Allelochemicals: Role in Agriculture and Forestry 9780841209923, 9780841211674, 0-8412-0992-8

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ACS SYMPOSIUM SERIES 330

Allelochemicals: Role in Agriculture and Forestry George R. Waller,

EDITOR

Oklahoma State University

Developed from a symposium sponsored by the Division of Agricultural and F o o d Chemistry at the 190th Meeting of the American Chemical Society, Chicago, Illinois, September 8-13,

1985

American Chemical Society, Washington, DC 1987

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Library of Congress Cataloging-in-Publication Data Allelochemicals: role in agriculture and forestry. (ACS symposium series, ISSN 0097-6156; 330) Includes bibliographies and indexes. 1. Allelopathic agents—Congresses. 2. Allelopathy— Congresses. 3. Allelopathic agents—Industrial applications—Congresses. 4. Agriculture—Congresses. 5. Forests and forestry—Congresses. I. Waller, George R. II. American Chemical Society. Division of Agricultural and Food Chemistry III. American Chemical Society Chicago,Ill.)IV. Series. QK898.A43A45 1986 632'.5 86-26568 ISBN 0-8412-0992-8

Copyright © 1987 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 the first page 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 Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ACS Symposium Series M. Joan Comstock, Series Editor Advisory Board Harvey W. Blanch University of California—Berkeley

Donald E. Moreland USDA Agricultural Research Service

A l a n Elzerman Clemson University

J. T. Baker Chemical Company

J o h n W . Finley Nabisco Brands, Inc.

James C . R a n d a l l Exxon Chemical Company

Marye Anne Fox The University of Texas—Austin

W. D . Shults Oak Ridge National Laboratory

M a r t i n L . Gorbaty Exxon Research and Engineering Co.

Geoffrey K . S m i t h Rohm & Haas Co.

R o l a n d F. H i r s c h U.S. Department of Energy

Charles S.Tuesday General Motors Research Laboratory

Rudolph J . Marcus Consultant, Computers & Chemistry Research

Douglas B . Walters National Institute of Environmental Health

Vincent D . M c G i n n i s s Battelle Columbus Laboratories

C . Grant W i l l s o n IBM Research Department

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Foreword T h e A C S S Y M P O S I U M S E R I E S was founded i n 1974 to provide a m e d i u m for p u b l i s h i n g s y m p o s i a q u i c k l y in b o o k

form.

The

format o f the Series parallels that o f the c o n t i n u i n g A D V A N C E S IN C H E M I S T R Y

S E R I E S except that, i n order to save time, the

papers are not typeset but are reproduced as they are submitted by the authors i n camera-read the supervision o f the E d i t o r s w i t h the assistance o f the Series A d v i s o r y B o a r d and are selected to m a i n t a i n the integrity o f the symposia; however, verbatim reproductions of previously published papers are not accepted. B o t h reviews a n d reports

of

research are a c c e p t a b l e , because s y m p o s i a m a y e m b r a c e b o t h types o f presentation.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Division of Agricultural and Food Chemistry Symposium on Allelochemicals: Role in Agriculture, Forestry, and Ecology Steering Committee A n a Luisa Anaya

G . W a y n e Ivie

Nelson E . B a l k e

M a r t i n Jacobson

H . H . Cheng

H a n Sau K u

Chang Hung Chou

Donald E . Moreland

Jerry D . C o h e n

Frank E . Mumford

H o r a c e G . Cutler

E . F . Paschall

Thanh H . Dao

A l a n R. P u t n a m

James M . Davidson

Elroy L . Rice

Glen Fuller

F a l k R. Ritting

A . R. G i l m o r e

G e r a l d D . Rosenthal

Stephen R. Gliessman

W a r r e n C . Shaw

Paul Hedin

A l l a n E . Smith

Stephen B . Horsley

Alonzo E . Thompson

Ted L . Huller

A . Douglas W o r s h a m

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Preface ^N^OST

P E O P L E A R E A W A R E that s k u n k s a n d p o r c u p i n e s have

effective

w a y s to repel their enemies. W h a t they don't realize, and science is just n o w l e a r n i n g , is that plants also have defense systems. T h e o p h r a s t u s ( 2 8 5 B.C.) a n d , later, P l i n y r e c o g n i z e d the existence interference

among

plants and noted

its s i g n i f i c a n c e

of

in agriculture.

However, involvement of plant-produce tions was first suggested b In 1937, H a n s M o l i s c h c o i n e d the term allelopathy to i n c l u d e both h a r m f u l a n d b e n e f i c i a l b i o c h e m i c a l interactions between a l l types o f plants a n d interactions i n v o l v i n g m i c r o o r g a n i s m s . T h i s definition was later adopted by R i c e i n 1983 and is currently accepted. We w o u l d l i k e to i n c l u d e the p l a n t insect a n d the p l a n t - h i g h e r a n i m a l interactions i n the terms allelopathy a n d a l l e l o c h e m i c a l s for this book. The

nature o f a l l e l o c h e m i c a l s ; the m e c h a n i s m s a n d rates o f

their

e m i s s i o n f r o m the aggressive plant; their fate i n the soil; and their uptake, translocation, and mode

of

a c t i o n w i t h i n the

receptive plant are

all

processes that s h o u l d be studied. These processes w i l l be discussed i n the p l a n t - p l a n t , p l a n t - m i c r o o r g a n i s m , p l a n t - i n s e c t , a n d p l a n t - a n i m a l sections o f this book. In d e s c r i b i n g the allelopathic p h e n o m e n o n , understanding h o w the aggressive plant (the donor) avoids autotoxicity is also essential. In

indigenous

plant c o m m u n i t i e s , a l l e l o p a t h y

may

determine

the

d i s t r i b u t i o n patterns o f plants i n r e l a t i o n to their neighbors, whereas i n agriculture and forestry allelopathy m a y affect yields. F o r example, weeds w i t h allelopathic potential or crops that produce autotoxic aftereffects m a y reduce yields; conversely, u s i n g crops w i t h the a l l e l o p a t h i c p o t e n t i a l to decimate weeds m a y i m p r o v e yields. Agricultural Implications Incorporating allelopathy into a g r i c u l t u r a l management m a y reduce the use o f herbicides, fungicides, a n d insecticides; cause less p o l l u t i o n ; d i m i n i s h autotoxic hazards; etc. P l a n t s a n d soil w i t h their a l l e l o c h e m i c a l s or those a l l e l o c h e m i c a l s p r o d u c e d by associated m i c r o o r g a n i s m s , insects, or higher a n i m a l s c o u l d provide new strategies for m a i n t a i n i n g and increasing forest and a g r i c u l t u r a l p r o d u c t i o n i n the future. If the c h e m i c a l s are a l l e l o p a t h i c , they l e n d themselves

to b e c o m e

starting materials for the synthesis o f herbicides, pesticides, a n d fungicides xi

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

that are not based o n p e t r o l e u m c o m p o u n d s , w h i c h are a p u b l i c health concern. We try to be responsive to the needs o f the p u b l i c . T h e r e must be an interactive w o r k i n g group c o m p o s e d o f foresters, entomologists, botanists, agronomists, biochemists, plant pathologists, a n d a n i m a l scientists to solve these research p r o b l e m s . O r g a n i z i n g this g r o u p w o u l d create c o o r d i n a t i o n problems as w e l l as funding problems. In spite o f these obstacles, the n u m b e r s o f scientists w o r k i n g w i t h allelopathy i n its v a r i o u s forms are increasing. Acknowledgments We thank e a c h o f the p a r t i c i p a n t s , b o t h domestic a n d f r o m a b r o a d , for sharing w i t h us the results obtained i n their studies o n a l l e l o c h e m i c a l s . We also w o u l d l i k e to thank s y m p o s i u m upon w h i c h this b o o k is based because this is the third time they have honored this subject i n the past year a n d a half. GEORGE R.

WALLER

Department of Biochemistry Oklahoma Agricultural Experiment Station Oklahoma State University Stillwater, O K 74078 October 1985

xii

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Introduction .ALLELOCHEMICALS

have

already

been

shown

to i m p o s e

numerous

i m p a c t s i n c u l t i v a t e d a n d natural ecosystems. A l t h o u g h their influences were observed at least as far b a c k as the time o f Theophrastus ( 2 8 5 B.C.), the major progress i n this science has o c c u r r e d d u r i n g the past 2 5 years. O n l y d u r i n g that p e r i o d has the science gained credence a m o n g scientists of m a n y disciplines. T h e p h e n o m e n o n o f allelopathy m a y be unique i n that it p r o b a b l y

involves

mor

phenomenon. T h e diversity o f disciplines and w i d e g e o g r a p h i c a l area represented i n this b o o k

are i n d i c a t o r s o f the w o r l d w i d e

importance

o f allelopathy.

A l l e l o p a t h y i m p a c t s v i r t u a l l y a l l the plant science and pestology disciplines as w e l l as m i c r o b i o l o g y and natural products chemistry. O n e o f m y greatest pleasures has been to see the disciplines c o m e together to create the c r i t i c a l mass necessary to study this science. F o u r recent i n t e r n a t i o n a l s y m p o s i a have fostered this i n t e r d i s c i p l i n a r y effort. N u m e r o u s research teams have gelled as a result o f these meetings. E l r o y Rice's fine b o o k has also served as a n i m p o r t a n t focus for this science. T h e science has n o w clearly entered its l o g a r i t h m i c phase o f growth. Challenge to All Disciplines A l l e l o p a t h i c interactions are c o m p l e x . I ' m aware o f no case where one c h e m i c a l has been u n e q u i v o c a l l y p r o v e n to e x p l a i n the entire situation. A l m o s t a l l a l l e l o p a t h i c interactions i n v o l v e not o n l y products o f higher plants but also those o f m i c r o b e s , either as enhancers o r detoxifiers. A l l cases require c h e m i c a l c h a r a c t e r i z a t i o n w o r k f o l l o w e d b y intensive studies by plant physiologists. S e l d o m can a l l the w o r k be a c c o m p l i s h e d w i t h i n one group. S i m p l y , this means w e must w o r k together. We should encourage that the term allelopathy be used i n its broadest sense (as intended b y H a n s M o l i s c h ) . A l l e l o p a t h y w o u l d l o g i c a l l y i n c l u d e the c h e m i c a l s p r o d u c e d b y m i c r o b i a l plants (Actinomycetes,

algae, fungi,

etc.) and those that enhance g r o w t h as w e l l as inhibit growth. We must refine o u r methods to prove allelopathy. M e r e l y g r i n d i n g up a plant and o b t a i n i n g a p h y t o t o x i n is not p r o o f o f allelopathy. W e urge that protocols

s i m i l a r to K o c h ' s postulates be f o l l o w e d

to d e v e l o p

proofs.

I m p r o v e d techniques are needed i n m a n y phases o f the w o r k . F o r example, we must be careful not to produce c h e m i c a l artifacts d u r i n g our extractions and separations. W e must f i n d better ways o f c o l l e c t i n g a l l e l o c h e m i c a l s xiii

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

f r o m the rhizosphere. We have not yet identified p r o b a b l y 2 5 % o f the a l l e l o c h e m i c a l s p r o d u c e d by higher plants and microbes. E v e n i f we prove that allelopathy is i n v o l v e d i n plant interference, we should not forget that it is but one m e c h a n i s m that influences the eventual outcome i n plant c o m m u n i t i e s . Challenge to Crop and Forest Scientists T h e a p p l i e d aspects o f allelopathy should provide excitement for a g r i c u l tural scientists. A l l e l o c h e m i c a l s pose b o t h a p r o b l e m and an opportunity. A l l e l o c h e m i c a l s undoubtedly cost w o r l d agriculture b i l l i o n s o f dollars a n n u a l l y ; h o w e v e r , by g a i n i n g an understanding o f these natural m e c h a nisms, we c o u l d put them to w o r k to benefit agriculture. S e l d o m are plant rotations, tillage systems planned w i t h the idea o f r e d u c i n g adverse allelopathic effects, m u c h less to exploit beneficial impacts. We don't even k n o w the best way to design our h o m e vegetable gardens. In recent years, A m e r i c a n farmers have been c h a l l e n g e d to p r o d u c e crops w i t h a profit m a r g i n . A l t h o u g h higher c o m m o d i t y prices and higher yields c a n enhance p r o f i t a b i l i t y , reduced input into p r o d u c t i o n w i l l also produce s i m i l a r results. W h e r e v e r possible, we should g a i n an understandi n g o f n a t u r a l m e c h a n i s m s a n d try to put them to w o r k i n a g r i c u l t u r a l p r o d u c t i o n systems. T h i s m a y a l l o w us to reduce some costly fuel and c h e m i c a l expenditures. L i t t l e is k n o w n about the potential to exploit m u t u a l i s m i n a g r i c u l t u r a l systems. A l m o s t a l l the w o r k to date has concentrated o n s y m b i o t i c nitrogen-fixers a n d m y c o r r h i z a l associations. A v a i l a b i l i t y o f soil nitrogen and phosphorus is a severe p r o b l e m i n m a n y areas o f the w o r l d . A l l e l o c h e m i c a l s m a y i m p a c t the a v a i l a b i l i t y o f these nutrients through effects o n the symbiotic microbes. We have g a i n e d some understanding o f autotoxicity and replant p r o b l e m s , w h i c h are c o m m o n i n p e r e n n i a l c r o p p i n g systems, but m u c h remains to be learned. These problems c a n cause serious e c o n o m i c losses, and m a n y appear to involve allelopathy. P l a n t s and m i c r o b e s w i l l undoubtedly be a r i c h source o f c h e m i c a l s that are b e n e f i c i a l to plant g r o w t h , y i e l d , or quality. S o m e interesting developments are n o w under way i n this r e a l m . I've often wondered w h y we haven't done more to exploit allelopathic plants to manage vegetation on our right-of-way lands. Steve H o r s l e y and others have shown that selected herbaceous species c a n v i r t u a l l y eliminate tree g r o w t h for as l o n g as 8 0 years. We've also done little to exploit a l l e l o p a t h i c turf grasses, a l t h o u g h excellent weed-suppressing types have been reported i n Lolium and Festuca. T h e w o r k o n d w a r f spikerush gives hope that we m i g h t manage aggressive aquatic plants w i t h nonweedy xiv In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

allelopathic species. A l l these examples c o u l d greatly reduce c h e m i c a l input into systems. Challenge to Plant Physiologists We k n o w very little about the fundamentals o f a l l e l o c h e m i c a l p r o d u c t i o n and release. M a n i p u l a t i n g a l l e l o c h e m i c a l s by i m p o s i n g the stress o n the o r g a n i s m w i l l p r o b a b l y be possible. D o plants release c h e m i c a l s as a result o f exposure to an alien species? has been done regarding defense to insect attack, but v i r t u a l l y on p l a n t - p l a n t responses has been done.

appropriate produce or M u c h work no research

T h e l i m i t e d w o r k o n m o d e o f action o f a l l e l o c h e m i c a l s suggests that they affect a variety o f sites a n d b i o c h e m i c a l processes, m a n y o f w h i c h are s i m i l a r to those affected m e c h a n i s m s probably r e m a i P r o o f o f allelopathy c o u l d be strengthened i f more research was done to document uptake a n d fate o f a l l e l o c h e m i c a l s i n the recipient or suscept plant. We need to determine relative toxicities (selectivity) o f a l l e l o c h e m icals on the target species. W h a t m i g h t be the o u t c o m e o f a 3 0 % g r o w t h reduction d u r i n g only 2 weeks o f a plant's life cycle? P l a n t physiologists c a n contribute i m m e n s e l y i n the technology o f bioassay. Several i n vitro systems c o u l d prove useful w h e n more is k n o w n about the m o d e o f a c t i o n o f the c o m p o u n d s . These types o f assays m a y prove extremely useful i n m o n i t o r i n g t o x i c i t y t h r o u g h fractionations o f extracts or exudates. Challenge to Plant Ecologists A l t h o u g h ecologists have r e c o g n i z e d several possible m e c h a n i s m s for plant interference, they have s e l d o m determined the relative i m p a c t s o f v a r i o u s m e c h a n i s m s at different life stages or under different e n v i r o n m e n t a l conditions. A l l e l o c h e m i c a l s m a y be especially i m p o r t a n t for some species and at some stages o f g r o w t h , but have little i m p a c t on others. T h e y might be i m p o r t a n t under wet (anaerobic) environments a n d absent under dry conditions. A l t h o u g h some h i g h l y respected plant ecologists say it is virtually impossible to separate interference mechanisms i n the field, I say instead that we must be m o r e creative i n our a p p r o a c h to that p r o b l e m . Several years ago, C . H . M u l l e r made important progress o n this p r o b l e m . H i s papers c a n teach a l l o f us a lesson. P l a n t ecologists a n d a g r i c u l t u r a l scientists have s e l d o m agreed o n anything. It is i r o n i c that one general area o f agreement involves the notion that w h e n plants do not perform w e l l together it is because o f competition. I contend that this notion is false. In m y view, interference is the o u t c o m e and c o m p e t i t i o n for resources is but one m e c h a n i s m . A l l e l o p a t h y is another

xv In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

i m p o r t a n t m e c h a n i s m that produces interference. T h i s has been stated before by others, but I feel it needs repeating. M o r e needs to be learned about plant succession and w h y it proceeds as it does i n a n u m b e r o f different environments. C o n s i d e r a b l e evidence exists that a l l e l o c h e m i c a l s m a y have i m p a c t i n this area. We also need a better understanding o f m u t u a l i s m . U n d o u b t e d l y , m a n y mutualistic associations exist o f w h i c h we are not aware. F o r e x a m p l e , might associated microbes produce c h e m i c a l s that help plants defend their space? Challenge to Microbiologists S o m e o f the m o r e i m p o r t a n t a l l e l o c h e m i c a l interactions i n v o l v e soil microbes. T h e y m a y be either donors or recipients. M i c r o b i o l o g i s t s should m a k e an effort to identify they m a y damage crops i n the field or prove useful as b i o c o n t r o l agents or sources o f useful chemicals. We s h o u l d also learn w h i c h o r g a n i s m s and a l l e l o c h e m i c a l s adversely affect the m i c r o b i a l symbionts. S o m e have already been shown to suppress g r o w t h o f b a c t e r i a l nitrogen fixers a n d nitrifiers as w e l l as m y c o r r h i z a l fungi. C o m p o u n d s that inhibit n i t r i f i c a t i o n c o u l d prove to be i m p o r t a n t agriculturally. W e e d seed longevity is attributed, at least i n part, to inhibitors that protect the seed f r o m decay by microbes. O n e o f the reasons weeds pose such a serious p r o b l e m is because their seeds can persist for decades. T h i s p r o b l e m m i g h t be a t t a c k e d by either destroying the inhibitors or by developing strains o f microbes that can destroy the seeds. Challenge to Pestologists We s h o u l d determine w h i c h plant pests i n f l i c t their d a m a g e through production o f phytotoxins. It n o w appears that several pathogenic fungi m a y do this. N u m e r o u s w e e d species m a y impose interference on crop g r o w t h , at least i n part t h r o u g h a l l e l o c h e m i c a l s . M o r e than 7 0 species have n o w been alleged to have allelopathic potential. Perhaps the most e x c i t i n g concept is to use the natural p r o d u c t as a pest-regulating or pest-inhibiting c o m p o u n d . T h i s approach might w o r k on vertebrate pests as w e l l as insects, nematodes, plant pathogens, and weeds. O n e o f the most e x c i t i n g new n e m a t o c i d a l and m i t i c i d a l c o m p o u n d s is a v e r m e c t i n , a c o m p l e x natural product p r o d u c e d by a Streptomyces. A n exciting new herbicide w i t h glyphosate-like activity has also recently been discovered i n a Streptomyces culture. I a m confident that the pesticidep r o d u c i n g factory o f the future w i l l be a biosynthetic unit. M a n y o f our useful c o m p o u n d s w i l l be p r o d u c e d by actinomycetes, bacteria, or fungi using plant products as substrates. T h i s i n itself c o u l d produce another xvi In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

major m a r k e t for a g r i c u l t u r a l products. T h e c o m p o u n d s p r o d u c e d i n this manner might also pose less environmental hazard. A n obvious place for intensive w o r k on allelopathy is i n the w e e d science area. H e r e , plant interference is either our p r o b l e m or o u r opportunity. We should be clever enough to exploit allelopathy as a w e e d suppression strategy. T h i s c o u l d be a c c o m p l i s h e d w i t h crops that release a l l e l o c h e m i c a l s through exudation or by crop residues placed into sequential c r o p p i n g systems. M y research team and others have already developed some p r o m i s i n g leads i n this area. Challenge to Natural Product Chemists T h i s aspect has been the r a t e - l i m i t i n g step i n m a n y studies o f allelopathy. A l t h o u g h a l l signs m a y point to allelopathy, p r o o f requires positive i d e n t i f i c a t i o n o f the a l l e l o c h e m i c a l s a c c o m p l i s h this, and some s i m p l y go l o o k i n g for the same o l d c o m p o u n d s because they can buy standards f r o m the c h e m i c a l supply house. W h a t is needed is a cooperative effort w i t h plant physiologists and chemists w o r k i n g side-by-side on fractionation and bioassay. We need to isolate and identify the m o r e active c o m p o u n d s even i f they are present i n s m a l l quantities. We must be careful not to isolate c h e m i c a l s that prove to be artifacts. O u r i s o l a t i o n techniques s h o u l d begin w i t h steps that m i g h t be expected to operate i n nature. U s u a l l y , water w i l l be the appropriate solvent. We should perform isolation i n the absence and presence of microbes to see if they add new toxins. C h e m i s t s s h o u l d h e l p d e v e l o p better m e t h o d o l o g y for i s o l a t i n g c o m p o u n d s f r o m the environment, p a r t i c u l a r l y the soil environment. We need more breakthroughs a l o n g the line o f the trapping resin developed by Tang and Young. C h e m i s t s should continually develop i m p r o v e d separation and spectral analyses systems. T h e instruments n o w available are extremely powerful but, because o f cost, are available to only a few laboratories. W h e n n o v e l c h e m i c a l s are c h a r a c t e r i z e d , chemists s h o u l d synthesize s i m i l a r structures to search for useful analogs. C h e m i s t s should also consider more plant c o m p o u n d s as potential intermediates or even starting points i n production o f other useful products. T h e r e appears to still be a shortage o f natural product chemists. If m o r e n a t u r a l product chemists were available for postdoctoral positions, their appetites c o u l d be whetted for allelopathic research. Acknowledgments I must thank several colleagues w h o have contributed a great deal to m y success. I w i s h to first thank m y department c h a i r m a n , J a c k K e l l y , w h o has most i m p o r t a n t l y g i v e n me the f r e e d o m to pursue m y research on xvii In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

allelopathy. I w i s h to especially thank the allelopathy graduate

students

Jane Barnes, Joe D e F r a n k , R o n L o c k e r m a n , Tracy Sterling, A n n e H a r t u n g , and L e s l i e Weston; a n d postdoctorates

R o d Heisey, F r e d L e h l e , a n d Saroj

M i s h r a for a l l their contributions. I p a r t i c u l a r l y appreciate the technical help o f B i l l C h a s e a n d C u r t W h i t e n a c k a n d the fine secretarial assistance o f Jackie

Schartzer.

In a d d i t i o n , I w i s h to thank

Bill

Duke

of Cornell

U n i v e r s i t y a n d the entire W e e d Science Staff at the U n i v e r s i t y o f C a l i f o r n i a ( D a v i s ) for the i n v i g o r a t i o n they s u p p l i e d d u r i n g m y s a b b a t i c a l leaves. F i n a l l y , I must thank Stan R i e s for t e a c h i n g m e some i m p o r t a n t

lessons

about intensity a n d creativity. T h i s i n t r o d u c t i o n w a s o r i g i n a l l y presented

as the 1985 S t e r l i n g B .

H e n d r i c k s M e m o r i a l L e c t u r e d u r i n g the 190th A m e r i c a n C h e m i c a l Society National Meeting in Chicago. ALAN R. PUTNAM

Department of Horticultur Michigan State Universit East Lansing, M I 48824

d Pesticid Research Cente

xviii

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 1

The Potential of Allelochemicals Opportunities for the Future Orville G. Bentley U.S. Department of Agriculture, Washington, DC 20250

How to deal with pests that attack cultivated plants is a continuing challenge to the food and agricultural production system. Natural resistance or tolerance to pests has proven to be one of the safest and least costly ways to protect plants A identif th specific plant component it will be easie incorporat plan capacity to produce the desired chemical. There is the further possibility of identifying additional natural chemicals that may be useful as pest control materials, through the use of natural products or products of industrial synthesis patterned after the natural products. It is clear that allelochemicals are involved in these complex processes and they hold promise for even greater applications. Expanded research in the new biotechnologies offers great potential for further development of allelochemicals. I a p p r e c i a t e the o p p o r t u n i t y to make the opening p r e s e n t a t i o n at t h i s o u t s t a n d i n g symposium on a l l e l o c h e m i c a l s and t h e i r r o l e i n a g r i c u l t u r e , f o r e s t r y and e c o l o g y . It involves multiple d i s c i p l i n e s , i t s r e c e n t advances have been f a c i l i t a t e d by modern i n s t r u m e n t a t i o n , and i t i s moving i n the f u l l continuum from b a s i c m o l e c u l a r b i o l o g y through to p r a c t i c a l a p p l i c a t i o n s . I t i s an i m p o r t a n t emerging a r e a . I commend t h e American C h e m i c a l S o c i e t y and the D i v i s i o n o f A g r i c u l t u r a l and Food C h e m i s t r y f o r p u t t i n g t o g e t h e r t h i s i m p r e s s i v e s e t o f r e v i e w s , t e c h n i c a l r e p o r t s , and p o s t e r s e s s i o n s . One cannot h e l p but be i m p r e s s e d by r e a d i n g t h r o u g h the program t h a t r e s e a r c h i n a l l e l o c h e m i c a l s i s coming i n t o i t s own. I would f i r s t l i k e to g i v e my p e r c e p t i o n o f the c o n c e p t u a l s e t t i n g f o r food and f i b e r i n the U n i t e d S t a t e s t o d a y . In the h i s t o r y o f t h e E a r t h t h e r e has never been a s p e c i e s so s u c c e s s f u l as Homo s a p i e n s i n e s t a b l i s h i n g and m a i n t a i n i n g i t s e l f . M e e t i n g the e s s e n t i a l r e q u i r e m e n t s o f e v e r i n c r e a s i n g numbers o f human b e i n g s b o t h i n t h i s c o u n t r y and now g l o b a l l y has been an u n r e m i t t i n g challenge f o r a g r i c u l t u r a l research f o r decades. You a r e a l l T h i s chapter not subject to U.S. copyright. P u b l i s h e d 1987 A m e r i c a n C h e m i c a l Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1.

BENTLEY

The Potential of

Allelochemicals

3

f a m i l i a r w i t h how w e l l the c h a l l e n g e has been met: fewer people on the l a n d f e e d i n g more Americans than ever b e f o r e , h i g h e x p o r t l e v e l s of a g r i c u l t u r a l p r o d u c t s , and the p r o v i s i o n o f l i f e s a v i n g food to l e s s f o r t u n a t e people around the w o r l d . It c o u l d not have been done w i t h o u t advances i n knowledge t h r o u g h r e s e a r c h . A successful, c o m p e t i t i v e i n d u s t r y i s dependent upon s u s t a i n e d , h i g h q u a l i t y r e s e a r c h and the development o f new and i n n o v a t i v e t e c h n o l o g y . In such a s e t t i n g the t r a n s f e r and the r a t e o f a d o p t i o n o f new t e c h n o l o g y takes on g r e a t s i g n i f i c a n c e . T h i s means t h a t the p r i v a t e s e c t o r - u n i v e r s i t y - F e d e r a l l a b o r a t o r y i n t e r f a c e must be dynamic and responsive. Now to some g e n e r a l comments on a l l e l o p a t h i c c h e m i c a l s . I n t e r a c t i o n s among p l a n t s and o t h e r organisms have l o n g been r e c o g n i z e d and d e s c r i b e d . F o r example, g a r d e n e r s l o n g ago observed t h a t tomatoes do p o o r l y under b l a c k walnut t r e e s . Sorghum p l a n t r e s i d u e s i n h i b i t the growth of many weeds. Pyrethrum and neem p l a n t s have d e f i n i t e i n s e c t i c i d a l p r o p e r t i e s . C e r t a i n v a r i e t i e s of crop p l a n t s e x h i b i t r e s i s t a n c t h a t many such r e l a t i o n s h i p c h e m i c a l s , produced by the p l a n t s or o t h e r o r g a n i s m s . On a c h e m i c a l and m o l e c u l a r b a s i s , we are now b e g i n n i n g to u n r a v e l why these i n t e r a c t i o n s among v a r i o u s organisms o c c u r . B a l a n d r i n e t a l . (J_) have p r o v i d e d us w i t h an i m p r e s s i v e sampling o f the wide a r r a y o f chemical constituents i n p l a n t s . T h e i r main i n t e r e s t i s i n f i n d i n g u s e f u l p l a n t p r o d u c t s , and one must be impressed by b o t h the number and the c o m p l e x i t y of c o n s t i t u e n t s p r e s e n t . Some of these have i d e n t i f i a b l e f u n c t i o n s i n the p l a n t or e f f e c t s on o t h e r o r g a n i s m s . We a r e l i k e l y to l e a r n t h a t more of these c o n s t i t u e n t s have as yet u n r e c o g n i z e d and s o p h i s t i c a t e d i n t e r a c t i o n s b i o c h e m i c a l l y and p h y s i o l o g i c a l l y i n b i o l o g i c a l systems. D r . Putnam's r e v i e w i n C&E News two y e a r s ago (_2) p r o v i d e d an e x c e l l e n t s u r v e y of where we were i n t h i s a r e a and I am p l e a s e d to see t h a t he w i l l be g i v i n g the 1985 S t e r l i n g B . H e n d r i c k s Memorial L e c t u r e l a t e r t h i s week. From the a b s t r a c t s f o r the p r e s e n t a t i o n s t h a t f o l l o w t h i s m o r n i n g , I know t h a t o u t s t a n d i n g speakers w i l l p r e s e n t an e x c e l l e n t r e v i e w o f the s t a t u s of knowledge and of e x c i t i n g developments i n t h i s emerging a r e a . How to d e a l w i t h the p e s t s t h a t a t t a c k p l a n t s under c u l t i v a t i o n i s a c o n t i n u i n g c h a l l e n g e to the food and a g r i c u l t u r a l p r o d u c t i o n system. N a t u r a l r e s i s t a n c e or t o l e r a n c e to p e s t s has proven to be one o f the s a f e s t and l e a s t c o s t l y ways to p r o t e c t p l a n t s . As we i d e n t i f y the s p e c i f i c p l a n t components i n v o l v e d and t h e i r a c t i o n s , i t w i l l be e a s i e r to i n c o r p o r a t e the c a p a c i t y to produce the d e s i r e d c h e m i c a l i n t o the p l a n t of i n t e r e s t . There i s the f u r t h e r p o s s i b i l i t y to i d e n t i f y a d d i t i o n a l n a t u r a l c h e m i c a l s t h a t may be u s e f u l as pest c o n t r o l m a t e r i a l s . The l a t t e r might be t h r o u g h the use o f n a t u r a l p r o d u c t s or i t might be p r o d u c t s of i n d u s t r i a l s y n t h e s i s p a t t e r n e d a f t e r the n a t u r a l p r o d u c t s . It i s c l e a r that a l l e l o c h e m i c a l s are i n v o l v e d i n these complex p r o c e s s e s and they h o l d promise f o r even a g r e a t e r r o l e . B e t t e r u n d e r s t a n d i n g of a l l e l o c h e m i c a l s i n p l a n t p o p u l a t i o n dynamics i n ecosystems w i l l p r o v i d e a b a s i s f o r improved management d e c i s i o n s f o r managers of r a n g e l a n d s and f o r e s t s . T h i s w i l l be e s p e c i a l l y s i g n i f i c a n t f o r ecosystems undergoing d r a s t i c changes such as a f o r e s t t h a t has burned or has been h a r v e s t e d .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4

ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY

H i g h performance l e v e l s f o r s p e c i f i c crops a t t a i n e d i n some s i t u a t i o n s are o f t e n d i f f i c u l t to reproduce even though a l l r e c o g n i z e d c o n d i t i o n s are d u p l i c a t e d . There may be answers to some of the unknowns i n a l l e l o c h e m i c a l s . P r e v i o u s c r o p s and s o i l m i c r o o r g a n i s m s are p o s s i b l e s o u r c e s o f such d i f f e r e n c e s . Another major b e n e f i t t h a t w i l l flow from the more b a s i c r e s e a r c h o f a l l e l o c h e m i c a l mechanisms i s an improved b a s i s f o r u n d e r s t a n d i n g and d e a l i n g w i t h e n v i r o n m e n t a l i s s u e s t h a t are a l s o a concern of a g r i c u l t u r e . As we have become more s o p h i s t i c a t e d i n our view o f the i n t e r a c t i o n s o c c u r r i n g i n the n a t u r a l environment we have a l s o become more s e n s i t i v e to p o s s i b l e s i d e e f f e c t s from p r o d u c t i o n p r a c t i c e s i n a g r i c u l t u r e and f o r e s t r y . This i s p a r t i c u l a r l y true for the use of a g r i c u l t u r a l c h e m i c a l s . The study o f a l l e l o c h e m i c a l s i s h e l p f u l b o t h i n p r o v i d i n g new knowledge about p l a n t and pest i n t e r a c t i o n s and a b a s i s f o r u s i n g s p e c i f i c c h e m i c a l s that produce d e s i r e d r e a c t i o n s w i t h a minimum o f u n d e s i r a b l e s i d e e f f e c t s . Improving knowledge o f the f u n c t i o n s of a l l e l o c h e m i c a l s i n b i o l o g i c a l systems c o n t r i b u t e i m p o r t a n t a r e a : r i s k assessmen of r i s k assessment and r i s k management are e s s e n t i a l elements i n d e v e l o p i n g sound e n v i r o n m e n t a l p o l i c y but i n p r a c t i c e t h e r e has been f r u s t r a t i o n due to l a c k o f adequate d a t a and proven methodology. The c o n c e p t s are s t i l l a t t r a c t i v e and w i t h more complete knowledge t h e y w i l l become l e s s f r u s t r a t i n g and more u s e f u l . I t h i n k i t i s a p p r o p r i a t e i n t h i s s e t t i n g to d i s c u s s the e x c i t i n g p o s s i b i l i t i e s opened up by new developments i n m o l e c u l a r b i o l o g y and f r e q u e n t l y r e f e r r e d to i n terms o f the new biotechnologies. The a p p l i c a t i o n o f the s c i e n c e o f g e n e t i c s t o a g r i c u l t u r e i s not new, but c o m p a r a t i v e l y r e c e n t d i s c o v e r i e s have c a t a p u l t e d our u n d e r s t a n d i n g o f the h e r e d i t a r y a p p a r a t u s o f l i v i n g organisms i n t o a new e r a . The t r a n s f e r o f g e n e t i c m a t e r i a l from one organism to a n o t h e r t h a t i s not even c l o s e l y r e l a t e d to the o r i g i n a l donor i s now commonplace. Recombinant DNA t e c h n i q u e s now f a c i l i t a t e gene identification, characterization, splicing, replication, regulation, and t r a n s f e r , a l l unknown " a r t s " a g e n e r a t i o n ago. The p u r s u i t o f p r o m i s i n g l e a d s may r e q u i r e the e x p e r t i s e o f m i c r o b i o l o g i s t s , p h y s i o l o g i s t s , and b i o c h e m i s t s who may have to c o l l a b o r a t e w i t h plant p a t h o l o g i s t s , entomologists, agronomists, h o r t i c u l t u r i s t s , f o r e s t e r s , and g e n e t i c i s t s . Expanded r e s e a r c h i n the new b i o t e c h n o l o g i e s i s moving ahead w i t h s u p p o r t from many s o u r c e s . P r i v a t e i n d u s t r y has shown a v e r y s i g n i f i c a n t i n t e r e s t t h r o u g h a number of new s p e c i a l i z e d companies as w e l l as o l d e r e s t a b l i s h e d f i r m s . Many S t a t e s a r e p u t t i n g up funds to e s t a b l i s h biotechnology centers. A p a r t of the new s u p p o r t comes from an i n i t i a t i v e by the L a n d - G r a n t U n i v e r s i t i e s t h a t was c a r r i e d forward by USDA f o r a new program i n the c o m p e t i t i v e r e s e a r c h g r a n t s program f o r b i o t e c h n o l o g y . We a r e i n the f i n a l award s t a g e s f o r the f i s c a l y e a r 1985 program. N e a r l y $20,000,000 o f new f u n d i n g was made a v a i l a b l e f o r f i s c a l year 85 i n t h r e e broad program a r e a s : m o l e c u l a r b i o l o g y , m o l e c u l a r and c e l l u l a r mechanisms o f growth and development ; and g e n e t i c and m o l e c u l a r mechanisms c o n t r o l l i n g r e s p o n s e s to p h y s i c a l and biological stress. A p p r o x i m a t e l y 890 p r o p o s a l s were s u b m i t t e d , o f

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1.

BENTLEY

The Potential of

Allelochemicals

5

which 165 can be funded from the funds a v a i l a b l e . Our 1986 budget i s not y e t f i n a l , but we a n t i c i p a t e a s i m i l a r program f o r next y e a r and p l a n to have an announcement p u b l i s h e d i n the F e d e r a l R e g i s t e r i n the near f u t u r e . The new t e c h n i q u e s a v a i l a b l e i n g e n e t i c e n g i n e e r i n g , m o l e c u l a r b i o l o g y , t i s s u e c u l t u r e , and so f o r t h o f f e r g r e a t p o t e n t i a l f o r i d e n t i f y i n g a l l e l o c h e m i c a l s and t h e i r f u n c t i o n . They a l s o o f f e r o p p o r t u n i t i e s to more r e a d i l y i n c o r p o r a t e the c a p a c i t y to produce s p e c i f i c a l l e l o c h e m i c a l s into given plants for a desired e f f e c t . One a s p e c t o f g e n e t i c e n g i n e e r i n g t h a t i s not y e t r e s o l v e d i s the q u e s t i o n o f a p p r o p r i a t e p r e c a u t i o n s and g u i d e l i n e s f o r recombinant DNA e x p e r i m e n t s . In p a r t i c u l a r , t h e r e i s c o n t i n u i n g d i a l o g r e g a r d i n g the r e l e a s e o f p r o d u c t s of recombinant DNA i n t o the environment. D i s c u s s i o n s at the symposium on e n g i n e e r e d organisms i n the environment o r g a n i z e d by the American S o c i e t y f o r M i c r o b i o l o g y l a s t June and the r e c e n t exchange o f l e t t e r s between W. J . B r i l l O ) and R. K. C o l w e l l (4_) p r o v i d e a good sense of the concerns and r e s p o n s e s t for a g r i c u l t u r e broadly f o r a p p l i c a t i o n o f a l l e l o c h e m i c a l r e s e a r c h r e s u l t s would be a f f e c t e d . Because much o f the r e l e a s e i n q u e s t i o n i s o f d i r e c t c o n c e r n to a g r i c u l t u r e , the a g r i c u l t u r a l r e s e a r c h community has developed a proposed assessment system t h a t would supplement e x i s t i n g r e g u l a t o r y and o v e r s i g h t programs. It would i n s u r e t h a t adequate s a f e t y and e n v i r o n m e n t a l c o n s i d e r a t i o n s are f u l l y addressed b e f o r e m o d i f i e d organisms a r e r e l e a s e d i n t o the open e n v i r o n m e n t . Before I get i n t o the d e t a i l s of the p r o p o s a l , I want to r e v i e w the background of how we got where we a r e t o d a y . There has been f o r many y e a r s a v e r y h e a l t h y , open debate on e t h i c s and s a f e t y q u e s t i o n s r e l a t e d to experiments i n v o l v i n g recombinant DNA. S i n c e i t s c r e a t i o n i n 1974, the N a t i o n a l I n s t i t u t e s o f H e a l t h Recombinant DNA A d v i s o r y Committee (NIH-RAC) has been the p r i m a r y forum f o r t h i s d e b a t e . Guidelines for recombinant DNA r e s e a r c h developed by the NIH-RAC became b i n d i n g on a l l F e d e r a l l y s u p p o r t e d r e s e a r c h and i n p r a c t i c e have guided a l l r e s e a r c h i n the U n i t e d S t a t e s . In the USDA, an A g r i c u l t u r a l Recombinant DNA R e s e a r c h Committee (ARRC) has been i n p l a c e f o r many y e a r s i n c l o s e a s s o c i a t i o n w i t h the NIH-RAC and has a s s i s t e d the NIH-RAC i n i t s h a n d l i n g of a g r i c u l t u r a l l y r e l a t e d p r o p o s a l s . The NIH-RAC g u i d e l i n e s have e v o l v e d as new knowledge has become a v a i l a b l e and they have served the c o u n t r y w e l l . However, r e s e a r c h and e x p e r i m e n t a t i o n have p r o g r e s s e d from c o n t a i n e d f a c i l i t i e s to the p o i n t t h a t t r i a l s i n the open environment are a p p r o p r i a t e and p r o d u c t s f o r commercial use are at hand. For a c o o r d i n a t e d approach t o the r e g u l a t i o n and o v e r s i g h t o f recombinant DNA p r o d u c t s and a c t i v i t i e s , a government-wide C a b i n e t C o u n c i l Working Group on B i o t e c h n o l o g y was e s t a b l i s h e d t h r o u g h the P r e s i d e n t ' s O f f i c e o f S c i e n c e and Technology P o l i c y . The Working Group p r e p a r e d a p r o p o s a l f o r a c o o r d i n a t e d framework f o r r e g u l a t i o n of b i o t e c h n o l o g y t h a t was p u b l i s h e d i n the F e d e r a l R e g i s t e r (5) f o r p u b l i c comment. A r e v i s e d d r a f t t a k i n g i n t o account the v a r i o u s r e a c t i o n s and comments i s i n p r e p a r a t i o n . I p o i n t out these a c t i v i t i e s to l e t you know t h a t we are working w i t h i n a c o o r d i n a t e d o v e r a l l framework. Now I want to go back to the p r o p o s a l t h a t i s p a r t o f our b i o t e c h n o l o g y i n i t i a t i v e and g i v e some d e t a i l s on the proposed

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

6

ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY assessment system and what i t would a c c o m p l i s h . The proposed system i s r e f e r r e d t o as the N a t i o n a l B i o l o g i c a l Impact Assessment Program. It w i l l not assume a r e g u l a t o r y r o l e , but i s i n t e n d e d to complement e x i s t i n g r e g u l a t o r y and o v e r s i g h t a c t i v i t i e s . It i s a r e s e a r c h - b a s e d program, u s i n g the e x i s t i n g n a t i o n a l network of e x p e r t i s e and e x p e r i e n c e i n l o n g - t e r m assessments o f a g r i c u l t u r a l biota. And the e x i s t i n g n a t i o n a l network I am r e f e r r i n g to i s the S t a t e A g r i c u l t u r a l Experiment S t a t i o n System and the f i e l d l o c a t i o n s o f the A g r i c u l t u r a l R e s e a r c h S e r v i c e . The program w i l l e v a l u a t e the impacts o f b i o l o g i c a l changes o c c u r r i n g i n our N a t i o n , i n c l u d i n g the needs brought on by the new b i o t e c h n o l o g y r e s e a r c h i n a g r i c u l t u r a l systems. C u r r e n t l y , we a s s e s s the impact o f p l a n t , a n i m a l , and m i c r o b i a l b i o t a i n a g r i c u l t u r e i n a wide range of c r o p p i n g and animal p r o d u c t i o n systems. T h i s i s done through growth chambers, greenhouse s t u d i e s , and r e s e a r c h p l o t s i n the f i e l d . In the case o f a l l major c r o p s , a wide range of p r o c e s s e s are used i n which new v a r i e t i e s and s p e c i e s are a s s e s s e d f o r t h e i r i m p a c t . The need to know th our N a t i o n has i n c r e a s e recombinant DNA and c l o s e l y r e l a t e d t e c h n i q u e s i n the e a r l y 1 9 7 0 s . It a l s o has changed c o n s i d e r a b l y i n c h a r a c t e r i s t i c s s i n c e those discoveries. On the one hand, recombinant DNA t e c h n i q u e s r e q u i r e e n t i r e l y new concepts of a s s e s s m e n t , w h i l e on the o t h e r they p r o v i d e w h o l l y new t o o l s f o r a s s e s s m e n t s . A r e s e a r c h - b a s e d system i s e s s e n t i a l f o r s u c c e s s f u l involvement o f the e x p e r t i s e , f a c i l i t i e s , and i n s t r u m e n t a t i o n t h a t are now a v a i l a b l e i n the s c i e n c e and e d u c a t i o n system n a t i o n a l l y . F o r t u n a t e l y , the emergence of t h i s need can be s u p p o r t e d by a r a p i d l y growing system of new h i g h - s p e e d e l e c t r o n i c assessment and p r o c e s s i n g t e c h n i q u e s , which are e s s e n t i a l t o the development and a p p l i c a t i o n o f b i o t e c h n o l o g y i n a g r i c u l t u r e and f o r e s t r y . A g a i n , I want to emphasize t h a t the N a t i o n a l B i o l o g i c a l Impact Assessment Program w i l l not assume a r e g u l a t o r y role. However, i t w i l l p r o v i d e e x p e r t i s e and t e c h n i c a l backup to the r e g u l a t o r y a g e n c i e s . For m a t e r i a l s produced by the modern b i o t e c h n o l o g i e s such as recombinant DNA, t h e r e w i l l be a n a t i o n a l system o f p r o j e c t s f o r a s s e s s i n g the p o t e n t i a l impact of these m a t e r i a l s i n a s t e p - b y - s t e p manner p r i o r to t h e i r r e l e a s e i n t o the e n v i r o n m e n t . We w i l l draw h e a v i l y on the NIH-RAC g u i d e l i n e s and the l o c a l i n s t i t u t i o n a l b i o s a f e t y committees. The program w i l l o p e r a t e as a h i g h l y p a r t i c i p a t o r y system t h a t w i l l a p p r a i s e b i o l o g i c a l changes o f importance t o a g r i c u l t u r e , f o r e s t r y , and n a t u r a l r e s o u r c e s . There w i l l be c o n t i n u o u s c y c l i n g o f d a t a as assessments are made. The program w i l l m a i n t a i n a r o s t e r o f knowledgeable s c i e n t i s t s to a s s i s t i n r e v i e w i n g b i o t e c h n o l o g y p r o p o s a l s f o r s a f e t y and e f f i c a c y , i n f o r m a t i o n on s a f e t e s t s i t e s , and an i n v e n t o r y and ongoing assessment of m a t e r i a l s under t e s t . The t h i n g s I have been t a l k i n g about are a l l p a r t o f the " b i g p i c t u r e " f o r the food and a g r i c u l t u r a l s c i e n c e s . In r e c e n t y e a r s we have devoted more a t t e n t i o n to s t a n d i n g back from time to time to view the l a r g e r p i c t u r e so t h a t we can expect to do a b e t t e r j o b of i d e n t i f y i n g the c r i t i c a l elements of the system and t h e r e b y " f i t " them t o g e t h e r i n t o an e f f e c t i v e comprehensive program. This i n v o l v e s b e t t e r a r t i c u l a t i o n among the F e d e r a l - S t a t e p a r t n e r s and f

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1.

BENTLEY

The Potential of

Allelochemicals

7

the p r i v a t e s e c t o r r e s e a r c h . Much more needs to be done. The Congress o f the U n i t e d S t a t e s encouraged us i n t h i s d i r e c t i o n and i n the 1977 Farm B i l l c a l l e d f o r e s t a b l i s h m e n t o f a J o i n t C o u n c i l on Food and A g r i c u l t u r a l S c i e n c e s to f o s t e r c o o r d i n a t i o n o f the r e s e a r c h , e x t e n s i o n , and t e a c h i n g a c t i v i t i e s o f a l l p u b l i c and p r i v a t e o r g a n i z a t i o n s and i n d i v i d u a l s . The 1981 Farm B i l l c a l l e d on the J o i n t C o u n c i l to do a comprehensive needs assessment f o r food and a g r i c u l t u r a l s c i e n c e s , which was p u b l i s h e d i n J a n u a r y 1984.(6) The 1981 Farm B i l l a l s o c a l l e d f o r an annual p r i o r i t i e s r e p o r t to promote c o o r d i n a t i o n and j o i n t p l a n n i n g . The e d i t i o n f o r FY 1987 (7) l i s t s the f o l l o w i n g broad p r i o r i t i e s : 1. 2. 3. 4. 5.

I n c r e a s e a g r i c u l t u r a l p r o f i t a b i l i t y t h r o u g h management. Improve water q u a l i t y and management. Expand b i o t e c h n o l o g y e f f o r t s on p l a n t s , a n i m a l s , and m i c r o b e s . Develop n e c e s s a r y s c i e n t i f i c and p r o f e s s i o n a l human c a p i t a l . Improve human n u t r i t i o n and the u n d e r s t a n d i n g o f d i e t / h e a l t h relationships.

B r i n g i n g the e n t i r e system t o g e t h e r i n a s e t of p r i o r i t i e s p r o v i d e s a framework f o r a l l o f us and demonstrates t o our s u p p o r t e r s t h a t we can make statements about what we t h i n k i s i m p o r t a n t .

Literature Cited 1. 2. 3. 4. 5. 6. 7.

Balandrin, M. F.; Klocke, J. Α.; Wurtele, E. S.; Bollinger, W. H. Science 1985, 228 (4704), 1154-60. Putnam, A. R. Chemical and Engineering News 1983, 61, 34-45. Brill, W. J. Science 1985, 229 (4709), 115-17. Colwell, R. K.; Norse, Ε. Α.; Pimentel, D.; Sharpies, F. E.; Simberloff, D. Science 1985, 229 (4709), 111-12. Federal Register 1984, 49 (252), 50856-50907. "Summary: Needs Assessment for the Food and Agricultural Sciences," Joint Council on Food and Agricultural Sciences, U.S. Department of Agriculture, January 1984. "FY 1987 Priorities for Research, Extension, and Higher Education," Joint Council on Food and Agricultural Sciences, U.S. Department of Agriculture, June 1985.

RECEIVED December 17,1985

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 2

Allelopathy: An Overview Elroy L. Rice

Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019

Our increasing knowledge of allelopathy is aiding greatly in our understanding of many ecological phenomena. Our increasing awareness of conditions under which certain crop residues cause allelopathic effects on subsequent crops should enable us soon to guard against detrimental effects and to manage rotatio tak advantag f stimulator effects. Availabl possible, through breeding/o biotechnology, p p cultivars that will inhibit growth of the chief weeds in a given area through allelopathic action and thus decrease the need for synthetic weed killers. We are already able to use allelopathic companion crops or residues of allelopathic crop plants and weeds to control weed growth in some crops and orchards. Our understanding of allelopathic interactions between various plant species has been used advantageously in reforestation, and future developments are encouraging. Considerable information is available concerning types of chemicals involved in allelopathy, and some information is available concerning movement of the chemicals from plants and factors determining their effectiveness after egression from plants. Nevertheless, these areas of allelopathy are probably the ones that merit the strongest research emphasis in the near future. T h e o p h r a s t u s (1), a b o u t 3 0 0 B . C . , s t a t e d t h a t c h i c k p e a ( C i c e r a r i e t i n u m ) does not r e i n v i g o r a t e t h e ground as other r e l a t e d p l a n t s (legumes) do b u t " e x h a u s t s " i t instead. H e pointed out also that c h i c k p e a destroys weeds. P l i n y (2) r e p o r t e d i n t h e 1st c e n t u r y A . D . t h a t c h i c k p e a , b a r l e y ( H o r d e u m vulgare), fenugreek (Trigonella foenum-graecum), a n d bitter vetch (Vicia ervilia) all "scorch up" cornland. In s p i t e o f t h e e a r l y s u g g e s t i o n s c o n c e r n i n g a p p a r e n t a l l e l o p a t h i c e f f e c t s , no s o l i d s c i e n t i f i c e v i d e n c e w a s o b t a i n e d t o s u p p o r t t h e s u g g e s t i o n s until t h e present c e n t u r y . T h e t e r m a l l e l o p a t h y w a s c o i n e d by M o l i s c h i n 1937 t o r e f e r t o b i o c h e m i c a l i n t e r a c t i o n s b e t w e e n a l l t y p e s o f p l a n t s , i n c l u d i n g m i c r o o r g a n i s m s t r a d i t i o n a l l y p l a c e d i n t h e p l a n t k i n g d o m (3). H i s discussion i n d i c a t e d that he meant the t e r m to cover both inhibitory a n d stimulatory biochemical interactions. 0097-6156/87/0330-0008$06.00/0 © 1987 A m e r i c a n C h e m i c a l Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2.

RICE

Allelopathy:

An

Overview

A v e r y i m p o r t a n t p o i n t c o n c e r n i n g a l l e l o p a t h y is t h a t i t s e f f e c t d e p e n d s o n a c h e m i c a l c o m p o u n d b e i n g a d d e d t o t h e e n v i r o n m e n t . It is t h u s s e p a r a t e d f r o m c o m p e t i t i o n w h i c h i n v o l v e s the r e m o v a l or r e d u c t i o n of s o m e f a c t o r f r o m t h e e n v i r o n m e n t t h a t is r e q u i r e d by s o m e o t h e r p l a n t o r m i c r o o r g a n i s m sharing the habitat. M u l l e r (4) s u g g e s t e d the term interference to refer to the overall influence of one plant (or m i c r o o r g a n i s m ) on a n o t h e r . I n t e r f e r e n c e would thus encompass both allelopathy and c o m p e t i t i o n . E v i d e n c e i n d i c a t e s t h a t a l l e l o p a t h i c c o m p o u n d s get out of plants by v o l a t i l i z a t i o n , e x u d a t i o n f r o m r o o t s , l e a c h i n g f r o m plants or residues by r a i n , or d e c o m p o s i t i o n o f r e s i d u e s (5). The goals of this paper are to discuss some of the major g e n e r a l i z a t i o n s t h a t c a n be m a d e a b o u t a l l e l o p a t h i c i n t e r a c t i o n s , p r o v i d e s o m e e x a m p l e s of suggested r o l e s , a n d focus on d e s i r a b l e f u t u r e r e s e a r c h applications. O n l y a few of the p e r t i n e n t i n v e s t i g a t i o n s are c i t e d in i l l u s t r a t i n g major p r i n c i p l e s . Allelopathy in Plant Patholog Spores of most p a r a s i t i c fungi r e m a i n ungerminated while l o c a t e d in their s i t e o f p r o d u c t i o n (6). T h i s c a n be due t o s e v e r a l f a c t o r s , o n e o f w h i c h i s p r o d u c t i o n by t h e s p o r e s o f f u n g i s t a t i c a g e n t s t h a t a r e e x c r e t e d i n t o t h e w a t e r around the spores. These s e l f - i n h i b i t o r s generally assure dispersal of viable ungerminated spores. Endogenous germination stimulators that c o u n t e r a c t i n h i b i t i o n by s e l f - i n h i b i t o r s o c c u r i n m a n y s p o r e s . N o n a n a l a n d 6 - m e t h y l - 5 - h e p t e n - 2 - o n e w e r e i s o l a t e d f r o m uredospores of U r o m y c e s and P u c c i n i a (7). T h e s e c o m p o u n d s s t i m u l a t e g e r m i n a t i o n of s t e m rust spores that contain m e t h y l f e r u l a t e as t h e i r i n h i b i t o r , b u t t h e y do n o t s t i m u l a t e g e r m i n a t i o n o f s p o r e s t h a t c o n t a i n a d i m e t h o x y c i n n a m a t e as i n h i b i t o r . M o s t p a r a s i t e s have to s u r v i v e prolonged periods of t i m e a p a r t f r o m the host p l a n t . C o n s e q u e n t l y , the f o r m a t i o n of r e s t i n g p r o p a g u l e s , such as s c l e r o t i a , c o n s t i t u t e s a c r i t i c a l p a r t of the p a r a s i t e ' s l i f e c y c l e . Several o b s e r v a t i o n s i n d i c a t e t h a t f o r m a t i o n o f s c l e r o t i a m a y be s t i m u l a t e d b y a l l e l o c h e m i c a l s (8,9). B r a n d t a n d R e e s e (10) c o n c l u d e d t h a t V e r t i c i l l i u m dahliae produces a diffusible morphogenetic factor that stimulates p r o d u c t i o n of m i c r o s c l e r o t i a . When low c o n c e n t r a t i o n s of the diffusible f a c t o r w e r e added to c u l t u r e s of the p a t h o g e n , the hyphae s w e l l e d and became c o n s t r i c t e d , septation was increased, and c e l l walls became thickened. K e r r (11) g r e w s t e r i l e s e e d l i n g s i n s i d e c e l l o p h a n e b a g s b u r i e d i n s o i l i n o c u l a t e d w i t h P e l l i c u l a r i a f i l a m e n t o s a and found an intense d e v e l o p m e n t o f t h e p a t h o g e n on t h e c e l l o p h a n e o p p o s i t e t h e r o o t s o f t h e t w o s u s c e p t i b l e h o s t s , l e t t u c e a n d r a d i s h , b u t no s t i m u l a t i o n o p p o s i t e t o m a t o r o o t s , w h i c h a r e n o t s u s c e p t i b l e . B u x t o n (12) a l s o d e m o n s t r a t e d a d e f i n i t e s p e c i f i c i t y i n r e l a t i o n t o t h e g e r m i n a t i o n o f s p o r e s o f F u s a r i u m o x y s p o r u m f. p i s i i n t h e e x u d a t e s of t h r e e pea v a r i e t i e s d i f f e r i n g in s u s c e p t i b i l i t y to this p a t h o g e n . E x u d a t e f r o m a w i l t - r e s i s t a n t v a r i e t y i n h i b i t e d spore g e r m i n a t i o n , whereas exudate from a susceptible plant stimulated such germination. In s o i l t h a t h a s n o t h a d r e c e n t a d d i t i o n s o f p l a n t r e s i d u e o r o t h e r organic m a t e r i a l , m i c r o b i a l respiration proceeds at a low rate (13). M o r e o v e r , f u n g i a p p a r e n t l y e x i s t m o s t l y as s p o r e s i n a s t a t e o f f u n g i s t a s i s . T h i s m i c r o f l o r a u s u a l l y r e s p o n d s t o t h e a d d i t i o n o f p l a n t r e s i d u e by s p o r e germination, increased respiration, and growth. These responses w e r e i n d u c e d by v o l a t i l e c o m p o n e n t s f r o m a l f a l f a t o p s , c o r n l e a v e s , w h e a t s t r a w , bluegrass clippings, tea leaves, and tobacco leaves, even when the residue

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was s e p a r a t e d f r o m the soil by a 5 - c m a i r gap. T h e r e was a r a p i d o u t g r o w t h of hyphae f r o m the soil surface t o w a r d the residue before any g r o w t h of f u n g i c o u l d be s e e n i n t h e p l a n t m a t e r i a l . V a p o r s f r o m d i s t i l l a t e s o f w a t e r e x t r a c t s of the various plant residues m e n t i o n e d had s i m i l a r e f f e c t s on g r o w t h of fungi and m a r k e d l y i n c r e a s e d n u m b e r s of b a c t e r i a and the respiratory rate of m i c r o o r g a n i s m s in soil samples. W i t c h w e e d ( S t r i g a a s i a t i c a ) is a n e c o n o m i c a l l y i m p o r t a n t r o o t p a r a s i t e affecting many warm-season grasses, including such important crop plants as c o r n , g r a i n s o r g h u m , a n d s u g a r c a n e . V i a b l e w i t c h w e e d s e e d s m a y r e m a i n d o r m a n t i n t h e s o i l f o r m a n y y e a r s (l*f). The seeds w i l l usually not g e r m i n a t e unless p r e t r e a t e d in a w a r m , m o i s t e n v i r o n m e n t for several days before exposure to a c h e m i c a l c o m p o u n d exuded f r o m the roots of a host p l a n t or s o m e non-host p l a n t s . O n e such c o m p o u n d , s t r i g o l , was i s o l a t e d f r o m t h e r o o t e x u d a t e o f c o t t o n a n d h a s p r o v e d t o be a p o w e r f u l s t i m u l a n t o f w i t c h w e e d s e e d g e r m i n a t i o n . J o h n s o n , R o s e b e r y a n d P a r k e r (15) r e p o r t e d the synthesis and t e s t i n g of s e v e r a l analogs of s t r i g o l , and some w e r e powerful seed g e r m i n a t i o n s t i m u l a n t s for species of b o t h S t r i g a and O r o b a n c h e . C o t t o n is n o noteworthy that the structure t h e r o o t s o f t h e h o s t p l a n t s r e m a i n u n k n o w n (16). T h e h a u s t o r i a o f t h e p a r a s i t e s do n o t f o r m w h e n t h e p l a n t s a r e g r o w n a x e n i c a l l y , but are rapidly i n d u c e d in the presence of the host roots or host r o o t e x u d a t e s (17). S e v e r a l h a u s t o r i a l - i n d u c i n g compounds have now been c h a r a c t e r i z e d . Xenognosin A and Β were identified in gum t r a g a c a n t h , an e x u d a t e of A s t r a g a l u s g u m m i f e r , a n d s o y a s a p o g e n o l Β w a s i d e n t i f i e d i n r o o t s of L e s p e d e z a sericea. A l l e l o p a t h y in N a t u r a l Ecosystems P a t t e r n i n g of v e g e t a t i o n . C u r t i s a n d C o t t a m (18) o b s e r v e d t h e f a i r y - r i n g p a t t e r n o f t h e p r a i r i e s u n f l o w e r H e l i a n t h u s r i g i d u s , w h i c h is due t o a pronounced r e d u c t i o n in plant numbers, s i z e , and i n f l o r e s c e n c e s in the c e n t e r of the c l o n e . T h e y subsequently d e m o n s t r a t e d t h a t the p a t t e r n was due to a u t o t o x i n s p r o d u c e d by d e c a y of dead parts of the s u n f l o w e r . Prostrate knotweed, Polygonum aviculare, rapidly encroaches into b e r m u d a g r a s s lawns and the grass dies in p a t c h e s of p r o s t r a t e k n o t w e e d w h i l e b e r m u d a g r a s s at the edges of the k n o t w e e d patches turns y e l l o w . Soil minus l i t t e r was c o l l e c t e d under a P . a v i c u l a r e stand and under a b e r m u d a g r a s s stand and used to g r o w b e r m u d a g r a s s (19,20). S o i l c o l l e c t e d i n M a r c h under k n o t w e e d m a r k e d l y i n h i b i t e d seed g e r m i n a t i o n and seedling g r o w t h of bermudagrass c o m p a r e d w i t h soil f r o m under b e r m u d a g r a s s . D e c a y i n g roots and shoots of p r o s t r a t e k n o t w e e d reduced seed g e r m i n a t i o n and seedling g r o w t h of bermudagrass. A d d i t i o n a l l y , root exudates of k n o t w e e d r e d u c e d s e e d l i n g g r o w t h of b e r m u d a g r a s s . E l e v e n a l l e l o c h e m i c a l s i n h i b i t o r y to g r o w t h o f b e r m u d a g r a s s w e r e i s o l a t e d f r o m s o i l u n d e r p r o s t r a t e k n o t w e e d , w h e r e a s none of these o c c u r r e d in soil under b e r m u d a g r a s s (20,21). F o u r w e r e p h e n o l i c s and seven w e r e l o n g - c h a i n f a t t y a c i d s . V e g e t a t i o n under the trees in Japanese red pine, Pinus d e n s i f l o r a , f o r e s t s is s p a r s e d e s p i t e t h e f a c t t h a t t h e i n t e r i o r o f t h e s e f o r e s t s is o n e o f the b r i g h t e s t a m o n g f o r e s t s (22,23). Many other forests have dense undergrowths of herbs in spite of m u c h l o w e r l i g h t i n t e n s i t i e s . V a r i o u s p a r t s of red pine and the soil under i t c o n t a i n e d c h e m i c a l s t o x i c to m a n y p o t e n t i a l understory plants. Thus, it was concluded that allelopathy probably plays an i m p o r t a n t role in r e t a r d i n g understory g r o w t h .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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L y c o r i s r a d i a t a is a d o m i n a n t s p e c i e s a l o n g r o a d s i d e s a n d s l o p e s i n J a p a n , and i t appears to p r e v e n t some other plant species f r o m e m e r g i n g a n d g r o w i n g n e a r i t (24). U e k i and T a k a h a s h i found t h a t the bulbs of L . r a d i a t a exuded two a l l e l o c h e m i c a l s that m a r k e d l y reduced root growth of several weedy species usually o c c u r r i n g in the same general areas w i t h L y c o r i s . M o r e o v e r , the same compounds w e r e found in the soil adjacent to Lycoris bulbs. P l a n t s u c c e s s i o n . In t h e t a l l g r a s s p r a i r i e r e g i o n o f O k l a h o m a a n d K a n s a s , there are four m a i n successional stages when fields that are i n f e r t i l e are abandoned f r o m c u l t i v a t i o n : a pioneer w e e d stage t h a t persists for only 2-3 y e a r s , a n a n n u a l - g r a s s s t a g e t h a t l a s t s f o r 9 t o 13 y e a r s , a p e r e n n i a l b u n c h g r a s s s t a g e t h a t r e m a i n s f o r 30 y e a r s or l o n g e r a f t e r a b a n d o n m e n t , a n d t h e c l i m a x p r a i r i e (25). T h e e v i d e n c e is s t r o n g t h a t t h e p i o n e e r w e e d s t a g e disappears rapidly because the species are e l i m i n a t e d through strong a l l e l o p a t h i c i n t e r a c t i o n s (5). A r i s t i d a o l i g a n t h a , p r a i r i e t h r e e a w n , the d o m i n a n t o f t h e s e c o n d s t a g e , i n v a d e s n e x t a p p a r e n t l y b e c a u s e i t is n o t i n h i b i t e d by the a l l e l o c h e m i c a l t h a t is s t i l l t o o l o w i n n i t r o g e s u c c e s s i o n (26). A . oligantha and several pioneer species produce allelochemicals that inhibit g r o w t h of R h i z o b i u m and f r e e - l i v i n g n i t r o g e n - f i x i n g organisms, and n o d u l a t i o n a n d h e m o g l o b i n f o r m a t i o n i n l e g u m e s (5). T h i s i n d i r e c t e v i d e n c e suggested that b i o l o g i c a l nitrogen f i x a t i o n was slowed in the first two stages o f s u c c e s s i o n . K a p u s t k a a n d R i c e (27) m e a s u r e d n i t r o g e n f i x a t i o n r a t e s i n soils of the pioneer w e e d stage, the a n n u a l grass stage, and the c l i m a x p r a i r i e using the a c e t y l e n e r e d u c t i o n t e c h n i q u e . The rate was about four t i m e s as h i g h i n t h e c l i m a x s o i l as i n t h e p i o n e e r w e e d s t a g e a n d a b o u t f i v e t i m e s as h i g h i n t h e c l i m a x as i n t h e a n n u a l g r a s s s t a g e , t h u s s u b s t a n t i a t i n g the i n d i r e c t evidence. T h e s l o w i n g of n i t r o g e n f i x a t i o n i n the f i r s t t w o successional stages probably gives A r i s t i d a oligantha a selective advantage in c o m p e t i t i o n w i t h species h a v i n g higher n i t r o g e n r e q u i r e m e n t s and causes it to r e m a i n for a l e n g t h y p e r i o d . T h e r e is a s t r o n g e v i d e n c e t h a t n i t r i f i c a t i o n is s l o w e d i n t h e l a t e r s t a g e s o f s u c c e s s i o n c a u s i n g a v a i l a b l e n i t r o g e n t o be p r e s e n t c h i e f l y a s a m m o n i u m n i t r o g e n (28,29,30). T h i s should help to conserve nitrogen b e c a u s e t h e a m m o n i u m i o n is a d s o r b e d by t h e n e g a t i v e l y c h a r g e d m i c e l l e s i n t h e s o i l a n d is n o t r e a d i l y l e a c h e d b e l o w t h e d e p t h o f r o o t i n g o r w a s h e d away into streams. T h e r e is e v i d e n c e that tannins, phenolic acids, f l a v o n o i d s , a n d c o u m a r i n s m a y be i m p o r t a n t i n h i b i t o r s o f n i t r i f i c a t i o n (30, 31). The nitrogen c o n c e n t r a t i o n gradually increases to the point where some l a t e r species c a n i n v a d e . T h i s a p p a r e n t l y results in less i n h i b i t i o n of n i t r o g e n f i x a t i o n and m o r e i n h i b i t i o n of n i t r i f i c a t i o n . T h u s , the rate of a d d i t i o n o f n i t r o g e n is i n c r e a s e d a n d t h e r a t e of l o s s o f n i t r o g e n is decreased. E v e n t u a l l y t h e c o n c e n t r a t i o n o f n i t r o g e n is i n c r e a s e d t o t h e point where c l i m a x species can invade. U r b a n i z a t i o n a r o u n d l a r g e c i t i e s in J a p a n has g r e a t l y changed the p l a n t c o m m u n i t i e s because of the c r e a t i o n of bare areas or serious d i s t u r b a n c e of n a t u r a l e c o s y s t e m s (32-34). W e e d s u c c e s s i o n on urban w a s t e l a n d is s i m i l a r t o t h a t i n o l d - f i e l d s i n s o m e p a r t s o f t h e U . S . A . w i t h A m b r o s i a a r t e m i s i i f o l i a b e i n g t h e f i r s t - y e a r d o m i n a n t f o l l o w e d by S o l i d a g o a l t i s s i m a a n d E r i g e r o n s p p . f o r a f e w y e a r s , a n d n e x t by M i s c a n t h u s s i n e n s i s . N u m a t a a n d his c o l l e a g u e s f o u n d t h a t S . a l t i s s i m a a n d E r i g e r o n a n n u u s b o t h p r o d u c e p o l y a c e t y l e n i c m e t h y l e s t e r s (one i n S o l i d a g o a n d t h r e e i n E .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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a n n u u s ) t h a t i n h i b i t s e e d g e r m i n a t i o n o f A . a r t e m i s i i f o l i a , M. s i n e n s i s a n d a species of T a g e t e s , and g r o w t h of r i c e seedlings. T h e p h y t o t o x i n found i n Solidago was also e x t r a c t e d f r o m soil in a stand of the species, and the concentration present was sufficient to regulate germination and growth of associated species. A v e r y d i l u t e s o l u t i o n (5 p p m ) o f t h r e e o f t h e phytotoxins inhibited growth of A . a r t e m i s i i f o l i a in soil. K o b a y a s h i e t a l . (35) f o u n d t h a t t h e r o o t s o f S o l i d a g o a l t i s s i m a c o n t a i n 250-400 p p m o f t h e C . - p o l y a c e t y l e n e , c i s - d e h y d r o m a t r i c a r i a e s t e r ( c i s D M E ) . T h e y found t h a r soil under a stand of the Solidago c o n t a i n e d 6 ppm of c i s - D M E plus t r a n s - D M E . B o t h compounds were found to i n h i b i t g r o w t h of rice seedlings. Three C . «-polyacetylenes were i d e n t i f i e d f r o m m e t h a n o l extracts of Erigeron annuus, E . canadensis, E . floribundus, and E . philadelphicus. These were the c i s - and t r a n s - m a t r i c a r i a ester and the c i s l a c h n o p h y l l u m ester. A l l w e r e i n h i b i t o r y to seed g e r m i n a t i o n of A m b r o s i a a r t e m i s i i f o l i a a n d s e e d l i n g g r o w t h of r i c e a t a c o n c e n t r a t i o n of 5 p p m or above. Kobayashi et a l . concluded that the dominance of Solidago altissima a n d E r i g e r o n s p p . i n t h e s e c o n d s t a g e o f s e c o n d a r y s u c c e s s i o n is p r o b a b l y due t o t h e i r p r o d u c t i o n o growth of many other plan s h o r t p e r i o d o f o c c u p a t i o n by S. a l t i s s i m a a n d E r i g e r o n s p p . m a y be a consequence of the a c c u m u l a t i o n of such polyacetylenes in the soil to the point where they are t o x i c to these species also. n

Allelopathy in Manipulated Ecosystems A l l e l o p a t h y i n f o r e s t r y . W a l t e r s a n d G i l m o r e (36) n o t e d t h a t h e i g h t g r o w t h of s w e e t g u m , L i q u i d a m b a r s t y r a c i f l u a , w a s less i n plots c o n t a i n i n g f e s c u e , F e s t u c a arundinacea, than in adjacent plots without fescue. C h e m i c a l and physical soil factors did not appear to explain the differences. G r o w t h of sweetgum was c o r r e l a t e d w i t h residual phosphorus and magnesium, but this correlation was achieved across a l l experimental plots without respect to the presence or absence of f e s c u e . S e e d i n g of fescue into pots c o n t a i n i n g sweetgum seedlings resulted in a reduction in dry weight increment of s w e e t g u m u p t o 9 5 % . E l i m i n a t i o n o f c o m p e t i t i o n t h r o u g h use o f a s t a i r s t e p apparatus suggested that an allelopathic mechanism was involved. L e a c h a t e s f r o m the rhizosphere of live fescue, dead fescue roots, and dead f e s c u e l e a v e s c a u s e d r e d u c t i o n s i n d r y w e i g h t i n c r e m e n t s o f s w e e t g u m up t o 6 0 % . C h e m i c a l analysis of sweetgum seedlings from the stairstep experiment suggested that fescue leachates decreased absorption of phosphorus and nitrogen. T u b b s (37) f o u n d t h a t s u g a r m a p l e s e e d l i n g s i n h i b i t e d g r o w t h o f seedlings of yellow birch despite the apparent absence of c o m p e t i t i o n i n nursery e x p e r i m e n t s . R o o t e l o n g a t i o n of b i r c h w a s r e t a r d e d by exudates of a c t i v e l y g r o w i n g root tips of sugar m a p l e . When seedlings of these species were grown together in aerated nutrient solution, the number of actively growing root tips of b i r c h f o r m e d each day w a s inversely c o r r e l a t e d w i t h the a c t i v i t y o f t h e a l l e l o c h e m i c a l p r o d u c e d b y m a p l e , as i n d i c a t e d b y t h e retardation of elongation of yellow birch roots. A l d e r species are often i m p o r t a n t in forests because of the f i x a t i o n of n i t r o g e n by F r a n k i a i n nodules on t h e i r roots. J o b i d o n a n d T h i b a u l t (38) observed growth depression of alders near balsam poplar, Populus b a l s a m i f e r a , stands. Water e x t r a c t s of leaf l i t t e r and buds, and fresh leaf l e a c h a t e s of b a l s a m poplar i n h i b i t e d seed g e r m i n a t i o n and r a d i c l e a n d hypocotyl growth of green alder, Alnus crispa v a r . mollis, seedlings. There was m a r k e d i n h i b i t i o n of root hair d e v e l o p m e n t and necrosis of the r a d i c l e

