Roles of natural products for biorational pesticides in agriculture 9780841233546, 0841233543, 9780841233553

Table of contents :
Content: Roles of natural products for biorational pesticides in agriculture --
Role of the IR-4 project in the regulatory approval of biopesticides for specialty crops --
Insect antifeedant activities and preparation of dihydrobenzofurans from cyperus spp --
Movement of thymol in citrus plants --
Use of omics methods to determine the mode of action of natural phytotoxins --
Pesticides on the inside: exploiting the natural chemical defenses of maize against insect and microbial pests --
Integrated pest management strategies for phytoplasma diseases of woody crop plants: possibilities and limitations --
Pear ester: from discovery to delivery for improved codling moth management --
Strategies for the manipulation of root knot nematode behavior with natural products in small scale farming systems --
Quantitative assessment of nectar microbe-produced volatiles --
Investigating host plant-based semiochemicals for attracting the leaffooted bug (hemiptera: coreidae), an insect pest of california agriculture.

Citation preview

Roles of Natural Products for Biorational Pesticides in Agriculture

ACS SYMPOSIUM SERIES 1294

Roles of Natural Products for Biorational Pesticides in Agriculture John J. Beck, Editor U.S. Department of Agriculture, Agricultural Research Service Gainesville, Florida

Caitlin C. Rering, Editor U.S. Department of Agriculture, Agricultural Research Service Gainesville, Florida

Stephen O. Duke, Editor U.S. Department of Agriculture, Agricultural Research Service Oxford, Mississippi

Sponsored by the ACS Division of Agrochemicals

American Chemical Society, Washington, DC Distributed in print by Oxford University Press

Library of Congress Cataloging-in-Publication Data Names: Beck, John J. (John James), 1965- editor. Title: Roles of natural products for biorational pesticides in agriculture / John J. Beck, editor, U.S. Department of Agriculture, Agricultural Research Service, Gainesville, Florida, Caitlin C. Rering, editor, U.S. Department of Agriculture, Agricultural Research Service, Gainesville, Florida, Stephen O. Duke, editor, U.S. Department of Agriculture, Agricultural Research Service, Oxford, Mississippi ; sponsored by the ACS Division of Agrochemicals. Description: Washington, DC : American Chemical Society, [2018] | Series: ACS symposium series ; 1294 | Includes bibliographical references and index. Identifiers: LCCN 2018033949 (print) | LCCN 2018041075 (ebook) | ISBN 9780841233546 (ebook) | ISBN 9780841233553 (alk. paper) Subjects: LCSH: Natural pesticides. | Agricultural pests--Control. | Pesticides--Formulation. Classification: LCC SB951.145.N37 (ebook) | LCC SB951.145.N37 R65 2018 (print) | DDC 632/.95--dc23 LC record available at https://lccn.loc.gov/2018033949

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48n1984. Copyright © 2018 American Chemical Society Distributed in print by Oxford University Press All Rights Reserved. Reprographic copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Act is allowed for internal use only, provided that a per-chapter fee of $40.25 plus $0.75 per page is paid to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Republication or reproduction for sale of pages in this book is permitted only under license from ACS. Direct these and other permission requests to ACS Copyright Office, Publications Division, 1155 16th Street, N.W., Washington, DC 20036. 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

Foreword The ACS Symposium Series was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of the series is to publish timely, comprehensive books developed from the ACS sponsored symposia based on current scientific research. Occasionally, books are developed from symposia sponsored by other organizations when the topic is of keen interest to the chemistry audience. Before agreeing to publish a book, the proposed table of contents is reviewed for appropriate and comprehensive coverage and for interest to the audience. Some papers may be excluded to better focus the book; others may be added to provide comprehensiveness. When appropriate, overview or introductory chapters are added. Drafts of chapters are peer-reviewed prior to final acceptance or rejection, and manuscripts are prepared in camera-ready format. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previous published papers are not accepted.

ACS Books Department

Contents Dedication: In Memoriam of Dr. Agnes M. Rimando ................................................. ix 1.

Introduction .............................................................................................................. 1 John J. Beck, Stephen O. Duke, and Caitlin C. Rering

2.

Role of the IR-4 Project in the Regulatory Approval of Biopesticides for Specialty Crops ......................................................................................................... 5 Jerry J. Baron, Michael P. Braverman, William P. Barney, Krista D. Coleman, and Daniel L. Kunkel

3.

Insect Antifeedant Activities and Preparation of Dihydrobenzofurans from Cyperus spp. ............................................................................................................ 11 Masanori Morimoto

4.

Movement of Thymol in Citrus Plants ................................................................. 23 Colin R. Wong and Joel R. Coats

5.

Use of Omics Methods To Determine the Mode of Action of Natural Phytotoxins ............................................................................................................. 33 Stephen O. Duke, Zhiqiang Pan, Joanna Bajsa-Hirschel, Adela M. Sánchez-Moreiras, and Justin N. Vaughn

6.

Pesticides on the Inside: Exploiting the Natural Chemical Defenses of Maize against Insect and Microbial Pests ............................................................ 47 Shawn A. Christensen, Charles T. Hunter, and Anna Block

7.

Integrated Pest Management Strategies for Phytoplasma Diseases of Woody Crop Plants: Possibilities and Limitations .......................................................... 69 Wolfgang Schweigkofler, Silvia Schmidt, and Christian Roschatt

8.

Pear Ester – From Discovery to Delivery for Improved Codling Moth Management ........................................................................................................... 83 Alan L. Knight, Douglas M. Light, Gary J. R. Judd, and Peter Witzgall

9.

Strategies for the Manipulation of Root Knot Nematode Behavior with Natural Products in Small Scale Farming Systems .......................................... 115 Baldwyn Torto, Hillary Kirwa, Ruth Kihika, and Lucy K. Murungi

10. Quantitative Assessment of Nectar Microbe-Produced Volatiles .................... 127 Caitlin C. Rering, John J. Beck, Rachel L. Vannette, and Steven D. Willms

vii

11. Investigating Host Plant-Based Semiochemicals for Attracting the Leaffooted Bug (Hemiptera: Coreidae), an Insect Pest of California Agriculture ............................................................................................................ 143 John J. Beck, Wai S. Gee, Luisa W. Cheng, Bradley S. Higbee, Houston Wilson, and Kent M. Daane Editors’ Biographies .................................................................................................... 167

Indexes Author Index ................................................................................................................ 171 Subject Index ................................................................................................................ 173

viii

Dedication: In Memoriam of Dr. Agnes M. Rimando

We dedicate this book to memory of Dr. Agnes M. Rimando who died on July 12, 2018 after a short illness. She was born in the Philippines on Oct. 17, 1957. She received a B.S. and a M.S. in Pharmacy from the University of the Philippines in 1980 and 1985, respectively, and a Ph.D. in Pharmacognosy from the University of Illinois at Chicago in 1993. She was an instructor at the University of the Philippines from 1981–1985 and a Research Trainee at Hiroshima University School of Medicine in 1985–1987. She worked as a research chemist for USDA, Agricultural Research Service from 1994 until her death. From 1996, she was located at the USDA Natural Products Utilization Research Unit in Oxford, Mississippi, USA. She was a world famous natural products chemist, authoring almost 200 scientific papers and acting as editor of several books on the chemistry of plants. Agnes was the recipient of many prestigious awards, including: Fellow of the American Chemical Society, Fellow of the Agricultural and Food Division of the American Chemical Society, the Kenneth A. Spencer Award for outstanding achievement in food and agricultural chemistry, the Federal Laboratory Consortium Excellence in Technology Transfer Award, the USDA, ARS Mid South Area Technology Transfer Award, and the USDA, ARS Mid South Area Senior Scientist of the Year Award. She was elected to the Philippine American Academy of Science and Engineering. Agnes was an invited speaker to many scientific meetings throughout the world, often serving as the keynote or plenary speaker. Her many contributions to the American Chemical Society included Chair of the Agricultural and Food Division of the American Chemical Society, American Chemical Society Councilor, the International Activities Committee, as well as service in many other capacities. She served as President of the American Council for Medicinally Active Plants. ix

Her expertise on the chemistry of plants was sought out by many. For example, she served as a consultant all over the world for the USDA and the US State Department (e.g., in Rwanda, and Colombia). She was fearless about going anywhere or tackling any problem. The research for which she is best known is her extensive work on the health benefits of pterostilbene, a constituent of grapes and blueberries. Her findings were extensively covered by the popular press, and this publicity gave a boost to blueberry production worldwide. Several of her discoveries related to pterostilbene were patented. Products based on these patents are sold throughout the world. She is survived by her mother, five sisters, two brothers, and a large extended family. Her many friends, co-workers, and collaborators will greatly miss her.

x

Chapter 1

Introduction John J. Beck,*,1 Stephen O. Duke,2 and Caitlin C. Rering1 1Chemistry Research Unit, Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, 1700 SW 23rd Drive, Gainesville, Florida 32608, United States 2Natural Products Utilization Research Unit, Agricultural Research Service, U.S. Department of Agriculture, University, Mississippi 38677, United States *E-mail: [email protected]

Chemical products from nature have been used to control pests since the beginnings of agriculture. Since these early years, natural products have played a direct role in controlling weeds, insects, plant pathogens, and nematodes. Topics of this AGRO symposium at the 254th ACS meeting highlighted the use and importance of natural products as biorational pesticides as they relate to agricultural commodities. Presented papers focused on the isolation of and applied use of natural products to agricultural systems, comprising topics such as: host plant volatiles as attractants of herbivorous insects; synthetic formulations of semiochemicals as insect monitoring tools; sensitive collection techniques for in situ or in-field analyses of plant volatiles; plant-insect, plant-microbe interactions that influence insect pests or beneficial insects; plant- or microbe-produced natural products that influence insects, pathogens, nematodes, or weeds; and plant-plant interactions that influence plant defense systems; and, plant-incorporated protectants for crop pest management. Scientific experts presented original research results and review of important topics from a number of diverse disciplines, including, analytical and environmental chemists, chemical ecologists, entomologists, and plant physiologists.

© 2018 American Chemical Society

Natural products have been used for centuries for pest management. Approximately 70 years ago, modern agriculture started to become dependent on a continual stream of synthetic chemical pesticides. These products were widely adopted due to their high level of efficacy, ease of use, and relatively low immediate price. However, an increase in evolved resistance, environmental issues, and toxicological problems with synthetic pesticides have created a desire to return to products that use a more natural-based means of managing pests. This trend has fueled resurgence in basic and applied research in this area, resulting in the potential for utilization of natural compounds for pest management more effectively than before the age of synthetic pesticides. The eclectic and expert compilation of chapters included in this volume represents a sampling of some of this research as it continues to evolve. We hope to see contributions such as the research within someday help to solve agricultural pest management problems. Regulation of biopesticides is a complex process. Chapter 2 describes the role that the IR-4 project has had in the regulatory approval of biopesticides in the USA. IR-4 is a project funded by the U.S. Department of Agriculture to facilitate the regulatory approval of pesticides for which there is insufficient industry support to achieve registration. This can be for new products or for new uses for established products. Much of this effort is with “minor crops” that are a relatively small market for pesticides. There are usually few pest management options for farmers of these crops. Biopesticides are a growing part of the pesticide market and also a growing portion of the IR-4 portfolio of projects. IR-4 has contributed to the use of: 1) microbial biopesticides (e.g., Bacillus thuringensis and the codling moth granulosis virus), 2) biochemical biopesticides (e.g., pheromones for insect pest management and acetic acid as a herbicide), and 3) plant-incorporated protectant biopesticides (e.g., transgenic viral coat protein to protect plums from plum pox virus). Some insect antifeedants are also biopesticides and can play an important role in pest management. In Chapter 3, Morimoto expands his work on compounds from the genus Cyperus (sedges) for insect management. He describes the antifeedant activity of natural dihydrobenzofurans and aurones from these plants, as well that of synthetic analogs inspired by these natural chemical structures. His structure-activity analysis of the biological activity of these compounds points the way for further improvement of this chemistry for a potential commercial product. Thymol is a common monoterpene found in many essential oils. It is serendipitous that this simple compound is effective against both the microbe that causes citrus greening and the insect vector of this disease that has caused huge economic damage to the citrus industry worldwide. In Chapter 4, Wong and Coats provide evidence that thymol applied to citrus, either as a foliar spray or via root uptake, translocates to untreated parts of the plant, potentially improving its efficacy as an antimicrobial and/or an insect management compound. Evolution of resistance to herbicides has become a major problem in pest management. A favored method of resistance management is alternation of herbicides with different modes of action. However, a new herbicide mode of action has not been introduced for 30 years and weeds have evolved resistance to most of the herbicides currently used. Natural compounds are not only a good source of new pesticide chemistry, but also provide clues for new modes of 2

action. Mode of action discovery is not trivial, as there are thousands of potential pesticide molecular targets. In Chapter 5, Duke et al. review omics methods (transcriptomics, proteomics, metabolomics, and physionomics) for herbicide mode of action discovery, discussing both the difficulties and limitations of these powerful methods as well as their strengths. Recent successes are discussed. Pesticidal substances produced by plants, as well as the genes necessary for the plants to produce these substances are known as plant-incorporated-protectants (PIPs). The most widely adopted biopesticides are the PIPs found in Bt crops. These crops resist insect pests via the insertion of a gene native to the bacterium Bacillus thuringiensis that produces an insecticidal protein. Inserting a transgene into plants for synthesis of pesticidal compounds may require extensive optimization and modification in order to provide effective control. In the case of Bt crops, early transgenics failed to express sufficient protein quantities to elicit acute toxicity to pests in the field and substantial modifications were necessary to increase expression levels (100 x) in plants from the original bacterial gene. Rather than inserting transgenes, other approaches to genetically improving pest resistance may rely on exploiting and improving the inherent pest resistance means of a crop or restoring defense that were lost during plant domestication. In Chapter 6, Christensen and colleagues review known defense mechanisms of maize, one of the world’s most abundantly produced crops and discuss how they may be exploited for improved pest control. Domestication of maize has improved yield and taste, but in many cases, reduced pest resistance relative to wild types. Plant growth regulators also have an important role to play in pest management. In Chapter 7, Schweigkofler et al., examine the effects of plant growth regulators and resistance inducers towards phytoplasma disease control. Phytoplasma infections cause significant economic loss in woody crops like apple and grape and are poorly controlled using conventional methods. Another source of effective biorational pesticides is the elucidation of chemical signals between organisms. Semiochemicals (also called infochemicals) are message-bearing signals (chemical compounds) that influence the behavior of organisms. When these signals occur within a same species (intraspecies) they are called pheromones, and when between different species (i.e., plant-insect, plant-microbe, or insect-microbe) they are called allelochemicals (interspecies). Investigations among plants, insects, and microbes have shown that semiochemicals enable many insect behaviors (i.e., aggregation, host location, predation, parasitism, etc.). Careful study of these systems can provide important natural products that provide efficacious pest management without associated environmental or ecological problems. Semiochemicals from host plant volatiles provide many different types of signals to insects. In Chapter 8, Knight and coauthors provide a comprehensive review of the chemical signaling involved with a major insect pest of apples, the codling moth (Cydia pomonella). In particular, they review the role of the attractive plant odor kairomone, (E,Z)-2,4-decadienoate (the pear ester), as well as its interaction with codling moth pheromone and other important factors included in the trapping of codling moth in various orchards. What makes this chapter particularly distinctive is the impressive cumulative years (ca. 104 years) 3

of experience and number of publications (ca. 120 combined) the authors have dedicated to the study of the codling moth and pear ester. Root knot nematodes are a serious plant parasite for many agricultural products. In Chapter 9, Torto and co-workers provide an elegant appraisal of natural products used for control of root knot nematode, a major pest of several crops grown by small-scale farmers in Africa, as well as crops worldwide. The authors provide overviews of numerous methods for control of root knot nematodes, including sections on crop rotation, soil amendments, intercropping, trap crops, push-pull systems, and offer insightful future directions for this field of study. Many aspects of these strategies are dependent on phytochemicals of the plants involved. The analysis of discrete volatile signals is a challenging endeavor. In Chapter 10, Rering and colleagues present the application of quantitative measurements of nectar microbe-produced semiochemicals, which represent an understudied and relatively new field of study of chemical signaling of plant pollinators. This timely report discusses the challenges of studying discrete emissions from small sources of plant-insect systems, particularly when the ratio and/or amounts of volatiles emitted may be important aspects of their attractiveness. Emerging pests or sudden population increases of minor pests require rapid responses from a multitude of disciplines. In Chapter 11, scientists comprising the recently formed (and self-named) “Leaffooted Bug Workgroup” provide an overview of the California agriculture insect pest Leptoglossus spp., and their combined efforts toward the use of host plant volatiles for control of this insect pest. Included in the chapter is a background of the history and biology of the Leptoglossus spp. affecting California agriculture, as well as a brief description of current control methods. The chapter also offers some original research results from the evaluation of several California host plants of the leaffooted bug, and offers some insight, including electrophysiological data, into what classes of compounds may be of interest to researchers in this field. Due to their selectivity, relatively low toxicity, and low environmental persistence, biopesticides represent an attractive alternative management approach to conventional synthetic pesticides. As public attitudes towards synthetic pesticides become increasingly critical, high-value crops, such as organic produce, have increased the potential market share of biopesticides and hastened their adoption. However, the successful deployment of these agents relies on a deep, fundamental understanding of natural ecological interactions between plants, insects and microorganisms in the field. The conventional synthetic pesticide revolution taught us that “silver bullet” solutions do not exist. New agricultural innovations are constantly needed in order to keep pace with resistance development and emerging pests. Natural products will continue to provide a source of inspiration for novel crop protection strategies.

4

Chapter 2

Role of the IR-4 Project in the Regulatory Approval of Biopesticides for Specialty Crops Jerry J. Baron,* Michael P. Braverman, William P. Barney, Krista D. Coleman, and Daniel L. Kunkel IR-4 Project, Rutgers University, 500 College Road East Suite 201W, Princeton, New Jersey 08540, United States *E-mail: [email protected]

The IR-4 Project was established in 1963 by U.S. Department of Agriculture and the State Land Grant Universities to help farmers of fruits, vegetables, nuts, herbs, ornamentals and other specialty crops gain legal access to safe and effective pest management products. The IR-4 Project develops required data and facilitates the registrations of chemical pesticides as well as biopesticides with the U.S. Environmental Protection Agency. IR-4’s efforts with biopesticides involve regulatory support and data development. With regulatory support, IR-4 serves as a consultant, helping public sector scientists and small business achieve registrations with new products/new uses. IR-4 also coordinates biopesticide product performance research. Since 2015, data development has focused on priority needs for biopesticides in fruit crops, vegetable crops, ornamental crops, organic markets, as well as the use of biopesticides to mitigate residues of chemical pesticides. IR-4 expects that the demand for biopesticide use in specialty crops will continue to increase as well as the demand for IR-4 services.

Farmers of organically and conventionally produced specialty crops must protect these high value commodities from economic damage caused by insects, plant diseases, weeds and other crop pests. For the past 60 plus years, a significant amount of pest management practices involved the spraying or application of a chemical or of a biological pesticide product to prevent damages and food waste. © 2018 American Chemical Society

The U.S. Environmental Protection Agency (EPA) regulates most products used to protect crops whether they are conventional pesticides or biopesticides. The private sector invests significant resources in research to meet EPA and other regulatory data requirements, which ensure that products are safe to humans and the environment. Because of the research costs, the crop protection industry concentrates their registration efforts on large acreage “major” crops like corn, soybean, wheat, and cotton where potential sales support an acceptable return on investment. Small acreage, specialty crops are deemed orphan crops and often lack availability of crop protection products. Due to limited potential sales, it is economically unfeasible for the crop protection industry to commercialize pest control products for specialty crops. Recognizing this dilemma, the IR-4 Project was created in 1963 to help America’s specialty crop growers and food processers with their pest control needs. IR-4 operates under a unique partnership formed between the USDA National Institute of Food and Agriculture (NIFA), the USDA-Agriculture Research Service (ARS), the State Agricultural Experiment Station/Land Grant University system, the crop protection industry, the commodity and grower groups and the Environmental Protection Agency. The mission of the IR-4 Project is to facilitate the regulatory approval of sustainable pest management technology for specialty crops and specialty uses to promote public well-being. Since its start, the IR-4 Project has supported over 48,000 national registrations of pest management uses to the benefit of agriculture in all states, providing a great value to the public. IR-4 Project infrastructure consists of multiple, independent units cooperating to accomplish the goals and priorities articulated by U.S. stakeholders. IR-4 maintains three core objective programs: Food Crops, Ornamental Horticulture, and Biopesticide & Organic Support. Historically, the Food Crops Program has developed Magnitude of the Residue data, studies that determine the amount of conventional chemical pesticide remaining in or on the crop at harvest when following the proposed registration use rates and timing for application and harvest. The EPA uses the residue data from IR-4 submissions to establish pesticide tolerances (also known as Maximum Residue Levels or MRLs), to set appropriate standards. More recently, IR-4 has been providing additional efficacy data or crop safety data to show that the proposed chemical is safe on the crop and effective in managing the target pest(s). Also under the Food Crops Program, is a sub-goal to expand and enhance the domestic and international use of residue data with crop grouping. Crop groupings are established for the purpose of extrapolation models that allow the regulated community to develop data on a few “representative” crops. If the data meet certain criteria, then the MRL and registrations can be extended to many similar “member” crops. This process has been extremely important for efficiently addressing specialty crop grower needs. IR-4’s Food Crops Program is also involved in efforts to support international harmonization of MRLs and to remove pesticide residues as a trade barrier. U.S. specialty crop growers/exporters want access to the lucrative international markets. Often, MRL standards established in the U.S. do not match what is allowed in export market countries. Harmonizing MRLs are key to expanding trade and markets. 6

The objectives of the IR-4 Project were expanded in 1977 to include registration of pest control products for the protection of ornamental horticultural crops (nursery, floral, forestry, and Christmas trees). Because of the large number of ornamental plant species, plant production systems and pests, the pesticide companies were pursuing only a small number of uses. Companies holding the registrations of pesticides products for ornamental crops typically need product performance (efficacy and crop safety) data to add specific new plant and/or specific new pests to product registrations and to protect them from liability. IR-4 was further expanded in 1982 with the establishment of the Biorational Program (later renamed IR-4 Biopesticide and Organic Support Program) that would assist in the registration of microbial and biochemical products. The primary objective of the IR-4 Biopesticide and Organic Research Program is to further the development and registration of biopesticides for use in pest management systems for specialty crops or for minor uses on major crops. It should be noted that even before the establishment of the IR-4 Biorational Program, IR-4 was involved with biopesticides. In 1970, IR-4 supported the registration of Bacillus thuringensis on all crops. The initial IR-4 Biorational Program supported the registrations of microbial products such as fungi, bacteria, and viruses in addition to low toxicity biochemicals, pheromones, insect and plant growth regulators, and genetically modified plants (plant incorporated protectants). Macro biologicals (insect parasites, predators, predacious nematodes, etc.) are not regulated under FIFRA and do not fall under the IR-4 biopesticide activities. Initially, IR-4’s Biorational Program supported public sector (typically USDA-Agriculture Research Service and Land Grant Universities) and small business biopesticide discoveries by providing regulatory support. Typically, IR-4 served as an intermediary between the group that has the technology and USDA and/or EPA to determine the requirements for registration of proposed uses. IR-4 would also perform literature searches in an effort to use information in the public domain to address regulatory data needs. IR-4 would submit the studies/reports to the regulatory authority. More recently, IR-4 expanded its regulatory support activities to facilitate approval for biopesticides and other products to be used in USDA certified organic production systems. Please note, it is estimated that only 10% of biopesticides are used in organic production; the remaining amount are used in the production of conventional agriculture and food products. Some assume that all biopesticides can be used in organic production. To be used in organic production, biopesticides must be first registered by EPA and have all active and inert ingredients appear on USDA’s National Organic Program (NOP) National List of Allowed and Prohibited Substances (National List) for use in organic production. EPA will then allow the language on the registration such as “For Organic Production” and/or show the triple leaf logo. A copy of this logo along with the specifics is found in EPA’s Pesticide Registration Notice 2003-1, the Labeling of Pesticide Products under the National Organic Program (1). Listing by Organic Materials Research Institute (OMRI), Washington State Department of Agriculture, Oregon Tilth, and other national certification organizations are not required. However, recognition of certification organizations is deemed desirable because end users recognize the logos. 7

In 1994, IR-4 expanded its biopesticide activities by providing small grants to assist in the development of biopesticides (proof of concept, efficacy data etc.). These grants funded work on biopesticide products that were in the “early” stage of regulatory development. That is, products whose core data packages had not yet been submitted to EPA. In some cases, IR-4 combined it efforts involving regulatory support and grants for early stage products. IR-4’s regulatory experience was used to develop research protocols, assist with Experimental Use Permits, coordinate and fund field and greenhouse research, assist in the development of Tier I toxicology and non-target organism waivers, as well as prepare and submit data packages to the EPA. The grant funding for this aspect of the program was increased in 1999 to develop efficacy and performance data on biopesticides currently being commercialized, with the goal of encouraging and expediting use of these newer, lower-risk technologies to a broader range of crops. This expanded grant program was called “Advanced Stage.” The focus was on the development of product performance data for products that had been registered by EPA or were in the registration process, where additional data was needed to assist with expansion of the registration to new crops or pests. The IR-4 Project’s Biopesticide Program was further expanded in 2004 with the establishment of a jointly funded project with EPA’s Biopesticide and Pollution Prevention Division (BPPD) to support large-scale demonstration studies. This program was referred to as “Demonstration” grants, with the intent to gather information and provide outreach to show that biopesticides could be useful tools in IPM/pest management systems. These grants were instrumental in sparking the adoption of biopesticides into integrated programs by growers. On occasion, the IR-4 Biopesticide and Organic Support Program takes on projects that require residue data. In these cases, the IR-4 Food Crops Program works in close cooperation with the IR-4 Biopesticide and Organic Support Program to complete data development efforts. A recent example of such a project was the development of residue data for potassium phosphite to support export markets. The residue data on this biopesticide was required by the European Union and/or Japan to maintain registrations, and allow U.S. grown crops to enter those markets. IR-4’s definition of a biopesticide has changed with the evolution of BPPD. As BPPD has expanded their regulatory oversight into various new technologies, IR-4 has been involved in regulatory support and/or funding research with progressive technology. This includes Plant Incorporated Protectants/Genetically Modified Crops and RNA interference technology. IR-4 expects to be involved in Gene Editing technologies once a regulatory frame work is in place for this technology. IR-4’s mechanism in deciding how to distribute research funds with biopesticides continues to evolve. Originally, IR-4 managed fund distribution through a competitive grant process. IR-4 would solicit grant applications and fund the proposals deemed most worthy. Proposals were classified and funded based on three categories: Early, Advanced, and Demonstration. Every year, IR-4 set aside $400,000 and funded between 20 and 30 proposals. A database of 8

grant-funded projects from 1983 to 2014 can be searched and viewed at the IR-4 Project website (2). In 2015, IR-4 modified the funding distribution process changed to reflect open and transparent priority setting process associated with having a public workshop. This process mirrored the process in the Food Crops and the Ornamental Horticulture Programs. The first major step in the new process occurred in September 2014, when IR-4 convened its first Biopesticide Workshop. Approximately 140 participants attended the inaugural session. Before and during the workshop, IR-4 received over 80 new requests for pest management assistance. These requests were considered pest management voids, where a farmer/grower was looking for a biopesticide solution. This first workshop included priorities for biopesticides for use in: • • • • • •

Conventional vegetable production Conventional fruit production Conventional ornamental production Organic production Public health Other

The priority areas have stayed relatively consistent since that first workshop, except that the IR-4 Project no longer allocates resources to public health. IR-4 has added on the new priority area “Residue Mitigation”, which involves utilizing biopesticides close to harvest as a replacement of a conventional chemical pesticide. The idea is that this action will result in similar pest management while reducing the chemical residues in the harvested food and ease trade barriers. A complete overview of the IR-4 Biopesticide Priority setting process was presented at the 2014 Biopesticide and Organic Support workshop (3). The research projects that IR-4 has funded since 2015 under the new system can be found at IR-4 Priority Need Funded Projects Database (4). Agriculture has benefited from IR-4’s involvement in biopesticides. IR-4 has contributed new active ingredient registrations, new use registrations and experimental use permits. Some notable contributions include the approval of: Bacillus thuringensis on all crops; codling moth granulosis virus; AF-36, a non-aflatoxin producing strain of Aspergillus flavus that is a competitive inhibitor of Aspergillus flavus that produces aflatoxin; a pheromone to disrupt the mating of the carob moth and oriental beetle; bacteriophage of Clavibacter michiganensis subsp. Michiganensis and Xanthomonas in tomato; acetic acid herbicide in organic crop production; and C5 HoneySweet plum. The C5 plum is resistant to plum pox virus because it produces the viral coat protein due to a transgene that encodes the protein, thereby preventing the virus from replicating. IR-4 has also been very active in facilitating products to manage varroa mites in honeybee colonies. A historical IR-4 Regulatory Database containing all of the IR-4 biopesticide successes can be searched (5). The IR-4 Project continues to see great growth opportunities in the future with biopesticides in specialty crop pest management systems. The crop protection industry has invested heavily with companies like Bayer Crop Science, the world’s 9

number one company in pesticide sales volume. Bayer Crops Science’s purchase of a company like AgraQuest for nearly $500 million dollars demonstrates the increasing enthusiasm of these products. BASF purchased Becker Underwood for $1 billion, getting them into biologicals. Growers/farmers are also seeing that more effective biopesticide products are being sold and used. There are products that meet or exceed the performance of conventional chemical products. As such, there is also great opportunity for biopesticides to be used in association with conventional products. We call this the R3 system, which means Rotation to manage Resistance and Residues. In other words, conventional chemical pesticides and biopesticides are used in a planned and monitored system. The idea is to rotate the products to prevent the pests from evolving and developing resistance to products. The system also calls for the use of the biopesticide close to harvest to minimize the amount of chemical pesticides that enter the food. A fair bit of IR-4 effort is utilized to support this approach. IR-4 continues to examine the potential to incorporate biopesticide research into the programs of Food Crops and Ornamental Horticulture. In this model, the Biopesticide Regulatory Support would stay separate and unattached. However, the research work with biopesticides would become integrated into product performance testing across programs. The positive of such an approach is that IR-4 has the resources to manage biopesticide research that would become more efficient, thus allowing IR-4 to synergize programs under existing research funding allocations. Biopesticides have come a long way, and the IR-4 Project is proud to be an important factor in these lower risk products becoming more valued and accepted by the farm community. It is IR-4’s desire that the acceptance of biopesticides will continue to grow as proper data and information is developed and shared.

References 1.

2.

3.

4. 5.

US Environmental Protection Agency Pesticide Registration (PR) Notice 2003-1. 2003. Labeling of Pesticide Products under the National Organic Program. https://www.epa.gov/sites/production/files/2014-04/documents/ pr2003-1.pdf (accessed June 14, 2018). IR-4 Project Biopesticide Grant Funded Projects Database (1983-2014). http://ir4app.rutgers.edu/biopestPub/grantFundedProj.aspx (accessed June 14, 2018). Braverman, M. P. Welcome, Goals and Process of the IR-4 Biopesticide Workshop. 2014. http://ir4.rutgers.edu/Biopesticides/ workshoppresentations/Overview%20of%20new%20process.pdf (accessed June 14, 2018). IR-4 Project Biopesticide Priority Need Funded Projects Database. http:// ir4app.rutgers.edu/biopestPub/pnnProjects.aspx (accessed June 14, 2018). IR-4 Project Biopesticide Regulatory Database. http://ir4app.rutgers.edu/ biopestPub/RegulatorySearch.aspx (accessed June 14, 2018).

10

Chapter 3

Insect Antifeedant Activities and Preparation of Dihydrobenzofurans from Cyperus spp. Masanori Morimoto* Department of Applied Biological Chemistry, School of Agriculture, Kindai University, Nakamachi 3327-204, Nara 631-8505, Japan *E-mail: [email protected]

The alien species Cyperus eragrosits produces the insect antifeedant quinones, cyperaquinones, and its dihydrobenzofuran precursor remirol also exhibited similar biological activity. Congeners of remirol were easy to prepare via organic synthesis and an electrolytic reaction. Aurones, a type of flavonoid, also possess a benzofuran moiety, and the tropical sedge, C. radians, produces large amounts of these compounds. Aurone derivatives were synthesized from various phenols via coumaranones using a coupling reaction. A structure-activity relationship (SAR) study on dihydrobenzofurans and aurones was then conducted using the common cutworm (Spodoptera litura) with a dual choice-type feeding test. The results of the insect antifeedant activity test revealed that acetophenone-type dihydrobenzofurans, which have an acetyl group at the 7-position on the benzene ring, exhibited strong insect antifeedant activities. Furthermore, lignan-like derivatives performed better than simple dihydrobenzofurans in the insect antifeedant assay. The most effective compounds against the common cutworm were o-dimethoxyphenyl acetophenone-type and methylenedioxy acetophenone-type dihydrobenzofurans. Aurone derivatives also showed similar tendencies, the coumaranones, lacking a phenyl group from aurone, decreased insect antifeedant activity. The introduction of methoxyl and methylenedioxy groups on the B-ring was advantageous for this biological activity. Consequently, natural aurones exhibited significantly stronger biological activities than the synthetic derivatives tested. The © 2018 American Chemical Society

results of these insect antifeedant activity evaluations revealed that the dihydrobenzofuran moiety is highly effective and easily manipulated to provide novel phytophagous insect behavioral control agents.

Introduction The plant defense system comprises physical and chemical defenses, with a hard cuticle, thorns, and trichomes for physical defense and the production and storage of biologically active compounds for chemical defense. A number of chemical defense systems against herbivores involve antifeedants and insecticidal compounds. These natural compounds can be used for the development of new pesticide. Also, they can be directly utilized for pest control. Cyperus species belong to Cyperaceae and are distributed globally. Some of these species, e.g. C. rotundus and C. esculentus, are noxious weeds and, thus, are subject to control by weed management. However, they have also been used as traditional medicines and spices. Recently, the alien sedge, C. eragrostis is widely distributed throughout Japan. Allelochemical, e.g. glucosinolate can be utilized to expand habitats where alien plant species invade. A relevant case of garlic mustard (Alliaria petiolata) has been reported, where the production of alleochemicals is reduced after the expansion of habitats (1). Since allelochemicals including phytotoxins and antifeedants are components of the plant chemical defense system, they allow alien plant species to expand their habitats. A previous study reported that Cyperus spp. also produced insect antifeedants (2). Insect antifeedants include the difuranobenzoquinones, cyperaquinones, and the precursor dihydrobenzofuran, remirol (Figure 1). The tropical sedge found in the sea shore, C. radians, produces large amounts of aurones. Aurones are a type of flavonoid that exhibit various biological activities including anticancer, antiprotozoal, and insect antifeedant activities (3–6).

Figure 1. Insect antifeedant natural phenolics and synthetic compounds for the SAR study.

Biologically active natural dihydrobenzofurans from various plants have been extensively examined and have been shown to exhibit insecticidal and phytotoxic activities (7–9). Therefore, the commercial insecticide, carbofuran and its prodrug, benfuracarb, which include the dihydrobenzofuran moiety, have also been developed for pest control (Figure 2). 12

Figure 2. Commercial insecticides that include the dihydrobenzofuran moiety in their chemical structures.

Insect Antifeedants in Cyperus spp. Cyperus species produce secondary metabolites as insect antifeedants and insecticidal compounds for chemical defence. C. iria produces an insect hormone, JH III, which exerts lethel effects (abnormal metamorphosis) on insects when ingested (10). The concentration of JH III in C. iria was found to be maintained at a higher level in premature plant tissue than in mature plant tissue (11). These suggest a chemical defense system in Cyperus species. However, unique natural products in Cyperus, called cyperaquinones and remirol, which is a precursor dihydrobenzofuran, exhibited insect antifeedant activities against common cutworm (Figure 1) (2). The introduction of acetyl and/or methoxyl groups on the benzene ring was shown to enhance insect antifeedant activity based on a SAR study using synthetic derivatives (12).

Synthesis of Lignan-like Dihydrobenzofurans by Electrochemical Oxidation The test lignan-like dihydrobenzofuran, stilbene was synthesized by the cycloaddition of an electrically generated phenoxonium cation to an inactivated alkene (Figure 3). Coupling reactions were performed by initially mixing all starting substances with the relevant electrolytes in a solvent using two bare glassy carbon felts between two poles. An appropriate charge was passed using a reference electrode in the reaction vessel. The solvent and electrolyte used were nitromethane and lithium perchlorate, respectively, as described by Chiba et al. (13).

Figure 3. Synthesis of lignan-like dihydrobenzofurans by electrochemical oxidation. 13

Electrochemically prepared lignan-like dihydrobenzofurans were designed based on previous SAR findings obtained from synthetic remirol congeners (12). The introduction of an acetyl group at the 7-position of the benzene ring and the presence of a methoxy group in the dihydrobenzofuran moiety were advantageous for insect antifeedant activity against the common cutworm. Therefore, the prepared dihydrobenzofuran series had hydrogen, acetyl, and t-butyl groups at the 7-position (Figure 4). Additionally, the introduction of a methylenedioxy moiety was adopted because it has been found in many insecticidal and enzyme inhibitory compounds. The prepared dihydrobenzofurans were tested in a dual choice-type insect antifeedant test against the common cutworm (S. litura) and diamond back moth (Plutella xylostella) larvae (DBM) (14).

Figure 4. Test dihydrobenzofurans prepared by electrochemical oxidation in the present study.

Insect Antifeedants for a SAR Study on Lignan-like Dihydrobenzofurans against S. litura (common cutworm) and P. xylostella (DBM) Larvae The results of the SAR study using the test dihydrobenzofurans and remirol congeners showed that acetophenone-type dihydrobenzofurans with an acetyl group at the 7-position on the benzene ring, namely, 2, 5, 8, 11, and 17, but not 14, exhibited stronger insect antifeedant activities in both test insect species than their respective congeners. The most effective compounds against the common cutworm were the o-dimethoxyphenyl-attached acetophenone-type dihydrobenzofuran (8) and methylenedioxy-attached acetophenone-type 14

dihydrobenzofuran (17), whereas that for DBM larvae was the phenyl group-eliminated acetophenone-type dihydrobenzofuran (2) (Figure 4, Table 1).

Table 1. Insect antifeedant activities of dihydrobenzofurans prepared by electrochemical synthesis against the common cutworm (S. litura) and DBM (P. xylostella) larvae S. litura

P. xylostella

ED50 (μmol/cm2)

ED95 (μmol/cm2)

ED50 (μmol/cm2)

ED95 (μmol/cm2)

1

0.48

3.5

Not tested

Not tested

2

0.031

0.18

0.015

0.090

3

1.9

>10

0.080

0.83

4

0.15

0.74

0.054

0.8

5

0.017

0.41

0.071

0.48

6

0.032

0.08

Not tested

Not tested

7

0.049

0.061

Not tested

Not tested

8

0.0056

0.035

>10

>10

9

0.14

0.89

Not tested

Not tested

10

0.14

0.79

Not tested

Not tested

11

0.028

0.50

0.42

9.7

12

0.082

0.45

Not tested

Not tested

13

Inactive

Inactive

Not tested

Not tested

14

Inactive

Inactive

Inactive

Inactive

15

Inactive

Inactive

Not tested

Not tested

16

0.030

1.7

0.17

>10

17

0.0049

0.035

Inactive

Inactive

18

0.13

>10

Not tested

Not tested

Correspondingly, the neolignan, kusunokinin, possessing a methylenedioxy and o-dimethoxyphenyl from Aristolochia malmeana, has been reported to exhibit insecticidal activity against velvet bean caterpillar moth (Anticarsia gemmatalis) larvae (15). This activity of dihydrobenzofuran (8) was weaker against DBM larvae and was absent for 17 (Table 1). Additionally, none of the 3′-acetoxy derivatives exhibited insect antifeedant activity. In a comparison between non-stilbene types, the phenyl group was eliminated (1-3) and stilbene, C6-C2-C6, types (4-18), stilbene-type dihydrobenzofurans exhibited slightly stronger activities than non-stilbenes. In a comparison of insect antifeedant activities between phenylpropanoids and lignans, lignans were more advantageous 15

against common cutworm than phenylpropanoids. The neolignans from Piper decurrence also exhibited insect antifeedant activities against European corn borer (Ostrinia nubilalis) larvae (16). The trimethoxy derivatives 7-9 exhibited the strongest antifeedant activities against the common cutworm in the present study. As reported previously, epimagnolin A from magnolia, a polymethoxy lignan with a similar structure, exhibited strong growth inhibitory activity against fruit fly larvae (17). The o-dimethoxyphenyl derivative (8) exhibited stronger insect antifeedant activity than the corresponding o-methoxyhydroxy derivative (11) against the common cutworm (Table 1). Wukirsari, T. et al. also reported that the strongest insecticidal lignan was the o-dimethoxyphenyl derivative in this SAR study using the systemic synthetic lignan series against Musca domestica (18). The neolignan, ococymosin from Ocotea cymosa, possessing the same moiety as these compounds, exhibited insecticidal activity and antiprotozoal activity against Aedes aegypti and Plasmodium falciparum, respectively (19). Compound 17, which was the most effective against the common cutworm, did not exhibit activity against DBM larvae (Table 1). Therefore, insect antifeedant activity is not universal, it depends on the target insect species. Flavonoids function as chemical defense secondary metabolites that avoid various stresses including feeding damage and the invasion of phytopathogens. Many flavonoids have been shown to exhibit insect antifeedant activities in various plants (20–22). These aurones are structurally similar to 2-phenyldihydrobenzofurans (lignan-like dihydrobenzofuran, i.e., stilbene). In aurones, one carbon was inserted between the furan and phenyl and was oxidized at the C3-position to a carbonyl group (Figure 5).

Figure 5. Structural similarities between 2-phenyl dihydrobenzofuran and aurone.

Synthesis of Aurones The general procedure for preparing test aurones in the present study was as follows: various phenols in ether were added to sodium metal at room temperature. The solution was maintained under the same conditions with stirring until hydrogen was no longer generated. 2-Bromoethylacetate was added to the mixture and reflux conditions were maintained for several hours. After the starting material had disappeared, as confirmed by a TLC analysis, the solution 16

was cooled, ethanol was then added to completely dissolve sodium metal, and 0.1 N NaOH aq. was poured into the solution. The synthetic ester was hydrolyzed at 80°C. The solution was adjusted to pH 8-9 and washed with chloroform to remove residual esters from the solution. The solution was then adjusted to below pH 3, extracted with chloroform, and phenoxyacetic acid was obtained, which was used directly in the next reaction without further purification. These phenoxy acetic acids were obtained at a yield of 23.9-86.0 % in this reaction. Phenoxy acetic acids were obtained by polyphosphoric acid (PPA)-mediated cyclization. Various phenoxyacetic acids were added to PPA and the mixture was stirred using a mechanical stirrer at 100°C for 8 hr. The mixture was cooled, followed by addition of water. The reaction product was extracted with chloroform to obtain the corresponding coumaranones. These coumarans and benzaldehydes were reacted with basic active alumina for chromatography (Chameleon Special reagent for chromatography, 300 mesh, Kishida Chemical Co. Ltd., Japan) in dichloromethane solution, and then left to stand overnight at room temperature. The E-Z isomer was assessed by examining the chemical shift value in the C2′ aryl proton based on the 1H-NMR spectrum. Each aurone was prepared by a known method and only the Z form was obtained (Figure 6) (6).

Figure 6. General procedure for the synthesis of test aurones.

Insect Antifeedants in the SAR Study of Aurones against Common Cutworm Among the B-ring-eliminated compounds, coumaranones (19-22), compound 22 did not exhibit insect antifeedant activity; however, the introduction of methoxyl and methyl groups to the benzene ring induced insect antifeedant activity, with the 4,5,6-trisubstituted compound exhibiting the strongest activity at an ED50 of 0.41 μmol/cm2. This activity was similar to that of 2, with both possessing similar molecular sizes and properties (Figures 4 and 7). Aurone 23, which had no substituent on its benzene rings, also did not exhibit insect antifeedant activity; however, the introduction of a methoxyl group to the Aand/or B-ring(s), namely, compounds 28, 29, 34, 36, and 37, induced insect 17

antifeedant activities with ED50 of 2.03, 0.86, 3.45, 0.85, and 0.12 μmol/cm2, respectively. The 4,5,6- and 3′,4′,5′-trisubstituted compounds 35 and 38 did not exhibit this activity in this test (Figures 8 and 9). This result differed from that with coumaranone, but was similar to previous findings obtained using flavones and chromones (22). Although the majority of commercial pesticides have halogens, two halogenated aurones, 26 and 27, exhibited no activity or were moderately active, ED50 4.21 μmol/cm2, respectively (Figure 8). Since the lignan-like dihydrobenzofuran 16 was more effective than aurone 30, ED50 of 1.61 μmol/cm2, which has a methylene dioxy moiety, the introduction of a methylenedioxy moiety to the benzene ring was advantageous for increasing insect antifeedant activity (Figures 4 and 8, Table 1).

Figure 7. Test coumaranones in the present study.

Figure 8. Test aurones with substituents on the B-ring in the present study.

Figure 9. Test aurones in the present study. 18

Conclusions Test dihydrobenzofurans and its 2-substituted diphenyl derivatives were prepared and their insect antifeedant activities were evaluated. Since these natural product analogs can be prepared with simple procedures, the results can be readily applied to agrochemical drug development. Insect antifeedant activity is a repelling effect without insecticidal activity. Therefore, such biologically active ingredients maybe delay the development of pesticide resistance. This property is explained by the fact that repellents do not kill susceptible organisms of the targets. The isoflavonoid derivatives found in the phytoalexin of legumes need to be discussed. Pterocarpans are a kind of flavonoids, with a 2-phenyldihydrobenzofuran moiety when viewed from a different angle (Figure 10). The natural pterocarpans, 42-43 from Pterocarpus macrocarpus also exhibited insect antifeedant activities against the common cutworm, with ED50 of 0.14, 0.91, and 0.10 μmol/cm2, respectively (23). These activities of pterocarpans were similar to that of the structural analogue 7 (Figure 4) and were slightly stronger than those of the test aurones.

Figure 10. Insect antifeedant pterocarpans in the Pterocarpus tree.

Consequently, the substituent effects of the phenyl, lignan-like dihydrobenzofuran series and benzyl, aurone series at the C2-position of dihydrobenzofurans are advantageous for increasing insect antifeedant activities against test insects. Similar findings were obtained in antiprotozoal activity evaluations using the same compounds against Leishmania donovani and Plasmodium falciparum (24).

References 1.

2. 3.

Lankau, R. A. Coevolution between invasive and native plants driven by chemical competition and soil biota. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (28), 11240–11245. Morimoto, M.; Fujii, Y.; Komai, K. Antifeedants in Cyperaceae: Coumaran and quinones from Cyperus spp. Phytochemistry 1999, 51, 605–608. Kayser, O.; Kiderlen, A. F.; Folkens, U.; Kolodziej, H. In vitro leishmanicidal activity of aurones. Planta Med. 1999, 65, 316–319. 19

4.

5.

6.

7.

8.

9.

10. 11.

12.

13.

14.

15.

16. 17.

18.

