Rothamsted Research - Field evolved polygenic … · Web viewPenicillium digitatum and probably...

Abstractsof oral

presentations

1

What does resistance mean in the field?

Jon Knight

The talk will highlight the impact that the development of pesticides resistance across pests, weeds and diseases is having on the ability of farmers and growers to remain financially viable and deliver high quality and safe food to the public. It is, of course, only one of the pressures acting on businesses but it may be less visible than some others, more complex to understand and manage, and possibly more tempting to ignore.

On the one hand, the loss of actives, especially in minor use crops, makes it increasingly difficult to implement successful resistance management strategies, whilst on the other, the increase, albeit somewhat slow in some parts of the world, of new biologically derived actives offer new modes of action that present significant opportunities for robust strategies to be developed and implemented.

Since the farmer is the last link in the chain in resistance management it is imperative that the management strategies are fit for purpose in that they can be easily understood and implemented into production systems without undue cost either in terms of money or time.

2

Putting Resistance Management into Practice: The Industry Perspective

Andrew Ward CropLife International and its associations work with the global herbicide, fungicide, insecticide and rodenticide Resistance Action Committees (RACs) to catalyze the deployment of resistance management by farmers and growers across the globe. Key elements of this are i) the activities of the RACs, 2) advocacy work to promote the inclusion of Mode of Action (MoA) classification information on crop protection product labels and 3) the effective promotion of resistance management information to farmers.

The purpose of the global RACs are to ensure cross industry monitoring of the resistance situation and to provide resistance management guidelines to prolong the effectiveness of crop protection agents. They achieve this through identifying resistance management knowledge requirements and then generting technical resistance management information which is shared within the industry and broader research groups involved in resistance management. The RACs promote resistance management information by themselves but also through the CropLife Resistance Management Project Team who promote resistance management as part of global stewardship activities. These target both farmers and policy makers. MoA information provision simplifies the advice provided to farmers on pesticide resistance management. The six CropLife International member companies have committed to including MoA information on their produce labels by 2023. However, for farmers to make informed decisions, they will need all products, manufactured by all companies, to display MoA information and this MoA information needs to be from the same classification scheme. Meanwhile farmers need to be made aware of how to use the MoA information for resistance management and to be clear on the benefits of implementing resistance management strategies. CropLife International is also looking for effective resistance management and therefore is seeking to increase the scale and sustainability of resistance management knowledge promotion, through working with appropriate partners, e.g. CropLife stewardship initiatives, extension services, Farmers’ Unions, NGOs etc. to promote and reinforce messaging on resistance management.

3

Non target-site resistance mechanisms against agricultural fungicides, the tip of the iceberg?

Sabine Fillinger

UMR BIOGER, INRA, AgroParisTech, Université Paris-Saclay, Thiverval-Grignon, France

E-mail: [email protected]

Most (if not all) plant pathogenic fungi have evolved resistance to agricultural fungicides world-wide. Over decades target site modifications were reported as major resistance mechanisms against these drugs, conferring specific resistance to a single mode-of-action. Especially against demethylation inhibitors (DMIs), several fungal species have evolved multiple, successive alterations in the target Cyp51 protein. Also target site overexpression was shown to reduce susceptibility to specific fungicides. In recent years, however, evidence for more complex fungicide resistance mechanisms has increased, their elucidation facilitated by genomic approaches.

Thus, increased drug efflux, affecting several unrelated modes of action, has been detected in the fungal pathogens Botrytis cinerea, Sclerotinia homoeocarpa, Oliculimacula yallundae, Zymoseptoria tritici, suspected in Penicillium digitatum and probably other fungal species. This mechanism, also referred to as multidrug resistance (MDR), is well known in the clinical sector where it impacts antiviral, antibacterial, antifungal and anti-cancer therapies. Its relevance was underestimated in the agricultural sector for a long time. Constitutive overexpression of membrane-transporters is at the orgin of MDR; its responsible mutations in fungal field strains may reside in the transporter genes themselves or in transcription factor genes. While MDR on its own does not compromise fungicide efficacy at field-rates, the combination with target site modification and other resistance mechanisms increases the respective resistance levels and thereby reduces fungicide efficacy. Evidence of fungicide detoxification through metabolization is rather scarce. In the genus Botrytis fungicide metabolization by a cytochrome P450 mono-oxygenase is partially responsible for the selectivity of the hydroxyanilide fenhexamid. The same type of mechanism was shown to be involved in multidrug resistant field isolates of the turf grass pathogen S. homoeocarpa.

These few, relatively new cases underline the strong adaptive potential of plant pathogenic fungi to agricultural fungicides. More discoveries are to come, as pointed out by some recent whole genome association studies. Which type of mechanism may be more threatening to disease control, how they will evolve and recombine remains then to be established.

4

mailto:[email protected]

Resistance to synthetic pyrethroids in Helicoverpa armigera worldwide via an unusual cytochrome P450 enzyme

David G. Heckel, Nicole Joussen and Jeffrey Choon Wei Wee

Department of Entomology, Max Planck Institute for Chemical Ecology, Jena, Germany


Fenvalerate resistance in Helicoverpa armigera on Australian cotton was reported in 1983. Over the next 15 years, resistance levels steadily increased within and decreased between growing seasons, producing a sawtooth-like pattern with an overall increasing trend. Contemporaneous studies identified the main mechanism as detoxification by one or more cytochromes P450. Our more recent mapping studies have identified a single P450 gene Cyp337B3. Sequencing of BAC clones showed that Cyp337B3 is a chimeric gene with a shorter 5' portion originating from Cyp337B2 and a greater 3' portion from Cyp337B1, produced by unequal crossing-over. Two haplotypes have been found in Australian populations: one with Cyp337B2 and Cyp337B1 in a tandem array, and the other with Cyp337B3 only. Heterologous expression of the three enzymes has shown that CYP337B3 but neither CYP337B1 nor CYP337B2 detoxifies fenvalerate by hydroxylation at the 4' position. Systematic site-directed mutagenesis of the N-termini of CYP337B1 and CYP337B3 has shown that no single amino acid substitution is responsible for the difference in activity, suggesting that formation of a chimeric gene with several substitutions was a necessary preadaptation for this type of resistance. Remarkably, CYP337B3 is found in most populations of H. armigera worldwide, including the recent invasion into Brazil.

5


The use of genome-wide association to identify mutations associated with DMI resistance in Cercospora beticola

Rebecca E. Spanner, Viviana Rivera-Varas, Linda L. Young, Gary A. Secor and Melvin D. Bolton

Department of Plant Pathology, North Dakota State University and U.S. Dept. Agriculture, United States


Cercospora leaf spot (CLS) caused by Cercospora beticola is one of the more destructive foliar diseases of sugarbeet worldwide. In the United States, losses to CLS approached $200 million in the 2018 growing season alone. Management strategies for CLS rely on timely fungicide application. The CLS management fungicide repertoire often includes the application of fungicides in the sterol demethylation inhibitor (DMI) class. The reliance on DMIs has led to the emergence of resistance in many C. beticola populations. Previously, we showed that DMI-resistant strains of C. beticola have high expression levels of Cyp51, which are induced further upon DMI exposure. However, no mutations in the Cyp51 coding or promoter regions appeared to correlate with DMI resistance. To identify mutations responsible for DMI resistance, a genome-wide association approach was undertaken using 194 isolates harvested from local sugarbeet fields. Approximately half of the isolates were DMI-sensitive (EC50 value <1.0 µg mL-1) while the other half were DMI-resistant (EC50 value ≥1.0 µg mL-1). After whole genome resequencing, genome wide association identified two loci highly correlated with DMI-resistance. Additionally, one of the two loci was also highly correlated in strains capable of inducible over-expression of Cyp51. The results of this study will be reported.

6


Overexpression of GSTs is associated with metabolic herbicide resistance to several pre-emergence herbicides in blackgrass

Anita Küpper1, Evlampia Parcharidou2, Rebecka Dücker2, Veronika Brabetz1 and Roland Beffa1

1Bayer Crop Science, Frankfurt, Germany, 2University of Göttingen, Frankfurt, Germany


The increasing resistance pattern to post-emergence herbicides (e.g. ACCase and ALS inhibitors) in grasses leads to an increase in reliance on pre-emerging herbicides and requires more complex weed management strategies. Especially the pre-emergent herbicide flufenacet has gained in importance to control multiple-resistant blackgrass (Alopecurus myosuroides Huds.) in European winter wheat. Screening of several blackgrass populations from various European countries generally showed control at the registered field rate of flufenacet. However, a few populations showed survivors. The observed reduced sensitivity correlated with glutathione-S-transferase (GST)-mediated enhanced metabolism. The same populations also showed reduced sensitivity to the pre-emergence herbicides pendimethalin, prosulfocarb, S-metolachlor, and pethoxamid. The use of flufenacet in combinatione with diflufenican or particularly flurtamone or metribuzin again restored sensitivity in blackgrass populations less sensitive to flufenacet. Transcriptome analyses revealed five GSTs to be overexpressed in flufenacet-resistance plants. The genes differed from the GSTs found in flufenacet-resistant ryegrass populations. It is crucial to apply best weed management practices (e.g. application of full dose rates and herbicide mixtures and wide crop rotations) while there is low practical resistance to reduce selection pressure and to protect the efficacy of one of the few herbicides still effective in the control of multiple-resistant blackgrass.

7


Rapid adaptive evolution of herbicide resistance via extrachromosomal DNA amplification

Mithila Jugulam

Kansas State University, Manhattan, KS, USA

Email: [email protected]

Evolution of glyphosate resistance in weeds is a major crop constraint globally. Several mutations including amplification of enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the molecular target of glyphosate bestow weed resistance to this herbicide. Our molecular cytogenetic analyses revealed facinating information related to fundamental, evolutionary mechanisms of glyphosate resistance in Amaranthus palmeri and Lolium multiflorum. The EPSPS gene amplification in glyphosate-resistant A. palmeri was driven by extra-chromosomal, circular DNA (eccDNA) molecules. Each eccDNA can multiply rapidly during the growth of the sporophyte and produce copy number variation in somatic cells. The somatic cells with amplified EPSPS survive the herbicide treatment, and this acquired trait is transmitted to the germ cells and the progeny. Similarly, it appears that in glyphosate-resistant L. multiflorum also the amplified copies of the EPSPS gene may not have integrated into the chromosome but exist as extra-chromosomal (i.e., ecDNA) elements. Based on our results, it is likely that in response to glyphosate stress, the extrachromosomal DNAs drive the amplification of EPSPS gene resulting in the rapid evolution of glyphosate resistance. This is a completely novel mechanism of evolution of glyphosate resistance and has vast implications for the development and management of glyphosate resistance.

8


Exploring receptor/insecticide interactions of the nicotinic acetylcholine receptor gene family using CRISPR/CAS9

Trent Perry1, Wei Chen1, Razi Ghazali1 Danielle Christesen1, Ying Ting Yang1, Hang Ngoc Bao Luong1,2

and Philip Batterham1

1School of BioSciences and Bio21 Institute, The University of Melbourne, AUSTRALIA2Institute Molecular Biology Biotechnology FORTH, Heraklion, Crete


The nicotinic acetylcholine receptors (nAChRs) are a gene family found from worms to humans, that facilitate synaptic signalling through their conversion of a chemical to an electrical signal. Insect nAChRs have been a regular target of insecticides and the loss of receptor function or structural changes can lead to high levels of resistance. The functional nAChR is a pentamer of subunits encoded by multiple genes, hence many combinations are possible, yet these are not well characterised. With the capacity to efficiently use CRISPR/CAS9 gene editing in Drosophila melanogaster, we systematically deleted or modified nAChR genes to identify those targeted by insecticides. Five nAChR subunits were identified as targets of at least one of the neonicotinoid, sulfoximine or spinosyn insecticide classes. Negative cross resistance was also found, suggesting compensatory changes in nAChR expression occur in some receptor deletion mutants. Further analysis is beginning to uncover the many roles these receptors play in insect behaviour, with subunit mutations impacting critical behaviours such as sleep and courtship. Our findings define the receptor subunits targeted by insecticides, providing a greater understanding of possible target site resistance mechanisms, their likelihood of evolving in the field and should help to inform resistance monitoring and management programs.

9


Resistance to ametoctradin, cyazofamid and amisulbrom in French populations of Plasmopara viticola: new resistances and increasing occurrence

Séverine Fontaine, Florent Remuson, Laetitia Caddoux and Benoit Barrès

USC CASPER, Anses Laboratoire de Lyon, France


Complex III inhibitors are key compounds in the control of Plasmopara viticola, the causal agent of downy mildew of grapes. But they are prone to evolution of resistance against them, as demonstrated by the emergence of resistance to QoIs in the past. By using a combination of bioassays and molecular methods, we monitored sensitivity to ametoctradin, cyazofamid and amisulbrom in P. viticola populations in French vineyards from 2012 to 2018. Two type of resistance mechanisms were identified: AOX-related resistance and target-site resistance to both ametoctradin and cyazofamid. We found that the AOX-related resistance mechanism was common in French P. viticola populations and its occurrence in populations was increasing over the years. Target-site resistance to ametoctradin was first detected in 2015 and is likely caused by a single point mutation in cytochrome b gene, leading to the S34L substitution. To date, the frequency of this mutation is low in P. viticola population. Target-site resistance to cyazofamid was first detected in 2016 and was found to be associated with the E203-DE-V204 insert in cytochrome b. This latter resistance seems to cause local loss of efficacy. These two types of resistance mechanisms could involve different management strategies and should be monitored carefully.

10


A triple target-site mutation confers high levels of resistance to glyphosate in an Amaranthus quitensis population from Argentina

Sarah-Jane Hutchingsa, Eddie McIndoea, Ryan Carlinb, Wenjin Yub, Anushka Howella, Weining Gub, Wenling Wangb, Mike Langforda, Jo Mattocksa and Shiv-Shankar Kaunduna

a Syngenta, Jealott’s Hill International Research Centre, RG42 6EY Bracknell, UKb Syngenta, Research Triangle Park, NC USA

E:mail: [email protected]

Resistance to glyphosate has evolved and is quickly spreading in Amaranthus quitensis due to excessive use of the EPSPS-inhibiting herbicide in Argentinian soybean production systems. Here, we confirmed resistance to glyphosate and determined the mechanism involved in an A. quitensis population (AMAQU-R) from Santa Fe Province, Argentina. AMAQU-R plants survived glyphosate rates as high as 48 kg ai/ha, whereas individuals from the sensitive populations (AMAQU-S1 and AMAQU-S2) were killed at 750 g ai/ha or below. Differential glyphosate uptake, movement or metabolism was not associated with resistance in AMAQU-R and only a small average relative increase (1.94) in EPSPS gene copy number/expression was detected in AMAQU-R. Three linked EPSPS mutations were identified around the binding site of glyphosate in AMAQU-R. These comprised T102I and P106S amino acid substitutions documented to confer high levels of glyphosate resistance, as well as a novel A103V target-site mutation. A 3.5-fold more tolerance to glyphosate was observed for the TIAVPS mutant compared to the double TIPS strain following heterologous gene expression and enzyme analysis. The triple glyphosate resistance mutations may explain the rapid spread of A. quitensis in Argentina and could be a real threat for neighbouring countries under similar glyphosate selection pressure.

11


The molecular innovations underlying resistance to natural and synthetic xenobiotics in the aphid Myzus persicae

Chris Bass

University of Exeter, College of Life and Environmental Sciences, Penryn, UK


The process by which genetic novelty is created and drives the evolution of key innovations required for ecological adaptation is still relatively poorly understood. In this talk I will outline recent work investigating the mutational events driving host-range expansion and xenobiotic resistance in the peach potato aphid, Myzus persicae, the most economically important aphid pest worldwide. M. persicae is globally distributed and highly polyphagous with a host range of over 400 species including many important crop plants. Relatively recently this species host-shifted to tobacco, and we have previously demonstrated that the constitutive overexpression of a cytochrome P450, CYP6CY3, allows tobacco-adapted races of M. persicae to efficiently detoxify nicotine and has preadapted them to resist neonicotinoid insecticides. Our recent work has employed a suite of genomic, transcriptomic and post-genomic functional approaches to demonstrate the role of chromosomal rearrangements and transposable elements in creating the genetic novelty required for M. persicae to resist xenobiotics and exploit a new ecological niche.

12

The molecular genetic mechanisms underlying acaricide resistance in the spider mite Tetranychus urticae

Thomas Van Leeuwen1 and Richard M. Clark2

1Department of Plants and Crops, Ghent University2School of Biological Sciences, University of Utah


The spider mite Tetranychus urticae is a global pest known to feed on 1,100 different hosts from 140 plant families, including most major crops. Due to a very strong adaptation potential, it is one of the most notorious organisms for insecticide/acaricide resistance development. With experimental advances and new tools developed for T. urticae, we are now poised for fundamental advances in understanding the molecular genetic make-up of this extreme adaption.

We have generated a large collection of fully inbred and resistant mite strains and describe the sampled genomic variation in the context of selection and adaptation. Patterns of linkage disequilibrium and long haplotype blocks are consistent with a modest but recent bottleneck that may be associated with spread as an agricultural pest. Multiple striking examples of selective sweeps are apparent, including at loci associated with pesticide resistance. Next, using dedicated crosses between these inbred lines, we have studied gene regulation mechanisms on a genome-wide scale. We report here on a number of striking examples where gene copy number variation is associated with acaricide resistance, but also start to unravel the global architecture of gene expression variation in a generalist herbivore.

13


Relevance of pathogens´ mutant patterns and resulting molecular aspects of resistance for sustainable fungicide resistance management

Andreas Mehl and Jürgen Derpmann

Bayer AG, Division Crop Science, 40789 Monheim, Germany


Molecular detection of fungicide resistance is a growing field in agrochemical research due to increasing reports of resistance or insufficient performance of fungicide applications. As it is today quite challenging for agrochemical companies to develop and introduce new fungicides with novel modes of action, the management of fungicide resistance evolution in plant pathogens is an important issue not only to the agrochemical industry, but also to government officials, academia, advisers, and finally to farmers.

Therefore, particularly for SBI-, QoI-, and SDHI-fungicides meanwhile a multitude of mutants have been described of major pathogens and crops inside and outside of Europe, partly showing incomplete or even no cross-resistance between active ingredients of the same mode of action class, while others clearly impact all compounds to the same degree or are less competitive to wild-type isolates due to obvious fitness penalties.

Examples of recent findings with mutants of important fungal pathogens in soybean, cereals, or oilseed rape are discussed, including cases of incomplete cross-resistance and their potential impact on future disease- and resistance management strategies.

14


Novel herbicide resistance mechanisms to synthetic auxins in weeds

Neeta Soni1, Marcelo Rodrigues Alves de Figueiredo1, Olivia Todd1, Dean Pettinga1, Eric Patterson2, Cristiana Argueso1, Anita Kuepper3, Christopher Preston4, Jenna Malone4 Phil Westra1, Franck Dayan1, and

Todd Gaines1

1Bioagricultural Sciences and Pest Management, Colorado State University, US; 2 Department of Plant, Soil and Microbial Sciences, Michigan State, US; 3Weed Control Research, Bayer Crop Science;

4Agriculture, Food and Wine, University of Adelaide, Australia.


Synthetic auxin herbicides mimic the endogenous plant hormone indole-3-acetic acid (IAA). Commercialization of synthetic auxin tolerant crops is expected to increase the evolutionary resistance frequency in weed species. Here we showed our most recent work understanding target site (TS) and non-target site (NTS) molecular mechanisms in 3 weed species. Further studies on cross resistance in the Bassia scoparia population containing the G127N mutation suggested that is not sufficient to resist 2,4-D and fluroxypyr. We are currently investigating the association between this TS mutation and the NTS reported mechanism related to chalcone synthase in more susceptible and dicamba resistant populations. We identified a different B. scoparia dicamba resistant population that shows a novel mutation in an auxin co-receptor. It is hypothesized that a 9 amino acid deletion in the auxin co-receptor IAA-2 confers resistance to 2,4-D in Sisymbrium orientale. A 2,4-D resistant Amarantus tuberculatus population showed a higher metabolism rate. It was identified a 2,4-D hydroxylation mediated by P450. This is the first case where a common monocot metabolism pathway is identified in a dicot species. Currently, we are conducting further functional validation and heterologous expression to confirm if the identified TS and NTS mechanisms confer synthetic auxin resistance.

15


The role of sexual reproduction in pathogen adaptation to fungicide mixtures

Alexey Mikaberidze1,2 and Hildegard Uecker3

1ETH Zurich, Institute of Integrative Biology, Universitaetstrasse 2,ETH Zentrum, LFW B28 CH-8092 Zurich Switzerland

2University of Reading, School of Agriculture, Policy and Development,Whiteknights, Reading, RG6 6AR, UK

3Max Planck Institute for Evolutionary Biology, Department of Evolutionary Theory,August-Thienemann-Strasse 2, 24306 Plön, Germany


Fungicides are often applied as mixtures of two components belonging to different modes of action. This exerts a directional selection for pathogen strains resistant to both components of the mixture (double resistance). Since, many important fungal pathogens of crop plants undergo ample sexual reproduction, sexual recombination may accelerate the emergence of double resistance by bringing together mutations conferring resistance to each of the individual components of the mixture. Sex has been previously found to speed up adaptation of yeast populations in vitro [McDonald et al., Nature 2016]. Does a similar effect occur in populations of crop pathogens exposed to fungicide mixtures? To address this question, we formulated a stochastic eco-evolutionary model of pathogen populations that incorporates sexual reproduction and parameterised it for Zymoseptoria tritici, an important wheat pathogen. According to our model, sex does accelerate emergence of double resistance across wide ranges of the strength of selection and the mutation rate. Thus, fungicide mixtures may prove to be an inappropriate long-term strategy to control crop diseases caused by sexually-reproducing pathogens. More studies on specific pathosystems and fungicide modes of action are needed to investigate further the durability of fungicide mixtures with respect to emergence to double resistance.

16


Insecticide resistance in Myzus persicae: can it be slowed?