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m e r i s t e m s . T h e a v e r a g e n u m b e r of nodules on a l d e r p l a n t s t r e a t e d w i t h any one of the three b a l s a m e x t r a c t s d e s c r i b e d a b o v e was o n l y 5 1 % of t h a t of c o n t r o l p l a n t s (39). A c e t y l e n e reduction (nitrogen fixation) was decreased 6 2 % by green a l d e r p l a n t s t r e a t e d w i t h the m o s t c o n c e n t r a t e d bud a n d l e a f litter extracts. C e r t a i n t r e e s p e c i e s s u c h as B e t u l a p e n d u l a a n d P i c e a a b i e s f a i l t o d e v e l o p in a s s o c i a t i o n w i t h h e a t h e r , C a l l u n a v u l g a r i s (40,41). This a p p a r e n t l y r e s u l t s f r o m t h e p r o d u c t i o n by h e a t h e r o f a n a l l e l o c h e m i c a l t o x i c to g r o w t h o f m y c o r r h i z a e o f B e t u l a a n d P i c e a . F r u t i c o s e s o i l l i c h e n s a r e o f t e n a l l e l o p a t h i c to the g r o w t h of m y c o r r h i z a e and f o r e s t t r e e seedlings a l s o (42). R e m o v a l o f r e i n d e e r m o s s (a l i c h e n ) i n f i e l d t e s t s r e s u l t e d i n a c c e l e r a t e d g r o w t h of pine and s p r u c e . A l l e l o p a t h y in a g r i c u l t u r e . S c h r e i n e r and his associates published s e v e r a l p a p e r s s h o r t l y a f t e r 1900 w h i c h i n d i c a t e d t h a t c e r t a i n c r o p p l a n t s p r o d u c e c o m p o u n d s i n h i b i t o r y t o g r o w t h o f t h e s a m e a n d o t h e r c r o p p l a n t s (5). M c C a l l a a n d D u l e y (43,44) r e p o r t e d t h e a l l e l o p a t h i c e f f e c t s o f d e c a y i n g wheat residues in 1948-1949 crop plants have been publishe The unharvested parts of r i c e plants are generally m i x e d w i t h the soil b e c a u s e t h i s h a s b e e n t h o u g h t t o be b e n e f i c i a l . It h a s b e e n observed h o w e v e r , t h a t p r o d u c t i v i t y of the second c r o p of r i c e in a paddy is less than t h a t o f t h e f i r s t c r o p . C h o u a n d L i n (45) f o u n d t h a t a q u e o u s e x t r a c t s o f d e c o m p o s i n g r i c e residues in soil r e t a r d e d r a d i c l e g r o w t h of r i c e seedlings and g r o w t h of rice plants. M a x i m u m t o x i c i t y o c c u r r e d in the first m o n t h of decomposition and declined thereafter. Some t o x i c i t y persisted for four months in the paddies. F i v e inhibitory phenolic acids were identified from decaying rice residues and several unidentified allelochemicals were isolated. In t h e s o u t h e r n p a r t o f T a i w a n , a c r o p o f r i c e is o f t e n followed i m m e d i a t e l y by a l e g u m e c r o p . Y i e l d s o f s o y b e a n s h a v e b e e n i n c r e a s e d b y s e v e r a l h u n d r e d k i l o g r a m s p e r h e c t a r e by b u r n i n g t h e r i c e s t r a w p r i o r t o planting the soybeans. R i c e e t a l . (46) h y p o t h e s i z e d t h a t t h e d e c r e a s e d y i e l d s in unburned f i e l d s m a y r e s u l t f r o m an i n h i b i t i o n of n i t r o g e n f i x a t i o n by R h i z o b i u m i n t h e n o d u l e s o f t h e s o y b e a n p l a n t s . T h e f i v e p h e n o l i c a c i d s i d e n t i f i e d by C h o u a n d L i n a n d s t e r i l e e x t r a c t s o f d e c a y i n g r i c e s t r a w i n s o i l m a r k e d l y i n h i b i t e d g r o w t h of R h i z o b i u m . T h e phenolics also reduced nodule n u m b e r s and h e m o g l o b i n c o n t e n t of the nodules of t w o bean v a r i e t i e s . M o r e o v e r , e x t r a c t s of decomposing rice straw in soil reduced n i t r o g e n f i x a t i o n (acetylene reduction) in Bush B l a c k Seeded beans. It h a s b e e n o b s e r v e d f o r s o m e t i m e i n S e n e g a l i n w e s t A f r i c a t h a t g r o w t h o f s o r g h u m is d e c r e a s e d m a r k e d l y f o l l o w i n g s o r g h u m i n s a n d y s o i l s b u t n o t i n s o i l s h i g h i n m o n t m o r i l l o n i t e (47). S i m i l a r r e s u l t s o c c u r r e d i n t h e g r o w t h of s o r g h u m seedlings w h e n roots or tops of s o r g h u m w e r e added to sandy soils in l a b o r a t o r y e x p e r i m e n t s . N o i n h i b i t i o n r e s u l t e d , h o w e v e r , when the residues w e r e added to soil h i g h in m o n t m o r i l l o n i t e . W a t e r e x t r a c t s of roots or tops r e t a r d e d g r o w t h of sorghum seedlings in sandy soils s i m i l a r l y . I n o c u l a t i o n w i t h T r i c h o d e r m a v i r i d e or an u n k n o w n species of A s p e r g i l l u s e l i m i n a t e d the i n h i b i t o r y e f f e c t s of aqueous e x t r a c t s of sorghum roots in a short t i m e . Several weeks were required, however, to detoxify nonsterile f i e l d s o i l a f t e r a d d i t i o n o f r o o t r e s i d u e s o f s o r g h u m . It w a s c o n c l u d e d t h a t the m i c r o f l o r a in the sandy soils of S e n e g a l w e r e not able to d e t o x i f y the soil fast enough to p r e v e n t i n h i b i t i o n of subsequent crops of s o r g h u m . Some c r o p residues and weeds appear to s t i m u l a t e g r o w t h of other plants. C h o p p e d a l f a l f a added to soil s t i m u l a t e d the g r o w t h of t o m a t o ,

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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cucumber, lettuce, and several other p l a n t s (48). The stimulatory a l l e l o c h e m i c a l w a s i d e n t i f i e d as 1 - t r i a c o n t a n o l . S u b s e q u e n t t e s t s w i t h t h i s c o m p o u n d h a v e given v a r i a b l e r e s u l t s , but a d d i t i o n of c a l c i u m or l a n t h a n u m salts to the t r i a c o n t a n o l s o l u t i o n appears to m a k e the s t i m u l a t o r y a c t i v i t y c o n s i s t e n t (49). A s t e r o i d , b r a s s i n o l i d e , has b e e n i s o l a t e d f r o m rape ( B r a s s i c a n a p u s ) a n d a l d e r ( A l n u s ) p o l l e n (49). O n e nanogram applied to a bean plant causes significant growth increases. Weeds versus crop plants. V e l v e t l e a f , A b u t i l o n t h e o p h r a s t i , is a s e r i o u s w e e d of s e v e r a l c r o p s in the U n i t e d S t a t e s and C a n a d a . A v e r a g e y i e l d r e d u c t i o n s of soybeans under a v a r i e t y of v e l v e t l e a f densities, p l a c e m e n t s , a n d d u r a t i o n o f i n t e r f e r e n c e r a n g e d f r o m 14 t o 4 1 % (50-52). R e d u c t i o n s i n c o t t o n y i e l d s r a n g e d f r o m 44 t o 1 0 0 % ( 5 3 , 5 4 ) . A l l t h e c i t e d r e s e a r c h e r s a t t r i b u t e d the r e d u c t i o n s in c r o p yields to c o m p e t i t i o n a l t h o u g h none p e r f o r m e d e x p e r i m e n t s t o d e t e r m i n e w h e t h e r a l l e l o p a t h y m i g h t be i n v o l v e d . Numerous other researchers have found velvetleaf to have marked a l l e l o p a t h i c p o t e n t i a l (55-58). W a t e r e x t r a c t s of v e l v e t l e a f residues w e r e slightly allelopathic (5-24 c o r n a n d to h y p o c o t y l g r o w t highly a l l e l o p a t h i c (50% or m o r e i n h i b i t i o n ) to height g r o w t h and fresh w e i g h t i n c r e a s e of shoots of b o t h c o r n and soybeans in double pot experiments. P u r p l e n u t s e d g e , C y p e r u s r o t u n d u s , w a s l i s t e d by H o l m (59) as o n e o f t h e t e n w o r s t w e e d s i n t h e w o r l d , a n d i n t e r f e r e n c e by t h i s w e e d c a u s e d r e d u c t i o n s i n y i e l d s of v a r i o u s c r o p s r a n g i n g f r o m 2 3 t o 8 9 % (5). It is n o t e w o r t h y t h e r e f o r e t h a t n u m e r o u s w o r k e r s have found purple nutsedge to b e s t r o n g l y a l l e l o p a t h i c . S o i l p r e v i o u s l y i n f e s t e d w i t h t h i s w e e d f o r 9 t o 12 w e e k s s i g n i f i c a n t l y reduced g e r m i n a t i o n of m u s t a r d , b a r l e y , and c o t t o n seeds; and soil infested for only 6 weeks s i g n i f i c a n t l y reduced g e r m i n a t i o n o f m u s t a r d a n d c o t t o n s e e d s (60). E t h a n o l e x t r a c t s of the p r e v i o u s l y i n f e s t e d s o i l i n h i b i t e d r a d i c l e g r o w t h of b a r l e y a l s o . D e c o m p o s i n g t u b e r s of p u r p l e n u t s e d g e r e d u c e d r o o t a n d t o p g r o w t h o f b a r l e y (61), s o r g h u m (62), a n d s o y b e a n s (62). S o m e p o l y p h e n o l s (63) a n d s e s q u i t e r p e n e s (64,65) w e r e i s o l a t e d f r o m tubers and other parts of purple nutsedge. Seven sesquiterpenoids were i d e n t i f i e d in the s t e a m d i s t i l l a t e of soil in w h i c h purple nutsedge was g r o w i n g (65) a n d t h e s a m e c o m p o u n d s w e r e i s o l a t e d f r o m e s s e n t i a l o i l i n p u r p l e n u t s e d g e (66). S e v e r a l of the compounds i d e n t i f i e d were previously shown to i n h i b i t e l o n g a t i o n of w h e a t c o l e o p t i l e segments in the presence of i n d o l e a c e t i c a c i d a n d s e c o n d l e a f s h e a t h g r o w t h of r i c e s e e d l i n g s i n t h e p r e s e n c e o f g i b b e r e l l i n A - (64). D e c a y i n g g r o u n d - i v y ( G l e c h o m a h e d e r a c e a ) l e a v e s (2 g p e r k g o f s o i l ) m a r k e d l y s t i m u l a t e d both root and shoot growth of downy brome (Bromus t e c t o r u m ) a n d r a d i s h ( R a p h a n u s s a t i v u s ) (67). R a d i s h root g r o w t h was s t i m u l a t e d 1354% in one e x p e r i m e n t . M o r e o v e r , root e x u d a t e s of g r o u n d - i v y s i g n i f i c a n t l y s t i m u l a t e d b o t h r o o t a n d s h o o t g r o w t h of r a d i s h . In f a c t , t h e r o o t s a t t a i n e d t a b l e s i z e i n o n l y 14 d a y s . C r o p plants versus w e e d s . B o t h t h i n and dense f i e l d stands of K e n t u c k y - 3 1 f e s c u e w e r e o b s e r v e d by P e t e r s (68) t o be r e l a t i v e l y f r e e o f w e e d s . E x t r a c t s of fescue, sand c u l t u r e s , and s p l i t - r o o t - s y s t e m e x p e r i m e n t s d e m o n s t r a t e d that fescue produced t o x i c c h e m i c a l s w h i c h exuded f r o m the roots and i n h i b i t e d g r o w t h of w i l d m u s t a r d a n d b i r d s f o o t t r e f o i l . T h r e e t h o u s a n d a c c e s s i o n s of the U S D A c o l l e c t i o n of o a t , A v e n a , g e r m p l a s m were screened for their a b i l i t y to exude s c o p o l e t i n , a c o m p o u n d

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2.

RICE

Allelopathy:

An

Overview

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known to have root-growth-inhibiting properties (69). Twenty-five accessions exuded more blue-fluorescing material (characteristic of scopoletin) f r o m t h e i r roots than a standard oat c u l t i v a r (Garry). Four a c c e s s i o n s e x u d e d u p t o t h r e e t i m e s as m u c h s c o p o l e t i n a s G a r r y o a t s . W h e n o n e o f t h e s e w a s g r o w n i n s a n d c u l t u r e f o r 16 d a y s w i t h a w i l d m u s t a r d , g r o w t h of t h e m u s t a r d w a s s i g n i f i c a n t l y less t h a n t h a t o b t a i n e d when the weed was grown with Garry oats. Moreover, plants grown in close association with the toxic accession were chlorotic, stunted, and twisted i n d i c a t i v e o f c h e m i c a l e f f e c t s r a t h e r t h a n c o m p e t i t i o n . It a p p e a r s p o s s i b l e t h e r e f o r e to breed a l l e l o p a t h i c genes into standard c u l t i v a r s to a i d i n w e e d control. A l l e l o p a t h i c crop plants have already been used e x p e r i m e n t a l l y in weed control. L e a t h e r (70) f o u n d o n e o f t h i r t e e n g e n o t y p e s o f t h e c u l t i v a t e d s u n f l o w e r t e s t e d t o b e v e r y a l l e l o p a t h i c t o s e v e r a l w e e d s . In a 5 year field study w i t h oats and sunflower grown in r o t a t i o n , the weed density was s i g n i f i c a n t l y less than i n c o n t r o l plots w i t h oats only. P u t n a m a n d D e F r a n k (71) t e s t e d r e s i d u e s o f s e v e r a l f a l l - a n d s p r i n g planted crops for weed contro the herbicides glyphosate o B a l b o a r y e r e s i d u e s r e d u c e d w e e d g r o w t h b y up t o 8 8 % . M u l c h e s o f s o r g h u m or s u d a n g r a s s a p p l i e d t o a p p l e o r c h a r d s i n e a r l y s p r i n g r e d u c e d w e e d b i o r n a s s by 9 0 % a n d 8 5 % , r e s p e c t i v e l y . In a 3 - y e a r s e r i e s o f f i e l d t r i a l s , s o r g h u m residues reduced populations of c o m m o n purslane by 7 0 % and of s m o o t h c r a b g r a s s b y 9 8 % (72). C h e m i c a l N a t u r e of A l l e l o p a t h i c C o m p o u n d s A l l e l o p a t h i c compounds consist of a wide variety of c h e m i c a l types w h i c h a r i s e t h r o u g h e i t h e r t h e a c e t a t e o r t h e s h i k i m i c a c i d p a t h w a y (5). T h e s e c o m p o u n d s range f r o m v e r y s i m p l e gases a n d a l i p h a t i c compounds to complex multi-ringed aromatic compounds. Only a few examples are mentioned below. A c e t i c and butyric acids were among the toxins produced during d e c o m p o s i t i o n o f r y e r e s i d u e s (73), a n d s a l t s o f a c e t i c , p r o p i o n i c , a n d b u t y r i c acids were the chief p h y t o t o x i n s produced in d e c a y i n g wheat straw (74). A s i m p l e l a c t o n e , parasorbic a c i d , f r o m the fruit of m o u n t a i n a s h , i n h i b i t s s e e d g e r m i n a t i o n a n d a l s o h a s a n t i b a c t e r i a l a c t i o n (75). Another s u c h c o m p o u n d , p a t u l i n , is p r o d u c e d b y s e v e r a l f u n g i , i n c l u d i n g P é n i c i l l i u m u r t i c a e , w h i c h produced large a m o u n t s of the substance when growing on w h e a t s t r a w (76). L o n g - c h a i n f a t t y a c i d s have l o n g been r e p o r t e d to be i m p o r t a n t a l l e l o c h e m i c a l s p r o d u c e d by a l g a e (77). These compounds were recently r e p o r t e d t o be p o t e n t t o x i n s i n d e c a y i n g r e s i d u e s o f a h i g h e r p l a n t , P o l y g o n u m a v i c u l a r e (21). P o l y a c e t y l e n e s a r e a p p a r e n t l y d e r i v e d f r o m l o n g c h a i n f a t t y a c i d s (78), a n d e v i d e n c e is i n c r e a s i n g t h a t t h e y a r e i m p o r t a n t a l l e l o p a t h i c c o m p o u n d s (35,79). α-Terthienyl p r o d u c e d by roots of m a r i g o l d , Tagetes e r e c t a , caused 5 0 % m o r t a l i t y in seedlings of four test species in c o n c e n t r a t i o n s f r o m 0 . 1 5 t o 1.93 p p m (79). J u g l o n e is t h e o n l y q u i n o n e i d e n t i f i e d as a n a l l e l o p a t h i c c o m p o u n d f r o m h i g h e r p l a n t s (5). It i s p r o d u c e d b y w a l n u t t r e e s a n d is a p o t e n t i n h i b i t o r . N u m e r o u s a n t i b i o t i c s p r o d u c e d by m i c r o o r g a n i s m s a r e quinones, i n c l u d i n g t h e t e t r a c y c l i n e a n t i b i o t i c s s u c h a s a u r e o m y c i n (80). Simple phenols, phenolic acids derived from benzoic a c i d , and phenolic acids derived from c i n n a m i c a c i d have been the most c o m m o n l y identified

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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a l l e l o p a t h i c c o m p o u n d s p r o d u c e d by h i g h e r p l a n t s . The most common a l l e l o p a t h i c compounds identified in soil under a l l e l o p a t h i c plants are £hydroxybenzoic, v a n i l l i c , £-coumaric, and f e r u l i c acids. C o u m a r i n s are l a c t o n e s of o - h y d r o x y c i n n a m i c acids in w h i c h side c h a i n s o f t e n a r e i s o p r e n o i d (78). C o u m a r i n , e s c u l i n , a n d p s o r a l e n (a f u r a n o c o u m a r i n ) a l l strongly i n h i b i t seed g e r m i n a t i o n . Such i n h i b i t o r s are p r o d u c e d by a v a r i e t y of l e g u m e s and c e r e a l g r a i n s . F l a v o n o i d s are widespread in higher plants and a few have been i m p l i c a t e d i n a l l e l o p a t h y . P h l o r i z i n i n a p p l e r o o t s is t o x i c t o y o u n g a p p l e trees and often causes d i f f i c u l t y in replanting old apple orchards. N u m e r o u s f l a v o n o i d s a n d t h e i r g l y c o s i d e s a r e p r o d u c e d by s p e c i e s f r o m t h e t a l l g r a s s p r a i r i e and post oak/blackjack oak forest and are i n h i b i t o r y to n i t r i f y i n g b a c t e r i a a n d t o s e e d g e r m i n a t i o n (31). Several hydrolyzable and condensed tannins have been i m p l i c a t e d in a l l e l o p a t h y (5). T h e y h a v e b e e n i d e n t i f i e d as g r o w t h a n d g e r m i n a t i o n i n h i b i t o r s i n d r y f r u i t s (81), a s g r o w t h r e t a r d e r s of n i t r o g e n - f i x i n g a n d n i t r i f y i n g b a c t e r i a i n s e v e r a l p l a n t s , a n d as r e d u c e r s o f s e e d l i n g g r o w t h i n s e v e r a l p l a n t s (5). Higher plants produc of these have been i m p l i c a t e d in a l l e l o p a t h y . The monoterpenoids are the major c o m p o n e n t s of e s s e n t i a l oils of plants and they are the p r e d o m i n a n t terpenoid inhibitors that have been identified from higher plants. Many f u n g i (82) a n d a l g a e (83) p r o d u c e t e r p e n o i d a l l e l o c h e m i c a l s a l s o . There are only a few instances in w h i c h amino acids have been i m p l i c a t e d in a l l e l o p a t h y and in most cases the s p e c i f i c a m i n o acids have not been i d e n t i f i e d . R h i z o b i t o x i n e is p r o d u c e d b y c e r t a i n s t r a i n s o f R h i z o b i u m j a p o n i c u m a n d is a n o n p r o t e i n a m i n o a c i d (84). S e v e r a l of the p h y t o t o x i n s p r o d u c e d by p a t h o g e n i c m i c r o o r g a n i s m s a r e p o l y p e p t i d e s a n d r e l a t e d g l y c o p e p t i d e s (82). Many alkaloids have been i m p l i c a t e d in p l a n t - a n i m a l c h e m i c a l i n t e r a c t i o n s b u t f e w h a v e b e e n a s s o c i a t e d w i t h a l l e l o p a t h y (85). Several a l k a l o i d s w e r e d e m o n s t r a t e d by E v e n a r i (75) t o be s t r o n g i n h i b i t o r s o f s e e d germination. L i t t l e r e c e n t w o r k has been done on a l k a l o i d s e x c e p t for c a f f e i n e (78). α - P i c o l i n i c a c i d is a m i c r o b i a l a l k a l o i d w i t h t o x i c a c t i o n o n p l a n t s (82). O n e o f t h e m o r e a c t i v e s y n t h e t i c h e r b i c i d e s on t h e m a r k e t , p i c l o r a m ( D o w ' s T o r d o n ) , is a c h l o r i n a t e d p i c o l i n i c a c i d d e r i v a t i v e . C y a n o h y d r i n s have been i m p l i c a t e d in a l l e l o p a t h y in several instances. D h u r r i n occurs in grain sorghum seedlings and the seedlings c o n t a i n e n z y m e s t h a t h y d r o l y z e d h u r r i n to g l u c o s e , H C N (hydrogen c y a n i d e ) , a n d j>h y d r o x y b e n z a l d e h y d e (86). T h e s i t u a t i o n is s i m i l a r i n J o h n s o n g r a s s , S o r g h u m h a l e p e n s e , a v e r y a l l e l o p a t h i c w e e d (87). B o t h the H C N and £hydroxybenzaldehyde are potent a l l e l o c h e m i c a l s . H C N and benzaldehyde a r e p r o d u c e d by t h e h y d r o l y s i s o f a m y g d a l i n p r e s e n t i n p e a c h r o o t r e s i d u e s (88) . H C N a n d b e n z a l d e h y d e a r e i n h i b i t o r y t o g r o w t h o f p e a c h s e e d l i n g s a n d apparently cause the peach replant problem in old peach orchards. M u s t a r d o i l s , s u c h as a l l y l i s o t h i o c y a n a t e , a r e p r o d u c t s o f t h e h y d r o l ­ y s i s o f m u s t a r d o i l g l y c o s i d e s (78). M u s t a r d o i l s a r e p r o d u c e d by a l l o r g a n s o f p l a n t s b e l o n g i n g t o t h e C r u c i f e r a e ( m u s t a r d f a m i l y ) (75), a n d a r e s t r o n g i n h i b i t o r s of seed g e r m i n a t i o n and m i c r o b i a l g r o w t h . M a n y a n t i b i o t i c s p r o d u c e d by v a r i o u s m i c r o o r g a n i s m s a r e n u c l e o s i d e s (5). A m o n g these are nebularine, c o r d y c e p i n , and n u c l e o c i d i n . The only k n o w n p u r i n e s i n h i g h e r p l a n t s s h o w n t o be i n v o l v e d i n a l l e l o p a t h y a r e c a f f e i n e , t h e o p h y l l i n e , p a r a x a n t h i n e , and theobromine f r o m the c o f f e e t r e e (89) .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Allelopathy:

An

Overview

17

F a c t o r s D e t e r m i n i n g E f f e c t i v e n e s s of A l l e l o c h e m i c a l s S o m e a l l e l o c h e m i c a l s have been shown to be bound by the h u m i c m a t e r i a l i n t h e s o i l a n d p r e s u m a b l y i n a c t i v a t e d (90). When k n o w n a m o u n t s of t a n n i c a c i d w e r e a d d e d t o a p r a i r i e s o i l t h a t c o n t a i n e d no t a n n i c a c i d , a m i n i m u m o f 4 0 0 p p m h a d t o be a d d e d b e f o r e a n y c o u l d be r e c o v e r e d i m m e d i a t e l y . It is n o t e w o r t h y , t h e r e f o r e , t h a t as s m a l l a c o n c e n t r a t i o n as 30 p p m a d d e d t o the same soil r e d u c e d the nodule number of h e a v i l y i n o c u l a t e d legumes growing in the soil. O b v i o u s l y , some of the bound t a n n i c a c i d r e m a i n e d b i o l o g i c a l l y a c t i v e (91). S o m e plants e x e r t g r e a t e r a l l e l o p a t h i c e f f e c t s in f i n e - t e x t u r e d than in c o a r s e - t e x t u r e d soils and evidence indicates that the g r e a t e r r e t e n t i o n c a p a c i t y o f t h e f i n e t e x t u r e d s o i l s f o r a t l e a s t s o m e a l l e l o c h e m i c a l s m a y be i m p o r t a n t in the a c c u m u l a t i o n of physiologically a c t i v e c o n c e n t r a t i o n s of these c h e m i c a l s (92-94). M a n y a l l e l o c h e m i c a l s a r e d e c o m p o s e d i n s o i l , e i t h e r a b i o t i c a l l y (37) o r by microorganisms (95-100). Obviously, the attainment of active c o n c e n t r a t i o n s of a l l e l o c h e m i c a l addition and i n a c t i v a t i o n . d e c o m p o s i t i o n of a l l e l o c h e m i c a l s does not n e c e s s a r i l y r e s u l t in a decrease in a l l e l o p a t h i c a c t i v i t y . In f a c t , t h e r e v e r s e m a y be t r u e . H y d r o j u g l o n e is o x i d i z e d i n s o i l t o j u g l o n e , a q u i n o n e t h a t is i n h i b i t o r y t o s o m e s p e c i e s a t a 10" M c o n c e n t r a t i o n (101). Isoflavonoids p r o d u c e d by red c l o v e r a r e d e c o m p o s e d t o e v e n m o r e t o x i c p h e n o l i c c o m p o u n d s (95); a n d t o r e p e a t , amygdalin from peach roots is c h a n g e d to hydrogen cyanide and b e n z a l d e h y d e w h i c h c a u s e t h e p e a c h r e p l a n t p r o b l e m (88), a n d p h l o r i z i n f r o m a p p l e r o o t s is d e c o m p o s e d t o s e v e r a l p h e n o l i c c o m p o u n d s t h a t a p p e a r t o be r e s p o n s i b l e f o r t h e a p p l e r e p l a n t p r o b l e m ( 1 0 0 ) . It is a l s o i m p o r t a n t t o u n d e r s t a n d t h a t m o s t a l l e l o p a t h i c e f f e c t s apparently result f r o m the c o m b i n e d actions of several a l l e l o c h e m i c a l s , o f t e n w i t h e a c h b e l o w a t h r e s h o l d c o n c e n t r a t i o n f o r i m p a c t . In a l l e l o p a t h i c situations w h i c h i m p l i c a t e phenolic acids, soil concentrations have ranged f r o m b e l o w 10 t o a b o v e 1000 p p m f o r e a c h c o m p o u n d . T h e l o w e r e n d o f t h e s p e c t r u m is b e l o w a c o n c e n t r a t i o n r e q u i r e d f o r a n e f f e c t i n c u r r e n t bioassays. A d d i t i v e and synergistic e f f e c t s have been demonstrated, h o w e v e r , f o r c o m b i n a t i o n s o f c i n n a m i c a c i d s (102), b e n z o i c a c i d s (103), b e n z o i c a n d c i n n a m i c a c i d s (104), a n d £ - h y d r o x y b e n z a l d e h y d e w i t h c o u m a r i n ( 1 0 5 ) . It a p p e a r s t h a t s u c h c o m b i n e d i n t e r a c t i o n s m a y b e v e r y i m p o r t a n t under field conditions. It is r e v e a l i n g t o c o n s i d e r m i c r o b i a l d e c o m p o s i t i o n o f a l l e l o p a t h i c c o m p o u n d s i n r e l a t i o n to s y n e r g i s m . As discussed above, partial d e c o m p o s i t i o n of one c o m p o u n d may result in the presence of s e v e r a l a c t i v e compounds, which may exert synergistic allelopathic effects. Thus, partial decomposition could increase allelopathic a c t i v i t y , rather than decrease it. The D i r e c t i o n of F u t u r e R e s e a r c h in A l l e l o p a t h y M o s t of our present knowledge c o n c e r n i n g a l l e l o p a t h y has been o b t a i n e d in the past three decades. T h u s , i t is a v e r y y o u n g f i e l d o f s c i e n c e a n d r e s e a r c h s h o u l d be c o n t i n u e d i n a l l a r e a s o f a l l e l o p a t h y i n v e s t i g a t e d i n t h e past. T h e point has been r e a c h e d , h o w e v e r , w h e r e c e r t a i n areas need special emphasis. E v e n though many types of c h e m i c a l compounds have been i m p l i c a t e d in allelopathy, there are probably many highly important ones t h a t have been o v e r l o o k e d . T e c h n i q u e s are now a v a i l a b l e to i d e n t i f y a l l e l o c h e m i c a l s m u c h more rapidly and a c c u r a t e l y than in the past, and

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

18

ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY

many more chemists are doing research in allelopathy. Therefore, there s h o u l d be s p e c i a l e m p h a s i s i n t h i s a r e a . There have been many d e m o n s t r a t e d i n s t a n c e s of a l l e l o p a t h i c a c t i o n on the p a r t of m a n y p l a n t s p e c i e s w h e r e no a l l e l o c h e m i c a l s w e r e i d e n t i f i e d . M o s t species for w h i c h g o o d e v i d e n c e e x i s t s o f a l l e l o p a t h i c p o t e n t i a l , s h o u l d p r o b a b l y be e x a m i n e d again also w i t h new techniques and e x p e r t i s e . It is i m p o r t a n t t o i d e n t i f y t h e a l l e l o p a t h i c c o m p o u n d s i n t h e s u b s t r a t e (soil or w a t e r ) of the a l l e l o p a t h i c p l a n t and to d e t e r m i n e w h e t h e r these c o m p o u n d s h a v e c o m e f r o m t h e p l a n t , a r e p r o d u c e d by p a r t i a l d e c o m p o s i t i o n o f o t h e r c o m p o u n d s , o r a r e s y n t h e s i z e d by m i c r o o r g a n i s m s u s i n g c a r b o n s o u r c e s f r o m t h e p l a n t . It is i m p o r t a n t t o k e e p i n m i n d t h a t t h e a l l e l o p a t h i c c o m p o u n d s p r o d u c e d b y b a c t e r i a , f u n g i , a n d a l g a e a r e j u s t as m u c h a p a r t o f t h e s c i e n c e o f a l l e l o p a t h y as a r e t h o s e p r o d u c e d d i r e c t l y by p l a n t s . T h e r e is a l a r g e b o d y o f i n d i r e c t e v i d e n c e , b u t o n l y a r e l a t i v e l y s m a l l body of direct evidence, c o n c e r n i n g the movement of allelopathic compounds f r o m plants that produce them and the uptake and t r a n s l o c a t i o n o f t h e s e c o m p o u n d s b y n e i g h b o r i n g p l a n t s . T h i s is no d o u b t t h e w e a k e s t l i n k in our c h a i n of i n f o r m a t i o T h e r e is a n u r g e n t n e e d P o t e n t i a l a l l e l o p a t h i c c o m p o u n d s n e e d t o be t a g g e d ( w i t h r a d i o i s o t o p e s ) i n s u s p e c t e d a l l e l o p a t h i c p l a n t s , a n d p a t h s of t h e c o m p o u n d s s h o u l d be t r a c e d out of the donor plant and i n t o and t h r o u g h a f f e c t e d a c c e p t o r p l a n t s . S u c h investigations should i n c l u d e studies of the possible movement of a l l e l o p a t h i c c o m p o u n d s f r o m donor to a c c e p t o r t h r o u g h n a t u r a l root or s t e m g r a f t s , m y c o r r h i z a l fungi, and h a u s t o r i a l c o n n e c t i o n s of p a r a s i t i c plants (106-109). After allelochemicals have been identified in the substrate, c o n c e n t r a t i o n s s h o u l d be c a l c u l a t e d , a n d t h r e s h o l d c o n c e n t r a t i o n s for a c t i v i t y s h o u l d be d e t e r m i n e d a g a i n s t t e s t p l a n t s u s i n g c o m b i n a t i o n s o f compounds present in the substrate, in addition to individual ones. U n d o u b t e d l y , many i m p o r t a n t a l l e l o p a t h i c e f f e c t s have been overlooked b e c a u s e o f t h e use o f s i n g l e a l l e l o c h e m i c a l s i n d e t e r m i n i n g t h r e s h o l d c o n c e n t r a t i o n s for a c t i v i t y . A m o d e r a t e a m o u n t o f i n f o r m a t i o n is a v a i l a b l e c o n c e r n i n g t h e f a c t o r s a f f e c t i n g c o n c e n t r a t i o n s of phenolics in plants, and a l i t t l e research has been c o m p l e t e d c o n c e r n i n g f a c t o r s a f f e c t i n g c o n c e n t r a t i o n s of a l k a l o i d s and terpenoids. L i t t l e i n f o r m a t i o n is a v a i l a b l e c o n c e r n i n g f a c t o r s affecting c o n c e n t r a t i o n s of o t h e r types of a l l e l o p a t h i c c o m p o u n d s ; thus, r e s e a r c h is urgently needed in this a r e a . There is a c r i t i c a l n e e d f o r more study of f a c t o r s affecting i n a c t i v a t i o n and e f f e c t i v e n e s s of a l l e l o c h e m i c a l s a f t e r they m o v e out of d o n o r p l a n t s . V e r y l i t t l e is k n o w n c o n c e r n i n g t h e b i n d i n g o f t h e s e c h e m i c a l s i n s o i l a n d t h e e f f e c t s o f t h e b i n d i n g o n t h e i r a c t i v i t y . V i r t u a l l y n o t h i n g is k n o w n c o n c e r n i n g the role of t e x t u r e in the a c c u m u l a t i o n of a l l e l o c h e m i c a l s to p h y s i o l o g i c a l l y a c t i v e c o n c e n t r a t i o n s . T e m p e r a t u r e stress markedly a c c e n t u a t e s the a l l e l o p a t h i c e f f e c t s of f e r u l i c a c i d on g r o w t h of s o r g h u m a n d s o y b e a n s (110). T h e r e a r e o b v i o u s l y m a n y s t r e s s f a c t o r s w h i c h c o u l d a f f e c t response of a p l a n t or m i c r o o r g a n i s m to a g i v e n a l l e l o c h e m i c a l or combination of allelochemicals, and such interactions should be investigated. T h e s u r f a c e h a s j u s t b e e n s c r a t c h e d i n d e t e r m i n i n g t h e m e c h a n i s m s by w h i c h the d i f f e r e n t kinds of a l l e l o p a t h i c compounds e x e r t t h e i r a c t i o n s . T h e r e f o r e , i t is i m p o r t a n t t h a t m u c h m o r e r e s e a r c h be d o n e i n t h i s a r e a o f allelopathy.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2.

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Allelopathy:

An Overview

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I have e m p h a s i z e d to this point the need for research i n a l l the basic areas of allelopathy. Such work c o u l d open up new horizons for applied research in the field of allelopathy; in fact, the results form the foundation of t h e e n t i r e f i e l d . T h i s emphasis o n basic r e s e a r c h should i n no w a y d e t r a c t f r o m the value and need for more progress in the various applied areas of a l l e l o p a t h y . In r e a l i t y , o n l y a r e l a t i v e l y s m a l l a m o u n t o f r e s e a r c h h a s b e e n c a r r i e d out concerning the roles of allelopathy i n natural or any of the m a n made or m a n - a l t e r e d ecosystems. O n l y a f e w of t h e more obvious areas i n need of a t t e n t i o n will be mentioned here. M u c h research is needed on t h e q u a n t i t a t i v e e f f e c t s on crop yields of i n t e r f e r e n c e by most of our serious weeds, and on the relative c o n t r i b u t i o n s of a l l e l o p a t h y a n d c o m p e t i t i o n to the t o t a l i n t e r f e r e n c e by e a c h w e e d species. C r o p - c r o p relationships need t o be i n v e s t i g a t e d m u c h m o r e thoroughly to d e t e r m i n e w h i c h crops c a n follow others w i t h the least i n h i b i t o r y o r most s t i m u l a t o r y e f f e c t s . M o r e emphasis' should be p l a c e d on investigations of s t i m u l a t o r y a l l e l o p a t h i c e f f e c t s , because these e f f e c t s have been largely ignored i n the past. Possible a u t o t o x i c i t y should be i n v e s t i g a t e d also to d e t e r m i n continuously without rotation R e s e a r c h i n t h e use of a l l e l o p a t h y i n b i o l o g i c a l w e e d c o n t r o l should be vigorously p u r s u e d . T h i s should i n c l u d e t h e use o f m u l c h e s o f a l l e l o p a t h i c p l a n t s ; r o t a t i o n o f c r o p s i n w h i c h o n e o r m o r e o f t h e c r o p p l a n t s is a l l e l o p a t h i c to major weeds; use of a l l e l o p a t h i c cover crops; underplanting of a l l e l o p a t h i c c o m p a n i o n crops in orchards, vineyards, e t c . ; a n d the development (through breeding or genetic engineering) of crop c u l t i v a r s w h i c h c a n c o n t r o l major weeds in a given a r e a through a l l e l o p a t h i c a c t i v i t y . M o r e r e s e a r c h is n e e d e d o n t h e p o s s i b l e u s e o f c e r t a i n a l l e l o c h e m i c a l s a s herbicides or as s t r u c t u r a l models for h e r b i c i d e d e v e l o p m e n t . The very broad area of allelopathic interactions between microorganisms and plants has been largely ignored by researchers. There has been some study of e f f e c t s o f s e l e c t e d w e e d y species on f r e e - l i v i n g a n d symbiotic nitrogen fixers and on nitrifiers in natural ecosystems but v i r t u a l l y n o t h i n g has been done on these r e l a t i o n s h i p s i n other e c o s y s t e m s . M u c h more r e s e a r c h needs to be done also on t h e a n t a g o n i s t i c e f f e c t s o f plants on soil-borne plant pathogens, and on the e f f e c t s of a l l e l o c h e m i c a l s i n the p r e d i s p o s i t i o n of plants to i n f e c t i o n by pathogens (111-113). T h e r e is a pressing need also for better understanding of the production by m i c r o o r g a n i s m s of a l l e l o c h e m i c a l s in soil or w a t e r that a f f e c t g r o w t h of plants. This extends also to the partial decomposition of allelochemicals from plants, which produces more active compounds or simply more compounds which can increase allelopathic effects through additive or synergistic action. Obviously these suggestions for future research i n allelopathy a r e only a few of t h e large numbers that could be given. H o p e f u l l y , however, they may give some impetus to progress i n some v i t a l areas of a l l e l o p a t h y .

Literature Cited 1. Theophrastus. (ca 300 B.C.) "Enquiry into plants and Minor Odours and Weather Signs". 2 Vols.; transi, to English by Hort, Α.; W. Heinemann: London, 1916. 2. Plinius Secundus, C. (First Century A.D.) "Natural History". 10 Vols., transi, to English by Rackam, H.; Jones, W.H.S.; Eichholz, D. E. Howard University Press: Cambridge, Mass., 1938-1963.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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3. Molisch, H. "Der Einfluss einer Pflanze auf die andere—Allelopathie"; Gustav Fischer: Jena, 1937. 4. Muller, C. H, Vegetatio 1969, 18, 348-57. 5. Rice, E. L. "Allelopathy"; 2d ed.; Academic Press: Orlando, Florida, 1984. 6. Bell, A. A. In "Report of the Research Planning Conference on the Role of Secondary Compounds in Plant Interactions (Allelopathy)"; McWhorter, C. G.; Thompson, A. C.; Hauser, E. W. Eds.; USDA, Agricultural Research Service: Tifton, Georgia, 1977; pp. 64-69. 7. French, R. C.; Graham, C. L.; Gales, A. W.; Long, R. K. J. Agric. Food Chem. 1977, 25, 84-88. 8. Chet, J.; Henis, Y. Ann. Rev. Phytopathol. 1975, 13; 169-92. 9. Willetts, H. J. Biol. Rev. 1972, 47, 515-36. 10. Brandt, W. H.; Reese, J. E. Am. J. Bot. 1964, 51, 922-27. 11. Kerr, A. Aust. J. Biol. Sci. 1956, 9, 45-52. 12. Buxton, E. W. Trans. Brit. Mycol. Soc. 1957, 40, 145-54. 13. Menzies, J. D.; Gilbert R G Soil Sci Soc Am Proc 1967 31 49596. 14. Pepperman, A. B., Allelopathy"; Thompson, A. C. Ed.; American Chemical Society: Washington, D.C., 1985, pp. 415-25, 15. Johnson, A. W.; Rosebery, G.; Parker, C. Weed Res. 1976, 16, 223. 16. Dailey, O. D., Jr.; Vail, S. L. In "The Chemistry of Allelopathy"; Thompson, A.C., Ed.; American Chemical Society: Washington, D.C.; 1985; pp. 427-35. 17. Lynn, D. G. In "The Chemistry of Allelopathy"; Thompson, A. C., Ed.; American Chemical Society: Washington, D.C., 1985; pp. 55-81. 18. Curtis, J. T.; Cottam, G. Bull. Torrey Bot. Club 1950, 77, 187-91. 19. AlSaadawi, I. S.; Rice, E. L. J. Chem. Ecol. 1982, 8, 993-1009. 20. AlSaadawi, I. S.; Rice, E. L. J. Chem. Ecol. 1982, 8, 1011-23. 21. AlSaadawi, I. S.; Rice, E. L.; Karns, T. Κ. B. J. Chem. Ecol. 1983, 9, 761-74. 22. Lee, I. K.; Monsi, M. Bot. Mag. (Tokyo) 1963, 76, 400-13. 23. Kil, B.S. Ph.D. Dissertation, Chung-Ang University, Iri, Korea, 1981. 24. Ueki, K.; Takahashi, M. Intern. Chem. Congr. Pacific Basin Soc. Honolulu Hawaii, 1984, Abstract 02F11. 25. Booth, W. E. Am. J. Bot. 1941, 28, 415-22. 26. Rice, E. L.; Penfound. W. T.; Rohrbaugh, L. M. Ecology 1960, 41, 224-28. 27. Kapustka, L. Α.; Rice, E. L. Soil Biol. Biochem. 1976, 8, 497-503. 28. Rice, E. L. Ecology 1964, 45, 824-37. 29. Rice, E. L.; Pancholy, S. K. Am. J. Bot. 1972, 59, 1033-40. 30. Rice, E. L.; Pancholy, S. K. Am. J. Bot. 1973, 60, 691-702. 31. Rice, E. L.; Pancholy, S. K. Am. J. Bot. 1974, 61, 1095-1103. 32. Numata, M.; Kobayashi, Α.; Ν. Ohga. In "Fundamental Studies in the Characteristics of Urban Ecosystems"; Numata, M. Ed.; 1973; pp. 59-64. 33. Numata, M.; Kobayashi, Α.; Ohga, N . In "Studies in Urban Ecosystems"; Numata, M., Ed.; 1974; pp. 22-25. 34. Numata, M.; Kobayashi, Α.; Ohga, N . In "Studies in Urban Ecosystems"; Numata, M., Ed.; 1975; pp. 38-41. 35. Kobayashi, Α.; Morirnoto, S.; Shibata, Y.; Yamashita, K., Numata, M. J. Chem. Ecol. 1980, 6, 119-31. 36. Walters, D. T.; Gilmore, A. R. J. Chem. Ecol. 1976, 2, 469-79. 37. Tubbs, C. H.Forest Sci. 1973, 19, 139-45. 38. Jobidon, R.; Thibault, J. R. Bull. Torrey Bot. Club 1981, 108, 413-18. 39. Jobidon, R.; Thibault, J. R. Am. J. Bot. 1982, 69, 1213-23.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2. RICE Allelopathy: An Overview

40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83.

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Handley, W. R. C. Bull. Forest Comm., London, 1963, No. 36. Robinson, R. K. J. Ecol. 1972, 60, 219-24. Brown, R. T.; Mikola, P. Acta Forest. Fenn. 1974, 141, 1-22. McCalla, T. M.; Duley, F. L. Science 1948, 108, 163. McCalla, T. M.; Duley, F. L. Soil Sci. Soc. Amer. Proc. 1949, 14, 196-99. Chou, C. H.; Lin, H. J. J. Chem. Ecol. 1976, 2, 353-67. Rice, E. L.; Lin, C. Y.; Huang, C. Y. J. Chem. Ecol. 1981, 7, 333-44. Burgos-Leon, W.; Ganry, F.; Nicou, R.; Chopart, J. L.; Dommergues, Y. Agron. Trop. 1980, 35, 319-34. Ries, S. Κ.; Went, V.; Sweeley, C. C.; Leavitt, R. A. Science 1977, 195, 1339-41. Maugh, T. H., II. Science 1981, 212, 33-34. Oliver, L. R.; Weed Sci. 1979, 27, 183-8. Staniforth, D. W. Weeds 1965, 13, 191-3. Hagood, E. S., Jr.; Bauman, T. T.; Williams, J. L., Jr.; Schreiber, M. M. Weed Sci. 1980, 28, 729-34. Chandler, J. M. Weed Sci 1977 25 151-58 Robinson, E. L. Weed Elmore, C. D. Weed Sci Colton, C. E.; Einhellig, F. A. Am. J. Bot. 1980, 67, 1407-13. Bhowmik, P. C.; Doll, J. D. Proc. North Central Weed Cont. Conf. 1979, 34, 43-45. Bhowmik, P. C.; Doll, J. D. Agron. J. 1982, 74, 601-6. Holm, L. Weed Sci. 1969, 17, 113-18. Friedman, T.; Horowitz, M. Weed Sci. 1971, 19, 398-401. Horowitz, M.; Friedman, T. Weed Res. 1971, 11, 88-93. Lucena, J. M.; Doll, J. Revista Comalfi 1976, 3, 241-56. Komai, K.; Ueki, K. Weed Res. (Japan) 1975, 20, 66-71. Komai, K.; Iwamura, J.; Ueki, K. Weed Res. (Japan) 1977, 22, 14-18. Komai, K.; Ueki, K. Weed Res. (Japan) 1980, 25, 42-47. Komai, K.; Sato, S.; Ueki, K. Mem. Fac. Agr. Kinki Univ. 1982, 15, 3341. Rice, E. L. In "Advances in Allelopathy"; Putnam, A. R.; Tang, C. S., Eds.; John Wiley: New York (In press). Peters, Ε. J. Crop Sci. 1968, 8, 650-53. Fay, P. K.; Duke, W. B. Weed Sci. 1977, 25, 224-28. Leather, G. R. Weed Sci. 1983, 31, 37-42. Putnam, A. R.; DeFrank, J. Proc. IX Int. Cong. Plant Protection, 1979, pp. 580-82. Putnam, A. R.; DeFrank, J. Crop Prot. 1983, 2, 173-81. Patrick, Z. A. Soil Sci. 1971, 111, 13-18. Tang, C. S.; Waiss, A. C., Jr. J. Chem. Ecol. 1978, 4, 225-32. Evenari, M. Bot. Rev. 1949, 15, 153-94. Norstadt, F. Α.; McCalla, T. M. Science 1963, 140, 410-11. Spoehr, Η. Α.; Smith, J. H. C.; Strain, H. H.; Milner, H. W.; Hardin, G. J. "Fatty Acid Antibacterials from Plants," Carnegie Institution of Washington, 1949, Pub. 586. Robinson, T. "The Organic Constituents of Higher Plants"; 5th ed.; Cordus Press: North Amherst, Mass., 1983. Campbell, G.; Lambert, J. D. H.; Arnason, T.; Towers, G. H. N . J. Chem. Ecol. 1982, 8, 961-72. Whittaker, R. H.; Feeny, P. P. Science 1971, 171, 757-70. Varga, M.; Koves, E. Nature 1959, 183,401. Owens, L. D. Science 1969, 165, 18-25. Fenical, W. J. Phycol. 1975, 11, 245-59.

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84. Owens, L. D.; Thompson, J. F.; Fennessey, P. V. J. Chem. Soc., Chem. Commu. 1972, 1972, 715. 85. Rice, E. L. "Pest Control with Nature's Chemicals: Allelochemicals and Pheromones in Gardening and Agriculture"; University of Oklahoma Press: Norman, 1983. 86. Conn, E. E.; Akazawa, T. Fed. Proc. 1958, 17,205. 87. Abdul-Wahab, A. S.; Rice, E. L. Bull. Torrey Bot. Club 1967, 94, 486-97. 88. Patrick, Z. A. Can. J. Bot. 1955, 33, 461-86. 89. Chou, C. H.; Waller, G. R. J. Chem. Ecol. 1980, 6, 643-54. 90. Wang, T. S. C.; Yeh, K. L.; Cheng, S. Y.; Yang, T. K. In "Biochemical Interactions among Plants"; U.S. Nat. Comm. for IBP, Ed.; National Academy Sciences: Wash. D.C., 1971; pp. 113-20. 91. Blum, U.; Rice, E. L. Bull. Torrey Bot. Club 1969, 96, 531-44. 92. Ahshapanek, D. C. Ph.D. Dissertation, University of Oklahoma, Norman, 1962. 93. Muller, C. H.; del Moral, R. Bull. Torrey Bot. Club 1966, 93, 130-37. 94. del Moral, R.; Muller, C. H. Am. Midi. Natur. 1970, 83, 254-82. 95. Chang, C. F.; Suzuki, 33, 398-408. 96. Bonner, J. Bot. Gaz. 1946, 107, 343-51. 97. Henderson, Μ. Ε. Κ.; Farmer, V. C. J. Gen. Microbiol. 1955, 12, 37-46. 98. Kunc, F. Folia Microbiol. 1971, 16, 41-50. 99. Turner, J. Α.; Rice, E. L. J. Chem. Ecol. 1975, 1, 41-58. 100. Borner, H. Contrb. Boyce Thompson Inst. 1959, 20, 39-56. 101. Rietveld, W. J. J. Chem. Ecol. 1983, 9, 295-308. 102. Einhellig, F. Α.; Schon, M. K.; Rasmussen, J. A. J. Plant Growth Regul. 1982, 1, 251-58. 103. Einhellig, F. Α.; Rasmussen, J. A. J. Chem. Ecol. 1978, 4, 425-36. 104. Rasmussen, J. Α.; Einhellig, F. A. Plant Sci. Letters 1979, 14, 69-74. 105. Williams, R. D.; Hoagland, R. E. Weed Sci. 1982, 30, 206-12. 106. Bjorkman, E. Physiol. Plant. 1960, 13, 308-27. 107. Graham, B. F., Jr.; Bormann, F. H. Bot. Rev. 1966, 32, 255-92. 108. Woods, F. W.; Brock, K. Ecology 1964, 45, 886-89. 109. Atsatt, P. R. In "Biochemical Coevolution"; Chambers, K. L . Ed.; Biol. Colloquium #29, Oregon State University Press: Corvallis; pp. 53-68. 110. Einhellig, F. Α.; Eckrich, P. C. J. Chem. Ecol. 1984, 10, 161-70. 111. Patrick, Ζ. Α.; Toussoun, Τ. Α.; Snyder, W. C. Phytopathology 1963, 53, 152-61. 112. Toussoun, Τ. Α.; Patrick, Z. A. Phytopathology 1963, 53, 265-70. 113. Patrick, Ζ. Α.; Koch, L. W. Can. J. Bot. 1963, 41, 747-58. RECEIVED

June 9, 1986

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 3

Japanese Contributions to the Development of Allelochemicals Horace G. Cutler Richard B. Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA 30613

The Japanese people l i v e i n a delicate microecosystem that can be easily polluted by industrial chemical accidents and the use of persistent agricultural chemicals. An intensive s c i e n t i f i c effort has led to the isolation and identificatio for potential us microbial metabolites that have activity against plants, microorganisms, nematodes, and insects. The chemical structures range from complex to simple and represent diverse classes of compounds. In addition, compounds isolated by non-Japanese researchers have been assigned specific uses by Japanese scientists for potential agricultural use. I f a s c i e n t i s t who knew n o t h i n g about t h e geography o r demography o f Japan v i s i t e d t h a t c o u n t r y he would b e g i n t o a r r i v e a t c e r t a i n c o n c l u s i o n s about t h e n a t u r e o f the c o u n t r y q u i t e q u i c k l y . Visits to r e s t a u r a n t s i n t h e c i t i e s a n d s u r r o u n d i n g c o u n t r y s i d e w o u l d indicate that vegetables a n d f i s h a r e q u i t e p l e n t i f u l and r e l a t i v e l y c h e a p ; t h a t t h e m a i n g r a i n f o r f o o d and b e v e r a g e i s r i c e ; t h a t r e d meat i s b o t h e x p e n s i v e a n d d i f f i c u l t t o o b t a i n . V i s i t s to shops and o p e n - a i r m a r k e t s t a l l s would c o n f i r m t h e s e observations. S t a c k s upon s t a c k s o f f r e s h vegetables i n c l u d i n g some t h a t a r e o n l y j u s t f i n d i n g t h e i r way i n t o w e s t e r n m a r k e t s , such as d a i k o n (Raphanus s a t i v u s l o n g i p i n n a t u s ) » greet the e y e . P l a s t i c pans o f a s s o r t e d f i s h i n a l l s h a p e s a n d s i z e s , a n d an abundance o f s h e l l f i s h , a r e c o m m o n p l a c e . While t r a v e l l i n g along t h e h i g h w a y s o u r s c i e n t i s t would n o t e t h a t a r a b l e l a n d i s used t o i t s maximum, even t o t h e edge of t h e r o a d , and t o some e x t e n t o n e i s reminded o f the a l l o t m e n t gardens o f World War II E n g l a n d , where a l l a v a i l a b l e l a n d was used t o produce v e g e t a b l e s t o supplement t h e f o o d r a t i o n i n g program. Even r i c e p a d d i e s extend to t h e r o a d edge and one becomes s u d d e n l y q u i t e aware of the v u l n e r a b i l i t y o f t h i s s t a p l e c r o p d u r i n g t h e a q u a c u l t u r a l s t a g e o f i t s l i f e c y c l e . One c h e m i c a l a c c i d e n t , o r one m a l i c i o u s a c t , can s p e l l d i s a s t e r f o r t h e T h i s chapter not subject to U.S. copyright. P u b l i s h e d 1987 A m e r i c a n C h e m i c a l Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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r i c e c r o p b e c a u s e o f t h e r a p i d d i f f u s i o n o f agents i n w a t e r . In a d d i t i o n , our s c i e n t i s t would see t h e b a c k b o n e o f m o u n t a i n s , t h e o m n i p r e s e n t s e a t h a t c a n be r e a c h e d m o d e r a t e l y e a s i l y , and one m a j o r c o n c l u s i o n w o u l d be t h a t J a p a n i s a c o u n t r y t h a t c a n n o t a f f o r d to h a v e c h e m i c a l p o l l u t i o n problems from e i t h e r i n d u s t r y or agriculture. F u r t h e r m o r e , our o b s e r v e r w o u l d have b e e n s t r u c k b y t h e p o p u l a t i o n d e n s i t y and the s m a l l , compact houses t h a t are b u i l t q u i t e c l o s e l y t o g e t h e r and have s m a l l , i f any, g a r d e n s . Any e f f e c t on the food c h a i n i s immediately f e l t by the l a r g e p o p u l a t i o n . Our s c i e n t i s t would not be s u r p r i s e d i f t h e map o f J a p a n was shown t o h i m . The c o u n t r y i s b o w - s h a p e d , l o n g , and c o n s i s t s o f f o u r main i s l a n d s , H o k k a i d o , H o n s h u , K y u s h u , and S h i k o k u , p l u s several smaller islands. The l a n d mass i s 377,748 s q u a r e k i l o m e t e r s , o r about 4% the s i z e of the U n i t e d S t a t e s o f A m e r i c a , and s u p p o r t s a p o p u l a t i o n o f 119.6 m i l l i o n p e o p l e (_1 ). Mountains c o m p r i s e 71% o f t h e l a n d w i t h 29% p l a i n s and b a s i n s o f w h i c h a p p r o x i m a t e l y 15% i s s u i t a b l e f o r crop p r o d u c t i o n . The m o u n t a i n s s e r v e as a watershed an as a s o u r c e of t i m b e r . Compare d o e s h a v e a d e n s e p o p u l a t i o n , e s p e c i a l l y when those f i g u r e s are compared on an a r a b l e l a n d r a t i o , but i t i s i n t e r e s t i n g t o s e e t h e demographic f i g u r e s for other countries. For example, the p o p u l a t i o n per square k i l o m e t e r i n 1983 was 616 f o r B a n g l a d e s h , 388 f o r S o u t h K o r e a , 346 f o r the N e t h e r l a n d s , 323 f o r B e l g i u m , 317 f o r J a p a n , 100 f o r C h i n a , and n o t s u r p r i s i n g l y , 24 f o r the USA (_1 ). A l l t h e d e m o g r a p h i c f i g u r e s and g e n e r a l o b s e r v a t i o n s l e a d one to the i n e v i t a b l e c o n c l u s i o n t h a t J a p a n has a v e r y delicate m i c r o e c o s y s tern t h a t c a n be e a s i l y damaged by i n d u s t r i a l o r pesticide s p i l l s . And J a p a n i s a h i g h l y c o m p e t i t i v e i n d u s t r i a l n a t i o n w h i l e , a t the same t i m e , an i n t e n s e l y a g r i c u l t u r a l o n e . No one i s more aware of the e f f e c t s o f p o l l u t i o n t h a n t h e J a p a n e s e . In 1 9 8 4 , t h e E n v i r o n m e n t A g e n c y o f the Government o f Japan p u b l i s h e d t h e " I l l u s t r a t e d W h i t e P a p e r on t h e E n v i r o n m e n t i n J a p a n " , i n w h i c h t h e a t t i t u d e s and law c o n c e r n i n g p o l l u t i o n and waste c o n t r o l are d i s c u s s e d . The word " i m p e r a t i v e " occurs throughout t h e t e x t and i t i s q u i t e o b v i o u s t h a t t h e c o u n t r y i n t e n d s to implement a r i g o r o u s s e t of s t a n d a r d s f o r i n d u s t r y and the p r i v a t e c i t i z e n . The v u l n e r a b i l i t y o f the environment and the p o p u l a t i o n i s v i v i d l y e x p r e s s e d i n the examples of i n d u s t r i a l spills. T h e f i r s t i n v o l v e d c a d m i u m , w h i c h a f f e c t s b o n e s and k i d n e y , t h a t s p i l l e d i n t o the J i n t s u r i v e r i n the Toyama P r e f e c t u r e to produce the i t a i - i t a i d i s e a s e . The s e c o n d , w h i c h i s b e t t e r known because of a p o i g n a n t s e t o f p i c t u r e s t h a t were p u b l i s h e d o f a mother b a t h i n g her s o n , was a methylmercury d i s c h a r g e t h a t caused b r a i n d i s o r d e r s and damage to t h e n e r v o u s s y s t e m . This occurred a l o n g t h e Y a t s u s h i r o c o a s t , Kumamoto P r e f e c t u r e , and i n the Agano R i v e r B a s i n i n the N i i g a t a P r e f e c t u r e ; the symptoms were d e s c r i b e d as t h e M i n a m a t a d i s e a s e . A g a i n , one n o t e s the r a i n washing down the m o u n t a i n s , sweeping through the i n d u s t r i a l and agrarian p i e d m o n t , c a r r y i n g p o l l u t a n t s out i n t o the bays and s e a . In s p i t e of the p o p u l a t i o n p r è s s u r e s , t h e s h o r t a g e o f a r a b l e l a n d , t h e c o n c e n t r a t i o n of i n d u s t r y , and the r e l a t i v e i s o l a t i o n o f the c o u n t r y , the J a p a n e s e have done a r e m a r k a b l e j o b i n k e e p i n g t h e i r c o u n t r y i n an o r d e r l y f a s h i o n . Parks abound and t h e r e i s a r e s p e c t f o r n a t u r e and a r t t h a t i s q u i t e e x t r a o r d i n a r y . B u t aware

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o f what has happened i n the p a s t , and aware o f what might happen i n the f u t u r e , Japanese s c i e n t i s t s have asked themselves two i m p o r t a n t questions: "What s o r t o f c h e m i c a l s c a n we u s e on o u r c r o p s as p e s t i c i d e s t h a t have h i g h s p e c i f i c a c t i v i t y a g a i n s t target o r g a n i s m s ? " and "How b i o d e g r a d a b l e a r e t h e s e c h e m i c a l s ? " The a n s w e r t o t h e f i r s t q u e s t i o n may be m i c r o b i a l metabolites (allelochemicals) w h i c h a r e o r g a n i c n a t u r a l p r o d u c t s and to the second, n a t u r a l products are i n t r i n s i c a l l y b i o d e g r a d a b l e . In a d d i t i o n , a t h i r d q u e s t i o n i n v o l v e s the n a t u r e o f p h y t o p a t h o g e n i c m i c r o o r g a n i s m s and the p h y t o t o x i n s t h a t t h e y p r o d u c e . F o r i f one i s t o c o n t r o l t h e s e i n v a d e r s , t h e b i o c h e m i c a l pathways by which p h y t o t o x i n s a r e p r o d u c e d by t h e p a t h o g e n s must be e l u c i d a t e d . B e s i d e s , p h y t o p a t h o g e n s may p r o d u c e t o x i n s t h a t c a n be used to c o n t r o l crop p e s t s . We s h a l l s e e , i n t h i s b r i e f r e v i e w , t h a t Japanese work has been i n t e n s e i n 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 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 from m i c r o o r g a n i s m s . T h i s i n t e n s i t y has been marked by the f o l l o w i n g s t r a i n s of m i c r o o r g a n i s m s s t r u c t u r e of a m e t a b o l i t e , and thorough t e s t i n g on p e s t s and c r o p s t h a t a r e o f economic importance to Japan when s u f f i c i e n t q u a n t i t i e s o f t h e n a t u r a l p r o d u c t a r e a v a i l a b l e , and s y n t h e s i s ( w h e n e v e r p o s s i b l e ) o f t h e m e t a b o l i t e so t h a t f u r t h e r s c r e e n i n g may be carried out. In some i n s t a n c e s m e t a b o l i t e s have been r e d i s c o v e r e d and new a p p l i c a t i o n s f o u n d f o r them t h a t were o v e r l o o k e d by the primary d i s c o v e r e r . W h i l e the Japanese c o n t r i b u t i o n s t o t h e a r e a 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 have been q u i t e e x t e n s i v e , I have chosen a few examples to i l l u s t r a t e t h e w i d e d i v e r s i t y and a c t i v i t y o f m i c r o b i a l m e t a b o l i t e s against p l a n t s , microorganisms ( i n c l u d i n g a s e l f - i n h i b i t o r ) , nematodes, i n s e c t s , and o t h e r zoological species. C y l i n d r o c l a d i u m s c o p a r i u m i s an u b i q u i t o u s phytopathogenic fungus t h a t causes d i s e a s e s i n a wide v a r i e t y o f p l a n t s , e s p e c i a l l y o r n a m e n t a l s , a n d , more i m p o r t a n t l y , r i c e (Oryza s a t i v a L . ) , where i t induces sheath n e t - b l o t c h . H i r o t a and coworkers f i r s t p u b l i s h e d on the n a t u r e of the t o x i n s produced by t h i s o r g a n i s m , i n c u l t u r e , i n 1973 ( 2 . 3 ) and d u r i n g the course of the next e l e v e n y e a r s they c a r e f u l l y a n a l y z e d t h e s t r u c t u r e o f two m e t a b o l i t e s possessing b i o l o g i c a l a c t i v i t y w h i c h t h e y d e s i g n a t e d as c y l - 1 and c y l - 2 . F i n a l l y , i n 1984, the proposed s t r u c t u r e s f o r these compounds were p u b l i s h e d (4.) and a l l t h e e v i d e n c e p o i n t e d to two o l i g o p e p t i d e s , specifically t e t r a ρ e ρ t i d e s arranged i n the sequence of D - . O - m e t h y l t y r o s i n e , L - i s o l e u c i n e , L - p i p e c o l i c a c i d ( i n c y l - 2 ) or p r o l i n e i n ( c y l - 1 ) , and 2 - a m i n o - 8 - o x o - 9 , 1 0 - e p o x y d e c a n o i c a c i d (Figure 1). Both m e t a b o l i t e s were a c t i v e a g a i n s t p l a n t s p e c i e s b u t t h e m o r e a c t i v e o f t h e two m e t a b o l i t e s was c y l - 1 , w h i c h was i s o l a t e d i n r a t h e r s m a l l amounts. C y l - 1 , f o r example, i n h i b i t e d l e t t u c e r o o t e l o n g a t i o n 50% a t 0 . 5 ppm w h i l e c y l - 2 i n h i b i t e d e x t e n s i o n 50% o n l y at 1.0 ppm (2.). I n o t h e r t e s t s , u s i n g Avena s a t i v a L . c v . R u s s e l l c o l e o p t i l e s , c y l - 2 d i d not i n h i b i t the growth o f c o l e o p t i l e s b u t when i n d o l e - 3 - a c e t i c a c i d was a d d e d t o t h e i n c u b a t i o n medium at 1.0 ppm the e x t e n s i o n n o r m a l l y i n d u c e d by t h a t s u b s t a n c e d i d not o c c u r . That i s , c y l - 2 at 10-100 ppm a c t e d as an a n t a g o n i s t i c agent to i n d o l e - 3 - a c e t i c a c i d and w h i l e t h e m e c h a n i s m o f a c t i o n was n e i t h e r r e p o r t e d n o r suggested the o b s e r v a t i o n i s , n e v e r t h e l e s s , an i n t e r e s t i n g o n e .

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ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY

The c y c l i c t e t r a p e p t i d e s a r e a f a s c i n a t i n g group o f compounds and the f i r s t o b s e r v a t i o n t h a t one makes on v i e w i n g the s t r u c t u r e s o f t h e c y l g r o u p i s t h e s e q u e n c i n g o f t h e D and L amino a c i d s . T h i s l e a d s to some t h o u g h t s a b o u t t h e s y n t h e t i c p e r m u t a t i o n s by s u b s t i t u t i n g D and L s p e c i e s a n d , o f c o u r s e , t h e c o n c o m i t a n t b i o l o g i c a l a c t i v i t y . But an even g r e a t e r s u r p r i s e i s t h a t w h i l e t h e c y l s t r u c t u r e s a r e unique they are p a r t o f a g r e a t e r c l a s s of t o x i n s , a l l o f w h i c h p o s s e s s t h e c h a r a c t e r i s t i c s of h a v i n g p i p e c o l i c a c i d (or p r o l i n e ) and the 2 - a m i n o - 8 - o x o - 9 , 10-epoxydecanoic acid residue. Among t h e s e a r e c h l a m y d o c i n (Diheterospora c h l a m y d o s p o r i a ) (5.) , HC t o x i n ( H e l m i n t h o s p o r i u m carbonum) ( 6 , 7 ) , and WF-3161 ( P e t r i e l l a g u t t u l a t a ) (8.), t h e l a t t e r b e i n g a n o t h e r Japanese c o n t r i b u t i o n . A l l t h e s e i s o l a t i o n s and i d e n t i f i c a t i o n s are m i l e s t o n e s i n c a r e f u l l y c o n s t r u c t e d and m e t i c u l o u s work. It i s e s t i m a t e d t h a t a p p r o x i m a t e l y f i f t e e n y e a r s were spent p u t t i n g t o g e t h e r the c y l - 1 and 2 d a t a . O t h e r c y c l i c t e t r a p e p t i d e s have a l s o been i s o l a t e d by Japanese w o r k e r s and AM t o x i n s mali« are extremely t o x i are c o n s t r u c t e d of L - A - h y d r o x y i s o v a l e r i c a c i d , L - a l a n i n e , * - a m i n o a c r y l i c a c i d a n d , i n AM t o x i n I , L-o-methoxyphenyl)v a l e r i c a c i d . The p h e n y l r e s i d u e i n AM t o x i n I I is L - 0 C - a m i n o - i - p h e n y l v a l e r i c a c i d , w h i l e i n AM t o x i n I I I , it is L-

^ Ο I π CH (CH ) -C-R 2

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4

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2

1

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( Cylindrocladium s c o p a r i u m ) Figure

1.

C y l - 1 and c y l 2.

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- a n i s y l o x y p r o p i o n a t e was s y n t h e s i z e d a n d shown t o h a v e t h e same n e m a t i c i d a l p r o p e r t i e s as the n a t u r a l product L - 3 . Another m e t a b o l i t e from A s p e r g i l l u s n i g e r i s n i g r a g i l l i n , a p i p e r a z i n e c l o s e l y r e s e m b l i n g n i g e r a z i n e Β (from A . n i g e r 1 - 6 3 9 ) , t h e s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e two m o l e c u l e s b e i n g a terminal p h e n y l group in nigerazine B. Nigragillin, N - m e t h v l - t r a n s - 2 . 5 - d i m e t h y l - j T - s o r b y l p i p e r a z i n e , was o r i g i n a l l y i s o l a t e d b y C a e s a r e t a l . i n 1969 from A s p e r g i l l u s u s t u s but t h e compound was n o t t e s t e d i n b i o l o g i c a l s y s t e m s (22.) ( F i g u r e 8 ) . When s i l k w o r m , Bombyx m o r i L . , were o r a l l y dosed w i t h n i g r a g i l l i n i n c o r p o r a t e d i n t o t h e i r d i e t , 40 ppm proved t o be t o x i c w i t h i n 48 h and 40% o f t h e l a r v a e , w h i c h had been t r e a t e d a f t e r t h e t h i r d m o l t , died. A t 72 h , 70% h a d d i e d . Treatment w i t h 80 ppm proved t o be more l e t h a l and 100% of t h e l a r v a e d i e d w i t h i n 48 h . H o w e v e r , i t s h o u l d be p o i n t e d o u t t h a t w h i l e t h e m e t a b o l i t e was i n c o r p o r a t e d i n t o t h e media a t these r a t e s i t i s most p r o b a b l e t h a t o n l y s m a l l amounts w e r e i n g e s t e d b y t h e l a r v a e . A further d i l u t i o n effect o c c u r r e d because t e n l a r v a e were i n c l u d e d i n each d i e t assay. O t h e r symptoms n o t e d d u r i n g t h e course o f t h e experiments i n c l u d e d v o m i t i n g , c o n v u l s i o n s , and swooning ( 2 3 ) . The e f f e c t s o f t o p i c a l a p p l i c a t i o n o f n i g r a g i l l i n i n e t h y l a c e t a t e t o s i l k w o r m were n o t i c e a b l e almost i n s t a n t a n e o u s l y . D o s e s o f 5 yg/g caused i m m e d i a t e k n o c k d o w n a n d p o i s o n i n g , t h o u g h d e a t h d i d n o t always follow. S y n t h e t i c d _ l - n i g r a g i l 1 i n was made a f t e r t h e m e t h o d o u t l i n e d b y Caesar (22) and i t had t h e same b i o l o g i c a l a c t i v i t y as the n a t u r a l p r o d u c t . A n o t h e r compound w i t h marked i n s e c t i c i d a l p r o p e r t i e s i s

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L - a l a n o s i n e ( F i g u r e 9 ) , i s o l a t e d from a Streptomyces s p e c i e s , which i n h i b i t e d l a r v a l e c d y s i s when a d m i n i s t e r e d to f o u r t h - i n s t a r l a r v a e o f the common armyworm, L e u c a n i a s e p a r a t a , i n a r t i f i c i a l d i e t (24). The m o l e c u l e i s q u i t e s i m p l e , L-2-amino-3-(hydroxynitrosamino)p r o p i o n i c a c i d , and the r e a d e r q u i c k l y c a l l s to mind t h e number o f ' s i m p l e ' amino compounds t h a t h a v e been d i s c o v e r e d d u r i n g r e c e n t y e a r s , t h a t range from a r t i f i c i a l s w e e t e n e r s ( a s p a r t a m e ) t o t h e herbicide glyphosate. Doubtless, w i t h a l l the possible p e r m u t a t i o n s f o r amino a c i d d e r i v a t i v e s , many more w i l l f i n d t h e i r way t o t h e m a r k e t p l a c e . The a c t i o n o f L - a l a n o s i n e appears t o be q u i t e s p e c i f i c on e c d y s i s and r a t e s as low as 5 ppm i n d i e t s c a u s e d i n h i b i t i o n o f head c a p s u l e removal i n 50% of l a r v a e . W i t h 40 ppm, not o n l y was head c a p s u l e removal t o t a l l y i n h i b i t e d , b u t c u t i c l e shed d i d n o t t a k e p l a c e . I f the m e t a b o l i t e was f e d to the i n s e c t i m m e d i a t e l y f o l l o w i n g e c d y s i s , t h e n l a r v a l g r o w t h was slightly delayed. T h e same e f f e c t s w e r e a l s o o b s e r v e d i n t h e c a b b a g e armyworm, Mamestra b r a s s i c a e . L - A l a n o s i n e was o r i g i n a l l y i s o l a t e d i n 1966 by M u r t h y and m e t a b o l i t e from Streptomyce i n h i b i t e d r e p r o d u c t i o n i n the h o u s e f l y , Musca d o m e s t i c a ( 2 7 ) . Its m e c h a n i s m o f a c t i o n may i n v o l v e b l o c k i n g RNA adenine s y n t h e s i s , thereby i n h i b i t i n g p r o d u c t i o n o f t h e c u t i c u l a r p r o t e i n t h a t i s e s s e n t i a l f o r s c l e r o t i z a t i o n i n the m o l t i n g p r o c e s s ( 2 4 ) . Again, L - a l a n o s i n e i s an e x c e l l e n t example o f how p r e v i o u s l y d i s c o v e r e d n a t u r a l p r o d u c t s have been r e - i s o l a t e d by Japanese r e s e a r c h e r s and t e s t e d i n systems t h a t have p r a c t i c a l , n a t i o n a l a p p l i c a t i o n . In a d d i t i o n to c o n t r o l l i n g p l a n t g r o w t h and d e v e l o p m e n t and c e r t a i n i n s e c t p e s t s there i s great i n t e r e s t i n e l i m i n a t i n g plant pathogens. One o f t h e s e i s V a l s a c e r a t o s p e r m a , a f u n g u s that p r o d u c e s a p p l e c a n k e r and ranks among the most s e v e r e problems i n a p p l e c u l t i v a t i o n . M e c h a n i c a l l y i n j u r e d p a r t s of t r e e s are r e a d i l y a t t a c k e d by t h e f u n g u s , as a r e n e c r o t i c a r e a s , and i n v a s i o n proceeds m e t h o d i c a l l y u n t i l the t r u n k i s damaged. Infected parts become cankered by the fungus and e v e n t u a l l y d i e . In an attempt to c o n t r o l V . c e r a t o s p e r m a s e v e r a l a n t i b i o t i c s w e r e e v a l u a t e d and m i c r o b i a l products t e s t e d . Micromonospora c h a l c e a produced a n o v e l a n t i b i o t i c , d e s i g n a t e d p r o p a n o s i n e ( K - 7 6 ) , w h i c h had s p e c i f i c a c t i v i t y a g a i n s t V . c e r a t o s p e r m a (28) ( F i g u r e 1 0 ) . The compound w h i c h was l i k e L - a l a n o s i n e i n UV s p e c t r a l p r o p e r t i e s , was o f relatively simple s t r u c t u r e . D i s k a s s a y s i n p e t r i d i s h e s were conducted a g a i n s t s e v e r a l microorganisms. G r e a t e r t h a n 800 jigfmL were n e c e s s a r y to c o n t r o l C o l l e t o t r i c h u m l a g e n a r i u m . F u s a r i u m oxvsporum. f. l y c o p e r s i c i . G i b b e r e l l a f u i i k u r o i , P e l l i c u l a r i a f i l a m e n t o s a , and S a p r o l e g n i a p a r a s i t i c a . But 200 jig/mL c o n t r o l l e d A . k i k u c h i a n a , B o t r y t i s c i n e r e a . C o c h l i o b o l u s miyabeanus, D i a p o r t h e c i t r i , G r o m e r e l l a c i n g u l a t a , P y r i c u l a r i a o r v z a e . and R h i z o c t o n i a solani. In excess o f 100 jig/mL were needed to c o n t r o l J i . s u b t i l i s , A T C C 6 6 3 3 , J3. s t e a r o t h e r m o p h i l u s , M y c o b a c t e r i u m p h l e i 607 , S t a p h y l o c o c c u s a u r e u s 2 0 9 P , E . c o l i NIHJ, Pseudomonas a e r u g i n o s a M8152, S e r r a t i a m a r c e s c e n s , V i b r i o p e r c o l e n s ATCC 8461, and C a n d i d a a l b i c a n s M9001. O n l y 25 yg/mL were n e c e s s a r y to i n h i b i t S a c c h a r o m v c e s c e r e v i s i a e Y 2 1 - 1 , b u t most i m p o r t a n t l y , o n l y 1.0 μg/mL o f t h e a n t i b i o t i c was needed to c o n t r o l V a l s a c e r a t o s p e r m a . S i n c e o n l y 45 mg of the Na s a l t were i s o l a t e d i n i t i a l l y , t h e r e was insufficient material for f i e l d t r i a l s . S y n t h e t i c m a t e r i a l was

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

32

ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY

H Ο L-1 Η

Η

Η Ο L-2

H CO

OCH CH COOCH

3

2

2

3

L-3

F i g u r e 7.

