Edited by Peter Jeschke, Wolfgang Kramer,

Ulrich Schirmer, and Matthias Witschel

Modern Methods in Crop ProtectionResearch

Related Titles

Kramer, W., Schirmer, U., Jeschke, P.,Witschel, M. (eds.)

Modern Crop ProtectionCompounds

2012

ISBN: 978-3-527-32965-6

Filho, V. C.

Plant Bioactives and DrugDiscoveryPrinciples, Practice, and Perspectives

2012

ISBN: 978-0-470-58226-8

Walters, D.

Plant DefenseWarding off attack by pathogens,herbivores and parasitic plants

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ISBN: 978-1-4051-7589-0

Tadros, T. F. (ed.)

Colloids in AgrochemicalsColloids and Interface Science

Volume 5 of the Colloids and Interface Science

Series

2009

ISBN: 978-3-527-31465-2

Edited by Peter Jeschke, Wolfgang Kramer, Ulrich Schirmer,and Matthias Witschel

Modern Methods in Crop ProtectionResearch

The Editors

Dr. Peter JeschkeBayer CropScience AGBCS AG-R&D-CPR-PC-PCC-Chemistry 2Bldg. 6510Alfred-Nobel-Str. 5040789 MonheimGermany

Dr. Wolfgang KramerRosenkranz 2551399 BurscheidGermany

Dr. Ulrich SchirmerBerghalde 7969126 HeidelbergGermany

Dr. Matthias WitschelBASF SEGVA/HC-B00967056 LudwigshafenGermany

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V

Contents

Preface XV

List of Contributors XIX

Part I Methods for the Design and Optimizationof New Active Ingredients 1

1 High-Throughput Screening in Agrochemical Research 3Mark Drewes, Klaus Tietjen, and Thomas C. Sparks

1.1 Introduction 31.2 Target-Based High-Throughput Screening 61.2.1 Targets 61.2.2 High-Throughput Screening Techniques 91.3 Other Screening Approaches 131.3.1 High-Throughput Virtual Screening 131.4 In Vivo High-Throughput Screening 131.4.1 Compound Sourcing and In-Silico Screening 151.5 Conclusions 17

Acknowledgments 18References 18

2 Computational Approaches in Agricultural Research 21Klaus-Jurgen Schleifer

2.1 Introduction 212.2 Research Strategies 212.3 Ligand-Based Approaches 222.4 Structure-Based Approaches 262.5 Estimation of Adverse Effects 332.6 In-Silico Toxicology 342.7 Programs and Databases 342.7.1 In-Silico Toxicology Models 36

VI Contents

2.8 Conclusion 39References 40

3 Quantum Chemical Methods in the Design of Agrochemicals 43Michael Schindler

3.1 Introduction 433.2 Computational Quantum Chemistry: Basics, Challenges,

and New Developments 443.3 Minimum Energy Structures and Potential Energy Surfaces 473.4 Physico-Chemical Properties 513.4.1 Electrostatic Potential, Fukui Functions, and Frontier Orbitals 533.4.2 Magnetic Properties 553.4.3 pKa Values 573.4.4 Solvation Free Energies 593.4.5 Absolute Configuration of Chiral Molecules 603.5 Quantitative Structure-Activity Relationships 603.5.1 Property Fields, Wavelets, and Multi-Resolution Analysis 613.5.2 The CoMFA Steroid Dataset 633.5.3 A Neonicotinoid Dataset 643.6 Outlook 66

References 67

4 The Unique Role of Halogen Substituents in the Designof Modern Crop Protection Compounds 73Peter Jeschke

4.1 Introduction 734.2 The Halogen Substituent Effect 754.2.1 The Steric Effect 764.2.2 The Electronic Effect 784.2.2.1 Electronegativities of Halogens and Selected Elements/Groups

on the Pauling Scale 784.2.2.2 Effect of Halogen Polarity of the C–Halogen Bond 794.2.2.3 Effect of Halogens on pKa Value 794.2.2.4 Improving Metabolic, Oxidative, and Thermal Stability

with Halogens 804.2.3 Effect of Halogens on Physico-Chemical Properties 824.2.3.1 Effect of Halogens on Molecular Lipophilicity 824.2.3.2 Classification in the Disjoint Principle Space 844.2.4 Effect of Halogens on Shift of Biological Activity 844.3 Insecticides and Acaricides Containing Halogens 864.3.1 Voltage-Gated Sodium Channel (vgSCh) Modulators 864.3.1.1 Pyrethroids of Type A 864.3.1.2 Pyrethroids of Type B 89

