p a i r e d comparison t - t e s t was used t o a n a l y z e t h e food preference experiments. 50

Results

and

50

5 0

Discussion

I. A c u t e T o x i c i t y . The u t i l i t y o f an i n s e c t i c i d e has o f t e n been judged by i t s immediate and a c u t e a c t i o n s on p e s t s p e c i e s o f insects. T e r p e n o i d s can e f f e c t t o x i c i t y symptoms v e r y r a p i d l y v i a c o n t a c t o r v a p o r e x p o s u r e s , i n c l u d i n g h y p e r a c t i v i t y and t r e m o r s . T h e i r degree of potency i s s i g n i f i c a n t l y l e s s than c o n v e n t i o n a l s y n t h e t i c o r g a n i c i n s e c t i c i d e s , o f t e n by o r d e r s o f magnitude. However, t h e i r a c t i o n s can be v e r y e f f e c t i v e under c i r c u m s t a n c e s t h a t a l l o w b r i e f h i g h - c o n c e n t r a t i o n uses of g e n e r a l l y s a f e

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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20.

COATS E T A L .

Toxicity and Neurotoxic Effects of Monoterpenoids

pulegone

Fig.

1 - Structures of 4

monoterpenoids

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

307

308

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PEST

BIOREGULATORS

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c h e m i c a l s ( e . g . , g r e e n h o u s e s , a n i m a l shampoos and d i p s , fumigations). R e s e a r c h has been l i m i t e d t o a few i n s e c t s p e c i e s and a few terpenoids, but the r e s u l t s of the t o p i c a l treatments, fumigations, and r e p e l l e n c y s t u d i e s i n d i c a t e t h a t t h e m o n o t e r p e n o i d s c a n e x e r t s u b s t a n t i a l t o x i c i t y a l o n e , o r w i t h a s y n e r g i s t , and d e m o n s t r a t e c o n s i d e r a b l e r e p e l l e n t a c t i v i t y as w e l l . V e r y l i t t l e i s known about t h e i r mode o f a c t i o n . T o p i c a l Exposure. D o s i n g o f female house f l y f e m a l e s w i t h f i v e m o n o t e r p e n o i d s y i e l d e d t o x i c e f f e c t s when a p p l i e d a l o n e a t h i g h doses. d-Limonene was t h e most a c t i v e o f t h e f i v e ( T a b l e I ) . Use o f t h e s y n e r g i s t p i p e r o n y l b u t o x i d e enhanced t h e a c t i v i t y o f dlimonene, p u l e g o n e , and l i n a l o o l c o n s i d e r a b l y , by 17, 21, and >14 f o l d , r e s p e c t i v e l y . These r e s u l t s i n d i c a t e t h a t t h o s e t h r e e t e r p e n o i d s ' i n s e c t i c i d a l a c t i v i t y i s e x p r e s s e d more f u l l y when t h e o x i d a t i v e d e t o x i f i c a t i o n process i s i n h i b i t e d . I t i s not s u r p r i s i n g t h a t f l i e s can d e t o x i f y them r a p i d l y , c o n s i d e r i n g t h e r e l a t i v e l y simple hydrocarbon s t r u c t u r e s of the monoterpenoids. In t h e male German c o c k r o a c h t r i a l s , p u l e g o n e was shown t o be t h e most p o t e n t compound t e s t e d , b u t i t r e q u i r e d 260 ug/insect at t h e median l e t h a l dose. d-Limonene and l i n a l o o l a l s o d e m o n s t r a t e d s l i g h t t o x i c i t y ( T a b l e I ) . Myrcene, a - t e r p i n e o l and I - l i m o n e n e (not shown) were t h e l e a s t t o x i c . d-Limonene was s l i g h t l y s y n e r g i z e d , w h i l e l i n a l o o l was n o t .

Table I.

A c u t e t o x i c i t y o f m o n o t e r p e n o i d s t o t h e house f l y , Musca domestica, and t h e German c o c k r o a c h , Blattella germanica, by t o p i c a l a p p l i c a t i o n (24-h m o r t a l i t y d a t a ) , a l o n e o r w i t h p i p e r o n y l b u t o x i d e (p.b.)

Treatment

8

LDc«(/ig/insect) female house t l v mal e German c o c k r o a c h

d-Limonene 1 d-Limonene:5 p.b. Pulegone 1 Pulegone: p.b. Linalool 1 Linalool: p.b. 1 Linalool: p.b. Myrcene a-Terpineol

8

95%

confidence

90(70-130) 5.2(3.7-7.5) 166(131-201) 7.7(5.4-11) >100 32(23-44) 7.2(4.1-12) 360(300-430) 310(260-380)

700(610-810) 300(180-560) 260(230-300) 550(410-730) 610(530-700) >1,580 1,070(680-1,690)

intervals

C o n t a c t t o x i c i t y t r i a l s w i t h d-limonene a g a i n s t a d u l t c a t f l e a s i l l u s t r a t e d e x t r e m e l y f a s t knockdown t i m e and m o r t a l i t y . A s y n e r g i s t i c r a t i o o f 3.2 was o b s e r v e d when p i p e r o n y l b u t o x i d e was a l s o used on t r e a t e d f i l t e r p a p e r s ( 5 ) . S o u t h e r n p i n e b e e t l e s , Dendroctonus frontalis, were a s s a y e d f o r s u s c e p t i b i l i t y t o 16 t e r p e n o i d s from p i n e o l e o r e s i n ( 1 5 ) . The LD^ o f d-limonene was 0.47 / i g / i n s e c t , w h i l e i - l i m o n e n e was s l i g h t l y l e s s t o x i c (0.55 / j g / i n s e c t ) , as was myrcene (0.62 jig/insect). The most t o x i c c h e m i c a l t e s t e d was limonene d i o x i d e (0.24 / i g / i n s e c t ) , i n d i c a t i n g t h a t o x i d a t i o n o f limonene may be an a c t i v a t i o n process (15). D o s e - m o r t a l i t y s t u d i e s w i t h c o n s t i t u e n t s o f l y o p h i l i z e d lemon o i l d e m o n s t r a t e d n o t a b l e t o x i c i t y t o t h e a d u l t cowpea w e e v i l , Callosobruchus maculatus. The L D o f t h e o i l was 16.4 / i g / i n s e c t , 0

5 Q

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

20.

COATS E T AL.

Toxicity and

Neurotoxic Effects of Monoterpenoids

w h i l e t h r e e p o t e n t compounds i s o l a t e d from t h e o i l by t h i n - l a y e r chromatography had L D ' s from 4.73 t o 2.66 / i g / i n s e c t ( 1 6 ) . N e m a t i c i d a l a c t i v i t y o f some e s s e n t i a l o i l s and t h e i r major c o n s t i t u e n t s has been r e p o r t e d (17.) • E u g e n o l showed t h e most e f f i c a c y a g a i n s t 3 s p e c i e s o f nematodes, w h i l e l i n a l o o l , g e r a n i o l , and menthol were more e f f e c t i v e a g a i n s t t h e r o o t - k n o t nematode. 50

Fumigation. The m o n o t e r p e n o i d s , as a f a m i l y , a r e v o l a t i l e which makes them p o t e n t i a l l y q u i t e u s e f u l as f u m i g a n t s . Most a r e a l s o p l e a s a n t l y o d i f e r o u s and o f low t o x i c i t y t o mammals, p r o p e r t i e s w h i c h a l s o a r e c o n s i s t e n t w i t h f u m i g a t i o n usages on p r o d u c e , g r a i n , c l o t h i n g , b u i l d i n g s , s h i p s and s o i l . Laboratory t r i a l s against a d u l t r i c e weevils i l l u s t r a t e the r e l a t i v e potencies of f i v e monoterpenoids i n Table I I .

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Table I I .

Treatment

cf-Limonene Pulegone

Linalool Myrcene a-Terpineol

A c u t e t o x i c i t y o f monoterpenoids t o t h e r i c e w e e v i l (Sitophilus oryzae) and t h e German c o c k r o a c h (Blattella germanica) by f u m i g a t i o n (24-h m o r t a l i t y data) LC r i c e weevil

a

5 0

D

(ppm) ' German c o c k r o a c h

0

23(17-31) 9.6(1-113) 9 17(10-30) 6 4.5(0.6-36) 12 >100 >100

19(13-27) 3.1(2.7-3.5)

14 >100 >100

a

95% c o n f i d e n c e i n t e r v a l s ^g/liter b o t h sexes i n c l u d e d (50:50) e x c e p t where n o t e d c

The most t o x i c o f t h e f i v e i n a v a p o r form was p u l e g o n e w i t h an LC o f 3.1 ppm ( m g / l i t e r o f a i r ) . L i n a l o o l and cf-limonene were a l s o e f f e c t i v e , w h i l e myrcene and a - t e r p i n e o l were i n e f f e c t i v e . A g a i n s t German c o c k r o a c h e s , p u l e g o n e was, a g a i n , t h e most e f f i c a c i o u s , f o l l o w e d by l i n a l o o l and limonene ( T a b l e I I ) . Male c o c k r o a c h e s were f o u r t i m e s as s u s c e p t i b l e as t h e f e m a l e s t o pulegone. V a p o r - e x p o s u r e a s s a y s f o r a d u l t c a t f l e a s a l s o showed t h a t cflimonene was e f f e c t i v e a t i n d u c i n g r a p i d knockdown and m o r t a l i t y as a fumigant (5) • The l a r v a e were a l s o r e l a t i v e l y s u s c e p t i b l e t o cflimonene v a p o r s , w h i l e t h e eggs were l e s s s u s c e p t i b l e and pupae were r e l a t i v e l y t o l e r a n t o f t h i s c h e m i c a l . V a p o r s o f monoterpenes from p i n e were e v a l u a t e d f o r b i o a c t i v i t y a g a i n s t t h e Western p i n e b e e t l e , Dendroctonus brevicomis. The f o u r - d a y b i o a s s a y d e t e r m i n e d t h a t limonene was the most t o x i c among t h e f i v e compounds t e s t e d ( 7 ) . 5 0

L a r v i c i d a l and O v i c i d a l A c t i v i t y . Acute t o x i c i t y t e s t i n g of t e r p e n o i d s on immature s t a g e s o f i n s e c t s has been l i m i t e d t o a few studies. L a r v a e ( t h i r d i n s t a r ) o f t h e w e s t e r n c o r n rootworm, Diabrotica vergifera vergifera, were a s s a y e d i n t r e a t e d s o i l . The 48-h L C C Q f o r cf-limonene was 12.2 /ig/g s o i l ( 4 ) , which i s a p p r o x i m a t e l y 1 0 - f o l d l e s s p o t e n t t h a n t h e s t a n d a r d organophosphorus and carbamate compounds c u r r e n t l y used f o r rootworm c o n t r o l ( 1 9 ) . The eggs o f t h a t s p e c i e s were exposed t o t r e a t e d b l o t t e r paper t o assess contact o v i c i d a l a c t i v i t y . The L C ' s a t 28 days f o r cflimonene and l i n a l o o l were 1.8% a c t i v e i n g r e d i e n t ( a . i . ) and 0.26% a . i . , r e s p e c t i v e l y . These 2 monoterpenoids were about 3 o r d e r s o f magnitude l e s s p o t e n t t h a n c h l o r d i m e f o r m ( L C = 0.0006% a . i . ) . 5 0

5

0

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

309

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310

C i t r u s o i l s and s e v e r a l i n d i v i d u a l components o f them were t e s t e d a g a i n s t l a r v a e o f t h e C a r i b b e a n f r u i t f l y , Anaatrepha auapenaa (6_). C i t r a l was t h e most e f f i c a c e o u s monoterpenoid, f o l l o w e d by limonene, t h e n a - p i n e n e and a - t e r p i n e o l . One-hour immersion i n a 40% ( a . i . ) s o l u t i o n o f t h e most p o t e n t compound r e s u l t e d i n 50% m o r t a l i t y d u r i n g t h e l a r v a l development p e r i o d . The eggs o f t h e C a r i b b e a n f r u i t f l y have a l s o been e v a l u a t e d f o r o v i c i d a l e f f e c t s of t e r p e n o i d s ( 6 ) . a - T e r p i n e o l was t h e most e f f e c t i v e o f t h o s e t e s t e d (1% a . i . caused 100% m o r t a l i t y ) , f o l l o w e d by c i t r a l and limonene. a - P i n e n e had no e f f e c t . S t u d i e s on t h e house f l y , Musea domes tica, revealed the considerable acute l a r v i c i d a l a c t i v i t y of s e v e r a l terpenoids, e s p e c i a l l y c a r v a c r o l and cf-limonene (19). S e v e r a l metamorphosis i n h i b i t i o n e f f e c t s were a l s o o b s e r v e d , i n c l u d i n g i n h i b i t i o n o f p u p a l e c d y s i s , u n e c l o s e d pupae, and deformed a d u l t s . Numerous compounds e x h i b i t e d p o t e n c y i n t h e s e d e v e l o p m e n t a l m o r t a l i t i e s : camphene, c a r v a c r o l , c a r v o n e , c i n e o l e , c i t r a l , c i t r o n e l l a l , c i t r o n e l l o l , e u g e n o l , f a r n e s o l , g e r a n i o l , limonene, l i n a l o o l , 13p h e l l a n d r e n e , and a - p i n e n e . Egg h a t c h was a l s o i n h i b i t e d by e x p o s u r e t o t h e t e r p e n o i d s ; t h e most e f f e c t i v e o v i c i d a l compounds were c a r v a c r o l , c i t r o n e l l a l and B - p h e l l a n d r e n e , w i t h no a c u t e l y t o x i c e f f e c t s n o t e d t o t h e embryos, but r a t h e r show i n h i b i t i o n o f development o f t h e embryo and i n a b i l i t y t o e c l o s e from t h e egg (19). The l a r v a e o f t h e c a t f l e a , Ctenocephalidea felia, were s u s c e p t i b l e t o cf-limonene ( L D o f 226 /ig/enr o f f i l t e r p a p e r ) , and some modest s y n e r g i s m was o b s e r v e d when p i p e r o n y l b u t o x i d e was employed ( L C o f 157 uq/cxx?). The v a p o r s were a l s o t o x i c t o t h e f l e a larvae. Pupae were l e s s s u s c e p t i b l e t h a n eggs, l a r v a e , o r adults. Eggs o f t h e c a t f l e a were exposed t o cf-limonene a t 65 ug/ctvr and m o r t a l i t y was 100%. A t e s t o f i t s t o x i c i t y t o them i n a v a p o r chamber showed 60% m o r t a l i t y a t 130 uq/ctt? (5) . I t i s e v i d e n t t h a t some t e r p e n o i d s p o s s e s s e f f i c a c y a g a i n s t i n s e c t eggs, but t h e degree o f p o t e n c y i s modest a t b e s t . I t was r e p o r t e d t h a t l a r v a e o f 3 s p e c i e s o f m o s q u i t o e s were s u s c e p t i b l e t o cf-limonene but no d a t a were p r o v i d e d (5.). 5Q

5 Q

Repellencv. A number o f o t h e r p l a n t - d e r i v e d t e r p e n o i d s have been d e m o n s t r a t e d t o be r e p e l l e n t t o v a r i o u s i n s e c t s . Many compounds i n t h i s c l a s s have been p r o v e n t o be a t t r a c t a n t s t o c e r t a i n i n s e c t s (1). Limonene a t 1 o r 10 mg/box r e p e l l e d t h e c o c k r o a c h e s , t o t h e u n t r e a t e d box, i n s i g n i f i c a n t l y (p

[3]

H0 ' 2°2

+

4

°2 + 2

>

0

>

HO* + "OH + F e

2

+ Fe

I1 [5] + 3

[6]

N o t e , i n E q u a t i o n s 4 - 6 , t h r e e O2"' a r e r e q u i r e d f o r e a c h HO* produced. S u p e r o x i d e i s d e r i v e d by a one e l e c t r o n r e d u c t i o n o f O2 (16). Trace q u a n t i t i e s of t r a n s i t i o n metals are p o t e n t i a l c a n d i d a t e s to a c t as r e d u c t a n t s i n t h i s r e a c t i o n ( i . e . E q u a t i o n 7 ) . 0

2

+ Fe

2 +

>

0 "* 2

+ Fe

3 +

[7]

T h u s , SOD i s n o t e x p e c t e d to p r e v e n t the d e c o m p o s i t i o n o f dHG; the p r o d u c t o f i t s r e a c t i o n w i t h 02"' i s H2O2 w h i c h c a n be a s o u r c e o f HO* ( e . g . E q u a t i o n 6 ) . However, c a t a l a s e would p r e v e n t the d e c o m p o s i t i o n o f dHG s i n c e i t d e s t r o y s H2O2. Baker and G e b i c k i (15) o b s e r v e d a s i m i l a r phenomena when t h e y s t u d i e d the F e n t o n r e a c t i o n by gamma i r r a d i a t e d Fe -EDTA s o l u t i o n s . Catalase c o m p l e t e l y i n h i b i t e d s y n t h e s i s o f HO*, b u t o n l y 43% was i n h i b i t e d w i t h SOD. The f o r m a t i o n o f HO* was a t a maximum a t pH 4 . 8 ; t h i s d e c r e a s e d to 42% a t pH 7 . 4 . S t r o n g r e d u c i n g agents a r e e x p e c t e d to i n t e r c e p t the d e c o m p o s i t i o n o f dHG a t t h r e e p o i n t s : 1) by decomposing H2O2, 2) by r e d u c i n g [dHG]*, and 3) by k e e p i n g t r a n s i t i o n m e t a l s i n t h e i r r e d u c e d s t a t e and thus s l o w i n g the f o r m a t i o n o f O2"* (Equation 7). One might e x p e c t the HO* s c a v e n g e r , b e n z o a t e , a l s o to slow the d e c o m p o s i t i o n because HO* i s c r u c i a l t o i n i t i a t i o n o f the c h a i n r e a c t i o n shown i n F i g u r e 4. However, once HO* a b s t r a c t s a h y d r o g e n atom from dHG, the i n i t i a t e d c h a i n r e a c t i o n w i l l p r o c e e d unimpeded by s i m p l e HO* s c a v e n g e r s . Benzoate i s s p e c i f i c , a c t i n g o n l y t o scavenge HO* (.15) , and n o t o t h e r f r e e r a d i c a l s . Thiourea i s a weak r e d u c i n g agent as compared to g l u t a t h i o n e ( 1 7 ) . Thus, t h i o u r e a i s a b l e to scavenge HO*, b u t a p p a r e n t l y i s a p o o r s c a v e n g e r o f [dHG]*. The weak r e d u c i n g a g e n t , t h i o u r e a , when combined w i t h SOD s i g n i f i c a n t l y r e d u c e s the d e c o m p o s i t i o n o f dHG, b u t the benzoate-SOD c o m b i n a t i o n does n o t . Two r o u t e s e x i s t f o r the f o r m a t i o n o f HO*:

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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23.

STIPANOVIC E T A L .

the l a s t s t e p i n the F e n t o n r e a c t i o n ( i . e . Haber-Weiss r e a c t i o n , Equation 8 (18). 0 "* 2

+ H

+

345

Desoxyhemigossypol, A Cotton Phytoalexin

+ H 0 2

>

2

H 0 2

E q u a t i o n 6)

+ 0

2

and

+ HO*

the

[8]

T h i o u r e a c a n a c t as a weak r e d u c i n g agent t o d e s t r o y H 0 slowly and t o keep t r a c e q u a n t i t i e s o f t r a n s i t i o n m e t a l s s u c h as i r o n i n t h e i r r e d u c e d ( e . g . f e r r o u s ) s t a t e and thus r e d u c e the r a t e o f 0 " ' formation (Equation 7). As o b s e r v e d by Baker and G e b i c k i ( 1 5 ) , the o p t i m i z a t i o n o f E q u a t i o n 6 i s dependent on the r a t e o f formation of 0 ~*. Thus any r e a c t a n t t h a t slows E q u a t i o n 7 w i l l u l t i m a t e l y slow E q u a t i o n 6. SOD i n t e r c e d e s i n E q u a t i o n 8, by d e s t r o y i n g 0 ~* but produces H 0 which i s r e q u i r e d i n E q u a t i o n 6. When a c t i n g i n c o n c e r t , t h i o u r e a slows the f o r m a t i o n o f 0 ~ * and i s a b l e t o r e a c t w i t h the l i m i t e d amount o f H 0 p r o d u c e d by SOD. T h i s sequence o f e v e n t s a g a i n i m p l i c a t e s H 0 as a c r i t i c a l agent o f d e c o m p o s i t i o n . The F e n t o n r e a c t i o n s ( E q u a t i o n s 4-6) and the f o r m a t i o n o f 0 " * by r e d u c t i o n o f 0 ( i . e . E q u a t i o n 7 ) , r e q u i r e the i n t e r v e n t i o n o f a t r a n s i t i o n m e t a l such as f e r r o u s i o n . DTPA i s a s t r o n g enough c h e l a t o r o f f e r r o u s i o n to s i g n i f i c a n t l y d e t e r the f o r m a t i o n o f 0 ~ * ( i . e . E q u a t i o n 7 ) , w h i l e EDTA i s n o t ( 1 9 , 2 0 ) . We f o u n d DTPA s i g n i f i c a n t l y r e d u c e d the r a t e o f d e c o m p o s i t i o n o f dHG and was more e f f e c t i v e t h a n EDTA ( F i g u r e 3 d ) . 2

2

2

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2

2

2

2

2

2

2

2

2

2

2

2

Structure-Activity Relationship. The s i t e and mode o f a c t i o n o f p h y t o a l e x i n s have been i n v e s t i g a t e d . E l e c t r o n and l i g h t m i c r o s c o p y s t r o n g l y i m p l i c a t e t h e i r i n v o l v e m e n t i n d i s r u p t i o n o f the plasmalemma ( 2 1 - 2 3 ) . O t h e r symptoms o f membrane d y s f u n c t i o n i n pathogens t r e a t e d w i t h p h y t o a l e x i n s a r e : l e a k a g e o f e l e c t r o l y t e s and m e t a b o l i t e s ( 1 2 , 2 4 , 2 5 ) , l o s s i n m y c e l i a d r y w e i g h t ( 2 4 , 2 6 , 2 7 ) and i n h i b i t i o n o f oxygen uptake ( 2 4 ) . Free r a d i c a l s are c y t o t o x i c e n t i t i e s t h a t r e a c t w i t h and d e s t r o y c e l l membranes. Apostol et a l . (28) p r o p o s e d t h a t H 0 may be i n v o l v e d i n r e s i s t a n c e o f soybeans to V. dahliae, and Sun e t a l . (29) a l s o i m p l i c a t e d H 0 i n the p h o t o a c t i v a t i o n o f the f o l i a r c o t t o n p h y t o a l e x i n , 2,7-dihydroxycadalene. These o b s e r v a t i o n s s u g g e s t a r o l e f o r f r e e radicals in disease resistance. 2

2

2

2

Our e x p e r i m e n t s on the s t a b i l i t y o f dHG s t r o n g l y s u g g e s t t h a t dHG decomposes by a f r e e r a d i c a l mechanism. We h y p o t h e s i z e t h a t dHG d e r i v e s i t s t o x i c i t y from i t s r e a d y a b i l i t y to form f r e e r a d i c a l s . E x t r a p o l a t i o n s from our o b s e r v a t i o n s have a l l o w e d us t o p r o b e the nature of t h i s t o x i c i t y . We have d e v e l o p e d a c o l o r i m e t r i c method to a s s a y v i a b l e c o n i d i a , w h i c h employs the t e t r a z o l i u m s a l t , MTT ( 3 0 ) . U s i n g t h i s method we measured the t o x i c i t y o f dHG to V. dahliae, and d e t e r m i n e d an LD5Q v a l u e o f 3.5 / i g / m l . The e f f e c t s on the t o x i c i t y o f dHG o f the r e d u c i n g a g e n t s , HO* s c a v e n g e r s , and enzymes examined i n the d e c o m p o s i t i o n s t u d i e s were a s s a y e d by t h i s method. As p r e d i c t e d from the d e c o m p o s i t i o n s t u d i e s , n e i t h e r sodium b e n z o a t e (0.1 mM), t h i o u r e a ( 1 . 0 mM), n o r SOD (900 U/ml) a p p r e c i a b l y a f f e c t s the t o x i c i t y o f dHG ( F i g u r e 6 ) . Sodium b e n z o a t e was a s s a y e d a t 0.1 mM b e c a u s e i t i s t o x i c to V. dahliae at higher c o n c e n t r a t i o n . The e f f e c t s o f the r e d u c i n g a g e n t s g l u t a t h i o n e and a s c o r b i c a c i d , and

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

too

i —

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1

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23.

STIPANOVIC ET AL.

Desoxyhemigossypol, A Cotton Phytoalexin

347

the enzyme c a t a l a s e a l s o a r e shown i n F i g u r e 6. When 1.0 mM a s c o r b i c a c i d o r g l u t a t h i o n e was added t o the growth m e d i a , the t o x i c i t y o f dHG was s i g n i f i c a n t l y r e d u c e d ; c a t a l a s e (1700 U/ml) a l s o reduced i t s t o x i c i t y . The a c t i o n o f a s c o r b i c a c i d , g l u t a t h i o n e and c a t a l a s e i s c o n s i s t e n t w i t h our h y p o t h e s i s . The p h y t o a l e x i n dHG i s r a p i d l y a b s o r b e d by V. dahliae c o n i d i a , 75% o f the dHG i n a 6.5 / i g / m l s o l u t i o n b e i n g a b s o r b e d from the b i o a s s a y media by V. dahliae c o n i d i a (5 X 10 c e l l s / m l ) i n one m i n u t e . Each V. dahliae c e l l would t h e r e f o r e c o n t a i n 3.2 X 10 m o l e c u l e s o f dHG. When t h e s e c e l l s were washed r e p e a t e d l y w i t h m e d i a , -70% o f the dHG was removed i n s i x washes; subsequent washes removed s m a l l e r and s m a l l e r amounts. R a p i d uptake from s o l u t i o n a l s o has been i m p l i c a t e d f o r the phytoalexin, kievetone. Damage to the plasmalemma o f Rhizoctonia solani h y p h a . as shown by e l e c t r o n m i c r o g r a p h s and i n c r e a s e d leakage of C - l a b e l e d m e t a b o l i t e s , o c c u r s w i t h i n 90 minutes a f t e r exposure to k i e v i t o n e (31). We h y p o t h e s i z e dHG i s r a p i d l y a b s o r b e d by V. dahliae conidia and m y c e l i a . An i n i t i a t o r ( e . g . HO*) b e g i n s a c h a i n r e a c t i o n i n which a s e r i e s o f h i g h l y r e a c t i v e , c y t o t o x i c f r e e r a d i c a l s are generated. One o r more o f t h e s e r a d i c a l s r e a c t s w i t h v i t a l p r o c e s s e s i n c e l l membranes ( p r o b a b l y the plasmalemma) l e a d i n g u l t i m a t e l y to d e a t h o f c o n i d i a and m y c e l i a . The HO* does n o t appear to be the t o x i c e n t i t y s i n c e HO* s c a v e n g e r s do n o t d e c r e a s e t o x i c i t y . The d i m i n u t i o n o f t o x i c i t y by r e d u c i n g agents and c a t a l a s e a p p a r e n t l y r e s u l t s from t h e i r ability t o p r e v e n t i n i t i a t i o n o f the f r e e r a d i c a l r e a c t i o n , a n d / o r i n t e r r u p t i o n o f the c h a i n r e a c t i o n p r o c e s s . S t e r o l s do n o t appear to be i n v o l v e d i n the i n t e r a c t i o n o f p h y t o a l e x i n s because s t e r o l i n c o r p o r a t i o n i n the media does n o t p r o t e c t Rhizoctonia solani a g a i n s t the p h y t o a l e x i n s k i e v i t o n e (31) o r p h a s e o l i n ( 2 4 ) . A l i k e l y s i t e of i n t e r a c t i o n i s p o l y u n s a t u r a t e d f a t t y a c i d e s t e r s i n membranes w h i c h r e a d i l y undergo f r e e r a d i c a l a u t o x i d a t i o n s , and the t h r e e d i m e n s i o n a l c o n f o r m a t i o n o f t h i s a u t o x i d a t i o n p r o d u c t i s s i g n i f i c a n t l y d i f f e r e n t from the starting material. Such c o n f o r m a t i o n a l changes a r e e x p e c t e d to d i s r u p t membranes. O x i d a t i o n o f i n e r t c e l l u l a r components such as p r o t e i n s by the c o o x i d a t i o n mechanism p r o p o s e d by P r y o r (32) i s a l s o possible. Peroxy r a d i c a l s (ROO*), and e s p e c i a l l y the a l k o x y r a d i c a l (RO*) which r e s u l t from l i p i d o x i d a t i o n a r e p r o p o s e d as the m e d i a t o r s t h a t r e a c t w i t h t h e s e r a t h e r i n e r t c e l l u l a r components (32) . The r o l e o f a c t i v e oxygen and the r e s u l t i n g f r e e r a d i c a l s from p h y t o a l e x i n s may be i m p o r t a n t a s p e c t s i n the d e f e n s e r e s p o n s e . For example, the p r o d u c t i o n o f H 0 2 (28) and O2"' (33) in r e s p o n s e to a pathogen d e r i v e d e l i c i t o r has been c o n f i r m e d i n soybean c e l l s u s p e n s i o n c u l t u r e s . Tobacco l e a f d i s c i n f e c t e d w i t h t o b a c c o mosaic v i r u s showed a marked i n c r e a s e i n O2"' g e n e r a t i n g activity (34)• The p t e r o c a r p a n s and the e x t e n s i v e r e s e a r c h on t h e i r t o x i c i t y to p l a n t pathogens p r o v i d e an e x c e l l e n t example o f compounds w h i c h have the p o t e n t i a l to r e a d i l y form f r e e r a d i c a l s . VanEtten c a r e f u l l y s t u d i e d the t o x i c i t y o f s i x p t e r o c a r p a n s , t h r e e 6 a , l l a - d e h y d r o p t e r o c a r p a n s and two coumestans to Aphanomyces 2

American Chemical Society Library 1155 16th St., N.w. In Naturally Occurring Pest D.C. Bioregulators; Washington, 20036Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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euteiches and Fusarium solani (3j>). The g e n e r a l s t r u c t u r e s f o r t h e s e i s o f l a v o n o i d s a r e shown i n F i g u r e 7. A l l o f the p t e r o c a r p a n s have an e t h e r e a l b e n z y l i d e n e p r o t o n a t C ^ w h i c h i s part of a dihydrofuran r i n g . This proton should r e a d i l y d i s s o c i a t e t o form a f r e e r a d i c a l . The f r e e r a d i c a l , w h i c h i s an sp c a r b o n , i s p l a n a r w i t h and s t a b i l i z e d b y t h e benzene r i n g , a s i t u a t i o n a n a l o g o u s t o t h a t f o u n d i n dHG*. The d e h y d r o p t e r o c a r p a n s l a c k a p r o t o n a t C - ^ ^ , b u t p r o t o n s a r e a v a i l a b l e a t C g . These p r o t o n s , which a r e not p a r t o f a f u r a n r i n g , are capable o f forming a s t y r y l free r a d i c a l . E x a m i n a t i o n o f D r i e d i n g models shows t h e p y r a n r i n g i s s l i g h t l y p u c k e r e d when Cr i s an sp c a r b o n . Thus t h e s e p r o t o n s a r e e x p e c t e d t o be l e s s r e a c t i v e t h a n t h e p r o t o n a t C - ^ i n the p t e r o c a r p a n s . The coumestans l a c k p r o t o n s a t b o t h C - ^ and C g , and c o n t a i n no c a r b o n s w h i c h would r e a d i l y form f r e e r a d i c a l s . a

a

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a

a

I n g e n e r a l t h e p t e r o c a r p a n s a r e more t o x i c t o F. solani than the d e h y d r o p t e r o c a r p a n s , and t h e same i s t r u e f o r most o f t h e p t e r o c a r p a n s t o A. euteiches. The c o u m e s t r o l s a r e n o t t o x i c t o e i t h e r o f these organisms. Thus t h e p r o p e n s i t y t o form f r e e r a d i c a l s c o u l d be u s e d t o p r o v i d e some g u i d a n c e as t o t h e p r e d i c t e d t o x i c i t y o f t h e s e compounds. There a r e g l a r i n g e x c e p t i o n s t o t h i s o b s e r v a t i o n s u c h as t h e l o s s o f t o x i c i t y when t h e p t e r o c a r p a n , pisatin - OCH3, R - 0-, R3 - 0CH -, R4 - OH), i s demethylated; the demethylated product i s s i g n i f i c a n t l y l e s s t o x i c than p i s a t i n . The r e a s o n s f o r t h e l a r g e d i f f e r e n c e s i n t o x i c i t y among r e l a t e d p h y t o a l e x i n s may depend on s o l u b i l i t y c o n s i d e r a t i o n s . As s u g g e s t e d by V a n E t t e n (3j>) , s o l u b i l i t y i n t h e b i o a s s a y medium i s i m p o r t a n t i n determination of t o x i c i t y . Indeed some t o x i c i t y r e s u l t s i n vitro c o u l d be a f f e c t e d b y m i c r o s c o p i c p r e c i p i t a t i o n o f t h e phytoalexin. However, we s u s p e c t l i p i d s o l u b i l i t y i n t h e plasmalemma o r o t h e r membranes i s a l s o i m p o r t a n t . Thus a c a r e f u l b a l a n c e between s o l u b i l i t y i n t h e aqueous xylem f l u i d and l i p i d s o l u b i l i t y i n t h e plasmalemma must be m a i n t a i n e d f o r optimum phytoalexin toxicity. T h i s p r o p e r b a l a n c e i n s o l u b i l i t i e s and a p r o p e n s i t y t o form f r e e r a d i c a l s a r e each e x p e c t e d to, p l a y an important r o l e i n p h y t o a l e x i n t o x i c i t y . The s t r u c t u r e o f dHG seems t o be i d e a l l y s u i t e d t o a c t as an e f f e c t i v e p h y t o a l e x i n i n c o t t o n stems. The water s o l u b i l i t y o f dHG a l l o w s r a p i d d i f f u s i o n from p a r a v a s c u l a r c e l l s i n t o xylem v e s s e l s . L i g h t i s n o t n e c e s s a r y f o r t h e t o x i c i t y o f dHG, w h i c h i s c o n s i s t e n t w i t h i t s s i t e o f a c t i o n i n t h e xylem v e s s e l s . A p p a r e n t l y , the only c o n s t i t u e n t s n e c e s s a r y t o s t a r t t h e d e c o m p o s i t i o n o f dHG, and a c t i v a t e i t as a t o x i n , a r e oxygen and a t r a n s i t i o n m e t a l which a r e b o t h i n adequate s u p p l y i n t h e xylem f l u i d s . I n v a s i o n o f t h e xylem by t h e p a t h o g e n t r i g g e r s a s e r i e s o f e v e n t s , one o f w h i c h i s t h e b i o s y n t h e s i s o f dHG. The dHG r e a c t s w i t h oxygen t o form a f r e e r a d i c a l that t h e o r e t i c a l l y should react i n d i s c r i m i n a t e l y with both p a t h o g e n and p l a n t t i s s u e . To overcome dHG t h e p a t h o g e n c o u l d d i m i n i s h i t s e f f e c t i v e n e s s by p r o d u c i n g a s t r o n g r e d u c i n g e n v i r o n m e n t o r o x i d i z i n g i t a t a d i s t a n c e b e f o r e c o n t a c t i s made. Factors i n f l u e n c i n g t h i s aspect of v i r u l e n c e require a d d i t i o n a l investigations. 2

2

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Literature Cited: 1. 2.

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3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.

Snyder, W. C.; Smith, S. H. In Fungal Wilt Diseases of Plants; Mace, M. E., Bell, A. A.; Beckman, C. H . , Eds.; Academic: New York; 1981, pp. 25-50. Schnathorst, W. C. In Fungal Wilt Disease of Plants; Mace, M. E . ; Bell, A. A.; Beckman, C. H . , Eds.; Academic: New York, 1981; pp. 81-111. Gerik, J . S.; Huisman, O. C. Phytopathology 1988, 78, 1174-78. Beckman, C. H . ; Talboys, P. W. In Fungal Wilt Diseases of Plants; Mace, M. E.; Bell, A. A.; Beckman, C. H . , Eds.; Academic: New York, 1981; pp. 487-21. Mace, M. E. Physiol. Plant Pathol. 1978, 12, 1-11. Mace, M. E.; Bell, A. A.; Beckman, C. H. Can. J. Bot. 1976, 54, 2095-99. Mace, M. E. New Phytol. 1983, 95, 115-19. Bell, A. A . ; Stipanovic, R. D.; Howell, C. R.; Fryxell, P. A. Phytochemistry. 1975, 14, 225-31. Stipanovic, R. D.; Bell, A. A.; Howell, C. R. Phytochemistry. 1975, 14, 1809-11. Mace, M. E.; Stipanovic, R. D.; Bell, A. A. Physiol. Plant Pathol. 1985, 26, 209-18. Daly, J . M. Phytopathology 1972, 62, 392-400. Smith, D. A. In Phytoalexins; Bailey, J . A.; Mansfield, J . W., Eds.; John Wiley: New York, 1982; p. 247. Mace, M. E.; Stipanovic, R. D.; Bell, A. A. New Phytol. 1989, 111, 229-32. Ghosh, J . C.; Rakshit, P. C. Biochem. Z. 1937, 294, 330-35. Baker, M. S.; Gebicki, J. M. Arch. Biochem. Biophy. 1984, 234, 258-64. Taube, H. In Oxygen, Proc. Symp. Spons. New York Heart Association; Little Brown: New York, 1965; pp. 29-52. Randall, L. O. J . Biol. Chem. 1946, 164, 521-27. Haber, F . ; Weiss, J . J. Proc. Roy. Soc. London, Ser. A. 1934, 147, 332-51. Marklund, S.; Marklund, G. Eur. J. Biochem. 1974, 47, 469-74. Cohen, G.; Sinet, P. M. FEBS Letters. 1982, 138, 258-60. Harris, J . E . ; Dennis, C. Physiol. Plant Pathol. 1976, 9, 155-65. Smith, D. A.; Bull, C. H. Proc. 3rd Int. Cong. Plant Pathol. 1978, p. 245. Rossall, S.; Mansfield, J . W.; Hutson, R. A. Physiol. Plant Pathol. 1980, 16, 135-46. Van Etten, H. D.; Bateman, D. F. Phytopathology. 1971, 61. 1363-1372. Higgins, V. J. Phytopathology. 1978, 68, 339-45. Bailey, J . A.; Deverall, B. J. Physiol. Plant Pathol. 1971, 1, 435-49. Smith, D. A. Physiol. Plant Pathol. 1976, 9, 45-55. Apostol, I.; Heinstein, P. F . ; Low, P. S. Plant Physiol. 1989, 90, 109-16. Sun, T. J.; Melcher, U.; Essenberg, M. Physiol. Mol. Plant Pathol. 1988, 33, 115-26.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

23.

30.

31. 32. 33. 34.

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35.

STIPANOVIC E T AL.

Desoxyhemigossypol, A Cotton Phytoalexin

351

Stipanovic, R. D . ; Mace, M. E.; E l i s s a l d e , M. H. In Beltwide Cotton Production Research Conference; J. M. Brown, E d . ; National Cotton Council, Memphis: 1989; p. 39 (Abstract). B u l l , C. A. Ph.D. Thesis, University of H u l l , U . K . , 1981, and micrographs shown i n Ref. 12. pp. 223-28. Pryor, W. A. Fed. Proceed. 1973, 32, 1862-68. Lindner, W. A.; Hoffmann, C.; Grisebach, H. Phytochemistry. 1988, 27, 2501-03. Doke, N . ; Ohashi, Y. Physiol. Mol. Plant Pathol. 1988, 32, 163-75. VanEtten, H. D.