Paul A. Umina*, Garrick McDonald*, James Maino*, Owain Edwards#, Ary A. Hoffmann*

* School of BioSciences, The University of Melbourne, Parkville, Victoria, AUSTRALIA#CSIRO Land & Water Flagship, Perth, WA, AUSTRALIA


Chemicals are too often used as the major tool for arthropod pest management, ignoring evolutionary forces that lead to resistance. Considerable research funds are spent understanding insecticide resistance (e.g. field surveillance, testing methodologies, exploring evolutionary processes and molecular mechanisms). This knowledge should inform the development of Insecticide Resistance Management Strategies (IRMSs), but these are often not widely adopted by farmers.

In Australia, an IRMS has recently been developed for Myzus persicae in grain crops. M. persicae is a global agricultural pest with a high propensity to evolve resistance. The IRMS is underpinned by principles relating to the judicious use of insecticides and IPM. It was first provided to growers in 2014 and has since been updated based on surveillance data. Unfortunately, the IRMS appears to have been unsuccessful in reducing selection for further resistance. Metabolic resistance to neonicotinoids has spread over the last 4 years to now encompasses the whole continent. Carbamate, pyrethroid and organophosphate resistance continue to be common and widespread. Low-level resistance to sulfoximines has also very recently emerged in some fields. A successful IRMS will likely require a much greater emphasis on the social and political context contributing to current chemical application patterns.

17


Fungicide resistance evolution in global populations of the wheat pathogen Zymoseptoria tritici

Guilherme Rossato Augusti, Michael W. Shaw, Nichola J. Hawkins and Bart A. Fraaije

Department of Biointeractions and Crop Protection, Rothamsted Research, UK


The wheat pathogen Zymoseptoria tritici, causal agent of septoria tritici blot (STB), has a great risk of developing fungicide resistance. In NW-Europe, methyl benzimidazole carbamates (MBC) and quinone outside inhibitors (QoI) are no longer used for STB control because of complete resistance, leaving azoles (demethylation inhibitors-DMI) and succinate dehydrogenase inhibitors (SDHI) as options for disease management. Decline of fungicide effectiveness has been observed in other countries where STB also poses as a threat for wheat production. This research has assessed the fungicide sensitivity of 10 globally distributed populations of Z. tritici. European isolates appear to be replacing the local genetic background in South America and New Zealand and for this reason populations from these continents are less sensitive for azole fungicides and are evolving convergently towards the more resistant NW-European genotypes. Nonetheless, the North American populations are slowly evolving azole resistance independently to European populations. QoI and MBC resistance are widespread all over the globe, although at lower frequency for non-European populations. Mutants with altered sensitivity to SDHI fungicides are already present in European field populations. No shifts in bixafen sensitivity were detected outside Europe, but 60% of Oregon isolates are insensitive to up to 1ppm of fluopyram.

18


Population genomics of herbicide resistance in Alopecurus myosuroides

Sonja Kersten, Fernando A. Rabanal, Ulrich Lutz and Detlef Weigel

Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany


Unintendedly, humans have been carrying out a series of –often alarming– evolutionary “experiments” that have led to the selection for resistance to herbicides in agricultural weeds over the past several decades. The repeated exposure of Alopecurus myosuroides (Alomy), a major weed in cereal crops of the temperate climate zone, to high selective pressures through herbicide applications gives us the opportunity to look into the underlying evolutionary processes of rapid adaptation. Because of the large size of Alomy's genome (~4.6 Gb), we have exploited two complementary methods, amplicon sequencing (Amplicon-Seq) and Restriction site Associated DNA Sequencing (RAD-Seq), to characterize genetic variation at different genomic scales in field populations across Europe. At a scale of specific loci, variation in and around three known Target Site Resistant genes (ACCase, ALS, psbA) obtained by Amplicon-Seq will tell us how and how often resistance alleles –or entire haplotypes– have appeared. At the genome-wide scale, RAD-Seq markers will reflect whether spread of resistance alleles is due to an overtake of entire genotypes or the introgression of specific haplotypes. Furthermore, to improve our ability to detect genetic markers and place them in their right genomic context, we are assembling a reference genome from PacBio long-reads.

19


Evolution of herbicide resistance in waterhemp (Amaranthus tuberculatus)

Patrick J. Tranel

Department of Crop Sciences, University of Illinois, USA


Amaranthus tuberculatus is a dioecious, broadleaf weed that has evolved resistances to herbicides spanning seven sites of action. Most recently, A. tuberculatus with resistance to inhibitors of very-long-chain fatty acid synthesis (Group 15/K3) was reported. Historically, herbicide resistance in A. tuberculatus was largely attributed to target-site mutations, including point mutations, a codon deletion, and gene amplification. More recently, however, herbicide resistance in this species is increasingly attributed to non-target-site modifications. Investigations of these modifications—and their evolutionary origins—are greatly benefitting from “omics” approaches. Transcriptomics, for example, coupled with genome mapping, has revealed genomic hot spots of co-regulated gene expression changes as a potential source of variation on which herbicide selection acts. In addition, a recently obtained draft genome of A. tuberculatus is already being used for fine mapping of herbicide resistance genes and in a population-genetics approach to shed light on the origins and evolution of glyphosate resistance. Herbicide resistance in A. tuberculatus is both a growing threat to U.S. crop production and an emerging model system to study rapid adaptation in plants.

20


Two decades of insecticide resistance monitoring in the fall armyworm in Brazil

Celso Omoto

University of Sao Paulo, Luiz de Queiroz College of Agriculture, Piracicaba, São Paulo, Brazil


The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a serious cross-crop pest in Brazil. Because of intensive farming with successive plantings of soybean, cotton and maize crops, the problem of insecticide resistance represents a growing threat to the sustainability of Brazilian agriculture. We have been conducting insecticide resistance monitoring of the fall armyworm populations collected from major Brazilian agri-ecosystems for more than two decades. This monitoring program has been partially sponsored by IRAC Brazil. Baseline susceptibility data were established to major insecticides including carbamates (1A), organophosphates (1B), pyrethroids (3A), spynosins (5), Bacillus thuringiensis (11A), pyrroles (13), benzoylureas (15), diacylhydrazines (18), oxadizines (22A), semicarbazones (22B), diamides (28) and baculovirus (31). Our insecticide resistance monitoring program is based on diagnostic concentration/dose bioassays and F2 screeening to some insecticides. A stair-step increase in the frequency of resistance to some insecticides (e.g. pyrethroids, benzoylureas and others) has been detected throughout years, reaching values greater than 0.7 at some locations. After the commercial release of Bt maize in Brazil in 2008, resetting to insecticide susceptibility was detected to some insecticides (e.g. benzoylureas, spynosins) due to fitness costs associated with the resistance. Because of the high adoption of Bt crops in Brazil, field-evolved resistance of fall armyworm to Cry1 proteins has already been documented which are affecting the durability of current pyramided Bt maize hybrids. Our research also demonstrated a high risk of resistance evolution of fall armyworm to Vip3A and diamide insecticides. Therefore, urgent actions of IRM strategies need to be implemented in Brazil. However, with the invasion of the fall armyworm in Africa, India and Asia in recent years, global solutions will need to be discussed to manage this pest.

21


Impact on QoI sensitivity by application of Pavecto®

Yuichi Matsuzaki, So Kiguchi, Haruka Suemoto and Fukumatsu Iwahashi

Health and Crop Sciences Research Laboratory, 4-2-1, Takatsukasa, Takarazuka, Japan


QoI resistance conferred by target site mutation is well known to be spread in many pathogen populations. Major mechanisms of resistance are G143A mutation for highly resistant strains and F129L mutation for moderately resistant strains, respectively. Pavecto® (metyltetraprole) is a first tetrazolinone-type QoI which is significantly less affected by both G143A and F129L mutations compared to existing QoIs.

We collected field population of Zymoseptoria tritici and Pyrenophora teres from West Europe in 2015. As a result of sensitivity tests, there was no statistically significant difference in EC50 values of Pavecto®

between wild types and G143A strains in Z. tritici. On the other hand, statisitically significant difference was observed in EC50 values of Pavecto® between wild types and F129L strains in P. teres although the resisntace factor was small (the medians of each types were different by approximately 1.5 times).

Further study indicated the application of Pavecto® does not increase the proportion of G143A strains in Z. tritici while increase of F129L strains was observed in P. teres. We should take these results into account when we consider the adoption of Pavecto® into resistant management programs.

22


Glutathione-S-transferases confer pre-emergence metabolic herbicide resistance in various ryegrass populations

Roland S. Beffa1, Rebecka Duecker1,2, Veronika Brabetz1, Peter Zoellner1, Susanne Ries1, A. Collavo1 and P. Luemmen1

1Bayer Crop Science, Industriepark Hoechst, 65926 Frankfurt, Germany; 2Georg-August Universität Göttingen, Section of General Plant Pathology and Crop Protection, Grisebachstraße 6, 37077 Göttingen,

Germany


Pre-emergence herbicides are one of the last solutions to increase diversity from the chemical perspective and mitigate the evolution of resistance in grass weeds in cereal crops. Metabolic resistance to herbicides has been a growing problem for successful weed management and also poses a threat to herbicides that will be discovered in the future due to the potential for cross-resistance to a broad spectrum of chemical classes. Through the identification of metabolites, the detoxification pathway of flufenacet (HRAC, K3

group, VLCFAs) was confirmed to happen via glutathione conjugation. Transcriptome analyses identified eleven glutathione-S-transferase (GST) genes to be overexpressed in resistant plants. The expression of four recombinant GST genes in E. coli showed that one tau class GST detoxifies flufenacet fast while one phi class GST detoxified it weakly. The tau class GST was also able to detoxify S-metolachlor at a slow rate but not pyroxasulfone, both K3 inhibitors. No SNPs were found to co-segregate with resistance suggesting that overexpression of GST genes is responsible for enhanced detoxification. The characterization of the molecular mechanisms of flufenacet resistance will help to develop accurate resistance diagnostic to optimize the use of the herbicide, select the right mixtures, and thus mitigate the evolution of resistance.

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The tomato borer Tuta absoluta: pest management in the era of insecticide resistance

Emmanouil Roditakis1, Chis Bass2, Anastasia Tsagkarakou1 and John Vontas3,4

1 Institute of Olive Tree, Subtropical Crops and Viticulture, Hellenic Agricultural Organisation - 'Demeter', Heraklion, Crete, Greece;

2College of Life and Environmental Sciences, Biosciences, University of Exeter, Penryn Campus, Penryn, Cornwall, UK,

3Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, Crete, Greece,

4Department of Crop Science, Agricultural University of Athens, Athens, Greece

E-mail: [email protected] & [email protected]

The tomato borer Tuta absoluta is a major pest of tomato crops globally. Tuta absoluta invaded Europe in 2006 and has since spread worldwide, affecting local economies and global tomato production. Management of this pest has largely relied on insecticide applications which inopportunely contributed to the development of insecticide resistance. Resistance to diamide insecticides is a striking example of this; a potent new chemical class was practically compromised within just four years of use. Diamide resistance, associated with mutations at the ryanodine receptor, is extremely stable and widespread in Europe and Asia. Resistance to additional chemical classes, such as indoxacarb, spinosad and emamectin benzoate have been recently reported in distinct but still restricted cases, thus elucidation of the underlying resistance mechanisms is of key importance for proactive resistance management. At the same time, the implications of resistance development in the control of the pest are considerable. Established pest management schemes may gradually become ineffective with significant socio-economic impact. As insecticide resistance is continuously evolving, any modifications to existing pest management schemes have to be rapid, effective and sustainable. Such major adaptations should be steered by academia and in consultation with other stakeholders and policy makers.

24



A novel mechanism confers high levels of coumaphos resistance in Varroa destructor

John Vontas1,2, Spyros Vlogiannitis1, Kostas Mavridis1, Wannes Dermauw3, Simon Snoeck3, Eva Morou1, Eva Katsavou2, Paschalis Harizanis2, Luc Swevers4 and Thomas Van Leeuwen3,5

1Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, 100 N. Plastira Street, GR-700 13 Heraklion, Crete, Greece

2Department of Crop Science, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece

3Department of Crop Protection, Faculty of Bioscience Engineering, Coupure Links 653, Ghent University, B-9000 Ghent, Belgium

4Insect Molecular Genetics and Biotechnology, Institute of Biosciences and Applications, National Centre for Scientific Research ‘Demokritos’, Athens, Greece

5Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam (UvA), Science Park 904, 1908 XH, Amsterdam, The Netherlands.


Varroa destructor and the viruses it transmits is one of the main problems in modern beekeeping. The use of acaricides has been the main practice to control these mites since their introduction in Europe and the US. Although more alternative control strategies become available, chemical control of Varroa continues until today in many regions of the world.

One of the main acaricides used for Varroa control is the organophosphate coumaphos, which has excellent selective toxicity. The frequent use of this acaricide has in some regions placed a strong selection pressure with the development of resistance as a consequence.

We report here on a population that exhibits extreme high levels of coumaphos resistance. Sequencing the acetylcholinesterases gene, the target-site of coumaphos, did not reveal any candidate resistance mutation, nor did biochemical tests revealed different binding kinetics. Coumaphos is thought to be metabolized to comaphos-oxon the active principle, but chlorferon is considered the main non-toxic metabolite. Although we could clearly identify chlorferon as the principle metabolite after incubation with radio-labeled coumaphos, we could not detect different rates of metabolite formation between susceptible and resistant strains. Surprisingly, the amount of comaphos-oxon was barely detected in resistant mites. All data together points towards a novel resistance mechanism, a hypothesis supported by deep RNA sequencing and functional assays.

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Resistance to the SDHI fungicides boscalid and fluopyram in Podosphaera xanthii from commercial cucurbit fields in Spain

Alejandra Vielba-Fernández1,2, Antonio de Vicente1,2, Alejandro Pérez-García1,2 and Dolores Fernández-Ortuño1,2

1- Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain2- Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora"-Universidad de Málaga-

Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Departamento de Microbiología, Campus de Teatinos, 29071 Málaga, Spain


Powdery mildew elicited by Podosphaera xanthii is a devastating disease of cucurbits worldwide and one of the most important diseases affecting these crops in Spain. Application of fungicides is the main control practice for managing P. xanthii; however, isolates resistant to multiple classes of site-specific fungicides have been recently reported in the Spanish cucurbit powdery mildew population. Succinate dehydrogenase inhibitors (SDHIs) constitute a relatively novel class of fungicides registered for powdery mildew control representing new alternatives for cucurbit growers. In the present study, 30 P. xanthii isolates were used to determine the effective concentration that reduces mycelial growth by 50% (EC50) to boscalid and fluopyram. The present study was also conducted to obtain discriminatory doses to monitor SDHI fungicide resistance in 180 P. xanthii isolates collected from several commercial cucurbit fields in Spain during 2017-2018. Three SDHI resistance patterns were observed in our population, which include patterns I (resistance to boscalid), II (resistance to fluopyram), and III (resistance to boscalid and fluopyram). The amino acid changes associated with these resistance patterns in the Sdh protein were also examined. Based on our results, SDHI fungicides are good alternatives for cucurbit powdery mildew control, although they should be applied with caution.

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Present pesticide discovery paradigms promote the evolution of resistance

Jonathan Gressel

Plant and Environmental Science, Weizmann Institute of Science, Rehovot, Israel


The emphasis of industry discovery programs has been for many years on finding new target sites for pesticides and finding pesticides that control pests by inhibiting a single target. Thus, there has been an emphasis on genomics in finding targets for potential pesticides. There is also the feeling that registration of single target pesticides is simpler as one can state that its mode of action is known. Conversely, if one looks at with an epidemiological view at which pesticides have been the most recalcitrant to evolutionary forces, it is those that have multiple targets of actions: the thiocarbamates, the long-chain fatty acid biosynthesis inhibitors and sterol biosynthesis inhibitors that affect more than one target, the heavy metals of old, etc. Metabolic or other non-target site resistances can evolve to multisite inhibitors, but these resistances can be overcome by structural modification of the pesticide. Industry has looked at pest-toxic natural products as pesticide leads but abandons those where they can find no single target of action. Perhaps nature has been intelligent and evolved natural products that are multi-site inhibitors and that is why they have been active for millennia? Perhaps we should be learning from nature?

27


Integrated approaches to manage gray mold and fungicide resistance with focus on producer education and involvement

Schnabel, G1., Dowling, M1. and Peres, N.2

1Clemson University, Department of Plant and Environmental Sciences, Clemson SC 2University of Florida, Gulf Coast Research and Education Center, Wimauma, FL

Commercial strawberry producers in the United States use plasticulture to produce high quality fruit. This annual production system starts with nurseries producing tips (mostly in Canada and California), bare roots, and plugs (often outsourced to local nurseries). Both levels of nurseries chemically control diseases and thus preselect for resistance before the plants even gets into the fruit producer fields. Consequently, fruit producers may be confronted with fungicide resistance problems before they have even applied their first spray of the season. Efforts are made to provide nurseries and fruit producers with tools and guidelines to manage diseases and fungicide resistance concerns. Through regionwide resistance monitoring, improved producer understanding of the biology of the fungus and spread of the disease, and development of modern tools for producer education, we involve producers in our efforts to manage resistance and improve disease management. This presentation highlights some impacts of our monitoring program, features an animated and narrated disease cycle that discusses management approaches, and explains new features in the MyIPM smartphone app used by specialists, county agents, consultants, and producers to more easily navigate active ingredients, trade names, and resistance management options.

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Field evolved polygenic resistance against diamide insecticides in the soybean looper, Chrysodeixis includens

Christoph T. Zimmera, Miriam Danielsb,1, Eve Dauma, Liz Hirstb, Alicia Lerouxa, Phillip Milnesb, Reza Nouranib, Philip Süessa, Fergus Earleyb, Russell Slaterc and Jan Eliasa

aSyngenta Crop Protection, Schaffhauserstrasse, CH-4332 Stein, SwitzerlandbSyngenta Crop Protection, Jealott's Hill International Research Centre, Bracknell, RG42 6EY, UK

cSyngenta Crop Protection, Schwarzwaldallee 215, CH-4002 Basel, Switzerland1TSG Consulting, London, EC3N 2EX, UK


Chrysodeixis includens syn. Pseudoplusia (Lepidoptera: Noctuidae: Plusiinae), is a polyphagous species and an abundant crop pest in soybean and cotton in North and South America. The primary means of controlling this pest is via synthetic insecticides and genetically modified crops expressing B.t. toxins. Diamide insecticides as well as the macrocyclic lactone emamectin-benzoate are highly active insecticides with an exceptional potency against lepidopteran pests including C. includens. A field strain collected from Puerto Rico showed decreased susceptibility to a range of diamides but not emamectin-benzoate. Resistance ratios for diamides based on artificial diet feeding/contact bioassays ranged between 30-300 fold when compared to a fully susceptible long-term lab colony that was collected in the 1980s in the USA. Toxicokinetics of chlorantraniliprole vs. emamectin-benzoate were investigated in vivo and confirmed faster and distinct metabolism of chlorantraniliprole in the resistant strain when compared to the susceptible one while there was no significant difference for emamectin-benzoate. The diversity of chlorantraniliprole metabolites provide evidence for a polygenic resistance. The underlying resistance mechanisms are currently investigated using genome wide association studies (GWAS), the contribution of individual mechanisms will be deciphered using state of the art functional genomics tools.

29


Inatreq™ - a new tool for control of Septoria leaf blotch in cereals

Gregory Kemmitt, Andrew Leader and David Young

Corteva Agriscience, Agriculture Division of DowDupont, UK


Fenpicoxamid (Inatreq™ active) belongs to a new chemical class of fungicide (picolinamide) for control of Septoria (Zymoseptoria tritici) and other diseases. Fenpicoxamid is derived from the natural product UK-2A, produced by fermentation of an actinomycete. It undergoes a minor synthetic modification post-fermentation for stabilization, then in plants or fungi is metabolized to release UK-2A as the active species. Fenpicoxamid will be the first product in the cereals market to act at the Qi site of the cytochrome bc1 complex in mitochondria. Because it has a novel mode of action, there is no target site-based cross-resistance between fenpicoxamid and current fungicides. Target site-based resistance to fenpicoxamid has been explored through isolation and characterization of resistant mutants in the laboratory. Baseline sensitivity testing of European Z. tritici isolates has been conducted to characterize sensitivity of the existing population to fenpicoxamid and facilitate early detection of resistance after product introduction through continued monitoring. Resistance to existing products and the likely loss of some current solutions has created an urgent need for a compound such as fenpicoxamid with a new mode of action to control Z. tritici. However, implementation of a sound resistance management strategy will be critical to maintain its long-term effectiveness.

30


Epidemiology and agronomic predictors of herbicide resistance in rice at a large scale

Maurizio Sattin1, Elisa Mascanzoni2, Alessia Perego3, Laura Scarabel1, Silvia Panozzo1, Aldo Ferrero4 and Marco Acutis3

1Institute for Sustainable Plant Protection (IPSP-CNR), Legnaro, Italy

2DAFNAE, Legnaro, Italy3DISAA University of Milano, Milano, Italy

4DISAFA, University of Torino, Grugliasco, Italy


The identification of concise, yet informative, agronomic predictors of herbicide resistance diffusion can significantly facilitate effective management and improve sustainability. The study analyses the impact in the main Italian rice area (2000,000 ha) of major agronomic factors such as seeding type, rotation rate and soil texture on the epidemiology of herbicide resistance and generates a large-scale resistance risk map. The Italian Herbicide Resistance Working Group database (www.resistenzaerbicidi.it) was used to generate herbicide resistance maps. The distribution of resistant weed populations resulted as not homogeneous, with two pockets where resistance had not been detected. Random sampling was then done in these pockets and samples tested for herbicide resistance. Through the integration of complaint monitoring, mapping and neural network analyses, we prove that a high risk of resistance evolution is associated with traditional rice cropping systems with intense monoculture rates and where water-seeding is widespread. resistance is present in areas where it had never been reported through extensive complaint monitoring. However, these resistant populations cause medium-low density infestations, likely not alarming rice farmers. This highlights the importance of integrated agronomic techniques at cropping system level to prevent the diffusion and impact of herbicide resistance or limit it to an acceptable level.