L-l L-2 L-3

: 5-pentylfuraldehyde, : 5 - ( 4 - p e n t y l ) - 2 - f u r a l d e h y d e , and : methyl 3 - j > - a n i s y l o x y p r o p i o n a t e .

Ο

NIGRAGILLIN ( Aspergillus niger ) Figure 8.

Nigragillin.

0 = N-N-CH -CH-COOH ι ι OH NH 2

2

L-ALANOSINE (Streptomyces sp.) Figure 9.

L-Alanosine.

CH -CH-CH OH 3 3

"Ο

2

N-OH

PROPANOSINE ( K - 7 6 ) ( Micromonospora chalcea ) F i g u r e 10.

Propanosine

(K-76).

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Contributions

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o b t a i n e d (.28) b u t i t was a r a c e m i c m i x t u r e and had o n l y h a l f the a c t i v i t y o f the n a t u r a l p r o d u c t . The c a t a l o g o f n a t u r a l p r o d u c t s t h a t c o n t r o l m i c r o o r g a n i s m s i s extensive. That i s , a f t e r a l l , one of the e a r l y a r e a s o f m e d i c i n a l r e s e a r c h and r e a l l y marks the b e g i n n i n g of a n t i b i o t i c s d e r i v e d from microbes. But l e s s common are n a t u r a l p r o d u c t s from m i c r o o r g a n i s m s t h a t a r e s e l f - i n h i b i t o r s a t low l e v e l s o f a p p l i c a t i o n . While h i g h e r p l a n t s c o n t a i n a p p a r e n t s e l f - i n h i b i t o r s and p l a n t growth regulators, s p e c i f i c a l l y a b s c i s i c a c i d , t h e r o l e of these m e t a b o l i t e s i n the p r o d u c e r o r g a n i s m i s n o t c l e a r l y u n d e r s t o o d . Aspermutarubrol, bis(5-methy1-2,3-dihydroxypheny1) e t h e r , from A s p e r g i l l u s svdowi i s such a compound ( F i g u r e 1 1 ) . The p r e s e n c e o f t h e o r g a n i s m on t h e s u r f a c e o f o l d shoe p o l i s h i s something o f a m y s t e r y b u t t h e c u r i o s i t y o f S a t o m u r a i s t o be commended I The f i r s t r e a c t i o n on d i s c o v e r i n g a fungus on any household i t e m i s to discard i t with a l a c r i t y . D u r i n g the c u l t u r i n g of the o r g a n i s m i t was o b s e r v e d t h a t p i g m e n t p r o d u c t i o n c o u l d be c o r r e l a t e d w i t h m y c e l i a l i n h i b i t i o n and t h a both responses. Eventually c o l o r l e s s c r y s t a l s f r o m c h l o r o f o r m (1 mg/L) (29) but i n aqueous s o l u t i o n the m e t a b o l i t e o x i d i z e d q u i t e r e a d i l y to give red products. T h e m e t a b o l i t e p r o v e d t o be i n e f f e c t i v e against S a c c h a r o m y c e 8 c e r e v i s i a e . P é n i c i l l i u m no t a turn, A s p e r g i l l u s n i g e r , A . o r v z a e . M u c o r m u ç e d o , R h i z o p u s i a p o n i c u s . and v e r y s l i g h t l y a c t i v e a g a i n s t JS. c o l i . but i t was v e r y a c t i v e a t 50, 100, and 200 ppm a g a i n s t t h e g r a m - p o s i t i v e jB. s u b t i l i s . S t a p h y l o c o c c u s a u r e u s , and M i c r o c o c c u s l y s o d e i k t i c u s . A t 1 2 . 5 , 2 5 , 50, and 100 ppm i t m o d e r a t e l y i n h i b i t e d A . svdowi and c o m p l e t e l y i n h i b i t e d at 200 ppm (.29.) · The a u t h o r s o f t h i s w o r k were q u i c k to p o i n t out t h a t the s e c r e t i o n o f a n t i b i o t i c s u b s t a n c e s as a c o m p e t i t i v e mechanism f o r the s u r v i v a l and b e n e f i t of the p r o d u c e r o r g a n i s m i s well e s t a b l i s h e d , but the p u z z l e posed by a s p e r m u t a r u b r o l remains to be explained. D u r i n g t h e c o u r s e o f t r y i n g t o i s o l a t e a s e l f - i n h i b i t o r from b e n o m y l - r e s i s t a n t s t r a i n s o f the c h e r r y brown r o t f u n g u s , M o n i l i n i a f r u e t i c o l a . two new m e t a b o l i t e s t h a t h a d a n t i m i c r o b i a l and p h y t o t o x i c p r o p e r t i e s , though not s e l f - i n h i b i t o r y c h a r a c t e r i s t i c s , were i s o l a t e d . These were m o n i l i d i o l and d e c h l o r o m o n i l i d i o l ( 3 0 ) , " e a l i c y l a l d e h y d e t y p e o c t a k e t i d e s (30.)", which are s t r u c t u r a l l y r e l a t e d t o t h e p h y t o t o x i n s p y r i c u l o l (31.) and p y r i c u l a r i o l (32) from P y r i c u l a r i a o r y z a e (Figure 12). M o n i l i d i o l , when a p p l i e d to c h e r r y l e a v e s a t 2 - 5 ^ g , f o l l o w e d by p i n p r i c k s , i n d u c e d d a r k necrotic spots. It a l s o i n h i b i t e d the growth of r i c e seedlings though h a r d d a t a a r e not a v a i l a b l e (30). D e c h l o r o m o n i l i d i o l was n o t as a c t i v e as t h e c h l o r i n a t e d compound i n e i t h e r c h e r r y or r i c e plants. O t h e r t e s t s were c a r r i e d o u t w i t h m o n i l i d i o l a g a i n s t s e l e c t e d o r g a n i s m s b u t g r e a t e r t h a n 100 ppm w e r e n e c e s s a r y t o i n h i b i t p l a n t p a t h o g e n s ( u n n a m e d ) , i n c l u d i n g M. f r u c t i c o l a . The compounds w e r e s y n t h e s i z e d a n d t h e p r o c e d u r e s were p u b l i s h e d i n 1983 ( 3 3 ) . No s y s t e m i s p e r f e c t and sometimes events become q u i t e t a n g l e d in research. What a p p e a r s t o be a p l a c i d a r e a o f e n d e a v o r is s u d d e n l y e n t e r e d b y s e v e r a l w o r k e r s who a r e , unknowingly, i n d e p e n d e n t l y p u r s u i n g common g o a l s . Such was t h e case w i t h the j > - t e r p h e n y l s , a c u r i o u s group o f compounds t h a t a r e expected to be

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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34

A L L E L O C H E M I C A L S : ROLE IN AGRICULTURE A N D FORESTRY

p r o d u c t s of a chemical r e a c t i o n i n v i t r o r a t h e r than n a t u r a l products. I n 1 9 7 5 , T a k a h a s h i was i n the p r o c e s s o f i s o l a t i n g and i d e n t i f y i n g t e r p h e n y l l i n ( F i g u r e 13) from A . c a n d i d u s when, as he states, " A t t h i s s t a g e o f t h e work M a r c h e l l i and V i n i n g ( 3 4 ) r e p o r t e d t h e i s o l a t i o n o f t h e t e r p h e n y l assumed to be i d e n t i c a l w i t h compound A . . . " ( 3 5 ) . The compounds were i d e n t i c a l . Takahashi h a d n o t e d t h e e f f e c t o f t e r p h e n y l l i n a g a i n s t HeLa c e l l s , where 3 . 2 ppm p r o d u c e d s l i g h t c e l l u l a r damage and h i g h e r c o n c e n t r a t i o n s i n d u c e d g r e a t e r c h a n g e s so t h a t a t 100 ppm t h e r e was c o m p l e t e cytolysis. C e l l s a l s o had v a r y i n g d e g r e e s o f R t y p e changes, s l i g h t l y e n l a r g e d c e l l s , e v e n l y d i s t r i b u t e d c h r o m a t i n , and s m a l l n u c l e o l i (.35). In 1 9 7 8 , C u t l e r , et a l . , d i s c o v e r e d h y d r o x y t e r p h e n y l l i n , a l s o f r o m A . c a n d i d u s . u s i n g t h e e t i o l a t e d wheat c o l e o p t i l e b i o a s s a y and showed t h a t i t had p l a n t growth r e g u l a t o r y a c t i v i t y (36) (Figure 1 4 ) . Furthermore, t e r p h e n y l l i n s i g n i f i c a n t l y i n h i b i t e d c o l e o p t i l e s 35% a t 1 0 " 3 , w h i l e h y d r o x y t e r p h e n y 1 1 i n i n h i b i t e d 1 0 0 , 4 2 , and 8%, a t 1 0 - 3 , i o ~ 4 IQ-5 . Peracetylt i o n of the m o l e c u l e r e n d e r e a c t i v i t y a p p e a r e d to b Four years l a t e r , Kobayashi et a l . , p u b l i s h e d t h e s t r u c t u r e s for c a n d i d u e i n s A a n d Β f r o m A . c a n d i d u s ( F i g u r e s 15 and 1 6 ) . Both were a n a l o g s o f t e r p h e n y l l i n and h y d r o x y t e r p h e n y 1 1 i n , r e s p e c t i v e l y . Each b l o c k e d i n i t i a l c l e a v a g e i n s e a u r c h i n e m b r y o s when a d d e d 5 m i n a f t e r f e r t i l i z a t i o n a t 1 χ 1 0 " ^ and 5 χ 10""* M, and i n h i b i t e d ! · 8 u b t i l i s a t 50 yg/mL (37). F u r t h e r experiments were conducted w i t h c a n d i d u s i n Β t o determine i t s e f f e c t s on DNA, RNA and p r o t e i n s y n t h e s i s i n t h e e a s t r u l a s t a g e o f sea u r c h i n embryos, uptake of [^H]-thymidine, [ ^ H ] - u r i d i n e , and [ 3 R ] - L - l e u c i n e was o b s e r v e d and percent i n h i b i t i o n noted. A t 0 pg/mL o f c a n d i d u s i n Β t h e r e was 0 i n h i b i t i o n o f u p t a k e o f these r a d i o i s o t o p e s , but a t 1 Mg/mL t h e r e was 8 3 , 5 8 , and 0% i n h i b i t i o n o f uptake o f r a d i o l a b e l e d t h y m i d i n e , u r i d i n e and L - l e u c i n e , r e s p e c t i v e l y . A t 10 jug/mL these f i g u r e s were 9 6 , 86 and 0%, r e s p e c t i v e l y . T h e r e f o r e , c a n d i d u s i n Β was n o t c o n s i d e r e d t o be a r e s p i r a t o r y i n h i b i t o r b e c a u s e i t h a d b e e n determined, i n sea u r c h i n embryos, t h a t r e s p i r a t o r y i n h i b i t o r s s u p p r e s s p r o t e i n s y n t h e s i s , and a l s o DNA and RNA s y n t h e s i s ( 3 7 ) . I n 1 9 8 5 , d i h y d r o x y t e r p h e n y l l i n was i s o l a t e d from A . c a n d i d u s and was f o u n d t o be a p p r o x i m a t e l y t w i c e a s a c t i v e as h y d r o x y t e r p h e n y l l i n i n b l o c k i n g f i r s t c l e a v a g e and i n d u c i n g i r r e g u l a r l y s h a p e d o r o d d - n u m b e r e d c e l l s i n s e a u r c h i n e m b r y o s (.38) . The laboratory synthesis of these terphenyl d e r i v a t i v e s is e x c e p t i o n a l l y d i f f i c u l t and has n o t y e t been a c c o m p l i s h e d , but m i c r o o r g a n i s m s appear t o make them w i t h r e l a t i v e e a s e . J

A

N

D

M

These few examples o f r e s e a r c h i n d i c a t e , at l e a s t i n p a r t , the i n t e n s i t y and p e r s e v e r a n c e w i t h w h i c h Japanese s c i e n t i s t s approach t h e i r work w i t h m i c r o b i a l m e t a b o l i t e s . We have seen t h a t , i n many i n s t a n c e s , e f f o r t s a r e made t o d u p l i c a t e , by s y n t h e s i s , these s u b s t a n c e s a n d i t may o n l y b e a q u e s t i o n o f t i m e b e f o r e a m a r k e t a b l e product i s developed. D o u b t l e s s , what we are s e e i n g i n the l i t e r a t u r e i s o n l y a s m a l l p o r t i o n of the energy b e i n g expended to s u c c e s s f u l l y produce biodegradable a g r o c h e m i c a l s b a s e d on n a t u r a l product templates. Japan i s c o m m i t t e d to p r o d u c i n g abundant crops on s m a l l p a r c e l s o f l a n d and to h a v i n g an u n p o l l u t e d environment.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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35

Allelochemicals

ASPERMUTARUBROL ( Aspergillus sydowi ) Figure

11.

Aspermutarubrol.

HQ,

R MONILIDIOL

CI

DECHLOROMONILIDIOL

H

( Monilinia f r u c t i c o l a ) Figure

12.

M o n i l i d i o l and d e c h l o r o m o n i l i d i o l .

OCH

3

OH

TERPHENYLLIN ( Aspergillus candidus ) Figure

13.

Terphenyllin.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY

OCH

3

OH

HYDROXYTERPHENYLLIN ( Aspergillus candidus ) Figure

CANDIDUSIN A ( Aspergillus candidus ) Figure

15·

Candidusin A.

CANDIDUSIN Β ( Aspergillus candidus ) Figure

16.

Candidusin B.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

3. CUTLER Japanese Contributions to Allelochemicals

37

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. 25.

Demographic and statistical data were kindly supplied by the Japanese Consulate, Atlanta, Georgia. Figures are given for 1983. The author thanks Masami Mera for these services. Hirota, Α.; Suzuki, Α.; Suzuki, H.; Tamura, S. Agric. Biol. Chem. 1973, 37, 643. Hirota, Α.; Suzuki, Α.; Aizawa, K.; Tamura, S. Agric. Biol. Chem. 1973, 37, 955. Takayama, S.; Isogai, Α.; Nakata, M.; Suzuki, H.; Suzuki, A. Agric. Biol. Chem. 1984, 48, 839. Closse, Α.; Huguenin, R. Helv. Chim. Acta 1974, 57, 533. Walton, J.D.; Earle, E.D.; Gibson, B.W. Biochem. Biophys. Res. Commun. 1982, 107, 785. Gross, M.L.; McCrery, D.; Crow, F.; Tomer, K.B.; Pope, M.R.; C i u f f e t t i , L.M.; Knoche, H.W.; Daly, J.M.; Dunkle, L.D. Tetrahedron Lett. 1982, 23, 5381. Umehara, K.; Nakahara M.; Tanaka, H . Antibiot. 1983, 36, 478. Ueno, T.; Nakashima, T.; Hayashi, Y.; Fukami, H. Agric Biol. Chem. 1975, 39, 1115. Ueno, T.; Nakashima, T.; Hayashi, Y.; Fukami, H. Agric Biol. Chem. 1975, 39, 2081. Iwamoto, T.; Shima, S.; Hirota, Α.; Isogai, Α.; Sakai, H. Agric. Biol. Chem. 1983, 47, 739. Hamasaki, T.; Nakajima, H.; Yokota, T.; Kimura, Y. Agric. Biol. Chem. 1983, 47, 891. El-Rayyes, N.R.; Al-Hajjar, F.H. J. Prakt. Chem. 1977, 319, 927. Isogai, Α.; Washizu, M.; Kondo, K.; Murakoshi, S.; Suzuki, A. Agric. Biol. Chem. 1984, 48, 2607. McCorkindale, N . J . ; Blackstone, W.P.; Johnstone, G.A.; Ray, T.R.; Troke, R.A. 11th IUPAC Int. Symp. Chem. Nat. Prod. Vol. I, 1978, p. 151. McCorkindale, N . J . ; Wright, J . L . C . ; Brian, P.W.; Clarke, S.M.; Hutchinson, S.A. Tetrahedron Lett. 1968, 727. Sassa, T.; Tomizuka, K.; Ikeda, M.; Miura, Y. Agric. Biol. Chem. 1973, 37, 1221. Sassa, T.; Tomizuka, K.; Ikeda, M.; Miura, Y. Tetrahedron Lett. 1973, 2333. Suzuki, Α.; Gohbara, M.; Kosuge, Y.; Tamura, S.; Ohashi, Y.; Sasada, Y. Agric. Biol. Chem. 1976, 40, 2505. Gohbara, M.; Kosuge, Y.; Yamasaki, S.; Kimura, Y.; Suzuki, Α.; Tamura, S. Agric. Biol. Chem. 1978, 42, 1037. Hayashi, M.; Wada, K.; Munakata, K. Agric. Biol. Chem. 1981, 45, 1527. Caesar, F . ; Jansson, K.; Mutschler, E. Pharm. Acta Helv. 1969, 44, 676. Isogai, Α.; Horii, T.; Suzuki, Α.; Murakoshi, S.; Ikeda, K.; Sato, S.; Tamura, S. Agric. Biol. Chem. 1975, 39, 739. Matsumoto, S.; Sakuda, S.; Isogai, Α.; Suzuki, A. Agric. Biol. Chem. 1984, 48, 827. Murthy, Y.K.S.; Thiemann, J . E . ; Coronelli, C.; Sensi, P. Nature 1966, 211, 1198.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Coronelli, C.; Pasqualucci, C.R.; Tamoni, G.; Gallo, G.G. II Farmaco. Ed. Sci. 1966, 21, 269. Kenaja, E.E. J. Econ. Entomol. 1969, 62, 1006. Abe, Y . ; Kadokura, J.; Shimazu, Α.; Seto, H.; Otake, N. Agric. Biol. Chem. 1983, 47, 2703. Taniguchi, M.; Kaneda, N. Shibata, K.; Kamikawa, T. Agric. Biol. Chem. 1978, 42, 1629. Sassa, T.; Nukina, M.; Sugiyama, T.; Yamashita, K. Agric. Biol. Chem. 1983, 47, 449. Iwasaki, S.; Nozoe, S.; Okuda, S.; Sato, Z . ; Kozaka, T. Tetrahedron Lett. 1969, 3977. Nukina, M.; Sassa, T.; Ikeda, M.; Umezama, T.; Tasaki, H. Agric. Biol. Chem. 1981, 45, 2161. Sugiyama, T.; Watanabe, M.; Sassa, T.; Yamashita, K. Agric. Biol. Chem. 1983, 47, 2411. Marchelli, R.; Vining, L.C. J. Chem. Soc. Chem. Commun. 1973, 555. Takahashi, C.; Yorhihira Pharm. Bull. 1976 Cutler, H.G.; Lefiles, J . H . ; Crumley, F . G . ; Cox, R.H. J. Agric. Food Chem. 1978, 26, 632. Kobayashi, Α.; Takemura, Α.; Koshimizu, K.; Nagano, H.; Kawazu, K. Agric. Biol. Chem. 1982, 46, 585. Kobayashi, Α.; Takemoto, Α.; Koshimizu, K.; Kawazu, K. Agric. Biol. Chem. 1985. 49, 867.

RECEIVED February 4, 1986

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 4

Allelopathy in the Soviet Union A. M. Grodzinsky Central Republic Botanical Garden, Ukrainian Academy of Sciences, Kiev, Union of Soviet Socialist Republics Allelopathy has been developed in several main directions throughout Russia, which has a long and distinguished record in this research area. Soil sickness under wheat, oats, corn, rye, alfalfa, peas, sugar beets, clover, flax, woody plants and shrubs has received much attention. A n understanding of recultivation-regulation of microbial activity and breeding of new plant varieties with less allelopathic activity are among the main objectives. Mechanism various types of ecosystem Research on isolatio produced by plants and/or microorganisms and testing them are discussed. Allelopathy is an o l d tradition i n the Soviet Union. In the middle of the last century the botanist Levakovskii (1) from Kazan published his observations about interactions between forest trees and forest grasses. H e suggested that forest litter chemically influences plant seedlings. A t the beginning o f the present century Periturin (2) investigated the causes of soil fatigue under common cereals — oats, wheat, and barley. That found toxins accumulated i n soil, w h i c h c o u l d be extracted with alcohol; after this the soil regained its fertility. A special direction i n research on chemical interaction among plants began i n 1926, when T o k i n (2) discovered the presence of volatile protective substances of plants, w h i c h he called phytoncides. Besides their influence on pathogenic organisms, w h i c h cause illnesses o f men and animals, the most important function of phytoncides is the protection of plants against herbivorous animals and parasite damage and against fungal and bacterial infection. Independently of those researches K h o l o d n y i (4) at the end of the 1930s noted the ability of some microorganisms and the roots of higher plants to absorb volatile substances from air and to use them for growth. O n the basis of these investigations he developed the idea o f interaction among plants and between plants and microorganisms mediated by volatile compounds, such as terpenes and other hydrocarbons. In 1956 C h e r n o b r i v e n k o (5.) summarized many such observations and field experimental data. Some interesting observations on the biological effects o f volatile substances were also published by Sanadze (6). A t the end of the 1950s allelopathy was already w e l l known among botanists and plant physiologists, but it was considered rather a m i n o r and rare phenomenon that had no great ecological importance. W e have screened many plant exudates, and reached the conclusion that many species are allelopathically active; indeed, practically any plant under certain conditions so affects other plants 0097-6156/87/0330-0039$06.00/0 © 1987 A m e r i c a n C h e m i c a l Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY

(7). Thus allelopathy is indeed very important ecologically, although skeptics remain. M a n y investigations i n c h e m i c a l interactions i n different types o f phytocenoses (natural, artificial, forest, steppe, aquatic ecosystems) are currently conducted by specialists: botanists, plant physiologists, m i c r o b i o l o g i s t s , biochemists, soil scientists, agronomists, etc. B y drawing analogies with other sciences, we can mark several stages of allelopathy development and paradigms that change and add to each other. Originally, allelopathy was considered only as the harmful influence of certain active plant species on adjacent plants. This definition was extended by the author of the term allelopathy, M o l i s c h (&). M o l i s c h indicated that allelopathy included stimulatory as w e l l as inhibitory growth effects. M a n y of the phenomena he discussed i n v o l v e d growth stimulation or a combination of stimulation and inhibition e.g., effects of ethylene. M o s t researchers now accept allelopathy. A c t i v e substances can be likened to herbicides and their high specificity is accepted without question. For example, a very allelopathicall crucifer Crambe tataria Sebeôk (9). It is a biennial or perennial herbaceous plant with a well-developed root and large spherical bushlike stems with many dry fruits. After ripening, the stem tears loose from the root and is rolled throughout the steppe by wind. E n route the fruits are lost i n the grass. The fruits contain very strongly inhibiting substances, a mixture of many phenolic acids, amino acids, and some sulfur-organic substances. W e still do not know the whole chemical constitution of fruit coats of Crambe tataria. but that plant first gave us the idea of chemical interaction between plants. The water-soluble inhibitors from the fruits go into the surroundings, suppress other plants, and make free places for germination and growth of Crambe. Such plants as Crambe are not numerous -- they may constitute less than 1% o f the general flora. However, allelochemicals can be not only harmful, but favorable, particularly at low concentration. Chernobrivenko (5) and other Soviet scientists assumed the possibility of positive chemical influence of adjacent plants. American authors, Rice QQ) among them, took this position much later. The notion of action by specific allelochemical compounds is also unjustified. Detailed study of some allelochemicals i n active species has shown the presence of phenolic acid mixtures and other phenolic derivatives or terpenes. I think that we can never talk about the action of a single substance; everywhere many compounds having different biological activity act simultaneously, perhaps mutually increasing their activity. A s a rule, such allelochemicals are the intermediate products of soil humus, synthesis, or the ground detritus i n aquatic ecosystems (11). H i g h concentrations of these substances are lethal, moderate ones inhibit growth processes, and low concentrations stimulate them. Accordingly, the second paradigm of allelopathy was formed. In this pattern the chemical mutual influence is manifested as a cycling of physiologically active substances, which play the role of regulators of internal and external interrelations — of initiation, development, and change of plant cover i n biocenosis (ecosystem) (12). Allelopathy is part of the whole recycling of organic substances i n the ecosystem; it involves low-molecular-weight carbon compounds, which are either mineralized or polymerized into large humic molecules. Such molecules do not penetrate into plants and thus have no allelopathic effect. This means that we are discussing the intermediate products in humus formation and decomposition.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4.

GRODZINSKY

Allelopathy

in the Soviet

Union

41

This paradigm provided our basic notion of allelopathic soil fatigue (13). A s a broad ecological notion it includes accumulation of toxic products o f the vital activity o f plants and heterotrophic organisms; this adversely affects the productivity of f o l l o w i n g plants. A s a consequence o f specialization and concentration of the agricultural industry, the s o i l fatigue problem became particularly serious and urgent. W e managed to devise an isolation method for allelochemicals by using ion-exchange resins, which permitted us to obtain those substances without destruction of humus complexes that could not be absorbed by living plant roots. O u r method simulates plant root absorption of allelochemicals. It was shown that cinnamic, g-coumaric, £- hydroxybenzoic, and other phenolic acids accumulate under monocultures of wheat, rye, and other cereals. The concentrations o f these acids increase two- or threefold under permanent wheat culture, while that of neutral humus decreases. A t the same time we observe changes in microflora; the diversity and number of bacteria decrease and the mass of soil fungi increases (14). To relieve allelopathic soil fatigue we can use either the o l d tested method of crop rotation or, i f we know the chemical basis o f the problem, the agrotechnical method, which accelerates mineralization/polymerizatio allelopathically inactive specie think that the last method is the most reliable. This second paradigm includes both negative and positive allelochemical effects on plant growth and physiological processes. This new view of allelopathy, its new paradigm, conveys the notion of chemical information exchange among plants and other organisms. Current plant physiology makes it possible to suggest that plants are able to "perceive" a chemical environment and respond with appropriate reaction. This can be shown in changes of their life strategy and tactics. In this case an allelochemical plays the role of a signal; its effect does not depend on concentration, but releases a trigger connected with a genetic program. For example, seeds and bulbs of many herbal plants may rest i n the soil many years under cover of a forest and germinate only after the trees are removed; humidity, temperature, and extent of aeration often changed and several times there were apparently perfect conditions for germination, except that the seeds were under l i v i n g dominant plants. Obviously, the signal of mature trees holds the plant embryos i n the resting stage. In other cases, root exudates, for example, those of oats, stimulate germination of weeds i n the field. The research of G a j i c ' (15) from Yugoslavia found that the weed Agrostemma githago stimulates germination and growth of wheat. After many years of research she identified the allelochemicals, which is a mixture of amino acids, and includes allantoin and tryptophan. B y analogy G a j i c ' created the biostimulator Agrostemin, commercially marketed, which accelerates growth and germination, and increases productivity of many cultivated plants and native meadows by 10-15%. Agrostemin is used at remarkably low concentrations ~ about 10 g/ha. F o r a long time phenomena of this k i n d have attracted Soviet researchers' attention. In the 1920s G u r v i t c h (16) suggested the existence of so-called mitogenetic rays, which were supposed to stimulate cell division of yeast, lower plants, protozoa, and so on. But no such physical rays could be found and in the 1950s Moiseeva (17) showed that the signal for mitosis is indeed chemical i n nature. However, such signal substances have not been discovered yet. In recent years i n the agricultural high school i n Kharkov, Naumov and his students (18) demonstrated that a water extract from 2 k g of grain (wheat, rye,

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

42

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

oats, barley) when added to 700 k g o f the same or other cereal grain before sowing stimulates all vital functions o f the growing plants and reliably increases the harvest. Moreover, reciprocally soaking the seeds o f wheat i n seed extract of rye, and o f rye seeds i n wheat extract a few times, is claimed to facilitate fructification by hybridization between the two genuses. The soaking o f seeds i n extracts from some weeds made the new plants more resistant to the weeds used. These investigations are i n progress and sometimes may be o f doubtful validity, but suggest that allelochemicals of a plant can act on the expression of the heredity program i n another plant. In other words, chemical signals coming from l i v i n g plants causes recipient plants to follow a suitable life strategy — for example, resting. Perhaps the signal is other than chemical; i n particular maybe it is slow (circadian) vibrations of electric fields near l i v i n g plants (for example mature trees) and this is what prevents the germination o f resting seeds and b u l b s . Although concentration does not change essentially the character o f the recipient reaction, the signal is operativ the substance does not conve observation on birch and pine trees by Marchenko (19) i n Bryansk. W h e n a coniferous tree and birch grow together, their needles and twigs deviate on opposite sides. M a r c h e n k o calculated that the forces required to cause such deviation amount to a few hundred or thousand horsepower. T o show the dimensions o f the allelochemical effect with a l l three abovementioned paradigms: before the era o f mineral fertilizers, production of a crop required application o f 20 tons of manure per hectare; later, w i t h mineral fertilizers only several centner per hectare are required, with regulators such as herbicides, 2 to 20 k g , and regulators such as Agrostemin only a few grams per hectare. O f course, the above-mentioned paradigms do not exclude or contradict each other; each o f them concerns its o w n circle o f phenomena, but a l l these phenomena apply to allelopathy. I think that Soviet researchers contribute much to the development o f this science, w h i c h is very important for understanding the nature o f vegetation development and species evolution and especially for increasing production of consecutive crops.

Literature Cited 1. 2. 3. 4. 5. 6.

Levakovskii, N. Trudy Obshch. Estestv. Pri. Kazan Univ. 1971, 33-52. (Cited by Gortinskii, G. B. Bull. Mosk. Obshch. Ispyt. Prir. Otd. Biol. 1966, 71, (5), 128-133). Periturin, F. T. Izv. Mosk. s.-kh. Inst. 1913, kn. 4. Tokin, B. P. "Salubrious Poisons of Plants: Story of Phytoncides"; Publ. Leningrad University: Leningrad, 3d ed., 1980. Kholodnyi, N. G. Izv. AN. Arm. SSR 1944, (3), 31-42. Chernobrivenko, S. I. "Biological Role of Plant Excretions and Interspecific Interrelation in Mixed Culture"; Nauka: Moscow. Sanadze, G. Α. Izd. An. Gruz. SSR. (Tbilisi), 1961.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4. GRODZINSKY Allelopathy in the Soviet Union 43

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Grodzinsky, A. M . "Allelopathy in Life of Plants and Their Communities"; Naukova Dumka: Kiev, 1985 Molisch, H. "Der Einflus seiner Pflanzen auf die andere -- Allelopathie"; G. Fischer: Jena, 1937. Grodzinsky, A. M.; Kuznetsova, G. O. Ukr. Bot. Zh. 1960, 17, (1), 2930. Rice, E. L. "Allelopathy", 2nd ed; Academic Press: Orlando, Florida, 1984. Khailov, K. J. "Ecological Metabolism in the Sea"; Naukova Dumka: Kiev, 1971. Grodzinsky, A. M . "Principles of Chemical Interaction of Plants"; Naukova Dumka: Kiev, 1973. Grodzinsky, A. M., Bogdan, G. P., Golovko, Ε. Α., Dzyubenko, N . N.; Moroz, P. Αp.; Prutenskaya, Ν. I. "Allelopathic Soil Sickness"; Naukova Dumka: Kiev, 1984 Golovko, E. A. "Microorganisms In Allelopathy of Higher Plants"; Naukova Dumka: Kiev 1984 Gajic', D. Fragmenta Herbol Gurvitch, A. G. Moscov Moiseeva, M . M . Ukr. Bot. Zh. 1960, 17, (4), 29-33. Naumov, G. F.; Teteryatchenko, K. G.; Moskienko, N . F. Sborn. Nauch. Truji. Khark. s.-kh. Inst. (Kharkov) 1982, 288, 31-39. Marchenko, I. S. Br' anski: Izd. Tekhnol. Inst. 1973, 91 pp.

RECEIVED

June 9, 1986

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 5

Allelopathy Involving Microorganisms Case Histories from the United Kingdom J. M. Lynch Glasshouse Crops Research Institute, Littlehampton, West Sussex, BN17 6LP, United Kingdom Plant residues can provide substrates for the production of phytotoxic metabolites by soil microorganisms but they can also support the growth of pathogens and other deleterious micro-organisms This is illustrated by establishing crop residues and of decaying weed and grass residues that have been previously killed with herbicides. Short-chain acids accumulate,under anoxic conditions, which favor fermentative metabolism of bacteria. Such phytotoxins may damage the plant directly or predispose plants to infection by pathogens. However, plant residues may also be used as substrates for beneficial micro-organisms to produce plant nutrients, soil conditioners, and plant protection chemicals. There is scope to promote the beneficial microbial effects against the harmful by soil management and by inoculation. M i c r o - o r g a n i s m s produce a v a s t range o f m e t a b o l i t e s t h a t c a n p o t e n t i a l l y i n f l u e n c e p l a n t growth (_1/ 2, 3). T h i s a c t i o n c a n be positive or negative. Pathogens produce a n e g a t i v e e f f e c t b y p r o d u c i n g s p e c i f i c m e t a b o l i t e s o r enzymes. B e n e f i c i a l organisms may a c t d i r e c t l y on the p l a n t by p r o d u c i n g c h e m i c a l s t h a t s t i m u l a t e p l a n t growth o r enhance the uptake o f n u t r i e n t s . Indirect effects o f b e n e f i c i a l organisms i n c l u d e the s u p p r e s s i o n o f harmful organisms and the improvement o f s o i l s t r u c t u r e . T h i s d i v e r s e range o f m i c r o b i a l a c t i v i t i e s f a l l s w i t h i n the phenomenon o f a l l e l o p a t h y a s d e f i n e d by R i c e ( 4 ) . The d e s c r i p t i o n o f t h i s phenomenon i s u s e f u l / b u t the p r o c e s s e s c a n a l s o be d e s c r i b e d under t h e heading o f plant/microbe i n t e r a c t i o n s . Whereas a wide range o f p o t e n t i a l a l l e l o p a t h i c a g e n t s h a s been i d e n t i f i e d from s o i l m i c r o - o r g a n i s m s / i t has seldom been p r o v e n t h a t they a r e o f true e c o l o g i c a l s i g n i f i c a n c e . The minimum n e c e s s a r y c r i t e r i o n f o r t h i s i s t h a t the p r o d u c t s h o u l d o c c u r i n the form and

0097-6156/87/0330-0044$06.00/0 © 1987 A m e r i c a n C h e m i c a l Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

5.

LYNCH

Allelopathy

Involving

45

Microorganisms

c o n c e n t r a t i o n that i n f l u e n c e p l a n t growth. To demonstrate t h i s u s u a l l y i n v o l v e s v e r y m i l d e x t r a c t i o n p r o c e d u r e s because/ f o r example/ even m i l d a c i d s o r a l k a l i s can d e p o l y m e r i z e l i g n i n t o y i e l d p h e n o l s t h a t would o t h e r w i s e be i n e r t because i n the p o l y m e r i c state. A n o t h e r f a c t o r r a r e l y c o n s i d e r e d i s the zone o f the r o o t system s u b j e c t e d t o the m i c r o b i a l m e t a b o l i t e . I t i s l i k e l y t h a t the m e t a b o l i t e s w i l l o n l y be formed i n p a r t i c u l a r r e g i o n s o f the s o i l where t h e r e a r e s u i t a b l e s u b s t r a t e s f o r p r o d u c e r m i c r o - o r g a n i s m s and i t i s u n l i k e l y t h a t the e n t i r e r o o t system w i l l come under the influence of a metabolite. F o r example/ a c e t i c a c i d i s a common m i c r o b i a l f e r m e n t a t i o n p r o d u c t o f c e l l u l o s e and i s p h y t o t o x i c (j>/ 6). However/ i n ^ t r e a t i n g a s i n g l e r o o t t i p w i t h a s m a l l c o n c e n t r a t i o n (5 mol m )/ r o o t and s h o o t growth were s t i m u l a t e d . This was n o t o b s e r v e d when a g r e a t e r number o f t i p s were t r e a t e d o r g r e a t e r c o n c e n t r a t i o n s o f the a c i d were used ( T a b l e I ) . Lengths o f r o o t s were more s e n s i t i v e than t i p s t o i n h i b i t o r y c o n c e n t r a t i o n s o f the a c i d . Compensator response t o t r e a t m e n t o

Table

I.

Response o f B a r l e y Root E l o n g a t i o n t o Treatment w i t h 10 mol m

Region

treated

2 cm t i p 2 cm t i p 2 cm s e c t i o n 2 cm s e c t i o n Control*

No. o f roots treated

1 3 1 3 _

Acetic Acid

Mean l e n g t h o f non-treated root + s.e.m.

11.6 15.8 16.5 17.5 11.2

Mean l e n g t h o f treated root + s.e.m.

8.2 9.1 12.9 7.4 12.4

+ 1.7 + 0.2 + 0.5 + 1.3 + 0.7

+ + + + +

1.1 0.6 2.6 0.8 0.7

* Means o f p l a n t s where e i t h e r one o r t h r e e t i p s o r l e n g t h s were t r e a t e d w i t h p l a n t c u l t u r e s o l u t i o n o r where no r o o t s were t r e a t e d . Source: The New

Reproduced w i t h p e r m i s s i o n from K e f . Phytologist.

7.

Copyright

1982

Whereas a l i p h a t i c a c i d s can produce permanent s h o o t and t i l l e r damage/ when the a c i d s a r e removed from the growth medium/ r o o t growth can be promoted (8)/ presumably by compensatory a c t i o n . Q u i t e commonly m i c r o - o r g a n i s m s and t h e i r p r o d u c t s a r e b i o assayed together. When l e a v e s o f Anthoxanthum odoraturn were decomposed a e r o b i c a l l y the t o t a l s u s p e n s i o n c o n t a i n e d g r o w t h i n h i b i t o r y m i c r o - o r g a n i s m s b u t no c e l l - f r e e p h y t o t o x i c m e t a b o l i t e s (Table I I ) . By c o n t r a s t wheat straw degraded a n a e r o b i c a l l y y i e l d e d p h y t o t o x i c m e t a b o l i t e s b u t no g r o w t h - i n h i b i t i n g m i c r o - o r g a n i s m s .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

46

ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY T a b l e I I . E f f e c t o f M i c r o - o r g a n i s m s and T h e i r M e t a b o l i t e s Formed D u r i n g 14 Days D e c o m p o s i t i o n o f P l a n t R e s i d u e s on Longest Root L e n g t h (mm) o f B a r l e y S e e d l i n g s

Control (distilled water)

Residue

Micro­ organisms

Total suspension Filtrate

Anthoxanthum l e a v e s / a e r o b i c

75

79

74

59*

Wheat s t r a w / a n a e r o b i c

84

35***

37***

7

Significantly different

r e s u l t s i n d i c a t e d by * * *

Source: Reproduced w i t h p e r m i s s i o n from R e f . M a r t i n u s N i j h o f f Β. V .

Straw

9·

6

Ρ < .001; * Ρ ^ N

Copyright

.05

1984

Residues

In the d i r e c t d r i l l i n g ( n o - t i l l s e e d i n g ) p r a c t i c e i n the U n i t e d Kingdom/ s t r a w r e s i d u e s from the p r e c e d i n g c r o p a r e u s u a l l y b u r n t because poor c r o p e s t a b l i s h m e n t and y i e l d s c a n r e s u l t / p a r t i c u l a r l y on heavy s o i l s i n wet y e a r s ( 1 0 ) . S i m i l a r problems can o c c u r i n the c o n s e r v a t i o n t i l l a g e systems o f the P a c i f i c Northwest ( L . F . E l l i o t t and H . - H . Cheng/ t h i s v o l u m e ) . The o l d e r a g r i c u l t u r a l t e x t b o o k s i n d i c a t e t h a t t h i s i s due t o the s t r a w h a v i n g a h i g h C : N r a t i o ( c . 100:1) compared w i t h the decomposer m i c r o - o r g a n i s m s ( c . 5:1) and t h a t Ν o t h e r w i s e a v a i l a b l e to p l a n t s i s immobilized i n t o m i c r o b i a l biomass. It i s quite easy t o demonstrate t h i s e f f e c t i n p o t experiments/ where s e e d l i n g s show o b v i o u s s i g n s o f Ν d e f i c i e n c y i n the p r e s e n c e o f s t r a w . However/ i t i s l i k e l y t h a t i n the f u l l c r o p p i n g season the i m m o b i l i z e d Ν w i l l s u b s e q u e n t l y become a v a i l a b l e t o the c r o p / a l t h o u g h l i t t l e f i r m e x p e r i m e n t a l e v i d e n c e has been o b t a i n e d t o s u p p o r t t h i s h y p o t h e s i s . Indeed Ν i m m o b i l i z e d i n m i c r o b i a l biomass d u r i n g w i n t e r c o u l d p r e v e n t w i n t e r l e a c h i n g and t h e r e f o r e s t r a w c o u l d even be b e n e f i c i a l t o the Ν c y c l e . C e r t a i n l y e x t e n s i v e t r i a l s i n the UK show t h a t a p p l i c a t i o n o f seedbed Ν has l i t t l e o r no b e n e f i c i a l e f f e c t on c r o p p r o d u c t i v i t y compared w i t h normal a p p l i c a t i o n t i m e s . There i s a need f o r f u r t h e r s t u d i e s on t h i s t o p i c . One model l a b o r a t o r y s t u d y has demonstrated t h a t the N - i m m o b i l i z a t i o n p o t e n t i a l i s g r e a t l y r e d u c e d when f e r t i l i z e r Ν i s p l a c e d s e v e r a l c e n t i m e t r e s below the s o i l s u r f a c e ( 1 1 ) . The Ν t i e - u p i s much s m a l l e r when r e s i d u e s a r e l e f t on the s o i l s u r f a c e a s opposed t o b e i n g mixed i n the s o i l . In the USA some e v i d e n c e (12) has been r e p o r t e d f o r Pythium s p p . i n c r e a s i n g a s a consequence o f d i r e c t - d r i l l i n g i n t o s t r a w b u t i n the UK l i t t l e e v i d e n c e has been r e p o r t e d a s y e t f o r pathogens b u i l d i n g - u p on straw/ a l t h o u g h t h i s i s l i k e l y t o v a r y g r e a t l y between l o c a t i o n s and c o n d i t i o n s . Straw i s a f a v o r a b l e s u b s t r a t e f o r p a t h o g e n i c Fusarium s p p . and Pythium s p p . Growth-inhibitory b a c t e r i a may a l s o be a p a r t o f the problem ( L . F . E l l i o t t and H . - H . Cheng, t h i s v o l u m e ) .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

5.

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47

Microorganisms

A l l the present evidence p o i n t s t o p h y t o t o x i c s t e a m - v o l a t i l e f a t t y a c i d s / p a r t i c u l a r l y a c e t i c , b e i n g a major m i c r o b i o l o g i c a l f a c t o r r e s p o n s i b l e f o r t h e c r o p damage, and t h e c o n d i t i o n s o f e c o l o g i c a l s i g n i f i c a n c e r e f e r r e d t o e a r l i e r have been s a t i s f i e d . However, the t o x i n i s produced o n l y i n the s t r a w t i s s u e and i t s c o n c e n t r a t i o n d e c l i n e s e x p o n e n t i a l l y w i t h d i s t a n c e from t h e s t r a w (13). A c o r r e l a t i o n o f s o i l a c e t i c a c i d content with p h y t o t o x i c i t y i s therefore n e i t h e r expected n o r found. Weed R e s i d u e s When dense i n f e s t a t i o n s o f weeds a r e k i l l e d w i t h h e r b i c i d e s , a s i t u a t i o n a n a l o g o u s t o the s t r a w problem c a n o c c u r because a l a r g e amount o f r e a d i l y d e g r a d a b l e s u b s t r a t e becomes a v a i l a b l e t o t h e saprophytic microbial population of s o i l . With h e r b i c i d e s t h a t a r e t r a n s l o c a t e d , such a s g l y p h o s a t e , t h e r e i s a chance t h a t t h e h e r b i c i d e i t s e l f would be r e l e a s e d t o the s o i l , b u t t h i s has n o t been found t o be the cas p r o p i o n i c and b u t y r i c a c i d grass rhizome. However c r o p damage i s u s u a l l y o b s e r v e d i n d r y (50% water s a t u r a t i o n ) s o i l s and t h i s c o u l d be r e p e a t e d i n g l a s s h o u s e t r i a l s (15). Thus i t appeared u n l i k e l y t h a t t h e n e c e s s a r y a n a e r o b i c c o n d i t i o n s f o r b a c t e r i a l f e r m e n t a t i v e metabolism would n o r m a l l y exist. Dry s o i l s f a v o r the development o f the pathogen Fusarium culmorum (16) and l a r g e p o p u l a t i o n s o f t h i s fungus have been found on decomposing rhizomes o f the weed. However, even under d r y c o n d i t i o n s s m a l l c o n c e n t r a t i o n s o f the o r g a n i c a c i d c a n form and u n l e s s the pathogen p o p u l a t i o n i s v e r y l a r g e the a c i d a p p e a r s t o p r o v i d e a compounding s t r e s s on the h o s t p l a n t ( T a b l e I I I ) .

Table I I I .

E f f e c t o f 5 mM A c e t i c A c i d and Fusarium culmorum on t h e Growth o f B a r l e y S e e d l i n g s

Mean l e n g t h o f f i r s t t h r e e l e a v e s (mm) 12 days a f t e r g e r m i n a t i o n Inoculum d e n s i t y (spores/ml)

7 10

0

S e e d l i n g s t r e a t e d w i t h 5 mM a c e t i c a c i d No a c i d t r e a t m e n t Results with d i f f e r e n t (P < 0.05)

b 120 140

a

letters are significantly

c 98Γ 118

Ζ 10

D

cd 89, 114

~7~ 10°

cd 83 , 78

different

N

Source: Reproduced w i t h p e r m i s s i o n from R e f . Blackwell S c i e n t i f i c Publications Ltd.

17.

Copyright

American Chemical Society Library 1155 16th St, N.W. Washington, D.C 20038

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1982

ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY

48

Permanent N e u t r a l

Grassland

I t c a n be d i f f i c u l t t o e s t a b l i s h new g r a s s e s i n t o permanent g r a s s ­ land. T h i s may be an example o f a l l e l o p a t h y and the r e a s o n why some s p e c i e s dominate o l d g r a s s l a n d . Newman (18) p r e p a r e d a r e v i e w on whether a l l e l o p a t h y i s e c o l o g i c a l a d a p t a t i o n o r a c c i d e n t . His r e s e a r c h team a t B r i s t o l U n i v e r s i t y i n v e s t i g a t e d a l l e l o p a t h y w i t h i n permanent g r a s s l a n d u s i n g p o t e x p e r i m e n t s w i t h ' d o n o r or ' t r e a t m e n t and ' r e c e i v e r ' o r ' t e s t ' s p e c i e s o f d i f f e r e n t g r a s s e s (19/ 20/ 21/ 22/ 2 3 ) . These s t u d i e s showed/ f o r example, t h a t the decomposing r o o t s o f Rumex a c e t o s a had the g r e a t e s t i n h i b i t i n g e f f e c t on f o u r s p e c i e s ( T a b l e I V ) . When the n u t r i e n t c o n t e n t o f Loiiurn perenne a s ' t e s t ' s p e c i e s was a n a l y z e d / R^ a c e t o s a r e s i d u e s gave r i s e t o a s i m i l a r Ρ c o n t e n t i n the t e s t s p e c i e s a s the P - d e f i c i e n t s o i l a l o n e ; t h e r e was no s u c h e f f e c t on Ν c o n t e n t ( T a b l e V). They c o n c l u d e d t h a t the a l l e l o p a t h y a c t e d by the r e s i d u e s o f the t r e a t m e n t s p e c i e s f a i l i n g t o make Ρ a v a i l a b l e t o the t e s t s p e c i e s / an e f f e c t whic 1

1

T a b l e IV. Dry Weights (mg) o f Shoots o f ' T e s t ' P l a n t s o f G r a s s l a n d S p e c i e s Grown on S o i l s C o n t a i n i n g the Decomposing Roots o f 'Treatment' Species

'Test'

'Treatment'

species

Anthoxanthum odoratum (Ao) L o l i u m perenne (Lp) Plantago l a n c e o l a t a (Pi) Rumex a c e t o s a (Ra) Nil

species

Ao

Lp

Pi

61b 155a 199a 47b 44b

122a 180a 216a 38b 62b

215a 456a 326a 18c 77b

Ra

174a 73ab 41c 17c 103ab

V a l u e s n o t s h a r i n g the same s m a l l l e t t e r s i n each column d i f f e r s i g n i f i c a n t l y (P < 0.05) Source: The New

Reproduced w i t h p e r m i s s i o n from R e f . Phytologist.

24.

Copyright

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1981

5.

LYNCH

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Table V. N i t r o g e n and Phosphorus i n Shoots o f ' T e s t Plants Grown on S o i l s C o n t a i n i n g the Decomposing Roots o f T r e a t m e n t Species 1

1

1

L o l i u m perenne

•Treatment

1

N%

species

P%

0.217a 0.195a 0.201a 0.140b 0.157b

2.95ab 2.19c 2.39bc 2.51bc 3.22a

Anthoxanthum odoratum L o l i u m perenne Plantago l a n c e o l a t a Rumex a c e t o s a Nil (wet)

V a l u e s n o t s h a r i n g the sam s i g n i f i c a n t l y (P < 0.05) Source: Reproduced w i t h p e r m i s s i o n from R e f . Blackwell S c i e n t i f i c Publications Ltd.

23.

Copyright

1979

In p a r a l l e l s t u d i e s i t was demonstrated t h a t the r h i z o s p h e r e p o p u l a t i o n s of g r a s s l a n d s p e c i e s c o u l d a f f e c t each other/ there b e i n g a l a r g e i n c r e a s e i n f u n g a l biomass ( T a b l e V I ) . The s i g n i f i c a n c e of t h i s observation i s s t i l l unclear. There have been r e l a t i v e l y few q u a n t i t a t i v e s t u d i e s o f m i c r o - o r g a n i s m s on p l a n t r o o t s i n m o n o c u l t u r e l e t a l o n e i n mixed s t a n d s . Such a p p r o a c h e s s h o u l d prove u s e f u l i n a s s e s s i n g the p o t e n t i a l magnitude o f microbial metabolic processes i n a l l e l o p a t h i c i n t e r a c t i o n s .

Table VI. B a c t e r i a l Cover and Fungal Mycelium Length on Root S u r f a c e s o f L o l i u m perenne (Lp) and P l a n t a g o l a n c e o l a t a (Pi)

B a c t e r i a l cover (%)

Separate Together

Lp

Pi

4.3 6.3

5.6 5.8

Fungi (mm mm" )

Mean p l a n t w e i g h t (g)

2

Lp

Pi

Lp

Pi

0.7 2.1

1.8 2.9

0.71 1.01

0.69 0.69

S o u r c e : Reproduced w i t h p e r m i s s i o n from R e f . Macmillan Journals L t d .

19·

Copyright

1974

Reseeding O l d G r a s s l a n d When g r a s s l a n d becomes u n p r o d u c t i v e because o f p o o r s p e c i e s c o m p o s i t i o n / the o l d sward c a n be k i l l e d o f f w i t h h e r b i c i d e and new g r a s s e s r e s e e d e d by d i r e c t - d r i l l i n g i n t o the t r e a t e d s w a r d . This

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY

50

p r e s e n t s a s i t u a t i o n o f m i c r o b i a l d e c o m p o s i t i o n which i s a n a l o g o u s t o the d e c o m p o s i t i o n o f s t r a w and weed r e s i d u e s . Shoots o f a range o f g r a s s s p e c i e s were t o x i c t o o t h e r g r a s s e s and c l o v e r when decomposed a n a e r o b i c a l l y ( T a b l e V I I ) . The photot o x i c i t y seemed t o be caused by o r g a n i c a c i d s and was l e s s a f t e r 20 days o f d e c o m p o s i t i o n than a f t e r 1 0 . F e s t u c a r u b r a , A g r o s t i s s t o l o n i f e r a / and A l o p e c u r u s p r a t e n s i s r e s i d u e s were the most t o x i c ; which t o an e x t e n t i s c o n s i s t e n t w i t h f i e l d o b s e r v a t i o n s t h a t r e s i d u e s o f the former two s p e c i e s a r e p a r t i c u l a r l y d i f f i c u l t t o seed i n t o . When the s h o o t s were decomposed a e r o b i c a l l y , some were t o x i c a f t e r 10 days b u t t h i s t o x i c i t y d i s a p p e a r e d a f t e r 20 days when some r e s i d u e s c o u l d s t i m u l a t e p l a n t growth ( T a b l e VII).

T a b l e VII.

E f f e c t o f S o l u t i o n s Produced a f t e r 10 Days D e c o m p o s i t i o n o f P l a n t R e s i d u e s on Root E x t e n s i o n

Residue

Alopecurus myosuroides

Fr

Hi

Lp

Poa annua

Pt

Trifolium repens

(a) Aerobic Agrostis stolonifera Alopecurus p r a t e n s i s Anthoxanthum odoraturn Festuca rubra (Fr) H o l c u s l a n a t u s (Hi) L o l i u m perenne (Lp) Poa t r i v i a l i s (Pt) Control

13 13 13 13 13 11 11 11

7 6 7 11 6 6 6 6

11 10 10 10 9 10 11 11

28 35 25 28 25 24 26 30

8 9 10 10 9 10 11 8

4 5 6 7 3 6 7 5

38 42 34 30 32 24 32 46

(b) Anaerobic Agrostis stolonifera Alopecurus p r a t e n s i s Anthoxanthum odoratum Festuca rubra (Fr) Holcus l a n a t u s ( H i ) L o l i u m perenne (Lp) Poa t r i v i a l i s (Pt) Control

0 0 15 0 15 6 11 10

0 5 7 0 9 8 4 7

3 0 14 4 21 10 7 10

9 3 25 5 36 26 16 32

5 0 10 0 12 10 7 10

0 0 6 0 11 7 3 5

2 0 33 0 37 22 21 48

C o n t r o l c o n t a i n e d s o i l and water o n l y Source: The New

Reproduced w i t h p e r m i s s i o n from R e f . Phytologist.

24.

Copyright

1981

Even though t o x i n s c o u l d a t l e a s t i n p a r t be i n v o l v e d , F u s a r i u m culmorum a g a i n seemed t o be r e s p o n s i b l e f o r the damage ^25). Whereas the f u n g i c i d e s carbendazim and d r a z o x o l o n were e f f e c t i v e i n c o n t r o l l i n g the d i s e a s e , c a l c i u m p e r o x i d e was a l s o e f f e c t i v e (26). T h i s compound had the added advantage o f r e l e a s i n g a l k a l i t o

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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n e u t r a l i s e o r g a n i c a c i d t o x i n s and oxygen t o m i n i m i z e o r g a n i c formation (27).

C o n c l u s i o n : The Scope f o r S o i l

acid

Biotechnology

The p o t e n t i a l o f m a n i p u l a t i n g s o i l m i c r o - o r g a n i s m s / e s p e c i a l l y f o r the u t i l i z a t i o n o f c r o p r e s i d u e s , has been o u t l i n e d ( 2 8 ) . F o r example, a c c e l e r a t i n g s t r a w breakdown c a n reduce the "time p e r i o d i n which o r g a n i c a c i d t o x i n s a r e produced ( 2 9 ) . By i n o c u l a t i n g s t r a w w i t h a c o n s o r t i u m o f a c e l l u l o l y t i c fungus and a n a n a e r o b i c N ^ f i x i n g b a c t e r i u m i n the l a b o r a t o r y , s t r a w breakdown has been a c c e l e r a t e d and the r e s u l t i n g r e s i d u e i s e n r i c h e d i n Ν ( T a b l e V I I I ) . In o t h e r s i m i l a r a s s o c i a t i o n s the c e l l u l o l y t i c fungus has b i o c o n t r o l p o t e n t i a l a g a i n s t r o o t d i s e a s e , and a s s o c i a t e d p o l y s a c c h a r i d e p r o d u c i n g b a c t e r i a c a n a s s i s t w i t h the s t a b i l i z a t i o n o f s o i l s t r u c t u r e (211). I f such approaches c o u l d be c a r r i e d t o p r a c t i c e i n the f i e l d , a new e r a o f presents a great challeng c e r t a i n l y n o t be r e a l i s e d u n t i l the e c o l o g i c a l a s p e c t s o f a l l e l o ­ pathy a r e c l e a r l y understood.

Table V I I I .

D e c o m p o s i t i o n o f N o n - S t e r i l e Straw C o n t a i n e d i n G l a s s Columns a t 25°C f o r 8 Weeks

Ν gain

Treatment

Non-inoculated Pénicillium corylophilum + C l o s t r i d i u m butyricum

Decomposition r a t e c o n s t a n t , k (d~ )

Per g straw lost

(mg)

Per g original straw

0.0096

8.8

2.8

0.0139

11.5

5.0

Source: Reproduced w i t h p e r m i s s i o n from R e f . 30. Society f o r General Microbiology.

Copyright

1983

Acknowledgments The comments o f Dr L . F . E l l i o t t and Dr E . I . Newman a r e much appreciated. P e r m i s s i o n s t o p u b l i s h d a t a i n the t a b l e s i s a s f o l l o w s : The New P h y t o l o g i s t T r u s t ( T a b l e s I , IV and V I I ) , M a r t i n u s N i j h o f f (Table I I ) , Blackwell S c i e n t i f i c P u b l i c a t i o n s (Tables I I I and V ) , M a c m i l l a n J o u r n a l s ( T a b l e VI) and S o c i e t y f o r G e n e r a l M i c r o b i o l o g y (Table V I I I ) .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

52

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

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

Lynch, J.M. CRC Crit. Rev. Microbiol. 1976, 5, 67-107. Lynch, J.M. In "Soil Organic Matter and Biological Activity"; Vaughan, D.; Malcolm, R.E., Eds.; Martinus Nijhoff: The Hague, 1985; pp. 151-74. McCalla, T.M.: Norstadt, F.A. Agric. Ehviron. 1974, 1, 153-74. Rice, E.L. "Allelopathy"; Academic: Orlando, 1984, 2nd edition. Lynch, J.M. J . Appl. Bact. 1977, 42, 81-7. lang, C.S.; Waiss, A.C. J . Chem. Ecol. 1978, 4, 225-32. Gussin, E.J.; Lynch, J.M. New Phytol. 1982, 92, 345-8. Cochran, V.L.; Bikfasy, D.; Elliott, L.F.; Rapendick, R.I. Plant Soil 1983, 74, 369-77. Chapman, S.J.; Lynch, J.M. Plant Soil 1984, 74, 457-9. Lynch, J.M.; Ellis, F.B.; Harper, S.H.T.; Christian, D.G. Agric. Environ. 1980, 5, 321-8. Cochran, V.L.; Elliott Amer. S. 1980, 44 Cook, R.J.; Sitton, J.W.; Waldher, J.T. Plant Disease 1980, 64, 102-3. Lynch, J.M.; Gunn, K.B.; Ranting, L.M. Plant Soil 1980, 56, 93-8. Penn, D.J.; Lynch, J.M. New Phytol. 1982, 90, 51-5. Penn, D.J.; Lynch, J.M. J . Appl. Ecol. 1981, 18, 669-74. Papendick, R.J.; Cook, R.J. Phytopathology 1974, 64, 358-63. Penn, D.J.; Lynch, J.M. Plant Pathol. 1982, 31, 39-43. Newman, E.I. "Biochemical Aspects of Animal and Plant CoEvolution"; Barbourne, J.B., Ed.; Academic: London, 1978, pp. 327-42. Christie, P.; Newman, E.I.; Campbell, R. Nature (Lond.) 1974, 250, 570-1. Newman, E.I.; Rovira, A.D. J . Ecol. 1975, 63, 727-37. Newman, E.I.; Miller, M.H. J . Ecol. 1977, 65, 399-411. Newbery, D.McC.; Newman, E.I. Oecologia (Berl.) 1978, 33, 361-80. Newbery, D.McC. J . Appl. Ecol. 1979, 16, 613-22. Gussin, E.J.; Lynch, J.M. New Phytol. 1981, 89, 449-57. Gussin, E.J.; Lynch, J.M. J . Gen. Microbiol. 1983, 129, 271-5. Gussin, E.J.; Lynch, J.M. Trans. Brit. Mycol. Soc. 1983, 81, 426-9. Lynch, J.M.; Harper, S.H.T.; Sladdin, M. Curr. Microbiol. 1981, 5, 27-30. Lynch, J.M. "Soil Biotechnology. Microbiological Factors in Crop Productivity", Blackwell Scientific Publications: Oxford, 1983. Lynch, J.M.; Elliott, L.F. Soil Biol. Biochem. 1983, 15, 221-2. Lynch, J.M.; Harper, S.H.T. J . Gen. Microbiol. 1983, 129, 251-3. Lynch, J.M.; Harper, S.H.T. Phil. Trans. R. Soc. Lond. 1985, B310, 221-6.

RECEIVED

June

20,1986

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 6

Allelopathy in Desert Ecosystems Jacob Friedman Department of Botany, George S. Wise Faculty of Life Sciences, Tel Aviv University, 69978, Israel

Allelopathy and autotoxicity in desert ecosystems in both hemispheres generalizations ar allelopathic potential are often adult perennials, members of the Compositae or Labiatae, that are capable of reducing germination and/or growth of various annuals or of their own seedlings; (b) allelochemicals emanated from aggressive plants are either common secondary metabolites (terpenes, terpenoids, or phenolic compounds), or inorganic salts. Allelochemicals, although frequently nonspecific, do not constitute general phytocides. They may reach susceptible plants through the soil in different ways, either washed off from the fresh or dried shoots by rainfall, released as volatile substances later absorbed by the s o i l , or released in part from the mature or decomposed roots; (c) some ecological factors in the desert favor production of allelochemicals, e.g. water or mineral stresses, or grazing, whereas other factors improve the preservation of the allelochemicals, i . e . low rates of leaching, or reduced activity of the soil microflora. Wide desert areas are covered with sandy soils containing small amounts of organic material and this may account for a slow release of allelochemicals. Dependent upon such interactions, allelopathy may be manifested in a certain area and not at a l l in a similar one despite the presence of very similar plant populations in both areas. It is likely that further observations in desert areas that show a gradient of aridity will allow exploration of additional allelopathic effects.

0097-6156/87/0330-0053$06.00/0 © 1987 A m e r i c a n C h e m i c a l Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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The s p a r s e v e g e t a t i o n i n a r i d environments p r o v i d e s an e x c e l l e n t model f o r s t u d y i n g g e r m i n a t i o n , p r o g r e s s i v e growth, and m o r t a l i t y o f i n d i v i d u a l p l a n t s . No wonder, t h e n , t h a t p i o n e e r i n g work on a l l e l o p a t h y was p e r f o r m e d i n v a r i o u s d e s e r t s of the w o r l d , and some o f t h i s work w i l l be d e s c r i b e d h e r e i n . F i r s t , however, I raise the c o n j e c t u r e t h a t a r i d c o n d i t i o n s may f a v o r p l a n t s p e c i e s endowed w i t h a l l e l o c h e m i c a l p o t e n t i a l , and t h i s more o f t e n than c u r r e n t l y r e a l i z e d . I b e l i e v e the a l l e l o c h e m i c a l e f f e c t i n t e r a c t s w i t h o t h e r e n v i r o n m e n t a l f a c t o r s and i s d i m i n i s h e d o r enhanced i n a c c o r d a n c e w i t h l o c a l changes i n the a r i d i t y . Because of the s p a t i a l and t e m p o r a l dependence of p l a n t i n t e r a c t i o n s , i n c o n s i s t e n c i e s i n the o b s e r v a t i o n s made a t d i f f e r e n t l o c a l i t i e s a r e i n e v i t a b l e , and f o r t h o s e who s t r u g g l e t o p r o v e a l l e l o p a t h y an element of u n c e r t a i n t y i s t h u s i n t r o d u c e d . I t i s t o be hoped t h a t an e x a c t i n g c o m b i n a t i o n o f e c o l o g i c a l o b s e r v a t i o n s w i t h b i o c h e m i c a l p r o c e d u r e s w i l l e n a b l e the t r a c i n g o f pathways t a k e n by p h y t o t o x i n s from the p r o d u c e r p l a n t t o the s u s c e p t i b l e one, and w i l l e v e n t u a l l y a l l o w a q u a n t i f i c a t i o n o f the a l l e l o p a t h i c e f f e c t r e v i e w some o f the work d e s e r t ecosystems i n the w o r l d , t o p o i n t out f a c t o r s t h a t may modify the e f f e c t of a g g r e s s i v e p l a n t s o r the response o f s u s c e p t i b l e ones, and t o e l a b o r a t e on the methodology employed t o a s s e s s a l l e l o p a t h y or a u t o t o x i c i t y . A l l e l o p a t h y i n D e s e r t s o f the Western Hemisphere The b e s t documentation o f p l a n t a l l e l o p a t h y d e r i v e s from the U n i t e d S t a t e s . For i n s t a n c e , i n the Borego V a l l e y of the Mojave D e s e r t , where the a n n u a l r a i n f a l l i s 200-255 mm, Went (1) n o t e d t h a t o n l y a few a n n u a l s were a s s o c i a t e d w i t h l i v i n g shrubs of E n c e l i a farinosa ( C o m p o s i t a e ) , whereas the d e n s i t y of a n n u a l s i n the v i c i n i t y o f dead E n c e l i a s h r u b s , as w e l l as near l i v i n g shrubs o t h e r than E n c e l i a , was much h i g h e r . Annuals n o t a s s o c i a t e d w i t h l i v i n g E n c e l i a were : Malacothrix californica, Emmenanthe p e d u l i f l o r a , Rafinesquia neomexicana ,and H i l a r i a r i g i d a . I t was f i r s t supposed t h a t t o x i c i t y i s i n d u c e d by the r o o t s o f E n c e l i a , but Gray and Bonner (2) were u n a b l e t o show any i n h i b i t i o n by such r o o t s , whereas e i t h e r f r e s h o r d r i e d l e a v e s o f E n c e l i a , when added t o sand-grown c u l t u r e s of tomato, i n h i b i t e d growth o f the s e e d l i n g s . Aqueous e x t r a c t o f E n c e l i a l e a v e s (0.025% on d r y weight b a s i s ) was e x t r e m e l y t o x i c t o c o r n and pepper, b u t b a r l e y , o a t s and s u n f l o w e r were o n l y s l i g h t l y a f f e c t e d . The p h y t o t o x i c p r i n c i p l e , c o l o r l e s s n e e d l e s w i t h a p l e a s a n t odor, was c r y s t a l l i z e d f r o m e x t r a c t s o f p l a n t s growing w i l d i n the C o l o r a d o D e s e r t , C a l i f o r n i a and i d e n t i f i e d as 3 - a c e t y l - 6 methoxybenzaldehyde ( F i g u r e 1) ( 3 ) . At a c o n c e n t r a t i o n of 1.4 mM, t h i s compound k i l l e d , w i t h i n 24 h, 100% o f tomato s e e d l i n g s grown i n sand c u l t u r e s . When t e s t p l a n t s were grown i n f e r t i l e garden s o i l , the e f f e c t was s m a l l e r , but Gray and Bonner have p o i n t e d out t h a t , i n n a t u r e , E n c e l i a i s common on sandy s o i l s and the i n h i b i t o r y e f f e c t t h e r e f o r e i s t o be e x p e c t e d . These a u t h o r s , however, d i d not a s s e s s the r e s p o n s e t o the p h y t o t o x i n of such a n n u a l s as were a b s e n t from the v i c i n i t y o f E n c e l i a . Nor d i d t h e y attempt t o i s o l a t e the

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

FRIEDMAN

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0 1. 8 - c i n e o l e

a-pinene

camphor

COCH.,

CHO OCH 0-pinene

Figure

j.H

C0 H 2

3

3-acetyl-6-methoxybenzaldehyde

1·

W

trans-cmnamk a c i d

A l l e l o c h e m i c a l s from V a r i o u s D e s e r t P l a n t s .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY

p h y t o t o x i n from s o i l s around t h e s e s h r u b s . I n t e r e s t i n g l y , l e a v e s o f E n c e l i a c o l l e c t e d i n A r i z o n a y i e l d e d a d i f f e r e n t , as y e t unidentified toxin (3). Guayule ( P a r t h e n i u m argentatum, Compositae), a r u b b e r p r o d u c i n g p l a n t , i s common i n t h e Chihuahua D e s e r t (Mexico and South T e x a s ) , where s p a r s e p o p u l a t i o n s of t h i s shrub grow a t an a l t i t u d e of 700-3500 m above sea l e v e l , r e c e i v i n g 250 mm of a n n u a l r a i n f a l l ( 4 ) . When t h i s p l a n t was grown under n u r s e r y c o n d i t i o n s , m a r g i n a l rows p r o d u c e d l a r g e r p l a n t s t h a n rows i n the c e n t e r , and r o o t s o f one p l a n t d i d not i n t e r m i n g l e w i t h those o f n e i g h b o r i n g plants· From g r a v e l c u l t u r e s w i t h Hoagland s o l u t i o n , Bonner and G a l s t o n (5) i s o l a t e d t r a n s - c i n n a m i c a c i d ( F i g u r e 1), which was found a u t o t o x i c t o g u a y u l e s e e d l i n g s grown i n s i m i l a r g r a v e l c u l t u r e . The a u t o t o x i c 3

e f f e c t was s t i l l e v i d e n t even a t c o n c e n t r a t i o n s of 6.7 χ 10~ mM, and a t 1 mM i t caused an 80-90% r e d u c t i o n i n growth of guayule s e e d l i n g s . Subsequent attempts t o i s o l a t e t h i s o r o t h e r p h y t o t o x i c compounds from v a r i o u s s o i l s s u p p o r t i n g guayule p r o v e d u n s u c c e s s f u l (6,7) . In a s e r i e s of p a p e r (8-11) r e p o r t e d t h a t i n a r e a s around shrubs o f S a l v i a leucophylla, S. apiana, S. m i l l i f e r a ( L a b i a t a e ) , or A r t e m i s i a californica (Compositae), g r a s s e s and h e r b s a r e s u p p r e s s e d . The p e r t i n e n t o b s e r v a t i o n s were made i n the Santa Ynez V a l l e y o f Santa B a r b a r a County, C a l i f o r n i a , a r e g i o n w i t h an average a n n u a l r a i n f a l l o f 200-250 mm. Zones e n t i r e l y d e v o i d o f a n n u a l p l a n t s o c c u r r e d w i t h i n 60-90 cm from the canopy of each shrub, whereas f u r t h e r o u t , t o about 6 m, v a r i o u s g r a d a t i o n s of i n h i b i t i o n were o b s e r v e d . V o l a t i l e m a t e r i a l s from the c r u s h e d l e a v e s o r t w i g s of t h e named shrubs i n h i b i t e d r o o t growth i n s e e d l i n g s o f cucumber o r o a t s , as w e l l as of some a n n u a l s common i n the a r e a . H i g h e s t i n h i b i t i o n was e x e r t e d by A r t e m i s i a c a l i f o r n i c a , w h i l e no i n h i b i t i o n was o b t a i n e d w i t h m a c e r a t e d young o r mature r o o t s o f S a l v i a l e u c o p h y l l a or t h e i r l e a c h a t e s (10) · S e v e r a l t e r p e n e s and t e r p e n o i d s , e.g. a-pinene, β - p i n e n e , camphor, and c i n e o l e , r e l e a s e d from t h e shrubs canopy were i d e n t i f i e d ( F i g u r e 1 ) . Of t h e s e , camphor d i s p l a y e d the h i g h e s t t o x i c i t y . The agent t r a n s p o r t i n g the p h y t o t o x i n s i n t o the s o i l was f i r s t assumed t o be dew (9) but s u b s e q u e n t l y i t was shown t h a t d r y , r a t h e r t h a n wet, s o i l s a b s o r b e d more o f the v o l a t i l e p h y t o t o x i n s and t h e s e p r o v e d t o be t o x i c f o r the s e e d l i n g s o f a n n u a l s common i n the r e g i o n s s t u d i e d (11) · T h i s l e d t o the c o n j e c t u r e t h a t v o l a t i l e compounds accumulate i n the s o i l d u r i n g the l o n g dry summer and i n the w i n t e r , when g e r m i n a t i o n commences, t o be r e l e a s e d by r a i n f a l l i n t o the s o i l m i c r o s p h e r e , where they i n h i b i t growth o f the a n n u a l s (12) . A l l e l o p a t h y i n Deserts

o f the E a s t e r n Hemisphere

In t h e Negev D e s e r t o f I s r a e l , near Sede Boquer, w i t h i n a r e g i o n b o a s t i n g up t o 100 mm of r a i n f a l l p e r y e a r ( F i g u r e 2 ) , Friedman e t a l . (13) o b s e r v e d t h a t on s o u t h - f a c i n g s l o p e s the y i e l d of a n n u a l s was 6-8 times t h a t on a d j a c e n t n o r t h - f a c i n g s l o p e s ( F i g u r e 3 ) . T h i s was c o n f i r m e d b o t h by the a n n u a l - p l a n t s d e n s i t y and by the dry m a t t e r y i e l d (g/m ), and p r o v e d t r u e d u r i n g 4 y e a r s , d e s p i t e the 2

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Figure

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Map showing L o c a t i o n o f the Study Area Isohyetal Lines.