Kayser, O.; Kiderlen, A. F.; Brun, R. In vitro activity of aurones against Plasmodium falciparum strains K1 and NF54. Planta Med. 2001, 67, 718–721. Lawrence, N. J.; Rennison, D.; McGown, A. T.; Hadfield, J. A. The total synthesis of an aurone isolated from Uvaria hamiltonii: Aurones and flavones as anticancer agents. Bioorg. Med. Chem. Lett. 2003, 13, 3759–3763. Morimoto, M.; Fukumoto, H.; Nozoe, T.; Hagiwara, A.; Komai, K. Synthesis and insect antifeedant activity of aurones against Spodoptera litura larvae. J. Agric. Food Chem. 2007, 55 (3), 700–705. Ishibashi, F.; Satasook, C.; Isman, M. B.; Neil, T. G. H. Insecticidal 1Hcyclopentatetrahydro[b]benzofurans from Aglaia odorata. Phytochemistry 1993, 32 (2), 307–310. Céspedes, C. L.; Uchoa, A.; Salazar, J. R.; Perich, F.; Pardo, F. Plant growth inhibitory activity of p-hydroxyacetophenones and tremetones from Chilean endemic Baccharis species and some analogous: A comparative study. J. Agric. Food Chem. 2002, 50, 2283–2292. Schneider, C.; Bohnenstengel, F. I.; Nugroho, B. W.; Wray, V.; Witte, L.; Hung, P. D.; Kiet, L. C.; Proksch, P. Insecticidal rocaglamide derivatives from Aglaia spectabilis (Meliaceae). Phytochemistry 2000, 54, 731–736. Toong, Y. C.; Schooley, D. A.; Baker, F. C. Isolation of insect juvenile hormone III from a plant. Nature 1988, 333 (12), 170–171. Bede, J. C.; Goodman, W. G.; Tobe, S. S. Developmental distribution of insect juvenile hormone III in the sedge, Cyperus iria L. Phytochemistry 1999, 52, 1269–1274. Morimoto, M.; Urakawa, M.; Fujitaka, T.; Komai, K. Structure-activity relationship for the insect antifeedant activity of benzofuran derivatives. Biosci. Biotechnol. Biochem. 1999, 63 (5), 840–846. Chiba, K.; Fukuda, M.; Kim, S.; Kitano, Y.; Tada, M. Dihydrobenzofuran synthesis by an anodic [3 + 2] cycloaddition of phenol and unactivated alkenes. J. Org. Chem. 1999, 64, 7654–7656. Morimoto, M.; Urakawa, M.; Komai, K. Electrochemical synthesis of dihydrobenzofurans and evaluation of their insect antifeedant activities. J. Oleo Sci. 2017, 66 (8), 857–862. Messiano, G. B.; Vieira, L.; Machado, M. B.; Lopes, L. M. X.; de Bortoli, S. A.; Zukerman-Schpector, J. Evaluation of insecticidal activity of diterpenes and lignans from Aristolochia malmeana against Anticarsia gemmatalis. J. Agric. Food Chem. 2008, 56 (8), 2655–2659. Chauret, D. C.; Bernard, C. B.; Arnason, J. T.; Durst, T. Insecticidal neolignans from Piper decurrens. J. Nat. Prod. 1996, 59, 152–155. Miyazawa, M.; Ishikawa, Y.; Kasahara, H.; Yamanaka, J.-i.; Kameoka, H. An insect growth inhibitory lignan flower buds of Magnolia fargesii. Phytochemistry 1994, 35 (3), 611–613. Wukirsari, T.; Nishiwaki, H.; Hasebe, A.; Shuto, Y.; Yamauchi, S. First discovery of insecticidal activity of 9,9′-epoxylignane and dihydroguaiaretic acid against houseflies and the structure–activity relationship. J. Agric. Food Chem. 2013, 61 (18), 4318–4325. 20

19. Rakotondraibe, L. H.; Graupner, P. R.; Xiong, Q.; Olson, M.; Wiley, J. D.; Krai, P.; Brodie, P. J.; Callmander, M. W.; Rakotobe, E.; Ratovoson, F.; Rasamison, V. E.; Cassera, M. B.; Hahn, D. R.; Kingston, D. G. I.; Fotso, S. Neolignans and other metabolites from Ocotea cymosa from the Madagascar rain forest and their biological activities. J. Nat. Prod. 2015, 78 (3), 431–440. 20. Morimoto, M.; Kumeda, S.; Komai, K. Insect antifeedant flavonoids from Gnaphalium affine D. Don. J. Agric. Food Chem. 2000, 48, 1888–1891. 21. Harborne, J. B.; Grayer, R. J. Flavonoids and insects. In The flavonoids; Harborne, J. B., Ed.; Chapman & Hall: London, 1993; pp 589−618. 22. Morimoto, M.; Tanimoto, K.; Nakano, S.; Ozaki, Y.; Nakano, A.; Komai, K. Insect antifeedant activity of flavones and chromones against Spodoptera litura. J. Agric. Food Chem. 2003, 51 (2), 389–393. 23. Morimoto, M.; Fukumoto, H.; Hiratani, M.; Chavasiri, W.; Komai, K. Insect antifeedants, pterocarpans and pterocarpol, in heartwood of Pterocarpus mactocarpus Kruz. Biosci. Biotechnol. Biochem. 2006, 70 (8), 1864–1868. 24. Morimoto, M.; Cantrell, C. L.; Khan, S.; Tekwani Babu, L.; Duke Stephen, O. Antimalarial and antileishmanial activities of phytophenolics and their synthetic analogues. Chem. Biodivers. 2017, 14 (12), e1700324.

21

Chapter 4

Movement of Thymol in Citrus Plants Colin R. Wong and Joel R. Coats* Pesticide Toxicology Laboratory, Department of Entomology, Iowa State University, Ames, Iowa 50011, United States *E-mail: [email protected]

Citrus greening or Huanglongbing is a devastating disease of citrus trees and cause for major concern across the citrus-growing areas of the United States. Disease management has been difficult because the bacterium, Liberibacter asiaticus, can be harbored within an infected tree while it is efficiently vectored by the Asian citrus psyllid, Diaphorina citri, to spread to naive trees. Traditional pesticides have not been able to effectively control the vector nor the pathogen. We investigate the possibility that the naturally occurring monoterpenoid, thymol, can act as a control measure against both the bacterium and the vector simultaneously. Using tritium-labeled thymol, we found that the terpenoid could enter plants after simulated pesticide applications. This could inform the use of plant essential oils containing monoterpenoids that are both anti-microbial and insecticidal to combat citrus greening.

Introduction Since its introduction into Florida in 2005, citrus greening or Huanglongbing (HLB) has been spreading through orange groves across the United States (1). The disease sickens the trees and prevents the fruit from ripening properly and causing it to fall to the ground prematurely (2). The disease causing bacteria, Liberibacter asiaticus, is unable to move between trees on its own. Instead, it is vectored by the Asian Citrus Psyllid (ACP), Diaphorina citri (Liviidae) (2). Control attempts have mostly targeted the insect with insecticides, whereas, treatment of the tree © 2018 American Chemical Society

with antimicrobials to kill the pathogen is still being investigated (3). These control attempts are expensive, and one study estimated that since the introduction of HLB, the cost of production for sweet oranges increased by $1,600/ha (4). Costs could continue to increase because insecticide resistance has been found in ACP against common insecticide classes used in citrus including neonicotinoids, organophosphates and pyrethroids (5). Insecticide resistance and the difficulty of applying antibiotics necessitate the development of new control measures that can be used safely within commercial citrus. Plant-based compounds and their biorational derivatives are underutilized tools for pest control of herbivorous arthropods; concurrently, consumer demand is growing for safer and greener chemistries throughout society, including in agricultural pest control (6). A natural pesticide currently in use is the monoterpenoid, thymol. Natural monoterpenoids, such as thymol, are compounds made by plants (7). The efficacy of monoterpenoids and their derivatives against arthropod pest species has been studied previously (8–10). Thymol from the garden thyme plant (Thymus vulgaris, Lamiaceae) is used in both insecticidal and anti-microbial products, as well as a number of foods and fragrances (6, 11–13). Unfortunately it is difficult to run direct tests for antimicrobial activity against the HLB bacterium because of the inability to culture L. asiaticus in the laboratory (14). Therefore, it is not known if thymol is specifically active against L. asiaticus. It is possible that thymol may act as an insecticide or repellent and contribute toward controlling the psyllid. It is also possible that thymol may be serving as an antimicrobial and could help control the causal bacterium. Radiotracers were utilized to determine if thymol could be taken up by an orange tree when co-applied with a commercial adjuvant, insecticide or plant nutrient mix. Uptake into the plant would be necessary to combat existing infections of L. asiaticus because the bacterium resides in the phloem. After treatment with the radiolabeled thymol, the leaf tissues were extracted to determine the amount of thymol remaining on the leaf surface, amount in the waxy layer, and the amount present inside the leaf (15). The plant nutrient mixes used, Motivate by KeyPlex (Winter Park, FL) and 1000DP by KeyPlex contain metal chelators such as EDTA. These cation chelating compounds have been shown to facilitate the uptake of metals by plants by following radiolabeled metal ions through the roots (16). This study examined root uptake of organic compounds which could enter the plant through a mechanism similar to how EDTA is transported, however, determination of the mechanism of entry was beyond the scope of this study due to the complex nature of plant nutrient procurement. Foliar uptake is complex, with leaf surfaces composed of a mosaic of cell types, three-dimensional structures and layers of non-living protective materials (17). Adjuvants and oils can potentially help organic compounds spread and enter through the stoma or diffuse through the waxy cuticle (18). This study showed that thymol can enter a plant under normal environmental conditions. The knowledge that small molecules from natural products can penetrate into orange plants after external application opens a new avenue for control of both arthropod pests and the plant diseases that they can vector.

24

Materials and Methods Radiolabeled thymol (175 mCi) was dissolved in 5 mL of ethanol after its synthsis in February 2000. Commercial adjuvant and micronutrient formulations were provided by Keyplex, Winter Park, FL; these were KP 1000*DP, Ecotrol EC and Ecotrol Plus for the foliar uptake studies; and KP Motivate for the root uptake investigation. The thymol was singly 3H-labeled on the aliphatic carbons of the isopropyl group (Carbons 8 and 9; Figure 1). The radiolabeled thymol was purified using thin layer chromatography. Purifications were performed throughout the study prior to use of the radiotracer. Radioactivity was measured using a Packard Tri-Carb 2900TR liquid scintillation analyzer.

Figure 1. Structure of thymol with tritium-labeled carbon positions numbered. The compound is only singly labeled and is a mixture of thymol labeled at the 8 or 9 carbon. Orange trees (var, Hamlin) were provided by KeyPlex. Fine-grain #70 sand was purchased from Carolina Biological Supply. Sand was washed with tap water three times to remove organic matter and dust. Solutions of commercial adjuvants were mixed according to rates suggested on the label. Solutions of KP 1000*DP were made at 2.5% (v/v) in deionized water. Ecotrol EC and Ecotrol Plus were mixed at 4% (v/v) in deionized water. KP Motivate solution was mixed at 2.5% (v/v) in Hoagland’s solution (19). Uptake by Direct Foliar Application Leaves were selected and marked with tape on the abaxial side before application of the solution. Leaves were wiped with a damp tissue paper to remove any dust. The solution containing radiolabeled thymol was pipetted onto the adaxial surface of the leaf and gently spread using the pipette tip. Each leaf received 50 µL of Ecotrol EC solution, spiked with 0.045 µCi of 3H-labeled thymol, for the experiment. Leaves were labeled at the time of budding and the young cohort was determined to be leaves less than 40 days old. These leaves were still a lighter green and soft and waxy to the touch compared to the older leaves. Leaves used in the old cohort were over 1 year old. Applications were made while the leaves were still growing on the tree and kept under greenhouse conditions. Leaves were removed from the plant at specified time point and immediately rinsed with one portion of 10 mL of ethyl acetate to remove 3H-labeled thymol from the surface. This ethyl acetate was 25

kept and counted. The leaves were then dipped in 10 mL chloroform to dissolve the waxes on the cuticle, extracting any thymol present in the lipophilic matrix. The leaf was rolled and placed in the test tube for 15 seconds while shaking gently, and then the leaf was rolled the other direction and submerged again to expose both surfaces. The chloroform with the dissolved waxes was kept and counted. Leaves were frozen for storage for between 2 hours and 1 week. Leaf contents were then extracted in ethyl acetate using a Teflon homogenizer driven by a drill press. Each leaf was homogenized 3 times for 5 minutes each with a new portion of ethyl acetate each time. The ethyl acetate was filtered, combined and concentrated to 10 mL in volume and then stored and counted. Control samples consisted of leaves taken from the tree prior to application of any radiolabeled material. Leaves were extracted in the same manner as experimental samples. Each experiment used 2 leaves for a control for any tree tested.

Leaf to Leaf Movement after Foliar Application Trees were placed within a polyethylene chamber that separated the leaves and branches into three separate sections; top, middle and bottom (Figure 2). The chamber was constructed of a wooden frame covered with two layers of high density polyethylene (HDPE). The sections dividing the tree branches was made of single sheets of HDPE taped to the tree in the center. Foliar treatment using Ecotrol Plus solution spiked with radiolabeled thymol (0.225 µCi per treated leaf) was applied to 10 leaves and their branches in the middle section only and this section was closed for the duration of the experiment. Leaves from the top and bottom sections were randomly collected at specified intervals after the initial application of radiolabeled material and extracted. Leaf ages were selected randomly for treatment because there were not enough leaves on each tree that met the young criteria used in the direct uptake study.

Vertical Movement after Root Zone Application Trees of the same variety but smaller were provided by Keyplex for the root uptake investigation. The young orange trees had already been grafted, but were less than 1 inch in trunk diameter. The roots were washed to remove soil and repotted in clean aquarium sand in a 10 x 10 x 33 cm pot. A plastic sleeve was placed inside the pot to make a water tight seal around the sand. Trees were allowed at least 3 days to acclimate to the new soil conditions. Pre-treatment control leaves were removed from the plant and analyzed immediately before application of the radiolabeled thymol treatment. For each tree, 100 mL of Motivate solution spiked with radiolabeled thymol (0.901 µCi)was added to the sand. The plastic sleeve was tied at the top to stop thymol vapors from leaving the system. Leaves from the tree were sampled at random, frozen and analyzed at the end of the experiment. 26

Figure 2. Containment unit for testing leaf-to-leaf movement within the entire orange tree. The box was completely enclosed in polyethylene sheeting and the internal space was separated into three compartments the top (A), middle (B) where the application was made, and bottom (C). Statistical Analysis Treatment differences were obtained using R version 3.4.3. Differences were calculated using a least squares means regression corrected for multiple comparisons with the Tukey method for adjusting the significance threshold. A 2-Way ANOVA was used to find differences for individual parameters.

Results Direct Foliar Uptake Comparing the Ages of Leaves Figure 3 shows that at 0 minutes post-treatment (treatment and immediate recovery from the plant) much of the thymol is still on the surface of the leaves. This shows that the experimental system was able to account for all of the radioactive material that was introduced to the experiment when not allowing time for dissipation. Levels of radioactivity drop quickly from the rinse data over the hour experiment and only some of that then appears in the leaf extract or waxes. At one hour, the young leaves have taken up an average 15.5% of the thymol introduced whereas the old leaves have only taken up 4.7%. The control leaves taken at the beginning had a background reading equaling 1.09% of the dose applied to treatment leaves. The leaf extract data for either treatment was not significantly different from the control at our Tukey adjusted significance level. By one hour the proportion found in the leaf extract and the ethyl acetate rinse are similar, suggesting that an equilibrium has been reached for thymol to cross the waxy leaf cuticle. Not all of the thymol is accounted for at the times after the 0-minute time point. Thymol is slightly volatile under normal greenhouse conditions, and the proportion of thymol not included in the two rinses or the leaf extract is assumed to have sublimated. 27

Figure 3. Age comparison of the ability of leaves to internalize topically applied thymol. “Leaf” data is the radioactivity found within the homogenized leaf tissue. “Chlor” data is the radioactivity found within the waxes dissolved in chloroform. “Rinse” data is the radioactivity found in the ethyl acetate used to rinse the surface of the leaves. n = 4.

Leaf-to-Leaf Movement The whole tree sectioned into three parts showed that small amounts of thymol does move upward and downward through the tree after uptake by a leaf (Figure 4). More thymol appeared to move upward to leaves in the top section, however this was not significantly more than that which moved downward (P > 0.366). The leaf extracts together were significantly different from the ethyl acetate and chloroform rinses (P < 0.001). The most radioactivity was found in the leaves at the 72 hour time point, however the variability is high due to a reduced sample size (Figure 4). The 96 hour time point had lower recovery of the tritium label than previous time points, suggesting that by 96 hours the compound had fully diffused throughout the tree tissues and had begun to dissipate.

28

Figure 4. Movement of thymol from treated leaves to untreated leaves. Note: a data point was removed from the test “Rinse – Top” at 72 hours because it was an outlier and suspected to be a contaminated sample. Sample size varied due to carrying out the test longer after the first trial; 1 hour n = 6, 24 hours n = 6, 48 hours n = 6, 72 hours n = 2, 96 hours n = 4.

Root Uptake Radioactivity found within the leaves of plants exposed at the roots increased for the first few days, but then steadily declined after 144 hours (Figure 5). At time points 48 and 144 hours, the leaf extract radioactivity was significantly different from the ethyl acetate and chloroform rinses and the control (P = 0.0155 or less for all comparisons). A greater portion of the radioactivity was found in each leaf in the root uptake experiment (average approx. 0.2%) than in the leaf-toleaf movement experiment (average approx. 0.025%). This could suggest that the roots are more capable of acquiring thymol from the environment, however, the plants used in the root uptake experiment were smaller and had fewer leaves. When multiplied by an estimate for the number of total leaves on the tree, the difference is much less, with an estimated 4.0-5.0% total uptake to the leaves in both experiments.

29

Figure 5. Root uptake of thymol. Radioactivity found in the leaf extract is shown. n = 6.

Discussion These data show that the monoterpenoid thymol can be taken up by a plant through several routes of entry. The proportions recovered are small for the amounts of thymol we used, but this allows us to consider a new method of plant protection using monoterpenoid compounds. Plant essential oils rich in monoterpenoids are already popular as a natural or organic alternative for plant protection from insects, bacteria and fungi (6). This observation of living plants mobilizing the monoterpenoid thymol created by a different plant may enable the use of natural products for more modes of control than current practice. The use of a radiotracer allows us to identify and quantify the labeled molecules throughout the plant tissue, however there are limitations to the method. Due to cost and safety restraints, the absolute mass of thymol used was small, and it is unclear if there is some point where the uptake of thymol would plateau and no longer enter a plant. Furthermore, we did not determine whether the 3H-labeled material found within the plant was the parent thymol that we added or if some of it was a metabolite. Further explorations could employ chromatography paired with an HPLC radiodetector to analyze the plant tissue extract for labeled metabolites in addition to the parent thymol. This approach could inform how the molecule enters the plant and what is happening to it over time as the signal dissipates, even at very low levels. The toxicological significance of the concentrations of thymol in the orange tree is not currently known, and is beyond the scope of this project. The thymol in the tree may be sufficient to cause mortality to the Asian citrus psyllid, or cause a feeding non-preference; it could also be at a high enough concentration in certain 30

tissues within the plant to combat the bacterial pathogen. Thymol and some other monoterpenoids have been shown to be antimicrobial, which gives the potential for uptake of these compounds to combat bacterial diseases, such as HLB, within a plant (11). Co-application of thymol or other terpenoids with a nutrient mix product instead of a conventional insecticide could kill or repel the psyllid, but could also have the alternative benefit of enabling the tree to combat the causative agent of the HLB disease. Growers currently use soil amending micronutrient products because symptoms of HLB can include deficiencies of essential nutrients (4). It is also possible that because thymol is distributing throughout the plant, there could be a feeding deterrent effect. Thymol is known to be repellent to many insects which could imply that it would deter feeding if detected at some level (6, 9). Whether through anti-microbial activity or stopping the psyllid from acquiring the bacteria, uptake of thymol or other monoterpenoids from a soil drench could help interrupt the transmission cycle.

Acknowledgments We would like to thank the support of the Iowa Agricultural Experiment Station. This study was funded by KeyPlex, Winterpark, FL. We thank the editors and reviewer for their helpful feedback and revisions.

References 1.

2.

3.

4.

5.

6. 7.

Manjunath, K. L.; Halbert, S. E.; Ramadugu, C.; Webb, S.; Lee, R. F. Detection of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and its importance in the management of citrus huanglongbing in Florida. Phytopathology 2008, 98, 387–396. Halbert, S. E.; Manjunath, K. L. Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: a literature review and assessment of risk in Florida. Fl. Entomologist 2004, 87, 330–353. Blaustein, R. A.; Lorca, G. L.; Teplitski, M. Challenges for Managing Candidatus Liberibacter spp. (Huanglongbing Disease Pathogen): current control measures and future directions. Phytopathology 2018, 108, 424–435. Stansly, P. A.; Arevalo, H. A.; Qureshi, J. A.; Jones, M. M.; Hendricks, K.; Roberts, P. D.; Roka, F. M. Vector control and foliar nutrition to maintain economic sustainability of bearing citrus in Florida groves affected by Huanglongbing. Pest Manage. Sci. 2014, 70, 415–426. Tiwari, S.; Mann, R. S.; Rogers, M. E.; Stelinski, L. L. Insecticide resistance in field populations of Asian citrus psyllid in Florida. Pest Manage. Sci. 2011, 67, 1258–1268. Isman, M. B. Plant essential oils for pest and disease management. Crop Protection 2000, 19, 603–608. Croteau, R. Biosynthesis and catabolism of monoterpenoids. Chem. Rev. 1987, 87, 929–954. 31

8.

9.

10.

11.

12.

13. 14.

15.

16.

17. 18.

19.

Norris, E. J.; Gross, A. D.; Dunphy, B. M.; Bessette, S.; Bartholomay, L.; Coats, J. R. Comparison of the insecticidal characteristics of commercially available plant essential oils against Aedes aegypti and Anopheles gambiae (Diptera: Culicidae). J. Medic. Entomol. 2015, 52, 993–1002. Lee, S.; Tsao, R.; Peterson, C.; Coats, J. R. Insecticidal activity of monoterpenoids to western corn rootworm (Coleoptera: Chrysomelidae), twospotted spider mite (Acari: Tetranychidae), and house fly (Diptera: Muscidae). J. Econ. Entomol. 1997, 90, 883–892. Rice, P. J.; Coats, J. R. Insecticidal properties of monoterpenoid derivatives to the house fly (Diptera: Muscidae) and red flour beetle (Coleoptera: Tenebrionidae). Pestic. Sci. 1994, 41, 195–202. Didry, N. p; Dubreuil, L.; Pinkas, M. Activity of thymol, carvacrol, cinnamaldehyde and eugenol on oral bacteria. Pharm. Acta Helvetiae 1994, 69, 25–28. Didry, N. p; Dubreuil, L.; Pinkas, M. Antibacterial activity of thymol, carvacrol and cinnamaldehyde alone or in combination. Die Pharmazie 1993, 48, 301–304. Burt, S. Essential oils: their antibacterial properties and potential applications in foods-a review. Int. J. Food Microbiol. 2004, 94, 223–253. Fagen, J. R.; Leonard, M. T.; McCullough, C. M.; Edirisinghe, J. N.; Henry, C. S.; Davis, M. J.; Triplett, E. W. Comparative genomics of cultured and uncultured strains suggests genes essential for free-living growth of Liberibacter. PLoS One 2014, 9, e84469. Casterline Jr, J. L.; Barnett, N. M.; Ku, Y. Uptake, translocation, and transformation of pentachlorophenol in soybean and spinach plants. Environ. Res. 1985, 37, 101–118. Athalye, V. V.; Ramachandran, V.; D'Souza, T. J. Influence of chelating agents on plant uptake of 51Cr, 210Pb and 210Po. Environ. Pollut. 1995, 89, 47–53. Koranda, J. J.; Robison, W. L. Accumulation of radionuclides by plants as a monitor system. Environ. Health Perspect. 1978, 27, 165–179. McKay, I.; Sharma, S. D.; Kirkwood, R. C.; Frank, R.; Retzlaff, G. Bentazone-surfactant interactions in three selected weed species of differing leaf characteristics. Weed management: balancing people, planet, profit. 14th Australian Weeds Conference, Wagga Wagga, New South Wales, Australia, 6-9 September 2004: papers and proceedings; Weed Society of New South Wales: 2004; pp 236−240. Hoagland, D. R.; Broyer, T. C. General nature of the process of salt accumulation by roots with description of experimental methods. Plant Phys. 1936, 11, 471–507.

32

Chapter 5

Use of Omics Methods To Determine the Mode of Action of Natural Phytotoxins Stephen O. Duke,1,* Zhiqiang Pan,1 Joanna Bajsa-Hirschel,1 Adela M. Sánchez-Moreiras,2 and Justin N. Vaughn3 1United

States Department of Agriculture, University of Mississippi, Oxford, Mississippi 38677, United States 2Dept. Plant Biology & Soil Sci., Univ. of Vigo, 36310, Vigo, Spain 3United States Department of Agriculture, Athens, Georgia, United States *E-mail: [email protected]

Technology has greatly increased the power of omics methods to profile transcription, protein, and metabolite responses to phytotoxins. These methods hold promise as a tool for providing clues to the modes of action of such compounds. However, to date, only two putative modes of action have been found with these methods; one with proteomics and the other with metabolomics. As with traditional physiological methods (physionomics) for mode of action discovery, differentiating between primary and secondary and tertiary effects is problematic. This problem can partially be overcome by careful experimental design. More powerful tools for metabolic pathway analysis are making transcriptome data easier to interpret with regard to the potential phytotoxin target site. Stable isotope measurement of metabolite pool turnover (fluxomics) has the potential to improve the insight into metabolomics for mode of action discovery.

Introduction Like all other living things, largely based on their genomes, plants have a complex array of responses to anything that affects them. Many of these responses begin with transcriptional changes that cascade to the proteome and metabolome, © 2018 American Chemical Society

resulting in an array of biochemical and physiological changes. Advances in capabilities in sequencing mRNA and proteins and identifying components of metabolite extracts, as well as determining the quantity of each component, have resulted in the rise of “omics” technologies to analyze responses of plants to anything that affects them at the transcriptome, proteome, and metabolome levels in great detail. The physiological profile of a plant that results from changes at these more fundamental levels has been termed physionomics. The “omics” suffix has been use to describe several subsets of each of the three basic omics levels (e.g., glycomics and lipidomics under metabolomics), but we only discuss the main omics categories in this short chapter. The topic of using omics to find herbicide and natural phytotoxin modes of action has been reviewed before (1, 2). This short review updates our earlier review. A herbicide with a new mode of action has not been introduced for over thirty years (3). Rapidly evolving herbicide resistance has made the value of a new herbicide mode of action greater than at any time since the advent of the age of herbicides in agriculture (4). Natural products are good sources of phytotoxic compounds with both proven and putative novel modes of action that could be used for herbicides (5). The modes of action of many of these promising compounds is unknown, largely because the determination of the molecular target of a phytotoxin is not a trivial pursuit. Omics technologies offer powerful tools in mode of action discovery research. Any one of these “omics” technologies can be used to develop profiles of plant responses to herbicides or phytotoxins with known specific modes of action. The omic response of a plant to a compound with an unknown mode of action can be compared to a library of responses to compounds with known modes of action. At a minimum, this approach should be able to identify the modes of action that are similar to those in the library. If the compound has a novel mode of action, the effect on the omics profile should not be in the library, but it might provide a hint as to the molecular target. Several herbicide discovery companies have tried this approach to new target site discovery, but only BASF has published papers that provide their methods in detail and a description of their limited success (2, 6). Like any tool, omics technologies have their weaknesses. The omics response to a toxin is dependent on the dose of the toxin and the time after exposure. Although all commercial herbicides appear to have one primary molecular target site, at high doses, a phytotoxin might directly affect secondary targets, and thereby complicate the results. At low doses, the plant might compensate too rapidly to see an effect or even stimulate growth, as low doses of phytotoxins commonly cause hormesis – the stimulatory effect of a non-toxic dose of a toxicant (7). So, intermediate doses such as concentrations needed to inhibit growth by 50 to 80% (ED50 or ED80) are preferred. After the target site is inhibited, there can be many secondary and/or tertiary effects of a herbicide. In fact, at higher doses, most everything is eventually affected. Stress and detoxification responses can be dramatic with herbicides, athough these effects are not generally mode of action specific. Thus, early time points after exposure are preferred to avoid a cascade of effects that can mask effects more closely tied to the primary target. In whole plants or plant organs (e.g., the leaf), there can be tissue to tissue and cell type to cell type variation in response to a herbicide, even if each cell 34

gets the same dose. To make things more complicated, the distribution of a herbicide in a plant can vary dramatically between organs, tissues and cell type, depending on many factors (8). Compounds with different modes of action can have most of their effects in different cell types. For example, compounds that inhibit photosynthesis or production of carotenoids or chlorophyll should act primarily on green, photosynthesizing cells, whereas mitotic inhibitors act mostly in meristematic cells. Furthermore, the mRNA, proteins, and metabolites can vary dramatically between different cells types even before being affected by a phytotoxin. Although, there are methods for sampling specific cell types (especially for transcriptomics), if the mode of action is unknown, this approach may not be useful (9). Thus, in most studies, the mRNA, protein, or metabolites measured are a mixture from different tissues and cell types. Less affected tissues and cell types can mask what might be dramatic effects in more affected tissues and cell types. Another consideration is whether a compound might have more than one molecular target site. This may be unlikely, as the twenty or so commercial herbicide mechanisms of action involve only one target at the doses used for weed management (3). However, this may not be the case with some natural phytotoxins. For example, the allelochemical sorgoleone targets several molecular targets (10). Evolution of target site resistance to a compound with several molecular targets is much less likely than to a compound with a single target, a fact that has plagued agriculture in recent decades. Thus, results from these omics technologies must be interpreted with several qualifications. Omics results alone cannot prove a mode of action, but can only provide clues. Clues from omics studies must be followed up with in vitro assays of effects on putative molecular targets and/or genetic alteration of the putative target for unequivocal proof of the target site. Nevertheless, the unprecedented power of these methods to provide detailed information on effects at several levels promises to provide considerable new information about mode of action and effects of herbicides and phytotoxins on the biochemistry and physiology of plants.

Transcriptomics After genomics provided complete genomes of Arabidopsis thaliana with reasonably good annotation of the genes, especially those of primary metabolism, transcriptomic responses of plants to herbicides and phytotoxins via DNA microarray chips became possible. Because Arabidopsis has genes for all primary metabolism enzymes, and most all herbicide targets are enzymes of primary metabolism, transcriptomics of Arabidopsis seemed like a good approach for probing the mode of action of herbicides. Our earlier review covered what was had been achieved with herbicides and transcriptomics up until 2012 (1). A review earlier than this mostly hypothesized how this technology could be used to study herbicide modes of action (11). To summarize, despite the promise of this technology, no new modes of action have been discovered with this method. Tresch summarized the results of this methodology up to about 2012: 35

“So far, transcriptomics techniques were used to describe the similarities of new compounds with well characterized ones, but a substantial contribution to the description of a new target site has rarely been reported” (12). From the published literature, the term ‘rarely’ may be generous, but there may be successes in the herbicide discovery and development industry that are unreported. However, these early studies provided some good information about the limitations of transcriptome studies of herbicide action. Very soon after exposure of a plant to a phytotoxins with an unknown mode of action, genes involved in stress responses and detoxification are strongly upregulated (13). Thus, there is no “smoking gun” in the large number of upregulated or downregulated genes. For example, our earlier work with yeast transcriptomics, as affected by inhibitors of enzymes of the ergosterol pathway, showed that even when a molecular target site is known, the gene for the target enzyme may be less affected than genes of other enzymes in the same metabolic pathway (14). Likewise, Zhu et al. (15) did not find any indication that 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of glyphosate from glyphosate-susceptible soybean transcriptome responses to glyphosate. In the case of the natural phytotoxin cantharidin, transcription of as much as 10% of the approximately 24,000 genes of Arabidopsis is significantly affected within 24 h of treatment by a dose that inhibits growth by 30% (16). This compound and its commercial herbicide analog, endothall, inhibit all of the several serine/threonine protein phosphatases (PPPs) of the plant, thereby affecting many different processes simultaneously (17). The transcriptome response fits the mode of action, but, as with the yeast example, it is doubtful that the transcriptome results would point to PPPs as the target site if the target was not known. Expression of the genes encoding both catalytic and regulatory A subunits of PPPs was not effected by cantharidin. Expression of the B regulatory subunits was significantly, but not dramatically, upregulated at 2 h, but not at 10 and 24 h after treatment. A drawback to DNA chip transcriptomics technology is inaccurate measurements of low abundance transcripts. RNA-Seq has become the preferred method for transcriptome analysis due to its much improved efficiency for low abundance transcripts (18). RNA-Seq has a 9000-fold range of sensitivity, versus a few hundred-fold ranges for microarray methods. RNA-Seq has not been used so far for phytotoxin mode of action studies, but it has been used extensively in studies of mechanisms of herbicide resistance (19). However, all of this resistance work has been done with weed species for which we do not have the full genome and for which gene annotation is less developed than that of Arabidopsis. It has been mostly used to determine nontarget site-based resistance changes in expression of multiple genes. RNA-Seq can also reveal mutations that might be associated with the evolved resistance, whether target site or non-target site based. There are serious problems in much of this work related to the lack of genetic uniformity within the populations studied (19). This is not an issue in the use of RNA-Seq for mode of action studies. Despite the superiority of RNA-Seq over DNA chip technologies for transcriptomics research, there have been essentially no mode of action studies published on this topic using RNA-Seq methods. This is unfortunate, as the massive amount of detailed data generated with RNA-Seq allows for detection of gene transcription effects, even 36

for genes with a low transcription rate. There is evidence that the best herbicide target sites are those that are present in low abundance, thus requiring less herbicide to poison an effective percentage of the target (3). The only publications using RNA-Seq to probe herbicides and plant responses other than resistance studies have been on stress responses (20, 21). The improved resolution of a wide range of gene expression with RNA-seq allows better analyses of effects of a toxin on genes of a complete biochemical pathway. Most herbicides have modes of action associated with enzymes or energy transduction proteins of primary metabolism. One of the analytical tools for analyzing effects on genes associated with primary metabolism is the R software (keggseq) associated with the Kyoto Encyclopedia of Genes and Genomes (KEGG). This software will determine which pathways and processes are significantly affected and will provide a detailed image of an entire pathway, showing effects on all of the genes. For example, the effects of an IC50 concentration of t-chalcone, a demonstrated phytotoxin, on the genes of the phenylpropanoid pathway of Arabidopsis thaliana are shown in Figure 1 (22). KEGG analysis is extremely helpful in identifying what pathways might be first and/or most affected by a phytotoxin, thereby indicating a potential mode of action. Other pathway analysis software for transcriptomics data is available (e.g., GAGE and Pathview). In summary, although there have been huge improvements in the technology of transcriptomics and in the software for analysis of the massive amounts of data produced, this approach to determination of new modes of action of phytotoxins has not yet been successful. Other omics approaches that are at a level more closely related to the target site might be more appropriate.

Proteomics Proteomics is less exact than transcriptomics, partly because low abundance proteins are often missed with current methods. There are significantly fewer proteins than genes to deal with, simplifying analysis. However, post-translational modification of proteins can complicate interpretation of results. Our previous review covered the small amount of use of this technology for herbicide and phytotoxin mode of action research through 2012 (1). Little has been done since then, with herbicide related proteomics studies being almost entirely focused on herbicide resistance (23). An exception is the publication of Bajsa et al. (24) of the proteomics study that came from the same experiment that we discuss above regarding the effects of cantharidin on the transcriptome (16). The samples for protein analysis were from the same experiment, but there was little correspondence between affected gene transcripts and proteins, other than with glutathione-S-transferases and enzymes involved in xenobiotic detoxification. The 2D gel analysis of the proteins could not resolve low abundance proteins, such as PPP subunits. By immunochemistry, the catalytic subunits of PP2Ac, one of the cantharidin target sites, were upregulated at 2 and 10 h after treatment, even though the transcription of these genes was not affected (see above). These effects were too subtle to have lead to identification of the target site of this potent 37

phytotoxin. Lack of correlations between transcriptome and proteome effect is not unusual and can be due to multiple factors (1, 25, 26). There has been one paper in which proteomics has been used to identify a putative target of a natural phytotoxin. Zhao et al. (27) found α-terthienyl to affect sixteen proteins associated with energy transduction in A. thaliana. In particular, transketolase protein was signficantly reduced, even though there was higher mRNA expression for the transketolase gene. A mutant with an altered transketolase was less affected by the phytotoxin, and the in vitro activity of the enzyme from the mutant was less affected by the phytotoxin than that from the wild type (Figure 2). Nevertheless, the very weak effect of the toxin on the in vitro enzyme activity is hard to reconcile with a primary target site.

Figure 1. Effects of 21 μM t-chalcone on glutathione metabolism transcripts of Arabidopsis seedling roots 3 h after treatment. Red = upregulation; Green = downregulation. (see color insert) 38

Figure 2. Effects of α-terthienyl on the growth (photographs) of wild type (Col-0) and transketolase mutant (attkl1) A. thaliana plants and on in vitro transketolase activity from the plants. Reproduced with permission from Zhao et al. (27). (see color insert)

In a less definitive study, Monazzah et al. (28) found the broad spectrum phytotoxin oxalic acid to cause differential expression of 17 proteins in sunflower. Upregulated proteins were involved in carbon fixation, and photosynthesis, as well as stress responses such as apoptosis, heat shock proteins, flavonol synthesis, and antioxidant enzymology. Downregulated proteins included actin, an ATP synthase subunit, the cupin family, and ketol-acid reducto-isomerase. There was no clear indication of a primary target. Similarly, Xie et al. (29) examined changes on the proteome of cotton plants caused by the toxin produced by Verticillum dahlia, and observed upregulation of stress-related protein and downregulation of proteins involved in cell structure and primary metabolism, but no indication of the toxin’s molecular target was discerned. Protein site identification based on interaction of the inhibitor or affector with the protein (chemoproteomics) is being used to find target sites of pharmaceuticals (30). This approach has recently been used to find the target site of the natural product-based herbicide cinmethylin (see discussion below).

Metabolomics Metabolomic profiling has been a method of choice for BASF for new mode of action discovery of herbicides (2, 6, 31). This method generally involves measuring the levels of relatively small number of primary plant metabolites – 39

compounds with molecular weights of 500 or less. Few of these compounds are clear biomarkers of a particular mode of action. Exceptions are large increases in shikimic acid, protoporphyrin IX, and sphingoid bases in the cases of inhibitors of EPSPS, protoporphyinogen IX oxidase, and ceramide synthase, respectively (32–34). Such clear examples of biomarker metabolites for particular modes of action are exceptions. The BASF group claimed that metabolite and physionomic (see below) profiling were critical to their determination that the cineole analog herbicide cinmethlin was an inhibitor of tyrosine amino transferase (6). However, as with the case of transketolase inhibitor discussed above, the relatively high amount of compound needed for in vitro enzyme inhibition was hard to reconcile with the herbicidal activity of the compound. Most herbicides are active at micromolar or lower concentrations at the enzyme level. More recent work by BASF, using chemoproteomics, found that acyl-ACP thioesterases, enzymes involved in fatty acid biosynthesis, are the actual target sites of cinmethylin (35). Cinmethylin is an old herbicide based on natural cineole structure, but the MOA was not confirmed before this recent work. Since our review of 2013, most papers on metabolomics and herbicide effects on plants deal with differentiating herbicide-resistant from wild type weeds (1, 36–38). There are many more papers on the effect of herbicides on the metabolome of mammals. Nevertheless, Pederson et al.,examined the effects of glyphosate and two phytochemical phytotoxins (biochanin A and catechin) on the metabolome of A. thaliana (39). Growth-reducing doses of the three compounds affected 72 to 80% of the plant metabolites. Shikimate increases in response to glyphosate was the only clear indication of a mode of action. Kim et al. (40) provided a metabolomic analysis of the effects of the natural phytotoxin coronatine on duckweed. The concentrations of this jasmonic acid analog that were used were not phytotoxic, so the results could not be used to study phytotoxicity. A problem with all studies based on pool sizes, whether it be mRNA, proteins, or metabolites is that the pool size does not give an indication of the pool turnover rate. The turnover rate in two pools of the same size can be quite different. An effector, whether it is environmental or chemical, can have a profound effect on pool size and/or pool turnover rate. For example, 24 h of light increases the phenylalanine pool flux rate in dark-grown maize seedling roots more than three fold, even though the pool size was reduced by 40% (41). Pool size alone would have indicated that phenyalanine synthesis was reduced when it was actually increased. This early work was done with 14C pulse chase experiments. With GC/MS or LC/MS, similar studies can now be done in metabolomics with stable isotopes (usually 13C or 15N), an approach termed fluxomics (42). Using stable isotope-resolved metabolomics, glyphosate was found to increase de novo amino acid synthesis in glyphosate-susceptible and -resistant Amaranthus palmeri (Figure 3) (37). This more sophisticated approach to metabolomics is more likely to provide a clearer insight into the effects of a phytotoxin on plant metabolism.

40

Figure 3. 15N isotopologue enrichment of amino acids in glyphosate-treated sensitive (S)- and resistant (R)-biotypes of Amaranthus palmeri at 36 h after treatment. Asterisks designate a significantly enhanced enrichment in the R biotype. Reproduced with permission from Maroli et al. (37). (see color insert)

Araniti et al. analyzed the phytotoxic effects of coumarin through a metabolomic, proteomic, and morpho-physiological approach in mature Arabidopsis plants (43). Metabolomic analysis found increases of certain amino acids and a high accumulation of soluble sugars, although no definitive mode of action could be established for this phytotoxic secondary metabolite. Proteomic analysis provided no clues to the mode of action.

Physionomics Physionomics is the use of a battery of physiological assays and whole plant bioassays to profile the physiological effects of a phytotoxin (2). Coupled with metabolomics, BASF has used this approach to profile the physiological effects of all known herbicide modes of action as well as the mode of action of several phytotoxins. A recent example of the profile of an antimalarial compound with good herbicidal activity is shown in Figure 4 (44). Such physiological data can be used to focus on aspects of the transcriptome, proteome, or metabolome that are related to observed physiological effects.

41

Figure 4. Physiological profile of the effects of MMV007978 in a series of bioassays compared to an untreated control. See Corral et al. (43) for details. Reproduced with permission of Wiley press.

Conclusions Omics methods are powerful tools; however, like any tool, they have be used properly. To date, we cannot point to any purely omics study that has clearly found a new phytotoxin mode of action. However, the studies that have been done provide a wealth of information on secondary and tertiary effects of natural phytotoxins. At best, omics can suggest a mode of action, after which it has to be verified by biochemical, physiological, and/or genetic means.

References 1. 2.

3. 4.

Duke, S. O.; Bajsa, J.; Pan, Z. Omics methods for probing the mode of action of natural and synthetic phytotoxins. J. Chem. Ecol. 2013, 39, 333–347. Grossmann, K.; Christiansen, N.; Looser, R.; Tresch, S.; Hutzler, S.; Pollman, S.; Ehrhardt, T. Physionomics and metabolomics – two key approaches in herbicide mode of action discovery. Pest Manage. Sci. 2012, 68, 294–304. Duke, S. O. Why have no new herbicide modes of action appeared in recent years? Pest Manage. Sci. 2012, 68, 505–512. Heap, I.; Duke, S. O. Overview of herbicide-resistant weeds worldwide. Pest Manage. Sci. 2018, 74, 1040–1049. 42

5. 6.

7. 8. 9.

10.

11.

12. 13.

14.

15.

16.

17.

18.

Dayan, F. E.; Duke, S. O. Natural compouns as next generation herbicides. Plant Physiol. 2014, 166, 1090–1105. Grossmann, K.; Hutzler, J.; Tresch, S.; Christiansen, N.; Looser, R.; Ehrhardt, T. On the mode of action of the herbicide cinmethylin and 5-benzyloxymethyl-1,2-isoxazolines: Putative inhibitors of plant tyrosine aminotransferase. Pest Manage. Sci. 2012, 68, 482–492. Belz, R. G.; Duke, S. O. Herbicides and plant hormesis. Pest Manage. Sci. 2014, 70, 698–707. Duke, S. O. Pesticide dose – A parameter with many implications. Am. Chem. Soc. Symp. Ser. 2017, 1249, 1–13. Sakai, K.; Taconnat, L.; Borrega, N.; Yanouni, J.; Brunaud, V.; Paysant-LeRoux, C.; et al. Combining laser-assisted microdissection (LAM) and RNAseq allows to perform a comprehensive transcriptomic analysis of epidermal cells of Arabidopsis embryo. BMC Plant Methods 2018, 14, 10. Dayan, F. E.; Rimando, A. M.; Pan, Z.; Baerson, S. R.; Gimsing, A.-L.; Duke, S. O. Molecules of interest: Sorgoleone. Phytochemistry 2010, 71, 1032–1039. Eckes, P.; Busch, M. Fast identification of the mode of action of herbicides by DNA chips. In Methods in Crop Protection Research, Jeschke, P., Krämer, W., Schirmer, U., Witschel, M., Eds.; Wiley-VCH: Weinheim, Germany, 2007; pp 161−174. Tresch, S. Strategies and future trends to identify the mode of action of phytotoxic compounds. Plant Sci. 2013, 212, 60–71. Baerson, S. R.; Sánchez-Moreiras, A.; Pedrol-Bonjoch, N.; Schulz, M.; Kagan, I. A.; Agarwal, A. L.; Reigosa, M. J.; Duke, S. O. Detoxification and transcriptome response in Arabidopsis seedlings exposed to the allelochemical benzoxazolin-2,(3H)-one (BOA). J. Biol. Chem. 2005, 280, 21867–21881. Kagan, I. A.; Michel, A.; Prause, A.; Scheffler, B. E.; Pace, P.; Duke, S. O. Gene transcription profiles of Saccharomyces cerevisiae after treatment with plant protection fungicides that inhibit ergosterol biosynthesis. Pestic. Biochem. Physiol. 2005, 82, 133–153. Zhu, J.; Patzoldt, W. L.; Shealy, R. T.; Vodkin, L. O.; Clough, S. J.; Tranel, P. J. Transcriptome response to glyphosate in sensitive and resistant soybean. J. Agric. Food Chem. 2008, 56, 6355–6363. Bajsa, J.; Pan, Z.; Duke, S. O. Transcriptional responses to cantharidin, a protein phosphatase inhibitor, in Arabidopsis thaliana reveal the involvement of multiple signal transduction pathways. Physiol. Plantarum 2011, 143, 188–205. Bajsa, J.; Pan, Z.; Dayan, F. E.; Owens, D. K.; Duke, S. O. Validation of serine/threonine protein phosphatase as the herbicide target site of endothall. Pestic. Biochem. Physiol. 2012, 102, 38–44. Wang, C.; Gong, B.; Bushel, P. R.; Thierry-Mieg, J.; Thierry-Mieg, D.; Xu, J.; et al. The concordance between RNA-seq and microarray data depends on chemical treatment and transcript abundance. Nat. Biotechnol. 2014, 32, 926–932. 43

19. Giacomini, D. A.; Gaines, T.; Beffa, R.; Tranel, P. J. Optimizing RNA-seq studies to investigate herbicide resistance. Pest Manage. Sci. 2018, 74; DOI: 10.1002/ps.4822. 20. Li, R. T.; Chen, L.; Jing, J. Z.; Luo, F.; Yang, H. A collection of cytochrome P450 monooxygenases genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants. Ecotoxicol. Environ. Safety 2015, 119, 25–34. 21. Wright, A. A.; Sasiharan, R.; Koski, L.; Rodriguez-Carres, M.; Peterson, D. G.; Nandula, V. K; et al. Transcriptomic changes in Echinochloa colona in response to treatment with the herbicide imazamox. Planta 2018, 247, 369–379. 22. Díaz-Tielas, C.; Sotelo, T.; Graña, E.; Reigosa, M. J.; Sánchez-Moreiras, A. M. Phytotoxic potential of trans-chalcone on crop plants and model species. J. Plant Growth Regul. 2013, 33, 181–194. 23. Gonzalez-Torralva, F.; Brown, A. P.; Chivasa, S. Comparative proteomic analysis of horseweed (Conyza canadensis) biotypes identifies candidate proteins for glyphosate resistance. Sci. Rep. 2017, 7, 42565. 24. Bajsa, J.; Pan, Z.; Duke, S. O. Cantharidin, a protein phosphatase inhibitor with broad effects on the the transcriptome, strongly upregulates glutathioneS-transferase in the Arabidopsis proteome. J. Plant Physiol. 2015, 173, 33–40. 25. Payne, S. H. The utility of protein and mRNA correlation. Trends Biochem. 2015, 40, 1–3. 26. Narayananan, R.; Van de Ven, W. J. M. Transcriptome and proteome analysis: A perspective on correlation. MOJ Proteomics Bioinformatics 2014, 1, 00027; DOI: 10.15406/mojpb.2014.01.00027. 27. Zhao, B.; Huo, J.; Liu, N.; Zhang, J.; Dong, J. Transketolase is identified as a target of herbicidal substance α-terthienyl by proteomics. Toxins 2018, 10, 41; DOI: 10.3390/toxins10010041. 28. Monazzah, M.; Enferadi, S.; Sattar, T.; Soleimani, M. J.; Rabiei, Z. An unspecific oxalic acid and its effect on sunflower proteome. Aust. J. Bot. 2016, 64, 219–226. 29. Xie, C.; Wang, C.; Wang, X.; Yang, X. Proteomics-based analysis reveals that Verticillium dahliae toxin induces cell death by modifying the synthesis of host proteins. J. Gen. Plant Pathol. 2013, 79, 335–345. 30. Bantscheff, M.; Drewes, G. Chemoproteomic appoaches to drug target identification and drug profiling. Bioorganic Med. Chem. 2012, 20, 1973–1978. 31. Grossmann, K.; Niggeweg, R.; Christiansen, N.; Looser, R.; Ehrhardt, T. The herbicide saflufenacil (Kixor™) is a new inhibitor of protoporphyrinogen IX oxidase activity. Weed Sci. 2010, 58, 1–9. 32. Duke, S. O.; Baerson, S. R.; Rimando, A. M. Herbicides: Glyphosate. In Encyclopedia of Agrochemicals; Plimmer, J. R., Gammon, D. W., Ragsdale, N. N., Eds.; John Wiley & Sons: New York, 2003. https://onlinelibrary.wiley.com/doi/full/10.1002/047126363X.agr119 (accessed April 15, 2018). 44

33. Dayan, F. E.; Duke, S. O. Herbicides: Protoporphyrinogen oxidase inhibitors. In Encyclopedia of Agrochemicals; Plimmer, J. R., Gammon, D. W., Ragsdale, N. N., Eds.; John Wiley & Sons: New York, NY, 2003; Vol. 2, pp 850−863. 34. Abbas, H. K.; Duke, S. O.; Sheir, W. T.; Duke, M. V. Inhibition of ceramide synthesis in plants by phytotoxins. In Advances in Microbial Toxin Research and its Biochemical Exploitation; Upadhyay, R. K., Ed.; Kluwer Academic/ Plenum: London, 2002; pp 211−219. 35. Campe, R.; Hollenbach, E.; Kämmerer, L.; Hendriks, J.; Höffken, H. W.; Kraus, H.; Lerchl, J.; Mietzner, T.; Tresch, S.; Witschel, M.; Hutzler, J. A new herbicidal mode of action: Cinmethylin binds acyl-ACP thioesterae and inhibits plant fatty acid biosynthesis. Pestic. Biochem. Physiol. 2018; DOI: 10.1016/j.pestbp.2018.04.006. 36. Maroli, A. S.; Nandula, V. K.; Dayan, F. E.; Duke, S. O.; Gerard, P.; Tharayil, N. Metabolic profiling and enzyme analyses indicate a potential role of antioxidant systems in complementing glyphosate resistance in an Amaranthus palmeri biotype. J. Agric. Food Chem. 2015, 63, 9199–9209. 37. Maroli, A. S.; Nandula, V. K.; Duke, S. O.; Tharayil, N. Stable isotope resolved metabolomics reveals the role of anabolic and catabolic processes in glyphosate-induced amino acid accumulation in Amaranthus palmeri biotypes. J. Agric. Food Chem. 2016, 64, 7040–7048. 38. Maroli, A. S.; Nandula, V. K.; Duke, S. O.; Gerard, P.; Tharayil, N. Comparative metabolomic analysis of two Ipomoea lacunosa biotypes with contrasting glyphosate tolerance elucidates the glyphosate-induced differential perturbations in cellular physiology. J. Agric. Food Chem. 2018, 66, 2027–2039. 39. Pederson, H. A.; Kudsk, P.; Fomsgaard, I. Metabolic profiling of Arabidopsis thaliana reveals herbicide- and allelochemical-dependent alterations before they become apparent on plant growth. J. Plant Growth Regul. 2015, 34, 96–107. 40. Kim, J.-Y.; Kim, H.-Y.; Jeon, J.-Y.; Kim, D.-M.; Zhou, Y.; Lee, J. S.; Lee, H.; Choi, H.-K. Effects of coronatine elicitation on growth and metabolic profiles of Lemna pauscicostata culture. PLoS One 2017; DOI: 10.1371/journal.pone.0187622. 41. Duke, S. O.; Naylor, A. W. Light effects on phenylalanine ammonia-lyase substrate levels and turnover rates in maize seedlings. Plant Sci. Lett. 1976, 6, 361–367. 42. Srivastava, A.; Kowalski, G. M.; Callahan, D. L.; Meikle, P. J.; Creek, D. J. Strategies for extending metabolomics studies with stable isotope labeling and fluxomics. Metabolites 2016, 6, 32/1–32/13. 43. Araniti, F.; Scognamiglio, M.; Chambery, A.; Russo, R.; Esposito, A.; D'Abrosca, B.; Fiorentino, A.; Lupini, A.; Sunseri, F.; Abenavoli, M. R. Highlighting the effects of coumarin on adult plants of Arabidopsis thaliana (L.) Heynh. by an integrated -omic approach. J. Plant Physiol. 2017, 213, 30–41.