Contents VII

4.3.1.3 Pyrethroids of Type C 904.3.2 Voltage-Gated Sodium Channel (vgSCh) Blockers 904.3.3 Inhibitors of the γ -Aminobutyric Acid (GABA) Receptor/Chloride

Ionophore Complex 914.3.4 Insect Growth Regulators (IGRs) 934.3.5 Mitochondrial Respiratory Chain 964.3.5.1 Inhibitors of Mitochondrial Electron Transport at Complex I 964.3.5.2 Inhibitors of Qo Site of Cytochrome bc1 – Complex III 974.3.5.3 Inhibitors of Mitochondrial Oxidative Phosphorylation 974.3.6 Ryanodine Receptor (RyR) Effectors 984.4 Fungicides Containing Halogens 994.4.1 Sterol Biosynthesis Inhibitors (SBIs) and Demethylation Inhibitors

(DMIs) 994.4.2 Mitochondrial Respiratory Chain 1014.4.2.1 Inhibitors of Succinate Dehydrogenase (SDH) – Complex II 1014.4.2.2 Inhibitors of Qo Site of Cytochrome bc1 – Complex III 1044.4.2.3 NADH Inhibitors – Complex I 1074.4.3 Fungicides Acting on Signal Transduction 1074.5 Plant Growth Regulators (PGRs) Containing Halogens 1084.5.1 Reduction of Internode Elongation: Inhibition of Gibberellin

Biosynthesis 1084.6 Herbicides Containing Halogens 1094.6.1 Inhibitors of Carotenoid Biosynthesis: Phytoene Desaturase (PDS)

Inhibitors 1094.6.2 Inhibitors of Acetolactate Synthase (ALS) 1114.6.2.1 Sulfonylurea Herbicides 1114.6.2.2 Sulfonylaminocarbonyl-Triazolone Herbicides (SACTs) 1154.6.2.3 Triazolopyrimidine Herbicides 1164.6.3 Protoporphyrinogen IX Oxidase (PPO) 1174.7 Summary and Outlook 119

References 119

Part II New Methods to Identify the Mode of Actionof Active Ingredients 129

5 RNA Interference (RNAi) for Functional Genomics Studiesand as a Tool for Crop Protection 131Bernd Essigmann, Eric Paget, and Frederic Schmitt

5.1 Introduction 1315.2 RNA Silencing Pathways 1315.2.1 The MicroRNA (miRNA) Pathway 1335.2.2 The Small Interfering Pathway (siRNA) 1345.3 RNAi as a Tool for Functional Genomics in Plants 134

VIII Contents

5.4 RNAi as a Tool for Engineering Resistance against Fungiand Oomycetes 138

5.5 RNAi as a Tool for Engineering Insect Resistance 1405.6 RNAi as a Tool for Engineering Nematodes Resistance 1425.7 RNAi as a Tool for Engineering Virus Resistance 1445.8 RNAi as a Tool for Engineering Bacteria Resistance 1495.9 RNAi as a Tool for Engineering Parasitic Weed Resistance 1505.10 RNAi Safety in Crop Plants 1535.11 Summary and Outlook 153

References 153

6 Fast Identification of the Mode of Action of Herbicidesby DNA Chips 161Peter Eckes and Marco Busch

6.1 Introduction 1616.2 Gene Expression Profiling: A Method to Measure Changes

of the Complete Transcriptome 1626.3 Classification of the Mode of Action of an Herbicide 1646.4 Identification of Prodrugs by Gene Expression Profiling 1656.5 Analyzing the Affected Metabolic Pathways 1696.6 Gene Expression Profiling: Part of a Toolbox for Mode of Action

Determination 171References 172

7 Modern Approaches for Elucidating the Mode of Actionof Neuromuscular Insecticides 175Daniel Cordova

7.1 Introduction 1757.2 Biochemical and Electrophysiological Approaches 1767.2.1 Biochemical Studies 1767.2.2 Electrophysiological Studies on Native and Expressed Targets 1797.2.2.1 Whole-Cell Voltage Clamp Studies 1797.2.2.2 Oocyte Expression Studies 1807.2.3 Automated Two-Electrode Voltage-Clamp TEVC Recording

Platforms 1827.3 Fluorescence-Based Approaches for Mode of Action Elucidation 1837.3.1 Calcium-Sensitive Probes 1837.3.2 Voltage-Sensitive Probes 1867.4 Genomic Approaches for Target Site Elucidation 1877.4.1 Chemical-to-Gene Screening 1877.4.2 Double-Stranded RNA Interference 1907.4.3 Metabolomics 191