Phytochemistry

1976, 15, 655-59.

RECEIVED May 16, 1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 24

Bioregulation of Preharvest Aflatoxin Contamination of Peanuts Role of Stilbene Phytoalexins 1

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3

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National Peanut Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 1011 Forrester Drive, SE, Dawson, G A 31742 School of Pharmacy, Hebrew University, Jerusalem, Israel Department of Biotechnology, Instituttet for Bioteknologi, Lyngby, Denmark 2

3

A r i c h and varied assortment of phytoalexins i s produced by legumes. Peanuts (Arachis hypogaea L . ) have been shown to produce various stilbene phytoalexins in response to fungal i n f e c t i o n . Recent studies have been conducted to elucidate the role of these stilbenes i n the bioregulation of preharvest aflatoxin contamination of peanuts by Aspergillus flavus and A. parasiticus. Stilbene phytoalexins appear to provide s o i l borne peanut seed with a degree of resistance to aflatoxin contamination by i n h i b i t i n g fungal growth. Under conditions of prolonged, late-season drought stress that lead to aflatoxin contamination, phytoalexin biosynthesis breaks down, presumably due to dehydration of the peanut seed. However, seed moisture under such conditions remains adequate to support A. flavus growth and aflatoxin production. The chemistry and b i o l o g i c a l activity of stilbene phytoalexins as related to preharvest aflatoxin contamination of peanuts is described. P h y t o a l e x i n s , a n t i m i c r o b i a l s u b s t a n c e s p r o d u c e d by p l a n t s i n r e s p o n s e t o i n f e c t i o n o r s t r e s s , a r e important i n the n a t u r a l defense of plants against disease. Phytoalexins a r e extremely diverse c h e m i c a l l y , and a l t h o u g h t h e i r u b i q u i t y t h r o u g h o u t t h e p l a n t kingdom i s open t o q u e s t i o n , c e r t a i n p l a n t f a m i l i e s have been found t o c o n t a i n many s p e c i e s c a p a b l e o f p h y t o a l e x i n p r o d u c t i o n ( 1 ) . Among t h o s e i s t h e Leguminosae, an e c o n o m i c a l l y i m p o r t a n t f a m i l y o f p l a n t s t h a t i n c l u d e s t h e peanut (Arachis hypogaea L . ) ( 2 ) . Peanuts a r e grown i n many a r e a s o f t h e w o r l d and have a h i g h nutritive value. However, peanuts are subject to aflatoxin c o n t a m i n a t i o n under c e r t a i n e n v i r o n m e n t a l c o n d i t i o n s , and when t h i s happens, i t n u l l i f i e s t h e i r u s e f u l n e s s a s f o o d o r f e e d ( 3 ) . I n 1972 i t was r e p o r t e d t h a t peanut k e r n e l s c a n s y n t h e s i z e p h y t o a l e x i n s i n r e s p o n s e t o f u n g a l c h a l l e n g e ( 4 ) , and s u b s e q u e n t l y s e v e r a l o f these phytoalexins were c h e m i c a l l y c h a r a c t e r i z e d a s s t i l b e n e s (5-8). S i n c e t h a t time, s t u d i e s have shown t h a t t h e s e compounds p o s s e s s b i o l o g i c a l activity against fungi, including Aspergillus flavus , one o f t h e s p e c i e s t h a t p r o d u c e s a f l a t o x i n ( 9 ) . Thus, i t has been s p e c u l a t e d t h a t s t i l b e n e p h y t o a l e x i n s might be important i n t h e n a t u r a l d e f e n s e o f peanuts a g a i n s t aflatoxinproducing fungi.

This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

24.

DORNER ET AL.

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The p u r p o s e s o f t h i s c h a p t e r a r e t o r e v i e w t h e f a c t o r s i n v o l v e d i n a f l a t o x i n c o n t a m i n a t i o n o f peanuts, review the chemistry of s t i l b e n e p h y t o a l e x i n s from peanuts, d i s c u s s e v i d e n c e s u p p o r t i n g t h e involvement of these s t i l b e n e s i n the b i o r e g u l a t i o n of a f l a t o x i n c o n t a m i n a t i o n , and e x p l o r e approaches t o e x p l o i t o r enhance s u c h a b i o r e g u l a t i v e c a p a c i t y t o reduce or e l i m i n a t e p r e h a r v e s t a f l a t o x i n contamination of peanuts.

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Aflatoxin A f l a t o x i n s a r e s e c o n d a r y f u n g a l m e t a b o l i t e s p r o d u c e d by A. flavus L i n k and A. parasiticus Speare and a r e r e c o g n i z e d f o r t h e i r p o t e n t h e p a t o t o x i c , t e r a t o g e n i c , and c a r c i n o g e n i c e f f e c t s i n some a n i m a l s (10). The f o u r n a t u r a l l y o c c u r r i n g a f l a t o x i n s a r e d e s i g n a t e d B B, G and G~. O t h e r known a f l a t o x i n s a r e c h e m i c a l o r b i o l o g i c a l p r o d u c t s or t h e f o u r n a t u r a l l y o c c u r r i n g compounds. Under c e r t a i n e n v i r o n m e n t a l c o n d i t i o n s t h e a f l a t o x i n - p r o d u c i n g f u n g i can i n v a d e v a r i o u s a g r i c u l t u r a l commodities, and subsequent p r o l i f e r a t i o n by these fungi contaminates the commodity with aflatoxin. The commodities most a f f e c t e d i n t h e U n i t e d S t a t e s a r e p e a n u t s , c o r n , and cottonseed. S t r i c t r e g u l a t o r y a c t i o n l e v e l s f o r a f l a t o x i n must be met f o r t h e s e commodities t o be used as food or feed (10). T h e r e f o r e , s e v e r e economic l o s s e s can o c c u r when t h e s e commodities become c o n t a m i n a t e d and a r e d i v e r t e d from e d i b l e markets. 1#

2

1#

Preharvest A f l a t o x i n Contamination

o f Peanuts

C o n t a m i n a t i o n o f peanuts w i t h a f l a t o x i n can o c c u r d u r i n g v a r i o u s phases of production, storage, handling, and marketing (11). C o n t a m i n a t i o n t h a t o c c u r s a f t e r peanuts a r e h a r v e s t e d i s p r e v e n t a b l e i f p r o p e r s t e p s a r e t a k e n t o e n s u r e t h a t t h e m o i s t u r e o f peanuts i s maintained at a l e v e l that i s u n f a v o r a b l e f o r growth o f the a f l a t o x i n - p r o d u c i n g f u n g i . However, c o n t a m i n a t i o n t h a t o c c u r s i n t h e f i e l d p r i o r t o h a r v e s t ( p r e h a r v e s t ) i s much more d i f f i c u l t t o c o n t r o l and u s u a l l y r e s u l t s i n t h e most s e v e r e a f l a t o x i n c o n t a m i n a t i o n o f peanuts. P r e h a r v e s t a f l a t o x i n c o n t a m i n a t i o n o f peanuts o c c u r s when peanuts a r e s u b j e c t e d t o severe, prolonged drought s t r e s s d u r i n g the l a s t f o u r t o s i x weeks o f t h e growing season (12-17) . E l e v a t e d s o i l temperatures t h a t u s u a l l y accompany such p e r i o d s o f l a t e - s e a s o n d r o u g h t e x a c e r b a t e t h e problem and produce optimum c o n d i t i o n s f o r p r e h a r v e s t c o n t a m i n a t i o n (12, 1 8 ) . When c o n t a m i n a t i o n o c c u r s , i t i s n o t homogeneous t h r o u g h o u t a p o p u l a t i o n o f p e a n u t s . Immature peanuts a r e more l i k e l y t o be c o n t a m i n a t e d t h a n mature p e a n u t s , and k e r n e l s t h a t a r e damaged, p a r t i c u l a r l y by i n s e c t s , can c o n t a i n e x t r e m e l y h i g h c o n c e n t r a t i o n s o f a f l a t o x i n (13, 15, 18-20). I n v a s i o n o f peanuts by the a f l a t o x i g e n i c f u n g i does n o t n e c e s s a r i l y r e s u l t i n t h e i r b e i n g contaminated w i t h a f l a t o x i n . In t h e absence o f d r o u g h t s t r e s s , samples o f peanuts have been shown t o be c o l o n i z e d by A. flavus/parasiticus a t p e r c e n t a g e s as h i g h as 25% of t h e k e r n e l s w i t h o u t d e t e c t a b l e l e v e l s o f a f l a t o x i n (18, 19). T h i s i n d i c a t e s t h a t i n t h e absence o f d r o u g h t c o n d i t i o n s t h e f u n g i a r e a b l e t o i n v a d e peanuts, but t h e peanuts a r e p r o t e c t e d from e x t e n s i v e f u n g a l p r o l i f e r a t i o n by some i n h e r e n t d e f e n s e mechanism(s) . However, when exposed t o t h e s t r e s s e s o f d r o u g h t and h e a t , a breakdown i n t h i s d e f e n s e mechanism(s) a l l o w s f o r f u n g a l p r o l i f e r a t i o n and subsequent a f l a t o x i n contamination. S t i l b e n e P h y t o a l e x i n s from

Peanuts

The f i r s t r e p o r t o f p h y t o a l e x i n p r o d u c t i o n by peanuts appeared i n 1972 when V i d h y a s e k a r a n et al. found t h a t s e v e r a l s p e c i e s o f f u n g i , i n c l u d i n g A. flavus, i n d u c e d p r o d u c t i o n o f an i n h i b i t o r y p r i n c i p l e by

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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peanut pods (4). A l t h o u g h t h e p r i n c i p l e was n o t c h e m i c a l l y c h a r a c t e r i z e d , i t was deemed t o be a p h y t o a l e x i n because i t was p r o d u c e d by t h e peanut o n l y a f t e r i n t e r a c t i o n between t h e f u n g i and the peanut. I n t h a t s t u d y , immature p e a n u t s p r o d u c e d a h i g h e r q u a n t i t y o f t h e p h y t o a l e x i n t h a n mature p e a n u t s , and t h e a u t h o r s s u g g e s t e d t h a t r e s i s t a n c e o f immature peanut pods t o f u n g i was b a s e d on t h e i r c a p a c i t y t o p r o d u c e p h y t o a l e x i n s i n r e s p o n s e t o i n f e c t i o n . I n 1975 Keen (21) r e p o r t e d t h a t n a t i v e m i c r o f l o r a s t i m u l a t e d p r o d u c t i o n o f two a n t i f u n g a l compounds by peanut seeds t h a t were soaked i n water, s l i c e d i n t o s e c t i o n s , and i n c u b a t e d f o r 3-5 d a y s . These compounds were judged t o be p h y t o a l e x i n s and were s u b s e q u e n t l y i d e n t i f i e d a s c i s - and t r a n s - i s o m e r s o f 4 - ( 3 - m e t h y l - b u t - 2 - e n y l ) 3 , 5 , 4 ' - t r i h y d r o x y s t i l b e n e [1](5)(Figure 1). S i m u l t a n e o u s l y , Ingham (6) r e p o r t e d t h e i s o l a t i o n o f c i s - and t r a n s - r e s v e r a t r o l ( 3 , 5 , 4 t r i h y d r o x y s t i l b e n e ^ ] ) from peanut h y p o c o t y l s . Additional stilbenes have been shown t o be p r o d u c e d by peanut seeds i n r e s p o n s e t o wounding, and t h e s e i n c l u d e 4 - ( 3 - m e t h y l - b u t - l - e n y l ) - 3 , 5 , 3 , 4 ' - t e t r a hydroxystilbene [3](7), 4-(3-methyl-but-l-enyl)-3,5,4'-trihydroxystilbene [4](7), and 3 - i s o p e n t a d i e n y l - 4 , 3 ' , 5 - t r i h y d r o x y s t i l b e n e [5](8).

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1

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F i g u r e 1. C h e m i c a l s t r u c t u r e s o f s t i l b e n e p h y t o a l e x i n s from p e a n u t s .

To stimulate peanut seeds t o produce phytoalexins f o r l a b o r a t o r y s t u d i e s , t h e d r y seeds a r e t y p i c a l l y a l l o w e d t o imbibe water f o r a p p r o x i m a t e l y 24 h o u r s . The seeds a r e t h e n s l i c e d i n t o 1-3 mm s e c t i o n s o r g e n t l y chopped t o cause e x t e n s i v e c e l l u l a r damage, and t h e s e e d s a r e e i t h e r i n o c u l a t e d w i t h a fungus and i n c u b a t e d i n t h e d a r k f o r s e v e r a l days o r i n c u b a t e d u s i n g t h e n a t i v e peanut m i c r o f l o r a t o i n d u c e p h y t o a l e x i n p r o d u c t i o n (2, 2 1 , 2 2 ) . The s t i l b e n e s a r e t y p i c a l l y e x t r a c t e d from p e a n u t s w i t h 95% e t h a n o l , and f o l l o w i n g p a r t i a l p u r i f i c a t i o n t h e samples a r e s u b j e c t e d t o e i t h e r t h i n - l a y e r c h r o m a t o g r a p h i c (TLC) o r l i q u i d c h r o m a t o g r a p h i c (LC) a n a l y s e s . On TLC p l a t e s , t h e s t i l b e n e s f l u o r e s c e b l u e under 254 nm UV l i g h t o r t h e y c a n be d e t e c t e d i n LC a n a l y s i s by UV d e t e c t i o n r a n g i n g from 290-335 nm ( 5 £ 7 , 2 1 , 2 2 ) .

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

24.

DORNER ET AL.

Preharvest Aflatoxin Contamination of Peanuts

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Evidence Supporting Preharvest A f l a t o x i n

Stilbene Involvement Contamination

in

Bioregulation

355 of

N a t u r a l O c c u r r e n c e o f S t i l b e n e s i n P e a n u t s . S t i l b e n e s a r e n o t found i n sound, undamaged p e a n u t s . However, t h e compounds a r e e a s i l y d e t e c t e d i n p e a n u t s t h a t have been s u b j e c t e d t o some t y p e o f damage i n t h e f i e l d when they were a t h i g h water a c t i v i t i e s (unpublished data). I n peanut s h e l l i n g p l a n t s , damaged p e a n u t s a r e removed r o u t i n e l y by e l e c t r o n i c c o l o r s o r t i n g machines, and t h e s e machines e f f i c i e n t l y e l i m i n a t e d i s c o l o r e d k e r n e l s d u r i n g the p r o c e s s i n g of peanuts. D i s c o l o r a t i o n i s u s e d t o i d e n t i f y p e a n u t s t h a t have been damaged ( u s u a l l y i n the f i e l d , but a l s o d u r i n g s t o r a g e ) and would severely detract from the overall quality of the peanuts, p a r t i c u l a r l y w i t h r e g a r d t o f l a v o r . In many c a s e s t h i s d i s c o l o r a t i o n i s v e r y s i m i l a r t o t h e d i s c o l o r a t i o n t h a t o c c u r s when imbibed p e a n u t s a r e s t i m u l a t e d t o produce p h y t o a l e x i n s i n t h e l a b o r a t o r y . When t h e s e d i s c o l o r e d p e a n u t s were a n a l y z e d f o r s t i l b e n e p h y t o a l e x i n s , the a n a l y s e s i n v a r i a b l y showed the p r e s e n c e o f the compounds, s u g g e s t i n g t h a t they a r e n a t u r a l l y p r o d u c e d i n t h e f i e l d i n r e s p o n s e t o damage ( u n p u b l i s h e d d a t a ) . S i n c e d i s c o l o r a t i o n and s t i l b e n e p r o d u c t i o n does n o t o c c u r when peanuts a r e d r y , i t i s a p p a r e n t t h a t some minimum water a c t i v i t y i s r e q u i r e d f o r the enzyme-mediated s y n t h e s i s o f t h e stilbenes. A c l o s e e x a m i n a t i o n o f damaged peanuts grown under adequate m o i s t u r e c o n d i t i o n s r e v e a l s l i t t l e , i f any, A. flavus proliferation. Likewise, i t i s unusual t o d e t e c t even low concentrations of a f l a t o x i n i n s u c h p e a n u t s . However, s t i l b e n e p h y t o a l e x i n s a r e e a s i l y detected i n such peanuts. When s i m i l a r p e a n u t s were s u r f a c e s t e r i l i z e d and p l a t e d out t o d e t e r m i n e c o u n t s and t y p e s o f f u n g a l c o l o n i z a t i o n , t h e p e r c e n t a g e s o f k e r n e l s c o l o n i z e d by A. flavus was as h i g h as 25% ( 1 9 ) . S i n c e c o l o n i z a t i o n had o c c u r r e d and p h y t o a l e x i n s had been p r o d u c e d w i t h an absence o f a f l a t o x i n c o n t a m i n a t i o n , p h y t o a l e x i n s presumably i n h i b i t e d A. flavus growth and aflatoxin production. C o n v e r s e l y , a c l o s e e x a m i n a t i o n o f damaged p e a n u t s t h a t were s u b j e c t e d t o l a t e - s e a s o n drought c o n d i t i o n s u s u a l l y r e v e a l s s e v e r a l k e r n e l s w i t h p r o l i f i c A. flavus growth and a f l a t o x i n c o n c e n t r a t i o n s t h a t can be e x t r e m e l y h i g h . P h y t o a l e x i n c o n c e n t r a t i o n s a r e t y p i c a l l y much lower i n t h e s e peanuts compared t o damaged, non-stressed peanuts. T h e r e f o r e , t h e f a c t t h a t peanuts produce s t i l b e n e p h y t o a l e x i n s n a t u r a l l y i n r e s p o n s e t o damage i n the f i e l d but do n o t become c o n t a m i n a t e d w i t h a f l a t o x i n ( i n d i c a t i v e o f A. flavus growth) u n t i l s u b j e c t e d t o p r o l o n g e d d r o u g h t s t r e s s p o i n t s toward a p r e s u m p t i v e r o l e f o r t h e s e compounds i n t h e n a t u r a l b i o r e g u l a t i o n of a f l a t o x i n contamination. B i o l o g i c a l A c t i v i t y o f S t i l b e n e s A g a i n s t A. flavus and O t h e r F u n g i . Further evidence supporting a r o l e f o r s t i l b e n e phytoalexins i n i n h i b i t i n g f u n g a l growth i n peanuts i n v o l v e s the b i o l o g i c a l a c t i v i t y of t h e s e compounds a g a i n s t A. flavus and o t h e r f u n g i . Wotton and S t r a n g e t e s t e d [ 1 ] , [ 3 ] , and [4] f o r i n h i b i t i o n o f A. flavus spore g e r m i n a t i o n and h y p h a l e x t e n s i o n ( 9 ) . The EDgQ v a l u e s f o r i n h i b i t i o n of s p o r e g e r m i n a t i o n were 12.7, 12.8, and 8.9 ug/ml, r e s p e c t i v e l y , i n V o g e l ' s medium. S i m i l a r l y , germ tube e x t e n s i o n was a l s o i n h i b i t e d w i t h E D ^ v a l u e s o f 6.8, 4.9, and 9.7 ug/ml, r e s p e c t i v e l y , f o r [ 1 ] , [ 3 ] , ana [ 4 ] . Cooksey e t a l . r e p o r t e d E D ^ v a l u e s o f 14.0 and 11.3 ug/ml f o r i n h i b i t i o n of s p o r e g e r m i n a t i o n and h y p h a l e x t e n s i o n o f A. flavus, r e s p e c t i v e l y , by [5] ( 8 ) . I n one l a b o r a t o r y , a t i m e - c o u r s e s t u d y was c a r r i e d out t o d e t e r m i n e t h e r e l a t i o n s h i p o f growth and a f l a t o x i n p r o d u c t i o n by A. flavus t o p h y t o a l e x i n a c c u m u l a t i o n by i n t a c t peanut k e r n e l s . Wotton and S t r a n g e (23) r e p o r t e d t h a t f o l l o w i n g i n o c u l a t i o n , A. flavus grew l o g a r i t h m i c a l l y f o r 2 days. However, by t h e t h i r d day when t h e

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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p h y t o a l e x i n c o n c e n t r a t i o n had exceeded 50 ug/g o f k e r n e l s , f u n g a l growth e s s e n t i a l l y c e a s e d . This provided evidence f o r both the e l i c i t a t i o n o f p h y t o a l e x i n p r o d u c t i o n by A. flavus and t h e i n h i b i t i o n o f A. flavus growth by t h e p h y t o a l e x i n s . The i n h i b i t i o n o f A. parasiticus growth by [4] was d e t e r m i n e d i n our l a b o r a t o r y u s i n g a m o d i f i c a t i o n o f the a s s a y r e p o r t e d by A r n o l d i and coworkers ( 2 4 ) . The s t i l b e n e was d i s s o l v e d i n a c e t o n e and a p p r o p r i a t e amounts were added t o s t e r i l e p o t a t o d e x t r o s e a g a r (PDA) a t 50°C t o g i v e c o n c e n t r a t i o n s o f 10, 20, 50, and 100 ug/ml. The medium was dispersed i n 60 mm t i s s u e culture dishes and i n o c u l a t e d w i t h 4 mm PDA p l u g s o f a c t i v e l y growing A. parasiticus cultures. P l a t e s were i n c u b a t e d i n t h e d a r k a t 30°C and colony d i a m e t e r s were measured d a i l y . A f t e r t h r e e days t h e percent i n h i b i t i o n o f A. parasiticus growth a t 10, 20, 50, and 100 ug/ml was 15, 26, 37, and 33%, r e s p e c t i v e l y ( u n p u b l i s h e d d a t a ) . O t h e r s t u d i e s were c o n d u c t e d i n our l a b o r a t o r y t o d e t e r m i n e t h e i n h i b i t o r y e f f e c t s o f [4] a g a i n s t A. parasiticus and s e v e r a l s p e c i e s o f Penicillium ( C h r i s t i a n s e n , u n p u b l i s h e d d a t a ) . The g e r m i n a t i o n o f f u n g i was d e t e r m i n e d i n a p r o c e d u r e t h a t was s i m i l a r t o t h a t r e p o r t e d by Wotton and S t r a n g e ( 9 ) , and r a d i a l growth a l s o was d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d (24). R e s u l t s p r e s e n t e d i n T a b l e I show t h a t both spore germination and growth o f most t e s t e d s p e c i e s were inhibited by [4], although the degree of inhibition varied c o n s i d e r a b l y among s p e c i e s . Table

I.

E f f e c t o f [4] on s p o r e g e r m i n a t i o n several fungal species.

and

Spore g e r m i n a t i o n ED^Q (ug/ml)

Fungus Aspergillus parasiticus Penicillium commune P. crustosum P. verrucosum P. aurantiogriseum P. echinulatum var. d i s c o l o r

94 132 18000 42 70 77

radial

growth

of

R a d i a l growth % inhibition 25 ug/ml 50 ug/ml 54 36 46 33 25 13

54 55 61 44 50 63

Taken t o g e t h e r , t h e s e s t u d i e s show t h a t peanut k e r n e l s t i l b e n e s p o s s e s s b i o l o g i c a l a c t i v i t y a g a i n s t s p e c i e s o f b o t h Aspergillus and Penicillium. T h i s , c o u p l e d w i t h t h e f a c t t h a t t h e s e compounds a r e p r o d u c e d in vivo as a r e s u l t o f damage, p r o v i d e s e v i d e n c e that s t i l b e n e s p l a y a p a r t i n the n a t u r a l defense of peanuts a g a i n s t fungi. Occurrence of A f l a t o x i n Contamination A f t e r C e s s a t i o n of P h y t o a l e x i n Production. I n view o f t h e f a c t t h a t s t i l b e n e p h y t o a l e x i n s are n a t u r a l l y p r o d u c e d i n peanut k e r n e l s i n r e s p o n s e t o f u n g a l i n v a s i o n and that these s t i l b e n e s possess antifungal activity against a f l a t o x i g e n i c f u n g i , t h e q u e s t i o n o f how p e a n u t s become c o n t a m i n a t e d with a f l a t o x i n remains. Simply s t a t e d , how does A. flavus overcome t h i s a p p a r e n t n a t u r a l d e f e n s e mechanism o f p e a n u t s ? Because of the association of preharvest aflatoxin c o n t a m i n a t i o n o f p e a n u t s w i t h l a t e - s e a s o n d r o u g h t s t r e s s , a s t u d y was undertaken to determine the relationship among aflatoxin c o n t a m i n a t i o n , d r o u g h t , and p h y t o a l e x i n p r o d u c t i o n ( 2 2 ) . The s t u d y i n v o l v e d s a m p l i n g o f F l o r u n n e r p e a n u t s s u b j e c t e d t o and n o t s u b j e c t e d t o l a t e - s e a s o n d r o u g h t and d e t e r m i n i n g t h e peanut k e r n e l water activity (a ), phytoalexin-producing capacity, and aflatoxin c o n c e n t r a t i o n s i n a l l m a t u r i t y s t a g e s o f t h e sampled p e a n u t s . I t was r e p o r t e d t h a t t h e a and p h y t o a l e x i n - p r o d u c i n g c a p a c i t y o f p e a n u t s n o t exposed t o d r o u g h t s t r e s s remained h i g h t h r o u g h o u t t h e s t u d y w

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Preharvest Aflatoxin Contamination of Peanuts

period. These p e a n u t s d i d n o t become c o n t a m i n a t e d w i t h a f l a t o x i n even though t h e f i n a l s a m p l i n g took p l a c e 184 days a f t e r p l a n t i n g , a p p r o x i m a t e l y 40 days beyond t h e o p t i m a l h a r v e s t d a t e . The a o f peanuts grown under l a t e - s e a s o n drought c o n d i t i o n s d e c r e a s e d d u r i n g t h e s t u d y (22). The m o i s t u r e c o n t e n t was not u n i f o r m i n a sample, i n d i c a t i n g t h a t p e a n u t s s u b j e c t e d t o drought s t r e s s d i d n o t d r y a t a u n i f o r m r a t e . T h i s appeared t o be a s s o c i a t e d w i t h i n d i v i d u a l p l a n t s i n t h a t t h e peanuts on c e r t a i n p l a n t s l o s t m o i s t u r e more e a s i l y t h a n t h o s e on o t h e r p l a n t s . However, t h e o v e r a l l e f f e c t o f drought s t r e s s was t o r e d u c e t h e m o i s t u r e o r a o f peanut k e r n e l s . As peanuts became d e h y d r a t e d d u r i n g t h e drought p e r i o d , t h e y l o s t the c a p a c i t y t o produce s t i l b e n e p h y t o a l e x i n s . This lost c a p a c i t y was n o t d i r e c t l y due t o t h e d u r a t i o n o f t h e s t r e s s , but i t was directly a s s o c i a t e d w i t h the drop in a of the peanuts. R e g a r d l e s s o f drought t r e a t m e n t s o i l temperature o r peanut m a t u r i t y , t h e p h y t o a l e x i n - p r o d u c i n g c a p a c i t y o f peanuts d e c r e a s e d as t h e a d e c r e a s e d , w i t h e s s e n t i a l l y no p h y t o a l e x i n p r o d u c t i o n below a k e r n e l a o f 0.95 (Figure 2). The o n s e t o f a f l a t o x i n c o n t a m i n a t i o n d i d not o c c u r u n t i l peanut k e r n e l s had l o s t t h e c a p a c i t y t o produce p h y t o a l e x i n s as an a p p a r e n t r e s u l t of drought-induced moisture l o s s . However, mature p e a n u t s r e t a i n e d a h i g h e r degree o f r e s i s t a n c e t o a f l a t o x i n c o n t a m i n a t i o n t h a n immature p e a n u t s even a f t e r t h e l o s s o f p h y t o a l e x i n - p r o d u c i n g capability. T h i s i n d i c a t e d t h a t immature k e r n e l s r e l y more h e a v i l y on a phytoalexin-based r e s i s t a n c e than mature k e r n e l s , which a p p a r e n t l y have some a d d i t i o n a l r e s i s t a n c e not based on s t i l b e n e phytoalexins. The e v i d e n c e c l e a r l y s u p p o r t s t h e h y p o t h e s i s t h a t s t i l b e n e p h y t o a l e x i n s i n peanuts a r e an i m p o r t a n t n a t u r a l b i o r e g u l a t o r o f p r e h a r v e s t a f l a t o x i n c o n t a m i n a t i o n . That e v i d e n c e i n c l u d e s t h e f a c t s t h a t : (1) s t i l b e n e s a r e n a t u r a l l y produced i n f i e l d - d a m a g e d p e a n u t s ; (2) s t i l b e n e s p o s s e s s b i o l o g i c a l a c t i v i t y a g a i n s t A. flavus and A. parasiticus; and (3) a l t h o u g h i n v a s i o n o f p e a n u t s by A. flavus and A. parasiticus c a n o c c u r under any c o n d i t i o n s , a f l a t o x i n c o n t a m i n a t i o n does n o t o c c u r u n t i l peanuts l o s e t h e c a p a c i t y f o r p h y t o a l e x i n p r o d u c t i o n as a r e s u l t o f d r o u g h t - i n d u c e d k e r n e l d e h y d r a t i o n .

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w

w

w

T a k i n g Advantage o f t h e N a t u r a l Defense Mechanism The g o a l o f our r e s e a r c h today i s t o g r e a t l y r e d u c e o r e l i m i n a t e p r e h a r v e s t a f l a t o x i n contamination of peanuts. As t h e demand f o r more wholesome f o o d w i t h l e s s r i s k o f e x p o s u r e t o t o x i n s and c a r c i n o g e n s i n c r e a s e s , t h e c o n t i n u e d use o f p e a n u t s and peanut p r o d u c t s as f o o d becomes more dependent on e f f e c t i v e management o f t h e a f l a t o x i n problem. Many approaches a r e b e i n g t a k e n t o s o l v e t h e a f l a t o x i n problem i n peanuts. An i m p o r t a n t q u e s t i o n i s whether t h e p h y t o a l e x i n - b a s e d n a t u r a l d e f e n s e mechanism o f peanuts a g a i n s t f u n g i c a n be e x p l o i t e d i n some way t o p r o v i d e a s o l u t i o n ; and i f so, how. A l l data i n d i c a t e t h a t growing a l l peanuts w i t h adequate l a t e - s e a s o n i r r i g a t i o n would e s s e n t i a l l y s o l v e t h e p r e h a r v e s t problem, but c u r r e n t l y t h i s i s n o t a f e a s i b l e approach. T h e r e f o r e , t a k i n g advantage o f any n a t u r a l d e f e n s e mechanism must i n v o l v e i t s e f f e c t i v e n e s s d u r i n g p e r i o d s o f l a t e - s e a s o n drought s t r e s s . Two a p p r o a c h e s t o m a i n t a i n i n g p h y t o a l e x i n - p r o d u c i n g c a p a c i t y d u r i n g drought a r e a p p a r e n t . The f i r s t would be t o i d e n t i f y peanut genotypes t h a t c o u l d c o n t i n u e p r o d u c i n g p h y t o a l e x i n s as t h e m o i s t u r e of the k e r n e l s decreased. I n F l o r u n n e r p e a n u t s i t appears t h a t t h e a p p r o x i m a t e lower a l i m i t for phytoalexin production i s 0.95. Peanuts t h a t c o u l d m a i n t a i n p r o d u c t i o n o f p h y t o a l e x i n s as t h e a approached 0.90 might p o s s e s s much g r e a t e r p r o t e c t i o n from A. flavus growth and a f l a t o x i n p r o d u c t i o n . A l t h o u g h t h e lower a limit for a f l a t o x i n p r o d u c t i o n i n peanuts i s about 0.85, a practical solution w

w

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991. w

0.96 a

w

0.94

0.92

F i g u r e 2. R e l a t i o n s h i p o f p h y t o a l e x i n p r o d u c t i o n t o peanut k e r n e l water a c t i v i t y (a ). T o t a l p h y t o a l e x i n s were d e t e r m i n e d by c o m b i n i n g a r e a s under p h y t o a l e x i n peaks from l i q u i d chromatograms.

0.98

0.90

» « • — * ~ ^|—i—i—i—i—i—i—i—i—i—|—i—i—i—i—i—i—i—i—i—|—i—i—i—i—i—i—i—i—i—|—i—i—i—i—i—i—i—i—i—|—i—i—i—i—i—i—i—i—i—p

1.00

50000

60000

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Preharvest Aflatoxin Contamination of Peanuts

t o t h e problem might n o t r e q u i r e p h y t o a l e x i n p r o d u c t i o n down t o s u c h a low a l e v e l . T h i s i s because a s t h e a c o n t i n u e s t o d e c r e a s e , s o does t h e r a t e o f f u n g a l growth and a f l a t o x i n p r o d u c t i o n . Whether o r n o t a genotype e x i s t s t h a t c a n produce p h y t o a l e x i n s a t lower a i s unknown a t t h i s t i m e . T h e r e f o r e , a r i g o r o u s s c r e e n i n g program would have t o be u n d e r t a k e n t o i d e n t i f y such a g e n e t i c c a p a b i l i t y i n peanuts. A second approach t o m a i n t a i n i n g p h y t o a l e x i n - p r o d u c i n g c a p a c i t y d u r i n g d r o u g h t would be t o i d e n t i f y d r o u g h t - t o l e r a n t g e n o t y p e s t h a t can m a i n t a i n a h i g h k e r n e l a f o r a s i g n i f i c a n t l y l o n g e r p e r i o d during drought stress. I f e i t h e r o f these approaches were s u c c e s s f u l , b i o t e c h n o l o g y t e c h n i q u e s might be used t o i n c o r p o r a t e t h e d e s i r a b l e t r a i t ( s ) i n t o c o m m e r c i a l l y - d e s i r a b l e c u l t i v a r s , such as Florunner. I t i s u n l i k e l y t h a t any s i n g l e a p p r o a c h w i l l p r o v i d e a s o l u t i o n t o t h e problem o f p r e h a r v e s t a f l a t o x i n c o n t a m i n a t i o n o f peanuts. However, a m u l t i f a c e t e d a p p r o a c h t h a t c o u l d i n c l u d e enhancement o f t h e n a t u r a l b i o r e g u l a t i v e p r o p e r t i e s o f s t i l b e n e p h y t o a l e x i n s might u l t i m a t e l y y i e l d t h e s o l u t i o n t o a s e r i o u s and complex problem. w

w

w

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w

Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Deverall, B. J. In Phytoalexins; Bailey, J . A.; Mansfield J. W., Eds.; John Wiley and Sons, Inc.: New York, 1982; pp 1-20. Ingham, J . L. In Phytoalexins; Baily, J . A.; Mansfield, J . W., Eds.; John Wiley and Sons, Inc.: New York, 1982, p 21. Cole, R. J.; Sanders, T. H.; Blankenship, P. D.; H i l l , R. A. In Xenobiotics in Foods and Feeds; Finley, J . W.; Schwass, D. E . , Eds.; ACS Symposium Series No. 234; American Chemical Society: Washington, DC, 1983; pp 233-239. Vidhyasekaran, P.; Lalithakumari, D.; Govindaswamy, C. V. Indian Phytopathol. 1972, 25, 240-45. Kenn, N. T . ; Ingham, J . L. Phytochemistry 1976, 15, 1794-95. Ingham, J . L. Phytochemistry 1976, 15, 1971-93. Aguamah, D. G.; Langcake, P.; Leworthy, D. P.; Page, J . A.; Pryce, R. J.; Strange, R. N. Phytochemistry 1981, 20, 1381-83. Cooksey, C. J.; Gaviatt, P. J.; Richards, S. E . ; Strange, R. N. Phytochemistry 1988, 27, 1015-16. Wotton, H. R.; Strange, R. N. J. Gen. Microbiol. 1985, 131, 487-94. Diener, U. L.; Cole, R. J.; Sanders, T. H.; Payne, G. A.; Lee, L. S.; Klich, M. A. Ann. Rev. Phytopathol. 1987, 25, 249-70. Diener, U. L.; Pettit, R. E.; Cole, R. J . In Peanut Science and Technology; Pattee, H. E.; Young, C. T . , Eds.; American Peanut Research and Education Society, Inc.: Yoakum, Texas, 1982; pp 486-519. Blankenship, P. D.; Cole, R. J.; Sanders, T. H.; Hill, R. A. Mycopathologia 1984, 85, 69-74. Cole, R. J.; Hill, R. A.; Blankenship, P. D.; Sanders, T. H.; Garren, K. H. Dev. Ind. Microbiol. 1982, 23, 229-36. Dickens, J . W.; Satterwhite, J . B.; Sneed, R. E. J. Am. Peanut Res. Educ. Soc. 1973, 5, 48-58. H i l l , R. A.; Blankenship, P. D.; Cole, R. J.; Sanders, T. H. Appl. Microbiol. 1983, 45, 628-33. Pettit, R. E.; Taber, R. A.; Schroeder, H. W.; Harrison, A. L. Appl. Microbiol. 1971, 22-629-34. Wilson, D. M.; Stansell, J . R. Peanut Sci. 1983, 10, 54-56. Cole, R. J.; Sanders, T. H.; Hill, R. A.; Blankenship, P. D. Mycopathologia 1985, 91, 41-46. Sanders, T. H.; Cole, R. J.; Blankenship, P. D.; H i l l , R. A. Peanut Sci. 1985, 12, 90-93. Sanders, T. H.; Hill, R. A.; Cole, R. J.; Blankenship, P. D. J. Am. Oil Chem. Soc. 1981, 58, 966A-70A. Keen, N. T. Phytopathology 1975, 65, 91-92.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

360 22. 23. 24.

NATURALLY OCCURRING PEST BIOREGULATORS

Dorner, J . W.; C o l e , R. J.; Sanders, T. H.; B l a n k e n s h i p , P. D. Mycopathologia 1989, 105, 117-28. Wotton, H. R.; S t r a n g e , R. N. Appl. Environ. Microbiol. 1987, 53, 270-73. A r n o l d i , A.; C a r u g h i , M.; F a r e i n a , G.; Merlini, L.; P a r r i n o , M. G. J. Agric. Food Chem. 1989, 37, 508-12.

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RECEIVED May 16, 1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 25

Phototoxic Metabolites of Tropical Plants 1

2

Lee A. Swain and Kelsey R. Downum

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1

Department of Biological Sciences, Florida International University, Miami, FL 33199 Fairchild Tropical Gardens, 10901 Old Cutler Road, Miami, FL 33156

2

Over 400 species of tropical plants from 76 families were assayed for phototoxic activity. Furanocoumarins, an important class of phototoxins, were identified from three genera of the Moraceae (fig family).Dorstenia,an herbaceous member of this family, was particularly rich in these metabolites. The distribution of furanocoumarins in the Moraceae as well as evolutionary and ecological aspects are discussed.

Phototoxic phytochemicals, or "photosensitizers", exhibit broad spectrum biocidal activity against a range of organisms including viruses, bacteria, fungi, nematodes, insects and other plants (1-12). They are also responsible for causing serious health problems in range animals and man (13-191 After absorbtion of light (usually in the UV-A range, 320-400nm), photosensitizers undergo a transition to an excited state. Some of them (type II phototoxins) can transfer the excitation energy to molecular oxygen to form singlet oxygen, which is capable of oxidizing many biomolecules. Cell membranes and cell wall components are particularly vulnerable to these types of phototoxins (20). Others (photogenotoxins) react directly with DNA and RNA, though reactions with other cellular components are known (21-25). Although most of the information concerning the biological activity of these phototoxins has been demonstrated jn vitro, it is likely that they provide a viable defensive mechanism against nonadapted organisms. Phototoxins in the wild parsnip, for example, are effective in killing or deterring generalist insects such as the armyworm (Spodoptera eridania). while they have little effect on swallowtail butterfly larvae (Papilio polyxenes) which utilize the wild parsnip as a food plant (26-31). Swallowtail caterpillars are able to detoxify the chemicals and excrete them as harmless waste products (32-37).