45

44. Corral, M. G.; Leroux, J.; Tresch, S.; Newton, T.; Stubbs, K. A.; Mylne, J. S. Structure-activity analysis of an antimalarial lead compound against Arabidopsis thaliana. Pest Manage. Sci. 2018, 74; DOI: 10.1002/ps.4872.

46

Chapter 6

Pesticides on the Inside: Exploiting the Natural Chemical Defenses of Maize against Insect and Microbial Pests Shawn A. Christensen,* Charles T. Hunter, and Anna Block Chemistry Research Unit, Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, 1700 SW 23rd Drive, Gainesville, Florida 32608, United States *E-mail: [email protected]

Maize (Zea mays) is one of the most significant and abundantly grown crop plants in the United States with yields exceeding $50 billion annually. Despite major improvements in productivity, it is estimated that billions of bushels are lost per year due to the combined effects of herbivory and disease. The use of insecticides/fungicides has been a long-standing strategy to manage pests; however, broad public recognition of the environmental and human-health concerns associated with synthetic pesticides has heightened interest in the use of genetic tools to provide immunity. Breeding programs continue to work towards optimizing maize lines for effective resistance, but insufficient knowledge of the molecular mechanisms and specific defense chemicals that regulate pest resistance limits rapid progress in this area. To address this issue, efforts have been made to elucidate and characterize important maize defense chemicals including benzoxazinoids, terpenoid phytoalexins, free fatty acids, hormones, signaling peptides, inducible volatiles, and phenylpropanoids. In this chapter, we review the existing knowledge of these maize defense chemicals and discuss current and future strategies to exploit them for improved resilience to biological threats.

© 2018 American Chemical Society

Introduction Maize (Zea mays) is a key agricultural commodity both in the United States and throughout the world, accounting for the majority of animal feedstocks, while also being important for biofuel production, human consumption, and dozens of industrial applications. While advances in maize breeding and biotechnology have resulted in unprecedented yields, biotic challenge from pathogens and insect pests continue to cause major losses. Recent surveys show that disease pressure alone accounts for approximately 10% of reduced production in the United States, averaging 1.5 billion bushels (valued at over $5 billion) in losses annually (1). Common fungal pathogens responsible for these losses include mycotoxigenic ear-rotting (e.g. Fusarium verticillioides and Aspergillus flavus), stalk-rotting (e.g. Fusarium graminearum and Colletotrichum graminicola), root-rotting (e.g. F. verticillioides and Pythium aristosporum) and leaf blight-causing (e.g. Cochliobolus heterostrophus and C. graminicola) fungi that devastate plant organs and contaminate seed with carcinogenic mycotoxins. Under recent climate conditions, post-harvest losses due to fungal derived mycotoxin contamination has been endemic, averaging an additional 1.1 billion bushels per year (1). In addition to fungal diseases, insect pests continue to cause significant damage to maize crops. Fall armyworm (Spodoptera frugiperda) and beet armyworm (Spodoptera exigua) defoliate plants, whereas the corn earworm (Helicoverpa zea) targets the developing cob, causing extensive damage to the seeds. Stem-boring insects such as the European corn borer (Ostrinia nubilalis) lead to structural damage (i.e. lodging) and microbial colonization through the creation of humid and contaminated tunnels conducive to stalk rot infections (2, 3). Also problematic are phloem-feeding insects such as the corn leaf aphid (Rhopalosiphum maidis), which causes wilting and curling of leaves. Moreover, below-ground herbivores like the western corn rootworm (Diabrotica virgifera virgifera) damage roots and lead to poor water and nutrient uptake. In an attempt to control losses from pathogen and insect attack, billions of dollars are spent annually on insecticides and fungicides. Despite these investments, insect and pathogen damage continues to be a widespread problem. Ineffective insect/disease management strategies coupled with environmental and human health concerns associated with the use of fungicides/insecticides has led to a heightened interest in the use of genetic tools to develop more pest resistant plants. The most successful and widely adopted technology for insect resistance in maize has been the engineering of Bt toxins to provide protection against Lepidoptera larvae (caterpillars) (4). These natural toxins, originally isolated from the bacteria Bacillus thuringiensis, are selectively operative to avoid harming non-Lepidopteran insects (5), and their use has led to dramatic reductions in the application of broad-spectrum insecticides (6). While historically an effective strategy for maize resistance to earworms, the emergence of Bt resistance has reduced its effectiveness against certain maize pests (7). Considering the potential for further Bt resistance and the widespread reluctance to grow and consume genetically modified plants in some parts of the world, there is a pressing need for new multipronged approaches to pest management. One strategy is to exploit the naturally occurring chemical defenses of maize to defend against attacking 48

organisms. In this perspective we will give a brief overview of the predominant chemical defenses of maize and discuss possible strategies for utilizing them.

Maize Defense Chemicals Benzoxazinoids One of the first chemical defenses discovered in maize was a class of compounds collectively referred to as Benzoxazinoids (BXs). They were identified in the 1950s as important components of host resistance against Fusarium rot in rye (8) and O. nubilalis herbivory in maize (9). They have since been characterized as anti-insect and antimicrobial hydroxamic acids found in a variety of grasses and other plant species (10). In maize, BXs are constitutively produced in immature tissues such as young seedlings (11, 12), but are also inducible by pathogen infection (13, 14), insect herbivory (15, 16), aphid feeding (14), and wounding in mature tissue. The BXs are composed of a class of related metabolites whose biosynthesis, beginning with indole, is modulated by a series of enzymes, denoted as BX1 through BX14 (17–19). The BX cascade produces an array of compounds with varying levels of toxicity (20, 21), the most widespread of which in maize is DIMBOA (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one) (Figure 1).

Figure 1. Structures of select chemical defense compounds from maize. Glycosylation by BX8 and BX9 to convert the toxic aglycones into less-toxic glucosides allows for storage of BXs in vacuoles (22). When plant cells are damaged and vacuoles disrupted, glucosidases convert the glucosides back to 49

the biocidal aglycone toxins (23), whereupon they delay growth and potentially kill attacking pests (20, 21, 24, 25). Interestingly, the BX biosynthetic genes are clustered in maize, with Bx1 through Bx8 all localized to the short arm of chromosome 4 (18), perhaps allowing for co-regulation of gene expression and co-inheritance of the functionally-related loci. The BXs and their breakdown products also play important roles in allelopathy by suppressing growth of competing plants (26), and have thus attracted attention for utilization in weed control (27, 28). Terpenoid Phytoalexins Nonvolatile acidic terpenoids constitute a more recently discovered class of maize anti-insect and antimicrobial compounds. Initially observed in maize stems infested with O. nubilalis, six acidic diterpenoids were identified as ent-kaurane-related structures, termed kauralexins. Common maize pathogens such as C. heterostrophus, F. graminearum, and Rhizopus microsporus elicit transcript accumulation of the kauralexin biosynthesis gene ent-copalyl diphosphate synthase known as Anther Ear 2 (An2), leading to significant increases in kauralexin concentrations (29, 30). In vitro studies provided initial evidence for the antimicrobial activity of select kauralexins against leaf-, ear-, and stalk-rot pathogens (29, 30). This activity was later confirmed in vivo using null mutations in An2, demonstrating that kauralexins play an important role in maize defense against pathogens (29). In addition to kauralexins, novel acidic sesquiterpenoids, termed zealexins, were also recently identified (31, 32). Zealexins are pathogen-elicited β-macrocarpene derivatives that appear to be ubiquitous in maize. Like kauralexins, zealexins have demonstrated strong anti-fungal growth activity against pathogens such as R. microsporus, A. flavus, and F. graminearum, supporting a broad role for nonvolatile terpenoid defenses in maize (29, 31, 32). The comparative accumulation of phytoalexins can vary widely and be organ- and stress-dependent (29–32). For example, the nonvolatile oxygenated sesquiterpene β-costic acid was recently characterized as one of the predominant root phytoalexins, promoting below-ground fungal and herbivore resistance (33). β-costic acid is produced by cyclization of farnesyl diphosphate to β-selinene by Terpene Synthase 21 (TPS21), and can accumulate to concentrations >100 µg·g-1 fresh weight in pathogen-challenged roots. Such concentrations were sufficient to inhibit the in vitro growth of several fungal pathogens and the corn root worm (Diabrotica balteata) (33). An even more recent discovery in maize root phytoalexins revealed non-acidic diterpenoids, termend dolabralexins (34). These newly identified derivatives of An2 and kaurene synthase-like 4 are elicited by pathogen attack and oxidative stress, demonstrating yet another class of maize phytoalexins with functional roles in defense. Free Fatty Acids Free fatty acids (FAs) and their oxygenated and cyclized derivatives represent a broad class of lipid constituents that play protective roles against biotic stress in dicot species (35–37), however, comparatively less is known about their function 50

in maize and other monocots. Plant FAs largely consist of long hydrophobic unbranched chains of hydrocarbons with hydrophilic carboxylic acid functional groups on one end of the molecule. Some FAs have been shown to have a role in pathogen resistance. For instance, transgenic eggplant overproducing palmitoleic acid (16:1) was more resistant to the fungus Verticillium dahliae (38). C18 FAs are involved in both basal resistance (39) and in resistance-gene mediated responses via stimulation of NADPH-oxidase activity and subsequent reactive oxygen species production (40). Very long chain FAs (VLCFAs; C ≥ 20) play roles in structural defenses such as cuticle formation, as well as sphingolipid generation, cell signaling, and pathogen resistance (36). While correlations between VLCFAs and plant defense have been reported, it is not clear whether they or their derivatives are directly involved in defense. As very little is known about the role of VLCFAs in maize defense, we refer to the following reviews that discuss their potential defensive functions in other plant species (35–37). Phenylpropanoids Phenylpropanoids are induced in maize in response to biotic attack. For instance, maize roots or leaves infected with C. graminicola accumulate high levels of flavonoids such as naringenin chalcone, apigenin and genkwanin as well as other phenylpropanoids including 3-caffeoyl-quinic acid, N-p-coumaryltryptamine and feruloyl-feruloyl-glycerol (41). The functional role (if any) of many of these compounds in pest resistance remains to be elucidated, however, bioactivity has been determined for select phenylpropanoids. Flavonoids, for instance, are small molecule derivatives of 2-phenyl-benzylγ-pyrone that form more than 9,000 isoforms in the plant kingdom (42). A class of flavonoids, termed flavones (distinguished by a 2-phenylchromen-4-one backbone), has several constituents that have demonstrated defensive roles against maize insects and pathogens. One example is the C-glycosyl flavone, maysin (2′′-O-α-rhamnosyl-6-C-(6-deoxy-xylo-hexos-4-ulosyl)-luteolin), which is constitutively produced in maize silks and provides resistance against H. zea (43). The biosynthetic pathway for the production of maysin in maize has recently been elucidated (44). Its production is highest in silk tissue where it can reach up to 2% of silk dry weight. Maysin is also produced to lower levels in leaves where its accumulation and that of it’s precursor rhamnosylisoorentin can be induced in response to UV-B exposure (45). Other C-glycosyl flavones isolated from maize silks (e.g. apimaysin and methoxymaysin) and phenylpropanoids, such as chlorogenic acid have also demonstrated important herbivore resistance properties (46–48). Mechanistically, flavones and other phenylpropanoids can be oxidized to quinones by polyphenol oxidase in tissues wounded by insect feeding. These quinones act as antinutritive compounds by binding proteins and thus reducing bioavailability to insects (49, 50). An unfortunate side effect of this process is the commercially-undesirable trait of silk browning (51, 52), which has led to the deselection of these compounds during sweetcorn breeding and increased insect susceptibility. Although less is known about the functional benefit of flavonoids in antipathogenic responses, both maysin and maysin-3′-methyl ether have demonstrated selective antimicrobial 51

activity against several bacterial pathogens (53). One could speculate that the antimicrobial activity of these C-glycosyl flavones may also be due to their protein binding activities. Another class of phenylpropanoids that have defensive roles are phenolic esters that function in the peroxidase-catalyzed cross-linking of cell wall polysaccharides, lignin, and proteins (54). These compounds are thought to strengthen the cell walls to restrict entry of stem boring caterpillars and movement of fungal pathogens. The levels of these compounds, such as dehydrodiferulate isomers, negatively correlates with O. nubilalis damage in maize (55, 56). The amount of diferulic acid in the pericarp and aleurone tissues of maize kernels negatively correlates with both F. graminearum disease serverity and fungal growth (57). Interestingly the production of phenylpropanoids can be benifical for pests. For instance p-coumaroyltyramine can be metabolized by Spodoptera littoralis and leads to increased larval growth, possibly by acting as a nitrogen source (58). Additionally, the maize bacterial pathogen Pantoea stewartii injects the effector protein WtsE into maize cells that leads to the accumulation of coumaroyl tyramine and heightened pathogen susceptibility (59). Similarly, the biotrophic fungus Ustilago maydis uses the effector protein Tin2 to induce anthocyanin production and reduce liginin production in maize to enhance U. maydis pathogenicity (60).

Herbivore-Induced Plant Volatiles Herbivore-induced plant volatiles (HIPVs) are volatile compounds produced in response to attack by plant-chewing insects. These metabolites are generally released as complex blends that can include green leaf volatiles (GLVs), monoterpenes, sesquiterpenes, homoterpenes, and indole. HIPVs have been shown to play important roles in indirect defense by helping parasitic wasps cue in on their lepidopteran hosts (61, 62) and in plant-plant signaling (63, 64). The genetic characterization of several HIPV regulatory and biosynthetic pathways has extended our knowledge regarding their defensive roles against herbivory. For instance, mutations in a maize 13-lipoxygenase (LOX) caused significant reductions in production of both GLVs and the plant hormone jasmonic acid (JA), leading to attenuated HIPV emissions and attractiveness to parasitoid wasps (65). Expression profiling, correlation of gene and metabolite presence, and recombinant protein analysis have revealed several other enzymes involved in HIPV production. For monoterpenes, recombinant TPS26 was reported to make limonene and myrcene in vitro (66). The biosynthesis pathway for the homoterpenes (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and (3E,7E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene (TMTT) were also recently discovered in maize, resulting from the sequential action of TPS2 and cytochrome p450s (67). Moreover, for sesquiterpenes, TPS23 is responsible for synthesis of (E)-β-caryophyllene (68), while TPS1, TPS3, TPS4, and TPS10 all function in β-farnesene biosynthesis (69–71). While many studies have demonstrated a role for HIPVs in above-ground interactions, their function in below-ground 52

defense is also important. For example, Köllner et. al., (2008) showed that Tps23 is induced in maize roots by D. v. virgifera feeding and that the resulting (E)-β-caryophyllene it produces attracts entomopathogenic nematodes, the natural enemies of D. v. virgifera (68).

Plant Hormones and Regulation of Defense Chemistry Most maize defense chemicals are either produced or activated in response to biotic attack. This requires tight regulation and is necessary both to conserve plant resources and to limit unintended self-harm due to the toxic nature of many of these compounds. A major stratagem that plants use to control the appropriate production or activation of these defense metabolites is regulation by hormones. These plant hormones form a complex network of synergistic or antagonistic signals, each with different rates and strengths of signal perception, promulgation, and downstream responses that govern resistance (Figure 2).

Figure 2. A generalized view of the complex network of plant hormone synergies (solid arrows) and antagonisms (dashed lines). Abbreviations: ABA-abscisic acid; Aux-Auxin; GA-Gibberellic acid; CK-cytokinin; BR-brassinosteroid; SA-salicylic acid; ET-ethylene; JA-jasmonic acid.

53

The core biosynthetic and primary response elements for some plant hormones and their signaling pathways have been determined in model plants like Arabidopsis thaliana, but many of the regulatory processes and downstream signaling components remain unclear. This is especially true for maize, where comparatively little is known about the signaling networks that regulate the perception and response of maize to biotic challenge. It is important to understand how these responses are regulated, as responses to infection or insect attack tend to vary widely between different plant species. Nearly all known plant hormones have been shown to participate in regulating plant stress responses to some degree and are, therefore, likely to impact the control of plant defense chemistry.

Jasmonates Jasmonates are peroxidation products of α-linolenic (18:3) acid produced by 13-LOXs. They include the well-studied products of the 13-LOX pathway 12-oxo-phytodienoic acid (12-OPDA), the hormone JA, and their derivatives. Unlike biotrophic pathogens that induce SA-mediated responses, necrotrophic pathogens elicit host responses that promote the accumulation of jasmonates. For example, C. heterostrophus-infected maize plants showed a rapid accumulation of 12-OPDA and JA in both infected tissues and in healthy tissues adjacent to infection sites (72). Evidence for JA regulation in direct antimicrobial chemical defenses has also been observed with the induction of the maize phytoalexins, kauralexins and zealexins (30, 32). Using Mutator transposon insertions in Lox10 and Lox12 and the JA biosynthesis genes, Lox8, Opr7, and Opr8, genetic evidence demonstrated that JA is required for pathogen-infected maize to survive under field and laboratory conditions, and further established its necessity for senescence, herbivore resistance, and reproductive development (65, 73–75). While research pertaining to the 13-LOX pathway has been extensive, particularly in the study of jasmonates, knowledge of the 9-LOX pathway has been more elusive. In the year 2000, analysis of potato (Solanum lycopersicum) homogenate revealed two inefficiently cyclized 9-allene oxide cyclase products, 10-oxo-11,15-phytodienoic acid (10-OPDA) and the 18:2-derived 10-oxo-11-phytoenoic acid (10-OPEA). Although structurally similar to the jasmonate 12-OPDA, the predictable downstream derivatives remained intangible for more than a decade until a recent study in maize helped elucidate their structures and functions. While profiling JA and other known FAs in fungal infected maize tissues, high levels of 10-OPEA and a novel series of related analytes were detected. Large scale purification and structure elucidation of these metabolites revealed 9-LOX derived 12-, 14- and 18-carbon cyclopente(a)none FAs that conceptually parallel jasmonates (Figure 3) (72). Intitial studies of these novel cyclopente(a)none FAs demonstrated that they exceed jasmonates in abundance within infected tissues, display signaling properties that mediate defense gene expression, activate programmed cell death, and promote direct phytoalexin activity against both insects and pathogens (72). Whether or not any of these newly identified molecules would specifically be classified as hormones remains to be determined. 54

Figure 3. Working model for the biosynthesis pathway of novel cyclopente(a)none fatty acids (FAs) in comparison to jasmonates. Superscript letters and symbols indicate the following: 1 = 18:3 derived; 0 = 18:2 derived; S = saturated cyclopente(a)none ring; and U = unsaturated cyclopente(a)none ring. Ethylene Ethylene functions synergistically with JA to promote defense responses in plants, particularly against insects and necrotrophic pathogens. The interconnected and mutually-stimulating signaling properties of ethylene and JA have made teasing apart the precise roles for each hormone difficult. In maize, ethylene has been shown to be important for promoting phytoalexin production (30, 32), inducing HIPV emissions (76), and in regulating resistance to R. maidis and S. frugiperda (77, 78). Salicylic Acid Salicylic acid (SA) also has well-documented roles in plant defense responses to biotrophic and hemibiotrophic pathogens, where it induces pathogenesis-related genes and activates systemic acquired resistance (79). SA engages in cross-talk with other hormones, including JA and ethylene. These interactions have been demonstrated to be either antagonistic or synergistic, depending on the specific plant-biotic interaction (80–82). The roles of SA have been extensively studied in 55

dicots and rice, but very little is known about them in maize. SA does appear to be involved in defense against fungal pathogens, as SA application induces cysteine proteases (83), PR-1 gene expression (83), and trypsin and lectin inhibitors (84). Additionally, SA is suppressed by the biotrophic pathogen U. maydis (85). On the other hand, application of methyl salicylate results in higher recruitment of Diabrotica speciosa to maize plants, suggesting a possible role for SA in conferring susceptibility to below-ground herbivory (86). Abscisic Acid Abscisic acid (ABA) is well-known for its roles in abiotic stress responses, particularly for drought and salinity stresses. More recently, ABA has been shown to be involved in regulation of chemical defense pathways as well (87). Studies in maize show that ABA appears to regulate the production of DIMBOA and certain phenolic acids including chlorogenic acid, caffeic acid, and ferulic acid (88). It is also induced by exposure to HIPVs, including indole (89). Application of exogenous ABA to maize roots elicited terpenoid phytoalexins (90) and resulted in increased resistance to foliar pathogens and insects, including C. graminicola (91), S. littoralis and Setosphaeria turcia (88), suggesting a possible role for ABA in systemic acquired resistance (91). Conversely, above ground exogenous ABA application led to increased susceptibility to C. graminicola (92). While ABA likely has a role in defense against biotic attack in maize, our current understanding is based solely on metabolite profiling or exogenous chemical applications. Studies using loss-of-function mutants in ABA biosynthesis or perception genes will help to better determine the role of ABA in maize defense. Other Plant Hormones and Enogenous Maize Signals Brassinosteroids, gibberellic acid, cytokinins, and auxins are all important for plant growth and development, but also appear to contribute to plant defense. In the maize relatives barley and brachypodium, brassinosteroids were found to be negative regulators of resistance to necrotrophic pathogens (93, 94). In rice, mutants of a gibberellic acid receptor accumulate gibberellic acid and have enhanced resistance to the blast fungus Magnaporthe grisea (95), though gibberellic acid appears to play a negative role in basal disease resistance (96). In maize, brassinosteroid activity has been shown to be important for antioxidant defense, but a role against biotic stress has not been determined. Evidence for a function of cytokinins in maize defense is also limited; however, recent evidence demonstrated a curious signaling role for DIMBOA in regulating cytokinin concentrations. The oxidative cleavage of DIMBOA via laccase and peroxidase generates transitional free radicals that mediate the activity of a dehydrogenase, leading to cytokinin degredation (97). Finally, the growth-promoting hormone auxin also appears to influence defense responses in plants, although results thus far have been contradictory (98, 99). Strongly integrated in plant hormone signaling are endogenous short-chain amino acid signals such as plant elicitor peptides (Peps) that effectively regulate herbivore and disease resistance. Similar to the potent signal systemin found in 56

the Solanaceae family (100), maize Peps activate defense responses including the production of jasmonic acid, ethylene, and increased expression of genes encoding proteins associated with herbivore and disease resistance (101, 102). In addition to Peps, the recently identified Z. mays immune signaling peptide (Zip1) functions as a biotroph-elicited signal that activates SA defense signaling (103), further demonstrating the interconnected role of petide signals in hormone-mediated maize resistance.

Functional Characterization of Defense Chemistry Maize makes a staggering array of compounds when faced with biotic challenge, many with unknown functions. Various approaches can be taken to determine whether a compound contributes to pest resistance. As an initial test, in vitro bioassays can be a helpful tool in determining biological activity. For example, recent analyses showed that a maize 9-oxylipin (i.e. 10-OPEA) had significant and dose-dependent insect and pathogen growth inhibitory activity (72), whereas diterpenoid phytoalexins demonstrated only minimal growth impairment against insects but had strong antifungal activity against ear, stem, and root pathogens of maize (29–32). While in vitro bioassays provide evidence for potential molecular function, in vivo analysis using genetic confirmation is needed to demonstrate a biological role. The identification of target genes involved in the biosynthesis of defense molecules can be accomplished by association mapping, co-expression analysis, and/or forward and reverse genetic approaches. A recent example in maize demonstrated the effectiveness of several of these approaches (33). Examination of transcript accumulation in response to F. graminearum found strong elicitation of the An2 gene, which was later genetically shown to be required for kauralexin production (90) and resistance to ear, stem, and leaf pathogens (29). In another study, metabolite-based genetic mapping using biparental populations, genome wide association studies (GWAS), and near-isogenic lines identified TPS21 to be essential for β-costic acid formation. Through both in vitro and genetic analyses, it was determined that β-costic acid plays a strong chemical defensive role against root pathogens and herbivores (33). The aforementioned studies provide proof of function, yet to engineer or incorporate the production of a particular compound into maize breeding lines, it is preferable to identify all of the genes involved in its production. As mentioned, one approach for identifying candidate genes of chemical defense pathways can be accomplished using co-expression network analysis. This approach works on the principal that genes with common/shared function will be co-regulated over a wide range of conditions as their products need to be present at the same time in the same tissue. This strategy was used in A. thaliana to identify P450s that co-expressed with geranyllinalool synthase that makes the substrate for TMTT synthase (104). Subsequent functional characterization of the candidates led to the identification of a bifunctional DMNT/TMTT homoterpene synthase (104). While not yet widely used in maize, this approach is promising for the elucidation of maize chemical defenses. Several online databases exist that perform co-expression analyses for maize genes. These include ATTED-ii (http://atted.jp/) (105), 57

CORNET (https://bioinformatics.psb.ugent.be/cornet/versions/cornet_maize1.0// main/precalc) (106) and PLANEX (http: //planex.plantbioinformatics.org/) (107). Collectively, these studies highlight current approaches to elucidate the function of maize chemical defenses and the pathways that produce them.

Engineering for Enhanced Chemical Defense Defense metabolites are differentially elicited in response to a variety of biological threats. While some plants are able to deploy effective tactics that result in immunity, pests have also evolved the ability to manipulate plant chemistry and suppress defense systems. Such chemical warfare can be observed in maize. For example, Figure 4 demonstrates that C. graminicola elicits far less production of many common maize defense metabolites than F. graminearum and C. heterostrophus, despite having demonstrated patterns of significantly greater fungal growth (29).

Figure 4. Heat map (n=4) displaying the relative abundance of maize defense metabolites in a B73-Mo17 hybrid under no treatment (control) or 24 h after damage or infection with C. graminicola (C. gram), F. graminearum (F. gram), and C. heterostrophus (C. het). Metabolites measured include salicylic acid (SA); jasmonic acid (JA); auxin (IAA); abscisic acid (ABA); 12-oxo-phytodienoic acid (12-OPDA); kauralexin A1 (KA1)-KA3 and KB1-KB3; zealexins A1 (ZA1), ZB1, and ZA4; and 6-methoxy-2-benzoxazoline (MBOA). Given the diversity of biological threats and the complexity of defense regulation, the challenge to engineer plants for broad resistance is substantial. Identification of defense-related quantitative trait loci (QTL) continues to inform breeding programs for enhanced resistance phenotypes; however, specific knowledge of chemical defenses has, until relatively recently, been unavailable. Recent studies in maize have demonstrated that targeted enhancement of chemical defenses can have significant impacts on plant-biotic interactions. For example, 1) overexpression of TPS10, responsible for a series of sesquiterpene HIPVs, resulted in increased recruitment of the parasitic wasp, Cotesia marginiventris (70); 2) upregulation of β-caryophyllene and α-humulene by introduction of an oregano TPS resulted in improved resistance against the root herbivore Diabrotica 58

v. v. (108), but led to lower yield, higher above-ground herbivory (109), and increased susceptibility to C. graminicola (110); 3) expression of a sorghum MYB transcription factor involved in the production of 3-deoxyanthocyanidin phytoalexins resulted in improved resistance to C. graminicola (111); and 4) downregulation of ethylene-response factors by targeted disruption using CRISPR/Cas9 resulted in increased resistance to M. oryzae (112). These examples and others serve to demonstrate the potential for manipulating or enhancing maize chemistry to improve pest resistance. Still in its infancy, the engineering of enhanced resistance will require a greater understanding of the genetics, chemistry, and regulatory pathways of plant defense. As many defense chemicals are phytotoxic or require plant resources for their production, a means to control the production of defense chemicals to reduce potential negative impacts on growth, development, and yield will also be required.

Future Directions One of the perpetual challenges of developing crop resistance is the continuing evolution and adaptation of plant pests against engineered defense mechanisms. These evolutionary impacts are similar to those seen in the development of resistance to pesticides, where exposure to a particular compound limits the survival and reproductive capacity of a given organism only until natural seletion overcomes the treatment. Examples of acquired resistance to certain maize-produced defense chemicals already exist. For instance, F. verticillioides can effectively detoxify the benzoxazolinones, 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA) (113). Many other Fusarium species do not display such tolerance to these compounds (113). Insects have also developed resistance to certain BXs, as both S. littoralis and S. frugiperda are able to detoxify DIMBOA via glycosylation (21). Interestingly neither species can detoxify the related compound HDIMBOA (2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one) (21). Such examples reveal the biochemical warfare currently underway between maize and its pests. Enhancing maize lines to produce controlled amounts of diverse defense chemicals that have antimicrobial and anti-herbivore activity, without compromising growth, development, and yield, could facilitate a more prolific agro-economic industry. Rapid progress and advancement of these intiatives will reduce the need for external pesticide application, reducing growing costs and increasing the safety of farm workers and consumers alike.

References 1.

Mueller, D. S.; Wise, K. A.; Sisson, A. J.; Allen, T. W.; Bergstrom, G. C.; Bosley, D. B.; Bradley, C. A.; Broders, K. D.; Byamukama, E.; Chilvers, M. I.; Collins, A.; Faske, T. R.; Friskop, A. J.; Heiniger, R. W.; Hollier, C. A.; Hooker, D. C.; Isakeit, T.; Jackson-Ziems, T. A.; Jardine, D. J.; Kinzer, K.; Koenning, S. R.; Malvick, D. K.; McMullen, M.; Meyer, R. F.; Paul, P. A.; Robertson, A. E.; Roth, G. W.; Smith, D. L.; Tande, C. A.; Tenuta, A. U.; Vincelli, P.; Warner, F. Corn yield loss estimates due to diseases in the United 59

2.

3.

4.

5. 6. 7. 8. 9. 10.

11. 12.

13.

14.

15.

16.

States and Ontario, Canada from 2012 to 2015. Plant Health Prog. 2016, 17, 211–222. Keller, N. P.; Bergstrom, G. C.; Carruthers, R. I. Potential yield reductions in maize associated with an anthracnose European corn-borer pest complex in New-York. Phytopathology 1986, 76, 586–589. Gatch, E. W.; Munkvold, G. P. Fungal species composition in maize stalks in relation to European corn borer injury and transgenic insect protection. Plant Dis. 2002, 86, 1156–1162. Vaughn, T.; Cavato, T.; Brar, G.; Coombe, T.; DeGooyer, T.; Ford, S.; Groth, M.; Howe, A.; Johnson, S.; Kolacz, K.; Pilcher, C.; Purcell, J.; Romano, C.; English, L.; Pershing, J. A method of controlling corn rootworm feeding using a Bacillus thuringiensis protein expressed in transgenic maize. Crop Sci. 2005, 45, 931–938. de Maagd, R. A.; Bosch, D.; Stiekema, W. Toxin-mediated insect resistance in plants. Trends Plant Sci. 1999, 4, 9–13. Brookes, G.; Barfoot, P. Global impact of biotech crops: environmental effects, 1996-2010. GM Crops Food 2012, 3, 129–37. Tabashnik, B. E.; Brevault, T.; Carriere, Y. Insect resistance to Bt crops: lessons from the first billion acres. Nature Biotechnol. 2013, 31, 510–21. Virtanen, A. I.; Heitala, P. K. 2(3)-Benzoxazolinone, an Anti-Fusarium Factor in Rye Seedlings. Acta Chem. Scand. 1955, 9, 1543–1544. Sissman, E.; LaPidus, J.; Beck, S. Corn plant resistance factor. New Phytol. 1957, 22, 220. Niemeyer, H. M. Hydroxamic acids derived from 2-hydroxy-2h-1,4benzoxazin-3(4h)-one: key defense chemicals of cereals. J. Agric. Food Chem. 2009, 57, 1677–1696. Niemeyer, H. M. Hydroxamic acids (4-hydroxy-1,4-benzoxazin-3-ones), defence chemicals in the gramineae. Phytochemistry 1988, 27, 3349–3358. Ebisui, K. I. A.; Hirai, N.; Iwamura, H. Occurrence of 2,4-dihydroxy-7methoxy-1,4-benzoxazin-3-one (DIMBOA) and a b-glucosidase specific for its glucoside in maize seedlings. Z. Naturforsch. 1998, 53, 793–798. Basse, C. W. Dissecting defense-related and developmental transcriptional responses of maize during Ustilago maydis infection and subsequent tumor formation. Plant Phys. 2005, 138, 1774–1784. Ahmad, S.; Veyrat, N.; Gordon-Weeks, R.; Zhang, Y.; Martin, J.; Smart, L.; Glauser, G.; Erb, M.; Flors, V.; Frey, M.; Ton, J. Benzoxazinoid metabolites regulate innate immunity against aphids and fungi in maize. Plant Phys. 2011, 157, 317–327. Gutierrez, C.; Castañera, P.; Torres, V. Wound‐induced changes in DIMBOA (2, 4 dihydroxy‐7‐methoxy‐2H‐1, 4 benzoxazin‐3(4H)‐one) concentration in maize plants caused by Sesamia nonagrioides (Lepidoptera: Noctuidae). Annals Appl. Biol. 1988, 113, 447–454. Dafoe, N. J.; Huffaker, A.; Vaughan, M. M.; Duehl, A. J.; Teal, P. E.; Schmelz, E. A. Rapidly induced chemical defenses in maize stems and their effects on short-term growth of Ostrinia nubilalis. J. Chem. Ecol. 2011, 37, 984–991. 60

17. Frey, M.; Chomet, P.; Glawischnig, E.; Stettner, C.; Grun, S.; Winklmair, A.; Eisenreich, W.; Bacher, A.; Meeley, R. B.; Briggs, S. P.; Simcox, K.; Gierl, A. Analysis of a chemical plant defense mechanism in grasses. Science 1997, 277, 696–699. 18. Frey, M.; Schullehner, K.; Dick, R.; Fiesselmann, A.; Gierl, A. Benzoxazinoid biosynthesis, a model for evolution of secondary metabolic pathways in plants. Phytochemistry 2009, 70, 1645–1651. 19. Wouters, F. C.; Blanchette, B.; Gershenzon, J.; Vassão, D. G. Plant defense and herbivore counter-defense: benzoxazinoids and insect herbivores. Phytochem. Rev. 2016, 15, 1127–1151. 20. Cambier, V.; Hance, T.; De Hoffmann, E. Effects of 1,4-benzoxazin-3-one derivatives from maize on survival and fecundity of Metopolophium dirhodum (Walker) on artificial diet. J. Chem. Ecol. 2001, 27, 359–370. 21. Glauser, G.; Marti, G.; Villard, N.; Doyen, G. A.; Wolfender, J. L.; Turlings, T. C. J.; Erb, M. Induction and detoxification of maize 1,4-benzoxazin-3-ones by insect herbivores. Plant J. 2011, 68, 901–911. 22. von Rad, U.; Huttl, R.; Lottspeich, F.; Gierl, A.; Frey, M. Two glucosyltransferases are involved in detoxification of benzoxazinoids in maize. Plant J. 2001, 28, 633–642. 23. Oikawa, A.; Ebisui, K.; Sue, M.; Ishihara, A.; Iwamura, H. Purification and characterization of a β-glucosidase specific for 2,4-dihydroxy- 7-methoxy1,4-benzoxazin-3-one (DIMBOA) glucoside in maize. Z. Naturforsch. 1999, 54, 181. 24. Campos, F.; Atkinson, J.; Arnason, J. T.; Philogene, B. J. R.; Morand, P.; Werstiuk, N. H.; Timmins, G. Toxicity and toxicokinetics of 6methoxybenzoxazolinone (MBOA) in the European corn-borer, OstriniaNubilalis (Hubner). J. Chem. Ecol. 1988, 14, 989–1002. 25. Martyniuk, S.; Stochmal, A.; Macias, F. A.; Marin, D.; Oleszek, W. Effects of some benzoxazinoids on in vitro growth of Cephalosporium gramineum and other fungi pathogenic to cereals and on Cephalosporium stripe of winter wheat. J. Agric. Food Chem. 2006, 54, 1036–1039. 26. Barnes, J. P.; Putnam, A. R. Role of benzoxazinones in allelopathy by rye (Secale cereale L.). J. Chem. Ecol. 1987, 13, 889–906. 27. Tabaglio, V.; Gavazzi, C.; Schulz, M.; Marocco, A. Alternative weed control using the allelopathic effect of natural benzoxazinoids from rye mulch. Agron. Sust. Dev. 2008, 28, 397–401. 28. Kruidhof, H. M.; van Dam, N. M.; Ritz, C.; Lotz, L. A. P.; Kropff, M. J.; Bastiaans, L. Mechanical wounding under field conditions: A potential tool to increase the allelopathic inhibitory effect of cover crops on weeds? Eur. J. Agron. 2014, 52, 229–236. 29. Christensen, S. A.; Sims, J.; Vaughan, M.; Hunter, C.; Block, A.; Willett, D.; Alborn, H. T.; Huffaker, A.; Schmelz, E. A. Commercial hybrids and mutant genotypes reveal complex protective roles for inducible terpenoid defenses. J. Exp. Bot. 2018, 69, 1693–1705. 30. Schmelz, E. A.; Kaplan, F.; Huffaker, A.; Dafoe, N. J.; Vaughan, M. M.; Ni, X. Z.; Rocca, J. R.; Alborn, H. T.; Teal, P. E. Identity, regulation, and 61

31.

32.

33.

34.

35. 36.

37. 38.

39.

40. 41.

42. 43.

44.

activity of inducible diterpenoid phytoalexins in maize. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 5455–5460. Christensen, S. A.; Huffaker, A.; Sims, J.; Hunter, C. T.; Block, A.; Vaughan, M. M.; Willett, D.; Romero, M.; Mylroie, J. E.; Williams, W. P.; Schmelz, E. A. Fungal and herbivore elicitation of the novel maize sesquiterpenoid, zealexin A4, is attenuated by elevated CO2. Planta 2017, 247, 863–873. Huffaker, A.; Kaplan, F.; Vaughan, M. M.; Dafoe, N. J.; Ni, X. Z.; Rocca, J. R.; Alborn, H. T.; Teal, P. E. A.; Schmelz, E. A. Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize. Plant Phys. 2011, 156, 2082–2097. Ding, Y. Z.; Huffaker, A.; Kollner, T. G.; Weckwerth, P.; Robert, C. A. M.; Spencer, J. L.; Lipka, A. E.; Schmelz, E. A. Selinene volatiles are essential precursors for maize defense promoting fungal pathogen resistance. Plant Phys. 2017, 175, 1455–1468. Mafu, S.; Ding, Y.; Murphy, K. M.; Yaacoobi, O.; Addison, J. B.; Wang, Q.; Shen, Z.; Briggs, S. P.; Bohlmann, J.; Castro-Falcon, G.; Hughes, C. C.; Betsiashvili, M.; Huffaker, A.; Schmelz, E. A.; Zerbe, P. Discovery, biosynthesis and stress-related accumulation of dolabradiene-derived defenses in maize. Plant Phys. 2018, 176, 2677–2690. Kachroo, A.; Kachroo, P. Fatty acid-derived signals in plant defense. Annu. Rev. Phytopathol. 2009, 47, 153–176. Raffaele, S.; Leger, A.; Roby, D. Very long chain fatty acid and lipid signaling in the response of plants to pathogens. Plant Signaling Behav. 2009, 4, 94–99. Walley, J. W.; Kliebenstein, D. J.; Bostock, R. M.; Dehesh, K. Fatty acids and early detection of pathogens. Curr. Opin. Plant Biol. 2013, 16, 520–526. Xue, H. Q.; Upchurch, R. G.; Kwanyuen, P. Ergosterol as a quantifiable biomass marker for Diaporthe phaseolorum and Cercospora kikuchii. Plant Dis. 2006, 90, 1395–1398. Koo, A. J. K.; Chung, H. S.; Kobayashi, Y.; Howe, G. A. Identification of a peroxisomal acyl-activating enzyme involved in the biosynthesis of jasmonic acid in Arabidopsis. J. Biol. Chem. 2006, 281, 33511–33520. Yaeno, T.; Matsuda, O.; Iba, K. Role of chloroplast trienoic fatty acids in plant disease defense responses. Plant J. 2004, 40, 931–941. Balmer, D.; de Papajewski, D. V.; Planchamp, C.; Glauser, G.; Mauch-Mani, B. Induced resistance in maize is based on organ-specific defence responses. Plant J. 2013, 74, 213–225. Buer, C. S.; Imin, N.; Djordjevic, M. A. Flavonoids: new roles for old molecules. J. Int. Plant Biol. 2010, 52, 98–111. Waiss, A. C.; Chan, B. G.; Elliger, C. A.; Wiseman, B. R.; Mcmillian, W. W.; Widstrom, N. W.; Zuber, M. S.; Keaster, A. J. Maysin, a flavone glycoside from corn silks with antibiotic-activity toward corn earworm (Lepidotera: Noctuidae). J. Econ. Entomol. 1979, 72, 257–258. Casas, M. I.; Falcone-Ferreyra, M. L.; Jiang, N.; Mejia-Guerra, M. K.; Rodriguez, E.; Wilson, T.; Engelmeier, J.; Casati, P.; Grotewold, E. 62

45.

46.

47.

48.

49.

50.

51. 52.

53.

54. 55.

56.

57.

58.

Identification and characterization of maize salmon silks genes involved in insecticidal maysin biosynthesis. Plant Cell 2016, 28, 1297–309. Casati, P.; Walbot, V. Differential accumulation of maysin and rhamnosylisoorientin in leaves of high‐altitude landraces of maize after UV‐B exposure. Plant Cell Environ. 2005, 28, 788–799. Cortes-Cruz, M.; Snook, M.; McMmullen, M. D. The genetic basis of Cglycosyl flavone B-ring modification in maize (Zea mays L.) silks. Genome 2003, 46, 182–194. Bushman, B. S.; Snook, M. E.; Gerke, J. P.; Szalma, S. J.; Berhow, M. A.; Houchins, K. E.; McMullen, M. D. Two loci exert major effects on chlorogenic acid synthesis in maize silks. Crop. Sci. 2002, 42, 1669–1678. Guo, B. Z.; Zhang, Z. J.; Butron, A.; Widstrom, N. W.; Snook, M. E.; Lynch, R. E.; Plaisted, D. Lost P1 allele in sh2 sweet corn: quantitative effects of p1 and a1 genes on concentrations of maysin, apimaysin, methoxymaysin, and chlorogenic acid in maize silk. J. Econ. Entomol. 2004, 97, 2117–2126. Wiseman, B. R.; Carpenter, J. E. Growth-inhibition of corn-earworm (Lepidoptera: Noctuidae) larvae reared on resistant corn silk diets. J. Econ. Entomol. 1995, 88, 1037–1043. Felton, G. W.; Donato, K.; Delvecchio, R. J.; Duffey, S. S. Activation of plant foliar oxidases by insect feeding reduces nutritive quality of foliage for noctuid herbivores. J. Chem. Ecol. 1989, 15, 2667–2694. Levings, C. S.; Stuber, C. W. A maize gene controlling silk browning in response to wounding. Genetics 1971, 69, 491–498. Guo, B. Z.; Widstrom, N. W.; Wiseman, B. R.; Snook, M. E.; Lynch, R. E.; Plaisted, D. Comparison of silk maysin, antibiosis to corn earworm larvae (Lepidoptera : Noctuidae), and silk browning in crosses of dent x sweet corn. J. Econ. Entomol. 1999, 92, 746–753. Nessa, F.; Ismail, Z.; Mohamed, N. Antimicrobial activities of extracts and flavonoid glycosides of corn silk (Zea mays L). Int. J. Biotechnol. Wellness Ind. 2012, 1, 115–121. Santiago, R.; Malvar, R. A. Role of dehydrodiferulates in maize resistance to pests and diseases. Int. J. Mol. Sci. 2010, 11, 691–703. Bergvinson, D. J.; Arnason, J. T.; Hamilton, I. R.; Mihm, J. A.; Jewell, D. C. Determining leaf toughness and its role in maize resistance to the European com borer (Lepidoptera: Pyralidae). J. Econ. Entomol. 1994, 87, 1743–1748. Bergvinson, D. J.; Hamilton, R. I.; Arnason, J. T. Leaf profile of maize resistance factors to European corn borer, Ostrinia nubilalis. J. Chem. Ecol. 1995, 21, 343–354. Bily, A. C.; Reid, L. M.; Taylor, J. H.; Johnston, D.; Malouin, C.; Burt, A. J.; Bakan, B.; Regnault-Roger, C.; Pauls, K. P.; Arnason, J. T.; Philogene, B. J. Dehydrodimers of ferulic acid in maize grain pericarp and aleurone: resistance factors to Fusarium graminearum. Phytopathology 2003, 93, 712–719. Marti, G.; Erb, M.; Boccard, J.; Glauser, G.; Doyen, G. R.; Villard, N.; Robert, C. A.; Turlings, T. C.; Rudaz, S.; Wolfender, J. L. Metabolomics 63

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

reveals herbivore-induced metabolites of resistance and susceptibility in maize leaves and roots. Plant Cell Environ. 2013, 36, 621–639. Asselin, J. E.; Lin, J.; Perez-Quintero, A. L.; Gentzel, I.; Majerczak, D.; Opiyo, S. O.; Zhao, W.; Paek, S. M.; Kim, M. G.; Coplin, D. L.; Blakeslee, J. J.; Mackey, D. Perturbation of maize phenylpropanoid metabolism by an AvrE family type III effector from Pantoea stewartii. Plant Phys. 2015, 167, 1117–1135. Tanaka, S.; Brefort, T.; Neidig, N.; Djamei, A.; Kahnt, J.; Vermerris, W.; Koenig, S.; Feussner, K.; Feussner, I.; Kahmann, R. A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize. eLife 2014, 3, e01355. Turlings, T. C.; Tumlinson, J. H.; Heath, R. R.; Proveaux, A. T.; Doolittle, R. E. Isolation and identification of allelochemicals that attract the larval parasitoid, Cotesia marginiventris (Cresson), to the microhabitat of one of its hosts. J. Chem. Ecol. 1991, 17, 2235–2251. Turlings, T. C.; Tumlinson, J. H.; Lewis, W. J. Exploitation of herbivoreinduced plant odors by host-seeking parasitic wasps. Science 1990, 250, 1251–1253. Engelberth, J.; Alborn, H. T.; Schmelz, E. A.; Tumlinson, J. H. Airborne signals prime plants against insect herbivore attack. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1781–1785. Erb, M.; Veyrat, N.; Robert, C. A. M.; Xu, H.; Frey, M.; Ton, J.; Turlings, T. C. J. Indole is an essential herbivore-induced volatile priming signal in maize. Nat. Commun. 2015, 6, 6273. Christensen, S. A.; Nemchenko, A.; Borrego, E.; Murray, I.; Sobhy, I. S.; Bosak, L.; DeBlasio, S.; Erb, M.; Robert, C. A.; Vaughn, K. A.; Herrfurth, C.; Tumlinson, J.; Feussner, I.; Jackson, D.; Turlings, T. C.; Engelberth, J.; Nansen, C.; Meeley, R.; Kolomiets, M. V. The maize lipoxygenase, ZmLOX10, mediates green leaf volatile, jasmonate and herbivore-induced plant volatile production for defense against insect attack. Plant J. 2013, 74, 59–73. Lin, C. F.; Shen, B. Z.; Xu, Z. N.; Koellner, T. G.; Degenhardt, J.; Dooner, H. K. Characterization of the monoterpene synthase gene Tps26, the ortholog of a gene induced by insect herbivory in maize. Plant Phys. 2008, 146, 940–951. Richter, A.; Schaff, C.; Zhang, Z.; Lipka, A. E.; Tian, F.; Kollner, T. G.; Schnee, C.; Preiss, S.; Irmisch, S.; Jander, G.; Boland, W.; Gershenzon, J.; Buckler, E. S.; Degenhardt, J. Characterization of biosynthetic pathways for the production of the volatile homoterpenes DMNT and TMTT in Zea mays. Plant Cell 2016, 28, 2651–2665. Kollner, T. G.; Held, M.; Lenk, C.; Hiltpold, I.; Turlings, T. C.; Gershenzon, J.; Degenhardt, J. A maize (E)-b-caryophyllene synthase implicated in indirect defense responses against herbivores is not expressed in most American maize varieties. Plant Cell 2008, 20, 482–494. Schnee, C.; Kollner, T. G.; Gershenzon, J.; Degenhardt, J. The maize gene terpene synthase 1 encodes a sesquiterpene synthase catalyzing the formation 64

70.