Contents IX

7.5 Conclusion 191References 192

8 New Targets for Fungicides 197Klaus Tietjen and Peter H. Schreier

8.1 Introduction: Current Fungicide Targets 1978.2 A Retrospective Look at the Discovery of Targets for Fungicides 1998.3 New Sources for New Fungicide Targets in the Future? 1998.4 Methods to Identify a Novel Target for a Given Compound 2008.4.1 Microscopy and Cellular Imaging 2008.4.2 Cultivation on Selective Media 2008.4.3 Incorporation of Isotopically Labeled Precursors

and Metabolomics 2018.4.4 Affinity Methods 2018.4.5 Resistance Mutant Screening 2018.4.6 Gene Expression Profiling and Proteomics 2028.5 Methods of Identifying Novel Targets without Pre-Existing

Inhibitors 2028.5.1 Biochemical Ideas to Generate Novel Fungicide Targets 2038.5.2 Genomics and Proteomics 2038.6 Non-Protein Targets 2138.7 Resistance Inducers 2138.8 Beneficial Side Effects of Commercial Fungicides 2148.9 Concluding Remarks 214

References 214

Part III New Methods to Improve the Bioavailabilityof Active Ingredients 217

9 New Formulation Developments 219Rolf Pontzen and Arnoldus W.P. Vermeer

9.1 Introduction 2199.2 Drivers for Formulation Type Decisions 2239.3 Description of Formulation Types, Their Properties, and Problems

during Development 2259.3.1 Pesticides Dissolved in a Liquid Continuous Phase 2259.3.2 Crystalline Pesticides in a Liquid Continuous Phase 2289.3.3 Pesticides in a Solid Matrix 2329.4 Bioavailability Optimization 2359.4.1 Spray Formation and Retention 2369.4.2 Spray Deposit Formation and Properties 2389.4.3 Cuticular Penetration 2409.4.3.1 Cuticular Penetration Test 242

X Contents

9.4.3.2 Effect of Formulation on Cuticular Penetration 2439.5 Conclusions and Outlook 246

References 247

10 Polymorphism and the Organic Solid State: Influenceon the Optimization of Agrochemicals 249Britta Olenik and Gerhard Thielking

10.1 Introduction 24910.2 Theoretical Principles of Polymorphism 25010.2.1 The Solid State 25010.2.2 Definition of Polymorphism 25110.2.3 Thermodynamics 25110.2.3.1 Monotropism and Enantiotropism 25110.2.3.2 Energy Temperature Diagrams and the Rules 25210.2.4 Kinetics of Crystallization: Nucleation 25410.3 Analytical Characterization of Polymorphs 25510.3.1 Differential Thermal Analysis and Differential Scanning

Calorimetry 25610.3.2 Thermogravimetry 25810.3.3 Hot-Stage Microscopy 25910.3.4 IR and Raman Spectroscopies 26110.3.5 X-Ray Analysis 26510.4 Patentability of Polymorphs 26810.5 Summary and Outlook 270

Acknowledgments 270References 270

11 The Determination of Abraham Descriptors and Their Applicationto Crop Protection Research 273Eric D. Clarke and Laura J. Mallon

11.1 Introduction 27311.2 Definition of Abraham Descriptors 27411.3 Determination of Abraham Descriptors: General Approach 27511.3.1 V and E Descriptors 27611.3.2 A, B, and S Descriptors 27711.3.3 A, B, S, and L Descriptors 27711.3.4 LSER Equations for Use in Determining Descriptors 27811.3.5 Prediction of Abraham Descriptors 28011.4 Determination of Abraham Descriptors: Physical Properties 28111.5 Determination of Abraham Descriptors: Examples 28311.5.1 Herbicides: Diuron (1) 28411.5.2 Herbicides: Simazine (2) and Atrazine (3) 28511.5.3 Herbicides: Acetochlor (4) and Alachlor (5) 288

Contents XI

11.5.4 Insecticides: Fipronil (6) 28911.5.5 Insecticides: Imidacloprid (7) 29011.5.6 Insecticides: Chlorantraniliprole (8) 29211.5.7 Insecticides: Thiamethoxam (9) 29311.5.8 Fungicides: Azoxystrobin (10) 29411.5.9 Plant Growth Regulator: Paclobutrazol (11) 29511.6 Application of Abraham Descriptors: Descriptor

Profiles 29611.7 Application of Abraham Descriptors: LFER Analysis 29711.7.1 LFERs for RP-HPLC Systems 29711.7.2 LFERs for Soil Sorption Coefficient (KOC) 29911.7.3 LFERs for Partitioning into Plant Cuticles 30011.7.4 LFERs for Root Concentration Factor (RCF) 30011.7.5 LFER for Transpiration Stream Concentration Factor 30111.8 Application of Abraham Descriptors: Generality of