0097-6156/91/0449-0361$06.00/0 © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Distribution of phototoxins Various biosynthetic classes of phototoxic compounds have been isolated from over thirty plant families (5). Some plant families produce more than one type of phototoxin. Three different types of phototoxins have been isolated from the Apiaceae ( celery family), for example, and eight different types have been identified so far from the Rutaceae (citrus family). Among other families that contain phototoxins are the Asteraceae (sunflower family), Euphorbiaceae (spurge family), Fabaceae (pea family) and Moraceae (fig family) (£). It was suggested that photosensitizers would be most effective in plant species that evolved under high-light environments and that the incidence of phototoxin-containing plants might be greater in these areas (2). A recent survey for phototoxins in one high-light environment, the desert southwestern United States, suggested that light-activated phytochemicals were relatively common in families in which phototoxins had previously been described (38). Over 35% of the extracts from members of the Asteraceae exhibited phototoxic activity, with many of these belonging to a limited number of tribes of this family. Almost half of the members of the tribe Heliantheae, for example, tested positive for lightactivated toxins. All of the extracts of the Pectidinae, a subtribe within the Heliantheae, were phototoxic toward the test organisms. Photobiocides from tropical plants We recently surveyed a cross-section of plants from many tropical regions of the world in a search for photosensitizers to further test the above hypothesis. The methods used to test for phototoxic phytochemicals are described in detail elsewhere (22). Briefly, methanolic extracts were spotted onto sterile filter-paper discs and allowed to dry. The dried discs were placed onto replicate nutrient agar plates that had been spread with E . coli B/r (a U V resistant bacterium). The plates were incubated in the dark at 37°C for 30 min. Half of the plates were irradiated for 60 min. with eight Sylvania F40BLB U V A lamps (18W m' ), while the other half were kept in the dark. All plates were incubated overnight in the dark at 37°C, after which the zones of inhibition surrounding the filter paper discs were measured. Over 400 tropical/subtropical species representing 76 families were collected at either Fairchild Tropical Gardens or Chapman Field, USDA Plant Induction Station in Miami, F L Table I lists the number of genera and species of the families that were examined. Only five of the 76 families tested elicited phototoxic responses from E.coli. These families are listed in boldface caps in Table I . The Asteraceae has been studied extensively in regards to its phototoxic components (38). as have members of the Rutaceae (citrus family; 5.7)and the Moraceae (fig family: 40). The compound(s) responsible for phototoxicity in the Sapotaceae (sapodilla family) is not known and is the only instance of lightenhanced toxicity from this family that has been reported to date (41). Elucidation of the structure of this phytochemical is in progress and we are also examining other members of the Sapotaceae for phototoxicity. 2

Biocidal compounds of the Moraceae In contrast to the Asteraceae and the Rutaceae, the Moraceae is almost

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

25.

SWAIN AND DOWNUM

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Table I.

Phototoxic Metabolites of Tropical Plants

Tropical plants examined for phototoxic antimicrobial properties (#genera,#species examined).

Acanthaceae (6,9) Amaryllidaceae (2,2) Angiopteridaceae (1,1) Annonaceae (4,5) Apocynaceae (9,11) Aquifoliaceae (1,1) Araceae (1,1) Araucariaceae (1,1) Arecaceae (1,1) Aristolochiaceae (1,3) Asclepiadaceae (1,1) •ASTERACEAE (4,5) Barringtoniaceae (1,1) Bignoniaceae (13,15) Bombacaceae (3,3) Bromeliaceae (2,2) Buddlejacaceae (1,1) Burseraceae (1,3) Cactaceae (1,1) Capparidaceae (1,1) Caryophyllaceae (1,1) Celastraceae (2,2) Chrysobalanaceae (1,1) Cistaceae (1,2) Clusiaceae (2,2) Combretaceae (5,11) Convolvulaceae (2,2) Costaceae (1,1) Cupressaceae (2,3) Cyperaceae (1,1) Ehretiaceae (2,9) Eleagnaceae (1,1) Empetraceae (1,1) Ericaceae (1,1) Euphorbiaceae (4,4) Fabaceae (20,36) Flacourtiaceae (7,9) Heliconiaceae (1,1)

* HYPERICACEAE (1,2) Iridaceae (1,1) Lamiaceae (2,2) Lecythidaceae (1,1) Liliaceae (1,1) Lythraceae (1,1) Malphigiaceae (3,5) Malvaceae (1,1) Meliaceae (5,6) Menispermiaceae (2,2) •MORACEAE (7,86) Myrtaceae (1,1) Onagraceae (1,1) Oxalidaceae (1,2) Phileiaceae (1,1) Piperaceae (1,1) Pittosporaceae (1,4) Poaceae (1,1) Podocarpaceae (1,5) Polygalaceae (1,2) Polygonaceae (4,9) Rhamnaceae (2,3) Rosaceae (1,1) Rubiaceae (6,6) •RUTACEAE (23,57) Sapindaceae (3,4) •SAPOTACEAE (7,21) Selaginellaceae (1,1) Simaroubaceae (3,3) Solanaceae (4,8) Sterculiaceae (1,1) Taxaceae (1,1) Theophrastinaceae (1,3) Ulmaceae (1,1) Urticaceae (2,4) Verbenaceae (4,4) Zamiaceae (2,2) Zygophyllaceae (3,4)

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exclusively a tropical/subtropical family (42). Many members of this family are economically important (42-44). Besides the edible fig (mainly Ficus carica). the jakfruit (Artocarpus heterophyllus) and breadfruit (A. atilis) are important food sources throughout the tropics. In addition to their importance as food plants, many members of this family are widely used in treating diseases and other health problems (43-45). Species of Dorstenia are used for everything from mouthwash and hangover cures to emetics and diuretics. Other members provide paper (Broussonetia papyrifera) and rubber (Castilloa elastica). while still others are popular ornamentals. A summary of phototoxicity in the various genera of the Moraceae is shown in Table II (40). Of the eight genera tested, only nine

Table II.

Genus

Distribution of phototoxic activity in extracts of various members of the Moraceae.

#species assayed

Artocarpus Brosimum Cecropia Cudrania Dorstenia FatQua Ficus Morus

4 3 2 1 5 1 69 2

#species with phototoxic activity 0 0* 0 0 5 1 2"

0

* 1 species listed by Murray, £ i .al, 1982. ** 5 additional species listed by Murray, £ l .al, 1982.

species from three genera elicited phototoxic responses from E.coli. Although 72 species of Ficus were assayed, only two, or about 3%, showed phototoxic activity. In contrast, all five species of Dorstenia were phototoxic. Only one species of Fatoua was assayed, which was the third genus that tested positive for photosensitizers. HPLC analysis was performed on the extracts of the Moraceae, and a number of furanocoumarins were identified including psoralen (I) and 5methoxypsoralen (It) (5-MOP). Furanocoumarins are potent photosensitizers and their presence in a small number of Ficus species has already been reported (4642). Table III lists the distribution of furanocoumarins in Ficus. One important point that should be noted is the limited number of Ficus species from which furanocoumarins have been identified. The actual number of Ficus species tested for these compounds is unknown, but furanocoumarins have been detected in only seven species from a genus with roughly 1000 members. Furanocoumarins were also detected in Dorstenia and Fatoua. the only

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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25.

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Phototoxic Metabolites of Tropical Plants

(IX R = H (IIX R = O C H (IIIX R = OOCCH(CH )C^CHCH CH=(CH )

365

3

3

Table III.

Species

F. palmata

£. pymila F. religiosa F. salicifolia

£. sywmorus

3

2

Distribution of furanocoumarins in Ficus determined by HPLC.

psoralen

F. asprima F. carica

2

+ + + +

5-MOP

8-MOP

+ + + + + + +

•

+ detected from species; - not detected from species.

two herbaceous genera in the Moraceae. In addition to psoralen and 5-MOP, a new furanocoumarin was detected. After NMR and mass spectral analysis, the compound was identified as the furanocoumarin 5-EDOP (III) (4g). Unlike psoralen and 5-MOP which are highly phototoxic, 5-EDOP was only slightly antibiotic, and the activity was not enhanced by U V A irradiation. Table IV displays the distribution of furanocoumarins in Dorstenia and Fatoua. Psoralen and 5-MOP, the two highly phototoxic furanocoumarins, are mainly found in root and flowers, particularly in roots. 5-EDOP, the nonphototoxic furanocoumarin is the major furanocoumarin in Dorstenia and is particularly concentrated in leaf tissue. In addition, 5-EDOP was identified in all species of Dorstenia but was absent from Fatoua.

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Table IV. Distribution of furanocoumarins in Dorstenia and Fatoua.

Taxon

psoralen

Dorstenia contrajerva leaves flowers roots Dorstenia foetida leaves roots Dorstenia zanzibrica leaves flowers roots Dorstenia sp. (FTG 80-207) leaves flowers roots Dorstenia sp. (FTG 80-506) leaves flowers roots Fatoua villosa leaves flowers roots

5-MOP

5-EDOP

+ ++

++ +++

++ + ++ ++ +

+

+ +

++ +

+ + ++

++ ++ +++

++ ++ ++

+

+ ++ +++

++ + ++ + ++

+

+ ++

++ + ++ + ++

-

+ ++

- = not detected; + = < 10 ug/gdw; + + = 10-100 ug/gdw; + + + = > 100 ug/gdw.

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The distribution of furanocoumarins in the Moraceae raises an important point. While all of the species of Dorstenia produce furanocoumarins, this ability is found in only a small percentage of Ficus species. The evolutionary relationships within the Moraceae are still in question (42), but it is generally agreed that Dorstenia is an evolutionary advanced genus in the family. It is possible and even probable that the ancestor to this genus possessed the ability to synthesize furanocoumarins. It may even be possible that Dorstenia evolved from a furanocoumarin-producing species of Ficus. From an ecological perspective, the presence of furanocoumarins in Dorstenia and Fatoua is interesting as well. In contrast to the rest of the Moraceae, both Dorstenia and Fatoua are herbaceous. This might suggest that furanocoumarins serve a more important purpose in small, herbaceous plants than they do in large, woody plants. Even limited herbivory would have a severe effect on plants with small leaf areas such as Dorstenia and Fatoua due to loss of limited nutrients and photosynthetic ability. Although 5-EDOP does not demonstrate the phototoxic ability of either psoralen or 5-MOP, it may serve as feeding deterrent to potential herbivores. We are currently preparing to test this hypothesis. A final observation brought to light by these data involves the distribution of furanocoumarins throughout the individual Dorstenia plants. The phototoxic furanocoumarins, psoralen and 5-MOP are more concentrated in the roots of Dorstenia than above ground parts while the highest concentrations of the nonphototoxic furanocoumarin, 5-EDOP, are in the leaves. A first glance, it would seem logical for phototoxic chemicals to be concentrated in an environment where light was present in order to utilize the full potential of their toxicity. Although some light is transmitted from leaves to other parts of the plant (49). the levels of activating wavelengths are probably too low to elevate furanocoumarins to their reactive state. Recently, however, it was suggested that mechanisms other than light may activate these phototoxins (5Q). Photochemicaltype reactions can occur in the absence of light (51-52^ and it has been suggested that enzymes such as peroxidase may be capable of catalyzing such reactions. Peroxidase is a common enzyme in plants and levels of peroxidase increase drastically in the roots of many plants that have been infected or wounded by invaders. This enzyme may provide the means by which furanocoumarins could be activated to their most toxic state in these tissues. Conclusions Plants are capable of producing a seemingly limitless number of compounds, many of which have proven to be toxic to one or more types of organisms. Phototoxic metabolites are more limited in their distribution, but have a broad spectrum of biocidal activity and appear to provide their hosts with formidable chemical defenses against potential invaders. Certain plant families such as the Rutaceae, Asteraceae, and Apiaceae are well known for their phototoxic abilities while this characteristic is less common in others. The distribution of phototoxic furanocoumarins in the Moraceae provides us with an opportunity to study what role these chemicals may have played in the evolution of this family as well as what function they may serve today. The

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concentration of phototoxins in the roots of Dorstenia is by no means unique. Phototoxic thiophenes and polyacetylenes are commonly found in the roots of members of the Asteraceae, so similar roles for these compounds appears plausible. Further investigations are needed to clarify their importance to the host plants, and plants such as Dorstenia can serve as useful tools in this venture. Acknowledgments We thank Dr. J.M.E. Quirke, Dr. Stephen A. Winkle, Dr. Mehrzad Mehran, and Lavina Faleiro for their technical assistance. We also thank Dr. Robert Knight and the USDA Plant Induction Station at Chapman Field for many of the plant specimens. This work was supported by grants from the Whitehall Foundation and NSF (BBS-8613863) to KRD. Literature cited 1. 2. 3. 4.

5.

6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

18.

Towers, G.H.N. J. Chem. Ecol. 1986, 12, 813-821. Downum, K.R.; Rodriguez, E. J. Chem. Ecol. 1986, 12, 823-834. Towers, G.H.N. Can. J. Bot. 1984, 62, 2900-2911. Arnason, T.; Swain, T.; Wat, C.-K.; Graham, E.A.; Partington, S.; Towers, G.H.N. Biochem. Svst. Ecol. 1981, 9, 63-68. Downum, K.R. In Natural Resistance of Plants to Pests: Roles of Allelochemicals; Green, M.; Hedin, P., Eds.; ACS Symposium Series No. 296; American Chemical Society: Washington D.C., 1986, pp.197205. Knox, J.P.; Dodge, A.D.Phytochem.1985,24,889-896. Downum, K.R.; Nemec, S. In Light-Activated Pesticides; Heitz, J.R.; Downum, K.R., Eds.; ACS Symposium Series No. 339; American Chemical Society: Washington D.C., 1987, pp. 281-294. Tuveson, R.W.; Berenbaum, M.R.; Heininger, E.E. J. Chem. Ecol. 1986,12,933-948. Marston, A.; Hostettmann, K.Phytochem.1985, 24, 639-652. Hanson, C.V.; Riggs, J.L.; Lennette, E.H. J. Gen. Virol. 1978, 40, 345. Specht, K.G.; Kittler, L.; Midden, W.R. Photochem. Photobiol. 1988, 47, 537. Hudson, J.B. Antiviral Compounds fromPlants;CRC Press: Boca Raton, 1990, pp. 67-81. Oertli, E.H.; Rowe, L.D.; Lovering, S.L.; Ivie, G.W.; Bailey, E.M. Am. J. Vet. Res. 1983, 44, 1126-1129. Clare, N.T. Adv. Vet. Sci. 1955, 2,182-211. Martins, J.E.C.; Puzetti, G.L.; Sodre, M. Derm. Int. 1974, 13, 124. Birmingham,D.J.;Key, M.; Tubich, G.E.; Perone, V.B. Arch. Dermatol.1961,83,73. Ivie, G.W. In Effects of Poisonous Plants onLivestock;Keeler, R.; Van Kampen, K.; James, L., Eds.; Academic Press: New York, 1977, pp.475485. Fitzpatrick, T.B.; Pathak, M.A. J. Invest. Dermatol. 1959, 32, 229.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

25.

SWAIN AND DOWNUM

Phototoxic Metabolites of Tropical Plants

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19.

369

Mitchell, J.; Rook, A. Botanical Dermatology; Greengrass: Vancouver, 1979, pp.471-482. 20. Girotti, A.W. Photochem. Photobiol. 1983, 38, 745-751. 21. Musajo, L.; Rodighiero, G. In Photophysiology, VII: A.C.Giese, Ed.; Academic Press: New York, 1972, pp.115-147. 22. Song, P.; Tapley, K.J. Photochem. Photobiol. 1979, 29, 1177-1197. 23. Scheel, L.D.; Perone, V.B.; Larkin, R.L.; Kupel, R.E. Biochemistry 1963, 2, 1127. 24. Veronese, F.M.; Schiavon, O.; Bevilacqua, R.; Bordin, F.; Rodighiero, G. Photochem. Photobiol. 1982, 36, 25-30. 25. Granger, M.; Helene, C. Photochem. Photobiol. 1983, 38, 563-568. 26. Berenbaum, M.Science,1978,201,532-534. 27. Berenbaum, M. Ecol. Entomol. 1981, 6, 345-351. 28. Berenbaum, M. Ecology 1981, 62, 1254-1266. 29. Berenbaum, M. Evolution 1983, 37, 163. 30. Berenbaum, M. In Light-ActivatedPesticides;Heitz, J.R.; Downum, K.R., Eds. ACS Symposium Series No. 339; American Chemical Society: Washington D.C., 1987, pp. 206-216. 31. Berenbaum, M.; Feeny, P. Science 1981, 212, 927-929. 32. Ivie, G.W.; Bull, D.L.; Beier, R.C.; Pryor, N.W. J. Chem. Ecol. 1986, 12, 871-884. 33. Bull, D.L.; Ivie, G.W.; Beier, R.C.; Pryor, N.W. J. Chem. Ecol. 1986, 12, 885-892. 34. Ivie, G.W.; Bull, D.L.; Beier, R.C.; Pryor, N.W. Science 1983, 221, 374376. 35. Bull, D.L.; Ivie, G.W.; Beier, R.C.; Pryor, N.W.; Oertli, E.H. J. Chem. Ecol. 1984,10,893-911. 36. Ivie, G.W. In Light-ActivatedPesticides;Heitz, J.R.; Downum, K.R., Eds. ACS Symposium Series No. 339; American Chemical Society: Washington D.C., 1987, pp. 217-230. 37. Larson, R.A. J. Chem. Ecol. 1986, 12, 859-870. 38. Downum, K.R.; Villegas, S.; Rodriguez, E.; Keil, D.J. J. Chem. Ecol. 1989, 15, 345-355. 39. Downum, K.R.; Hancock, R.E.W.; Towers, G.H.N. Photochem. Photophys.1983.6,145-152. 40. Swain, L.A.; Downum, K.R. Biochem. Syst. Ecol. 1990. (in press). 41. Downum, K.R.; Swain, L.A. Fairchild Tropical Gard. Bull. 1988, 43, 611. 42. Corner, E.J.H. Gardens Bulletin (Singapore). 1962, 19, 187-252. 43. Morton, J. Fruits from Warm Climates; J.Morton: Miami, 1987, pp.4764. 44. Usher, G. A Dictionary of Plants Used by Man; Constable & Co., Ltd.: London, 1974, pp. 62-63, 254-257. 45. Ayensu, E.S. Medicinal Plants of the WestIndies;Reference Publications: Algonac, MI, 1981, pp.127-129. 46. Murray, R.D.H.; Mendez, J.; Brown, S.A. The Natural Coumarins; J.Wiley & Sons: New York, 1982.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

370

47. 48. 49. 50.

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51. 52.

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Yarosh, E.A.; Nikonov, G.K. Khim. Prir. Soedin.1973,9,269-270. Swain, L.A.; Quirke, J.M.E.; Winkle, S.A.; Downum, K.R. Phytochem. 1990, (in review). Mandoli, D.F.; Briggs, W.R. Photochem. Photobiol.1984,39,709-715. Gommers, F.J.; Bakker, J. In Chemistry and Biology of Naturally­ -occurring Acetylenes and RelatedCompounds;Lam J.; Breteler, H.; Arnason, T.; Hansen, L., Eds. Elsivier: Amsterdam, 1988, pp. 61-69. Cilento, G. Photochem. Photobiol. Rev. 1980, 5, 199-228. Cilento, G. Pure Appl. Chem.1984,56,1179-1190.

RECEIVED July31,1990

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Chapter 26

Photosensitizing Porphyrins as Herbicides 1

Stephen O . Duke, Josfe M. Becerril , Timothy D . Sherman, and Hiroshi Matsumoto 2

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Southern Weed Science Laboratory, Agricultural Research Service, U . S . Department of Agriculture, P.O. Box 350, Stoneville, M S 38776

Several porphyrin intermediates of heme and/or chlorophyll biosynthesis are potent photosensitizers which generate high levels of singlet oxygen in the presence of molecular oxygen and light. Many compounds that affect the heme and/or chlorophyll pathways are strongly herbicidal due to accumulation of phytotoxic levels of these porphyrins in response to the chemical. For instance, several commercial and experimental herbicides inhibit protoporphyrinogen oxidase, the enzyme that converts protoporphyrinogen to protoporphyrin IX (PPIX). This leads to uncontrolled autooxidation of the substrate and results in massive accumulation of PPIX. In plants treated with these herbicides, damage is light dependent and closely correlated with the level of PPIX that accumulates. PPIX accumulation is apparently largely extraplastidic. Treatment with the porphyrin precursor δ-aminolevulinic acid (ALA), in combination with the heme and chlorophyll pathway inhibitor 2,2'-dypyridyl (DP), results in the accumulation of toxic levels of primarily Mg-PPIX monomethylester. DP deregulates porphyrin synthesis and ALA provides additional substrate. DP and other chlorophyll synthesis modulators in combination with ALA can increase the selectivity as well as enhance the efficacy of ALA as a herbicide. Exogenously applied porphyrins are far less effective as herbicides than treatment with compounds that cause plants to accumulate their own porphyrins. P h o t o d y n a m i c compounds, i n c l u d i n g many n a t u r a l p r o d u c t s , h a v e b e e n p r o p o s e d f o r u s e a s h e r b i c i d e s b y many r e s e a r c h e r s ( e . g . , 1 - 3 ) . Current address: Universidad del Pais Vasco-EHU, Facultad de Ciencias, Dept. Fisiologia Vegetal y Ecologia, Apartado 644, '18080 Bilbao, Spain 2

Current address: Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

NATURALLY OCCURRING PEST BIOREGULATORS

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However, a problem w i t h t h e s e c h e m i c a l s i s t h e i r i n d i s c r i m i n a t e t o x i c i t y i n t h e p r e s e n c e o f l i g h t and m o l e c u l a r oxygen. This t o x i c i t y p r e c l u d e s t h e i r use a s p e s t i c i d e s . S a f e a l t e r n a t i v e s are to t r e a t t a r g e t organisms with chemicals that e i t h e r are s e l e c t i v e l y m e t a b o l i z e d t ophotodynamic compounds o r cause t h e t a r g e t o r g a n i s m t o produce t o x i c l e v e l s o f n a t u r a l photodynamic compounds w i t h i t s own b i o c h e m i c a l m a c h i n e r y . T h i s r e v i e w examines one a s p e c t o f t h e l a t t e r a l t e r n a t i v e - t r e a t m e n t o f p l a n t s w i t h compounds t h a t cause the accumulation o fh e r b i c i d a l l e v e l s o f photodynamic p o r p h y r i n s . C h l o r o p h y l l i s a p h o t o d y n a m i c compound when i t i s n o t a c o m p o n e n t o f t h e p h o t o s y n t h e t i c a p p a r a t u s . One w a y i n w h i c h t h e p l a n t p r o t e c t s i t s e l f from the photodynamic p r o p e r t i e s o f c h l o r o p h y l l i s through l i n k i n g c h l o r o p h y l l t oa biochemical complex that d i s s i p a t e s the energy o fl i g h t - e n e r g i z e d c h l o r o p h y l l through s p l i t t i n g o f w a t e r and e n e r g i z i n g o f e l e c t r o n s f r o m w a t e r f o r e n e r g y t r a n s d u c t i o n and p h o t o s y n t h e t i c r e d u c i n g power. I n t e r m e d i a t e s o f c h l o r o p h y l l b i o s y n t h e s i s are photodynamic a l s o . Since they cannot be u t i l i z e d i n p h o t o s y n t h e s i s and a r e p h o t o d y n a m i c , t h e r e i s s t r o n g s e l e c t i o n p r e s s u r e a g a i n s t accumulation o f these compounds. M u t a t i o n s t h a t cause the a c c u m u l a t i o n o f t h e s e compounds are d e l e t e r i o u s t othe p l a n t . For i n s t a n c e , y e l l o w mutants of maize have been d e s c r i b e d t h a t accumulate h i g h l e v e l s o fp r o t o p o r p h y r i n IX (PPIX) (4). A l t h o u g h t h e s e mutants have i m p a i r e d c h l o r o p h y l l s y n t h e s i s , p a r t o f t h e p h y t o t o x i c e f f e c t o f t h e m u t a t i o n i s due t o the photodynamic e f f e c t o f PPIX. Two a p p r o a c h e s t o s t i m u l a t i o n o f p o r p h y r i n a c c u m u l a t i o n i n p l a n t s have been t a k e n . The f i r s t i s t o s u p p l y t h e p l a n t w i t h t h e p o r p h y r i n p r e c u r s o r 6 - a m i n o l e v u l i n c a c i d (ALA) a l o n g w i t h compounds t h a t a f f e c t t h e p o r p h y r i n pathway. The second i s t o b l o c k porphyrin synthesis a tthe protoporphyrinogen oxidase step i n the pathway, t h e r e b y d e r e g u l a t i n g t h e pathway and c a u s i n g a c c u m u l a t i o n p r i m a r i l y o f PPIX. ALA a s a H e r b i c i d e R e b e i z e t a l . (5) i n t r o d u c e d t h e c o n c e p t o f ALA i n c o m b i n a t i o n w i t h various c h l o r o p h y l l synthesis modulators as a h e r b i c i d e . Previous l i t e r a t u r e had d e m o n s t r a t e d t h a t ALA t r e a t m e n t o f p l a n t t i s s u e s could cause the accumulation o fabnormally high l e v e l s o f c o p r o p o r p h y r i n , p r o t o c h l o r o p h y l l i d e ( P C h l i d e ) , PPIX, a n d M g - p r o t o p o r p h y r i n IX monomethyl e s t e r (MgPPIXME) (6, 7 ) . I n cucumber s e e d l i n g s s p r a y e d w i t h 1 0 t o 2 0 mM A L A , a n d t h e n p l a c e d i n t h e d a r k f o r 17 h t o a l l o w c h l o r o p h y l l p r e c u r s o r s t o a c c u m u l a t e , a two- t o f o u r - f o l d i n c r e a s e i n t o t a l p o r p h y r i n s ( p r i m a r i l y PChlide) was o b s e r v e d ( 5 ) . T h i s l e d t o 95 % photodynamic damage t o t h e s e e d l i n g s a f t e r t h e y were p l a c e d i n t h e l i g h t . The e f f e c t i v e l e v e l o f a p p l i e d ALA c o u l d b e r e d u c e d b y s p r a y i n g i t i n c o m b i n a t i o n w i t h 2 , 2 ' - d i p y r i d y l (DP), a r e l a t i v e l y i n e x p e n s i v e s y n t h e t i c compound t h a t s t i m u l a t e s p o r p h y r i n s y n t h e s i s b y p r e v e n t i n g heme s y n t h e s i s t h r o u g h c h e l a t i n g i r o n ( 8 ) . The p o r p h y r i n s y n t h e s i s pathway i s u n d e r s t r o n g f e e d b a c k c o n t r o l b y heme ( 9 , 1 0 ) . I n a d d i t i o n t o s t i m u l a t i n g p o r p h y r i n s y n t h e s i s , DP b l o c k s c o n v e r s i o n o f MgPPIXME t o P C h l i d e (8, 9 ) ( F i g . 1 ) . Thus, both t h e q u a n t i t y and t y p e o f porphyrins that accumulate are a f f e c t e d .

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A L A p l u s DP a c t e d s y n e r g i s t i c a l l y a s h e r b i c i d e s , d e s p i t e t h e f a c t t h a t t h e r e was g e n e r a l l y o n l y an a d d i t i v e e f f e c t o n t o t a l p o r p h y r i n a c c u m u l a t i o n ( T a b l e I ) . T h e s e r e s u l t s i n d i c a t e one o r more o f t h e f o l l o w i n g : (a) one o f t h e e a r l i e r c h l o r o p h y l l i n t e r m e d i a t e s (MgPPIXME o r PPIX) i s more h e r b i c i d a l t h a n P C h l i d e , (b) t h e p o r p h y r i n s a c t s y n e r g i s t i c a l l y , o r (3) t h a t t h e s y n e r g i s m o f A L A p l u s DP i s n o t b a s e d o n t h e i r e f f e c t s o n p o r p h y r i n s . T a b l e I . E f f e c t s o f A L A a n d DP, a l o n e o r i n c o m b i n a t i o n , o n p o r p h y r i n a c c u m u l a t i o n and h e r b i c i d a l damage. Cucumber s e e d l i n g s (6-day-old) were assayed f o r p o r p h y r i n s a f t e r being sprayed w i t h t h e h e r b i c i d e s a n d i n c u b a t e d i n d a r k n e s s f o r 17 h . H e r b i c i d a l d a m a g e was a s s e s s e d 10 d a y s a f t e r p o r p h y r i n s w e r e a s s a y e d a n d d u r i n g which they were exposed t o greenhouse l i g h t c o n d i t i o n s . Taken from r e f . ( 5 ) . Treatment Control 5 mM A L A 15 mM DP 5 mM A L A + 15 mM a * ,

P o r p h y r i n s ( n m o l e s / 1 0 0 mg PChlide MaPPIXME PPIX 0.0 17.3 0.6 0.0 100.7 1.6 24.0 2.6 12.3 DP 121.1 8.1 26.3

protein) D a m a a e (%) Total 0 17.9 30 101.3 10 38.9 80 155.5

The f i r s t and s e c o n d p o s s i b i l i t i e s a r e c o m p l i c a t e d by t h e p o s s i b i l i t y that these porphyrins are a l l d i f f e r e n t i a l l y p h o t o l a b i l e a n d p h o t o d e g r a d a t i o n p r o d u c t s may b e i n v o l v e d i n t h e i r p h o t o d y n a m i c action. For instance, PChlide disappears i n green t i s s u e r a p i d l y a f t e r e x p o s u r e t o l i g h t (5) ( F i g . 2 ) , a l t h o u g h t h e p r o p o r t i o n s c o n v e r t e d t o c h l o r o p h y l l o r p h o t o d e g r a d e d a r e n o t k n o w n . Some e v i d e n c e i n d i c a t e s t h a t most o f the A L A - s t i m u l a t e d P C h l i d e a c c u m u l a t i o n i s p h o t o d e g r a d e d ( 6 ) , a l t h o u g h t h i s may n o t a l w a y s b e t h e c a s e . MgPPIXME l e v e l s i n cucumber c o t y l e d o n s d e c l i n e l e s s r a p i d l y i n l i g h t t h a n do t h o s e o f P C h l i d e ( F i g . 2 ) . In normal e t i o l a t e d p l a n t s , complete PChlide phototransformation of chlorophyllide (Chlide) in bright light i s very rapid (usually less t h a n a m i n u t e ) and s u b s e q u e n t p h y t y l l a t i o n o f C h l i d e t o f o r m c h l o r o p h y l l ( C h i ) i s c o m p l e t e i n 30 m i n o r l e s s ( e . g . , H ) . The p r o l o n g e d d e c a y o f P C h l i d e i n t i s s u e t r e a t e d w i t h A L A p l u s DP c o u l d be due t o s y n t h e s i s o c c u r r i n g more r a p i d l y t h a n n o r m a l r a t e s o f c o n v e r s i o n t o C h i , t o slowed p h o t o t r a n s f o r m a t i o n and p h y t y l l a t i o n , o r t o a l a r g e component o f n o n - p h o t o t r a n s f o r m a b l e (NPTF) P C I i d e . Gassman (12) f o u n d t h a t extended t r e a t m e n t o f e t i o l a t e d bean l e a v e s w i t h 10 mM A L A r e s u l t e d i n a g r e a t e r p o r p o r t i o n o f N P T F P C I i d e a c c u m u l a t i o n t h a n i n u n t r e a t e d l e a v e s and t h a t NPTF P C I i d e i n h i b i t s a c c u m u l a t i o n o f p h o t o t r a n s f o r m a b l e (PTF) P C I i d e . T h u s , NPTF P C I i d e may p l a y a r o l e a s a p h o t o d y n a m i c p i g m e n t i n t h i s s y s t e m , a l t h o u g h R e b e i z et a / . ( 5 ) d i d n o t d i f f e r e n t i a t e b e t w e e n P T F a n d N P T F PCIide. I n t h e o r i g i n a l s y s t e m o f R e b e i z et al. ( 5 ) , a r a t h e r l o n g p o s t - s p r a y d a r k p e r i o d was r e q u i r e d f o r s u f f i c i e n t a c c u m u l a t i o n o f p o r p h y r i n s f o r h e r b i c i d a l a c t i v i t y o c c u r . The h e r b i c i d a l e f f e c t o f A L A p l u s DP was a g e a n d s p e c i e s d e p e n d e n t ; h o w e v e r , t h e r e was n o t a l w a y s a s t r o n g c o r r e l a t i o n b e t w e e n t h e e f f e c t on p o r p h y r i n

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

374

NATURALLY OCCURRING PEST

BIOREGULATORS

Gtutamate

Gabacullne

^ ALA

DHA

Levullnate

Membrane lipid peroxidation

Porphobilinogen Photobleaching

^

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herbicides -

^ Protoporphyrin IX

Protoporphyrinogen-^

^

r

oxygen

'2 Protoporphyrin IX

8

Cytochromes

M g

+

^ ^

Light +

0

2

Mg Protoporphyrin IX M g PPIX M E ^^2,2-dlpyridyl Protochlorophyllide Chlorophyllide Chlorophyll a

F i g u r e 1. T h e p o r p h y r i n s y n t h e s i s p a t h w a y a n d s i t e s o f i n h i b i t i o n o f v a r i o u s i n h i b i t o r s and m o d u l a t o r s . Inhibitors are u n d e r l i n e d and s i t e s o f i n h i b i t i o n a r e i n d i c a t e d

F i g u r e 2. T i m e c o u r s e o f P C h l i d e a n d M g - P P I X M E d i s a p p e a r a n c e from ALA p l u s D P - t r e a t e d cucumber s e e d l i n g s i n d a y l i g h t a f t e r a 17-h a c c u m u l a t i o n p e r i o d . (Reprinted with permission from ref. 5. Copyright 1984 Butterworth Scientific, Ltd.)

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

26.

DUKEETAL.

375

Photosensitizing Porphyrins as Herbicides

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s y n t h e s i s a n d h e r b i c i d a l d a m a g e . No h e r b i c i d a l d a m a g e was o b s e r v e d w h e n t h e r e was l i t t l e o r no e f f e c t o n c h l o r o p h y l l p r e c u r s o r l e v e l s b u t , i n some t i s s u e s o f s o m e s p e c i e s , a c c u m u l a t i o n o f p o r p h y r i n s d i d not r e s u l t i n h e r b i c i d a l damage ( T a b l e I I ) . G e n e r a l l y , t h e y found g r a s s e s , i n c l u d i n g m a i z e , b a r l e y , o a t , and w h e a t t o be t o l e r a n t t o t h e s e h e r b i c i d e s , w h i l e a l l d i c o t weeds t e s t e d were h i g h l y s e n s i t i v e . Thus, t h i s h e r b i c i d e c o m b i n a t i o n showed promise f o r b r o a d l e a f weed c o n t r o l i n g r a i n c r o p s . T a b l e I I . E f f e c t s o f A L A (5mM) p l u s DP ( 1 5 mM) o n p o r p h y r i n accumulation in seedling tissues of several plant species. Plant were assayed f o r p o r p h y r i n s a f t e r being sprayed with the h e r b i c i d e s a n d i n c u b a t e d i n d a r k n e s s f o r 17 h . H e r b i c i d a l d a m a g e was a s s e s s e d 10 d a y s a f t e r p o r p h y r i n s w e r e a s s a y e d a n d s u b s e q u e n t e x p o s u r e t o greenhouse l i g h t c o n d i t i o n s . Taken from r e f . (5). Species ( S e e d l i n g age)

Porphyrins ( n m o l e s / 1 0 0 mg p r o t e i n ) Herbicidal PChlide MgPPIXME PPIX D a m a g e (%) Con T r e a t e d Con T r e a t e d Con T r e a t e d 90 ( 1 2 ) 30 24 29 36 201 12

Mustard leaves Cotton cotyledon (14) 18 C o t t o n stem (14) 4 Kidney bean l e a f (9) 117 Kidney bean stem (9) 37 G i a n t f o x t a i l (6) 8 M a i z e (9) 79 small n e c r o t i c areas

37 4

4 1

9 1

0 0

0 0

63 0

439

3

430

5

22

100

3 0 12

14 14 0

0 S.N. S.N.

82 79 85

4 0. ,4 5

76 12 15

a

R e b e i z e t a7. (13) c l a s s i f i e d s p e c i e s i n t o f o u r d i f f e r e n t g r e e n i n g g r o u p s , b a s e d on t h e i r C h i s y n t h e s i s h e t e r o g e n e i t y . The f o u r groups were c a t e g o r i z e d a c c o r d i n g t o the predominance o f d i v i n y l o r m o n o v i n y l p o r p h y r i n s y n t h e s i s and under what c o n d i t i o n s ( d a r k o r l i g h t ) s y n t h e s i s o f each p o r p h y r i n t y p e o c c u r r e d . The f o u r groups are described with r e p r e s e n t a t i v e species i n Table I I I . Table I I I . Greening groups

and r e p r e s e n t a t i v e p l a n t s p e c i e s .