31

http://www.resistenzaerbicidi.it/


Herbicide resistance management: Recent developments and trends

Hugh J. Beckie

Australian Herbicide Resistance Initiative (AHRI), School of Agriculture andEnvironment, The University of Western Australia, Crawley, WA 6009, Australia


Agronomic field crop growers in Australia have been at the forefront of many of the recent developments in herbicide-resistant (HR) weed management because of having to deal with multiple-HR weed populations the longest, traditionally narrow crop profit margins, and dedicated innovative research, development, and extension efforts since the 1990s. In Australia or other countries where input-intensive agriculture is practiced, these developments and trends over the past decade include renewed efforts by the agrichemical industry in herbicide discovery, crops with combined (stacked) HR traits, increasing reliance on preemergence vs. postemergence herbicides, breeding for weed-competitive crop cultivars, harvest weed seed control, and site-specific or precision weed management. The unifying framework or strategy underlying these developments and trends is mitigation of viable weed seeds into the soil seed bank and maintaining low weed seed banks. The key question going forward is how much weed control is enough to consistently achieve this goal? The vision for future HR weed management programs must be sustained crop production and profitability with reduced herbicide (particularly glyphosate) dependency.

32


Insect resistance management in European winter oilseed rape: squaring the circle

Ralf Nauen

Bayer AG, Crop Science Division R&D, 40789 Monheim, Germany


Insect resistance management in winter oilseed rape is jeopardized by a steady decrease in the number of insecticide modes of action to control some of the most important pests. Those insecticides surviving regulatory measures are further exhausted by the development of resistance. New classes of insecticides are urgently necessary to prevent the spread of insecticide resistance against established compounds such as pyrethroids in a number of highly destructive pests, thus helping to manage pest insects by mode of action rotation and to sustain yields.

Winter oilseed rape is under attack by a number of serious pests throughout the cropping cycle, starting early season with flea beetles and aphids (a new phenomenon since the ban of neonicotinoid seed treatment in EU), followed in spring by other coleopteran pests such as stem weevils, pollen beetle and seedpod weevil. The control of many of these pests relies on a few modes of action, or even a single mode of action in some countries. The presentation highlights the dilemma by reviewing the development of resistance and providing an update on the latest resistance monitoring data and mechanistic findings with special reference to cabbage stem flea beetle, green peach aphid and pollen beetle.

The implications of the findings for resistance management and future control of insect pests in winter oilseed rape will be discussed.

33


Overcoming resistance of plant pathogenic fungi with the use biological-synthetic pesticide combinations

Lee Simmons

AgBiome Inc., Durham, NC, USA


Resistance to fungicides has been observed in plant pathogenic fungi, especially in high value crops where treatment options are limited, and multiple applications are required. Providing novel fungicides to reduce resistance development has been limited by the lack of discovery of new modes of action (MOA), regulatory issues, development costs, and consumer preference.

There is a trend towards combining several synthetic fungicides to attain additional modes of action to delay resistance to at-risk MOAs, provide ease of use to end users, and to decrease the use rates of active ingredients (AIs) under regulatory pressure.

AgBiome is leading the way to address resistance development by combining our proprietary biological pesticides with synthetic fungicides. Biological pesticides have multiple MOAs, are under less regulatory pressure, and consumers approve of a greener approach to pest management. Our research trials in controlled environments and the field provide support for this concept.

This research is a vital step in providing additional solutions to growers in their fight to prevent resistance of plant pathogenic fungi. Our field testing results have shown that combining a biological pesticide with 0.5x rates of Mefenoxam or Chlorothalonil provides comparable control of Phytophthora capsici and Podosphaera xanthii to 1x rates, respectively.

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Herbicide diversity and the evolution of generalist herbicide resistance traits in Alopecurus myosuroides

David Comont1, Claudia Lowe1, Richard Hull1, Laura Crook1, Helen Hicks2, Nawaporn Onkokesung3, Roland Beffa4, Dylan Childs2, Robert Edwards3, Robert Freckleton2 and Paul Neve1

1Rothamsted Research, UK, 2University of Sheffield, UK, 3Newcastle University, UK, 4Bayer AG, Germany


Using herbicide mixtures, rotations and sequences to diversify herbicide selection is advocated to delay resistance evolution, however the efficacy of these practices in the field is difficult to assess. Here, we use epidemiological data from over 100 UK field populations of Alopecurus myosuroides to directly examine the effects of historical herbicide intensity and diversity on the evolution of herbicide resistance. Our results demonstrate widespread resistance to the acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) herbicides with >90%, 75% and 60% resistance to fenoxaprop-p-ethyl, mesosulfuron and cycloxydim, respectively. Assessment of resistance mechanisms confirmed that target-site mutations (TSR) and non-target-site resistance (NTSR) co-occur in this species. Analysis of field management histories reveals that the intensity of herbicide use was the main driver for observed phenotypic resistance levels, with herbicide mixtures and rotations (diversity) causing no consistent reduction in selection for resistance. Moreover, epidemiological analysis of the relationship between resistance mechanisms and selection histories identified a catch-22 scenario; whereby use of herbicide mixtures reduced selection for TSR but promoted selection for NTSR mechanisms. These results conform with eco-evolutionary theories of selection for ‘specialist’ and ‘generalist’ adaptations, and highlights that the success of herbicide mixtures is contingent on the type of resistance mechanism present.

35


Field-evolved resistance to Bt maize by western corn rootworm: patterns of resistance, potential causes and management approaches

Aaron J. Gassmann

Department of Entomology, Iowa State University, Ames, IA

email: [email protected]

In the United States, the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is among the most serious pests of maize. Current management of this pest relies heavily on planting of transgenic maize that produces insecticidal toxins derived from the bacterium Bacillus thuringiensis (Bt). However, in some regions of the United States, populations of western corn rootworm have evolved resistance to Bt maize. The first cases of Bt resistance involved maize that produced Bt toxin Cry3Bb1. Over time, the magnitude of Cry3Bb1 resistance detected in field populations has increased, and Cry3Bb1 resistance has been found to extend to additional Bt toxins, in particular mCry3A and eCry3.1Ab, through the presence of cross-resistance. More recently, evidence has emerged of resistance to Cry34/35Ab1 maize in some populations, and of widespread resistance to Cry3Bb1 maize. Resistance management for corn rootworm targeted by Bt maize has focused on non-Bt refuges and pyramiding of multiple Bt toxins. However, non-recessive inheritance of resistance coupled with minimal fitness costs affecting resistant genotypes have likely diminished the resistance-management benefits of refuges. Furthermore, use of Bt maize traits singly before pyramiding has likely reduced the resistance-management benefit of pyramiding toxins. Other management options for western corn rootworm include crop rotation and use of soil-applied insecticides at planting. Available data indicate that both of these strategies may influence the level of Bt resistance within populations of corn rootworm, however, depending on the context in which these management approaches are applied resistance may either increase or decrease. In general, it is clear that better use of integrated pest management, in particular a more diversified approach, will be essential to achieve more sustainable management of western corn rootworm with current and future transgenic technologies.

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Optimal insecticide resistance management strategies: mixtures or alternations?

Joe Helpsa, Neil Paveleyb, Sacha Whitec and Frank van den Boscha

aDepartment of Computational and Systems Biology, Rothamsted Research, UKbCrop Protection, ADAS, UK

cSoils, Agriculture and Water, ADAS, UK


Strategies by which resistance can be slowed are necessary to prolong the effectiveness of the remaining modes of action. Here we use a flexible mathematical model of resistance evolution to compare three insecticide application strategies: (i) mixing two insecticides each at their full label doses, (ii) rotating (alternating) two insecticides at full label dose, or (iii) mixing two insecticides at a reduced dose (with each mixture component at half the full label dose). The model represents target-site resistance.

The analysis shows that mixing two insecticides at their label (or registered) dose normally results in the fastest selection for resistance, whereas a reduced-dose mixture frequently results in the slowest.

While a reduced-dose mixture is most often the best strategy, either a label-dose mixture or a label-dose rotation are, under certain conditions, the best strategy. The primary determinant of the optimal strategy is related to the relative efficacy of the two insecticides, and whether the insect reproduces sexually or asexually. The life history of the pests in question does not have a large influence on which strategy best prolongs the durability of the insecticides.

37


Fungicide resistance development and management in Cercospora beticola

Gary Secor, Mohamed Khan and Viviana Rivera-Varas

Department of Plant Pathology, North Dakota State University, Fargo, ND, USA


Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola (Cb) continues to be the most important foliar disease of sugar beet worldwide. Management has been dependent on fungicides and is hampered by widespread resistance to multiple fungicides that has developed in most sugar beet production areas. Despite improvements in fungicide application, it will be important to develop a more integrated approach to manage CLS that includes variety resistance and cultural practices. Fungicide practices have evolved to include new fungicides, fungicide alternations and combinations, and additions of copper and EBDC. Sugar beet varieties with higher resistance to CLS are being grown, and minimum variety resistance scores have decreased in recent years. It will be important to consider and emphasize cultural practices to reduce initial inoculum. Examples include inoculum barriers on field edges to prevent or delay infection such as early fungicide application to field edges, deep plowing to bury inoculum, early application of chemicals directly on the soil to kill spores and ringing fields with a highly resistant variety. Widespread resistance of Cb to multiple fungicides appears to be a major issue for continued sugar beet production and requires new strategies and tactics for sustained CLS management.

38


Herbicide resistance in ‘focus farms’ adopting IWM

Roberto Busi and Hugh Beckie

Australian Herbicide Resistance Initiative, School of Agriculture and EnvironmentUniversity of Western Australia


A study was conducted to assess herbicide resistance in annual ryegrass seed samples collected from “focus farms” in Western Australia practicing harvest weed seed control. Farmers’ perceptions of herbicide resistance were collected before herbicide testing. Herbicide resistance was determined by treating germinating seeds or seedlings with 18 treatments including different dosages of PRE and POST herbicides, binary mixtures, sequences and new herbicides (not commercialized). Plant survival > 6% indicated a developing level of herbicide resistance. Plant survival to POST herbicides was > 25% denoting substantial herbicide resistance, whereas 1% survival to tested PRE herbicides reflects effective control of ryegrass field populations. High level resistance to POST herbicides corresponds to farmers’ perceptions of resistance and it has been the driving force for widespread adoption of harvest weed seed control techniques. Herbicide mixtures of PRE herbicides are effective to control resistant ryegrass. Adoption of harvest weed seed control in combination with mixtures of PRE herbicides should be adopted to reduce population size and lower the risk of herbicide resistance by increasing the heterogeneity of selection pressures on weeds. A centre for herbicide resistance testing in Western Australia will support growers’ decisions/investments on weed control, encourage herbicide stewardship and help develop new solutions to control herbicide-resistance prone weeds.

39


Posterswith

2-minutetalks

40

Poster 1

Survey of the prevalence of the Rdl resistance mutation in mosquitoes from Laos

Andrew K. Jones, Sebastien Marcombe, Phoutmany Thammavong, Phonesavanh Luangamath, Somsanith Chonephetsarath, Nothasin Phommavan, Khaitong Lakeomany, Numpherng Xayavong, Somphat Nilaxay, Zuhal Rahmani, Penelope J. Saverton, Omobolanle H. Abdullateef, Jordan Forward, Anna E. Jacob, Safina

Khadam, Wlaa Ali, Chloè Boer, Hayato Kakinuma, Joseph Hawkins, Rosie Longstreeth, Natalie M. Portwood, Madeleine Smee, Natasha Brown, Nursu C. Kuyucu, Susannah Lechmere, Gabriela Stieger,

Santi Maithaviphet, Simone Nambanya and Paul T. Brey

Department of Biological and Medical Sciences, Oxford Brookes University, Headington, Oxford, OX30BP, UK

Institut Pasteur du Laos, Ministry of Health, Vientiane, Lao PDR


A mutation, commonly A296S, in the insect GABA receptor, RDL, underlies resistance to several insecticides such as cyclodienes. Even though the use of cyclodienes has been banned, the occurrence of mutations substituting A296 is notably high in mosquitoes from several countries. Here we report a survey investigating the prevalence of the Rdl mutant allele in mosquitoes from Laos, a country where malaria and dengue fever are health concerns. Anopheles and Aedes mosquitoes were collected from twelve provinces in Laos. Adult bioassays showed that all populations tested were susceptible to dieldrin (4%). From a total of 791 mosquitoes tested, only one, Anopheles maculatus rampae from Attapeu, carried the mutant allele, being heterozygous for A296S.

We found a lack of the Rdl mutant allele indicating that the presence of A296S is driven by selection pressure rather than being a natural polymorphism and that mosquitoes from Laos are exposed to lower levels of insecticides that act on the GABA receptor than mosquitoes in other countries. Identifying the prevalence of the Rdl mutation may help inform the use of alternative insecticides should there be a need to replace pyrethroids in order to prevent/manage resistance.

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Poster 2

SAP2, a chemosensory protein, is a key player in pyrethroid resistance in African malaria vectors

V A Ingham1, A Anthousi1, V Douris2, N J Harding3, G Lycett1, M Morris1, J Vontas2,4 and H Ranson1*

1 Vector Biology Department, Liverpool School of Tropical Medicine, UK2 Foundation for Research and Technology - Hellas (FORTH), Institute of Molecular Biology and

Biotechnology, Greece3 The Big Data Institute, University of Oxford, UK

4 Pesticide Science Laboratory, Department of Crop Science, Agricultural University of Athens, 11855 Athens, Greece


Insecticide based vector control strategies represent the most important malaria intervention tool, accounting for >80% of the reduction in malaria cases since 2000. Recently the steep reductions in malaria related morbidity and mortality have started to plateau and case numbers are rising across sub-Saharan Africa, corresponding with the spread of insecticide resistance across the continent. These statistics demonstrate the need for new intervention strategies and a greater understanding of insecticide resistance. Majority of studies on insecticide resistance in mosquitoes focus on three mechanisms: target site mutations, metabolic clearance and cuticular resistance. Here, we describe a novel pyrethroid resistance mechanism based on insecticide sequestration by a chemosensory protein, SAP2. SAP2 is both induced by pyrethroid exposure and constitutively overexpressed in pyrethroid resistant populations of Anopheles gambiae s.l. Attenuation of SAP2 expression leads increases mortality after exposure to pyrethroid insecticides. Furthermore, overexpression of SAP2 in a susceptible strain increases resistance to permethrin. SAP2 expression is highly enriched in the legs and the recombinant protein strongly binds to pyrethroids, suggesting it may play a role in sequestering the insecticide following penetration through the cuticle. Whole genome sequence data shows a selective sweep at this locus across Western Africa, confirmed by longitudinal sequence data.

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Poster 3

The importance of host specialization in insecticide resistance in the green peach aphid Myzus persicae

C. Plantamp, S. Benhamou, C. Mottet, E. Segura, L. Caddoux and B. Barrès

USC CASPER, ANSES Laboratoire de Lyon, France


Host-parasite interaction leads to dynamic arm race between host and parasite. A possible outcome of this co-evolutionary arm race is the emergence of host races within parasite species. In agroecosystems, the interaction between host and parasite might be impacted by additional selection pressure such as the use of pesticides. In our study, we focused on Myzus persicae, a polyphagous pest that have evolved resistance against the main insecticide classes. Despite extensive use of neonicotinoids on both peach tree and rapeseed in Europe, M. persicae populations with a target site resistance to neonicotinoids have only been identified in peach orchards. Differentiation between aphids’ populations coming from different host species (peach tree, rapeseed and tobacco) is further confirmed by population genetics analyses. We investigated whether plant specialization could explain the genetic compartmentalization and whether insecticide resistance could interfere. Populations were sampled on different host species in two areas in South-Eastern France. We compared their performance on different host plant in the lab. Our results suggest that aphid’s fitness depends on host plant. The role of the detoxifying genes of the CYP6 family is confirmed on tobacco. These results offer interesting avenue of research on why resistance is specific on each host.

43


Poster 4

Genetic basis of codling moth resistance to (bio) insecticides

Bertrand Gauffre1, Mathieu Gautier2, Jérome Olivares1 and Myriam Siegwart1

1 UR PSH, INRA, Avignon, France 2 UMR CBGP, INRA, CIRAD, IRD, Montpellier SupAgro, University of Montpellier, Montpellier, France


Pesticide resistance is a key example of rapid evolution under strong selective pressures and provides an interesting case to address fundamental questions about the repeatability of evolution. Also, the identification of genetic markers associated with resistances may allow to cost effectively screen resistances in natural populations. The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is one of the major insect pests in apple, walnut and pear orchards. Since chemical and organic insecticides are massively used to maintain its populations at a low level, C. pomonella evolved resistance to numerous (bio)insecticides. The major aim of our study was to characterize the genetic bases of resistance in C. pomonella, in particular to a biopesticide, the C. pomonella granulovirus (CpGV-M). First, we identified in the recently sequenced genome of C. pomonella 662 candidate genes, which have been identified to confer insecticide resistance in insects. Second, we investigated CpGV-M resistance in three regions of France. In each region, one susceptible and one resistant population to CpGV-M were identified using biotests. We then applied a GWAs approach to identify genetic markers associated with resistance to CpGV-M and to test for the repeatability of resistance evolution across these regions.

44


Poster 5

Proposal for a simple and unified classification of pesticides

R4P network (A-S Walker)INRA BIOGER, Avenue Lucien Brétignières, 78 850 Thiveraval-Grignon, France


Resistance management relies on the knowledge of selection pressures. Simple classifications are therefore needed to distinguish and understand pesticides modes of action.

The current classifications (FRAC, HRAC, IRAC, WSSA) legitimately use the biochemical mode of action, and to a lesser extent, the chemical classes of active ingredients to classify them. The historical separation of these classifications according to pesticide use has led to the assignment of a separate code to pesticides sharing the same mode of action but targeting different categories of pests.

In order to facilitate education about pesticides and the monitoring and management of their unintended effects, and in particular of resistance, the R4P network proposes a new classification of pesticides aggregating the previous ones, so that identical codes are assigned to active ingredients of the same mode of action. This unified classification:

• Is simple, comprehensive, integrated, “All-in-One”, intuitive,• Is compatible with existing classifications,• Integrates biocontrol solutions,• Provides a description of the uses and regulatory status of active ingredients in France,• Provides a description of the status of resistance, resistance mechanisms and cross-

resistance associated with the modes of action,• Is adaptable to future innovations and national variations in registration and resistance,• Is collaborative and freely available: https://osf.io/ubhr5 /

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https://osf.io/ubhr5/

https://osf.io/ubhr5/

Poster 6

Development of a LAMP method for detecting MBC-resistant isolates of the cucurbit powdery mildew pathogen Podosphaera xanthii.

Alejandra Vielba-Fernández1,2, Antonio de Vicente1,2, Alejandro Pérez-García1,2, Dolores Fernández-Ortuño1,2

1- Departamento de Microbiología, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain2- Instituto de Hortofruticultura Subtropical y Mediterránea "La Mayora"-Universidad de Málaga-

Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Departamento de Microbiología, Campus de Teatinos, 29071 Málaga, Spain


Powdery mildew, caused by Podosphaera xanthii, is one of the most economically important diseases affecting cucurbit crops in Spain. Chemical control is the most efficient management of the disease; however, P. xanthii isolates resistant to multiple classes of site-specific fungicides have been reported in Spain. In previous studies, resistance to the Methyl Benzimidazole Carbamates (MBCs) fungicides was found to be caused by the amino acid substitution E198A on β-tubulin. To detect MBC-resistant isolates in a faster, efficient and specific way than the traditional methods, a loop-mediated isothermal amplification (LAMP) system was developed. Three sets of LAMP primers were designed and tested; however, only one set was optimized specifically to distinguish the E198A mutant genotype. The amplification products were visualized using gel electrophoresis and hydroxynaphtol blue (HNB), an azo dye that turn from violet to sky blue if the results were negative (E198) and positive (A198), respectively. In addition, to confirm that the amplified product corresponded to the β-tubulin gene, the LAMP product was digested with the enzyme LweI and sequenced. Our results have shown that the LAMP technique is a specific and reproducible method that could be used to monitoring MBC-resistance of P. xanthii in the field.

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Poster 7

A method to translate field trial efficacy data into model parameters representing the effect of fungicides on pathogen life cycles

Isabel Corkleya,b,d, Frank van den Boschb, Joseph Helpsb, Alice E Milneb, David J Skirvina, Helge Sierotzkic and Neil D Paveleya

a ADAS SCW, RSK ADAS Ltd, UKb Department of Biointeractions and Crop Protection, Rothamsted Research, UK

c Syngenta Crop Protection, Switzerlandd Department of Agriculture, Policy and Development, University of Reading, UK


Models of fungal pathogens on crops can inform sound tactics for resistance management. These tactics have the best chance of success if deployed immediately following the release of a new fungicide. The relative effectiveness of fungicides is vital information to guide resistance management tactics: fungicides used as mixture partners to an at-risk fungicide must have sufficient activity against the target pathogen to reduce its growth rate on the crop and so reduce selection for strains resistant to the at-risk fungicide. Model parameters are used to represent the effect of fungicides on elements of the pathogen life cycle that affect its growth rate, such as latent period and infection efficiency. Commercial field efficacy trials are often the earliest source of data on the effects of new fungicides on pathogens, but model parameterisation is not typically the primary focus in the design of these trials. We present a method to extract information on the effectiveness of fungicides using disease severity data from commercial field trials and demonstrate this using a model of Phakopsora pachyrhizi infection of soybean crops. We show how this information can be used, through modelling, to predict and compare the effectiveness of alternative resistance management strategies.