Including

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

F i g u r e 3.

Annual Y i e l d o f Annuals on Southern and N o r t h e r n S l o p e s i n Sede Boquer, i n t h e Years 1964-1967.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

6.

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f a c t t h a t the s o u t h - f a c i n g s l o p e s a r e much more a r i d t h a n t h e n o r t h - f a c i n g ones (due t o h i g h s o l a r r a d i a t i o n , h i g h e r s a l i n i t y , and h i g h e r water run-off)· I t was n o t e d , however, t h a t t h e n o r t h - f a c i n g s l o p e s are dominated by the a r o m a t i c semi-dwarf shrub A r t e m i s i a h e r b a - a l b a (Compositae) ( F i g u r e s 4a & b ) , whereas t h e s o u t h - f a c i n g ones are dominated by a nonaromatic shrub ( Zygophyllum dumpsurn , Z y g o p h y l l a c e a e ) ( F i g u r e 5 ) . One y e a r f o l l o w i n g removal o f p e r e n n i a l s from b o t h s l o p e s , the y i e l d o f a n n u a l s on the n o r t h e r n s l o p e i n c r e a s e d s i g n i f i c a n t l y ( F i g u r e s 6a & b ) , a l b e i t i t d i d n o t match t h a t on the p e r e n n i a l - f r e e s o u t h e r n s l o p e . Counts made d u r i n g g e r m i n a t i o n time showed t h a t d e n s i t y of the s e e d l i n g s o f a n n u a l s i n the v i c i n i t y o f A r t e m i s i a h e r b a - a l b a was o n l y h a l f t h a t o b s e r v e d 100 cm from the canopy. On the s u s p i c i o n t h a t v o l a t i l e a l l e l o c h e m i c a l s were r e s p o n s i b l e f o r the d e c i m a t i o n o f a n n u a l s on the n o r t h - f a c i n g s l o p e s , l a b o r a t o r y t e s t s were u n d e r t a k e n u s i n g s m a l l p l a s t i c b e a k e r s ( F i g u r e 7 ) . These t e s t s c o n f i r m e d t h a t Artemisia h e r b a - a l b a , l i k e A. c a l i f o r n i c a , produces v o l a t i l e phytotoxins. Thus, one gra s e a l e d 50-ml f l a s k a r r e s t e s p e c i e s common i n the s t u d i e d a r e a , whereas no such i n h i b i t i o n was i n d u c e d by the l e a v e s o f Zygophyllum used as a c o n t r o l ( 1 3 ) . Of the s p e c i e s examined f o r g e r m i n a t i o n i n h i b i t i o n , S t i p a c a p e n s i s and Helianthemum l e d i f o l i u m were s t r o n g l y i n h i b i t e d , Zygophyllum dumosum l e s s so, and the two v a r i e t i e s o f Medicago l a c i n i a t a not a t a l l . Major v o l a t i l e i n h i b i t o r s t u r n e d out t o be t e r p e n e s and t e r p e n o i d s , such as α - p i n e n e , camphor, and c i n e o l e ( F i g u r e 1 ) . We then p o s t u l a t e d t h a t chemical i n h i b i t i o n i s mainly r e s p o n s i b l e f o r the absence or s c a r c i t y of s e n s i t i v e s p e c i e s i n the v i c i n i t y o f A r t e m i s i a and t h a t the y i e l d o f a n n u a l s on p l o t s f r e e o f p e r e n n i a l s on the l e s s a r i d n o r t h - f a c i n g s l o p e s does not exceed the y i e l d on the s o u t h e r n s l o p e s owing t o p e r s i s t e n c e o f r e s i d u a l p h y t o t o x i n s i n the s o i l . A l l our attempts t o demonstrate s o i l t o x i c i t y by sampling o f s o i l s i n p o t s f a i l e d , p r o b a b l y because o f the v o l a t i l e n a t u r e o f the phytotoxins· When shoots of A r t e m i s i a herba-alba c o l l e c t e d i n the d e s e r t were p l a c e d near seeds o f v a r i o u s a n n u a l p l a n t s , g e r m i n a t i o n was i n h i b i t e d and such i n h i b i t i o n was h i g h l y reproducible. However, when p l a n t s of A. h e r b a - a l b a were t r a n s p l a n t e d i n a more humid r e g i o n i n T e l A v i v , s i m i l a r i n h i b i t o r y e f f e c t s were o b t a i n e d o n l y when 3-4 times as many s h o o t s were applied. Water s t r e s s , h i g h sun r a d i a t i o n , and h i g h t e m p e r a t u r e s a r e b e l i e v e d sometimes t o f a v o r h i g h e r p r o d u c t i o n and r e l e a s e o f v o l a t i l e t e r p e n e s and t e r p e n o i d s . T h i s i s a common, a n c i e n t b e l i e f among m i n t growers which g a i n e d s u p p o r t from the work o f C l a r k and Manary (14) . I n t e r e s t i n g l y , our o b s e r v a t i o n s on p o p u l a t i o n s of Artemisia h e r b a - a l b a i n l e s s a r i d r e g i o n s i n I s r a e l ( a l b e i t w i t h no more t h a n 350 mm of a n n u a l r a i n f a l l ) not o n l y f a i l e d t o show any r e d u c t i o n i n the number o f nearby a n n u a l s , but a c t u a l l y r e v e a l e d t h a t a n n u a l s may a g g r e g a t e around t h e A r t e m i s i a p l a n t s . In g e n e r a l , one would e x p e c t the degree o f i n h i b i t i o n t o depend b o t h on the s u s c e p t i b i l i t y o f d i f f e r e n t p l a n t s p e c i e s as w e l l as on the r e a c t i v i t y of the i n h i b i t o r s , t h e i r c o n c e n t r a t i o n and t h e i r p r o x i m i t y t o the s u s c e p t i b l e p l a n t . P o p u l a t i o n s of _Aj_ herba-alba 2

a r e r e l a t i v e l y dense (about 3 p l a n t s / m ) and the shrubs a r e e x t r e m e l y a r o m a t i c , p a r t i c u l a r l y d u r i n g the l o n g summer

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D FORESTRY

F i g u r e 4.

Artemisia (b) .

herba-alba

, Branch (a) and

Inflorescences

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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F i g u r e 5.

Figure

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A r t e m i s i a h e r b a - a l b a on a N o r t h - f a c i n g S l o p e (bottom) and Zygophyllum dumpsum on a S o u t h - f a c i n g S l o p e (top).

a. A f l a s h o f a n n u a l s on a n A r t e m i s i a - f r e e p l o t ( l e f t ) compared w i t h t h o s e o f an u n d i s t u r b e d p l o t u s e d as a c o n t r o l ( r i g h t ) , 8 months a f t e r removal o f t h e A r t e m i s i a shrubs. ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m r e f e r e n c e 13. C o p y r i g h t 1977 B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s L t d . )

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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F i g u r e 6.

b . Mature P l a n t s o f I f l o g a s p i c a t a Sampled from t h e A r t e m i s i a - f r e e P l o t ( l e f t ) and from t h e U n d i s t u r b e d Plot (right).

F i g u r e 7.

P o l y t h e n e Beaker Used i n G e r m i n a t i o n Experiments : A, P l a s t i c c o v e r ; B, Seeds; C, F i l t e r Paper; D, Open b e a k e r employed i n t h e " v o l a t i l e s method" (13) (by p e r m i s s i o n o f B l a c k w e l l S c i e n t i f i c Publications)·

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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(May-October), when the upper s o i l l a y e r may a d s o r b i n h i b i t o r s t o the p o i n t of s a t u r a t i o n . In such a c a s e , a r a i n f a l l of 100 mm p e r annum, such as o c c u r s on the s l o p e s and p a r t o f which (10-30%) d i s a p p e a r s q u i c k l y as r u n - o f f , would be u n l i k e l y t o d e p l e t e the s o i l of i n h i b i t o r s by l e a c h i n g . V a r i a b l e e c o l o g i c a l c o n d i t i o n s may t r i g g e r a l l e l o p a t h y o f A. h e r b a - a l b a i n one h a b i t a t and a b r o g a t e i t i n the n e x t one. The wide g l o b a l d i s t r i b u t i o n o f A. herba-alba from N o r t h A f r i c a t o the I r a n i a n d e s e r t s s u g g e s t s t h a t the a l l e l o p a t h i c phenomenon a s s o c i a t e d w i t h t h i s s p e c i e s may m a n i f e s t a l s o i n geographic regions other than i n I s r a e l . In the r e g i o n s t u d i e d , n e a r Sede Boquer, dwarf shrubs o f Artemisia h e r b a - a l b a d i s p e r s e 85% of t h e i r d i a s p o r e s (achenes) under the canopy and y e t most o f the g e r m i n a t i o n t a k e s p l a c e o u t s i d e the s h r u b s canopy (15) · T h i s i s t r u e even though the canopy of A. h e r b a - a l b a p r o v i d e s b o t h shade and l i t t e r d u r i n g the g e r m i n a t i o n p e r i o d , so t h a t h u m i d i t y under the shrubs i s b e l i e v e d t o be h i g h e r t h a n o u t s i d e i t . P o p u l a t i o n r e g u l a t i o n by a u t o t o x i c i t y i s t h u s s u g g e s t e d . Our f i e l d o b s e r v a t i o n s i m i l a r i n t e r a c t i o n s betwee s e e d l i n g s ( p r o b a b l y by the s h r u b s ' l i t t e r ) i n the case of the sand-dune-located non-aromatic A r t e m i s i a monosperma. 1

Tamarix a p h y l l a (Tamaricaceae) i s a t r e e of moderate h e i g h t (8-11 m). In I s r a e l i t i s p r e v a l e n t i n the c o a s t a l p l a i n and i n t h e Negev d e s e r t . I t n o r m a l l y grows i n x e r i c a r e a s w i t h 100 mm of a n n u a l r a i n f a l l (16) and i s r e c o g n i z e d as a s a l t - e x c r e t i n g t r e e ( 1 7 ) . L i t w a k i n 1957 (18) s t u d i e d the i n f l u e n c e of t h i s t r e e on soil salinization. At t h a t time he n o t i c e d t h a t i n a r i d l o c a l i t i e s w i t h l e s s t h a n 200 mm of a n n u a l r a i n f a l l no p l a n t s o f any k i n d grew under the c a n o p i e s o f the l a r g e t r e e s , even i n r a i n y y e a r s . At the p e r i p h e r y and s t i l l under the p a r t i a l i n f l u e n c e of the l i t t e r and water d r i p p i n g from the canopy, some h a l o p h y t i c and r u d e r a l s p e c i e s appeared, e.g. B a s s i a muricata, Mesembryanthemum n o d i f l o r u m , o r Chenopodium o p u l i f o l i u m ( F i g u r e s 8a, b ) . Compared w i t h s o i l samples from the open a r e a , t h o s e from under the canopy c o n t a i n e d t w i c e as many s o l u b l e s a l t s ( T a b l e I ) . TABLE I . T o t a l S o l u b l e S a l t s under the Canopy o f O l d T r e e s of Tamarix a p h y l l a and out i n the Open (average v a l u e s , Depth (cm) 0 40 80

Under the (a) 1201 1198 859

+ + +

canopy

345 216 187

In the open (b) 480 512 490

+ + +

Source: Reproduced w i t h p e r m i s s i o n from R e f . The tfeizmann S c i e n c e P r e s s o f I s r a e l ,

120 102 78 18.

ppm)

a/b

2.5 2.3 1.7 Copyright

1957

Under s m a l l t r e e s o f Ί\_ a p h y l l a (3-5 m i n h e i g h t ) , a n n u a l s were not e n t i r e l y a b s e n t . To e v a l u a t e the e f f e c t of the t r e e s on a n n u a l s , a l o n g a d i s t a n c e g r a d i e n t from the stem, we d e t e r m i n e d b o t h the d e n s i t y o f

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

F i g u r e 8.

F i g u r e 8.

a . A n n u a l s - f r e e Areas around T r e e s o f Tamarix aphylla, 15 km South o f Beer Sheva ( c f . F i g . 2 ) .

b . H a l o p h y t e s and R u d e r a l s (HR) i n t h e P e r i p h e r y o f an A n n u a l s - f r e e Area around Tamarix aphylla.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Ecosystems +

a n n u a l s and the c h l o r i d e ( C l ~ ) and sodium (Na ) c o n c e n t r a t i o n s i n t h e upper s o i l l a y e r (0.5 cm), p r o c e e d i n g s e r i a t i m from the stem towards t h e p e r i p h e r y of the t r e e . The d a t a p r e s e n t e d i n F i g u r e 9 show t h a t c l o s e t o t h e stem t h e C l ~ c o n c e n t r a t i o n i s h i g h e s t , and the a n n u a l s d e n s i t y l o w e s t , but the s i t u a t i o n r e v e r s e s towards the +

tree's periphery. A s i m i l a r p a t t e r n was o b s e r v e d a l s o f o r t h e N a c o n c e n t r a t i o n i n the upper s o i l l a y e r v i s - a - v i s the a n n u a l s d e n s i t y . Sodium c h l o r i d e i s the major s a l t e x c r e t e d by Tamarix aphylla. However, the c o m p o s i t i o n o f the e x c r e t e d s a l t s i s t o a c e r t a i n e x t e n t i n f l u e n c e d by the c o m p o s i t i o n o f t h e s a l t s e n c o u n t e r e d by t h e r o o t s (19) . The r a t e o f e x c r e t i o n of sodium c h l o r i d e , when measured i n r e l a t i o n t o i t s c o n c e n t r a t i o n around t h e r o o t system, shows an optimum p a t t e r n , i . e . h i g h e s t a t a 0.2 M c o n c e n t r a t i o n and d i m i n i s h i n g when the r o o t s a r e i r r i g a t e d w i t h e i t h e r lower o r h i g h e r c o n c e n t r a t i o n s o f sodium c h l o r i d e (19) · The marked a l l e l o p a t h i c e f f e c t i n the more a r i d r e g i o n s i s p r o b a b l y due t o a c o m b i n a t i o n o f the f o l l o w i n g : the e c o l o g i c a i n these areas are s u f f i c i e n t l e x c r e t i o n by the t r e e s , t o wash t h e e x c r e t e d s a l t s i n t o lower s a l t l a y e r s . I t seems t h a t t h i s a l l e l o p a t h i c e f f e c t i s a s e c o n d a r y e v e n t r e s u l t i n g from the e x c r e t i o n mechanism o f t a m a r i s k s which removes t h e s a l t s from t h e r o o t s , s u b s e q u e n t l y e l i m i n a t i n g them by the s a l t g l a n d s and f i n a l l y c o n c e n t r a t i n g them i n the upper s o i l l a y e r around t h e c a n o p i e s . The c o n t r i b u t i o n of t h i s a l l e l o p a t h i c e f f e c t t o the s u r v i v a l o f the species i s doubtful. T r e e s o f T. a p h y l l a were f o u n d t o be s u s c e p t i b l e t o sodium c h l o r i d e even when i r r i g a t e d w i t h as l i t t l e as 0.1 M N a C l (19) · I t i s n o t s u r p r i s i n g , t h e r e f o r e , t h a t i n t h e a n n u a l - f r e e c o n c e n t r i c a r e a s under the c a n o p i e s , we c o u l d not f i n d any r o o t s o f t a m a r i s k c a p a b l e o f e x p l o i t i n g the n o n - u t i l i z e d w a t e r . Nevertheless, i t i s c l e a r that inorganic minerals of p l a n t o r i g i n can i n d u c e a l l e l o p a t h y . Indeed, i n the case o f C e r a t o p h y l l u m demersum e l e m e n t a l s u l f u r was r e c e n t l y found a c c o u n t a b l e f o r the f a c t t h a t v e r y few e p i p h y t e s a s s o c i a t e w i t h t h i s water p l a n t (20) · I t i s p o s s i b l e t h a t o t h e r t r e e s o r shrubs a c t i v e i n t h e s a l i n i z a t i o n p r o c e s s o f s o i l s o r a f f e c t s o i l pH, e. g. S a r c o b a t u s vermiculatus (Chenopodiaceae) i n E s c a l e n t e D e s e r t , Utah (21) may under s p e c i f i c e c o l o g i c a l c o n d i t i o n s induce a l l e l o p a t h i y . Discussion

and

Conclusions

From t h e accumulated i n f o r m a t i o n on a l l e l o p a t h y o r a u t o t o x i c i t y i n a r i d r e g i o n s of b o t h hemispheres, some g e n e r a l i z a t i o n s may be drawn: (a) a g g r e s s i v e p l a n t s w i t h a l l e l o p a t h i c p o t e n t i a l are a d u l t p e r e n n i a l s o r young s e e d l i n g s o f such p e r e n n i a l s ; (b) the a l l e l o c h e m i c a l s a r e e i t h e r common s e c o n d a r y m e t a b o l i t e s or i n o r g a n i c s a l t s . They a r e o f t e n n o n s p e c i f i c , n o t g e n e r a l p h y t o c i d e s . They r e a c h the s u s c e p t i b l e p l a n t t h r o u g h t h e s o i l i n v a r i o u s ways - by b e i n g washed o f f d u r i n g r a i n f a l l from t h e f r e s h o r d r i e d l e a v e s and stems, by e s c a p i n g as v o l a t i l e s u b s t a n c e s from t h e s h o o t s t o be a d s o r b e d l a t e r by the s o i l , o r by l i b e r a t i o n from t h e mature o r decomposed r o o t s t o r e a c h t h e r o o t l e t s o f t h e young p l a n t ;

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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canopy

F i g u r e 9.

—>j

radius

Annuals D e n s i t y (o) and C h l o r i d e C o n c e n t r a t i o n ( A ) a t V a r i o u s D i s t a n c e s from Stem t o P e r i p h e r y o f Tamarix a p h y l l a (mean v a l u e s o f 5 t r e e s ) . D i s t a n c e s e x p r e s s e d as f r a c t i o n o f t h e t r e e s radii. 1

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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(c) the a l l e l o c h e m i c a l e f f e c t i s s u b j e c t t o l o c a l s m a l l - s c a l e changes as w e l l as t o l a r g e g e o g r a p h i c a l ones. V e r y few a n n u a l s , f o r i n s t a n c e , a r e a s s o c i a t e d w i t h shrubs of Artemisia h e r b a - a l b a on the h i l l s l o p e s o f the Negev D e s e r t , but t h i s i s not so i n the r u n n e l s or i n l e s s a r i d n o r t h e r n r e g i o n s , f o r t r a n s p l a n t a t i o n of t h e p l a n t s from t h e dry d e s e r t c l i m a t e t o the w e t t e r M e d i t e r r a n e a n , one r e d u c e s the p r o d u c t i o n o f v o l a t i l e p h y t o t o x i n s . I t i s t h e r e f o r e s u g g e s t e d t h a t a r i d r e g i o n s t h a t m a n i f e s t an a r i d i t y g r a d i e n t a r e f i t t e d f o r the e x p l o r a t i o n of new a l l e l o p a t h i c e f f e c t . C l e a r l y , a l l e l o p a t h y i s more common under d e s e r t c o n d i t i o n s t h a n i n humid e n v i r o n m e n t s . T h i s i s not because the wide s p a c i n g s between the p l a n t s i n the d e s e r t a l l o w an e a s i e r d e t e c t i o n of the a l l e l o p a t h i c e f f e c t , b u t because t h e r e a r e e c o l o g i c a l c o n d i t i o n s t h a t f a v o r a l l e l o p a t h y , such as t h o s e a f f e c t i n g the r a t e o f p r o d u c t i o n o f a l l e l o c h e m i c a l s o r d e t e r m i n i n g the e f f e c t i v e n e s s o f the a l l e l o c h e m i c a l s a l r e a d y i n the s o i l . Among f u r t h e r f i n d i n g s o f the p r e s e n t study a r e t h 1· Water and n u t r i e n production. In a r e c e n t r e v i e w , Gershenzon (22) has p o i n t e d o u t t h a t numerous a l l e l o c h e m i c a l s o f v a r i o u s c h e m i c a l groups a r e o f t e n p r o d u c e d and s t o r e d i n much h i g h e r c o n c e n t r a t i o n s by p l a n t s under water o r n u t r i e n t s t r e s s t h a n by p l a n t s growing under o p t i m a l conditions· 2. There i s some e v i d e n c e t h a t g r a z i n g can a l s o t r i g g e r the p r o d u c t i o n o f a l l e l o c h e m i c a l s (23-25) , a l b e i t t h i s was r e p o r t e d f o r o n l y a few p l a n t s and was n o t s t u d i e d a t a l l i n d e s e r t p l a n t s . F u t u r e i n v e s t i g a t i o n may perhaps r e v e a l t h a t the g r a z i n g e f f e c t i m p o r t a n t l y a g g r a v a t e s a l l e l o p a t h y i n the d e s e r t s . 3. A l l e l o c h e m i c a l s i n d e s e r t s o i l s a r e p r e d i c t a b l y l e s s prone t o l e a c h i n g and r a p i d biodégradation t h a n a r e s o i l s i n humid environments· 4. Sandy s o i l s , w i t h low amounts o f o r g a n i c m a t t e r , a r e most common i n d e s e r t s . As such, t h e y may r e l e a s e the a d s o r b e d c h e m i c a l s more r e a d i l y t h a n w i l l heavy s o i l s r i c h i n o r g a n i c m a t t e r . As i n o t h e r e n v i r o n m e n t s , so a l s o i n a r i d r e g i o n s , a l l e l o p a t h y i s a s s o c i a t e d w i t h p l a n t - p l a n t c o m p e t i t i o n , but here the p a u c i t y of r e s o u r c e s may l e a d t o c o n s i d e r a b l e mutual i n t e r f e r e n c e r e s u l t i n g not o n l y i n d i m i n u t i o n i n s i z e o r number of the p l a n t s , but a l s o i n t o t a l e x t i n c t i o n of a v u l n e r a b l e s p e c i e s . T h i s does not mean t h a t a n a l y s i s o f a l l e l o p a t h y i n an a r i d environment s h o u l d be done d i f f e r e n t l y from t h a t which i s customary i n a humid environment; y e t i t i s i m p o r t a n t t o e s t i m a t e the e x t e n t t o which i n o r g a n i c s a l t s ( e x c r e t e d by the p l a n t o r r e l e a s e d from i t s l i t t e r ) a r e i n v o l v e d i n the a l l e l o p a t h i c e f f e c t . So f a r as s e c o n d a r y m e t a b o l i t e s a r e c o n c e r n e d , i t s h o u l d be o f i n t e r e s t t o compare t h e i r p r o d u c t i o n under humid and s t r e s s e d c o n d i t i o n s . It i s s u g g e s t e d t h a t f o r the e v a l u a t i o n o f the a l l e l o c h e m i c a l e f f e c t , s p e c i e s s u p p r e s s e d i n t h e i r n a t u r a l h a b i t a t s h o u l d be p r e f e r r e d o v e r any o t h e r s t a n d a r d seeds commonly u s e d f o r e v a l u a t i n g g e r m i n a t i o n i n h i b i t o r s . A l s o , e f f o r t s t o i s o l a t e a l l e l o c h e m i c a l s from s o i l s w i l l a s s i s t i n the e s t a b l i s h m e n t o f a l l e l o p a t h y on a more c o n c r e t e b a s i s than i s a v a i l a b l e at present.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

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. 25.

Went, F. W. Bull. Torrey Bot. Club, 1942, 39, 100-114. Gray, R.; Bonner, J . Am. J . Bot., 1948, 35, 52-57. Gray, R.; Bonner, J . J . Am. Chem. Soc. 1948, 70, 1249-1253. Lloyd, F. E. "Guayule ( Parthenium argentatum Gray); A rubber-plant of the Chihuahuan Desert". Carnegie Institute of Washington, 1911, 213 pp. Bonner, J.; Galston, A. W. Bot. Gaz. (Chicago) 1944, 106, 185-198. Bonner, J . Bot. Gaz. (Chicago) 1946, 107, 343-351. Bonner, J . Bot. Rev., 1950, 16, 51-65. Muller, C.-H.; Chou, C. H. In "Phytochemical Ecology"; Harborne, J. B., Ed.; Academic Press: London, 1972; pp. 201-216. Muller, C. H.; Muller, W. H.; Haines, B. L. Science, 1964, 143, 471-473. Muller, W. H.; Muller, C. H. Bull. Torrey Bot. Club, 1964, 91, 327-330. Muller, C. H.; de 93, 130-136. Halligan, J . P. Am. Midi. Nat. 1976, 95, 406-421. Friedman, J.; Orshan, G.; Ziger-Cfir, Y. J . Ecol. 1977, 65, 413-426. Clark, R. J.; Menary, R. C. Aust. J . Agricul. Res. 1980, 31, 489-498. Friedman, J., Orshan, G. J. Ecol. 1975, 63, 627-632. Friedman, J., Waisel, Y. La-Yaaran (The Forester) 1964, 13, 156-161. Volkens, G. "Die Flora der Aegyptisch-Arabischen Weste auf lage Grundanatomisch-physiologischen Untersuchungen", Berlin, Gebr. Borntraeger: Berlin, 1887; pp. 156, tab 18. Litwak, M. Bull. Res. Counc. Israel, 1957, 6D, 38-45. Waisel, Y. Plant and Soil, 1961, 13, 356-364. Wium-Andersen, S.; Antoni, U.; Houen, G. Phytochemistry, 1983, 22(11), 2613. Fireman, M.; Hayward, H. E. Utah Bot. Gaz. 1952. 114, 2. Gershenzon, J . In "Recent Advances in Phytochemistry"; Timmermann, N.; Steelnik, G.; Loewus, F. Α., Eds.; Plenum Press: New York, London, 1984; pp. 273-320. Green, T. R.; Rayan, C. A. Science, 1972, 175, 776-777. Loper, G. M. Crop Sc., 1968, 8, 104-106. Schultz, J . C.; Baldwin, I. T. Science, 1982. 217, 149-151.

RECEIVED

December 23,1985

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 7

Improving Crop Productivity in India: Role of Allelochemicals S. J. H. Rizvi and V. Rizvi Department of Botany and Plant Pathology, Rajendra Agricultural University, Pusa, Samastipur 848125, Bihar, India

The possible role of allelochemicals has been explored for pest control, crop rotation, and agroforestry. Various allelochemicals were screened for herbicibal and fungicidal activities against Amaranthus spinosu completely inhibite mM, respectively, and similarly inhibited mycelial growth of the test fungus at 10.3 and 2.5 mM. In a tobacco-maize rotation system, nicotine at 5 m M significantly increased the growth of maize. Leucaena leucocephala is widely recommended for agroforestry, so the effect of its most important allelochemical, mimosine, on several crops was studied. It appreciably inhibited growth of rice and wheat at 1 to 5 m M concentrations. These observations establish in principle that allelochemicals can be important in improving pest control, crop rotation, and agroforestry programs and thereby increase crop production.

B o t h the harmful and the beneficial effects o f plant-plant, plantmicroorganism, and plant-insect interaction must be considered aspects o f allelopathy, as advocated by R i c e (1). The role o f allelopathy i n natural and manipulated ecosystems i n poorly recognized. In the past decade we have been concerned mainly with exploring the frontiers of applied allelopathy that may lead to increased crop production. There are two ways o f this k i n d by which crop production may be improved: allelopathy can be exploited by developing new crop management systems and i m p r o v i n g existing ones, and p u r i f i e d allelochemicals may be used commercially as agrochemicals. Management Systems Different types o f management may be developed for weed, insect, and disease control, for crop rotation, and for agroforestry employing allelopathy. In the following paragraphs these are discussed separately. In weed management systems involving allelopathy, crop varieties may be screened or new varieties developed for their potential for controlling weeds. Such varieties may be left as residues i n the field, or be incorporated i n every rotation system, and/or used as a companion crop. Similarly, i f crop varieties allelopathic to pathogens can be found, their residues can be used similarly for disease control. Research groups of Putnam at M i c h i g a n State University and of Gliessman at the University o f California are two o f many that are involved i n research of this kind. 0097-6156/87/0330-0069$06.00/0 © 1987 A m e r i c a n C h e m i c a l Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

70

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Crop rotation is a "cropping system i n which two or more crops are grown i n a fixed sequence" (2). A n y crop may release allelochemicals either directly i n the form o f exudate or vapors, or its residue/litter can produce them afterward. Irrespective o f their source, allelochemicals may affect the next crop i n the rotational sequence either positively or negatively. In India, many farmers grow two or three crops/year on the same field, and there are definite recommendations on the sequence o f crops. The rotational sequences are based on maintenance o f s o i l fertility, s o i l structure, and plant nutrients, etc., but they have been formulated with little or no consideration for allelopathy, although it is unlikely that when crops are continuously supplying the soil with allelochemicals, these would not affect the following crop. This thought persuaded us to test our views experimentally, with two locally practiced rotational sequences: tobacco and maize, and tobacco and rice. O n a very conservative estimate the leaf litter from a chewing tobacco crop adds about 133 kg/ha o f organic matter to the soil (2), and the average nicotine content i n leaves o f tobacco is aproximately 4%. Thus every harvested crop to tobacco adds about 5.32 k g nicotine/ha merely through its leaf litter. O f course, roots also contribute some nicotine. Therefore we studied the effect o f nicotine on germinatio technique o f R i z v i et al. (4) plumule length o f maize, but decreased all the parameters studied i n rice; the radical length was most affected at 5 m M concentration (Table I). These results demonstrated the differential effect o f nicotine, and suggest that careful consideration o f allelopathic interactions o f crops of a rotation management can improve its productivity, either by eliminating deterimental interactions or by exploiting beneficial ones. Agroforestry. Agroforestry may be defined simply as intercropping o f woody plants with food or forage crops i n order to maintain or increase total yields (5.). W h i l e agroforestry has a potential to increase yield, it has its own limitations: competition o f trees with food crops, damage to food crops during tree harvesting, etc. The literature remains silent on allelopathic interactions between trees recommended for agroforestry and food crops, and no suggestion has been made to consider such effects before recommending food/forage crops for agroforestry programs (É). Hence we worked with mimosine, and allelochemical produced by leguminous trees - Leucaena sp. The concentrations of mimosine i n air-dried leaves o f such species range from 2.35% to 6.37% (2). O u r studies showed that mimosine is toxic for rice and wheat. Seed germination and radicle and plumule length o f both rice and wheat were adversely affected by mimosine (Table II). Earlier reports of Smith and Fowden ( £ ) and K u o et al. (2) o n the injurious effect o f mimosine on mung bean and rice, respectively, support our results. Therefore, possible allelopathic interactions between trees and food/fiber crops should be studied to improve agroforestry management. Allelochemicals as Sources of Agrochemicals N o w we come to the other potential application o f allelochemicals: the development o f new, safer and effective agrochemicals as pesticides and growth promoters. However, we restrict the discussion o f pesticides to herbicides, fungicides, and broad-spectrum pesticides. It is obvious that synthetic pesticides are important i n controlling weeds and plant-pathogenic fungi, but i n judging the efficacy of a method, it is essential to assess its non target toxicity, including health and environmental hazards. The literature abounds with reports that synthetic pesticides affect nontarget plants, their consumers and the environment (2.). Thus, plant protection needs

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

7.

RIZVI A N D RIZVI

Improving

Crop Productivity

71

in India

Table L Change Caused by Nicotine i n Seed Germination, Radicle Length and Plumule Length over Control in T w o Crops Plants

Change

Concentration (mM) Gennination

(%)

Radicle

Plumle

For Zea mays (var. Diarra composite): increase 2.5

2.73 ± 0.98

43.56* ± 1.10

25.82* ± 1.38

5.0

2.63 ±0.88

46.20* ± 0.98

33.11* ± 1 . 2 0

For Orvz 2.5

4.72 ± 0 . 2 1

14.99* ±0.98

3.10* ± 0 . 9 6

5.0

11.02 ± 0.82

57.64* ± 1.08

26.54* ± 1.01

* Significant at 5 % level.

Table II. Reduction of Seed Germination, Radicle Length and Plumule Length over Control i n T w o Crop Plants by M i m o s i n e

% Reduction

Concentration (mM)

Germination

1.0 2.0

2.44 ± 0.32 9.76 ± 0.91

Plumule

Radicle Oryza s a t i v a

( v a r . Saket)

5.48 ±0.18 11.39 ± 0 . 2 5

4.21 ± 0.68 15.72 ± 1.01

3.0

13.42* ± 1.01

19.40* ± 0 . 8 6

32.01* ± 1.20

4.0

21.96* ± 1.32

84.38* ± 1.20

83.15* ± 1.82

5.0

25.62* ± 1.42

100.00* ± 0.63

85.08* ± 1.82

Triticum vulgare (var. Sonalika) 2.27 ± 0 . 1 2

11.33 ± 1.10

14.84 ± 0.96

30.60* ± 0.98

20.20* ± 1.02

15.90* ±0.93

57.12*± 1.11

43.84* ± 0.98

4.0

19.88* ± 1.01

65.51* ± 1.01

70.44* ± 0.68

5.0

23.32* ± 1.32

80.93* ± 1.20

63.05* ± 1.20

1.0 2.0

12.72 ± 0.36

3.0

* Significant at 5 % level.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

72

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

improvement. It is important to note that many allelochemicals are k n o w n to inhibit the germination and growth of weeds and fungi (1), which is an essential feature o f all herbicides and fungicides, respectively. Therefore, allelochemicals can be an alternative to synthetic pesticides, and, being natural products, are safer to use and easily biodegradable (9-11). Herbicides. The idea o f using allelopathy i n weed control was conceived i n the late seventies (12). and several workers have considered this possibility, as already mentioned (13.-16). However, characterization and possible use o f allelochemicals as selective herbicides received attention only recently, and our demonstrations o f the selective herbicidal activity o f caffeine are among the pioneer ones (17-21). T o explore the potential of allelochemicals as herbicides, we tested some monoterpenes and terpenoids for suppression o f germination and growth o f the test weed Amarathus spinosus. using the bioassay o f R i z v i et al. (4). A m o n g the monoterpenes tested, geraniol was most potent. It inhibited radicle growth by 100% and seed gennination by 9 0 % at a concentration o f 2 m M , and completely inhibited plumule growth a geraniol indicates that i t an synthetic herbicides.. Fungicides. Since most commercial fungicides are synthetic products, almost all demerits o f synthetic pesticides i n general are associated with them. T o look for an alternative to such synthetics, some allelochemicals were tested i n vitro by a technique o f ours (17). Surprisingly, geraniol again proved the best. It inhibited mycelial growth o f Alternaria solani. the test fungus, by 9 2 % at a concentration of 2 m M , and proved fungicidal above 2 m M (Table IV), Thus geraniol i n particular and allelochemicals i n general may be o f use as fungicides. Multipurpose pesticides. Occurrence o f more than one pest-controlling property in a single allelochemical has persuaded us to propose using allelochemicals as multipurpose pesticides (IS). Such use would be beneficial i n several ways: (i) Reduction i n cost o f pesticides. Use o f a single compound for the control of several pests may reduce the total expenditure on crop protection. (ii) Reduction i n cost o f production and research. The cost o f production o f single multipurpose compound would probably be less than the total cost o f product o f several pesticides. (iii) Improved quality o f life. Being plant products, most allelochemicals should affect fewer nontarget organisms, and improve quality o f farm produce. Further, multipurpose pesticides can be very important i n developing integrated pest management systems (IPMS). A n y I P M S is designed to minimize losses o f crop y i e l d and quality due to pests, through integration o f various approaches to pest control so as to get m a x i m u m benefits w i t h m i n i m u m disadvantages. In spite o f their hazardous nature, synthetic pesticides are often among the dominating components of an I P M S . Moreover, an I P M S often requires simultaneous use of several synthetic pesticides, which, harmless singly, may become poisonous through interaction among themselves or their metabolites (22.23). Therefore, the number o f chemicals used i n any I P M S should be minimized. A viable approach to this may be to find multipurpose pesticides. A multipurpose allelochemical pesticide for I P M S would not only reduce the chances o f synergistic toxicity but also, being plant-derived, have all the merits of natural products. O u r studies with geraniol indicate its ability to control more than one agriculturally important pest, a weed and a pathogenic fungus. Moreover, up to

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

RIZVI A N D RIZVI

Table ΠΙ.

Crop Productivity

in India

Amaranthus spinosus: Reduction of Seed Germination, Radicle Length, and Plumule Length over Control by Geraniol

Concentration (mM)

1.0

Improving

Germination

% Reduction Radicle

Plumule

2.86 ±

2.0

90.90* ± 1.60

100.00* ± 1.10

35.00* ± 1.2

3.0

91.60* ± 0.98

100.00* ± 0.52

100.00* ± 1.4

* Significant at 5%

Table I V .

level.

Alternaria solani: Inhibition of M y c e l i a l Growth over Control by Geraniol

Concentration (mM)

Inhibition of Mycelial Growth %

1.0

53.20* ±1.10

2.0

92.80* ±1.68

3.0

100.00* ± 0.09

* Significant at 5%

level.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

2.5 m M geraniol exerted no visible adverse effect o n a test crop, tomato (Lycopersicon esculentum). in which both the test pests are problems. Thus, the possibility of exploiting allelochemicals to develop multipurpose pesticides has promise. G r o w t h promoters. The beneficial effects o f allelochemicals have been recognized only recently (1). Our studies with nicotine (Table I) suggest that nicotine and other allelochemicals with such potential may be used as growth promoters. Conclusions A n y improvement or new development in crop management based on allelopathic studies could not only increase production, but reduce expenditures on farm labor and agrochemicals, and reduction in use of synthetic agrochemicals would lead to an improved quality of life. If allelochemicals can be developed as botanical pesticides, they would be better than synthetic ones owing to their smaller non target toxicity, easy biodegradability possible that allelochemica into cultivars, to provide an inexpensive, safe and permanent means of biological pest control. Acknowledgments Financial assistance by the International Foundation for Science (Sweden) i n the form of a research grant N o . A/745-1/1984 is gratefully acknowledged. The first author is thankful to the authorities o f Rajendra Agricultural University for providing necessary facilities. W e acknowledge a gift of geraniol from George R. Waller.

Literature Cited 1. 2.

Rice, E. L. "Allelopathy"; Academic Press: New York, 1984. Lockerhart,J. Α.; Wiseman, A. J. "Introduction to Crop Husbandry"; Pergamon Press: Oxford, 1970. 3. Srivastava, R. P. Central Tobacco Research Station, Pusa, India, personal communication, 1985 4. Rizvi, S. J. H., Mukerji, D. J., Mathur, S. N. Indian J. Exp. Biol. 1980, 18, 777-8. 5. Vergara, Ν. T. "New Directions In Agroforestry: The Potential of Tropical Legume Trees" East West Centre: Hawaii, 1982. 6. Labelle, R. (International Council for Research in Agroforestry Nairobi, Kenya), private communication, 1984. 7. Kuo, Y.-L., Chou, C.-H, Hu, T-W. In "Allelochemicals and Pheromones". Waller, G. R.; Chou, C.-H., Eds. Institute of Botany, Academia Sinica: Taipei, R. O. C., 1982; pp. 107-119. 8. Smith, I. K.; Fowden, C. J. Exp. Bot. 1966, 17, 750-61. 9. Mathur, S. N.; Mukerji, D.; Rizvi, S. J. H.; Jaiswal, V. In "Current Trends in Life Sciences"; Chauhan et al., Eds.; Today's and Tomorrow's Publications: New Delhi, 1982; pp. 287-300. 10. Fawcett, C. H.; Spencer, D. M . Annu. Rev. Phvtopathol. 1970, 3, 403418. 11. Beye, F. Plant Res. Dev. 1978, 7, 13-31. 12. Putnam, A. R.; Duke, W. B. Science 1974, 185, 370-372.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

7. RIZVI AND RIZVI Improving Crop Productivity in India 75

13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Robson, T. O. Aquatic Bot. 1977, 3, 125. Szezepanski, A. Hidrobiologia 1977, 12, 193-7. Lockerman, R. H.; Putnam, A. R., Weed Sci. 1979, 27, 54-7. Putnam, A. R., DeFrank, J. Proc. 9th Int. Cong. Plant Protection 1979, 580-2. Rizvi, S. H. J.; Jaiswal, V.; Mukerji, D.; Mathur, S. N . Naturwissenschaften 1980, 67, 459-460. Rizvi, S. H. J., Jaiswal, V., Mukerji, D., Mathur, S. N., Indian J. Mycol. Plant Pathol. 1980, 10, 72. Rizvi, S. J. H.; Mukerji, D.; Mathur, S. N. Agric. Biol. Chem. 1981, 54, 1255-1256. Rizvi, S. J. H.; Rizvi, V. Proc. 10th Internat. Cong. Plant Protection 1983, 1, 234. Rizvi, S. J. H.; Rizvi, V. Proc. 1st Trop. Weed Sci. Conf. 1984, 2, 393400. Samersov, V. F.; Prishchepa, I. A. Khim. Sel'sk. Khoz. 1978, 16, 75861; Chem. Abstr. 1978 84 141771 Ramakrishna, N.; Ramachandran 1978, 15, 77.

RECEIVED

June 9, 1986

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 8 Variation of Root and Microflora Rhizosphere Exudates in Genotypes of Barley 1

1

Gunnar Stenhagen , Hans Alborn , and Tomas Lundborg

2

1

Department of Chemical Ecology, University of Göteborg, Kärragatan 6, S-431 33 Mölndal, Sweden Department of Crop Genetics and Breeding, University of Agricultural Sciences, S-268 00 Svalöv, Sweden 2

The potentia ling their rhizospher agricultural systems. Variation between cultivars in rhizosphere exudates can be observed, provided that the right analytical techniques are used. Rhizospheres from two barley cultivars with different adaptations to acidic s o i l s were investigated. Plants of the two species were cultivated under ident i c a l conditions in s o i l with a high peat concentration. Volatile components in the s o i l were sampled after three weeks by sucking air from the pots through adsorption tubes. A l l samples were taken at the same time in a greenhouse and then analyzed by capillary gas chromatography. The large amount of data from the GC analyses was then transferred to a computer for calibration and multiple component analyses (SIMCA). The results show differences in the occurrence of volatiles in the rhizosphere between the two cultivars. This is discussed in relation to p o s s i b i l i t i e s of the genetic capacity of plants to control their own environment.

0097-6156/87/0330-0076$06.00/0 © 1987 A m e r i c a n C h e m i c a l Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

8.

STENHAGEN ET AL.

Rhizosphere

Exudates

in Barley

11

It i s o f g r e a t i n t e r e s t t o know more a b o u t t h e capacity of crop p l a n t s i n c o n t r o l l i n g t h e i r root environment in o r d e r t o i n c r e a s e c r o p p r o d u c t i o n and a d a p t p l a n t s t o new a g r i c u l t u r a l systems. B e s i d e s , c r o p improvement by p l a n t breeding needs genetic v a r i a t i o n of the character of interest and one s u c h c h a r a c t e r i s t h e p o t e n t i a l o f the plant to c o n t r o l the s o i l microflora, especially the rhizosphere f l o r a . One p o s s i b i l i t y t o r e c o r d d i f f e r e n c e s between plant genotypes i n t h e i r root environment and microflora i s t o a n a l y z e the p a t t e r n of chemical compounds p r e s e n t i n t h e r o o t z o n e . The a d v a n t a g e o f s u c h an a n a l y s i s i s t h e p o s s i b i l i t y t o c a r r y o u t t h e measurements in an intact r o o t - s o i l system with unstressed plant m a t e r i a l . I f v o l a t i l e compounds a r e c h o s e n f o r i n v e s t i g a tion, sampling can even be made i n a undisturbed root zone. The aim o f t h e p r e s e n t s t u d y was t o find whether differences could b m i x t u r e o f v o l a t i l e compound of two different cultivars of barley. To have a reasonable base f o r a r e l e v a n t g e n e t i c v a r i a t i o n i n the plant material, two c u l t i v a r s w i t h d i f f e r e n t adaptation to a c i d s o i l s were s e l e c t e d . The s a m p l i n g was done from young plants, as t h e e s t a b l i s h m e n t o f the rhizosphere microflora i s of importance i n e a r l y stages of plant development. P l a n t M a t e r i a l and

Soil

System

Seeds of two b a r l e y (Hordeum vulgare L.) cultivars, Tellus ( n o t t o l e r a n t t o a c i d s o i l s ) and E t u ( t o l e r a n t t o acid soils), were sown i n s t a n d a r d p l a s t i c (PVC) pots ( h e i g h t 15 cm; u p p e r d i a m e t e r 14 cm) c o n t a i n i n g 800 g o f a soil mixture. B e f o r e use t h e p o t s were purified by heating i n b o i l i n g w a t e r f o r 10 m i n u t e s t o a v o i d l e a k i n g of compounds from t h e p l a s t i c m a t e r i a l . The soil had a h i g h c o n c e n t r a t i o n o f p e a t and was mixed w i t h P e r l i t e ( a l u m i n u m - s i l i c o n m a t e r i a l ) and Leca (burned c l a y ) p a r t i c l e s t o make t h e m i x t u r e more p o r o u s . The soil had a pH o f a b o u t 6, contained 45 mg/1 of nitrate nitrogen, 119 mg/1 o f p h o s p h o r u s and 284 mg/1 of p o t a s s i u m , and was mixed c a r e f u l l y b e f o r e t r a n s f e r t o t h e pots. The plants were t h i n n e d t o f o u r p e r pot and no a d d i t i o n a l f e r t i l i z a t i o n was g i v e n . E a c h p o t was w a t e r e d w i t h t h e same volume o f t a p w a t e r . The p l a n t s were grown in a greenhouse i n n a t u r a l l i g h t supplemented with l i g h t from m e t a l h a l i d e lamps (HQI-E 400W/DW) t o a d a y l e n g t h o f 18 h. The t e m p e r a t u r e was 20-25°C i n t h e day and 5-12 °C in the n i g h t .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

78

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

The experiments were c a r r i e d o u t 21 days after sowing. Two days b e f o r e the experiments started the plants were transferred to a small greenhouse for sampling. They were c u l t i v a t e d i n f o u r p a r a l l e l p o t s f o r e a c h c u l t i v a r and f o u r p o t s w i t h o u t p l a n t s were included as a control and were t r e a t e d i n t h e same way as the other pots. Sampling

Method

At t h e D e p a r t m e n t f o r C h e m i c a l E c o l o g y we have d e v e l o p e d adsorption techniques for sampling and a n a l y z i n g of volatile components i n a i r . T h e s e gas chromatographic (GC) methods have been u s e d i n a l l e l o c h e m i c a l research, i.a. f o r a n a l y s e s o f v o l a t i l e s e m i t t e d from p l a n t l e a v e s (1,2) . We b e l i e v e d t h a t s u c h an a d s o r p t i o n method c o u l d be a d a p t e d f o r s a m p l i n g v o l a t i l e s i n t h e s o i l by a l l o w i n g for the high humidit adsorbent. Because o f i t s low a f f i n i t y f o r w a t e r we have used TENAX TA, poly-(2,6-diphenyl-p-phenylene oxide). This v e r y p o r o u s p o l y m e r has a h i g h t h e r m o s t a b i l i t y and may be used with a thermal d e s o r p t i o n technique. It is also p o s s i b l e t o d r y i t t o a v o i d p l u g g i n g t h e GC column, with o n l y minimum l o s s e s o f o t h e r c o l l e c t e d components. The s a m p l i n g t u b e i s made o f h e a t - r e s i s t a n t glass and one end i s drawn o u t t o f o r m a capillary injection needle ( F i g u r e 1). The o t h e r end o f t h e t u b e i s a g l a s s cone (Quickfit 7/16) w h i c h a l l o w s a clean connection, w i t h o u t any g a s k e t , t o t h e vacuum pump o r gas s u p p l y . The a d s o r b e n t , 0.3 g o f TENAX TA, 35-60 mesh, i s k e p t between p l u g s of g l a s s wool. A d a p t e r s ( F i g u r e 1) were d e s i g n e d t o p l u g i n t o the drain holes a t the bottom of the c u l t i v a t i o n pot. The adapters were made o f a c e t a l p l a s t i c and had caps with silicone septa of the same type as used in gas chromatography. The needle of the sampling tube was i n s e r t e d t h r o u g h e a c h septum and t h e o t h e r end connected t o a f l o w r e g u l a t o r and vacuum pump. To make sure of i d e n t i c a l background and other c o n d i t i o n s a l l s a m p l i n g were done a t t h e same t i m e i n t h e greenhouse. Twelve p o t s , four w i t h p l a n t s of the b a r l e y c u l t i v a r Etu, f o u r w i t h b a r l e y c u l t i v a r T e l l u s , and f o u r w i t h s o i l o n l y , were p l a c e d on a t a b l e w i t h h o l e s f o r t h e pots. The b o t t o m o f t h e p o t was t h e n e a s i l y accessible for a l l the necessary connections. Two samples f r o m e a c h pot and i n a l l t w e n t y - f o u r samples i n each set were o b t a i n e d . A f t e r s a m p l i n g , a l l t u b e s were s t o r e d i n s c r e w capped g l a s s t u b e s u n d e r i n e r t c o n d i t i o n s (low temperature and helium atmosphere) u n t i l gas chromatographic analysis.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

8.

STENHAGEN ET AL.

Rhizosphere

79

Exudates in Barley

TO FLOW REGULATOR AND VACUUM PUMP

Figure

1.

Assembly

f o r Sampling

of

Volatiles

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

80

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Sample I n j e c t i o n

System

Gas chromatographic a n a l y s i s of the a d s o r p t i o n samples were made on a c a p i l l a r y gas c h r o m a t o g r a p h (Carlo Erba 2900) equipped with a modified injection system c o n s t r u c t e d a t our d e p a r t m e n t (_3 ) . It

consists

o f t h r e e main p a r t s

(Figure

1. d e s o r p t i o n oven 2. o r d i n a r y s p l i t / s p l i t l e s s 3. h e a t a b l e c o l d t r a p .

2):

injector

(Grob

type)

The s a m p l i n g t u b e i s p u t i n t o t h e d e s o r p t i o n oven and t h e gas s u p p l y i s c o n n e c t e d . I f the sampling tube i s v e r y wet, d r y i n g i s p e r f o r m e d by f l u s h i n g d r y h e l i u m gas through the t u b e a t low ( a m b i e n t ) t e m p e r a t u r e for ten min. The tube i s the substances are c a r r i e c o o l e d w i t h l i q u i d n i t r o g e n . The sample i s t r a p p e d i n t h e first part of t h e c a p i l l a r y column which is located inside t h e g l a s s - l i n e d s t e e l t u b e (GLT t u b e ) . When the desorption step is finished ( i t t a k e s about t e n min), the t r a p i s h e a t e d r a p i d l y t o 2 5 0 C i n 30 s by applying an e l e c t r i c c u r r e n t (10 A) t h r o u g h t h e GLT t u b e . This i s made a u t o m a t i c by t h e use o f a t e m p e r a t u r e r e g u l a t o r . The starting time of h e a t i n g of the c o l d t r a p is also the s t a r t i n g time of the chromatographic run. The integrator (Hewlett Packard 3385) is automatically started and a l l r e t e n t i o n times are referred to this t i m e . The i n t e g r a t o r i s a l s o u s e d f o r t i m e programming o f various events. With this injection technique a very rapid and d i s t i n c t i v e s t a r t of the s e p a r a t i o n i s o b t a i n e d which is particularly i m p o r t a n t f o r t h e most v o l a t i l e components. The most s i g n i f i c a n t p r o p e r t y o f t h e i n l e t s y s t e m i s i t s a b i l i t y t o y i e l d r e p r o d u c i b l e r e t e n t i o n time v a l u e s . e

Data P r e - P r o c e s s i n g F i g u r e 3 shows a gas c h r o m a t o g r a m o b t a i n e d from s o i l w i t h plants o f one b a r l e y c u l t i v a r , Etu. It i s possible to c o u n t o v e r 400 p e a k s , and t h e f i g u r e i l l u s t r a t e s t h e v e r y complex p a t t e r n of v o l a t i l e s i n the r h i z o s p h e r e . I f we compare chromatograms obtained from samples of Etu, T e l l u s and s o i l o n l y , no s p e c i f i c d i f f e r e n c e can be s e e n . Most of the peaks seem t o be present in a l l three chromatograms, although the i n t e n s i t i e s of the peaks vary. The n e c e s s i t y o f computer h e l p w i t h the pattern recognition i s obvious. Before we can use any computerized method for pattern r e c o g n i t i o n t h e c o n s i d e r a b l e amount o f d a t a from t h e GC a n a l y s i s must be p r e p r o c e s s e d .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

8.

STENHAGEN ET AL.

Figure

2.

Rhizosphere

Gas

81

Exudates in Barley

Chromatographic

Inlet

System

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

82

A L L E L O C H E M I C A L S : R O L E IN A G R I C U L T U R E

AND

— ιι

FORESTRY

C Τ5 Ο Ο 0) in

•Η Λ

3

'·

·

Ο Μ-Ι · Η Ο CP m

ο -Ρ υ ο — α ε (ΰ ο n ω u α) ·Η tP^d Ο fC ο 0

-Ρ

•Ρ Λ

ο

u

to

··

ω ω α « ε σ> ω

•Η -Ρ

υ ε

Ο Χ3 οο m M en c οο-ρ "Η ε c •—ι -Ρ

•—I (Ο -Ρ •Η ε α,

•Η

ο Q_i Ο V-l CN ε ίΟ Μ Ο u

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

8.

Rhizosphere

STENHAGEN ET AL.

Exudates in Barley

83

The r e p o r t s from t h e i n t e g r a t o r c o n s i s t o f retention time and sample amount f o r e a c h i n t e g r a t e d peak. T h e s e a r e t r a n s m i t t e d t o a s m a l l c o m p u t e r . The i n t e g r a t o r has the c a p a c i t y t o p r o c e s s up t o 250 p e a k s i n a run. However, b e c a u s e o f the limited memory space of the computer, we had to decrease the number of peaks processed. C h r o m a t o g r a p h i c r u n s w i t h more t h a n 150 p e a k s were r e d u c e d t o 150 p e a k s by e l i m i n a t i o n o f those with the smallest area. The r e d u c e d r e p o r t s were t h e n s t o r e d on t a p e . Retention time c a l i b r a t i o n . In s p i t e o f a l l e f f o r t to obtain r e p r o d u c i b l e r e t e n t i o n time v a l u e s these varied f o r t h e same component between d i f f e r e n t chromatographic runs, mainly because o f d i f f e r e n t sample amounts. To solve this problem a l l retention time values were calibrated. Values f o r a l i m i t e d number o f p e a k s , that c o u l d e a s i l y be f o u n were manually entere these reference p e a k s t h e mean v a l u e M ( j ) o v e r a l l t h e r u n s was c a l c u l a t e d . New r e t e n t i o n t i m e s , Rtcal(i), for the peaks i n t h e d a t a s e t were t h e n c a l c u l a t e d by the straight-line expression: Rtcal(i)

=

(Rt(i)-A(i))/B(i)

The coefficients A ( i ) and B ( i ) a r e b a s e d on values M ( j ) o f t h e n e a r e s t r e f e r e n c e p e a k s on o f t h e peak t o be calibrated.

the both

mean sides

Reference vector. A f t e r c a l i b r a t i o n a reference vector i s created. T h i s v e c t o r i s u s e d t o match a l l t h e same f r a c tions together in a data matrix and i s created by ordering of a l l existing calibrated retention time values. The number o f t h e v a l u e s i s r e d u c e d by r e p l a c i n g each value that falls into a small retention time "window" w i t h i t s mean v a l u e . The "window" was s e t wide enough t o r e d u c e t h e number o f v a l u e s t o 250. M a t c h i n g t o form a d a t a m a t r i x . Each value of reference vector i s compared w i t h the retention values for the f r a c t i o n s i n every chromatographic Only a s m a l l v a r i a t i o n i n the v a l u e s i s t o l e r a t e d ; no d o u b l e x m a t c h i n g s h o u l d o c c u r .

the time run. thus

The result of the p r e - p r o c e s s i n g i s a data matrix with information a b o u t t h e amount o f e a c h f r a c t i o n , for a l l runs, on a l i n e . T h i s m a t r i x i s the base f o r the m u l t i v a r i a t e data a n a l y s i s ( F i g u r e 4).

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

84

A L L E L O C H E M I C A L S : ROLE IN A G R I C U L T U R E A N D FORESTRY

OBJECT 1 VARIABLE Y11

Y12 Y1

Y21

Y22 Y23

Y2K

Y2N

Y31

Y32 Y32

Y3K

Y3N

Y41

Y42 Y43

Y4K

Y4N

YM1

YM2 YM3

YMK

YMN

ν CLASS ETU

CLASS

TRAINING SET

TELLUS

NON

CLASSIFIED SOIL y TEST SET

Figure 4 . Available Data i n the P a t t e r n R e c o g n i t i o n P r o b l e m Form a M a t r i x o f D i m e n s i o n s M Times N.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

8.

STENHAGEN ET AL.

Pattern

Rhizosphere

Exudates

in

Barley

85

Recognition

In the nineteen-seventies, new methods for pattern r e c o g n i t i o n have been d e v e l o p e d by means o f quantitative analogy models. The a n a l o g y a n a l y s i s i m p l i e s l o o k i n g f o r regularities in t h e o b s e r v a t i o n s made. The individual items t h a t are analyzed are c a l l e d o b j e c t s . The objects are a l i k e t h a t w i l l be b r o u g h t i n t o t h e same class. In our case t h e number o f c l a s s e s w o u l d be two, the two b a r l e y c u l t i v a r s ( E t u , T e l l u s ) . The s a m p l e s o b t a i n e d f r o m s o i l a r e u s e d as t e s t o b j e c t s . One q u e s t i o n we want an answer t o i s : i s t h e r e any difference between t h e gas chromatographic separation p a t t e r n of the t h r e e o b j e c t s ? A method s u c c e s s f u l l y u s e d f o r c h r o m a t o g r a p h i c d a t a and c a p a b l e t o answer t h i s and r e l a t e d q u e s t i o n s i s the SIMCA method ( S t a t i s t i c a l I s o l i n e a r M u l t i p l e Component Analysis). It has bee Svante Wold and h i Sweden. The SIMCA method and t h e p r i n c i p a l components (PC) analysis, a common method f o r o b t a i n i n g a v i e w o f m u l t i variate data, have been d e s c r i b e d i n d e t a i l elsewhere ( 4 , 5 ) ; t h u s o n l y a s h o r t p r e s e n t a t i o n w i l l be g i v e n h e r e . P r i n c i p a l v e c t o r p l o t (PC p l o t ) . The p r i n c i p l e o f t h e PC plot i s shown i n F i g u r e 5 . The p l a n e t h a t b e s t a p p r o x i mates the d a t a s e t ( i n the sense of l e a s t squares) is calculated. The c o o r d i n a t e s i n t h e p l a n e o f t h e projection o f e a c h p o i n t a r e c a l c u l a t e d and t h e p o i n t plotted in the diagram. A l r e a d y i n t h i s s i m p l e p l o t we see t h a t t h e t h r e e c l a s s e s a r e s e p a r a t e d f r o m e a c h o t h e r and that o b j e c t s 1 - 3 ( s o i l ) l i e f a r from t h e o t h e r s . SIMCA ( e a c h c l a s s d e s c r i b e d by a PC m o d e l ) . The basic idea of the SIMCA method is that multivariate data measured on a g r o u p o f s i m i l a r o b j e c t s , a proper class a r e w e l l a p p r o x i m a t e d by a s i m p l e PC m o d e l . The d i m e n s i o n a l i t y o f t h e m o d e l , a, i s e s t i m a t e d so as to g i v e t h e model as good p r e d i c t i v e p r o p e r t i e s as possible. G e o m e t r i c a l l y , t h i s corresponds t o the f i t t i n g of an a - d i m e n s i o n a l h y p e r p l a n e t o t h e o b j e c t points in the measurement space. The f i t t i n g i s made using the least squares c r i t e r i o n , i . e . t h e sum o f s q u a r e d r e s i duals i s minimized f o r the c l a s s data s e t . The class b e l o n g i n g can be determined when the d i s t a n c e o f an o b j e c t t o t h e c l a s s model i s compared w i t h the typical d i s t a n c e of the c l a s s o b j e c t s to the same model. In Figure 6 models are f i t t e d s e p a r a t e l y t o each class and the distances f o r each o b j e c t to the two c l a s s e s are p l o t t e d . The two c l a s s e s a r e w e l l separated and the o b j e c t s corresponding t o the s o i l samples are l o c a t e d c l o s e t o the dashed l i n e , i n d i c a t i n g equal c l a s s distance.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

86

Λ

! I

I ETU

Ο

(BARLEY)

Θ SOIL

Φ

(REFERENCE)

TELLUS (BARLEY)

Figure 5. Eigenvector Projection (Principal Vector Plot). A plane i s l e a s t squares f i t t e d t o a l l the data. This plane c o n s t i t u t e s a two-dimensional window into the multi-dimensional measurement space. The p r o j e c t i o n s o f t h e o b j e c t p o i n t s down t o t h e p l a n e a r e visualized in this plot.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

8.

STENHAGEN ET AL.

DIST.

Rhizosphere

87

Exudates in Barley

TO CLASS 2

,1

/ /

/ /

/ / / /

/ /

/

/

DIST.

TO CLASS 1

Figure 6 . C l a s s D i s t a n c e P l o t (Coomans P l o t ) . M o d e l s are fitted separately t o each class (Etu resp. Tellus). The distances f o r e a c h o b j e c t t o t h e two classes are plotted. The d a s h e d l i n e i n d i c a t e s equal class distance. The s o i l samples ( o b j e c t 1-3) are located close to this line.

Discussion The r e s u l t s show a s i g n i f i c a n t d i f f e r e n c e i n c o m p o s i t i o n of t h e v o l a t i l e s f r o m t h e r o o t z o n e s o f t h e two barley cultivars. The v o l a t i l e s a m p l e s i n c l u d e s e v e r a l h u n d r e d s of d i f f e r e n t compounds and t h e d i s t i n c t i o n between t h e cultivars was possible only by using the technique presented. The p r e s e n t a n a l y s e s d i d n o t r e v e a l w h i c h compounds v a r i e d , o r i f t h e v a r i a t i o n was p r e d o m i n a n t l y q u a l i t a t i v e o r q u a n t i t a t i v e . However, f u r t h e r SIMCA a n a l y s i s of the p r e s e n t data can g i v e i n f o r m a t i o n of t h i s k i n d .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ALLELOCHEMICALS: ROLE IN AGRICULTURE A N D

88

FORESTRY

The origin o f t h e v o l a t i l e compounds t h a t differ between t h e two b a r l e y c u l t i v a r s i s n o t known. Three sources f o r t h e d i f f e r i n g compounds are possible. The first one i s the o r i g i n a l root exudate (6,7). In this case the r e s u l t s r e f l e c t s c u l t i v a r v a r i a t i o n i n plant metabolism and t h u s the p o t e n t i a l of the plant in c o n t r o l l i n g i t s environment. A s e c o n d s o u r c e m i g h t be t h e p r o d u c t s o f t h e soil microflora, which then d i r e c t l y i n d i c a t e a v a r i a t i o n i n the composition of the m i c r o f l o r a i n the root zone f o r the two cultivars. A third possible source f o r the observed variation i s d e g r a d a t i o n o f t h e components i n the whole s o i l system. In t h i s c a s e d i f f e r e n t l e v e l s o r kinds of m i c r o b i o l o g i c a l a c t i v i t y are recorded. Probably we have a c o m b i n a t i o n o f t h e t h r e e s u g g e s t e d s o u r c e s . In conclusion, we have found that even young u n s t r e s s e d p l a n t s o f d i f f e r e n t c u l t i v a r s show a v a r i a t i o n in the chemical compositio zones. We suggest tha potential of the plant i n i t s c o n t r o l of the root zone environment and e s p e c i a l l y o f i t s r h i z o s p h e r e microflora. We s u s p e c t t h a t t h i s g e n e t i c p o t e n t i a l w i l l be o f importance i n s e l e c t i o n and a d a p t a t i o n of a g r i c u l t u r a l crop plants i n the future.

Literature

Cited

1. Andersson,B.Å.; Holman,R.T.; Lundgren,L.; Stenhagen,G. J . Agric. Food Chem., 1980, 28, 985. 2. Lundgren,L; Stenhagen,G Nordic Journal of Botany, in press. 3. A n d e r s s o n , Β . Å . ; Lundgren,L.; Stenhagen,G. In "Biochemical Applications of Mass Spectrometry"; Waller,G.R.; Dermer,O.C., Eds.; Vol II, Wiley Interscience: New York, 1980; pp. 855-894. 4. Wold,S.; Albano,C.; Dunn,W.J. III; Esbensen,K.; Hellberg,S.; Johansson,Ε.; Sjöström,M. In "Food Research and Data Analysis": Martens,H.; Russwarm,H.Jr., Eds.; Elsevier: Amsterdam, 1983. 5. Lundgren,L.; Norelius,G.; Stenhagen,G. Hereditas, 1981, 95, pp. 173-179. 6. Rovira,A.D.; Foster,R.C.; Martin,J.K. In "The S o i l Root Interface"; Harley,J. Academic Press: London, 1979; pp. 1-4. 7. Lundborg,T. Sveriges Utsädesförenings T i d s k r i f t , 1984, 94, pp. 111-121. RECEIVED

December 23, 1985

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 9 Allelopathy in Mexico A . L . Anaya, L . Ramos, J . G . Hernandez, and R . Cruz Institute de Fisiología Celular, Departamento de Bioenergética, U N A M . Apdo., Postal 70-600, 04510 Mexico, D. F . Mexico

Studies of allelopathy in Mexico were initiated in 1970 within the project entitled Recovery of Tropical Rain Forests. This paper summarizes the main results obtained from: research about the allelopathic potential of some tropical secondary plants in Veracruz; studies in coffee plantations (herbs discovery of the allelopathi and the use of this plant as fertilizer in the "chinampas"; the relationships of crops and weeds in "chinampas" and the allelopathic properties of corn pollen. Likewise studies on Helietta parvifolia and Piqueria trinervia are mentioned and finally research that is in progress in temperate and tropical agroecosystems in order to permit more efficient agricultural and forest management of the agroecosystems in Mexico is mentioned, mainly the biological control of weeds and pests, the use of green manures and composts, and the management of water and b i o l o g i c a l d i v e r s i t y . The studies on allelopathy i n Mexico were i n i t i a t e d i n 1970 within the project e n t i t l e d Recovery of Tropical Rain Forests, as a suggestion of i t s d i r e c t o r , Dr. Arturo Gomez-Pompa, at the Institute of Biology of the National Autonomous University of Mexico. Studies of Secondary Vegetation The main objective of this project was to study some of the ecological processes that occur during secondary succession i n warm a n d h u m i d tropics. This process i s triggered a f t e r a perturbation i n the t r o p i c a l rain forest or the abandonment of crop land (1_»2) . The quantity and quality of leached plant metabolites, i n warm and humid regions, suggest that there exists a great variety of complex interactions among plants and microorganisms. In 1970, studies on allelopathy i n the t r o p i c a l zones were scarce, p a r t i c u l a r l y i n Mexico. The contributions of McPherson (3), F r e i and Dodson (4), Quarterman C5), Webb, Tracey and Haydock (6), Marinero (7) , and Gliessman (8) are some important antecedents for the study 0097-6156/87/0330-0089$06.00/0 © 1987 A m e r i c a n C h e m i c a l Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

90

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

that was carried out concerning the a l l e l o p a t h i c potential of the secondary vegetation i n Veracruz, Mexico (9) at the b i o l o g i c a l station of the National Autonomous University of Mexico located at Los Tuxtlas. In t h i s place we selected the most abundant species i n order to detect their a l l e l o p a t h i c p o t e n t i a l . The species were: Piper auritum, Piper hispidum, Croton pyramidalis, Siparuna nicaraguensis, and Cecropia o b t u s i f o l i a . The aqueous extracts of roots and leaves, the leachates of a e r i a l parts, aqueous extracts of s o i l s and i n some cases organic s extracts of leaves, the essential o i l s , and isolated pure compounds were tested for their e f f e c t s upon germination and growth of several test species from the same area. Likewise, bioassays of simultaneous germination were carried out with some of the available seeds. The test seeds used for the bioassays were: Mimosa pudica, Achyranthes aspera, Bidens p i l o s a , and Crusea calocephala (herbaceous species); Ochroma lagopus and Heliocarpus donell-smithii (arboreal species). F i r s t the tolerance o determined i n order to avoi results (10). Then we tested the following aqueous extracts of leaves, made with 1 and 4 g of dried leaves at 30°C , and 100 mL of d i s t i l l e d water i n a blender. Root extracts were made with 15 g of plant i n 100 mL of d i s t i l l e d water. The a e r i a l parts were leached by soaking 100 g of fresh plants i n 100 mL of d i s t i l l e d water. S o i l extracts were prepared i n a 2:1 proportion. The organic extracts of leaves were obtained with the following solvents: hexane, ethyl acetate, chloroform, benzene, acetone, and methanol. The essential o i l s were obtained by steam d i s t i l l a t i o n and the pure substances with several extraction techniques (11, 12, 14). A l l materials were tested upon seeds i n P e t r i dishes with agar (1%) or f i l t e r paper as substrate, at 27°C and a 12-h photoperiod. Lengths of roots and stems were measured and the percent of germination was calculated. A l l results were s t a t i s t i c a l l y analyzed with an F^ test. The bioassays showed the wide a l l e l o p a t h i c potential of these plants as well as the phytotoxicity of some s o i l s extracts and essent i a l o i l s (Tables I, I I , and I I I ) . A l l species studied inhibited the growth of certain test species. This confirms the s e l e c t i v i t y of the a l l e l o p a t h i c compounds (15). The species with the higher a l l e l o p a t h i c potentials were: Piper auritum, Piper hispidum, Croton pyramidalis, and Siparuna nicaraguens i s . The essential o i l s of the Piperaceae were highly i n h i b i t o r y while that of Croton pyramidalis was less i n h i b i t o r y and even produced stimulations (Table I I I ) . A very interesting result was the i s o l a t i o n of safrole from the essential o i l of Piper auritum. This compound i s abundant i n the Monimiaceae and Lauraceae families. It was found to constitute 60 to 70% of the essential o i l of P.auritum (16). The benzenic extract from leaves of Croton pyramidalis was highly i n h i b i t o r y . From t h i s extract we isolated a flavone and a diterpene (Figure 1) but there are other compounds not yet i d e n t i f i e d i n this extract

that

a r e much more t o x i c

( V\_) .

The tests of simultaneous germination with seeds of Siparuna n i c a -

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Allelopathy

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91

Mexico

T a b l e I . E f f e c t s o f the E x t r a c t s o f Leaves and Roots of P i p e r a u r i t u m , _P. h i s p i d u m , C r o t o n p y r a m i d a l i s , C e c r o p i a o b t u s i f o l i a , and S i p a r u n a n i c a r a g u e n s i s on R a d i c l e Growth o f Some Secondary Species

1

Treatments

Inhibition/Stimulation (%) Piper Pipe auritum hispidu L e a f Root L e a f Root L e a f Root L e a f Root

Leaf

Root

Species : Mimosa pudica

59

2

27

A.aspera

42

2

(44)

B.pilosa

68

2

28

2

(75)

Crusea calccephalf 3 5 H.donne11 47 smithii 0.lagopus

mean of

3

2

11

five

2

2

2

67

2

21

(67 Ϋ

49

2

(22)

(11)

56

2

17

2

79 76

2

_

21

56

2

2

2

100 86

2

24

2

2

3

(42)

2

_

18

3

25

15

2

66

2

56

2

37

2

2

3

(49)

2

_

31

2

51

2

2

(13)

3

54

(29)

2

52

0

64

(11)

5

4

(8)

_

51

41

0

(3)

2

t o the 1% l e v e l .

significant

t o the 5% l e v e l .

11

(9) 2

repetitions.

significant

8 (7)

2

(3)

2

2

Numbers i n p a r e n t h e s i s a r e s t i m u l a t i o n s .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

_

8

92

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Table I I . Effects of the Aqueous Extracts of Soils Associated with Piper auritum, P_. hispidum, Croton pyramidalis, Cecropia o b t u s i f o l i a , and Siparuna nicaraguensis on the Radicle Growth of Some Secondary Species

Treatments

Inhibition/Stimulatio Piper Pipe auritum hispidu pyramidali

njcaraguensi

Species : Mimosa pudica

3

12.4

10.7

0

11

A.aspera

(6)

24

2

(16)

B.pilosa

9.4

21

2

18

25

2

(3)

24

2

34

Crusea calocephala

19.2

H. donnellsmithii

20

0.lagopus

Mean of f i v e

2

2

3

10

3

2

6

3

(7)

10

(4) 15

3

11 (20)

10

14

3

(77)

25

2

repetitions.

2

s i g n i f i c a n t to the 1% level.

3

s i g n i f i c a n t to the 5% level.

Numbers i n parenthesis are stimulations.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2

2

(87)

2

9.

Allelopathy

ANAYA ET AL.

Table

III.

E f f e c t s of the E s s e n t i a l O i l s of Piper auritum, Piper hispidum, and C r o t o n p y r a m i d a l i s ( 1 0 0 ppm), on t h e Growth ( R a d i c l e and Stem) o f Some Secondary S p e c i e s

inhibition/stimulation

Treatments Species

93

in Mexico

^^"^^^

1

(%)

Pipe Root 3

(55)

2

90

2

100

2

100

2

100

2

100

2

84

2

34

2

2

100

2

100

2

100

2

49

2

27

2

100

2

100

2

(5)

15

3

Heliocarpus donnell-smithii

91

2

100

2

16

3

Ochroma lagopus

74

2

100

2

100

2

100

2

Mimosa

pudica

Achyranthes aspera Bidens

pilosa

Crusea calocephala

Solanum n i t e n s

Mean from f i v e

3

87

2

81

2

100

91

-

_

100

2

2

-

100

100

2

100

2

1 9

(22)

2

(26)

2

-

repetitions.

significant

t o t h e 1% l e v e l .

significant

t o t h e 5% l e v e l .