Approach 301Acknowledgments 302References 302

Part IV Modern Methods for Risk Assessment 307

12 Ecological Modeling in Pesticide Risk Assessment: Chancesand Challenges 309Walter Schmitt

12.1 Introduction 30912.2 Ecological Models in the Regulatory Environment 31112.2.1 Consideration of Realistic Exposure Patterns 31212.2.2 Extrapolation to Population Level: The Link to Protection

Goals 31312.2.3 Extrapolation to Organization Levels above Populations 31412.3 An Overview of Model Approaches 31512.3.1 Toxicokinetic Models 31612.3.2 Population Models 31912.3.2.1 Differential Equation Models 31912.3.2.2 Matrix Models 32012.3.2.3 Individual-Based Models 32212.3.3 Ecosystem or Food-Web Models 32512.4 Regulatory Challenges 328

References 331

XII Contents

13 The Use of Metabolomics In Vivo for the Developmentof Agrochemical Products 335Hennicke G. Kamp, Doerthe Ahlbory-Dieker, Eric Fabian, Michael Herold,Gerhard Krennrich, Edgar Leibold, Ralf Looser, Werner Mellert, AlexandreProkoudine, Volker Strauss Tilmann Walk, Jan Wiemer, and Bennard vanRavenzwaay

13.1 Introduction to Metabolomics 33513.2 MetaMap®Tox Data Base 33613.2.1 Methods 33613.2.1.1 Animal Treatment and Maintenance Conditions 33613.2.1.2 Blood Sampling and Metabolite Profiling 33613.3 Evaluation of Metabolome Data 33713.3.1 Data Processing 33713.3.1.1 Metabolite Profiling 33713.3.1.2 Metabolome Patterns 33713.3.1.3 Whole-Profile Comparison 33813.4 Use of Metabolome Data for Development of Agrochemicals 33913.4.1 General Applicability 33913.4.2 Case Studies 33913.4.2.1 Liver Enzyme Induction 33913.4.2.2 Liver Cancer 34213.4.3 Chemical Categories 34413.5 Discussion 34513.5.1 Challenges and Chances Concerning the Use of Metabolite Profiling

in Toxicology 34513.5.2 Applicability of the MetaMap®Tox Data Base 34713.6 Concluding Remarks 347

References 348

14 Safety Evaluation of New Pesticide Active Ingredients: Enquiry-LedApproach to Data Generation 351Paul Parsons

14.1 Background 35114.2 What Is the Purpose of Mammalian Toxicity Studies? 35414.3 Addressing the Knowledge Needs of Risk Assessors 35814.4 Opportunities for Generating Data of Direct Relevance to Human

Health Risk Assessment within the Existing Testing Paradigm 36214.4.1 Dose Selection for Carcinogenicity Studies 36214.4.2 Integrating Toxicokinetics into Toxicity Study Designs 36514.5 Enquiry-Led Data Generation Strategies 36714.5.1 Key Questions to Consider While Identifying Lead Molecules 36914.5.2 Key Questions to Consider When Selecting Candidates for Full

Development 370

Contents XIII

14.5.3 Key Questions to Consider for a Compound in FullDevelopment 371

14.6 Conclusions 371References 378

15 Endocrine Disruption: Definition and Screening Aspects in the Lightof the European Crop Protection Law 381Susanne N. Kolle, Burkhard Flick, Tzutzuy Ramirez, Roland Buesen,Hennicke G. Kamp, and Bennard van Ravenzwaay

15.1 Introduction 38115.2 Endocrine Disruption: Definitions 38215.3 Current Regulatory Situation in the EU 38215.4 US EPA Endocrine Disruptor Screening Program and OECD

Conceptual Framework for the Testing and Assessment ofEndocrine-Disrupting Chemicals 384

15.5 ECETOC Approach 38515.6 Methods to Assess Endocrine Modes of Action and Endocrine-Related

Adverse Effects in Screening and Regulatory Contexts 38815.6.1 In-Vitro Assays 38815.6.2 In-Vivo Assays 39115.7 Proposal for Decision Criteria for EDCs: Regulatory Agencies 397

References 397

Index 401

XV

Preface

Today, modern agriculture is facing an enormous challenge – namely, that it ensurethat sufficient high-quality food is available to meet the needs of a continuouslygrowing population.

In 2011, the world’s population exceeded seven billion people, and a prognosisby the United Nations has suggested that by the year 2050 – assuming moderatebirth rates – this will increase to as many as 9.1 billion.