Greening Group 1. D a r k d i v i n y l / 1 i g h t d i v i n y l

Representative

Species

(DDV/LDV)

Cucumber, mustard, common p u r s l a n e 2. D a r k m o n o v i n y l / 1 i g h t d i v i n y l ( D M V / L D V ) Maize, wheat, b a r l e y , common b e a n , s o y b e a n , pigweed 3. D a r k d i v i n y l / 1 i g h t m o n o v i n y l ( D D V / L M V ) Ginkgo 4. D a r k m o n o v i n v l / 1 i g h t m o n o v i n y l (DMV/LMV) A p p l e , . i o h n s o n q r a s s T h e y h y p o t h e s i z e d t h a t t h e s e n s i t i v i t y o f a s p e c i e s t o ALA p l u s c h l o r o p h y l l m o d u l a t o r s i s due t o both e x t e n t o f p o r p h y r i n s y n t h e s i s and t h e c h e m i c a l n a t u r e o f t h e a c c u m u l a t e d p o r p h y r i n s . T h u s , t h e greening group to which a s p e c i e s belongs c o u l d s t r o n g l y i n f l u e n c e

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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376

NATURALLY OCCURRING PEST BIOREGULATORS

i t s s u s c e p t i b i l i t y . I t f o l l o w e d t h a t s i n c e some s p e c i e s a c c u m u l a t e r e l a t i v e l y l a r g e amounts o f p o r p h y r i n s i n r e s p o n s e t o ALA, b u t d i s p l a y l i t t l e h e r b i c i d a l damage, some p o r p h y r i n s m i g h t b e r e l a t i v e l y poor p h o t o s e n s i t i z e r s . T h i s hypothesis was t e s t e d by u s i n g ALA i n c o m b i n a t i o n w i t h c h l o r o p h y l l s y n t h e s i s m o d u l a t o r s o t h e r t h a n DP ( 1 3 ) . Although the r e s u l t s of these extensive experiments are not e a s i l y i n t e r p r e t e d , t h e h y p o t h e s e s w e r e made (a) t h a t P C h l i d e i s t h e most i m p o r t a n t and u b i q u i t o u s p h o t o d y n a m i c s p e c i e s c a u s e d t o a c c u m u l a t e b y A L A - b a s e d t r e a t m e n t s , ( b ) t h a t MV P C h l i d e i s a m o r e e f f e c t i v e p h o t o d y n a m i c p i g m e n t t h a n DV P C h l i d e i n D D V / L D V a n d DMV/LDV s p e c i e s , a n d ( c ) t h a t b o t h D D V / L D V a n d DMV/LDV s p e c i e s a r e h i g h l y s u s c e p t i b l e t o a m i x t u r e o f Mg-PPIX and Mg-PPIXME ( 1 4 ) . T h e r e s u l t s are d i f f i c u l t t o i n t e r p r e t because equimolar l e v e l s o f d i f f e r e n t p o r p h y r i n s were n o t p r o d u c e d and t h e c o m b i n a t i o n s o f porphyrins produced by d i f f e r e n t modulators v a r i e d with s p e c i e s . P o t e n t i a l d i f f e r e n c e s i n t o l e r a n c e to t o x i c oxygen s p e c i e s between s p e c i e s were not c o n s i d e r e d . Others have attempted t o e x p l a i n d i f f e r e n t i a l s e n s i t i v i t y to p o r p h y r i n - g e n e r a t i n g h e r b i c i d e s between s p e c i e s (15) and between h e r b i c i d e - s e n s i t i v e b i o t y p e s w i t h i n s p e c i e s (16) b y d i f f e r e n c e s i n a b i l i t y t o d e t o x i f y t o x i c oxygen s p e c i e s . A s w i t h o t h e r h e r b i c i d e s , p e n e t r a t i o n o f t h e l e a f c u t i c l e b y ALA a n d / o r DP c a n a l s o p l a y a r o l e i n d i f f e r e n c e s i n e f f i c a c y o f t h i s h e r b i c i d e combination (17). The Chi s y n t h e s i s m o d u l a t o r s t h a t R e b e i z e t a/. (13, 1 4 ) u s e d i n c o n j u n c t i o n w i t h ALA c o u l d b e d i v i d e d i n t o t h r e e c a t e g o r i e s : A) e n h a n c e r s o f ALA c o n v e r s i o n t o p o r p h y r i n s ( 2 - p y r i d i n e a l d o x i m e , 2-pyridine aldehyde, p i c o l i n i c acid, 2,2'dipyridyl d i s u l f i d e , 2 , 2 ' - d i p y r i d y l amine, 4 , 4 ' d i p y r i d y l , and p h e n a n t h r i d i n e ) , B) i n d u c e r s o f ALA b i o s y n t h e s i s and p o r p h y r i n a c c u m u l a t i o n ( 2 , 2 ' - d i p y r i d y l a n d 1 , 1 0 - p h e n a n t h r o l i n e ) , a n d C ) i n h i b i t o r s o f MV PChlide synthesis (2,3-dipyridyl, 2,4-dipyridyl, 1,7-phenanthroline, and 4 , 7 - p h e n a n t h r o l i n e ) . Compounds i n group A d i d not cause s i g n i f i c a n t p o r p h y r i n accumulation alone; however, they enhanced d a r k c o n v e r s i o n o f e x o g e n o u s ALA t o p o r p h y r i n s . T h i s g r o u p w a s f u r t h e r s u b d i v i d e d i n t o compounds t h a t e n h a n c e d c o n v e r s i o n o f ALA t o MV P C h l i d e ( 2 - p y r i d i n e a l d o x i m e , 2 - p y r i d i n e a l d e h y d e , p i c o l i n i c a c i d , and 2 , 2 ' - d i p y r i d y l d i s u l f i d e ) and t h o s e t h a t s t i m u l a t e d c o n v e r s i o n t o DV P C h l i d e ( 4 , 4 ' d i p y r i d y l , 2 , 2 ' d i p y r i d y l a m i n e , a n d p h e n a n t h r i d i n e ) . T o q u a l i f y a s a n ALA b i o s y n t h e s i s and p o r p h y r i n a c c u m u l a t i o n i n d u c e r ( c a t e g o r y B ) , t h e compound had t o c a u s e t h e s e e f f e c t s i n t h e a b s e n c e o f ALA. Compounds i n c a t e g o r y C had t o i n h i b i t a c c u m u l a t i o n o f MV P C h l i d e w i t h o r w i t h o u t A L A . In most c a s e s , i n c o n j u n c t i o n w i t h A L A , t h e c o m p o u n d s s t i m u l a t e d DV P C h l i d e accumulation compared t othe ALA-treated c o n t r o l . With knowledge o f g r e e n i n g group c h a r a c t e r i s t i c s and m o d u l a t o r t y p e , one can t h e o r e t i c a l l y m a n i p u l a t e t h e s e l e c t i v i t y o f ALA-modulator combinations. T h i s approach was used to d e s i g n a c o m b i n a t i o n f o r c o n t r o l o f c r e e p i n g c h a r l i e (DDV/LDV) i n K e n t u c k y b l u e g r a s s ( D M V / L D V ) ( 1 3 ) . S i n c e t h e g r e e n i n g t y p e o f t h e two s p e c i e s d i v e r g e d a t n i g h t , a " d a r k s p r a y " a p p l i e d n e a r d u s k was t h e o r i z e d t o be m o s t s e l e c t i v e . H o w e v e r , a f i n a l c h o i c e o f t h e ALA p l u s DP c o m b i n a t i o n w a s made s i n c e t h i s c o m b i n a t i o n l e d t o t h e g e n e r a l l y l e t h a l a c c u m u l a t i o n o f DV M g P P I X M E a n d DV M g P P I X i n m o s t s p e c i e s .

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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26.

D U K E E T AL.

Photosensitizing Porphyrins as Herbicides

377

H o w e v e r , K e n t u c k y b l u e g r a s s w a s g e n e r a l l y much l e s s s e n s i t i v e , despite these accumulations. The mechanism o f t o l e r a n c e was not explained. R e c e n t l y , A v e r i n e t a/. (18) found t h a t more A L A s u p p l i e d t o barley accumulated as porphobilinogen, r a t h e r than being converted t o p o r p h y r i n s , than i n bean. A l s o , the p o r p h y r i n s i n b a r l e y were d e g r a d e d i n l i g h t much more r a p i d l y t h a n i n b e a n . T h u s , some o f t h e s e l e c t i v i t y o f A L A p l u s DP may b e i n f l u e n c e d b y many f a c t o r s o t h e r t h a n g r e e n i n g t y p e . T h e u l t r a s t r u c t u r a l d e v e l o p m e n t o f damage from t r e a t m e n t w i t h A L A p l u s DP i n b e a n c o t y l e d o n s o c c u r s r a p i d l y ( 1 9 ) . W i t h i n 1 h o f exposure t o l i g h t c h l o r o p l a s t s s w e l l e d a n d became s p h e r i c a l . Subsequently, grana andstromal t h y l a k o i d s swelled and d e s t r u c t i o n o f i n t r a c h l o r o p l a s t membranes was o b s e r v e d b y 2 h . W i t h i n 6 t o 24 h , c h l o r o p l a s t s and m i t o c h o n d r i a were b r o k e n . T o d a t e , o n l y s i x p u b l i c a t i o n s ( 5 , 13, 1 4 > 1Z-12) e x i s t o n A L A a s a h e r b i c i d e . A n o t h e r paper has been p u b l i s h e d o n A L A a s a n i n s e c t i c i d e (20). Although most o f t h e s e are h i g h l y s u b s t a n t i a l papers, several questions remain regarding r e s u l t s o f these s t u d i e s . The a c t u a l r e l a t i v e p h y t o t o x i c i t y o f v a r i o u s p o r p h y r i n s i s not clear. The i n t r a c e l l u l a r s i t e ( s ) o fporphyrin accumulation are also n o t known. F u r t h e r m o r e , t h e complex i n t e r a c t i o n s between g r e e n i n g t y p e , t o l e r a n c e t o t o x i c oxygen s p e c i e s , and c a p a c i t y t o s y n t h e s i z e porphyrins i s poorly understood. Although A L A i n combination with v a r i o u s Chi s y n t h e s i s modulators has been p a t e n t e d f o r h e r b i c i d e use, none o f t h e c o m b i n a t i o n s i s p r e s e n t l y c o m m e r c i a l l y a v a i l a b l e . However, a l a r g e number o f synthetic herbicides that act by causing the accumulation o f photodynamic porphyrins are sold throughout the world. Synthetic Herbicide-Induced Accumulation

o f Porphyrins

H i s t o r i c a l background. Diphenyl ethers o f the general s t r u c t u r e shown b e l o w were i n t r o d u c e d a s commercial h e r b i c i d e s i n t h e 1960's (21) a n d s i n c e t h a t t i m e many members o f t h i s h e r b i c i d e c l a s s h a v e been commercialized (22). A l l o f the commercialized v e r s i o n s have been p a r a - n i t r o s u b s t i t u t e d . _ ni

™ CF3,

CI

R

2

= CI. NC*

R

3

-

R4 -

OCH3. COOCH3. OCjHs. H NO2. CI. I. NO

T h e s e h e r b i c i d e s c a u s e r a p i d b l e a c h i n g and d e s s i c a t i o n o f g r e e n t i s s u e s , s i m i l a r t o the e f f e c t s o fparaquat. Like paraquat, l i g h t i s r e q u i r e d f o r a c t i v i t y (22), however, u n l i k e paraquat, p h o t o s y n t h e s i s i s n o t a r e q u i r e m e n t f o r a c t i v i t y ( 2 3 - 2 7 ) , e x c e p t when p h o t o s y n t h e s i s i s i n d i r e c t l y r e q u i r e d f o r s u b s t r a t e (28) o r g e n e r a t i o n o f oxygen f o r l i p i d p e r o x i d a t i o n ( 2 7 ) . The d e v e l o p m e n t o f i n j u r y t o p l a n t t i s s u e s a f f e c t e d b y t h e s e compounds i s much l i k e t h a t c a u s e d b y p h o t o d y n a m i c pigments. Thef i r s t measureable e f f e c t i s c e l l u l a r leakage, followed s e q u e n t i a l l y by i n h i b i t e d photosynthesis, ethylene e v o l u t i o n , ethane and m a l o n d i a l d e h y d e e v o l u t i o n , a n d f i n a l l y b l e a c h i n g o f c h l o r o p l a s t

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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p i g m e n t s - a l l c h a r a c t e r i s t i c o f p h o t o d y n a m i c membrane l i p i d peroxidation (29). Furthermore, a f t e r a s u f f i c i e n t l ylong incubation in darkness, the h e r b i c i d a l a c t i v i t y i s almost e n t i r e l y independent o f temperature, l i k e a photodynamic dye (30). I t was obvious from several s t u d i e s t h a t the h e r b i c i d e i t s e l f i s not the photodynamic dye. The most c o m p e l l i n g e v i d e n c e f o r t h i s i s t h a t t h e a c t i o n s p e c t r u m i n d i c a t e d t h a t t h e p h o t o r e c e p t o r f o r p h o t o d y n a m i c damage i s a v i s i b l e pigment (31-33). Diphenyl ether h e r b i c i d e s absorb i n the u l t r a v i o l e t r a t h e r than the v i s i b l e spectrum. Furthermore, t h e r e was no strong evidence that the diphenyl ether herbicide acted a s a l i p i d p e r o x i d i z i n g r a d i c a l a s the r e s u l t o f energy t r a n s f e r from a p h o t o r e c e p t o r , e v e n t h o u g h some n i t r o d i p h e n y l e t h e r h e r b i c i d e s c a n b e photoreduced t on i t r o r a d i c a l anions by ^-carotene (34). This, coupled with apparent evidence that carotenoids are involved in the m o d e o f a c t i o n o f t h e s e h e r b i c i d e s ( e . g . , 2 3 , 24, 2 6 , 3 5 - 3 7 ) , l e d some t o h y p o t h e s i z e that a carotenoid-diphenyl ether exciplex might be i n v o l v e d i n t h e m e c h a n i s m o f a c t i o n o f t h e s e h e r b i c i d e s ( 3 8 ) . I n f a c t , o x y f l u o r f e n - t r e a t e d t h y l a k o i d membranes w i l l g e n e r a t e s i n g l e t o x y g e n when e x p o s e d t o l i g h t ( 3 9 ) . A l t h o u g h some o f t h e s e c o m p o u n d s can form r a d i c a l s , t h e r e are d i p h e n y l e t h e r h e r b i c i d e s t h a t d o not form r a d i c a l s t h a t are q u i t e e f f e c t i v e a s 1 i p i d - p e r o x i d i z i n g h e r b i c i d e s (40-42). D e s p i t e i n v e s t i g a t i o n s b y many l a b o r a t o r i e s , t h e n a t u r e o f t h e p h o t o r e c e p t o r f o r t h e p h o t o d y n a m i c damage r e m a i n e d a n enigma f o r more t h a n two d e c a d e s . S t u d i e s d e m o n s t r a t i n g t h a t t h e r e w a s a m e t a b o l i c requirement before the herbicide could cause e f f e c t s l i k e a p h o t o d y n a m i c d y e ( 2 4 , 28, 3 0 , 4 3 ) s h o u l d h a v e p r o v i d e d a c l u e t o t h e actual mechanism - the induction of the accumulation o fa natural photodynamic compound. S i t e o f a c t i o n . M a t r i n g e a n d S e a l l a (44> 4 5 ) , f o l l o w e d c l o s e l y b y o t h e r s (46, 47) r e p o r t e d t h a t d i p h e n y l e t h e r h e r b i c i d e - t r e a t e d t i s s u e s accumulated abnormally high l e v e l s o f PPIX. Furthermore, s p e c i f i c i n h i b i t o r s o fporphyrin synthesis could completely o r almost c o m p l e t e l y p r e v e n t h e r b i c i d a l damage from d i p h e n y l e t h e r h e r b i c i d e s ( 4 4 - 4 8 ) ( F i g s . 1 and 3 ) and t h e a b s o r p t i o n s p e c t r u m o f PPIX r o u g h l y f i t t h e a c t i o n s p e c t r a f o r t h e l i g h t - i n d u c e d damage b y t h e s e h e r b i c i d e s (31-33, 4 4 ) . N o n - d i p h e n y l e t h e r h e r b i c i d e s t h a t had been observed t oact in a s i m i l a r fashion to diphenyl ethers (oxadiazon, the p y r i d i n e d e r i v a t i v e LS 82-556, the novel phenylpyrazole T N P P - e t h y l , and t h e c y c l i c i m i d e c h l o r o p h t h a l i m - see b e l o w ) a l s o c a u s e d t r e a t e d p l a n t s t o a c c u m u l a t e h i g h l e v e l s o f PPIX (44-46> 4 8 - 5 2 ) . The c y c l i c i m i d e s , d i p h e n y l e t h e r s , and o x a d i a z o l e s had p r e v i o u s l y been demonstrated t o i n h i b i t c h l o r o p h y l l s y n t h e s i s (53-56), however, the connection t ot h e i r l i p i d - p e r o x i d i z i n g a c t i o n was n o t c l e a r . T o d a t e , common s t r u c t u r e / a c t i v i t y r e l a t i o n s h i p s between these d i v e r s e h e r b i c i d e groups have not been determined. P r o t o p o r p h y r i n IX-magnesium c h e l a t a s e s y n t h e s i z e s Mg-PPIX from PPIX. Thus, i t seemed l i k e l y t h a t i n h i b i t i o n o f t h i s enzyme would l e a d t o a c c u m u l a t i o n o f P P I X ( 4 7 , 4 9 ) . H o w e v e r , M a t r i n g e et a / . ( 5 7 , 5 8 ) f o u n d t h a t t h e a c c u m u l a t i o n o f P P I X was d u e , i n f a c t , t o s t r o n g i n h i b i t i o n o f p r o t o p o r p h y r i n o g e n o x i d a s e ( P r o t o x ) , the enzyme t h a t c o n v e r t s p r o t o p o r p h y r i n o g e n t o PPIX ( F i g . 1). T h e s e r e s u l t s

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were c o n f i r m e d by Witkowski and H a i l i n g ( 5 9 ) . A p p a r e n t l y , b l o c k a g e at t h i s s i t e l e a d s t o a u t o o x i d a t i o n o f t h e s u b s t r a t e t o form PPIX, a s has b e e n o b s e r v e d when t h i s enzyme i s i n a c t i v e d u e t o g e n e t i c l e s i o n s i n y e a s t and humans ( 6 0 - 6 2 ) .

OXADIAZON

M ft B 39279

US 82-556

CHLOROPHTHAUM

TNPP—ETHYL

A c i f l u o r f e n - m e t h y l and LS 8 2 0 3 4 0 , r j - n i t r o a n d p _ - c h l o r o d i p h e n y l e t h e r s , r e s p e c t i v e l y , h a d U n ' s o f l e s s t h a n 1 /iM f o r t h e s y n t h e s i s o f Mg-PPIX f r o m ALA i n a m a i z e e t i o p l a s t p r e p a r a t i o n ( 5 7 ) . A h e r b i c i d a l l y i n a c t i v e a n a l o g o f a c i f l u o r f e n - m e t h y l (RH 5 3 4 8 ) h a d a n I q almost three orders o f magnitude higher than t h a t o f a c i f l u o r f e n - m e t h y l . None o f t h e compounds i n h i b i t e d Mg-PPIX f o r m a t i o n from PPIX. The I c q ' S f o r maize e t i o p l a s t P r o t o x were a b o u t 1 0 nM f o r t h e h e r b i c i d a l d i p h e n y l e t h e r . S i m i l a r r e s u l t s w e r e o b t a i n e d w i t h p o t a t o , y e a s t , and mouse l i v e r m i t o c h o n d r i a . No s i g n i f i c a n t i n h i b i t i o n o f t h e PPIX f e r r o c h e l a t a s e was m e a s u r e d . S i m i l a r r e s u l t s w e r e o b s e r v e d w i t h o x a d i a z o n , L S 8 2 - 5 5 6 , a n d M&B 3 9 2 7 9 ( 5 8 ) . M&B 3 9 2 7 9 ( s e e a b o v e ) i s a p h e n y l p y r a z o l e t h a t a p p e a r s to have a mechanism o f a c t i o n s i m i l a r t o t h a t o f d i p h e n y l e t h e r s ( 6 3 ) . The r e s u l t s o f Witkowski and H a i l i n g (59) w i t h a c i f l u o r f e n - m e t h y l o ncucumber Protox were s i m i l a r , a l t h o u g h they f o u n d a n I q o f a b o u t 3 0 nM. T o d a t e , n o s t u d i e s h a v e b e e n p u b l i s h e d on t h e t y p e ( s ) o f i n h i b i t i o n o f P r o t o x c a u s e d b y t h e s e h e r b i c i d e s o r on w h e t h e r t h e b i n d i n g s i t e s f o r t h e d i f f e r e n t c h e m i c a l types overlap. 5

5

Mode o f a c t i o n . PPIX i s a p h o t o l a b i l e compound, s o t h e q u e s t i o n o f w h e t h e r s u f f i c i e n t l e v e l s o f i t c a n e x i s t in vivo f o r i t t o e x e r t i t s e f f e c t i s i m p o r t a n t . We f o u n d t h e h a l f - l i f e o f P P I X i n a c i f l u o r f e n - t r e a t e d cucumber t i s s u e d u r i n g exposure t o b r i g h t l i g h t t o b e a b o u t 2.5 h ( 4 1 ) - s u f f i c i e n t t i m e f o r i t t o b e a n e f f e c t i v e h e r b i c i d e . F u r t h e r m o r e , PPIX a c c u m u l a t e d r a p i d l y i n b r i g h t l i g h t and d i d n o t b e g i n t o d e c r e a s e u n t i l c e l l u l a r damage was n e a r l y c o m p l e t e . Others have found l i t t l e c o r r e l a t i o n between t h e h e r b i c i d a l e f f e c t s and t h e amount o f PPIX a c c u m u l a t e d b y P r o t o x - i n h i b i t i n g h e r b i c i d e s (50, 6 4 ) - In cucumber ( F i g . 4 ) , pigweed, and v e l v e t l e a f , we f o u n d a s t r o n g c o r r e l a t i o n b e t w e e n t h e a m o u n t o f P P I X a c c u m u l a t e d i n r e s p o n s e t o a c i f l u o r f e n and t h e amount o f e n s u i n g h e r b i c i d a l d a m a g e ( 4 1 ) . A l s o , t h e r e was a n e x c e l l e n t c o r r e l a t i o n b e t w e e n t h e PPIX and t h e r e s u l t i n g h e r b i c i d a l damage c a u s e d b y a v a r i e t y o f d i p h e n y l e t h e r and o x a d i a z o l e h e r b i c i d e s ( 4 1 ) .

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10 /xM AF & 1 mM DA O - control A - 1 mM DA • - 10 /iM AF -

A

-O control

A -

O - • 10 /zM AF -A - A 1 mM gabaculine •

E o 300 -

-a

#

/

AF + gabaculine

E

3.

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/ o

-

100

c o o

5

6

0

1

2 3

4

5

time (h)

F i g u r e 3. E f f e c t s o f g a b a c u l i n e and d i o x o h e p t a n o i c a c i d (DA) o n e f f i c a c y o f a c i f l u o r f e n (AF) on c e l l u l a r leakage a smeasured by e l e c t r o l y t e i n c r e a s e i n t h e bathing media o f cucumber c o t y l e d o n d i s c s i n c u b a t e d i n t h e v a r i o u s treatment s o l u t i o n s f o r 20 h i n darkness before exposure t o l i g h t (time 0 ) . (Reprinted with permission from ref. 46. Copyright 1988 Academic Press.)

E o o E

"o

D TJ C o o

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

protoporphyrin IX ( n m o l e s / 5 0 discs)

F i g u r e 4. R e l a t i o n s h i p between c e l l u l a r damage i n g r e e n cucumber c o t y l e d o n d i s c s and PPIX a c c u m u l a t i o n caused b y e x p o s u r e t o v a r i o u s c o n c e n t r a t i o n s o f a c i f l u o r f e n . C e l l u l a r damage was a s s a y e d a s i n F i g . 3, 1 h a f t e r exposure t o l i g h t . PPIX was assayed j u s t before exposure t o l i g h t and a f t e r a 20 h i n c u b a t i o n in darkness. (Reprinted with permission from ref. 41. Copyright 1989 Plant Physiology.)

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Some l a b o r a t o r i e s ( 6 4 - 6 6 ) h a v e f o u n d P C h l i d e t o b e t h e p r i m a r y p o r p h y r i n t o a c c u m u l a t e i n d i p h e n y l e t h e r - t r e a t e d t i s s u e s . In o u r g r e e n c u c u m b e r c o t y l e d o n d i s c s y s t e m , we h a v e f o u n d o n l y P P I X l e v e l s t o be i n c r e a s e d by t h e s e h e r b i c i d e s ( 6 7 ) , h o w e v e r , i n i n t a c t c u c u m b e r s e e d l i n g s ( F i g . 5) a n d t e n t o x i n - a f f e c t e d c u c u m b e r c o t y l e d o n d i s c s ( 6 8 ) we f o u n d d i p h e n y l e t h e r - e n h a n c e d P C h l i d e l e v e l s . P P I X l e v e l s a c c u m u l a t e t o many ( a s much as s e v e r a l h u n d r e d ) t i m e s t h e c o n t r o l l e v e l s i n h e r b i c i d e - t r e a t e d t i s s u e s , w h e r e a s t h e maximum P C h l i d e accumulation i s only f o u r - f o l d that of the c o n t r o l . Since the m e t a b o l i c b l o c k i s a t P r o t o x , t h e r e i s no i n h i b i t i o n o f c o n v e r s i o n o f P C h l i d e t o Chi a f t e r e x p o s u r e t o l i g h t . T h e r e f o r e , i t seems u n l i k e l y that PChlide plays a s i g n i f i c a n t p a r t i n the mechanism of a c t i o n of t h e s e h e r b i c i d e s . Indeed, o f the Chi p r e c u r s o r s assayed, o n l y PPIX s i g n i f i c a n t l y c o r r e l a t e d w i t h h e r b i c i d a l damage c a u s e d by a s e v e r a l d i f f e r e n t acifluorfen/DP/ALA treatment combinations (67). No s i g n i f i c a n t c o r r e l a t i o n s were found with accumulated P C h l i d e , Mg-PPIX, o r Mg-PPIXME. F u r t h e r m o r e , n e i t h e r o f t h e p h o t o d y n a m i c p r e c u r s o r s o f PPIX, c o p r o p o r p h y r i n o g e n nor uroporpophyrinogen ( e x t r a c t e d as c o p r o p o r p h y r i n I I I and u r o p o r p h y r i n I I I ) a c c u m u l a t e i n diphenyl ether herbicide-treated plant tissues (Table IV). A l l of our d a t a a r e c o n s i s t e n t w i t h t h e view t h a t PPIX i s t h e p r i m a r y photodynamic pigment i n v o l v e d i n the mechanism o f a c t i o n o f these herbicides. T a b l e I V . E f f e c t o f 10 /xM a c i f l u o r f e n o n a c c u m u l a t i o n o f P P I X i t s i m m e d i a t e p r e c u r s o r s i n g r e e n c u c u m b e r c o t y l e d o n d i s c s d u r i n g 20 h o f d a r k i n c u b a t i o n . P r e v i o u s l y u n p u b l i s h e d d a t a o f Matsumoto and Duke. Porphyrin Uroporphyrin III Coproporphyrin III P r o t o D o r D h v r i n IX

Control -(nmoles/g 0.016 ± 0 . 0 0 5 0 . 0 0 4 ± 0.001 0 . 0 0 8 ± 0.001

Treated fresh weight)0 . 0 2 5 ± 0..001 0.014 ± 0,,004 0 . 2 0 7 ± 0,,015

Our l a b o r a t o r y (46) and t h a t o f Yanase and Andoh (52) f o u n d t h e h e r b i c i d a l e f f e c t o f ALA t o be s h o r t - l i v e d i n t h e l i g h t c o m p a r e d t o t h a t o f P r o t o x - i n h i b i t i n g h e r b i c i d e s . T h i s i s p r o b a b l y due t o t h e f a c t t h a t P C h l i d e l e v e l s a r e r a p i d l y r e d u c e d by c o n v e r s i o n t o C h i o r by p h o t o d e s t r u c t i o n i n t h e l i g h t i n A L A - t r e a t e d p l a n t t i s s u e s . The h a l f - l i f e o f P C h l i d e i n c u c u m b e r i n l i g h t was a h a l f h o u r o r l e s s ( F i g . 2 ) , whereas, the h a l f - l i f e o f PPIX i n cucumber c o t y l e d o n s d i s c s was m o r e t h a n 2 h ( 4 1 ) . The t o t a l amount o f p o r p h y r i n s t h a t accumulate i n d i p h e n y l e t h e r - t r e a t e d t i s s u e s i s c o n s i d e r a b l y higher than t h a t which accumulates in untreated t i s s u e s (67). Therefore, these h e r b i c i d e s a p p e a r t o i n c r e a s e c a r b o n f l o w i n t o t h i s p a t h w a y . Heme i s k n o w n t o f e e d b a c k i n h i b i t t h i s p a t h w a y a n d P P I X i s r e q u i r e d f o r heme s y n t h e s i s ( 9 ) . F e e d i n g heme t o d i p h e n y l e t h e r - t r e a t e d p l a n t c e l l s r e d u c e s t h e amount o f p o r p h y r i n - c a u s e d l i p i d p e r o x i d a t i o n o f c e l l h o m o g e n a t e s as m e a s u r e d by o x y g e n c o n s u m p t i o n (10). Why P P I X f o r m e d b y n o n e n z y m a t i c o x i d a t i o n o f p r o t o p o r p h y r i n o g e n d o e s n o t i m m e d i a t e l y r e e n t e r t h e c h l o r o p h y l l pathway i s p r o b a b l y due to a requirement f o r accumulation of a threshold level or saturation o f a n o n - a v a i l a b l e pool before r e e n t r y v i a a non-pathway r o u t e can

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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o c c u r . I f s u b s t r a t e c h a n n e l i n g , a s i n heme s y n t h e s i s i n m o u s e mitochondria (69), occurs with conversion of protoporphyrinogen to P P I X , P P I X f o r m e d b y a u t o o x i d a t i o n may b e o u t s i d e t h e n o r m a l m e t a b o l i c c h a n n e l . Thus, o n l y a f t e r a s u f f i c i e n t c o n c e n t r a t i o n o f PPIX b u i l d s up o u t s i d e t h e normal m e t a b o l i c channel w i l l i t r e e n t e r t h e pathway. The s i m u l t a n e o u s k i n e t i c s o f PPIX and P C I i d e accumulation in acifluorfen-treated, yellow (tentoxin-treated) cucumber t i s s u e s s u p p o r t t h i s h y p o t h e s i s ( 6 8 ) . High l e v e l s o f PPIX accumulate before the h e r b i c i d e enhances PCIide accumulation. P r o t o x i s thought t o b e a membrane-bound enzyme; p r o b a b l y i n o r on t h e p l a s t i d e n v e l o p e ( F i g . 6 ) . F l u o r e s c e n c e m i c r o s c o p y o f achlorophyllous t i s s u e s treated with acifluorfen in darkness, results i n s t r o n g p o r p h y r i n f l u o r e s c e n c e i n both p l a s t i d s and c y t o p l a s m , w h e r e a s , f l u o r e s c e n c e was l o c a l i z e d a l m o s t e x c l u s i v e l y i n p l a s t i d s o f u n t r e a t e d c e l l s ( 6 8 ) . PPIX c o n c e n t r a t i o n s were a l m o s t 2 0 0 - f o l d g r e a t e r i n t r e a t e d than untreated t i s s u e s . These data suggest t h a t PPIX l e a k s from p l a s t i d s o r p l a s t i d e n v e l o p e s o f t r e a t e d t i s s u e s and t h a t t h i s l e a k a g e i s i n d e p e n d e n t o f membrane damage due t o l i p i d p e r o x i d a t i o n . T h u s , a s i n o u r p r e v i o u s model (38)> t h e c e l l u l a r s i t e o f a c t i o n o f t h e s e h e r b i c i d e s may b e t h e p l a s t i d e n v e l o p e . The a c t i o n s p e c t r a f o r h e r b i c i d a l damage c a u s e d b y P r o t o x i n h i b i t o r s has a s t r o n g component i n t h e r e d (31, 32, 7 0 ) , w h e r e a s , PPIX a b s o r b s r e l a t i v e l y weakly i n t h i s r e g i o n o f t h e spectrum. Sato (70) has s p e c u l a t e d t h a t , i n g r e e n cucumber t i s s u e , i n a c t i v a t i o n o f p h o t o s y n t h e s i s a s a s e c o n d a r y e f f e c t o f p e r o x i d a t i v e damage r e s u l t s in c h l o r o p h y l l s being i n v o l v e d i n the photodynamic process. Sato (70) found a p o r p h y r i n - p r o t e i n complex i n c h l o r o p l a s t s o f cucumber t i s s u e s t r e a t e d w i t h S-23142, a c h l o r o p h t h a l i m a n a l o g . The complex was n o t f o u n d i n u n t r e a t e d p l a n t s . T h e p r o t e i n was a 6 3 - 6 6 k D m e m b r a n e p r o t e i n a n d P P I X was i d e n t i f i e d a s o n e o f t w o p o r p h y r i n s t h a t complex i t . A p p a r e n t l y t h i s p r o t e i n i s not P r o t o x , s i n c e i t has been d e t e r m i n e d t o h a v e a m o l e c u l a r mass o f 3 6 kD ( H ) . The r o l e o f t h i s p o r p h y r i n - p r o t e i n c o m p l e x i n t h e mode o f a c t i o n o f P r o t o x - i n h i b i t i n g h e r b i c i d e s has y e t t o b e d e t e r m i n e d . A p p l i e d a l o n e , PPIX i s r e l a t i v e l y i n e f f e c t i v e a s a h e r b i c i d e ( 4 6 ) . Even i f i t were h e r b i c i d a l l y e f f e c t i v e , i t s c o s t and t o x i c o l o g i c a l p r o p e r t i e s would p r o b a b l y p r o h i b i t s i t s use. The low a c t i v i t y o f PPIX s u p p l i e d e x o g e n o u s l y c o u l d b e due t o p o o r c e l l u l a r a b s o r p t i o n o f t h i s r e l a t i v e l y complex molecule. The s e l e c t i v i t y o f P r o t o x - i n h i b i t i n g h e r b i c i d e s i s p r o b a b l y due t o many f a c t o r s , i n c l u d i n g : d i f f e r e n t i a l d e g r a d a t i o n o f t h e h e r b i c i d e s (72), d i f f e r e n t i a l s e n s i t i v i t y t o t o x i c oxygen s p e c i e s (15, 16, 73), and, perhaps, d i f f e r e n t i a l s u s c e p t i b i l i t y a t t h e s i t e o f a c t i o n . No d a t a a r e a v a i l a b l e t o s u p p o r t t h e l a t t e r p o s s i b i l i t y . However, mutants o f the u n i c e l l u l a r green a l g a Chlamvdomonas r e i n h a r d t i i . produced bys e l e c t i o n with a Protox i n h i b i t o r a f t e r mutagenesis, were c r o s s r e s i s t a n t to a v a r i e t y o f P r o t o x - i n h i b i t i n g h e r b i c i d e s , but not t o PSII i n h i b i t o r s or t o paraquat, i n d i c a t i n g resistance a the molecular s i t e of action (70). Summary and

Conclusions

P o r p h y r i n s c a n n o t b e used d i r e c t l y as p h o t o s e n s i t i z i n g h e r b i c i d e s b e c a u s e o f t h e c o s t and p o s s i b l e t o x i c o l o g i c a l d a n g e r s . However, a

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D U K E ET AL.

383

Photosensitizing Porphyrins as Herbicides

2I

. 10

• 100 Acifluorfen (/zM)

1

— 1000

F i g u r e 5. E f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f a c i f l u o r f e n o n PPIX and P C h l i d e a c c u m u l a t i o n i n c o t y l e d o n s o f i n t a c t , g r e e n cucumber s e e d l i n g s . The p l a n t s were sprayed t o r u n o f f w i t h t h e h e r b i c i d e i n 0.5 % T w e e n 8 0 ( v / v ) a n d i n c u b a t e d i n d a r k n e s s f o r 12 h b e f o r e a s s a y s w e r e c o n d u c t e d . Previously unpublished data o f B e c e r r i l and Duke. INSIDE /

-4—» |

ALA

PROTOXJ

^

HERBICIDE INHIBITION

O

PLASTID ENVELOPE

MG-CHELATASE ~

S

W

E

PROTOPORPHYRINOGEN IX AUTOOXIDATION J ^ — 0 PPIX

2

SINGLET OXYGEN LIPID PEROXIDATION

I MEMBRANE DESTRUCTION

F i g u r e 6. H y p o t h e t i c a l m o d e l o f t h e p h y t o t o x i c m e c h a n i s m o f action f o r herbicides that inhibit Protox. Protoporphyrinogen which accumulated as a r e s u l t o f Protox i n h i b i t i o n l e a v e s t h e membrane-bound, c h a n n e l e d p o r p h y r i n pathway and i s a u t o o x i d i z e d to PPIX. Redrawn from ( 5 9 ) .

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v a r i e t y o f b o t h n a t u r a l l y - o c c u r r i n g and s y n t h e t i c compounds can e f f e c t i v e l y s t i m u l a t e p l a n t s t o s y n t h e s i z e l e t h a l amounts o f photosensitizing porphyrins. ALA, a p o r p h y r i n p r e c u r s o r , i s r e a d i l y a b s o r b e d and c o n v e r t e d t o p o r p h y r i n s i n p l a n t t i s s u e s . Compounds t h a t modulate the c h l o r o p h y l l s y n t h e s i s pathway can b e used t o s y n e r g i z e ALA's e f f e c t s onporphyrin accumulation. Several classes o f c o m m e r c i a l h e r b i c i d e s ( o x a d i a z o l e s , /V-phenyl i m i d e s , a n d diphenyl ethers) i n h i b i t Protox, causing deregulation o f the p o r p h y r i n p a t h w a y and a u t o o x i d a t i o n o f p r o t o p o r p h y r i n o g e n t o f o r m p h o t o s e n s i t i z i n g , d e s t r u c t i v e l e v e l s o f PPIX. These compounds are much more e f f i c i e n t a s h e r b i c i d e s t h a n ALA p l u s m o d u l a t o r s . Future r e s e a r c h o n s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s between these h e r b i c i d e s and i n h i b i t i o n o f P r o t o x c o u l d i m p r o v e t h e a c t i v i t y a n d / o r s e l e c t i v i t y of these herbicides. Acknowledgments We t h a n k Rohm a n d H a a s C o . f o r p r o v i d i n g t e c h n i c a l g r a d e c h e m i c a l s used i n our s t u d i e s d e s c r i b e d h e r e . The t e c h n i c a l a s s i s t a n c e o f Al Lane was i n v a l u a b l e i n the our e x p e r i m e n t s r e p o r t e d h e r e . J . L . W i c k l i f f , S.H. D u k e , a n d G.W. E l z e n p r o v i d e d h e l p f u l c r i t i c i s m s . T h i s w o r k was s u p p o r t e d i n p a r t b y t h e F u l b r i g h t / M i n i s t r y o f E d u c a t i o n and S c i e n c e , M a d r i d , S p a i n and t h e J a p a n e s e M i n i s t r y o f E d u c a t i o n , S c i e n c e , a n d C u l t u r e . Ryo S a t o g e n e r o u s l y p r o v i d e d a c o p y o f h i s Ph.D. Thesis. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Towers, G. H. N.; Arnason, J . T. Weed Technol. 1988, 2, 545-9. Knox, J . P.; Dodge, A. D. Plant Cell Environ. 1985, 8, 19-25. Knox, J . P.; Dodge, A. D. Plant Sci. Lett. 1984, 37, 3-7. Mascia, P. N.; Robertson, D. S. Planta 1978, 143, 207-11. Rebeiz, C. A . ; Montazer-Zouhoor; Hopen, H. J.; Wu, S.-M. Enzyme Microb. Technol. 1984, 6, 390-401 Sisler, E. C.; Klein, W. H. Physiol. Plant. 1963, 16, 315-22. Rebeiz, C. A . ; Haidar, A . ; Yaghi, M.; Castelfranco, P. A. Plant Physiol. 1970, 46, 543-9. Duggan, J.; Gassman, M. Plant Physiol. 1974, 53, 206-15. Castelfranco, P. A . ; Beale, S. I. Annu. Rev. Plant Phvsiol. 1983, 34, 241-278. Masuda, T . ; Kouji, H . ; Matsunaka, Pestic. Biochem. Physiol. 1990, 36, In Press. Wickliff, J . L . ; Duke, S. O.; Vaughn, K. C. Physiol. Plant. 1982, 56, 399-406. Gassman, M. A. Plant Physiol. 1973, 52, 590-594. Rebeiz, C. A . ; Montazer-Zouhoor; Mayasich, J . M.; Tripathy, B. C.; Wu, S.-M.; Rebeiz, C. C. Amer. Chem. Soc. Symp. Ser. 1987, 339, 295-328. Rebeiz, C. A . ; Montazer-Zouhoor; Mayasich, J . M.; Tripathy, B. C.; Wu, S.-M.; Rebeiz, C. C. CRC Crit. Rev. Plant Sci. 1988, 6, 385-436. Finckh, B. F . ; Kunert, K. J. J . Agric. Food Sci. 1985, 33, 574-7 Shaaltiel, Y.; Glazer, A . ; Bocion, P. F . ; Gressel, J . Pestic. Biochem. Physiol. 1988, 31, 13-23.

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DUKEETAL.