Numbers i n p a r e n t h e s i s

are stimulations.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

(35)

-

2

2

94

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND

FORESTRY

raguensis showed that they are i n h i b i t o r y to the germination and growth of other test seeds. Rodriguez-Hahu (personal communication) mentioned that this e f f e c t i s due to a terpene, a rhamnoside and a flavonol, among other substances that are not yet i d e n t i f i e d . As part of the same project a study of one of the most common weeds i n some disturbed habitats from many regions i n Los Tuxtlas (Veracruz) was carried out. Ambrosia cumanensis i s found as an important species of the ruderal vegetation. It grows vigorously and i n almost pure stands. We decided to assess i t s a l l e l o p a t h i c p o t e n t i a l i n order to evaluate this phenomenom as a determining factor for the structure of the community as well as i n the secondary succession process. Root and leaf aqueous leachates of Ambrosia cumanensis did indeed produce a strong i n h i b i t i o n on the growth of weed species. Aqueous extracts of s o i l c o l l e c t e d under A.cumanensis i n July (during i t s flowering) were strongly a l l e l o p a t h i c to weed growth. Decomposition of leaves and roots i n pots caused i n h i b i t i o n of some weeds also. Microorganisms have a major r o l e i n this process, as shown by results from s t e r i l e and nonsterile s o i l Bioassays with severa showed that these compounds produce d i f f e r e n t e f f e c t s (stimulatory and i n h i b i t o r y ) on the germination and growth of several species of the secondary vegetation (18). Therefore, i t i s possible that the a l l e l o p a t h i c p o t e n t i a l of A. cumanensis contributes to the autocontrol of i t s population by preventing the growth of seedlings of i t s own species (Figure 2). The information obtained at Los Tuxtlas shows that the studied species from the secondary vegetation produce one or more a l l e l o p a t h i c substances, mainly i n leaves or through the decomposition of t h e i r organic matter, that can i n h i b i t growth or have deleterious e f f e c t s on plants and may cause p a r a l l e l effects that are related to the r o l e of auxins and to tropisms and other metabolic processes. The production of a l l e l o p a t h i c compounds i n t r o p i c a l zones, p a r t i c u l a r l y i f they are continuously released into the environment, may contribute to the elimination of secondary species already established and to the s e l e c t i o n of those that are beginning to e s t a b l i s h i n the habitat. Studies i n Coffee

Plantations

In 1979, we decided to extend our studies to one of the agroecosystems of greater importance i n Mexico: the coffee plantations. These studies were r e a l i z e d within the Program of Agroecosystems at the-Instituto Nacional de Investigaciones sobre Recursos B i o t i c o s . We worked at the coffee plantations i n Coatepec, Veracruz, which are characterized by the presence of shade trees which resemble the structure of the deciduous temperate f o r e s t s , with three well defined s t r a t a : the herbaceous layer, the shrub layer represented by coffee plants and the tree layer. The main objective of this study was to assess the a l l e l o p a t h i c i n t e r actions among the species that constitute this community, i n p a r t i c u l a r the coffee plants (19). Figure 3 shows the e f f e c t of the s o i l extracts from the coffee plantation. Waller et a l . mention that these effects might be explained by the accumulation of caffeine and other alkaloids i n s o i l i n old coffee plantations (20). The greatest a l l e l o p a t h i c e f f e c t s were produced by plants from the

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

9.

Allelopathy

ANAYA ET AL.

in

95

Mexico

MeO

OAc CH OAc 2

Diterpene

F i g u r e 1.

S t r u c t u r e s of P y r a m i d o l a c t o n e ( F u r o l a c t o n e D i t e r p e n e ) of the N o r c l e r o d a n e group ( A ) , and 3 , 5 - d i h y d r o x y - 7 , 4 dimethoxyflavone (B) i s o l a t e d from Croton pyramidalis. f

H

U

0 H

Cumambrin A

Cumambrin Β

Psilostachyin Β

HO

Psilostachyin C Figure 2.

Peruvin

Cumanin

S e s q u i t e r p e n e l a c t o n e s i s o l a t e d from cumanensis-psilo stachya complex.

the

Ambrosia

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

96

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND

FORESTRY

herbaceous layer (Figure 4), p a r t i c u l a r l y from various species of Commeliriaceae. These results led us to the study of three of the most abundant species i n the coffee orchards: Commelina d i f f u s a , Tripogandra serrulata, and Zebrina sp. A l l species, fresh, dried, and chopped, as well as their l i t t e r , exerted a s i g n i f i c a n t i n h i b i t i o n i n the growth of Bidens p i l o s a (21). Studies on Water Hyacinth Simultaneous to the studies at the coffee plantation was the study of the a l l e l o p a t h i c potential of the water hyacinth (Eichornia crassipes). This aquatic plant, introduced i n Mexico at the beginning of this century, invades many of the water reservoirs and streams and i s considered as one of the worst aquatic weeds i n our country. Its capacity to establish i t s e l f i n several water habitats and i t s extensive vegetative growth suggested a strong mechanism of invasion, perhaps of a l l e l o p a t h i c nature. Several bioassays were used for testing aqueous extracts from leaves, roots plants. Results showed a leaves and flower leachates (Figure 5). Water hyacinth i s widely used as a green f e r t i l i z e r i n the Valley of Mexico, mainly i n the ancestral t r a d i t i o n a l agroecosystems known as "chinampas". These are long narrow s t r i p s of land surrounded on at least three sides by water. Once the a l l e l o p a t h i c potential of water hyacinth was demonstrated i n laboratory assays, we decided to study the e f f e c t of this plant upon the a g r i c u l t u r a l production and growth of weeds i n a chinampa where turnip, radish, lettuce, and cabbage were cultivated. The s o i l i n the chinampa was prepared i n the t r a d i t i o n a l way of peasants at Xochimilco, by making a seed bed with mud from the bottom of the channels that surround the chinampa. When the mud was dry, i t was cut i n small cubes where the seeds were planted. The seedbed was then covered with s o i l and twigs. Once the seedlings reached 10-15 cm they were transplanted to a plot previously weeded and plowed. Treatments were placed randomly and covered with mud. These were: 1. control (without f e r t i l i z e r ) ; 2. inorganic f e r t i l i z e r (10:10:15), 250 g/m ; 3. cow manure; 4. water hyacinth (whole plant); 5. water hyacinth (roots); 6. water hyacinth ( a e r i a l p a r t ) . Treatments 3,4,5, & 6 were added i n a proportion of aproximately 2kg/m . Figure 6 shows that a l l plots with water hyacinth exhibited a higher crop y i e l d than the other treatments. This was mainly due to: 1) improvement of the s o i l texture; 2) a decreased i n the s a l i n i t y of the s o i l due to a reduced water evaporation and s a l t deposition on the s o i l surface. Leaves and whole plants added to the s o i l caused the greatest i n h i b i t i o n s to weeds i n turnip, radish, and cabbage plots (Figure 7). This effect might be due to: 1) a selective a l l e l o p a t h i c effect of water hyacinth and vegetables upon weeds, and/or 2) competition with crops. Water hyacinth has been widely used as a f e r t i l i z e r i n the chinampas because i t improves the physical and chemical properties of s o i l and i t exerts a certain control of weeds through itsdecomposition i n the s o i l . 2

2

Studies on Crop-Weed Relationships As p a r t of the Program of Agroecosystems from INIREB

(Instituto

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

9.

ANAYA ET AL.

Allelopathy

in

97

Mexico

-Bidens pilosa

j

j _ Mimosa pudica_|

60-Γ-

c o

50—

5 .c c

40—

'S

30— 20— 10 — 0 — 10- 1

stem

root Figure

3.

E f f e c t o f the aqueous e x t r a c t s o f s o i l s o f the shrubs s t r a t u m ( c o f f e e ) on weed growth. ( 1 ) S o i l o f Typica c o f f e e . ( 2 ) S o i l o f Bourbon c o f f e e .

too

100

50

50

ο 3*

3

7

4

TREES 1 2 3 4 5 Figure

. . . .

4.

Inga v e r a Inga j i n i c u i l Inga l e p t o l o b a Grevillea robusta L e u c a e n a pulverulenta

JZL 8

COFFEE 6 7 8 9

. . .

10

11

JO

HERBS

10 . Typica 11 . P. Bourbon Mundo Novo Ca t u r r a

aquilinum

E f f e c t o f the aqueous e x t r a c t s of d r y l e a v e s o f t r e e s , c o f f e e and herbs on the g e r m i n a t i o n and growth o f Rumex sp. (· non s i g n i f i c a n t ) .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

98

ALLELOCHEMIC ALS: ROLE IN AGRICULTURE AND FORESTRY -r-100

100-r

504-

δ

8

I i ο

.Turnip

l_Mimosa pudica_l

I

I

Beans_ leaves IZ! roots I.

flowers & F i g u r e 5.

E f f e c t o f aqueous l e a c h a t e s o f Eichomia orassipes ( l e a v e s , r o o t s and f l o w e r s ) on t h e g e r m i n a t i o n and growth o f t h r e e s p e c i e s ( · non s i g n i f i c a n t ) .

^Control ESZÎ Inorganic f e r t i l i z e r f v T l Manure K-ggE-3 Water hyacinth (whole plant) recai Water hyacinth (rhizome and root) ggggj Water hyacinth ( l e a f and bulb)

TREATMENTS Figure 6.

Y i e l d of t u r n i p , r a d i s h , l e t t u c e and cabbage w i t h t h e s i x t r e a t m e n t s ( · non s i g n i f i c a n t ) .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ANAYA ET AL.

Figure

7.

Allelopathy

in

Mexico

Y i e l d and d i v e r s i t y of weeds w i t h ( · non s i g n i f i c a n t ) .

the s i x

treatments

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Nacional de Investigaciones sobre Recursos B i o t i c o s ) , we conducted a study of the relationships between cultivated plants and weeds i n a "chinampa" at San Andres Mixquic, D.F., i n the southeastern Valley of Mexico. In t h i s study i t was found that leachates from c u l t i v a t e d plants (corn, squash, and beans) stimulated the growth of their own seedlings and inhibited that of weeds. Also, we found that corn production i s less affected by the presence of weeds when i t i s associated with Cucurbita f i c i f o l i a (Jimenez et a l . , i n preparation). F i n a l l y , i t was demonstrated that corn pollen has a strong a l l e l o p a t h i c p o t e n t i a l . These results led us to the study of several organic extracts of such pollen tested upon Cassia jalapensis. The hexanic and methanolic f r a c tion proved to be very i n h i b i t o r y to Cassia jalapensis seedlings(22)and the ethanolic extract was found to act as an i n h i b i t o r of electron transport i n isolated mitochondria from watermelon seedlings (Cruz,R., i n preparation). Studies on H e l i e t t a p a r v i f o l i a . In the northern arid region of Mexico, Rovalo e_t a l . (23) carrie H e l i e t t a p a r v i f o l i a . The H e l i e t t a acts as a fungicide upon Pénicillium, Rhizopus, Fusarium, and Aspergillus and also acts as an i n s e c t i c i d e upon Anastrepha ludens ( f r u i t f l y ) . The a l l e l o p a t h i c potential of H e l i e t t a leaves was demonstrated upon a common weed: Convolvulus arvense. Studies on Piqueria t r i n e r v i a . A very i n t e r e s t i n g study i s that of the a l l e l o p a t h i c p o t e n t i a l of Piqueria t r i n e r v i a and i t s piquerols A and B, by Gonzalez de l a Parra et a l . (14). It was found that this widely d i s t r i b u t e d weed i n the Valley of Mexico has a wide b i o l o g i c a l a c t i v i t y upon other plants. Present studies i n warm and temperate region. At present, we are assessing the a l l e l o p a t h i c p o t e n t i a l of weeds from a t r o p i c a l region of the country (Uxpanapa, Veracruz), as a complement to the project e n t i t l e d Recovery of Tropical Rain Forests from INIREB. This information i s necessary to permit more e f f i c i e n t a g r i c u l t u r a l and forest management of the secondary vegetation i n the t r o p i c s . At the same time we are studying the a l l e l o p a t h i c interactions among crops, weeds, and microorganisms and some aspects of the t r a d i t i o n a l agroecosystems known as "camellones" i n Tlaxcala, a central state i n Mexico. Those are distinguished by their b i o l o g i c a l d i v e r s i ty, the presence of water channels along the border of the crop lands, and the t r a d i t i o n a l management system, which involves b i o l o g i c a l cont r o l of pests and the use of green manure i n mono cultures of corn and i n mixed c u l t i v a t i o n with beans and squash. Our f i n a l goal i s to help generate a multiple model of production and to maintain our natural resources.

Literature Cited 1. Gómez-Pompa, Α. ; Anaya, A.L. ; Golley, F. ; Hartshorn, G. ; Janzen, D. ; Kellman, M. ; Nevling, L. ; Penalosa, J. ; Richards, P. ; Vazquez, C. ; Zinke, P. ; Guevara, S. ; In "Fragile Ecosystems" ; Farnsworth, E.G. ; Golley, F.B., Eds. ; Springer-Verlag ; New York, Heidelberg, Berlin, 1974 ; pp. 113-138. 2. Gómez-Pompa, A. ; Vázquez-Yañes, C. ; Amo R., S. del ; Butanda, Α., Eds. ; "Regeneración de Selvas" ; Cia. Editorial Continental, S.A. México, 1976. In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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3. McPherson, J.K. Bull. Torrey Bot. Club 1972, 99, 293-300. 4. Frei, Sister J.K., O.P. ; Dodson, C.H. Bull. Torrey Bot. Club 1972, 99, 301-307. 5. Quaterman, E . , J. Tennessee Acad. Sci. 1973, 48, 147-150. 6. Webb, L . J . ; Tracey, J.G. ; Haydock, K.P. J. Appl. Ecol. 1967, 4, 13-25. 7. Marinero, R.M. "Influencia de Melinis minutiflora Beauv. en el Crecimiento de Cordia alliodora (R y Ρ) Cham". Tésis de Maestría. Instituto de Ciencias Agrícolas, Turrialba, Costa Rica, 1962. 8. Gliessman, S.R. Bot. J. Linn. Soc. 1976, 73, 95-104. 9. Anaya, A.L. "Estudio sobre el Potencial Alelopático de Algunas Plantas Secundarias de una Zona Cálido-Húmeda de México". Tésis Doctoral. Facultad de Ciencias, Universidad Nacional Autónoma de México, 1976. 10. Anaya, A.L. ; Rovalo, M. In "Regeneración de Selvas". Gómez-Pompa, A. ; Vázquez-Yanes, C. ; Amo R., S. del ; Butanda, Α., Eds. ; Cia. Editorial Continental S.A México 1976 ; pp 388-427 11. Rodríguez-Hahu, L. ; G. Rev. Latinoam. Quim 12. Haro-Guzmán, L. ; Silva de Esquivel, Y. Perfumería Moderna, México 1975, 6, 37-40. 13. Romo, J. ; Romo delVivar, A. ; Díaz, Ε. ; Vélez, A. ; León, Ε. ; Urbina, Ε. ; Amo, S. del. Rec. Adv. Phytochem. 1970, 3, 249-254. 14. González de la Parra, M. ; Anaya, A.L. ; Espinosa, F. ; Jiménez, M.; Castillo, R. J. Chem. Ecol. 1981, 7, 509-515. 15. Anaya, A.L. In "Regeneración de Selvas". Gómez-Pompa, A. ; VázquezYanes, C. ; Amo, R., S. del ; Butanda, A. Eds. ; Cia. Editorial Continental, S.A. México, 1976 ; pp. 428-445. 16. Collera Zúñiga, O. "Estudio del Aceite Esencial de Piper auritum". Tésis de Licenciatura. Facultad de Ciencias Químicas, Universidad Nacional Autónoma de México, 1956. 17. Anaya, A.L. ; Amo, S. del; J. Chem. Ecol. 1978, 4, 289-304. 18. Amo, S. del; Anaya, A.L. J. Chem. Ecol. 1978, 4, 305-313. 19. Anaya, A.L. ; Roy-Ocotla, G. ; Ortíz, L.M. ; Ramos, L. In "Estudios Ecológicos en el Agroecosistema Cafetelero"; Jiménez Avila, E. ; Gómez-Pompa, Α., Eds. ; Simposio del Instituto Nacional de Investigaciones sobre Recursos Bióticos, Xalapa, Veracruz; Cía. Editorial Continental, S.A. México, 1982; pp. 83-92. 20. Waller, G.R.; Friedman, J. ; Chou, C.-H. ; Suzuki, T. ; Friedman, Ν. Proceedings of the Seminar on Allelochemicals and Pheromones, Taipei, R.O.C., 1982 ; pp. 230-260. 21. Ramos, L. ; Anaya, A.L. ; Nieto de Pascual,J. J. Chem. Ecol. 1983, 9, 1079-1097. 22. Jiménez, J . J . ; Schultz, K. ; Anaya, A.L. ; Nieto de Pascual, J. ; Espejo, O. J. Chem. Ecol. 1983, 9, 1011-1025. 23. Rovalo, M. ; Graue, B. ; González, M.E. ; González, L. ; Rojas, D.B. Covarrubias, M.L. ; Magallanes, E. Cuaderno de Divulgación 11, 1-19. Instituto Nacional de Investigaciones sobre Recursos Bióticos, Xalapa, Veracruz, México, 1983. RECEIVED

June 9, 1986

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 10 Allelopathy in Subtropical Vegetation and Soils in Taiwan Chang-Hung Chou Institute of Botany, Academia Sinica, Taipei, Taiwan 11529, Republic of China Allelopathy plays an important role in subtropical vegetation and soils, regulating the formation of plant dominance, succession, population dynamics of understory plants and the productivity of many crops in Taiwan findings on autointoxicatio plants, sugar cane plantation, asparagus plants, and pangola grass (Digitaria decumbens), and on allelopathy in relation to agricultural practice, forest plantation, and environmental stresses. Allelopathy even plays an appreciable role in plant adaptation in many natural vegetation and plantations. The responsible phytotoxins reported here are phenolics, flavonoids, alkaloids, and other unidentified compounds. Since the 1960s allelopathy has been increasingly recognized as one of the important ecological factors i n plant interactions and has been regarded as impossible to single out from an environmental complex (1 ). Koeppe et a l . (2, 3) also reported several a l l e l o p a t h i c studies, i n which the tested plants were placed under conditions of environmental stresses. Duke and Putnam (4) introduced the concept into a g r i c u l t u r a l practice to select a crop variety with high phytotoxic potential i n order to avoid using herbicides. In the l a s t decade, a tremendous growth of publication on allelopathy has occurred i n the world (5, 6, 7, 8, 9 ) . Wang and his associates described methods of extraction and i d e n t i f i c a t i o n of phytotoxins i n s o i l (10, 11, 12), and subsequently studied the behaviors of phytotoxic phenolics i n s o i l (13, 14, 15). Since 1972, Chou and his co-workers have conducted such research i n a subtropical humid zone of Taiwan and accumulated substantial information concerning allelopathic interactions i n vegetation and s o i l s (5). These findings of a l l e l o p a t h i c studies are of great significance to understand the role of allelopathy i n the natural and a g r i c u l t u r a l ecosystems i n Taiwan. 0097-6156/87/0330-0102$06.00/0 © 1987 American Chemical Society

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A g r i c u l t u r a l Productivity

Autointoxication, i n which an organism releases a toxic chemical that suppresses i t s own growth, i s one phase of allelopathy. Autointoxication can also be important in i n t r a s p e c i f i c interactions, such as the regulation of population size by self-thinning. Several case studies conducted i n Taiwan are described below. Autointoxication as the cause of low y i e l d of the second crop of r i c e . Rice (Oryza sativa), the most important crop i n Taiwan, i s planted twice a year by a continuous monoculture system. For nearly a century, the y i e l d of the second crop there has been generally lower by 25% than that of the f i r s t crop (a reduction of about 1000 kg/ha). This reduction of r i c e productivity has been p a r t i c u l a r l y pronounced i n areas of poor water drainage. The cropping system of r i c e i n Taiwan i s different from that of other countries. For example crop and the second cro week period elsewhere. In growth of the f i r s t crop (from March to July) the temperature increases gradually from 15 "C to 30 "C but for the second crop (August to December) i t decreases from 30 C to 15 C. Between these two crops, the farmers always leave r i c e stubble i n the f i e l d after harvesting, and submerge these residues in the s o i l for decomposition during the fallowing time. During the second crop season, the typhoon (or monsoon) brings a great amount of r a i n f a l l , leading to a high water table i n some areas where water drainage i s rather poor. Chou and h i s associates therefore conducted a series of experiments to elucidate the reason f o r the low y i e l d of r i c e i n the second crop season. Aqueous extracts of paddy s o i l collected i n Nankang were bioassayed and found to be phytotoxic. In pot experiments, a r i c e straw-soil mixture (100 g: 3 kg) was saturated with d i s t i l l e d water and allowed to decompose for 1, 2, and 4 weeks under greenhouse conditions. S o i l alone was treated i n the same manner, as a control. At the end of each decomposition time, 5 r i c e seedlings (3 weeks old) were transplanted into a pot containing straw-soil mixture or into the control s o i l . After one month, r i c e seedlings grown under control conditions were normal and usually over 66 cm t a l l , while the seedlings grew poorly (about 36 cm t a l l ) i n the straw-soil mixture. The roots of retarded plants were dark brown and the root c e l l s were abnormal and enlarged. Further experimental results showed that when the amount of r i c e straw mixed was increased to 100 g/3 kg s o i l , the phytotoxicity increased with the increase of straw added. The t o x i c i t y was s t i l l persistent after 16 weeks of decomposition. The r i c e straws o i l mixture with d i f f e r e n t i n t e r v a l s of decomposition was extracted with ethanol, the ethanol evaporated, and the residue reextracted with ethyl ether; then the phytotoxins present i n the ether extract were i d e n t i f i e d by chromatography. The compounds i d e n t i f i e d were p-coumaric, p-hydroxybenzoic, syringic, v a n i l l i c , o-hydroxyphenylacetic, and T e r u l i c acids (.16), and propionic, acetic, and butyric acids (17). P a r t i c u l a r l y , o-hydroxyphenylacetic acid, f i r s t reported to be a phytotoxin by us, was toxic to

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f i x e growth at a concentration of 1.64 χ 10 M. We found tha£ the concentration of o-hydroxyphenylacetic acid reached about 10 M i n the s o i l containing decomposing r i c e residues. The additional evidences of phytotoxic effects a r i s i n g from the study w i l l be described l a t e r i n this paper. Inter- and i n t r a - s p e c i f i c interactions between Oryza perennis and Leersia hexandra. The wild r i c e , Oryza perennis Moench, distributed throughout the humid tropics, i s considered to be a progenitor of cultivated 0^_ sativa (18). The Asian race shows a perennial-annual continuum, varying greatly i n various lifehistory t r a i t s among i t s v a r i e t i e s (19, 20). Leersia hexandra Sw. i s a perennial grass with short rhizomes, commonly found i n marshy habitats i n Taiwan and other t r o p i c a l Asian countries. It i s a companion of (h_ perennis i n about 40% of the habitats observed i n India and Thailand (21). In Taiwan, three small populations of 0. perennis, hybrid with 0^ sativa (18), had existed i n marshes along natural streams at Patu around 1975, displace introduction of CL_ perennis populations into different habi.tats indicated that L^_ hexandra was a key determining the b i o t i c enviionment of the former. To look into the interaction mechanisms of the two species, their a l l e l o p a t h i c interrelations were examined by several methods, such as bioassay of the effects of aqueous leachates and extracts of the two species on the radicle growth of r i c e and lettuce and on the growth of adventitious roots from nodes of cuttings of the two species, and the effects of powdered plant material added to s o i l s on the root development of cuttings. Both grass species showed phytotoxic effects on the radicle growth of r i c e and lettuce, i n t r a - and inter-specifically. L,^ hexandra showed i n many cases higher phytotoxicity than Oryza although the pattern of variations was complex. The concentrations of many phytotoxins i d e n t i f i e d were higher i n Leersia extract than i n that from Oryza. Observation of plants growing from buried seed pool i n s o i l s to which powdered plan,: materials were added also showed higher phytotoxicity of Leersia than Oryza. Probably, allelopathy plays an appreciable role in the successional replacement of the two species (22). Autointoxication of sugar cane plantation. Inadequate germination and growth of ratoon cane have been found to be the two major problems i n the farms of Taiwan Sugarcane Corporation (TSC). The y i e l d of monoculture sugar cane has declined i n many sugar cane f i e l d s . The causes of t h i s y i e l d reduction have been investigated, but no single factor causing the reduction can be found. Wang et al. (23J demonstrated by f i e l d and laboratory experiments that phytotoxic effects are one of the important factors involved. Five phenolic acids (p-hydroxybenzoic, ferulic, p-coumaric, syringic, and v a n i l l i c ) and formic, acetic, oxalic, malonic, t a r t a r i c , and malic acids were i d e n t i f i e d i n t^e decomposing sugar cane leaves i n water-logged s o i l . At 3 χ 10 M solution of these phenolic acids i n water culture, the growth of young sugar cane root was i n h i b i t e d . The a l i p h a t i c acids w^ere also found to i n h i b i t the growth of ratoon sugar cane at 10 M. Furthermore, Wu et

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al.(17) found that the population of Fusarium oxysporum associated with the rhizosphere s o i l of poor ratoon cane roots was much greater than that of good growing ratoon or of newly planted sugar cane roots. They found that fusaric acid, a secondary metabolite of the organism, was toxic to the growth of young sugar cane plants i n v i t r o (17). Autointoxication of Asparagus officinalis L. Asparagus o f f i c i n a l i s i s a perennial ratoon crop widely planted i n many plantations of Taiwan. A s i g n i f i c a n t reduction of y i e l d and quality of asparagus often occurs i n old plantation s o i l . The wilting of asparagus plants has been found to be due to monoculture of the crop. Young (2Λ) indicated that there was about 40% of asparagus seedlings missing from the plantation. Young further showed that the root exudates of asparagus retarded the seedling growth of asparagus c u l t i v a r s , namely Mary Washington, C a l i f o r n i a 309 and C a l i f o r n i a 711 (24). Exudate collected by use of th s i g n i f i c a n t l y retarded seedlings (25). Six phytotoxic phenolics, namely 3,4-dihydroxybenzoic, 3,4-dimethoxybenzoic, 2,5-dihydroxybenzoic, 3,4dihydroxyphenylacetic, and p-(m-hydroxyphenyl)propionic acid, and 3,4-dimethoxyacetophenone were found i n the extracts and exudates of asparagus plant parts. The amount of phytotoxins i d e n t i f i e d was s i g n i f i c a n t l y higher i n the stem than i n the root, and was well correlated to phytotoxicity (25). I t i s concluded that the reduction of asparagus productivity i n old asparagus f i e l d s i s due primarily to phytotoxins released from the plant parts and those produced from the decomposition of residues remaining i n s o i l . Allelopathy And A g r i c u l t u r a l Practice Allelopathy of native and pasture grasses. Miscanthus floridulus, widely distributed i n Taiwan, i s a native and predominant grass and often occurs i n poor s o i l on h i l l s i d e s and /or channels. A f i e l d experiment conducted at a Nankang h i l l s i d e showed that the botanical composition i n Miscanthus stands i s about 65% for M. f l o r i d u l u s , 17% for Lactuca indica, and less than 4% for Eupatorium formosanum, Brachiaria distachys, Sporobolus fertilis, Pouderia scandens, Cyperus pilosus, Digitaria violascens, and three unknown grasses (26). A successional trend of botanical composition was caused by the aggressive nature of Miscanthus f l o r i d u l u s . For example, i n an experiment i n which M. f l o r i d u l u s was cleared, the dominance of Miscanthus recurred after three years. The associated species found i n the Miscanthus stands were again found to be suppressed by the Miscanthus (5,). Furthermore, aqueous extracts and leachate of Miscanthus leaves caused a s i g n i f i c a n t reduction of radicle growth of tested species (26). Additionally, the extracts of s o i l s collected from the Miscanthus rhizosphere, between stands, under the canopy of Miscanthus, and i n open ground control area adjacent to the Miscanthus stands were also bioassayed for their phytotoxicity. Of these, the extract of root s o i l of Miscanthus exhibited the highest i n h i b i t i o n of the tested plants (26).

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Some 12 subtropical introduced species of forage grasses gave aqueous leaf extracts evaluated for their phytotoxicity on tested species. Acroceras macrum, Cynodon dactylon, Chloris gayana,· D i g i t a r i a decumbens, Eragrostis curvula, Panicum repens, and P. maximum always caused s i g n i f i c a n t i n h i b i t i o n of radicle growth of test plants. Of them, D i g i t a r i a decumbens had the highest phytotoxicity upon the tested species at 10 milliosmols, i n which the osmotic i n h i b i t i o n i s zero (27, 22). Chou furthermore found that D^ decumbens was also an autotoxic species, and the productivity was s i g n i f i c a n t l y depressed after several years of planting (Chou, unpublished data). The aqueous leachate and exudates of D i g i t a r i a plants showed a s i g n i f i c a n t reduction of growth of this species. Selection of weed control grass for pasture. An increased amount of a l l e l o p a t h i c research on grassland species has been conducted i n many parts of the world during recent decades (5, 9.) Most of the studies have bee a l l e l o p a t h i c phenomen employed the a l l e l o p a t h i c effect as a p r a c t i c a l means of d i r e c t l y c o n t r o l l i n g weeds. In Taiwan, many grasses have been introduced into pasture but only a few v a r i e t i e s can be established as forage pasture. As already mentioned, among 12 species studied (28), pangola ( D i g i t a r i a decumbens) exhibited the highest toxic effect on test species. Under sufficient nitrogen fertilizer application, pangola grass forms a pure stand where almost no other weeds can grow. We also found that d i f f e r e n t v a r i e t i e s of pangola had different growth performance and competitive ability. Liang et a l . (29) thus selected eight v a r i e t i e s of pangola for f i e l d t r i a l s and laboratory assays. These showed that the invasion a b i l i t y of c u l t i v a r s A65, A255, and A254 were highest i n Hsinhwa, Hengchun, and Hwalien station, respectively; while c u l t i v a r s A79 and A80 were i n f e r i o r i n a l l stations. Cultivars A84, A254, and A255 possessed the highest t o x i c i t y , which was due to phytotoxins, of which nine phytotoxic phenolics were identified. The interference of grasses i n the f i e l d i s very complicated, and allelopathy alone cannot account for the complicated phenomena. Further f i e l d and laboratory experiments thus need to be performed i n order to c l a r i f y the role of allelopathy i n grassland ecosystems. Phytotoxic e f f e c t of cover crops on orchard plants. Wu et a l . (30) compared the phytotoxic e f f e c t s of some cover crops, namely Centrocema sp., Indigofera sp., and Paspalum notatum (Bahia grass), on the growth of pea, mustard, cucumber, cauliflower, rape, Chinese cabbage, mungbean, watermelon, tomato, and r i c e . They found that rape was most sensitive to the extracts of these cover crops. Among them, Centrocema and Indigofera exhibited the greater phytotoxic e f f e c t ; moreover, the leachate of Centrocema inhibited the growth of banana. More recently, several cover crops including Bromus catharticus, Pennisetum cladestinum, Lolium multiflorum (both chromosome 4X and 2X c u l t i v a r s ) , Paspalum notatum, and white clover are now under investigation for a l l e l o pathic effects on the productivity of apple and peach plantations

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in the Lishan area of central Taiwan. A vast area of apple plantations has been situated on the h i l l s i d e s of the Central mountain since the 1950s. The productivity of these plantations was exceedingly high i n the f i r s t decade after planting but has gradually decreased i n recent years. In fact, t h i s problem has been encountered i n many European countries and Northern America as well. Forest-pasture intercropping system. Taiwan i s an island, with two thirds of the land occupied by mountains, and i t s forests are extremely important for water conservation. The limited amount of a g r i c u l t u r a l land for crops and pasture forces farming a c t i v i t i e s to move upward to h i l l s i d e s and higher elevations. A forestpasture intercropping system has been thought to be a possible way to increase livestock production. Recently we have conducted several experiments i n the forest area of Hoshe Experiment Station of National Taiwan University located at an elevation of about 1200 meters. An are was cleaned by removin 'Cunninghamia lanceolata), and part was l e f t unchanged to serve as control. The cleaned and unchanged plots were planted with kikuyu grass (Pennisetum cladestinum) or l e f t open. The experiment was designed to determine the reciprocal interaction of f i r l i t t e r and kikuyu grass, and to evaluate the a l l e l o p a t h i c potential of the two plants on weed growth under natural condition. Results indicated that the biomass of kikuyu grass i n the cleaned plot was signifcantly higher than that i n the control plot. In addition, the number of weeds that grew i n the plot planted with kikuyu grass was lower than that i n the control plot, indicating that the kikuyu grass may compete with and suppress weeds. The seedlings of f i r regenerated i n the deforested area grew well and seemed to not be affected by the neighoring newly planted kikuyu grass. However, the growth of kikuyu grass was inhibited by the f i r l i t t e r l e f t on the unchanged plot i n the f i r s t three months after deforestation. Furthermore, bioassay of aqueous extracts showed that the f i r l i t t e r extract exhibited higher phytotoxicity than the kikuyu grass. Nevertheless, four months after deforestation the kikuyu grass growth i n the f i e l d was luxuriant, indicating that the phytotoxicity of f i r l i t t e r disappeared (Chou et a l . , 1985 unpublished data). Forest intercropping system. On the h i l l s i d e s of mountainous d i s t r i c t i n Taiwan, there i s an increasing area of deforestation. Forest regeneration of the area i s very important to ecological conservation. Many highly valuable forest species have been planted i n a forest intercropping system, such as bamboo, conifers, Acacia confusa, Leucaena leucocephala, Liquidambar formosana, Casuarina glauca, Alnus formosana, and Pinus taiwanensis. We have evaluated the s u i t a b i l i t y of intercropping systems among the aforementioned species. The f i r s t experiment was conducted with leucocephala, an a l l e l o p a t h i c plant, intercropped with other species mentioned above. Pinus taiwanesis grew very well and could tolerate the leachate of leucocephala, but the remaining species were damaged by the leachate to some

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND

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extent. As mentioned e a r l i e r , we found several phytotoxic phenolics and mimosine produced by L^ leucocephala. It i s notable that the growth of Mimosa pudica was suppressed by Leucaena leaf leachate, even though the leaf juice of M^_ pudica contains a r e l a t i v e l y high amount of mimosine. Among 84 seedlings of M^_ pudica tested only 2 seedlings survived, showing that mimosine can be p r a c t i c a l l y useful to control a notorious weed such as M. pudica i n the f i e l d . Allelopathy And Forestry Plantation Allelopathic nature of some bamboos. On many h i l l s i d e s of mountainous d i s t r i c t s i n Taiwan, there i s a vast area of bamboo plantations, and i n the Chitou area we often found Cryptomeria japonica (conifer) and Phyllostachys edulis (bamboo) growing adjacent to one another. However, the P. edulis often encroaches on the C_j_ japonica area, resulting i n the gradual decline of productivity and ultimatel and Yang (31) found tha possesses phytotoxic phenolics, which suppress the growth of i t s understory. The f l o r i s t i c composition of the two vegetations showed that the understory species are d i f f e r e n t . For example, f i v e predominant species of the understory i n the ί\_ edulis community are Ageratum conyzoides, Cornmelina undulata, P i l e a funkikensis, Pratia nummuaria, and Tetrastigma formosana; while i n the C^ japonica community, they are Ficus pumila, Pellionia scabra, Pilea funkikensis, Piper arboriola and Urtica thunbergiana. These species respond d i f f e r e n t l y either to l i g h t intensity or to the phytotoxic leachates, so that there i s a d i f f e r e n t d i s t r i b u t i o n of species density and biomass under the canopy of the two tree species. The t o t a l number and dry weight of seedlings per square meter were much higher i n the conifer community than i n the bamboo forest, although the l i g h t intensity, s o i l moisture, and nutrient contents were s i g n i f i c a n t l y higher i n the bamboo habitat than i n the conifer. Further experimental r e s u l t s indicated that the aqueous extracts and leachates of bamboo leaves were more phytotoxic than those of conifer leaves, r e f l e c t i n g that allelopathy plays a s i g n i f i c a n t role i n the regulation of species diversity and production under the canopy of at least these two forests. Nevertheless, the difference i n potential for species exclusion between the two forests may be due partly to an anatomic factor, such as the rhizome. There are two types of rhizomes, sympodial rhizocauls and horizontal rhizomes with l a t e r a l culms. edulis has the l a t t e r type, which grow rapidly. Thus, the invasion of P_;_ edulis to t e r r i t o r y of C. japonica may be due to (a) the fast-growing rhizomes, which may possibly release phytotoxic root exudates, and (b) a l l e l o p a t h i c substances produced by the bamboo leaves and decomposing l i t t e r . The continuous release of water-soluble phytotoxins from F\_ edulis and accumulation of these i n the s o i l may result i n suppression of the growth of understory or i n elimination of neighboring plants. In addition, the aqueous leaf extracts of 14 bamboo species were evaluated for a l l e l o p a t h i c potential. The bioassay results showed that Sinocalamus l a t i f l o r u s possessed the highest phytotoxicity

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

10.

CHOU

Allelopathy

in Subtropical

Vegetation and Soils in

Taiwan

109

for lettuce, rye grass, and r i c e plants, but Bambusa oldhami, B. pachinensis, B. ventricosa, Phyllostachys edulis, and ί\_ makinoi also showed s i g n i f i c a n t phytotoxicity. Aqueous extracts obtained from the associated bamboo s o i l s also exhibited some i n h i b i t i o n , which i n most extracts was correlated to that of leaf extracts (32). Allelopathic e f f e c t of Leucaena leucocephala. Leucaena leucocephala trees have been widely planted in Taiwan because of i t s high economic value for producing n u t r i t i o u s forage, firewood, and timber. Generally, after a few years of growth, the f l o o r s of these plantations are r e l a t i v e l y bare of understory plants, except Leucaena seedlings. This pattern of weed exclusion beneath Leucaena trees i s p a r t i c u l a r l y pronounced i n areas having a drought season. Chou and Kuo (33) therefore undertook a series of experiments conducted i n f i e l d s , greenhouse, and laboratory. F i e l d data showed that the phenomenon was not due primarily to physical competition, involvin nutrients. Instead, aqueou litter, s o i l , and seed exudate showed s i g n i f i c a n t l y phytotoxic effects on many test species, including r i c e , l e t t u c e , Acacia confusa, Alnus formosana, Casuarina glauca, Liquidambar formosana, and Mimosa pudica. However, the extracts were not toxic to Leucaena seedlings. Decomposing leaves of Leucaena also suppressed the growth of the aforementioned plants grown i n pots but did not i n h i b i t that of Leucaena plants. 3y means of paper and thin-layer chromatography, UV-visible spectrophotometry, and high performance l i q u i d chromatography, 10 phytotoxins were identified. They included mimosine, quercetin, and gallic, protocatechuic, p-hydroxybenzoic, p-hydroxyphenylacetic, v a n i l l i c , f e r u l i c , c a f f e i c , and p-coumaric acids. The mature leaves of Leucaena contain about 5% (dry weight) of mimosine, the amount varying with v a r i e t i e s . Seed germination and r a d i c l e growth of lettuce, r i c e , and rye grass were s i g n i f i c a n t l y i n h i b i t e d by aqueous mimosine solutions at a concentration of 20 ppm while that of the forest species mentioned was suppressed by mimosine solution at 50 ppm or above. Hov/ever, the growth of Miscanthus f l o r i d u l u s and Pinus taiwanensis was not suppressed by a mimosine solution at 200 ppm. Seedlings of Ageratum conyzoides died i n mimosine solution at 50 ppm within 7 days and wilted at 300 ppm within 3 days. It i s concluded that the exclusion of understory plants i s due to the a l l e l o p a t h i c effect of compounds produced by Leucaena. The a l l e l o p a t h i c pattern was most c l e a r l y shown i n the area with a heavy accumulation of Leucaena leaf l i t t e r , which was a r e s u l t of drought and heavy winds. Allelopathic potential of V i t e x negundo. Vitex negundo i s a dominant component of coastal vegetation and widely distributed i n the southern parts of Taiwan. Chou and Yao (34) found that the biomass and density of i t s associated understories are relatively lower than i n adjacent pasture. F i e l d results showed that the natural leachate of V_^_ negundo s i g n i f i c a n t l y retarded the growth of D i g i t a r i a decuabens but stimulated the growth of Andropogon nodosus as compared to the r a i n f a l l control. The growth of

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

110

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND

FORESTRY

decumbens grown i n pots under greenhouse conditions was s i g n i f i c a n t l y retarded by watering with a 1% aqueous extract of V. negundo, but the growth of Andropogon nodosus and Mimosa pudica was stimulated. The aqueous extract was phytotoxic to lettuce and rye grass seeds. The aqueous effluents obtained from a polyamide column chromatograph were also bioassayed. Some fractions inhibited r a d i c l e growth of lettuce and r i c e seedlings, whereas other f r a c t i o n s had a stimulatory e f f e c t . The responsible substances were isolated and i d e n t i f i e d . These included phenolic acids, p-hydroxybenzoic, f e r u l i c , p-coumaric, v a n i l l i c , and syringic acids, and 10 flavonoidsT One flavonoid, 3hydroxyvitexin, and nine other flavonoids were i d e n t i f i e d (34). f

Allelopathy And Environment Relationship The actions of many a l l e l o p a t h i c compounds produced by plants are often affected by environmental factors, such as water potential of the environment moisture, nutrient, an released to the environment by means of v o l a t i l i z a t i o n , leaching, decomposition of residues, and root exudation (_1, 5, 7_, 9). Firstly, the terpenoids, such as 0 are common constituents of Eucalyptus o i l s . These and other compounds may contribute to a l l e l o p a t h i c manifestations by Eucalyptus baxteri ssp. (9), E. regnans F.Muell. (1^3) and E. globulus ssp. (8"). Allelopathy and Introduced Species Examples of allelopathy between weeds and crop or pasture species i n Australia have been documented within the past decade. Lovett and Lynch (17) discussed the association of Salvia reflexa Hornem. (mintweed), an annual member of the family Lamiaceae introduced to Australia from North America (35)· This species produces aromatics as well as water-soluble compounds that may be i n h i b i t o r y to species such as wheat and sorghum. Kloot and Boyce (_21) documented i n h i b i t o r y effects of allelochemicals produced by Polygonum aviculare L. (wireweed) on the annual Medicago species, important leguminous components of annually regenerating pastures i n southern Australia.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

(12)

Ashton and W i l l i s

Willis

Lange and Reynolds

(13)

(11)

1982

198Ο

Eucalyptus regnans

Eucalyptus microcarpa

Nothof agus ninghami i and other r a i n f o r e s t species

Eucalyptus b i c o s t a t a

Pinus sp., A r a u c a r i a sp. F l i n d e r s i a sp.

C a l l i t r i s calcarata Eucalyptus crebra E. dawsoni i E. m e l l i o d o i a Not elaea microcarpa

Casuarina leuhmanii

Acacia pendula

Eucalyptus mol] uccana

Eucalyptus p i l u l a r i s

A l l e l o p a t h i c species

H I i s et al . (10)

19 S3

1976

1 *•>02

Eucalyptus b a x t e r i i

(7)

(2)

repens

Seedlings of E. regnans

Gonocarpus elatus

Seedlings of E. regnans

Leptospermum myrsinoides Casuarina

Festuca rubra var. fa"l lax

Trifolium

Bothriochloa ambigua Eragrosti s J eptostachya Sporobolus elongatus

Seedlings of E. pi 1 u l a r i s

Species a f f e c t e d

Lipids

Lipids

Gentisic,ellagic g a l l i c , sinapic, caffeic acids Phenolic aglycones Glycosides Terpenoids

Alleiochemicals present

Chemicals from f o l i a r and l i t t e r leachates i n h i b i t ­ ory i n bioassays

Frass from Paropsis atomaria r e s u l t e d i n growth inhibition

Sensitive to s o i l type

'Halo' e f f e c t v i s i b l e on a e r i a l photographs

Microorganism antagonism implicated

NH3/NO4 balance c r i t i c a l f o r seed­ ling survival. Mature t r e e s strongly mycorrhizal competition w i t h microorganisms f o r NH3. A n t a g o n i s t i c s o i l f a c t o r s may be a f f e c t e d by root exudates

Suppression of G. e l a t u s under E. microcarpa

Dieback of mature trees; i n h i b i t i o n of r e g e n e r a t i o n by seedlings

Suppression of L. myrsinoides and C. p u s i l l a Feneath canopy of E. b a x t e r i i

E x c l u s i o n from zone around E. b i c o s t a t a

Species compos­ i t i o n within 'circle' effect of t r e e s d i f f e r ­ ent t o o u t s i d e

Grasses statistically sparser beneath t r e e canopies

"Prima f a c i e " evidence

Difference i n F a i l u r e of seedabundance of r h i 20- l i n g s t o r e sphere fungus, generate i n Cylindrocarpon absence of f i r e destructans, between healthy and unhealthy roots of s e e d l i n g s

I n h i b i t i o n of growth

I n h i b i t i o n of growth

Blackening of radicle tips

Seedling t i p necrosi s, chlorosi s

A f f e c t s root and root h a i r development and shoot growth

Effects

Reports of Allelopathic Phenomena i n A u s t r a l i a — Native Species

Del Moral et a l . (9)

(8)

and S i lander

S i l a n d e r et a l .

Trenbath

A uthor* s ) Florence and Crocker

Table I I .

70

Ά

m

70

m > Z D *n Ο

70

n c r H G

> ο

ο

70

>

m g ο

χ

r r m r Ο ο

>

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

(20)

K l o o t and Boyce (21)

Lovett et a l .

1982 Polygonum a v i c u l a r e

1981 Datura stramonium

Medicago

truncatula

Linum u s i t a t i s s i m u m

Linum u s i t a t i s s i m u m

198l Camelina s a t i v a

Lovett and D u f f i e l d (19)

T r i t i c u m aestivum

1979 S a l v i a r e f l e x a

T r i t i c u m aestivum

Tropane alkaloids

benzlyamine

Carduus pycnocephalus Cirsium vulgare C. arvense Hordeum d i s t i c h u m LOT ium perenne T r i f o l i u m subterraneum Silybum marianum

T r i t i c u m aestivum

T r i t i c u m aestivum

T r i t i c u m aestivum Avena s a t i v a

Lovett and Speak (18)

1975 C i r s i u m arvense

1973b T r i t i c u m aestivum (residuesl

Secale c e r e a l e Pisurn sativum (residues )

1973a Hordeum v u l g a r e Avena s a t i v a

1967 T r i t i c u m aestivum (residuesl

Year A l l e l o p a t h i c species Species a f f e c t e d

1979 S a l v i a r e f l e x a

(3)

Allelochemicals present

Reduced germination and i n h i b i t i o n of r a d i c l e growth Causes m o r p h o l o g i c a l deformities i n germinating medic s e e d l i n g s by i n t e r ­ fering with c e l l d i v i s i o n and e a r l y growth o f meristems

High c o n c e n t r a t i o n s of benzylamine inhibited radicle elongation

I n h i b i t i o n o f germin­ a t i o n and e a r l y growth

I n h i b i t i o n o f germin­ a t i o n and e a r l y growth

I n h i b i t i o n o f seed germination and s e e d l i n g growth

Reduction o f germin­ a t i o n , growth and ultimate y i e l d

I n h i b i t i o n o f root growth

Root growth reduced

Water s o l u b l e allelochemicals present i n green l e a v e s and stems

Phyllosphere bact­ e r i a - Pseudomonas f l u o r e s c e n s and Enterobacter cloacae convert benzyl i s o t h i o c y a n ate t o benzylamine and hydrogen sulphide

Continued on next page

F a i l u r e o f medic pastures to s e l f - r e g e n e r a t e i n paddocks dominated by Polygonum

A noxious weed, e s p e c i a l l y of summer crops

F i e l d e f f e c t s noted by e a r l i e r workers

Trichomes i m p l i c a t ­ ed as r e s e r v o i r s for allelochemicals

S o i l type shown t o a f f e c t expression of a l l e l o p a t h y

C. arvense i s autot o x i c - both r o o t s r o o t s and f o l i a g e i n h i b i t growth o f seeds and s e e d l i n g s

E f f e c t diminished w i t h time. Ν immobilization may be i n v o l v e d

Toxic e f f e c t i s o f an ephemeral nature

Phytotoxic effect d i m i n i s h e d with time, depended on previous weathering and v a r i e d w i t h v a r i e t y of wheat

Comments

Species

Observed c o m p e t i t i v e a b i l i t y i n the f i e l d

F a i l u r e o f C. seedlings

Proximity o f new wheat straw r e s i d u e s t o sown wheat seed

Germination, p l a n t growth and u l t i m a t e y i e l d affected

"Prima f a c i e " evidence

Reports of A l l e l o p a t h i c Phenomena i n Australia—Introduced

Lovett and Lynch ( .17)

Kimber

Table I I I .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1985 Brassica napus Sorghum bicolor Pisum sativum Helianthus annuus Triticum aestivum (residuesl

Imperata cylindrica (L.) Beauv.

1984 Datura stramoni'

Avena fatua Avena ludoviciana

subterraneum Calopogonium mucunoides

nthus annuus

et al. (30)1986 Brassica campestris Triticum aestivum B. juncea B. napus B. nigra

Purvis et al.

Levitt et al. (27)

Helianthus annuus

Calotropis procer;

1984 Cenchrus ciliaris

Levitt and Lovett (26) 1984 Datura stramonium

Triticum aestivum

1983 Salvia reflexa

Songlei

Inhibition of radicle growth

Suppression of root, stem and leaf growth of Calotrope seedlings Inhibition of radicle growth

Germination and seed­ ling growth adversely affected

A noxious weed which colonizes large areas to the virtual exclusion of other species

Calotrope seedlings fail to establish in well grown buffel grass areas

Crop residues may have a deleterious affect on the following crop and may also affect weed growth

"Prima facie" evidence

Variable inhibition of Effects persisted through growth to final grain yield

£-hydroxyDelay of germination benzoic acid and inhibition of vanillic acid radicle growth £-coumaric acid ferulic acid Germination and growth of wild oats differ­ entially affected by crop residues

Tropane alkaloids

Tropane alkaloids

Monoterpenes

Crop residues affect early growth of wheat by affecting germination emergence, coleoptile height and length of longest seminal root

Allelochemicals present Year Allelopathic species Species affected Inhibitioi of early 1982 Camelina sativ, Linum usitatissimum Benzylamine growth

Continued

Lovett and Jessop (23) 1982 Pisui Vicia faba c Glycine max Lupinum angustifolius Cicer arietinum Carthamus kinctorius Helianthus annuus Brassica napus Sorghum bicolor Avena sativa Hordume vulgare Triticum aestivum

Table I I I .

Effects documented in the field

Implications for crop rotation development

Primary effect of allelochemicals on metabolism of food reserves. Effect documented in the field Soil type shown to affect expression of allelopathy

Unidentified but active watersoluble compound(s) also present

Comments Indirect effect of benzylamine through creation of hydro­ phobic conditions in soil Phytotoxic effect increased when crop residues were incorporated into the soil

70

H 70

tn C/3

70

D Ο

> z

ο c r H G 70 m

> ο

ο

70

>

m g

χ

r m r Ο π

ON Ο

15.

LOVETT

Grazing

Allelopathy

Capacity

in Australia:

Bacterial

Table IV Loss Due to Shrub Invasion on Located West of Wanaaring (33)

Lost grazing capacity Property A Β C D Mean a

161

Mediation

C were inspected

Properties

Lost grazing capacity

(1978X56)

(1968-70)(36)^ 1 4 6 9 5

Properties A, Β and

Four

8

10 22 15 14 i n 1968

and

property

D in

1970.

An example of allelopath grass against a weed i the effects of Cenchru (buffe grass) Calotropi procera (Ait.) W.T. A i t (calotrope), also an import to Australia LMeadly (37)]. Sixand 9-week old b u f f e l grass plants s i g n i f i c a n t l y suppressed the growth and development of calotrope seedlings, which were also i n h i b i t e d when grown i n s o i l which previously supported a b u f f e l grass stand (Table V). Cheam (36, 38) considers that planting of b u f f e l grass w i l l prevent ingress by calotrope i n areas that are now free of the weed, that introduction of b u f f e l grass on land already infested should lead to a steady decline i n the calotrope population, and that the bioactive compound may prove useful as a 'natural herbicide' for C. procera and other species. Table V Germination and Growth of Calotrope i n S o i l i n which Buffel Grass Had Grown for 6 Weeks Previously [The treated s o i l was free from b u f f e l grass roots.] (25) Cumulative germination(%) Growth response Weeks a f t e r sowing Plant F i r s t pair of true leaves Treatment 1 week 2 weeks height(mm) Length(mm) Breadth(mm) Control s o i l EÔ7Ô W7j 49.6 3075 Γ8Τ5 Treated s o i l 83.3 90.0 32.8 21.7 12.8 Level of significance N.S. N.S. Ρ = 0.001 Ρ = 0.001 Ρ = 0.001 This example illustrates the benefits and costs of introducing plants to new l o c a l i t i e s . Over the mere two hundred years of European settlement i n Australia many of the World's most important crop and pasture plants and a l l of the World's worst weeds (Table I) have been introduced to the continent. The l a t t e r have attained problem status and several other introductions, such as Echium plantagineum L. (Paterson's Curse) from the Mediterranean region, freed from those organisms which maintain them i n balance i n their native communities, have posed threats to agriculture.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

162

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND

FORESTRY

Parthenium hysterophorus L. (parthenium weed), native to North and Central America and introduced into Queensland as recently as I960 (39)> is contemporary example. It i s aggressive, persistent and lowers crop y i e l d through interference, a component of which i s a l l e l o p a t h i c (40). a

Allelopathy and Microorganisms The production of compounds which may act as allelochemicals i s not r e s t r i c t e d to higher plants. For example, Heisey, DeFrank and Putnam (41) have discussed substances produced by soil microorganisms which may have h e r b i c i d a l a c t i v i t y . Bacteria may also mediate a l l e l o p a t h i c a c t i v i t y i n economically important situations such as forest regeneration (Line, personal communication); crop/weed associations (42), and reduced c u l t i v a t i o n systems where plant residues are retained (43). Cruciferous species, i n which the glucosinolates are b i o l o g i c a l l y active compound the latter categories associated with introduced c r u c i f e r s such as Brassica t o u r n e f o r t i i Gouan (wild turnip) and a more complete study has been made of Camelina sativa (L.) Crantz (false f l a x ) . Grummer and Beyer (45) reported allelopathy between Linum usitatissimum L. and Camelina species i n the f i e l d , providing that r a i n f e l l during a c r i t i c a l (unspecified) period of growth. Lovett and Sagar (42), working i n the United Kingdom, established that the presence of bacteria i n the phyllosphere of C. sativa was necessary for allelopathy to be manifested. The organisms were f r e e - l i v i n g , motile, Gram-negative rods representative of the bacteria which tend to predominate i n the phyllosphere (46) and were i d e n t i f i e d as Enterobacter cloacae (Jordan) Hormaeche and Edwards. In subsequent work, carried out i n A u s t r a l i a , Pseudomonas fluorescens (Trevisan) Migula has been i d e n t i f i e d as playing a similar role (47)· Typically, bacteria are recovered from aqueous washings of fresh f o l i a g e of C. sativa. I f foliage washings are incubated at +23 C for 24 h the washings become strong smelling and cloudy i n appearance. I f bacteria are removed from the washings a f t e r c o l l e c t i o n , by f i l t r a t i o n , no change i s noted. GC/MS analyses have shown that organic acids of the c i t r i c acid cycle are present i n fresh washings but are much depleted a f t e r 24 h incubation with bacteria (Figure 1, Figure 2). Similar results are obtained by inoculation of l e a f washings, freed of bacteria by M i l l i p o r e f i l t r a t i o n , with either of the two bacteria i d e n t i f i e d (19)» Lovett and Jackson (47) observed that C. sativa l e a f washings exhibited allelopathic activity in bioassay after incubation for as l i t t l e as 12 h and demonstrated that during this period of time there was exponential growth of the b a c t e r i a l population (Figure 3). Benzyl isothiocyanate was i d e n t i f i e d i n aqueous extracts of C. sativa f o l i a g e by Lovett and D u f f i e l d (19). Tang, Bhothipaksa and Frank (48) showed that E. cloacae was capable of degrading benzyl isothiocyanate to hydrogen sulfide and benzylamine. Tests were, accordingly, carried out with incubated l e a f washings of Camelina and showed the presence of hydrogen sulfide and

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

LOVETT

Allelopathy

in Australia:

Bacterial

163

Mediation

100"

50-

-l 100

1 2 0 0

1

1

1

I

3 0 0

4 0 0

500

6 0 0

Scan number

Figure 1. Total i o n current of η-butyl esters of the a c i d i c f r a c t i o n from non-sterile leaf washings of C. sativa. Key: 1 = oxalic acid; 2 = malonic acid; 3 = maleic acid; 4 = succinic acid; 5 = fumaric acid; 6 = alpha-ketoglutaric acid; 7 = c i s - a c o n i t i c acid; 8 = c i t r i c acid (19)«

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

ι oo-i

50-

Scan number

F i g u r e 2. T o t a l i o n c u r r e n t o f n - b u t y l e s t e r s o f t h e a c i d i c f r a c t i o n f r o m s t e r i l e l e a f w a s h i n g s o f C. s a t i v a . Key: 1 = oxalic acid; 2 = malonic acid; 3 = maleic acid; 4 = succinic acid; 5 = fumaric acid; 6 = malic acid; 7 = alpha-ketoglutaric a c i d ; 8 = c i s - a c o n i t i c a c i d ; 9 = c i t r i c a c i d . (Reproduced w i t h p e r m i s s i o n f r o m r e f e r e n c e 19. C o p y r i g h t 1981 B l a c k w e l l Scientific Publications Ltd.)

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

15.

LOVETT

A llelopathy

in A ustralia: Bacterial

165

Mediation

F i g u r e 3. I n c r e a s e i n b a c t e r i a l c o l o n y numbers incubation period. (Reproduced w i t h permission C o p y r i g h t 1980 The New P h y t o l o g i s t . )

d u r i n g a 24-h from reference

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

47.

166

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND

FORESTRY

benzylamine (19). The l e v e l of the l a t t e r component, i n p a r t i c u l a r , i s variable depending on age of the plant and season of the year. This observation accords with the report of Griimmer and Beyer (45) that allelopathy i n the f i e l d occurred only at a p a r t i c u l a r time of the year. Our data (43) have demonstrated that bacteria are most p r o l i f i c on senescent leaves, a f i n d i n g which agrees with that of E t t l i n g e r and Kjaer (49), namely that i n j u r y to plants containing glucosinolates r e s u l t s i n the l i b e r a t i o n of isothiocyanates from those substances. Thus, the presence of available quantities of isothiocyanate and large numbers of bacteria during senescence of the weed could explain f i e l d observations of allelopathy. Two e f f e c t s of benzylamine as an allelochemical have been documented. When L. usitatissimum i s used i n bioassay, germination i s impaired but only at r e l a t i v e l y high concentrations of the allelochemical ( i n excess of 500 ppm). Radicle length of germinating L. usitatissimum i n bioassay i s increased at low concentrations of benzylamine (less than 200 ppm) but is increasingly i n h i b i t e d however, i n h i b i t i o n may 4), probably as a result of improved contact between germinating seedlings and substrate. In addition to direct effects on the plant, benzylamine may induce hydrophobic (water repellent) conditions i n s o i l (Figure 4 ) . These data indicate a l i n e a r increase i n moisture content as benzylamine content increases, a t t r i b u t a b l e to the development of a lower unsaturated hydraulic conductivity i n the surface s o i l , which thus became less able to transfer water from depth i n response to evaporative demand. McGhie (50) suggests that poor germination of crop and pasture plants may be related to the development of hydrophobic conditions, the affected s o i l being unable to supply water to the germinating seed. Strains of E. cloacae are known to f i x nitrogen (51). An additional component of the complex Camelina/Linum/bacteria association was the f i n d i n g by Lovett and Sagar (42) that E. cloacae cultured from C. sativa f o l i a g e washings gave indications (through acetylene-ethylene assays) of a nitrogen-fixing c a p a b i l i t y . It i s not known whether such nitrogen contributes to the nitrogen economy of the plant, although Jones (52) suggests several means by which nitrogen fixed i n the phyllosphere of conifers may aid the growth of these trees. Both E. cloacae and P. fluorescens are capable of a c t i v i t y i n the phyllosphere and i n s o i l . This may be of significance i n r e l a t i o n to the findings of E l l i o t t and Lynch (53) and Lynch and Clarke (54) that pseudomonads, i n large numbers, are a dominant feature of the microflora of the rhizosphere and straw of some temperate cereals. Among the plant growth-promoting rhizobacteria that are being tested for commercial applications, Burr and Caesar (55) have found the most e f f e c t i v e strains to be fluorescent Pseudomonas species. Lynch and Clarke (54) suggested that some strains of pseudomonads stimulate root and shoot extension and dry matter production of barley, a species that shows variable s e n s i t i v i t y to benzylamine (Figure 5)· However, E l l i o t t and Lynch (53) indicated that pseudomonads antagonistic towards seedling growth colonize roots of wheat, their i n h i b i t o r y effects also

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Allelopathy in Australia: Bacterial Mediation

LOVETT

CONCENTRATION (ppm, A

WATER

•

LENGTH

CONTENT

OF

OF RADICLE

l o g

e

OF

BENZYLAMTNE

scale)

SOIL (r

0.*Q2;

ρ


Μ· The same phenomenon has recently been determined to occur in the Drosophila-cactus-yeast coevolved system (5,6) and may be present in the Danaiid-Asclepias and other systems Glycosides themselves are generally regarded as representing simple storage products, accumulable derivatives of the aglycone moiety (8). The fact of glycosidation is often disregarded as irrelevant in terms of potential biological a c t i v i t y , except in reducing same (9). It is certainly appreciated that glycosidasemediated enzymatic hydrolysis of glycosides (8J releases the agly­ cone whereby these glycosides may become active plant toxic principles, but this process is also regarded as essentially a storage-release phenomenon. 3-Glucosidases and other glycosidases have been determined to often show an exceedingly high degree of s p e c i f i c i t y toward a par­ ticular substrate (J_0,rj_). In recent work (_Γ2,_Π) specificity has been shown to be attributable to the structure of the aglycone. t n i s

0097-6156/87/0330-0275$06.00/0 © 1987 American Chemical Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

276

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Several recent reviews (14-16) have i n d i c a t e d t h a t one can gener­ a l l y expect t o f i n d a s p e c i f i c g l y c o s i d a s e c o r r e s p o n d i n g t o a spe­ c i f i c g l y c o s i d e s t r u c t u r e being p r e s e n t i n t h e same p l a n t . How­ e v e r , t h i s necessary c o e l a b o r a t i o n has been f r e q u e n t l y o v e r l o o k e d as a f u n c t i o n a l mechanism o f e v o l u t i o n a r y change i n d i v e r s i f i e d systems o f c o e v o l v e d p l a n t s and i n s e c t s . I w i l l argue i n t h i s c h a p t e r t h a t t h e very p r o c e s s o f h y d r o l y ­ s i s i s an event under t h e e v o l u t i o n a r y c o n t r o l o f t h e competing i n t e r e s t s o f p l a n t and i n s e c t s p e c i e s , and t h a t " t o x i c " p l a n t g l y ­ c o s i d e s should be regarded as such o n l y i n terms o f an i n s e p a r a b l e , t a r g e t e d g l y c o s i d e - g l y c o s i d a s e system. I w i l l extend t h i s argument t o encompass t h e d i v e r s i f i c a t i o n o f such systems i n l i n e a g e s o f p l a n t s i n an e f f o r t t o c o r r e l a t e enzyme-mediated g l y c o s i d e t o x i c i t y w i t h t h e e v o l u t i o n o f host p l a n t s p e c i f i c i t y and t h e c o e v o l u t i o n o f p l a n t s and i n s e c t s . The

Passiflora-Heliconius

Interaction

The s p e c i f i c i t y o f 3 - g l u c o s i d a s e c o n f i r m e d i n t h e p r o d u c t i o n o f c y a n i d e from cyanogenic g l y c o s i d e s of Passiflora and i t s r e l a t i v e s (Table i ) . While e c o l o g i c a l l y s i g n i f i c a n t q u a n t i t a t i v e v a r i a t i o n i n s p e c i f i c i t y e x i s t s (J_>2.)> t h e more o b v i o u s degree o f s p e c i f i c i t y shown by these enzymes toward cyanogenic g l y c o s i d e s w i l l s u f f i c e f o r t h e present d i s c u s s i o n . The s p e c i f i c i t y i s such t h a t even a c r u d e l y p u r i f i e d enzyme p r e p a r a t i o n (2,15) can be used t o i d e n t i f y b i o s y n t h e t i c and s t r u c t u r a l types o f cyanogens. The p e c u l i a r o b s e r v a t i o n t h a t p a r t i c u l a r c o m b i n a t i o n s o f 3g l u c o s i d a s e s from v a r i o u s r e l a t e d s p e c i e s , o r c o m b i n a t i o n s o f 8glucosidases and s u b s t r a t e substitutes, frequently prevented expected h y d r o l y s i s (Table n ) , gave t h e f i r s t i n d i c a t i o n t h a t t h e i n t e r a c t i o n o f p l a n t enzymes c o u l d be o f importance i n t h e Passiflora-Heliconius system. The same i n h i b i t o r y i n t e r a c t i o n has s i n c e been observed i n i n s e c t - p l a n t g l u c o s i d a s e c o m b i n a t i o n s ( 1 7 ) . The c y c l o p e n t e n o i d cyanogenic g l y c o s i d e s undergo h y d r o l y s i s a c c o r d i n g t o t h e r e a c t i o n i l l u s t r a t e d i n F i g u r e 1. T h i s two-step p r o c e s s i s e n t i r e l y c o n s i s t e n t w i t h t h a t determined f o r o t h e r cyan­ ogenic g l y c o s i d e s ( T8). The second step i s t h e r m o d y n a m i c a l l y f a v o r e d , and o c c u r s r a p i d l y a t normal c e l l pH even i n t h e absence o f α - h y d r o x y n i t r i l e lyase. The p r o d u c t i o n o f a 2-cyclopenteny1 ketone i s unique t o c y c l o ­ p e n t e n o i d h y d r o l y s i s . T h i s f a c t i s o f b i o l o g i c a l importance, as the ketone i s an a,β-unsaturated compound, and has been determined t o be a powerful a l k y l a t i n g agent ( F i g u r e 2) ( 6 , ^ 9 ) . While HCN i s a g e n e r a l t o x i n which reduces f i t n e s s i n many organisms (6,J_7,20_ and r e f e r e n c e s t h e r e i n ) , i t i s t h e ketone moiety which c o n f e r s s p e c i f i c t o x i c i t y upon t h e Passiflora cyanogenic g l y c o s i d e s . Preliminary data i n d i c a t e t h a t t h e a l k y l a t i o n r e a c t i o n i s so r a p i d and non­ s p e c i f i c as t o t h e o r e t i c a l l y p r e c l u d e t h e s u c c e s s f u l development o f a s p e c i f i c p o s t - h y d r o l y s i s r e s i s t a n c e i n an h e r b i v o r e . The i n s e c t s p e c i e s i s t h e r e f o r e h i g h l y induced t o produce a d e f e n s i v e c a p a b i l ­ i t y that prevents h y d r o l y s i s a l t o g e t h e r . This s e l e c t i v e pressure i s not a t a l l n e c e s s a r i l y t h e same as would induce t h e development of a defense a g a i n s t HCN.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

25.

SPENCER

Table I.

Specificity

S p e c i f i c i t y of

of Action

of

β-Glucosidases

o f Passif'lora

Enzyme P r e p a r a t i o n

Species

eu

ω

ulmifolïa

-

-

-

+++

foetida

+++

Passiflora

and R e l a t e d

Compound

ω Turnera

277

Allelochemicab

+ -

+

-

-

-

-

-

-

-

-

-

+++

-

-

+++

P. caeruJea

-

-

+++

Ρ·

trifasciata

-

-

-

-

-

-

Ρ·

suberosa

-

-

-

-

-

-

-

-

+++

+

P. coriacin

-

-

-

-

-

-

-

-

+

+++

Emulsin

-

+++

-

+++

-

P. X alatocaerulea

(Sigma)

+++

-

+ +

-

+

+

-

-

+++

-

-

-

L i namarase Gynocardia

-

odorata

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

+++

278

Table

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

II.

Inhibition

o fNatural

β-Glucosidase A c t i v i t y by A d d i t i o n o f S i m i l a r of Competing Substrates

C o m b i n a t i o n s o f Enzyme P r e p a r a t i o n s and C y a n o g e n i c Compounds

Compound

T. ulmifolia

+ P. X alatocaerulea

T. ulmi folia

+ emu 1 s i η

T. ulmifolia

+ linamarase

(+)

T. ulmifolia

+ P. foetida

-

P. X alatocaerulea

+

+ P. foetida

P. foetida

+ emulsin

P. biflora

+ P. trifasciata

(+)

-

(+)

P. coriacea

-

Emulsin

G.

+ P. trifasciata + amygdalin

+ tetraphyllin

Β

-

H

-

+ prunasin

^

HO*

C

N

(+)

-

odorata + l i n a m a r i n

P. X alatocaerulea

-

-

ulmifolia

-

-

P. suberosa + P. biflora

T.

Glucosidases

-

Plant

ff-glucosid^se

glucose

'O-glucose

N>

(Unstable)

Toxic

H o J

LCN

α-hydroxynitrile^

H

0

J

Ί

Q

+

Toxic

Figure

1.

3-Glucosidase-mediated cyanogenic

hydrolysis

HCN

T

of

o

x

i

c

cyclopentenoid

glycosides.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

25.

SPENCER

Specificity

of Action of

Allelochemicab

279

in vitro s t u d i e s i n t h i s l a b o r a t o r y have e s t a b l i s h e d t h a t s p e c i f i c r e s i s t a n c e s to t o x i f i c a t i o n by c y c l o p e n t e n o i d cyanogenic g l y c o s i d e s e x i s t i n Heliconius and i t s r e l a t i v e s , and t h a t the process i n v o l v e s the s p e c i f i c i n h i b i t i o n of the h y d r o l y s i s of the compounds present i n the host p l a n t ( ] _ , 2 j . While the a c t u a l mechanism has not been s a t i s f a c t o r i l y q u a n t i t a t i v e l y demonstrated, our data a r e c o n s i s t e n t w i t h the models i l l u s t r a t e d i n F i g u r e 3 · In the f i r s t r e a c t i o n , the Heliconius 8-g1ucosidase b i n d s t o the p l a n t substrate-enzyme complex, e i t h e r d u r i n g or a f t e r complex f o r m a t i o n , in a c o m p e t i t i v e manner. The o n e - s u b s t r a t e , two-enzyme complex p r e c i p i t a t e s out o f s o l u t i o n . In the second r e a c t i o n , the Heliconius 8 - g l u c o s i d a s e and/or o t h e r g l y c o s i d a s e s a c t u a l l y a t t a c k and h y d r o l y z e the p l a n t 8 - g l u c o s i d a s e . Both r e a c t i o n s have been measured in vitro and appear t o occur i n vivo ( 6 , 1 7 ) » Both r e a c t i o n s a r e h i g h l y s u b s t r a t e s p e c i f i c , and both r e s u l t i n the i n a c t i v a t i o n of the p l a n t 8 - g l u c o s i d a s e and prevent h y d r o l y s i s . The s p e c i f i c i t y o f the r e a c t i o n i s such t h a t the p r o d u c t i o n o f Heliconius i n h i b i t o r y enzyme response t o the s p e c i f i g l y c o s i d e p l u s 8 - g l u c o s i d a s e ) which i s t a r g e t e d a g a i n s t i t . The i n t e r a c t i o n o f the Passiflora 8 - g l u c o s i d a s e and cyanogenic g l y c o s i d e w i t h a s p e c i a l i z e d Heliconius h e r b i v o r e i s summarized i n Figure 4. Glucose i s assumed as the model sugar moiety. Here, i n response t o the development o f an i n s e c t 3-g'lucosidase c a p a b l e o f i n a c t i v a t i n g the p l a n t t o x i f i c a t i o n syndrome, the p l a n t s p e c i e s may e v o l v e any one or more o f the f o l l o w i n g changes: 1) m o d i f i c a t i o n o f aglycone s t r u c t u r e . T h i s o c c u r s i n Passiflora through the a t t a c h ment o f d i f f e r e n t s u b s t i t u e n t s or replacement of the double bond w i t h a s i n g l e bond or e p o x i d e , or through changes i n symmetry. 2) M o d i f i c a t i o n o f the sugar m o i e t y , through a change i n number, type or l i n k a g e of sugar s u b s t i t u e n t s . 3) Change i n cyanogenic g l y c o s i d e s k e l e t a l type through an a l t e r a t i o n i n the b i o s y n t h e t i c pathway t o another p r e c u r s o r t o y i e l d an a l t e r n a t e type cyanogenic g l y c o s i d e ( i . e . c y c l o p e n t e n o i d becomes a r o m a t i c as 2 - c y c l o p e n t e n y l g l y c i n e i s r e p l a c e d w i t h p h e n y l a l a n i n e as p r e c u r s o r ) , k) P r o duction of ionically destabilized cyanogenic g l y c o s i d e s ; in Passiflora by attachment o f a s u l f a t e a t C-4. 5) P r o d u c t i o n o f c y a n o h y d r i n s through the o m i s s i o n of the f i n a l g l y c o s y l a t i o n s t e p in b i o s y n t h e s i s . T h i s r e s u l t s i n an e x c e e d i n g l y u n s t a b l e form o f cyanogenic compound ( a - h y d r o x y n i t r i l e ) which, having no sugar m o i e t y , no longer r e q u i r e s a 8 - g l u c o s i d a s e f o r h y d r o l y s i s . 6) Change i n s t r u c t u r e of the p l a n t 8 - g l u c o s i d a s e complement: a) t o f a c i l i t a t e h y d r o l y s i s o f an a l t e r e d s t r u c t u r e , b) t o r e s i s t b i n d i n g and i n a c t i v a t i o n by a g i v e n i n s e c t 8 - g l u c o s i d a s e , c) t o h y d r o l y z e i n s e c t 8 - g l u c o s i d a s e s (not y e t o b s e r v e d ) . The

Pierid-Cruciferae

Interaction

Given the r a p i d r a d i a t i o n o f many s p e c i f i c p l a n t g l u c o s i d a s e - s u b s t r a t e systems and the c o r a d i a t i o n o f s p e c i f i c i n s e c t a d a p t a t i o n s in the Passiflora-Heliconius c o e v o l u t i o n a r y i n t e r a c t i o n , i t seems q u i t e r e a s o n a b l e t o expect t h a t such a process would occur i n o t h e r systems. As the d i s c o v e r y of the d i v e r s i f i c a t i o n o f cyanogenic g l y c o s i d e s i n Passiflora was the p r e r e q u i s i t e f o r i n t e r p r e t i n g the co-

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

280

RN 2

R C:" 3

H

H

if

R"

R RN 2

R" F i g u r e 2.

3 - A l k y l a t i n g property of 2-cyclopentenones derived from h y d r o l y s i s o f c y c l o p e n t e n o i d cyanogenic g l y c o s i des.

Hel iconius /3-gl ucosidase

P a s s i f l o r a /3-glucosidase (may be glycoprotein)

HO,

HO,

1.

+

glucose

+

HCN

p r e c i p i t a t i o n , no hydrolysis

HO H

H

=0

d-glu

H

° ^ k > H^

i n a c t i v a t i o n , no hydrolysis C

N

O-glu

F i g u r e 3·

Two h y p o t h e t i c a l mechanisms by which Heliconius may p r o t e c t i t s e l f from t o x i f i c a t i o n by Passiflora cyanogen i c g l y c o s i d e s .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

SPENCER

Figure 4 .