Beyond that, losses of agriculturally usable land, climate change, and changes inthe eating habits of the peoples of newly industrialized countries will require majorimprovements to be made in agricultural productivity. In addition to the increasingdemand for food in general, people are today requesting a greater protein intake,especially in countries undergoing transition, and this in turn will lead to a higherconsumption of the cereals required as feed used for meat production. Coinciden-tally, these changing food demands are meeting new requests for bioenergy to beproduced via agriculture. Climatic changes that influence the distribution of weeds,pests, and diseases, and their prospective consequences for agriculture, representa further challenge for crop protection. Change in seed breeding and geneticallymodified (GM) crops demonstrate progressive solutions for better supplies offood by employing technological innovations from both biochemistry and biotech-nology. Nevertheless, the traditional research and development of crop protectioncompounds remains the most effective method for combating losses in agriculturalyields. Currently, such losses are in the range of 14% due to competition by weeds,13% due to damage by fungal pathogens, and 15% by insect damage.

Another very important reason for employing crop protection compounds isto improve the quality of food. For example, mycotoxins produced by species ofFusarium (a fungus that causes damage to the ears of wheat) lead to increasingproblems in food production. In addition, changes in rainfall, temperature, andrelative humidity can each favor the growth of fungi that produce mycotoxins, so thatcrops such as groundnuts, wheat, maize, rice, and coffee may become unsuitable forconsumption by both humans and animals. Thus, the need for effective researchinto new crop protection compounds can be fulfilled only by introducing newscientific approaches within the methodology of seeking new active ingredients,by improving the identification process of new targets, by studying aspects of

XVI Preface

bioavailability, and by improving the tools applied to risk assessment studies oftoxicological and ecotoxicological aspects, utilizing new technologies.

This book, which is based partly on Part IV: New Research Methods of the FirstEdition of the textbook Modern Crop Protection Compounds (Wiley-VCH, 2007),provides details of the progress that has been made during the past few yearstowards new methods in modern crop protection research. This includes progressnot only in chemical synthesis but also in physico-chemical research, the useof biological research progress and the knowledge and application of geneticsand proteomics, and the use of mathematical methods in the design and riskassessment of new active ingredients. Consequently, this book will reflect theexclusively broad field of research in the areas of chemistry, biology, biochemistry,formulation research, toxicology, and ecotoxicology that have been used to identifyand develop new chemical tools, such that ‘‘green’’ technology can enjoy furthersuccess.

The book, which provides a broad overview of a range of current methods usedin modern crop protection research, is divided into four Parts that incorporate15 chapters, each written by renowned experts at the R&D divisions of majoragrochemical companies.

Part I presents methods for the design and optimization of new active ingredients.By using modern research techniques and serendipitous, highly specific biologicalscreening systems, significant progress has been achieved during the past 25years in computational methods for lead identification and optimization, based onmolecular structure information and/or quantum chemistry. Additionally, in-silicotoxicology approaches to estimate specific risk profiles of agrochemicals will havean emerging impact in the future. In the search for a so-called ‘‘optimal product’’ inmodern crop protection in terms of efficacy, environmental safety, user friendliness,and economic viability, the halogen substitution of active ingredients is increasinglyrecognized as a very important tool.

In Part II are described new methods for identifying the modes of action of activeingredients. Reverse-genetic approaches such as RNA interference (RNAi) offeruseful tools to elucidate modes of action, to identify novel targets for exploitation,or to help create new generations of crop protection technologies. For severalyears, the rapid identification of herbicidal modes of action has been possible viagene expression profiling, using DNA chips. An elucidation of the target sites ofneuromuscular insecticides at an early stage in their discovery and development canplay an important role in the prioritization of selected candidates. However, despitegreat technological progress having been made, the targeted discovery of novelfungicides remains an immense challenge because of the restrictions that havebeen posed on new active ingredients by the obligatory physico-chemical propertiespermitting a sufficient bioavailability that will, in turn, guarantee fungicidal activity.

In Part III, new methods are examined to improve the bioavailability of the activeingredients. According to novel trends in application technologies, an innovativeformulation comprises a mixture of various molecules, besides the active ingredient.In this context, the influence of polymorphism and the organic solid state onthe quality and efficiency of agrochemicals plays an important role. Molecular

Preface XVII

descriptors, as defined by Abraham, can be used to set up linear free energyrelationships (LFERs) of relevance to agrochemical research and environmentalfate.