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Poster 8

Evidence for rapid Z. tritici evolution and SDHI cross-resistance in Oregon, USA

Christina H. Hagerty1, Catherine Reardon2, Duncan R. Kroese1, Ann Klein2, and Christopher C. Mundt1

1 Department of Botany and Plant Pathology, Oregon State University, USA 2 Columbia Plateau Conservation Research Center, United States Department of Agriculture, USA

Email: [email protected] Zymoseptoria tritici is the causal agent of septoria tritici blotch (STB), a disease of wheat (Triticum aestivum) that causes significant yield loss worldwide. Z. tritici’s life cycle, reproductive system, effective population size, and gene flow put it at high risk for developing fungicide resistance. In the Willamette Valley of Oregon, fungicide use has increased due to increased grain prices and reduced fungicide cost. Succinate dehydrogenase inhibitor (SDHI) fungicides (Group 7) were not widely used to control STB in the Willamette Valley until 2016. We utilized a Z. tritici collection spanning SDHI adoption (2015-2017) to calculate EC50 values of four SDHI active ingredients (benzovindiflupyr, pethiopyrad, fluxapyroxad, and fluindapyr). Our results indicate a significant annual increase in benzovidiflupyr EC50 values following the adoption of SDHI fungicides in Oregon. Additionally, cross resistance among SDHI active ingredients was also observed between benzovindiflupyr and pethiopyrad (p = 0.0002), and pethiopyrad and fluxapyroxad (p = 0.0482). No significant correlation was found between benzovindiflupyr and fluxapyroxad EC50 values. Fluindapyr EC50 values were not correlated with benzovindiflupyr, pethiopyrad, or fluxapyroxad. Results highlight the importance of managing increasing resistance and cross-resistance of Z. tritici to SDHI active ingredients.

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Poster 9

Enhanced metabolism to ALS inhibitors and synthetic auxin herbicides in Papaver rhoeas

Joel Torra1*, Antonia María Rojano-Delgado2, Julio Menéndez3, Rafael de Prado2 and Marisa Salas4

1Universitat de Lleida, 25198 Lleida, Spain; 2Universidad de Córdoba, 14071 Córdoba, Spain; 3Universidad de Huelva, 21071 Huelva, Spain; 4 Corteva Agriscience™, Agriculture Division of

DowDuPont™, 78067 Saint Quentin Yvelines Cdx, France

*E-mail: [email protected]

Papaver rhoeas populations with multiple herbicide resistance to synthetic auxin herbicides and acetolactate synthase (ALS) inhibitors are frequent in winter cereals fields in Spain. Both target-site resistant (several Pro197 amino acid substitutions) and non-target site resistant (NTSR) mechanisms can contribute to resistance to ALS inhibitors in this species, as metabolites have been already detected in one population. On the other hand, impaired transport and enhanced metabolism have been reported as being involved in the resistance to synthetic auxin herbicides in two populations, though their relationship and relative importance as NTSR mechanisms is yet to be understood.

In this research, several P. rhoeas populations from winter cereal fields in Spain with different resistance profiles were assessed in metabolism studies to further confirm the presence of enhanced rates of detoxification to ALS inhibitors and/or synthetic auxin herbicides.

The presence of enhanced rates of metabolism to these two modes of action was confirmed in more resistant P. rhoeas populations from Spain. Moreover, additional metabolism studies are underway to unravel the potential role of the cytochrome P450 enzyme family in the detoxification mechanism as NTSR mechanism of both synthetic auxin herbicides and ALS inhibitors.

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Poster 10

Resistance benefit endowed by a double EPSPS glyphosate resistance mutation (TIPS) in Eleusine indica

Martin M. Vila-Aiub1,2, Federico García1, Heping Han2, Adam Jalaludin2, Qin Yu2 and Stephen B. Powles2

1IFEVA - CONICET – Faculty of Agronomy, Department of Ecology, University of Buenos Aires (UBA), Argentina

2Australian Herbicide Resistance Initiative (AHRI) - School of Agriculture & Environment, University of Western Australia (UWA), Australia


A known single (P106S) and novel double (TIPS) glyphosate resistance point mutations have been identified in Eleusine indica. Experiments were conducted to assess the plant resistance benefit endowed by these specific TIPS (homozygous RR vs heterozygous Rr) and P106S mutations.

The P106S mutation gives modest 50% survival under glyphosate field dose with surviving plants showing a significant reduction in vegetative and reproductive growth, compared to untreated plants. The RR TIPS variant endows 100% survival under glyphosate treatment and no significant impact on reproductive growth is observed. An even higher resistance fitness benefit is associated with the compound heterozygous Rr TIPS plants. Although both RR and Rr TIPS plants show no plant mortality under glyphosate treatment, vegetative and reproductive growth is significantly higher in Rr than in RR TIPS plants. Rapid herbicide resistance evolution is expected when resistance mutations endow high resistance benefits under herbicide selection.

Based on these results, the homozygous RR TIPS resistance alleles are expected to rapidly increase and be fixed in E. indica populations under glyphosate selection. Whereas, the homozygous P106S alleles are expected to increase at a lower rate given the low/moderate fitness benefit when glyphosate challenged. This hypothesis is currently under empirical analysis.

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Poster 11

Modelling the sustainability and economics of residual herbicides vs. stacked herbicide-resistant traits

Chun Liu*, Paul Neve†1, Les Glasgow‡, R. Joseph Wuerffel§, Micheal D.K. Owen‖ and Shiv S. Kaundun*

*Herbicide Bioscience, Syngenta, UK†School of Life Sciences, University of Warwick, UK

‡Product Management Herbicides, Syngenta, US§Weed Control, Syngenta, Vero Beach, FL 32967, US‖Department of Agronomy, Iowa State University, US

1Current address: Department of Agroecology, Rothamsted Research, UK


Diversity is key for sustainable weed management and can be achieved in different ways. Genetically modified (GM) crops with two-way or three-way stacked herbicide-resistant traits allow use of herbicide mixtures which would otherwise be phytotoxic to the crop if applied post-emergence. Alternatively, Early Weed Management (EWM) strategies promote the use of pre-emergence herbicides with residual activity to keep the field free of weeds for successful crop establishment early in the season. In order to compare and contrast the respective sustainability and practicality of these two chemical-based management tactics, we modelled the waterhemp populations and simulated the evolution of herbicide resistance in this key weed species in Midwestern U.S. soybean agroecosystems. The model tested application scenarios with a varying number of herbicides, application timings (PRE and POST) and pre-existing levels of resistance. The model output showed that both tactics could provide good control of susceptible as well as some resistant weed populations. In general, each pass over the field should include at least two effective herbicide sites of action. Additionally, economic calculations suggested that, in the long term, these diversified programs could be twice more profitable than conventional systems, because of better yield and grain quality. The decision of which exact tactic to follow also involved consideration of herbicide cost, seed traits, formulations and labour. Ultimately, the essence of a successful herbicide program is to be proactive. Nevertheless, with the increasing presence of weed populations with multiple resistances, finding herbicides to which weeds are still susceptible is becoming increasingly difficult, and thus the necessity of re-introducing non-herbicide practices to support herbicides must be recognised.

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Posters

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Poster 12

Cercospora beticola is not cross-resistant to all DMI Fungicides

Viviana Rivera-Varas, Ana Heilman-Morales, Melvin Bolton and Gary Secor

Plant Pathology and Plant Sciences Departments, North Dakota State University, and USDA-ARS, Fargo, ND, USA


Cercospora leaf spot (CLS) caused by Cercospora beticola, is the most important foliar disease of sugar beet in the world. CLS is managed by crop rotation, resistant varieties and fungicide applications. C. beticola has developed resistance to multiple fungicides in the United States and other locations. Fungicide resistance is present in all fungicide groups used to manage CLS including the commonly used QoI and DMI fungicides. As part of our continuing work to manage fungicide resistance, we evaluated sensitivity of 60 C. beticola isolates collected from three commercial sugar beet fields in North Dakota and Minnesota USA to seven DMI fungicides. The isolates collected ranged in sensitivity to tetraconazole from sensitive to highly resistant based on EC50 values calculated from radial growth on fungicide-amended medium. Pairwise comparison data show differential sensitivity to the seven DMI fungicides with R values ranging from 0.06 to 0.94. We conclude that DMI fungicides differ in their interaction with DMI resistant isolates in the CLS system, and this data may be useful to identify DMI fungicide rotations or mixtures that could help manage fungicide resistance. In order to assess whether in vitro assays can be extrapolated in planta, greenhouse studies are in progress.

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Poster 13

Mechanism of multidrug resistance and risk assessment towards fungicide in Zymoseptoria tritici

Anne-Sophie Walker and Gwilherm Gazeau

INRA BIOGER, Avenue Lucien Brétignières, 78 850 Thiveraval-Grignon, France


Multidrug resistance (MDR) is characterized in Zymoseptoria tritici by low resistance levels to DMIs and SDHIs. MDR is associated to the overexpression of the membrane transporter MFS1, inducing a more effective efflux of these fungicides. Three insertions of nucleotide sequences (types I to III), varying according to their size and position in the promoter of mfs1, are responsible for the MDR phenotype. This resistance mechanism is independent from the more frequent fungicide target modification mechanism, and in Z. tritici, sexual reproduction allows the combination of these mechanisms.

Crosses between isolates carrying either MDR or target site resistance revealed semi-isogenic progeny used to characterize resistance. We show a significant increase in resistance levels towards DMIs and SDHIs, compatible with practical resistance, when the two mechanisms are combined. Moreover, the in vitro and in planta growth of isolates bearing combined mechanisms doesn’t seem affected, suggesting the maintenance of a fitness identical to that of sensitive individuals or presenting only one resistance mechanism. Field trials from the “Performance network” in France between 2004 and 2017 identified the programs most likely to be involved in MDR selection. Finally, we discuss management measures to limit the selection of different resistance mechanisms.

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Poster 14

Eurowheat – screening for azoles’ efficacy for control of Zymoseptoria tritici across Europe

Jørgensen LN1, Matzen N1, Havis N2, Holdgate S3, Clark B3, Blake J4, Glazek M5, Korbas M6, Jakub Danielewicz6, Maumene C7, Rodemann B8, Weigand S9, Kildea S10, Bataille C11, Treikale O12, Gulbis K12,

Ban R13, Semaskiene R14 and Hess M15

1Aarhus University, Flakkebjerg, 4200 Slagelse, Denmark, 2 SRUC, Edinburgh EH9 3JG, UK, 3NIAB, Cambridge CB3 0LE, UK, 4ADAS Rosemaund, Preston Wynne, Hereford HR1 3PG, UK, 5Institute of Plant Protection, Sosnicowice 44-153, Poland, 6Institute of Plant Protection – National Research Institute, 60-

318 Poznań, Poland, 7Arvalis Institut du végétal, Station Expérimentale, 91720 Boigneville, France, 8JKI, 38104 Braunschweig, Germany, 9Institut für Pflanzenschutz, Bayerische Landesanstalt für Landwirtschaft, 85354 Freising-Weihenstephan, Germany,10Teagasc, Oak Park, Carlow, Ireland, 11CRA-W, Protection des plantes et écotoxicologie, 5030 Gembloux, Belgium, 12Latvian Plant Protection Research Centre, LV1039

Riga, Latvia,13Plant Protection Institute, St. István University, 2103 Gödöllö, Hungary, 14Lithuanian Research Centre for Agriculture and Forestry, 58344 Akademija, Kėdainiai Distr., Lithuania,

15Phytopathology, TUM School of Life Sciences, 85354 Freising-Weihenstephan, Germany.


During four seasons from 2015-2018 the efficacies of different azoles for control of Septoria tritici blotch (STB) have been screened in field trials of winter wheat across Europe in a project including many European partners. For control of STB a general drop and a big variation in efficacy has been seen for the tested azoles. Particularly the best ranked azoles varied markedly between regions. Based on samples with STB from the trial sites, the sensitivity of Zymoseptoria tritici populations has been tested in vitro and the EC50 values calculated. Furthermore, the samples have been investigated for six specific CYP51 mutations using pyrosequencing and qPCR. During the 4 seasons, the populations have shifted in the different regions and more mutations have developed. A major gradient in sensitivity and presence of mutations has been seen going from eastern to western Europe, where Ireland and UK represent the most insensitive region and Hungary and Poland and the Baltic region represent the most sensitive region. Mixtures of azoles have generally performed better than single azoles exploiting the none-complete cross-resistance seen between azoles. During the last two seasons, mefentrifluconazole was included for comparison and outperformed the efficacy of all the old azoles in all regions of Europe.

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Poster 15

Efficacy of different group of fungicides on Sclerotinia sclerotiorum and their potential for control of sclerotinia stem rot in oilseed rape cultivation

Nazanin Zamani-Noor and Bernd Rodemann

Institute for Plant Protection in Field Crops and Grassland; Messeweg 11-12, 38104 Braunschweig, Germany


Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is a major disease of oilseed rape in Germany. Crop management practices and cultivar selection contribute to the control of SRR, but the effectiveness of these tactics can be modified by inoculum potential.

At the present study, field trials with artificial inoculation were conducted to assess the effects of different groups of fungicides (SDHI, QoI, DMI, Inhibitor of MAP) alone or in combinations and a biological compound (Bacillus subtilis strain QST 713) on development of SSR and crop yield parameters.

The results showed that the occurrence of SSR is strongly depending on presence of the inoculum and proper weather conditions. Significant differences in disease severity were observed between artificial inoculated plots and non-inoculated ones. Disease severity index in non-inoculated plots was 4.2%, while in inoculated controls it was up to 82%. All fungicide treatments as well as the biological agent significantly reduced the disease severity (36-87 %) and glucosinolate content (14-25 %) and increased yield (55-99 %), TGW (1.5-9.8 %), and oil content (1.4-5.5 %) as compared with the untreated control. Among all treatments, applications of fludioxonil and boscalid+pyraclostrobin combination have the best effect on the reduction of disease and yield loss.

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Poster 16

Fungicide resistance mechanisms in field isolates of Botrytis spp. from strawberry in Norway

Katherine A. G. Nielsen1, 2, Gunn Mari Strømeng1, Magne Nordang Skårn1, Kari Ørstad1, Arne Stensvand1, 2 and May Bente Brurberg1, 2

1NIBIO, Norwegian Institute of Bioeconomy Research, Ås, Norway 2NMBU, Norwegian University of Life Sciences, Ås, Norway

E-mail: [email protected] Botrytis fruit rot, or gray mold, is a major challenge in open-field production of strawberry in Norway. Unexpectedly high yield losses in 2016 prompted a study was to characterize and identify molecular mechanisms of fungicide resistance in the gray mold pathogen. Isolates of Botrytis spp. were collected from 19 strawberry fields in southern Norway in 2016. We used a germination assay to assess fungicide resistance and PCR assays to identify species of Botrytis, mating type, the multidrug resistance MDR1h genotype, and mutations known to confer resistance to boscalid, pyraclostrobin, and fenhexamid. High levels of resistance to boscalid (89.7%), pyraclostrobin (86.0%), and fenhexamid (65.0%) were detected in the gray mold pathogen, and identification of known target-site mutations in a selection of isolates corroborated these results. The most frequently detected mutations conferring resistance to boscalid, pyraclostrobin and fenhexamid were H272R, G143A, and F412S, respectively. Identification of the MDR1h genotype in isolates from 10 of 19 locations indicate that efflux pump overexpression also contributes to fungicide resistance in the gray mold pathogen population. Resistance management techniques for Botrytis must be employed to attempt to postpone resistance development to the single-site fungicides which still exhibit field efficacy.

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Poster 17

Influence of selected fungicides on the control of Ramularia collo-cygni considering sensitivity adjustments of the pathogen

Bernd Rodemann and Nazanin Zamani-Noor

Institute for Plant Protection in Field Crops and Grassland; Messeweg 11-12, 38104 Braunschweig, Germany

E-Mail: [email protected]

Ramularia collo-cygni is responsible for the disease Ramularia leaf spot (RLS) in barley causing rapid leaf senescence late in the season, which can result in extensive quality and yield losses.

For controlling RLS, the efficacy of various fungicides from the groups of azoles (DMI), carboxamides (SDHI), strobilurins (QoI) and "multi sites" were tested in a field trial. The application was carried out in GS 49/51 as a protective application.

The results showed a very good effect of chlorothalonil with values of 82.81%. By strobilurin-containing fungicide the infestation could hardly not be reduced. The DMIs, epoxiconazole, prothioconazole and the multi site folpet showed moderate efficiencies about 60.0%. The efficacy of carboxamides varied between 48.44% (benzovindiflupyr) and 71.09% (fluxapyroxad).

Another aim of the study was sensitivity test with Ramularia isolates after pretreatment with chlorothalonil, folpet, prothioconazole and pyraclostrobin. In the chlorothalonil screening, all pretreated isolates were well controlled. The mean ED50 varied between 0.27 and 0.66 mg/L. Isolates pretreated with folpet were more difficult to control with prothioconazole or folpet.

Depending on the results of the field trial it can be assumed that the known G143A mutation was present, especially in the isolates pretreated with pyraclostrobin in the field.

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Poster 18

Control of ramularia leaf spot of barley under Irish growing conditions

Mulhare J1, Creissen H1,2 and Kildea S1

1Department of Crop Science, CELUP, Teagasc, Oak Park, Ireland2 Crop and Soils Systems, Scotland’s Rural College, Edinburgh, UK


Ramularia leaf spot of barley (RLS), caused by the fungal pathogen Ramularia collo-cygni can reduce yields of Irish winter and spring barley by up 15%. Currently fungicides are the most effective means of control. Unfortunately, as the disease often only exhibits symptoms post-anthesis, decisions on control, such as product choice and/or application rate are required in the absence of disease levels or risk indicators. In most instances growers will be risk adverse and routinely apply fungicides for its control. Leaf surface wetness during stem extension has been tentatively suggested as a risk indicator for RLS. To assess if this can be used as means of determining risk and subsequently how RLS treatments can be tailored to reflect this risk, spring barley field trials were conducted at two locations in Ireland during the 2016-2018 seasons. Fortunately, each season presented different levels of risk and subsequently disease levels. Depending on risk levels as determined by minutes of leaf wetness during stem extension, it was possible to reliably reduce both the dose or fungicide product, without adversely impacting control of RLS. However, the multisite fungicide chlorothalonil remained central to each fungicide treatment and its withdrawal from the EU will adversey impacts RLS control.

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Poster 19

Sensitivity monitoring of Plasmopara viticola to oxathiopiprolin (Zorvec ®) in Italy

Irene M. Nanni, Michela Taccioli and Marina Collina

Department of Agricultural and Food Sciences, University of Bologna - IT


Oxathiopiprolin is the first member of a new class of piperidinyl thiazole isoxazoline fungicides and it is classified under the FRAC code 49. This fungicide inhibits an oxysterol binding protein (OSBP) homologue. OSBP are lipid-binding proteins implicated in many cellular processes including signaling, vesicular trafficking, lipid metabolism, and non-vesicular sterol transport [1]. OSBP represents a novel target for oomycete disease control. The aim of this work was to evaluate the sensitivity of oxathiopiprolin (Zorvec ® – Corteva) on Plasmopara viticola samples coming from different area of Northern Italy in 2018 (first year of authorization). Bioassays on leaf discs were carried out, and the results showed the complete sensitivity of P. viticola populations to the active ingredient.

The availability of oxathiapiprolin may now improve significantly the downy mildew disease control in field. Unfortunately, the fungicide is prone to resistance phenomenon then it has to be monitored and follow the guidelines provided each year by Fungicide Resistance Action Committee (FRAC) is essential.

[1]. Raychaudhuri S, Prinz WA (2010) The Diverse Functions of Oxysterol-Binding Proteins. In: Schekman R, Goldstein L, Lehmann R, editors. Annual Review of Cell an Developmental Biology, Vol 26. pp.157–177.

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Poster 20

Resistance of Penicillium expansum to the SDHI fungicide boscalid and identification of mutations in sdh gene

P. Ntasiou, Α. Samaras, C. K. Myresiotis and G. S. Karaoglanidis

Aristotelian University of Thessaloniki, Faculty of Agriculture, Forestry and Natural Environment, School of Agriculture Laboratory of Plant Pathology, POB 269, 54124, Thessaloniki, Greece


Blue mold caused by Penicillium spp. is considered as the major postharvest disease of apple fruit. Disease control is based on fungicide use, applied either in the orchard or in the packinghouses after harvest. This study was initiated to determine resistance frequency to the SDHI fungicide boscalid in fungal populations originating from apple orchards in Greece and identify mutations in sdh gene, associated with resistance to SDHIs. In total, 260 isolates of P. expansum were obtained from fruit collected in different apple growing regions. For sdh gene mutations identification 5 boscalid-resistant and 5 sensitive isolates were selected. The sdhB, C and D subunits were amplified and sequenced. Sequencing results revealed the presence of 4 different mutations in sdhB (S106L/S175L) and sdhD subunits (L36P/N52I). To confirm the effect of these mutations in the resistance to SDHIs, sdh mutants were constructed and fuction analysis was performed. The boscalid-resistant isolates were further characterized in terms of fitness parameters, mycotoxigenic ability and cross-resistance relationships among several SDHIs. The above-mentioned data represent the first report of resistance development to SDHIs in field isolates of P. expansum, worldwide.

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Poster 21

Optimal mixtures of high risk fungicides for resistance management

Nick P. Taylor1*, Frank van den Bosch2 and Nik J. Cunniffe1

1 Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA, UK2 Rothamsted Research, Harpenden, AL5 2JQ

* E-mail: [email protected]

Fungicide resistant crop pathogens are widespread and prolonging the effective lifetime of those few chemicals which remain effective is imperative. The remaining chemicals are often so-called “high risk” fungicides, to which some resistance is already present in pathogen populations, or against which resistance can readily be evolved. With the availability of low risk fungicides (e.g. chlorothalonil) decreasing, due to environmental and regulatory pressures, understanding how to best utilise high risk fungicides is increasingly important. However, most modelling studies focus on the simpler case of combining a low risk with a high-risk chemical.

We use a carefully parameterised epidemiological model to determine the optimal way in which to combine pairs of high risk fungicides. Our model is parameterised for Zymoseptoria triciti, an economically-important disease of winter wheat. We show how an optimal mixture maximising the lifetime crop yield can be defined. The optimal strategy is to use the minimal doses providing economically-acceptible yields, which exert equal selection rates on the two fungicides. This allows the precise spray concentrations to be determined using the fungicide dose response curves.