Specificity

of Action

ofAllelochemicals

Chemical responses o f Passif'lora t o s p e c i a l i z a t i o n by Heliconius . See t e x t f o r e x p l a n a t i o n o f numbered responses.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

282

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

e v o l u t i o n o f Passiflora and Heliconius, a s i m i l a r coevolved system w i t h a d i v e r s i f i e d g l y c o s i d e c h e m i s t r y should p r o v i d e a l o g i c a l comparison o f t o x i f i c a t i o n p r o c e s s e s . The c r u e i f e r - P i e r i d i n t e r a c t i o n i s p a r t i c u l a r l y w e l l s t u d i e d (2>ÎL>iLL) and i s thought t o r e p r e s e n t an i n t r i c a t e l y coevolved system. Species i n t h e C r u c i f e r a e c h a r a c t e r i s t i c a l l y e l a b o r a t e g l u c o s i n o l a t e s ( t h i o g l u c o s i d e s ) . A l a r g e number ( > 7 5 ) have been d e s c r i b e d from t h e f a m i l y ( 2 2 , 2 3 _ ) , r e p r e s e n t i n g a c o n s i d e r a b l e d i v e r s i f i c a t i o n i n s t r u c t u r e when compared t o g l u c o s i n o l a t e prod u c t i o n in o t h e r f a m i l i e s ( 4 ) . These compounds are b i o s y n t h e s i z e d i n a pathway e n t i r e l y a n a l ogous t o t h a t o f cyanogenic g l y c o s i d e s ( 2 4 ) , and a r e h y d r o l y z e d t o t o x i c (25) i s o t h i o c y a n a t e s by 3 - t h i o g l u c o s i d a s e i n a p r o c e s s a n a l o gous t o t h e h y d r o l y s i s o f cyanogenic g l y c o s i d e s ( 2 6 ) . The subs t r a t e and enzyme may a l s o be compartmentalized w i t h i n t i s s u e s i n the same way as cyanogenic g l y c o s i d e s and g l u c o s i d a s e ( 2 8 - 3 1 ) . $ T h i o g l u c o s i d a s e (myrosinase) has long been c o n s i d e r e d , a t l e a s t de facto* t o c o n s i s t o f o n l myrosinase has been determine glycoproteins) ( 2 7 ) . While t h e host s p e c i f i c i t y o f Pieris and i t s r e l a t i v e s t o c r u c i f e r s p e c i e s has been i n t e r p r e t e d as a stepwise r e c i p r o c a l s e l e c t i v e response t o e v o l u t i o n a r y changes i n g l u c o s i n o l a t e chemi s t r y ( 3 2 ) , f u r t h e r s y n t h e s i s has proved d i f f i c u l t . Because o f t h e i n t e r p r e t a t i o n o f t h e t o x i c a c t i v i t y as r e s i d i n g i n t h e g l u c o s i n o l a t e s a l o n e , as r e l e a s e d by a n o n s p e c i f i c a c t i v a t i n g enzyme, a n a l y s e s become compounded by t h e a p p a r e n t l y widespread d i s t r i b u t i o n o f many compounds i n v a r i o u s c o m b i n a t i o n s . The d e f e n s i v e v a l u e o f the g l u c o s i n o l a t e a r r a y s , and t h e i r importance as s e l e c t ive agents i n t h e c o e v o l u t i o n o f t h e C r u c i f e r a e and t h e P i e r i d a e , have been w e l l e x p l o r e d ( 3 3 - 3 5 ) . However, a p l a u s i b l e mechanism f o r r e c o g n i t i o n and t o l e r a n c e o f these compounds remains e l u s i v e . I s o l a t i o n s were made o f t h i o g l u c o s i d a s e f r a c t i o n s from e i g h t p l a n t s i n the C r u c i f e r a e and s e v e r a l o t h e r s known t o produce g l u c o s i n o l a t e s . Each showed a number o f s p e c i f i c t h i o g l u c o s i d a s e a c t i v i t i e s t o be present upon s e p a r a t i o n by g e l e l e c t r o p h o r e s i s and assay f o r SCN~ r e l e a s e a f t e r treatment w i t h a v a r i e t y o f s u b s t r a t e s Combinations o f enzymes i n h i b i t e d expected h y d r o l y s i s o f a s u b s t r a t e i n an experiment c o n s t r u c t e d a c c o r d i n g t o Table I I . Enzyme f r a c t i o n s from d i f f e r e n t p l a n t s p e c i e s r e l e a s e SCN~ a t d i f f e r e n t r a t e s when s i n i g r i n , s i n a l b i n , benzyl g l u c o s i n o l a t e , and g l u c o s i n o l a t e - c o n t a i n i n g f r a c t i o n s o f each p l a n t were used as substrate. The spécificités showed were s i g n i f i c a n t b u t were n o t as r e s t r i c t e d as was observed f o r g l u c o s i d a s e s m Table I . This i s t o be expected as a f a r g r e a t e r number o f g l u c o s i d e s and enzymes appear t o be present in the l a t t e r samples than i n the p r e v i o u s experiment. Enzyme p r e p a r a t i o n s o f t h r e e Pieris s p e c i e s each c o n t a i n e d 3t h i o g l u c o s i d a s e a c t i v i t y , a f a c t p r e v i o u s l y r e p o r t e d ( 3 6 . ) , and each i n h i b i t e d h y d r o l y s i s i n one o r more c o m b i n a t i o n s w i t h a p l a n t enzyme-substrate system. These data a r e being q u a n t i f i e d and extended t o i n c l u d e i n s e c t / p r e f e r r e d h o s t - p l a n t p a i r s . The Pieris-crucifer i n t e r a c t i o n t h e r e f o r e seems t o i n v o l v e t h e same b i o c h e m i c a l parameters as t h e Passiflara-Heliconius intera c t i o n , and i t i s proposed t h a t t h e e v o l u t i o n o f host p l a n t s p e c i f i c i t y has proceeded i n an analogous f a s h i o n i n both systems.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

25.

SPENCER

Specificity

of Action

The Drosophila-Cactus-Yeast

of

Allehchemicab

283

Interaction

The i n t e r a c t i o n between Drosophila, y e a s t s and columnar c a c t i o f the Sonoran Desert has been the s u b j e c t o f much recent i n t e r e s t (37)« As a c o e v o l v e d system, perhaps more i s known about t h i s i n t e r a c t i o n than any o t h e r . The c h e m i s t r y o f the c a c t i (70 spp.) has been p o s t u l a t e d t o p l a y a s i g n i f i c a n t r o l e i n the e s t a b l i s h m e n t of t h i s system (38), but t h i s was based upon r e p o r t s o f a r e l a t i v e l y small number o f r e l a t i v e l y s i m p l e a l k a l o i d s , and a small number o f t e r p e n o i d compounds. Only r e c e n t l y , the d i v e r s i f i c a t i o n o f p l a n t compounds has been d i s c o v e r e d t o be much g r e a t e r (5.). In t h e i r s e c t i o n on a l k a l o i d s i n t h e above work, B a j a j and McLaughlin r e p o r t the presence o f some t h i r t y - f i v e s t r u c t u r e s . In a d d i t i o n , I have been a b l e t o i s o l a t e some s i x t y t r i t e r p e n o i d g l y c o s i d e s ( s t r u c t u r e s were not determined) and more were d e t e c t e d but not i s o l a t e d . P r e v i o u s l y (39.), some s i x t e e n d i s t i n c t t r i t e r p e n e s k e l e t o n s i n two c l a s s e s were i s o l a t e d and r e p o r t e d from t h i s group o f c a c t i Standard i s o l a t i o n procedur s i s of sugars, so th d e s c r i b e d . As the data p r e s e n t e d p r e v i o u s l y imply, i n d i v i d u a l g l y c o s i d e s a r e t h e compounds o f e c o l o g i c a l i n t e r e s t . A l a b o r a t o r y study was undertaken t o determine the l i k e l i h o o d t h a t c a c t u s t r i t e r p e n o i d g l y c o s i d e s a r e important f a c t o r s i n t h e h o s t - p l a n t c h o i c e o f d e s e r t Drosophila (6). A f e e d i n g experiment was conducted u s i n g f i e l d c o n c e n t r a t i o n s of t h e a l k a l o i d a l f r a c t i o n and the t o t a l t r i t e r p e n o i d g l y c o s i d e f r a c t i o n o f t h i r t y r e l a t e d s p e c i e s o f columnar c a c t i (most i n t h e Pachycereeae). S u r v i v o r s h i p was measured as + o r - and i n d i c a t e s s u c c e s s f u l development, p u p a t i o n and emergence a f t e r eggs were l a i d by s e v e r a l D. melanogaster o r D . mojavensis f e m a l e s . The l a t t e r s p e c i e s i s a d e s e r t f l y known t o s p e c i a l i z e on s e v e r a l s p e c i e s o f Pachycereeae; t h e former i s a n o n s p e c i a l i z e d , nondesert s p e c i e s . Heliothis zea l a r v a e were a l s o used i n a separate b i o a s s a y o f t o x i c i t y where compounds were added t o commercial d i e t . T r i t e r p e n o i d g l y c o s i d e s are hydrolyzed to y i e l d aglycone ketones and d i o l s by 3 - g l u c o s i d a s e s . Assays o f p l a n t m a t e r i a l s showed these enzymes t o be p r e s e n t . It i s known t h a t c a c t o p h i l i c y e a s t s a r e a b l e t o h y d r o l y z e t e r p e n o i d g l y c o s i d e s (38J. I t was determined t h a t p l a n t s and y e a s t s h y d r o l y z e d i f f e r e n t g l y c o s i d e s a t different rates. In the p r e s e n t experiment, commercial baker's y e a s t was u t i l i z e d f o r Drosophila f e e d i n g t r i a l s . I t was p o s s i b l e t o d e t e c t h y d r o l y s i s o f g l y c o s i d e s and a r r a y s o f g l y c o s i d e s i n t r i t e r p e n o i d f r a c t i o n s through the p r o d u c t i o n o f f r e e sugars and the d e g r a d a t i o n o f i n d i v i d u a l compounds as r e v e a l e d by s p e c i f i c c o l o r reagents and TLC. Many t r i t e r p e n o i d s underwent h y d r o l y s i s , but a l k a l o i d s d i d n o t . S p e c i f i c d i f f e r e n c e s i n t o x i c i t y toward each t e s t s p e c i e s were d i s c o v e r e d between t h e v a r i o u s p l a n t chemical a r r a y s . Table I I I l i s t s s u r v i v o r s h i p f o r these species f o r a l k a l o i d - p r o d u c i n g c a c t i . A d d i t i o n a l l y , data was o b t a i n e d f o r 20 s p e c i e s i n which a l k a l o i d s were not d e t e c t e d . The t h r e e i n s e c t s p e c i e s showed c o n s i s t e n t d i f f e r e n c e s i n t o l e r a n c e toward g i v e n p l a n t s p e c i e s , w i t h no c l e a r phylogenetic pattern accounting f o r t h i s . I n s e c t s a l s o responded d i f f e r e n t l y t o a l k a l o i d a l f r a c t i o n s versus t r i t e r p e n o i d g l y c o s i d e f r a c t i o n s . A l k a l o i d s were found t o be g e n e r a l l y not t o x i c t o D.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

284

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Table I I I . S u r v i v o r s h i p o f Drosophila and Heliothis Upon A l k a l o i d a l and T r i t e r p e n o i d G l y c o s i d e E x t r a c t s o f A l k a l o i d - P r o d u c i n g Columnar C a c t i

D mel

Stenocereus

D moj

D moj

stellatus treleasei

+

beneckei

+

quevedonis

+

duwortieri

+

Pola skia

chende

+

Escontria

chiotilla

+

Lemaireocereus

H zea D mel

hollianus

+

humilis Pterocereus

gauneri

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

H zea

25.

SPENCER

Specificity of Action of Allebchemicals

285

mojavensis ( t o l e r a t e d 90$ o f p l a n t s p e c i e s ) , y e t t r i t e r p e n o i d g l y ­ c o s i d e s from 60$ o f t h e a l k a l o i d - p r o d u c i n g and from 80$ o f nona l k a l o i d - p r o d u c i n g p l a n t s were l e t h a l . D. melanogaster grew suc­ c e s s f u l l y upon 40$ o f a l l t r i t e r p e n o i d f r a c t i o n s , and somewhat l e s s (30$) o f a l k a l o i d f r a c t i o n s . H. zea performed c o n s i s t e n t l y b e t t e r on t r i t e r p e n e f r a c t i o n s (60$+) than on a l k a l o i d f r a c t i o n s (40$+). In terms o f c o m p a r a t i v e performance upon e x t r a c t s from i n d i v i ­ dual p l a n t s p e c i e s , D· melanogaster and D. mojavensis showed d i f ­ f e r e n t t o l e r a n c e on 80$ o f p l a n t s p e c i e s whether t e s t e d a g a i n s t t r i t e r p e n o i d s or a l k a l o i d s . From t h e s e p r e l i m i n a r y d a t a , i t can be c o n c l u d e d t h a t : 1) t r i t e r p e n o i d g l y c o s i d e s are h y d r o l y z e d by y e a s t s t o y i e l d a g l y c o n e s t h a t a r e s e l e c t i v e l y t o x i c t o D· mojavensis a t f i e l d l e v e l s . 2) Cactus a l k a l o i d a r r a y s a r e not g e n e r a l l y t o x i c t o D. mojavensis a t f i e l d levels. 3) Non-adapted and n o n - s p e c i a l i z e d i n s e c t s do not e x h i b i t s e l e c t i v e t o l e r a n c e toward any g i v e n compound a r r a y s , and are s u s c e p t i b l e t o t o x i f i c a t i o n by e i t h e r a l k a l o i d s o r t r i t e r p e n o i d g l y c o s i d e s or both. 4 t a r g e t e d a g a i n s t Drosophila may be l e s s s p e c i a l i z e d t o x i n s . T h i s would t h e r e f o r e be analogous t o t h e s e p a r a t i o n o f s p e c i f i c ( a g l y c o n e ) and general (HCN) t o x i c p r i n c i p l e s i n the Passiflora-Heliconius interaction. F u r t h e r work i s being conducted u s i n g s p e c i a l i z e d y e a s t s and other s p e c i e s o f Drosophila and q u a n t i f y i n g d i f f e r e n c e s i n h y d r o l y ­ s i s and t o x i c i t y o f g l y c o s i d e s under these c o n d i t i o n s . We may h y p o t h e s i z e f o r the p r e s e n t t h a t columnar c a c t i produce a l k a l o i d s as a general d e t e r r e n t t o h e r b i v o r y , and t r i t e r p e n o i d g l y c o s i d e s and a s s o c i a t e d h y d r o l y t i c g l y c o s i d a s e s as a s p e c i f i c t o x i f i c a t i o n mechanism a g a i n s t s p e c i a l i s t Drosophila species. R a d i a t i o n and d i v e r s i f i c a t i o n o f t r i t e r p e n o i d s may have o c c u r r e d i n response t o c o n t i n u e d i n t e r a c t i o n between the c a c t i and Drosophila · T h i s p r o c e s s i s dependent upon c o e v o l u t i o n w i t h s p e c i a l i z e d y e a s t s which may i n t e r f e r e w i t h h y d r o l y s i s o f t r i t e r p e n o i d s o r h y d r o l y z e i n d i v i d u a l compounds s e l e c t i v e l y . Other Systems G l y c o s i d e d i v e r s i f i c a t i o n a l s o has o c c u r r e d i n t h e c o e v o l u t i o n o f monarch b u t t e r f l i e s and milkweeds (7.). I t may be d e s i r a b l e t o r e l a t e the t o x i c i t y of cardenolides t o the h y d r o l y t i c c a p a b i l i t i e s o f s u s c e p t i b l e and n o n s u s c e p t i b l e insects. Cardenolides from Asclepias s p e c i e s can be h y d r o l y z e d by 3 - g l u c o s i d a s e s p r e s e n t i n the p l a n t (β), y e t s p e c i a l i z e d Danaus s p e c i e s are a b l e t o s e q u e s t e r these compounds, a process which r e q u i r e s c o n t r o l o f h y d r o l y s i s . P l a n t 3 - g l u c o s i d a s e s a l s o cause h y d r o l y s i s o f i r i d o i d g l y c o ­ s i d e s , and a r e h i g h l y s p e c i f i c i n a c t i v i t y (6). S e v e r a l i n s e c t s p e c i e s t h a t a r e a b l e t o t o l e r a t e i r i d o i d s have been found t o con­ t a i n i n h i b i t o r y 3-glucosidases. These and o t h e r systems a r e under continuing investigation in this laboratory. Ecoregulatory

Processes

Recent work (kQ) has shown t h a t p l a n t t a n n i n s are c a p a b l e o f i n a c t i ­ v a t i n g p l a n t 3-glucosidases in vitro. I t has been determined (6) t h a t i n s e c t 3 - g l u c o s i d a s e s are a l s o i n a c t i v a t e d by p l a n t t a n n i n s a t

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

286

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

f i e l d l e v e l s ( F i g u r e 5 ) . Heliconins 3-glucosidase f r a c t i o n s have been assayed f o r t h e i r a b i l i t y t o i n h i b i t in vitro Passiflora 3g l u c o s i d a s e h y d r o l y s i s o f e y e l o p e n t e n o i d cyanogenic g l y c o s i d e s (£, 17) » A f t e r treatment w i t h t a n n i n s , t h e i n s e c t enzyme f r a c t i o n s were d i a l y z e d t o recover s o l u b l e g l u c o s i d a s e a c t i v i t y . The remain­ ing i n h i b i t o r y a b i l i t y was determined through q u a n t i t a t i v e measure­ ment o f HCN r e l e a s e i n t h e t e s t r e a c t i o n . It i s p o s s i b l e t h a t t h e s e e c o r e g u l a t o r y enzymes, used by t h e i n s e c t t o i n h i b i t t a r g e t e d p l a n t t o x i f i c a t i o n systems, may them­ s e l v e s be t h e p r i n c i p a l t a r g e t o f t h e p r o d u c t i o n o f t a n n i n s by plants. The d i s c o v e r y o f o t h e r examples o f secondary chemical i n t e r a c t i o n s w i t h enzymes d e d i c a t e d t o t h e r e g u l a t i o n o f t o x i f i c a ­ t i o n and d e t o x i f i c a t i o n mechanisms may be expected as our knowledge o f c h e m i c a l l y mediated ρ I a n t - i n s e c t i n t e r a c t i o n expands.

8.0-

7-oH

Relative 66.0 r a t e of hydrolysis of 55.0H Passiflora cyanogens as 44.0H HCN r e l e a s e d (yg * 10) 3.0H

2.0H

0

0.01

0.1

Tannin

1.0

10.0 100.0

(mg/mL)

(Quebracho, w a t t l e , see R e f . 40) F i g u r e 5.

Tannin i n h i b i t i o n o f Heliconius i n t e r f e r e w i t h cyanogènes is o f

chestnut,

3-glucosidases Passiflora.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

that

SPENCER

25.

Specificity of Action of Allelochemicals

287

Summary Plant lineages exhibiting diversification of glycosides should more properly be regarded as having produced a diverse set of glycoside/glycosidase systems. These are generally toxic, owing their toxicity to the structures of one or more of the hydrolysis products and to the fact of successful hydrolysis. The effectiveness of the glycoside-derived toxin also depends upon inhibitory glycosidases present in the target insect digestive system which can inhibit hydrolysis through competitive interaction with the substrateenzyme complex, or through direct hydrolytic action against the plant enzyme. Plant tannins may interact directly with the enzymes responsible for hydrolysis, and may therefore be targeted against insect ecoregulatory enzymes. This underscores the importance of regarding plant allelochemicals as one part of complex targeted toxification systems. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Spencer, K.C. In "Chemical Mediation of Coevolution"; Spencer, K.C., Ed.; Pergamon Press: New York, 1986; in preparation. Spencer, K.C. Ph.D. Thesis, University of Illinois, Urbana, 1984. Feeny, P. Ann. Missouri Bot. Gdn. 1977, 64, 221-234. Rodman, J.E. In "Phytochemistry and Angiosperm Phylogeny"; Young, D.A.; Seigler, D.S., Eds.; Praeger Press: New York, 1981; p. 43. Gibson, A.C.; Spencer, K.C.; Bajaj, R.; McLaughlin, J.L. The Everchanging Landscape of Cactus Systematics. Ann. Missouri Bot. Gdn., in press. Spencer, K.C, in preparation. Brower, L.P. In "Chemical Mediation of Coevolution"; Spencer, K.C., Ed.; Pergamon Press: New York, 1986; in preparation. Hosel, W. In "the Biochemistry of Plants"; Stumpf, P.K.; Conn, E.E., Eds.; Academic Press: New York, 1981 ; Vol. 7, p. 725. Pridham, J.B. Ann. Rev. Plant Physiol. 1965, 16, 13-36. Hosel, W.; Nahrstedt, A. Hoppe Seyler's Z. Physiol. Chem. 1975, 356, 1265-1275. Nahrstedt, Α.; Hosel, W.; Walther, A. Phytochemistry 1979, 18, 1137-1141. Hosel, W.; Conn, E.E. Trends Biochem. Sci. 1982, 7, 219-221. Dale, M.P.; Emsley, H.E.; Kern, K.; Sastry, K.A.R.; Byers, L.D. Biochemistry 1985, 24, 3530-3539. Nisizawa, K.; Hashimoto, J. In "The Carbohydrates"; Pigman, W.; Horton, D., Eds.; Academic Press: New York, 1970; 2nd ed., Vol. 2A, Ch. 33. Spencer, K.C.; Seigler, D.S. Phytochem. Bull. 1984, 16, 13-21. Conn, E.E. In "Herbivores: Their Interaction with Secondary Plant Metabolites"; Rosenthal, G.A.; Janzen, D.H., Eds.; Academic Press: New York, 1979; p. 387. Spencer, K.C.; Smiley, J.T., in preparation. Hosel, W. In "Cyanide in Biology"; Vennesland, B.; Conn, E.E.; Knowles, C.J.; Westley, J.; Wissing, F., Eds.; Academic Press: New York, 1981; p. 217.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

288

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

39. 40.

A L L E L O C H E M I C A L S : R O L E IN A G R I C U L T U R E A N D F O R E S T R Y

H a l l , I.H.; Lee, K.H.; Mar, E.C.; Starnes, C.O.; Waddell, T.G. J. Med. Chem. 1977, 20, 333-337. Jones, D.A. In "Cyanide in Biology"; Vennesland, B.; Conn, E.E.; Knowles, C.J.; Westley, J . ; Wissing, F., Eds.; Academic Press: New York, 1981; p. 509. Chew, F.S.; Rodman, J.E. In "Herbivores: Their Interaction with Secondary Plant Metabolites"; Rosenthal, G.A.; Janzen, D.H., Eds.; Academic Press: New York, 1979; p. 271. Kjaer, Α.; Larsen, P.O. Biosynthesis 1973, 2, 71-105. Kjaer, Α.; Larsen, P.O. Biosynthesis 1976, 4, 179-203. Conn, E.E. Naturwissenschaften 1979, 66, 28-34. Van Etten, C.H.; Tookey, H.L. In "Herbivores: Their Interac­ tion with Secondary Plant Metabolites"; Rosenthal, G.A.; Janzen, D.A., Eds.; Academic Press: New York, 1979; Ρ. 471. U n d e r h i l l , E.W.; Wetter, L.R.; Chisholm, M.D. Biochem. Soc. Symp. 1973, 38, 303-326. Bjorkman, R. In "The Biology and Chemistry of the Cruciferae"; Vaughan Eds.; Academic Press H o l l e r , R.A.; Jones, J.D. Can. J. Bot. 1985, 63, 521-526. Kojima, M.; Poulton, J.E.; Thayer, S.S.; Conn, E.E. Plant Physiol. 1979, 63, 1022-1028. Fahn, A. In "Secretory Tissues in Plants"; Academic Press: New York, 1979; p. 147. Pihakaski, K.; Iversen, T.H. J. Exper. Bot. 1976, 27, 242258. E h r l i c h , P.R.; Raven, P.H. Evolution 1965, 18, 586-6Ο8. Erickson, J.M.; Feeny, P. Ecology 1974, 55, 103-111. Blau, P.Α.; Feeny, P.; Contardo, L.; Robson, D.S. Science 1978, 200, 1296-1298. Larsen, P.O. In "The Biochemistry of Plants"; Stumpf, P.K.; Conn, E.E., Eds.; Academic Press: New York, 1981; Vol. 7, p. 502. MacGibbon, D.B.; A l l i s o n , R.M. N.Z.J. S c i . 1971, 14, 134-140. Barker, J.S.F.; Starmer, W.T. "Ecological Genetics and Evolu­ t i o n " ; Academic Press: New York, 1982. Kircher, H.W. In "Ecological Genetics and Evolution"; Barker, J.S.F.; Starmer, W.T., Eds.; Academic Press: New York, 1982; p. 143. Djerassi, C. In "Festschr. Arthur S t o l l " ; Birkhauser-Verlag: B e r l i n , 1957; p. 330. Goldstein, W.S.; Spencer, K.C. J . Chem. Ecol. 1985, 7, 847858.

RECEIVED December 23, 1985

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 26

Purine Alkaloids in Tea Seeds During Germination Takeo Suzuki and George R. Waller 1

2

Faculty of Textile Science, Kyoto Kogei-Sen-i University, Matsugasaki, Kyoto 606, Japan Department of Biochemistry, Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, OK 74078 1

2

During imbibition of whole tea seeds (6 days) two purine alkaloids, caffein seed coats and with and after the breaking seed coats there was a gradual release of caffeine from coats of germinating seeds. By contrast, when the seed was freed from the outer seed coat and soaked, imbibition of the seed required only two days and simultaneously caffeine was released from the inner seed coat. In such seeds, but not in whole seeds, growth of embryonic tissues (roots and shoots) was inhibited after the breaking of the inner seed coats. Nevertheless, caffeine increased more in such roots of the seedlings of decoated seeds than in roots of normal seedlings. Studies of caffeine (1,3,7-trimethylxanthine; Figure 1) in the coffee plant have shown that it undergoes a variety of metabolic changes (1-31 and has an ecological role rather than one as a nitrogen reserve (éz2.)> We (10) reported that two purine alkaloids, caffeine and theobromine (3,7-dimethylxanthine; Figure 1), in leaves and shoots of tea (Camellia sinensis) decreased significantly in August, October, and November in Japan. This suggests that the alkaloids have no role in the storage of nitrogen in tea leaves during winter months. The biosynthesis of caffeine in coffee and tea plants proceeds through the steps: purine nucleotides in the nucleotide pool (AMP and/or GMP) —> X M P —> xanthosine —> 7methylxanthosine —» 7-methylxanthine -» theobromine —» caffeine (3.11.12). In contrast with the seed caffeine of Coffea species, relatively little attention has been paid to that of tea. This is in part because the fruit of tea, including the seeds, is of minor economic importance compared with that of coffee; moreover earlier studies revealed little caffeine in the tea seed (13.14). Recently we (15) found that the pericarp contains the greatest concentrations of alkaloids in the dry fruit of tea, and that appreciable amounts occur in the seeds, especially in the coats. Thus, from physiological and ecological viewpoints, our concerns are the roles of purine alkaloids and seed coats of tea during fruit development (seed formation) and seed germination. Caffeine in Coffea arabica seed is synthesized in the pericarp, transported to the seed, and accumulated there during fruit 0097-6156/87/0330-0289$06.00/0 © 1987 American Chemical Society

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development (16). whereas the caffeine i n both the pericarp and seed coats o f tea is synthesized and accumulated i n the same tissues (15). Allelopathic reactions o f coffee seeds and fruits containing these alkaloids have been described (7.17.18). The behavior o f two purine alkaloids i n tea during germination, and their physiological and ecological functions are described. Materials and Methods Seeds of tea (Camellia sinensis L.) were surface-sterilized in a saturated solution of calcium hypochlorite for 30 min, and soaked for 30 min i n running water. The whole seeds, or else seed that had been decorticated, i.e. removed from the hard testae, were sown in moist sea sand and allowed to germinate and grow i n dark at 28 °C. The mixture o f alkaloids present was extracted and analyzed as described previously (2.3). Results After the flowering i n October spring, and then proceeds progressively until the fruit is full-ripened and dried (15). The seeds are then shed i n November, and lose viability after several months. The contents o f purine alkaloids in the seeds of coffee and tea plants are given in Table I. W h e n the whole tea seed was used, imbibition required about 6 days, and then the seed broke out o f the outer seed coat 6-10 days after soaking (Figures 2 & 3). During imbibition alkaloid concentrations were unchanged i n the seed coat although caffeine is readily water-soluble (Figure 4). D u r i n g and after the breaking o f seed coats caffeine was gradually released from the coats o f germinating seeds (Figure 3). B y contrast, when the seed was removed from the outer seed coat and soaked, imbibition required only 2 days, and concurrently caffeine was released from the inner seed coat (Figures 2 & 3). In such seeds, but not i n whole ones, growth inhibition o f embryonic tissues (roots and shoots) occurred after the breaking o f the inner seed coats. Nevertheless, caffeine increased more in roots o f the decoated seed than i n normal seedling roots. Table II shows results on growth inhibition and increments o f caffeine in the roots o f 5week-old tea seedlings, when the seeds were freed from the outer seed coats, allowed to imbibe, and incubated i n water extracts from seed coats. Discussion F r o m these studies and the work o f others (4-9.17.18). we conclude that caffeine found in the seed coats of tea seeds (Table I) has no nutritive function and that it is phytotoxic and autotoxic i.e., inhibits growth o f germinating tea seedlings (Table II). T h e seed coats are the barriers between the embryo and its immediate environment. A s dead tissues, the seed coats o f the mature seed protect the enclosed embryo. Equally important are the nutritive and regularatory functions of the living seed coats during embryo development (19-23). The developing tea seed has a more highly developed seed coat than the coffee seed (15). The coat functions as a good reservoir o f the toxic alkaloids (caffeine and theobromine) (Table I), but prevents autotoxic hazards from occurring during fruit development in the tea plant. The seed coat o f tea regulates imbibition o f tea seed (Figure 2) and releases caffeine during germination (Figure 3). These processes may be dependent upon

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

26.

SUZUKI AND WALLER

Xanthine 1,3-Dimethylxanthine 3,7-Dime thy lx an thine 1,7-Dimethylxanthine 1,3,7-Trimethylxanthine

Pwrine Alkaloids

in Tea Seeds

Trivial name

Rl

Theophylline Theobromine Paraxanthine Caffeine

H CH H CH CH

R

291

R3

H CH CH H CH

3

3

3

3

3

3

3

Figure 1. Some Naturally Occurring Methylated Xanthines.

Table I. Caffeine (Cf) and Theobromine (Tb) Contents of Coffee and Tea Seeds

C , arabica seed

C . sinensis seed

a

Seed coat

Tb

Cf

Cf

b

Cotyledon

Tb

Cf

Tb

^g/g fresh wt.) 3893

54

2343

143

a

S u z u k i and Waller Q5).

b

F r o m the ripened dry fruits almost ready to drop.

100

F 0.0001, R^ = 0.92. SFW = 0.30 + 0.002 (Time); F = 492.1, P>F 0.0001, R = 0.95 SRFW = -0.06 + 0.005 (Time); F = 183.9, P>F 0.0001, R = 0.87. D

c

Figure 1. The relationship between barnyardgrass t o t a l fresh weight (TFW), seed fresh weight (SFW), and shoot-plus-root fresh weight (SRFW) over time.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

A Rapid Seedling Bioassay

SHILLING AND YOSHIKAWA

)\

Ο

. Sx

I

I

I

I

.

I

.

I

10 20 3 0 40 5 0 6 0 7 0 80 9 0 100

? TFW = 0 . 4 9 + 0.02 ( T i m e ) ; F = 1 , 4 6 1 . 7 , P>F 0 . 0 0 0 1 , R = 0 . 9 8 . SFW = 0 . 5 + 0.02 (Time) - 0.000161 (Time) ; F = 9 2 . 1 , P> 0.0001, R = 0.87. SRFW = - 0 . 1 4 + 0.01 ( T i m e ) ; F = 4 6 9 . 8 , P>F 0 . 0 0 0 1 , R = 0 . 9 4 . C

F i g u r e 2. The r e l a t i o n s h i p between hemp s e s b a n i a t o t a l f r e s h weight (TFW), seed f r e s h weight (SFW) and s h o o t - p l u s - r o o t f r e s h weight (SRFW) o v e r t i m e .

» -Or

0.0 0.2 0.4 0.6 0.8 TOTAL

a

F = 2,532.8,

P>F 0 . 0 0 0 1 , R

1.0 1.2

1.4 1.6

FRESH WEIGHT (g)

2

= 0.99.

F i g u r e 3 . Model f o r t h e d e t e r m i n a t i o n o f p r e d i c t e d s h o o t - p l u s - r o o t f r e s h weight (PSRFW) from t o t a l f r e s h weight (TFW) f o r barnyardgrass.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

340

caused t h i s t y p e o f r e s p o n s e i n hemp s e s b a n i a . Root l e n g t h was i n h i b i t e d by 82% b u t PSRFW o n l y 12%. I n p l a n t s t h a t were exposed t o t h e s e t y p e s o f compounds, t h e l o n g i t u d i n a l growth ( r o o t l e n g t h ) was a f f e c t e d more t h a n t h e o v e r a l l f r e s h weight o f t h e r o o t . Therefore, when a q u a l i t a t i v e assessment i n d i c a t e s t h i s t y p e o f a c t i v i t y , a more a p p r o p r i a t e growth measurement, such as r o o t l e n g t h , s h o u l d be used t o q u a n t i t a t e p h y t o t o x i c i t y . I t was e v i d e n t from t h i s s t u d y t h a t a whole p l a n t b i o a s s a y i s more a p p r o p r i a t e t h a n a s p e c i f i c b i o c h e m i c a l b i o a s s a y f o r t h e e v a l u a t i o n o f d i v e r s e c h e m i c a l t y p e s and/or e x t r a c t s c a u s i n g unknown l.8r

0.0

0.5

10

1.5

2.0

2.5

3.0

T O T A L F R E S H W E I G H T (g)

a

F = 3 6 8 . 7 , P>F 0 . 0 0 0 1 , R

2

= 0.93.

F i g u r e 4 . Model f o r t h e d e t e r m i n a t i o n o f p r e d i c t e d - r o o t f r e s h weight (PSRFW) from t o t a l f r e s h weight sesbania.

shoot-plus (TFW) f o r hemp

T a b l e I I . C o r r e l a t i o n c o e f f i c i e n t s between growth parameters a f f e c t e d by α - p h e n y l l a c t i c a c i d a n d ^ - e t h o x y b e n z o i c a c i d i n the l i g h t * a

Growth parameter Shoot f r e s h w e i g h t Root f r e s h weight Actual shoot-plusr o o t f r e s h weight Shoot l e n g t h Root l e n g t h Average s h o o t - p l u s root length T o t a l f r e s h weight

Barnyardgrass PSRFW TFW 0.82 0.69 0.92 0.95

Hemp s e s b a n i a TFW PSRFW 0.79 0.79 0.77 0.77

0.93 0.62 0.81

0.97 0.62 0.84

0.91 0.74 0.87

0.91 0.74 0.87

0.84

0.86 0.90

0.88

0.88 0.66

—

—

^Average f o r b o t h c h e m i c a l s a t 0 . 5 , 1.0 and 2 . 0 mM. A l l c o e f f i c i e n t s s i g n i f i c a n t at the 0.05 l e v e l . ^ T o t a l fresh weight. Predicted s h o o t - p l u s - r o o t fresh weight.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

31.

341

A Rapid Seedling Bioassay

SHILLING AND YOSHIKAWA

b i o l o g i c a l a c t i v i t y . Numerous q u a l i t a t i v e observations were made which indicated varying modes of action for the compounds tested. Juglone caused blackening of the tips of barnyardgrass shoots and roots. Flavone caused severe bleaching i n barnyardgrass shoots and coumarin caused root swelling. Some of these observations would not have been made i f a s p e c i f i c biochemical bioassay were used. Although there are l i m i t a t i o n s , as indicated previously, the described bioassay appears to be an e f f i c i e n t method to evaluate the phytotoxicity of various samples, both q u a l i t a t i v e l y and quantitatively. Table I I I . The effect of 27 compounds on four growth parameters of barnyardgrass and hemp sesbania Chemical 2 mM

SL

Barnyardgrass RL PSRFW

a

*e

Benzoic acid p_-Ethoxybenzoic acid 1 £-Hydroxybenzoic acid 1 £-Aminobenzoic acid 0 o-Ethoxybenzoic acid 16 Vanillin 12 V a n i l l i c acid 4 G a l l i c acid 7* Shikimic acid * Protocatechuic acid 20 2,3-Dihydroxy* benzaldehyde 44 3-Ethoxy-4-hydroxybenzaldehyde _t-Cinnamic acid 37 3-Phenyllactic acid * Caffeic acid * F e r u l i c acid 27 o-Hydroxycinnamic * acid 27 Coumarin 100* Scopoletin * Umbelliferone * 4-Hydroxycoumarin 17 Flavanone * Flavone 55 2-Carbethoxy-5,7dihydroxy-4-methoxy* 2 isoflavone Quercetin Juglone 68 α-Phenyllactic acid 12

37 27 0 0 7 0 18

1 8

*

!5* * * * 23 4 2

2 0

1 3

*

2 0

4 0

9

* * * * * * * 51

°* *

*

*

18 3 0 0 16 10

12 0 30 0 0 3 15 12

* 81 0 0 0 10 0

20 0 0 0 3 1

47 0 0 0 4 1

51

25

17

2

3

27°* 0 0 8

91°* 0

23°* 3 0 0

34°* 4 0 0

*

19 5 6

* * * 30 2 6

1 6

*

0

0

°*

2 5

**

40 4* 67

39°*

82

7 9

* * 66

* * 74 1* 52 6 4 0

50°* 24 4 0

0 9 8 0 5

34

24

13

7.

12

*3*

3 2

1 0 8 9

2 0

*

0

°*

* 40

97 9 0 12

U

Hemp sesbania RL TFW PSRFW

*

12 2 0 0 12 8

45

1 2

SL

*

2 9

5 7

9 6

* *

3 7

4 9

5 1

67

2 8

3 6

3 0

3 9

*

10 12 0 23 11 0 7

* 0

V "*

26 5 2

* 22

57

*

33

13*

46

7

°* 56

9. * * * 34 9

22

*

22

C

3 5

Shoot length. Root length. T o t a l fresh weight. Predicted shoot-plus-fresh weight. Values followed by an asterisk are s i g ­ n i f i c a n t l y d i f f e r e n t from the control at the 0.05 l e v e l according to general l i n e a r model procedure. e

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE INAGRICULTURE A N D

FORESTRY

Table IV. Correlation c o e f f i c i e n t s between growth parameters of barnyardgrass and hemp sesbania averaged for a l l 27 compounds Hemp sesbania

Barnyar•dgrass Growth parameter Total fresh weight Root length Shoot length Average root-plusshoot length

TFW

PSRFW : 1

0.71 0.90

—

0.93 0.84 0.87

0.86

0.94

J

TFW

PSRFW

0.80 0.47

—

0.97 0.81 0.43

0.73

0.72

Compared a t 2 mM o n l y . ^ T o t a l f r e s h w e i g h t . °Predicted s h o o t - p l u s f r e s h weight. A l l c o e f f i c i e n t s s i g n i f i c a n t a t the 0.05 l e v e l . a

Literature Cited 1.

Putnam, A.R.; Duke Phytopathol 1978 16 431-51. 2. Stevens, K.L.; Merrill, Agric , , 644-46. 3. Nicollier, G.F.; Pope, D.F.; Thompson, A.C. J. Agric. Food Chem. 1983, 31, 744-48. 4. Liebl, R.A.; Worsham, A.D. J. Chem. Ecol. 1983, 9, 1027-43. 5. Lehle, F.R.; Putnam, A.R. Plant Physiol. 1982, 69, 1212-16. 6. Fay, P.K.; Duke, W.B. Weed Sci. 1977, 25, 224-28. 7. Williams, R.D.; Hoagland, R.E. Weed Sci. 1982, 30, 206-12. 8. Jankay, P.; Muller, W.H. Am. J. Bot. 1976, 63(1), 126-32. 9. Tang, C.C.; Young, C.C. Plant Physiol. 1982, 69, 155-60. 10. Muller, W.H.; Muller, C.H. Bull. Torrey Bot. Club 1964, 91, 327-30. 11. Leather, G.R.; Einhellig, F.A. In "The Chemistry of Allelopathy: Biochemical Interactions Among Plants"' American Chemical Society: Washington, D.C., 1985; pp. 197-218. 12. Kida, T; Takaro, S.; Ishikawa, T.; Shibai, H. Agric. Biol. Chem. 1985, 49, 1299-1303. 13. Esahi, Y.; Leopold, A.C. Plant Physiol. 1969, 44, 618-67. 14. Chrispeels, M.J.; Varner, J.E. Nature 1966, 212, 1066-67. 15. Jones, R.L.; Varner, J.E. Planta 1967, 72, 255-61. 16. Helwig, J.T.; Council, K.A. (Eds) "SAS User's Guide"; SAS Institute, Inc.: Cary, N.C., 1979. 17. Shilling, D.G.; Liebl, R.A.; Worsham, A.D. In "The Chemistry of Allelopathy: Biochemical Interactions Among Plant"; American Chemical Society: Washington, D.C., 1985; pp 243-71. 18. Shettel, N.L.; Balke, N.E. Weed Sci. 1983, 31, 293-98. 19. Rice, E.L. "Allelopathy"; Academic Press: New York, N.Y., 1974. RECEIVED December 23, 1985

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

C h a p t e r 32

Interactions Among Allelochemicals and Other Stress Factors of the Plant Environment F. A. Einhellig Department of Biology, University of South Dakota, Vermillion, SD 57069

Current evidence indicates allelopathic inhibition most often result different chemicals be present at a growt inhibition threshold and s t i l l affect growth. Several combinations of allelochemicals have been shown to have either additive or synergistic action. Other work demonstrates that the action of phenolic acids is interrelated with nutrient conditions and temperature, moisture, and herbicide stress. Grain sorghum and soybean seedlings grown under relatively hot conditions exhibited a ferulic acid inhibition threshold at only one-half the concentration required under moderate temperatures, indicating stress interactions. Additive inhibition occurred when phenolic acids were tested in conjunction with moisture stress. Recent work showed that the growth of seedlings subjected to ferulic acid and atrazine together was suppressed more than with either alone. Allelochemicals may also promote damage from disease organisms. Hence, associated physical and chemical stress conditions may either enhance the inhibitory action of allelochemicals or result in an additive incremental detriment to plant growth.

It has been difficult to make an absolute connection between a suspected allelochemical inhibitor and the reduction in plant germination, growth, or function that characterizes a particular allelopathic situation. One reason this cause-effect relationship has been hard to establish is that the quantity of a biologically active compound recovered from the environment typically has been below the level required for inhibition in bioassays. Thus, a constant concern and argument against allelopathy has been that the level of an allelochemical in a natural setting is inadequate to be effective in growth regulation. However, the literature on allelopathy is replete with situations where several different 0097-6156/87/0330-0343$06.00/0 © 1987 American Chemical Society

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chemicals have been i d e n t i f i e d and i n f e r r e d as substances which could cause interference. In f a c t , i n most of the cases where the putative chemicals have been sought a number of compounds with b i o l o g i c a l a c t i v i t y have been found. This suggests the p o s s i b i l i t y that a l l e l o p a t h i c interference may be the result of the simultaneous action of several compounds. Receiving plants often contact allelochemicals through the s o i l medium, yet the information on a v a i l a b i l i t y from the s o i l i s minimal (1). Much of what i s known concerning allelochemicals i n the s o i l references phenolic acids and closely related phenolic structures. Because these compounds have received more scrutiny than others, they w i l l be a central focus i n the subsequent discussion. Reported concentrations of p-coumaric, f e r u l i c , p-hydroxybenzoic, v a n i l l i c , and other phenolic acids i n the s o i l have varied according to what a l l e l o p a t h i c species colonized the area, abundance and duration of plant residue, s o i l type, environmental factors, time of year, and method of extraction. Although i n d i v i d u a l phenolic acids i n the s o i l may exceed 1,000 jag/g o f r a c t i o n of t h i s contribute percentage may not be b i o l o g i c a l l y a c t i v e . For example, Whitehead et a l . (5) found that water extracts of p-coumaric, p-hydroxybenzoic, and v a n i l l i c acids from s o i l under quackgrass [Agropyron repens (L.) Beauv.] were equivalent to one micromolar or less for each compound in the s o i l solution. An extraction that might simulate limed conditions, 5% Ca(0H)2» gave values i n the 10 to 100 jiM range. These values are t y p i c a l , yet they are below concentrations that have been used i n tests for b i o l o g i c a l a c t i v i t y . A c h a r a c t e r i s t i c feature of allelopathy i s that the i n h i b i t o r y effects of a l l e l o p a t h i c compounds are concentration dependent. Dose-response curves with known compounds show an i n h i b i t i o n threshold. Below t h i s l e v e l either no measurable e f f e c t occurs, or stimulation may r e s u l t . Although the concentration of a compound required to exceed the i n h i b i t i o n threshold varies extensively according to d i f f e r e n t s e n s i t i v i t i e s among species and also among phases of the growth cycle for higher plants, the concept of an i n h i b i t i o n threshold seems consistent. Thus, i t i s reasonable to evaluate how, and i f , a subthreshold concentration of an allelochemical may contribute to a l l e l o p a t h i c interference. Also i n need of evaluation i s how environmental conditions may influence the deleterious action of an allelochemical and the concentration required for an e f f e c t . Such interactions are e s p e c i a l l y pertinent for those environmental situations that place some degree of stress on plant functions. This paper w i l l review the l i t e r a t u r e on the cooperative action of known allelochemicals. It w i l l also focus on the increasing evidence that the b i o l o g i c a l importance of these substances, especially i n low concentrations, depends on associated environmental conditions. Inhibition by Combinations of Allelochemicals The a l l e l o p a t h i c p o t e n t i a l of plants has often been evaluated from tests of the b i o l o g i c a l a c t i v i t y of v o l a t i l e s , leachates, and root exudates, or from aqueous extracts of the tissue. A l t e r n a t i v e l y , assessment of i n h i b i t o r s i n the s o i l associated with a suspected

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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a l l e l o p a t h i c plant has been routine. Such evaluations almost always deal with a complex matrix of biochemicals. When the bioassays have demonstrated effects on germination or growth, subsequent work on i d e n t i f i c a t i o n of the responsible allelochemicals has often followed. Although these searches have seldom been exhaustive, they have consistently revealed more than one compound with b i o l o g i c a l a c t i v i t y . I t has not been uncommon to i s o l a t e ten or more compounds which may include several different chemical classes (6-9). However, i t has been d i f f i c u l t to determine the quantity of each that might be functional i n the environment, and generally the r e l a t i v e contribution of each allelochemical to growth i n h i b i t i o n has not been evaluated. ι Some investigators have tested equimolar mixtures of chemicals they have isolated as the agents i n a l l e l o p a t h i c situations (10-12) , and a few have attempted to simulate combinations from f i e l d situations. Glass (13) grew plants hydroponically i n a solution which reproduced the phenolic acid conditions found i n the s o i l associated with Pteridium 39 uM p-hydroxybenzoic acid p-hydroxycinnamic acid, and 4 μΜ f e r u l i c acid altered the root growth of barley (Hordeum vulgare L.) and several other species. In a study of hackberry (Celtis laevigata L.) allelopathy, Lodhi (3) found that the combined e f f e c t of p-coumaric, f e r u l i c , and c a f f e i c acids at the concentrations found i n s o i l underneath these trees was much more i n h i b i t o r y to seed germination than the e f f e c t of each chemical (at i t s s o i l concentration) tested separately. Weaver and K l a r i c h (14) reported an increase i n r e s p i r a t i o n rate i n wheat plants that were exposed i n the f i e l d to v o l a t i l e substances, presumably monoterpenes, from Artemisia tridentata Nutt., but the r e l a t i v e amount of d i f f e r e n t terpenes was not ascertained. A recent study of Lupinus albus L. showed the a l l e l o p a t h i c e f f e c t s of a mixture of q u i n o l i z i d i n e alkaloids which approximated that excreted from germinating seeds and seedlings (15). More d e f i n i t i v e e f f o r t s have been made to ascertain the concerted action of allelochemicals by quantitatively comparing the action of a mixture of substances with the a c t i v i t y of each component part (Table I ) . Although most of these studies have been with derivatives of benzoic acid, cinnamic acid, and coumarin, some evaluations of other compounds have occurred. Asplund (16) reported that the phytotoxic monoterpenes, camphor, pulegone, and borneol, exhibited marked synergistic action on root growth. Up to 100-fold enhancement was found using two compounds simultaneously, demonstrating that b i o l o g i c a l a c t i v i t y could occur with concentrations two orders of magnitude below the threshold f o r a single compound. Wallace and whitehead (17) showed the synergistic action of v o l a t i l e fatty acids, and their work demonstrated the value of recognizing that s i m i l a r compounds may have d i f f e r e n t a c t i v i t i e s . I t took ten times as much acetic acid to i n h i b i t wheat (Tritiown aestivum L.) as butyric or propionic acid. We have investigated the concurrent action of some of the more commonly reported phenolic allelochemicals by testing these compounds at, or below, their i n h i b i t i o n threshold (18-21). Our f i r s t experiments showed that a combination of 5 mM each of p-coumaric and f e r u l i c acids reduced grain sorghum [Sorghum bicotor (L.) Moench.] germination appreciably more than separate treatments

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Table I .

Quantitative Assessment of E f f e c t s of Combinations of Allelochemicals a

Chemical C l a s s C o n e , of S i n g l e B i o a s s a y E f f e c t Cpd. i n Mixture

5

Ref.

Monoterpenes

0.017-0.68 μΚ/L

G

Syn

16

Fatty Acids

0.27 - 3.2 mM

RE

Syn

17

2.5 5.0 M 0.125-0.2

G

Syn

G SG

Syn Syn

Phenolic Acids, etc. FA,pCA

VA,pHB

2.5 - 5.0 mM 0.5 mM

FA,VA,pCA

G,SG

3.3 mM

£CnA,pCA,FA,CA

CA,FA,pCA CA,FA,VA CA,FA,pCA,pHB, PRO,SIN,SYR,VA

G = germination;

Syn Syn Syn

21

1.0 mM

G

Several Add

22

1.0 - 3.0 mM

G

Add,Ant

23

0.5 mM

RE

Ant

24

0.125- 0.5 mM

SG

Syn,Add,Ant

25

RE = root elongation;

^Syn = s y n e r g i s t i c ; Add = additive; CA = c a f f e i c ;

20

G RE SE

1.0 - 2.5 mM 0.25 - 1.0 mM 0.04 - 0.1 mM

Coumarin,CGA,FA, HCnA,p CA,pHBAL,PYR

Syn,Ant

19

CGA = chlorogenic;

SG = seedling growth

Ant = antagonistic

FA = f e r u l i c ;

HCnA = hydrocinnamic; pCA = p-coumaric; pHB = p-hydroxybenzoic; pHBAL = p-hydroxybenzaldehyde; PRO = protocatechuic; PYR = pyrocatechol; SIN = sinapic; SYR = s y r i n g i c ; VA = v a n i l l i c ; £CnA = t-cinnamic

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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of these chemicals (18). In tests with sorghum seedlings grown i n nutrient solution amended with p-coumaric and f e r u l i c acids, the threshold for growth reduction was less than l/20th the l e v e l required to reduce germination. Cooperative i n h i b i t o r y action of these phenolics was obvious since seedlings grown with 0.125 mM p-coumaric or f e r u l i c acids were s i g n i f i c a n t l y stimulated, whereas growth of plants i n a combination of the two was i n h i b i t e d . Similar studies showed the three-way i n t e r a c t i o n of p-coumaric, f e r u l i c , and v a n i l l i c acids on seed germination was s y n e r g i s t i c , while v a n i l l i c acid seemed to antagonize some of the i n h i b i t i o n of the other two on shoot elongation (20). Colby's (26) analysis was used as an index for judging potential interactions among four cinnamic acids (21). Based on this c r i t e r i o n , concentrations of 0.04 mM t-cinnamic acid and 0.1 mM f e r u l i c , p-coumaric, and c a f f e i c acids had synergistic effects on sorghum growth when applied i n combinations of two, three, and a l l four. Similar cooperative effects may also occur with mixtures of allelochemicals of d i f f e r e n t chemical categories. Work now i n progress (unpublishe of sorghum germination an flavonoid ( r u t i n ) , a coumarin (umbelliferone), and a benzoic acid ( s a l i c y l i c acid) . Antagonism among these three occurred i n Lernna minor L . bioassays, demonstrating that species vary i n their response. Investigations using above-threshold concentrations also indicate that several phenolic compounds i n a mixture can have at least a cumulative e f f e c t . Duke et a l . (23) concluded from probit analysis of data on lettuce seed germination that p-coumaric and f e r u l i c acids produced additive i n h i b i t i o n . Blum et a l . (25) tested eight phenolics i n various combinations of two or three on cucumber (Cucumis sativa L . ) leaf expansion, and applied regression analysis. The e f f e c t s of mixtures tested ranged from s y n e r g i s t i c to antagonistic, and the authors concluded that the nature of the response depended on the magnitude of i n h i b i t i o n associated with each compound, the compounds i n the mixture, and the factor measured. Methods for assessing the j o i n t action of i n h i b i t o r s have many d i f f i c u l t i e s , especially when used for studying the r e l a t i v e l y low concentrations that are most probable i n a f i e l d s i t u a t i o n . As pointed out by Morse (27) and Nash (28), nearly a l l methods have t h e i r shortcomings and even an unambiguous d e f i n i t i o n of terms such as synergism has remained elusive. However, these shortcomings and problems i n communication should not be allowed to detract from the b i o l o g i c a l importance of the j o i n t action of chemicals functioning in allelopathy. The preponderance of evidence indicates: (a) an allelochemical seldom acts alone, (b) concentrations considerably below i n h i b i t i o n thresholds i n bioassays may be b i o l o g i c a l l y active, and (c) the j o i n t action of several compounds can influence plant growth and functions. Viewed i n this manner, an a l l e l o p a t h i c substance that i s released into the environment can j u s t l y be considered one of several stresses that may influence plant d i s t r i b u t i o n and vigor of plant growth. Interaction Between Allelopathy and Mineral N u t r i t i o n An early observation that a l l e l o p a t h i c e f f e c t s might be subject to other environmental conAmg^t^^Ctl6ilfôâJiS06^q^f luence noted

Library 1155 16th St., N.W. Washington, D.C. 20038 Waller, G.; In Allelochemicals: Role in Agriculture and Forestry; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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from a d d i n g n u t r i e n t s (29). S e v e r a l i n v e s t i g a t i o n s i n the l a s t decade have demonstrated t h a t p h e n o l i c a l l e l o p a t h y may be more s e v e r e under low f e r t i l i t y , and r a i s i n g the n u t r i e n t l e v e l can s u p p r e s s some o f t h e a l l e l o c h e m i c a l e f f e c t (13,30). Stowe and Osborn (30) r e p o r t e d t h a t t o x i c i t y of v a n i l l i c (25 and 50 ppm) and p - c o u m a r i c (5 and 10 ppm) a c i d s t o b a r l e y p l a n t s depended i n t i m a t e l y on n u t r i e n t c o n c e n t r a t i o n s . Two-way a n a l y s i s of v a r i a n c e showed a d e f i n i t e i n t e r a c t i o n between p h e n o l i c t r e a t m e n t s and n i t r o g e n and phosphorus l e v e l s . V a n i l l i c acid inhibited barley growth i n a manner dependent upon phosphorus s u p p l y , and p - c o u m a r i c a c i d e f f e c t s were dependent upon n i t r o g e n l e v e l s . At low n u t r i e n t l e v e l s b o t h p h e n o l i c s were s i g n i f i c a n t l y i n h i b i t o r y , s u g g e s t i n g soil f e r t i l i t y might be v e r y i m p o r t a n t i n p h e n o l i c a l l e l o p a t h y . H a l l e t a l . (31) found pigweed (Amaranthus retroflexus L.) grown i n s o i l amended w i t h c h l o r o g e n i c a c i d was s t u n t e d and the p l a n t s had a r e d u c e d phosphorus c o n t e n t , but t h e s e e f f e c t s were overcome by a d d i n g a n i t r o g e n - p h o s p h o r u s - p o t a s s i u m supplement. Indeed, case studies i n d i c a t e inputs allelopathic inhibition t a l l f e s c u e (Festuca arundinacea S c h r e b . ) , and s u n f l o w e r (Helianthus

annuus L.) (31-33). S e v e r a l p h e n o l i c a c i d s and many n o n s p e c i f i c a l l e l o p a t h i c c o n d i t i o n s have been shown to a l t e r the m i n e r a l c o n t e n t of p l a n t s , and c e r t a i n l y p h e n o l i c a l l e l o c h e m i c a l s may p e r t u r b c e l l u l a r f u n c t i o n s i n a number of ways t h a t a r e o f importance t o p l a n t n u t r i t i o n (34 ,_35) . However, r a i s i n g f e r t i l i t y does not always s u p p r e s s a l l e l o p a t h i c i n h i b i t i o n , and t h e i n t e r r e l a t i o n s h i p s between t h e s e two f a c t o r s a r e s t i l l not c l e a r . Bhowmik and D o l l (36) showed t h a t a l l e l o p a t h i c i n h i b i t i o n o f c o r n and soybeans by r e s i d u e s o f f i v e a n n u a l weeds was n o t a l l e v i a t e d by s u p p l e m e n t a l n i t r o g e n o r phosphorus. S i m i l a r l y , an i n c r e a s e i n f e r t i l i z e r d i d not overcome i n h i b i t i o n o f c o r n by q u a c k g r a s s o r c i r c u m v e n t the a u t o t o x i c i t y of berseem c l o v e r (Trifolium alexandrium L.) (37,38). Even when r a i s i n g n u t r i e n t l e v e l s r e l e a s e s i n h i b i t i o n , i t does not mean t h a t a l l e l o p a t h y was i n o p e r a t i v e under the o r i g i n a l c o n d i t i o n s . These i n s t a n c e s s i m p l y i l l u s t r a t e the i m p o r t a n c e o f t h e i n t e r a c t i o n o f the two s t r e s s c o n d i t i o n s . Enhancement of A l l e l o c h e m i c a l E f f e c t s by

Temperature

Stress

I o f t e n o b s e r v e d t h a t under greenhouse c o n d i t i o n s t h e r e was c o n s i d e r a b l e v a r i a t i o n i n the e f f e c t a p a r t i c u l a r c o n c e n t r a t i o n o f a p h e n o l i c i n h i b i t o r had on the growth of s e e d l i n g s . Since p r o c e d u r e s used i n t h e s e b i o a s s a y s were q u i t e u n i f o r m , i t was l o g i c a l t h a t e n v i r o n m e n t a l f a c t o r s were i n f l u e n c i n g the r e s u l t s . T h i s was v e r i f i e d when we t e s t e d the h y p o t h e s i s t h a t t e m p e r a t u r e o f the growth environment m o d i f i e d a l l e l o c h e m i c a l a c t i o n (39). Grain sorghum and soybean [Glycine max (L.) M e r r . ] s e e d l i n g s were t r e a t e d w i t h s e v e r a l l e v e l s of f e r u l i c a c i d , t h e n each t r e a t m e n t group was s u b d i v i d e d and t h e two s u b s e t s h e l d a t d i f f e r e n t t e m p e r a t u r e s w i t h l i g h t i n t e n s i t y the same f o r t h e two e n v i r o n m e n t s . The two t e m p e r a t u r e regimes i n each experiment were w i t h i n the normal range the s e e d l i n g s might e x p e r i e n c e under f i e l d c o n d i t i o n s . However, the h i g h e r t e m p e r a t u r e s would g e n e r a l l y be c o n s i d e r e d more stressful.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Seedling response over a 10-day treatment period showed a s i g n i f i c a n t interaction e f f e c t (two-way analysis of variance) between temperature and f e r u l i c acid. Although several morphological features were d i f f e r e n t , the dry weights of control plants i n the two temperature regimes were equivalent at the end of each experiment. Effects of equimolar concentrations of f e r u l i c acid were more severe at the higher temperatures. The threshold concentration f o r i n h i b i t i o n of sorghum growth was 0.2 mM f e r u l i c acid at 37° C average day temperature, while 0.4 mM f e r u l i c acid was required f o r i n h i b i t i o n with an average day temperature of 29° C. Both shoot and root weight reductions evidenced t h i s difference i n an i n h i b i t i o n threshold. Soybeans were more sensitive than sorghum to both temperature and f e r u l i c acid, but a s i m i l a r interaction between temperature and f e r u l i c acid occurred i n these experiments. Soybeans grown with a day temperature of 34° C and 0.1 mM f e r u l i c acid were s i g n i f i c a n t l y i n h i b i t e d , weighing 63% as much as control plants, while at 23° C even 0.25 mM f e r u l i c acid-treated plants were not stunted as severely r e l a t i v e l y hot environmenta inhibition. Several other evidences that temperature v a r i a t i o n can a l t e r the extent of a l l e l o p a t h i c i n h i b i t i o n have been reported. Glass (13) subjected barley seedlings to a mixture of phenolic acids with subgroups at 5, 10, 15, 20, 25, and 30° C. The phenolic acid mixture suppressed root growth (fresh weight) i n each environment over a 14-day growth period, but the extent of i n h i b i t i o n was more severe at the extremes of low and high temperature. Steinsiek et a l . (40) reported leachates from wheat straw caused a more marked i n h i b i t i o n of germination and growth of sensitive weeds, such as ivy leaf morningglory \_Ipomoea hederaoea (L.) Jacq.], when incubated at 35° C than at 30 or 25° C. Both temperature and photosynthetic photon f l u x density altered the a l l e l o p a t h i c e f f e c t s of residues of redroot pigweed (Amaranthus retro flexus L.) and yellow f o x t a i l \Setaria glauoa (L.) Beauv.] on corn (41). The i n h i b i t o r y e f f e c t s of these weed residues were less when corn was grown with moderate l i g h t and at 30/20° C light/dark conditions, as compared to a lower l i g h t l e v e l and temperature. However, a l l e l o p a t h i c effects of the weed residues on soybean were not overcome at the more moderate conditions. Concurrent Action of Allelochemicals and Moisture Stress Phenolic acids interfere with many major physiological processes of higher plants (35). These disruptions of function include an a l t e r a t i o n of plant water balance. We found depression of leaf water potential to be an early indicator of allelochemical stress from f e r u l i c and p-coumaric acids (42). Likewise one mechanism of a l l e l o p a t h i c action by cultivated sunflower, velvetleaf (Abutilon theophrasti M e d i c ) , Kochia [Kochia scoparia (L.) Schrad.], and several other weeds was water stress (43-45). Since some allelochemicals i n t e r f e r e with plant-water relationships, i t seemed l o g i c a l that t h e i r action might be most c r i t i c a l at times when plants are under water stress from other causes. We i n i t i a t e d several experiments to determine the impact of allelochemicals acting simultaneously with moisture stress (46).

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Seeds were germinated i n vermiculite for f i v e days, then transplanted to 80-ml opaque v i a l s containing a complete nutrient medium. The following day, seedlings were treated by transferring them to nutrient solutions amended with f e r u l i c acid (0.1 or 0.25 mM), an osmoticum (e.g., -0.2 MPa), or both. After ten days growth in the greenhouse, plant dry weights were compared by analysis of variance and Colby's (26) analysis was applied for evaluating potential interactions. A l l osmotic agents may create some a n c i l l a r y e f f e c t s , but at the l e v e l s used i n these experiments equivalent results were obtained with either polyethylene g l y c o l 4000, KC1, or NaCl. The results of these experiments c l e a r l y established that the simultaneous actions of water stress and an allelochemical are more deleterious than either alone. This combination e f f e c t was especially evident with r e l a t i v e l y minor stresses from each source, as shown with data from one experiment (Table I I ) . Treatment of 0.1 mM f e r u l i c acid had no e f f e c t on growth, while nutrient media with an osmotic adjustmen sorghum seedlings. Thes below the e f f e c t caused by the NaCl alone. Apparently moisturestressed plants were more sensitive to f e r u l i c acid. Dry weights of plants grown i n the combination of 0.25 mM f e r u l i c acid and -0.2 MPa were s i g n i f i c a n t l y below those observed i n the separate treatments, and Colby s analysis suggested the combined action at these levels was more than additive. Replicate experiments gave similar r e s u l t s . We found comparable cooperative effects i n studies of seed germination, except that both a lower water potential and higher phenolic acid concentration were required to achieve a germination i n h i b i t i o n threshold. Experiments using a matrix of four levels of f e r u l i c acid and four levels of moisture stress demonstrated that the combined action was additive under more s t r e s s f u l levels of the i n d i v i d u a l factors than i n the previous t e s t s . Duke et a l . (23) tested the germination of lettuce seeds treated with phenolic acids (1 mM) at water potentials (D-mannitol) of 0, -0.2, -0.4, and -0.6 MPa. The combined action of low water p o t e n t i a l and exposure to phenolic acids resulted i n an additive detriment to germination, and the authors concluded from probit analysis that the mechanism of action from these sources was s i m i l a r . Whatever their mechanisms, moisture stress and phenolic acids appear to work together i n l i m i t i n g growth of plants. Although i n d i r e c t and probably quite rare, another route has been reported for allelochemical interference with plant-water relationships. Lovett and D u f f i e l d (47) i d e n t i f i e d benzylamine as an allelochemical i n the leaf washings from the cruciferous weed Cconelina sativa (L.) Crantz. Subsequent work showed benzylamine induced hydrophobic conditions i n the s o i l , and these conditions could reduce water a v a i l a b i l i t y for plant growth (48). Thus, i n d i r e c t action through changes in s o i l structure could be p a r t i a l l y responsible for adverse e f f e c t s on linseed (Linseed usitatissirnvon L.) and could enhance more d i r e c t a l l e l o p a t h i c e f f e c t s . 1

Joint Action of Herbicides

and

Allelochemicals

Opportunity exists i n agroecosystems for two sources of chemical interference, natural and synthetic. The o r i g i n of allelochemicals

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987. +8..7 65.4

63..6 56..7

300.6+25.9bc 267.9+20.3c

171. .1+12.9cd 155, ,6+11.9d

129, .5+13.5de

112, .3+ 8.9e

1

!

Positive difference from predicted = synergism.

product of % of control f o r single treatments

Means (N = 15) i n a column not followed by the same l e t t e r are s i g n i f i c a n t l y d i f f e r e n t , P

Adsorption Desorption Polymerization

SOIL SOLUTION

PHENOLIC COMPOUNDS

de novo m i c r o b i a l synthesis

(CO2

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

A g r i c u l t u r a l Conservation

Production

Systems

C o n s e r v a t i o n agroecosystems developed i n t h e Great P l a i n s of t h e U.S. t o c o n t r o l s o i l e r o s i o n a r e c h a r a c t e r i z e d by the presence o f v a r y i n g q u a n t i t i e s o f p l a n t r e s i d u e s on the s o i l s u r f a c e . This r e s i d u e mulch p r o t e c t s the s o i l from the e r o s i v e f o r c e s o f wind and water, r e s u l t i n g i n improved stream water q u a l i t y and s o i l c o n s e r v a tion. C o n s e r v a t i o n t i l l a g e systems a l s o h e l p m a i n t a i n s o i l product i v i t y and reduce energy requirements of crop p r o d u c t i o n ( 1 5 ) . Howe v e r , c r o p y i e l d r e d u c t i o n has been observed w i t h c o n s e r v a t i o n wheat p r o d u c t i o n i n some areas o f t h e U.S. (16-18) and w i t h r i c e c u l t u r e i n t h e F a r E a s t (_19, 20) . Unger and M c C a l l a have p o i n t e d out f a c t o r s (21) which c o u l d c o n t r i b u t e t o t h e observed y i e l d r e d u c t i o n s i n c l u d i n g l a c k of p r o p e r e x p e r t i s e and equipment t o manage s u r f a c e r e s i d u e s , l a c k o f pest and d i s e a s e c o n t r o l , inadequate n u t r i e n t a v a i l a b i l i t y , p a r t i c u l a r l y n i t r o g e n , major a l t e r a t i o n s i n b i o l o g i c a l p r o p e r t i e s o f s o i l , and production of phytotoxi W h i l e much r e s e a r c been c o n d u c t e d , many q u e s t i o n about t h e r a t e o f p h y t o t o x i n p r o d u c t i o n and a c c u m u l a t i o n , localized c o n c e n t r a t i o n , t h r e s h o l d s o i l c o n c e n t r a t i o n f o r e x p r e s s i o n of b i o a c t i v i t y , d u r a t i o n o f b i o a c t i v i t y , s t a b i l i t y i n s o i l , and environmental redistribution. Although the f a t e o f s p e c i f i c c h e m i c a l s under d e f i n e d e x p e r i m e n t a l c o n d i t i o n s i s d i s c u s s e d , i t i s hoped t h a t the p r i n c i p l e s i n v o l v e d can be extended to p r o v i d e a b a s i c u n d e r s t a n d i n g of t h e f a t e o f the wide range o f a l l e l o c h e m i c a l substances i n the soil. The

Complex S o i l

Environment

O r g a n i c c h e m i c a l s i n t h e s o i l p a r t i c i p a t e i n many i n t e r a c t i o n s and t r a n s f o r m a t i o n s between the gaseous, l i q u i d , s o l i d , and b i o l o g i c a l phases. The c h e m i c a l f i n d s i t s environment c o n s i s t i n g o f s o l i d p a r t i c l e s r a n g i n g i n s i z e from one to s e v e r a l m i l l i m e t e r s down to subm i c r o s c o p i c c o l l o i d a l m a t e r i a l s . The s o l i d m a t r i c e s may be coated w i t h aqueous f i l m s o r be p a r t o f g a s - s o l i d i n t e r f a c e s . Microorganisms are p r e s e n t i n the v o i d s and t h e f l u i d medium, forming t h e l i v i n g phase o f the complex environment (22-24). The m i n e r a l phase. M i n e r a l c o l l o i d s are composed o f l a y e r e d s i l i c a t e s and amorphous m e t a l h y d r o x i d e s . The two b a s i c b u i l d i n g l a y e r s o f t h e s i l i c a t e s are ( i ) a t e t r a h e d r a l s i l i c o n d i o x i d e l a y e r modif i e d by o c c a s i o n a l s u b s t i t u t i o n by A l ^ and ( i i ) an o c t a h e d r a l A l o x y h y d r o x i d e l a y e r w i t h o c c a s i o n a l s u b s t i t u t i o n by Mg2 , F e ^ , o r Fe3 . The two types o f l a y e r s can be found i n a 1:1 arrangement as i n k a o l i n i t e and h a l l o y s i t e c l a y m i n e r a l s , o r i n a 2:1 arrangement as i n m o n t m o r i l l o n i t e , v e r m i c u l i t e , i l l i t e , and c h l o r i t e . Dissociat i o n o f edge h y d r o x y l groups and the s u b s t i t u t i o n o f S i ^ and/or Al^ by l o w e r - v a l e n c y c a t i o n s w i t h i n t h e t e t r a h e d r a l / o c t a h e d r a l l a y e r s r e s u l t i n a net n e g a t i v e charge. Moreover the l a y e r e d s i l i c a t e s are o f t e n h y d r a t e d by a t h i n f i l m o f water. The water molec u l e s i n t h i s f i l m a r e h i g h l y s t r u c t u r e d and i n c o n j u n c t i o n w i t h c o u n t e r - i o n s i n t h e b u l k s o l u t i o n g i v e r i s e t o t h e v e r y low s u r f a c e pH. T h i s h i g h a c i d i t y o f c l a y s u r f a c e s p l a y s an important r o l e i n +

+

+

+

+

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

+

33.