Finally, modern methods for risk assessment are addressed in Part IV. Today,many tools are available that can be used to assimilate the knowledge required toevaluate human health and environmental safety, such as exposure modeling, invitro models to evaluate phenotypic and gene expression changes, computationaltoxicology, bioinformatics, and systems biology. Despite its complexity and a lackof experience of its use, environmental effect modeling has a great potential forregulatory risk assessments with modern crop protection products, although atpresent its use is not yet fully accepted. In Chapter 14, entitled Safety Evalua-tion of New Pesticide Active Ingredients: Enquiry-Led Approach to Data Generation,attention is focused heavily on advances in molecular biology and biotechnology,and how these may be used in conjunction with computational toxicology andbioinformatics to make toxicity testing more relevant to low-level human expo-sures, to reduce the need for in-vivo testing in animal models, and to make thewhole process of hazard data generation quicker and less expensive. In parallel,an evaluation of the endocrine disruption definition and screening aspects in lightof the European Crop Protection Law has led to a proposal for decision crite-ria for endocrine-disrupting compound (EDC) regulatory agencies. This aspect isdiscussed, taking into consideration the scientific needs of the near future.

We hope that this book will prove to be an invaluable source of information forall of those people working in crop protection science – whether as governmentalauthorities, as researchers in agrochemical companies, scientists at universities,conservationists, and/or managers in organizations and companies involved withmaking improvements in agricultural production – to help nourish a continuouslygrowing world population, and to advance the production of bioenergy.

Note

Within this book the authors have named the products/compounds preferablyby their common names. Although, occasionally, registered trademarks are cited,their use is not free for everyone. In view of the number of trademarks involved, itwas not possible to indicate each particular case in each table and contribution. Weaccept no liability for this.

May 2012 Peter JeschkeWolfgang Kramer

Ulrich SchirmerMatthias Witschel

XIX

List of Contributors

Doerthe Ahlbory-DiekerMetanomics GmbHTegeler Weg 3310589 BerlinGermany

Roland BuesenBASF SEExperimental Toxicologyand EcologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Marco BuschBayer CropScience SASite La Dargoire14-20 rue Pierre BaizetB.P. 916369263 Lyon Cedex 09France

Eric D. ClarkeSyngenta LtdJealott’s Hill InternationalResearch CentreBracknellBerkshire RG42 6EYUK

Daniel CordovaDuPont Crop ProtectionChemical Genomics GroupStine Haskell Research Center1090 Elkton Rd.Newark, DE 19714USA

Mark DrewesBayer CropScienceBCS-R & D Bldg 6250Alfred-Nobel-Str. 5040789 Monheim am RheinGermany

Peter EckesBayer CropScience AGBiology Weed ControlIndustriepark HoechstBldg. H872N65926 FrankfurtGermany

Bernd EssigmannBayer CropScience SASite La Dargoire14-20 rue Pierre BaizetB.P. 916369263 Lyon Cedex 09France

XX List of Contributors

Eric FabianBASF SEExperimental Toxicologyand EcologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Burkhard FlickBASF SEExperimental Toxicologyand EcologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Michael HeroldMetanomics GmbHTegeler Weg 3310589 BerlinGermany

Peter JeschkeBayer CropScienceBCS AG-R&D-CPR-PC-PCCChemistry 2Bldg. 6510Alfred-Nobel-Str. 5040789 Monheim am RheinGermany

Hennicke G. KampBASF SEExperimental Toxicology andEcologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Susanne N. KolleBASF SEExperimental Toxicologyand EcologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Gerhard KrennrichBASF SEExperimental Toxicologyand EcologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Edgar LeiboldBASF SEExperimental Toxicologyand EcologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Ralf LooserMetanomics GmbHTegeler Weg 3310589 BerlinGermany

Laura J. MallonSyngenta LtdJealott’s Hill InternationalResearch CentreBracknellBerkshire RG42 6EYUK

List of Contributors XXI

Werner MellertBASF SEExperimental Toxicologyand EcologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Britta OlenikBayer HealthCare AGFriedrich-Ebert-Str. 33342069 WuppertalGermany

Eric PagetBayer CropScience SASite La Dargoire14-20 rue Pierre BaizetB.P. 916369263 Lyon Cedex 09France

Paul ParsonsSyngenta LimitedToxicology and Health ScienceJealott’s Hill InternationalResearch CentreBracknellBerkshire RG42 6EYUK

Rolf PontzenBayer CropScience AGFormulation TechnologyAlfred-Nobel-Straße 5040789 Monheim am RheinGermany