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Poster 22

Insensitivity to pyraclostrobin in Peyronellaea pinodes affecting field pea

Julie S. Pasche, Dimitri L. Fonseka, Robin S. Lamppa, and Kimberly Zitnick-Anderson

Dept. of Plant Pathology, North Dakota State University, Fargo, ND. USA


Ascochyta blight is one of the most severe diseases of field peas when cool, wet weather persists. This host-specific disease is caused by a complex of fungal pathogens, including Peyronellaea pinodes, P. pinodella, and Ascochyta pisi. These pathogens infect all above-ground parts of the pea plant including stems, leaves, pods, and seeds. Cultural practices alone can be insufficient to manage the disease, thus application of foliar fungicides is necessary. Reduced efficacy of QoI fungicides was first detected in isolates of P. pinodes from North Dakota in 2017. Fourteen and 28 isolates collected from two locations in 2017 and 2018, respectively, exhibited resistance to pyraclostrobin in mycelial growth assays. In vitro resistance factors ranged from approximately 5 to >1,400 when compared to sensitive isolates. Disease control of some resistant isolates was less than half that of sensitive isolates at 100 µg/ml pyraclostrobin in greenhouse efficacy tests. The frequency and distribution of QoI resistant P. pinodes isolates in North Dakota is under investigation. High frequencies and vast distribution of resistant isolates will result in the fungicide to be of no value to the growers. Implementing new resistance management tactics may delay, or prevent, the development of a fully resistant P. pinodes population.

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Poster 23

The Pathogen-Host Interactions phenotype database, PHI-base: Harnessing community expertise to fight microbial incited diseases

Martin Urban1, Alayne Cuzick1, James Seager1, Kim Rutherford2, Valerie Wood2 and Kim E. Hammond-Kosack1

1Department of Biointeractions and Crop Protection, Rothamsted Research, Harpenden, AL5 2JQ, UK and 2Cambridge Systems Biology and Department of Biochemistry, University of Cambridge, CB2 1GA,

UK

The multi-species pathogen-host interactions database PHI-base, www.phi-base.org, is a gold-standard database storing phenotypes on genes implicated in virulence for 266 pathogens tested on 199 hosts. Information is also given on the target sites of commercial and experimental anti-infective chemistries. It is a primary information source for researchers studying plant, animal and human pathogens. Manually curated information from more than 3,200 peer reviewed research articles are made accessible and searchable to provide relevant molecular and biological facts on pathogenicity, wild-type/mutant genes and fungicide target sites. High-level phenotypes are used to describe the overall experimental interaction outcomes enabling comparative analysis of different pathosystems. More detailed information including the effect of fungicides are captured using free text manual curation and controlled vocabularies.

Here we describe our new PHI-base Version 4.7 release (May 2019). Our data platform includes advanced search functions, extended data displays and a protein-to-phenotype BLAST capability. A search for keyword ‘anti-infective’ will retrieve all genes where chemistry targets were experimentally tested. We will also demonstrate a new web-based community curation tool called PHI-Canto for authors to capture how fungicide applications have affected the outcome of host-microbe interactions reported in their own peer reviewed articles. PHI-Canto supports the curation of resistance phenotypes.

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Poster 24

FRAC - working together globally to manage fungicide resistance

Juergen Derpmann1) and Dietrich Hermann2)

1) Bayer AG, Division CropScience, Germany2) Syngenta Crop Protection AG, Switzerland

Fungicide resistance is a long standing and expanding problem for disease control. Effective resistance management is essential to ensure long term efficacy of fungicides for sustainable agriculture. Established in 1981, the Fungicide Resistance Action Committee (FRAC) serves as a specialist technical group within CropLife International, the association of crop protection companies. The FRAC Mode of Action (MoA) Expert-Panel is continuously reviewing information on cross-resistance and MoA of new and established fungicides. The FRAC Code List and the corresponding FRAC Poster provides the latest classification scheme based on the MoA and cross-resistance pattern of fungal control agents. This knowledge serves as basis for developing appropriate resistance management strategies. FRAC-Working-Groups, Expert-Fora and Task-Forces are platforms to annually review available resistance monitoring data to unanimously agree on resistance management guidelines for all fungicides within respective MoA/cross-resistance group in relevant crops. Guidelines are published on the FRAC website and are regularly updated. Dissemination and implementation of advice is done in efficient ways by FRAC regional and national organizations. Also, information is passed freely between FRAC and national action groups (FRAGs) including members of research institutions, advisory bodies and other experts involved. This forms a strong network in providing fact-based recommendations to growers and advisors.

65

Poster 25

How to mitigate development of resistance to pesticides in Alternaria solani, the causal agent of potato early blight?

Åsa Lankinen, Linnea Almqvist, Hadis Mostafanezhad, Erik Alexandersson, Laura Grenville-Briggs and Erland Liljeroth

Department of Plant Protection Biology, Swedish University of Agricultural Sciences (SLU), Sweden


Potato suffers from several serious diseases, leading to large economic losses and dependence of pesticides. Early blight, caused by the fungus Alternaria solani, is currently increasing in Sweden and other parts of Europe due to rising temperatures but also due to increased presence of pesticide resistance in Alternaria populations. There is a clear need for new and innovative plant protection strategies. An underused possibility is to take advantage of agricultural practices that create disruptive, frequency-dependent selection on the pathogen, thereby stabilizing its evolutionary potential. In an ongoing project involving potato field trials, we are studying how an IPM approach with combinations of biological control agents, induced resistance and cultural practices could reduce the pesticide dependence and delay pesticide resistance in A. solani without mitigating crop yield. We also investigate the impact of soil quality on survival of A. solani during the inter-epidemic phase of the pathogen life cycle as well as whether the main source of inoculum comes from the soil or the air, using spore traps. Moreover, we explore a potential impact of wild Solanum species growing naturally within or close to potato fields. The results could help us understand which factors mitigate pesticide resistance in A. solani.

66


Poster 26

Confirmation of Bromus rubens resistance to glyphosate in perennial crops in Spain

José G. Vázquez-García1, Sajedeh Golmohamadzadeh 2, Candelario Palma-Bautista1, Antonia M Rojano-Delgado1, Joel Torra2 and Rafael De Prado1

1 Department of Agricultural Chemistry and Soil Science, University of Cordoba, 14071 Cordoba, Spain2Gorgan University of Agricultural and Natural Resource, Iran

3Department of Hortofruticulture, Botany and Gardening, University of Lleida, 5198 leida, Spain


As the only herbicide used, resistance to glyphosate is a major concern in perennial crops in Southern Spain, in. At the end of 2017 Bromus spp had begun to spread through olive and almond orchards due to the low glyphosate control, while previously it was well controlled at a field dose of 1080 g ae ha-1.

In a field screening carried out on these crops treated with glyphosate during 2017 and 2018, 14 populations of Bromus spp were collected and their levels of resistance were studied in greenhouse and laboratory experiments. Early studies showed that every population belonged to the species Bromus rubens. Of those, two populations were glyphosate susceptible (GS), with LD50 values lower than the field dose, while the 12 remaining were glyphosate resistant (GR) with values greater than that dose. The GR50

values were in agreement with the previous results and the resistance factors (RF) were between 2.46 and 6.25. Posterior laboratory tests showed that the two GS populations accumulated 4-fold more shikimic acid than the respective GR populations, with values ranging between 187 and 264 μg g-1 fresh weight. This is the first confirmation case of glyphosate-resistance in B. rubens worldwide.

67


Poster 27

Glyphosate-resistant goosegrass confirmed in Tolima, Colombia

Javier Ramírez1, Verónica Hoyos2, Guido Plaza3 and Rafael De Prado4

1Market development, Bayer Crop Science 2Facultad de Ingeniería, Universidad del Magdalena, Colombia

3Departamento de Agronomía, Universidad Nacional de Colombia-Bogotá, Colombia 4Departamento de Química Agrícola y Edafología, Universidad de Córdoba, España

e-mail: [email protected]

Goosegrass is a highly competitive weed, with a wide distributon throughout the country. In the last years, poor goosegrass control have been observed after commercial glyphosate applications over Roundup Ready corn fields in Tolima department, suggesting this species resistance to the herbicide. The objective of this study was to evaluate the existence of goosegrass populations resistant to glyphosate. Sampling was carried out in commercial crops of cotton, rice, and maize GMO. The experiment was conducted in a greenhouse in a completely randomized design with four replications. Glyphosate dose-response curve experiments were performed using the following doses: 0, 195, 391, 781, 1563, 3125, 6251 and 12502 g ae ha-1. Herbicide application occurred in the 3-4 leaf stage of the weed and the variables analyzed were control, fresh and dry matter 21 days after treatment (DAT). Dose-response assays confirmed that all assessed accessions were resistant to glyphosate, showing GR50 higher than susceptible biotype “La Isla” (147.7 g ae ha-1) and resistance index (RI) higher than 2. The accesion “Horizonte” had higher resistant index (61.4) and GR50 5905 g ae ha-1. By contrast, the accession "Providencia" presented lower levels of resistance (RI 6.04) and GR50 561.3 g ae ha-1.

68


Poster 28

Physiological and biochemical studies in Conyza canadensis populations, multiple-resistant to tribenuron-methyl and glyphosate

Candelario Palma-Bautista1, Nikolina Cheimona2, Antonia M. Rojano-Delgado1*, María D. Osuna-Ruiz3, Rafael De Prado1

1Department of Agricultural Chemistry and Soil Science, University of Cordoba, 14071 Cordoba, Spain2 Laboratory of Agronomy, Agricultural University of Athens, 118 55 Athens Greece

3 Center for Scientific and Technological Research of Extremadura (CICYTEX), 06187 Badajoz, Spain

E-mail: qe1pramarco.es

The physiological and biochemical bases for multiple resistance to acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor herbicides were studied in two populations of Conyza canadensis (RTG and STG), harvested in southern Spain.

Using whole plant dose-response and enzymatic activity studies, the results showed only cross- resistance to the sulfonylureas group, but not to the other four ALS chemical groups. Concerning glyphosate, the dose-response studies showed that the RTG population was 11.8 times more resistant than the STG population, while the inhibition of the EPSPS enzyme (I50) showed similar values for both populations. Moreover, absorption and translocation were not the mechanisms responsible for tribenuron-methyl resistance in horseweed, while in the case of glyphosate, they were the main resistance mechanisms. Metabolic studies showed differences between both populations for tribenuron-methyl, being higher the rate of metabolism in the RTG compared to the STG population; however, for glyphosate, no metabolite appeared in either populations of C. canadensis. These results document the first case in the world of C. canadensis with multiple-resistance and it has been demonstrated that NTSR mechanisms (absorption/translocation and metabolism) are involved in the multiple-resistance to sulfonylureas and glyphosate, respectively.

69

Poster 29

First case of multiple resistance to glyphosate and synthetic auxin herbicides in Parthenium hysterophorus in citrus orchards from Dominican Republic

Andrés D. Mora1, Jesús Rosario2, Antonia M Rojano-Delgado1, Candelario Palma-Bautista1, Joel Torra3*, Julio Menéndez4 and Rafael De Prado1

1Universidad de Córdoba, 14071 Córdoba, Spain; 2SODIAF, Santo Domingo, Dominican Republic; 3Universitat de Lleida, 25198 Lleida, Spain; 4Universidad de Huelva, 21071 Huelva, Spain

*Email: [email protected]

Glyphosate has been widely used in citrus groves from the Dominican Republic. Unfortunately, Parthenium hysterophorus has developed glyphosate-resistance, and farmers have started to apply synthetic auxin herbicides (SAH) as the single alternative. Field assays carried out in 2017 in a field located in Balsima (Villa Altagracia province) showed that this P. hysterophorus population survived field rates of glyphosate, 2,4-D, dicamba and picloram (with efficacies of 17.5, 1.5, 31.5 and 43.6, respectively). In in vivo assays, the resistance factors (RF) using doses causing 50% reduction in the above ground biomass showed high levels of glyphosate resistance (RF= 49.75) and moderate to low levels of resistance to the SAH picloram (RF=6.81), dicamba (RF=5.34) and 2,4-D (RF=5.10). Firstly, the EPSPS gene sequencing showed that a Pro-106-Ser aminoacid substitution was conferring resistance to glyphosate. Secondly, more in vivo assays were carried out to study the synergistic effects of the cytochrome P450 (P450) inhibitor malathion with SAH. RF fell by about 50%, which demonstrated that P450 could be involved in the resistance mechanism to these three SAH. To explain the overall resistance levels observed to SAH, we hypothesized that another mechanism could be contributing to the resistance in this multiple herbicide resistant P. hysterophorus population.

70


Poster 30

Target and non-target site resistance to ALS inhibiting herbicides in Gleobonis coronaria L.

Zeineb Hada*1, Joel Torra2, Julio menendez3, Yosra Menchari1, 4, Thouraya Souissi 1

1: Département de Santé Végétale et Environnement, Institut National Agronomique de Tunisise Université de Carthage, Tunis, Tunisia 2: Department d’Hortofructicultura, Botànica i Jardineria,

Agrotecnio, Universitat de Lleida, Lleida, Spain 3: Departamento de Ciencias Agroforestales, Escuela Politécnica Superior, Campus 11 Universitario de La Rábida, Huelva, Spain 4 : Higher Institute of

Biotechnology of Beja, University of Jendouba, Tunisia

* E-mail: [email protected]

Chrysanthemum (Glebionis coronaria L. Cass. Ex Spach) is an annual dicot broadleaf weed very common in cereal crops in north Tunisia. The chemical control using common ALS inhibiting herbicides is declared ineffective by Tunisian farmers in last years. This control failure could be attributed to the development of herbicide resistance in chrysanthemum in the north of the country.

This research was conducted to confirm the resistance of Tunisian chrysanthemum to ALS inhibiting herbicides and the potential mechanisms involved in this resistance, investigating both target site and non-target site mechanisms.

ALS gene sequencing revealed different amino acid replacements at three positions which confer high resistance levels to tribenuron methyl in chrysanthemum as target-site mechanism.

The mobility of tribenuron-methyl was also studied in two resistant populations of chrysanthemum compared to two susceptible ones. The data showed no differences between the resistant and susceptible plants at all sampling times, as herbicide was always moving from treated leaves to the rest of the plant. These results indicate that absorption and translocation are not involved as resistance mechanisms to tribenuron-methyl in chrysanthemum. However, we are still investigating the presence of enhanced metabolism mechanism to be sure about the non-target site mechanisms to ALS inhibiting herbicides in this species.

71


Poster 31

Target and non-target site resistance mechanisms in a Poa annua biotype from Tennessee

J.T. Brosnan, M. LaForest, S.L. Boggess, J.J. Vargas and R.N. Trigiano

A weedy biotype of annual bluegrass (Poa annua) selected from a stand of bermudagrass (Cynodon spp.) on a golf course (Memphis, TN USA) was confirmed to have target site resistance to photosystem II inhibiting herbicides, as well as a novel Ala205Phe substitution conferring resistance to inhibitors of acetolactate synthase. Transcriptomic analyses explored non-target site resistance in this P. annua biotype (POAAN-R3). POAAN-R3 and an herbicide susceptible control were treated with trifloxysulfuron (28 g a.i. ha-1) after the three-tiller growth stage. Aboveground biomass was harvested from all five replicates of each biotype at 0, 2, 6, 12, 24 and 48 hours after treatment and mRNA was isolated and sequenced. Five differentially expressed genes were identified and verified via RT-qPCR, including an ABC Type-2 transporter that was constitutively overexpressed in POAAN-R3 compared to the herbicide susceptible control. Complimentary DNA transformations in rice (Oryza sativa, cv. ‘Kitaake’) callus will be completed to validate the role of this ABC Type-2 transporter in conferring resistance to multiple herbicidal modes of action.

72

Poster 32

Prevalence and mechanisms of resistance to PSII inhibitors in Chenopodium album in the Czech Republic

Pavlina Kosnarova, Pavel Hamouz, Jaromir Suk and Josef Soukup

Department of Agroecology and Plant Production, Czech University of Life Sciences Prague, Prague 165 00, Czech Republic


Seeds from 35 putatively resistant populations of Chenopodium album were collected from weed survivals in sugar beet, maize and potato fields across the country. Registered doses of terbuthylazine and metamitron were applied in small pots assays to identify resistant (R) populations and subsequently the dose-response test to determine the GR50R and GR50S values and resistance factor (RF) was conducted. To describe the mechanism of resistance, the nucleotide changes at positions known to confer resistance to PSII-inhibiting herbicides were analysed on surviving plants. Fluorescence measurements were performed with 10 plants from each R biotype using pulse amplitude modulation fluorometer Imaging-PAM Maxi and Imaging Win software.

Two resistant populations were identified in the greenhouse dose-response study. RF value for terbuthylazine was higher than 4514 for both R biotypes and the RF value for metamitron was 10.6 and 30.4 respectively. A Ser-264-Gly mutation at the psbA gene was detected in 100% of the analysed plants which survived the terbuthylazine and metamitron treatment. Approximately half of the tested plants of these two biotypes showed only marginal decrease of QY PSII after terbuthylazine application. Significant differences in QY PSII were observed between resistant and susceptible biotypes 12 hours after application.

This study work was supported by Syngenta Research Project.

73


Poster 33

Resistance to ALS inhibiting herbicides in a Spanish Echinochloa cruss-galli population from a corn field

Joel Torra1*, José María Montull1, Aritz Royo-Esnal1, Andreu Taberner1 and Marisa Salas2

1 Universitat de Lleida, Avda. Rovira Roure 191, 25198, Lleida, Spain; 2 Corteva Agriscience™, Agriculture Division of DowDuPont™, 78067 Saint Quentin Yvelines Cdx, France

*E-mail: [email protected]

Acetolactate synthase (ALS) inhibiting herbicides are widely used in corn in Spain for grass weed control in post-emergence. Monocrop and recurrent use has resulted in the evolution of several resistant (R) Echinochloa crus-galli populations. So far, only the Target-site resistance mechanism by means of a Pro-197-Ser aminoacid substitution has been reported as being responsible of the resistance to ALS inhibiting herbicides in Spain.

A potential E. crus-galli population R to nicosulfuron-methyl was studied after farmer complaints of lack of control in the province of Lleida. The initial frequency of R plants was assessed by sequencing the ALS gene. After sequencing 245 plants in positions 197 and 574, 28% of the plants were all heterozygous for the Trp-574-Leu amino acid substitution. After sequencing more than 500 plants in these and further samplings, no single plant had the amino acid substitution in position 197.

Experiments are underway to further characterise the resistance profile of this population to ALS inhibiting herbicides with dose-response tests. This is the first report of the Trp-574-Leu amino acid substitution in this species from Spanish corn fields.

74


Poster 34

First Czech case of herbicide resistance to tribenuron and florasulam in Stellaria media

Kateřina Hamouzová, Pavlína Košnarová, Josef Soukup, Pavel Hamouz and Jaromír Šuk

Czech University of Life Sciences, Faculty of Agrobiology, Food and Natural Resources, Dpt. Of Agroecology and Crop Production, Kamýcká 125, Prague 165 00, Czech Republic


Resistance to ALS inhibitors, PSII inhibitors and synthetic auxins in Stellaria media has been reported in 15 countries worldwide, but no case has been found in the Czech Republic so far. Stellaria media belongs to the weed species that is considered to have a medium inherent risk of evolving resistance. Studies showed that resistance was conferred by point mutation in Trp-574, associated with resistance to sulfonylurea and triazolopyrimidine herbicides.

Seeds from Czech biotype (R) that survived an application of sulfonylureas in field and sensitive standard (S) were grown and registered doses of florasulam and tribenuron were applied in small pots assays to confirm resistance. Subsequently the dose-response test to calculate the resistance factor (RF) was conducted. The nucleotide changes at positions known to confer resistance to ALS-inhibiting herbicides in Stellaria media were analysed on surviving plants.

RF values for both active ingredients were very high (>3000) in R biotype. Resistant biotype carried target-site mutation previously reported in many weeds. A Trp‐574‐Leu mutation of the als gene was detected in 100% of the analysed plants which survived the herbicide treatment, no plant possessed a point mutation in Pro-197 position. This particular R- biotype may be cross-resistant to other B2 group herbicides.

75


Poster 35

Derived Polymorphic Amplified Cleaved Sequence (dPACS): a novel PCR-RFLP procedure for detecting known single nucleotide and deletion-insertion polymorphisms

Shiv Shankhar Kaundun*, Elisabetta Marchegiani, Sarah-Jane Hutchings and Ken Baker

Syngenta Ltd, Jealott’s Hill International Research Centre, Bracknell RG42 6EY, United Kingdom


Most methods developed for detecting known single nucleotide and deletion-insertion polymorphisms are dependent on sequence conservation around the SNP/DIP and are therefore not suitable for application to heterogeneous organisms. Here we describe a novel, versatile and simple PCR-RFLP procedure baptised ‘derived Polymorphic Amplified Cleaved Sequence’ (dPACS) for genotyping individual samples. The peculiarity of the method is that it employs a pair of primers that cover the entire fragment to be amplified except for one or few diagnostic bases around the SNP/DIP being investigated. As such, it provides greater opportunities to include mismatches in one or both of the 35-55 bp primers for creating a restriction site that unambiguously differentiates wild from mutant sequences, following PCR-RFLP and horizontal Metaphor gel electrophoresis. Selection of effective restriction enzymes and primers is aided by the newly developed dPACS 1.0 software. The highly transferable dPACS procedure is exemplified here with the positive detection in up to 24 diverse grass and broadleaf weed species tested, of wild type proline106 of 5-enolpyruvylshikimate-3-phosphate synthase and its serine, threonine and alanine variants that confer resistance to glyphosate, and serine264 and isoleucine2041 which are key target-site determinants for weed sensitivities to some photosystem II and acetyl-CoA carboxylase inhibiting herbicides, respectively.