Photosensitizing Porphyrins as Herbicides

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17. Yaronskaya, E. B.; Shalygo, N. V . ; Averina, N. G. Vestsi Akad. Navuk BSSR, Ser. Biayal. Navuk 1989, (4), 38-40. 18. Averina, N. G.; Shalygo, N. V.; Yaronskaya, E. B.; Rassadina, V. V. Vestsi Akad. Navuk BSSR, Ser. Biyal. Navuk 1989, 100-2. 19. Averina, N. G.; Radyuk, M. S. Dokl. Akad. Nauk BSSR 1989, 33, 471-4. 20. Rebeiz, C. A . ; Juvik, J . A . ; Rebeiz, C. C. Pestic. Biochem. Physiol. 1988, 30, 11-27. 21. Matsunaka, S. in Herbicides: Chemistry, Degradation, and Mode of Action, Vol. 2.; Kearney, P. C.; Kaufman, D. D., Eds.; Marcel Dekker: New York, 1976; pp. 709-39. 22. Kunert, K. J.; Sandmann, G.; Böger, P. Rev. Weed Sci. 1987, 3, 35-55. 23. Matsunaka, S. J . Agric. Food Chem. 1969, 17, 171-5. 24. Duke, S. O.; Vaughn, K. C.; Meeusen, R. L. Pestic. Biochem. Physiol. 1984, 21, 368-76. 25. Ensminger, M. P.; Hess, F. D. Plant Physiol. 1985, 78, 46-50. 26. Duke, S. O.; Kenyon, W. H. Plant Physiol. 1986, 81, 882-8. 27. Bowyer, J . R.; Hallahan, B. J.; Camilleri, P.; Howard, J. Plant Physiol. 1989, 89, 674-80. 28. Nurit, F . ; Ravanel, P.; Tissut, M. Pestic. Biochem. Physiol. 1988, 31, 67-73. 29. Kenyon, W. H . ; Duke, S. O.; Vaughn, K. C. Pestic. Biochem. Physiol. 1985, 24, 240-50. 30. Kenyon, W. H . ; Duke, S. O.; Paul, R. N. Pestic. Biochem. Physiol. 1988, 30, 57-66. 31. Ensminger, M. P.; Hess, F. D. Plant Physiol. 1985, 77, 503-5. 32. Sato, R.; Nagano, E . ; Oshio, H . ; Kamoshita, K.; Furuya, M. Plant Physiol. 1987, 85, 1146-50. 33. Gaba, V . ; Cohen, N.; Shaaltiel, Y.; Ben-Amotz, A; Gressel, J . Pestic. Biochem. Physiol. 1988, 31, 1-12. 34. Rao, D. N. R.; Mason, R. P. Photochem. Photobiol. 1988, 47, 791-5. 35. Kenyon, W. H . ; Duke, S. O. Plant Physiol. 1985, 79, 862-6. 36. Fadayomi, O.; Warren, G. F. Weed Sci. 1976, 24, 598-600. 37. Orr, G. L.; Hess, F. D. Plant Physiol. 1982, 69, 502-7. 38. Duke, S. O.; Kenyon, W. H. Z. Naturforsch. 1987, 42c, 813-8. 39. Haworth, P.; Hess, F. D. Plant Physiol. 1988, 86, 672-6. 40. Orr, G. L.; Elliott, C. M.; Hogan, M. E. Plant Physiol. 1983, 73, 939-44. 41. Becerril, J . M.; Duke, S. O. Plant Physiol. 1989, 90, 1175-81. 42. Ensminger, M. P.; Hess, F. D.; Bahr, J. T. Pestic. Biochem. Physiol. 1985, 23, 163-70. 43. Matringe, M.; Scalla, R. Pestic. Biochem. Physiol. 1987, 27, 267-74. 44. Matringe, M.; Scalla, R. Proc. Brit. Crop Protect. Conf. 1987, 9B, 981-8. 45. Matringe, M.; Scalla, R. Plant Physiol. 1988, 86, 619-22. 46. Lydon, J.; Duke, S. O. Pestic. Biochem. Physiol. 1988, 31, 74-83. 47. Witkowski, D. A . ; Halling, B. P. Plant Physiol. 1988, 87, 632-7. 48. Matringe, M.; Scalla, R. Pestic. Biochem. Physiol. 1988, 32, 164-72. 49. Duke, S. O.; Lydon, J.; Paul, R. N. Weed Sci. 1989, 37, 152-60. 50. Sandman, G.; Böger, P. Z. Naturforsch. 1988, 43c, 699-704.

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NATURALLY OCCURRING PEST BIOREGULATORS

51. Nicolaus, B.; Sandmann, G.; Watanabe, H . ; Wakabayashi, K.; Böger, P. Pestic. Biochem. Physiol. 1989, 35, 192-210. 52. Yanase, D.; Andoh, A. Pestic. Biochem. Physiol. 1989, 35, 70-80. 53. Sandmann, G.; Reck, H.; Böger, P J. Agric. Food Chem. 1984, 32, 868-72. 54. Wakabayashi, K.; Matsuya, K.; Teraoka, T . ; Sandmann, G.; Böger, P. J . Pestic. Sci. 1986, 11, 635-40. 55. Teraoka, T . ; Sandmann, G.; Böger, P.; Wakabayashi, K. J. Pestic. Sci. 1987, 12, 499-504. 56. Halling, B.P.; Peters, G. R. Plant Phvsiol. 1987, 84, 1114-20. 57. Matringe, M.; Camadro, J . - M . ; Labbe, P.; Scalla, P. Biochem. J. 1989, 260, 231-5. 58. Matringe, M.; Camadro, J . - M . ; Labbe, P.; Scalla, P. FEBS Lett. 1989, 245, 35-8. 59. Witkowski, D. A . ; Halling, B. P. Plant Physiol., 1989, 90, 1239-42. 60. Camadro, J. M.; Urban-Grimal, D.; Labbe, P. Biochem. Biophys. Res. Commun. 1982, 106, 724-30. 61. Brenner, D. A . ; Bloomer, J . R. New Engl. J . Med. 1980, 302, 765-8. 62. Deybach, J . C.; de Verneuil, H . ; Nordmann, Y. Hum. Genet. 1981, 58, 425-8. 63. Derrick, P. M.; Cobb, A. H.; Pallett, K. E. Pestic. Biochem. Physiol. 1988, 32, 153-63. 64. Mayasich, J . M.; U. B. Nandihalli; R. A. Leibl; C. A. Rebeiz WSSA Abstr. 1989, 29, 90. 65. Kouji, H.; Masuda, T . ; Matsunaka, S. J . Pestic. Sci. 1988, 3, 495-9. 66. Kouji, H.; Masuda, T . ; Matsunaka, S. Pestic. Biochem. Physiol. 1989, 33, 230-8. 67. Beccerril, J . M.; Duke, S. O. Pestic. Biochem. Physiol. 1989, 35, 119-26. 68. Lehnen, L. P.; Sherman, T. D.; Becerril, J . M.; Duke, S. O. 1990, Pestic. Biochem. Physiol. - In Press. 69. Ferreira, G. C.; Andrew, T. L . ; Karr, S. W.; Dailey, H. A. J. Biol. Chem. 1988, 263, 3835-9. 70. Sato, R., Ph.D. Thesis, University of Tokyo, Tokyo, Japan, 1989. 71. Jacobs, J . M.; Jacobs, N. J. Biochem. J . 1987, 244, 219-224. 72. Frear, D. S.; Swanson, H. R.; Mansager, E. R. Pestic. Biochem. Physiol. 1983, 20, 299-310. 73. Sandmann, G.; Böger, P. Amer. Chem. Soc. Symp. Ser. 1990, 421, 407-18. Received May16,1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 27

Black Shank Disease Fungus Inhibition of Growth by Tobacco Root Constituents and Related Compounds 1

1

2

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Maurice E . Snook , Orestes T . Chortyk , and Alex S. Csinos 1

Russell

Research Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 5677, Athens, G A 30613 Department of Plant Pathology, University of Georgia, Coastal Plain Experiment Station, Tifton, G A 31793

2

Tobacco root phenolics were investigated for their possible role in resistance of tobacco toPhytophthoraparasitica var. nicotianae (black shank). Chlorogenic acid (CA), scopolin, and scopoletin were found to be significantly increased in apparently healthy root tissue, adjacent to infected tissue. In contrast, C A and scopolin concentrations remained relatively constant throughout the length of control roots, while scopoletin increased slightly from the proximal to the distal end. Roots of resistant and susceptible varieties showed similar trends in concentrations of phenolics. Chlorogenic acid, scopolin, scopoletin, and structurally related compounds were evaluated for inhibition of growth of black shank fungus in a laboratory bioassay. At a 4000 ppm dosage level, C A produced 25% inhibition of fungal growth, while scopoletin gave 39% inhibition at 1000 ppm dosage. The activity of C A was shown to reside in the caffeic acid portion of the molecule. Free phenolic acids were also investigated and were found to be very active against black shank growth, with mono-hydroxycinnamic acids being more active than the 3,4-di-hydroxy acid and the o-hydroxy acid being the most active. Dihydro-cinnamic acids were slightly more active than the corresponding cinnamic acids. While scopoletin and esculetin were active in the laboratory bioassay, their glucose-derivatives were found to be completely inactive.

Phytophthora parasitica var. nicotianae (black shank) is a fungus that only attacks tobacco and also causes extensive physical damage, resulting in large economic losses for farmers. It affects both flue-cured and burley types of tobaccos. The fungus attacks the roots of the plant and eventually blocks water and nutrient transport, resulting in the death of the plant. Several varieties of tobacco are known to be highly resistant to the black shank fungus. Several reports have shown that phenolic compounds of tobacco increase in tissues that have been exposed to pathogens, such as tobacco mosaic virus (1), blue mold (Peronospera tabacina) (2), and black root rot (Thielaviopsis This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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basjcoja) (3). Recently, Gasser, et al. (3) reported that chlorogenic acid (3-O-caffeoylquinic acid, C A ) and scopolin (6-methoxy-7- glucosylcoumarin) increased in callus tissue cultures of black root rot-resistant tobacco varieties. They also reported that C A and scopoletin (6-methoxy-7-hydroxycoumarin), phenolics present in tobacco root tissue, were toxic to the black root rot fungus in laboratory bioassays. We, therefore, have investigated the levels of the major tobacco phenolics in roots of susceptible and resistant varieties grown in black shank infected and disease free fields. Also, a number of tobacco root phenolics and related compounds were tested for inhibition of growth of the black shank fungus in a laboratory bioassay. MATERIALS AND METHODS Tobacco was grown in 1989 at the University of Georgia Coastal Plain Experiment Station, Tifton, G A , under standard cultural practices. Varieties ( N C 2326, N C 82, McNair 944, Coker 371, and V A 509) were grown in both a black shank infected field and in a disease free field. Susceptible varieties were sampled in July, while resistant varieties were sampled in August. The soil was gently washed from the roots. Single roots, attached to the main tap root and possessing visible signs of disease incidence (as indicated by brown discoloration), were detached. Root hairs were removed and the entire root length was cut into 1-cm segments and each segment was immediately frozen in dry-ice. Roots ranged from 4-7 mm in diameter at their point of attachment to 2-3 mm at their tip end (distal end). The segments were freeze-dried and chopped with a sharpened spatula. Each segment was extracted with 2.5 m L M e O H (containing 0.14 mg 5,7-dimethoxycoumarin as ISTD) by ultrasonication for 10 min. Then, 2.5 m L water were added and the solution ultrasonicated for an additional 10 min. The samples were filtered and analyzed by high performance liquid chromatography, as described before (4). Briefly, a Waters uBondapak C18 column was used with a concave gradient solvent program from 13% M e O H / H O (containing 0.08 M K H P 0 buffer adjusted to p H 4.45) to 50% M e O H / H 0 . A Hewlett-Packard 1040 Diode Array Detector, set at 340 nm, was employed. A typical chromatogram of a tobacco root extract is shown in Figure 1. The laboratory bioassay of Phvtophthora parasitica var. nicotianae was similar to that already described (5). Stock P. parasitica var. nicotianae was grown on V - 8 juice agar. Additional V-8 juice agar was prepared, autoclaved, cooled to 45°C, and the test compounds were added in quantities to prepare 250 m L solutions of each concentration of test compound. The agar and chemicals were mixed well, dispensed into 60X15 mm plastic petri plates, and allowed to solidify. Each concentration of test material was replicated ten times. Plugs of the fungus culture, cut out with a 5mm #2 cork borer, were placed with fungal culture side down on the edge of the test plates. Plates were placed in Ziploc bags and incubated at 27°C for 14 days or until fungal growth had reached the opposite edge of the agar control plates. Radial mycelial growth was measured and the percent inhibition of growth determined. Scopolin was isolated from freeze-dried, flue-cured tobacco roots. The ground roots were extracted with M e O H and the extract was separated by silicic acid column chromatography, eluted first with ethyl acetate and then with acetone. The acetone eluent contained the scopolin, which was purified by preparative reverse-phase C18 chromatography on a Waters PrepPak 500 C18 column, using a 35% M e O H / H 0 solvent. 2

4

2

2

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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NATURALLY OCCURRING PEST BIOREGUIATORS

Q U J o Q. 0

U

01

10 T 1 me

Figure 1.

20 (min,

30

High Performance Liquid Chromatogram of Tobacco Root Phenolics.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

40

27. SNOOK ET AL.

Black Shank Disease Fungus

R E S U L T S A N D DISCUSSION

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Tobacco Root Chemistry Versus Fungal Attack The major phenolic compound in tobacco roots (Figure 1) is chlorogenic acid ( C A ) , correctly termed 3-O-caffeoylquinic acid (3-O-CQA). Only minor to trace amounts of the 4- and 5 - O - C Q A isomers are found in the roots, but occur in larger quantities in the leaves. Two coumarin derivatives are found in major amounts in root tissue: scopoletin (6-methoxy-7-hydroxycoumarin) and its glucoside, scopolin. Both C A and scopolin can reach levels of 1% of the dry weight of the root. Very little scopolin or scopoletin is found in the leaves. Roots from resistant and susceptible plants, grown in disease free and infected fields were analyzed for their phenolic contents. Whole roots were removed from the tap root, cut into 1-cm segments and analyzed for C A , scopolin, and scopoletin (Figures 2 and 3). It was determined that C A and scopolin remained relatively constant throughout the entire length of a root, from point of attachment at the tap root to the distal end (Figures 2a and 2b). Scopoletin however, tended to increase towards the distal end of the root. Both resistant and susceptible tobacco roots gave these same trends. The fungus attacks the roots of the plant by first entering the root hairs and then advancing towards the tap root. The disease will eventually engulf the tap root and lower stalk, becoming visible as a brown to black coloration on the stalk surface; hence, the name "black shank". Frequently, one observes apparently healthy roots attached to an infected tap root with infection advancing from the tap root outward to the root tip. Roots, with varying degrees of fungal attack (as indicated in Figures 2c and 3a-3d), were analyzed for their phenolics. Roots, under pressure of fungal attack, snowed different patterns of phenolics in their tissues, than roots grown in disease-free fields. Chlorogenic acid, scopolin, and scopoletin all increased significantly in tissue adjacent to visibly-infected tissue. This trend was consistent whether the infection was progressing from the tip end (Figure 2c), from the tap root outward (Figures 3a,3b,3c), or had entered a root hair at the midsection of the root (Figure 3d). Both susceptible (Figures 2c,3a) and resistant (Figures 3b-3d) varieties showed similar increases in these phenolics in root tissues adjacent to infection. Also, there was no difference in flue-cured (Figures 3b,3c) versus burley (Figure 3d) resistant varieties. Gasser et al. (3) found that callus cultures of resistant tobaccos gave an increase in phenolics, while susceptible tobaccos gave a decrease, when challenged by black root rot. In contrast, both resistant and susceptible whole root tissue showed increases in phenolics, when challenged by black shank. Perhaps, callus cultures infected with black shank would show the same effect observed by Gasser for black root rot. Laboratory Bioassays of Tobacco Root Compounds The reported activity of C A and scopoletin in laboratory bioassays of black root rot (3) prompted us to examine these and related compounds for activity towards black shank. C A and scopoletin were tested for inhibition of growth of black shank in our laboratory bioassay (Table I). Chlorogenic acid produced a 25% inhibition of growth at the highest level tested (4000 ppm), while scopoletin gave 39% inhibition of growth at a 1000 ppm dosage rate. The constituent parts of chlorogenic acid (caffeic and quinic acids) were also tested (Table I) and showed that the activity of chlorogenic acid lies in the caffeoyl moiety of the molecule.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

391

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392

NATURALLY

1

2

3

4

5

6

7

8

9

OCCURRING PEST BIOREGULATORS

10

12

11

ROOT SECTION # (HIGHEST # DISTAL END) - G A (Y1) •SCOPOLIN (Y1)

McNair 944 Root Healthy Disease Free Field

•SCOPOLETIN (Y2)

% DRY WGT. (Y2)

% DRY WGT. (Y1)

0.07

0.4

2b

0.06

0.3

0.06 0.04

0.20.03 0.02

0.1 -

0.01 0 1

2

3

4

6

6

7

8

9

10

ROOT SECTION # (HIGHEST # DISTAL END) NC 2326 Root A Black Shank Nursery % DRY WGT. (Y1)

% DRY WGT. (Y2)

CA (Y1) S C O P O L E T I N (Y2) SCOPOLIN (Y1) DISEASE AREA

0.6 -

0

2

4

6

8

10

12

14

16

18

20

22

24

ROOT SECTION # (HIGHEST # DISTAL END) Figure 2.

Root Levels of Phenolics in Healthy Resistant Varieties (2a-2b) and a Black Shank Infected Susceptible Tobacco Variety (2c).

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

CA (Y1)

Figure 3.

-e-

SCOPOLIN (Y1)

S C O P O L E T I N (Y2)

-*-

DISEASE A R E A

ROOT SECTION # (HIGHEST # DISTAL END)

Coker 371 Black Shank Nursery

Levels of Phenolics in Black Shank Infected Tobacco Roots of Different Varieties.

ROOT SECTION # (HIGHEST # DISTAL END)

N C 2326 Root B Black Shank Nursery

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^

394

NATURALLY OCCURRING PEST BIOREGULATORS

TABLE I

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Dosage (ppm) 31.25 62.5 125 250 500 1000 2000 4000 a

% I N H I B I T I O N O F GROWTH O F B L A C K SHANK FUNGUS ( R a c e 0 ) BY T O B A C C O ROOT P O L Y P H E N O L S

Chlorogenic Acid

Quinic Acid

1 1 2 2 6 11 14 25

1 2 0 4 11 4 11 NT

Caffeic Acid 0 0 0 25 59 71 74 NT

Scopoletin

0 0 0 17 38 39 NT NT

a

NT - n o t t e s t e d

Laboratory Bioassavs of Phenolic Acids The high activity observed for caffeic acid was further investigated by studying the activity of a number of related compounds, in order to determine structure-activity relationships. Table II compares the activities of mono-hydroxycinnamic acids to caffeic acid (3,4-dihydroxy-cinnamic acid). A l l of the mono-hydroxy acids were more active than caffeic acid. o-Hydroxycinnamic (o-coumaric) acid was the most active and gave 91% inhibition of fungal growth at only 62.5 ppm. As p-hydroxy-cinnamic acid can exist in either the cis or trans configuration, both were tested. The data (Table III) showed that the cis-isomer was more active than the trans-isomer. Saturation of the double bond in the aliphatic portion of the molecule was investigated by testing the dihydro-derivatives of p-hydroxycinnamic and 3,4-dihydroxycinnamic acids. The dihydro-acids were slightly more active than the corresponding cinnamic compounds. Laboratory Bioassavs of Coumarins The activities of scopoletin (6-methoxy-7-hydroxycoumarin) and esculetin (6,7-dihydroxycoumarin) were compared to each other and to those of their respective glucosides, scopolin and esculin (Table IV). The dihydroxy-coumarin was found to be just as active as the methoxy-hydroxy-compound. Thus, the presence of a free hydroxyl group at position-6 is not required for activity. Surprisingly, substitution of a glucose on the hydroxyls of both compounds resulted in the complete loss of activity. Gasser (3) found similar results with the activity of scopolin towards black root rot. SUMMARY We have found compounds (chlorogenic acid and scopoletin) in tobacco roots that inhibit the growth of the black shank fungus in a laboratory bioassay.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

37 57 75 100 100

14 39 87 100 100

62.5

125

250

500

1000

R= - C H = C H - C 0 0 H

17

m-Coumaric

0

p-Coumaric

100

100

100

96

91

65

o-Coumaric

% I N H I B I T I O N O F GROWTH O F B L A C K SHANK FUNGUS BY H Y D R O X Y - C I N N A M I C A C I D S

31.25

Dosage (ppm)

TABLE I I .

78

75

22

2

0

0

Caffeic

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In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

R-

% I N H I B I T I O N O F GROWTH OF B L A C K SHANK FUNGUS BY H Y D R O X Y - C I N N A M I C A C I D S

-CH-CH-COOH

TABLE I I I .

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In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

T A B L E I V . % I N H I B I T I O N O F GROWTH O F B L A C K SHANK FUNGUS BY COUMARINS

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NATURALLY OCCURRING PEST BIOREGULATORS

398

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However, levels of these compounds in roots of disease-free, resistant, and susceptible varieties were comparable. Although levels of these compounds have been shown to increase in roots in response to infection, the levels of increase were similar in resistant and susceptible roots. Free phenolic acids were shown to be very active in inhibiting the growth of black shank. Free phenolic acids do not occur in tobacco, but could play a role in resistance. Perhaps, they are produced at the narrow point of attack of the fungus. Because of their high activity, only small amounts would be needed to block the advance of the fungus through the root. Further research is needed to determine if this is a viable mode of resistance and to search for other chemicals that may be responsible for the observed black shank resistance of certain tobacco varieties. L I T E R A T U R E CITED 1. 2. 3. 4. 5.

Fritig, B.; Hirth, L. ActaPhytopathol.Acad. Sci. Hung. 1971, 6, 21-29. Cohen, Y.; Kuc, J. J. Phytopathology 1981, 71, 209. Gasser, R.; Kern, H.; Defago, G. J. Phytopathology 1988, 123, 115-123. Snook, M . E.; Mason, P. F.; Sisson, V. A. Tob. Sci. 1986, 30, 43-49. Csinos, A. S.; Fortnum, B. A.; Gayed, S. K.; Reilly, J. J.; Shew, H . D. In Methods for Evaluating Pesticides for Control of Plant Pathogens; Hicky, K. D., Ed.; APS Press: St. Paul, MN; pp 231-236.

RECEIVED July 18, 1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 28

A Search for Agrochemicals from Peruvian Plants 1

1

1

1

D. Howard Miles , Jorge Meideros , Liza Chen , Vallapa Chittawong , Colin Swithenbank , Zev Lidert , Allen Matthew Payne , and Paul A. Hedin 1

2

1

3

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1

Department of Chemistry, University of Central Florida, Orlando, FL 32826 Rohm and Haas Company, Spring House, PA 19477 Crop Science Laboratory, U.S. Department of Agriculture, Mississippi State, M S 39762 2

3

The objective of this study was the isolation and identification of the antifeedant, antifungal, and antibacterial active constituents from Peruvian plants. 5,7-Dihydroxy-6,8-dimethyl-4'-methoxyflavanone (matteucinol) was isolated from the root of Miconia cannabina. This is the first report of the spectral data of matteucinol. Matteucinol showed significant antifungal activity (Pythium ultimum 135%). Also reported is the isolation of sitosterol and medicarpin from the toluene extract of the wood of Dalbergia monetaria. Medicarpin possessed high antifungal (Rhizoctonia solani, 145% and Helminthosporium teres, 100%) and antibacterial activity (Xanthomonas campestris, 100%) along with high antifeedant activity against the cotton boll weevil (Anthonomus grandis, 98%). As long as mankind continues to cultivate and grow plants for food, feed, and fiber, crops will be threatened by various enemies and agents such as insects, fungi, and bacteria. Prevention and control of insect pests and plant diseases will remain an important objective of agrochemical research. In this study, the isolation and structure determination of several compounds showing insect antifeedant activity and antifungal activity from Peru plants are presented. The plant Miconia cannabina Markgr. (Melastomaceae) is common to Iquitos, Peru. No previous chemical study has been undertaken on this plant; however, examples of studies on related plants are known. A study of the protein and water contents of several parts of the plant M. theaezans was undertaken by Gaulin and Craker (1). In another study Marini-Bettolo (2) isolated primin and miconidin from the ethyl acetate extract of a nonidentified species of Miconia. Primin has biological activity against various Gram positive and negative microorganisms and fungi as well as antitumor activity against sarcoma of mice (Swiss). Miconidin is less active against common microorganisms, but inhibits Mucobacterium sp and Gibberella fulikuroi (2). These two compounds also showed antifeedant activity (3) against six insect species - desert locust Schistocerca gregaria, the migratory locust Locusta migratoria, the armyworms S. littoralis and S. extemota, the budworm Heliothis amigera and caterpillars of the cabbage white butterfly Pieris brassicae. Dalbergia, a wood climber, belongs to the family Leguminosae and subfamily Papilionaceae. Nearly 120 Dalbergia species are known to occur in nature (4). The chemical components of this genus of plants provides a great number of structures belonging to widely different chemical categories such as, neoflavonoids, isoflavonoids, flavonoids, furans, isoflavans, benzophenones, styrenes, sterols, and terpenoids. 0097-6156/91/0449-0399$06.00A) © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

400

NATURALLY OCCURRING PEST BIOREGULATORS

Materials and Methods Sample collection. — Roots of M. canabina (voucher number 7261) and wood of D. monetaria (voucher number 7000) were collected in Iquitos, Peru in 1982, and identified

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by Dr. Sidney McDaniel, Department of Biological Sciences, Mississippi State University. The plant material was air dried and ground in a Wiley mill before extraction. Extraction. — M. Canabina (6.8 kg dry weight) was extracted in a soxhlet apparatus with methylene chloride (16 h.) and then ethanol (16 h.). The solvent was evaporated under reduced pressure to yield 6.7 g of methylene chloride extract (fraction A , Figure 1) and 22.6 g of ethanol extract (fraction B , Figure 1). Plant Material 6.8 kg Soxhlet CH Cl (16h.) 2

2

Fraction A 6.7 g I P ++ R + H

Ins. Soxhlet EtOH(16h) Fraction B 22.6 g I P ++++ R H

Ins.

Silica gel column 1

1 20% C H -C H 7

8

6

1 2

4 30% Et 0-C H 2

1 1

P R H

7

8

3 100% CHC1

I

+ ++ -

p

R H

... , 4 100% 3

CH3OH

1

1

1

1

+

+

++++ +++

P R H

1 +++ -

P R H

+ --

Purification Compound 1 P - Pythium ultimum; R - Rhizoctonia solani; H - Helminthosporium teres diameter of the clear zone of inhibition around the disk: + - 1-2 mm; ++ - 2-5 mm; +++ - 5-10; ++++ - > 10 mm Figure 1. Fractionation Scheme for Miconia cannabina D. monetaria (25.6 kg dry weight) was extracted in sequence with methylene chloride (16 h.) and ethanol (16 h.) in a Soxhlet apparatus. The solvent was removed in vacuo to yield 211.1 g of methylene chloride extract (fraction A , Figure 2) and 174.4 g of extract ethanol (fraction B , Figure 2). Fraction A was sequentially extracted with hot hexane (fraction A l , Figure 2) and toluene (fraction A 2 , Figure 2). Fraction B was extracted with ether. The ether-insoluble fraction (72.2 g) (fraction C, Figure 2) was

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

28.

MILES E T A L .

401

A Search for Agrochemicals from Peruvian Plants

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sequentially extracted with hexane : ether (2:1), and chloroform. The ether-soluble fraction (60.9 g) was partitioned between chloroform and water (1:1). Fraction A 2 (73.8 g) was extracted with chloroform. The chloroform solution was extracted with a 5% hydrochloric acid solution. The aqueous layer was neutralized with a 5% sodium hydroxide solution and shaken several times with chloroform. The chloroform was removed in vacuo to yield 4 g of fraction A2-b (Figure 3). The original chloroform layer was partitioned between chloroform and 5% aqueous sodium hydroxide solution (1:1). After separation of the layers, the aqueous layer was neutralized with 5% hydrochloric acid and extracted repeatedly with chloroform. The combined chloroform extracts were then dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo to yield 12.0 g of residue (fraction A2-a). Plant Material 25.6 kg Soxhlet CH Cl2(16h) 2

Fraction A 211.1 g Hexane (hot) Fraction A l 54.1 g P R H

h

INSOLUB. I Soxhlet EtOH(16h)

Ins. 138.0 g

Fraction B 174.49 g I ether

Toluene Fraction C 72.2 g (Ins.) ++ |— 79 Fraction A2 73.8 g Fraction A3 Hexane: ether (Ins.) (Sol.) 79 (2:1) 64.2 g

Sol162.9 i —i Ins^ P R H Ii

1 Fraction D 4.8 g (Sol.)

P R H Ij

h

P R H I,

80 95

h

76 98

P R H Ij

h

93 98

h

Fraction E 58.5 g

^cLci3 Sol. 4.5 g P R H 89 I, h 93

Ins. ^3.61 P R H 11 1 2

-

diameter of the clear zone of inhibition around the disk: + -1-2 mm; ++ - 2-5 mm; +++ - 5-10 mm; ++++ - >10 mm Boll weevil antifeedant activity (I): Iflo = [(# holes blank - # holes sample) / (# holes blank)] x 100 n = # mg / cm paper 2

Figure 2. Fractionation Scheme for Dalbergia monetaria

Fraction A2-a was chromatographed on silica gel (60-200 mesh, 800 g) with eluents of increasing polarity from hexane through toluene to chloroform, and then to methanol. Fractions of 250 ml were collected, the solvent was removed in vacuo, and those fractions exhibiting similar T L C profiles were combined. Bioassay Antifungal and Antibacterial Bioassay. — Fractions were screened for activity against the fungi Helminthosporium teres, Pythium ultimum, and Rhizoctonia solani and the bacteria Xanthonomas campestris by the paper disc method (5). Each fraction to be tested was

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

NATURALLY OCCURRING PEST BIOREGULATORS

402

Fraction A2 73.8 g (1) CHC1 (2) HC1 (5%) 3

~B 0

cricIT

2

(1) 5%aq.NaOH

aq. NaOH (5%) CHC1

1—

"So

3

H 0

1

2

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(1) HC1 (5%)

HoO

(2) CHCI3 CHCI3 A2-b4g

(2) CHCI3 CiHCI3 A2-a 12.0 g Silica gel column

1— 3 n

6 12

T

50%

Compound 2

C7H0 ^

h

1

H R P h I9

1— 10 CHCl,

+++ +++ +++ 95.0 98.0 "T 11

12

CH3OH

++++ +++ +++ 97.5 99.0 Purification

Compound 3 diam. of the clear zone of inhibition around the disk: + -1-2 mm; ++ - 2-5 mm; +++ - 5-10 mm; ++++ - >10 mm Boll weevil antifeedant activity (I): In % = [l-(# holes sample / # holes blank)] x 100 n = # mg / cm paper 2

Figure 3. Purification of Fraction A2 of Dalbergia monetaria

dissolved in an appropriate solvent. In the preliminary tests of semi-purified plant extracts, a 100,000 ppm solution was prepared, while in the final test of pure compounds a 10,000 ppm solution was used. Filter paper discs (6 mm dia.) were soaked in the test solution and allowed to dry overnight. Petri plates containing solidified agar (DIFCO Potato-Dextrose for H. teres and R. solani, DIFCO cornmeal for P. ultimum, and DIFCO nutrient agar for X. campestris) were inoculated with fungi. The inoculation with H. teres and X. campestris was performed by mixing 100 ml of the appropriate agar with the filtrate of an aqueous suspension of their spores and then placing 5 ml of the mixture on top of the solidified agar layer. For R. solani and P. ultimun, pieces of the mycelium were cut from the culture plate and placed in the center of the bioassay plate. Three of the prepared paper disks were then placed on each Petri plate. A paper disk soaked only in the solvent used and dried by the usual method was also placed on each plate as a blank. As a control, a 10,000 ppm solution of Dithane M-45 (from Rhom & Hass Co.), a commercially available fungicide, was used. Inhibition was determined by measuring the diameter of the clear zone (if present) around the disc. Boll weevil antifeedant bioassay. — The bioassay was performed according to the procedure of Hedin et. al. (6, 7). Agar plugs were formed by boiling 3 grams of agar and

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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28.

MILES ET AL.

403

A Search for AgrochemicalsfromPeruvian Plants

3 grams of freeze-dried cotton bolls in 100 mis of distilled water to effect a viscous sol. The sol. was poured into 18 mm diameter tubing, and gelation occurred after cooling. These gelatinous rods were cut into individual 3.6 cm plugs. The extracts of the plant samples were applied to preweighed 4.5 x 4.5 cm squares of Whatman #1 chromatography paper by dipping the paper into a solution of the test sample. After air-drying, the paper was weighed. A blank was prepared by dipping one of the papers into the solvent used on the test sample. The papers were wrapped around the agar-cotton plugs and fastened with four staples. The ends of the plugs were sealed with corks. The plugs were then placed staple-side down in the petri dishes. Twenty newly emerged boll weevils were placed in 14 x 2 cm petri dishes containing the test and control plugs. The bioassay was carried out in the dark at 25°C for 4 hours. When the bioassay was finished the paper was removed from the plugs and the number of punctures counted. Antifeedant activity (IJ was expressed as: (IJ % = [l-(# holes sample/^ holes blank] x 100 where n indicates the amount used (in grams) of the compound per surface units (cm ) of the filter paper. 2

Results and Discussion The ethanolic extract of M. cannabina revealed the highest antifungal activity when tested against P. ultimum (P), R. solani (R), and H. teres (H)» using the preliminary "paper disc" method (5). Fractionation of the ethanolic extract was performed by column chromatography (Figure 1) with silica gel as the support. The resulting chromatographic fractions were monitored by the "paper disc" bioassay. The material which was eluted with 30% (v/v) ether in toluene was further purified by repetitive crystallization from methanol to yield the compound 1. The high resolution mass spectrum suggested a molecular formula of C H 0 for compound 1. This compound developed the characteristic color reactions of flavonoids (8,9). The U V spectrum of compound 1 in methanol, consisted of two major absorption maxima, one of which occurred at 294 nm. The other band at 328 nm was less intense and was suggestive of the presence of a flavonoid lacking conjugation between the A - and B-rings (9,10,11). A doublet of doublets at 5 2.83 and 3.08 in the H N M R spectrum of compound 1 suggested the presence of a flavanone. 1 8

1 8

5

l

7

r ^

6

^ O Skeleton of flavanones

Examination of the U V spectra of compound 1 in methanol with the shift reagents NaOMe, A1C1 , A1C1 + HC1, NaOAc, and NaOAc + H B 0 indicated that compound 1 had the basic skeleton of 5,7-dihydroxyflavanone. The absence of signals between 8 5.7 6.9 in the *H N M R spectrum indicated that the C-6 and C-8 positions in the A-ring were substituted. The presence of protons, at C-3', C-5', C-2', and C-6' was indicated by two pairs of ortho coupled doublets at 5 6.98 and 7.42 (J = 10 Hz). A mass spectral fragment at m/z 180, suggested that the two methyl groups observed in the *H N M R spectrum (8 2.06 and 2.08 were at C-6 and C-8; therefore, the methoxy group at 8 3.84 ppm in the *H N M R spectrum could be located at C-4'. These spectral properties are consistent with 3

3

3

3

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

404

NATURALLY OCCURRING PEST BIOREGULATORS

the assignment of the structure of compound \ as 5,7-dihydroxy-6,8-dimethyl-4'methoxyflavanone. A compound of this structure was first isolated from Matteucia orientalis by Munesada (12) and was named (-)-matteucinol. Later the isolation of matteucinol was reported from two species of Rhododendron, by Arthur and Hui (13) and by Tanabe, Kondo and Takahashi (14); however, the structural assignments were not unequivocal since no spectral data was reported. Thus, this paper provides the first report of the spectral data of (-)-matteucinol and confirms the structure assignment as 5,7-dihydroxy-oc,8dimethyl-4'-methoxyflavanone. Matteucinol demonstrated excellent antifungal and antibacterial activity (Table I). The highest antifungal activity was observed against R. solani for which a threshold concentration of 15.1 p,g/cm was determined by regression analysis. A threshold concentration of 7 x ltf u,g/cm was determined for the activity against the bacterium X. campestris.

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2

2

2

Table I. Antifungal and antibacterial activity* of Matteucinol and (-)-Medicarpin

P. ultimum

MICROORGANISM R. solani H. teres

X. campestris

Matteucinol

25

135

30

69

(-)-Medicarpin

75

145

100

80

'relative activity of compound (10,000 ppm) to the standard (10,000 ppm): Relative activity (%) =

diam. of clear zone for test compound diam. of clear zone for standard

X

100

In a continuing search for compounds with antifungal and insect antifeedant activity, the active constituents from the wood of D. monetaria were also examined. The toluene soluble material (fraction A-2) from the methylene chloride extract of the wood of this plant showed both potent insect antifeedant activity against the boll weevil (A. grandis Boheman) and high antifungal activity (Figure 2). Partitioning of fraction A-2 between chloroform and 5% aqueous hydrochloric acid resulted in the concentration of the basic components in the aqueous layer. The basic fraction (fraction A-2-b), obtained as shown in Figure 3 presented no antifungal or insect antifeedant activity.

[

O

Matteucinol

Partitioning the chloroform layer between chloroform and 5% aqueous sodium hydroxide (Figure 3) resulted in concentration of the acid components in the aqueous layer. T7ie acid fraction (fraction A-2-a) showed high antifungal activity (P. ultimum - +++;/?. solani - +++; H. teres - +++) and antifeedant activity against boll weevils (feeding inhibition activity of 95.0 and 98.0% at the dose level of 1 and 2 mg/cm of the filter 2

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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28. M I L E S E T A L .

405

A Search for AgrochemicalsfromPeruvian Plants

paper). Fraction A-2-a was fractionated on a silica gel column with eluants of increasing polarity from hexane through toluene to chloroform and then to methanol as shown in Figure 3. The material was eluted with toluene-hexane (1:1) and was further purified by recrystallization from benzene to produce compound 2. A more polar material was eluted with toluene and purified by flash chromatography using hexane-acetone (2:1) as the eluant. Fractions 13-18 were combined and further purified by preparative T L C using 10% methanol in chloroform (v/v) to obtain compound 3. Compound 2, m.p. 134 - 135°C, showed a NT at m/e 414. The *H N M R , U V , and IR spectra suggested the presence of a steroid. The color reactions developed with the Liebermann-Burchard and Carr-Price reagents were also in accord with a steroid structure. Compound 2 was identified as sitosterol by comparing its m.p. and *H N M R , IR, U V , and M S spectra data with those of an authentic sample (Aldrich Chemical Company, Inc., Milwaukee, WI). Compound 3 had a molecular formula of C H 0 , as determined by high resolution mass spectrum. The *H N M R spectrum was similar to those observed for pterocarpans (13). Compound 3 was identified as (-)-Medicarpin by comparing its m.p. and *H N M R , IR, U V , and M S with those of an authentic sample (Dr. H . D . Van Etton, Cornell University). (-)-Medicarpin showed excellent antifungal and antibacterial activity (Table I). The highest antifungal activity (14.5%) was observed against R. solani. 16

14

4

O

(-)-3 -Hydroxy-9-methoxypterocaipan [(-)-medicarpin]

Conclusion This is the first report of the isolation of matteucinol from Af. cannabina and (-)medicarpin from D. monetaria. Matteucinol demonstrated excellent antifungal activity (R. solani 135%). (-)-Medicarpin showed activity against the fungi P. ultimum (75%), R. solani (145%), H. teres (100%), the bacteria X. campestris (80%), and the boll weevil (100% antifeedant at a dose level of 2 mg/ml). Thus matteucinol and (-)-medicarpin have potential as new agrochemicals. Acknowledgments This study was supported by the Rohm & Haas Co. We are indebted to Drs. Catherine Costello and Thomas Dorsey of the M I T mass spectrometry facility for their invaluable assistance in obtaining the mass spectrum of compounds. We thank Dr. Jim Dechter and Vicki Farr of the University of Alabama N M R facility for their help in obtaining H N M R data. !