71.

72.

73.

74.

75.

76.

77.

78.

79. 80. 81.

of (E)-b-farnesene, (E)-nerolidol, and (E,E)-farnesol after herbivore damage. Plant Phys. 2002, 130, 2049–2060. Schnee, C.; Kollner, T. G.; Held, M.; Turlings, T. C.; Gershenzon, J.; Degenhardt, J. The products of a single maize sesquiterpene synthase form a volatile defense signal that attracts natural enemies of maize herbivores. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 1129–1134. Kollner, T. G.; Schnee, C.; Gershenzon, J.; Degenhardt, J. The variability of sesquiterpenes emitted from two Zea mays cultivars is controlled by allelic variation of two terpene synthase genes encoding stereoselective multiple product enzymes. Plant Cell 2004, 16, 1115–1131. Christensen, S. A.; Huffaker, A.; Kaplan, F.; Sims, J.; Ziemann, S.; Doehlemann, G.; Ji, L. X.; Schmitz, R. J.; Kolomiets, M. V.; Alborn, H. T.; Mori, N.; Jander, G.; Ni, X. Z.; Sartor, R. C.; Byers, S.; Abdo, Z.; Schmelz, E. A. Maize death acids, 9-lipoxygenase-derived cyclopente(a)nones, display activity as cytotoxic phytoalexins and transcriptional mediators. Proc. Natl. Acad. Sci. U.S.A. 2015, 112, 11407–11412. Christensen, S. A.; Nemchenko, A.; Park, Y. S.; Borrego, E.; Huang, P. C.; Schmelz, E. A.; Kunze, S.; Feussner, I.; Yalpani, N.; Meeley, R.; Kolomiets, M. V. The novel monocot-specific 9-lipoxygenase ZmLOX12 is required to mount an effective jasmonate-mediated defense against Fusarium verticillioides in maize. Mol. Plant-Microbe Interact. 2014, 27, 1263–1276. Yan, Y. X.; Christensen, S.; Isakeit, T.; Engelberth, J.; Meeley, R.; Hayward, A.; Emery, R. J. N.; Kolomiets, M. V. Disruption of Opr7 and Opr8 reveals the versatile functions of jasmonic acid in maize development and defense. Plant Cell 2012, 24, 1420–1436. Acosta, I. F.; Laparra, H.; Romero, S. P.; Schmelz, E.; Hamberg, M.; Mottinger, J. P.; Moreno, M. A.; Dellaporta, S. L. tasselseed1 is a lipoxygenase affecting jasmonic acid signaling in sex determination of maize. Science 2009, 323, 262–265. Schmelz, E. A.; Alborn, H. T.; Tumlinson, J. H. Synergistic interactions between volicitin, jasmonic acid and ethylene mediate insect-induced volatile emission in Zea mays. Physiol. Plant 2003, 117, 403–412. Louis, J.; Basu, S.; Varsani, S.; Castano-Duque, L.; Jiang, V.; Williams, W. P.; Felton, G. W.; Luthe, D. S. Ethylene contributes to maize insect resistance1-mediated maize defense against the phloem sap-sucking corn leaf aphid. Plant Physiol. 2015, 169, 313–324. Harfouche, A. L.; Shivaji, R.; Stocker, R.; Williams, P. W.; Luthe, D. S. Ethylene signaling mediates a maize defense response to insect herbivory. Mol. Plant-Microbe Interact. 2006, 19, 189–199. Grant, M.; Lamb, C. Systemic immunity. Curr. Opin. Plant Biol. 2006, 9, 414–420. Kunkel, B. N.; Brooks, D. M. Cross talk between signaling pathways in pathogen defense. Curr. Opin. Plant Biol. 2002, 5, 325–331. Glazebrook, J.; Chen, W.; Estes, B.; Chang, H. S.; Nawrath, C.; Metraux, J. P.; Zhu, T.; Katagiri, F. Topology of the network integrating salicylate and 65

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

jasmonate signal transduction derived from global expression phenotyping. Plant J. 2003, 34, 217–228. De Vos, M.; Van Oosten, V. R.; Van Poecke, R. M.; Van Pelt, J. A.; Pozo, M. J.; Mueller, M. J.; Buchala, A. J.; Metraux, J. P.; Van Loon, L. C.; Dicke, M.; Pieterse, C. M. Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Mol. Plant-Microbe Interact. 2005, 18, 923–937. van der Linde, K.; Hemetsberger, C.; Kastner, C.; Kaschani, F.; van der Hoorn, R. A.; Kumlehn, J.; Doehlemann, G. A maize cystatin suppresses host immunity by inhibiting apoplastic cysteine proteases. Plant Cell 2012, 24, 1285–1300. Molodchenkova, O. O.; Adamovskaya, V. G.; Levitskii, Y. A.; Gontarenko, O. V.; Sokolov, V. M. Maize response to salicylic acid and Fusarium moniliforme. Appl. Biochem. Microbiol. 2002, 38, 381–385. Djamei, A.; Schipper, K.; Rabe, F.; Ghosh, A.; Vincon, V.; Kahnt, J.; Osorio, S.; Tohge, T.; Fernie, A. R.; Feussner, I.; Feussner, K.; Meinicke, P.; Stierhof, Y. D.; Schwarz, H.; Macek, B.; Mann, M.; Kahmann, R. Metabolic priming by a secreted fungal effector. Nature 2011, 478, 395–398. Filgueiras, C. C.; Willett, D. S.; Pereira, R. V.; Moino, A.; Pareja, M.; Duncan, L. W. Eliciting maize defense pathways aboveground attracts belowground biocontrol agents. Sci. Rep. 2016, 6, 36484. Adie, B. A.; Perez-Perez, J.; Perez-Perez, M. M.; Godoy, M.; SanchezSerrano, J. J.; Schmelz, E. A.; Solano, R. ABA is an essential signal for plant resistance to pathogens affecting JA biosynthesis and the activation of defenses in Arabidopsis. Plant Cell 2007, 19, 1665–1681. Erb, M.; Gordon-Weeks, R.; Flors, V.; Camanes, G.; Turlings, T. C.; Ton, J. Belowground ABA boosts aboveground production of DIMBOA and primes induction of chlorogenic acid in maize. Plant Signaling Behav. 2009, 4, 636–638. Erb, M.; Veyrat, N.; Robert, C. A.; Xu, H.; Frey, M.; Ton, J.; Turlings, T. C. Indole is an essential herbivore-induced volatile priming signal in maize. Nat. Commun. 2015, 6, 6273. Vaughan, M. M.; Christensen, S.; Schmelz, E. A.; Huffaker, A.; Mcauslane, H. J.; Alborn, H. T.; Romero, M.; Allen, L. H.; Teal, P. E. A. Accumulation of terpenoid phytoalexins in maize roots is associated with drought tolerance. Plant Cell Environ. 2015, 38, 2195–2207. Balmer, D.; de Papajewski, D. V.; Planchamp, C.; Glauser, G.; Mauch-Mani, B. Induced resistance in maize is based on organ-specific defence responses. Plant J. 2013, 74, 213–225. Vargas, W. A.; Martin, J. M. S.; Rech, G. E.; Rivera, L. P.; Benito, E. P.; DiazMinguez, J. M.; Thon, M. R.; Sukno, S. A. Plant defense mechanisms are activated during biotrophic and necrotrophic development of Colletotricum graminicola in maize. Plant Phys. 2012, 158, 1342–1358. Ali, S. S.; Gunupuru, L. R.; Kumar, G. B. S.; Khan, M.; Scofield, S.; Nicholson, P.; Doohan, F. M. Plant disease resistance is augmented in uzu barley lines modified in the brassinosteroid receptor BRI1. BMC Plant Biol. 2014, 14. 66

94. Goddard, R.; Peraldi, A.; Ridout, C.; Nicholson, P. Enhanced disease resistance caused by BRI1 mutation is conserved between Brachypodium distachyon and barley (Hordeum vulgare). Mol. Plant-Microbe Interact. 2014, 27, 1095–1106. 95. Tanaka, N.; Matsuoka, M.; Kitano, H.; Asano, T.; Kaku, H.; Komatsu, S. gid1, a gibberellin-insensitive dwarf mutant, shows altered regulation of probenazole-inducible protein (PBZ1) in response to cold stress and pathogen attack. Plant Cell Environ. 2006, 29, 619–631. 96. Yang, D. L.; Li, Q.; Deng, Y. W.; Lou, Y. G.; Wang, M. Y.; Zhou, G. X.; Zhang, Y. Y.; He, Z. H. Altered disease development in the eui mutants and Eui overexpressors indicates that gibberellins negatively regulate rice basal disease resistance. Mol. Plant 2008, 1, 528–537. 97. Frebortova, J.; Novak, O.; Frebort, I.; Jorda, R. Degradation of cytokinins by maize cytokinin dehydrogenase is mediated by free radicals generated by enzymatic oxidation of natural benzoxazinones. Plant J. 2010, 61, 467–481. 98. Bari, R.; Jones, J. Role of plant hormones in plant defence responses. Plant Mol. Biol. 2009, 69, 473–488. 99. Naseenn, M.; Kaltdorf, M.; Dandekar, T. The nexus between growth and defence signalling: auxin and cytokinin modulate plant immune response pathways. J. Exp. Bot. 2015, 66, 4885–4896. 100. Pearce, G.; Strydom, D.; Johnson, S.; Ryan, C. A. A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor proteins. Science 1991, 253, 895–907. 101. Huffaker, A.; Dafoe, N. J.; Schmelz, E. A. ZmPep1, an ortholog of Arabidopsis elicitor peptide 1, regulates maize innate immunity and enhances disease resistance. Plant Phys. 2011, 155, 1325–1338. 102. Huffaker, A.; Pearce, G.; Veyrat, N.; Erb, M.; Turlings, T. C. J.; Sartor, R.; Shen, Z.; Briggs, S. P.; Vaughan, M. M.; Alborn, H. T.; Teal, P. E. A.; Schmelz, E. A. Plant elicitor peptides are conserved signals regulating direct and indirect antiherbivore defense. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 5707–5712. 103. Ziemann, S.; van der Linde, K.; Lahrmann, U.; Acar, B.; Kaschani, F.; Colby, T.; Kaiser, M.; Ding, Y.; Schmelz, E.; Huffaker, A.; Holton, N.; Zipfel, C.; Doehlemann, G. An apoplastic peptide activates salicylic acid signalling in maize. Nature Plants 2018, 4, 172–180. 104. Lee, S.; Badieyan, S.; Bevan, D. R.; Herde, M.; Gatz, C.; Tholl, D. Herbivore-induced and floral homoterpene volatiles are biosynthesized by a single P450 enzyme (CYP82G1) in Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 21205–21210. 105. Aoki, Y.; Okamura, Y.; Tadaka, S.; Kinoshita, K.; Obayashi, T. ATTED-II in 2016: A plant coexpression database towards lineage-specific coexpression. Plant Cell Phys. 2016, 57, 1. 106. De Bodt, S.; Carvajal, D.; Hollunder, J.; Van den Cruyce, J.; Movahedi, S.; Inze, D. CORNET: A user-friendly tool for data mining and integration. Plant Phys. 2010, 152, 1167–1179. 107. Yim, W. C.; Yu, Y.; Song, K.; Jang, C. S.; Lee, B-M. PLANEX: the plant co-expression database. BMC Plant Biol. 2013, 83. 67

108. Degenhardt, J.; Hiltpold, I.; Kollner, T. G.; Frey, M.; Gierl, A.; Gershenzon, J.; Hibbard, B. E.; Ellersieck, M. R.; Turlings, T. C. Restoring a maize root signal that attracts insect-killing nematodes to control a major pest. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 13213–13218. 109. Robert, C. A.; Erb, M.; Hiltpold, I.; Hibbard, B. E.; Gaillard, M. D.; Bilat, J.; Degenhardt, J.; Cambet-Petit-Jean, X.; Turlings, T. C.; Zwahlen, C. Genetically engineered maize plants reveal distinct costs and benefits of constitutive volatile emissions in the field. Plant Biotechnol. J. 2013, 11, 628–639. 110. Fantaye, C. A.; Köpke, D.; Gershenzon, J.; Degenhardt, J. Restoring (E)β-caryophyllene production in a non-producing maize line compromises its resistance against the fungus Colletotrichum graminicola. J. Chem. Ecol. 2015, 41, 213–223. 111. Ibraheem, F.; Gaffoor, I.; Tan, Q.; Shyu, C. R.; Chopra, S. A sorghum MYB transcription factor induces 3-deoxyanthocyanidins and enhances resistance against leaf blights in maize. Molecules 2015, 20, 2388–2404. 112. Wang, F.; Wang, C.; Liu, P.; Lei, C.; Hao, W.; Gao, Y.; Liu, Y. G.; Zhao, K. Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PloS One 2016, 11, e0154027. 113. Glenn, A. E.; Hinton, D. M.; Yates, I. E.; Bacon, C. W. Detoxification of corn antimicrobial compounds as the basis for isolating Fusarium verticillioides and some other Fusarium species from corn. Appl. Environ. Microbiol. 2001, 67, 2973–2981.

68

Chapter 7

Integrated Pest Management Strategies for Phytoplasma Diseases of Woody Crop Plants: Possibilities and Limitations Wolfgang Schweigkofler,*,1,2 Silvia Schmidt,1 and Christian Roschatt1 1Research

Centre Laimburg, Laimburg 6, 39040 Auer, Italy University of California, 50 Acacia Avenue, San Rafael, California 94901, United States *E-mail: [email protected]

2Dominican

Phytoplasmas causing diseases of woody plants in native environments and agricultural systems are responsible for considerably economic and environmental damage. In Central and Southern Europe, phytoplasma diseases of apple (Malus domestica: Apple proliferation, AP) and grapevine (Vitis vinifera: Bois noir, BN and Flavescence dorèe, FD) are widespread and impact quantity and quality of the fruits. The ecology of phytoplasma diseases is complex and involves one or more insect vectors and in some cases alternative host plants. Phytoplasma densities in infected plants and expression of symptoms can vary considerably among seasons, and remission of symptoms occurs frequently. Disease control by pesticide application generally is not very efficient, therefore a polyphasic control strategy using a mix of agronomical, chemical and biological strategies needs to be developed for each disease. In the case of Bois noir e.g., the population density of the vector Hyalesthes obsoletus is related to the presence of its main host plants Urtica dioica and Convulvulus arvensis in the understory of the vineyards, which can be managed by agronomical methods. Application of foliar fertilizer showed no significant effect on BN-infected grapevines. AP-infected apple trees were treated with four bio-active compounds (Acibenzolar-S-Methyl, Harpin protein, Prohexadione-Ca and Cyanamide) over a

© 2018 American Chemical Society

three-year period, but their effects on symptom expression were only limited and transient.

Introduction Phytoplasmas are a group of obligate biotrophic plant pathogens causing diseases of numerous agronomical important plants. Phytoplasmas belong to the bacterial class Mollicutes, which is characterized by the absence of a cell wall. Phylogenetically, Mollicutes are related to the Firmicutes (or ‘gram-positive’ bacteria), and are believed to be the product of extreme adaption and reductive evolution. Phytoplasmas have very small genomes (600-880 kb) with a very low GC content and lack the genes for many biochemical pathways, such as the tricarboxylic acid cycle, sterol biosynthesis, fatty acid biosynthesis, de novo nucleotide synthesis, the pentose phosphate pathway and biosynthesis of most amino acids (1); consequently they depend entirely on their host cells (both plants and insects) to supply them with the products of these pathways. Phytoplasmas have been isolated from several hundred host plants, most of them dicots, but also some monocots and conifers. In the USA, diseases caused by phytoplasmas were reported from corn (Zea mays) in several southern states; alfalfa (Medicago sativa), garlic (Allium sativum) and potatoes (Solanum tuberosum) in the Midwest; grapevines (V. vinifera) in the Eastern US and especially palm trees (several species in the family Arecaceae; lethal yellowing) in Florida. In Europe, phytoplasmas are mainly a concern as pathogens of highly valuable perennial fruit plants such as apples (M. domestica), grapevines (V. vinifera), apricots (Prunus armeniaca) and plums (Prunus domestica), but diseases of forest trees, both conifers and broad-leaved trees such as oaks (Quercus spp.), willows (Salix spp.), alders (Alnus spp.) and ash (Fraxinus spp.) are also widespread. Common symptoms of phytoplasma infection include growth deformations of fruits and branches (also known as witches’ broom), premature fruit fall, discoloration or irregular growth of leaves, abnormal flower development of flowers such as phyllody and uneven lignifications (Figure 1). Not all symptoms are present on every host plant, and symptom expression can follow a seasonal pattern. Phytoplasmas occur in the nutrient-rich phloem of both aerial plant parts and roots. The movement and distribution of phytoplasmas in the host plant is not well known, however, phytoplasma titres can fluctuate significantly over time and between different plant tissues (2, 3). In general passive movement occurs in the direction of the phloem stream from source to sink tissue. Phytoplasmas are non-motile and spread via insect vectors, mainly phloem-sucking cicada and plant hoppers of the order Hemiptera, families Cixiidae, Jassidae and Psyllidae (4). Both larvae and imagines can transmit phytoplasmas; different life stages of an insect might feed on different host plants and tissue (e.g. roots vs. aerial tissue). Transovarial transmission has been shown for several insect vectors (5). Many vectors are polyphagous and can transmit phytoplasmas to a wide range of phylogenetically distinct host plants, however host specificity has been found for several vectors. 70

Figure 1. Symptoms of Bois noir (A-C) and Apple proliferation (D-F): A) shriveled grape; B) yellowing and downward curling of leaf; C) uneven lignifications; D) left plant: growth deformation of branches (witches’ broom), right plant: healthy; E) enlarged stipulae; F) infected apple plantlets in a field tunnel at the Research Centre Laimburg, Italy. Host plants can be distinguished into three types based on insect feeding behavior: 1) Plants on which vectors feed throughout their complete life cycle (larvae and imagines) 2) Plants on which imagines (but not larvae) feed regularly 71

3) Plants on which imagines feed only arbitrarily. We studied two economically important phytoplasma diseases in Northern Italy which show significant differences in the ecology of the plant-vector-pathogen interaction, and consequently require very different control strategies. Apple Proliferation Apple proliferation (AP) is an important disease of apple trees in several European countries, including Austria, France, Germany and Italy (6). AP is caused by ‘Candidatus Phytoplasma mali’, also known as the AP phytoplasma, and transmitted mainly by the phloem-feeding hemiptera Cacopsylla melanoneura and Cacopsylla picta (Psyllidae) (7). Transmission can also occur by grafting plant material on infected rootstock in the nursery, and through root bridges (natural grafting) in the orchard. Typical symptoms are the production of small and tasteless apples, premature bud opening and leaf reddening, enlarged stipulae and growth abnormalities (witches’ broom) caused by changes in plant hormone levels. Similar to other phytoplasma diseases, recovery of AP infected apple trees has been observed in the field, and can occur either transiently or permanently (8, 9). Recovery has been defined as the spontaneous remission of disease symptoms in plants previously symptomatic. Recovered plants exhibit similar behaviour as healthy plants, with the advantage of resistance acquisition towards the disease (10). Little is known about the molecular mechanisms responsible for recovery, but reduced levels of reactive oxygen species (ROS) and scavenging enzymes, especially ascorbate peroxidases (APX) and catalases (CAT) were measured in recovered plants. Recovered asymptomatic plants might still be colonized by the AP phytoplasma, but can produce apples equal in size and quality to fruits of healthy plants (11). Bois Noir Bois noir (BN), caused by ‘Candidatus Phytoplasma solani’, is a grapevine yellow disease common in Italy, France, Germany, Austria and other European wine regions. Typical symptoms include leaf discoloration (yellow in white cultivars, deep red/purple in red cultivars), uneven lignifications, stunted shoots and shrivelling of the ripening fruits. Susceptibility and symptom expression can vary significantly between grapevine cultivars; Chardonnay, Riesling and Pinot Noir are especially susceptible. Virtually the same symptoms are caused by another widespread grapevine yellow disease, Flavescence dorèe (FD), caused by ‘Candidatus Phytoplasma vitis’ (12).

The Complex Ecology of Phytoplasma Diseases In general, the ecology of phytoplasma diseases consists of three essential components: the phytoplasma as the causal agent, a susceptible host plant and an 72

insect vector transmitting the microbe. However, the presence of multiple host plants and insect vectors as well as agronomic practices and other environmental parameters can result in more complex interactions. A simplified scheme of the ecology of the three phytoplasma diseases AP, BN and FD is shown in Figure 2. In the case of AP, the two main vectors C. picta and C. melanoneura use the apple tree as a major food source during a relatively short period in spring and early summer, and overwinter on alternative hosts, most probably conifers and hawthorn (Crataegus sp., Rosaceae). The effect of the winter hosts on population density of both phytoplasmas and vectors is unclear. The leafhopper Fieberiella florii (Cicadellidae) might be an alternative vector, as it has been shown to transmit the AP phytoplasma to apple seedlings under experimental conditions (13). F. florii is polyphagous and feeds on a wide variety of broad-leafed, woody trees, shrubs and vines, esp. members of the Rosaceae, which might act as additional reservoirs for the AP phytoplasma. Agronomic practice can play a major role in transmission and symptom expression of phytoplasma diseases. AP for example was present in orchards in Northern Italy for several decades; however symptoms appeared only on relatively few branches of the vigorous apple trees grown at that time, causing little economic damage. On modern small apple trees growing on dwarf rootstocks (such as M9), phytoplasma symptoms seem to be more severe. Tree spacing in the orchard might also play a role in pathogen transmission, because AP phytoplasmas can be transmitted by root bridges from an infected to a healthy tree (14). Other agronomic parameters which can influence the severity of AP outbreaks include cultivar, age of the apple tree, orchard location (hill vs. plain) and plant protection strategies (organic vs. integrated). The two grapevine diseases (BN and FD) differ by their vectors: BN is transmitted by the polyphagous Hyalesthes obsoletus (Cixiidae), whereas the strictly monophagous Scaphoideus titanus (Cicadellidae) transmits FD. H. obsoletus favorite host plant are the stinging nettle Urtica dioica (Urticaceae) and the field bindweed Convulvulus arvensis (Convolvulaceae). Juvenile stages of H. obsoletus acquire the phytoplasma by feeding on roots from perennial herbaceous plants after hibernation. The grapevine is only a secondary host, which serves as a supplementary food source for H. obsoletus adults, but is a ‘dead end’ host from an epidemiological point of view. Vineyards can support a surprisingly rich diversity of plant- and leafhoppers (Hemiptera; suborder: Auchenorrhyncha). During a survey of eleven vineyards with BN symptoms in South Tyrol (Northern Italy), a total of 57 Auchenorrhyncha species were identified, some of them with very high population densities (15). Most species are phloem-feeders, and many are known vectors or carriers of viruses and phytoplasmas, among them Reptalus cuspidatus (Cixiidae), a polyphagous species common in several European countries. It is yet not clear, if some of these insects play a role in BN-transmission in the field. More than forty herbaceous plant species were found in the understory of those vineyards, and the BN phytoplasma was identified from several plants (Convolvululus arvensis, Echium vulgare, Polygonum aviculare, Silene vulgaris, Taraxacum officinal, Urtica dioica and U. urens) (16). On the other hand, S. titanus completes its whole life cycle on the grapevine and no other vector is known to transmit FD (17). Long-distance spread of both BN and FD can occur through plant trade of infected material. 73

Figure 2. Simplified scheme of the ecology of three phytoplasma diseases: A) Apple proliferation, B) Bois noir and C) Flavescence dorèe. Every phytoplasma infects at least a host plant and insect vector; some phytoplasmas have additional host plants and vectors. 74

Integrated Polyphasic Approach for Disease Control Control strategies for phytoplasma diseases of grapevine and apple trees include a wide variety of physical, chemical and biological approaches, which target different components of the complex disease ecology (Table 1). In addition, strategies to minimize disease symptoms and induce recovery have also been tested in the field (18, 19) and under controlled conditions in an insect-proof field tunnel (3).

Table 1. Possible control strategies for phytoplasma diseases of woody plants. Control target Phytoplasmas

Control principle direct

chemical thermal physical

Insect vectors

Control method antibiotics (tetracycline) hot water treatment of nursery material pruning; clearing

direct

chemical mechanical biological

insecticides soil treatment biocontrol agents (entomopathogens)

indirect

reduction of host plants

herbicides; managing understory vegetation by planting non-hosts

Phytoplasma transmission

indirect

agronomical

using grow tubes to protect plantlings from insect feeding

Symptom reduction, recovery

direct

bio-chemical

bio-active compounds; phytohormones; nutrients

Symptom prevention

direct

agronomical

use of tolerant cultivars; breeding resistant rootstocks

Antibiotics interfering with protein synthesis, like tetracycline, have a bacteriostatic effect on phytoplasmas, whereas antibiotics targeting the cell wall are not effective. However, no antibiotics are registered for apple and grapevine production in Italy. Thermal treatment in a hot water bath (50°C for 45 min) is an efficient method to eliminate phytoplasmas and certain viruses from planting material, and is recommended by EPPO (the European and Mediterranean Plant Protection Organization) for dormant wood of both scions and rootstocks prior to grafting and for rooted grafted vines of V. vinifera before planting (20). In Italy clearing of apple trees showing symptoms of AP is mandatory since 2006 to reduce inoculum density and spread of the pathogen. In the Province of South Tyrol alone, which is the biggest apple producing area within the European Union, several hundred of thousands of apple trees were cleared and replaced with disease-free plantlets since the first major AP epidemic swept through around 1998, causing considerable financial loss for the growers. Despite these efforts, AP is still endemic in the area, and especially wide-spread on sun-exposed slopes, 75

which might be a favored habitat for the insect vectors. Insecticide treatments in general are not very efficient in reducing population densities of the main vectors of AP and BN, most probably because the polyphagous species C. picta, C. melanoneura and H. obsoletus are present in orchards and vineyards for a limited time only. However, a long-term insecticide trial in South Tyrol (Italy), which combined detailed monitoring of psyllids presence in the orchard with timed application of three different pesticides, resulted in significantly lower AP infection rates in the treated field block compared to the untreated control block (21). Spread of FD can be reduced by treating the monophagous vector S. titanus, the habitat of which is restricted to vineyards. Chemical control of FD is mandatory in Italy and other European countries, and several insecticides have shown to be effective, including pyrthrins for organic production. At least two annual treatments are recommended, the first one targeting nymphs and the second one mature insects, respectively.

Remission of Symptoms and Recovery Phytoplasma titres and symptom expression can vary significantly between, but also within plants infected with AP and BN phytoplasmas. Infected but asymptomatic plants can produce apples which are equal in size and quality to fruits of healthy plants (11). Healthy fruits can also be obtained from asymptomatic branches of a plant, which shows severe symptoms on other branches. Remission of symptoms can be observed regularly in orchards and vineyards, but the underlining biochemical and physiological mechanisms are only poorly understood. An extensive survey of BN-spread in several vineyards in South Tyrol with more than 50,000 grapevines monitored over a five year period (2005-2009) showed an average recovery rate of 51%. For this survey, recovery was defined when a grapevine after two consecutive years of symptom expression was asymptomatic for the next two consecutive years (Schweigkofler & Roschatt, unpublished).

Leaf Symptoms and Mineral Content in Grapevine Leaves Typical leaf symptoms of grapevines infested by the BN phytoplasma include chlorosis, reddening, curling and patchy necrosis. Symptom expression can differ significantly between V. vinifera cultivars, but in general show some similarities to abiotic diseases caused by nutrient deficiencies. Therefore, we determined the content of seven elements (Ca, K, Mg, Mn, N, P and F) in leaves of healthy and BN-infected field grown grapevines of two white (Chardonnay, Müller Thurgau) and three red cultivars (Pinot noir, Lagrein, Zweigelt). As expected, the infection status of the plants changed the mineral levels (Table 2).

76

Table 2. Effect of BN infection on mineral content of white and red cultivars of V. viniferaa Mineral element

a

V. vinifera cultivar Chardonnay

Pinot noir

MüllerThurgau

Lagrein

Zweigelt

Ca

--

-

-

-

-

Fe

-

-

-

+/-

-

K

+/-

+/-

+/-

+/-

-

Mg

-

+/-

+/-

+/-

-

Mn

-

+/-

-

-

-

N

--

+/-

-

-

-

P

-

+/-

+/-

-

+/-

--: strongly reduced; -: reduced; +/-: no difference

The most significant and consistent decrease was found for Calcium levels. Calcium plays an important role as a messenger for many physiological processes, but is also involved in stabilizing morphological structures. Calcium deficiency in older leaves can lead to downward curling, similar to symptoms in grapevines infected with BN (Figure 1 B). The strongest effect of BN infection on mineral contents was found on the cultivars Chardonnay and Zweigelt, which is agreement with observations on diseases severity in commercial vineyards. Details on the experiment were published previously (22). The effect of foliar application of two fertilizers on mineral content, symptom expression and recovery of V. vinifera cv. Chardonnay was tested. Two commercial products (a complex NPK fertilizer and a calcium foliar spray) were applied monthly from June until September of 2008. While the application resulted in a temporary increase of Ca levels (Figure 3), no significant differences in symptom expression, fruit yield and recovery rates at harvest time were measured. Calcium is involved in several defence processes triggered by phytoplasmas and other phloem-limited pathogens; and Ca2+ levels are increased in apple trees spontaneously recovered from apple proliferation (23). We think that changes of Ca2+ levels play an important role in phytoplasma-induced pathogenesis and symptom development of grapevine; but further studies are needed to reveal the benefits of foliar or soil application of mineral elements to reduce BN symptoms.

77

Figure 3. Calcium content in leaves of V. vinifera cv. Chardonnay. Healthy and BN-infected grapevines were sprayed with a calcium foliar spray (Ca) and a complex NPK fertilizer (NPK), respectively. Mineral contents were analyzed ten days post-application. C: untreated control. Statistical significance assessed by Student’s t-test. Lowercase letters indicates significant at 0.05 probability level. Effect of Resistance Inducers and Plant Growth Regulators on Infection Rates, Growth Rates and Symptom Expression of Apple Trees Infected by the AP Phytoplasma Several experiments were carried out recently to induce recovery in plants infected with phytoplasmas. Romanazzi et al. (18, 19) used resistance inducers in a field experiment to control BN, and found that numbers of symptomatic plants decreased significantly compared to the untreated control. Recovery of the periwinkle Catharanthus roseus (Apocynaceae) infected with ‘Ca. P. mali’ was achieved by using the plant growth regulators indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) (24). We studied the effects of four commercially available bio-active compounds on the infection rates, phytoplasma titres, symptom expression and growth rates of apple trees cv. Golden Delicious infected with the AP phytoplasma over a three-year period under controlled conditions in an insect-proof field tunnel (3). The tested products were: Bion® 50 WG (active ingredient: Acibenzolar-S-Methyl; Syngenta Crop Protection, Switzerland), Messenger® (Harpin protein; Eden Bioscience Corporation, USA), Regalis® (Prohexadione-Ca, BASF, Germany) and Dormex® (Cyanamide, SKW Trostberg AG, Germany). The products trigger different physiological reactions in plants. Bion and Messenger induce the systemic acquired resistance (SAR) and hypersensitive reactions (HR) (25, 26). Regalis inhibits gibberellins synthesis, interferes with the metabolism of flavonoids (27) and decreases host susceptibility (28). In apple orchard it is used to regulate shoot elongation. Dormex contains 78

hydrogen cyanamide, which upregulates IAA and cytokinin (CK) levels (29) and inhibits catalases (30). In production areas with warm winters and insufficient chill hours Dormex is used for breaking bud dormancy in several deciduous fruit crops, such as apples. The products, except Cyanamid, were dissolved in water according to manufacturer’s instructions and sprayed on the canopy of a plant block consisting of 90 trees each. Cyanamid was dissolved and applied to the pots of the plants. Four to eight applications of the compounds were done at 10 to 14 day intervals between May and July. The treatments had no significant effect on infection rates of the apple trees. AP infection increased significantly the terminal growth of apple trees. The only product which had a significant (inhibiting) effect on the growth of both infected and non-infected apple trees was Prohexadione-Ca. Effects on symptom expression was mixed: Prohexadione-Ca caused severe growth abnormalities masking AP symptoms for several months; Harpin and Acibenzolar-S-Methyl showed no significant effects on symptom expression. The seasonal appearance of AP symptoms was changed by treatments with Cyanamide: symptoms were delayed compared to the untreated control the first two years of the experiment (2008 and 2009), but symptoms appeared earlier the third year (2010). Differences in symptom expression levelled off later in the vegetative season, and no significant difference was found in fall (Figure 4). Effects of the bio-active compounds -with the exception of the growth regulator Regalis- on plant growth and symptom development were restricted to the application period from May to July; no long-lasting effect was induced by the treatments.

Figure 4. Effect of resistance inducers and plant growth regulators on symptom expression (enlarged stipulae or/and witches’ broom) of AP infected plants. 79

Phytoplasma concentrations in leaves of AP infected trees increased significantly from spring until fall, whereas concentrations in the roots showed less variability over time. Treatment with Cyanamide showed no significant effect on phytoplasma densities; the effect of the other compounds on phytoplasma densities was not measured (3). The results from our experiments in the field and in a controlled environment underline the difficulties to control phytoplasma diseases of woody plants using chemical treatment alone. A comprehensive analysis of the ecology of the phytoplasma disease complex however can lead to a polyphasic management strategy, which will not eliminate the disease from an infested area, but decrease its economic and environmental impact.

Acknowledgments The work on Bois noir was funded by the Autonomous Province of Bozen/Bolzano, Italy. The Strategic Project on Apple Proliferation (APPL) was initiated by Drs. J. Dalla Via and R. Zelger and funded by the Autonomous Province of Bozen/Bolzano, Italy, and the South Tyrolean Fruit Growers’ Co-operatives, in particularly VOG (Verband der Südtiroler Obstgenossenschaften) and VI.P (Verband der Vinschgauer Produzenten für Obst und Gemüse). We want to thank all colleagues who helped with this research over the years, especially Sanja Baric, Gernot Kunz, Matteo Biagetti, Manfred Wolf, Christian Cainelli, Christine Kerschbamer, Giovanni Peratoner, Jennifer Berger, Thomas Letschka, Maya Massenz, Anna Cassar, Valerie Vanas, Elmar Stimpfl, Manuel Pramsohler, Armin Morandell and Erwin Haas, and the owners of the vineyards used for the surveys.

References 1. 2.

3.

4. 5.

Oshima, K.; Maejima, K.; Namba, S. Genomic and evolutionary aspects of phytoplasmas. Front. Microbiol. 2013, 4 (230), 1–8. Baric, S.; Berger, J.; Cainelli, C.; Kerschbamer, C.; Letschka, T.; Dalla Via, J. Seasonal colonisation of apple trees by ‘ Candidatus Phytoplasma mali’ revealed by a new quantitative TaqMan real-time PCR approach. Eur. J. Plant Pathol. 2011, 129, 455–467. Schmidt, S.; Baric, S.; Massenz, M.; Letschka, T.; Vanas, V.; Wolf, M.; Kerschbamer, C.; Zelger, R.; Schweigkofler, W. Resistance inducers and plant growth regulators show only limited and transient effects on infection rates, growth rates and symptom expression of apple trees infected with ‘Candidatus Phytoplasma mali’. J. Plant Dis. Prot. 2015, 122, 207–214. Weintraub, P. G.; Beanland, L. Insect vectors of phytoplasmas. Annu. Rev. Entomol. 2006, 51, 91–111. Mittelberger, C.; Obkircher, L.; Oettl, S.; Oppedisano, T.; Pedrazzoli, F.; Panasitti, B.; Kerschbamer, C.; Anfora, G.; Janik, K. The insect vector Cacopsylla picta vertically transmits the bacterium ‘Candidatus Phytoplasma mali’ to its progeny. Plant Pathol. 2017, 66, 1015–1021. 80

6.

7.

8. 9.

10.

11.

12. 13. 14.

15.

16.

17. 18.

19.

20. 21.

Tedeschi, R.; Jarausch, B.; Delic, D.; Weintraub, P.-G. Actual distribution of fruit tree and grapevine phytoplasma diseases and their vectors in Europe and neighboring regions. Phyt. Moll. 2013, 2, 3–4. Tedeschi, R.; Alma, A. Transmission of Apple Proliferation Phytoplasma by Cacopsylla melanoneura (Homoptera: Psyllidae). J. Econ. Entomol. 2004, 97, 8–13. Carraro, L.; Ermacora, P.; Loi, N.; Osler, R. The recovery phenomenon in apple proliferation-infected apple trees. J. Plant Pathol. 2004, 86, 141–146. Seemüller, E.; Kunze, L.; Schaper, U. Colonization behavior of MLO, and symptom expression of proliferation-diseased apple trees and decline-diseased pear trees over a period of several years. Z. Pflanzenkr. Pflanzenschutz 1984, 91, 525–532. Musetti, R.; Ermacora, P.; Martini, M.; Loi, N.; Osler, R. What can we learn from the phenomenon of “recovery”? Phytopathogenic Mollecutes 2013, 3, 63–65. Bianchedi, P.; Deromedi, M.; Battocletti, I.; Gualandri, V.; Zorer, R.; Piffer, I. Interferenza di ‘Candidatus Phytoplasma mali’ nello sviluppo del frutto: applicazione del modello espolineare di crescita. Proceedings SMAP: Scopazzi del melo apple proliferation: atti del convegno; Fondazione Edmund Mach; San Michele all'Adige, Italy, 2008; pp 12. Maixner, M. Recent advances in Bois noir research. Petria 2011, 21, 17–32. Tedeschi, R.; Alma, A. Fieberiella florii (Homoptera: Auchenorrhyncha) as a vector of ‘Candidatus Phytoplasma mali’. Plant Dis. 2006, 90, 284–290. Baric, S.; Kerschbamer, C.; Vigl, J.; Dalla Via, J. Translocation of Apple Proliferation Phytoplasma via natural root grafts - a case study. Eur. J. Plant Pathol. 2008, 121, 207–211. Kunz, G.; Roschatt, C.; Schweigkofler, W. Biodiversity of planthoppers (Auchenorrhyncha) in vineyards infected by the Bois noir phytoplasma. Gredleriana 2010, 10, 89–108. Berger, J.; Schweigkofler, W.; Kerschbamer, C.; Roschatt, C.; Dalla Via, J.; Baric, S. Occurrence of Stolbur phytoplasma in the vector Hyalesthes obsoletus, herbaceous host plants and grapevine in South Tyrol (Northern Italy). Vitis 2009, 48, 185–192. Chuche, J.; Thiéry, D. Biology and ecology of the Flavescence dorée vector Scaphoideus titanus: a review. Agron. Sustain. Dev. 2014, 34, 381–403. Romanazzi, G.; D’Ascenzo, D.; Murolo, S. Field treatment with resistance inducers for the control of grapevine Bois noir. J. Plant Pathol. 2009, 91, 677–682. Romanazzi, G.; Murolo, S.; Feliziani, E. Effects of an innovative strategy to contain grapevine Bois noir: Field treatment with resistance inducers. Phytopathology 2011, 103, 785–791. Anonymous. Hot water treatment of grapevine to control Grapevine flavescence dorée phytoplasma. EPPO Bull. 2012, 42, 490–492. Rizzolli, W.; Acler, A. Einfluss der Blattsaugerabwehr auf das Auftreten der Apfeltriebsucht. Obstbau Weinbau 2014, 51, 194–197.

81

22. Schweigkofler, W.; Cassar, A.; Stimpfl, E. Reduced levels of calcium and other mineral elements in grapevine leaves affected by Bois noir (BN). Mitt. Klosterneuburg 2008, 4, 117–122. 23. Bendix, C.; Lewis, J. D. The enemy within: phloem-limited pathogens. Mol. Plant Pathol. 2018, 19, 238–254. 24. Curković Perica, M. 2008. Auxin-treatment induces recovery of phytoplasma-infected periwinkle. J. Appl. Microbiol. 2008, 105, 1826–1834. 25. Vallad, G. E.; Goodman, R. M. Systemic Acquired Resistance and Induced Systemic Resistance in conventional agriculture. Crop Sci. 2004, 44, 1920–1934. 26. Wei, Z.; Betz, F. S. Messenger®: An Environmentally Sound Solution for Crop Production and Protection. In Crop Protection Products for Organic Agriculture; Felsot, A. S., Racke, K. D., Eds.; ACS Symposium Series 947; American Chemical Society: Washington, DC, 2007; pp 195−211. 27. Spinelli, F.; Speakman, J. P.; Rademacher, W.; Halbwirth, H.; Stich, K.; Costa, G. Luteoforol., a flavan 4-ol, is induced in pome fruits by prohexadione-calcium and shows phytoalexin-like properties against Erwinia amylovora and other plant pathogens. Eur. J. Plant Pathol. 2005, 112, 133–142. 28. Bazzi, C.; Messina, C.; Tortoreto, L.; Stefani, E.; Bini, F.; Brunelli, A.; Andreotti, C.; Sabatini, E.; Spinelli, F.; Costa, G.; Hauptmann, S.; Stammler, G.; Doerr, S.; Marr, J.; Rademacher, W. Control of pathogen incidence in pome fruits and other horticultural crop plants with Prohexadione-Ca. Eur. J. Hortic. Sci. 2003, 68, 8–14. 29. Guevara, E.; Jimènez, V. M.; Herrera, J.; Bangerth, F. Effect of hydrogen cyanamide on the endogenous hormonal content of pea seedlings (Pisum sativum L.). Braz. J. Plant Physiol. 2008, 20, 159–163. 30. Amberger, A. Wirkung von Cyanamid auf Katalase und Peroxydase in Pflanzen. Zeitschrift für Pflanzenernährung, Düngung, Bodenkunde 1961, 95, 123–130.

82

Chapter 8

Pear Ester – From Discovery to Delivery for Improved Codling Moth Management Alan L. Knight,*,1 Douglas M. Light,2 Gary J. R. Judd,3 and Peter Witzgall4 1Temperate

Tree Fruit and Vegetable Research, Agricultural Research Service, U.S. Department of Agriculture, 5230 Konnowac Pass Road, Wapato, Washington 98951, United States 2Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany California 94710, United States 3Agriculture and Agri-Food Canada, Summerland Research and Development Centre, 4200 Highway 97, Summerland, British Columbia, Canada 4Division of Chemical Ecology, Department of Plant Protection Biology, Swedish University of Agricultural Sciences, Alnarp, Sweden *E-mail: [email protected]

The chemical ecology of codling moth, Cydia pomonella (L.), has been the subject of a worldwide research effort spanning five decades. The initial focus of this work was the characterization of codling moth sexual behavior and the identification of its sex pheromone, followed by the development of effective monitoring and management programs. Subsequently, a large body of work was dedicated to deciphering the chemical messaging systems that exist between both moth sexes and their apple host. However, it was from pear that a potent kairomone, pear ester, ethyl (E,Z)-2,4-decadienoate, was discovered, and surprisingly from field studies in walnut. Pear ester over the last decade has been the basis for the development of a range of commercial products that impact larval and adult behaviors and reduce levels of fruit injury. A review of codling moth and behavioral-active apple volatiles, the discovery of pear ester, and the development of useful technologies is provided here. A recounting of this story provides some considerations © 2018 American Chemical Society

for the reader. First, that single odorants from a host e.g. (E,E)-α-farnesene and pear ester and maybe not complex volatile blends that more thoroughly characterize a host odor, are fruitful targets to develop female attractants. However, practical concerns such as chemical stability and synthesis cost will limit the implementation of any discovery. Second, it is not clear what semiochemical evoked behaviors should be targeted to develop an effective lure, i.e. suitable host for sexual rendezvous, oviposition, or as a food source. Background odors from immature fruits and undamaged foliage are generally more dilute and less complex than from ripening fruits or damaged foliage. Thus, effective chemical signals need to be more intense and apparent to lure moths. Third, it appears that adding acetic acid to host plant volatile lures is effective in drawing moths into traps, perhaps as a short-range food cue. Fourth, it was a field bioassay with a pear volatile in a walnut grove that unveiled the power of pear ester. Only later did a series of physiological and molecular studies detail the evolved interplay of pear ester and sex pheromone in the brain of codling moth. It is possible that this more basic approach will in the future allow the purposeful discovery of new attractants which can aid pest management of tortricids and other pest species. But more likely, chemists and applied insect ecologists need to continue to identify, synthesize, and test the various semiochemicals that define the lives of insects.

Background Growers of commercial apple, Malus domestica Borkhausen, are engaged in a dynamic enterprise based on the production, storage, and shipping of high-quality fruits throughout the world. To achieve this market-mandated level of perfection, apple has become one of the most chemically-treated fruit crops; annually receiving multiple sprays of fungicides, growth regulators, and insecticides (1). A key driver in the international market is the enforcement of ever increasing regulatory restrictions on permitted post-harvest residues of crop protection chemicals (2). Typically, these standards restrict the types, timing, and rates of materials that can be applied during the season. Within this regulatory framework, producers are attempting to grow perfect fruit in a complex agricultural environment where an array of arthropod pests feed on the roots, wood, flowers, foliage, and fruits of the crop throughout the year (3, 4). Single tactic approaches, such as repeated applications of broad-spectrum insecticides have a documented history that demonstrates the complexity of the ecological associations among pests in apple orchards (evolution of resistance, outbreaks of secondary pests), their natural enemies, and the associated externalities within and surrounding orchards (5). Today, several consistent trends, including: lower 84

chemical residues, greater protection for workers and their families, containment of spray drift and surface water run-off, and suburban expansion are together all placing greater restrictions on pesticide usage that directly impact production of apple. To achieve successful production of fruit with judicious use of the available pesticides requires ongoing research and education (6). Conceptual development of an integrated pest management (IPM) strategy for apple was pioneered in the 1970’s, and widely showcased as an IPM success story (7). The development of predictive models and monitoring tools has allowed growers to reduce their reliance on pesticides, enhance biological control, and likely slow the evolution of pesticide resistance and loss of efficacy of existing materials (8, 9). Another key component of apple IPM has been the identification and development of natural products, such as semiochemicals, including sex pheromones and plant volatiles (kairomones) (10, 11). Various behaviorally active compounds have been identified and countless studies have evaluated the potential effectiveness of these compounds and suggested how they might be used. In some cases, innovative tools have been developed and adopted by industry to improve management of key pests with minimal supplemental use of pesticides (12). The major pest group attacking apples throughout its world-wide distribution are the larvae of moths (Family, Tortricidae) that feed on both the skin and internal tissues of fruit including the seed cavity (13). Codling moth, Cydia pomonella (L.), (Lepidoptera: Tortricidae), the most important species in this group, also attacks pear, Pyrus communis L., and walnut, Juglans regia (L.) (14). In addition to codling moth, it is common for one or more regionally endemic tortricid species to reach pest status in most apple growing districts, and these tortricid pests often require additional management practices, including insecticides (15, 16). The life history and several adaptive traits make management of codling moth problematic for IPM. Eggs are often laid near or on the fruit and neonate larvae enter fruits quickly, completing development in the seed cavity (17). Mature larvae leave the fruit and construct protective silken cocoon chambers under the bark of trees (18). Adults can emerge, mate, and begin to lay eggs, within 24 hours (19). Naturally-occurring biological control agents are unable to protect fruit in commercial orchards at levels demanded by the fresh market (20). Typically, codling moth is managed with a series of insecticide sprays that cover or blanket the crop with toxic residues (i.e., cover sprays) during the season (21). The switch from broad spectrum to more selective classes of “reduced-risk” insecticides has not always reduced the number of sprays applied, but has created new issues with several secondary pests and new invasive pests (22, 23). Development of natural products which might impact adult mating and oviposition or disrupt larval behaviors has been advocated as an important priority to reduce the repeated dependence on insecticides, and improve the efficacy of less-effective, but more selective materials, especially within organic production systems (24, 25). One of the major non-insecticide developments affecting management of codling moth and other tortricid pests of apple has been the identification, synthesis, and use of sex pheromones as management tools and direct control agents. Low-cost, effective sex pheromone lures in traps are widely used by 85

growers to monitor adult populations (26). Moth counts are used to track the seasonal occurrence of pests and to estimate the relative pest population activity and abundance within orchards (27). Unfortunately, the use of sex pheromones to monitor pests has several associated problems. There is an inherent uncertainty in establishing thresholds for what are primarily female-based behaviors based on male catches due to the likely occurrence of either false-negative or false-positive male catches, the errors associated with timing egg hatch based on male flight, and the inability to monitor the potential immigration of female moths into orchards (28). One approach to eliminate these inherent problems with sex pheromone-based male monitoring systems could be to develop similar tools based on kairomones that allow managers to directly track female moths. Key chemically-mediated behaviors of female moths include both detecting and following host signals that reveal suitable sites for mating, oviposition, and feeding (29). Numerous studies have characterized the volatiles emanating from tortricid host plants and an array of laboratory and field trials have attempted to develop effective management tools for codling moth and other tortricids. However, the development of effective kairomone-based monitoring lures for female moths, including codling moth, has been difficult. Sex pheromones have also been used to directly manage pests by disrupting moth mating, and a variety of products have been tested against codling moth (30–33). The development of mating disruption (MD) technologies for codling moth was initially concerned with formulation issues, i.e. chemical stability of the conjugated diene, (E,E)-8,10-dodecadien-1-ol and dispenser’s blend, emission rate, and longevity (34–37). Secondly, the possible mechanisms by which the different formulations of sex pheromones, i.e. sprayables, hand-applied, and aerosol dispensers, affect mating behaviors have been tested and detailed (38). MD was shown to work well when applied in an areawide program that included growers’ collective cooperation, careful pest monitoring, and elimination of unmanaged hosts surrounding treated areas (12). The effectiveness of MD for managing codling moth is impacted by aspects of its biology, especially the mating frequency of both sexes (19, 39) and the aggregation of overwintering larvae before pupation (40). Temporal delays in mating and subsequent reductions in fecundity and egg fertility were shown to be important in net population reductions (41–43). Unfortunately, levels of mating of female codling moth in populations under management with MD, has been shown to be high (44, 45). Female codling moths can detect sex pheromone (46), and exposure to its sex pheromone can impact female behaviors, such as calling and egg laying (47). Positive interactions between sex pheromones and plant volatiles were found with male codling moth (48–50). It is possible that further improvements in MD for codling moth could involve behavioral disruption of both sexes with kairomones (24, 51). The history of identifying host-plant volatiles and developing their applied uses for managing codling moth has been fraught with difficulty and punctuated by serendipitous discovery. This chapter briefly discusses critical aspects of this history and attempts to draw conclusions about the most productive approaches future scientists could use to identify additional compounds to 86

improve management of codling moth and other related pest species. As the key example, we will summarize the research that has led to the identification of pear ester and the development of its use for monitoring and managing codling moth. We are fortunate that following the extensive applied development of pear ester as a novel attractant the sensory perception, genetic basis, and molecular underpinning of the significance of pear ester in the biology of codling moth and related species has been revealed and will also be summarized.