76


Poster 36

Detection of resistance to PPO-inhibiting herbicides using the Syngenta Herbicide Resistance Leaf (HRL) test

Joe Downes, Lucy V Jackson, R. Joseph Wuerffel, Sarah-Jane Hutchings and Shiv S Kaundun

Syngenta Ltd, Jealott’s Hill International Research Centre, United Kingdom


Herbicide resistance is generally verified using seeds collected from field survivors at the end of the growing season in the laborious and time-consuming whole-plant pot test. The Syngenta Herbicide Resistance Leaf (HRL) assay, conducted in Petri-dishes or other suitable containers, offers an alternative method with resistance manifested by green and healthy meristematic leaves and sensitivity by bleaching and necrosis after 7-10 days of exposure to an informative dose rate of herbicide. The methodology is illustrated by the confirmation of resistance to PPO-inhibiting herbicides in Amaranthus spp. Preliminary studies showed that individuals containing the 210 target-site codon deletion or mutations (R to G or M) at codon 128 in the PPX2L gene could be clearly distinguished from wild type sensitive plants. Resistance to PPO-inhibiting herbicides in field survivors was successfully detected in a number of US Amaranthus tuberculatus/palmeri populations tested locally or at a central location alongside known sensitive and resistant standards. The Syngenta HRL test has also proved effective for detecting resistance to some other fast-acting herbicides and weed species. Wherever applicable, it could constitute a very simple and cost-effective methodology for detecting herbicide resistance at its very onset by targeting the initial plant survivors in the field.

77


Poster 37

Weak and strong target site ALS resistance mutations in Raphanus raphanistrum: correlation between resistance at the ALS and plant level

Federico García1, Mercedes Gil2, Graciela Nestares2, Pedro E. Gundel1 and Martin M. Vila-Aiub1

1IFEVA-CONICET – Facultad de Agronomía, Departamento de Ecología, Universidad de Buenos Aires (UBA), Argentina

2IICAR-UNR-CONICET, Facultad de Ciencias Agrarias, Universidad Nacional de Rosario (UNR), Argentina


Despite the largely known effect of target site ALS resistance mutations in endowing plant survival under treatment of ALS inhibiting herbicides in many weeds, it remains unknown whether plants with these resistance mutations can reproduce similarly to an environment without ALS treatment.

The resistance level of Raphanus raphanistrum (wild radish) with either the homozygous Ala-122-Tyr or Asp-376-Glu resistance mutations growing under chlorsulfuron treatment in a wheat crop was evaluated. The resistance level was estimated at both the plant and enzyme (ALS activity) scales.

Results showed a similar level of survival in plants exhibiting either of the mutations and a positive correlation between ALS activity and fecundity under chlorsulfuron treatment. Plants with the Ala-122-Tyr mutation showed higher ALS activity and siliques mass than plants with the Asp-376-Glu mutation. Plants with the Ala-122-Tyr mutation showed no reduction in the mass of siliques produced under chlorsulfuron herbicide and wheat competition compared to the control treatment (no chlorsulfuron, no wheat). However, plants with the Asp-376-Glu mutation significantly reduced the reproductive output under herbicide and wheat competition compared to an environment with no herbicide and crop.

The observed results enable to rank these ALS mutations in wild radish as ecologically strong (Ala-122-Tyr) and weak (Asp-376-Glu).

78


Poster 38

Prediction of PPO-inhibitor resistance development through sequence analysis

Abigail Barker1, Dawn Refsell2, John Pawlak2 and Franck Dayan1

1Bioagricultural Science and Pest Management, Colorado State University, USA. 2Valent U.S.A, USA


PPO-inhibiting herbicides were introduced in the 1960’s and became important tools for weed control in soybean fields. Lethal action of these herbicides is associated with the light-dependent catastrophic consequences of disrupting chlorophyll and heme synthesis. Resistance was first reported in 2001 in common waterhemp (Amaranthus tuberculatus) and has now been reported in 13 different species of weeds in six different countries including both monocots and dicots. Although chlorophyll is the more abundant product, all known cases of target-site resistance are on the mitochondrial isoform of the protein, credited with heme production. The mutations are a single SNP Arg98 to Leu, Met or Gly and a codon deletion of Gly210. There is evidence that this protein may then be dual targeted to both the chloroplast and the mitochondria. An in-depth sequence analysis was preformed to determine which species were capable of the Gly210 deletion, the Arg98 deletion, and in some cases the possible dual-targeting. These results identify a few troublesome weed species which should be monitored for rapid development of resistance to PPO inhibitors, but since the sequence data is not available for most weedy species we can only make general inferences based on related phyla.

79


Poster 39

Characterizing reduced sensitivity to HRAC group K3 herbicides in scentless mayweed

Dagmar Rissel and Lena Ulber

Institute for Plant Protection in Field Crops and Grassland, Julius Kühn Institute, Germany


To date, pre-emergence herbicides belonging to HRAC group K3 were considered to posses a low potential to evoke herbicide resistance. However, in the last four years the number of global resistance cases reported for HRAC group K3 had doubled. During a national resistance survey focusing on dicotyledonous weed species, a scentless mayweed (Tripleurospermum perforatum) population was detected showing reduced sensitivity to metazachlor, a HRAC group K3 active, under field conditions. In a repeated bioassay using the maximum registered dose rate of metazachlor (750 g/ha) individual surviving plants were found after herbicide treatment. These surviving plants were propagated to produce a F1 progeny. Using this F1 progeny, a dose-response pot bioassay using metazachlor at multiple dose rates (0.125N – 4N) was conducted. Again, individual plants surviving metazachlor treatments including the maximum registered dose rate were detected. Additionally, several plants also survived the field dose rate of dimethenamide, another HRAC group K3 active. To unravel the mechanisms of reduced sensitivity in this scentless mayweed biotype, F1 progeny plants were treated with inhibitors of cytochrome P450 and glutathione-S-transferase (malathion, NBD-Cl, ethacrynic acid). As the responses of the soil-grown plants to the tested inhibitors are ambiguous, agar-based bioassays including these inhibitors are under way.

80


Poster 40

Herbicides efficacy on classified Echinochloa spp. accesisons combining classic dichotomous keys and DNA barcoding

Mascanzoni E.1, Panozzo S.2, Hoste I.3, Verloove F.3, 2Scarabel L., Sattin M.2

1University of Padova, Italy, 2Research Institute on Terrestrial Ecosystems (IRET), CNR, Italy, 3Meise Botanic Garden, Belgium

Echinochloa is a complex genus that includes about 50 species for which classification is often difficult due to its high morphological variability and plasticity. The susceptibility to herbicides can differ among Echinochloa species and several of them have evolved resistant populations to multiple herbicides, threatening the sustainability of rice cropping systems in Italy. Identification of the species involved and their behavior is a key information for devising proper resistance management strategies. By combining the classification results of several Echinochloa accessions with a molecular approach - DNA barcoding - and classic dichotomous keys we aimed to a) clarify which Echinochloa species are present in Italian rice fields and if plants with an "intermediate" phenotypes should be considered as different species or morphological variations of already classified ones and b) verify the response of nine purified and classified susceptible Echinochloa accessions to herbicides with different site of action. Results obtained show that E. oryzicola and E. crus-galli are widespread in Italian rice fields and that growing environment influences phenotypic variability. Also, the different species of Echinochloa do not show a specific response to herbicides with different SoA, although E. crus-galli seems slightly more susceptible than the other species of Echinochloa identified.

81

Poster 41

Evolutionary genomics of herbicide resistant weeds

Bridgit Waithaka-Vasiljević, Ulrich Lutz, Ilja Bezrukov and Detlef Weigel

Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany


The evolution of herbicide-resistant weeds is a predictable consequence of natural selection. Mechanisms underlying herbicide resistance are classified into two broad categories: the wellstudied target-site resistance mechanisms (TSR), and non-target-site resistance mechanisms (NTSR) whose genetic determinants are poorly understood. Numerous studies have illustrated the quantitative nature of NTSR-associated traits. Owing to the high variability of NTSR, noncandidate-gene based approaches are necessary to elucidate the genetic basis underlying NTSR. In order to identify the natural modulators of glyphosate resistance, I am evaluating the differential response of 104 Arabidopsis thaliana accessions to 4 glyphosate doses for genomewide association studies (GWAS). Preliminary analyses have hinted at a continuous phenotypic distribution suggesting the polygenic control of resistance. I will then conduct quantitative trait locus (QTL) mapping on segregating F2 populations derived from parental lines exhibiting extreme phenotypic responses. Combining both approaches will aid in resolving the complexity of genetic architectures underlying NTSR mechanisms and sets the stage for identifying causal loci in the evolution of NTSR after recurrent herbicide selection.

82


Poster 42

Update to the Herbicide Resistance Action Committee classification on mode of action

Rex Liebl1, Jeffrey Epp2, Bernd Laber3, Hubert Menne3, James Morris4 and Matthias Witschel5

1BASF, RTP NC USA, 2Corteva Agriscience, Indianapolis IN USA, 3Bayer Crop Science, Frankfurt Germany, 4Syngenta, Jealotts Hill UK, 5BASF, Ludwigshafen Germany


The Herbicide Resistance Action Committee (HRAC) mode of action classification was last updated in 2010 and needed an overhaul to add new actives, rationalize chemical family naming and update modes of actions. HRAC sponsored a Working Group comprised of senior herbicide chemists and agronomists at Corteva, Bayer Crop Science, Syngenta and BASF to update the HRAC mode of action classification including “The World of Herbicides” poster. Changes included the addition of 10 recently launched actives and three new modes of action: inhibition of homogentisate solanesyltransferase, inhibition of solanesyl diphosphate synthase and inhibition of fatty acid thioesterase. Full details of all changes will be presented.

83


Poster 43

A matrix for herbicide resistance risk quantification

Lena Ulber1, Stephen Moss2 and Ingrid den Hoed3

1Julius Kühn-Institut, 38104 Braunschweig, Germany 2Stephen Moss Consulting, Harpenden, Hertfordshire, AL5 5SZ, UK

3Health and Safety Executive (HSE), Kings Pool, York, YO1 7PX, UK


Resistance risk assessment is a key part of the authorisation process for plant protection products in most European countries. EPPO Standard ‘PP 1/213 (4) Resistance risk analysis’ provides some guidance on the quantification of the resistance risks posed by herbicides. However, a risk matrix has now been developed, in cooperation with EPPO, to achieve greater harmonisation of herbicide resistance risk assessment across Europe.

In the matrix, the inherent, ‘unmodified’ risk is first assessed by ranking herbicide active ingredients and major target weed species on a scale from ‘low’ to ‘high’ resistance risk. The second step considers agronomic practices which can reduce the resistance risk, including both chemical and non-chemical management strategies (‘modifiers’). By assigning defined impact factors to possible agronomic modifiers, the overall resistance risk of a herbicide under defined use conditions can be quantified.

When tested with four contrasting scenarios, the matrix produced realistic assessments of the associated resistance risks. Although the matrix has a European legislative focus, the approach and principles are relevant in other parts of the world where knowledge on herbicide resistance and the evaluation of resistance risks may be more limited.

Reference: Moss, S., Ulber, L. & den Hoed, I. (2019) A herbicide resistance risk matrix. Crop Protection 115:13-19 (Open access).

84


Poster 44

Studies of rigid ryegrass (Lolium rigidum) resistance to glyphosate in perennial crops in Greece

Demosthenis Chachalis1, I. Travlos2, E. Tani3 and T. Gitsopoulos4

1Benaki Phytopathological Institute, Weed Science Laboratory, Athens 14561, GR2Agricultural University of Athens, Laboratory of Agronomy, Athens, GR

3Agricultural University of Athens, Laboratory of Breeding and Biometry, Athens, GR4Hellenic Agriculture Organazation-DEMETER, Institute of Plant Breeding and Genetic Resources,

Thessaloniki, GR


There has been an increasing concern about the low level of control of rigid ryegrass populations with glyphosate, across various regions in Greece. A total of eleven rigid ryegrass biotypes (1 susceptible, and 10 possibly resistant), were collected from olive trees in western Greece (Agrinion) and south Greece (Messinia) in order to test them for glyphosate resistance. Dose-response experiments were conducted in pots, as follows: 0.6X, 0.8X, 1X, 1,5X, 2X, (whereas 1X=720 g. a.i./ha). In all populations, a biochemical test (shikimate analysis) was done for further evidence or not of glyphosate resistance. The S-population demonstrated very low survival and growth rates (lower than 8% in both cases, even at 0.6X glyphosate dose) compared to the untreated control. In contrast, one population (Agr.0.38) was found to be resistant (approximately 40% survival and growth, even at 2X glyphosate dose). Three populations had an intermediate response, between to the S- and R-biotype, and they are considered “emerging or mixed resistant” populations. Shikimate analysis confirmed the status of the resistant and susceptible populations. Seeds of the survived plants in each population were collected for further analysis after plant protection to avoid inter-population contamination due to cross-pollination. Results from this study clearly indicated that the rigid ryegrass resistance to glyphosate could be an emerging problem in perennial crops (olives) thus a more widespread screening is necessary in Greece.

85


Poster 45

Evolution of black grass resistance to ACCase and ALS inhibitors in Wallonia (Belgium)

Pierre Hellin, Aurélie Clinckemailie, François Henriet and Maxime Duvivier

Walloon Agricultural Research Centre, Plant Protection and Ecotoxicology Unit, Gembloux, Belgium


Black-grass (Alopecurus myosuroides) is one of the major weed causing yield losses in wheat production. In Belgium, some areas were historically known for their black-grass management problems and herbicide resistance seemed to be restricted to those lands.

In 2018, greenhouse assays were conducted to evaluate the resistance levels to two ACCase and two ALS inhibitors. The monitoring was conducted on 42 populations sampled in wheat fields scattered in the Walloon wheat production area. As compared to previous monitoring made in 2008, the frequencies of resistant black-grass to pinoxaden and to the iodosulfuron and mesosulfuron mixture have drastically increased in the current populations. Moreover, the resistances are now widespread over the territory. Fortunately, the resistance levels to cycloxydim and foramsulfuron, which are not registered for wheat production, are still low, indicating that those herbicides could still have a role to play in the control of black-grass in other crops. Our results seem to indicate that in Wallonia, black-grass resistances to ALS and ACCase inhibitors are mostly governed by non-target site mechanisms.

Due to the loss of efficacy of ACCase and ALS inhibitors in wheat culture, fall herbicide applications could grow in popularity. Resistance monitoring regarding those herbicides are also needed.

86


Poster 46

Discovery and monitoring of knock-down resistance (kdr, L1014F) to pyrethroids in multiple oilseed rape pest species of the genus Ceutorhynchus

Eve Dauma, Meike Brandesb, Udo Heimbachb, Russell Slaterc, Christoph T. Zimmera and Jan Eliasa

aSyngenta Crop Protection, Schaffhauserstrasse, CH-4332 Stein, SwitzerlandbJulius Kühn-Institut, Federal Research Centre for Cultivated Plants, 38104 Braunschweig, Germany

cSyngenta Crop Protection, Schwarzwaldallee 215, CH-4002 Basel, Switzerland


Oilseed rape is the most important oilseed crop in Europe with a total annual cropping area exceeding 6 million hectares. Oilseed rape is attacked by many Coleopteran crop pests, with the majority of them belonging to the weevil genus Ceutorhynchus. This pest genus was successfully controlled by insecticide applications, primarily pyrethroids, for more than 30 years. Albeit a relatively low inherent risk for resistance development, i.e. all Ceutorhynchus species attacking oilseed rape are univoltine and not all species are particularly high abundant; the lack of alternative modes of action to control insect pests in oilseed rape has built and maintained a high selection pressure - inevitably resulting in the development and spread of pyrethroid resistance. The lack of alternatives for seed treatment after the recent ban of neonicotinoids in the EU has further increased selection pressure on the foliar applied pyrethroids. Here we report pyrethroid resistance in 4 species of the same genus: Ceutorhynchus picitarsis, Ceutorhynchus napi, Ceutorhynchus pallidactylus and Ceutorhynchus obstrictus. Resistance has been detected in vivo using a glass-vial bioassay based on the IRAC Susceptibility Test Method 11 and in vitro using pyrosequencing. Results will be presented and discussed.

87


Poster 47

Characterization and monitoring resistance to diamide insecticides in the tomato leafminer, Tuta absoluta, collected across the Mediterranean Basin

Christoph T. Zimmera, Eve Dauma, Liz Hirstb, Alicia Lerouxa, Philip Süessa, Agnes Stempniewicza, Bartlomiej J. Troczkad, Fergus Earleyb, Russell Slaterc and Jan Eliasa

aSyngenta Crop Protection, Schaffhauserstrasse, CH-4332 Stein, SwitzerlandbSyngenta Crop Protection, Jealott's Hill International Research Centre, Bracknell, RG42 6EY, UK

cSyngenta Crop Protection, Schwarzwaldallee 215, CH-4002 Basel, SwitzerlanddUniversity of Exeter, College of Life and Environmental Sciences, Treliver Road, Penryn, TR10 9FE, UK


Tuta absoluta is an invasive species on the Eurasian continent and Africa. Native to South America, it arrived in Europe (Spain) more than 10 years ago. It has since then kept spreading, being reported now across Africa, the entire Mediterranean, the Middle East and South Asia. The control of T. absoluta relies on integrated pest management strategies of which modern chemistry, such as the diamide insecticides, form a key pillar. The efficacy of chemical control measures is threatened by resistance development and just as the pest itself, resistance mechanism(s) may spread fast. In this study we investigated the impact of mutations in the diamide target-site, the ryanodine receptor. Moreover, we monitored the frequency of mutations in field populations across the Mediterranean Basin and linked them to resistant phenotypes.

88


Poster 48

Control of commonly occurring insecticide resistant hemipteran pests with spiropidion, a new ACCase inhibitor insecticide

Christoph T. Zimmera, Agnes Stempniewicza, Philip Süessa, Alicia Lerouxa, Jan Eliasa, Russell Slaterb, and Robert Sennb

aSyngenta Crop Protection, Schaffhauserstrasse, CH-4332 Stein, SwitzerlandbSyngenta Crop Protection, Schwarzwaldallee 215, CH-4002 Basel, Switzerland

E-mail: [email protected] Spiropidion is a novel insecticide, currently under development at Syngenta, targeting lipid biosynthesis through the inhibition of acetyl-CoA carboxylase in arthropods (Submitted for IRAC Group 23). Spiropidion offers excellent systemic properties, it is translocated in both the xylem and phloem system of the plant. It is a highly active compound against sucking pests including aphid and whitefly species. The activity of spiropidion was evaluated against a range of commonly occurring resistant hemipteran pests, including Aphis gossypii, Myzus persicae and Bemisa tabaci which carry well characterized resistance mechanisms. The study shows that spiropidion is not affected by the different metabolic and target-site based resistance which are known to reduce the effectiveness of neonicotinoids, sulfoximines and butenolides, as well as pyrethroids, carbamates, cyclodienes and organophosphates. These results emphasize the potential of spiropidion for controlling many of the hemipteran pests that have developed resistance to other insecticide modes of action. Spiropidion will play a pivotal role in the management of hemipteran pests across a wide range of agricultural crops and the lack of cross-resistance with other modes of action means it will play an important role in resistance management strategies and sustainable integrated pest management.

89


Poster 49

Impact of host range on the evolution of insecticide resistance in pest insects

Kristina Karlsson Green, Amit Roy and Peter Anderson

Dept of Plant Protection Biology, Swedish University of Agricultural Sciences, Sweden


An increased number of phytophagous pest insects have developed resistance to the available insecticides. According to the pre-adapted hypothesis, insecticide resistance easily evolves in herbivorous insects since they already are adapted to tolerate and resist chemical defenses of their host plants. Evolution of such so-called cross-resistance is expected to be more prominent in generalist insect species since they have the ability to detoxify or metabolise a wider array of plant chemical compounds. Recent research has suggested that insecticide resistance can evolve when insects expand their host plant range. This opens up for the mirroring hypothesis that evolution of insecticide resistance in turn could select for an increased degree of generalism, i.e. an extended use of host plant species, in the insect. In a new project, we aim to investigate the pre-adapted hypotheses in a generalist and a specialist moth species of genus Spodoptera, as well as the converse hypothesis that insecticide resistance increases host plant range. To study this, we will combine selection experiments and behavioural studies on preference and performance with molecular studies.

90


Poster 50

Risks of aphid adaptation to caffeic acid derivatives used as bioinsecticides

Siegwart M.1, Lecerf E.1, Mascle O.1, Salette M.1 and Poëssel J.L.2

1 INRA, UR 1115 Plantes et Systèmes de culture Horticoles, Agroparc, F-84914 Avignon Cedex 9, France2 INRA, UR 1052 Génétique et Amélioration des Fruits et Légumes, Domaine Saint Maurice, 67 allée des

chênes, CS 60094, 84143 Montfavet Cedex, France


Dicaffeoylquinic and dicaffeoyltartaric (chicoric) acids are currently being studied at INRA Avignon as natural substances of biocontrol. They have a specifically aphicidal effect that could be very useful in agriculture while presumably being harmless to auxiliary wildlife and human health. However, aphids are well known for quickly developing resistances to different insecticides used to control their populations. Although having a reputation for better sustainability, several bioinsecticides have already been bypassed. Mechanisms of resistance developed by pests for natural substances are often those used to resist chemical insecticides. We therefore use the knowledge acquired on this subject during the last 50 years to try to estimate the risks of appearance of resistance to these two molecules.

To test this, we followed four parallel approaches: (i) Experimentally evolve a strain of M. persicae by applying a low selection pressure (ii) Find wild populations of M. persicae that have already developed resistance to these natural molecules (iii) Test the susceptibility of M. persicae strains already resistant to chemical insecticides (iv) Find other aphids species feeding naturally on plants rich in these secondary compounds.

Our first results show a good durability of products based on these two molecules, making us optimistic for the future.

91


Poster 51

Base line susceptibility of Spodoptera frugiperda and Tuta absoluta to insecticides in South Africa

Hannalene du Plessis and Johnnie van den Berg

Unit for Environmental Sciences and Management, North-West University, South Africa

E mail: [email protected]

Two economically important pest species viz. the Fall armyworm (Spodoptera frugiperda) and the Tomato leafminer (Tuta absoluta) invaded South Africa during 2016. Following these invasions, several insecticides received emergency registration for control of both these pests. Insecticides in 11 and six different mode of action groupings have been registered for control of S. frugiperda and T. absoluta, respectively. These insecticides are currently intensively applied to maize against S. frugiperda and to tomato and potato against T. absoluta in South Africa. Both these species are known to be resistant to various insecticides in other countries of the world.