Alexandre ProkoudineMetanomics GmbHTegeler Weg 3310589 BerlinGermany

Tzutzuy RamirezBASF SEExperimental Toxicologyand EcologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Bennard van RavenzwaayBASF SEExperimental Toxicologyand EcologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Michael SchindlerBayer CropScience AGResearch Building 6500Alfred-Nobel-Str. 5040789 MonheimGermany

Klaus-Jurgen SchleiferBASF SEComputational Chemistryand BiologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Frederic SchmittBayer CropScience SASite La Dargoire14-20 rue Pierre BaizetB.P. 916369263 Lyon Cedex 09France

Walter SchmittBayer CropScience AGEnvironmental SafetyAlfred-Nobel-Str. 5040789 Monheim am RheinGermany

XXII List of Contributors

Peter SchreierBayer CropScience AGBCS-R-DCM Bldg. 6240Alfred-Nobel-Str. 5040789 Monheim am RheinGermany

Thomas C. SparksDow AgroSciences9330 Zionsville RoadIndianapolis, IN 46268USA

Volker StraussBASF SEExperimental Toxicologyand EcologyCarl-Bosch-Str. 3867056 LudwigshafenGermany

Gerhard ThielkingBayer CropScience AGAlfred-Nobel-Str. 5042117 WuppertalGermany

Klaus TietjenBayer CropScience AGBCS-R-DCM Bldg. 6240Alfred-Nobel-Str. 5040789 Monheim am RheinGermany

Arnoldus W.P. VermeerBayer CropScience AGFormulation TechnologyAlfred-Nobel-Straße 5040789 Monheim am RheinGermany

Tilmann WalkMetanomics GmbHTegeler Weg 3310589 BerlinGermany

Jan WiemerMetanomics GmbHTegeler Weg 3310589 BerlinGermany

1

Part IMethods for the Design and Optimizationof New Active Ingredients

Modern Methods in Crop Protection Research, First Edition.Edited by Peter Jeschke, Wolfgang Kramer, Ulrich Schirmer, and Matthias Witschel.© 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

3

1High-Throughput Screening in Agrochemical ResearchMark Drewes, Klaus Tietjen, and Thomas C. Sparks

1.1Introduction

Efficient and economical agriculture is essential for sustainable food productionfulfilling the demands for high-quality nutrition of the continuously growingpopulation of the world. To ensure adequate food production, it is necessary tocontrol weeds, fungal pathogens, and insects, each of which poses a threat ofyield-losses of about 13–15% before harvest (Figure 1.1). Although a broad rangeof herbicides, fungicides and insecticides already exists, shifts in target organismsand populations and increasing requirements necessitate a steady innovation ofcrop-protection compounds.

Weeds, fungal pathogens and insects belong to evolutionary distinct organ-ism groups (Figure 1.2), which makes it virtually impossible to have a singlecrop-protection compound capable of addressing all pest control problems. Oncloser examination, even the grouping of pests simply as insects, fungi and weedsis, in many cases, still an insufficient depiction. Although the term ‘‘insecticide’’is often used for any chemical used to combat insects, spider mites or nematodes,the differences between these organisms are so significant that it is more preciseto speak of insecticides, acaricides, and nematocides. Among plant pathogenicfungi, the evolutionary range is even much broader and oomycetes are not fungiat all, although oomyceticides commonly are also commonly referred to as ‘‘fungi-cides’’. Hence, the agrochemical screening of fungicides and insecticides requiresa substantial range of diverse species. The situation for herbicide screening is,in some ways, the reverse, but is no easier. Indeed, the close genetic similaritybetween crop and weed plants generates challenges with regards to the specificityof herbicidal compounds, in differentiating between crop and weed plants. Thisalso results in a need to use a range of different crop and weed plants in screeningprograms.

In light of the above circumstances, agrochemical screening has employed, inboth laboratory and glass-house trials, a wide spectrum of model and pest species.The recent developments described in this chapter, however, have allowed aneven higher throughput not only in glass-house tests on whole organisms, but

Modern Methods in Crop Protection Research, First Edition.Edited by Peter Jeschke, Wolfgang Kramer, Ulrich Schirmer, and Matthias Witschel.© 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

4 1 High-Throughput Screening in Agrochemical Research

Post-harvest losses

Pre-harvest losses

60 % of crop area worldwide

50

40

30

20

1015 % insects

13 % fungal pathogens

14 % weeds

10 %

Figure 1.1 Losses of potential agricultural harvest of major crops due to different pests,diseases, and weeds [1, 2]. Non-treated, approximately 50% of the harvest would belost.

also the exploitation of biochemical (in vitro) target tests. Not surprisingly, theimplementation of molecular screening techniques and the ‘‘omics’’ technolo-gies – functional genomics, transcriptomics and proteomics, etc. – into agrochem-ical research has been a major challenge due to the high diversity of the targetorganisms [5].