The Path to Pear Ester Codling moth larvae and adults have long been known to exhibit a strong olfactory and behavioral response to apple fruits (52, 53). But, it was not until the 1970’s that the (E,E) and (Z,E)- stereo-isomers of α-farnesene, were identified as the principal volatiles released from the skin of apple, pear, and quince fruits eliciting various behavioral responses (54–56). Unfortunately, Russ (57) showed that pear was less attractive than apple in his assays and assumed this response was directly related to the lower levels of α-farnesene. No other pear volatiles were considered as possible attractants. Also, due to the known chemical instability of α-farnesene (58). the potential to develop management tools based on α-farnesene was not discussed in these early papers, and over the next two decades no further work was reported on host-plant attractants for codling moth. During this period of inactivity on kairomones analytical chemistry based on headspace collections and gas chromatographic and mass spectrometric (GC-MS) analyses, became more widely available for applied laboratories to characterize the volatiles emitted by codling moth host plants (59–63), and to develop new attractants for other apple pests, including the apple maggot, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae (64, 65). By comparison, tortricid sex pheromones were identified with relative ease and this led to new monitoring tools and commercial formulations for mating disruption (66). The proliferation and availability of sex pheromones temporarily placed advances in kairomone chemistry in the background. During this same period, insecticide resistance in codling moth continued to develop to an ever-increasing number of classes of insecticides (67). Social and political trends led to further reductions in available effective insecticides in pest management, e.g. Food Quality and Protection Act 1996. Thus, new efforts were undertaken to characterize host volatiles in attempts to develop more effective management tools targeting the disruption of neonate searching behaviors and adult mating. This included looking at whether host plant volatiles could be used to improve the attractiveness of sex pheromones (48, 49); or whether plant volatiles could be used as oviposition deterrents (68). Yet, the largest part of this chemical ecology renaissance targeted a reconsideration of the activity of α-farnesene for larvae and adult codling moth (50, 69–71). These studies largely confirmed the previous body of work, but also continued to suggest that the release of α-farnesene alone did not fully explain the levels of larval or adult attraction elicited by fruit and this renewed the scientific curiosity to identify additional apple volatiles as attractants (24, 72–74). 87

In response to this challenge, two different research approaches were used to identify additional compounds that could account for the behavioral response of adult codling moth to apple. The first approach used a combination of GC-MS analyses of volatiles released by developing fruits and whole plants throughout their phenological season combined with electroantennographic detection (EAD) techniques to identify the most electrophysiologically sensitive compounds. Analysis of headspace volatiles from fruits and branches showed that terpenoids were most prevalent early and esters later in the apple phenology season (75). Among the early-season apple volatiles that codling moth antennae strongly responded to were six terpenoids, including β-linalool, β-caryophyllene, (E)-β-farnesene, germacrene D, (Z,E)-α-farnesene, and (E,E)-α-farnesene, the homoterpene 4,8-dimethyl-1,3,(E)7-nonatriene (DMNT), and the benzenoid methyl salicylate (76). Neither antennal response nor the strength of this depolarization response is an indication that chemicals are attractive, for example methyl salicylate is purported to be a codling moth repellent (77). Subsequent laboratory and field trials established some effectiveness for (E,E)-farnesol and (E)-β-farnesene as male attractants, but only in the wind tunnel or field, respectively (78, 79). The addition or combination of other volatiles eliciting antennal responses did not increase moth catches. Two additional studies were performed using volatile collections throughout the season and electroantennographic responses of codling moth to both apple and walnut volatiles (80, 81). Several compounds were found to elicit antennal depolarization responses, but the work was not extended to behavioral assays and no new attractants were identified. Instead, this work with its overwhelming numbers of identified compounds (e.g., > 80) is an excellent example of how limiting these types of large data sets are in unraveling the complexities of insect-host plant communication, where volatiles can be attractive or repellent, the magnitude of responses can often be primarily concentration-sensitive, and that volatiles always occur as components of natural blends that vary seasonally and among hosts. A second target for finding attractants for adult codling moth management focused primarily on the esters released by ripening fruits. These studies characterized the seasonal occurrence of various compounds from healthy and injured fruits (82–84), but behavioral bioassays to identify blends and individual attractants were only conducted in the laboratory (82, 85). This approach yielded two esters, hexyl hexanoate (82) and butyl hexanoate that attracted female moths (85). Interestingly, the former had elicited the strongest antennal response from females but the latter exhibited the second lowest antennal response among the seven esters evaluated previously (75). Field trials have not yet found these esters to be attractive to codling moth in orchard settings (78, 79, 86). The potential for host odour masking of putative attractants developed from electroantennogram (EAG) screening or laboratory olfactometer bioassays is a fundamental problem in identifying useful tools for pest management (78, 87). One assumption implicit in this approach is that the emission of a higher concentration above background of a ubiquitous volatile can create a chemical signal plume that would allow moths to orient to baited traps. Further, the effectiveness of any lure would need to be retained over the course of the season as the host odour profile evolves. Oligophagous and polyphagous pests have 88

evolved to respond to a variety of effective kairomones comprised of different volatiles derived from their uniquely different host plants e.g., apple, pear and walnut (80, 81). Thereby a different approach to discover new kairomones could be to identify volatiles from one host and test them for attraction in a different host context. Of course, this would be contrary to the assumption that any volatile attractant should be present in all hosts all the time (80, 81, 88). Yet, testing in a different host context is exactly how pear ester, ethyl (E,Z)-2,4-decadienoate, a well-known odorant of ripe pear (89), was discovered to be a potent bisexual attractant for codling moth.

Discovery of Pear Ester Chemists at the USDA laboratory in Albany, CA assembled an extensive library of volatile plant compounds which were available for testing with codling moth. Initially the focus of the work was to screen for possible synergists of the sex pheromone as had been done previously with green leaf volatiles (48). Ninety-two pome volatiles were organized into 23 blends based on their chemical structure, i.e. common carbon-chain length and/or alcohol, aldehyde, or ester moieties and tested as pheromone synergists in a walnut grove (90). It was assumed that the terpenoid odor profile of walnut (81) might only minimally mask these compounds. Six blends were found to significantly increase and synergize male attraction to the sex pheromone codlemone, (E,E)-8,10-dodecadien-1-ol. However, only one blend (Ester-10) a 4-compound blend of methyl and ethyl 10-carbon esters caught both moth sexes and both mated and virgin females. Field tests of these 23 host volatile blends without the presence of sex pheromone were repeated in walnut and expanded to pome fruits and this substantiated that the Ester-10 blend was the only blend attractive to females and males (86, 90). The most effective constituent of the Ester-10 blend was determined to be ethyl (E,Z)-2,4-decadienoate, “the pear ester” (86). GC-EAD studies confirmed that pear ester was the only pear volatile identified from among the 15 FID peaks that elicited an obvious and significant depolarization response (90). Laboratory behavioral assays with codling moth larvae also demonstrated the potent activity of pear ester, i.e. attractive at 1,000-fold lower concentration than α-farnesene (91). Surprisingly, pear ester exhibited a similar dose response threshold (10 µg dose per septum) as sex pheromone for adults, and within walnut caught similar numbers of moths as a sex pheromone lure (86, 90). Thus, pear ester for both adults and larvae was an effective attractant at very low concentrations and emission rates. Fortuitously, the combination of its low synthesis cost, good chemical stability, and a high level of potency quickly demonstrated pear ester’s potential commercial value, and its discovery was protected with two patents (92, 93). The attractiveness of pear ester to both larvae and adult codling moth was recognized as an opportunity to develop behaviorally-active management tools. The dose-response (0.01 µg – 50 mg) of loading pear ester in grey halobutyl septa was explored and the loading rate was found to be an important factor affecting 89

catch numbers, sex ratio, and mating status of females (94). Results from the initial studies in pome fruit and walnut comparing sex pheromone and pear ester lures found equivalence between lures in conventional apple and walnut orchards but lower attraction for pear ester in pear; while in both apple and walnut orchards treated with sex pheromone dispensers the pear ester lure attracted significantly more moths than the sex pheromone lure (90, 95). However, further studies showed that pear ester lures could be used in commercial pear orchards (96, 97). The effectiveness of pear ester was only compromised in ‘Bartlett’ pear orchards with high levels of codling moth injury, likely due to the release of pear ester and other volatiles from herbivore-injured fruits (98). An effort to improve monitoring of codling moth in pear by switching to propyl (E,Z)-2,4-decadienoate lures was not successful (99). The seasonal flight patterns of codling moth monitored with pear ester were compared with male monitoring with sex pheromone in apple orchards treated with sex pheromone and several interesting findings were found (100). For example, sex pheromone-baited traps caught moths before traps with pear ester, peak catch coincided between the two lures, while for pear ester-baited traps the sex ratio was only slightly skewed in favor of males over females, >80% of females were mated, and pear ester outperformed the sex pheromone lure when the density of sex pheromone MD dispensers was increased. The influence of various physical factors on the performance of pear ester-baited traps for male and female codling moths was examined in a range of early studies (101). For example, females were trapped with pear ester several hours before the start of male activity to sex pheromone (102). Moth catches in pear ester-baited traps were 6- to 14-fold higher in the late-season cultivar ‘Granny Smith’ than four other cultivars and this was supported with similar results from Australia (103). Trap size impacted the catch of moths, especially with greater numbers of females being caught on large sticky surfaces and always >15 cm from the lure, while males were often caught nearer or beside the lure. Similar results were found in Italy with larger traps catching more females (104). Trap height in the canopy was not a significant factor affecting the catch of females with pear ester, unlike males, caught mostly in the higher canopy, as similarly shown previously with males to sex pheromone (36). Also, the proximity of the pear ester-baited trap to sex pheromone dispensers placed in the canopy did not impact female catch unlike the interference previously shown with male codling moth and sex pheromone-baited traps (105). Interestingly, significantly more females were caught in pear ester-baited traps surrounded by foliage versus traps without adjoining foliage, and higher female counts occurred in traps placed adjacent to uninjured fruits compared with the absence of near-by fruits (101). It is important to emphasize that most of the factors that were shown in these studies to significantly impact female moth catches with pear ester are typically not considered and left uncontrolled by orchard managers utilizing traps to monitor codling moth. In response to increasing concerns among U.S. apple growers that the use of sex pheromone dispensers for mating disruption made monitoring with sex pheromone lures problematic and that moth catch was too low with pear ester alone, a “Combo” lure combining pear ester with sex pheromone was 90

developed (106). An optimal loading per septum was established, but studies also showed that suboptimal loadings could have significant effects on moth catch. For example, increasing the loading of pear ester above the optimum (1 mg) could decrease female catch and adding pear ester to traps with a high-load sex pheromone lure could significantly decrease male catch. This was confirmed in later studies with various experimental lure loadings in untreated orchards where combining sex pheromone and pear ester decreased male catches compare with a sex pheromone lure alone (107, 108). This competitive interference observed in traps combining pear ester with sex pheromone was also found in Italy; and a possible mechanism was suggested through saturation studies of antennal response to pear ester when the receptors were continuously stimulated by sex pheromone (109). However, later studies conducted with the commercial lure, Pherocon CM-DA Combo (“Combo” lure) loaded with equal amounts of both pear ester and sex pheromone, in MD apple orchards have been more consistent and clearly showed it out-performs sex pheromone lures (97, 110). Yet, the literature detailing the use of pear ester to monitor codling moth is somewhat variable. For example, some results are consistent with U.S. studies, e.g. higher catches in orchards treated with sex pheromone in Australia (103) and lower catches in orchards left untreated in Italy (104, 111); other results diverged. For example, traps with pear ester failed to catch any female moths in Bulgaria (112), while over a two-year period they caught 35 – 50% females in Italy (111) but in other cases females contributed only 1 and 4% of the total catch during the second moth flight in Italy (113) and Canada (108), respectively. Interestingly, the relatively low proportion of females caught with pear ester lures in one country was shown to be due to a male bias from inadvertent contamination of trapping materials with sex pheromone (unpublished results). The greatest excitement around the discovery of pear ester has primarily been its attractiveness to female moths (90). With this greater ability to monitor female moths, studies were conducted to develop new protocols to enhance detection of females and their potential immigration into sex pheromone-treated orchards. Dark colored sex pheromone-baited traps were found to catch more codling moth males and fewer honeybees than white traps (114). Orange traps had a lower spectral reflectance than white traps and were hypothesized to be less visually disruptive for the dusk-flying moths and this was supported by flight tunnel studies observing moth behaviors to traps (115). In other field trials there was no influence of trap color on female catches with pear ester, perhaps because they orient to pear ester-baited traps prior to sunset (101). However, related studies with pear ester lures found that female catch was 30-fold higher on clear horizontal interception traps than in white delta traps (116). Clear delta traps were developed and these caught 6-fold more females than similar orange delta traps when baited with pear ester (117). Similarly, clear delta traps baited with either the “Combo” lure or pear ester plus acetic acid lures caught 4-fold more female codling moths than orange traps (118), but these catches were still only about half as many as caught on the clear horizontal interception traps, suggesting that further improvements could be made to increase trap performance (116). Unfortunately, while clear traps are widely used in Hungary they have not been adopted in the U.S. or in other countries. 91

Various studies have been undertaken to improve the attraction of females to pear ester in both conventional and sex pheromone-treated orchards. Moth catches in traps baited with only pear ester is typically 40 – 60% female (90, 119). However, when pear ester has been used as a “Combo” lure with codlemone added females comprise only 5 to 10% of the catch in (97, 106) and 21% in walnut (119). Several research efforts were conducted to further improve the use of pear ester to monitor female codling moth and increase their catch. The addition of an acetic acid co-lure with pear ester was found to significantly increase male and female moth catches (120). Acetic acid is often a microbial-produced fermentation product and these authors suggested codling moth’s attraction to the acetic acid pear ester combination was a response to overripe or damaged fruit as a food source (74). Within orchards treated with sex pheromone dispensers this new combination of pear ester and acetic acid was much more effective than sex pheromone lures and caught 40% females in apple (121) and 62% in walnut (119). Clear or orange traps baited with pear ester and acetic acid performed similarly to orange traps baited with the “Combo” lure but with the advantage of a much higher proportion (>60%) of females (122, 123). Interestingly, the positional placement of the acetic lure within a delta trap (either hung from the inside roof of the trap or on the liner) was shown to be important factor affecting male but not female catches (124). The discovery that acetic acid synergized the activity of pear ester suggested that other volatile combinations should be reassessed. A review of the literature suggested that the damage-associated volatile DMNT could be another attractant for codling moth (i.e. evokes male and female antennal response and is present in both immature and ripening apples). Subsequent, laboratory and field studies demonstrated that it was attractive when used with acetic acid, but not as attractive as the combination of pear ester and acetic acid (125). DMNT was also found to be effective when used with sex pheromone (119, 126). Binary lures formulated with DMNT and pear ester marginally increased total and female moth catches compared to pear ester alone when both were used with acetic acid (127). Perhaps more importantly the use of combinational lures with DMNT either with or replacing pear ester plus sex pheromone and acetic acid demonstrated some utility in orchards treated with dispensers loaded with sex pheromone and pear ester (119, 123, 127, 128). Other plant volatiles purported to be attractive for codling moth were tested alone and in combination with acetic acid, including (E)-β-ocimene, butyl hexanoate, (E)-β-farnesene, Z-(3)-hexenyl acetate and farnesol and were found to be ineffective (123, 129). Similarly, neither the addition of (E)-β-farnesene or farnesol with sex pheromone and acetic acid were effective female attractants within orchards treated with sex pheromone and pear ester dispensers (123). Binary blends of pear ester with either DMNT or decanal significantly increased female catch compared with pear ester alone but comparable to pear ester with acetic acid (130). Unfortunately, this study did not test these binary blends in combination with acetic acid. One additional codling moth attractant blend has been reported that includes the use of N-butyl sulfide in combination with either pear ester or pear ester and acetic acid (131). These authors reported that adding N-butyl sulfide doubled the female catch; however, more extensive trials conducted in four countries over several years using the same lures have not shown it to be more effective than the “Combo” lure (132). 92

Monitoring with Pear Ester Practical use of pear ester to improve codling moth population monitoring has included both the establishment of action thresholds based on moth catches in traps and a consideration of predicting the phenology of egg hatch based on female moth flight instead of males. Action thresholds based on moth catches in traps baited with pear ester or sex pheromone were developed during a threeyear study in 102 apple blocks treated with sex pheromone dispensers and left largely unsprayed (28). Use of pear ester lures was found to be more effective than sex pheromone lures in predicting mid- and late-season codling moth fruit injury at levels >0.3%. However, neither lure was effective in predicting low levels of fruit injury, 50%. Similarly, effective action thresholds (10 total and 0.5 females) using the “Combo” lure were developed during a three year study in Utah (135). Similarly, pear ester has been used to improve the timing of insecticides targeting neonate larvae. A three-year study compared the prediction of egg hatch based on the timing of cumulative degree days following the start of sustained moth catch using either sex pheromone (males) or pear ester (males, females, total) lures (136). The cumulative degree-day totals differed between lures and sexes, yet both lures were shown to be effective. However, the associated variability with these predictions was found to be lowest using female moth catch in pear ester-baited traps. This work was expanded in Chile and the prediction of egg hatch using female instead of male moth catches provided a four-day improvement (137). Also, the addition of the acetic acid lure increased female catches and improved the use of the female-based model in this study.

Utility of Pear Ester The use of sex pheromone technologies to manage codling moth is purported to be due to mating disruption, and this implies that the success of any of the various commercial sex pheromone products could be measured by measuring female mating status. Prior to the development of pear ester lures, the mating success of female codling moth was evaluated using catches of moths in light traps (44), bait-pans, or passive oil-coated interception traps (45). Studies demonstrated that the proportion of unmated females was somewhat lower in traps with pear ester than with the passive interception traps suggesting some active bias of mated females for pear ester (138). The proportion of unmated females (ca. 40%) was higher in apple orchards with lower population densities (1-2 female moths per trap per season) than in orchards with 3 to >20 females per trap per season (ca. 20%). Surprisingly, the levels of mating were found to be similar 93

for populations in untreated or sex pheromone-treated orchards (138–140). These unexpected findings, however, were consistent with a previous study suggesting that a primary effect of sex pheromone disruption technology was to delay mating which reduced the fecundity of moths and fertility of eggs (41). The use of pear ester-baited traps also facilitated a more laborious research approach to evaluate the effectiveness of sex pheromone dispensers using dissections of mated females and counts of their remaining oocytes (45). An additional technique has been used to classify the size of deposited male spermatophores (39). However, perhaps a more useful approach to ascertain the effectiveness of sex pheromone dispensers has been to measure the reduction in the proportion of multiple-mated females caught in pear ester-baited traps (39, 141). Nevertheless, while apple growers have widely adopted the “Combo” lure to monitor codling moth, they largely ignore the additional information provided by the female moths caught in traps to assess the effectiveness of mating disruption technology. Several other useful pest management applications have followed the discovery of pear ester as an attractant for codling moth. Initially, pear ester was thought to be a specific attractant for codling moth because eight moth pests common in fruit and nut orchards in the western U.S., including the oriental fruit moth, Grapholita molesta (Busck), in peach were not caught in baited traps (142). However, authors of a later Italian study, using EAG responses and laboratory olfactometers hypothesized that male G. molesta might be attracted to pear ester within pome fruit but not peach, Prunus spp (143). Unfortunately, field trials were never conducted to support this claim. However, pear ester in combination with (E)-β-ocimene did not improve the performance of a sex pheromone lure for G. molesta in stone and pome fruit orchards (144). Another Italian study found that pear ester was attractive to three tortricid pests of chestnut, Castanea sativa Miller, but at a 10-fold higher lure loading than with codling moth (112). In comparison the polyphagous species, Hedya nubiferana Haworth was not attracted to pear ester, but the combination of pear ester and acetic acid was an effective lure for this species (145). Pear ester when used with acetic acid has also been shown to be attractive for apple clearwing moth, Synanthedon myopaeformis (Borkhausen) (146). The fact that neither of these pests infest fruits, even though they are associated with these pome host plants, and they only respond to pear ester in combination with acetic acid lends support to the hypothesis that this combination lure is attractive to these moths, including codling moth as a food cue. The attractiveness of pear ester has been useful in a range of other studies assessing the biology of codling moth. For example, pear ester has been used to study the distribution and dispersal of female codling moth using immunological techniques and marking dyes at various scales through typical agroecosystems (147, 148). Traps baited with acetic acid and pear ester were very effective at assessing the overflooding ratio of sterile codling moths released as part of the British Columbia sterile insect program (149). The sterile to wild ratio of male, female and total moth catches with acetic acid and pear ester lures were near identical, and all were better predictors of control and damage, than moth catches with either pheromone or CM-DA lures. Codling moth response to pear ester was shown to differ or not among populations with varying levels of resistance to 94

organophosphate insecticides (109, 150). The use of pear ester was influential in demonstrating the unrecognized sublethal effects of insecticides on both male and female behaviors (151, 152).

Disruption of Larvae with Pear Ester Codling moth neonate larvae were shown to orient to pear ester in several laboratory assays and this suggested that pear ester could be used in additional approaches to manage codling moth (91). Behavioral impacts observed with exposure to pear ester, including, increased larval wandering and longer arrestment times before entering fruits, would likely increase topical exposure of larvae to contact with surface toxicants (153, 154). Greater exposure to insecticides when combined with pear ester could allow managers to reduce rates of insecticides, boost the effectiveness of some classes of insecticides, and ameliorate the effects of poor spray coverage (91). Codling moth, in general, deposit eggs on foliage close to developing fruit early in the season and more often on fruits later in the season (17). Choice and non-choice bioassays demonstrated that pear ester stimulated oviposition (155), however, a practical method to develop an egg trap to monitor populations which could be standardized compared with mechanically injured fruits was not successful (156). The practical disruption of oviposition by applying pear ester to foliage was demonstrated in pear orchards where the distance between eggs and fruits increased by 50% leading to reduced fruit injury (157, 158). A microencapsulated (MEC) formulation of pear ester (CideTrak DA-MEC, 5% A.I.) was developed (received U.S. registrations for conventional and organic production systems), and has been carefully characterized, including capsule density, size range, emission rates, and a residual attractiveness of 14 days (153). Various field trials were conducted that added pear ester to different classes of insecticides which have a range of residual and per os route of exposures in both walnut (159, 160) and apple (161, 162). In general, pear ester significantly improved the performance of insecticides with residual effectiveness and also reduced nut injury by the navel orangeworm, Amyelois transitella Walker in walnut (160). However, more variable results were found between laboratory assays and field trials with the granulosis virus of codling (160, 163, 164), including when used in combination with sugar and various yeasts (Metschnikowia, Cryptocococcus and Aureobasidium) isolated from codling moth larval feeding (165), and with the commercially-available Saccharomyces cerevisiae Meyen ex E. C. Hansen (166).

Mating Disruption with Pear Ester The first field trials evaluating whether pear ester applied either as a MEC spray or as an experimental hand-applied dispenser could disrupt mating of codling moth were judged to be successful based on the shut-down of male capture in sex pheromone-baited traps and reduced nut and fruit injury (141). Field trials continued over a 7-year period to refine proprietary “Combo” MD dispensers with variable loadings and ratios of pear ester and sex pheromone (119, 167–170) 95

and to assess the addition of DA-MEC sprays to sex pheromone dispenser-based programs (171). All studies were conducted in replicated small plots (0.1 – 3.0 ha) and assessments of dispensers were conducted via male catches in sex pheromone-baited traps or in traps with “Combo” lures and/ or sampling of fruit injury. However, studies varied widely in their direct and indirect assessments of dispensers on actual disruption of sexual communication and included the frequency of mating of tethered females (170, 172) and moth catches in traps baited with virgin females, female-equivalent sex pheromone lures, and pear ester lures in screened cages (169). Female moths caught with pear ester lures were also dissected to determine their mating status. These studies demonstrated that neither male moth catch in sex pheromone-baited traps nor levels of fruit injury in small plots supplemented with seasonal spray programs were useful measures to assess the efficacy of adding pear ester to sex pheromone dispensers (168, 169). Instead, male catches in female-baited traps and the proportion of unmated wild females caught in pear ester-baited traps were more effective and a more direct measure to compare dispenser treatments (162, 168, 169). The use of tethered virgin females is an excellent direct measure of sexual disruption but is more laborious to deploy and provided little consistent discrimination between sex pheromone and “Combo” dispenser treatments, across generations and years (170, 172). Moreover, significant increases in the proportions of both unmated females and decreases in multiply-mated females were found when pear ester was used to augment MD (140, 162). Utilizing pear ester in an integrated program through repeated applications of the DA-MEC added to insecticides and the use of “Combo” dispensers was shown to be the most effective program to disrupt mating of codling moth in apple across studies conducted from 2006 to 2012 (162). Practical implementation of MD in walnuts due to their large canopy size required the formulation of ‘Meso’ PVC dispensers which were 10-fold larger and could be applied at 1/10th the rate (50 ha-1) of standard PVC dispensers (119). “Combo” ‘Meso’ dispensers have been shown to be effective in both walnut (119, 140) and apple (80 dispensers ha-1) (128). Similar to the results with the MEC formulation added to insecticides in walnut (160) ‘Meso’ dispensers were also effective in reducing nut injury by A. transitella (119).

Lure and Kill with Pear Ester Several earlier applications were developed to test the concept that sex pheromone formulations could be used to trap out or kill adult male codling moths (173, 174). Previous simulation models had suggested that in mating systems where males can mate more than once, control efforts that rely exclusively on male removal, would be ineffective unless pest populations were extremely low (175). The concept of using pear ester lures to remove male and female codling moth, either through mass trapping or attract and kill stations, was shown via similar simulation models to have some potential, especially if virgin females could be removed (176). A variety of trapping and killing approaches were considered and tested over a 5-year period, including sticky traps, and use of insecticide-coated traps, clear sticky panels, and screen netting (177, 178). 96

Following the identification of acetic acid as a synergist for pear ester new studies looked at low volume spot sprays of insecticide either alone or with MEC pear ester in the canopy surrounding acetic acid lures or stations baited with pear ester and acetic acid (unpublished data). Studies were conducted in replicated small plots (0.1 – 1.0 ha) with typically 60 traps or stations ha-1, and levels of fruit injury were never reduced > 50% compared with untreated plots. The key limitations of this method appeared to be the high labor and material costs associated with station and lure maintenance, and the short residual effectiveness of the toxicants. However, Washington growers remain interested in using lure and kill for troublesome hot-spots, including orchard borders. A recent registration of an insecticide-impregnated netting used for mosquito suppression and malaria control, has renewed interest in this approach in tree fruits (179).

Sensory Physiology and Perception of Pear Ester Neurophysiological studies have helped us understand the chemoreception of pear ester by codling moth. Motivating these studies were the observed behavioral responses to pear ester in both the field and laboratory demonstrating that both adult males and females, and larvae of codling moth respond with high specificity and sensitivity to this kairomone. In addition, male moths are more attracted to the combination of sex pheromone and pear ester than sex pheromone alone, suggesting that the sensory pathways dedicated to these two semiochemicals interact. At the antennal level, EAG screenings of host plant volatiles on female antennae found that pear ester elicited the largest depolarization responses of the 16 headspace volatiles captured from ripe Bartlett pear fruit (90). and the 37 and 25 synthetic apple volatiles (79, 80). In dose-response experiments, both male and female antennae exhibited the same response thresholds to low amounts of pear ester (109, 112, 180). No differences in antennal sensitivity were found among moths collected from apple, pear, or walnut orchards in Italy, and EAG amplitudes were not significantly different between unmated and mated males and females, respectively (109). Further, neither the occurrence of organophosphate insecticide resistance nor topical treatments of methoxyfenozide had any effect on EAG responses of moths when compared to untreated moths collected from an organic apple orchard or from a laboratory colony (109, 181). Electrophysiological recordings of action potentials from single sensilla recordings (SSR) have been conducted on several types of olfactory hairs – sensilla trichodea, pegs – s. basiconica, and shoehorn – rabbit-eared s. auricillica (109, 182). SSR studies have also demonstrated a variety of olfactory sensory neurons (OSN) specificities, some responding to a range of host-plant volatiles, others specifically tuned to codlemone, or pear ester, alone or combined. Male antennae have a large proportion and number of OSNs specifically responsive to codlemone and other pheromone components, while females have OSNs tuned to pheromone, but far fewer relative to OSNs responsive to host-plant volatiles (109, 182). Codlemone-OSNs, pear ester-OSNs, and those responsive to both compounds were present at ratios of 45:15:40% in males and 20:40:40% in 97

females (109). While the two morphological types of s. auricillica housed OSNs that are responsive to various host-plant volatiles, the dominate number of sensilla for both sexes had an affinity specific for pear ester alone, ranging from 50 to 78% of the regular shoehorn and rabbit-eared shoehorn sensilla, respectively (182). Availability of new molecular tools has enabled the discovery, identification, and characterization of the genes that code for olfactory receptor (OR) proteins (183, 184). Transcriptome analyses of male and female antennae have revealed 58 ORs in codling moths, with twelve of these belonging to the clade of lepidopteran pheromone receptors (183, 184). Among the ORs which are highly expressed in the adult moths, some were subsequently expressed in heterologous cell systems and functionally characterized by screening their affinity and sensitivity with a panel of semiochemicals (185, 186). One putative pheromone receptor, CpomOR3 was shown to selectively respond to pear ester only and not to any pheromone compounds found in codling moth and related species. The CpomOR3 gene is equally expressed in both males and females (184). Interestingly CpomOR3 chemoreceptors are 100-fold less sensitive to the natural pear-produced methyl (E,Z)-2,4-decadienoate analog of pear ester, which parallels its lower attractiveness relative to pear ester in field trapping trials (86). Olfactory neurons housing the ORs send their axons into the antennal lobe (AL) of the brain, where they converge and synapse with AL interneurons. The codling moth AL is comprised of 50 glomeruli, i.e., dense spherical entanglements of synaptic neurons (187). Most of these are ordinary glomeruli, the primary sites for integration of plant odors for both sexes. In addition, males also possess an enlarged macroglomerular complex (MGC) a dedicated neuropile where the neurons of pheromone ORs (from CpomOR1 to CpomOR6) project (183). Intracellular electrophysiological recordings from AL projection interneurons innervating either ordinary glomeruli or the MGC, showed that when antennae were exposed to single volatiles or two-component blends, a variety of integrated excitatory, inhibitory, and synergistic interaction responses was observed (187, 188). Moreover, synergistic excitatory interactions were found in certain projection interneurons when stimulated with blends of codlemone and pear ester, or pear ester and acetic acid (187). In males, a specific ordinary glomerulus (satellite 20), as seen through calcium imaging of neural activity, responds to pear ester and codlemone puffed onto the antennae, but not when both are puffed together (188). Moreover, and truly surprising, calcium imaging of the MGC-cumulus showed that when male antennae were exposed to pear ester alone no increased activity was apparent. Stimulation by codlemone activated approximately 30% of the cumulus volume, and stimulation by the pheromone-kairomone blend activated 70% of the cumulus (188). This synergistic activity of pear ester with codlemone observed in males has been attributed to the affinity of CpomOR3, that uniquely, among all host-plant volatiles, allows specifically pear ester to join in the initial integration of pheromone OSN afferent signals in the pheromone-dedicated neuropile of the MGC. The molecular phylogeny of codling moth OR groups like CpomOR3, together with receptors tuned to sex pheromone compounds (183–186), matches the behavioural, physiological and morphological evidence. It is also interesting that related Cydia species, such as the beech moth, Cydia fagiglandana (Zeller) 98

and G. molesta, are attracted to pear ester (112, 143), and that AL interneurons of both male and female G. molesta respond to pear ester (189), while adult C. fagiglandana possess a gene CfagOR3 that is a highly conserved ortholog of CpomOR3 (190). Thus the affinity for pear ester is evolutionarily conserved in these related species, even though their host preferences and host associations diverged. In aggregate, these studies suggest that pear ester is unique in influencing and adding to or completing the integration of the pheromone signal in adult codling moth. CpomOR3 and associated sensory circuitry allows pear ester to join the primary pheromonal afferent inputs and access the exclusive pheromone integration center (MGC), where otherwise OSNs for other plant volatiles do not directly arborize and synapse. The existence of a pheromone clade OR dedicated to pear ester underlines the biological significance of pear ester integration and interactions with sex pheromone reception. The molecular and neurophysiological synergistic integration of pear ester with pheromone confirms the demonstrated enhancement of male attraction to CM-DA Combo lures and the enhanced degree of mating disruption of both males and females by the CM-DA Combo dispensers (106, 119, 162, 168, 169). Thus, pear ester for male codling moth is likely perceived as a kairomone and perhaps as a unique parapheromone, enhancing both pheromone reception and behavioral activity (191).

Acknowledgments The authors would like to thank Bill Lingren, Trécé Inc., Adair, OK, for his unbridled enthusiasm to develop pear ester into a portfolio of successful commercial products. It is likely that this chapter would never have been written without his positive energy and unwavering support for the research. We would also like to thank Dr. Clive Henricks whose acute knowledge of synthetic and formulation chemistry provided the foundations for the various applications utilizing pear ester. A final point, this is a story of a remarkably successful public-private collaboration between a motivated industry and a cadre of scientists to foster advances toward sustainable pest management. We sincerely hope this success can be a template for other advances in applied insect chemical ecology.

References 1. 2. 3. 4.

Washington State University Tree Fruit Crop Protection Guide, 2017. http:// www.tfrec.wsu.edu/pages/cpg/TOC. Northwest Horticultural Council, 2017. http://nwhort.org/export-manual/. Croft, B. A.; Hoyt, S. C. Integrated management of insect pests of pome and stone fruits; Wiley: New York, NY, 1983. Beers, E. H.; Suckling, D. M.; Prokopy, R. J.; Avilla, J. Ecology and management of apple arthropod pests. In Apples, botany, production and uses; Ferree, D. C., Warrington, I. J., Eds.; CABI Publishing: Wallingford, United Kingdom, 2003; pp 489−519. 99

5.

6.

7. 8.

9.

10. 11. 12.

13.

14.

15. 16.

17.

18.

19.

20.

Prokopy, R. J.; Croft, B. A. Apple insect pest management. In Introduction to insect pest management; Metcalf, R. L., Luckman W. H., Eds.; Wiley: New York, NY, 1994; pp 543−585. Weddle, P. W.; Welter, S. C.; Thomson, D. History of IPM in California pears – 50 years of pesticide use and the transition to biologically intensive IPM. Pest Manage. Sci. 2009, 65, 1287–1292. Kogan, M. Integrated pest management: historical perspectives and contemporary developments. Annu. Rev. Entomol. 1998, 43, 243–270. Jones, V. P.; Unruh, T. R.; Horton, D. R.; Mills, N. J.; Brunner, J. F.; Beers, E. H.; Shearer, P. W. Tree fruit IPM programs in the western United States: the challenge of enhancing biological control through intensive management. Pest Manage. Sci. 2009, 65, 1305–1310. Damos, P.; Escudero-Colomar, L.-A.; Ioratti, C. Integrated fruit production and pest management in Europe: the apple case study and how far we are from the original concept? Insects 2015, 6, 626–657. Witzgall, P.; Stelinski, L.; Gut, L.; Thomson, D. Codling moth management and chemical ecology. Annu. Rev. Entomol. 2008, 53, 503–512. Ioratti, C.; Lucchi, A. Semiochemical strategies for tortricid moth control in apple orchards and vineyards in Italy. J. Chem. Ecol. 2016, 42, 571–583. Knight A. L. Areawide pest management, theory and implementation. In Codling moth areawide integrated pest management; Koul, O., Cuperus, G. W., Elliot, N., Eds.; CAB International: Wallingford, United Kingdom, 2008; pp 159–190. Barcenas, N. M.; Unruh, T. R.; Neven, L. G. DNA diagnostics to identify internal feeders (Lepidoptera: Tortricide) of pome fruits of quarantine importance. J. Econ. Entomol. 2005, 98, 299–306. Barnes, M. M. Codling moth occurrence, host race formation, and damage. In Tortricid pests of pome and stone fruits, Eurasian species; van der Geest, L. P. S., Evenhuis, H. H., Eds.; Elsevier: Amsterdam, The Netherlands, 1991; pp 313−328. Chapman, P. J. Bionomics of the apple-feeding Tortricidae. Annu. Rev. Entomol. 1973, 18, 73–96. Dickler, E. Tortricid pests. In Tortricid pests of pome and stone fruits, Eurasian species; van der Geest, L. P. S., Evenhuis, H. H., Eds.; Elsevier: Amsterdam, The Netherlands, 1991; pp 435–452. Wearing, C. H. Distribution characteristics of eggs and neonate larvae of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Int. J. Insect Sci. 2016, 8, 33–53. Audemard, H. Population dynamics of the codling moth. In Tortricid pests of pome and stone fruits, Eurasian species; van der Geest, L. P. S., Evenhuis, H. H., Eds.; Elsevier: Amsterdam, The Netherlands, 1991; pp 329−338. Howell, J. F. Reproductive biology. In Tortricid pests of pome and stone fruits, Eurasian species; van der Geest, L. P. S., Evenhuis, H. H., Eds.; Elsevier: Amsterdam, The Netherlands, 1991; pp 157−174. Mills, N. Selecting effective parasitoids for biological control introductions: codling moth as a case study. Biol. Cont. 2005, 34, 274–282. 100

21. Croft, B. A.; Riedl, H. Chemical control and resistance to pesticides of the codling moth. In Tortricid pests of pome and stone fruits, Eurasian species; van der Geest, L. P. S., Evenhuis, H. H., Eds.; Elsevier: Amsterdam, The Netherlands, 1991; pp 371−388. 22. Doerr, M. D.; Brunner, J. F.; Granger, K. R. Incorporating organophosphate alternative insecticides into codling moth management programs in Washington apple orchards. J. Integr. Pest Manage. 2012, 3, E1–E4. 23. Leskey, T. C.; Nielsen, A. L. Impact of the invasive brown marmorated stink bug in North America and Europe: history, biology, ecology, and management. Annu. Rev. Entomol. 2018, 63, 599–618. 24. Hughes, W. O.; Gailey, D.; Knapp, J. J. Host location by adult and larval codling moth and the potential for its disruption by the application of kariomones. Entomol. Exp. Appl. 2003, 106, 147–153. 25. Lacey, L. A.; Shapiro-Ilan, D. I. Microbial control of insect pests in temperate orchard systems: potential for incorporation into IPM. Annu. Rev. Entomol. 2008, 53, 121–144. 26. Riedl, H.; Howell, J. F.; McNally, P. S.; Westigard, P. H. Codling moth management: use and standardization of pheromone trapping systems; Univ. CA Div. Agric. and Nat. Resources Bulletin, 1918; Oakland, CA, 1986. 27. Knight, A. L. Modeling and prediction technology. In Tortricid pests of pome and stone fruits, Eurasian species; van der Geest, L. P. S., Evenhuis, H. H., Eds.; Elsevier: Amsterdam, The Netherlands, 1991; pp 301−312. 28. Knight, A. L.; Light, D. M. Developing action thresholds for codling moth (Lepidoptera, Tortricidae) with pear ester and codlemone-baited traps in apple orchards treated with sex pheromone mating disruption. Can. Entomol. 2005, 137, 739–747. 29. Landolt, P. J.; Phillips, T. W. Host plant influences on sex pheromone behavior of phytophagous insects. Annu. Rev. Entomol. 1997, 42, 371–391. 30. Knight, A. The impact of codling moth (Lepidoptera: Tortricidae) mating disruption on apple pest management in Yakima Valley, Washington. J. Entomol. Soc. B. C. 1995, 92, 29–38. 31. Knight, A. L. Testing an attracticide hollow fibre formulation for control of codling moth, Cydia pomonella (Lepidoptera: Tortricidae). J. Entomol. Soc. B. C. 2003, 100, 71–78. 32. Knight, A. L. Managing codling moth (Lepidoptera: Tortricidae) with an internal grid of either aerosol puffers or dispenser clusters plus border applications of individual dispensers. J. Entomol. Soc. B. C. 2004, 101, 69–77. 33. Knight, A. L.; Larsen, T. E. Improved deposition and performance of a microencapsulated sex pheromone formulation for codling moth (Lepidoptera: Tortricidae) with a low volume application. J. Entomol. Soc. B. C. 2004, 101, 79–86. 34. Brown, D. F.; Knight, A. L.; Howell, J. F.; Sell, C. R.; Krysan, J. L.; Weiss, M. Emission characteristics of a polyethylene pheromone dispenser for mating disruption of codling moth (Lepidoptera: Tortricidae). J. Econ. Entomol. 1992, 85, 910–917. 101

35. Millar, J. G. Degradation and stabilization of E8,E10-dodecadienol, the major component of the sex pheromone of the codling moth (Lepidoptera: Tortricidae). J. Econ. Entomol. 1995, 88, 1425–1432. 36. Knight, A. L. Evaluating pheromone emission rate and blend in disrupting sexual communication of codling moth (Lepidoptera: Tortricidae). Environ. Entomol. 1995, 24, 1396–1403. 37. Knight, A. L.; Howell, J. F.; McDonough, L. M.; Weiss, M. Mating disruption of codling moth (Lepidoptera: Tortricidae) with polyethylene tube dispensers: determining emission rates and the distribution of fruit injuries. J. Agric. Entomol. 1995, 12, 85–100. 38. Miller, J. R.; Gut, L. J.; de Lame, F. M.; Stelinski, L. L. Differentiation of competitive vs. non-competitive mechanisms mediating disruption of moth sexual communication by point sources of sex pheromone (Part I): theory. J. Chem. Ecol. 2006, 32, 2089–2114. 39. Knight, A. L. Multiple mating of male and female codling moth (Lepidoptera, Tortricidae) in apple orchards treated with sex pheromone. Environ. Entomol. 2007, 36, 157–164. 40. Duthie, B.; Gries, G.; Gries, R.; Krupke, C.; Derksen, S. Does pheromonebased aggregation of codling moth larvae help procure future mates? J. Chem. Ecol. 2003, 29, 425–436. 41. Knight, A. L. Delay of mating of codling moth in pheromone disrupted orchards. IOBC-WPRS Bull. 1997, 20, 203–206. 42. Vickers, R. A. Effect of delayed mating on oviposition pattern, fecundity and fertility in codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). Aust. J. Entomol. 1997, 36, 179–182. 43. Jones, V. P.; Wiman, N. G.; Brunner, J. F. Comparison of delayed female mating on reproductive biology of codling moth and obliquebanded leafroller. Environ. Entomol. 2008, 37, 679–685. 44. Howell, J. F.; Britt, R. IPM changes associated with using mating disruption to control codling moth in commercial apple production. Proc. Wash. State Hort. Assoc. 1994, 89, 258–264. 45. Knight, A. L. Monitoring codling moth (Lepidoptera: Tortricidae) with passive interception traps in sex pheromone-treated apple orchards. J. Econ. Entomol. 2000, 93, 1744–1751. 46. Barnes, M. M.; Millar, J. G.; Kirsch, P. A.; Hawks, D. C. Codling moth (Lepidoptera: Tortricidae) control by dissemination of synthetic female sex pheromone. J. Econ. Entomol. 1992, 85, 1274–1277. 47. Weissling, T. J.; Knight, A. L. Oviposition and calling behavior of codling moth (Lepidoptera: Tortricidae) in the presence of codlemone. Ann. Entomol. Soc. Am. 1996, 89, 142–147. 48. Light, D. M.; Flath, R. A.; Buttery, R. G.; Zalom, F. G.; Rice, R. E.; Dickens, J. C.; Jang, E. B. Host-plant green-leaf volatiles synergize the synthetic sex pheromone of the corn earworm and codling moth (Lepidoptera). Chemoecology 1993, 4, 145–152. 49. Schmera, D.; Guerin, P. M. Plant volatile compounds shorten reaction time and enhance attraction of the codling moth (Cydia pomonella) to codlemone. Pest Manage. Sci. 2012, 68, 454–461. 102

50. Yang, Z.; Bengtsson, M.; Witzgall, P. Host plant volatiles synergize response to sex pheromone in codling moth, Cydia pomonella. J. Chem. Ecol. 2004, 30, 619–629. 51. Hern, A.; Dorn, S. Sexual dimorphism in the olfactory orientation of adult Cydia pomonella in response to alpha farnesene. Entomol. Exp. Appl. 1999, 92, 63–72. 52. Hall, J. A. Six years’ study of the life history and habits of the codling moth (Carpocapsa pomonella L.). Ontario Entomol. Soc. 1929, 59, 96–105. 53. Geier, P. W. The life history of the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae) in the Australian Capital Territory. Aust. J. Zool. 1963, 11, 323–367. 54. Sutherland, O. R. W.; Hutchins, R. F. N. α-Farnesene, a natural attractant for codling moth larvae. Nature 1972, 239, 170–171. 55. Sutherland, O. R. W.; Hutchins, R. F. N. Attraction of newly hatched codling moth larvae (Laspeyresia pomonella) to synthetic stereo-isomers of farnesene. J. Insect Physiol. 1973, 19, 723–727. 56. Wearing, C. H.; Hutchins, R. F. N. α-Farnesene, a naturally occurring oviposition stimulant for the codling moth, Laspeyresia pomonella. J. Insect Physiol. 1973, 19, 1251–1256. 57. Russ, K. Investigations on the influence of fruit odor on the orientation of codling moth (Laspeyresia pomonella L.). Symp. Biol. Hung. 1976, 16, 237–240. 58. Anet, E. F. Autoxidation of α-farnesene. Aust. J. Chem. 1969, 22, 2403–2410. 59. Dimick, P. S.; Hoskin, J. C. Review of apple flavor – state of the art. CRC Crit. Rev Food Sci. Nutrit. 1983, 18, 387–409. 60. Umano, K.; Shoji, A.; Hagi, Y.; Shibamoto, T. Volatile constituents of peel of quince fruit, Cydonia oblonga Miller. J Agric. Food Chem. 1986, 34, 593–596. 61. Miller, R. L.; Bills, D. D.; Buttery, R. G. Volatile components from Bartlett and Bradford pear leaves. J. Agric. Food Chem. 1989, 37, 1476–1479. 62. Mattheis, J. P.; Fellman, J. K.; Chen, P. M.; Patterson, M. E. Changes in headspace volatiles during physiological development of Bisbee Delicious apple fruit. J. Agric. Food Chem. 1991, 39, 1902–1906. 63. Buchbauer, G.; Jirovetz, L.; Wasicky, M.; Nikiforov, A. Headspace and essential oil analysis of apple flowers. J. Agric. Food Chem. 1993, 41, 116–118. 64. Fein, B. L.; Reissig, W. H.; Roelofs, W. L. Identification of apple volatiles attractive to the apple maggot, Rhagoletis pomonella. J. Chem. Ecol. 1982, 8, 1473–1487. 65. Reissig, W. H.; Fein, B. L.; Roelofs, W. L. Field tests of synthetic apple volatiles as apple maggot (Diptera: Tephritidae) attractants. Environ. Entomol. 1982, 11, 1294–1298. 66. Cardé, R. T.; Minks, A. K. Control of moth pests by mating disruption: successes and constraints. Annu. Rev. Entomol. 1995, 40, 559–585. 67. Reyes, M.; Franck, P.; Olivares, J.; Margaritopoulos, J.; Knight, A.; Sauphanor, B. Worldwide variability of insecticide resistance mechanisms 103

68. 69. 70.

71. 72.

73.

74.

75.

76.

77.

78. 79.

80.