Literature Cited 1. Gaulin, S.J.C.; Carker, L.E. J. Agric. Food Chem.1974,27(4), 791. 2. Marini Bettolo, G.B.; Delle Monache, F.; Goncalves Da Lima, O.; de Barros Coelho, S. Gazz. Chim. Ital.1971,101, 41. 3. Benays, E.; Lupi, A.; Marini Bettolo, R.; Mastrofrancesco, C.; Tagliatesta, P. Experientia,1984,41, 1010. 4. Chawla, H.M.; Chibber, S.S. J. Scientific and Industrial Research1981,40, 313-325.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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NATURALLY OCCURRING PEST BIOREGULATORS

5. The Official Methods of Analysis of the Association of Official Analytical Chemist, Official First Action, Eleventh Ed., 1970, p. 843. 6. Hedin, P.A.; Thompson, A.C.; Minyard, J.P. J. Econ. Entomol.1966,59, 181. 7. Struck, R.F.; Frye, J.; Shealy, Y.F.; Hedin, P.A.; Thompson, A.C.; Minyard, J.P. J. Econ. Entomol.1961,61, 270. 8. Zweig, G.; Sherma, J., (Ed.s), Handbook of Chromatography, Vol. 2, The Chemical Rubber Co., Clevelent, 1972. 9. Mabry, T.J.; Markham, K.R.; Thomas, M.B. In: The Systematic Identification of Flavonoids, Springer-Verlag, New York, 1970. 10. Jurd, L., In: "The Chemistry of Flavonoid Compounds", Geissman, T.A., (Ed.), MacMillan, New York, 1962. 11. Harbone, J.B.; Mabry, T.J. In: "The Flavonoid Compounds", Academic: New York, 1975, Vol.1. 12. Munesda, J. Pharm. Soc. Japan, 1924, 505, 185. 13. Arthur, H.R.; Hui, W.K.; J. Chem.Soc.,1954,2782. 14. Yoshihisa, T.; Kondo, S.; Takahashi, K.; Kanazawa Daigaku Yakugakubu Kenkyu Nempo,1962,12, 7-14. RECEIVED May 16, 1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 29

Suppression of Fusarium Wilt of Adzukibean by Rhizosphere Microorganisms 1

2

2

Shinsaku Hasegawa , Norio Kondo , and Fujio Kodama 1

Hokkaido Institute of Public Health, North 19, West 12, North-ward, Sapporo 060, Japan The Hokkaido Central Agricultural Experiment Station, Naganuma 069-13, Japan

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2

During a study on the biological contorol for Fusarium wilt of adzukibean caused by Fusarium oxysporum f. sp. adzukicola, we isolated antagonistic microorganisms from the rhizoshere soil of adzukibean root by the improved triple layer method. Hemipyocianine, chrororaphin and phenazine-l-carboxylic acid were isolated from Pseudomonas aeruginosa S-7 and P. fluorescens S-2; pyrrolnitrin from P. cepacia B-17; antibiotic Y-1 (monazomycin like) from Streptomyces flaveus Y-1. These microbial cultures and antifungal agents strongly inhibited growth of F. oxysporum f. sp. adzukicola FA-3. In greenhouse, the treatment of adzukibean seed with P. cepacia B-17 in F. oxysporum FA-3-infested soil decreased the diseases incidence from 76.3 to 8.8%; St. flaveus Y-1, 9.0%; P. aeruginosa S-7, 11.6%; P. fluorescens S-2, 13.3%. At a crop filed, the incidence decreased from 88% to 58%, 38%, 63% and 59% respectively. The isolates may be useful as antagonists to Fusarium wilt of adzukibean.

Fusarium w i l t of the adzukibean caused by Fusarium oxysporum f. sp. adzukicola i s a serious and widespred disease i n Hokkaido, northern part of Japan. This pathogen i s a root-infecting fungus that spreads r e l a t i v e l y slowly along the vascular bundle and causes necrosis, yellowing and w i l t ( l - 3 ) . In our work on Fusarium wilt of adzukibean, we have isolated microorganisms antagonistic to F. oxysporum f. sp. adzukicola FA-3 from the adzukibean rhizosphere, and report here: the i s o l a t i o n and i d e n t i f i c a t i o n of antagonists and their producing antifungal agents; and the efficacy of isoates or these antifungal agents as seed treatment to Fusarium w i l t by F. oxysporum. f . sp. adzukicola(l,4-6).

0O97-6156/91/0449-O4O7$06.00/0 © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

408

NATURALLY OCCURRING PEST BIOREGULATORS

I s o l a t i o n and I d e n t i f i c a t i o n o f A n t a g o n i s t i c M i c r o o r g a n i s m s and t h e s e P r o d u c i n g A n t i f u n g a l Agents We i s o l a t e d a n t a g o n i s t s t o Fusarium oxysporum F A - 3 from the r i z o s phere o f a d z u k i b e a n and o t h e r m a t e r i a l s by t h e improved t r i p l e l a y e r

Table I. Isolated Antagonists and Their Producing Antifungal Agents

Pseudomonas aeruginosa S - l S-7 SH-6 f l u o r e s c e n s S-2 Y-15 NS-7371 c e p a c i a B-17 1218

Adzukibean(Rl) Adzukibean(Rl) Soil Adzukibean(Rl) Melone(Rl) liiy(Rl) Adzukibean(Rh) Beet(Rh)

Streptomyces sp. No. 2 sp. B-6 flaveus Y - l

Adzukibean(Rl) Adzukibean(Rl) R i v e r water

N

0

>luteorii

Source( )*1

Lororaph:

Hemipyocianinc

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Antagonist

1 -H 0) i H

rrolnitrj

i

a Antifungal agents

+

+ -

e + + + (+)

(+)*2 + + + + +

-

-

+

+ +

Water is o l u b l e Water is o l u b l e Antibiotic Y - l (monazomycin l i k e )

*1 Sample s o u r c e : ( R l ) , R h i z o p l a n e ; (Rh), Rhizosphere *2 A n t i f u n g a l agent: p r o d u c i n g , +; t r a c e , (+); no p r o d u c i n g , Hemipyocianine

|

H

}j

Pyrrolnitrin

CONH

2

Chlororaphin Pyoluteorin

Phenazine-1carboxylic acid

^1 OH

COOH

CI

O

HO

H

9H OH HO

Antibiotic Y - l (Monazomycin like)

HO OH

OH

OH OH

•CH3 OH

O H ^ H

CH3 C H g C H 3 OH >H 8H H

CH

HN

v

OH

OH

2

CH3 CH3

CH3 CH~

CH

3

CH

3

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

29.

HASEGAWA ET AL.

Suppression of Fusarium Wilt of Adzukibean 2

409

5

method(4). One ml of diluted(10 -10 w/v) rhizosphere s o i l of adzukibean were spread i n plates with 1.5% agar and added the spore suspension of F. oxysporum FA-3 on the plates. These plates were incubated at 28 C for 2 to 4 days, and then the colonies with clear zone were picked up. These i s o l a t e s were i d e n t i f i e d as Pseudomonas aeruginosa, P. fluorescens, P. cepacia, Streptomyces flaveus and Streptomyces spp.( 1,2,4-8, Table I). These produced hemipyocianine, chlororaphin, phenazine-l-carboxylic a c i d ( l , 6 ) , p y r r o l n i t r i n ( 1 , 4 ) , pyoluteorin(5,6), A n t i b i o t i c Y-l(monazomycin l i k e , 7) and water soluble unknowns(Table I ) .

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in vitro Antifungal A c t i v i t i e s of Microbial Cultures and A n t i fungal Agents against Fusarium oxysporum f. sp. adzukicola FA-3 Bioassay by reversed layer method(4, F i g . I) showed that 3 s t r a i n s of Pseudomonas and St. flaveus Y-l(8) were highly i n h i b i t o r y to F. oxysporum FA-3(Table II). On agar spot inoculation plates(4, F i g . I ) , P. aeruginosa S-7, St. flaveus Y-l and Streptomyces sp. No.2 showed highly i n h i b i t i o n and continuance of antifungal a c t i v i t i e s . The minimum i n h i b i t o r y concentration(MIC) of p y r r o l n i t r i n against F. oxysporum FA-3 was 3.13 ^ig/ml; A n t i b i o t i c Y - l , 6.25 pg/ml; hemipyocianine, 50 pg/ml; chlororaphin and phenazine-lcarboxylic acid, 100 ug/ml; and water soluble unknowns, 25-50 jug/ml (Table I I ) . Antifungal Spectrum of Microbial Cultures and Antifungal Agents The microbial cultures of i s o l a t e s and t h e i r antifungal agents strongly inhibited growth of F. oxysporum and also many other plant pathogenic fungi: F. solani, F. moniliforme, F. roseum, Biporaris sorokiniana, Alternaria alternata, Cladosporium cucumerinum, Pyricularia oryzae, Pythium graminicolum, Verticillium dahliae, Cylindrocarpon sp., Rhizoctonia solani(Table I I I , IV).

a. Reversed layer method Fig. I

b. Agar spot i n o c u l a t i o n method

Antifungal a c t i v i t i e s of antagonists against Fusarium oxysporum FA-3

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

NATURALLY OCCURRING PEST BIOREGULATORS

410

A n t i b i o t i c Y - l was an e f f e c t i v e i n h i b i t o r of a l l tested pathogens i n c u l t u r e . But p y r r o l n i t r i n was much l e s s e f f e c t i v e against Fusarium species than any other genera, and was t o t a l l y i n e f f e c t i v e s against F. oxysporum f.sp. cepae. Also, phenazines, such as hemipyocianine did not i n h i b i t strongly the growth of Fusarium species(Table IV). But the s t r a i n s which produced phenazines were a c t i v e against Fusarium species and continued these strength on the agar spot i n o c u l a t i o n method(Table I I I ) . The s p e c i f i c i t y of the s t r a i n s f o r s u s c e p t i b i l i t y against antifungal agents were recognized.

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Table I I The I n h i b i t i o n e f f e c t of i s o l a t e d antagonists and t h e i r a n t i fungal agents against Fusasrium oxysporum FA-3 Antagonist

I. Antagonist agar d i s c

I I . Antifungal agent

Reversed layer method*!, 2days (inhibitioni zone, mm)

Isolated antifungal agent

Pseudomonas aeruginosa S-7 (Brucella agar, 30 'C, 4days)

30.0

Pseudomonas cepacia B-17 (Brucella agar, 30 *C. 4days)

33.8

Pseudomonas fluorescens NS-7371 (Brucella agar, 30 *C. 4days)

31.5

Streptomyces sp. No. 2 (YM agar*3, 30*C, 7days)

24.0

Streptomyces sp. B-6 (PDA*4, 30 'C, 4days)

25.0

Streptomyces flaveus Y - l (GS agar*5, 30 C, 7days)

31.2

Agar spot inoculation method*2 (inhibition zone, mm)

9.5 +++(7days)

Hemipyocyanine Chlororaphin Phenazine-lcarboxylic a c i d

7.0 -H- (4days)

pyrrolnitrin

9.5 +++(4days)

3.5 +

3.13

Pyrrolnitrin Phenazine-lcarboxylic a c i d

3.13 200

(7days)

11.2 -H-K4days)

Crude water soluble

25

Crude water soluble

50

9.5 +++(7days) 4.7 ++ (4days) 0.0 -

(7days)

10.7 +++(4days)

{

#

100 200 200

(7days)

7.2 ++ (4days) 4.8 +

Minimum inhibitory concentration (ug/ml, 7days)

Antibiotic Y-l (Monazomycin like)

3.13

10.1 +++(7days)

*1 Reversed layer method: Fusarium oxvsprum FA-3 spores were used f o r bottom layer and 4 o r 7 days c u l t u r e agar d i s c s of antagonists were used. *2 Agar spot i n o c u l a t i o n method: 7 days culture agar d i s c s of Fusarium oxysporum FA-3 and 4 or 7 days culture agar discs of antagonists were used. I n h i b i t i o n zone between the pathogen and the antagonist: +++£ 9.0mm, $.0>-H->5.0, 5.0>+;>2.0, 2.0>(+)>0.0, — 0 . 0 *3 YM agar: Yeast-malt agar *4 PDA: Potato dextrose agar *5 GS agar: Glycerol-soybean meal agar

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991. +++ +++

+++ +++

++ ++ +++ +++ +++

++ ++ ++

+++ ++

-

++ ++ ++ ++ + +++ +++ +++ +++ +++

-

7

+++ +++

+

-

++

++ + + + ++

++ +

+++ +++

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++f +4+ +4+

++ ++ ++ ++ + ++

+4+ + + +

++

++ ++ ++ +++ +++ ++ ++ ++ +++ +++ +++ ++ +++ +++

4 ++ ++

+ +

7

4

4

3 O (V 3 I MHW PH 4-1 Z

+++ +++*5++ ++ ++ ++

7

Pu O CO

(0 V I fr, (0 to

3 (0

»iH O 0)

i

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«J c

(0 (0

7

++ ++ +++ ++ ++ + + + ++ +++ +++ ++ 4-4-4+4-4+4-+ ++

+++ +++ -H-f +++ ++ + + + ++ +++ +++ ++ +++ +++

+++ +++ +4+ +++

4

r 4J O

4

ND*6++

-

7

++ ++ ++4h +++

++ +

+

++ +-

+

++ +

7

+++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ +++ +++ 4h++ +++ NT* 7

4h++ 4h++ 4h++ 4h++ 4h4+ 4h++ 4-++ 4h++ 4-++ 4h++ 4h++ 4h++ 4h++ 4h++

Hh++ +++ 4h++ ++

4

0) > H (0

a 9.0mm, 9.0>++>5.0, 5.0>+>2.0, 2.0> (+)>0.0. — 0 . 0 ~ ~ *6 ND: not detected. *7 NT: not tested.

Fusarium oxysporum FA-3(adzukibean)*3 Fusarium oxysporum FGH(adzukibean) Fusarium oxysporum F-l(adzukibean) Fusarium oxysporum f.sp.*4 spinacea H-18 Fusarium oxysporum f.sp.spinacea H-29 Fusarium oxysporum H-32(green pepper) Fusarium oxysporum f . s p . f r a g a r i a e H-34 Fusarium oxysporum f.sp.melonis H-34 Fusarium oxysporum f . s p . l y c o p e r s i c i KF-244 Fusarium oxysporum f.sp.cepae KF-228 Fusarium oxysporum KF 8 5 4 ( l i l y ) Fusarium s o l a n i H-41(rice) Fusarium moniliforme H-24(rice) Fusarium moniliforme H-36(rice) Fusarium roseum H-35(wheat) B i p o r a r i s sorokiniana H-21(rice) A l t e m a r i a a l t e m a t a H-25(rice) Cladosporium cucumerinum H-23(melon) P y r i c u l a r i a oryzae H-22(rice) Pythium graminicolum H-30(rice) V e r t i c i l l u m d a h l i a e H-28(strawberry) Cylindrocarpon sp. KF 8 4 6 ( l i l y ) Rhizoctonia s o l a n i H-31(potato) Rhizoctonia s o l a n i KF 8 5 2 ( l i l y )

Culture days

O -H

09 C M

«J

Antagonists

Growth i n h i b i t i o n o f the plant pathogens by the m i c r o b i a l c u l t u r e s o f a n t a g o n i s t s * !

Phytopathgenic fungi*2

Table I I I

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412

NATURALLY OCCURRING PEST BIOREGULATORS

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In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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29.

HASEGAWA ET A L .

Supression of Fusarium Wilt of Adzukibean

E f f i c a c y of Seed Bacterization with M i c r o b i a l of Fusarium Wilt of Adzukibean

413

Cultures to Control

In greenhouse, treatment of the adzukibean seed with washed b a c t e r i a l c e l l s of P, cepacia B-17 i n F. oxysporum FA-3-infested s o i l ( F i g . I I ) decreased the disease incidence from 76.3 to 8.8% (Fig. I l l ) ; S t . flaveus Y - l , 9.0%; P. aeruginosa S-7, 11.6%;

Adzukibean

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ANTAGONIST

BACTERIZATION

NO TREATMENT

/

Dipping Incubation Fig. I I B i o l o g i c a l control of Fusarium w i l t

F i g . I l l E f f e c t o f seed b a c t e r i z a t i o n with Pseudomonas cepacia B-17 on the c o n t r o l of Fusarium w i l t o f adzukibean caused by Fusarium oxysporum FA-3*1 *1 Adzukibean: Hayate-syouzu, Treatment: Pathogenic f u n g i , Fusarium oxysporum FA-3(10 spores/g Antagonist, Pseudomonas cepacia B-17(10 cells/ml s o l u t i o n ) . 5

8

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

soil);

414

NATURALLY OCCURRING PEST BIOREGULATORS

Table V

E f f e c t s of seed b a c t e r i z a t i o n with antagonists on the control of adzukibean w i l t caused by Fusarium oxysporum i n green house and crop f i e l d * ! Disease incidence(Z) Green house (36days)

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Antagonist*2 Pseudomonas aeruginosa S-7 Pseudomonas cepacia B-17 Pseudomonas fluorescens NS-7371 Flavobacterium sp. AB7 Acinetobacter calcoaceticus B28 Streptomyces sp. No. 2 Streptomyces sp. B6 Streptomyces flaveus Y - l Without antagonist

Crop f i e l d (45days)

-

-

11.6 8.8 20.5 30.2 77.0 15.2 65.0 9.0 76.3

63 58 -*3

-

85 92 82 50 88

Me-cellulose 60 46

41-

-

38 88

*1 Adzuibean: Hayate-shozu i n green house; Takara-shozu i n crop field, Pathogenic fungi: Fusarium oxysporum FA-3, 10 spores/g s o i l i n green house; Fusarium oxysporum 10 c~ells/g s o i l i n crop f i e l d *2 Antagonist: 108cel?.s/ml s o l u t i o n *3 Not tested. 5

3

Streptomyces sp. No.2, 15.2%; and P. fluorescens NS-7371, 20.5% (Table V). In crop f i l e d , treatment with St. flaveus Y-l cultures decreased the disease incidence from 88 to 38%; Streptomyces sp. No.2, 41%; and P. cepacia B-17, 46%(Table V). Methyl c e l l u l o s e was used for i n j e c t i o n of microbial c e l l s at the time of seeding i n field. Growth of Microorganisms and Production of Antifungal Agents i n S o i l on the Control of Fusarium Wilt of Adzukibean In the case of low disease incidence, such as P. cepacia B-17 and St. flaveus Y - l , the increase of the number of antagonistic microorganisms and the decrease of the number of pathogen, F. oxysporum FA-3, i n the rhizosphere s o i l of adzukibean were recognized(Fig.IV, Table VI). The t i g h t l y relationship between the increase of a n t i fungal agents and the decrease of disease incidence was not recognized. Washed microbial c e l l s were as efficacious as whole cultures or culture f i l t r a t e s , even though no or a few amount of antifungal agents were present when the seeds were treated. None of the treatments was phytotoxic to adzukibean. The protective effect exhibited by treatment of adzukibean seed with cultures of the antagonistic microorganisms may be due to release of the antifungal agents by the gradual growth of the microbial c e l l s . Even though the concentration may be low, the lysing c e l l s may e f f e c t prolonged release and a v a i l a b i l i t y of the antifungal agents during the c r i t i c a l period of seeding growth.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

29.

HASEGAWA ET AL.

Pseudomonas cepacia 10

415

Suppression of Fusarium Wilt of Adzukibean

B-17

Streptomyces f l a v e u s Y - l

c

Without antagonist 10

;

3 8l0 ° »

w2

4

3 10 3

IS

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10 20

10

30

Culture days

F i g . IV

20

30

Culture days

Time course comparison of growth of microorganisms and production of a n t i f u n g a l agents on the c o n t r o l of Fusarium w i l t of adzukibean i n green house

Table VI Growth of microorganisms and production of antifungal agents i n rizoshere s o i l of adzukibean i n green house Number of c e l l s / g

soil

Antagonist Antagonist*l (days)O 10 20 30 36 Pseudomonas aeruginosa S-7

F. oxysporum*2 0 10 20 30 36 T

Production of antifungal agent (ug/g s o i l ) 0 10 20 30 36

Disease incidence

T~

Pseudomonas cepacia B-17 Pseudomonas fluorescens NS-7371 Acinetobacter calcoaceticus B-28 Streptomyces sp. No. 2

*1 10®cells/ml solutions of antagonists were used f o r seed bacterization. *2 10 c e l l s / g s o i l of Fusarium oxysporum FA-3 were used.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

NATURALLY OCCURRING PEST BIOREGULATORS

416

Conclusion These r e s u l t s suggested that the i s o l a t e s such as P. cepacia B-17, St. flaveus Y-l and Streptomyces sp. No.2 may be useful as antagonists to F. oxysporum f . sp. adzukicola and may f a c i l i t a t e establishment of c u l t i v a t i o n of healthy adzukibean. The antagonisms exhibited by the microorganisms are possibly the r e s u l t of production of antifungal agents, which are themselves e f f e c t i v e i n protecting agaist Fusarium w i l t of adzukibean. Literature Cited

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1. 2. 3. 4. 5.

6. 7. 8.

Hasegawa, S.; Kodama, F.; Kaneshima, H . ; Akai, J. J . Pharmacobio-Dyn. 1987, 10S, 57. Kodama, F.; Hasegawa, S. Kongetu no Nouyaku 1985, 30, 22-27 Kondo, N . ; Kodama, F. Shokubutu Boeki 1989, 43, 28-33. Hasegawa, S.; Kamneshima, H . ; Kodama, F.; Akai, J. Report of the Hokkaido Institute of Public Health 1986, 36, 16-23. Hasegawa, S.; Kondo, N . ; Kodama, F . Proceedings of 7th Symposium on the Development and Application of Naturally Occurring Drug Materials, 1989, 7, 67-70. Hasegawa, S.; Kondo, N . ; Kodama, F. J . Pharmacobio-Dyn. 1990, in press. Hasegawa, S.; Kamneshima, H. Report of the Hokkaido Institute of Public Health 1987, 37, 6-17. Hasegawa, S.; Kodama, F.; Nakajima, M . ; Akai, J.; Murooka, H. B u l l . Agr. & Vet. Med., Nihon Univ. 1987, 44, 80-86.

RECEIVED May 16, 1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 30

Soil-Borne Diseases in Japan Practical Methods for Biological Control 1

2

2

Shinsaku Hasegawa , Fujio Kodama , and Norio Kondo 1

Hokkaido Institute of Public Health, North 19, West 12, North-ward, Sapporo 060, Japan The Hokkaido Central Agricultural Experiment Station, Naganuma 069-13, Japan

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2

The interest and research in biological control have increased in this decade. The following are the pro­ mising procedures which will be adopted in the near future. 1. Sweet potato sprouts which were previously inoculated with non-pathogenic Fusarium oxysporum isolate showed high resistance against Fusarium wilt caused by F. oxysporum f.sp. betatas. 2. Pseudomonas gladioli suppressed some Fusarium diseases. Seedling of associate crops(Allium spp.) which had been dipped in suspension of this strain, and mix-cropped with commercial crops in Fusarium infected field. Fusarium diseases of tomato and bottle gourd(Lagenaria cicereria) were suppressed. 3. Edible lily root rot caused by Cylindrocarpon destructans was controlled when the mother bulbs were coated with P. fluorescens S-2 or P. aeruginosa S-7. These bulbs were much bigger than the non-treated ones. B i o l o g i c a l control of s o i l borne diseases i s keenly being promoted by the government and a g r i c u l t u r a l industries i n Japan. This i s i n part a response to public concern about hazards associated with chemical pesticides. Interest and research on the issue have increased t h i s decade i n Japan. B i o l o g i c a l control has been studied for over 65 years, but few successes have been made i n the commerc i a l f i e l d . The following are the promising a r t i c l e s which w i l l be adopted i n the f i e l d i n near future. The authors discuss three examples of b i o l o g i c a l control of s o i l borne diseases. The f i r s t i s the control of Fusarium w i l t of sweet potato by cross-protection that involves a prior inoculation of nonpathogenic Fusarium oxysporum(Ogawa et al., 1984)(1). The second i s the control of Fusarium w i l t of bottle gourd by mixedcropping with associate crops(Kijima et a l . , 1986)(2). The t h i r d i s the seed bulb bacterization for the control of root rot of edible l i l y ( H a s e g a w a et al., 1990)(3). 0O97-6156/91/0449-O417$O6.00/0 © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

418

NATURALLY

OCCURRING PEST BIOREGULATORS

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B i o l o g i c a l Control of Fusarium Wilt of Sweet Potato with Crossprotection by Prior Inoculation with Nonpathogenic Fusarium oxysporum The causal fungus of Fusarium w i l t of sweet potato i s Fusarium oxysporum f. sp. betatas. Some i s o l a t e s of F. oxysporum which are obtained from healthy sweet potato plants showed remarkable crossprotection against the disease, when they were previously inoculated in the sweet potato sprouts before being planted i n the infested s o i l . These i s o l a t e s of F. oxysporum were not pathogenic to sweet potato, and also not to other plants such as tomato, cucumber etc. In naturally infested experimental or commercial f i e l d s , crossprotection by pre-inoculation with nonpathogenic i s o l a t e s of F. oxysporum has always brought remarkable decreases i n w i l t incidence and increases i n the y i e l d of sweet potato. The effects were equivalent to those obtained with chemical treatment i n which cutends of the sprouts were dipped into a benomyl suspension(500 times of 50% w.p.) for 30 min., as shown F i g . I.

c CO

rH vO

++^ -°> -° > 2 -°> -° > 6

Phenazine-l-carbox y l i c acid 2 F i g . IV

6

Chlororaphin 3

t r a c e

>

-=

Hemipyocianine

Structure of phenazines.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

4

30.

HASEGAWA ET AL.

423

Soil-Borne Diseases in Japan

After dipping into the suspension of the biocontrol agent, i . e . s t r a i n S-2, the seed bulbs were transplanted into the f i e l d that was infested with Cylindrocarpon destructans. Table IV shows the e f f e c t of bacterization on the y i e l d of edible l i l y . The growth on the s o i l of the treated plants was obviously higher than the nontreated check. Yields of s t r a i n S-2 and S-7 bulbs treated by dipping were increased 167 and 145%, respectively. Strain S-2 was the most effective with an average bulb weight of 114.6 gm.

Table IV

Effect of bacterization on y i e l d of l i l y

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Weight Treatment

Pseudomonas aeruginosa S-7 Pseudomonas fluorescens S-2 Pseudomonas cepacia B-17 Streptomyces sp. B-2 Control

Y

1

^ ? o ?x (kg/4.8m^) 9.6 11.0 7.9 7.1 6.6

b*l a c cd d

o

Length

f

b u l b ( g

o

)

100.9 114.6 85.0 76.7 74.4

f

stem(cm) b a c d d

72.2 68.0 64.5 64.2 64.9

*1 Values within a column followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t according to Duncan's multiple range test(P=0.05).

In order to examine whether the antagonist played a role on the roots of the l i l y , we isolated microorganisms from l i l y roots treated with antagonists at harvest time(Table V). The t y p i c a l microflora was recognized. In the plot of s t r a i n S-2, no C. destructance and no Fusarium oxysporum were i s o l a t e d , and the plot of s t r a i n S-7 showed the same indication as s t r a i n S-2. Also, there i s a interesting phenomenon that the number of Trichderma sp. increased i n the plot of strains S-2 and S-7. The antagonists were re-isolated from the treated plants, but not from the non-treated one. The antagonisms exhibited by the treated microorganisms are possibly the result of the production of antifungal agents, which are themselves an e f f e c t i v e protectant against C. destructance. These results indicate that s t r a i n S-2 and s t r a i n S-7 may f a c i l i t a t e establishment of stands of healthy edible l i l y . We assume the mechanism of bacterization by antagonists showed in F i g . V, and expect that two types of e f f e c t s may occur. The f i r s t i s antifungal agents being produced by antagonists at the rhizosphere of plants. We have already isolated and i d e n t i f i e d antifungal agents such as phenazines(Fig. IV), and confirmed that these agents showed strong in vitro antagonistic a b i l i t i e s against Cylindrocarpon destructans(Table III). The other i s the production of growth promoting factors. We have not i d e n t i f i e d the growth promoting substance from strains S-7 and S-2. But, P. cepacia B-17 produced auxin, a t y p i c a l plant growth hormone. These actions are e f f e c t i v e i n protecting against root rot and growing bulbs of edible lily.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

NATURALLY OCCURRING PEST BIOREGULATORS

424

Table V

Isolation of microorganisms antagonistic bacteria

from l i l y roots treated with

Treatment

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Isolated

Pseudomonas Pseudomonas fluorescens aeruginosa S-2 S-7

microorganism

Fungi Cylindrocarpon destructans Fusarium oxysporum Trichoderma spp. Rhizoctonia solani Pseudomonas spp. Total Pseudomonas spp. Pseudomonas aeruginosa S-7 Pseudomonas fluorescens S-2

15/25 5/25 1/25 2/25

2/25*1 1/25 10/25 0/25

0/25 0/25 16/25 0/25

3.Axl0^ *2 5.0xl0 0

9.3xl0

2

o

Control

4

9

5.0xlO

z

z 1.5xl0 0 0

*1 Number of r o o t l e t s isolated/Number of r o o t l e t s tested. *2 Number of Pseudomonas spp./g of rhizosphere s o i l . Bacterization

No treatment Growth

Growth

A

GROWTH PROMOTING SUBSTANCES

Pseudomonas aeruginosa S-7 Pseudomonas fluorescens S-2 Pseudomonas cepacia B-17

Cylindrocarpon destructans

Fig.

V

ANTIFUNGAL AGENTS

Proposed mechanism of b i o l o g i c a l control of root rot of edible l i l y by Pseudomonas species

Conclusion In Japan, i t i s becoming more and more d i f f i c u l t for growers to achieve satisfactory crop yields using t r a d i t i o n a l b i o l o g i c a l a g r i c u l t u r a l treatments such as crop rotation, t i l l a g e and organic

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

4

30.

HASEGAWA E T AL.

Soil-Borne Diseases in Japan

425

amendments. On the other hand, chemical control of pests i s often too expensive. That i s the reason why new b i o l o g i c a l control procedures are being keenly demanded. The three types of b i o l o g i c a l control mentioned above w i l l surely be widely applied i n the near future. I t i s true that obviously, much more work remains to be done to c l a r i f y the mechanisms of disease suppression, but successful answers can be obtained from the f i e l d s . The authors w i l l continue to try several approaches, especially the effect of antagonists on edible l i l y root rot b i o l o g i c a l control. Literature Cited

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1. 2. 3. 4. 5. 6.

Ogawa, K . ; Komada, H. Ann. Phytopath. Soc. Japan 1984, 50, 1-9. Kijima, T . ; Tezuka, T; A r i e , T; Namba, S; Yamashita, S; Doi, Y Ann. Phytopath. Soc. Japan 1986, 52, 542. Hasegawa, S.; Kondo, N . ; Kodama, F . J . Pharmacobio-Dyn. 1990, 13, i n press. Hasegawa, S.; Kondo, N . ; Kodama, F. Ann. Phytopath. Soc. Japan 1990, i n press. Hasegawa, S.; Kaneshima, H . ; Kodama, F.; Akai, J. Report of the Hokkaido Institute of Public Health 1986, 36, 16-23. Hasegawa, S.; Kondo, N . ; Kodama, F. Naturally Occurring Pest Bioregulators; American Chemical Society: Washington, DC,

RECEIVED July 31, 1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

Chapter 31

Preparative Separation of Complex Alkaloid Mixture by High-Speed Countercurrent Chromatography

Downloaded by UNIV OF ARIZONA on August 1, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch031

Richard J . Petroski and Richard G. Powell Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604

Tall fescue (Festuca arundinacea), infected with the fungal endophyte Acremonium coenophialum, contains both ergot-type and saturated-pyrrolizidine ( l o l i n e ) alkaloids. These alkaloids are believed to be at least p a r t i a l l y responsible for the insect pest resistance of tall fescue and for periodic or seasonal t o x i c i t y of endophyte-infected tall fescue to grazing

cattle. Attempts to separate the relatively minor amounts of ergot-type alkaloids (1-10 ppm) from the much more abundant saturated-pyrrolizidine alkaloids (1000-6000 ppm) using ordinary chromatography systems, have been generally unsatisfactory. High-speed countercurrent chromatography was examined as an alternative separation method. A crude fescue alkaloid mixture (1 g) was separated, in less than 8 hours, using a two-phase solvent system composed of chloroform-methanol-water (5:4:3, v/v/v). Alkaloids such as N-methylloline, N-acetylloline, and N-formylloline were well separated from each other and recovery of these alkaloids was quantitative.

Relatively pure ergonovine was isolated from the mixture in one pass. Other minor alkaloids, including lysergic acid amide, were either separated from each other or were highly enriched in certain fractions.

This chapter not subject to U.S. copyright Published 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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31.

PETROSKI AND POWELL

Preparative Separation of Complex Alkaloids

T a l l f e s c u e , Festuca arundinacea Schreb, i s a c o o l season pasture g r a s s t h a t i s u s e d e x t e n s i v e l y i n the s o u t h e a s t e r n U n i t e d S t a t e s . Most e x i s t i n g p a s t u r e s a r e i n f e c t e d w i t h an e n d o p h y t i c f u n g u s , Acremonium c o e n o p h i a l u m M o r g a n - J o n e s and Gams ( 1 ) , t h a t has b e e n a s s o c i a t e d w i t h i n s e c t p e s t r e s i s t a n c e o f t a l l f e s c u e (2-5) . E n d o p h y t e - i n f e c t e d t a l l f e s c u e c o n t a i n s b o t h e r g o t - t y p e ( F i g u r e 1) and s a t u r a t e d - p y r r o l i z i d i n e ( l o l i n e - t y p e ) a l k a l o i d s ( F i g u r e 2 ) ; n e i t h e r type o f a l k a l o i d i s f o u n d i n t a l l f e s c u e when the endophyte i s absent ( 6 - 8 ) . These two c l a s s e s o f a l k a l o i d s have a l s o b e e n a s s o c i a t e d w i t h p r o d u c t i o n l o s s e s i n c a t t l e ( 9 ) , and economic l o s s e s t o c a t t l e p r o d u c e r s have been e s t i m a t e d a t $50 t o $200 m i l l i o n a n n u a l l y (10). E r g o t a l k a l o i d s p r e s e n t the g r e a t e r t o x i c danger ( 8 , 1 1 ) . The o b j e c t i v e o f t h i s s t u d y was t o i s o l a t e and i d e n t i f y the s i m p l e r amides o f l y s e r g i c a c i d p r e s e n t i n e n d o p h y t e - i n f e c t e d t a l l f e s c u e , i n c l u d i n g the two unknown compounds r e p o r t e d p r e v i o u s l y by Y a t e s e t a l . (8). A t t e m p t s t o s e p a r a t e e r g o t - t y p e a l k a l o i d s from the much more abundant l o l i n e - t y p e a l k a l o i d s u s i n g o r d i n a r y chromatography s y s t e m s , have been g e n e r a l l y u n s u c c e s s f u l . The number o f a l k a l o i d s p r e s e n t , the w i d e l y d i f f e r e n t c o n c e n t r a t i o n s and s t r u c t u r a l t y p e s o f a l k a l o i d s p r e s e n t , and t a i l i n g o f a l k a l o i d peaks i n t o one a n o t h e r , have been major problems p r e v e n t i n g i s o l a t i o n and c h a r a c t e r i z a t i o n o f the minor a l k a l o i d s i n t a l l f e s c u e . High-speed c o u n t e r c u r r e n t chromatography e n a b l e s r a p i d and e f f i c i e n t s e p a r a t i o n o f complex m i x t u r e s w i t h o u t a d s o r p t i v e l o s s o r d e g r a d a t i o n c a u s e d by s o l i d s u p p o r t s s u c h as a l u m i n a , s i l i c a , o r c e l l u l o s e (12-15). We r e p o r t the g r a m - s c a l e s e p a r a t i o n o f a complex f e s c u e a l k a l o i d m i x t u r e u s i n g a c o m m e r c i a l p r e p a r a t i v e c r o s s - a x i s high-speed countercurrent instrument. The p r o c e d u r e has a l l o w e d us t o i s o l a t e and t o i d e n t i f y s e v e r a l a l k a l o i d s p r e v i o u s l y n o t known t o o c c u r i n t a l l f e s c u e . Materials

and Methods

Apparatus. A Pharma-Tech ( B a l t i m o r e , MD, USA) Model CCC-600CX c r o s s - a x i s p r e p a r a t i v e c o u n t e r c u r r e n t chromatograph was u s e d i n our experiments. The complete i n s t r u m e n t i n c l u d e s a d u a l c o i l column ( 2 . 6 mm I . D . p o l y t e t r a f l o u r o e t h y l e n e t u b i n g ) p l a n e t c e n t r i f u g e , LDC M i l t o n Roy l i q u i d pump, d i g i t a l r e v o l u t i o n s p e e d m o n i t o r , Rheodyne HPLC i n j e c t o r , and a p r e s s u r e m o n i t o r . The a x i s o f the column h o l d e r i s p o s i t i o n e d p e r p e n d i c u l a r t o , and a t a f i x e d d i s t a n c e away from the c e n t r i f u g e a x i s t o promote more v i g o r o u s m i x i n g o f s t a t i o n a r y and m o b i l e p h a s e s . Replacement o f the c o u n t e r w e i g h t by a s e c o n d i d e n t i c a l column e l i m i n a t e s the need f o r t e d i o u s b a l a n c i n g o f the c e n t r i f u g e system and d o u b l e s the t o t a l column c a p a c i t y . The t o t a l column c a p a c i t y i s 1260 mL and the optimum r e v o l u t i o n a l speed o f t h i s a p p a r a t u s i s 600 rpra. P r e p a r a t i o n o f s o l v e n t system. A two-phase s o l v e n t s y s t e m composed o f C H C l / M e O H / H 0 ( 5 : 4 : 3 , v / v / v ) was u s e d . The s o l v e n t m i x t u r e was t h o r o u g h l y e q u i l i b r a t e d i n a s e p a r a t o r y f u n n e l a t 2 5 ° , and the two phases were s e p a r a t e d b e f o r e u s e . The upper phase was the s t a t i o n a r y phase and the bottom phase was the m o b i l e p h a s e . 3

2

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Ergonovine

F i g u r e 1.

Lysergic acid amide

Ergot-Type A l k a l o i d s T a l l Fescue.

Isolysergic acid amide

I s o l a t e d From

Endophyte-Infected

R

*1

F i g u r e 2.

2

Norloline

H

H

Loline

H

CH

3

N-Methyl Loline

CH

CH

3

N-Formyl Norloline

H

HCO

N-Acetyl Norloline

H

CHpD

N-Formyl Loline

CH

N-Acetyl Loline

CH

3

3

HCO

3

CH^CO

Saturated-Pyrrolizidine (Loline-Type) A l k a l o i d s Reported i n Endophyte-Infected T a l l Fescue.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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31.