The status of susceptibility to insecticides of the populations that initially invaded and are now present in South Africa, is unknown. Resistance development is a concern and base line susceptibility data of S. frugiperda and T. absoluta populations from various areas in South Africa were therefore determined. The base line susceptibility of all mode of action groupings is currently below the recommended dosages rates. However, base line data obtained for lambda cyhalothrin are of concern.

92


Poster 52

Spinosad resistance in the western flower thrips, Frankliniella occidentalis (Pergande)

Yanran Wan, Guangdi Yuan, Bingqing He and Qingjun Wu

Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China


The resistance of western flower thrips (WFT) to several insecticides, including spinosyns, was evaluated in Beijing and Yunnan province in China. Populations of WFT in parts of Beijing had developed 80- to 150-fold resistance to spinosad compared to a susceptible strain. The resistance ratio to spinetoram was as high as 7730-fold and the LC50 value to spinetoram had increased by a maximum of 258 times the initial value over the last five years. Populations of WFT in Kunming, Yunnan Province had developed 305-fold resistance to spinetoram. Nicotinic acetylcholine receptor (nAChR) is the molecular target for spinosyns. The resistance mechanisms of both the laboratory selected and field collected strains of WFT were investigated. We found that significantly more truncated forms of nAChR α6 subunit were detected in spinosad resistant strains, whereas, hardly any full-length form was found in the two highly resistant WFT strains (resistance ratio >104-fold). Under laboratory conditions, spinosad resistance was positively correlated with truncated α6 transcripts. The correlation was later confirmed under the field conditions using five field strains. As the molecular target of spinosad, the percentage of truncated nAChR α6 subunits can be used as a diagnostic tool to detect and quantify spinosad resistance in the field.

93


Poster 53

ABC transporter-mediated resistance mechanism to Bacillus thuringiensis Cry1Ac toxin in diamondback moth

Zhaojiang Guo and Youjun Zhang

Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China


Large-scale use of biopesticides and transgenic crops producing insecticidal Cry toxins from the gram-positive bacterium Bacillus thuringiensis (Bt) has resulted in episodes of field resistance in several lepidopteran pests. Unraveling these resistance mechanisms are of great importance for delaying insect field resistance evolution. The diamondback moth, Plutella xylostella (L.), was the first insect to evolve field resistance to Bt biopesticides and it is an excellent model insect to study Bt resistance mechanisms. Resistance to Bt Cry1Ac toxin in diamondback moth has been reported to be associated with cis-mutation of ABCC2 genes or MAPK signalling pathway trans-regulated differential expression of ABCC1, ABCC2, ABCC3, ABCG1 and ABCB1 genes. Hence, we can see that diverse ABC transporter genes in subfamily B, C and G can be involved in Cry1Ac resistance in P. xylostella. Although much progress has been achieved, how these ABC transporters work together and their precise roles in the molecular basis of P. xylostella Cry1Ac resistance still remains to be fully unveiled. Herein, we intend to deeply discuss and explore ABC transporter-mediated resistance mechanism to Cry1Ac toxin in P. xylostella, which will contribute to understanding the complex molecular mechanisms of Bt resistance in diverse insects.

94


Poster 54

Investigating insecticide resistance in UK populations of cabbage stem flea beetle, Psylliodes chrysocephala

Caitlin Willisa, Emyr Daviesa, Martin Williamsona, Stephen Fostera, Xianmin Changb, Jan Eliasc and Christoph Zimmerc

aRothamsted Research, West Common, Harpenden, Hertfordshire, AL5 2JQ, UKbRoyal Agricultural University, Stroud Road, Cirencester, GL7 6JS, UKcSyngenta, Werk Stein, Schaffhauserstrasse, Stein, CH4332, Switzerland


The cabbage stem flea beetle (CSFB), is a major pest of winter oilseed rape (OSR) in the UK and Germany, attacking emerging plants in the autumn and causing severe damage to the crop if left unchallenged. Prior to December 2013, control of CSFB relied on the protection of OSR seedlings by neonicotinoid seed treatments and foliar sprays using pyrethroids. However, conversion of the EU-imposed restriction to a complete ban on these neonicotinoids has left growers with just pyrethroids for CSFB control. The increased selection pressure caused by a lack of alternative control agents with different modes of action has led to the emergence of pyrethroid resistance in CSFB. As CSFB resistance to pyrethroids can cause significant yield losses in OSR crops and continued use of pesticides in their current format will only exacerbate this problem, there is a need to understand the mechanisms by which resistance is developed and sustained within CSFB populations. This knowledge will prove essential in improving our ability to effectively deploy pesticides. The aim of this project is therefore to monitor the extent and geographical spread of pyrethroid resistance in UK CSFB populations and identify the enzyme(s) involved in metabolic resistance using next generation transcriptomic approaches.

95

Poster 55

Resistance mechanisms and fitness costs related to avermectin resistance in the diamondback moth, Plutella xylostella L. (Lep: Plutellidae)

Maria Oplopoioua,b, Jan Eliasa, Marcus Guestc, Ricardo Kanitzd, Russell Slaterd, Chris Bassb and Christoph T. Zimmera

aSyngenta Crop Protection, Schaffhauserstrasse, CH-4332 Stein, SwitzerlandbUniversity of Exeter, College of Life and Environmental Sciences, Treliver Road, Penryn, TR10 9FE, UK

cSyngenta Crop Protection, Jealott's Hill International Research Centre, Bracknell, RG42 6EY, UKdSyngenta Crop Protection, Schwarzwaldallee 215, CH-4002 Basel, Switzerland


Plutella xylostella (Lepidoptera: Plutellidae) is an oligophagous species and an abundant pest in cruciferous crops. P. xylostella is highly migratory and is distributed globally. P. xylostella cannot overwinter in temperate areas where annual outbreaks can be attributed to migration. In contrast, in tropical and subtropical regions many continuous generations pose a threat to crop production throughout the year. P. xylostella is a high-risk pest concerning resistance development, having developed resistance against > 90 active ingredients that have been used to control this pest. Emamectin-benzoate is a potent lepidoptericide synthesized from the macrocyclic lactone abamectin which is derived from fermentation of the soil actinomycete Streptomyces avermitilis. Emamectin-benzoate is widely used to control Lepidopteran crop pests including P. xylostella. Reduced field performance of emamectin-benzoate in Plutella xylostella has been reported in Japan. This project aims to elucidate the resistance mechanism(s) resulting in reduced field performance and to investigate potentially related fitness costs. Successful resistance management relies heavily on resistance monitoring and carefully timed spraying regimes i.e. mode of action rotations. Knowledge about the mechanism, the mode of inheritance, the persistence of resistance under different treatment scenarios and the potential impact of resistance on fitness is critical for the design of a sustainable integrated pest management (IPM) program.

96


Poster 56

A chemical proteomic workflow for rapid identification of proteins responsible for metabolic insecticide resistance

Joshua Linfoota, Peter Kilbyb, Rüdiger Woscholskia, Jan Eliasc, Ed Tatea, Christoph T. Zimmerc and Alan Spiveya

aImperial College, Department of Chemistry, London, SW7 2AZ, UKbSyngenta, Jealott's Hill International Research Centre, Bracknell, RG42 6EY, UK

cSyngenta Crop Protection, Schaffhauserstrasse, CH-4332 Stein, Switzerland


Emerging resistance of organisms to different insecticide classes over recent decades has necessitated the development of new small molecules with novel modes of action. One such development is the diamide class, which includes chlorantraniliprole. Diamide insecticides act on the ryanodine receptor, a membrane-bound gated calcium channel protein present in insect muscle cells. However, certain lepidopteran pests, such as Plutella xylostella and Chrysodeixis includens, are developing resistance to chlorantraniliprole at an alarming rate. Whilst the most common resistance mechanism is target-site mutation, another prolific and potentially more easily countered mechanism is metabolic resistance, where resistant pests have evolved their portfolio of detoxifying enzymes, e.g. cytochrome P450s, to more rapidly render the insecticide inert. A chemical proteomic workflow, using a photoaffinity-labelled chlorantraniliprole analogue, has been successfully designed and implemented for the identification of the proteins responsible for chlorantraniliprole metabolism in Plutella xylostella. Further research is being performed to investigate the effects of the position and identity of the photoaffinity label in the probe molecules on the interactome, and workflow parameters will be optimised with the aim of extending it to other species and insecticides.

97


Poster 57

A comprehensive testing cascade to identify resistance breaking repurposed insecticides for next generation vector control tools: screening a panel of chemistries against Anopheles gambiae

Rosemary Lees1, Hilary Ranson1 and David Malone2

1Vector Biology Department and 2IVCC, Liverpool School of Tropical Medicine, UK


With insecticide resistance in malaria vectors spreading in geographical range and intensity, there is a need for compounds with novel modes of action to maintain the successes achieved by insecticide treated nets and indoor residual sprays. Repurposing existing insecticides, predominantly those developed for use in agriculture, is less risky than screening and chemical synthesis programmes for novel compounds and may be a rapid way to identify active ingredients of value to public health. Insecticides and acaricides from all IRAC classes including those with unclassified modes of action were considered for inclusion in a laboratory bioassay testing cascade against adult female Anopheles gambiae. A longlist of representative candidate compounds was selected, excluding those with safety concerns, unsuitable physiochemical properties, and those likely to be challenging to register for public health use. An initial screen using topical application eliminated compounds with insufficient intrinsic activity, and a tarsal assay identified those with activity at an appropriate concentration. Inclusion of an adjuvant enhanced the efficacy of several compounds, saving chemistries with great potential, given suitable formulation, from exclusion. The remaining shortlist of compounds of interest as vector control agents were ranked by relative potency using dose response assays and discriminating dose calculations.

98


Poster 58

Mechanisms of pyrethroid resistance and potential cross-resistance issues in clonal cultures of pea aphid, Acyrthosiphon pisum

Viola Müller, Bettina Lueke, Antje Rottmann and Ralf Nauen

Rheinische Friedrich-Wilhelms-Universität Bonn, 53113 Bonn, Germany; Bayer AG, Crop Science Division, R&D, 40789 Monheim, Germany

E-mail: [email protected]; [email protected] Insecticide resistance is quite common in green peach aphid, Myzus persicae, and cotton aphid, Aphis gossypii, a fact reflected by 750 documented cases found in the Arthropod Pesticide Resistance Database (https://www.pesticideresistance.org). In total this database lists 922 aphid resistance cases for 27 species, but strikingly not listing any entry for the pea aphid, Acyrthosiphon pisum. The pea aphid is known as a global sucking pest of leguminous plants including peas, broad beans and alfalfa. It is also a model aphid species and its genome has been recently sequenced and annotated. We recently collected a French population of A. pisum showing a significantly lower susceptibility to pyrethroids when compared to an insecticide susceptible reference strain. After separating and testing a number of clonal cultures we obtained a strain, PYR-R1, exhibiting high levels of pyrethroid resistance (RR>100-fold), but lacking any crossresistance to other chemical classes of insecticides. We investigated the molecular mechanism confering pyrethroid resistance in strain PYR-R1 by analysing the involvement of detoxification enzymes as well as possible target-site mutations in the voltage-gated sodium channel, known as the pyrethroid target site. Here we describe the molecular mechanism of resistance and its implications for resistance management.

99



Poster 59

The K+/Cl- cotransporter represents a putative target to reduce resistance to dieldrin (rdl)

Daniel R. Swale

Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA, USA 70803


The γ-aminobutyric acid (GABA) receptor and GABA-receptor-chloride-channel (GRCC) complex has been a long-standing target for insecticides, yet target site mutations in rdl, the gene encoding GABA-R, continue to threaten the utility of conventional GABAergic insecticides. We have focused on defining the functional interplay between the GABAA receptor and K+/Cl- cotransporter (KCC) as a putative mechanism to reduce rdl phenotypes. Although poorly defined in insects, mammalian KCCs concomitantly extrude K+

and Cl- from the cell to produce low intracellular chloride levels, which enables GRCC function and inhibitory synaptic signaling. Our data show that CNS-specific ablation or pharmacological inhibition of Drosophila KCC results in an increased CNS spike discharge frequency and significantly (P<0.05) reduces the CNS sensitivity to GABA. Further, KCC inhibitors at an EC10 were found to increase dieldrin activity in the Drosophila CNS by approximately 2-fold, which validates KCC is functionally coupled to GRCC and indicates KCC inhibition may rescue rdl phenotypes. Indeed, co-treatment of a KCC inhibitor (EC10) and dieldrin increased the CNS firing rate by 40% in flies carrying rdl mutations but did not alter the firing rate in susceptible flies. These data indicate KCC holds promise to provide a mechanism to mitigate resistance to GABAergics.

100


Poster 60

Effect of transfluthrin fluorination patterns on effectiveness and metabolic stability in in Anopheles funestus

Melanie Nolden1,2, Sebastian Horstmann1, Mark Paine2, Sarah Rees3 and Ralf Nauen1

Bayer AG, Crop Science Division,1 Liverpool School of Tropical Medicine (LSTM),2 International Vector Control Consortium (IVCC)3

Email to: [email protected]

Resistance in Anopheles-mosquitoes against common insecticide classes used in vector control is increasing and likely to compromise pest control interventions in the fight against malaria. In particular, pyrethroid resistance due to target site insensitivity and upregulated detoxification enzymes such as cytochrome P450 monooxygenases (P450s) are spreading fast. Particularly pyrethroid resistance in A. funestus has been shown to be conferred by over-expressed P450s. The volatile pyrethroid transfluthrin is a promising insecticide suggested to be much less affected by P450-mediated detoxification due to its high degree of fluorination. Recently bed nets impregnated with pyrethroids and the P450-inhibitor piperonylbutoxide (PBO) were launched to overcome P450 detoxification issues in mosquitoes due to resistance development against traditional pyrethroid scaffolds.

We reared two strains of A. funestus, FANG (susceptible) and FUMOZ-R (resistant) and tested them against transfluthrin and derivatives with different fluorination patterns. All derivates were tested alone and in combination with different P450 inhibitors by a newly developed contact assay to assess how the fluorination pattern affects P450-mediated metabolism.

Bioassay data and LC/MS-analysis on synergist uptake confirmed significant differences observed in mortality when mosquitoes were sequentially exposed to PBO and transfluthrin derivatives. Other P450 inhibitors show even stronger inhibitory potential against microsomal P450 involved in pyrethroid detoxification. Significant differences in dose-response data confirmed our hypothesis that the fluorination pattern has a strong impact on the efficacy of transfluthrin against A. funestus and other mosquito species. Our bioassay data are further supported by biochemical studies correlating with the observed efficacy of transfluthrin derivatives.

101


Poster 61

Understanding the impact of global warming on pest management: impacts of cross effects between temperature and three different insecticides on codling moth susceptibility

Marie Perrin 1,2 , Joffrey Moiroux 2, Thomas Delattre 1 and Myriam Siegwart 1

1 PSH, INRA, France 2 BES IMBE, Avignon University, France


Climate change may lead to increased applications of pesticides as pests are expected to persist almost all year long and expand their geographical distribution as temperature increases. Moreover, temperature is known to influence the absorption, penetration, translocation and detoxification of pesticides by organisms. It is therefore necessary to investigate interactions between pesticide and temperature to understand possible outbreaks of resistance in a climate change context.

We conducted such an experiment on the codling moth, Cydia pomonella, one of the main pests in orchards worldwide. Codling moth larvae mortality was assessed for three insecticides at different temperatures.

Preliminary results on a susceptible strain of codling moths show a decrease in the LD50 at 35°C compared to lower temperatures for one of the three insecticides tested. Such results have been reported in other pest species with other insecticides and highlight the need for research on the effects of temperature on the efficiency of pesticides in order to adapt their application to future climatic conditions.

Further research will be conducted to compare a susceptible and a resistant strain of codling moths in order to extrapolate effects of global warming on this adaptation.

102


Poster 62

Identification and functional characterization of BdGSTd9 involved in malathion resistance in Bactrocera dorsalis

Li-Wei Meng *, † Guo-Rui Yuan *, † and Jin-Jun Wang *, †

* Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, Southwest University, Chongqing 400715, P.R. China; † Academy of Agricultural Sciences, Southwest University,

Chongqing 400715, P.R. China


The oriental fruit fly, Bactrocera dorsalis (Hendel), as an important agricultural and widespread pest has evolved resistance to many insecticidal classes including organophosphorus pesticide. Glutathione S-transferases (GSTs) are involved in xenobiotics detoxification and insecticides resistance in insects. Here, the possible roles of BdGSTd9 in malathion detoxification in B. dorsalis were evaluated. BdGSTd9 was identified from B. dorsalis and highly expressed in malathion resistance strains (MR) with 1.67-fold. Although it abundant in female adult and its malpighian tubes, there was no obviously difference of silence efficiency of this gene between female and male. After BdGSTd9 was effective silenced, the susceptible to malathion in malathion susceptible strains (MS) showed no difference to control, but there was a significance increased post pesticide treatment 24 h till 72 h in both male and female of MR. The key residues of BdGSTd9 which docked with malathion/malaoxon was analysed and indicated GST might reacted with the pesticide directly. Subsequently, cytotoxicity assay of malathion was performed in Sf9 cells and showed BdGSTd9 could enhance cells resistance to malathion. In addition, HPLC analyses indicated that malathion could be significantly depleted by BdGSTd9, moreover, it could deplete malaoxon which was malathion metabolite and possessed the higher inhibition to acetylcholinesterase.

103


Poster 63

Monitoring for ketoenol resistance in Mediterranean populations of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)

Anastasia Tsagkarakou1, Marianna Stavrakaki1,2, Aris Ilias3, Jacques Lagnel4

Emmanouil Roditakis1, John Vontas2,3 and Ralf Nauen5

1 Institute of Olive Tree, Subtropical Crops and Viticulture, Hellenic Agricultural Organization “DEMETER", Greece

2Agricultural University of Athens, Department of Crop Science, GR-11855 Athens, Greece3Foundation for Research & Technology Hellas, Institute of Molecular Biology & Biotechnology, Greece

4INRA PACA UR 1052 GAFL, France5Bayer AG, Crop Science Division, R&D Pest Control Biology, D-40789 Monheim, Germany


Bemisia tabaci is among the most important pests worldwide. The efficacy of spirotetramat and spiromesifen, that belong to spirocyclic tetronic acid derivatives (ketoenols) to B. tabaci MED populations from the Mediterranean Basin and their role in the neonicotinoid resistance management was monitored in the present study.

Populations from Greece and Italy displayed the lower LC50 values, for both spirotetramat and spiromesifen. Variable LC50s ranging from low (LC50 = 1.8 mg/L) to high values (LC50 = 250 mg/L) for spirotetramat and from low (LC50 = 1.4 mg/L) to extremely high values (LC50 > 4000 mg/L) for spiromesifen were exhibited by Spanish populations. Among the populations that exhibited reduced susceptibility to imidacloprid none displayed reduced susceptibility to ketoenols, suggesting absence of cross resistance between the two groups of insecticides.

Genetic studies using a highly resistant spiromesifen strain revealed that resistance was inherited autosomally and incompletely recessive. Results from F1 back-crosses suggested that resistance is polygenic. Resistance mechanisms were investigated through a next generation sequencing approach (RNAseq).

Continuous monitoring of the ketoenol efficacy and of the presence of any target resistance mutations in field populations should improve the sustainable chemical control of this major pest.

104


Poster 64

Resistance in the non-target earwig Forficula auricularia to agricultural pesticides

Le Navenant A.(a)(b), Siegwart M.(b), Capowiez Y(c), Rault M.(a). and Suchail S. (a)

aAvignon University, Aix Marseille Univ, CNRS, IRD, IMBE, Pôle Agrosciences, 301 rue Baruch de Spinoza, BP 21239,84916 Avignon, France.

bINRA, UR 1115 Plantes et Systèmes de culture Horticoles, Agroparc, F-84914 Avignon Cedex 9, FrancecINRA UMR 114 EMMAH Domaine Saint Paul 84914 Avignon Cedex 9 - France


In the European context of reduced pesticide use, the development of biocontrol agents has to be promoted. The earwig Forficula auricularia, an effective generalist predator in pomefruit orchards, is exposed to repeated insecticide use which can increase the risk of resistance. Recently, we have shown that earwigs collected in conventional orchard exhibited lower mortality and higher enzyme activities involved in detoxification, compare to organic orchard. We then investigated possible side effects on energetic reserves and morphometric traits of earwigs. Earwigs were collected either in treated (organic, integrated pest management and conventional) or untreated orchards. Energy reserves are higher in earwigs from treated orchards. The increase in protein content support the adaptation of organisms to minimize the adverse toxic effects of pesticide treatments. Moreover, we observed a decrease in morphological traits in earwigs from treated compared to untreated orchards. Descendants of those earwigs bred in the laboratory under standard conditions (food ad libitum, absence of anthropic pressure) recovered similar traits whatever the orchard practices. In conclusion, the main impact on energetic reserves and morphological traits lies in the presence of anthropization rather than pesticide use. Resistance of earwigs from conventional orchard seems better related to phenotypic plasticity than a new genotype.

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Poster 65

Genetics, molecular and functional characterization of insecticide / acaricide resistance in Tetranychus urticae

Aris Ilias1, Maria Riga1,2, Kyriaki Maria Papapostolou1,2, Evaggelia Skoufa1,2, Dimitra Tsakireli2, Sabina Badja3, Vassilis Douris1, Vasileia Balabanidou1, Elena Vorgia1, Wannes Dermauw3, Thomas Van Leeuwen3

and John Vontas1,4

1Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas, Heraklion, Crete, Greece

2Dept. of Biology, University of Crete, Heraklion, Crete, Greece3Laboratory of Agrozoology, Dept. of Plants and Crops, Faculty of Bioscience Engineering, Ghent

University, Ghent, Belgium4Laboratory of Pesticide Science, Dept. of Crop Science, Agricultural University of Athens, Athens, Greece


The two-spotted spider mite (TSSM) Tetranychus urticae is one of the most notorious agricultural pests world-wide as it displays striking resistance levels in insecticides/acaricides used for its control. We have used a number of approaches, such as microarrays, functional expression and characterization of recombinant enzymes, immunohistochemical staining, as well as in vivo expression in Drosophila and marker-assisted backcrosses in T. urticae in order to better understand the role of individual genes and pathways in development of resistance against important acaricides/insecticides, such as abamectin, METIs and pyrethroids.