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Sorption

and Mineralization

of Plant Phenolic

Acids in Soil

361

the adsorption and c a t a l y t i c reactions of phenolic compounds as w i l l be shown l a t e r . The organic phase. S o i l organic matter that is in intimate contact with the mineral phase may be subdivided into two major fractions: ( i ) fresh or partly decayed plant or animal residues and ( i i ) humi­ fied or completely altered or resynthesized substances (24). The f i r s t group is referred to as nonhumic substances, and contains organic chemicals belonging to such classes as amino acids, carbohy­ drates, l i p i d s , pigments and other low-molecular weight compounds. The second group, known as humic substances, are high-molecular weight, dark-colored substances formed by secondary synthesis reac­ tions. Humic substances commonly are c l a s s i f i e d into three subcate­ gories on the basis of s o l u b i l i t y c h a r a c t e r i s t i c s : ( i ) humic acid, dark-colored substance soluble in a l k a l i but insoluble in acid, ( i i ) f u l v i c acid, the colored material which remains in solution or c o l ­ l o i d a l dispersion, after precipitation of humic acid by a c i d i f i c a ­ t i o n , and ( i i i ) humin, th variety of functional groups alcoholic hydroxyl, enolic hydroxyl, quinone, lactone, and ether have been shown to be present in the humic substances. Carboxyl and phenolic hydroxyl groups are the source of the cation exchange capacity of the organic matter. Differences in degree of humificat i o n of organic matter are reflected i n differences in the degree of r e a c t i v i t y and adsorptive behavior of the various fractions of the soil. Behavior and fate of phenolic compounds i n s o i l The behavior of phenolic compounds derived from decaying plant r e s i ­ dues, or released from degrading humic substances, is dictated by the physico-chemical processes of adsorption and desorption. E q u i l i b r i a between these processes determine the concentration of phenolic com­ pounds in the s o i l solution and consequently the b i o a c t i v i t y , move­ ment, and persistence of these substances in the s o i l . Surface interactions between phenolic compounds and c o l l o i d a l matrices may promote their polymerization (25, 26) or protect them from microbial degradation and mineralization. The nature of soil-phenolic acid interaction: adsorption-desorption. Adsorption of a solute from solution onto a solid matrix results i n a higher solute concentration at the f l u i d - s o l i d interface than in the solution. Huang and coworkers (27) observed a high sorption capacity of the mineral fraction of four latosols for phenolic acids. On the basis of their r e s u l t s , d i s t r i b u t i o n c o e f f i c i e n t s , K^, or the ratios of solution-phase solute concentration and adsorbed-phase concentration were calculated to estimate the r e l a ­ t i v e a f f i n i t y of the s o i l s for phenolic acids. The values for p-hydroxybenzoic acid, p-coumaric, v a n i l l i c , f e r u l i c , and syringic acids were 67, 75, 69, ^2 and 376, respectively for a 48-hr e q u i l i ­ bration of 0.1 μτηοΐ mL"* phenolic acid solution with a sample of an a l f i s o l preextracted in b o i l i n g water. The sorption capacity was greatly reduced by pretreatment of s o i l samples with sodium acetatehydrogen peroxide to remove organic matter and metal sesquioxides. values were 49, 32, 61, 37 and 92 respectively for the phenolic

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND

FORESTRY

a c i d s named, f u r t h e r treatment to remove more s e s q u i o x i d e reduced the v a l u e s to 16, 20, 39, 30, and 54 r e s p e c t i v e l y . Huang and coworkers p o s t u l a t e d t h a t c o m p l e x a t i o n of the p h e n o l i c a c i d s by n o n c r y s t a l l i n e s e s q u i o x i d e s was a t t r i b u t a b l e to the i n t e r a c t i o n of p h e n o l i c h y d r o x y l and c a r b o x y l groups with p o s i t i v e l y charged A1-0H ° and F e - 0 H ° ' sites. A p p r o x i m a t e l y 40 to 50% o f the t o t a l amount o f p h e n o l i c s sorbed was r e t a i n e d by the o r g a n i c matter f r a c t i o n ( 2 7 ) . In s u r f a c e s o i l l a y e r s , o r g a n i c m a t t e r i s f r e q u e n t l y i n t i m a t e l y a s s o c i a t e d w i t h the m i n e r a l components p r e s e n t , p r o v i d i n g a l a r g e s u r f a c e area and r e a c t i v e s i t e s for surface i n t e r a c t i o n . S o i l a c i d i t y has a major i n f l u ence on p h e n o l i c a d s o r p t i o n by the o r g a n i c carbon f r a c t i o n , s i n c e the degree of d i s s o c i a t i o n of the p h e n o l i c a c i d s i s pH-dependent. Whitehead and coworkers (28) observed that the e x t r a c t a b i l i t y o f s e v e r a l p h e n o l i c a c i d s was h i g h l y dependent upon the e x t r a c t a n t pH between pH 6 and 14. The amount e x t r a c t a b l e c o n t i n u a l l y i n c r e a s e d w i t h e x t r a c t a n t pH; thus the e x t r a c t e d a c i d s c o u l d not be r e a d i l y c l a s s i f i e d into d i s t i n c 5 +

2

5 +

2

The a d s o r p t i o n of cinnami sured i n our l a b o r a t o r y u s i n g the b a t c h e q u i l i b r a t i o n method and a l i q u i d chromatographic technique. Freundlich adsorption c o e f f i c i e n t s were 2.0, 1.7, and 0.98 (N = 0.92, 0.94, and 0.74) at s o i l s u s p e n s i o n pH o f 4.5, 5.0, and 5.5. Our d a t a agreed with the t r e n d of e x t r a c t a b i l i t y o b s e r v e d by Whitehead and co-workers .( 28) . The a d s o r p t i o n o f c i n n a m i c a c i d decreased w i t h i n c r e a s i n g pH o f the s o i l suspension. The magnitude of the p a r t i t i o n c o e f f i c i e n t s i n d i c a t e d a low to i n t e r m e d i a t e a d s o r p t i o n p o t e n t i a l f o r cinnamic a c i d . Howe v e r , the o b s e r v e d h i g h c a p a c i t y of s o i l s f o r r e t e n t i o n of p h e n o l i c compounds ( s t a b i l i z a t i o n ) may have i n f a c t been due to t h e i r s o r p t i o n on s o i l c o l l o i d s . The term s t a b i l i z a t i o n as used i n the humus literature i s to be i n t e r p r e t e d as the o v e r a l l d i s a p p e a r a n c e o f p h e n o l i c a c i d s d u r i n g c o n t a c t with the s o i l medium. Thus the phenomenon i n c l u d e s the e f f e c t s of a d s o r p t i o n , d e s o r p t i o n , s u r f a c e - i n duced p o l y m e r i z a t i o n , enzymatic p o l y m e r i z a t i o n , and i m m o b i l i z a t i o n i n m i c r o b i a l t i s s u e s . A d s o r p t i o n on c o l l o i d a l s u r f a c e s may promote the f o r m a t i o n of humic polymers by i n d u c i n g f a v o r a b l e c o n f o r m a t i o n a l arrangement f o r c a t a l y s i s by m e t a l s e s q u i o x i d e s (29) or by p a r t i c i p a t i o n of f r e e r a d i c a l groups of s o i l o r g a n i c m a t t e r (j30, 31). Because of the r e a c t i v i t y o f p h e n o l i c s u b s t a n c e s i n s o i l s , the p r o c e s s e s of o x i d a t i v e p o l y m e r i z a t i o n and d e g r a d a t i o n must be uncoupled from a d s o r p t i o n to p r o p e r l y assess the l a t t e r p r o c e s s . Several attempts have been made to o b t a i n d e t a i l e d k i n e t i c s of a d s o r p t i o n i n m i c r o b i a l l y a c t i v e systems. Ogram and co-workers (32) have shown t h a t the h e r b i c i d e 2,4-D can be m i c r o b i a l l y degraded o n l y i n the s o l u t i o n phase and by non-adsorbed d e g r a d e r s . Dao and Lavy (33) have o b s e r v e d t h a t a d s o r p t i o n of r e a c t i v e s o l u t e s such as p h e n o l , a n i l i n e , and b e n z o i c a c i d on s o i l was v e r y r a p i d ; e q u i l i b r i u m was a t t a i n e d i n a few m i n u t e s . D e g r a d a t i o n or s u r f a c e - i n d u c e d t r a n s f o r m a t i o n would l e a d to erroneous e s t i m a t e s of a d s o r p t i o n . Experiment a l and m a t h e m a t i c a l approaches to u n c o u p l i n g these two processes have been d e s c r i b e d (32, 33). A l t h o u g h not s p e c i f i c a l l y a p p l i c a b l e to p h e n o l i c a c i d s , h y d r o phobic a d s o r p t i o n o f many x e n o b i o t i c compounds has been r e p o r t e d , such a d s o r p t i o n b e i n g dependent on the o r g a n i c carbon content o f the s o r b i n g medium (34, 35). A l i q u i d - l i q u i d p a r t i t i o n model has been

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Acids in Soil

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extensively used to describe this partitioning of organic solutes between organic matter and an aqueous solution. The 1-octanol-water model was found to provide satisfactory preliminary indices of adsorption potential for a wide variety of organic chemicals in s o i l (36, 37). Recently, computation and predictive correlations using linear free-energy relationships have become increasingly accurate alternatives to experimental measurements of p a r t i t i o n c o e f f i c i e n t s (37, 38). Liquid chromatographic techniques have also been used to estimate the p a r t i t i o n i n g behavior of neutral as well as weakly ionizable chemicals (3^, 40). Therefore, varied mechanisms of adsorption, ranging from physical dipole-to-dipole interaction and hydrogen bonding to oxidation-reduction, can e f f e c t i v e l y dictate the behavior of allelochemicals in s o i l . Irrespective of the sources of phenolic compounds in s o i l , adsorption and desorption from s o i l c o l l o i d s w i l l determine their solution-phase concentration. Both processes are described by the same mathematical models, but they are not necessarily completely reversible. Complete r e v e r s i b i l i t desorption, an equilibriu with release as easy as retention. In non-singular adsorptiondesorption e q u i l i b r i a , the release of the adsorbate may involve a different mechanism requiring a higher activation energy, resulting in different reaction kinetics and desorption c o e f f i c i e n t s . This phenomenon is commonly observed with pesticides (41, 42). An acute need exists for experimental data on the adsorption, desorption, and e q u i l i b r i a for phenolic compounds to properly assess their environ­ mental chemistry in s o i l . Adsorption has a significant impact on the movement of a l l e l o ­ chemical substances in s o i l . Such movement in s o i l by water i s important from the standpoint of mechanism of phytotoxin a c t i v i t y in the receiving species at a s i t e remote from the donor plant. Adsorption reduces the solute concentration in the s o i l solution and consequently minimizes r e d i s t r i b u t i o n in the environment. Solute transport has been described by Fick's second law of d i f f u s i o n and the kinetic models for adsorption and degradation of reactive solutes (43, 44). The contribution of adsorption is measured and expressed as the retardation factor, R. R = 1 + ρ/θ

. Κ . NC

-1

(Ν )

where C = solution-phase solute concentration, Κ and Ν = Freundlich adsorption c o e f f i c i e n t s , ρ = s o i l bulk density, and θ = volumetric water content. For singular adsorption-desorption processes where Ν = 1, R becomes R = 1 + ρ/θ

. Κ

Thus unbound phenolic acids should be easily transported by convec­ t i o n . Shindo and Kuwatsuka (45) have observed this in leaching experiments. Phenolic acid learned about decomposition er, l i t t l e is

metabolism. During the last the production of phytotoxins in the laboratory and in the known about the fate of these

30 years, much has been during plant residue f i e l d (6-8, 18). Howev­ molecules in the s o i l .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Some c l u e s may be a v a i l a b l e from s t u d i e s o f the d e c o m p o s i t i o n of lignin. L i g n i n c o n s t i t u t e s the second most abundant carbon polymer on e a r t h a f t e r c e l l u l o s e ( 4 6 ) . The u n d e r s t a n d i n g o f b i o d e g r a d a t i v e pathways of l i g n i n and l i g n i n - c e l l u l o s i c polymers may e l u c i d a t e the problems of reduced p l a n t p r o d u c t i v i t y a s s o c i a t e d w i t h s u r f a c e r e s i d u e s i n c o n s e r v a t i o n p r o d u c t i o n systems. a. L i g n i n biodégradation as a source of p h e n o l i c a c i d s i n soils. P l a n t s s y n t h e s i z e p h e n o l i c a c i d s , which are then combined t o form polymers such as l i g n i n , l i g n i n - c e l l u l o s e s , f l a v o n o i d s , and tannins. The f l a v o n o i d s and t a n n i n s o c c u r i n p l a n t s as pigments. L i g n i n and l i g n i n - c e l l u l o s e s are important f o r the s t r u c t u r a l i n t e g r i t y of the p l a n t and impart r i g i d i t y to c e l l w a l l s , d e c r e a s e water p e r m e a t i o n a c r o s s c e l l w a l l s , and r e s i s t m i c r o b i a l i n v a s i o n of p l a n t t i s s u e s ( 4 6 ) . When the p l a n t senesces and decays, the p h e n o l i c polymers are a t t a c k e d by f u n g i o f v a r i o u s genera, i n c l u d i n g A s p e r g i l l u s , A u r e o b a s i d i u m , B a s i d i o m y c e t e s , Cephalosporium, Fusarium, Humicola, Neurospora polymers and t h e i r d e g r a d a t i o b a c t e r i a , most n o t a b l y the gram-negative s t r a i n s ( 4 7 ) . S o i l m i c r o o r g a n i s m s degrade the polymers by c l e a v i n g o f f subu n i t s c o n s i s t i n g o f one, two, o r at most t h r e e p h e n o l i c a c i d moieties. Fungal d e g r a d a t i o n of l i g n i n s appears to be e s s e n t i a l l y o x i d a t i v e and decayed l i g n i n s e x h i b i t e d t h r e e main changes from the parent l i g n i n : ( i ) o x i d a t i o n of the s u b s t i t u t e d s i d e c h a i n s , ( i i ) o x i d a t i o n o f the a l p h a - c a r b o n i n the p r o p a n o i d s i d e chains, and ( i i i ) c l e a v a g e of a r o m a t i c r i n g s s t i l l a t t a c h e d to the polymer ( 4 7 ) . C o l b e r g and Young (48) showed t h a t the e x t e n t o f d e g r a d a t i o n o f l i g n i n s i n a g i v e n time i n c r e a s e d as m o l e c u l a r s i z e d e c r e a s e d . R e l a t i v e l y l a r g e f r a c t i o n s h a v i n g an average m o l e c u l a r s i z e o f 1,000 to 1,400 l o s t 21% of the t o t a l a v a i l a b l e carbon as gaseous p r o d u c t s , w h i l e m o l e c u l e s h a v i n g an average m o l e c u l a r s i z e o f 400 to 1,000 l o s t 32% of the a v a i l a b l e c a r b o n . M o l e c u l e s of average m o l e c u l a r s i z e l e s s than 400 had 40% o f the c a r b o n o x i d i z e d t o C 0 . These r e s u l t s agreed w i t h those o f Crawford and co-workers (49) who found t h a t l a r g e r p h e n o l i c m o l e c u l e s , p a r t i c u l a r l y p o l y m e r i c ones, are more s t a b l e than s m a l l , f r e e monomeric p h e n o l i c a c i d s . Because of the s t a b i l i t y of l a r g e polymers, the r a t e - d e t e r m i n i n g s t e p s i n the b i o l o g i c a l t r a n s f o r m a t i o n of l i g n i n s to gaseous p r o d u c t s o c c u r at the s t a g e where l i g n i n s are broken down i n t o the monomeric p h e n o l i c acids (50). O f t e n t h i s d e c o m p o s i t i o n and i t s r a t e - d e t e r m i n i n g s t e p s are e x p r e s s e d i n terms of f i r s t - o r d e r k i n e t i c s ( 5 1 ) . Of c o u r s e the d e c o m p o s i t i o n r a t e c o e f f i c i e n t i s a f f e c t e d by v a r i o u s f a c t o r s such as t e m p e r a t u r e , pH, s o i l m o i s t u r e , and s u b s t r a t e c o n c e n t r a t i o n and composition. Many s t u d i e s have shown t h a t p l a n t r e s i d u e s are f i r s t decomposed r a p i d l y , then more s l o w l y . The s o l u b l e f r a c t i o n i s r a p i d l y m e t a b o l i z e d , f o l l o w e d by the c e l l u l o s e f r a c t i o n of the l i g n i n - c e l l u l o s e s , then the r e l a t i v e l y s l o w - d e g r a d i n g l i g n i n component. Crawford (52) showed t h a t the c e l l u l o s e component can decompose f o u r to t e n times as f a s t as the c o r r e s p o n d i n g l i g n i n . Since d e c o m p o s i t i o n i s an enzymatic p r o c e s s i n m i c r o o r g a n i s m s , f a c t o r s t h a t a d v e r s e l y a f f e c t the microorganisms or the enzymes decrease the d e c o m p o s i t i o n r a t e . F o r i n s t a n c e , as the temperature d e c r e a s e s , s o i l d r i e s , or pH changes from the optimum pH l e v e l f o r the enzyme system, d e c o m p o s i t i o n becomes slower ( 5 1 ) . 2

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The monomeric p h e n o l i c compounds r e l e a s e d d u r i n g l i g n i n d e g r a d a t i o n have been shown to c o n s i s t m a i n l y o f b e n z o i c and cinnamic a c i d s and t h e i r d e r i v a t i v e s , i n c l u d i n g the aldehyde forms (47, 53). C o l b e r g and Young (54) i s o l a t e d t e n monoaromatic compounds produced in l i g n i n m i c r o b i a l degradation, including catechol, phenylacetic, benzoic, 3-phenylpropionic, cinnamic, s y r i n g i c , v a n i l l i c , f e r u l i c and c a f f e i c a c i d s , and v a n i l l i n . Cinnamic, b e n z o i c , c a f f e i c , v a n i l l i c , and f e r u l i c a c i d s were found i n the l a r g e s t amounts. R e g a r d l e s s of the s o u r c e , p h e n o l i c a c i d s are u l t i m a t e l y b r o k e n down t o gaseous p r o d u c t s such as CO2 and methane. T h i s breakdown o c c u r s by t h r e e g e n e r a l methods: ( i ) aerobic r e s p i r a t i o n , using m o l e c u l a r oxygen as an e l e c t r o n a c c e p t o r , the end product b e i n g CO2, ( i i ) a n a e r o b i c r e s p i r a t i o n w i t h e l e c t r o n a c c e p t o r s such as n i t r a t e and ( i i i ) a n a e r o b i c f e r m e n t a t i o n w i t h p h o s p h o r y l a t i o n r e a c t i o n s i n v o l v i n g no e x t e r n a l e l e c t r o n a c c e p t o r (50). b. A e r o b i c c a t a b o l i s m o f p h e n o l i c compounds. The a e r o b i c breakdown o f p h e n o l i c s compound o u t l i n e d by G i b s o n (55) b e t a o x i d a t i o n r e a c t i o n w i t h the enzymatic removal o f two-carbonatom u n i t s . Thus p h e n o l i c a c i d s w i t h an odd number of carbon atoms i n the s i d e c h a i n u l t i m a t e l y y i e l d h y d r o x y b e n z o i c a c i d s , w h i l e those w i t h an even number of atoms i n the carbon s i d e c h a i n are c o n v e r t e d to h y d r o x y p h e n y l a c e t i c a c i d s . The p h e n o l i c a c i d i s then f u r t h e r h y d r o x y l a t e d and p o s s i b l y d e c a r b o x y l a t e d to form a r i n g c l e a v a g e p r e c u r s o r , such as c a t e c h o l or p r o t o c a t e c h u i c a c i d i n the case of h y d r o x y b e n z o i c a c i d , and 2 , 5 - d i h y d r o x y p h e n y l a c e t i c a c i d or 3,4 d i h y d r o x y p h e n y l a c e t i c a c i d i n t h e c a s e o f h y d r o x y p h e n y l a c e t i c a c i d . The h y d r o x y l a t e d b e n z e n e r i n g t h e n u n d e r g o e s f i s s i o n b y r i n g opening between the two h y d r o x y l groups w i t h a dioxygenase to y i e l d a l i p h a t i c a c i d s which are then o x i d i z e d t o C 0 (56). Once l i g n i n has been degraded i n t o monomeric p r o d u c t s , t h e d e g r a d a t i o n o f the i n d i v i d u a l aromatic monomers proceeds q u i t e rapidly. H a i d e r and M a r t i n (53) have shown t h a t ^ C - l a b e l e d b e n z o i c and cinnamic a c i d s and t h e i r d e r i v a t i v e s can be a e r o b i c a l l y m i n e r a l i z e d i n the f i r s t two weeks, and some of the compounds had o v e r 90% o f t h e i r carbon c o n v e r t e d to C 0 i n one week. S i n c e these r e a c t i o n s are r e l a t i v e l y r a p i d , i . e . , p h e n o l i c a c i d s are r a p i d l y degraded a e r o b i c a l l y , t h e i r presence i n the s o i l under these c o n d i t i o n s appears t r a n s i t o r y . It has been d i f f i c u l t t o d e t e c t unbound p h e n o l i c a c i d s i n the s o i l s o l u t i o n and the compounds do not appear to accumulate i n a p p r e c i a b l e amounts under a e r o b i c conditions. However, the s o i l i s a heterogeneous medium c o n s i s t i n g of l o c i or microenvironments t h a t are at times c o m p l e t e l y o p p o s i t e i n c h a r a c t e r , i . e . , a n e r o b i c m i c r o s i t e s i n a w e l l - a e r a t e d s o i l (57). The p h y t o t o x i c i t y problem should be viewed i n the c o n t e x t of a s p a c i a l l y v a r i a b l e environment. 2

2

c. Anaerobic catabolism. In s o i l , f r e e p h e n o l i c a c i d s may encounter a n a e r o b i c c o n d i t i o n s , p a r t i c u l a r l y when the s o i l has poor d r a i n a g e , has been t e m p o r a r i l y f l o o d e d , or i s i n the c e n t e r o f s o i l aggregates. Sommers and co-workers (51) note t h a t as s o i l water p o t e n t i a l d e c r e a s e s d i f f e r e n t groups of p h e n o l i c - d e g r a d i n g m i c r o o r ganisms become a c t i v e . They noted at s o i l water p o t e n t i a l s i n the

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range of plant growth (-0.03 to -1.5 MPa), microbial metabolism takes place under aerobic conditions with oxidation of carbon sources to C 0 . As the water content of the s o i l increases, the microbial population s h i f t s to facultative anaerobic organisms that degrade the phenolic compounds through fermentation reactions with production of organic acids, alcohols, and other p a r t i a l l y oxidized carbon compounds. At even lower water potential, as oxygen becomes l i m i t i n g , the microbial population s h i f t s to obligate anaerobic organisms. As the s o i l becomes more reduced, the electron acceptors used by the organisms change, usually following the sequence of ( i ) 0 , ( i i ) N 0 " , ( i i i ) Mn , ( i v ) F e , (v) S O 4 " , ( v i ) H , and ( v i i ) C 0 . The l a t t e r electron acceptors result i n the formation of gaseous products c h a r a c t e r i s t i c of anaerobic systems such as CH4, N , N 0, H , and H S. Evans (50) has shown that anaerobic fermentation i s often a two-stage reaction. A consortium of microorganisms mineralizes the aromatic compounds to methane and C 0 . F i r s t the benzene ring i s reduced to a cyclohexan such as adipate, heptanoat negative organisms. These acids are then broken down to form acetate and formate, which i n turn are mineralized by methagenic bact e r i a to C 0 and methane. Ferry and Wolfe (cited i n 50) showed that the conversion of phenolic acids to methane followed the reduct i v e pathway for benzoate: 2

3+

2

2

3 +

3

2

2

2

2

2

2

2

2

(1) 4 C H COOH + 24 H 0 = 12 CH3COOH + 4 HCOOH + 8 H 6

2

5

(2)

12 CH3COOH = 12 CH4 + 12 C 0

(3)

4 HCOOH = 4 C 0 + 4 H

(4)

3 C 0 + 12 H

2

2

2

2

2

2

= 3 CH4 + 6H 0 2

a t o t a l reaction of 4 C6H5COOH + 18 H 0 = 15 CH4 + 13 C 0 . Healy and Young (58) observed that the conversion of v a n i l l i c and f e r u l i c acids under anaerobic conditions to methane and C 0 was nearly stoichiometric. More than half of the organic carbon could p o t e n t i a l l y be converted to methane. This could have great importance i n studies where the degradation of phenolic compounds are studied by trapping the evolved C 0 . Under anaerobic condit i o n s , part of the normal C 0 evolution may be shifted to methane production with a subsequent low reporting of C 0 evolved, and an underestimation of microbial a c t i v i t y i n the s o i l (51). Colberg and Young (48) have also shown that there could be an effect on the degradation of l i g n i n i t s e l f , because under anaerobic conditions the methagenic consortium can break the beta-aryl bond, the most common linkage of aromatic monomers i n l i g n i n s , to release phenolic compounds for further degradation. These processes occur at a lower rate than under aerobic conditions, so some of these phenolic acids and t h e i r breakdown products may accumulate in the soil. for

2

2

2

2

2

2

d. Synthesis of humic acids by oxidative polymerization of phenolic acids. Phenolic acids and their polymers i n the s o i l are i n a continual state of flux, constantly being polymerized and part i a l l y degraded, broken down and resynthesized and adsorbed and released as they are eventually immobilized and mineralized by s o i l microorganisms.

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Phenolic compounds can be condensed forming a r y l - a r y l and aryl-oxygen-aryl (ether linkages) bonds to y i e l d d i a r y l and d i a r y l ether polymers (59) . These are in many ways similar to natural humic acids, confirming e a r l i e r research by others (60-62) that humic acids are formed from the copolymerization of phenolic compounds with amino acids, peptides, and amino sugars. The rate at which phenolic acid units are incorporated into humic fractions depends on many factors. Berry and Boyd (63) used a peroxidase enzyme from horseradish to study the oxidative coupling of phenols and anilines and found that the reaction rates were on the order of 10 to 185\|imoles/s for a number of methyl- and methoxy-phenols and anilines at 20°C. They suggested that the degree to which such compounds polymerize through enzymatic oxidative coupling reactions would be affected by substituent groups on the aromatic ring. Electron-withdrawing functional groups would i n h i b i t the polymerization whereas electron-donating groups (such as OCH3) that commonly occur on lignin-derived phenols would enhance it. The r e a c t i v i t y of substitute table from the positio reflected by the Hammett constant (64). The y i e l d of humic acid was found to be higher when the concent r a t i o n of phenolic acids was low; when the concentration of unbound acids was high, they were used as a substrate for microbial minerali z a t i o n , with a subsequent reduction in humic acid synthesis (65). Kassim and co-workers (31) further showed that a s i g n i f i c a n t portion of intact ^ C - l a b e l e d f e r u l i c acid was s t a b i l i z e d into s o i l humic substances. Two percent of the added carbon-14 remained i n the s o i l biomass after one year, representing 5 to 7% of the added f e r u l i c acid. The degree of s t a b i l i z a t i o n is related to the ease of free radical formation v i a the a c t i v i t y of phenolase or peroxidase enzymes. Solid humic acid was found to exhibit paramagnetic resonance due to the presence of unpaired electrons (30). Stable organic free radicals occur in humic acid on the order of 10^ radi c a l / g and appear to be an integral part of the humic acid structure. Their presence points to a humic acid biosynthetic pathway based on oxidative coupling of phenolic compounds v i a seraiquinone or quinhydrone-type free radicals (^0, 59). Phenolic compounds have also been oxidatively polymerized to humic substances by clay minerals (29) and by the mineral fraction of a l a t a s o l (66). After a 10-day e q u i l i b r a t i o n period, montmorill o n i t e and i l l i t e clay minerals yielded 44 to 47% of the t o t a l added phenolic acids as humic substances whereas quartz gave only 9%. Samples of a l a t a s o l yielded over 63% of the t o t a l amount, from mixtures in varied proportion, of mono-, d i - and trihydroxy phenolic compounds as humic substances (66). Extractions of the reaction products yielded humic, fulvic, and humin fractions that resembled s o i l natural fractions in color, in acid-base s o l u b i l i t y , and in infrared absorption spectra. Wang and co-workers (67) further showed that the c a t a l y t i c polymerization of catechol to humic substances was,enhanced by the presence of Al oxide and increased with pH i n the 5.0 to 7.0 range. Thus the normally very reactive products of l i g n i n degradation can be linked into very stable humic acid polymers which w i l l maintain a pool of p o t e n t i a l l y reactive phytotoxins in the s o i l .

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Summary and Conclusions In summary, much is known about the sources, the behavior, and fate of phenolic substances in soil. However, much of this information has been gained under relatively harsh and unnatural conditions. Improved extraction and isolation methodology must be developed for allelochemical studies in soil. Knowledge of the relationship between extracted and actual bioactive chemical entities is critical in assessing allelopathic interactions. Process models will also provide much insight for describing, predicting the presence and effective concentration of allelochemical substances, and relating them to the expression of a toxic response in higher plants. New understanding of the microbiology of the lignin-cellulosic and humic polymers in natural soil environment and the degradation of the lignin component in agricultural systems is needed. The productivi­ ty of millions of acres of rangelands and cultivated lands under conservation production practices hinges on the improved understand­ ing of the turnover of cycle. Research effort allelopathy involving plant residues in conservation production systems, crop-residue and weed-residue interactions, and the biocontrol potential in many of these agroecosystems. Acknow!edgments The author wishes to thank R. D. Meyer for his excellent assistance in the literature search and preparation of the manuscript. The suggestions of the reviewers are sincerely appreciated. Literature Cited 1. 2. 3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13.

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42. Koskinen, W. C.; O'Connor, G. Α.; Cheng, Η. H. Soil Sci. Soc. Am. J. 1979, 43, 871-874. 43. Letey, J.; Farmer W. In "Pesticides in Soil and Water"; Guenzi, W. D., Ed., Agron. Soc. Am.: Madison, WI, 1974; pp. 67-97. 44. Rao, P. S. C.; Davidson, J. M.; Jessup, R. E.; Selim, H. M. Soil Sci. Am. Soc. J. 1979, 43, 22-28. 45. Shindo, H.; Kuwatsuka, S. Soil Sci. Plant Nutr. 1975, 21, 227-238. 46. Zeikus, J. G. In "Lignin Biodegradation: Microbiology, Chemistry, and Potential Applications"; Kirk, T. K.; Higuchi, T.; Chang, Η., Eds.; CRC Press: Boca Raton, FL, 1980; pp. 101-109. 47. Cain, R. B. In "Lignin Biodegradation: Microbiology, Chemistry, and Potential Applications"; Kirk, T. K.; Higuchi, T.; Chang, Η., Eds., CRC Press: Boca Raton, FL, 1980; pp. 21-60. 48. Colberg, P. J.; Young 49, 345-349. 49. Crawford, D. L.; Floyd, S.; Pometto, A. L. III; Can. J. Microbiol. 1977, 23, 434-440. 50. Evans, W. C. Nature, 1977, 270, 17-22. 51. Sommers, L. E.; Gilmour, C. M.; Wildung, R. E.; Beck, S. M. In "Water Potential Relations in Soil Microbiology"; Soil Sci. Soc. Am. Spec. Pub. No. 9: Madison, WI, 1980; pp. 97-117. 52. Crawford, R. L. "Lignin Biodegradation and Transformation"; John Wiley and Sons: New York, NY, 1981; pp. 61-67. 53. Haider, K.; Martin, J. P. Soil Sci. Soc. Am. Proc. 1975, 39, 657-662. 54. Colberg, P. J.; Young, L. Y. Appl. Environ. Microbiol. 1985, 49, 350-358. 55. Gibson, D. T. Science. 1968, 161, 1093-1097. 56. Dagley, S. In "Soil Biochemistry"; McLaren, A. D.; Peterson, G. H., Eds.; Marcel Dekker: New York, NY, 1967; pp. 287-317. 57. Patrick, Z. A. Soil Sci. 1971, 111, 13-18. 58. Healy, J. B. Jr.; Young, L. Y. Appl. Environ. Microbiol. 1979, 38, 84-89. 59. Suflita, J. M.; Bollag, J.-M. Soil Sci. Soc. Am. J. 1981, 45, 297-302. 60. Verma, L.; Martin, J. P.; Haider, K. Soil Sci. Soc. Am. Proc. 1975, 39, 279-284. 61. Biederback, V.O.;Paul, E. A. Soil Sci. 1973, 115, 357-366. 62. Haider, K.; Frederick, L. R.; Flaig, W. Plant and Soil 1965, 22, 49-64. 63. Berry, D. F.; Boyd, S. A. Soil Sci. Soc. Am. J. 1984, 48, 565-569. 64. Brown, H. C.; Okamoto, Y. J. Am. Chem. Soc. 1958, 80, 1873-1882. 65. Martin, J. P.; Haider, K. Soil Sci. Soc. Am. J. 1980, 44, 983-988. 66. Wang, T. S. C.; Li, S. W.; Huang, P. M. Soil Sci. 1978, 126, 81-86. 67. Wang, T. S. C.; Wang, M. C.; Huang, P. M. Soil Sci. 1983, 136, 226-230. RECEIVED January 14, 1986

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

C h a p t e r 34

Allelopathic Influences on No Tillage Versus Conventional Tillage in Wheat Production George R. Waller , E. G. Krenzer, Jr. , James K. McPherson , and Steven R. McGown 1

2

3

4

Departments of Biochemistry , Agronomy , Botany , and Microbiology , Oklahoma State University, Stillwater, OK 74078 1

2

3

4

Incorporating allelopathy into agricultural management may reduce the use of herbicides, cause less pollution, and diminish autotoxic hazards. Authentic inhibitors isolated from plant material have been subjects for examination in vitro but attempts to compare their effect heterogeneous collectio Organic solvents and water extracts prepared from monoculture wheat soils under conventional tillage (CT) and no tillage (NT) indicated that both soils contain some inhibitory compounds. The CGC/MS/DA of some of the organics is presented. Selected organics from CT and NT as well as allelopathic and autotoxic effects are described and discussed. The relationship between the wheat yields in CT and NT and the possible biological stress is indicated. Conservation tillage practices in wheat production have increased steadily from about 15% in 1971 to 30% in 1985. These practices are attractive to growers for soil and moisture conservation and fuel savings, but there may be drawbacks. Variations in yields of forage and grain compared with those of conventional tillage differ according to rainfall conditions and geographic location. These erratic results are probably also partly attributable to biological factors such as diseases, insect damage, and allelopathy, which can vary substantially from season to season. Researchers elsewhere have generally shown that allelopathy from wheat residue reduces the subsequent wheat yield (1-3). There is reason to believe that the same phenomenon is occurring in the Great Plains Area as reported here, though under some circumstances it may be masked by the favorable effects of no-till growing such as greater retention of soil moisture. Allelopathic chemicals from soils, crop residues, and weeds are known to reduce the growth of several crops, and there are numerous examples of allelopathy among wild plants. We are studying the allelopathic effects of wheat residue and soil on the germination and growth of wheat under Oklahoma conventional-tillage and no-tillage conditions. We see this as basic research aimed at determining the presence and magnitude of any allelopathic effects of wheat on itself, as well as the identity of the chemicals causing them. This would, of course, be a necessary first step in remedying allelopathic effects and increasing wheat yield. Whittaker (4), Waller and Nowacki (5.), and Rabotnov (£) discussed the evolution of stable plant communities and species susceptible to allelopathic chemicals that were released by other plants. Such plants would have been eliminated by natural selection, and allelopathically neutral or allelopathically 0097-6156/87/0330-0371$06.00/0 © 1987 American Chemical Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

tolerant plant communities would result. Allelopathy is often more evident in disturbed plant communities, such as agricultural ones. M c C a l l a and coworkers (1-3. 7) did pioneer research on the wheat crop i n the eastern Nebraska area. They showed that water-soluble substances i n crop residues reduced the germination and growth o f seedlings o f wheat, c o r n , sorghum, and other crops. The water extracts o f the seeds had the least effect and the stem extracts had the greatest inhibitory effect on wheat seedlings. Wheat as well as other crops were shown to contain a number of phenolic acids and the five most dominant ones were: ferulic, p_-coumaric, syringic, v a n i l l i c , and p_hydroxybenzoic acids. These were quantitatively estimated i n the crop residues; e.g., the total amount o f phenolic acids from wheat left on the field was 1.5 tons/acre under no-tillage conditions. M c C a l l a and Norstadt (3) worked extensively o n the antibiotic patulin (CjHgO^; M . W . 156) produced by Pénicillium urticae Bainer, which was found i n wheat soil, and found that the severity o f visible symptoms o f phytotoxicity to winter wheat (no-tillage) corresponded to the concentration of patulin. Elliott et al. provided a review of phytotoxicity in 1978 (£) and Elliott et al. (9) showed that bacterial colonization of plant roots can cause a 2 5 % increas produced. Putman and DeFrank (10) made use o f phytotoxic plant residues for selective weed control. Lehle and Putman (11) used sorghum plants to show that the self-inhibitory activity (autotoxicity) varied widely depending upon the stage of development. Schilling et al. (12) snowed that compounds, some identified (βphenyllactic acid, β-hydroxybutyric acid) and some not identified, were effective in the suppression o f certain weeds by rye and wheat mulches in no-till crops. The data otherwise accumulated by the Oklahoma Agricultural Experiment Station so far indicate that for 6 years the wheat y i e l d obtained i n studies comparing conventional-tillage and no-tillage averages is 42 bu/acre for each. This is independent o f location. However, there is an indication that the soil systems have not stabilized with the change i n tillage practices; thus, one would not expect to see any real differences i n the soil as it relates to the wheat crop yields between the tillage treatments. Factors essential to maintain wheat crop yields are the availability o f water and nutrients, control o f insects, and disease. W e have found that the forage yield (hay) from the wheat crop taken at the early jointing stage for the conventional-tillage system is twice that for no-tillage. If this difference continues to be found i n future years then it provides some evidence for an allelopathic effect occurring early i n the growing season. O u r objective was to study i n detail these systems by identifying the allelochemicals involved, their primary modes o f action as mediated by soils, and as appropriate the microflora, microclimate, soil moisture and other factors that may influence allelopathy. Experimental Sampling o f the Soil. Representative soil samples were taken from conventional and no-tillage plots before planting and afterward at intervals o f 1 month since A p r i l , 1985. The soil samples were placed i n quart jars, frozen immediately by using dry ice, and stored at -18 °C. Extraction o f Organic Compounds from the S o i l . S o i l biochemicals that are free or absorbed loosely, but not bound to the humus, were extracted by the following procedures: A)

A 100-200-g sample o f soil was thawed, placed i n an extraction thimble, and extracted with redistilled isopropyl alcohol in a Soxhlet extractor for 48 h. Some of these alcohol extracts were analyzed for

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

34.

WALLER ET AL.

B)

C)

Tillage in Wheat

Production

373

their biological activity and others were evaporated and the residue was extracted with water at 60 °C, 3 times, and the mixture filtered through 7-mm Whatman 41 filter paper; the insoluble part was extracted with methyl alcohol at r o o m temperature, and those compounds that remained were redissolved i n isopropyl alcohol. Each extract was weighed and bioassayed. A 200-g sample of soil was loosely packed i n a 24 χ 40 c m chromatography column and sequentially extracted with redistilled organic solvents at room temperature and a flow rate of 0.5 m L per minute i n the following order; 200 m L hexane; 100 m L hexane + 100 m L methylene chloride; 200 m L methylene chloride; 100 m L methylene chloride + 100 m L ethyl acetate; 200 m L ethyl acetate; 100 m L ethyl acetate + 100 m L methyl alcohol; 200 m L methyl alcohol; 100 m L methyl alcohol + 1 0 0 m L triply distilled water; 800 m L triply distilled water. E a c h extract was taken to dryness over nitrogen gas except the aqueous one, which was evaporated with a rotary evaporator at 45 °C. E a c h residue was weighed and bioassayed. Steam distillation, extraction, and evaporation (Waller et al. [13. 14]) were completed and the resulting mixtures bioassayed.

Analysis of the M i x t u r e of Organic Compounds from the S o i l . The crude fractions were analyzed using a L K B - 2 0 9 1 capillary gas chromatograph/mass spectrometer/data analysis system ( C G C / M S / D A ) . The capillary column used was a J & W D B - 1 , 60 m χ 0.32 m m , connected directly to the ion source of the mass spectrometer. U p to 1.0 u L of a solution of the sample i n an appropriate solvent was injected directly onto the column at 40 °C, whereupon the column temperature was immediately raised to 100 °C for 4 min, and programmed to 310 °C at a rate of 10°/min. and held there for 30 min. Bioassay of Organic Compounds from the S o i l . Bioassay experiments measured the germination and early growth (generally the most sensitive time of any plant's life) of wheat. The methods are similar to those of McPherson and M u l l e r (15) and others i n the field, and are summarized below. Containers, media and seeds. Glass Petri dishes, 100 χ 15 cm, were used with two sheets of 75-mm Whatman 41 filter paper as the absorptive medium. Ten seeds of Τ Α Μ 105 wheat were placed i n a radial pattern with the micropyle end toward the center between the two sheets of filter paper. Seeds were hand-selected for normal size and absence of damage. TÀM105 was selected because it is the variety used i n the ongoing field research on conservation tillage practices. The bottom section of each Petri dish cover was covered with a square of kitchen-type plastic wrap to retard moisture loss before the l i d was pressed on. Allelopathic test materials and controls. Some 2.5 m L of aqueous or organic extracts were required for thorough saturation. Water-soluble or partially water-soluble extracts were applied directly to the filter paper. Distilled water controls were used. W i t h organic solvent-soluble extracts, the solution was applied to the filter paper and allowed to dry, then distilled water was added to support germination. Controls having pure solvent applied were similarly allowed to dry before the distilled water was added. Quantification of the amount of allelopathic material applied to each sample

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

374

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

was made by weighing the amount o f extract so that a consistent ratio could be maintained. Records o f the amounts were kept so that a consistent calculation o f concentrations could be made. Incubation conditions. Preliminary trials indicated that incubation at 20 °C for 72 h i n darkness is optimal. This relatively l o w temperature allows adequate wheat growth while retarding mold development. Replication. Six Petri dishes each containing ten seeds were used for each control and for each treatment Controls accompanied all experiments. Results, parameters, and measurements. Counts o f germinated vs. ungerminated seed, length and width of coleoptile, and length of central root and stem were recorded. Means per dish and per treatment (four to six dishes) were calculated and standard statistical tests were used i n the analysis. Results This paper reports the initial results of a new study. W e collected soil samples from the Agronomy Farm a extracted them with isopropy dried extracts o f soil from no-tillage vs conventional-tillage plots is shown i n Tables I, l i a and III. Suprisingly both types o f soils were inhibitory to the growth o f wheat. The Soxhlet extract o f June soil was dried i n a rotary evaporator and then subjected to successive methyl alcohol and water extractions. The bioassay results are shown in Table l i b and show that the conventional-tillage soil contained no more allelopathic material than did the no-tillage soil. In view o f a laboratory error that influenced these results, we believe that the extract o f notillage soil may have equalled or exceeded the conventional-tillage s o i l i n allelopathic potency. S o i l collected at other locations ( E l Reno and Altus) were extracted and steam distilled as described i n Experimental. No-tillage samples o f soil were used; however, they varied from Altus plots devoted to monoculture for 10 years previously, to soil that had been used to grow wheat only one previous year ( E l Reno) which had been part o f a virgin prairie until cultivation. The results, shown i n Table I V , again indicate that both these no-tillage soils are allelopathic toward wheat. Products o f steam distillation, although this is a severe treatment, showed slight growth inhibition by the initial (pH 6.0) fraction, whereas those form the highly basic soil suspensions were markedly inhibitory. Table V shows the number o f compounds obtained from T i l l m a n s o i l obtained by steam distillation as identified by the C G C / M S / D A system; the soil has quite an array of organic compounds ~ some quite complex (12,14). In the milder treatment by solvent extraction for E l Reno soil, shown in Table IVb, the aqueous fraction was more allelopathic than the ethyl acetate/methylene chloride fraction. The duplicate plots (Table IVb, no-till I & II) showed some difference in bioassay results which cannot be explained just now. A l s o shown i n Tables I-IV are the amounts o f crude organic extract and the amount o f soil extracted; each represents the quantity that was bioassayed per wheat seed. These amounts represent less than the soil mass i n the normal seedling environment. The quantity o f organic matter that is present i n the soil around the germinating seed and seedling is striking. It strongly suggests that this soil organic matter is a subject about which scientists should be concerned. In fact, solvent extraction (Table IVb) shows the quantity of organics in the soil and is representative o f what would be found i n nature. Presented i n Figures l a and 2a are reconstructed partial total ion current chromatograms obtained by the C G C / M S / D A run on the M a y , 1985 samples. Figures l b and 2b show mass spectra taken at a certain specified time and peak number. Figure l b shows the mass spectrum o f phthalate plasticizer i n the soil

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987. C

10

32 2

19

Significantly different from control at 9 5 % level of confidence or better (i-test).

6.5 ± 0.8

b

Not significantly different from control (t-test).

C

5.3 ± 0 . 7

Inhibition % Root Shoot

b

20.7 ± 4.2

C o n v - T i l l , A q . Ext.

b

6.6 ± 0.2

Shoot Length

c

15.5 ± 2.9

N o - T i l l , A q . Ext.

(mm)

23.1± 0.9

2

Root Length

Control, Dist. H 0

Experimental Soil

Collected A p r i l 9,1985

0.75

0.76

(mg/seed)

Amount of Crude Organic Extract

Table L Wheat Bioassay O f Soxhlet Extracts of Wheat Soil

3.6

3.5

(g/seed)

Amount of Soil Extracted

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

A

3.3 ± 0 . 5

C o n v - T i l l , A q . Ext. a

a

4.9 ± 1.3

N o - T i l l , A q . Ext.

{mm)

18.0 ± 1.7

2

Root Length

Control, Dist. H 0

Experimental Soil

a

2.9 ± 0 . 3

3.5 ± 0 . 5

5.7 ± 0 . 1

Shoot Length

a

92

83 49

39

Inhibition % Shoot Root

Collected June 10,1985

1.4

0.7*

(mg/seed)

Amount of Crude Organic Extract

Table Π. Wheat Bioassay O f Soxhlet Extracts of Wheat Soil

2.5

2.8

(g/seed)

Amount of S o i l Extracted

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Β

22.8 ± 1.7

18.2 ± 3 . 3

N o - T i l l , M e O H Ext.

Conv-Till,MeOHExt.

5.0 ± 0.9

6.2 ± 0.5 b

C

6.5 ± 0.7

Shoot Length

24

4 22

5

Inhibition % Root Shoot

c

b

a

*Lost about one-half of extract by accident. Significantly different from control at 99.9% level of confidence or better ft-test). Significantly different from control at 9 5 % level of confidence or better (i-test). Not significantly different from the control (t-test).

b

C

23.8 ± 4.6

2

Control, Dist. H 0 andMeOHExt.

(mm)

Root Length

Experimental Soil

1.4

0.7*

(mg/seed)

Amount of Crude Organic Extract

2.5

2.8

(g/seed)

Amount of Soil Extracted

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

18.7 ± 2 . 5

Conv-Till, A q . Ext.

b

16.6 ±1.9

N o - T i l l , A q . Ext. b

b

W

5.2 ± 0 . 6

5.2 ± 0 . 3

6.5 ± 0.7

Shoot Length

b

b

22

30 20

20

Inhibition % Root Shoot

Significantly different from control at 9 5 % level of confidence or better (t-test).

b

23.8 ± 4.6

2

Root Length

Control, Dist. H 0

Experimental Soil

Collected July 9,1985

0.58

0.45

(mg/seed)

Amount of Crude Organic Extract

Table ΠΙ. Wheat Bioassay O f Soxhlet Extract of Wheat Soil

2.0

1.8

(g/seed)

Amount of Soil Extracted

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987. Root Length (mm)

23.7 ± 1.7

3.9 ± 1.4a

No-Till Made at p H 5.9 (natural)

No-Till Made at p H 11

C

24.4± 0.7

2

Control, D i s t . H 0

Α-Steam Distillation, Extraction, Evaporation (2-kg sample)

Method of Obtaining Organics from Experimental Soil

2.7 ± 0 . 6

6.5 ± 0.3

7.2 ± 0.6

Shoot Length

a

C

85

10

10

10

Inhibition % Root Shoot

67 3.5

Continued on next page

67

Amount of Soil Extracted (g/seed)

2.9

Amount of Crude Organic Extract (mg/seed)

Table IV. Wheat Bioassay O f (A) Fractions O f Steam Distillates of Altus, O K and (B) Solvent Extracts of E l Reno, O K Wheat Soil

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Continued

Root Length

Significantly different from control at 9 5 % level of confidence or better (i-test).

0

Not significantly different from control ft-test).

11

0

13

e

e

17

39

Significantly different from control at 99.9% level of confidence or better (t-test).

7.2 ± 0.6

e

b

Inhibition % Root Shoot

b

e

7.3 ± 0.7

6.3 ± 0.7

7.2 ± 0.6

Shoot Length

a

2

23.4 ± 0.2

No-Till-Plot I EtOAc + CH C1

2

21.8 ± 5.2

No-Till-Plot Π A q . Ext.

b

e

16.2±2.5

No-Till-Plot I A q . Ext.

2

24.4 ± 0.7

(mm)

Control, Dist. H 0

B-Solvent E x t r a c t i o n and Evaporation (200-g s a m p l e )

Method of Obtaining Organics from Experimental Soil

Table IV.

2.5

2.5

Amount of Crude Organic Extract (mg/seed)

6.7

6.7

6.7

Amount of Soil Extracted (g/seed)

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

*A11 ethyl esters

Totals

Fatty acids Fatty acid esters Alcohols Aldehydes Ketones C - N and other N-contg, compounds S-contg. compounds Cl-contg. compounds Aromatics not otherwise included Aliphatics not otherwise included

39

8 0 1 3 1 4 1 2 7 11

Initial

75

0 0 3 8 2 17 2 0 23 16

20 3* 2 6 4 5 1 0 1 33

75

Basic

Acidic

Table V . Compound Groups Obtained From Soil B y Steam Distillation A s Identified B y C G C / M S / D A System

382

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

400 ' 500 ' 600 ' 760 860960' 100θ' ϊίββ' 1200" 1300' ΐ4θβ' 1*00 1600 Time,

Seconds

! b

' 2 5 ' W ' ' ''' 75^ i00 J

J,,

,,,ml

US • ' lié'" i?S " 260 " ÏH ' 256 " 1275 " 360

Figure 1. a) Reconstructed Part of the Total Ion Current Chromatogram of a No-Tillage Soil Extract (LKB-2091 C G C / M S / D A ) : Peaks Represent Compounds. Soil Sample: M a y 9,1985 b) Mass Spectrum o f a Phthalate Plasticizer Present i n the Soxhlet Soil Extract That Corresponds to Peak (750 s) Marked with the Cursor.

m/z

Figure 2. a) Reconstructed Part of the Total Ion Current Chromatogram of a Conventional-Tillage Soil Extract (LKB-2091 CGC/MS/DA): Peaks Represent Compounds. S o i l Sample: M a y 9,1985 b) Mass Spectrum of a Hydrocarbon Present i n the Soxhlet Soil Extract That Corresponds to Peak (1338 s) Marked with the Cursor.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

34.

WALLER ETAL.

Tillage in Wheat

Production

383

extract giving the peak at 749 s. We believe it to be an actual soil component and not an artifact of our work since our handling of the sample used all-glass equipment with Teflon stopcocks and closures without stopcock grease. We have not determined whether or not it is phytotoxic. Figures 2b is a mass spectrum of a hydrocarbon from the soil extract It is probably not an allelopathic compound. Allelopathic activity toward germinating wheat Was clearly demonstrated with extracts of Oklahoma soils. However, a convincing difference in activities in notill and conventional-till soil has not yet appeared. The total ion current chromatograms (Figures la and 2a) of the CGC/MS/DA illustrate the complexity of the soil extracts. Some of these compounds, if isolated, may serve as new effective biodegradable insecticides, herbicides, or fungicides. Acknowledgment The critical review of this manuscript by Otis C. Dermer is appreciated. This is Journal Article No. 4933 of the Oklahoma Agricultural Experiment Station, Oklahoma State University Stillwater Oklahoma 74078 We acknowledge with appreciation the asistance Research Laboratory, El Reno Literature Cited 1. McCalla, T. M.; Haskins, F. A. Bacteriol. Rev. 1964, 28, 181-207. 2. McCalla, T. M. In Biochemical Interactions Among Plants: Natl. Acad. Sci. U.S.A.: Washington, DC, 1971; pp. 39-43. 3. McCalla, T. M.; Norstadt, F. A. Agric. Environ. 1974, 1, 153-174. 4. Whittaker, R. N. "The Biochemical Ecology of Higher Plants. Chemical Ecology" Sondheimer; E.; Simeone, J. B., Eds.; Academic Press: New York, 1970; pp. 43-70. 5. Waller, G. R.; Nowacki, Ε. K. "Alkaloid Biology and Metabolism in Plants" Plenum Press: New York, 1978; 294 pp. 6. Rabotnov, Τ. Α., Soviet J. Ecol. 1981, 12, 127-131. 7. McCalla, T. M., Studies on Phytotoxic Residues from Soil, Microorganisms and Crop Residues, Lincoln, Nebraska, Nebr. Agric. Exp. Stn. Bull No. 2257, 1-8. 8. Elliott, L. F.; McCalla, T. M.; Waiss, Α., Jr. Crop Residue Management Systems: Am. Soc. Agron., Spec. Pub. No. 31, 1978; Chap. 7, pp. 131146. 9. Elliott, L. F.; Gilmour, C. M.; Lynch, J. M.; Titlemorre, D. Microbial-Plant Interactions: Am. Soc. Agron., Spec. Pub. No. 47, 1984; Chap. 1, pp. 124. 10. Putnam, A. R.; DeFrank, J. Crop Protect. 1983, 2, 173-181. 11. Lehle, F. R.; Putnam, A. R. Plant Physiol., 1982, 69, 1212-1216. 12. Schilling, D. G.; Liebl, R. Α.; Worsham, D. A. "The Chemistry of Allelopathy: Interaction Between Plants" Thompson, A. C., Ed.; ACS SYMPOSIUM SERIES No. 268, American Chemical Society: Washington, DC, 1985; pp. 243-71. 13. Waller, G. R.; Ritchey, C. R.; Krenzer, E. G., Jr.; Smith, G.; Hamming, M. 30th Ann. Conf. Am. Soc. Mass Spectrom. Allied Topics, San Antonio, TX, 1984; Abstract MPB14, pp. 144-145. 14. Waller, G. R.; McPherson, J. K.; Ritchey, C. R.; Krenzer, E. G., Jr. Smith, G.; Hamming, M. Abstracts of Papers, 190th American Chemical Society Meeting, Chicago, IL, September 9-14, 1985; AGFD 110. 15. McPherson, J. K.; Muller, C. H. Ecol. Monog. 1969, 39, 198 pp. RECEIVED June 16, 1986

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

C h a p t e r 35

Studies on the Fulvic and Humic Acids of Minnesota Peat Durga Kumari , S. A. Spigarelli , and George R. Waller 1,3

1

2

Center for Environmental Studies, Bemidji State University, Bemidji, M N 56601 Department of Biochemistry, Oklahoma State University, Stillwater, OK 74078

1

2

Fulvic and humic acids have been investigated with carbon-13 and proton nuclear magnetic resonance spectrometry, GC/MS, and IR spectroscopy. The fulvic and humic acids were found to be predominantly carboxylic and aromati with high proportio f d N-substituted carbo were also observed Humic substances constitute a very important class of natural products, especially fulvic and humic acids. These are present in soil, water, and coal throughout the world (1_) and participate in many significant agricultural, geochemical, and environmental processes (1-4). Examination of recent publications shows that there is an increasing interest in these materials by chemists, soil scientists, hydrologists, organic geochemists, and others in environmental sciences. Chemical investigations on humic substances have occupied the attention of scientists for more than 200 years (1,2); but relatively l i t t l e progress has been made in the elucidation of their chemical nature as compared to that of other natural products such as proteins, polynucleotides, and polysaccharides. However, this is not to imply that no progress has been made at all in the study of these substances. A vast amount of information has been accumulated on the chemical, physical, biological, geochemical and agricultural aspects of humic substances, but i t has not been possible to integrate this knowledge within a satisfactory conceptual framework of the nature of these substances. Although soil organic matter has been extensively studied by spectroscopic methods, very few investigators have studied peat as a source of humic substances (5-8). This paper presents carbon-13, proton nuclear magnetic resonance (^C-NMR and ^-H-NMR) spectra, 3

Current address: Department of Forest Products, University of Minnesota, St. Paul, MN 55108

0097-6156/87/0330-0384$06.00/0 © 1987 American Chemical Society

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p r o t o n n u c l e a r magnetic resonance (^c-NMR and ^H-NMR) s p e c t r a , GC/MS, and IR a b s o r p t i o n s p e c t r a of f u l v i c and humic a c i d s of M i n n e s o t a p e a t , and c o n c l u s i o n s about t h e n a t u r e of both t y p e s of acids.

Materials

and Methods

Materials. The p e a t , taken from S t . L o u i s C o u n t y , M i n n e s o t a , i s d e r i v e d from mosses of t h e genus Sphagnum. I t a l s o c o n t a i n s remains of some e r i c a c e o u s shrubs and few f o r b s and sedges. I t was a i r d r i e d t o a p p r o x i m a t e l y 15% m o i s t u r e , c o a r s e - s c r e e n e d t o remove woody p a r t i c l e s , and p u l v e r i z e d i n a l a b o r a t o r y W i l e y m i l l f i t t e d w i t h a 20-mesh s c r e e n . A l l s o l v e n t s were d i s t i l l e d b e f o r e u s e . P r e p a r a t i o n of F u l v i c and Humic A c i d s . Waxes, r e s i n s , and o t h e r s u b s t a n c e s s o l u b l e i n o r g a n i c s o l v e n t s were removed by s u c c e s s i v e e x t r a c t i o n s with petroleu acetate. These e x t r a c t i o n The r e s i d u a l peat was a i r - d r i e d t o remove s o l v e n t s . Wax and r e s i n f r e e peat (50 g) was s t i r r e d w i t h 1 l i t e r of 0.5N NaOH f o r 24 h. The i n s o l u b l e m a t e r i a l was removed by c e n t r i f u g i n g at 5000 rpm f o r 15 min i n a Beckman Model No J - 6 B c e n t r i f u g e . T h i s e x t r a c t i o n with a l k a l i was r e p e a t e d t w i c e more and t h e s o l u t i o n s of sodium s a l t s of a c i d s were c o l l e c t e d . Humic a c i d s were p r e c i p i t a t e d from the com­ b i n e d NaOH s o l u t i o n s by a d j u s t i n g the pH t o 1 w i t h 2N HC1 s l o w l y w i t h s t i r r i n g and t h e m i x t u r e was l e f t o v e r n i g h t . The p r e c i p i t a t e d humic a c i d s were c o l l e c t e d by f i l t r a t i o n through Whatman 1MM paper and washed w i t h 0.1N HC1. The f i l t r a t e s were e x t r a c t e d t h r e e t i m e s w i t h e t h y l a c e t a t e and the e x t r a c t s d r i e d over sodium s u l f a t e and e v a p o r a t e d , the r e s i d u e c o n s t i t u t i n g t h e f u l v i c a c i d s . Buth f u l v i c and humic a c i d s ( p r e c i p i t a t e s ) were a i r - d r i e d , and then d r i e d i n a vacuum d e s i c c a t o r over phosphorus p e n t o x i d e at room t e m p e r a t u r e . The y i e l d s were 3.5 g and 18.9 g r e s p e c t i v e l y . Samples of both f u l v i c and humic a c i d s were suspended i n methanol and m e t h y l a t e d w i t h diazomethane. Both *H and 13c s p e c t r a of t h e f r e e a c i d s were o b t a i n e d , at 299.94 MHz and 75.42 MHz r e s p e c t i v e l y , on a V a r i a n XL-300 s p e c t r o m e t e r h a v i n g a N i c o l e t TT-100 PET a c c e s s o r y . S p e c t r a were o b t a i n e d i n D2O, i n a 12-mm t u b e , w i t h d e u t e r a t e d TSP (sodium 3-(trimethylsilyl)propionate2 ^ , 3 , 3 - d 4 ) added as i n t e r n a l r e f e r e n c e . GC/MS of m e t h y l a t e d a c i d s was conducted on a H e w l e t t - P a c k a r d Model No 5995 GC/MS/DA system equipped w i t h a f u s e d s i l i c a c a p i l l a r y column (12 m χ .020 mm ID, Hewlett Packard) i n t e r n a l l y c o a t e d w i t h c r o s s l i n k e d methylene silicone. I n f r a r e d s p e c t r a were o b t a i n e d w i t h s o l i d samples d i s p e r s e d i n KBr p e l l e t s , by u s i n g a Beckman IR-33 s p e c t r o p h o t o ­ meter. The v a r i o u s a b s o r p t i o n peaks i n IR and NMR were i n t e r p r e t e d conventionally (9-10).

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Results The ^ C - N M R s p e c t r a of t h e f u l v i c and humic a c i d s are p r e s e n t e d i n F i g u r e s 1 and 2 r e s p e c t i v e l y . An i m p o r t a n t r e g i o n f o r a b s o r p t i o n was t h e 171-184 ppm r a n g e ; a p a r t i c u l a r l y sharp s i g n a l at 176 ppm i n f u l v i c a c i d i s c h a r a c t e r i s t i c of c a r b o x y l i c g r o u p s . The r e g i o n between 171 and 184 ppm i s r e p r e s e n t a t i v e of c a r b o n y l c a r b o n s . These can be c o n t a i n e d i n f r e e a c i d s , e s t e r s , s a l t s , amides, a l d e h y d e s , and k e t o n e s . The r e g i o n between 113 and 145 ppm r e p r e s e n t s a r o m a t i c , h e t e r o a r o m a t i c , and o l e f i n i c c o n s t i t u e n t s . A l t h o u g h o l e f i n i c carbons are g e n e r a l l y not major c o n s t i t u e n t s t o t h e s e a c i d s , a r o m a t i c ones are expected t o be major c o n t r i b u t o r s t o peaks i n t h i s r e g i o n . The l a r g e s t peaks are p r e s e n t i n t h e a r o m a t i c c a r b o n r e g i o n , which spans t h e r e g i o n from 119 t o 132 ppm i n both a c i d s ; t h u s they appear t o r e p r e s e n t p r e d o m i n a n t l y a r o m a t i c structures. The peaks at =58 ppm are most l i k e l y due t o methoxy g r o u p s , w h i l e t h o s e around 59-64 ppm would a l s o i n c l u d e c o n t r i b u ­ t i o n s from N - s u b s t i t u t e d show r e s o n a n c e s i n t h e 19-4 moieties. S i g n i f i c a n t d i f f e r e n c e s e x i s t between t h e two t y p e s of a c i d s , f u l v i c a c i d s p e c t r a showing more prominent r e s o n a n c e s . Both a c i d s , however, are r i c h i n carbon r e s o n a t i n g at - 3 0 ppm. These may i n c l u d e -CH2groups i n l o n g - c h a i n f a t t y a c i d s . The peak at 19 ppm i n f u l v i c a c i d ( F i g u r e 1) i s a s s i g n e d t o the t e r m i n a l methyl i n aliphatic chains. T h e r e i s l e s s a l i p h a t i c carbon than a r o m a t i c i n both a c i d s . The s p e c t r a , p a r t i c u l a r l y t h a t of f u l v i c a c i d ( F i g u r e 1 ) , a l s o show t h e p r e s e n c e of a c e t a l groups at 101 ppm. The 1-H-NMR spectrum of f u l v i c a c i d i s shown i n F i g u r e 3. The s i g n a l at 0.94 ppm i s t h e methyl *H r e s o n a n c e , w h i l e t h a t at 1.24 ppm i s due t o methylene p r o t o n s . The s t r o n g s i g n a l at - 1 . 9 7 ppm can a r i s e from t h e resonance of methylene groups to Ph, OH, - 0 - , OCOR, COPh, CHO, COOH, COOR, or groups. Although a l i p h a t i c c a r b o n s α t o c a r b o x y l , c a r b o n y l , or carboxamide groups y i e l d s i g n a l s t h a t are not s h i f t e d d o w n f i e l d of t h e a l i p h a t i c carbon r e g i o n , they produce a ^H-NMR s i g n a l at - 2 . 2 4 ppm t h a t i s c l e a r l y r e s o l v e d from t h a t of o t h e r a l i p h a t i c p r o t o n s , such as from amino acids. A wide r e g i o n between 2.99 ppm and 4 . 0 ppm shows t h e p r e s e n c e of methylene and methyl groups l i n k e d t o e l e c t r o n e g a t i v e atoms, v e r y l i k e l y oxygen, and/or the p r o t o n of a Ph-CH2 fragment t o c a r b o n y l and/or c a r b o x y l i c groups ( 1 2 - 1 3 ) . The broadness i s a t t r i b u t a b l e t o the l a r g e v a r i e t y of oxygen and n i t r o g e n compounds i n f u l v i c a c i d s , t h e most l i k e l y ones c o n t a i n i n g e t h e r , amino a c i d , and p e p t i d e g r o u p s . A r o m a t i c or c o n j u g a t e d o l e f i n i c p r o t o n s i g n a l s were p r e s e n t between 6.64 t o 8.54 ppm. The sharp s i g n a l at 8.54 ppm can be a t t r i b u t e d t o a r o m a t i c p r o t o n s . The prominent peak at a p p r o x i m a t e l y 5 ppm o b v i o u s l y shows t h e p r e s e n c e of OH p r o t o n s i n D 0.

COCH3,

β CONH2

Crl=CH2*

α

2

The 1 H - N M R spectrum of humic a c i d s i s shown i n F i g u r e 4. It i s s i m i l a r t o t h a t of f u l v i c a c i d s except t h e r e i s no sharp peak at 8.54 ppm c o r r e s p o n d i n g t o a r o m a t i c p r o t o n s . The peaks f o r a l i p h a t i c p r o t o n s were at 0 . 9 0 , 1.28, 1.34, 1.93, 1.94, 2 . 1 9 , 2 . 2 2 , 3.37 t o

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

^1

00

indicates (* Peat Minnesota 13

F i g u r e 1. C-NMR of F u l v i c A c i d of instrumental artifact)

I

500 m

i

2 500

2 000

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

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Peat

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

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Peat

3.83 ( b r o a d ) , and 3.97 ppm. O l e f i n i c and a r o m a t i c p r o t o n s were i n d i c a t e d by peaks at 6 . 0 5 , 6 . 2 4 , 6 . 3 0 , 6 . 6 5 , 7 . 2 0 - 7 . 5 2 and 7 . 7 2 - 7 . 7 7 ppm. The GC/MS s t u d y of m e t h y l a t e d f u l v i c a c i d showed t h e presence of ^ - h y d r o x y b e n z o i c a c i d (M * 166), v a n i l l i c a c i d ( M 196), a m e t h y l d i h y d r o x y b e n z o i c a c i d (NT "' 2 1 0 ) , coumaric a c i d (M * 192), s y r i n g i c a c i d (M * 2 2 6 ) , c a f f e i c a c i d ( M 2 2 2 ) , f e r u l i c a c i d (M * 2 2 2 ) , and s t e a r i c a c i d (M * 2 9 8 ) . The same compounds were p r e s e n t i n humic a c i d , a l o n g w i t h p a l m i t i c a c i d (M * 270). The p r e s e n c e of t h e s e compounds was c o n f i r m e d by T L C , u s i n g s t a n d a r d compounds. +

+e

4

+

+

+ e

+

+

+

A t y p i c a l IR spectrum of f u l v i c a c i d ( F i g u r e 5a) shows t h e following features: a wide and i n t e n s e band from s t r e t c h i n g of the Η-bonded OH groups at 3280 c m " , and a weak band near 2960 c m " due t o a l i p h a t i c C-H s t r e t c h i n g . A 1740 c m a b s o r p t i o n i s due t o C-0 s t r e t c h i n g i n c a r b o x y l i c and/or c a r b o n y l g r o u p s . The 1615 c m " band i s a t t r i b u t e d to v i b r a t i o n groups t h a t form hydroge with carbonyl groups. The a b s o r p t i o n at 1520 c m " i s p r o b a b l y due t o a r o m a t i c C=C s t r e t c h i n g , w h i l e the bands at 1445 c m " and 1380 c m " are l i k e l y due t o CH2 s c i s s o r d e f o r m a t i o n and CH3 symmetric deformation. Bands were a l s o e x h i b i t e d near 1215 c m " and 1025 c m " , l i k e l y due t o C-0 s t r e t c h i n g of e s t e r s and e t h e r s and OH deformation i n c a r b o x y l i c groups. 1

1

- 1

1

1

1

1

1

1

The IR spectrum of humic a c i d i s shown i n F i g u r e 5b. As f o r f u l v i c a c i d , i t shows broad bands i n t h e 3380 c m " r e g i o n (hydrogen-bonded OH g r o u p s ) , a weak band near 2920 c m " (aliphatic C-H s t r e t c h ) , a w e l l d e f i n e d peak near 1710 c m " (C=0 s t r e t c h i n g f r e q u e n c y of C00H; C=0 s t r e t c h of k e t o n i c c a r b o n y l ) , m e d i u m - s i z e d bands near 1650 c m " ( p r o b a b l y due t o a r o m a t i c C=C bonds c o n j u g a t e d w i t h C=0 and/or C00"), 1500 c m " ( a r o m a t i c C=C s t r e t c h i n g ) , and 1445 c m " (CH2 deformation). The bands at 1220 c m " and 1020 c m " are due t o C-0 s t r e t c h i n g of e s t e r s and e t h e r s and t o OH d e f o r m a t i o n of c a r b o x y l i c group; t h o s e at 825 c m " and 685 c m " might be a l i p h a t i c CH2 chain>or a r o m a t i c bands. 1

1

1

1

1

1

1

1

1

1

Di s c u s s i o n A number of i n v e s t i g a t o r s (14-20) have r e c o r d e d 1 3 -NMR s p e c t r a of humic a c i d and observed s i g n a l s from a r o m a t i c c a r b o n s , but o t h e r s (21-22) found no such s i g n a l s i n the *H and C - N M R s p e c t r u m . C

13

S i g n a l s i n t h e a r o m a t i c or c o n j u g a t e d o l e f i n i c r e g i o n between 6 and 8 . 5 ppm i n J-H-NMR and 101-145 ppm i n C - N M R are p r e s e n t . The presence of a r o m a t i c s was c o n f i r m e d by GC/MS of m e t h y l a t e d samples showing t h e p r e s e n c e of a number of p h e n o l i c a c i d s , and t h e p r e s e n c e of 1650, 1500, 1520 c m " bands i n the IR spectrum. Aromatic r e s o n a n c e s have been observed f o r both a c i d s and are predominant i n both C spectra. The c o n f i r m e d presence of p h e n o l i c a c i d s i s i n agreement w i t h t h e d i s c o v e r y of l i g n i n i n humic s u b s t a n c e s ( 2 3 ) . H resonances i n t h e 0 - 2 . 4 ppm r e g i o n c h a r a c t e r i z e a l i p h a t i c p r o t o n s , 13

1

1 3

1

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ACS In

Allelochemicals: Symposium

Series; Role

36£

AHisaaod QNV aannnDRiDV NI HIOH sivDM3HDOianv :

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while resonances at 0-50 ppm show the corresponding aliphatic carbons. The presence of fatty acids, palmitic and stearic, was shown by GC/MS and IR spectra. It is clear from the work of Schnitzer and Vendette (24) that fatty acids were present in arctic soil samples. Other spectra of soil humic acids obtained in NaOD solution (2J5) were quite informative, as both aromatic and aliphatic protons were shown present. No thorough study of fulvic and humic acids was conducted by Hatcher and associates (26-28) using *H and !3c-NMR. The experiments reported here show that there are more aromatic and less aliphatic components in fulvic and humic acids of Minnesota peat. Conclusion 13

Both *H and C NMR showed the presence of aromatic and aliphatic components. In 1C-NMR resonances at -58 ppm indicate the presence of many OCH syringic, vanillic, and presence of palmitic and stearic acids by GC/MS, IR, and NMR data. The fulvic and humic acids are predominantly made up of phenolic and fatty acid units. These are highly aromatic because lignin residues have been incorporated in the humification process. 3

Acknowledgments This work was supported by a grant from Minnesota Department of Natural Resources, St. Paul, Minnesota. Literature Cited 1. Schnitzer, M.; Khan, S.U. "Humic Substances in the Environment"; Marcel Dekker: New York, 1972. 2. Kononova, M. "Soil Organic Matter", 2nd ed.; Pergamon Press: Oxford, 1966. 3. Gjessing, E.T. "Physical and Chemical Characteristics of Aquatic Humus"; Ann Arbor Science: Ann Arbor, 1976. 4. Felbeck, G.T., Jr. In "Soil Biochemistry"; McLaren, A.D.; Skujins, J.J., Eds.; Marcel Dekker: New York, 1971; Vol. 2, pp. 36-59. 5. Arnold, C.L.; Lowy, Α.; Thiessen, R. Fuel 1935, 14, 107-112. 6. Lindberg, B.; Theander, O. Acta Chem. Scand. 1952, 6, 311-312. 7. Farmer, V.C.; Morrison, R.I. Sci. Proc. Roy. Soc. Dublin, Ser. A 1960, 1, 85-105. 8. Kosonogova, L.V.; Evdokimova, G.A.; Rakovski, V.E. Khim. Tverd. Topl. 1976, 10, (3), 97-101; Chem. Abstr. 86, 75698t. 9. Chamberlain, N.F. "The Practice of NMR Spectroscopy"; Plenum: New York, 1974. 10. Levy, G.C.; Lichter, R.L.; Nelson, G.L. "Carbon-13 Nuclear Magnetic Resonance Spectroscopy"; Wiley-Interscience, New York, 1980.

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11. Preston, C.M.; Mathur, S.P.; Rauthan, B.S. Soil Sci. 1981, 131, 344-352. 12. Neyroud, J.A.; Schnitzer, M. Can. J. Chem. 1974, 52, 4123-4133. 13. Sciacovelli, O.; Senesi, N., Solinas, V.; Testini, C. Soil Biol. Biochem. 1977, 9, 287-293. 14. Vila, F.J.G.; Lentz, H.; Lüdemann, H.D. Biochem. Biophys. Res. Commun. 1976, 72, 1063-1070. 15. Wilson, M.A.; Jones, A.J.; Williamson, B. Nature 1978, 276, 487-489. 16. Ogner, G. Soil Biol. Biochem. 1979, 11, 105-108. 17. Grant, D. Nature 1977, 270, 709-710. 18. Ruggiero, P.; Interesse, F.S.; Sciacovelli, O. Geochim. Cosmochim. Acta 1979, 43, 1771-1775. 19. Newman, R.H.; Tate, K.R.; Barron, P.F.; Wilson, M.A. J. Soil Sci. 1980, 31, 623-631. 20. Wilson, M.A.; Collins, P.J.; Tate, K.R. J. Soil Sci. 1983, 34, 297-304. 21. Schnitzer, M.; Barton 22. Wilson, M.A.; Goh, 23. Hurst, H.M.; Burges, N. In "Soil Biochemistry"; McLaren, A.D.; Peterson, G.H., Eds.; Marcel Dekker: New York, 1967. 24. Schnitzer, M.; Vendette, E. Can. J. Soil Sci. 1975, 55, 93-103. 25. Lentz, H.; Lüdemann, H.D.; Ziechmann, W. Geoderma 1977, 18, 325-328. 26. Hatcher, P.G.; Rowan, R.; Mattingly, M.A. Org. Geochem. 1980a, 2, 77-85. 27. Hatcher, P.G.; VanderHart, D.L.; Earl, W.L. Org. Geochem. 1980b, 2, 87-92. 28. Hatcher, P.G.; Macial, G.E.; Dennis, L.W. Org. Geochem. 1981, 3, 43-48. RECEIVED June 16,1986

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 36

Natural Plant Compounds Useful in Insect Control James A. Klocke NPI, University Research Park, Salt Lake City, UT 84108

Extracts of plants have been used as insecticides by humans since before the time of the Romans. Some of these extracts have yielded compounds useful as sources (e.g., pyrethrins, rotenoids, alkaloids), others as models (e.g., pyrethrins insecticides. Recen facilitate the isolation and identification of the bioactive constituents of plants should ensure the continued usefulness of plant compounds in commercial insect control, both as sources and models of new insect control agents and also as components in host plant resistance mechanisms. The focus in this paper will be on several classes of compounds, including limonoids, chromenes, ellagitannins, and methyl ketones, which were found to be components of the natural defenses of both wild and cultivated plants and which may be useful in commercial insect control. Insect resistance and environmental pollution due to the repeated application of persistent synthetic chemical insecticides have led to an increased interest in the discovery of new chemicals with which to control insect pests. Synthetic insecticides, including chlorinated hydrocarbons, organophosphorus esters, carbamates, and synthetic pyrethroids, will continue to contribute greatly to the increases in the world food production realized over the past few decades. The dollar benefit of these chemicals has been estimated at about $4 per $1 cost (I). Nevertheless, the repeated and continuous annual use in the United States of almost 400 million pounds of these chemicals, predominantly in the mass agricultural insecticide market (2), has become problematic. Many key species of insect pests have become resistant to these chemicals, while a number of secondary species now thrive due to the decimation of their natural enemies by these nonspecific neurotoxic insecticides. Additionally, these compounds sometimes persist in the environment as toxic residues, well beyond the time of their intended use. New chemicals are therefore needed which are not only effective pest 0097-6156/87/0330-0396$06.00/0 © 1987 American Chemical Society

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36.