81.

in the codling moth, Cydia pomonella L. (Lepidoptera: Tortricidae). Bull. Entomol. Res. 2009, 99, 359–369. Yokoyama, V. Y.; Miller, G. T. A plum volatile, 1-nonanal: an ovipositional deterrent for codling moth. Can. Entomol. 1991, 123, 711–712. Bradley, S. J.; Suckling, D. M. Factors influencing codling moth larval response to α-farnesene. Entomol. Exp. Appl. 1995, 75, 221–227. Landolt, P. J.; Hofstetter, R. W.; Chapman, P. S. Neonate codling moth larvae (Lepidoptera, Tortricidae) orient anemotactically to odor of immature apple fruit. Pan-Pacific Entomol. 1998, 74, 140–149. Yan, F.; Bengtsson, M.; Makranczy, G.; Löfqvist, J. Roles of α-farnesene in the behaviors of codling moth females. Z. Naturforsch. 2003, 58, 113–118. Yan, F.; Bengtsson, M.; Witzgall, P. Behavioral response of female codling moths, Cydia pomonella, to apple volatiles. J. Chem. Ecol. 1999, 25, 1343–1351. Reed, H. C.; Landolt, P. J. Attraction of mated female codling moths (Lepidoptera, Tortricidae) to apples and apple odor in a flight tunnel. Fla. Entomol. 2002, 85, 324–329. Landolt, P. J.; Guedot, C. Field attraction of codling moth (Lepidoptera, Tortricidae) to apple and pear fruit, and quantitation of kairomones from attractive fruit. Ann. Entomol. Soc. Am. 2008, 101, 675–681. Bengtsson, M.; Backman, A. C.; Liblikas, I.; Ramirez, M. I.; BorgKarlson, A. K.; Ansebo, L.; Anderson, P.; Lofqvist, J.; Witzgall, P. Plant odor anlysis of apple, antennal responses of codling moth females to apple volatiles during phenological development. J. Agric. Food Chem. 2001, 49, 3736–3741. Bäckman, A.-C.; Bengtsson, M.; Borg-Karlsson, A.-K.; Liblikas, I.; Witzgall, P. Volatiles from apple (Malus domestica) eliciting antennal responses in female codling moth Cydia pomonella (L.) (Lepidoptera: Tortricidae): effect of plant injury and sampling technique. Z. Naturforsch. 2001, 56, 262–268. Hern, A.; Dorn, S. Statistical modelling of insect behavioural responses in relation to the chemical composition of test extracts. Physiol. Entomol. 2001, 26, 381–390. Coracini, M.; Bengtsson, M.; Liblikas, I.; Witzgall, P. Attraction of codling moth males to apple volatiles. Entomol. Exp. Appl. 2004, 110, 1–10. Ansebo, L.; Coracini, M. D. A.; Bengtsson, M.; Liblikas, I.; Ramirez, M.; Borg-Karlson, A.-K.; Tasin, M.; Witzgall, P. Antennal and behavioral response of codling moth Cydia pomonella to plant volatiles. J. Appl. Entomol. 2004, 128, 488–493. Casado, D.; Gemeno, C.; Avilla, J.; Riba, M. Day-night and phenological variation of apple tree volatiles and electroantennogram responses in Cydia pomonella (Lepidoptera, Tortricidae). Environ. Entomol. 2006, 35, 258–267. Casado, D.; Gemeno, C.; Avilla, J.; Riba, M. Diurnal variation of walnut tree volatiles and electrophysiological responses in Cydia pomonella (Lepidoptera, Tortricidae). Pest Manage. Sci. 2008, 64, 736–747. 104

82. Hern, A.; Dorn, S. Induced emissions of apple fruit volatiles by the codling moth, changing patterns with different time periods after infestation and different larval instars. Phytochemistry 2001, 57, 409–416. 83. Hern, A.; Dorn, S. Monitoring seasonal variation in apple fruit volatile emissions in situ using solid-phase microextraction. Phytochem. Anal. 2003, 14, 232–240. 84. Vallat, A.; Dorn, S. Changes in volatile emissions from apple trees and associated response of adult female codling moths over the fruit-growing season. J. Agric. Food Chem. 2005, 53, 4083–4090. 85. Hern, A.; Dorn, S. A. Female-specific attractant for the codling moth, Cydia pomonella, from apple fruit volatiles. Naturwissenschaften 2004, 91, 77–80. 86. Light, D. M.; Knight, A. L. Specificity of codling moth (Lepidoptera, Tortricidae) for the host plant kairomone, ethyl (2E,4Z)-2,4-decadienoate, field bioassays with pome fruit volatiles, analogue, and isomeric compounds. J. Agric. Food Chem. 2005, 53, 4046–4053. 87. Knudsen, G. K.; Bengtsson, M.; Kobro, S.; Jaastad, G.; Hofsvang, T.; Witzgall, P. Discrepancy in laboratory ad field attraction of apple fruit moth Argyresthia conjugella to host plant volatiles. Physiol. Entomol. 2008, 33, 1–6. 88. Witzgall, P.; Ansebo, L.; Yang, Z.; Angeli, G.; Sauphanor, B.; Bengtsson, M. Plant volatiles affect oviposition by codling moths. Chemoecology 2005, 15, 77–83. 89. Jennings, W. G.; Creveling, R. K.; Heinz, D. E. Volatile esters of Bartlett pear. IV. Esters of trans:2-cis:4-decadienoic acid. J. Food Sci. 1964, 29, 730–734. 90. Light, D. M.; Knight, A. L.; Henrick, C.; Rajapaska, D.; Lingren, B.; Dickens, J. C.; Reynolds, K. M.; Buttery, R. G.; Merrill, G. B.; Roitman, J. N.; Campbell, B. C. A pear-derived kairomone with pheromonal potency that attracts male and female codling moth, Cydia pomonella (L.). Naturwissenschaften 2001, 88, 333–338. 91. Knight, A. L.; Light, D. M. Attractants from ‘Bartlett’ pear for codling moth, Cydia pomonella (L.), larvae. Naturwissenschaften 2001, 88, 339–342. 92. Light, D. M.; Henrick, C. A. Novel bisexual attractants, aggregants and arrestants for adults and larvae of codling moth and other species of Lepidoptera. U.S. Patent No. 6,264,939 B1, issued July 24, 2001. 93. Light, D. M.; Henrick, C. A. Bisexual attractants, aggregants and arrestants for adults and larvae of codling moth and other species of Lepidoptera, formulations. U.S. Patent No. 6,528,049 B2, issued March 4, 2003. 94. Knight, A. L.; Light, D. M. Dose-response of codling moth (Lepidoptera: Tortricidae) to ethyl (E,Z)-2,4-decadienoate in apple orchards treated with sex pheromone dispensers. Environ. Entomol. 2005, 34, 604–609. 95. Il’chev, A. L.; van de Ven, R.; Williams, D. G.; Penfold, N. Monitoring codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) in Victorian pome fruit orchards with pear ester. Gen. Appl. Entomol. 2009, 38, 57–64. 96. Il’chev, A. L. First Australian trials of ethyl (2E,4Z)-2,4-decandienoate for monitoring of female and male codling moth, Cydia pomonella (Lepidoptera, Tortricidae) in pome fruit orchards. Gen. Appl. Entomol. 2004, 33, 15–20. 105

97. Fernandez, D. E.; Cichon, L.; Garrido, S.; Ribes-Dasi, M.; Avilla, J. Comparison of lures loaded with codlemone and pear ester for capturing codling moths, Cydia pomonella, in apple and pear orchards using mating disruption. J. Insect Sci. 2010, 10, 1–12. 98. Knight, A. L.; Van Buskirk, P.; Hilton, R.; Zoller, B.; Light, D. M. Monitoring codling moth (Lepidoptera, Tortricidae) in four pear cultivars with the pear ester. Acta Hort. 2005, 594, 120–125. 99. Knight, A. L.; Light, D. M. Use of ethyl and propyl (E,Z)-2,4-decadienoates in codling moth management, improved monitoring in Bartlett pear with high dose lures. J. Entomol. Soc. B. C. 2004, 101, 45–52. 100. Knight, A. L.; Light, D. M. Seasonal flight patterns of codling moth (Lepidoptera, Tortricidae) monitored with pear ester and codlemone-baited traps in sex pheromone-treated apple orchards. Environ. Entomol. 2005, 34, 1028–1035. 101. Knight, A. L.; Light, D. M. Factors affecting the differential capture of male and female codling moth (Lepidoptera, Tortricidae) in traps baited with ethyl (E,Z)-2,4-decadienoate. Environ. Entomol. 2005, 34, 1161–1169. 102. Knight, A. L.; Weiss, M.; Weissling, T. J. Diurnal patterns of adult activity of four orchard pests measured by timing trap and actograph. J. Agric. Entomol. 1994, 11, 125–136. 103. Thwaite, W. G.; Hately, A. M.; Eslick, M. A.; Nicol, H. I. Evaluating pear ester lures for monitoring Cydia pomonella (L) (Lepidoptera, Tortricidae) in Granny Smith apples under mating disruption. Gen. Appl. Entomol. 2004, 33, 55–60. 104. Ioriatti, C.; Molinari, F.; Pasqualini, E.; De Cristofaro, A.; Schmidt, S.; Espinha, I. The plant volatile attractant (E,Z)-2,4-ethyl-decadienoate (DA2313) for codling moth monitoring. Bollet. Zool. Agrar. Bachicolt. 2003, 35, 127–137. 105. Knight, A. L.; Croft, B. A.; Bloem, K. A. Effect of mating disruption dispenser placement on trap performance for monitoring codling moth (Lepidoptera: Tortricidae). J. Entomol. Soc. B. C. 1999, 96, 95–102. 106. Knight, A. L.; Hilton, R.; Light, D. M. Monitoring codling moth (Lepidoptera, Tortricidae) in apple with blends of ethyl (E,Z)-2,4decadienoate and codlemone. Environ. Entomol. 2005, 34, 598–603. 107. Mitchell, V. J.; Manning, L. A.; Cole, L.; Suckling, D. M.; El-Sayed, A. M. Efficacy of the pear ester as a monitoring tool for codling moth Cydia pomonella (Lepidoptera, Tortricidae) in New Zealand apple orchards. Pest Manage. Sci. 2008, 64, 209–214. 108. Trimble, R. W.; El-Sayed, A. M. Potential of ethyl (2E,4Z)-2,4-decadienoate for monitoring activity of codling moth (Lepidoptera, Tortricidae) in eastern North American apple orchards. Can. Entomol. 2005, 137, 110–116. 109. De Cristofaro, A.; Ioriatti, C.; Pasqualini, E.; Anfora, G.; Germinara, G. S.; Villa, M.; Rotundo, G. Electrophysiological responses of Cydia pomonella to codlemone and pear ester, ethyl (E,Z)-2,4-decadienoate, peripheral interactions in their perception and evidences for cells responding to both compounds. Bull. Insect. 2004, 57, 137–144. 106

110. Joshi, N. K.; Hull, L. A.; Rajotte, E. G.; Krawczyk, G.; Bohnenblust, E. Evaluating sex-pheromone- and kairomone-based lures for attracting codling moth adults in mating disruption versus conventionally managed apple orchards in Pennsylvania. Pest Manage. Sci. 2011, 67, 1332–1337. 111. Schmidt, S.; Anfora, G.; Ioriatti, C.; Germinara, G.; Rotundo, G.; De Cristofaro, A. Biological activity of ethyl (E,Z)-2,4-decadienoate on different Tortricid species, electrophysiological responses and field tests. Environ. Entomol. 2007, 36, 1025–1031. 112. Kutinkova, H.; Subchev, M.; Light, D.; Lingren, B. Interactive effects of ethyl (2E,4Z)-2,4-decadienoate and sex pheromone lures to codling moth, apple orchard investigations in Bulgaria. J. Plant Protect. Res. 2005, 5, 49–52. 113. Trematerra, P.; Sciaretta, A. Activity of the kairomone ethyl (E,Z)-2,4decadienoate in the monitoring of Cydia pomonella (L.) during the second annual flight. Redia 2005, 88, 57–62. 114. Knight, A. L.; Miliczky, E. Influence of trap colour on the capture of codling moth (Lepidoptera: Tortricidae), honeybees, and non-target flies. J. Entomol. Soc. B. C 2003, 100, 65–70. 115. Knight, A. L.; Fisher, J. Increased catch of codling moth (Lepidoptera, Tortricidae) in semiochemical-baited orange plastic delta-shaped traps. Environ. Entomol. 2006, 35, 1597–1602. 116. Knight, A. L. Effect of sex pheromone and kairomone lures on catches of codling moth. J. Entomol. Soc. B. C. 2010, 107, 1–8. 117. Knight, A. L. Increased catch of female codling moth (Lepidoptera, Tortricidae) in kairomone-baited clear delta traps. Environ. Entomol. 2010, 39, 583–590. 118. Barros-Parada, W.; Knight, A. L.; Basoalto, E.; Fuentes-Contreras, E. An evaluation of orange and clear traps with pear ester to monitor codling moth (Lepidoptera, Tortricidae) in apple orchards. Cienc. Invest. Agrar. 2013, 40, 307–315. 119. Light, D. M. Control and monitoring of codling moth (Lepidoptera, Tortricidae) in walnut orchards treated with novel high-load, low-density “meso” dispensers of sex pheromone and pear ester. Environ. Entomol. 2016, 45, 700–707. 120. Landolt, P. J.; Suckling, D. M.; Judd, G. J. R. Positive interaction of a feeding attractant and a host kairomone for trapping the codling moth, Cydia pomonella (L.). J. Chem. Ecol. 2007, 33, 2236–2244. 121. Hári, K.; Penzes, B.; Josvai, J. K.; Ladanyi, M.; Toth, M. Performance of traps baited with pear ester-based lures vs. pheromone baited ones for monitoring codling moth, Cydia pomonella (L.), in Hungary. Acta Phytopath. Entomol. Hung. 2011, 46, 225–234. 122. Knight, A. L. Improved monitoring of female codling moth (Lepidoptera, Tortricidae) with pear ester plus acetic acid lures in sex pheromone-treated orchards. Environ. Entomol. 2010, 39, 1283–1290. 123. Knight, A.; Light, D.; Chebny, V. Monitoring codling moth (Lepidoptera, Tortricidae) in orchards treated with pear ester and sex pheromone combo dispensers. J. Appl. Entomol. 2013, 137, 214–224. 107

124. Barros-Parada, W.; Basoalto, E.; Fuentes-Contreras, E.; Cichon, L.; Knight, A. L. Acetic acid lure placement within traps affects moth catches of codling moth (Lepidoptera, Tortricidae). J. Appl. Entomol. 2016, 140, 786–795. 125. Knight, A. L.; Light, D. M.; Trimble, R. M. dentifying (E)-4,8-dimethyl1,3,7-nonatriene plus acetic acid as a new lure for male and female codling moth (Lepidoptera, Tortricidae). Environ. Entomol. 2011, 40, 420–430. 126. Knight, A. L.; Light, D. M. Monitoring codling moth (Lepidoptera, Tortricidae) in sex pheromone-treated orchards with (E)-4,8-dimethyl-1,3,7nonatrienene or pear ester in combination with codlemone and acetic acid. Environ. Entomol. 2012, 41, 407–414. 127. Knight, A. L.; Basoalto, E.; Katalin, J.; El-Sayed, A. M. A binary host plant volatile lure combined with acetic acid to monitor codling moth (Lepidoptera, Tortricidae). Environ. Entomol. 2015, 44, 1434–1440. 128. Basoalto, E.; Hilton, R.; Knight, A. Comparing mating disruption of codling moth with standard and meso dispensers loaded with per estar and codlemone. IOBC-WPRS Bull. 2014, 99, 33–37. 129. Knight, A. L.; Hilton, R.; Basoalto, E.; Stelinski, L. L. Use of glacial acetic acid to enhance bisexual monitoring of tortricid pests with kairomone lures in pome fruit. Environ. Entomol. 2014, 43, 1628–1640. 130. El-Sayed, A. M.; Cole, L.; Revell, J.; Manning, L. A.; Twidle, A.; Knight, A. L.; Bus, V. G. M.; Suckling, D. M. Apple volatiles synergize the response codling moth to pear ester. J. Chem. Ecol. 2013, 39, 643–652. 131. Landolt, P. J.; Ohler, B.; Lo, P.; Cha, D.; Davis, T. S.; Suckling, D. M.; Brunner, J. N-Butyl sulfide as an attractant and coattractant for male and female codling moth (Lepidoptera, Tortricidae). Environ. Entomol. 2014, 43, 291–297. 132. Basoalto, E.; Mujica, M. V.; Cichon, L.; Fuentes-Contreras, E.; Barros-Parada, W.; Knight, A. L. A binary (pheromone – host plant volatile) lure combined with acetic acid could enhance bisexual monitoring of codling moth (Lepidoptera: Tortricidae). Presented at the XXV International Congress of Entomology, Orlando, FL, 2016; DOI: 10.1603/ICE.2016.114996. 133. Knight, A.; Hawkins, L.; McNamara, K.; Hilton, R. Monitoring, managing codling moth clearly and precisely. Good Fruit Grower 2009, 60, 26–27. 134. Knight, A.; Hawkins, L.; McNamara, K.; Borman, M.; Hilton, R. Managing codling moth clearly and precisely with semiochemicals. IOBC-WPRS Bull. 2011, 54, 415–418. 135. Alston, D.; Murray, M. Validation and demonstration of trap thresholds for codling moth in mating disrupted apple orchards in Northern Utah. Proc. Utah State Hort. Assoc. 2009, 1–9. 136. Knight, A. L.; Light, D. M. Timing of egg hatch by early-season codling moth (Lepidoptera, Tortricidae) predicted by moth catch in pear ester- and codlemone-baited traps. Can. Entomol. 2005, 137, 728–738. 137. Barros-Parada, W.; Knight, A. L.; Fuentes-Contreras, E. Modeling codling moth (Lepidoptera, Tortricidae) phenology and predicting egg hatch in apple orchards of the Maule Region, Chile. Chilean J. Agric. Res. 2015, 75, 57–62. 108

138. Knight, A. L. Assessing the mating status of female codling moth (Lepidoptera, Tortricidae) in orchards treated with sex pheromone using traps baited with ethyl (E,Z)-2,4-decadienoate. Environ. Entomol. 2006, 35, 894–900. 139. Knight, A. L. Influence of within-orchard trap placement on catch of codling moth (Lepidoptera: Tortricidae) in sex pheromone-treated orchards. Environ. Entomol. 2007, 36, 425–432. 140. Light, D. M.; Grant, J.; Haff, R.; Knight, A. L. Addition of pear ester with sex pheromone enhances disruption of mating by female codling moth (Lepidoptera, Tortricidae) in walnut orchards treated with “meso” dispensers. Environ. Entomol. 2017, 46, 319–327. 141. Light, D. M.; Knight, A. L. Kairomone-augmented mating disruption control for codling moth in Californian walnuts and apples. IOBC-WPRS Bull. 2005, 28, 300–303. 142. Knight, A. L.; Light, D. M. Use of ethyl (E,Z)-2,4-decadienoate in codling moth management, kairomone species specificity. J. Entomol. Soc. B. C. 2004, 101, 61–67. 143. Molinari, F.; Anfora, G.; Schmidt, S.; Villa, M.; Ioriatti, C.; Pasqualini, E.; De Cristofaro, A. Olfactory activity of ethyl (E,Z)-2,4-decadienoate on adult oriental fruit moths. Can. Entomol. 2010, 142, 481–488. 144. Knight, A.; Cichon, L.; Lago, J.; Fuentes-Contreras, E.; Barros-Parada, W.; Hull, L.; Krawczyk, G.; Zoller, B.; Hansen, R.; Hilton, R.; Basoalto, E. Monitoring oriental fruit moth and codling moth (Lepidoptera, Tortricidae) with combinations of pheromone and kairomones. J. Appl. Entomol. 2014, 138, 783–794. 145. Jósvai, J. K.; Koczor, S.; Toth, M. Traps baited with pear ester and acetic acid attract both sexes of Hedya nubiferana (Lepidoptera, Tortricidae). J. Appl. Entomol. 2016, 140, 81–90. 146. Toth, M.; Landolt, P.; Szarukan, I.; Szollath, I.; Vitanyi, I.; Penzes, B.; Hari, K.; Josvai, J. K.; Koczor, S. Female-targeted attractant containing pear ester for Synanthedon myopaeformis. Entomol. Exp. Appl. 2011, 142, 27–35. 147. Basoalto, E.; Miranda, M.; Knight, A. L.; Fuentes-Contreras, E. Landscape analysis of adult codling moth (Lepidoptera, Tortricidae) distribution and dispersal within typical agroecosystems dominated by apple production in central Chile. Environ. Entomol. 2010, 39, 1399–1408. 148. Margaritopoulos, J. T.; Voudouris, C. C.; Olivares, J.; Sauphanor, B.; Mamuris, Z.; Tsitsipis, A.; Franck, P. Dispersal ability in codling moth, mark-release-recapture experiments and kinship analysis. Agric. Forest Entomol. 2012, 14, 399–407. 149. Judd, G. J. R. Potential for using acetic acid plus pear ester combination lures to monitor codling moth in an SIT program. Insects 2016, 7, E68. 150. Sauphanor, B.; Franck, P.; Lasnier, T.; Toubon, J.-F.; Beslay, D.; Boivin, T.; Bouvier, J.-C.; Renou, M. Insecticide resistance may enhance the response to host-plant volatile kairomone for the codling moth, Cydia pomonella. Naturwissen 2007, 94, 449–458. 109

151. Barrett, B. A. Exposure to methoxyfenozide-treated surfaces reduces the responsiveness of adult male codling moth (Lepidoptera, Tortricidae) to codlemone and pear ester lures in a wind tunnel. J. Econ. Entomol. 2010, 103, 1704–1710. 152. Knight, A. L.; Flexner, L. Disruption of mating in codling moth (Lepidoptera, Tortricidae) by chlorantranilipole, an anthranilic diamide insecticide. Pest Manage. Sci. 2007, 63, 180–189. 153. Light, D. M.; Beck, J. J. Characterization of microencapsulated pear ester, (2E,4Z)-ethyl-2,4-decadienoate, a kairomonal spray adjuvant against neonate codling moth larvae. J. Agric. Food Chem. 2010, 58, 7836–7845. 154. Light, D. M.; Beck, J. J. Behavior of codling moth (Lepidoptera, Tortricidae) neonate larvae on surfaces treated with microencapsulated pear ester. Environ. Entomol. 2012, 41, 603–611. 155. Knight, A. L.; Light, D. M. Use of ethyl (E,Z)-2,4-decadienoate in codling moth management, stimulation of oviposition. J. Entomol. Soc. B. C. 2004, 101, 53–60. 156. Zoller, B. G. Comparison of kairomone DA 2313 and pheromone lure trapping for codling moth with oviposition monitoring. Proc. Orchard Pest Disease Manage. Conf. 2003, 77, 1–6. 157. Pasqualini, E.; Villa, M.; Civolani, S.; Espinha, I.; Ioriatti, C.; Schmidt, S.; Molinari, F.; De Cristofaro, A.; Sauphanor, B.; Ladurner, E. The pear ester ethyl (E,Z)-2,4-decadienoate as a potential tool for control of Cydia pomonella larvae, preliminary investigation. Bull. Insect. 2005, 58, 65–69. 158. Pasqualini, E.; Schmidt, S.; Espinha, I.; Civolani, S.; Ioriatti, C.; De Cristofaro, A.; Molinari, F.; Villa, M.; Ladurner, E.; Sauphanor, B. Effects of the kairomone ethyl (2E,4Z)-2,4-decadienoate (DA 2313) on the oviposition behaviour of Cydia pomonella, preliminary investigations. Bull. Insect. 2005, 58, 119–124. 159. Light, D. M. Experimental use of the micro-encapsulated pear ester kairomone for control of codling moth, Cydia pomonella (L.), in walnuts. IOBC-WPRS Bull. 2007, 30, 133–140. 160. Light, D. M.; Knight, A. L. Microencapsulated pear ester enhances insecticide efficacy in walnuts for codling moth (Lepidoptera, Tortricidae) and navel orangeworm (Lepidoptera, Pyralidae). J. Econ. Entomol. 2011, 104, 1309–1315. 161. Knight, A. L.; Light, D. M. Adding microencapsulated pear ester to insecticides for control of Cydia pomonella (Lepidoptera, Tortricidae) in apple. Pest Manage. Sci. 2013, 69, 66–74. 162. Knight, A. L.; Light, D. M. Combined approaches using sex pheromone and pear ester for behavioural disruption of codling moth (Lepidoptera, Tortricidae). J. Appl. Entomol. 2014, 138, 96–108. 163. Arthurs, S. P.; Hilton, R.; Knight, A. L.; Lacey, L. A. Evaluation of the pear ester kairomone as a formulation additive for the granulovirus of codling moth (Lepidoptera, Tortricidae) in pome fruit. J. Econ. Entomol. 2007, 100, 702–709.

110

164. Schmidt, S.; Tomasi, C.; Pasqualini, E. The biological efficacy of pear ester on the activity of Granulosis virus for codling moth. J. Pest. Sci. 2008, 81, 29–34. 165. Knight, A. L.; Witzgall, P. Combining mutualistic yeast and pathogenic virus – a novel method for codling moth control. J. Chem. Ecol. 2013, 39, 1019–1026. 166. Knight, A. L.; Basoalto, E.; Witzgall, P. Improving the performance of the granulosis virus of codling moth (Lepidoptera, Tortricidae) by adding the yeast Saccharomyces cerevisiae with sugar. Environ. Entomol. 2015, 44, 252–259. 167. Bohnenblust, E.; Hull, L. A.; Krawczyk, G. A comparison of various mating disruption technologies for control of two internally feeding Lepidoptera in apples. Entomol. Exp. Appl. 2011, 138, 202–211. 168. Knight, A. L.; Stelinski, L. L.; Hebert, V.; Gut, L.; Light, D.; Brunner, J. Evaluation of novel semiochemical dispensers simultaneously releasing pear ester and sex pheromone for mating disruption of codling moth (Lepidoptera, Tortricidae). J. Appl. Entomol. 2012, 136, 79–86. 169. Knight, A. L.; Light, D. M.; Chebny, V. Evaluating dispensers loaded with codlemone and pear ester for disruption of codling moth (Lepidoptera, Tortricidae). Environ. Entomol. 2012, 41, 399–406. 170. Stelinski, L. L.; Gut, L. J.; Miller, J. R. An attempt to increase efficacy of moth mating disruption by co-releasing pheromones with kairomones and to understand possible underlying mechanisms of this technique. Environ. Entomol. 2013, 42, 158–166. 171. Kovanci, O. B. Co-application of microencapsulated pear ester and codlemone for mating disruption of Cydia pomonella. J. Pest Sci. 2015, 88, 311–319. 172. Stelinski, L. L.; Gut, L. J.; McGhee, P.; Miller, J. R. Towards highperformance mating disruption of codling moth, Cydia pomonella (L.). IOBC-WPRS Bull. 2007, 30, 115–122. 173. Madsen, H. F.; Vakenti, J. M.; Peters, F. E. Codling moth: suppression by male removal with sex pheromone traps in an isolated apple orchard. J. Econ. Entomol. 1976, 69, 597–599. 174. Charmillot, P. J.; Hofer, D.; Pasquier, D. Attract and kill: a new method for control of the codling moth Cydia pomonella. Entomol. Exp. Appl. 2000, 94, 211–216. 175. Potting, R. P. J.; Knight, A. L.; Losel, P. M.; Ebbinghaus, D. Predicting the efficacy of modified modes of action of a pheromone-based attracticide, a bisexual attractant and autosterilization. IOBC-WPRS Bull. 2002, 25, 215–223. 176. Knight, A. L.; Potting, R. P. J.; Light, D. M. Modeling the impact of a sex pheromone/kairomone attracticide for management of codling moth (Cydia pomonella). Acta Hort. 2002, 584, 215–220. 177. Light, D. M.; Knight, A. L.; Reynolds, K. M.; Brewer, M. Development of kairomone-based mass trapping control of codling moths in Californian walnuts and apples. Proc. Orchard Pest Disease Manage. Conf. 2003, 77, 85. 111

178. Knight, A. L. Ridding orchards of codling moth – one female at a time. Proc. Orchard Pest Disease Manage. Conf. 2003, 77, 21–22. 179. Kuhar, T. P.; Short, B. D.; Krawczyk, G.; Leskey, T. C. Deltamethrinincorporated nets as an integrated pest management tool for the invasive Halyomorpha halys (Hemiptera: Pentatomidae). J. Econ. Entomol. 2017, 110, 543–545. 180. Avilla, J.; Casado, D.; Varela, N.; Bosch, D.; Riba, M. Electrophysiological response of codling moth (Cydia pomonella) adults to semiochemicals. IOBC-WPRS Bull. 2003, 26, 1–7. 181. Barrett, B. A.; Keeesey, I. W.; Akbulut, S.; Terrell Stamps, W. Antennal responses of Cydia pomonella (L.) exposed to surfaces treated with methoxyfenozide. J. Appl. Entomol. 2013, 137, 499–508. 182. Ansebo, L.; Ignell, R.; Lofqvist, J.; Hansson, B. S. Responses to sex pheromone and plant odors by olfactory neurons housed in sensilla auricillica of the codling moth, Cydia pomonella (Lepidoptera, Tortricidae). J. Insect Physiol. 2005, 51, 1066–1074. 183. Bengtsson, J. M.; Trona, F.; Montagne, N.; Anfora, G.; Ignell, R.; Witzgall, P.; Jacquin-Joly, E. Putative chemosensory receptors of the codling moth, Cydia pomonella, identified by antennal transcriptome analysis. PLoS One 2012, 7, e10.1371. 184. Walker, W. B.; Gonzalez, F.; Garczynski, S. F.; Witzgall, P. The chemosensory receptors of codling moth Cydia pomonella – expression in larvae and adults. Sci. Rep. 2016, 6, 23518. 185. Bengtsson, J. M.; Gonzalez, F.; Cattaneo, A. M.; Montagne, N.; Walker, W. B.; Bengtsson, M.; Anfora, G.; Ignell, R.; Jacquin-Joly, E.; Witzgall, P. A predicted sex pheromone receptor of codling moth, Cydia pomonella, detects the plant volatile pear ester. Front. Ecol. Evol. 2014, 2, 33. 186. Cattaneo, A. M.; Gonzalez, F.; Bengtsson, J. M.; Corey, E. A.; Jacquin-Joly, E.; Montagné, N.; Salvagnin, U.; Walker, W. B.; Witzgall, P.; Anfora, G.; Bobkov, Y. V. Candidate pheromone receptors from the insect pest Cydia pomonella respond to pheromone and kairomone components. Sci. Rep. 2017, 7, 41–105. 187. Trona, F.; Anfora, G.; Bengtsson, M.; Witzgall, P.; Ignell, R. Coding and interaction of sex pheromone and plant volatile signals in the antennal lobe of the codling moth Cydia pomonella. J. Exp. Biol. 2010, 213, 4291–4303. 188. Trona, F.; Anfora, G.; Bengtsson, M.; Tasin, M.; Knight, A.; Janz, N.; Witzgall, P.; Ignell, R. Neural coding merges sex and habitat chemosensory signals in an insect herbivore. Proc. R. Soc. B 2013, 280, 20130267. 189. Varela, N.; Avilla, J.; Gemeno, C.; Anton, S. Ordinary glomeruli in the antennal lobe of male and female tortricid moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae) process sex pheromone and host-plant volatiles. J. Exp. Biol. 2011, 214, 637–645. 190. Gonzalez, F.; Witzgall, P.; Walker, W. B. Antennal transcriptomes of three tortricid moths reveal conserved chemosensory receptors for social and environmental olfactory cues. Sci. Rep. 2017, 7, 41821.

112

191. Renou, M.; Guerrero, A. Insect parapheromones in olfaction research and semiochemical-based pest controls trategies. Annu. Rev. Entomol. 2000, 48, 605–630.

113

Chapter 9

Strategies for the Manipulation of Root Knot Nematode Behavior with Natural Products in Small Scale Farming Systems Baldwyn Torto,*,1 Hillary Kirwa,1,2 Ruth Kihika,1 and Lucy K. Murungi2 1International

Centre of Insect Physiology and Ecology, P.O. Box 30772-00100, Nairobi, Kenya 2Department of Horticulture, Jomo Kenyatta University of Agriculture and Technology, P.O Box 62000-00200, Nairobi, Kenya *E-mail: [email protected]

Small scale farmers in sub-Saharan Africa rely on cultural and traditional methods based on natural products to control root knot nematodes (RKNs). A review of the literature shows that although most of these methods have been successfully tested in laboratory assays, there is limited knowledge on the identities of the bioactive compounds involved. Furthermore, these traditional methods are yet to be scientifically verified under field settings. This chapter examines some of these traditional strategies and explores science-based opportunities for effective control of RKNs for increasing agricultural production in small scale farmer holdings.

Introduction In Africa, small-scale subsistence farming contributes about 80% of food production from farm sizes which are typically 1 hectare or less. Production from these farms is severely constrained by abiotic (soil fertility, climate change, water resources etc.) and biotic factors (seed quality, pests and diseases, etc.). The average yield from a small scale farm is about 1t/ha against the potential of at least 4.5t/ha. Of the biotic factors, above- and below-ground pests and diseases severely impact on agricultural production, with the latter factor being the focus of this chapter. Plant parasitic nematodes constitute one of the most significant © 2018 American Chemical Society

below-ground pests (1) besides ants and termites, and the focus of this review. The symptoms of plant parasitic nematode infection are non-specific and include chlorosis, wilting, galling of roots and tubers, stunted growth, root lesion and yield loss (Figure 1). Root-knot nematodes (RKNs) have complex intimate interactions with their host plants throughout their life cycle, which includes an egg, juvenile stages (J1-J4) and an adult. The infective juvenile stage J2 is the motile and host seeking stage. The most damaging of plant parasitic nematodes, especially in the tropics and sub-tropics of the world, are RKNs (Meloidogyne spp.). Of the RKNs, Meloidogyne incognita is the most damaging and widespread (2, 3) especially in commercial production and may account for 15-60 % yield loss (4). In Kenya, losses have been reported to be principally due to M. incognita and M. javanica (5). They form synergy with plant pathogenic fungi and bacteria causing great yield loss (2, 6). Small scale farmers in Africa are often unaware of RKNs or the damage they cause but recognize reducing yields. As such, small scale farmers adopt various mitigation strategies to control RKNs and to improve crop yield. The chapter will provide highlights of some of these RKN mitigation strategies that are based on natural products and are derived from various plant sources applied in: crop rotation, organic soil amendments, intercropping, dead-end trap crops, and processed plant products. Additionally, this is followed by exploring novel ideas based on chemical ecological and biotechnological approaches for RKN control. Finally, possible future directions are discussed.

Figure 1. A) Second-stage infective juvenile of Meloidogyne incognita; B) infective juveniles puncturing the root tip of pepper; and, C) galled tomato roots.

Natural Products Use for Control of RKNs and How They May Function Crop Rotation Crop rotation involves the use of non-host crops that trap or are antagonistic to RKNs in a rotation program. The effectiveness of crop rotation for control of RKN is somewhat questionable. Firstly, RKNs are polyphagous, possessing relatively few non-host plants (7, 8). Secondly, RKN populations in the soil may not be uniformly distributed and because the infective juvenile stage is not highly 116

mobile, antagonist plants may impact only on infective juveniles at short distances in the soil environment. To ensure effective RKN control, some farmers practice intensive crop rotations with different crops in several rotation cycles. The mode of action of non-host crops in rotations are not well known, but it is believed that they release toxic compounds through the root exudate into the soil to control RKNs in various ways including suppressing hatching of eggs, interfering with the growth of juveniles (growth regulators), target the nervous system of juveniles or blocking the attraction of juveniles seeking a host (Table 1, Figure 2). Plants with large above-ground biomass that shed off their leaves frequently may also leach toxic compounds into the soil to supplement bioactivity of root exudates. Examples of crops that are used for rotation in sub-Saharan Africa include Crotalaria species (e.g. Crotalaria juncea) (Fabaceae) in Cote D’Ivoire (9), and Pyrethrum species (e.g. Chrysanthemum cinerarifolium), (Asteraceae), African Nightshades (Solanum nigrum, Solanum sarrachoides) (Solanaceae) and Bidens pilosa (Asteraceae) in Kenya (10). The mode of action of root exudates in the control RKNs not entirely clear. However, there is a high possibility that the blend of compounds from different natural product classes released with the root exudate may act additively or synergistically to control RKNs. Field trails are needed to demonstrate the effectiveness of crop rotation in reducing populations of RKNs and to identify the bioactive compounds.

Organic Soil Amendments This is probably the most common method used to control RKN, and it involves use of dried plant parts in a powder or cake form mixed with the soil before or after planting. Plant parts may be derived from the same plant or different plant species. Unlike root exudates which are continuously released into the soil as the plants mature, in organic soil amendments, either the moisture content in the soil or secretions of the RKNs could facilitate extraction of the bioactive components in the plant parts. The advantage in using this method is that it allows for a slow release and direct contact of the bioactive components over time with the RKNs. Additionally, microbial degradation of the plant parts could lead to changes in bioactive components that RKNs are exposed to and released into the soil over time. In this regard the likelihood of RKNs developing resistance to organic soil amendments is minimized. Studies suggest that although organic amendments are environmentally friendly, large quantities may be required per unit area to make them work effectively (7). Plant parts of Tagetes minuta (Asteraceae), Ricinus communis (Euphorbiaceae), Mucuna pruriens (Fabaceae), Datura stramonium (Solanaceae) and Tithonia diversifolia (Asteraceae) have been used as antagonistic plants with varying degrees of successes (4, 9, 10). These plants are considered to produce anti-nematode compounds with different modes of action either as repellents, hatching inhibitors or nematicidal effects. As discussed for crop rotation, well designed field experiments are necessary to provide proof of the effectiveness of organic soil amendments including knowledge of the identities of the bioactive compounds against RKNs. 117

Table 1. Examples of plants used for RKN management showing class of compounds with key compounds that have effect on nematodes. Plant

Family

Class of compound

Key compound

Sorghum Sudanese

Poaceae

cyanogenic glycoside

dhurrin (1)

Ricinus communis

Euphorbiaceae

pyridone alkaloid

ricinine (2)

Tagetes erecta

Asteraceae

thiophene

α-terthienyl (3)

Azadirachta indica

Meliaceae

limonoid

azadirachtin (4)

Lantana camara

Verbenaceae

triterpenoid

camarinic acid (5)

Brassica napus

Brassicaceae

glucosinolate

gluconasturtiin (6)

Crotaralia spp.

Fabaceae

pyrrolizidine alkaloid

monocrotaline (7)

Chrysanthemum cinerarifolium

Asteraceae

pyrethrin

pyrethrin I (8)

Solanum spp.

Solanaceae

steroidal glycoalkaloid

tomatine (9)

Mucuna pruriens

Fabaceae

amino acid

3-hydroxy-L-tyrosine (10)

Datura stramonium

Solanaceae

tropane alkaloid

atropine (11)

Tithonia diversifolia

Asteraceae

sesquiterpene lactone

tagitinin C (12)

Allium sativum

Amaryllidaceae

polysulphides

allicin (13)

Intercropping There is good evidence that some small-scale farmers intercrop to control RKNs. Intercropping involves use of a companion non-host crop which is planted alongside, typically in rows between a RKN-susceptible crop. Chemicals released in the rhizosphere by root exudate of the non-host crop can interfere with the host finding behavior of the RKN. It can also suppress RKN hatching or growth. However, because of the small farm sizes, farmers usually intercrop with food plants or plants that are economically valuable to the household. Examples of plants intercrop include African nightshades (Solanaceae) with spinach (Amaranthaceae), with some cultivars of the latter being susceptible to RKNs (11). Some studies have proved Tagetes erecta (Asteraceae) and Crotalaria juncea (Fabaceae) to be effective in offering protection to RKN-susceptible but high yielding banana (12) and tomato cultivars (13–15). 118

Figure 2. Key chemical components in plants used for the management of RKNs: 1, dhurrin; 2, ricinine; 3, α-terthienyl; 4, azadirachtin; 5, camarinic acid; 6, gluconasturtiin; 7, monocrotaline; 8, pyrethrin I; 9, tomatine; 10, 3-hydroxy-L-tyrosine; 11, atropine; 12, tagitinin; and, 13, allicin.

‘Dead-End’ Trap Crops The use of dead-end trap crops to control RKNs has been documented in several small-scale farming systems. Like economically valuable plants that are intercropped, ‘dead-end’ trap crops of economic value are typically preferred by small scale farmers. Dead-end trap crops allow for hatching and invasion of the host but the host’s defense system including cell wall reinforcement and production of toxic secondary metabolites prevents further development of the infective stage juvenile. This has been demonstrated in in vitro assays for the trap crops Amaranthus dubius (Amaranthaceae), Solanum scabrum (16), Solanum nigrum and S. sisymbriifolium (Solanaceae) (17) which are used as vegetables in Eastern Africa. However, the specific compounds exhibiting bioactivity have not been characterized or identified. Processed Plant Products Plant products can be used for organic amendments, seed coating and in bare-root dip treatments as a management strategy against nematodes. Compared to organic soil amendments, processed plant products such as plant extracts may contain high concentrations of the bioactive compounds thereby providing immediate action when applied. Bioactivity may depend on the concentration, stability of the compounds and substrate used for the formulation. Some commercial products have been developed from natural products for RKNs. For instance, allicin and polysulphides are biologically active ingredients derived from garlic Allium sativum (Amaryllidaceae) concentrate of NEMGUARD®, a commercially available product by Dudutech Kenya, that is applied as a soil 119

drench or spray for the control of RKNs. It was previously shown that garlic straw reduced hatchability and development of second stage juveniles of M. incognita (18). In addition, allicin has demonstrated additional benefits of antimicrobial activity against plant pathogenic bacteria and fungi (19). Seed treatment with plant-based products has been used to effectively suppress nematode populations in soil, reducing root-knot disease incidence in plants as well as other plant-parasitic nematodes, such as neem extracts and neem by-products (Meliaceae), with bioactivity associated with azadirachtin, the major compound and related limonoids in neem seeds (20–22). Other recommended approaches include seed coating with plant extracts before planting, which is cost effective as only small amounts are required unlike in soil fumigation or organic amendments where large quantities are required. In summary, this review although not exhaustive, suggests that only a limited number of plant species are used by small scale farmers in sub-Saharan Africa via different strategies to manipulate RKN behavior based on their phytochemicals. These plant species can be traced predominantly to the families Asteraceae, Euphorbiaceae, Fabaceae, Solanaceae, Amarantheceae, Amaryllidaceae and Meliaceae. The discovery of other plant species would require more detailed analysis and documentation of the cultural and farmer practices across Africa. The implication of this study is that follow-up field experimentation to prove the efficacies of these approaches and to identify the bioactive compounds would be critical to educating small scale farmers for adoption in their farming systems.

Potential of Chemical Ecological and Biotechnological Approaches to Control RKNs Chemical ecology is the study of naturally-occurring chemicals (semiochemicals) that mediate the interactions between organisms. Therefore, an understanding of their structure, function and origin is critical for the development of behavior-modifying chemicals for control of RKNs and other organisms. On the other hand, biotechnology employs biomolecular processes to develop technologies and products for improved wellbeing. There have been chemical ecological applications including use of attractants and repellents. Push-Pull In a cropping system, “push-pull” exploits behavior-modifying chemicals from a repellent (push) crop and an attractive (pull) trap plant. As the plants mature, they continuously release these naturally-occurring repellents and attractants to modify the behavior of the target pest. A recent chemical ecology study investigating the host-seeking behavior of M. incognita infective juveniles (J2) found that volatile organic compounds produced by pepper roots were important cues in either attracting or repelling the J2 (Figure 3) (23). In this study, methyl salicylate was found to induce the highest positive chemotaxis compared to limonene, alpha pinene and tridecane whereas thymol present in an accession, AVDRC PP0237, elicited avoidance and the highest dose tested had a toxic effect 120

to the nematodes. These results demonstrated a potential role for the use of thymol as a candidate repellent or nematicidal compound warranting further investigation in field experiments. A novel way to use this data is to develop a “push-pull” cropping system for control of RKN, utilizing RKN-tolerant/resistant cultivar as a repellent crop and a wild relative as a attractant trap crop in polycrop system.

Figure 3. Chemical components identified in root volatiles of different pepper cultivars and in tomato; 14, α-pinene; 15, limonene; 16, 2-methoxy-3-(1-methylpropyl)-pyrazine; 17, methyl salicylate; 18, tridecane; 19, thymol; 20, 2,6-Di-tert-butyl-p-cresol; 21, L-ascorbyl 2,6-dipalmitate; 22, dibutyl phthalate; 23, dimethyl phthalate. Similar results of attraction and repellence have been observed by our group working on volatiles and non-volatiles released by tomato roots (24) and tested against M. incognita. In the study by (24) several medium and high molecular weight hydrocarbons, ketones, esters and phenols were identified. Of these compounds, 2,6-Di-tert-butyl-p-cresol, L-ascorbyl-2,6-dipalmitate, dibutyl phthalate and dimethyl phthalate (Figure 3) suppressed egg hatch of M. incognita, with dibutyl phthalate elicting repellent activity on the infective stage juvenile J2. Using bioassay-guided isolation and identification of bioactive compounds in the root exudates of other tomato cultivars, we found a variety of flavonoids and alkaloids that elicit attraction and repellence in M. incognita infective stage juveniles, suggesting that bioactivity is associated with a blend of tomato-specific root exudate compounds. Biotechnology Plant breeding as well as engineering plants to produce thymol naturally can also offer benefits in nematode control. The previous study on pepper-RKN 121

interaction, did not carry out a genetic analysis of the four pepper cultivars, which could provide information on genes that code for the biosynthetic pathways leading to the production of thymol (23). Biosynthetically, thymol, a phenolic monoterpene is a derivative of cymene. The precursor for the formation of p-cymene is γ-terpinene which goes through aromatization to form p-cymene, which in turn gets hydroxylated to thymol or its isomer carvacrol (25, 26). In the study (23), γ-terpinene was not detected in the four pepper cultivars but p-cymene was detected in two hot pepper cultivars. Molecular tools could be used to identify the genetic variability that will inform crop improvement for nematode control. Other studies have shown that thymol can have both positive and negative impact on other soil microbes including other nematode species. For instance, in greenhouse experiments, thymol alone and in combitnation with benzaldehyde reduced the populations of the root-knot nematode, M. arenaria and the soybean cyst nematode, Heterodera glycines in the soil (27). Also, a study that investigated the effect of thymol in garlic white rot, found that it affects soil microflora by reducing fungal biomass and Gram negative bacteria even though it did not reduce the incidence of white rot (28). Thymol and its isomer carvacrol were also associated with reduction of pathogens and gas emissions in swine and cattle waste by inhibiting proliferation of fecal coliforms and anaerobic bacteria (29, 30). Elsewhere, in field experiments, thymol was found to reduce the bacteria wilt incidence in tomato (31). This shows that other than nematode suppression, thymol could have additional benefits such as dealing with the bacteria wilt which is currently a huge production constraint for small-scale agricultural production systems. Plant breeding and genetic engineering can be useful techniques for producing cultivars that are resistant to RKNs. Previously, resistance has been linked with disease incidence alone, where plants that recorded low galling and egg mass indices were associated with genes that confer resistance but the mechanisms of nematode suppression were not explained (32–34). Resistance breaking due to abiotic factors such as the Mi-1.2 gene that confers resistance to tomato is inactive at soil temperatures above 28 °C (35). This poses a challenge as such crops would not be useful in the tropics such as Africa. Other approaches that have been used for genetic engineering include the application of RNA interference (RNAi) where double stranded RNA (dsRNA) is delivered in vitro in PPNs and in planta for transgenic control (36–38). Plant-mediated RNAi led to down-regulation of target genes and also reduced parasitic success of nematodes infecting transgenic plants. In tomato, the transgenic expression of Mi-cpl-1 gene constructed using RNAi caused a reduction in infection and development of M. incognita (39). A recent review (40) also provides other transgenic approaches that include cloning of natural host-resistant genes from some plants to other susceptible plant species (41), and proteinase inhibitor coding genes as well as anti-nematode proteins that inhibit nematode development in plants. A novel way to protect plants from RKN could be to link biochemical processes with molecular techniques by engineering the biosynthesis of root compounds that disrupt chemotactic host finding signals by the infective juveniles.

122

Conclusions and Future Directions RKNs are pests of a wide range of crops, but a review of the literature shows that plants from a few families are utilized by small scale farmers to control them. This suggests that in-depth studies are required to catalogue all the traditional strategies based on natural products for RKN control in Africa. This calls for collaboration between taxonomists, nematologists and chemists to work together to close the gaps in our knowledge of the strategies for the manipulation of RKN behavior with natural products in small scale farming systems. A long-term study would require additional scientific disciplines to examine effects of climate change on plant-RKN interactions, and gene expressions which is starting to gain momentum for specific vegetables, microbial root mutualists on RKN and plant biological and physiological performance, and tri-trophic interactions between plants, RKNs and their natural enemies in the soil environment.

Acknowledgments We thank Dr. David Tchouassi of icipe for critical review of the manuscript.

References 1.

2.

3.

4.

5. 6.

7.

8.

Talwana, H.; Sibanda, Z.; Wanjohi, W.; Kimenju, W.; Luambano-Nyoni, N.; Massawe, C.; Kerry, B. R. Agricultural Nematology in East and Southern Africa: Problems, Management Strategies and Stakeholder Linkages. Pest Manage. Sci. 2016, 72, 226–245. Abad, P.; Gouzy, J.; Aury, M.-J. Genome Sequence of the Metazoan PlantParasitic Nematode Meloidogyne incognita. Nat. Biotechnol. 2008, 26, 909–915. Trudgill, D. L.; Blok, V. C. Apomictic, Polyphagous Root-Knot Nematodes: Exceptionally Successful and Damaging Biotrophic Root Pathogens. Annu. Rev. Phytopathol. 2001, 39, 53–77. Luc, M.; Sikora, R. A.; Bridge, J. Plant Parasitic Nematodes in Subtropical and Tropical Agriculture, 2nd ed.; CABI Publishing: Wallingford, UK, 2005; pp 1−11. Kanyagia, S. T. A Survey of Vegetable Nematodes in Kenya. East African Agric. For. J. 1980, 44, 178–182. Onkendi, E. M.; Kariuki, G. M.; Marais, M.; Moleleki, L. N. The Threat of Root-Knot Nematodes (Meloidogyne spp.) in Africa: A Review. Plant Pathol. 2014, 63, 727–737. Moens, M.; Perry, R. N.; Starr, J. L. Meloidogyne Species – A Diverse Group of Novel and Important Plant Parasites. In Root-knot Nematodes; Perry, R. N., Moens, M., Starr, J. L., Eds.; CAB International: Wallingford, UK, 2009; pp 1–17. Sikora, R. A.; Fernanduez, E. Nematode Parasites of Vegetables. In Plant Parasitic Nematodes in Subtropical and Tropical Agriculture, 2nd ed.; Luc, M., Sikora, R. A., Bridge, J., Eds.; CABI Publishing: Wallingford, UK, 2005; pp 319–392. 123

9.