We have functionally expressed several detoxification genes such as CYP392A16, a cytochrome P450 from T. urticae and characterized their ability to metabolize acaricides to a less toxic compound. In addition, ectopic expression of detoxification genes in Drosophila by employing the GAL4/UAS system aimed in validating the role of these enzymes in resistance in vivo. Immunohistochemical staining of detoxification enzymes, such as P450s and GSTds, in T. urticae may highlight the physiology of detoxification and help in identifying routes of insecticide uptake. Recently, marker-assisted backcrosses in T. urticae were applied in order to introgress target-site alleles bearing previously known mutations in a susceptible genetic background so as to study their relative contribution in acaricide resistance.

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Poster 66

Target mutation and expression level involved in insecticide resistance of earwigs

Thierry Fricaux1, Adrien Le Navenant2, Magali Rault2, Christine Coustau1 and Gaëlle Le Goff1

1UCA, INRA, CNRS, Institut Sophia Agrobiotech, France2Avignon université, Aix Marseille univ, CNRS, IRD, IMBE, France


Reducing pesticide treatments is an important challenge in apple orchards that are massively treated to control numerous insect pests and diseases. The use of earwigs, Forficula auricularia can contribute to control strategies, alternative to chemicals, as they effectively predate on pests. However, earwigs are exposed to the same treatments than pests and can develop resistance in the same manner as their preys. Because earwig resistance to pesticide has been poorly studied so far, here we started investigating potential genetic resistance mechanisms from earwigs collected in orchards subjected to three management strategies: conventional, integrated or organic strategies. The two major mechanisms of insecticide resistance characterized in other insect species rely either on a modification of the insecticide target or on a degradation of the insecticide by detoxification enzymes. We first identified and sequenced the major pesticide targets and pesticide detoxification enzymes in this non-model species. Then, we identified point mutations in target genes, and increased expression levels of resistance-related genes (pesticide targets or detoxification enzymes) in individuals that have been exposed to chemical and/or organic treatments. Altogether, these results pave the way for future ecotoxicological studies, further exploring the potentially diverse resistance-associated mutations and their occurrence in earwigs.

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Poster 67

Resistance to pyrethroids in predatory mites

Luis Benavent-Albarracin 1; Miquel Alonso-Valiente2, Jose Catalán2, Alberto Urbaneja2, T.G. Emyr Davies3, Martin S. Williamson3 and Joel González-Cabrera1

1Unidad Mixta Gestión Biotecnológica de Plagas UV-IVIA. Department of Genetics, Universitat de València. Estructura de Recerca Interdisciplinar en Biotecnología i Biomedicina (ERI-BIOTECMED),

Spain.2Instituto Valenciano de Investigaciones Agrarias (IVIA). Centro de Protección Vegetal y Biotecnología,

Unidad Mixta Gestión Biotecnológica de Plagas UV-IVIA. Spain. 3Biointeractions and Crop Protection Department, Rothamsted Research, UK


The implementation of IPM in agricultural practice is a very effective strategy to control pest populations. The use of Biological Control Agents (BCAs) is key for the success of such an approach. Predatory mites like Phytoseiulus persimilis and Amblyseius swirskii are widely used for the control of highly damaging pests like the two-spotted spider mite, white flies, thrips, etc. However, there are times where outbreaks of additional pests occur, and it is necessary to use conventional pesticides to save the crop. In these times, the combination of BCAs with selective resistance to certain pesticides can be a timely solution to maintain control of outbreaks, effectively reducing the input of conventional pesticides to the crop. We identified mutations located in the voltage gated sodium channel, the main target of pyrethroids, that correlate with the resistance to deltamethrin observed in populations of P. persimilis and A. swirskii. These mutations are located in a region of the channel previously proposed as the binding site for this group of pesticides. Our results suggest that it is possible to integrate the use of these predatory mites with pyrethroid applications in an IPM context to reduce the impact of damaging pests while protecting the BCAs.

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Poster 68

Deltamethrin resistance in the salmon louse, Lepeophtheirus salmonis (Krøyer): maternal inheritance and reduced apoptosis

Marit J. Bakke, Celia Agusti, Jo C. Bruusgaard, Arvind YM Sundaram and Tor E. Horsberg

Norwegian University of Life Science, Faculty of Veterinary Medicine, Sea Lice Research Centre, Ullevalsveien 72, 0454 Oslo, Norway


Resistance towards deltamethrin (DMT) in the crustacean ectoparasite Lepeophtheirus salmonis (Caligidae) is a problem on fish farms lining the North Atlantic Ocean. Two Norwegian strains with different susceptibility towards DMT were crossed in the parental generation (P0), females from a sensitive strain were crossed with males from a resistant strain and vice versa. Individual susceptibility towards DMT was assessed in the second filial generation (F2). DMT resistance was only found in F2 descendants when the P0 females were from the resistant strain, pointing to maternal inheritance. Since maternal inheritance might be linked to the mitochondrial (mt) genome, the nucleotide sequences and the gene expressions of mt-genes were analysed. Twenty non-synonymous single nucleotide polymorphisms (SNPs) were identified in mt-transcripts from resistant F2 parasites, including SNPs in two cytochrome C oxidase subunits (COX1 and COX3) and two subunits of the NADH dehydrogenase complex (ND1 and ND5) previously linked to DMT resistance in the salmon louse. Differential expression analysis between the sensitive and resistant strain revealed strain effect in seven out of twelve mt-genes. The current study also shows that DNA fragmentation (indicating apoptosis) was affected by DMT exposure in skeletal muscle tissue and that resistant parasites undergo less apoptosis than sensitive parasites.

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Poster 69

Functional genomic approaches in the cruciferous pest Plutella xylostella

Marcus Guest, Julia Bristow, Jim Goodchild, Stephanie Widdison, Grace Logan and Anthony Flemming

Syngenta, Jealott’s Hill research centre, Bracknell, Berkshire, RG42 6EY, UK


Forward genetic methodologies provide a non-hypothesis lead route to mode of action and mode of resistance elucidation. Linkage mapping of either field isolated or lab generated resistance provides a genetic link between active ingredient and resistance mutation and/or potential target sites. The use of such approaches has generally been limited to model organisms such as Caenorhabditis elegans and Drosophila melanogaster. However, Low cost, high throughput sequencing means that conducting such studies in crop pests is now much more achievable. This is due to the ability to generate high quality reference genomes for specific pests, and the feasibility of re-sequenceing genomes for linkge mapping studies. Reverse genetic studies in crop pests have been enhanced by the availability of genome editing with CRISPR/Cas9. Genome editing studies provide the opportunity to test candidate resistance mutations, weather identified in field resistant isolates or in lab generated mutant strains, enabling resistance phenotypes to be attributed to specific mutations.

Here we present two case studies where we have employed linkage mapping and genome editing methodologies. 1) We have utilised a near chromosome level genome assembly in combination with a whole genome sequencing mapping approach to map DDT resistance to a genomic region containing KDR. 2) We have utilised a CRISPR/Cas9 approach in Plutella xylostella, in which we have introduced the orthologous A301S mutation into PxGABARalpha1. The described approaches are important components required to realise the goal of non-hypothesis lead genetic mode of action and mode of resistance study in Plutella xylostella.

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Poster 70

‘Humanization’ of insect RyR sequence confers potent resistance to diamide insecticides

Ewan Richardson, T.G. Emyr Davies, Martin Williamson, Ulrich Ebbinghaus-Kinscher and Ralf Nauen

Biochemistry and Crop Protection, Rothamsted Research, UK and Bayer CropSciences, Germany

Corresponding Authors: [email protected]; [email protected]

As the dual pressures of legislative restriction and insecticide resistance combine to reduce available control options against agricultural pests, the requirement for safe, sustainable, and effective chemistry becomes increasingly urgent. In this context, diamide insecticides exhibit excellent activity against a range of insect pests, combined with exceptionally low toxicity toward humans and other vertebrates. It is thus pertinent to investigate how this class obtains such properties, in order to aid development of novel compounds. Diamides bind to the insect ryanodine receptor (RyR). Mutations to the membrane-spanning region of this channel have been shown to inhibit diamide binding, thereby causing resistance to the compound. Some of these resistance-causing mutations are homologous to the alterations seen between insect and vertebrate species. This finding raises the tantalising possibility that such alterations are also responsible for the exceptionally low toxicity shown toward vertebrates. ‘Humanization’ of an insect RyR is used here to investigate the impacts of these mutations individually and in combination. Results indicate that the large disparity in toxicity between insects and humans is conferred by just a small selection of alterations.

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Poster 71

Dissecting insecticide resistance via genome modification in Drosophila

Kyriaki M. Papapostolou1,2*, Rafaela Panteleri1,2, Mantha Lamprousi1,2 Thomas Van Leeuwen4, Ralf Nauen3, Vassilis Douris1 and John Vontas1,5*

1 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas (IMBB-FORTH), Greece; 2 Department of Biology, University of Crete (UoC), Greece; 3 Bayer AG, Crop

Science Division, R&D Pest Control, Monheim, Germany; 4 Lab of Agrozoology, Depart of Crop Protection, Ghent University, Belgium; 5 Pesticide Science Laboratory, Agricultural University of Athens,

Greece;

E-mail: [email protected] and [email protected]

The protection of agricultural production, as well as the prevention of vector borne diseases, largely relies on the control of insect populations with the use of insecticides. However, insects display an intriguing ability to develop resistance. Our research aims at the investigation of the mechanisms inducing resistant phenotypes using several experimental approaches; among these, genome modification in model species like Drosophila has played a pivotal role in the understanding of the role of individual alleles and the determination of specific insecticides’ mode of action. Certain examples include the investigation of the contribution of specific mutations in target-site resistance phenotypes (in genes like chitin synthase, ryanodine receptor and ACCase) using CRISPR/Cas9 genome modification. The versatility of this tool is demonstrated by the ability to devise ad hoc strategies in order to test hypotheses in the same genetic context, aiming to generate resistance phenotypes in a susceptible genetic background, taking advantage of CRISPR/Cas9 toolkit and standard Drosophila genetics. This “reverse genetics” approach greatly enhances our ability to investigate insecticide resistance in a fashion complementary to standard pipelines and classical forward genetics and can eventually be used to test the anti-resistance potential of novel candidate compounds and inform IRM strategies.

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Poster 72

Pyrethroid resistance in Italian populations of the mite Varroa destructor: a focus on the Lombardia region

Michela Panini1, Maria Cristina Reguzzi1, Olga Chiesa1, Filippo Cominelli1, Marco Quartieri1, Daniela Lupi2, Graham Moores3 and Emanuele Mazzoni1

1Department of Sustainable Crop Production (Di.Pro.Ve.S.), Università Cattolica del Sacro Cuore, Piacenza, Italy

2Department of Food, Environmental and Nutritional Science (DEFENS), Università di Milano, Italy3ApresLabs Ltd, Harpenden, Herts, UK


Varroa destructor is one of the major pests against honey bees. Beekeepers can rely on different approaches to try and prevent colony collapses, and the most relevant are acaricide treatments. Pyrethroid resistance was first reported in Lombardia in 1991 and is now spread worldwide. Recently, three different mutations (L925V/I/M) occurring in the voltage-gated sodium channel have been associated with tau-fluvalinate resistance. Furthermore, indirect evidence from laboratory bioassays indicated that high levels of esterases may be involved in mites resistant to tau-fluvalinate.

This study provides an update of the actual spread of target-site resistance to tau-fluvalinate in varroa samples collected in the Lombardia region. TaqMan assays showed that mutation L925V is present in this area, however only low frequencies of this resistant allele were detected. The majority of resistant mites were found in the homozygous form (11%), and only a small fraction possessed the heterozygous genotype (2%).Additionally, a protocol was set up to detect esterase activity directly in single mites to determine if metabolic resistance can occur. We observed slight variability among different populations, and piperonyl butoxide showed only very low inhibition of this activity in vitro. Additional evaluation of monooxygenases activity must also be explored in the future.

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Poster 73

Characterization of the cuticular hydrocarbon biosynthetic pathway in the malaria vector An. Gambiae

Linta Grigoraki, Gareth Lycett and Hilary Ranson

Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place L3 5QA, UK


The cuticle plays a critical role in the biology of mosquitoes and their response to control interventions. It protects from penetrating toxins like insecticides, prevents desiccation and influences communication. We are investigating the biosynthetic pathway of cuticular hydrocarbons, the main components of the upper cuticle layer that remains poorly understood. We used CRISPR/Cas9 to generate an An. gambiae strain with fluorescently tagged oenocytes, the cells synthesizing hydrocarbons. Fluorescent oenocytes were isolated with FACS for RNA seq, enabling us to predict genes involved in the hydrocarbon biosynthetic pathway. Key genes in this pathway are being silenced or over expressed to validate their role in CHC synthesis and determine the impact of perturbing this pathway on the mosquito phenotype.

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Poster 74

Proteomic and transcriptomic analysis of Varroa destructor populations resistant to organophosphates and amidines

C. Sara Hernández-Rodríguez1, Jody Johnson2, Steven Cook2 and Joel González-Cabrera1

1ERI BIOTECMED, Dep. Genética, Universitat de València, Spain.2USDA-ARS, Bee Research Lab, MD, USA


The varroosis, a severe disease caused by the parasitic mite Varroa destructor while infesting honeybee (Apis mellifera) colonies, is one of the main threats for current beekeeping. The mite feeds on the fat body of immature and adult bees and also vectors a complex array of viruses that decimates the colony in absence of an effective management approach. To control the mite, beekeepers are treating the hives intensively with a small set of acaricides belonging to three different groups: organophosphates (coumaphos), amidines (amitraz) and pyrethroids (tau-fluvalinate and flumethrin). As with other pesticides, the intensive treatment regime has resulted in the development of resistance to these compounds in several countries. Resistance to pyrethroids has been already correlated with amino acid substitutions in the mite’s voltage gated sodium channel, and high throughput diagnostics assays has been developed to detect them. However, the molecular basis of the resistance to organophosphates and amidines in this mite remain unknow. In this study, we used samples from V. destructor populations susceptible and resistant to coumaphos or amitraz to perform proteomic and transcriptomic analyses. We then assessed the differential expression profiles of proteins and genes to identify those candidates associated with the resistance to these compounds.

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Poster 75

The SCN coalition: a case study in increasing grower awareness when a management tool loses effectiveness

Sam Markell, Greg Tylka, George Bird, Albert Tenuta and Kaitlyn Bissonnette

Department of Plant Pathology, North Dakota State University, ND, USADepartment of Plant Pathology and Microbiology, Iowa State University, IA, USA

Department of Entomology, Michigan State University, MI, U.S.A.Ontario Ministry of Agriculture, Food & Rural Affairs, Ridgetown, Ont., Canada.

Division of Plant Sciences, University of Missouri, MO, USA.


The soybean cyst nematode (SCN), Heterodera glycines, is the most important yield-limiting biological factor in North American soybean production, and genetic resistance is a key pillar of SCN management. However, approximately 95% of the soybean varieties available to growers in the U.S.A. and Canada rely on a single source of resistance, PI 88788. In recent years, an increase in aggressive nematode populations, which feed and reproduce on genotypes with PI 88788, have been documented. An increase in yield loss to SCN and further loss of effective genetic resistance conferred by PI 88788 genetics is expected. Simultaneously, soybean growers are largely unaware of this evolving management crisis.

In response, a public-private partnership of Universities, agro-chemical companies and grower organizations launched ‘The SCN Coalition’ in February 2018. The goals of the SCN Coalition are to increase awareness and active management of SCN. Messaging is delivered through farm events, traditional and social media, and the resource center www.thescncoalition.com. Between the launch of the SCN Coalition and April 2019, the SCN Coalition generated 12.1M earned media impressions. Impacts will be measured by conducting market research in 2020 and comparing results to those generated from market research conducted prior to the launch of the project.

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http://www.thescncoalition.com/


Poster 76

Genome sequencing and transcriptome profiling of the greenhouse whitefly, Trialeurodes vaporariorum, reveals a link between host plant adaptation and insecticide sensitivity

Adam Pym, Kumar Saurabh Singh, Asa Nordgren, Emyr Davies, Christoph T. Zimmer, Jan Elias, Russell Slater and Chris Bass

College of Life and Environmental Sciences, University of Exeter, Penryn Campus, Penryn, Cornwall, TR10 9FE, UK

The greenhouse whitefly, Trialeurodes vaporariorum, is a damaging crop pest and an invasive generalist capable of feeding on a broad range of host plants. As such it has evolved mechanisms to circumvent the wide spectrum of anti-herbivore allelochemicals produced by its broad host range. T. vaporariorum has also evolved resistance to many synthetic insecticides used for control, with previous studies suggesting a link between these two factors. To gain insight into the underlying molecular mechanisms, we sequenced and assembled a reference genome for T. vaporariorum and curated gene families putatively involved in the detoxification of natural and synthetic xenobiotics. This revealed marked reductions in certain gene families, such as cytochrome p450s, when compared to another generalist whitefly, Bemisia tabaci. We also performed transcriptome profiling upon transfer of T. vaporariorum onto a range of host plants. This showed large scale changes in gene expression with a range of genes involved in regulation, signalling, detoxification and excretion differentially expressed. Observed changes were also associated with significant shifts in the tolerance of host-adapted T. vaporariorum lines to four different insecticide classes. In summary, whiteflies reared on traditionally more hostile hosts, such as nightshades, had increased numbers of differential gene expression which correlated to their increased insecticide tolerance.

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Poster 77

Vapor phase delivery of plant oils alters pyrethroid efficacy and detoxification enzyme activity in mosquitoes

Troy D. Anderson, Scott T. O’Neal, Ellis J. Johnson and Leslie C. Rault

Department of Entomology, University of Nebraska, Lincoln, NE 68583 USA


The use of synthetic insecticides to limit the spread of mosquito-borne disease faces significant challenges, including insecticide resistance, environmental impact of widespread insecticide use, and slowed development of new insecticide chemistries. One important alternative to broadcast insecticides is the use of personal protection strategies to limit contact with vector species, including the use of spatial repellents that can employ synthetic pyrethroids or plant-derived oils. A currently underexplored area of research involves the investigation of plant-derived oils for their potential to serve as insecticide synergists when delivered as a vapor. This presentation will describe the development and implementation of plant-derived oils delivered as a vapor for enhancement of deltamethrin efficacy in pyrethroid-susceptible and –resistant strains of the vector mosquito species Aedes aegypti. Deltamethrin efficacy was significantly increased following exposure to vapors of cinnamon, tagetes, and sage oils, while deltamethrin efficacy was significantly decreased following exposure to amyris oil vapor. Biochemical assays suggest these effects may be mediated by changes in cytochrome P450 monooxygenase activity. This work will demonstratate that plant-derived oil vapors are capable of increasing deltamethrin efficacy similar to classical synergists such as piperonyl butoxide, offering an alternative approach for the enhancement of insecticide efficacy using natural product chemistries.

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Poster 78

Pharmacological characterisation of amino acid substitutions (L925V, I and M) associated with resistance in the bee mite, Varroa destructor

Anabel Millán-Leiva1, Martin S. Williamson2, Ian R. Mellor3 and Joel González-Cabrera1

1ERI BIOTECMED, Department of Genetics, University of Valencia, Spain.2Biointeractions and Crop Protection Department, Rothamsted Research, UK

3School of Life Sciences, University of Nottingham, NG7 2UH, UK.


The ectoparasitic mite, Varroa destructor, is a major threat for modern apiculture. It feeds directly on immature and adult bees, but also vector a wide array of viruses causing a devastating impact on bee colonies. Pyrethroids like tau-fluvalinate and flumethrin have been used with success to control de mite but since the 1990s, many reports have described the evolution of resistance to these compounds in mite populations.

Further studies on the molecular characterisation of resistant and susceptible mites have found a significant correlation between certain amino acid substitutions at position 925 of the voltage gated sodium channel (L925V, I and M), the main target for pyrethroids and other pesticides, and the resistance detected in the field.

In the present work, aiming to understand the impact of these mutations on the biophysical properties of the channel, we have functionally characterized all four 925 variants for V. destructor VGSC (wild type and three mutants) using the Xenopus oocyte expression system and two-electrode voltage clamp. We have also tested the modification on the gating kinetics of all four variants in the presence of increasing concentration of mite selective (tau-fluvalinate and flumethrin) and non-mite selective (deltamethrin) pyrethroids.

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Poster 79

The aphid voltage-gated sodium channel is an unique target for species selective insecticides

T.G. Emyr Davies and Martin S. Williamson

Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK


Aphids represent a significant threat to crop productivity on a global scale. Aphids possess a unique heterodimeric voltage-gated sodium channel whereby domains I and II are encoded separately from domains III and IV, on two different genes. Furthermore, the ion selectivity filter of the aphid channel is DENS rather than DEKA as found in other eukaryotic sodium channels. To investigate how widespread this unique channel is within the insect kingdom we screened for the DENS filter motif. Hemipteran insects with voltage-gated sodium channels encoded by a single gene all have the classic DEKA selectivity filter. The unusual DENS channel is found to be restricted to the Aphidomorpha, which includes the Phylloxera, and the DENS selectivity filter was found in all of the aphids we have so far got sequence information for. We conclude that the aphid voltage-gated sodium channel specialisation must have occurred in the last 250 MY, possibly just prior to or around the time of acquisition of cyclical parthogenesis, and represents an unusual gene inversion event resulting in significant changes in the channels characteristics. Unique sequence motifs within the aphid channel present novel opportunities to control aphids, whilst protecting beneficial insects.

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