KLOCKE

Natural

Plant Compounds

Useful in Insect

Control

397

control agents, but which are safe, s e l e c t i v e , biodegradable, environmentally acceptable, economically v i a b l e , and suitable for use in programs of integrated pest management (3). One approach to the discovery of new i n s e c t i c i d e s which f u l f i l l the c r i t e r i a of e f f i c a c y , safety, s e l e c t i v i t y , etc., i s through the study of the natural chemical defenses of plants. Extracts of plants have been used as i n s e c t i c i d e s by humans since before the time of the ancient Romans, a practice that continues today with many of the 2000 species of plants known to have i n s e c t i c i d a l properties (4-5). The use of " i n s e c t i c i d a l plants i s e s p e c i a l l y prevalent among subsistence farmers since plants grown l o c a l l y are cheaper, and sometimes more accessible, than synthetic chemical p e s t i c i d e s . Commercially, however, only a few of these plants, including those containing pyrethrins, rotenoids, and a l k a l o i d s , have been used to any extent i n the United States as sources of i n s e c t i c i d e s (6-8). The most economically important group of natural plant i n s e c t i cides are the pyrethrins a group of s i x c l o s e l y related esters extracted from pyrethru heads (Figure 1). Pyrethru at least the early 1800*s i n Persia and Yugoslavia. By 1828 pyrethrum was being processed for commercial insect control, and by 1939 imports of pyrethrum into the United States reached a peak of 13.5 m i l l i o n pounds. Use of the natural product declined i n the early 1950's because of the advent of synthetic pyrethroid analogs (for example, a l l e t h r i n s ) , which were both more stable and more e f f e c t i v e i n the f i e l d . The present worldwide demand for pyrethrum flowers remains i n excess of 25,000 tons annually and i s s a t i s f i e d by the estimated 150 m i l l i o n flowers s t i l l hand-harvested d a i l y , predominantly i n natural stands and c u l t i v a t e d f i e l d s i n Kenya, Tanzania, and Ecuador (9). Rotenone and the rotenoids (Figure 2) have long been used as insecticides and p i s c i c i d e s ( f i s h poisons). By the early 1950's more than 7 m i l l i o n pounds of Leguminosae roots (Derris, Lonchocarpus, and Tephrosia spp.) containing these i n s e c t i c i d e s were imported annually into the United States. In 1972, about 1.5 m i l l i o n pounds of the roots were used i n the United States for pest control i n the home and garden markets and to control ectoparasites on animals (10). Among the most important of the natural alkaloids used i n insect control have been nicotine and the related compound nornicotine (Figure 3). The use of these i n s e c t i c i d a l alkaloids dates back to the 1600's and grew to 5 m i l l i o n pounds by the mid-1900's. Since then, the annual worldwide production of nicotine has dropped to about 1,250,000 pounds of nicotine sulfate and 150,000 pounds of nicotine a l k a l o i d because of the high cost of production, disagreeable odor, extreme t o x i c i t y to mammals, and l i m i t e d i n s e c t i c i d a l a c t i v i t y (10-11). The s t r u c t u r a l l y related compounds anabasine (neonicotine) (Figure 3) i s currently i n commercial use i n the Soviet Union (J5). Other less important i n s e c t i c i d a l alkaloids include veratrine [a mixture of alkaloids (cevadine, v e r a t r i d i n e , and s a b a c i l l i n e ) ] and ryanodine. Physostigmine (Figure 4), another alkal o i d that i s i s o l a t e d from the calabar bean (Physostigma venenosum), served as a model compound for the development of the carbamate insecticides (12-13)· 11

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

R=CH

R

3

'=

CH =

CH

2

Pyrethrin 1 Pyrethrin II

R=CH OCO

R'=

C H — C H

R =

R'=

CH

3

Cinerin 1

R = CH OCO

R = CH

3

Cinerin II

R=

R

3

CH

3

3

CH

3

R = CH OCO 3

2

'= C H

2

-CH

3

Jasmolin

1

R'= C H

2

-CH

3

Jasmolin

II

Figure 1. Structures Pyrethrum (Chrysanthemu

)

Figure 2 . Structure of Rotenone, an Insecticide and P i s c i c i d e Isolated from Leguminosae Roots

Ν R =CH R=H

3

NICOTINE NORNICOTINE

ANABASINE

Figure 3. Structures of Some Plant Alkaloids Used i n Insect Control

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Plant Compounds

Useful in Insect

Control

399

Insect growth regulators, including analogs and antagonists of endogenous hormones, have also been i d e n t i f i e d i n plants. Prominent among these are the analogs of two insect hormones (juvenile hormone and molting hormone) and the antagonist for juvenile hormone. Analogs of juvenile hormones found i n plants include the juvocimenes in Ocimum basilicum, juvabione i n Abies balsamea, and farnesol i n many plant o i l s (14). These natural plant products have never been used commercially as a source of i n s e c t i c i d e s , but they have served as model compounds for the development of synthetic juvenile hormone analogs such as kinoprene and methoprene (Figure 5). Chemicals s t r u c t u r a l l y similar or i d e n t i c a l to the insect molting hormone (ecdysterone) have been found i n many plants, especially in ferns and yews (15). One example i s ponasterone A, which d i f f e r s s t r u c t u r a l l y from the insect molting hormone only i n the absence of a hydroxyl group at C-25 (Figure 6). Ponasterone A caused severe disruption of the f i n a l stages of molting (termed ecdysis) when fed at 2 ppm i n a r t i f i c i a l diet to pink bollworm (Pectinophora gossypiella) larvae (16) _P. gossypiella fed 2 pp insect possessed three head capsules because i t underwent two f a i l e d molting cycles before death. Even though feeding became impossible after the f i r s t inhibited ecdysis (because the adhering second head capsule covered the mouth parts) the larva produced a third head capsule before i t s death. While the above observation i s interesting and could possibly have some implications for the control of the pink bollworm, the complexity of the steroid nucleus of ponasterone A and other molting hormone analogs and their weak i n s e c t i c i d a l e f f e c t when applied t o p i c a l l y or administered o r a l l y to most species of economically important insects may preclude their commercialization. The only commercial use of the molting hormone analogs thus f a r has been i n the s e r i c u l t u r a l industry f o r the synchronization of cocoon spinning of silkworm colonies (17)· Juvenile hormone antagonists were f i r s t isolated from the bedding plant Ageratum houstonianum (Asteraceae) (18). The active constituents, termed precocenes I and I I , were shown to be chromenes (Figure 8)· Although these compounds are highly active against several species of insects (e.g., the large milkweed bug, Oncopeltus f a s c i a t u s ) , the precocenes have been found to be e f f e c t i v e on r e l a t i v e l y few economically important species of insects, and their potential for commercialization may therefore be l i m i t e d . Other chromenes, which d i f f e r s t r u c t u r a l l y only i n the moieties attached to C-6 and C-7 (Figure 8), have been isolated from other species i n the Asteraceae, including xerophytic Encelia species (19) and Hemizonia f i t c h i i (20). The H. f i t c h i i chromenes, including encecalin, eupatoriochromene, and 6-vinyl-7-methoxy-2,2dimethylchromene (Figure 8), were moderately toxic to house mosquito (Culex pipiens) larvae, with 6-vinyl-7-methoxy-2,2-dimethylchromene being the most active, and to large milkweed bug (0. fasciatus) nymphs, with encecalin being the most active (Tables I and I I , r e s p e c t i v e l y ) . Although these compounds are i n s e c t i c i d a l , they showed no antijuvenile hormone a c t i v i t y . Apparently, the presence of a v i n y l or a methyl ketone moiety, such as found i n the Hemizonia chromenes, rather than a methoxy substituent, such as found i n the

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Ο II

Figure 4 . Structure of Physostigmine, an I n s e c t i c i d a l A l k a l o i d Isolated from Physostigma venenosum as a Model Compound f o r the Synthetic Carbamate Insecticide

Juvenile Hormone 3 (JH 3)

Figure 5. Structures of the Insect Juvenile Hormone 3 and a Commercially Available Analog, Methoprene

OH

ECDYSTERONE PONASTERONE A

Figure 6. Stereostructures of the Insect Molting Hormone, Ecdysterone, and a Plant Analog, Ponasterone A

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

36.

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Natural

Plant Compounds

Useful in Insect

Control

401

Figure 7. Electron Micrograph I l l u s t r a t i n g Ecdysis I n h i b i t i o n of a Larva of the Pink Bollworm, Pectinophora gossypiella after Ingestion of 2 ppm Ponasterone A i n an A r t i f i c i a l Diet (Magnification χ 100)

Jijl

Jl

2j>

C H

3

R_

2

Precocene 1 Precocene

II

Η OCH

3

Desmethoxyencecalin

COCH

Eupatoriochromene

COCH

Encecalin

COCH

6-Vinyl-7-methoxy2,2-dimethylchromene

CH = CH

OCH

3

OCH

3

Η

3

OH

3

OCH

3

2

3

OCH

3

Figure 8, Structures of some I n s e c t i c i d a l 2,2-Dimethylchromenes Isolated from Plant Species i n the Asteraceae

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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precocenes, r e s u l t s i n a loss of a n t i j u v e n i l e hormone a c t i v i t y . In f a c t , Bowers (21-22) found that alkoxy substitution of the chromene aromatic r i n g i n the C-6 and e s p e c i a l l y the C-7 positions was necessary for a n t i j u v e n i l e hormone a c t i v i t y . Although assays with other insects should be conducted, i t does not seem from an economic standpoint that the Encelia and Hemizonia chromenes themselves are of s u f f i c i e n t potency to warrant adaptation into pest management strategies. However, the r e l a t i v e ease of extraction of the H. f i t c h i i chromenes, as exemplified by their abundance (63%) i n the v o l a t i l e o i l (steam d i s t i l l a t e ) f r a c t i o n (Table I I I ) , coupled with the a v a i l a b i l i t y of the xerophytic Hemizonia plant material, make these compounds useful as models for new synthetic or semisynthetic i n s e c t i c i d e s . The a v a i l a b i l i t y and b i o l o g i c a l a c t i v i t y of xerophytic plants, such as H. f i t c h i i , make them a promising area of research for the discovery of new i n s e c t i c i d e s . Xerophytic plants are available on a large scale from marginal regions that generally cannot be used economically for food productio xerophytic plants has bee i n s e c t i c i d e s (19, 25) and as insect growth i n h i b i t o r s (26). E l l a g i c acid, a phenolic dilactone (Figure 9), was i s o l a t e d and spectrally i d e n t i f i e d as an insect growth i n h i b i t o r from the methanolic extracts of f i v e species of xerophytic plants, including Geranium viscosissimum var. viscosissimum, Erodium cicutarium, Tamarix chinensis, Quercus gambelii, and Cistus v i l l o s u s (26). Against the polyphagous herbivore, H e l i o t h i s virescens (tobacco budworm), the E C 5 0 (the e f f e c t i v e concentration for 50% growth i n h i b i t i o n ) was found to be 147 ppm or 0.49 mmol/kg diet wet weight (Table IV), well below the 0.77%-1.50% unbound e l l a g i c acid (dry weight basis) found i n the hot methanolic extracts of the a e r i a l parts of the f i v e plant species (26). Although the large amounts of unbound e l l a g i c acid could explain, for the most part, the a c t i v i t y of the methanol extracts against _H. virescens ( E C 5 0 = 0.0147%), i n the fresh plant very l i t t l e e l l a g i c acid i s found i n the unbound form, but i s instead bound into more complex molecules such as the ellagitannins (27-28). For instance, i n a number of species of Geranium, e l l a g i c acid i s predominantly bound i n an e l l a g i t a n n i n , geraniin (and a l l i e d compounds) (Figure 1 0 ) (29-30). A milder extraction methodology, that i s , one excluding heat, revealed this also to be the case for _G. viscosissimum var. viscosissimum. Geraniin was e a s i l y detected i n tissue which had been extracted at room temperature, while no geraniin was detected i n heated samples of s i m i l a r t i s s u e . Apparently, the large amounts of free (unbound) e l l a g i c acid detected, at least for £. viscosissimum var. viscosissimum, were a r t i f a c t s of the extraction methodology employed. Since e l l a g i c acid i s probably not encountered free i n large amounts i n fresh plant tissues, but rather as one or more bound forms, we i s o l a t e d and bioassayed geraniin. On a mmol/kg diet basis, geraniin was almost twice as active as a growth i n h i b i t o r of H. virescens larvae than was e l l a g i c acid ( E C 5 0 = 0.26 and 0.49, respectively) (Table IV). An increased a c t i v i t y of geraniin might be expected since complete hydrolysis of the compound would y i e l d , i n addition to e l l a g i c acid, equimolar amounts of b r e v i f o l i n (or other phenolic lactones), and g a l l i c acid (Figure 11) (31-32).

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Plant Compounds

Useful in Insect

Control

403

Table I. Bioassay of Hemizonia f i t c h i i Chromenes with Larvae of Culex pipens Compound Tested

Instar Tested

LC5o(ppm)

6-Vinyl-7-methoxy2,2-dimethylchromene Encecalin

1st 3rd 1st 3rd 1st 3rd

1.8 3.8 3.0 6.6 6.4 13.0

Eupatoriochromene

Table I I .

Topical Bioassay of Hemizonia f i t c h i i Chromenes with Nymphs of Oncopeltus f a s c i a t u s ^

Compound Tested

Instar Tested

LD (yg)

2nd 3rd 2nd 3rd 2nd 3rd

10 11 23 35 b c

Encecalin 6-Vinyl-7-methoxy2,2-dimethylchromene Eupatoriochromene

50

Assay period was 8-10 days, s u f f i c i e n t time for control insects to ^undergo two molts. No e f f e c t was observed with 100 yg. No e f f e c t was observed with 200 yg.

Table I I I . Constituents of V o l a t i l e O i l of Hemizonia f i t c h i i Compound Class

Peak Area (% of total)

V o l a t i l e f a t t y acids Monoterpenes Sesquiterpenes Chromenes Miscellaneous constituents Alkanes

0.56 29.04 2.50 62.70 3.10 1.70

a

b

The monoterpene f r a c t i o n was predominantly made up of 1,8-cineole. The chromene f r a c t i o n was predominantly made up of encecalin and eup a t ο r io chr omene.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND

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Figure 9. Structure of E l l a g i c Acid, an Insect Growth Inhibitor Isolated from the Extracts of a Number of Xerophytic Plant Species

Figure 10. Structure of Geraniin, an Insect Growth Inhibitor Isolated from Geranium Species

Table IV.

Growth-Inhibitory A c t i v i t y of Some Bioactive Constituents Derived from Geranium viscosissimum var. viscosissimum Fed i n an A r t i f i c i a l Diet to F i r s t - I n s t a r Larvae of H e l i o t h i s virescens EC a (mmol/kg diet) 50

Test Compound E l l a g i c acid Geraniin G a l l i c acid

0.49 0.26 7.40

EC (ppm)

a

5 0

147 250 1262

Confidence limits

b

Slope

103- 140 195- 320 931-3843

b

1.78 1.68 2.03

EC50 i s the e f f e c t i v e concentration of additive necessary to reduce l a r v a l growth to 50% of the control values. Units are given i n both mmol/kg diet wet weight and ppm of diet wet weight. Confidence l i m i t s and slope were determined using the method of L i t c h f i e l d and Wilcoxon (66).

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Natural Plant Compounds

Useful in Insect

405

Control

A l t h o u g h t h e b i o l o g i c a l a c t i v i t y of b r e v i f o l i n as an i n s e c t growth i n h i b i t o r i s unknown, g a l l i c a c i d e x h i b i t e d some g r o w t h - i n h i b i t o r y a c t i v i t y a g a i n s t H . v i r e s c e n s l a r v a e (EC50 = 7.40 mmol/kg d i e t ) (Table IV). I n l i g h t o f the s u s c e p t i b i l i t y of a t l e a s t one s p e c i e s o f i n s e c t ( i . e . , Iî. v i r e s c e n s ) t o i n g e s t i o n of the f r e e and bound forms of e l l a g i c a c i d , c o u p l e d w i t h t h e l a r g e amounts o f such forms found i n a number of p l a n t s p e c i e s , e l l a g i c a c i d may be a c o n s t i t u e n t o f the c h e m i c a l d e f e n s e s of c e r t a i n p l a n t s a g a i n s t some i n s e c t s . As s u c h , i t c o u l d become a c a n d i d a t e model f o r compound d e s i g n , i . e . , f o r t h e s y n t h e s i s and s e m i s y n t h e s i s o f new i n s e c t i c i d e s , e s p e c i a l l y i f i t s water s o l u b i l i t y and p o t e n c y can be enhanced. Fortunately, ellagic a c i d i s n e i t h e r mutagenic (33) n o r a c u t e l y t o x i c i n t e s t s w i t h e x p e r i m e n t a l a n i m a l s (34-35) and humans ( 3 6 ) . Of c o u r s e , more a c t i v e compounds m o d e l l e d a f t e r e l l a g i c a c i d would a l s o have to be t e s t e d f o r m u t a g e n i c i t y , mammalian t o x i c i t y , e t c . Perhaps t h e most p r o m i s i n g o f the p l a n t s p e c i e s y i e l d i n g i n s e c t i c i d a l compounds a r A z a d i r a c h t a i n d i c a (neem s p e c i e s y i e l d the p o t e n t i n s e c t i c i d e a z a d i r a c h t i n , a h i g h l y d é r i v â t i z e d t e t r a n o r t r i t e r p e n o i d o f the l i m o n o i d t y p e . Although the s k e l e t a l s t r u c t u r e and s t e r e o c h e m i s t r y o f a z a d i r a c h t i n have not been t o t a l l y r e s o l v e d ( F i g u r e 12) ( 3 7 ) , the p o t e n t and s p e c i f i c e f f e c t s o f t h i s n a t u r a l p r o d u c t have w a r r a n t e d i t s e v a l u a t i o n as a s o u r c e o f (38) and a model f o r (39) new c o m m e r c i a l i n s e c t c o n t r o l a g e n t s . A z a d i r a c h t i n has a v e r y p r o m i s i n g p o t e n t i a l because of i t s p o t e n c y (comparable to t h e most p o t e n t c o n v e n t i o n a l s y n t h e t i c i n s e c t i c i d e s ) , s p e c i f i c i t y ( a f f e c t i n g b e h a v i o r a l and b i o c h e m i c a l and d e v e l o p m e n t a l p r o c e s s e s p e c u l i a r to i n s e c t s ) , n o n - m u t a g e n i c i t y ( i n the Ames t e s t ) ( 4 0 ) , and s y s t e m i c a c t i v i t y i n plants (being t r a n s l o c a t e d throughout t h e p l a n t f o l l o w i n g a b s o r p t i o n through the r o o t s y s t e m ) . However, the accumulated i n f o r m a t i o n on a z a d i r a c h t i n , w h i l e p r o m i s i n g , i s p r e s e n t l y much l e s s t h a n t h a t needed f o r i n s e c t i c i d a l p r o d u c t commercialization (41). The mode o f a c t i 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 ( S A R ' s ) , f o r m u l a t i o n , and m e t a b o l i s m o f a z a d i r a c h t i n a r e not y e t w e l l u n d e r s t o o d . Furthermore, formulation studies are r e q u i r e d p r i o r to p r o d u c t development and c o m m e r c i a l i z a t i o n . C o n s e q u e n t l y , f u r t h e r i n v e s t i g a t i o n s a r e needed b e f o r e t h e f u l l p o t e n t i a l o f a z a d i r a c h t i n as an i n s e c t c o n t r o l agent o r i n s e c t i c i d e can be r e a l i z e d . I n o r d e r t o conduct s u c h i n v e s t i g a t i o n s , l a r g e amounts o f p u r i f i e d a z a d i r a c h t i n are needed. I s o l a t i o n schemes p r e v i o u s l y r e p o r t e d f o r a z a d i r a c h t i n have i n c l u d e d s o l v e n t e x t r a c t i o n and p a r t i t i o n f o l l o w e d by chromatography, e s p e c i a l l y open column and t h i n l a y e r chromatography (TLC) (42-44) a n d , more r e c e n t l y , h i g h performance l i q u i d chromatography (HPLC) ( 4 5 - 4 6 ) . While p r e p a r a t i v e HPLC i s an e x c e l l e n t t e c h n i q u e f o r g e n e r a t i n g a z a d i r a c h t i n of h i g h p u r i t y , a g e n e r a l method u t i l i z i n g the r a p i d and i n e x p e n s i v e t e c h n i q u e o f f l a s h chromatography (48) f o r the e f f i c i e n t p r e l i m i n a r y p u r i f i c a t i o n o f samples t h a t w i l l not r e a d i l y c o n t a m i n a t e e x p e n s i v e HPLC columns was d e v e l o p e d ( 4 7 ) . F u r t h e r p u r i f i c a t i o n ( g r e a t e r than 99%) was a c c o m p l i s h e d w i t h p r e p a r a t i v e HPLC. A z a d i r a c h t i n has s e v e r a l e f f e c t s on a number o f important species of i n s e c t p e s t s , i n c l u d i n g feeding

economically deterrency,

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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growth i n h i b i t i o n , and ecdysis i n h i b i t i o n (49-50). Azadirachtin i s a p a r t i c u l a r l y potent feeding deterrent (or antifeedant) against the f a l l armyworm, Spodoptera frugiperda, having a PC95 (the concen­ t r a t i o n resulting i n 95% "protection" of treated disks) i n a cotton leaf disk assay of 0.1 yg/1.0 cm^ leaf disk (Table V). Against the corn earworm, H e l i o t h i s zea, the PC95 was 6.2 yg/1.0 cm^, which was s t i l l a potency greater than that found with other b i o l o g i c a l l y active natural plant compounds such as quassinoids (51) and other limonoids (52-53)· The growth-inhibitory a c t i v i t y of azadirachtin fed i n a r t i f i c i a l diet to three species of a g r i c u l t u r a l pests, _P. gossypiella, II. zea, and S_. frugiperda, was compared to the a c t i v i t y of a number of limonoids isolated from plants i n the Meliaceae and the Rutaceae (Table VI). After azadirachtin, the most active limonoid was cedrelone (Figure 13). Cedrelone was unique among the compounds tested i n Table VI since i t was the only limonoid, besides azadirachtin, to cause an i n h i b i t i o n i n ecdysis ( L C 5 0 = 150 ppm) when fed to pink bollwor Nakatani (55) observe the 7-OH group rendered some Meliaceae limonoids ( i . e . , t r i c h i l i n s ) inactive as feeding deterrents. A similar phenomenon was observed when the growth-inhibitory a c t i v i t i e s of 7-deacetylgedunin, gedunin, and 7-ketogedunin, but not when the a c t i v i t i e s of azadiradione and deacetylazadiradione (Table VI and Figure 13), were compared. When comparing the a c t i v i t i e s of several Rutaceae limonoids, the most potent seem to have an a, ^-unsaturated 7-membered lactone (as i n obacunone) or at least have the p o t e n t i a l to form i t (as i n nomilin( (Figure 14). Apparently, at least i n v i t r o , nomilin eliminates i t s Α-ring a c e t y l group (forming obacunone) much more r e a d i l y than deacetylnomilin eliminates i t s Α-ring hydroxyl group (56). This fact might explain the much lowered growth i n h i b i t o r y a c t i v i t y of deacetylnomilin when compared to nomilin or obacunone (Table VI). L i t t l e i s yet known concerning the SAR's of b i o l o g i c a l l y active limonoids. However, many of the most potent of the growth-inhibitory limonoids from the Meliaceae and Rutaceae have one or more a l k y l a t i n g centers, including one almost invariably i n the Α-ring (e.g., a, 13-unsaturated ketone or lactone). Cedrelone has 2 a l k y l a t i n g centers, one i n the A-ring (α,β-unsaturated ketone) and one i n the B-ring (diosphenol) (Figure 13). Another example i s 6-O-acetylnimbandiol, which has an a,β-unsaturated ketone i n the Α-ring (Figure 13). 6-0-Acetylnimbandiol was i n s e c t i d i c a l ( E I 5 0 =21 ppm) when fed to the larvae of H. virescens, while the s t r u c t u r a l l y related salannin, which lacks the Α-ring ketone (Figure 13), was not (Table VII) (57). Nakanishi (58) has pointed out that natural products with e l e c t r o p h i l i c moieties tend to be cytotoxic and insect antifeedant. Possibly the growth-inhibitory a c t i v i t y of the limonoids may also be attributed to a nonspecific e l e c t r o p h i l i c e f f e c t . In addition to i t s a c t i v i t i e s of feeding deterrency and growth i n h i b i t i o n , azadirachtin disrupts the molting process of insects by i n h i b i t i n g ecdysis or the shedding of the " o l d " skin. The affected insects then die i n the pharate condition, unable to feed or locomote, The ecdysis-inhibitory a c t i v i t y of azadirachtin on three species of a g r i c u l t u r a l pests i s shown i n Table VIII. Although i t i s not known

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Plant Compounds

Useful in Insect

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OH Brevifolin

Gallic acid

Figure 11. Structures of Two Hydrolysis Products of Geraniin: B r e v i f o l i n and G a l l i c Acid

Figure 12. Upper: Azadirachtin (43). Azadirachtin (37)

Table V.

Previously Accepted Stereostructure of Lower: Recently Proposed Stereostructure of

Cotton Leaf Disk "Choice" Bioassay of Azadirachtin with Third-Instar Larvae of Two Species of A g r i c u l t u r a l Insect Pests Species

PC * (yg/leaf disk)

Spodoptera frugiperda H e l i o t h i s zea

0.1 6.2

95

*PC95 values are concentrationsjof azadirachtin r e s u l t i n g i n 95% "protection" of treated disks when compared to untreated disks.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

408

Table VI.

Insect Growth-Inhibitory A c t i v i t y of Some Meliaceae and Rutaceae Limonoids. Values are the Dietary Concentrations for 50% Growth I n h i b i t i o n (EC50)

Limonoid Azadirachtin Cedrelone Anthotheol Sendanin Methyl angolensate Nkolbisonin 7-Deacetylgedunin Gedunin Azadiradione 7-Ketogedunin Nomilin Obacunone Evodoulone 7-Deacetylproceranone Tecleanine Limonin 7-Deacetylazadiradione Deacetylnomilin

Pectinophora gossypiella 0.4 3 8 9 15 20 22 32 42 51

-96 175 210

290 950

Spodopter frugiperda 0.4 2 3 11 40 65 60 47 130 800 72 70 120 350 320 756 5000

zea 0.7 8 24 45 60 71 165 50 250 900 95 97 80 740

-

900 3500

*

*No a c t i v i t y was found with 2000 ppm.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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KLOCKE

Natural Plant Compounds Useful in Insect Control

409

CEDRELONE

R=Ac AZADIRADIONE R=H 7-DEACETYLAZADIRADIONE

6-O-ACETYLNIMBANDIOL

SALANNIN

Figure 13 Structures of Some Insect Growth Inhibitory Limonoids Isolated from Plant Species i n the Meliaceae 0

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

410

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Ο

R=Ac NOMILI R=H

OBACUNONE

1-DEACETYLNOMILIN

Figure 14. Structures of Some Insect Growth Inhibitory Limonoids Isolated from Plant Species i n the Rutaceae

Table VII.

Ecdysis-and Growth-Inhibitory A c t i v i t i e s of Some Neem O i l Limonoids Fed i n an A r t i f i c i a l Diet to F i r s t - I n s t a r Larvae of H e l i o t h i s virescens b

a

Test Compound Deacetylazadirachtinol

El50 (ppm)

EC (ppm) 5 Q

0.80 0.17

Azadirachtin

0.80 0.07

6-0-Acetylnimbandiol

21.0 4.4

Salannin

95% Confidence Limits 0.660.120.460.0515.2 2.8 -

0.97 0.23 1.39 0.10 29.0 7.0

c 170

138

-210

EI50 values are the concentrations r e s u l t i n g i n 50% ecdysis inhibition. EC50 values are the e f f e c t i v e concentrations resulting i n 50% growth inhibition. No mortality was observed at 400 ppm.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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how a z a d i r a c h t i n i n h i b i t s e c d y s i s , i t a p p a r e n t l y does n o t do so by i n h i b i t i n g c h i t i n synthetase (49). P o s s i b l y the d i s r u p t i o n o f the m o l t i n g hormone t i t r e i n s e v e r a l s p e c i e s o f i n s e c t s t o which a z a d i r a c h t i n was a d m i n i s t e r e d (59-61) p r e v e n t e d e c d y s i s . Ultimately, the d i s r u p t e d hormone t i t r e and the i n h i b i t e d e c d y s i s may have been caused by an i n t e r f e r e n c e w i t h t h e n e u r o e n d o c r i n e s y s t e m , i n v o l v i n g the p r o t h o r a c i c o t r o p i c and a l l a t o t r o p i c hormones w h i c h c o n t r o l t h e b l o o d t i t r e s o f m o l t i n g hormone and j u v e n i l e hormone, r e s p e c t i v e l y (45). A n o t h e r l i m o n o i d i s o l a t e d from neem seeds and d e t e r m i n e d t o be as p o t e n t as a z a d i r a c h t i n as an e c d y s i s i n h i b i t o r has been i d e n t i f i e d as 3 - d e a c e t y l a z a d i r a c h t i n o l ( F i g u r e 15) ( 5 7 ) . B o t h compounds were l e t h a l t o 50% o f the t r e a t e d H . v i r e s c e n s l a r v a e (EI50) a t 0 . 8 ppm i n a r t i f i c i a l d i e t (Table V I I ) . S t r u c t u r a l l y , t h e r e a r e two d i f f e r e n c e s between the compounds. In 3 - d e a c e t y l a z a d i r a c h t i n o l , t h e C - l l - O - C - 1 3 e t h e r l i n k a g e o f a z a d i r a c h t i n i s r e d u c t i v e l y c l e a v e d a t the 11 p o s i t i o n and t h e a c e t o x y l group a t C - 3 i s h y d r o l y z e d t o a h y d r o x y l group. The i d e n t i f i c a t i o n o f p l a n t s can be e x p l o i t e d by u t i l i z i n g t h e b i o l o g i c a l l y a c t i v e p l a n t c h e m i c a l c o n s t i t u e n t s as s o u r c e s and/or models o f new i n s e c t c o n t r o l agents. Examples h e r e i n c l u d e p y r e t h r i n s from Chry s an themum s p p . , chromenes from E n c e l i a and H . f i t c h i i , e l l a g i c a c i d and g e r a n i i n from CÎ. v i s c o s i s s i m u m v a r . v i s c o s i s s i m u m , and l i m o n o i d s from A . i n d i c a . A l t e r n a t i v e l y , a n a t u r a l c h e m i c a l d e f e n s e can be enhanced i n a p l a n t , such as i n an e c o n o m i c a l l y i m p o r t a n t c r o p p l a n t , i n o r d e r to a f f o r d endogenous p r o t e c t i o n from h e r b i v o r y . Such p r o t e c t i o n i s known as h o s t p l a n t r e s i s t a n c e . An example i s the w i l d tomato s p e c i e s L y c o p e r s i c o n h i r s u t u m f. g l a b r a t u m , w h i c h has been r e p o r t e d t o be r e s i s t a n t t o a wide range o f a r t h r o p o d p e s t s o f t h e c u l t i v a t e d tomato, L . e s c u l e n t u m . The t o x i c f a c t o r has been i d e n t i f i e d as 2 - t r i d e c a n o n e ( n - t r i d e c a n - 2 - o n e ) ( F i g u r e 16) (62) and r e l a t e d m e t h y l k e t o n e s (63) which accumulate i n g l a n d u l a r t r i c h o m e s on t h e l e a f s u r f a c e . In o r d e r t o enhance the low l e v e l s o f 2 t r i d e c a n o n e i n L^. e s c u l e n t u m , e x p i a n t t i s s u e d e r i v e d from s e g r e g a t i n g F - 2 p o p u l a t i o n s o f c r o s s e s between !L. e s c u l e n t u m and L^. h i r s u t u m f . g l a b r a t u m were c u l t u r e d and e v a l u a t e d f o r h i g h l e v e l s and accumul a t i o n of methyl ketones. C r o s s e s were e v a l u a t e d by a l e a f d i s k b i o a s s a y w i t h t h e t o b a c c o hornworm, Manduca s e x t a , and by gas c h r o m a t o g r a p h i c and s p e c t r o p h o t o m e t r y a n a l y s i s ( 6 4 ) . Several c r o s s e s have been i d e n t i f i e d thus f a r w h i c h accumulate h i g h l e v e l s of methyl ketones ( e s p e c i a l l y 2 - t r i d e c a n o n e ) . The c o r r e l a t i o n between the amount o f m e t h y l k e t o n e s and the r e s i s t a n c e to h e r b i v o r y by M. s e x t a i n t h e s e c r o s s e s was - 0 . 5 6 ( 6 5 ) . The g o a l o f t h i s r e s e a r c h i s t o g e n e r a t e a c o m m e r c i a l c u l t i v a r o f tomato which i s n a t u r a l l y r e s i s t a n t to i n s e c t a t t a c k . I n summary, n a t u r a l p l a n t compounds have been e x p l o i t e d c o m m e r c i a l l y as s o u r c e s ( e . g . , p y r e t h r i n s , r o t e n o i d s , a l k a l o i d s ) and models ( e . g . , p y r e t h r i n s , p h y s o s t i g m i n e ) o f i n s e c t i c i d e s . Other p l a n t compounds a r e c u r r e n t l y b e i n g e v a l u a t e d f o r s i m i l a r u s e s ( e . g . , chromenes, l i m o n o i d s ) . S t i l l o t h e r s a r e b e i n g e v a l u a t e d f o r use i n host p l a n t r e s i s t a n c e ( e . g . , l o n g - c h a i n methyl ketones). Such n o v e l c h e m i c a l s w i t h p o t e n t and o f t e n u n i q u e b i o l o g i c a l a c t i v i t i e s w i l l c o n t i n u e to be d i s c o v e r e d and e x p l o i t e d t h r o u g h b i o a s s a y and

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Table VIII.

Ecdysis-Inhibitory A c t i v i t y of Azadirachtin Fed i n an A r t i f i c i a l Diet to F i r s t - I n s t a r Larvae of Three Species of A g r i c u l t u r a l Insect Pests Species

El95(ppm)

H e l i o t h i s zea Spodoptera frugiperda Pectinophora gossypiella

2 1 10

vb EC50(ppm)

a

0.7 0.4 0.4

EI95 values are the concentrations r e s u l t i n g i n 95% ecdysis inhibition. EC50 values are the e f f e c t i v growth i n h i b i t i o n .

u

V

O C H l

OH

H

Figure 15. 3-Deacetylazadirachtinol, a Potent Insect Ecdysis Inhibitor Isolated from the Seeds of Azadirachta indica (57)

(n-Tridecan-2-one)

Figure 16. Structure of the Methyl Ketone, 2-Tridecanone, an Insecticide Found i n the Wild Tomato Species Lycopersicon hirsutum f. glabratum

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Useful in Insect

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through recent advances in isolation chemistry (chromatography) and structural analysis (spectroscopy). The benefits of isolating and identifying these plant chemicals have been proven in the past and plants will continue to be invaluable as renewable resources or chemical progenitors of new insecticides. Acknowledgments The author thanks Dr. M.F. Balandrin for helpful discussions. This work was supported in part by a grant awarded by the U.S. National Science Foundation (PCM-8314500). Literature Cited 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Pimentel, D. In "CRC Handbook of Natural Pesticides: Methods, Vol. I, Theory, Practice, and Detection"; Mandava, N.B., Ed.; CRC Press, Inc.: Boca Raton Florida 1985; pp 3-19 Storck, W.J. Chem Staal, G.B. Ann. Rev Crosby, D.G. In "Natural Pest Control Agents"; Crosby, D.G., Ed.; Marcel Dekker, Inc.: New York, 1966; pp. 177-242. Jacobson, M. "Insecticides from Plants, a Review of the Literature, 1954-1971"; Agricultural Handbook 461, U.S. Department of Agricultural-Agricultural Research Service: Washington, D.C., 1975. Jacobson, M. Econ. Bot. 1982, 36, 346-354. Matsumura, F. "Toxicology of Insecticides"; Plenum Press: New York, 1975. Secoy, D.E.; Smith, A.E. Econ. Bot. 1983, 37, 28-57. Levy, L.W. Environ. Exp. Bot. 1981, 21, 389-395. Tyler, V.E.; Brady, L.R.; Robbers, J.E. "Pharmacognosy (ed. 7)"; Lea and Febiger: Philadelphia, 1976. Schmeltz, I. In "Naturally Occurring Insecticides"; Jacobson, M.; Crosby, D.G., Eds.; Dekker: New York, 1971; pp. 99-136. Stedman, E. Biochem. J. 1926, 20, 719-734. Gysin, H. Chimia 1954, 8, 205-220. Menn, J.J.; Pallos, F.M. Environ. Lett. 1975, 8, 71-88. Jones, C.G.; Firn, R.D. J. Chem. Ecol. 1978, 4, 138-177. Kubo, I.; Klocke, J.A. In "Plant Resistance to Insects"; Hedin, P.Α., Ed.; ACS Symposium Series No. 208, American Chemical Society: Washington, D.C., 1983; pp. 329-346. Nakanishi, K. In "Natural Products and the Protection of Plants"; Marini-Bettolo, G.B., Ed.; Elsevier Scientific Publ. Co.: New York, 1977; pp. 185-210. Bowers, W.S.; Ohta, T.; Cleere, J.S.; Marsella, P.A. Science 1976, 193, 542-547. Rodriguez, E. In "Plant Resistance to Insects"; Hedin, P.A., Ed.; ACS Symposium Series No. 208, American Chemical Society: Washington, D. C., 1983; pp. 291-302. Klocke, J.A.; Balandrin, M.F.; Adams, R.P.; Kingsford, E. J. Chem. Ecol. 1985, 11, 701-712. Bowers, W.S. Ent. Exp. Appl. 1982, 31,3-14. Bowers, W.S. In "Insecticide Mode of Action"; Coates, J.R., Ed.; Academic Press: New York, 1982; pp. 403-427.

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23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

41.

42. 43. 44. 45. 46.

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Campos-Lopez, E.; Roman-Alemany, A. J. Agric. Food Chem. 1980, 28, 171-182. Myers, N. Impact Sci. Soc., 1984, 34, 327-333. Jermy, T.; Butt, B.A.; McDonough, L.; Dreyer, D.; Rose, A.F. Insect Sci. Application 1981, 1, 237-242. Klocke, J.A.; VanWagenen, B.; Balandrin, M.F. Phytochemistry, 1985, in press. Bate-Smith, E.C. In "Chemistry in Botanical Classification"; Bendz, G.; Santesson, J., Eds.; Academic Press: New York, 1973; pp. 93-102. Okuda, T.; Mori, K.; Hatano, T. Phytochemistry 1980, 19, 547-551. Haddock, E.A.; Gupta, R.K.; Al-Shafi, S.M.K.; Layden, K.; Haslam, E.; Magnolato, D. Phytochemistry 1982, 21, 1049-1062. Okuda, T.; Yoshida, T.; Nayeshiro, H. Tetrahedron Letters 1976, 3721-3722. Okuda, T.; Yoshida, T.; Kazuko, M. Phytochemistry 1975, 14, 1877-1878. Okuda, T.; Nayeshiro 4421-4424. Wood, A.W.; Huang, Μ.Τ.; Chang, R.L.; Newmark, H.L.; Lehr, R.E.; Yagi, H.; Sayer, J.M.; Jerina, D.M.; Conney, A.H. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 5513-5517. Blumenberg, F.W.; Claus, C.; Ennecker, C.; Kessler, F.J. Arzneim.-Forsch 1960,10, 223-226 (Chem. Abstr. 54, 16653). Cliffton, E.E. Am. J. Med. Sci. 1967, 254, 117-123. Girolami, Α.; Cliffton, E.E. Throm. Diath. Haemorrh. 1967, 17, 165-175. Dilton, J.N.; Broughton, H.B.; Ley, S.V.; Lidert, Z.; Morgan, E.D.; Rzepa, H.S.; Sheppard, R.N. J. Chem. Soc. Chem. Commun. 1985, 968-971. Larson, R.O.; personal communication. Lidert, Z.; personal communication. 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. Morgan, E.D. In "Natural Pesticides from the Neem Tree (Azadirachta indica A. Juss)"; Schmutterer, H.; Ascher, K.R.S.; Rembold, Η., Eds.; German Agency for Technical Cooperation: Eschborn, Germany, 1980; pp. 43-52. Butterworth, J.H.; Morgan, E.D. J. Insect Physiol. 1971, 17, 969-977. Zanno, P.R.; Miura, I.; Nakanishi, K.; Elder, D.L. J. Am. Chem. Soc. 1975, 97, 1975-1977. Uebel, E.C.; Warthen, J.D. Jr.; Jacobson, M. J. Liq. Chromatog. 1979, 2, 875-882. Rembold, H. In "Advances in Invertebrate Reproduction 3"; Engels, W.; Clark, W.H. Jr.; Fischer, Α.; Olive, P.J.W.; Went, D.F.; Elsevier Science Publishers: New York, 1984; pp. 481-491. Rembold, H.; Forster, H.; Czoppelt, Ch.; Rao, P.J.; Sieber, K.-P. In "Natural Pesticides from the Neem Tree (Azadirachta indica A. Juss) and Other Tropical Plants"; Schmutterer, H.; Ascher, K.R.S., Eds.; German Agency for Technical Cooperation: Eschborn, Germany, 1984; pp. 153-161.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

36.

47. 48. 49. 50.

51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66.

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Plant Compounds

Useful in Insect

Control

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Yamasaki, R.B.; Klocke, J.A.; Lee, S.M.; Stone, G.A.; Darlington, M.V., in preparation. S t i l l , W.C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 29232925. Kubo, I.; Klocke, J.A. Agric. Biol. Chem. 1982, 46, 1951-1953. Warthen, J.D. Jr. "Azadirachta indica: A Source of Insect Feeding Inhibitors and Growth Regulators"; Agricultural Reviews and Manuals ARM-NE-4, U.S. Department of Agriculture-Science and Education Administration: Beltsville, MD, 1979. Klocke, J.A.; Arisawa, M.; Handa, S.S.; Kinghorn, A.D.; Cordell, G.A.; Farnsworth, N.R. Experientia 1985, 41, 379-382. Klocke, J.A.; Kubo, I. Ent. Exp. Appl. 1982, 32, 299-301. Kubo, I.; Klocke, J.A. Les Colloques de L'I.N.R.A. 1982, 7, 117129. Klocke, J.A. Ph.D. Thesis, University of California, Berkeley, 1982. Nakatani, M.; James J.C.; Nakanishi K J Am Chem Soc 1981 103, 1228-1230. Dreyer, D.; persona Kubo, I.; Matsumoto, Α.; Matsumoto, T.; Klocke, J.A. Tetrahedron 1985, in press. Nakanishi, K. Abstr. No. 2, Proc. Joint Meeting of the American Society of Pharmacognosy and the Society for Economic Botany: Boston, Mass., 1981. Rembold, H.; Sieber, K.-P. Z. Naturforsch. Sect. C. Biosci. 1981, 36, 466-469. Sieber, K.-P.; Rembold, H. J. Insect Physiol. 1983, 29, 523-527. Barnby, M.A.; Darlington, M.V.; Klocke, J.A.; unpublished results. Williams, W.G.; Kennedy, G.G.; Yamamoto, R.T.; Thakcer, J.D.; Bordner, J. Science 1980, 207, 888-889. Dimock, M.B., Kennedy, G.G., Williams, W.G. J. Chem. Ecol. 1982, 8, 837-842. Nienhuis, J.; Klocke, J.A.; Locy, R.; Butz, Α.; Balandrin, M.F. Hortscience 1985, 20, 112. Nienhuis, J.; Klocke, J.A.; Balandrin, M.F.; unpublished results. Litchfield, J.T. Jr.; Wilcoxon, F. J. Pharmacol. Exp. Ther. 1949, 96, 99-113.

RECEIVED December 17,1985

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 37

Interactions Among Allelochemicals and Insect Resistance in Crop Plants M. R. Berenbaum and J. J . Neal Department of Entomology, University of Illinois, Urbana, IL 61801

Many crop plant cut production costs, not only of their own allelochemical defense systems against insect enemies, but also of the synthetic insecticide defense systems humans have devised for plant protection. Common among crop plants are allelochemicals which act as synergists, or chemicals that themselves lack toxicity to insects but can enhance the toxicity of a co-occurring chemical. Most widespread among these synergists are inhibitors of mixed-function oxidases, the membranebound metabolic enzymes responsible for the detoxification of a wide variety of xenobiotics. These inhibitors include methylenedioxyphenyl (MDP) compounds. Myristicin, a common constituent of many umbelliferous crops, is as effective a synergist for carbaryl as is piperonyl butoxide, a commercial synergist of synthetic insecticides. Also ubiquitous are synergists that inhibit glutathione-S-transferases, soluble enzymes involved in a number of xenobiotic transformations. These endogenous allelochemicals can synergize both cooccurring toxicants and exogenous synthetic organic insecticides. Such synergists in leaf tissue can significantly increase the toxicity of an insecticide; carbaryl toxicity is thus greatly influenced by the chemistry of the plant on which i t is applied. While there are possible complications involved in the use of endogenous synergists to potentiate chemical control of insects, there is great potential for using these chemicals to reduce the impact of synthetic insecticides on nontarget organisms and on the environment without compromising insect control. 0097-6156/87/0330-0416$06.00/0 © 1987 American Chemical Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

37.

B E R E N B A U M A N D NE A L

Insect Resistance in Crop

Plants

The term "insect-plant interaction" generally refers to plants of the botanical variety, as opposed to plants of the i n d u s t r i a l variety; however, weeds, wildflowers, and crop plants face many of the same problems that owners of chemical factories do in the process of manufacturing i n s e c t i c i d a l chemicals. One major consideration of i n s e c t i c i d e manufacturers i s to minimize costs of production; higher costs of manufacture are reflected by higher prices and reduced competitive a b i l i t i e s i n the marketplace. Costs are s i m i l a r l y important to plants that synthesize allelochemicals, substances with no known physiological functions which nonetheless possess ecological functions with respect to predators and pathogens of the plant. In the case of plants, "costs" represent biosynthetic costs—energy and material diverted into the formation of defensive chemicals that could otherwise be directed to the production of vegetative and reproductive tissues. That such costs exist and represen nutrient budges has bee measurements of these costs are infrequent. One case in which these costs have been estimated involves the wild parsnip, Pastinaca sativa, and i t s major insect enemy, the parsnip webworm, Depressaria p a s t i n a c e l l a (2_). Resistance to the parsnip webworm i s largely attributable to the measurable amounts and proportions of furanocoumarins, allelochemicals typical of many plants in the family Umbelliferae. Almost 75% of the variance in s u s c e p t i b i l i t y to the parsnip webworm can be attributed to the r e l a t i v e concentrations of bergapten and sphondin (two furanocoumarins) in the seeds and leaves; the proportion of bergapten in the seeds alone accounts for 36% of the variance. Each of these t r a i t s i s s i g n i f i c a n t l y heritable — that i s , a s i g n i f i c a n t amount of the phenotypic variance i s due to additive genetic factors that can respond to selection. Selection, however i s a two-edged sword; a response to selection i n one t r a i t can, by v i r t u e of linkage or pleiotropy, be correlated with an opposite response in a different t r a i t . A genetic correlation i s the correlation between the additive genetic variance for two t r a i t s measured on a single individual; as such, i t gives an indication of the response of that individual to selection on one t r a i t 03)· Three of the four resistance t r a i t s in parsnip are negatively g e n e t i c a l l y correlated with the number of secondary rays produced by the plant; the secondary rays each bear two seeds and thus r e f l e c t the genetic potential for seed production, the c l a s s i c fitness measure in a biennial plant such as wild parsnip. A negative genetic correlation between a resistance factor and secondary ray number indicates that those individuals that are most resistant to their major insect enemy are also less

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

417

8

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

competitive when the insect i s absent (2). Reduced fitness could well r e f l e c t the metabolic cost of producing the furanocoumarin resistance factors. Synergists That Inhibit Mixed-function Oxidases Insecticide manufacturers have responded to pressures to reduce costs in several ways that have d i r e c t in the plant world. One method i s to use synergists, compounds which may themselves lack t o x i c i t y to insects but which enhance the t o x i c i t y of co-occurring toxicants (4). Most synthetic synergists act by i n t e r f e r i n g with the a b i l i t y of the insect to metabolize the i n s e c t i c i d e ; synergists thus have the additional benefit of restoring potency of i n s e c t i c i d e s to insects resistant because of enhanced metabolic c a p a b i l i t i e s (_5). The most widely used commercial synergists are methylenedioxyphenyl (MDP) compounds such as sesamin; these chemicals interfere wit other i n s e c t i c i d e s b mixed-function oxidases (MFOs), the suite of enzymes responsible for a number of oxidative reactions that convert toxic l i p o p h i l i c materials into excretable hydrophilic materials (J3). The major commercial source of plantproduced pyrethrins, Tanacetum cinerariaefolium, also contains sesamin (7); the plant therefore produces both an i n s e c t i c i d e and a synergist. The widespread occurrence of MDPs and other MFO i n h i b i t o r s in plants grown commercially for food (Table I) raises the p o s s i b i l i t y that resistant c u l t i v a r s may owe their resistance not to v a r i a t i o n in the l e v e l s of toxicant but rather to v a r i a t i o n i n the l e v e l s of synergists. Again, the parsnip plant provides a good example. In at least two species of Lepidoptera (one, Spodoptera eridania, a noctuid generalist, and the other, P a p i l i o polyxenes, a p a p i l i o n i d s p e c i a l i s t pest on parsnip and related umbellifers), furanocoumarins are metabolized by midgut MFOs (27). However, parsnip plants vary considerably i n their content of m y r i s t i c i n , an MDP that i s a potent synergist of furanocoumarins i n the polyphagous H e l i o t h i s zea (Lepidoptera: Noctuidae) (28 (Table II). Inasmuch as only 100 mg/g m y r i s t i c i n can increase the t o x i c i t y of xanthotoxin (a co-occurring furanocoumarin) f i v e f o l d , the 100-fold v a r i a t i o n i n m y r i s t i c i n content i n the leaves of parsnip c u l t i v a r s (Table III) may make a substantial difference i n resistance not only to generalized feeders but also to an adapted pest species such as P. polyxenes.

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

37.

B E R E N B A U M A N D NE A L

Insect Resistance in Crop

419

Plants

Table I. Economically Important Plants Containing Methylenedioxyphenyl Compounds

Family

S c i e n t i f i c name

Common name

Ref. 8_

Ericaceae

Vacciniu

Myristicaceae

Myristic

Pedaliaceae

Sesamum indicum

sesame

10

Piperaceae

Piper betel Piper nigrum

betel black pepper

11

Anethum graveolens Anthriscus s y l v e s t r i s Apium graveolens Daucus carota Foeniculum vulgare Levisticum o f f i c i n a l e Pastinaca sativa Petroselinum crispum Pimpinella anisum Oenanthe japonica

dill 9 ,13,14 15 chervil 9 celery 9,16 carrot 9 fennel 9 lovage 17,18 parsnip 9,19 parsley 20 anise 2J_ water celery

Umbelliferae

fragran

g

11

Other MFO synergists i n economically important plants Benzothiazoles ( 2 2 ) : watercress ( 2 3 ) , coconut ( 2 4 ) , tomato ( 2 5 ) , soybean ( 2 6 ) · Benzimidazoles

( 2 2 ) : coffee ( 1 0 ) , tea ( 1 0 ) , cocoa ( 1 0 ) .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Table I I . Mortality (%) of F i r s t - i n s t a r Heliothis zea on A r t i f i c i a l Diets (28) Modified with Additives.

Myristicin

(% wet weight)

Xanthotoxin (% wet wt.)

0

0.000

1.1

0.0

0.0

0.0

0.100

0.0

6.7

0.0

6.7

0.250

20.0

13.3

43.3

86.7

0.375

23.3

40.0

38.5

93.3

0.500

16.7

30.0

60.0

86.7

1.500

50.0

100.0

96.7

2.000

86.7

0.57

0.38

LC

5 0

0.01

0.96

0.03

0.10

0.19

(±95% confidence) (0.79-1.17)(0.48-0.67)(0.32-0.46)(0.17-0.20)

Synergistic ratio

1.00

1.85

2.54

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4.97

37.

BERENBAUM AND NE AL

Insect Resistance in Crop

Synergists That Inhibit

421

Plants

Glutathione-S-transferases

The mixed-function oxidases are ubiquitous i n plant-feeding insects and are capable of a wide variety of metabolic conversions (4_, 29); they are by no means, however, the only metabolic system involved i n the detoxication of xenobiotics. Glutathione-S^transferases (GST) are soluble enzymes that are known to be involved i n some forms of organophosphate resistance (30, 31). There i s increasing evidence that these enzymes are also involved i n the metabolism of plant allelochemicals. Yu (32) demonstrated that several allelochemicals from plants induce ^e_ novo production of GST and suggested that GST i s involved i n their metabolism. Yu (33) also demonstrated that many ubiquitous plant chemicals can i n h i b i t the a c t i v i t y of GST and are thus p o t e n t i a l synergists. Quercetin (3,3 ,4',5,7pentahydroxyflavone) i s one such i n h i b i t o r (Table IV). Like the MDPs, quercetin an widely distributed amon within species. Resistant c u l t i v a r s may owe their resistance to v a r i a t i o n i n the levels of these synergists. A preliminary study (35) demonstrated that, i n H e l i o t h i s zea quercetin s i g n i f i c a n t l y increased the t o x i c i t y of s i n i g r i n , a co-occurring constituent of many c r u c i f e r crops. 1

Synergists of Synthetic Organic Insecticides In addition to improving the effect of endogenous toxicants, there i s increasing evidence that naturally occurring synergists i n plants can enhance the t o x i c i t y of synthetic organic i n s e c t i c i d e s . Marcus and Lichtenstein (34) demonstrated that many essential o i l constituents can activate insecticides when applied t o p i c a l l y to Drosophila melanogaster; anisaldehyde and m y r i s t i c i n , both MDPs, increase the t o x i c i t y of parathion. As mentioned previously, m y r i s t i c i n i s comparable to piperonyl butoxide, a commercial synergist, i n increasing the t o x i c i t y of carbaryl to Heliothis zea (35) (Table VI). That m y r i s t i c i n can synergize ingested carbaryl gives r i s e to the i n t r i g u i n g p o s s i b i l i t y that endogenous synergists i n crop plants can be used to enhance the e f f i c a c y of externally applied i n s e c t i c i d e s . If i n t e r n a l synergists can increase the e f f e c t i v e dose of an i n s e c t i c i d e , then lower amounts of i n s e c t i c i d e need be applied to effect equivalent control. Reduced applications are desirable not only i n terms of economic savings i n control costs but also i n terms of ecological impact; reduced applications mean reduced environmental contamination to affect nontarget species. Moreover, decreased environmental residues of i n s e c t i c i d e s may well

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

422

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Table I I I .

M y r i s t i c i n Content of Seeds of Cultivars of Pastinaca sativa L· (35)

Cultivar

M y r i s t i c i n (ppm)

Harris Early Model Harris Model Avon Resistor Offenham Tender and True A l l American Gladiator Hollow Crown Improved Fullback Short Thic

3.04 4.96 6.89 10.78 24.37 41.00 63.50 84.02

Leaf samples for m y r i s t i c i n analysis were weighed into 10-ml screw-top v i a l s . M y r i s t i c i n was extracted i n ca. 4 ml hexane for 24 h. Fifteen micrograms of octadecane was added as an internal standard. The hexane was decanted and the volume reduced to ca. 0.3 ml. M y r i s t i c i n was quantified by GLC-FID (3% OV-17, 2 m χ 4 mm ID, operated isothermally at 125° C) with a Varian 2700 instrument and a HewlettPackard 3390A integrator.

Table IV.

Effect of Plant Substances on Detoxifying Enzymes i n F a l l Armyworm Larvae (34)

Additive (0.27%) to A r t i f i c i a l Diet

None Indole-3-carbinol Quercetin Sinigrin

Glutathione ^-transferase (nmol DCNB conjugated/min/mg protein)

30.3 117.7 18.7 103.7

± 1.0 ±4.9 ± 0.6 ±3.4

a

Newly molted s i x t h - i n s t a r larvae were fed meridic diets containing the compounds for two days prior to enzyme assays·

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

37.

BERENBAUM AND NE AL

Table V.

Insect Resistance

in Crop

423

Plants

D i s t r i b u t i o n of Quercetin Glycosides i n Edible vs Nonedible Parts of Fruits and Vegetables (mg of aglycone/kg fresh weight) (47).

Quercetin (mg/kg) Species

Other parts of the same plant

Edible part

Small radish

Root

Radish

Root

0

Leaves

35

Rutabaga

Root

ca.0.1

Leaves

40

Horseradish

Root

0

Leaves

50

Scorzonera

Root

). T h e p h y t o t o x i n was c h a r a c t e r i z e d by s p e c t r o m e t r i c a n a l y s e s a n d c h e m i c a l c o n v e r s i o n a s (-)-dihydropyrenophorin ( V I ) , an i m p o r t a n t d i l a c t o n e m a c r o l i d e (15). However, the major p r o d u c t o b t a i n e d i n our e x t r a c t i o n procedure u s e d t o i s o l a t e ( - ) - d i h y d r o p y r e n o p h o r i η was t h e d i o l V I I (_^6) , w h i c h was n o t a c t i v e i n o u r b i o a s s a y t e s t s . We h a v e n o t y e t a s s i g n e d t h e s t e r e o c h e m i s t r y o f t h e s e c o n d a r y a l c o h o l c e n t e r s i n VII o r V I I I . The s i m p l i c i t y o f t h e NMR s p e c t r u m of VIII indicates a symmetrical molecule. T h i s w o u l d most p l a u s i b l y b e t h e m o l e c u l e w i t h t w o f o l d s y m m e t r y , b u t we c a n n o t y e t r u l e o u t t h e meso f o r m . Upon o x i d a t i o n V I I c o u l d b e c o n v e r t e d t o p y r e n o p h o r in ( V I I I ) . Pyrenophori b u t was n o t f o u n d i n avenae fungi (J7). B i o l o g i c a l l y , t h e most i n t e r e s t i n g a s p e c t o f (-)-dihydropyrenop h o r i n i s t h a t i t causes r e d d i s h l e s i o n s on Johnson g r a s s a t 10*6, 10~7, a n d 10~& M w h e r e i n no o t h e r p l a n t s p e c i e s t e s t e d shows a n y s e n s i t i v i t y whatever at these c o n c e n t r a t i o n s . Thus, i t would appear t h a t VI i s h o s t s e l e c t i v e . To o u r k n o w l e d g e J o h n s o n g r a s s i s n o t a h o s t o f D_. a v e n a e . Bipolaroxin (IX) i s t h e m o s t h o s t - s e l e c t i v e p h y t o t o x i n . It was i s o l a t e d f r o m B i p o l a r i s c y n o d o n t i s , a f u n g a l p a t h o g e n o n B e r m u d a g r a s s (18) . The t o x i n p r o d u c e s r e d d i s h l e s i o n s and r u n n e r s on t r e a t e d l e a v e s o f Bermuda g r a s s and J o h n s o n g r a s s ( h o s t p l a n t s o f t h e p a t h o g e n ) a t 10"5 M , b u t a t t h e s e c o n c e n t r a t i o n s no o t h e r p l a n t species tested is affected. B i p o l a r o x i n is a h i g h l y oxygenated member o f t h e e r e m o p h i l a n e f a m i l y . When t h e a l d e h y d e g r o u p i s r e d u c e d ( y i e l d i n g d i h y d r o b i p o l a r o x i n (X)) a l l p h y t o t o x i c i t y a p p e a r s t o b e l o s t e v e n a t c o n c e n t r a t i o n s a s h i g h a s 10~3 M ( 1 5 ) . Bermuda g r a s s i s one o f t h e most n o t o r i o u s weeds i n t h e g r a s s f a m i l y s i n c e i t h a s b e e n l i s t e d a s a p r o b l e m i n a t l e a s t 40 d i f f e r e n t crops. The z o n a t e l e s i o n s p r o d u c e d by B_. c y n o d o n t i s g r e a t l y r e s e m b l e t h o s e c a u s e d by b i p o l a r o x i n . Mode o f a c t i o n s t u d i e s o n b i p o l a r o x i n may be f a c i l i t a t e d by t h e u s e o f t h e ^ C - l a b e l e d compound. R e c e n t l y , we have learned t h a t [^C] mevalonate administered to c u l t u r e s of the f u n g u s s e r v e s a s a n e x c e l l e n t p r e c u r s o r t o b o t h IX a n d X . M

Future

Work

The m a j o r i t y o f t h e n o v e l p h y t o t o x i n s known f r o m w e e d p a t h o g e n s are summarized i n t h i s r e p o r t . S i n c e o n l y a few p a t h o g e n s h a v e b e e n s t u d i e d t o d a t e , t h e r e a r e many m o r e o r g a n i s m s y e t t o b e e x a m i n e d . F o r e x a m p l e , t h e r e i s no r e p o r t o f a p h y t o t o x i n f r o m a b a c t e r i a l w e e d p a t h o g e n , a l t h o u g h s u c h compounds s u r e l y e x i s t . I t seems u n l i k e l y that extremely h o s t - s e l e c t i v e (host-specific) phytotoxins w i l l b e r e a d i l y f o u n d f r o m weed p a t h o g e n s s i n c e t h e r e i s r e l a t i v e l y l i t t l e g e n e t i c s e l e c t i o n p r e s s u r e on t h e p a t h o g e n . This arises f r o m t h e h i g h l y d i v e r s e g e n e t i c b a c k g r o u n d o f any weed p o p u l a t i o n .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

, ;

522

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

46.

STROBEL ET AL.

Phytotoxins

from Plant Pathogens of Weedy Plants

523

Nevertheless, we are encouraged by the findings on bipolaroxin (18) to believe that host-selective compounds do exist. Thus far, there have been no biochemical/physiological studies showing the effects or modes of action of any of the phytotoxins from weed pathogens on their hosts. Also, chemical modification (5) of the toxins should shed light on the bioactive portion of the molecule. The intrinsic biological activity of these phytotoxins certainly warrants considerable study, since there is potential for them to serve as herbicides or models for herbicides, and because they possess somewhat unexpected bîological activities. Acknowledgments We thank Dr. E. S. Luttrell of the University of Georgia for supplying cultures of many of the weed pathogens. The work at Bozeman was funded by a grant from Rohm ε Haas Co. and the Montana Agricultural Experiment Station The work at Cornell was partially supported by a grant, NI Literature Cited 1. "Index of Plant Diseases in the United States"; USDA: Washington, DC, 1969. 2. Holm, L. G.; Plucknett, D.L.; Pancho, J.V.; Herberger, J.P. "The World's Worst Weeds, Biology and Distributions"; Univ. Press of Hawaii: Honolulu, 1977. 3. Charudattan, R.; Walker, H.L. "Biological Control of Weeds with Plant Pathogens"; Wiley-Interscience: New York, 1982. 4. Takematsu, T.; Konnai, M.; Tachibana, K.; Tsuruoka, T.; Inoue, S.; Watanabe, T. (Melji Seika Kaisha, Ltd.) U.S. 4,448,601 (1984). 5. Strobel, G.A. Ann. Rev. Biochem. 1982, 51, 309-329. 6. Pinkerton, F.; Strobel, G.A. Proc. Natl. Acad. Sci. USA, 1976, 73, 4007-4011. 7. Durbin, R.D. "Toxins in Plant Disease"; Academic Press: New York, 1981. 8. Strange, R.N.; Pippard, D.J.; Strobel, G.A. Physiol. Plant Pathol. 1982, 20, 359-364. 9. Tatum, L.A. Science 1971, 171, 1113-1115. 10. Robeson, D.J.; Strobel, G.A. Phytochemistry 1984, 23, 1597-1599. 11. Robeson, D.J.; Strobel, G.A. Agric. Biol. Chem. 1982, 46, 26812683. 12. Stevens , K.L.; Bader-Ud-Din, Α.Α.; Admad, M. Phytochemistry 1979, 18, 1579-1580. 13. Robeson, D.J.; Strobel, G.A.; Matsumoto, G.K.; Fisher, L.E.; Chen, M.H.; Clardy, J. Experientia 1984, 40, 1248-1250 and references therein. 14. Sugawara, K.; Sugawara, F.; Strobel, G.A.; Fu, Y.; Cun-Heng, H.; Clardy, J. J. Org. Chem. 1985, in press. 15. Sugawara, F.; Strobel, G.A. Plant Sci. 1985, in press. 16. Kis, Z.; Furgernard, P.; Sigg, H.P. Experientia 1969, 25, 123124. 17. Grove, J.F. Tetrahedron Lett. 1965, 4675-4677. 18. Sugawara, F.; Strobel, G.A.; Fisher, L.E.; Van Duyne, G.D.; Clardy, J. Proc. Natl. Acad. Sci. USA 1985, in press. RECEIVED January 3, 1986 In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 47

Chemical Ecology of Quinolizidine Alkaloids Michael Wink

1

Institut für Pharmazeutische Biologie der Technischen, Universität Braunschweig, MendelssohnstraBe 1, D-3300 Braunschweig, Federal Republic of Germany

The biochemistry and physiology of quinolizidine alkaloids is reviewed with respect to their role in lupin metabolism. Minor roles of the alkaloids may be nitrogen transport and nitrogen storage, but their main function is that of chemical defense. Alkaloid concentration same order or eve centrations against pathogens that have been es tablished experimentally. Alkaloid-free lupins are highly susceptible to herbivore predation, which shows that the alkaloids are obviously important for the survival of a lupin plant. Quinolizidine alkaloids (QA) are thought to be typical natural products of many Leguminosae (1-3) but a few isolated occurrences have been reported also in unrelated families, e.g. Chenopodiaceae (_1 ) , Berberidaceae (JO , Papaveraceae (JO , Scrophulariaceae (£) , Santalaceae (_5) , Solanaceae (2) , and Ranunculaceae (1) . These observations could indicate that the genes for QA biosynthesis are probably not restricted to the Leguminosae but are widely distributed in the plant kingdom; however, they are only rarely expressed in the other families. We could support this belief by recent experiments using plant cell suspension cultures. A short-term and transientQA formation could be detected after induction even in "QAfree" species, such as Daucus, Spinacia, Conium, and Symphytum (6). In order to understand the biological function of QA we have to analyze their physiology and biochemistry f i r s t . Biochemistry and Physiology of Quinolizidine Alkaloids In the first step lysine is decarboxylated to cadaverine. Then three cadaverine units are incorporated into the tetracyclic QA skeleton, such as in lupanine, which serves as a precursor for most of the other QA. Recent tracer experiments have been reviewed (_3, 1) . In y

Current address: Genzentrum der Universitât Munchen, D-8033 Martinsried, Federal Republic of Germany 0097-6156/87/0330-0524$06.00/0 © 1987 American Chemical Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

47.

WINK

525

Chemical Ecology of Quinolizidine Alkaloids

our l a b o r a t o r y we have c o n c e n t r a t e d on the enzymology o f QA forma­ t i o n and have been a b l e t o c h a r a c t e r i z e more t h a n f o u r d i s t i n c t new enzymes; a r e v i e w i s a v a i l a b l e ( 8 ) . S i t e o f a l k a l o i d f o r m a t i o n , t r a n s p o r t , and a c c u m u l a t i o n . QA are formed i n the a e r i a l green p a r t s o f legumes, e s p e c i a l l y i n the l e a v e s (9^) . In l u p i n l e a v e s we succeeded i n l o c a l i z i n g the key en­ zymes o f QA b i o s y n t h e s i s i n the c h l o r o p l a s t (K), _1J_) , where the f o r ­ m a t i o n o f the p r e c u r s o r l y s i n e a l s o t a k e s p l a c e . L i k e most o f the p r o c e s s e s t h a t are l o c a t e d i n the c h l o r o p l a s t , QA b i o s y n t h e s i s i s r e g u l a t e d by l i g h t (8) and QA f o r m a t i o n f o l l o w s a l i g h t - d e p e n d e n t d i u r n a l rhythm (12, 13). The a l k a l o i d s formed i n the l e a v e s are t r a n s l o c a t e d v i a the phloem (_1_3, 14) a l l o v e r a l u p i n p l a n t , so t h a t a l l p l a n t p a r t s c o n t a i n a l k a l o i d s . QA are accumulated and s t o r e d p r e f e r e n t i a l l y i n e p i d e r m a l and s u b e p i d e r m a l t i s s u e s o f stems and l e a v e s (15_, 16). E s p e c i a l l y r i c h i n a l k a l o i d s are the s e e d s , which may c o n t a i n up t o 5% (dry weight) a l k a l o i d ( e q u i v a l e n t t o 200 mmol/ kg) . T u r n o v e r o f q u i n o l i z i d i n e a l k a l o i d s . L i k e many o t h e r n a t u r a l p r o d ­ u c t s , e s p e c i a l l y n i t r o g e n - c o n t a i n i n g compounds, QA are not i n e r t end p r o d u c t s o f m e t a b o l i s m , but compounds w i t h a h i g h degree o f t u r n o v e r . T h i s phenomenon becomes e s p e c i a l l y e v i d e n t d u r i n g g e r m i ­ n a t i o n and s e e d l i n g development. Most o f t h e a l k a l o i d s are me­ t a b o l i z e d and t h e i r n i t r o g e n i s p r o b a b l y used f o r s e e d l i n g growth (Γ7) . R o l e o f Q u i n o l i z i d i n e A l k a l o i d s i n the M e t a b o l i s m o f

Lupins

P r i m a r y m e t a b o l i s m . Having b r i e f l y r e v i e w e d the p h y s i o l o g y and b i o ­ c h e m i s t r y o f QA i n the s e c t i o n s above, we can c o n s i d e r t h e q u e s t i o n as t o why do l u p i n s produce a l k a l o i d s . F i r s t o f a l l we can ask whether the QA may p l a y a r o l e i n the p r i m a r y m e t a b o l i s m o f a l u p i n plant. A l k a l o i d s a r e t r a n s l o c a t e d i n t h e phloem sap l i k e o t h e r p h o t o s y n t h a t e s , and QA c o n t r i b u t e about 8 % t o the o v e r a l l n i t r o g e n . S i n c e QA are r e a d i l y m e t a b o l i z e d by c e l l s , t h e a l k a l o i d s c o u l d t h u s p l a y a r o l e as a minor means o f n i t r o g e n t r a n s p o r t . S p e c i e s t h a t produce r a t h e r few and heavy seeds s t o r e up t o 5 % a l k a l o i d s i n a d d i t i o n t o c a . 30 % s t o r a g e p r o t e i n . We have e s ­ t i m a t e d t h a t QA c o n t r i b u t e c a . 10 % o f t h e t o t a l n i t r o g e n s t o r e d i n l u p i n s e e d s . T h e r e f o r e a n o t h e r minor r o l e o f QA c o u l d be n i t r o g e n s t o r a g e (_Γ7 , _18) . We c o u l d not f i n d o t h e r a r e a s o f p r i m a r y m e t a b o l i s m i n which QA might p l a y a p a r t . S i n c e we can assume t h a t a l l m e t a b o l i t e s found i n organisms have a d e f i n i t e f u n c t i o n (19) , we s u g g e s t t h a t t h e n i ­ t r o g e n s t o r a g e f u n c t i o n o f QA i s o f o n l y minor importance and p r o b ­ a b l y not s u f f i c i e n t t o e x p l a i n t h e complex p h y s i o l o g y o f QA. C h e m i c a l e c o l o g y . I t has been g e n e r a l l y a c c e p t e d t h a t many o f the s o - c a l l e d "secondary m e t a b o l i t e s " p l a y a r o l e i n the i n t e r r e l a t i o n ­ s h i p o f p l a n t - p l a n t s , p l a n t - m i c r o b e s , and p l a n t - h e r b i v o r e s (20-23). In a s e r i e s o f e x p e r i m e n t s we have sought t o d e t e r m i n e whether l u p i n a l k a l o i d s are i m p o r t a n t i n an e c o l o g i c a l c o n t e x t .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

526

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

What s h o u l d be t h e r e q u i r e m e n t s 1. 2. 3. 4.

o f a p l a n t defense

chemical?

I t s h o u l d d i s p l a y a s i g n i f i c a n t a c t i v i t y under e x p e r i m e n t a l conditions. I t s c o n c e n t r a t i o n i n t h e i n t a c t p l a n t s h o u l d e q u a l o r exceed the e x p e r i m e n t a l l y d e f i n e d i n h i b i t o r y c o n c e n t r a t i o n s . I t s h o u l d be p r e s e n t i n t h e p l a n t a t t h e r i g h t time and t h e right place. I t s h o u l d be e c o l o g i c a l l y r e l e v a n t .

We have i s o l a t e d pure a l k a l o i d s and s t u d i e d t h e i r e f f e c t on t h e m u l t i p l i c a t i o n o f p o t a t o - X - v i r u s ( F i g u r e 1 ) , on t h e growth o f gramp o s i t i v e and gram-negative b a c t e r i a ( 2 4 , T a b l e I) and o f f u n g i ( 2 4 , 2 5 , T a b l e I ) , t h e g e r m i n a t i o n o f l e t t u c e seeds (18_, 26), and t h e f e e d i n g o f i n s e c t s and m o l l u s c s (2_7, 2 8 , T a b l e I ) . F u r t h e r m o r e , QA are t o x i c f o r mammals, as has been r e v i e w e d ( 2 ^ 2j5, 27) . I t i s r e ­ markable t h a t QA a r e a c t i v e , n o t i n one, b u t i n a l l t h e i n t e r a c t i o n s s t u d i e d . The s t r u c t u r e e f f e c t o n l y t o some d e g r e e l u p a n i n e , such as 1 3 - t i g l o y l o x y l u p a n i n e , seemed t o be more t o x i c and r e p e l l e n t t h a n l u p a n i n e (_27, _28) . V e r t e b r a t e t o x i c i t y seemed t o be h i g h e s t i n t h e o ζ α

> Ο 73 η c r Η c 73

73 Ο

00

π > r

χ m

Ο Ο

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Asteraceae

H. o c c i d e n t a l i s

Isodon diterpenes Rhabdosia spp.

Isodons and derivatives

C i l i a r i c and angelylg r a n d i f l o r i c acids (-)-cis and (-)-trans ozic acids

Asteraceae

Helianthus spp.

Labiatae

Trachyloban-19-oic and kaur-16-en-19-oic acids

Asteraceae

annuus

Kauranes Helianthus

C, D, F,

Kalmitoxin-I, kalmitoxin-IV, grayanotoxin-III

Nagilactone podolide

Ericaceae

Podocarpaceae

Pinaceae

Grayanoid diterpenes Kalmia l a t i f o l i a

Norditerpenedilactones Podocarpus n i v a l i s , P. h a l l i i , P. g r a c i l i o r

Larix l a r i c i n a

p a l u s t r i c , levopimaric, neoabiètic acids A b i e t i c , neoabietic, dehydroabietic, iso^> pimaric, sandaracopimaric acids (43, 44) (37)

( 5 5 , 56)

(54) Lepidopterous larvae

Spodoptera exempta, S. l i t t o r a l i s

(52)

(50, 51)

(48)

Homoesoma electellum, H e l i o t h i s virescens, H. zea, Pefctinophora gossypiella Homoesoma electellum

Lymantria dispar

Pectinophora gossypiella,(45-47) H e l i o t h i s zea, Spodoptera frugiperda, Musca domestica, Laspeyresia pomonella, Epiphyas postvittana

Pristiphora erichsonii Schizaphis graminum

dubiosus, N. l e c o n t e i

538

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

OAc

ajuganptonsin

Figure 1.

Clerodanes with insect antifeedant a c t i v i t y .

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

48.

COOPER-DRIVER AND LE QUESNE

R = OH:

Diterpenoids

as Insect Antifeedants

18-hydroxygrindelic

R = 0C0CH CH C00H: 2

R

2

18-succinoyloxygrindelic

Figure 2.

acid

Grindelanes with insect antifeedant a c t i v i t y .

aa/3-4,8,13-duvatrien-1,3-diols

Labda-12,14-dien-8