10.

11.

12.

13.

14. 15.

16.

17.

18.

19.

20.

21.

22.

Coyne, D. L.; Fourie, D.; Moens, M. Current and Future Management Strategies in Resource-Poor Regions. In Root-knot Nematodes; Perry, R. N., Moens, M., Starr, J. L., Eds.; CAB International: Wallingford, UK, 2009; pp 444–475. Mwamba, S. Root Knot Nematodes (Meloidogyne incognita) Interaction with Selected Asteraceae Plants and their Potential Use for Nematode Management. MSc Thesis, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya, 2016. Mcsorley, R.; Gallaher, R. N. Comparison of Nematode Popualtion Densities on Six Summer Crops at Seven Sites in North Florida. J. Nematol. 1992, 24, 699–706. Hussain, M. A.; Mukhtar, T.; Kayani, M. Z. Efficacy Evaluation of Azadirachta indica, Calotropis procea, Datura stramonium and Tagetes erecta against Root-Knot Nematodes Meloigogyne incognita. Pakistan J. Bot. 2011, 43, 197–204. Murungi, L.; Kirwa, H.; Torto, B. Plant Volatiles Signal the Host Seeking Process in Root-Knot Nematodes. Presented at the International Society of Chemical Ecology Conference, Stockholm, Sweden, June 2015. Wang, K.; Hooks, C. R.; Ploeg, A. Protecting Crops from Nematode Pests. Plant Dis. 2007, PD-35. Taye, W.; Sakhuja, P. K.; Tefera, T. Root-Knot Nematode (Meloidogyne incognita) Management Using Botanicals in Tomato (Lycopersicon esculentum). Acad. J. Agric. Res. 2013, 1, 9–16. Chitambo, O.; Haukeland, S.; Fiaboe, K. K.; Grundler, F. M. African Nightshades and African Spinach Lures Plant Parasitic Nematodes to a Dead-end. Future Agriculture: Social-Ecological Transitions and Bio-Cultural Shifts; University of Bonn: Germany, 2017. Correia, M. C. C. P. Nematicidal Activity of Solanum nigrum and S. sisymbriifolium Extracts Against the Root-Lesion Nematode Pratylenchus goodeyi and Its Effects on Infection and Gene Expression. Ph.D thesis, Universidade de Madeira, Funchal, Portugal, 2014. Gong, B.; Bloszies, S.; Li, X.; Wei, M.; Yang, F.; Shi, Q.; Wang, X. Efficacy of Garlic Straw Application Against Root-Knot Nematodes on Tomato. Sci. Hortic. 2013, 161, 49–57. Curtis, H.; Noll, U.; Störmann, J.; Slusarenko, A. J. Broad-Spectrum Activity of the Volatile Phytoanticipin Allicin in Extracts of Garlic (Allium sativum L.) Against Plant Pathogenic Bacteria, Fungi and Oomycetes. Physiol. Mol. Plant Pathol. 2004, 65, 79–89. Tiyagi, S. A.; Alam, M. M. Efficacy of Oil-Seed Cakes Against Plant-Parasitic Nematodes and Soil-Inhabiting Fungi on Mungbean and Chickpea. Bioresour. Technol. 1995, 51, 233–239. Akhtar, M.; Mahmood, I. Control of Root-Knot Nematode Meloidogyne incognita in Tomato Plants by Seed Coating with Suneem and Neem Oil. J. Pestic. Sci. 1997, 22, 37–38. Javed, N.; Gowen, S. R.; Inam-ul-Haq, M.; Abdullah, K.; Shahina, F. Systemic and Persistent Effect of Neem (Azadirachta indica) Formulations 124

23.

24.

25. 26.

27.

28.

29.

30. 31.

32.

33.

34.

35.

Against Root-Knot Nematodes, Meloidogyne javanica and Their Storage Life. Crop Prot. 2007, 26, 911–916. Kihika, R.; Murungi, L. K.; Coyne, D.; Ng’ang’a, M.; Hassanali, A.; Teal, P. E. A.; Torto, B. Parasitic Nematode Meloidogyne incognita Interactions with Different Capsicum annum Cultivars Reveal the Chemical Constituents Modulating Root Herbivory. Sci. Rep. 2017, 7, 2903. Yang, G.; Zhou, B.; Zhang, X.; Zhang, Z.; Wu, Y.; Zhang, Y.; Lü, S.; Zou, Q.; Gao, Y.; Teng, L. Effects of Tomato Root Exudates on Meloidogyne incognita. PLoS One 2016, 11, e0154675. Mikio, Y.; Taeko, U. Biosynthesis of Thymol. Chem. Pharm. 1962, 10, 71–72. Thompson, J. D.; Chalchat, J. D.; Michet, A.; Linhart, Y. B.; Ehlers, B. Qualitative and Quantitative Variation in Monoterpene Co-occurence and Composition in the Essential Oil of Thymus vulgaris Chemotypes. J. Chem. Ecol. 2003, 29, 859–880. Soler-Serratosa, A.; Kokalis-Burele, N.; Rodriguez-Kabana, R.; Weaver, C. F.; King, P. S. Allelochemicals in Control of Plant-Parasitic nNematodes 1. In-vivo Nematicidal Efficacy of Thymol and Thymol/benzaldehyde Combinations. Nematropica 1995, 26, 57–71. Miñambres, G. G.; Conles, M. Y.; Lucini, E. I.; Verdenelli, R. A.; Meriles, J. M.; Zygadlo, J. A. Application of Thymol and Iprodione to Control Garlic White Rot (Sclerotium cepivorum) and Its Effect on Soil Microbial Communities. World J. Microbiol. Biotechnol. 2010, 26, 161–170. Varel, V. H.; Miller, D. N. Plant-Derived Oils Reduce Pathogens and Gaseous Emissions from Stored Cattle Waste. Appl. Environ. Microbiol. 2001, 67, 1366–1370. Varel, V. H. Carvacrol and Thymol Reduce Swine Waste Odor and Pathogens: Stability of Oils. Curr. Microbiol. 2002, 44, 38–43. Ji, P.; Momol, M. T.; Olson, S. M.; Pradhanang, P. M.; Jones, J. B. Evaluation of Thymol as Biofumigant for Control of Bacterial Wilt of Tomato Under Field Conditions. Plant Dis. 2005, 89, 497–500. Djian-Caporalino, C.; Farazi, A.; Arguel, M. J.; Vernie, T.; VandeCasteele, C.; Faure, I.; Brunound, G.; Abad, P. Root-Knot Nematode (Meloidogyne spp.) Me Resistance Genes in Pepper (Capsicum annuum L.) are Clustered on the P9 Chromosome. Theor. Appl. Genet. 2007, 114, 473–486. Thies, J. A.; Fery, R. L. Host Plant Resistance as an Alternative to Methyl Bromide for Managing Meloidogyne incognita in Pepper. J. Nematol. 2002, 34, 374–377. Thies, J. A.; Dickson, D. W.; Fery, R. L. Stability of Resistance to Root-Knot Nematodes in “Charleston belle” and “Carolina wonder” Bell Peppers in a Sub-Tropical Environment. HortScience 2008, 43, 188–190. Lilley, C. J.; Kyndt, T.; Gheysen, G. Nematode Resistant GM Crops in Industrialized and Developing Countries. In Genomics and Molecular Genetics of Plant-Nematode Interactions; Jones, J., Gheysen, G., Fenoll, C., Eds.; Springer: The Netherlands, 2011; pp 517−542. 125

36. Lilley, L. J.; Davies, L. J; Urwin, P. E. RNA Interference in Plant Parasitic Nematodes: A Summary of the Current Status. Parasitology 2012, 139, 630–640. 37. Atkinson, H. J.; Liley, C. J.; Urwin, P. E. Strategies for Transgenic Nematode Control in Developed and Developing World Crops. Curr. Opin. Biotechnol. 2012, 23, 251–256. 38. Dutta, T. K.; Banakar, P.; Rao, U. The Staus of RNAi-Based Transgenic Research in Plant Nematology. Front. Microbiol. 2015, 5, 1–7. 39. Dutta, T. K.; Papolu, P. K.; Banakar, P.; Choudhary, D.; Sirohi, A.; Rao, U. Tomato Transgenic Plants Expressing Hairpin Construct of a Nematode Protease Gene Conferred Enhanced Resistance to Root-Knot Nematodes. Front. Microbiol. 2015, 6, 260. 40. Ali, M. A.; Azeem, F.; Abbas, A.; Joyia, F. A.; Li, H.; Dababat, A. A. Transgenic Strategies for Enhancement of Nematode Resistance in Plants. Front. Plant Sci. 2017, 8, 750. 41. Milligan, S. B.; Bodeau, J.; Yaghoobi, J.; Kaloshian, I.; Zabel, P.; Williamson, V. M. The Root-Knot Nematode Resistance Gene Mi from Tomato is a Member of the Leucine Zpper, Nucleotide Binding, Leucine-Rich Repeat Family of Plant Genes. Plant Cell 1998, 10, 1307–1319.

126

Chapter 10

Quantitative Assessment of Nectar Microbe-Produced Volatiles Caitlin C. Rering,*,1 John J. Beck,1 Rachel L. Vannette,2 and Steven D. Willms1 1Chemistry

Research Unit, Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, 1700 SW 23rd Drive, Gainesville, Florida 32608, United States 2Department of Entomology and Nematology, University of California, Davis, Davis, California 95616, United States *E-mail: [email protected]

Nectar microbe-produced volatiles can contribute to floral blends and modify pollinator preference for flowers. Identifying and describing the compounds that may underlie this effect is a key goal. Semi-quantitative or quantitative data is often critical for chemical ecology investigations, given that biotic responses (i.e., insects or plants) can fluctuate with concentration, relative ratios, or composition of the emitted volatile profiles. However, field-based, in situ microbial volatile detection and quantification is difficult due to the small scale of the nectar microhabitat. Laboratory-based inoculations of bulk synthetic nectars are useful for screening weakly-abundant and difficult to analyze volatiles, which may be crucial in eliciting responses. Despite the limitations of this approach, it allows rapid identification of target molecules for further validation with bioassays. Targeted analytical methods to identify or quantify semiochemicals may then be developed and validated for field tests. Here, we report the first quantitative assessment of microbe volatile emission in laboratory-based tests with synthetic nectar. We also review briefly solid-phase microextraction collection and gas-chromatography data interpretation. Nectar microbe interactions with plants and insects offer opportunities for agricultural improvement, and a selection of potential uses are highlighted. © 2018 American Chemical Society

Introduction Visitor-mediated pollination is necessary or beneficial for the majority of the world’s food crops, yet reports indicate populations of managed bees are declining worldwide (1, 2). Rapidly deployable and flexible solutions to enhance pollination despite limited pollinator abundance and poor pollinator health are needed. Recent studies have suggested the importance of the nectar microbiome in plant-pollinator interactions (3–6). Tritrophic (plant-insect-microbe) interactions promise opportunities for agricultural improvement and a few potential applications are highlighted, though this chapter is not the first to outline their potential utility (7). Here, we offer a chemo-centric discussion of the interactions between nectar-inhabiting microbes, plants and pollinators, with a focus on the methods required to quantify volatile chemicals.

Materials and Methods Microbes and Inoculated Solutions Four microorganisms isolated from California wildflowers were selected for study and include the specialist yeast, Metschnikowia reukaufii and generalists, Aureobasidium pullans, a fungus, and the bacteria Neokomagataea sp. and Asaia astilbes (Genbank IDs MF319536, MF325803, MF340296, and KC677740, respectively). Sterile synthetic nectar (0.3% w/v sucrose; 0.6% w/v each: glucose and fructose; 0.1 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine), designed to mimic floral nectar of bee-pollinated flowers was inoculated with cells from actively growing subcultures (8, 9). Cells were grown on selective plate media (R2A agar with 100 mg L-1 cycloheximide for bacteria, YM agar with 100 mg L-1 chloramphenicol for fungi) for 4 d at 29 °C, then suspended in sterile synthetic nectar to create microbial stock solutions. Cell density in stock solutions was determined via UV absorbance (600 nm) for bacteria and a cell counter (Bio-Rad Laboratories, Inc, Hercules, CA) for yeast. Appropriate dilutions for stock solutions were delivered such that final cell densities in 20 mL synthetic nectar were 103 cells µL-1 (corresponding to 0.4 A UV600 for bacteria). Samples and sterile controls were incubated at 29 °C in sealed 118 mL Mason jars (n=3 replicates of each microbial species, M. reukaufii, A. pulluans, Neokomagataea, and Asaia and n=4 sterile controls). Lids were modified to accommodate two sampling ports by fitting gas chromatograph septa into pre-drilled holes.

Volatile Quantitation Mixed stock solutions of identified compounds were prepared in acetonitrile and used to prepare calibration standards under identical sterile conditions as microorganism inoculation samples: Stock solutions was spiked into 20 mL sterile synthetic nectar that had been previously incubated at 29 °C and analyzed 128

as described below. Calibration standards, prepared immediately before analysis, contained less than 0.1% v/v acetonitrile (carrier solvent) and spanned eight orders of magnitude (0.1 ng mL-1 − 10 mg mL-1).

Analysis by GC-MS Headspace volatiles from inoculated microbes and prepared calibration standards were collected onto solid-phase microextraction fibers (SPME; Supelco, Bellafonte, PA; 50/30 µm, 2 cm, divinylbenzene/ carboxen/ polydimethylsiloxane). Inoculated synthetic nectars were sampled at intervals of 0, 48 and 96 hr (±0.4 h). Standards were prepared immediately before analysis. Jar lids were vented under sterile conditions immediately prior to volatile collection, to allow dissipation of volatiles accumulated between sampling intervals and then sealed and returned to the incubator, set to 29 °C. Volatile collections used the following fiber parameters: accumulation of volatiles in the newly closed container, 15 min; exposure of fiber to adsorb volatiles, 15 min; storage time of volatiles on fiber, ≤ 1 min; and, thermal desorption of volatiles in gas chromatograph inlet, 6 min. The adsorbed volatiles were thermally desorbed in splitless mode onto an Agilent 7890A GC coupled to a quadrupole 5975C MSD in electron ionization mode (Palo Alto, CA) outfitted with a J&W Scientific (Folsom, CA) 60 m DB-Wax column. Volatiles were analyzed using parameters identical to those previously described except flow was set to 3 mL min-1 (10). Retention indices were calculated using a homologous series of n-alkanes and were used to assist with initial identification. Identities were further confirmed by comparison to retention times and fragmentation patterns of standards. Compound identities not verified with a commercial standard were marked as tentatively identified and are not discussed here. Peaks identified as background from the containers, fibers, column, and synthetic nectar that were found in sterile controls and blanks were not considered.

Results Microorganism volatile composition differed between species (Table 1). Volatiles were detectable immediately after inoculation (day 0, 30 min after inoculation into synthetic nectar with 15 min each permeation and exposure time), further diverging after 2 and 4 days of growth (11). Alcohols, esters, and ketones were more abundant in fungal solutions, while the volatile 2,5-dimethylfuran was characteristic of bacteria. All four microbes produced n-hexanol. The specialist nectar yeast M. reukaufii was characterized by esters, including ethyl butyrate, 2-methylpropyl acetate and 3-methylbutyl acetate, the alcohols 2-butanol and 3-ethoxy-1-propanol, and a relatively greater abundance of ethyl acetate, and the alcohols 3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-3-buten-1-ol, ethanol and 2-phenylethanol. 129

Table 1. Dissolved concentration of volatile compounds identified in single strain inoculations of synthetic nectar after 96 h growth, sorted by M. reukaufii concentration. Compound

M. reukaufii (μM)

A. pullulans (μM)

A. astilbes (μM)

Neokomagataea sp. (μM)

130

ethanol

197,155 (±9,983)a

22,461 (±4,991)a

150 (±50)

105 (±22)

2-methyl-1-butanol and 3-methyl-1-butanolb

511 (±9)a

16 (±1)

0.28 (±0.02)

0.31 (±0.01)

isobutanol

148 (±1)a

177 (±7)a

0.11 (±0.05)

0.15 (±0.01)

3-hydroxy-2-butanone (acetoin)

49.0 (±0.8)a

22 (±2)a

18 (±1)a

15.9 (±0.2)a

2-phenethyl alcohol

33 (±2)

9.0 (±0.8)

0.34 (±0.07)

0.33 (±0.03)

1-propanol

20 (±3)

101 (±5)

0

0

3-ethoxy-1-propanol

2.4 (±0.5)

0

0

0

2-butanol

1.52 (±0.03)

0

0

0

ethyl acetate

1.1 (±0.1)

0.16 (±0.02)

0

0

4-penten-1-ol

0.44 (±0.03)

0.44 (±0.02)

0

0

3-methyl-3-buten-1-ol

0.35 (±0.01)

0.23 (±0.01)

0

0

1-hexanol

0.06 (±0.01)

0.06 (±0.01)

0.059 (±0.001)

0.0489 (±0.0003)

2-methylpropyl acetate

0.0439 (±0.0009)

0

0

0

3-methylbutyl acetate

0.0323 (±0.0008)

0

0

0

2-ethyl-1-hexanol

0.025 (±0.003)

0.2 (±0.2)

0.13 (±0.02)

0.090 (±0.003)

ethyl butyrate

0.022 (±0.002)

0

0

0

M. reukaufii (μM)

Compound

A. pullulans (μM)

A. astilbes (μM)

Neokomagataea sp. (μM)

2-nonanone

0

9 (±1)

0

0

4-methyl-2-pentanone

0

0.4 (±0.4)

0

0

2,5-dimethylfuran

0

0

0.03 (±0.03)

0.031 (±0.001)

a Data is semi-quantitative due to poor linearity and/or saturation of MS signal. quantified due to co-elution and common fragmentation pattern.

b

2-methyl-1-butanol and 3-methyl-1-butanol could not be independently

131

Data Interpretation and the Need for Calibration Vapor-phase concentrations (Figure 1, Cg) of volatile abundance in the sealed jars are not reported or inferred by this approach. Instead, the correlation between dissolved concentration is correlated with the combined analytical factors: compound ionization efficiency in the GC-MS electron ionization source, chromatographic quality (i.e., co-elution, background interference), compound affinity for a given fiber type (Figure 1, Kd), partitioning between air and nectar, (Figure 1, KH) and aqueous concentration (Figure 1, Caq). Equilibrium based air-water partitioning (Figure 1, KH) depends on a compound’s water solubility limit and vapor pressure at a given temperature (12). The polar, low molecular weight volatiles produced by nectar microorganisms in this study have high aqueous solubility and volatility and so are well distributed between condensed and gas phases.

Figure 1. Partitioning behavior of volatiles in sealed jars.

Quantitative Method Evaluation Analytical column choice can affect peak shape and detection limits. In this study, samples were quantified on a polar analytical GC column because of improved retention and separation of analytes, relative to a non-polar column (data not shown). The co-eluting isomers 2-methyl-1-butanol and 3-methyl-1-butanol could not be independently quantified due to common fragmentation patterns (no unique ions for extraction ion analysis; Table 2). These analytes were quantified with a 1:1 mixture of both compounds and so concentrations reported here are only estimates of total 2- and 3-methyl-1-butanol content. Isomerism is known to impact bioactivity. Future chromatographic methods should be modified to incorporate independent quantitation of these volatiles.

132

Table 2. Volatile calibration details.

Compound

a

Calibration description

R2 valuea (calibration level, where applicable)

Calibration standard concentration range (ng mL-1)

ethanol

TIC, 8 standards, 2 curves

0.941 (high); 0.966 (low)

1E6-1E7 (high); 1,000-10,000 (low)

2- and 3-methyl-1butanol

TIC, 7 standards, 2 curves

0.716 (high); 0.999 (low)

2,000-10,000 (high); 10-2,500 (low)

isobutanol

TIC, 6 standards, 2 curves

0.920 (high); 0.991 (low)

500-25,000 (high); 25-100 (low)

3-hydroxy-2butanone (acetoin)

TIC, 4 standards

0.871

2,500-10,000

2-phenethyl alcohol

TIC, 7 standards

0.997

10-1,000

1-propanol

TIC, 4 standards

0.991

1,000-10,000

3-ethoxy-1propanol

EIC (59 m/z), 4 standards

0.969

50-1,000

2-butanol

EIC (45 m/z), 6 standards

0.993

1-1,000

ethyl acetate

TIC, 8 standards

0.988

1-100

4-penten-1-ol

TIC, 7 standards

0.993

10-1,000

3-methyl-3-buten1-ol

TIC, 5 standards

0.981

10-100

1-hexanol

TIC, 7 standards

0.991

1-100

2-methylpropyl acetate

TIC, 6 standards

0.998

1-1,000

3-methylbutyl acetate

TIC, 4 standards

0.998

0.1-100

2-ethyl-1-hexanol

TIC, 4 standards, blanks subtracted

>0.999

0.1-1,000

ethyl butyrate

TIC, 4 standards

0.999

1-100

4-methyl-2pentanone

EIC (100 m/z), 4 standards

>0.999

0.1-100

2,5-dimethylfuran

EIC (96 m/z), 4 standards

0.994

0.1-100

2-nonanone

TIC, 4 standards, blanks subtracted

0.999

5-1,000

Coefficient of determination. chromatogram.

TIC: total ion chromatogram, EIC: extracted ion

133

Table 3. M. reukaufii analyte concentration at 96 h growth vs. uncalibrated peak areas, sorted by mean dissolved concentration. M. reukaufii concentration at 96 h (mean ± SE, n=3) Compound

μM

Peak Area (x105)

ethanol

197,155 (±9,983)

6,800 (±200)

2-methyl-1-butanol and 3-methyl-1-butanol

511 (±9)

6,990 (±80)

isobutanol

148 (±1)

614 (±3)

3-hydroxy-2-butanone (acetoin)

49.0 (±0.8)

53 (±0.9)

2-phenethyl alcohol

33 (±2)

260 (±20)

1-propanol

20 (±3)

30 (±2)

3-ethoxy-1-propanol

2.4 (±0.5)

1.8 (±0.4)

2-butanol

1.52 (±0.03)

10 (±1)

ethyl acetate

1.1 (±0.1)

130 (±10)

4-penten-1-ol

0.44 (±0.03)

8.9 (±0.6)

3-methyl-3-buten-1-ol

0.35 (±0.01)

5.5 (±.2)

1-hexanol

0.06 (±0.01)

6 (±2)

2-methylpropyl acetate

0.0439 (±0.0009)

5.3 (±0.6)

3-methylbutyl acetate

0.0323 (±0.0008)

41 (±2)

2-ethyl-1-hexanol

0.025 (±0.003)

29 (±2)

ethyl butyrate

0.022 (±0.002)

6 (±1)

Analytes in microbial volatile headspaces collected at 96 h spanned seven orders of magnitude (1x10-8-0.1 M). To quantify this wide range of compounds, two calibration curves were sometimes necessary. Despite these efforts, poor linearity was observed for several compounds, particularly those produced at high concentrations (i.e. ethanol, 2- and 3-methyl-1-butanol, 3-hydroxy-2-butanone, Table 2). At 48 and 96 h sampling intervals, these volatiles saturated mass spectrometer response, as indicated by individual extracted ion chromatograms (EIC) traces. Semi-quantitative estimates (with poor percent accuracy; i.e. percent error >30%) of these compounds dissolved in nectar are presented in Table 1. Superficial method modifications, like shorter SPME fiber permeation and exposure times, would prevent saturation. Several compounds detected within microorganism replicate headspace were also present in sterile controls, at lower abundance. These compounds were quantified by subtracting mean sterile control peak area from all sample and calibration peak areas (Table 2, calibration description). Other compounds co-eluted near background peaks and in these cases unique fragmentation ions were used for calibration purposes (EIC, Table 2). 134

Care should be taken when interpreting peak area data of instruments with non-universal response, like mass spectrometry. Structural chemical diversity results in differing ionization efficiencies between compounds, particularly those with widely-variant structures. Therefore, relative peak areas do not always correspond directly to volatile abundance in the headspace. Furthermore, GC-MS peak areas do not correspond to dissolved concentrations (Table 3). For example, peak areas are similar between ethanol and 2- and 3-methyl-1-butanol, but dissolved ethanol concentrations are actually three orders of magnitude higher. Even if universal detection techniques are adopted, collected volatiles may not be representative of gas-phase volatile abundance due to differing sampling recoveries. The adoption of calibration curves prepared using identical conditions correlates the previously described variability directly to calibration standard concentration, but cannot directly describe volatile abundance in the sealed containers. In this case, dissolved concentration is still useful; informing solutions for field- and laboratory-based bioassays.

Discussion Nectar Microorganisms: Opportunities for Agriculture The presence of microbes in floral nectar has been known for decades, yet their distribution and effects in agriculture are not well understood, with the exception of globally-distributed floral pathogens like Erwinia amylovora, the causative agent in fire blight (13, 14). When flowers first open, nectar is often free of culturable microorganisms, but with successive visitation, cell densities increase, with some long-blooming floral nectars containing cell densities greater than 3.5x106 cells µL-1 (15, 16). Though fungi and bacteria are common inhabitants of floral nectar, a given nectar sample typically hosts few species (1-2 species/flower) (5, 15, 17–19). Certain species like the yeast, Metschnikowia reukaufii, are frequently observed and widely distributed (5, 18–20). All evidence suggests agricultural floral nectar regularly contains floral microorganisms (13, 21, 22), but their effects on plant reproduction and pollination are rarely considered. Reports in the literature are mixed: in wild herb populations, the presence of yeasts increased pollen transfer in one study, but reduced seed set in another (3, 6). We envision that with thoughtful application and stewardship of selected mutualist microorganisms in flowering crops, there may be the potential to affect yields through altered pollinator behavior, and thus reduce production costs and agrochemical application. Manipulation of Floral Visitation via Microbial Inoculation Floral volatiles may simultaneously attract and deter various visitors in what has been described as a “filtering” effect (23). Nectar microorgansims may also filter floral visitor species by enhancing or reducing affinity to nectar (24). Certain microbial species increase floral visitation and consumption of nectar, while others deter visitors (4, 5, 25, 26). The need for pollination services in agriculture is 135

outpacing supply (27). The ability to direct pollination services toward poorly attractive crops via inoculation of attractant species could alleviate consequences for supply limitations. The use of deterrent microbes may be useful in directing insect distribution in plants not dependent on visitor-mediated pollination. Floral microbiome manipulation could be directed toward repulsion of deleterious floral visitors, which consume or infect flowers. Alternatively, inoculation could protect beneficial or sensitive native insects from toxic crops. Co-application of deterrent microorganisms with insecticides could reduce pollinator exposure via ingestion and contact with treated flowers, reducing risk of toxicity (28). Volatiles identified to be generally insect-deterrent could be incorporated into pesticide blends, at concentrations informed by quantitative emission data. Environmental Remediation of the Nectar Microhabitat for Improved Pollinator Health Plant-produced compounds may be toxic or deterrent to pollinators or may impact bee learning, memory and behavior (29–32). Nectar secondary metabolites can also reduce disease incidence in bees but in other cases, may exacerbate infection (33–35). Application of beneficial nectar microbes capable of selectively degrading harmful compounds from nectar could influence pollinator health and increase pollination services. Nectar Microbes for Floral Pathogen Protection Priority effects, where early-arriving nectar microbes suppress the growth of later-arrivers, have been demonstrated in nectar communities and may be exploited for floral pathogen protection (36, 37). Rapid nectar resource depletion, particularly for amino acids, is hypothesized to drive competition between species (38). Intentional rapid establishment of selected microorganisms in crops may prevent subsequent colonization by pathogens. Biocontrol agents are already used for floral pathogen control in agriculture, but new, more effective strains are still sought (39, 40). Moreover, understanding the mechanisms by which biocontrol agents suppress pathogens (resource competition, antibiotic production, etc.) may be informed by a quantitative chemical approach. Quantitative Unbiased Chemical Assessment: The Need and Unattainable Goal Any assessment of a metabolome strives for a complete, unbiased, quantitative snapshot of all metabolites relevant to a particular organism, tissue or process with a single extraction procedure and instrumental analysis. This goal is unachievable using current technologies. Iterative metabolite sampling and detection strategies may be prohibitively expensive and will eventually yield diminishing returns in terms of new chemical data. Even routine volatile measurement using GC-MS has considerable instrument acquisition, operation, maintenance and technical support costs, putting it out 136

of reach of many non-chemistry specialists. Additionally, there are costs for purchasing or synthesizing large collections of pure chemicals needed to confirm chemical identity and prepare calibration curves (commonly >$100 for sub-gram quantities). A key tenet in chemical ecology is the dose-response relationship between semiochemicals and induced interactions: the magnitude and direction of an effect (i.e. attraction vs. deterrence) often depends on semiochemical concentration. Low abundance compounds may play crucial roles in biological responses and so exhaustive analysis of volatiles is needed. Volatile quantification is particularly necessary to assess microorganism contribution to floral scent in planta, since many microbial volatiles are also emitted by plants. The nectar metabolome poses unique analytical challenges. Nectar collection can be an arduous process, since many plants produce small (fractions of microliters) and intermittent nectar crops. The relevant biomolecules present in nectar samples are susceptible to abiotic degradation post-collection even when frozen, according to some reports (41, 42). The small scale of nectar also poses a challenge for detection. Volatile analysis performed on collected ex situ floral nectar has relied on pooling nectar to overcome analytical detection limits (43). Nectar chemistry can be highly variable even within nectaries of the same flower and so sample pooling may mask influential fluctuations between flowers and ideally should be avoided, where possible (17, 44). The physicochemical properties of some nectar volatiles also complicates detection. Microbial volatiles are typically polar, low molecular weight, high vapor pressure compounds which are poorly retained on many adsorbent materials. The range of concentration encountered in nectar samples spans many orders of magnitude, likely requiring sample preparation (dilution and concentration) of nectar samples for quantitative detection. Laboratory-based studies overcome many of these challenges by allowing essentially unlimited quantities of “nectar” to be uniformly prepared and analyzed. Specific nectar-solute effects can be rapidly reproduced, revealing important new chemistry. Laboratory experiments also are not restricted by limited and variable nectar plant production. Here, we describe an approach for assessing microbial volatiles in laboratory cultures. Additional details, including honey bee acceptance and electrophysiological tests related to this data is available (11). Reports of microbial-produced volatiles in nectar are very rare; however, several studies have inferred the contribution of microbe-produced volatiles to floral headspace. An early tentative detection was reported in a study contrasting volatile production in floral tissue and nectar of angiosperms (45). Headspace volatiles from excised flowers or nectar (5-10 µL) were analyzed by SPME-GC-MS. Nectar samples of two species contained unique compounds, including fermentation volatiles, which were tentatively ascribed to microbial contamination of nectar. The authors note that these plants are likely to host microorganisms, due to their extended flowering phase, large, stable nectar crop and frequent, diverse animal visitors. In other studies, the influence of the entire above-ground microbiome of a plant (i.e. phyllosphere and anthosphere) on floral aroma was investigated. Floral terpene abundance was reduced after whole-plant antibiotic fumigation (46). In 137

a complimentary approach, different volatile profiles were observed between Brassica rapa plants grown under sterile conditions and those inoculated with field-isolated bacteria (47). In both reports, the difficulty of parsing plant- vs. microbe-derived variations in floral volatile blends is discussed. The first direct measurement of microbe-produced volatiles in floral nectar was described in the weakly scented, insect-pollinated flower, Silene caroliana (43). Nectar (2 µL) was collected in sterilized autosampler vials from open flowers and transported back to the laboratory, where volatiles were allowed to accumulate for 24 h prior to analysis and then screened for yeasts. From the resulting cultures, four frequently observed nectar yeasts, including two M. reukaufii strains, were selected for further study in synthetic nectar under analougous conditions to real nectar samples. Due to the study of common nectar microbes and the use of the same SPME fiber type and analytical technique, data between these analyses can be compared to some extent. During method development, a dose-response approach to unknown volatile detection is advised, wherein increasingly large samples are pooled and analyzed, and the sample size at which no (or very few) new compounds are discovered is established. Golonka and colleagues adopted this approach to synthetic nectar inoculations: they compared volatile production in 2 and 200 µL synthetic nectar and found similar volatile emissions in both cases and so selected the more relevant 2 µL nectar volume (43). Many volatiles, including fermentation volatiles like ethanol, isobutanol, and 2- and 3-methyl-1-butanol, were detected in both floral and bulk and small-volume synthetic nectar analyses. In total, large-volume inoculated synthetic nectar revealed a broader suite of volatiles, probably due to higher cell counts. These volatiles had the largest relative peak areas and saturated detector response in bulk nectar (Table 1). Differential volatile emission could also be explained by genetic variation between the naturally-occurring M. reukaufii strains selected for study or could arise as a consequence of differing synthetic nectar chemistry. Different volatile blends were identified in collected S. caroliana nectar and inoculated synthetic nectar prepared by Golonka and colleagues, an observation which demonstrates the need to validate ex situ floral nectar and synthetic nectar data with in planta studies. Particularly in the case of synthetic nectar-based investigations, the interactions of nectar microorganisms with their plant hosts is entirely excluded. Plant-specific factors like nectary structure and genetics largely define nectar excretion and composition. It is well understood that microbial metabolites will vary depending on the nutritional quality of their growth substrate. However, a suite of environmental factors also have demonstrated effects, including: light quality, soil nutrients and moisture, herbivory, atmospheric carbon dioxide levels, humidity and temperature (48–53). Other epiphytic microorganisms can influence floral scent emissions (47). Even nectar removal by floral visitors may change nectar composition and excretion over time (54). Plants may also respond to nectar contamination by inducing defense strategies, or perhaps, supporting microbial mutualist colonization (55). Synthetic and ex situ studies of nectar microbe volatiles must be validated with in situ experiments, to parse these interconnected and cascading plant, microbe and environmental factors. 138

Conclusions Nectar microbes are an un-tapped resource in agriculture, and may lend themselves to a variety of applications. Here, we provide the first quantitative report of nectar microbe volatile emission in synthetic nectar. Quantitative chemical data should be validated with bioassays. Pollinator response between microorganism-inoculated and sterile, volatile-laced nectars could indicate whether key semiochemicals were detected and accurately quantified. Differing acceptance of these solutions may also point to non-volatile mediated attraction or deterrence. Bulk synthetic nectar inoculation proved to identify new, previously unknown volatiles that may mediate pollinator response. Unsurprisingly, some compounds were poorly quantified by this rather crude approach. In this case, saturation of some analytes was accepted in order to screen for low abundance volatiles. Parallel comparisons of synthetic and real nectars, like the experiment undertaken by Golonka and colleagues, may offer a compelling template for further investigations to evaluate the suitability of synthetic nectar-based screening of microbial semiochemicals (43).

References 1.

2.

3.

4.

5.

6. 7.

8.

Bailes, E. J.; Ollerton, J.; Pattrick, J. G.; Glover, B. J. How can an understanding of plant-pollinator interactions contribute to global food security? Curr. Opin. Plant Biol. 2015, 26, 72–79. Potts, S. G.; Biesmeijer, J. C.; Kremen, C.; Neumann, P.; Schweiger, O.; Kunin, W. E. Global pollinator declines: trends, impacts and drivers. Trends Ecol. Evolut. 2010, 25 (6), 345–353. Herrera, C. M.; Pozo, M. I.; Medrano, M. Yeasts in nectar of an early-blooming herb: sought by bumble bees, detrimental to plant fecundity. Ecology 2013, 94 (2), 273–279. Good, A. P.; Gauthier, M.-P. L.; Vannette, R. L.; Fukami, T. Honey bees avoid nectar colonized by three bacterial species, but not by a yeast species, isolated from the bee gut. PLoS One 2014, 9, e86494. Schaeffer, R. N.; Phillips, C. R.; Duryea, M. C.; Andicoechea, J.; Irwin, R. E. Nectar yeasts in the tall larkspur Delphinium barbeyi (Ranunculaceae) and effects on components of pollinator foraging behavior. PloS One 2014, 9, e108214. Schaeffer, R. N.; Irwin, R. E. Yeasts in nectar enhance male fitness in a montane perennial herb. Ecology 2014, 95 (7), 1792–1798. Beck, J. J.; Vannette, R. L. Harnessing insect-microbe chemical communications to control insect pests of agricultural systems. J. Agric. Food Chem. 2017, 65 (1), 23–28. Baker, H. G.; Baker, I. Chemical constituents of nectar in relation to pollination mechanisms and phylogeny. In Biochemical aspects of evolutionary biology; Nitecki, M. H., Ed.; University of Chicago Press: Chicago, IL, 1982; pp 131−171. 139

9.

10.

11.

12. 13.

14.

15. 16.

17.

18.

19.

20. 21. 22.

23. 24. 25.

Gardener, M. C.; Gillman, M. R. Analyzing variability in nectar amino acids: Composition is less variable than concentration. J. Chem. Ecol. 2001, 27, 2545–2558. Beck, J. J.; Willett, D. S.; Gee, W. S.; Mahoney, N. E.; Higbee, B. S. In situ volatile collection, analysis, and comparison of three Centaurea species and their relationship to biocontrol with herbivorous insects. J. Agric. Food Chem. 2016, 64, 9286–9292. Rering, C. C.; Beck, J. J.; Hall, G. W.; McCartney, M. M.; Vannette, R. L. Nectar-inhabiting microorganisms influence nectar volatile composition and attractiveness to a generalist pollinator. New Phytol. 2017. Schwarzenbach, R. P.; Gschwend, P. M.; Imboden, D. M. Environmental Organic Chemistry, 2nd ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2003. Gilliam, M.; Moffett, J. O.; Kauffeld, N. M. Examination of floral nectar of citrus, cotton, and Arizona desert plants for microbes. Apidologie 1983, 14, 299–302. Bubán, T.; Orosz-Kovács, Z.; Farkas, Á. The nectary as the primary site of infection by Erwinia amylovora (Burr.) Winslow et al.: A mini review. Plant Syst. Evol. 2003, 238, 183–194. Herrera, C. M.; de Vega, C.; Canto, A.; Pozo, M. I. Yeasts in floral nectar: A quantitative survey. Ann. Bot. 2009, 103, 1415–1423. de Vega, C.; Herrera, C. M.; Johnson, S. D. Yeasts in floral nectar of some South African plants: Quantification and associations with pollinator type and sugar concentration. S. Afr. J. Bot. 2009, 75, 798–806. Herrera, C. M.; Garcia, I. M.; Perez, R. Invisible floral larcenies: Microbial communities degrade floral nectar of bumble bee-pollinated plants. Ecology 2008, 89, 2369–2376. Schaeffer, R. N.; Vannette, R. L.; Irwin, R. E. Nectar yeasts in Delphinium nuttallianum (Ranunculaceae) and their effects on nectar quality. Fungal Ecol. 2015, 18, 100–106. Álvarez-Pérez, S.; Herrera, C. M. Composition, richness and nonrandom assembly of culturable bacterial-microfungal communities in floral nectar of Mediterranean plants. FEMS Microbiol. Ecol. 2013, 83, 685–699. Pozo, M. I.; Herrera, C. M.; Bazaga, P. Species richness of yeast communities in floral nectar of southern spanish plants. Microb. Ecol. 2011, 61, 82–91. Fridman, S.; Izhaki, I.; Gerchman, Y.; Halpern, M. Bacterial communities in floral nectar. Environ. Microbiol. Rep. 2012, 4, 97–104. Schaeffer, R. N.; Vannette, R. L.; Brittain, C.; Williams, N. M.; Fukami, T. Non-target effects of fungicides on nectar-inhabiting fungi of almond flowers. Environ. Microbiol. Rep. 2017, 9, 79–84. Junker, R. R.; Blüthgen, N. Floral scents repel facultative flower visitors, but attract obligate ones. Ann. Bot. 2010, 105, 777–782. Junker, R. R.; Tholl, D. Volatile organic compound mediated interactions at the plant-microbe interface. J. Chem. Ecol. 2013, 39, 810–825. Vannette, R. L.; Gauthier, M.-P. L.; Fukami, T. Nectar bacteria, but not yeast, weaken a plant-pollinator mutualism. Proc. R. Soc. B 2013, 280, 20122601.

140

26. Schaeffer, R. N.; Mei, Y. Z.; Andicoechea, J.; Manson, J. S.; Irwin, R. E. Consequences of a nectar yeast for pollinator preference and performance. Funct. Ecol. 2017, 31, 613–621. 27. Aizen, M. A.; Harder, L. D. The global stock of domesticated honey bees is growing slower than agricultural demand for pollination. Curr. Biol. 2009, 19, 915–918. 28. Sanchez-Bayo, F.; Goka, K. Pesticide residues and bees - A risk assessment. PLoS One 2014, 9, e94482. 29. Stevenson, P. C.; Nicolson, S. W.; Wright, G. A. Plant secondary metabolites in nectar: Impacts on pollinators and ecological functions. Funct. Ecol. 2017, 31, 65–75. 30. Vannette, R. L.; Fukami, T. Nectar microbes can reduce secondary metabolites in nectar and alter effects on nectar consumption by pollinators. Ecology 2016, 97, 1410–1419. 31. Wright, G. A.; Baker, D. D.; Palmer, M. J.; Stabler, D.; Mustard, J. A.; Power, E. F.; Borland, A. M.; Stevenson, P. C. Caffeine in floral nectar enhances a pollinator’s memory of reward. Science 2013, 339, 1202–1204. 32. Hurst, V.; Stevenson, P. C.; Wright, G. A. Toxins induce ‘malaise’ behaviour in the honeybee (Apis mellifera). J. Comp. Physiol. A 2014, 200, 881–890. 33. Manson, J. S.; Otterstatter, M. C.; Thomson, J. D. Consumption of a nectar alkaloid reduces pathogen load in bumble bees. Oecologia 2010, 162, 81–89. 34. Palmer-Young, E. C.; Sadd, B.; Irwin, R. E.; Adler, L. S. Synergistic effects of floral phytochemicals against a bumble bee parasite. Ecol. Evol. 2017, 7, 1836–1849. 35. Palmer-Young, E. C.; Alison, H.; Alexander, J. K.; Donnelly, D.; Jonathan, A.; Connon, S. J.; Weston, I.; Kimberly, S.; Irwin, R. E.; Adler, L. S. Context-dependent medicinal effects of anabasine and infection-dependent toxicity in bumble bees. PLoS One 2017, 12, e0183729. 36. Tucker, C. M.; Fukami, T. Environmental variability counteracts priority effects to facilitate species coexistence: Evidence from nectar microbes. Proc. R. Soc. B 2014, 281, 20132637. 37. Peay, K. G.; Belisle, M.; Fukami, T. Phylogenetic relatedness predicts priority effects in nectar yeast communities. Proc. R. Soc. B 2012, 279, 749–758. 38. Dhami, M. K.; Hartwig, T.; Fukami, T. Genetic basis of priority effects: Insights from nectar yeast. Proc. R. Soc. B 2016, 283 (1840). 39. Stockwell, V. O.; Johnson, K. B.; Sugar, D.; Loper, J. E. Mechanistically compatible mixtures of bacterial antagonists improve biological control of fire blight of pear. Phytopathology 2011, 101, 113–123. 40. Sharifazizi, M.; Harighi, B.; Sadeghi, A. Evaluation of biological control of Erwinia amylovora, causal agent of fire blight disease of pear by antagonistic bacteria. Biol. Control 2017, 104, 28–34. 41. Morrant, D. S.; Schumann, R.; Petit, S. Field methods for sampling and storing nectar from flowers with low nectar volumes. Ann. Bot. 2009, 103, 533–542. 42. Amato, B.; Petit, S. A review of the methods for storing floral nectars in the field. Plant Biol. 2017, 19, 497–503. 141

43. Golonka, A. M.; Johnson, B. O.; Freeman, J.; Hinson, D. W. Impact of nectarivorous yeasts on Silene caroliniana’s scent. Eastern Biologist 2014, 3, 1–26. 44. Herrera, C. M.; Perez, R.; Alonso, C. Extreme intraplant variation in nectar sugar composition in an insect-pollinated perennial herb. Am. J. Bot. 2006, 93, 575–581. 45. Raguso, R. A. Why are some floral nectars scented? Ecology 2004, 85, 1486–1494. 46. Penuelas, J.; Farre-Armengol, G.; Llusia, J.; Gargallo-Garriga, A.; Rico, L.; Sardans, J.; Terradas, J.; Filella, I. Removal of floral microbiota reduces floral terpene emissions. Sci. Rep. 2014, 4, 6727. 47. Helletsgruber, C.; Dotterl, S.; Ruprecht, U.; Junker, R. R. Epiphytic bacteria alter floral scent emissions. J. Chem. Ecol. 2017, 43, 1073–1077. 48. Gardener, M. C.; Gillman, M. P. The effects of soil fertilizer on amino acids in the floral nectar of corncockle, Agrostemma githago (Caryophyllaceae). Oikos 2001, 92, 101–106. 49. Samocha, Y.; Sternberg, M. Herbivory by sucking mirid bugs can reduce nectar production in Asphodelus aestivus Brot. Arthropod Plant Interact. 2010, 4, 153–158. 50. Rusterholz, H. P.; Erhardt, A. Effects of elevated CO2 on flowering phenology and nectar production of nectar plants important for butterflies of calcareous grasslands. Oecologia 1998, 113, 341–349. 51. Erhardt, A.; Rusterholz, H. P.; Stocklin, J. Elevated carbon dioxide increases nectar production in Epilobium angustifolium L. Oecologia 2005, 146, 311–317. 52. Bertsch, A. Nectar production of Epilobium angustifolium L. at different air humidities; nectar sugar in individual flowers and the optimal foraging theory. Oecologia 1983, 59, 40–48. 53. Muniz, J. M.; Pereira, A. L. C.; Valim, J. O. S.; Campos, W. G. Patterns and mechanisms of temporal resource partitioning among bee species visiting basil (Ocimum basilicum) flowers. Arthropod Plant Interact. 2013, 7, 491–502. 54. Ordano, M.; Ornelas, J. F. Generous-like flowers: Nectar production in two epiphytic bromeliads and a meta-analysis of removal effects. Oecologia 2004, 140, 495–505. 55. Harper, A. D.; Stalnaker, S. H.; Wells, L.; Darvill, A.; Thornburg, R.; York, W. S. Interaction of Nectarin 4 with a fungal protein triggers a microbial surveillance and defense mechanism in nectar. Phytochemistry 2010, 71, 1963–1969.

142

Chapter 11

Investigating Host Plant-Based Semiochemicals for Attracting the Leaffooted Bug (Hemiptera: Coreidae), an Insect Pest of California Agriculture John J. Beck,*,1 Wai S. Gee,2 Luisa W. Cheng,2 Bradley S. Higbee,3 Houston Wilson,4 and Kent M. Daane5 1Chemistry

Research Unit, Center for Medical, Agricultural and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, 1700 SW 23rd Drive, Gainesville, Florida 32608, United States 2Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, California 94710, United States 3Trécé Inc., P.O. Box 129, Adair, Oklahoma 74330, United States 4Department of Entomology, University of California, Riverside, California 92521, United States 5Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720, United States *E-mail: [email protected]

Leptoglossus species, commonly referred to as leaffooted bugs (LFB), inflict serious damage to a wide variety of hosts. Over the last few years, some California agricultural systems have experienced a marked increase in LFB populations and LFB-related issues, particularly in pomegranate, pistachio, and almond orchards. Leptoglossus clypealis, L. occidentalis, and L. zonatus are considered native to the southwestern US. Literature regarding Leptoglossus also indicates that LFB are capable of vectoring pathogenic microbes. While pheromones are known for LFB, there are currently no economical or reliable tools available to growers for monitoring LFB. Ongoing research in our laboratories has evaluated several California-based host plants of the polyphagous LFB. Using data from the collected headspace volatiles of several LFB hosts, as well as © 2018 American Chemical Society

electrophysiological and field trapping bioassays, our labs seek to formulate and develop a blend of host plant-based volatiles for use as an effective control tool for monitoring of LFB in pomegranate and tree nut orchards. This report provides an overview of the status of semiochemicals used for control of LFB species in California.