Molecular agrochemical research with biochemical high-throughput targetscreening commenced with several model species, each of which was chosenmainly because of their easy genetic accessibility or specific academic interests.These first favorite model organisms of geneticists and molecular biologists werelargely distinct from the most important pest species in agriculture, however.Nonetheless, recent progress in genome sequencing has led to a steadily growingknowledge about agronomically relevant organisms (Figure 1.3 and Table 1.1).

The situation is relatively simple for weeds, as all plants are closely related(Figure 1.2). The first model plant to be sequenced, Arabidopsis thaliana, is geneti-cally not very distinct from many dicotyledonous weeds, and the monocotyledonouscrops are closely related to the monocotyledonous weeds which, in turn– startingseveral thousand years ago – formed the foundation for today’s cereals species. Thefirst sequenced insect genome of Drosophila melanogaster, a dipteran insect, wasexploited extensively in both genetic and molecular biological research. To betterreflect relevant pest organisms such as lepidopteran pests or aphids, species such asHeliothis virescens (tobacco budworm) and Myzus persicae (green peach aphid) havebeen investigated by the agrochemical industry, while Bombyx mori, Acyrthosiphonpisum and Tribolium castaneum have been sequenced in public projects (Table 1.1).Baker’s yeast, Saccharomyces cerevisiae, has long been the most commonly usedmodel fungus, while the ascomycete Magnaporthe grisea and the ustilaginomyceteUstilago maydis have been the first sequenced relevant plant pathogens. It is certainthat, within the next few years, even the broad evolutionary range of the manydifferent plant pathogenic fungi and oomycetes (see Figure 1.2) will be included ingenome projects.

1.1 Introduction 5

Rough time scale in billion years before present

Oxy

gen-

free

Oxy

gen

3.6

Arche-bacteria

Eubac-teria

Ecdysozoa

Metazoa

Protostomia

Coelomata

Deuterostomia

MammalsMammals Fishes SpidersNematodesNematodes

InsectsInsectsButter-

fliesLice

Plants

Mono-cots

Pre-cellPre-cell

Mito-chondria Photosynthetic

Cyanobacteria

Oomy-

Fungi Proto-zoa

Nucleus

Yeast Basidiomycota Ferns

Brownalgae

Di-cots

Ascomycota

Plastids

Cyano-

O p h i s t o k o n t aO p h i s t o k o n t aArche-plastida

Chrom-alveolata

Amoebozoarhizaria

excavata

UstilaginomycetesUrediniomycetes

Animalia

ChordataArthropods

0

1

2

3

Oomy-

Red algae Greenalgae

Fungi

cetesBac-teria

Figure 1.2 Modern evolutionary tree of life. The view is based on Refs [3, 4]; for a more detailed view of fungi, seeRef. [5].

6 1 High-Throughput Screening in Agrochemical Research

Drosophila

Arabidopsis

Yeast Magnaporthe

Caenorhabditis

Common model organisms Relevant organisms

Ustilago

Heliothis Myzus

Oryza SetariaArabidopsis

Figure 1.3 Model organisms in molecular biology and agronomically relevant targetspecies.

1.2Target-Based High-Throughput Screening

1.2.1Targets

The progress of molecular biology of agronomically relevant organisms hasenabled the introduction of target-based biochemical (in vitro) high-throughputscreening (HTS), which has significantly changed the approach to the screening foragrochemicals during the past 15 years. Target-based HTS is a technology utilizedin the agrochemical industry to deliver new actives with defined modes of action(MoA) [6].

Most major research-based agrochemical companies have established biochemi-cal HTS, often conducted in cooperation with companies having special expertisein specific fields of biotechnology. The first wave of genomics – which includedgenome-wide knock-out programs of model organisms – indicated that aboutone-quarter of all genes are essential; that is, they were lethal by knock-out [6–8].The resulting high number of potential novel targets for agrochemicals must befurther investigated to clarify the genes’ functions (reverse genetics) and to betterunderstand their role in the organism’s life cycle. Although the technology ofgenome-wide knock-out itself was highly efficient and well established, it tran-spired that even the knock-out of some known relevant targets were not lethal,either because of genetic or functional redundancy, counter-regulation, or becausea knock-out does not perfectly mimic an agonistic drug effect on, for example, ionchannels. Consequently, knock-out data are today reviewed critically with respectto as many aspects as possible of the physiological roles of potential targets and,as a result, they are taken as just one argument for a gene to be regarded as