PETROSKI AND POWELL

Preparative Separation of Complex Alkaloids

P r e p a r a t i o n o f sample. A l t h o u g h t h e endophyte i n f e c t s t h e whole f e s c u e p l a n t , s e e d i s a r i c h s o u r c e o f b o t h l o l i n e - t y p e and e r g o t - t y p e a l k a l o i d s ( 7 , 8 ) , and p r e s e n t s fewer i s o l a t i o n p r o b l e m s . E n d o p h y t e - i n f e c t e d t a l l f e s c u e s e e d (Lambert Seed C o . , Camden, AL) was d e f a t t e d w i t h hexane (750 ml p e r 500 g s e e d ) , a i r - d r i e d , ground i n a m i l l i n g machine t o 2 mm mesh, and e x t r a c t e d w i t h methanol (3x, 1500 m L ) . The methanol e x t r a c t was c o n c e n t r a t e d i n vacuo a p p r o x . 5 0 - f o l d , t o 90 mL, and d i l u t e d w i t h 1% aqueous c i t r i c a c i d (500 m L ) . The a c i d i c s o l u t i o n was e x t r a c t e d w i t h CHCls (3x, 500 mL) and the CHCI3 e x t r a c t s were combined t o g i v e a f r a c t i o n t h a t was s a v e d for future study. This f r a c t i o n contained a mixture o f ergopeptine alkaloids, including ergovaline. The aqueous phase was c o l l e c t e d and t h e pH was a d j u s t e d t o 10 w i t h NaOH. The r e s u l t i n g a l k a l i n e s o l u t i o n was c a r e f u l l y e x t r a c t e d w i t h CHCI3 (5x, 500 ml) u s i n g t h o r o u g h b u t g e n t l e m i x i n g t o a v o i d t r o u b l e s o m e e m u l s i o n s . The CHCI3 e x t r a c t s were combined, c o n c e n t r a t e d i n vacuo t o 250 m l , and e x t r a c t e d w i t h 0.2N aqueous HC1 (3x, 80 m l ) . The CHCI3 phase was d i s c a r d e d because i t c o n t a i n e d o n l y s m a l l amounts o f r e s i d u a l , r e l a t i v e l y non-basic alkaloidal material. The aqueous HC1 p h a s e s , c o n t a i n i n g a l k a l o i d s as t h e i r HC1 s a l t s , were combined and a d j u s t e d to pH 10 w i t h NaOH. The r e s u l t i n g a l k a l i n e s o l u t i o n was e x t r a c t e d w i t h CHCI3 ( 5 x , 250 ml) t o a f f o r d a f r a c t i o n t h a t c o n t a i n e d b o t h l o l i n e - t y p e and e r g o t - t y p e a l k a l o i d s as f r e e b a s e s . The a l k a l o i d f r a c t i o n was c o n c e n t r a t e d i n vacuo t o 125 m l , and d r i e d o v e r anhydrous N a 2 S 0 . C h l o r o f o r m was removed i n v a c u o y i e l d i n g an o i l y r e s i d u e (1 g ) . The a l k a l o i d a l r e s i d u e was t h e n d i s s o l v e d i n a m i x t u r e o f m o b i l e phase (3 mL) and s t a t i o n a r y phase (1 m L ) . 4

C o u n t e r c u r r e n t Chromatography P r o c e d u r e . The e n t i r e column ( p a i r o f c o i l e d m u l t i l a y e r columns c o n n e c t e d i n s e r i e s ) was f i l l e d w i t h the s t a t i o n a r y p h a s e . The a p p a r a t u s was t h e n r o t a t e d c o u n t e r c l o c k w i s e a t 600 rpm i n p l a n e t a r y m o t i o n w h i l e t h e m o b i l e phase was pumped i n t o the i n l e t o f t h e column a t a f l o w - r a t e o f 2 . 2 mL/min (head t o t a i l e l u t i o n mode). Maximum p r e s s u r e a t t h e o u t l e t o f the pump measured 80 p s i . A f t e r a 1-hour e q u i l i b r a t i o n p e r i o d , the sample was l o a d e d i n t o t h e Rheodyne i n j e c t o r l o o p and injected. E f f l u e n t from t h e o u t l e t o f the column was c o n t i n u o u s l y m o n i t o r e d w i t h a Shimadzu UVD-114 d e t e c t o r a t 312 nm and f r a c t i o n s c o l l e c t e d w i t h a G i l s o n FC-100 f r a c t i o n c o l l e c t o r t o o b t a i n a p p r o x i m a t e l y 8.8 mL o f e l u a n t i n each tube ( d u r i n g a 4 - m i n interval). R e t e n t i o n o f the s t a t i o n a r y phase was e s t i m a t e d t o be 930 mL (74%) by m e a s u r i n g t h e volume o f s t a t i o n a r y phase e l u t e d from t h e column b e f o r e the e f f l u e n t changed t o m o b i l e phase (330 mL) and s u b t r a c t i n g t h i s volume from the t o t a l column c a p a c i t y o f 1260 mL. Analysis of fractions for l o l i n e alkaloids. Loline alkaloids p r e s e n t i n t h e v a r i o u s f r a c t i o n s were d e t e r m i n e d by t h e q u a n t i t a t i v e c a p i l l a r y gas c h r o m a t o g r a p h i c method o f Y a t e s e t a l . (7). P r i o r t o GC a n a l y s i s , 50-/iL o r IOO-/1L a l i q u o t s o f each f r a c t i o n t o be a n a l y z e d were d i l u t e d t o 0.98 mL w i t h MeOH, and p h e n y l m o r p h o l i n e was added as an i n t e r n a l s t a n d a r d (200 /ig i n 20 / i L MeOH); l - / i L i n j e c t i o n s were u s e d .

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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A n a l y s i s of f r a c t i o n s for ergot a l k a l o i d s . Ergot-type alkaloids were d e t e r m i n e d by HPLC w i t h f l u o r e s c e n c e d e t e c t i o n ( 8 ) . The HPLC system c o n s i s t e d o f a S p e c t r a - P h y s i c s SP8800 t e r n a r y s o l v e n t d e l i v e r y s y s t e m , Rheodyne i n j e c t o r , V a r i a n F l u o r i c h r o m d e t e c t o r , and a S p e c t r a - P h y s i c s SP4290 i n t e g r a t o r . HPLC a n a l y s i s was p e r f o r m e d w i t h a du Pont Zorbax ODS C-18 column (4.6 mm ID x 250 mm, 5 /im ODS) f i t t e d w i t h a S u p e l c o 5-8954 p r e p a c k e d d i s p o s a b l e 2-cm g u a r d column (LC-18 c a r t r i d g e ) . The chromatography s o l v e n t system was composed o f an 0.1 N ammonium a c e t a t e b u f f e r (pH 7.6) and a c e t o n i t r i l e , a t volume r a t i o s o f 65:35 o r 8 0 : 2 0 . A l l runs were i s o c r a t i c w i t h a f l o w - r a t e o f 0.8 m L / m i n . For fluorescence d e t e c t i o n o f e r g o t a l k a l o i d s , the e x c i t a t i o n w a v e l e n g t h was 310 nm; and the e m i s s i o n w a v e l e n g t h band p a s s e d , by V a r i a n 3-71 and 4-76 e m i s s i o n f i l t e r s , was between 375 and 460 nm. A l k a l o i d s were d e t e r m i n e d by measurement o f f l u o r e s c e n c e peak h e i g h t and c o m p a r i s o n w i t h ergotamine t a r t r a t e s t a n d a r d c u r v e s , and e r g o t a l k a l o i d amounts were e x p r e s s e d as /ig o f ergotamine t a r t r a t e . E r g o t a m i n e t a r t r a t e and e r g o n o v i n e were p u r c h a s e d from Sigma C h e m i c a l C o . , S t . L o u i s , MO. I s o l a t i o n of ergot a l k a l o i d s . P r e p a r a t i v e TLC o f c o u n t e r c u r r e n t f r a c t i o n s was c a r r i e d out on s i l i c a g e l 60 F-254 p l a t e s ( E . Merck) developed with CH Cl /MeOH ( 4 : 1 ) . R v a l u e s were 0 . 4 2 , 0 . 5 0 , and 0.68 f o r l y s e r g i c a c i d amide, e r g o n o v i n e , and i s o l y s e r g i c a c i d amide, r e s p e c t i v e l y . F l u o r e s c e n t bands on TLC p l a t e s were s e p a r a t e l y s c r a p e d from the p l a t e s , t r a n s f e r r e d to d i s p o s a b l e P a s t e u r p i p e t t e s , and e r g o t a l k a l o i d s were t h e n e l u t e d w i t h the same s o l v e n t s y s t e m . Mass s p e c t r a were o b t a i n e d w i t h a F i n n i g a n MAT 4535/TSQ i n s t r u m e n t e q u i p p e d w i t h a DEP p r o b e . 2

2

f

R e s u l t s and D i s c u s s i o n A l t h o u g h sample i s n o r m a l l y i n j e c t e d i n t o a c o u n t e r c u r r e n t chromatograph when the m o b i l e phase i s i n i t i a l l y pumped i n t o the column ( 1 2 - 1 5 ) , i t was f o u n d t h a t b e t t e r r e t e n t i o n o f s t a t i o n a r y p h a s e , a more s t a b l e a b s o r b e n c i e s b a s e l i n e , and b e t t e r r e s o l u t i o n o f peaks r e s u l t e d when samples were i n j e c t e d a f t e r a 1-hour equilibration period. U n s t a b l e b a s e l i n e s were o b s e r v e d d u r i n g the time i n w h i c h e f f l u e n t changed from s t a t i o n a r y phase t o m o b i l e p h a s e ; however, b a s e l i n e s s t a b i l i z e d by the time the f i r s t sample component a p p e a r e d i n the e f f l u e n t . An a l k a l o i d e x t r a c t from e n d o p h y t e - i n f e c t e d t a l l f e s c u e s e e d p r e p a r e d by a s e r i e s o f a c i d / b a s e e x t r a c t i o n s was i n j e c t e d i n t o the c o u n t e r c u r r e n t c h r o m a t o g r a p h , r a t h e r than a c r u d e e t h a n o l i c s e e d e x t r a c t , b e c a u s e i n j e c t i o n o f the l a t t e r r e s u l t e d i n e m u l s i o n s . When e m u l s i o n s were f o r m e d , the s t a t i o n a r y phase was n o t r e t a i n e d and s e p a r a t i o n s were unsuccessful. Attempts to p r o c e s s more t h a n 1 g o f a l k a l o i d c o n c e n t r a t e p e r i n j e c t i o n r e s u l t e d i n r e d u c e d r e s o l u t i o n o f sample components. The optimum f l o w - r a t e was 2.2 m L / m i n . A t y p i c a l c o u n t e r c u r r e n t chromatogram o f an a l k a l o i d e x t r a c t from e n d o p h y t e - i n f e c t e d t a l l f e s c u e s e e d i s shown i n F i g u r e 3. Two c u r v e s a r e s u p e r i m p o s e d ; the s o l i d l i n e r e p r e s e n t s mg o f l o l i n e - t y p e a l k a l o i d s d e t e r m i n e d by GC a n a l y s i s , and the d o t t e d l i n e r e p r e s e n t s a b s o r b e n c i e s a t 312 nm, w h i c h i s c h a r a c t e r i s t i c o f

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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31.

PETROSKI A N D POWELL

F i g u r e 3.

Preparative Separation of Complex Alkaloids

C o u n t e r c u r r e n t Chromatogram o f an A l k a l o i d C o n c e n t r a t e from E n d o p h y t e - I n f e c t e d T a l l F e s c u e , w i t h C H C l / M e O H / H 0 ( 5 : 4 : 3 ) Lower Phase M o b i l e . I d e n t i f i e d p e a k s : 1, l y s e r g i c a c i d amide (670 /ug) p l u s i s o l y s e r g i c a c i d amide (520 /xg); 2, N-methylloline (61 mg); 3, N - a c e t y l l o l i n e (305 mg); 4 , e r g o n o v i n e (55 / i g ) ; 5, N - f o r m y l l o l i n e (601 mg); 6, l o l i n e (31 mg) p l u s N - a c e t y l n o r l o l i n e (28 mg). Loline-type a l k a l o i d s d e t e r m i n e d by GC. E r g o t - t y p e a l k a l o i d s d e t e r m i n e d by HPLC w i t h f l u o r e s c e n c e d e t e c t i o n and e x p r e s s e d as /ig ergotamine t a r t r a t e . 3

2

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NATURALLY OCCURRING PEST BIOREGULATORS

the c r o s s - c o n j u g a t e d s t y r e n e - i n d o l e system o f l y s e r g i c a c i d d e r i v a t i v e s (16). A l t h o u g h 8.8-mL f r a c t i o n s were c o l l e c t e d , numbers (mL e f f l u e n t a f t e r sample i n j e c t i o n ) have b e e n rounded o f f to i n t e g e r s i n o r d e r t o s i m p l i f y the d i s c u s s i o n . The f i r s t sample component a p p e a r e d , a t an e f f l u e n t volume o f 250 mL, as a non-alkaloidal yellow material. A b r o a d peak a t 304-374 mL o f e f f l u e n t c o n t a i n e d two h i g h l y - f l u o r e s c e n t e r g o t a l k a l o i d s (670 /ig o f l y s e r g i c a c i d amide and 520 /ig o f i s o l y s e r g i c a c i d a m i d e ) ; N - m e t h y l l o l i n e (61 mg) was p r e s e n t a t 312-365 mL o f effluent. N - M e t h y l l o l i n e was d e t e r m i n e d by gas c h r o m a t o g r a p h y . P r e p a r a t i v e s e p a r a t i o n o f the f r a c t i o n a t 304-374 mL o f e f f l u e n t by T L C , y i e l d e d N - m e t h y l l o l i n e ( R 0 . 1 0 ) , l y s e r g i c a c i d amide (Rf 0.42) and i s o l y s e r g i c a c i d amide (Rf 0 . 6 8 ) . The s t r u c t u r e s o f l y s e r g i c a c i d amide and i s o l y s e r g i c a c i d amide were c o n f i r m e d by mass s p e c t r o m e t r y v i a c o m p a r i s o n w i t h p u b l i s h e d mass s p e c t r a (17). We c o u l d n o t d i f f e r e n t i a t e between l y s e r g i c a c i d amide and i s o l y s e r g i c a c i d amide on the b a s i s o f mass s p e c t r o m e t r y . The h i g h e r R f ( 0 . 6 8 ) band was a s s i g n e d to i s o l y s e r g i c a c i d amide b e c a u s e i s o l y s e r g i c a c i d d e r i v a t i v e s t y p i c a l l y have h i g h e r R f v a l u e s t h a n do the c o r r e s p o n d i n g l y s e r g i c a c i d d e r i v a t i v e s (18). A t 374-392 mL o f e f f l u e n t , a minor u n i d e n t i f i e d 312 nm a b s o r b a n c e peak was o b s e r v e d . A n o t h e r s u s p e c t e d e r g o t - a l k a l o i d peak (25 /xg) o c c u r r i n g a t 453-513 mL o f e f f l u e n t a l s o c o n t a i n e d N - a c e t y l l o l i n e (305 mg). A minor peak (312 nm, t r a c e amounts o f e r g o t a l k a l o i d ) was o b s e r v e d a t 513-557 mL o f e f f l u e n t . T h i s peak was f o l l o w e d by e r g o n o v i n e (55 /ig) a t 583-627 mL e f f l u e n t . E r g o n o v i n e was t e n t a t i v e l y i d e n t i f i e d by c o m p a r i s o n w i t h , and c o - c h r o m a t o g r a p h y w i t h an e r g o n o v i n e s t a n d a r d u s i n g two d i f f e r e n t HPLC c h r o m a t o g r a p h i c s o l v e n t s y s t e m s . E r g o n o v i n e was t h e n i s o l a t e d by T L C , and the s t r u c t u r e c o n f i r m e d by mass s p e c t r o m e t r y v i a comparison with authentic ergonovine. Subsequent f r a c t i o n s w i t h a b s o r b e n c i e s a t 312 nm c o n t a i n e d o n l y t r a c e amounts o f e r g o t a l k a l o i d s , e x p r e s s e d as /ig ergotamine t a r t r a t e . N-formylloline (601 mg) e l u t e d a t 627 t o 733 mL o f e f f l u e n t . Some s u s p e c t e d e r g o t - a l k a l o i d o v e r l a p o c c u r r e d from 654-750 mL o f e f f l u e n t . L o l i n e (31 mg) and N - a c e t y l n o r l o l i n e (28 mg) were f o u n d t o g e t h e r a t 733-812 mL o f e f f l u e n t . No a b s o r b e n c i e s a t 312 nm o r l o l i n e a l k a l o i d s were f o u n d a f t e r 812 mL o f e f f l u e n t .

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f

P a r t i t i o n e f f i c i e n c i e s o f the major l o l i n e - t y p e a l k a l o i d s were computed a c c o r d i n g t o the c o n v e n t i o n a l gas c h r o m a t o g r a p h i c f o r m u l a (13): N -

(4R/w)

2

where N denotes the p a r t i t i o n e f f i c i e n c y e x p r e s s e d i n terms o f t h e o r e t i c a l p l a t e number, R, the r e t e n t i o n volume (mL o f e f f l u e n t ) a f t e r i n j e c t i o n o f the peak maximum, and w, the peak w i d t h e x p r e s s e d i n the same u n i t s as R. The p r e s e n t s e p a r a t i o n y i e l d e d p a r t i t i o n e f f i c i e n c i e s o f 680, 1050, and 560 t h e o r e t i c a l p l a t e s f o r N - m e t h y l l o l i n e , N - a c e t y l l o l i n e , and N - f o r m y l l o l i n e , respectively. I n summary, N - m e t h y l l o l i n e , N - a c e t y l l o l i n e , and N - f o r m y l l o l i n e were w e l l s e p a r a t e d from each o t h e r u s i n g g r a m - s c a l e h i g h - s p e e d c o u n t e r c u r r e n t c h r o m a t o g r a p h y , and r e c o v e r y o f l o l i n e a l k a l o i d s was quantitative. E r g o t a l k a l o i d s were w e l l s e p a r a t e d from each o t h e r

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

31.

PETROSKI AND POWELL

Preparative Separation of Complex Alkaloids

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and were e n r i c h e d i n c e r t a i n f r a c t i o n s . The p r e p a r a t i v e TLC system was n o t s u f f i c i e n t , i n i t s e l f , t o i s o l a t e e r g o t a l k a l o i d s from t h e a l k a l o i d c o n c e n t r a t e u s e d i n t h i s s t u d y b u t was s u c c e s s f u l on t h e g r e a t l y s i m p l i f i e d mixtures contained i n countercurrent chromatographic f r a c t i o n s . T h i s i s the f i r s t r e p o r t e d occurrence o f l y s e r g i c a c i d amide and i s o l y s e r g i c a c i d amide i n e n d o p h y t e - i n f e c t e d t a l l f e s c u e , and HPLC a n a l y s i s showed t h a t t h e s e a r e t h e two unknown compounds r e p o r t e d p r e v i o u s l y ( 8 ) . The c o m b i n a t i o n o f p r e p a r a t i v e h i g h - s p e e d c o u n t e r c u r r e n t chromatography w i t h o t h e r s e p a r a t i o n methods, such as HPLC, and T L C , w i l l e n a b l e c h e m i s t s t o i s o l a t e minor components o f complex a l k a l o i d m i x t u r e s more e f f i c i e n t l y . This technique i s not l i m i t e d to a l k a l o i d s e p a r a t i o n s a n d , i n t h e o r y , o t h e r complex m i x t u r e s o f compounds h a v i n g o n l y minor d i f f e r e n c e s i n t h e i r partition c o e f f i c i e n t s s h o u l d be e f f i c i e n t l y s e p a r a t e d b y h i g h - s p e e d c o u n t e r c u r r e n t chromatography. Acknowledgment We thank R. D. P l a t t n e r technical assistance.

f o r mass s p e c t r a and T . W i l s o n f o r

M e n t i o n o f companies o r p r o d u c t s by name does n o t i m p l y t h e i r endorsement by the U . S . Department o f A g r i c u l t u r e o v e r o t h e r s not c i t e d .

Literature Cited 1. Morgan-Jones, G.; Gams, W. Mycotaxon. 1982, 15, 311. 2. Funk, C. R.; Halisky, P. M.; Johnson, C . ; Siegel, M. R.;, Stewart, A. V.; Ahmad, S.; Hurley, R. H . ; Harvey, I. C. Biotechnology 1983, 1(2), 189. 3. Hardy, T. D.; Clay, K.; Hammond, A. M. J r . Environ. Entomol. 1986, 15, 1083. 4. Johnson, M. C.; Dahlman, D. L.; Siegal, M. R.; Bush, L. P.; Latch, G. C.; Potter, D. A.; Varney, D. R. Appl. Environ. Microbiol. 1985, 49, 568. 5. Latch, G. C.; Christensen, M. J.; Gaynor, D. L. N. Z. J. Agr. Res. 1985, 28, 129. 6. Petroski, R. J.; Yates, S. G.; Weisleder, D.; Powell, R.G. J . Nat. Prod. 1989, 52(4), 810. 7 Yates, S. G.; Petroski, R. J.; Powell, R. G. J . Agr. Food Chem. 1990, 38, 182. 8. Yates, S. G.; Powell, R. G.; J . Agr. Food Chem. 1988, 36, 337. 9. Sanchez, D. Agr. Res. 1987, 35(8), 12. 10. Siegel, M. R.; Johnson, M. C.; Varney, D. R.; Nesmith, W. C . ; Buckner, R. C.; Bush, L. P.; Burrus, P. B.; Jones, T. A . ; Boling, J . A. Phytopathology 1984, 74, 932. 11. Berde, B., Schild, H. O., Eds. "Ergot Alkaloids and Related Compounds"; Springer-Verlag, New York, 1978. 12. Ito, Y; Oka, H; Slemp, J . L. J . Chromatogr. 1989, 463, 305. 13. Bhatnager, M.; Oka, H . ; Ito, Y. J . Chromatogr. 1989, 463, 317. 14. Zhang, T . - Z . ; Pannell, L. K.; Pu, Q . - L . ; Cai, D.-G.; Ito, Y. J. Chromatogr. 1988, 455. 15. Zhang, T . - Y . ; Pannell, L. K.; Cai, D.-G.; Ito, Y. J . Liq. Chromatogr. 1988, 11(8), 1661.

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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16. Scott, A. I. "Interpretation of the Ultraviolet Spectra of Natural Products"; Pergamon Press: Frankfurt, Germany, 1964; pp. 172-178. 17. Inoue, T.; Nakahara, Y,; Niwaguchi, T. Chem. Pharm. Bull. 1972, 20 (2), 409. 18. Stahl, E. Ed. "Thin-Layer Chromatography, A Laboratory Handbook"; Springer-Verlag, New York, 1969; pp. 452-453.

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RECEIVED May 16, 1990

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Author Index Alfatafta, A l i A., 278 Balandrin, Manuel F., 293 Barnby, Mark A., 293 Bartelt, Robert J., 27 Becerril, Jose M . , 371 Bell, A . A . , 336 Bird, T. G.,78 Blum, Murray S., 14 Chen, Liza, 399 Chittawong, Vallapa, 317,399 Chortyk, Orestes T.,388 Christensen, Allyson, 87 Christiansen, Benedikte, 352 Coats, Joel R., 305 Cole, Richard J., 352 Collins, L . , 224 Costello, C. E.,251 Coudron, Thomas A . , 41 Craig, R., 224 Csinos, Alex S., 388 de la Cruz, Armando A., 317 Dorner, Joe W., 352 Dowd, Patrick F., 27 Downum, Kelsey R., 361 Drewes, Charles D., 305 Duffey, SeanS., 166 Duke, Stephen 0.,371 Elissalde, M . H . , 336 Elliger, Carl A . , 210 Everett, Daniel M . , 14 Everett, Susan L . , 278 Fales, Henry M . , 14 Farrar, Jr., Robert R., 150 Felton, Gary W., 166 Gomez, Edgardo,317 Gueldner, R. C , 251 Gustine, David L., 324 Harman, Jody L . , 278 Hasegawa, Shinsaku, 407,417 Hayes, D . K . , 78 Hedin, Paul A., 1,399 Hesk, D., 224 Himmelsbach, D. S., 251 Hopkins, T. L . , 87 Jackson, D. M . , 264 Johnson, A . W., 264

Jones, Tappey H., 14 Karr, Laura L . , 304 Kashyap, R. K . , 150 Kennedy, George G., 150 Klocke, James A., 293 Kodama, Fujio, 407,417 Kondo, Norio, 407,417 Kramer, K . J., 87 Lee, S. Mark, 293 Lidert, Zev, 407 Mace, M . E . , 336 Matsumoto, Hiroshi, 371 McDonough, Leslie M . , 106 Meideros, Jorge, 399 Miles, D. Howard, 399 Morgan, T. D., 87 Mullin, Christopher A., 278 Mumma, R. O., 224 Paxton, Jack D., 198 Payne, Allen Matthew, 317,399 Petroski, Richard J., 426 Plattner, Ronald D., 27 Powell, Richard G., 426 Raina, A . K., 78 Schaefer, Jacob, 87 Serino, Anthony A . , 278 Severson, R. F., 264 Sherman, Timothy D., 371 Sisson, V . A., 264 Snook, Maurice E . , 251,388 Steffens, John C , 136 Stephenson, M . G., 264 Stipanovic, R. D., 336 Swain, Lee A., 361 Swithenbank, Colin, 399 Teal, Peter E. A., 66 Tingey, Ward M . , 126 Tumlinson, James H . , 66 Waiss, Jr., Anthony C , 210 Walters, Donald S., 136 Widstrom, N . W.,251 Wiseman, B . R.,251 Witham, Francis H . , 324 Yagen, Boris, 352 Yamasaki, R. Bryan, 293

437 In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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N A T U R A L L Y OCCURRING PEST BIOREGULATORS

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Affiliation Index Cornell University, 126,136 Fairchild Tropical Gardens, 361 Florida International University, 361 Haryana Agricultural University, 150 Hebrew University, 352 The Hokkaido Central Agricultural Experiment Station, 407,417 Hokkaido Institute of Public Health, 407,417 Instituttet for Bioteknologi, 352 Iowa State University, 305 Massachusetts Institute of Technology, 251 Mississippi State University, 317

National Institutes of Health, 14 National Product Sciences, Inc., 293 Pennsylvania State University, 224,278,324 Rohm and Haas Company, 399 U.S. Department of Agriculture, 1,27,41,66, 78,87,106,210,251,264,336,352,371,388,426 University of California—Davis, 166 University of Central Florida, 317,399 University of Georgia, 14,388 University of Illinois, 198 University of the Philippines, 317 Washington University, 87

Subject Index A p-Acaridial natural product of a mite, 17 structure, 17/ Accessory gland functions, 78 pheromone production, 78 Accessory gland factor, associated with mating, 72 Acetates half-life heat of evaporation, 114r relationship to structure, 113-114 stability in rubber septa, 121 Acetolactate synthetase, inhibition, 146-147 Acifluorfen, effect on accumulation of protoporphyrin IX, 38If Adduct formation, one-electron oxidative pathway, 90f Adzukibean antifungal agents in soil, 414-415 effects of seed bacterization, 413-414 suppression of Fusarium wilt by rhizosphere microorganisms, 407-416 Aflatoxin contamination, 352-360 Aggregation pheromone, nitidulid beetles, 27-40 A i r speed, effect on half-life, 118-120

Alcohols half-life, 112-114 stability in rubber septa, 121 Aldehydes half-life heat of evaporation, 115f relationship to structures, 112-115 temperature, 117-118 stability in rubber septa, 121 Alfalfa weevil, parasitoid, 42 Alkaloid(s) countercurrent chromatogram of endophyte-infected tall fescue, 431/ ergottype analysis, 430 endophyte-infected tall fescue, 428/ isolation, 430 experimental methods, 427-433 lolinetype analysis, 429 endophyte-infected tall fescue, 428/ partition efficiencies, 432 problems in chromatographic separation, 427 Alkaloid mixture, preparative separation by high-speed axmteiuirrent chromatography, 426-434 Alkaloid-rich venoms ants, 20-24

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N A T U R A L L Y OCCURRING PEST BIOREGULATORS

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Affiliation Index Cornell University, 126,136 Fairchild Tropical Gardens, 361 Florida International University, 361 Haryana Agricultural University, 150 Hebrew University, 352 The Hokkaido Central Agricultural Experiment Station, 407,417 Hokkaido Institute of Public Health, 407,417 Instituttet for Bioteknologi, 352 Iowa State University, 305 Massachusetts Institute of Technology, 251 Mississippi State University, 317

National Institutes of Health, 14 National Product Sciences, Inc., 293 Pennsylvania State University, 224,278,324 Rohm and Haas Company, 399 U.S. Department of Agriculture, 1,27,41,66, 78,87,106,210,251,264,336,352,371,388,426 University of California—Davis, 166 University of Central Florida, 317,399 University of Georgia, 14,388 University of Illinois, 198 University of the Philippines, 317 Washington University, 87

Subject Index A p-Acaridial natural product of a mite, 17 structure, 17/ Accessory gland functions, 78 pheromone production, 78 Accessory gland factor, associated with mating, 72 Acetates half-life heat of evaporation, 114r relationship to structure, 113-114 stability in rubber septa, 121 Acetolactate synthetase, inhibition, 146-147 Acifluorfen, effect on accumulation of protoporphyrin IX, 38If Adduct formation, one-electron oxidative pathway, 90f Adzukibean antifungal agents in soil, 414-415 effects of seed bacterization, 413-414 suppression of Fusarium wilt by rhizosphere microorganisms, 407-416 Aflatoxin contamination, 352-360 Aggregation pheromone, nitidulid beetles, 27-40 A i r speed, effect on half-life, 118-120

Alcohols half-life, 112-114 stability in rubber septa, 121 Aldehydes half-life heat of evaporation, 115f relationship to structures, 112-115 temperature, 117-118 stability in rubber septa, 121 Alfalfa weevil, parasitoid, 42 Alkaloid(s) countercurrent chromatogram of endophyte-infected tall fescue, 431/ ergottype analysis, 430 endophyte-infected tall fescue, 428/ isolation, 430 experimental methods, 427-433 lolinetype analysis, 429 endophyte-infected tall fescue, 428/ partition efficiencies, 432 problems in chromatographic separation, 427 Alkaloid mixture, preparative separation by high-speed axmteiuirrent chromatography, 426-434 Alkaloid-rich venoms ants, 20-24

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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INDEX

439

Alkaloid-rich venoms—Continued repellency by species, 23f,24 rcpellency of individual compounds, 24/ n-Alkenyl acetate, half-life, 112-113 n-Alkyl acetate, half-life, 112-113 AHelochemicals, 3 control of insects and other animals, 209-322 plant disease control agents, 387-434 screening for properties, 1 Allelopathic agents, definition, 6 Allomones, 3 defense against predatory species, 14 evolutionary advantage, 24 Amino acids biosynthesis of novel glucose esters, 146 branched fatty acids, 146 dietary protein, 174 function in insects, 187-188 0-Aminoacetophenone honeybee queens, 19 structure, 19/ 6-Aminolevulinic acid chlorophyll synthesis modulators, herbicide, 373-377 conversion to porphyrins, 376 duration of herbicidal effect, 381 plus DP, ultrastructural development of damage in bean cotyledons, 377 porphyrin accumulation, 372,373r with modulator combinations, selectivity manipulation, 376 Anacardic acids analysis of geraniums, 228-243 biosynthesis, 247 C23 anteiso saturated, *H N M R spectrum, 242/* C24 iso saturated, *H N M R spectrum, 244/ dimethylated, mass spectral fragmentation, 235/ D M D S derivative, mass spectral fragmentation, 237/ fatty acids, selectively synthesized, 246 geranium glandular trichomes, 225 glandular trichome exudate mass spectrometric analysis, 233f N M R analysis, 234f percentage composition, 245r H P L C and G C retention times, 230* mass spectral and N M R analysis, 232-243

Anacardic acids—Continued odd chain length and branched chain, 247-248 saturated and unsaturated, 245-246,248 Antibiosis, host plant resistance, 167 Antibiotics, plant disease control, 7 Antidigestive properties, proteinase inhibitors, 173 Antifeedant(s) Canadian goldenrod, 278 chrysomelids, 279 identification and use, 3-4 sunflower, 278 tropical and subtropical plants, 7 Antifeedant sesquiterpenes interactions with other plant allelochemicals, 287 neurotoxicity, 287-288 Antifungal agents Fusarium wilt, 409 in soil, 414-415 inhibition effect, 41 Or Antinutritive resistance breeding problems, 190-191 definition, 167 host plant resistance, 166-167 rationale, 167-172 tomato plant, 172 Ants deterred by defensive secretions, 15 predators, 14 repellent efficacy of thrips anal exudate, 16 venomous alkaloids, 15,20-24 Aphids abnormal insect behaviors, 129-130 entrapment, 127-129,139 feeding behavior and glandular trichomes, 130r genetic manipulation applications, 132-133 glandular trichome-mediated resistance, 128/ immobilization, 127-129 impact of oxidative enzymes, 179 insect resistance in wild potato species, 126-135 potato virus Y incidence, 130f resistant and susceptible geraniums, 224-250 sugar esters as feeding deterrent, 139

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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440

N A T U R A L L Y OCCURRING PEST B I O R E G U L A T O R S

Army worm wild parsnip, 361 See also Beet army worm, Bertha army worm, Fall army worm, Southern army worm Arthropod(s), 14 defensive secretion, 15 host plant chemistry, 151 resistant and susceptible geraniums, 224-250 Arthropod natural products, insect repellents, 14-26 Arthropod-resistant crop varieties, pest management, 150 Artificial induction studies, P B A N , 68-72 Ascorbic acid antinutritive effects, 185 lepidopterans, 185 Attractants, 3 Autointoxication, induced, 5 Aversive learning, Petunia ,211 Azadirachtin activity of analogues, 296 effective concentration, 301 hormonal disturbances, 301 insect control agent, 296 insect growth and development, 298 insecticidal and antifeedant, 293 structure-activity relationships, 301-303

B Bacterization edible lily seed bulbs, 423 production of antifungal agents at rhizosphere of plants, 423 production of growth-promoting factors, 423 Barley, A L A accumulation, 377 Bay leaves, cockroaches, 310 Beans, A L A plus DP, 377 Beet army worm cotton exposure to spider mites, 205 impairment of detoxicative ability, 185 negative resistance, utilization of dietary plant nitrogen, 168 polyphenol oxidase, 173 proteinase inhibitors vs. relative growth, 174r

Beetles aggregation pheromone, 27-40 See also Colorado potato beetles, Flea beetles, Flour beetles, Mexican bean beetles, Nitidulid beetles, Southern pine beetles, Western pine beetles Behavioral disturbance, glandular trichomes of S. berthaultii, 129-130 Bertha army worm, petuniolides, 220 Bioassays, beetle pheromone, 28-32 Biocidal compounds, Moraceae, 362-368 Biological control future strategies, 59 selection and genetic manipulation of species, 57 soil-borne diseases, 417-425 Biological control agents, potential incompatibility, 180-182 Biological pest control host plant resistance, 151 transfer of desirable bioregulators, 58 Biologically active natural products environmental concerns, 2 isolation and identification, 1 Bioregulation, insect behavior and development, 13-124 Bioregulators elicitors of phytoalexins, 8 levels required for activity, 2 search, 57-58 sources and mechanisms, 8 understanding of physiology and behavior, 8 Biosynthesis energy commitment, 141 pheromones, 38-39 sugar esters, 141-143 Biosynthetic pathways, Petunia steroids, 217-218 Black shank description of infected tobacco plant, 391 disease fungus, inhibition of growth, 388-398 effects, 388 inhibition by coumarins, 397f inhibition by hydroxy-cinnamic acids, 395f,396f inhibition by tobacco root polyphenols, 394*

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

INDEX

441

Boll weevil, cadinane sesquiterpene, 319 Botanical insecticides, control of insect pests, 305 Bottle gourd, Fusarium wilt, 410 Brain-subesophageal ganglion homogenate, effect of injection, 75/ Brine shrimp, petuniolides, 220 Brussels sprout, odd chain length and branched-chain fatty acids, 248 Bursa copulatrix, release of PBSF, 73 Bursa factor, associated with mating, 72

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C Cadinane sesquiterpene boll weevil, 319 isolated from mangrove plant, 318 structure, 319/-321/ Calling period, volatile pheromone components, 68 Calyx region, secretory nature, 51 Caribbean fruit fly citrus oil, 310 parasitoid, 42 Carotenoids, diphenyl ether herbicides, 378 Catalase defensive role of plant POD, 183 impact of peroxidase on food quality, 176 larval growth and foliar oxidative activities, 178f Catechol, inhibition of enzymes, 185 Catecholamine, two-electron oxidative pathway, 89/ Catecholamine metabolites, tanned cuticles, 101 Caterpillars, experiments on food-aversion learning, 210-211 Centipede grass, maysin, 251-263 Chemical deterrents, plant defenses, 198 Chemical exudate, variability within plant lines, 246-247 Chinaberry tree insecticidal constituents, 293-304 insecticidal potency, 298 Chitin, elicitors, 8 Chlorogenic acid corn, 252 corn earworm, 252 fall army worm, 262

Chlorogenic acid—Continued fungal growth inhibition, 391 growth inhibition, 252 location in tobacco plant, 391 Chlorophyll biosynthesis intermediates, 372 energy dissipation in plants, 372 Chlorophyll pathway, protoporphyrin DC, 381 Chlorophyll precursor levels, herbicidal damage, 375 Chlorophyll synthesis modulators categories, 376 effectiveness of o^aminolevulinic acid, 375 Choice tests, budworm or hornworm oviposition, 269 Chrysomelids antifeedants, 279 cucurbitacins, 279 Citrus, terpene biosynthesis, 5 Citrus oil, red scale, 312 Cockroaches bay leaves, 310 hedgeapple, 310 insect reproduction, monoterpenoids, 313 monoterpenoids acute toxicity, 308f,309f growth and development, 312 neurotoxic effects, 313 reproduction, 313 pyrethrins, 310 repellency of hedgeapple, bay leaves, spearmint gum, and pyrethrins, 311/ spearmint chewing gum, 310 terpenoids, 310 Codling moth, sex pheromone dispensers, 107 Colorado potato beetles abnormal behaviors, 130-131 activity on excised leaflets, 132* genetic manipulation applications, 132-133 glandular trichomes of S. berthaultii, 1311 insect resistance in wild potato species, 126-135 nature of resistance, 130-131 petuniolides, 220 tansy, 312 toxicity of Petunia plants, 210 2-tridecanone-glandular trichome-mediated resistance, 150 vital statistics on S. berthaultii and S. tuberosum, 133

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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442

N A T U R A L L Y OCCURRING PEST BIOREGULATORS

Competitive advantage, species that generate venoms, 22 Compositae interactions with com rootworm, 282-283 neurotoxic antifeedants, 288 terpenoids, 279 Computer simulation applications, 332 phytoalexin elicitor-DNA complex, 324-335 Conformers, definition, 41 Confusion, generated by pheromones, 3 Controlled-release substrates applications of sex pheromones of insect pests, 106-124 first-order mechanism, 107-108 natural rubber, 107 requirements, 107 Conversion and blocking factors, 95-99 Conversion factors, dopa metabolism in insects, 98/ Corn chemical resistance, 251-252 luteolin derivatives, 259 maysin, 251-263 mechanical resistance to corn earworm, 251 methods of analysis, 252-255 mutants with nonbrowning silks, 259 nitidulid beetles, 27 structures of three insect-inhibitory compounds, 253/ Corn earworm activity from accessory glands, 78-86 bioassay, 81 chlorogenic acid, 252 growth inhibition by petuniasterones, 218/ growth inhibition by petuniolides, 219/ hydroxamic acid, 252 leaf feeding sites, 262 maysin, 251-263 Petunia steroids, 218 petuniolides, 220 pheromone biosynthesis, 84/ receptivity termination factor, 84 Corn rootworm floral feeding deterrents, 285/ floral terpenoid and phenolic factors, 278-292 d-limonene, 309 sunflower petals, 283/ susceptibility to aldrin, 288 susceptibility to neurotoxins, 288/

Corn silk H P L C profile of extracts, 263/ mechanical resistance to corn earworm, 251 nonbrowning, 259 Cotton plant pathogenic fungus, 336 proposed resistance response, 337 Cotton phytoalexins structure^), 339/ structure-activity relationship, 336-351 Coumarins, laboratory bioassays, 394 Cross-link structures identifying bonds, 99 manducin in presence of mushroom tyrosinase, 101 polymers in insect exoskeleton, 102 side chain, 88 Cross-protection previously inoculated sprouts, 418-419 resistance products, 419 Cucumber, ^aminolevulinic acid and photodynamic damage, 372 Cuticle plant defenses, 198 stages of growth and development, 87 Cuticle tanning, insects, 87-105 Cuticular chemistry, resistance ratings, tobacco budworm and hornworm, 267/ Cuticular components Nicotiana spp. tobacco budworm ovipositional frequency, 276 tobacco hornworm oviposition, 276 quantitation, isolation, and characterization, 265-268 Cuticular diterpenes, Nicotiana spp., 266/ Cuticular glucose and sucrose esters, Nicotiana spp., 267/ Cuticular oxidases, 91-93 Cyclic sesquiterpenes, insect antifeedants, 280/-281/

D Dalbergia monetaria, 401/402/ Dark spray, selectivity manipulation, 376

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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INDEX