Introduction In California nut and pomegranate orchards, the leaffooted bug complex is comprised of three economically important species – Leptoglossus zonatus (Dallas), L. clypealis Heidemann and L. occidentalis Heidemann (Hemiptera: Coreidae). While L. occidentalis is primarily a forest pest that feeds on conifers (1), early descriptions of L. zonatus and L. clypealis invariably note their polyphagy and status as a potential pest of multiple orchard crops, including pomegranates, almonds, stone fruit and citrus (2–4). The pest status of L. clypealis was significantly elevated in the 1980s when it was discovered that its feeding in almonds and pistachios was a primary cause of epicarp lesion, premature nut abortion and kernel necrosis on pistachios (5, 6). Later work also revealed that L. clypealis aided the dispersion of Botryosphaeria dothidea, the causal agent of panicle and shoot blight (7). Subsequent evaluations identified critical time-periods during which pistachio shells are soft enough to be vulnerable to Leptoglossus feeding (8, 9). While early-season feeding on pistachio nuts prior to fruit set is negated when these damaged nuts are dropped from the cluster (with no negative impact on overall fruit load) (8, 10), feeding at mid-season (mid-May-July) can cause significant damage to pistachio nuts (8). Later in the season, after shell hardening, the kernel is mostly protected from Leptoglossus feeding, although it has been shown that this large bug can still penetrate nuts at the peduncle, the veritable “Achilles Heel” of this fruit (11, 12). The need for soft-tissue fruits is thought to be what drives the seasonal movement of Leptoglossus between multiple host crops, which also includes almond and pomegranate, where similar damage to nut meats and fruit seeds, respectively, can cause economic reductions in crop yield and quality. Tests in which Leptoglossus zonatus were caged on tree branches (Higbee, unpublished observations) have demonstrated that nymphs do not fully develop on almonds. This observation suggests nutritional deficiency and may also corroborate the idea of soft-tissue fruit preference driving Leptoglossus movement. The seasonal ecology and life-history of L. clypealis and L. zonatus are similar and have been described by Daane et al. (13). In the late fall, both L. clypealis and L. zonatus seek out shelter and overwinter in large aggregations. As the adults become reproductively active in the spring, these aggregations begin to break up as individuals and leave to seek out both food and oviposition sites. Adults will lay their brown, barrel shaped eggs in narrow rows primarily on leaves and twigs, but eggs have also been observed on fruits. Nymphs emerge from the eggs and then pass through five instars before molting into adults. A single adult can lay up to 144

200 eggs and over the course of a year the Leptoglossus populations in California’s San Joaquin Valley will complete 3-4 full generations if good food sources are available. Generally, Leptoglossus first migrate into almond and pistachio orchards in the spring, then move to other feeding hosts in late summer, and often arrive in pomegranates in the fall. As pomegranate trees senesce in late fall and winter, the Leptoglossus will then seek out shelter nearby and form their overwintering aggregations. This seasonal cycle of movement between orchards is thought to be driven by phenological differences across these three crops, which provide adequate soft-tissue fruits for Leptoglossus to feed on at different times of the year. It is important to note that this seasonal movement is not strictly limited to the three aforementioned crops and both L. clypealis and L. zonatus can reside on alternate host plants outside of the orchard and/or migrate in and out of orchards at any time during the season (13). Leptoglossus in California are attacked by egg parasitoids, primarily Gryon pennsylvanicum (Hymenoptera: Scelionidae), but Anastatus sp. (Hymenoptera: Eupelmidae), Brasema sp. (Hymenoptera: Eupelmidae), and Trissolcus sp. (Hymenoptera: Scelionidae) can also attack LFB. There is a range of generalist predators that includes green lacewings (Chrysopa and Chrysoperla spp.), assassin bugs (Zelus spp.), damsel bugs (Nabis spp.), ants (Dorimyrmex and Solenopsis spp.), earwigs (Forficula auricularis) and spiders (13). Unfortunately, none of these natural enemies has ever exhibited any significant biological control of this pest, either due to a lack of prey specificity (i.e. generalist predators) or temporal lags between the pest and parasitoid (i.e. Leptoglossus adults arrive in orchard and damage crop before their eggs are ever present for G. pennsylvanicum to attack). Currently, orchard management strategies for Leptoglossus are quite limited. Recommended monitoring practices include visual inspection of fruit clusters for signs of damage, sampling the lower canopy for adults using a beating tray, and/or disturbing the upper canopy with a long pole to look for adults that fly out. No economic thresholds have been established for this pest and therefore many growers base their treatment decisions on previous experience, relating perceived pest densities to crop damage at a given site or within a region (13). Insecticidal sprays, primarily pyrethroids, are the most commonly used tool to control Leptoglossus. These products are known to have negative impacts on beneficial organisms and can cause outbreaks of secondary pests, such as mites. Additionally, there are few known cultural controls beyond surveying for, and destroying, the overwintering aggregations. Pheromones are known to play a large role in the reproduction and aggregation of both L. clypealis and L. zonatus. The summer-form males are thought to produce a sex pheromone to attract female mates and the overwintering aggregations are possibly initiated and maintained through some type of aggregation cue. While some work has been conducted to characterize and synthesize these compounds (14, 15), no field applications have been developed. Far less well understood is the role of kairomones and herbivore induced plant volatiles, which could alone or in combination with pheromones be used as part of either an improved monitoring tool or attract-and-kill control strategy. 145

Leptoglossus are an increasingly important pest of tree nuts in California, particularly as more growers transition away from the use of pyrethroids for control of navel orangeworm (Amyelois transitella), which is the key insect pest of pistachio and almond. Pyrethroid applications targeting Leptoglossus usually have the secondary effect of also helping control A. transitella (and vice versa); however, mounting pressure on growers to reduce the use of this chemical class, especially for navel orangeworm, will likely result in increased frequency and/or intensity of Leptoglossus outbreaks. As such, improved monitoring tools, clear treatment thresholds and additional non-chemical control options, possibly based in the use of pheromones and/or kairomones, will all be necessary to maintain adequate management of this pest in the near future.

California Host Plants Surveyed Our laboratories’ involvement in this project began in 2014 as an observation conveyed to us (private communication to J. Beck) from a California pomegranate grower. The grower noticed an unusually large increase of LFB (Figure 1) on split pomegranates. Talking with other pomegranate growers, it became apparent that this was a trend not just for pomegranate, but that LFB were also sited more often and in greater numbers in pistachio and almond orchards. With this information in hand, we set out to address the immediate concern of the pomegranate, pistachio and almond growers by surveying the ex situ volatile emissions of these hosts and investigating the possibility of certain host plant volatiles acting as attractant cues for the LFB. As of this writing, we have collected and analyzed the volatile profiles of the following hosts: pomegranate (mechanically damaged, split, moldy); almond oil stock; pistachio oil stock (Figure 2); orange; and, tomato. From these seven various hosts, 161 peaks were detected, 147 tentatively identified (Table 1) to the general chemical class, and 14 unknown peaks. Work is ongoing to authenticate as many peaks as possible using commercial or other available standards. However, we will prioritize blend formulation based on the use of electrophysiological results from adult LFB antennae and evaluation of the common emissions among the hosts (Table 2). As seen in Table 1, with volatiles delineated by their primary functional groups, a wide variety of chemical classes were emitted by the surveyed host plant tissues, which included acids, esters, aldehydes, ketones, alcohols, aromatics (benzenoid and non-benzenoid aromatic compounds), monoterpenes, and sesquiterpenes. While the emission profiles from these tissues are likely different than their respective in situ tissues (16–18) that the insect may encounter in the orchards, it was our goal to collect as many volatiles as possible, including potential microbial influences (19, 20) for electrophysiological screening (21, 22) and/or inclusion into formulations. Since Leptoglossus are polyphagous (generalists), we are anticipating that a blend of host plant volatiles will likely be required for attractancy versus an individual compound (23–27).

146

Figure 1. An adult leaffooted bug.

Figure 2. Nymph leaffooted bugs in wing traps baited with almond oil stock, which were used as a general attractant in pomegranate orchards.

147

Table 1. Classes and relative amounts of volatile compounds detected in LFB host plant matrices surveyed Compound #

Class

Occurrencea and Relative Amountb

1

acid

A1

2

acid

A1, P1

3

acid

A1, P1

4

acid

A3, P3, Pd2, Ps1, Pm4, T1

5

acid

A1, Pd1, Ps1

6

acid

A3

7

acid

A1

8

acid

Pm1

9

alkanal

A3, P1

10

alkanal

A4, P3

11

alkanal

A3, P1

12

alkanal

A3

13

alkanal

A3

14

alkane

A1, O3

15

alkanol

Pm1

16

alkanol

Pd3, Ps3

17

alkanol

A3, P1, O3

18

alkanol

A1, O3

19

alkanone

A1, P1, Pm3, O2

20

alkanone

T1

21

alkanone

A1

22

alkanone

A1

23

alkanone

Pd3, Ps1, O3, T1

24

alkanone

T1

25

alkanone

Ps3

26

alkanone

A1, Pd1, Ps1, Pm1

27

alkanone

A1, Pd1, Pm3, T1

28

alkanone

A1, P1, Pd1, Ps3, Pm1, T1

29

alkenal

T2

30

alkenal

A1

31

alkene

O4 Continued on next page.

148

Table 1. (Continued). Classes and relative amounts of volatile compounds detected in LFB host plant matrices surveyed Compound #

Class

Occurrencea and Relative Amountb

32

alkene

T1

33

alkenol

A1, P1, Pd1, Pm1, O2, T1

34

alkenol

A1, O1

35

alkenol

A1, P1

36

alkenol

A1, Pm1

37

alkenone

A1, P1

38

alkenone

Ps1, T1

39

alkenone

O3, T1

40

aromatic

A1, P1, Pd2, Ps1, Pm1, T1

41

aromatic

P4

42

aromatic

A1

43

aromatic

A1, P1

44

aromatic

P1

45

aromatic

A1, P1

46

aromatic

P1

47

aromatic

Ps1

48

aromatic

A1, P2, Pd1, Ps2, Pm1, T1

49

aromatic

A1

50

aromatic

A1, P1, Ps1, Pm1

51

aromatic

Pm1

52

aromatic

Ps3

53

aromatic

Ps1

54

aromatic

Ps1

55

aromatic

P1

56

aromatic

Pm1

57

aromatic

A2, P1, Ps1, Pm4

58

ester

T1

59

ester

Ps1, Pm3, T1

60

ester

O1

61

ester

Pm3

62

ester

O3 Continued on next page.

149

Table 1. (Continued). Classes and relative amounts of volatile compounds detected in LFB host plant matrices surveyed Compound #

Class

Occurrencea and Relative Amountb

63

ester

O2

64

ester

O3

65

ester

O4

66

ester

O3

67

ester

O4

68

ester

Pd1, Pm1

69

ester

Pm1

70

ester

Pd1, Pm1, O3

71

ester

Pd1, Pm1, O4

72

ester

O4

73

ester

Pm2

74

ester

O3

75

ester

O4

76

ester

O4

77

ester

O3

78

ester

O3

79

ester

A1, P1, Pm1, O3

80

ester

O4

81

ester

O3

82

ester

Pm1

83

ester

O2

84

ester

Pm3

85

ester

O2

86

GLV

Pm1

87

lactone

A1, P2

88

lactone

A1

89

mono

P3, O4

90

mono

O3

91

mono

A2, P4, Pd1, Ps2, Pm3, O4, T1

92

mono

P3, Pm1, O3

93

mono

O3 Continued on next page.

150

Table 1. (Continued). Classes and relative amounts of volatile compounds detected in LFB host plant matrices surveyed Compound #

Class

Occurrencea and Relative Amountb

94

mono

A3, P4, Pd1, Ps1, Pm3, O5, T3

95

mono

A1, O3

96

mono

P1, Pd1, Pm1

97

mono

A1, P3, Pd1, Ps1, Pm3, O3

98

mono

P3, Pd1, Ps3, Pm4, O4

99

mono

P3, Pm1, O3

100

mono

A1, P4

101

mono

A1, P1, O4

102

mono

P3

103

mono

O3

104

mono

P1, Ps1, Pm3

105

mono

P1

106

mono

Pd1, Ps2, Pm3, O4

107

mono

P3, Pd1, Ps3, Pm4, O4

108

mono

P1, Ps1, Pm3, O3

109

other

A1

110

other

P2, T3

111

other

P1

112

sca

Pd2, Ps1, Pm2

113

sca

P1, Pm3

114

sca

A1, P2, Ps1, Pm4, O3

115

sca

P1

116

sca

A1, P2, Pm3, T1

117

sca

A1, Ps1, Pm1

118

sca

Pm3, T2

119

sesq

Pd4, Pm1, O4

120

sesq

Pm2

121

sesq

Pm2

122

sesq

P1

123

sesq

Pd1

124

sesq

Pd1, Ps1, Pm3 Continued on next page.

151

Table 1. (Continued). Classes and relative amounts of volatile compounds detected in LFB host plant matrices surveyed Compound #

Class

Occurrencea and Relative Amountb

125

sesq

Pm1

126

sesq

P1, O4

127

sesq

O3

128

sesq

O3

129

sesq

Pm1

130

sesq

Pm1

131

sesq

O3

132

sesq

Pm1

133

sesq

O3

134

sesq

Pm1

135

sesq

O4

136

sesq

O3

137

sesq

O3, T1

138

sesq

O3

139

sesq

O3

140

sesq

O4

141

sesq

O4, T1

142

sesq

Pd1, O3

143

sesq

Pm1

144

sesq

Pm2, O3

145

sesq

P1, Ps1, Pm3

146

sesq

O4

147

sesq

Pm1

a Occurrence: A = almond; P = pistachio; Pd = pomegranate, damaged; Ps = pomegranate, split; Pm = pomegranate, moldy; O = orange; and, T = roma tomato. b Relative amounts as detected by GCMS and peak surface area reported (not corrected): 1 = 0 – 500k; 2 = 500k – 1M; 3 = 3M – 10M; 4 = 10M – 200M; and, 5 = > 1,000M.

152

Table 2. Volatile compounds detected in at least four of the LFB host plant matrices surveyed. Compound numbers bolded were detected in the surveyed split pomegranate, pistachio and almond tissues

a

Compound

Class

Occurrence

EAG Response

4

acida,b

6

w

19

alkanonea

4

w

23

alkanonea

4

w

26

alkanonea

4

w

27

alkanonea

4

w

28

alkanonea,b

6

s

33

alkenola

6

s

40

aromatica,b

6

w

48

aromatica,b

6

s

50

aromatica,b

4

m

57

aromatica,b

4

w

71

estera

4

s

79

estera

4

m

91

monoa,b

7

m

94

monoa,b

7

m

97

monoa,b

6

w

98

monoa

5

w

106

monoa

4

m

107

monoa

5

m

108

monoa

4

w

114

scaa,b

5

w

116

scaa

4

w

b

Authenticated. Compounds detected in pomegranate, pistachio and almond; EAG responses: w = weak; m = medium; s = strong, relative responses of antennae to 50 μg of pure compound.

153

With such a broad range of hosts available to the leaffooted bug in California (28), we set out to survey the ex situ headspace of readily available commodities. Evaluation of all California hosts will be an ongoing survey, but Table 1 does offer some insight into host tissues surveyed to date. To initially prioritize the hosts and volatiles to concentrate on, we turned to an elegant study performed by Joyce and co-workers (28). In their report, they documented that L. clypealis and L. zonatus were present in the Central Valley of California, with L. zonatus found in pomegranate, pistachio and almond orchards, and L. clypealis collected from just pistachio and almond trees. Accordingly, Table 2 highlights nine compounds (footnote 3; 1 acid, 1 ketone, 4 aromatics, 3 monoterpenes, and 1 short chain alcohol) that were specifically detected in split pomegranate, pistachio and almond tissue. Even if only described at the class level, many of these host plant volatilebased compounds are similar to insect-based emissions of leaffooted bugs. For instance, aromatic, acid, and alcohol classes have been identified from the tissues of several Leptoglossus species (29–31), as well as alarm pheromones comprised of varying oxidation states of green leaf volatiles (alcohol, aldehyde, and acid) (14). Despite the wide host range of leaffooted bugs, limited literature exists for host plant volatiles as semiochemicals of Leptoglossus. For example, the crops wheat and oats, among others, were studied for use in peaches as trap crops of leaffooted bugs (32), yet no specific host plant volatiles were studied as attractants. In their study of L. occidentalis, Blatt and Borden (33) hypothesized that host plant volatiles mediate preference for aggregation pheromones. Given the scarcity of literature on host plant volatiles used as attractants for Leptoglossus species, it is conceivable that for such a broadly polyphagous insect, a synthetic blend of volatiles may not be an easy target as an attractant. This scarcity may be a result of the heretofore classification of the leaffooted bug being considered a minor insect pest and typically controlled by insecticides (32, 34, 35), and thus its control not a high priority for agriculture. However, this is not to say that host plant volatiles can not be a beneficial tool for the control or monitoring of LFB. The concept of host plant volatiles as effective semiochemicals of polyphagous insects is not new (25–27), and other examples of host plant volatile blends as attractants of polyphagous insects do exist in the literature. For example, host plant volatiles from mango were found to attract a polyphagous insect pest, the oriental fruit fly (Bactrocera dorsalis), which is known to have a very broad range of fruit hosts (36). Additionally, synthetic blends were developed from volatiles emitted by the chaste tree that attracted male and female planthoppers (Hyalesthes obsoletus) (22). The continued evaluation of volatiles from the varying hosts of the leaffooted bug and subsequent comparison of profiles is warranted and is currently ongoing in our laboratories. The highest priority will be the continuation of the evaluation of host plant emissions, and the subsequent electrophysiological and behavioral bioassays. Whether a single blend of volatiles will be efficacious across the various hosts, or if different blends will be necessary for specific host orchards is yet to be determined. This issue was also an obstacle encountered with the generalist insect pest, navel orangeworm in almond and pistachio orchards (37). 154

Electrophysiological Studies with the Leaffooted Bug The potential semiochemical importance of the majority of compounds detected and identified in Table 1 has not been ascertained via behavioral studies. However, initial electroantennographic (EAG) studies in our laboratories (Figure 3) have provided some preliminary results (Table 2) for comparison to known classes of electrophysiologically or behaviorally bioactive compounds, and inclusion in field or soon-to-be-developed lab-based bioassays. Interestingly, reports of electroantennographic studies of Leptoglossus species are few. Our EAG protocols were based on similar studies performed by Blatt and co-workers that evaluated the antennal responses of L. occidentalis to alarm pheromones (38). The primary difference in methods was our laboratory’s use of the fork method (21, 39) for antennal mounting (see experimental design below), where Blatt and co-workers used an electrode immersed in salt solutions in contact with the antennae nerves at the proximal end, and the distal end of the antennae pierced with an electrode.

Figure 3. Antennae of leaffooted bug (left) being prepared for electrophysiological experiment, and the resultant display (EAD - top right) showing elicited antennal responses to individual host plant volatiles of the corresponding peaks shown in the GC-FID chromatogram (bottom right). Using the initial assumption that similar classes of compounds may elicit the same antennal responses (i.e., the aldehyde functional group and its elicitation of responses from navel orangeworm antennae) (39, 40), it could be assumed that aldehydes or acetates from host plant tissues would elicit an antennal response but could likely provoke an inhibitory or dispersal behavioral response from the LFB. It is notable that Table 1 contains few instances of alkanal or alkenal compounds, and that these compounds were primarily detected from the almond tissues. The second most dominant class of compounds from the host tissues, second only to sesquiterpenes, were esters (total of 28 compounds, across all seven hosts surveyed). These esters were mainly detected from oranges and moldy pomegranates, and had a wide range of alkanes at both sides of the ester functional group. Interestingly, only two compounds with an ester moiety (type of ester cannot be disclosed at this time) were detected in four of the seven hosts, yet one of the esters tested did provide a strong antennal response in the EAG studies. This electrophysiological response warrants exploration via behavioral studies. However, given the wide range of functional groups in the alarm pheromones 155

(i.e., hexanal, hexanol, hexyl acetate) of L. occidentalis and L. zonatus (14, 38), care should be given to the interpretation of the EAG results, and any subsequent inclusion of these types of compounds into blends. Converse to the cautionary results of alkyl esters, alcohols, and aldehydes, an interesting result from our initial round of EAG studies was the relatively strong antennal response to the aromatic compounds. In their 1976 report, Aldrich and co-workers (29) hypothesized that the series of benzenoid compounds isolated from male L. phyllopus could serve as long-range attractant odors for the attraction of female LFB. Interestingly, the benzenoids isolated from L. phyllopus also had numerous functional groups, including alcohols, aldehydes, esters, and ketones, thus the cautionary note above may or may not be applicable to benzenoids. Ultimately, the careful consideration of EAG-active compounds for inclusion into attractive blend formulations will be dictated by behavioral studies, whether they be field- or laboratory-based.

Material and Methods for Electrophysiological Studies Volatile preparation for electroantennography (EAG) (21): each volatile solution was prepared at a concentration of 5 mg/mL in pentane. With a 50 μL glass syringe, 10 μL of each volatile solution was loaded onto a 6.0 mm assay disc (Whatman, Sigma-Aldrich St. Luis, MO). The pentane solvent was allowed to evaporate for 2 min, the assay disc inserted into a 14.6 cm (53/4 in) Pasteur pipet (VWR Radnor, PA), and the pipet ends sealed with Parafilm (VWR Radnor, PA) until the sample is needed for assay testing (usually within a few hours). Antennae preparation: individual adult insects were isolated in a lidded plastic container and sexed. Various sizes of pipet tips were used to enable exposure of the insect head via adjustment of the opening size by cutting small portion of the tip. Using a method similar to that used for Lepidoptera studies (21, 39), just prior to each EAG or GC-EAD experiment the insect was transferred head first into a pipet tip, the insect was secured from behind with a second pipet tip, and the antenna teased out using a wire-tipped tool (see Figure 3 on left). A bead of electrode gel (Parker, Fairfield, NJ) was applied to the blades of the electrode forks (Syntech Kirchzarten, Germany). Under a dissecting microscope, both antennae were excised, then individually transferred onto the electrode fork. This process is repeated for the transferring of the second antenna so both antennae are mounted symmetrically side by side. Excess antennae were excised at the base and a small portion of the tip so that nerves at both excised ends of the antenna were in contact with the electrode and fully inserted into the electrode gel, which was inspected to ensure no bubbles were present near the antennae. The fork electrode was inserted to the pre-amplifier and placed under constant stream of humidified air at a flow rate of 200 mL/min. GC-EAD experiments were first run on the collected volatile bouquets from each host plant tissue (i.e., Figure 4A), and then individual compounds verified or tested further using EAG puff experiments on known amounts of candidate compounds (i.e., Figure 4B). 156

Figure 4. Examples of a GC-EAD experiment using the pistachio tissue extract to elicit responses from adult LFB antennae (A), and EAG puffs of pure candidate compounds to measure LFB antennal response to standardized amounts (B).

GC-EAD: volatile solutions for testing (extracts and standards) were prepared in hexane. The test solutions were injected into the GC 5 min prior to antennae prep to allow time for solvent to elute before connecting the pre-amplifier containing the excised antennae on the fork electrode. A 2 μL injection of each trial was analyzed on a J&W Scientific DB-Wax column (30 m x 0.32 mm i.d. x 0.25 μm) installed on a HP-6890 GC with FID detector (Palo Alto, CA). The instrument method parameters were as follow: inlet temperature, 250 °C; pulsed splitless mode; constant flow, 2.0 mL/min; oven settings, initial temperature, 50 °C; hold time, 1.0 min; ramp 1, 10 °C/min; final temperature, 240 °C; hold time, 10 min; FID settings, temperature, 250 °C; constant makeup flow, 16.0 mL/min; GC-EAD interface temperature, 200 °C. The test volatile solution was also analyzed on a J&W DB-Wax column (60 m x 0.32 i.d. x 0.25 μm) installed on a HP-6890 GC coupled to HP-5972 mass spectrometer (MS). The GC method was same as above. MSD parameters: source temperature, 230 °C; MS source temperature, 150 °C; EI mode, 70 eV; solvent delay, 4.65 min; scan group 1, 40 – 400 amu. Wiley and NIST databases were used for fragmentation pattern identification. The relative peak positions and retention indices (RI) values of EAD-active compounds were compared to the corresponding GC-MS injections for tentative identification, and further verified by injection of authentic samples on GC-EAD and comparison to retention times and antennal responses. Individual compound EAG experiments: antennal responses to individual test volatiles (50 μg) were recorded on a 4-channel IDAC-4 data acquisition controller (Syntech), similar to previously published experiments, but on LFB antennae. Each antennae pair was exposed to sets of 4-6 individual test volatiles. 157

Laboratory-Based LFB Behavioral Bioassays As with many insect semiochemical investigations, an appropriate laboratorybased bioassay is an important component of the formulation of an attractive blend (16, 41). However, finding an efficacious bioassay that is representative of the insect’s behavior is typically a non-trivial task. Laboratory-based bioassays have been utilized previously, yet not all have been fully successful, or applicable for our studies. For example, Aldrich and co-workers (29) tested natural products from L. phyllopus in a small, round glass assay container (25 cm x 1 cm) and spotted the treatments at opposite ends. They were met with results that were of questionable significance. Wang and Millar (15) were able to obtain positive results for the testing of LFB sex pheromones using a larger wooden cage and glass top. Their treatments were placed in small metal cylinders and they measured activity near the treatments. Finally, Blatt and co-workers (38) used a straight glass tube to test their isolated LFB alarm pheromones. However, all of these bioassays were performed on pheromones, which are generally considered more bioactive than host plant volatiles in assays. Using variations of the laboratory-based bioassays mentioned above, we attempted a series of assay methods (Figure 5) to try and ascertain any bioactivity of our identified host plant volatiles. Using the premise of Wang and Millar (15), we tried a larger area and released LFB from a central container with sticks to simulate stems (Figure 5 on left), and the treatments (blank control and tissue or candidate volatile/blend) in the bottom of larger containers. However, similar to the results obtained by Aldrich and co-workers (29), this assay was met with inconclusive results. In a similar setup as used by Blatt and co-workers (38), we also evaluated the use of Y-tube olfactometry (Figure 5 on right) to determine attractancy of various host plant tissues and volatiles of LFB adults and nymphs. While we did obtain some success with this bioassay, the results were also not conclusive. Several iterations of tissue, air flows, and LFB age and times were evaluated. Modifications to this bioassay are ongoing.

Figure 5. Laboratory-based behavioral bioassays used to investigate a method to determine adult LFB responses to host plants or host plant volatiles. On the left is a dual-choice hood assay with a control and attractant. On the right is a Y-tube olfactometer system prior to introducing an adult LFB. 158

Material and Methods for Behavioral Studies Lab rearing of LFB: a 3.78 liter glass jar was cleaned with a 10% bleach solution to minimize microbial infection and the bottom lined with 2 layers of paper towel (Figure 6 on left). A colony size of 25 – 30 adults of both sexes were maintained in the jar. Alternatively, a bug dorm can be utilized (Figure 6 on right), and was preferred over the glass jar for maintaining larger colony populations of 100 or more insects. If larger colonies are required, the advantage of using a bug dorm was the higher incidence of mating and egg laying. The diets consisted of any combination fresh fruits/vegetables of carrots, whole green beans, cherry tomato, grapes (depending on availability) and pistachio or almond kernels. A source of water was also added by saturating a cotton pad placed in a petri dish. Fresh fruits/vegetables were rinsed and dried to reduce possible pesticide exposure. The diets were replenished twice weekly. Placed in the jar or bug dorm were bamboo skewers embedded into a floral foam block splay in a Vpattern providing LFB climbing and oviposition opportunities. The colonies were maintained at temperature of 25 °C, relative humidity of 60% and with ambient fluorescent lights of day:night cycle 14:10 hours. Freshly oviposited eggs were removed and placed into a clean container with diet. As the nymphs hatched and matured they were swept once a week and segregated according to their instar stage (number of cast skins, body size, and coloration).

Figure 6. Laboratory-based rearing of LFB for behavioral bioassays. On the left is a 3.78 liter glass container used for rearing relatively small populations of LFB (25-30). On the right are bug dorms for rearing larger populations of LFB adults/nymphs (larger bug dorm) and for LFB eggs while waiting for nymphs to emerge (smaller bug dorm). Y-tube olfactometry: behavioral studies with Y-tube olfactometry utilized Pyrex glass with a tube length of 28 cm on each of the three arms, the two odor source arms were at a 65° angle, an inside diameter of 19 mm, and ground glass joint fittings of 19/38 (see Figure 5, on right). Both odor-source arms were connected to an odor chamber via Teflon tubing. The odor chamber comprised a 500 mL Mason jar and a screw top lid fitted with two ports. Compressed medical-grade air was passed through distilled water for humidity before entering 159

a manifold, and then into each odor chambers at a flow rate 50 mL/min. For the odor treatment, plant tissue (i.e., split pomegranate, 2-3 slices), individual candidate compounds, or blends of volatiles (50 μg) were placed inside one odor chamber and the other odor chamber left blank as a control. An insect was placed inside the 24/40 to 19/22 glass adapter and the 24/40 fitting end covered with a mesh screen to keep the insect from exiting. The adapter was then connected to the entrance arm of the Y-tube at 19/22 fitting end, and the air turned on. During each experiment, a red light was used to observe the insect behavior and all surfaces covered with black cloth or black cardboard. All the glassware was rinsed with hot water, then 95% ethanol, and dried in a 100 °C oven between each experiment. Two Y-tubes were alternatively used, and the arm sides and treatment sides randomized to remove any insect bias. Each insect was observed for a total of 10 min and their location within the Y-tube or their choice recorded (Figure 7).

Figure 7. An adult Leptoglossus at the choice juncture during a Y-tube choice experiment.

Hood-based, open-environment choice bioassay: hood bioassays were performed in a stainless-steel hood with the dimensions 107 cm x 91 cm x 56 cm, W x H x D (42 in x 36 in x 22 in) with the front of the hood enclosed with a 0.32 cm (1/8 in) thick Plexiglas®. To allow air movement from the outside of the hood, into, then up through the hood area past the treatment containers, 42 vent holes (2 mm diameter, spaced 25 mm apart and in a 6 across x 7 row alternating grid pattern) were drilled into the lower left and right corners of the Plexiglas. The room containing the fume hood contained a humidifier, portable heater, and air purifier. An exhaust fan was connected on the outside top center of the hood, the exhaust port inside the hood was covered with a mesh screen and the exhaust 160

flow rate of the fan set to 113 – 170 L/min (4-6 ft3/min). Two funnel traps were placed approximately 76 cm (30 in) apart in the hood (Figure 5 on left), and either a candidate lure or blank placed inside. Two moistened, paper towels were placed in the corners of the hood to provide water for the insects. To provide vertical climbing surfaces, bamboo skewers embedded into a cardboard disk was placed in the center of the hood, as well as skewers suspended over each trap (Figure 5 on left). For each experiment, 10 males and 10 females were placed into a 1 liter glass jar, the glass jar transferred to the inside the hood, the insects allowed to escape into the enclosed hood arena and consider the two treatments. Each experiment was run for 48 h.

LFB Control – Where To from Here? Over the last decade in California agriculture, the leaffooted bug has transitioned from a relatively minor insect pest to a major insect pest that is causing significant damage to a number of crops. The reason for this increase in pest status is not yet fully known. One possibility could be the overall increase of temperatures in California (42), with warmer winters leading to reduced mortality and therefore increased populations of Leptoglossus emerging from overwintering aggregations (43). Tamburini and co-workers (44) have noted a correlation between threshold temperatures and the timing of when L. occidentalis go in to and come out of overwintering. Another possible explanation is the transition of many growers away from the use of pyrethroids for control of navel orangeworm (NOW). Pyrethroids sprays for NOW in the late spring tend to have secondary, negative impacts on LFB populations. As growers begin to adopt alternate chemistries for NOW management, LFB may become more of an issue for growers. California growers affected by LFB must not only deal with the direct negative economic impacts of this pest on crop production, but also need to manage the potential environmental impacts associated with the need for increased insecticide use specifically for LFB. In the absence of effective monitoring tools and precise economic thresholds, some growers are likely to resort to prophylactic, revenge, or fear-based insecticide applications. Items that are needed to maintain adequate management of this pest in the near future include: improved monitoring tools using either pheromones, kairomones, or combinations of both; clear treatment thresholds and additional non-chemical control options, which may include future biocontrol agents, whose populations more closely align with the transient nature of LFB; and finally, an attractant whose efficacy is such that it could be used either as a trap lure or as part of an attract and kill strategy. This last option may be dependent upon a pheromone whose attractancy is further enhanced or put in proper background odor context by combining it with host plant volatiles. While the known chemical and cultural practices currently available are of some use, more reliable and efficacious methods for monitoring LFB along with additional non-chemical control options would be of great benefit to both growers and the environment – and the use of semiochemicals will likely play a key role in the development of such strategies. 161

Acknowledgments The authors thank N. Mahoney, B. Reynolds (USDA-ARS), S. Paris, E. Higuera (Wonderful Orchards), A. DeMattei (D&D Farms, LLC), and numerous collaborators for their valuable contributions. Research was conducted under USDA-ARS CRIS Project 6036-22000-028-00D and 2030-42000-039-00D, and agreement SCB15054 (58-2030-6-003) with the California Department of Food and Agriculture.

References 1.

2. 3. 4. 5.

6. 7. 8.

9.

10. 11. 12.

13.

Koerber, T. W. Leptoglossus occidentalis (Hemiptera, Coreidae), a newly discovered pest of coniferous seed. Annu. Entomol. Soc. Am. 1963, 56, 229–234. Heidemann, O. New species of Leptoglossus from North America (Hemiptera: Coreidae). Proc. Entomol. Soc. Wash. 1910, 12, 191–197. Essig, E. O. Insects and mites of western North America, 2nd ed.; McMillan Press: New York, NY, 1958. Allen, R. C. A revision of the genus Leptoglossus Guerin (Hemiptera: Coreidae). Entomol. Am. 1969, 45, 35–140. Bolkan, H. A.; Ogawa, J. M.; Rice, R.; Bostock, R. M.; Crane, J. C. Leaffooted bug implicated in pistachio epicarp lesion. Calif. Agric. 1984, 38, 16–17. Rice, R.; Uyemoto, J. K.; Ogawa, J. M.; Pemberton, W. M. New findings on pistachio problems. Calif. Agric. 1985, 39, 15–18. Michailides, T. J.; Morgan, D. P.; Felts, D. Spread of Botryosphaeria dothidea in central California pistachio orchards. Acta Hort. 1998, 470, 582–591. Daane, K. M.; Yokota, G. Y.; Krugner, R.; Steffan, S. A.; da Silva, P. G.; Beede, R. H.; Bentley, W. J.; Weinberger, G. B. Large bugs damage pistachio nuts most severely during midseason. Calif. Agric. 2005, 59, 95–102. Uyemoto, J. K.; Ogawa, J. M.; Rice, R. E.; Teranishi, H. R.; Bostock, R. M.; Pemberton, W. M. Role of several true bugs (Hemiptera) on incidence and seasonal development of pistachio fruit epicarp lesion disorder. J. Econ. Entomol. 1986, 79, 395–399. Purcell, M.; Welter, S. C. Effect of Calocoris norvegicus (Hemiptera: Miridae) on pistachio yields. J. Econ. Entomol. 1991, 84, 114–119. Michailides, T. J. The ‘Achilles heel’ of pistachio fruit. Calif. Agric. 1989, 43, 10–11. Michailides, T. J.; Rice, R.; Ogawa, J. Succession and significance of several hemipterans attacking a pistachio orchard. J. Econ. Entomol. 1987, 80, 398–406. Daane, K. M.; Millar, J. G.; Rice, R. E.; da Silva, P. G.; Bentley, W. J.; Beede, R. H.; Weinberger, G. Stink bugs and leaffooted bugs. In Pistachio Production Manual; Ferguson, L., Haviland, D. R., Eds.; University of California: Oakland, CA, 2016; pp 225−238. 162

14. Leal, W. S.; Panizzi, A. R.; Niva, C. C. Alarm pheromone system of leaffooted bug Leptoglossus zonatus (Heteroptera: Coreidae). J. Chem. Ecol. 1994, 20, 1209–1216. 15. Wang, Q.; Millar, G. G. Mating behavior and evidence for male-produced sex pheromones in Leptoglossus clypealis (Heteroptera: Coreidae). Ann. Entomol. Soc. Am. 2000, 93, 972–976. 16. Beck, J. J. Addressing the complexity and diversity of agricultural plant volatiles: a call for the integration of laboratory- and field-based analyses. J. Agric. Food Chem. 2012, 60, 1153–1157. 17. Niinemets, Ü.; Kännaste, A.; Copolovici, L. Quantitative patterns between plant volatile emissions induced by biotic stresses and the degree of damage. Front. Plant Sci. 2013, 4, 262. 18. Beck, J. J.; Smith, L.; Baig, N. An overview of plant volatile metabolomics, sample treatment and reporting considerations with emphasis on mechanical damage and biological control of weeds. Phytochem. Anal. 2014, 25, 331–341. 19. Beck, J. J.; Vannette, R. L. Harnessing insect-microbe chemical communications to control insect pests of agricultural systems. J. Agric. Food Chem. 2017, 65, 32–28. 20. Beck, J. J.; Torto, B.; Vannette, R. L. Eavesdropping on plant-insect-microbe chemical communications in agricultural ecology: a virtual issue on semiochemicals. J. Agric. Food Chem. 2017, 65, 5101–5103. 21. Beck, J. J.; Light, D. M.; Gee, W. S. Electroantennographic bioassay as a screening tool for host plant volatiles. J. Vis. Exp. 2012, 63, e3931. 22. Riolo, P.; Minuz, R. L.; Peri, E.; Isidoro, N. Behavioral responses of Hyalesthes obsoletus to host-plant volatiles cues. Arthropod Plant Interact. 2017, 11, 71–78. 23. Bruce, T. J. A.; Wadhams, L. J.; Woodcock, C. M. Insect host location: a volatile situation. Trends Plant Sci. 2005, 10, 1360–1385. 24. Mitchell, P. L. Polyphagy in true bugs: a case study of Leptoglossus phyllopus (L.) (Hemiptera, Heteroptera, Coreidae). Denisia 2006, 19, 1117–1134. 25. Gripenberg, S.; Mayhew, P. J.; Parnell, M.; Roslin, T. A meta-analysis of preference-performance relationships in phytophagous insects. Ecol. Lett. 2010, 13, 383–393. 26. Bruce, T. J. A.; Pickett, J. A. Perception of plant volatile blends by herbivorous insects – finding the right mix. Phytochemistry 2011, 72, 1605–1611. 27. Cunningham, J. P. Can mechanism help explain insect host choice? J. Evol. Biol. 2012, 25, 244–251. 28. Joyce, A. L.; Higbee, B. S.; Haviland, D. R.; Brailovsky, H. Genetic variability of two leaffooted bugs, Leptoglossus clypealis and Leptoglossus zonatus (Hemiptera: Coreidae) in the central valley of California. J. Econ. Entomol. 2017, 110, 2576–2589. 29. Aldrich, J. R.; Blum, M. S.; Duffey, S. S.; Fales, H. M. Male specific natural products in the bug Leptglossus phyllopus: chemistry and possible function. J. Insect Physiol. 1976, 22, 1201–1206. 163

30. Aldrich, J. R.; Blum, M. S.; Fales, H. M. Species-specific natural products of adult male leaf-footed bugs (Hemiptera: Heteroptera). J. Chem. Ecol. 1979, 5, 53–62. 31. Aldrich, J. R.; Waite, G. K.; Moore, C.; Payne, J. A.; Lusby, W. R.; Kochansky, J. P. Male-specific volatiles from Neartic and Australasian true bugs (Heteroptera: Coreidae and Alydidae). J. Chem. Ecol. 1993, 19, 2767–2781. 32. Akotsen-Mansah, C.; Balusu, R. R.; Anikwe, J.; Fadamiro, H. Y. Evaluating potential trap crops for managing leaffooted (Hemiptera: Coreidae) and phytophagous stink bug (Hemiptera: Pentatomidae) species in peaches. Agric. Forest Entomol. 2017, 19, 332–340. 33. Blatt, S. E.; Borden, J. H. Evidence for a male-produced aggregation pheromone in the western conifer seed bug, Leptoglossus occidentalis Heidemann (Hemiptera: Coreidae). Can. Entomol. 1996, 128, 777–778. 34. Xiao, Y. F.; Fadamiro, H. Y. Evaluation of damage to satsuma mandarin (Citrus unshiu) by the leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae). J. Appl. Entomol. 2010, 134, 694–703. 35. Chi, A. A.; Mizell, R. F. Western leaffooted bug – Leptoglossus zonatus (Dallas). http://entnemdept.ufl.edu/creatures/citrus/Leptoglossus_ zonatus.htm (accessed April 10, 2018). 36. Jayanthis, P. D. K.; Woodcock, C. M.; Caulfield, J.; Birkett, M. A.; Bruce, T. J. A. Isolation and identification of host cues from mango, Mangifera indica, that attract gravid female Oriental fruit fly, Bactrocera dorsalis. J. Chem. Ecol. 2012, 38, 361–369. 37. Beck, J. J.; Mahoney, N. E.; Higbee, B. S.; Gee, W. S.; Baig, N.; Griffith, C. M. Semiochemicals to monitor insect pests – future opportunities for an effective host plant volatile blend to attract navel orangeworm in pistachio orchards. In Biopesticides: State of the Art and Future Opportunities; Gross, A. D., Seiber, J. N., Coats, J. R., Duke, S. O., Eds.; American Chemical Society: Washington, DC, 2014; pp 191−210. 38. Blatt, S. E.; Borden, J. H.; Pierce, H. D.; Gries, R.; Gries, G. Alarm pheromone system of the western conifer seed bug, Leptoglossus occidentalis. J. Chem. Ecol. 1998, 24, 1013–1031. 39. Beck, J. J.; Light, D. M.; Gee, W. S. Electrophysiological responses of male and female Amyelois transitella antennae to pistachio and almond host plant volatiles. Entomol. Exp. Appl. 2014, 153, 217–230. 40. Liu, Z.; Vidal, D. M.; Syed, Z.; Ishida, Y.; Leal, W. S. Pheromone binding to general odorant-binding proteins from the navel orangeworm. J. Chem. Ecol. 2010, 36, 878–794. 41. Ali, J. G.; Agrawal, A. A. Specialist versus generalist insect herbivores and plant defense. Trends Plant Sci. 2012, 17, 293–302. 42. EPA. What climate change means for California; EPA 430-F-16-007. https://www.epa.gov/sites/production/files/2016-09/documents/climatechange-ca.pdf (accessed April 10, 2018). 43. UC IPM. US Pest Management Guidelines. Leaffooted Plant Bugs. http:// ipm.ucanr.edu/PMG/r605300311.html (accessed April 10, 2018). 164

44. Tamburini, M.; Maresi, G.; Salvadori, C.; Battisti, A.; Zottele, F.; Pedrazzoli, F. Adaptation of the invasive western conifer seed bug Leptoglossus occidentalis to Trentino, an alpine region (Italy). Bull. Insectol. 2012, 65, 161–170.

165

Editors’ Biographies John J. Beck John J. Beck (Ph.D., Colorado State University) is Research Leader for the Chemistry Research Unit in the U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS), Center for Medical, Agricultural, and Veterinary Entomology (CMAVE) in Gainesville, FL. His research interests include the study of chemical communications between plants and insects. Over the last five years his interests have expanded to include microbial emissions and their contributions to plant-insect interactions. He has authored or co-authored over 55 peer-reviewed journal articles, book chapters, and four patents. He is on the Editorial Advisory Board for Journal of Agricultural and Food Chemistry and Phytochemistry Letters.

Caitlin C. Rering Caitlin C. Rering (Ph.D., University of California, Davis) is a Research Chemist and postdoctoral fellow at the Chemistry Research Unit in the U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS), Center for Medical, Agricultural and Veterinary Entomology (CMAVE) in Gainesville, FL. Her research is on chemically-mediated interactions between the plants, the floral microbiome, and pollinators. Her Ph.D. research investigated pesticide toxicity and environmental fate. She is an active member of ACS AGRO, co-organizer of AGRO symposia and was a finalist for the ACS AGRO New Investigator Award in 2017.

Stephen O. Duke Stephen O. Duke (Ph.D., Duke University) is Research Leader of the Natural Products Utilization Research Unit of the U.S. Department of Agriculture in Oxford, Mississippi. His research is on discovery and development of natural product-based pest management products. He has been Chair of the Agrochemicals Division (AGRO) of ACS, and President of the Weed Science Society of America (WSSA), the International Weed Science Society, and the International Allelopathy Society. He has authored or co-authored more than 400 journal articles and book chapters, co-edited 10 books, and co-authored one book. His honors include Fellow of AAAS, ACS, AGRO, and WSSA, and an Honorary Doctorate from the University of the Basque Country. He is Editor-in-Chief of Pest Management Science. © 2018 American Chemical Society

Indexes

Author Index Bajsa-Hirschel, J., 33 Barney, W., 5 Baron, J., 5 Beck, J., 1, 127, 143 Block, A., 47 Braverman, M., 5 Cheng, L., 143 Christensen, S., 47 Coats, J., 23 Coleman, K., 5 Daane, K., 143 Duke, S., 1, 33 Gee, W., 143 Higbee, B., 143 Hunter, C., 47 Judd, G., 83 Kihika, R., 115 Kirwa, H., 115

Knight, A., 83 Kunkel, D., 5 Light, D., 83 Morimoto, M., 11 Murungi, L., 115 Pan, Z., 33 Rering, C., 1, 127 Roschatt, C., 69 Sánchez-Moreiras, A., 33 Schmidt, S., 69 Schweigkofler, W., 69 Torto, B., 115 Vannette, R., 127 Vaughn, J., 33 Willms, S., 127 Wilson, H., 143 Witzgall, P., 83 Wong, C., 23

171

Subject Index B

introduction, 12 chemical structures, commercial insecticides that include the dihydrobenzofuran moiety, 13f SAR study, insect antifeedant natural phenolics and synthetic compounds, 12f lignan-like dihydrobenzofurans, synthesis, 13 electrochemical oxidation, synthesis, 13 test dihydrobenzofurans, 14f

Biopesticides, role of the IR-4 project, 5

C Citrus plants, movement of thymol discussion, 30 thymol or other terpenoids, co-application, 31 introduction, 23 materials and methods, 25 foliar application, leaf to leaf movement, 26 leaf to leaf movement, containment unit for testing, 27f thymol with tritium-labeled carbon positions numbered, structure, 25f results, 27 ability of leaves to internalize topically applied thymol, age comparison, 28f thymol, movement, 29f thymol, root uptake, 30f

D Dihydrobenzofurans, insect antifeedant activities and preparation, 11 aurones, synthesis, 16 synthesis of test aurones, general procedure, 17f common cutworm, SAR study of aurones, 17 B-ring, test aurones with substituents, 18f present study, test coumaranones, 18f test aurones in the present study, 18f conclusions, 19 pterocarpus tree, insect antifeedant pterocarpans, 19f insect antifeedants, 14 dihydrobenzofurans, insect antifeedant activities, 15t 2-phenyl dihydrobenzofuran and aurone, structural similarities, 16f

I Introduction, 1

L Leaffooted bug, insect pest of California agriculture, 143 California host plants surveyed, 146 adult leaffooted bug, 147f host plant volatiles, scarcity of literature, 154 LFB host plant matrices surveyed, volatile compounds detected, 153t nymph leaffooted bugs, 147f volatile compounds, classes and relative amounts, 148t introduction, 144 laboratory-based LFB behavioral bioassays, 158 adult Leptoglossus, 160f behavioral bioassays, laboratory-based rearing of LFB, 159f host plants or host plant volatiles, adult LFB responses, 158f leaffooted bug, electrophysiological studies, 155 electrophysiological studies, material and methods, 156 GC-EAD experiment, examples, 157f leaffooted bug, antennae, 155f LFB control, 161

173

M Maize, natural chemical defenses, 47 defense chemistry, functional characterization, 57 defense chemistry, plant hormones and regulation, 53 jasmonates, 54 novel cyclopente(a)none fatty acids (FAs), working model for the biosynthesis pathway, 55f other plant hormones and enogenous maize signals, 56 plant hormone synergies, generalized view of the complex network, 53f enhanced chemical defense, engineering, 58 maize defense metabolites, heat map, 58f future directions, 59 introduction, 48 maize defense chemicals, 49 herbivore-induced plant volatiles, 52 phenylpropanoids, 51 select chemical defense compounds from maize, structures, 49f terpenoid phytoalexins, 50

amino acids in glyphosate-treated sensitive (S)- and resistant (R)-biotypes, 15N isotopologue enrichment, 41f plants, metabolomics and herbicide, 40 physionomics, 41 effects of MMV007978, physiological profile, 42f proteomics, 37 α-terthienyl on the growth, effects, 39f 21 μM t-chalcone, effects, 38f transcriptomics, 35

P Pear ester, 83 background, 84 path to pear ester, 87 pear ester, discovery, 89 pear ester, disruption of larvae, 95 pear ester, lure and kill, 96 pear ester, mating disruption, 95 pear ester, monitoring, 93 pear ester, sensory physiology and perception, 97 pear ester, utility, 93 pear ester, attractiveness, 94

N Nectar microbe-produced volatiles, quantitative assessment, 127 conclusions, 139 discussion, 135 introduction, 128 materials and methods, 128 results, 129 M. reukaufii analyte concentration, 134t volatile calibration details, 133t volatile compounds, dissolved concentration, 130t volatiles in sealed jars, partitioning behavior, 132f

O Omics methods, use conclusions, 42 introduction, 33 metabolomics, 39

R Root knot nematode behavior with natural products, strategies for the manipulation conclusions and future directions, 123 control of RKNs, natural products use, 116 organic soil amendments, 117 plants used for the management of RKNs, key chemical components, 119f RKN management, examples of plants used, 118t control RKNs, potential of chemical ecological and biotechnological approaches, 120 plant breeding and genetic engineering, 122 root volatiles, chemical components identified, 121f introduction, 115 Meloidogyne incognita, second-stage infective juvenile, 116f

174

W Woody crop plants, phytoplasma diseases, 69 disease control, integrated polyphasic approach, 75 BN infection on mineral content, effect, 77t control phytoplasma diseases, difficulties, 80 resistance inducers and plant growth regulators, effect, 79f symptoms and recovery, remission, 76

175

V. vinifera cv. Chardonnay, calcium content in leaves, 78f woody plants, possible control strategies for phytoplasma diseases, 75t introduction, 70 bois noir (A-C) and apple proliferation (D-F), symptoms, 71f phytoplasma diseases, complex ecology, 72 three phytoplasma diseases, simplified scheme of the ecology, 74f