443

y-Decalactone, 16,16/ Defense response, active oxygen and free radicals from phytoalexins, 347 Defensive biology, Solarium berthaultii, 127-131 Defensive secretions, arthropodous species, 15 Desoxyhemigossypol chemistry, 338-345 decomposition, 339/,34qf,341/ decomposition mechanism, 338 effectiveness in cotton, 348 effects of reducing agents on toxicity, 346/ proposed steps in autoxidation, 342/ solubility, effectiveness of fungicides, 337 stabilizing effect of substrate-specific enzymes, 338 strong reducing agents, 343 structure-activity relationship, 336-351 structure for proposed intermediates, 344/ toxicity, 345-347,348 Detoxication, sulfur amino acid, 185 Developmental abnormalities, nonparalyzing venomous substances, 55-57 Developmental synchrony of parasitoid and host, ecdysteroid levels in host hemolymph, 49 Dihydroxyindole blocking factor, 98 Diphenolic compounds, one-electron oxidative pathway, 90f Diphenolic radicals, experimental problems, 94 Diphenyl ethers, 377/377-378 2,2'-Dipyridyl, 372,373/ Disease control, fungicides, 7 Disease resistance in plants,324 Diterpenes, insect antifeedants, 282/ D N A , phytoalexin elicitor complex, 324-335 Dopa quinone imine conversion factor, 98,99 Dopachrome conversion factor, decolorization of dopachrome, 95,98 Double bonds, effect on half-life, 115-116 Dufour's gland, Hymenoptera, 42-43

E Earthworm d-limonene, 315/ monoterpenoids, neurotoxic effects, 313-314 toxicity of monoterpenoids, 305-316 Earworm, See Corn earworm

Ecdysis, insects treated with azadirachtin, 298 Ecdysone, effect of parasitoids on synthesis, 48 Ectoparasitoids, venoms as sole source of host regulatory factors, 43 Edible lily effect of bacterization on yield, 423/ root microflora, 423 Egg development, parasitism, 55 Eggs laid per night, effect of age, 74/ Elicitor(s), 8 computer molecular model, configuration, 326 disease resistance, model of binding to D N A , 334 Elicitor-DNA complex, phytoalexins, 324-335 Embryotoxic effect, monoterpenoids, 313/ Emergence of parasites, effect of plant resistance, 154/ Encapsulation, suppression mechanisms, 52 Endocrine interactions, parasitoid-host, 47-49 Endoparasitoids, 42,43 Enzymatic defenses advantages of use, 189-190 antinutritive, tomato plant, 166-197 cost, 189 metabolic drain on plant, 189 oxidative plants, 190 resistance to digestion, 190 Enzymatic oxidation, laccase activity, 92 Enzyme(s) catalysis in cuticle, 88 chemical reactivity of products, 168,171 depreciation of nutritional quality, 171 induction by feeding damage, 178-179 phototoxin activation, 367 plant enzymes as insect-control, 190 plants, oxidants, and reductants-antioxidants, 187/ Enzyme systems, evolutionary modification, 147-148 European corn borer, hydroxamic acid, 252 Evaporation rate additivity of parameters, 118 from rubber septa, 120-121 instantaneous, 109 pheromone components, effect of air speed, 118-120 prediction, 118

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

444

N A T U R A L L Y OCCURRING PEST BIOREGULATORS

Evaporative loss, mechanistic model, 110-112 Exocrine compounds, thrips, 15 Exocrine glands, defensive secretion, 15 Exogenous hormone, effect on parasitoids, 49 Exoskeleton, 87-88 See also Cuticle

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F Fall army worm grasses, 251 acids, 252 leaf feeding sites, 262 maysin, 251-263 Petunia steroids, 218 petuniolides, 219 pulegone, 312 teosinte, 251 See also Army worm Fatty acid(s) insect nutrition, 188 produced by thrips, 18 synthesis of anacardic acids, 247 Fatty acid synthesis, synthesis of L. pennellii glucose esters, 147 Feeding deterrents, isolation and characterization, 283-287 Fenton reactions, 343,345 Fish toxins, Philippine mangrove plant, 317-322 Flavanones, skeleton, 403/ Flavonoid glycosides, maysin, 252 Flea(s) d-limonene, 309,310 monoterpenoids, neurotoxic effects, 313 terpenoids, 308 Flea beetles, insect resistance in wild potato species, 126-135 Flight activity of insects, rubber septa, 122 Floral chemistry, antiherbivore actions, 279 Floral ethyl acetate residues, composite chromatogram, 284/ Flour beetles, pigment structures, 99-101 Food preference tests, Petunia ,211 Formicid natural products, activities as repellents, 20 Free radicals cytotoxic, 347 disease resistance, 345 pterocarpans, 347-348

Fruit fly, 5 See also Caribbean fruit fly Fungal growth in peanuts, stilbene phytoalexins, 355-356 Fungal pathogens, 204 Fungi, aflatoxin-producing, 352 Fungicides, 7 Furanocoumarins, 365f,366f,367 Fusarium wilt, 407 antifungal agents and antagonists, 408* antifungal agents in soil, 414-415 biological control, 413/ bottle gourd, 419,420 effect of seed bacterization, 413/ inhibition effect, 410* microorganisms associated with Welsh onion, 419 proposed mechanism of biological control, 42 suppression by rhizosphere microorganisms, 407-416 sweet potato, 418

G

Gene(s) encoding insecticidal proteins, 58 insertion into crop plants, 2 Gene transfers, resistance factors, 222 Genetic manipulation applications, 132-133 molecular biotechnology, 58 pest management, 132-133 Solatium berthaultiU 131-132 Genetic theory, selective gene expression, 325 Geranium arthropod-resistant and -susceptible, 224-250 chemical characterization, 225-243 dimethyl disulfide derivative of dimethylated compound, mass spectrum, 2AQf dimethylated compound, mass spectrum, 239/ glandular trichomes, 225 H P L C profile of glandular trichome exudate, 229/231/

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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INDEX

445

Glandular trichome(s) adaptation by pests, 133 adhesive entrapment mechanism, 137-139 cocoon-spinning larvae, 157 cross-breeding inheritance, 132 definition, 137 effect on consumption of prey, 159,160* geranium, 225 natural enemy effectiveness, 151 parasite mortality, 153-156 regulatory gene sequences, 139 secretion of sugar esters, 139-143 Solanum berthaultii, 127 tobacco cultivars, 265 wild Bolivian potato species, 126-135 wild Solanaceae, 136-149 Glandular trichome exudates, sugar ester composition, 140* Glucans, elicitors, 8 Glucose esters biosynthesis, 141-143 biosynthesis of novel compounds, 146 biosynthetic pathway in L. pennellii, 142/ mass spectra, 144/ proposed biosynthesis pathway, experimental procedure, 143 See also Sugar esters Glutathione, lepidopterans, 185 Glyceollin, 203 Glycosides, maysin, 251-263 Gossypol, cotton plants, 199 Grasses, See Bermuda grass, Centipede grass, Kentucky bluegrass, Tall fescue Greening groups, 375*,375-376 Growth inhibition, parasitism, 55

H Half-life airspeed, 118-120 double bonds, 115-116 evaporation rate effect on rubber septa, 120-121 methodology for determining, 109-110 pheromones in rubber septa, 119* relationship to structure, 112-115 temperature, 116-118 Heat of vaporization, n-alkyl and n-alkenyl acetates, 117 Hedgeapple, cockroaches, 310

Heme synthesis, protoporphyrin IX, 381 Hemolymph proteins, decrease because of parasite, 46 2-Heptanone, 19 Herbicidal activity, accumulation of porphyrins, 373-375 Herbicide(s) photosensitized porphyrins, 371-386 phytotoxic mechanism of action, 383/ Herbicide concentration, cucumber seedlings, 383/ Herbicide concentration and cellular damage, cucumber cotyledon discs, 38Qf Herbicide groups, structure-activity relationships, 378 Herbivory, terpenoids as regulators, 279 Heritability, glandular trichomes, 132 Heritonin and heritianin cadinane sesquiterpenes, 317-322 structure, 3 1 ^ - 3 2 1 / toxicity, 319 Hexadecyl acetate, ant repellent, 16 Honeybee, pheromones as repellents, 19 Honeybee queens, deterrent compounds, 15 Hormonal agents from insects and plants, 4-5 Hormonal hypothesis, parasitoid response to host, 49 Host defense reactions composition of host tissues, 47 parasitoid resistance, egg texture and composition, 47 role of symbiotic viruses in inactivation, 51 synergism between symbiotic viruses and venomous material, 52 Host endocrine system, virus-induced degeneration, 53 Host hormonal milieu, development of parasitoid, 49 Host immune system, suppression by parasitoids, 52 Host metamorphosis, effect of viruses, 53 Host nutritional physiology, effect of viruses, 53 Host plant chemistry, 151 morphological features, trichomes, 151 resistance antibiosis, 167 antinutritive resistance, 166-167 interference with effectiveness of natural enemies, 151-152

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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446

NATURAL

Host plant, resistance—Continued selective toxicity, 153 utilization of nitrogen by insects, 167 Host-regulating factors, parasitic Hymenoptera, 41-65 Housefly activity from accessory ducts, 78-86 bioassay, 81 mating factors, experimental results, 81-82 mating inhibition, 82r,83f monoterpenoids acute toxicity, 308r growth effects, 312 insect reproduction, 313 neurotoxic effects, 313 photosensitization, 5 terpenoids, 310 Humoral defense response, parasitoids, 47 Hydrogen bonding, active molecule to D N A , 329-332 Hydrogen peroxide formed in autoxidation of desoxyhemigossypol, 343 joint antinutritive action, 183-185 nonenzymatic generation, 183 reduction by enzymes, 183 Hymenoptera adaptation to stages of the host, 43 parasitic, host-regulating factors, 41-65 physiology and metabolism, 46

I

Ichthyotoxicity, cadinane sesquiterpenes, 317-322 Immunological response, suppression in parasitoid-host systems, 56 Inducible plant defenses feeding inhibitor, 200-201 phytoalexins, 201-205 proteinase inhibitor-inducing factor, 200 resource conservation, 199 Inhibitory factor, associated with mating, 72 Insects) nutritional requirements, 172 plant defenses, 199-200 toxicity of monoterpenoids, 305-316

r

OCCURRING PEST BIOREGULATORS Insect behavior and development, bioregulation, 13-124 Insect behavioral responses to pheromones, rubber septa, 122 Insect control, 3-4 Insect cuticle, catecholamine metabolites, 101 Insect cuticle tanning, enzymes and cross-link structures, 87-105 Insect entrapment, glandular trichomes, 137-139 Insect feeding, plant responses, 204-205 Insect growth regulators, effect on parasitoids, 49 Insect molting hormone, ecdysterone, 301 Insect parasitoids, 46 Insect pest control, phytoalexins, 198-207 Insect population control, rubber septa, 121-122 Insect predators, host plant chemistry, 151 Insect repellents arthropod natural products, 14-26 defensive secretion, 15 Insect reproduction, monoterpenoids, 312-313 Insect resistance biochemical aspects, 136-149 chemical composition of plants, 225 geraniums, 224-250 induced by natural products, 5 potential solution, 58 wild Solanum germ plasm, 126 Insect resistance factors, Petunia, 210-223 Insect sex pheromones, controlled release from natural rubber substrate, 106-124 Insect viruses, use as vectors of alien genes, 182 Insect-resistant cultivars, wild Bolivian potato species, 126-135 Insecticidal activity, meliacins, 301 Insecticidal tetranortriterpenoids, 287/,296 Interactive factors, ability of oxidized phenolics to damage protein, 185 Isoflavonoid phytoalexins and analogues, structure, 349/ Isolysergic acid amide, endophyte-infected tall fescue, 433

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

INDEX

447 J

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Juglone ant repellent, 18 antifeedant, 199 structure, 18/ Juvenile hormone effect of parasitoid, 48 host events under endocrine control, 48 pheromone production and release regulation, 67-68 superparasitized host, 49

Luteolin analogues of maysin, 26 If biosynthetic pathways, 262 browning of corn silk, 259 derivatives in com, 259 U V spectra, 257/ Lycopersicon pennellii glandular trichome-based insect resistance traits, 137 insect feeding deterrents, 136 Lyophilized lemon oil, dose-mortality studies, 308-309 Lysergic acid amide, endophyte-infected tall fescue, 433

K Kairomones, definition, 3 Kentucky bluegrass, ALA-modulator combination, 376-377

L Laccase, 91-93 Larvae acquired tolerance, 152 toxicity of Petunia plants, 210 toxicity of tomato foliage, 152 Larval parasitism, effect of plant resistance, 156r Larval parasitoids, tomato fruitworm, 156-159 Leaf extracts, H P L C profile, 26Qf Leaf hoppers abnormal insect behaviors, 129-130 entrapment, 127-129 genetic manipulation applications, 132-133 immobilization, 127-129 insect resistance in wild potato species, 126-135 trichome removal and feeding behavior, 129f Leaf miner flies, insect resistance in wild potato species, 126-135 Lepidopteran larvae, oxidative plants, 190 Lignification of cell walls, plant defenses, 198 d-Limonene contact toxicity trials, 308 insecticidal activity, 306 relative conduction velocity, 315/ Lipoxygenase, antinutritive effects, 178

M Mammalian toxicity, gene products, 59 Manducin, cross-linked multimers of protein, 101 Mangrove plant description, 318 fish toxicity, 317-322 fractionation scheme, 318/ plant chemistry, 318-321 saline environment, 318 Mating factors, experimental procedure, 79-81 Mating inhibition factors, insects, 78-86 Matteucinol, 404f Maysin C N M R spectra, 258/ com silk, 262 fall army worm, 262 flavonoid glycosides, 252 H P L C profiles of leaf extracts, 256/ in com, teosinte, and centipede grass, 251-263 leaves of teosinte, corn, and centipede grass, 255 levels of leaf number, 262/ teosinte leaves, 262 U V spectra, 257/ Medicarpin antifungal and antibacterial activity, 404r structure, 405/ Melanin, biosynthesis, 95 Melanization development in cuticle, 88 1 3

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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448

N A T U R A L L Y OCCURRING PEST BIOREGULATORS

Melanization—Continued dopa quinone imine conversion factor, 9 8 enzymes in insect cuticle, 9 3 flour beetles, 9 9 - 1 0 1 Meliacins insecticidal activity, 301 plant-derived insecticidal compounds, 2 9 6 Membrane disruption, free radical autoxidations, 3 4 7 Membrane dysfunction, phytoalexins, 3 4 5 Membrane-bound enzyme, 3 8 2 Metamorphosis, developmental arrest, 5 3 Mexican bean beetle induced resistance, 2 0 4 - 2 0 5 soybean phytoalexins, 2 0 3 Miconia cannabina, fractionation scheme, 4 0 Q f Miconidin, insect antifeedant activity, 3 9 9 Microbial agents, control of insect pests, 5 Microbial cultures, antifungal activities, 4 0 9 Microorganisms from lily roots treated with antagonistic bacteria, 4 2 4 r Mites induction of tomato plant foliar oxidative enzymes, 179f insect resistance in wild potato species, 126-135 resistant and susceptible geraniums, 2 2 4 - 2 5 0 See also Spider mites Mixed-cropping technique, mechanism of biological control, 4 2 1 Molting inhibition, parasitism, 5 6 - 5 7 Monogamicity effect, housefly females, 8 2 Monogamicity factor accessory gland, 7 8 two distinct peptides, 7 9 Monoterpene, produced by thrips, 17 Monoterpenoids cockroaches, 312,313 embryotoxic effect, 3 1 3 r fumigation, 3 0 9 housefly, 3 1 2 insect growth and development, 3 1 2 insect reproduction, 3 1 2 - 3 1 3 insecticidal activity, 3 1 4 neurotoxic effects, 3 1 3 - 3 1 4 structures, 3 0 7 / sublethal effect in earthworm, 314* topical exposure, toxic effects, 3 0 8 - 3 0 9 toxicity and neurotoxic effects in insects and earthworms, 3 0 5 - 3 1 6 See also Terpenoids

Moraceae analysis, 3 6 4 - 3 6 5 biocidal compounds, 3 6 2 - 3 6 8 edible and medicinal plants, 3 6 4 evolutionary relationships, 3 6 7 phototoxic activity, 364* Mosquitoes implanted male accessory gland, 7 9 rf-limonene, 3 1 0 phenolics, 3 1 0 terpenoids, 3 1 0 Moth pheromone production, 6 6 - 7 7 See also Codling moth Multienzyme approach, noctuid larvae,

182-187 Mutation, phytotoxic effect, 3 7 2 Mycotoxins, nitidulid beetles, 2 7

N Natural defense mechanism, applications,

357-359 Natural products commercial use, 9 new pesticides, 8 research trends in utilization of pest control, 1-11 understanding of physiology and behavior, 8-9 Necrosis of host tissue, parasitism, 5 5 Neem seeds, 2 9 4 - 2 9 6 , 2 9 5 / Neem tree, insecticidal constituents, 2 9 3 - 3 0 4 Negative resistance adverse effect on ability to utilize food, 167-168 deficit of essential nutrients, pea, rice, maize, 167 impairment of protein quality,

169/,17(y utilization of dietary plant nitrogen, 168 Neurohormones, levels required for activity, 2 Neuropeptides, release of precursors, 71 Neurophysiological symptoms, earthworms exposed to monoterpenoids, 3 1 4 Nicotiana tabacum germ plasm collection, resistance to tobacco hornworm and tobacco budworm, 2 6 4

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

INDEX

449

Niudulid beetles aggregation pheromone, 2 7 - 4 0 infestation, 2 7 pest management potential of pheromones,

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39-40 Noctuid larvae antinutritive bases of resistance, 182 antinutritive plant systems, 1 8 7 - 1 8 9 multienzyme approach, 182-187 sterol intake reduction, 189 Nutrients destroyed by plant chemical or enzymatic interactions, 188f onslaughts against acquisition, 1 8 7 - 1 8 9 Nutritional quality, negative plant resistance, 171

O Oak wilt disease, nitidulid beetles, 2 7 Oligosaccharides binding to model DNA, 3 3 2 conformation, 3 2 6 construction of model molecules, 3 3 2 elicitor activity, 3 2 6 hydrogen bonding to DNA, 3 3 3 / positioning to phosphates of DNA,

329-332 structural features, 3 2 6 Oviposition choice tests, 2 6 9 parasitism, 5 5 Oviposition factor, associated with mating, 7 2 Oviposition stimulation factor, accessory gland, 7 9 Ovipositional bioassays, cuticular isolates from N. tabacum, 2 6 8 Ovipositional nonpreference, tobacco budworms, 2 6 5 Oxidative enzyme(s) incompatibility with biological control agents, 180 nutritional consequences, 171 resistance against noctuid larvae, 182 resistance to aphids, 179 Oxidative enzyme activity, growth of larval tomato fruitworm, 177f Ozone, soybean-induced resistance, 2 0 4

P Paragonial factor, mating activity, 7 9 Paralysis, wasp venom, 5 4 Parasite emergence, effect of methyl ketones in host diet, 159f mortality, 154r,155r,157r Parasitic Hymenoptera host-regulating factors, 4 1 - 6 5 secretions from glands, 5 4 Parasitism, effect on host, 4 6 eggs effect of plant resistance mechanisms, 154r tomato fruitworm, 1 5 3 - 1 5 6 relationship to glandular trichome density, 153 synthesis of host proteins, 4 6 - 4 7 Parasitization of tomato fruitworm, field cage studies, 157f Parasitoid(s) adult female gland secretions, 5 4 parasitoid-derived viruses, 5 1 - 5 4 venomous substances, 5 4 - 5 7 conformers and regulators, 4 1 - 4 2 factors associated with development composition of host, 4 3 - 4 7 host endocrine system, 4 7 - 4 9 teratocytes and serosa, 4 9 — 5 1 host-regulating factors, 44t-A5t host plant chemistry, 151 host response, 4 7 limitations to development, 4 2 nutrition, 4 3 physiological ecology of the host, 41 response to host, 4 9 restricted activity, 181 Parasitoid-host synchrony, mechanism, 4 2 Pathogens, phytoalexin elicitation, 2 0 4 P B A N , See pheromone biosynthesis activating neuropeptide PBSF, See Pheromone biosynthesis suppression factor Peanut(s), aflatoxin contamination, 3 5 2 - 3 6 0 capacity to produce stilbene phytoalexins, 3 5 7 damage and stilbene production, 3 5 5 discoloration, 3 5 5

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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450

N A T U R A L L Y OCCURRING PEST BIOREGULATORS

Peanut(s), — Continued drought-tolerant genotypes, 359 favorable genotypes, 357-359 fungal colonization, 355 late-season drought stress, 356-357 maturity vs. anatoxin contamination, 357 maturity vs. phytoalexin production, 354 relationship of phytoalexin and aflatoxin concentrations, 355 water and stilbene production, 355 Peanut seeds, production of phytoalexins, 354 Pentadecane, ant repellent, 16 Peptides, origin and effects, 5 Perillene, 17,17/ Peroxidase component of enzyme-based defense, 176 evidence for antinutritive action, 176 oxidation of tyrosyl residues of proteins, 93 phototoxin activation, 367 Peroxidative damage, inactivation of photosynthesis, 382 Peruvian plants search for agrochemicals, 399-406 experimental methods, 400-403 Pest control natural products, 1-11 phytoalexins, 198-207 Pest control materials, specific for insects, 4 Pest management arthropod-resistant crop varieties, 150 genetic manipulation applications, 132-133 Pesticides, 2 Pests, classification, 2 Petunia, 210-223 Petunia steroids, 212-216 biogenesis and interconversions, 217-218 biological activity, 218-220 growth of com earworm, 218-219 occurrence in the plant, 220-222 Petuniasterones, 212-215 fresh Petunia leaves, 2211 growth inhibition of corn earworm, 218* Petuniolides growth inhibition of corn earworm, 219* pests, 219-220

Petuniolides—Continued plant organs, 221-222 selective feeding, 221-222 structures, 215-216 Phenazines fungal inhibition, 410 structure, 422/ Phenolic(s) mosquitoes, 310 tobacco root levels, 392/ 393/ Phenolic acids, laboratory bioassays, 394 Phenolic compounds of tobacco, tissues exposed to pathogens, 388-389 Phenolic-insect associations, Chrysomelidae, 282 Phenoloxidases, oxidation of phenols to quinones, 91-93 Pheromone(s), 3 analysis by mass spectrometry, 3If apparatus for determination of half-life, 111/ applications for pest control, 106-107 comparison with tetraenes, 34-37 controlled release from natural rubber substrate, 106-124 controlled-release substrate, 107 during the photophase, 68 effectiveness as trap bait, 38 evaporation rate, 108 evaporation rate half-life, 109-110 honeybee workers, 19 hydrogenation of purified compound, 30-32 injection of P B A N , 68-71 isolation and spectra, 30 levels required for activity, 2 moth, production and release, 67 nitidulid beetles, 27-40 possible biosynthetic origin, 39f rate of evaporation, 109 temperature, effect on half-life, 117 Pheromone biosynthesis, insects, 78-86 Pheromonettosynthesisactivating neuropeptide artificial induction studies, 68-72 corn earworm, 78 diel periodic release, 68 effect of injection, 75* production in moths, 66 structure and mechanism, 67

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

INDEX

451

Pheromone biosynthesis suppression factor,

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66,73 Pheromone collections, nitidulid beetles, 2 8 Pheromone components, effect of air speed on evaporation rate, 118 Pheromone gland extract, 69/,7(y Pheromone production effect of age, 7 2 - 7 3 endogenous factors, 7 6 induction, 6 8 - 7 2 inhibition, 7 2 - 7 6 moths, endogenous regulation, 6 6 - 7 7 neural or neuroendocrine signal, 7 1 - 7 2 physiological and biochemical events, 6 8 precise timing, 7 6 regulation of induction, 6 7 - 6 8 Philippine mangrove plant, fish toxins,

317-322 Photobiocides, tropical plants, 362 Photodegradation products A L A plus DP-treated cucumber seedlings,

374/ efficacy on cucumber cotyledon discs, 3 8 Q f Photodynamic compounds herbicides, 3 7 1 - 3 8 6 toxicity in presence of light and molecular oxygen, 3 7 2 Photodynamic membrane lipid peroxidation, diphenyl ethers, 3 7 8 Photodynamic pigment, mechanism of action of herbicides, 381 Photogenotoxins, D N A and R N A , 361 Photoreceptor for photodynamic damage, visible pigment, 3 7 8 Photosensitization, houseflies, 5 Photosensitizers biocidal activity, 361 porphyrins, synthesis, 3 8 4 tropical plants, 3 6 1 - 3 7 0 Phototoxic activity, Moraceae, 3 6 4 f Phototoxic antimicrobial properties, tropical plants, 363r Phototoxic phytochemicals biocidal activity, 361 tropical plants, 3 6 1 - 3 7 0 Phototoxins activation mechanisms other than light,

367 distribution, 3 6 2 plant pest control, 3 2 3 - 3 8 6

Phytoalexin(s), 5,7-8 chemical characterization, 201 cotton vascular tissue, 3 3 7 criteria for effectiveness, 3 3 7 - 3 3 8 differences in toxicity, 3 4 8 elicitation, 7-8 by fungal pathogens, 204 by stresses, 2 0 4 insect feeding deterrents, 2 0 1 - 2 0 3 interaction between fungi and peanut, 354 maturity of peanuts, 3 5 4 membrane dysfunction, 3 4 5 peanut seeds, 3 5 4 plant pest control, 3 2 3 - 3 8 6 potential role in insect pest control, 1 9 8 - 2 0 7 response to fungal challenge, 3 5 2 solubility, effectiveness of fungicides, 3 3 7 soybean, 2 0 2 / 2 0 3 stilbene, 3 5 2 - 3 6 0 structure-activity relationship, 3 3 6 - 3 5 1 synthesis, 5 toxicity, 3 3 7 use, 2 0 5 vestitol, 201 Phytoalexin elicitors binding sites, 3 3 4 binding to D N A , 3 2 4 - 3 3 5 computer model with rotated residues,

33QT CPK space-filling model, bound to model DNA, 3 3 1 / interaction with D N A nucleotides, computer and C P K models, 3 2 9 oligosaccharide, 3 2 6 oligosaccharide V , computer model of conformation, 3 2 8 / structural requirements for optimum interaction, 3 3 4 structural similarities, 3 2 7 / Phytoalexin production relationship to peanut kernel water activity, 3 5 8 / soybeans, 3 2 4 - 3 2 5 Phytochemicals effect on infectivity, 182r enhancement of pollination, 2 7 8 - 2 7 9 feeding deterrency, 2 7 8 Phytotoxicity, porphyrins, 3 7 7 Pigment structures, 9 9 Pigmentation, development in cuticle, 8 8

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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452

N A T U R A L L Y OCCURRING PEST BIOREGULATORS

Pink bollworm, 218,219 Planidia mortality, tomato foliage with and without trichomes, 158; Plant antibiotics, phytoalexins, 198-207 Plant breeding, insect resistance, 131-132,222 Plant breeding programs, registering genetically modified organisms, 189 Plant defenses, 198-200 Plant enzymes as insect control measures, 190 oxidants and reductants-antioxidants, 187r resistance against noctuid larvae, 190 Plant growth regulators, categories of natural products, 6 Plant nitrogen, insect population dynamics, 167 Plant oxidative enzymes, antinutritive bases of resistance against insects, 189 Plant pathogenic fungus, cotton, 336 Plant pathogens, growth inhibition, 411/,412/ Plant resistance to insects, mechanisms, 125-207 Plant response to wounding, 168-171 Plastids, PPIX leakage, 382 Polyketide, origin of pheromone, 38-39 Polymeric substrates, release mechanism, 109 Polyphenol oxidase, 173 Polyphenol oxidase enzymes, 138 Polysaccharides, elicitors, 8 Porphyrin(s) cost and dangers, 382 herbicide-induced accumulation, 377-382 photodegradation products, 373 photosensitization, 371-386 Porphyrin accumulation artificial, 372 seedling tissues, 375/ Porphyrin synthesis pathway feedback control by heme, 372 inhibitors and modulators, 374/ Porphyrin-protein complex, herbicidal role, 382 Postinfectional defense, proteinase inhibitor production, 173 Potato, Petunia genetic material, 222

Potato glandular trichomes, defensive activity against insect attack, 126-135 Potato tuber moth complex, insect resistance in wild potato species, 126-135 Potato virus Y , 130 Preformed plant defenses, 198-200 Preinoculation, effect on disease incidence of Fusarium wilt, yield of sweet potato, 418f Preparative separation, complex alkaloid mixture, 426-434 Primin, 399 Protective interactions, pathogens and insects on plants, 204 Protein(s) antinutritive form of resistance, 171 dietary level vs. antinutritive effect, 175 nutritive value and relative growth rate, 175* quinone-mediated depreciation of nutritive quality, 175/ reduction of nutritive quality, 174 Proteinase inhibitors, 173 impact on bovine trypsin, 181/ potential incompatibility, 180 Protochlorophyllide, 381 Protoporphyrin I X accumulation, 378-379 effect vs. amount accumulated, 379 half-life, 381,379 herbicidal damage, 381 mode of action, 379-382 substrate autoxidation, 379 Pseudomonas gladioli, 419/,420 Pseudomonas spp., antifungal activity, 422/ Pterocarpans, 347-348,349/ Pulegone, fall army worm, 312 Pyrethrins, cockroaches, 310 Pyrethroids, structure, 4 Pyrrolnitrin, structure, 420/*

Q Quinone(s) interrelationship of oxidative and reductive processes, 184/ spontaneous oxidation in basic media, 183

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

INDEX

453

Quinone methides, evidence for existence, 93-94 Quinone production, ascorbic acid, 185 Quinonoid compounds, isomerization, 93-95 Quinonoid isomerases, 94-95,96r-97r

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R Receptivity termination factor, pheromone production, 78 Red scale, 312 Reducing power, quinones, ascorbic acid, and glutathione, 186/ Regulatory factors, parasitic Hymenoptera, 58-59 Repellency plant-derived terpenoids, 310-312 queen-containing colonies of ants, 20-24 Repellents, identification, 3 Resistance factors, petuniolides, 222 Resistance traits, wild and crop plants, 136-137 Resistant varieties, crop plant selection, 1 Rhizosphere microorganisms, suppression of Fusarium wilt, 407-416 Rice weevil acute toxicity of monoterpenoids by fumigation, 309f insect reproduction, monoterpenoids, 313 Root rot, edible lily, 421-425 proposed mechanism of biological control, 424/ growth inhibition, 421-422 Rootworm feeding stimulants, cucurbitacins, 278 See also Corn rootworm Rose furan, 17,17/ Rotenone, fish toxicity and crop protection, 317-318 Rubber septa applications, 121-122 evaporation rate of a pheromone, 108 factors controlling pheremone evaporation rates, model, 106-124 first-order evaporative loss, model, 110 heat of evaporation, 114r,l 15f limitations on uses, 120-121 mechanism of release, 107-109 pheromone stability, 121

Rubber septa—Continued proportionality between dose and evaporation rate, 108 research value, 107 temperature in field studies, 118

S Sclerotization N-acylated catecholamines, 88 definition, 87,88 enzymes in insect cuticle, 93 laccases as critical enzymes, 93 Scopoletin, location in tobacco plant, 391 Secretions from glands, parasitic Hymenoptera, 54 Seed bacterization Fusarium wilt of adzukibean, 413-414 root rot, 421 Selective feeding, petuniolides, 221-222 Selective gene expression, genetic theory, 325 Senescing virgins, pheromone production, 72-73 Sensory disturbance, glandular trichomes of S. berthaultiU 129-130 Serosa, 49,50 Sesquiterpene(s) fish toxin, 317-322 isolated from mangrove plant, 318 lactones, sunflower moth, 279 Sex pheromones, moths, 66-77 Social insects, deterrent compounds, 15 Soil-borne disease, methods for biological control, 417-425 Solanaceae, glandular trichome-mediated insect resistance, 136-149 Solanum berthaultii abnormal insect behaviors, 129-130 defensive biology, 127-131 genetic improvement of the potato, 133 genetic manipulation, 131-132 glandular trichomes aphid resistance, 128/ insect resistance traits, 137 resistance to insects and mites, 127 insect entrapment, 136 resistance to Colorado potato beetle, 130-131 resistance to insects, 126-135

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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454

N A T U R A L L Y OCCURRING PEST BIOREGULATORS

Southern army worm, insect reproduction, monoterpenoids, 3 1 3 Southern pine beetles, terpenoids, 308 Soybean(s) induced plant defense, 2 0 4 - 2 0 5 phytoalexin production, 3 2 4 - 3 2 5 Soybean looper, 2 0 3 - 2 0 5 Steroidal materials, lepidopteran larvae, 2 1 0 - 2 2 3 Stilbene(s), 3 5 5 - 3 5 7 Stilbene phytoalexins, 3 5 2 - 3 6 0 field-damaged peanuts, 3 5 7 from peanuts, 3 5 3 - 3 5 4 preharvest aflatoxin contamination of peanuts, 3 5 2 - 3 6 0 spore germination and radial growth of fungal species, 3 5 6 f structures, 3 5 4 / Stresses elicitation of phytoalexins, 2 0 4 plant defenses, 1 9 8 - 1 9 9 Structures of compounds, elucidation, 2 Substrate channeling, 3 8 2 Sucrose esters, ovipositional activity, 2 7 6 Sugar esters analysis of labeled acyl groups, 1 4 3 - 1 4 6 biosynthesis, 1 4 1 - 1 4 3 cuticular sugar fraction of Nicotiana species, 2 7 3 r - 2 7 4 r entrapment of spider mites, 139 metabolic investment in biosynthesis, 141 Nicotiana species, 2 6 9 proposed pathway of biosynthesis in L. pennelliU 142/ secretion by glandular trichomes, 1 3 9 - 1 4 3 structure, 139 See also Glucose esters, Sucrose esters Sulfur amino acid, detoxication, 185 Sunflower, 283-287,284/ Superoxide ion, formation, 183 Swallowtail butterfly larvae, wild parsnip, 361 Sweet potato, 4 1 8 Symbiotic viruses interaction of parasitoid with host, 4 2 parasitoid-derived, 5 1 - 5 4 structures and interaction with host, 5 3 - 5 4 Synergistic activity, pheromones and food volatiles, 3 8 Synergistic herbicides, 5-aminolevulinic acid plus 2,2'-dipyridyl, 373

Synergistic toxicity, tomato foliage components, 1 5 2 - 1 5 3 Synthetic compounds bioassays, 3 7 - 3 8 field bioassay of pheromones, 38r pesticides, 2

T Tall fescue, 4 2 6 - ^ 2 7 Tall fescue seeds, endophyte-infected,

429-433 Tanning agents, definition, 8 7 Tansy, Colorado potato beetle, 3 1 2 Temperature, effect on half-life, 1 1 6 - 1 1 8 Template modifications, elicitor binding to D N A , 335 Teosinte, maysin, 2 5 1 - 2 6 3 Teratocytes endocrine role, 5 0 functions that benefit parasitoid, 5 0 inhibition of host immune response, 5 0 interaction of parasitoid with host, 4 2 parasitoid-host interactions, 4 9 — 5 1 production and secretion of regulator molecules, 5 0 Terpenes, red scale, 312 Terpenoids floral concentrations, 2 7 9 insecticidal and antifeedant, 2 7 9 larvicidal and ovicidal activity, 3 0 9 — 3 1 0 pests, 3 1 0 toxicity, 3 0 6 - 3 0 8 See also Monoterpenoids Tetraenes comparison of synthetic compounds, 3 4 r comparison with pheromones, 3 4 - 3 7 mass spectra and diagnostic fragmentation, 3 6 / synthesis of model, 3 2 - 3 4 synthetic scheme, 3 3 / Tetranortriterpenoid(s) growth-inhibitory and larvicidal effects,

299f,300f limonoids, neem and chinaberry trees, 2 9 6 Thrips, 15-18 Tobacco leaf trichome types, 2 6 4 tobacco mosaic virus vs. green peach aphid, 2 0 4

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

455

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INDEX Tobacco budworm cage choice tests for ovipositional activity, 276 articular components from Nicotiana species, effect on ovipositional behavior, 264-277 ovipositional nonpreference, 265 percentages of eggs deposited in choice tests, 270t-272r percentages of Nicotiana spp. plants infested, 272r resistance ratings, cuticular chemistry, 267f Tobacco hornworm N M R spectra of pupal exuviae, lOOf cuticular components from Nicotiana species, effect on ovipositional behavior, 264-277 percentages of eggs deposited in choice tests, 270f-272f percentages of Nicotiana spp. plants infested, 272* petuniolides, 219 resistance ratings, cuticular chemistry, 267f toxicity of Petunia plants, 210 2-tridecanone-glandular trichome-mediated resistance, 150 Tobacco root phenolics analysis, 391 chromatogram, 390f experimental methods, 389 laboratory bioassays, 391 resistance to black shank, 388-398 vs. fungal attack, 391 Tomato acute toxicity of foliage, 152-153 negative resistance, 172 Petunia genetic material, 222 proteinase inhibitor-inducing factor, 200 resistance against insects, 166 substrates for enzymes, 172 2-tridecanone-glandular trichome-mediated resistance, 150 See also Wild tomato Tomato fruitworm digestive effect of plant antinutritive defense, 176 impairment of detoxicative ability, 185 induction of tomato plant foliar oxidative enzymes, 179f negative resistance, dietary plant nitrogen, 168

Tomato—Continued parasitoids and predators, 150 plant resistance egg parasitoid, 153-156 larval parasitoids, 156-159 polyphenol oxidase, 173 proteinase inhibitors vs. relative growth, 174* 2-tridecanone-glandular trichome-mediated resistance, 150 See also Fruitworm Tomato plant, enzymatic antinutritive defenses against insects, 166-197 Toxic oxygen species, degree of susceptibility, 376 Toxicants, origin, 4 Transfer of resistance factors, identity of allelochemical agents, 222 Transgenic crop plants, resistance traits of wild species, 137 Transgenic plants and microbials, applications, 58 Trap crops, levels of insect ovipositional stimuli, 276 Trichome(s), entrapping properties, 179 Trichome exudates budworm oviposition, 269 resistance to insect damage, 265 2-Tridecanone-glandular trichome-mediated insect resistance in tomato, effect on parasitoids and predators of Heliothis zea 150-165 Tropical plants biological activity, 7 phototoxic antimicrobial properties, 363f phototoxic metabolites, 361-370 Tyrosinase, larval cuticle, 91 t

V Vallapin,319,319/,321 Variegated cutworm, petuniolides, 219-220 Venom, genes encoding for fast action, 59 See also Ants, Fire ants, Wasp venom Venom gland, Hymenoptera, 42-43 Venomous substances ants, 15,20-24 nonparalyzing, 54-57 paralyzing, 54

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Vestitol, phytoalexins, 201 Viral agents control efficacy reduction, 181-182 control of insect pests, 5 Viral genomes, 51 Viral nucleic acid, synthesis of new protein, 53 Viruses endocrine regulation of development, 53 host synthesis of new proteins, 52-53 parasitoid-derived, 51-54 structures and interaction with host, 53-54 susceptibility of specific host tissues, 53-54 transcription of parasitoid-derived viruses, 52-53

W Walnut trees, hydrojuglone glucoside, 200 Wasp venom, 54

Weed control, definition, 6 Weevil, See Alfalfa weevil, Boll weevil, Rice weevil Welsh onion, associate crop with bottle gourd, 419 Western pine beetles, vapors of monoterpenes from pine, 309 Wild parsnip, 361 Wild tomato, acute toxicity of foliage, 152-153 Wind tunnel bioassay data, 29r bioassays with synthetic tetraenes, 37t laboratory pheromone bioassays, 28 Worms, See Army worm, Beet army worm, Bertha army worm, Corn earworm, corn rootworm, Earthworm, Fall army worm, Pink boll worm, Rootworm, Southern army worm, Tobacco budworm, Tobacco

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In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.