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The wheat dwarf disease is among the most damaging diseases in cereals. Its aetiological agent is theWheat dwarf virus(WDV), which is exclusively transmitted from plant to plant by leafhoppers from the genusPsammotettix(Hemiptera, Cicadellidae). The parameters linked to the WDV/Psammotettixpathosystem are still poorly understood. We studiedPsammotettixindividuals collected in wheat and barley fields in France and, as a comparison, from grassland at agroecological interface in West Slovenia. Species identity of males and females has been determined using multiple criteria. In the first step, the characterization of the collected individuals included recordings of vibrational signals used in mating behaviour and morphometric analyses. In addition, a 442 nt sequence of the mitochondrial cytochrome oxydase I (COI) gene was obtained for some individuals and compared to COI sequences of thePsammotettixleafhoppers available in public databases. In the cereal fields in France,Psammotettix alienuswas the most numerous species; however, it sometimes occurred together withPsammotettix confinis, while in the grasslands in Slovenia, the third syntopic species inPsammotettixcommunity wasPsammotettix helvolus. The temporal parameters of theP. alienusmale calling song that were measured in this study were very similar to those measured in a previous study. The local biotic and/or abiotic parameters most likely influence the life history ofPsammotettixleafhoppers, and the proportion of viruliferous individuals collected in cereal fields was 14.9%, while leafhoppers collected in Slovenia were virus-free. Taken together, results show that more detailed information on population structure ofPsammotettixleafhoppers is crucial for providing an insight into the epidemiology of wheat dwarf disease.

National audience

A novel process for preparing 3-fluoroalkyl-5-pyrazolecarboxylates and carboxylic acids is hereby presented. Easily accesible α-fluorinated ketimines were condensed with oxalyl monochloride derivatives, and the obtained vinamides underwent acid-catalyzed cyclization with substituted hydrazines. This highly efficient protocol can also be used for non-fluorinated C-3 and C-5 substituents.

International audience; The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium-mediated transformation to transiently express them as N-terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana, and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C-terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.

A new strategy was developed using fluorinated acetoacetates, malononitrile, and fluoroalkyl amino reagents (FARs) to access unprecedented 4,6-bis(fluoroalkyl)pyrimidine-5-carboxylates, their carboxylic acid analogues, and 4-amino-6-(fluoroalkyl)pyrimidine-5-carbonitriles. An efficient cyclization step using suitable amidines was developed under microwave irradiation, providing the desired pyrimidines rapidly and efficiently. Standard saponification conditions were applied from carboxylate derivatives to access to the corresponding carboxylic acids. These new valuable building blocks, bearing either a single or two emergent fluorinated substituents, hold strong potential for medicinal and agrochemical research.

Ce numéro comprend les articles correspondant aux présentations du Colloque Casdar 2018.; National audience; In field crops, by the destruction of seeds and young seedlings, slug attacks are recognized as very harmful. This damage is often amplified in climatic conditions favorable to slugs and in the implementation of cultivation practices reducing the intensity of tillage. A large network of observations of more than 80 farmer plots and crop plant trials provided many data that were compared with information from other studies and literature. Slugs sampled from this network were studied in the laboratory in order to specify their thermotolerance and their biochemical composition during their growth. Data analysis provided details concerning the harmfulness of slugs, their life traits in fields cultivated during the seasons; the effects of different agricultural practices, beneficial organisms and weather conditions. This project contributed to the construction of a multicriteria assessment tool for slug risk and a conceptual model of slug population dynamics taking into account climatic factors. Nevertheless, their computer development remains to be made to make them available.; En grandes cultures, de par la destruction des semences et des jeunes plantules, les attaques de limaces sont reconnues comme très nuisibles. Ces dégâts sont souvent amplifiés lors de conditions climatiques favorables aux limaces et lors de la mise en oeuvre de pratiques culturales réduisant l’intensité du travail du sol. La mise en place d’un réseau conséquent d’observations de plus de 80 parcelles et au sein d’essais avec différents systèmes de cultures a fourni de nombreuses données qui ont été mises en regard avec des informations issues d’autres études et de la bibliographie. Des limaces extraites de ce réseau ont fait l’objet d’études au laboratoire permettant de préciser leur thermotolérance et leur composition biochimique au cours de leur croissance. L’analyse de l’ensemble des données apporte des précisions concernant : la nuisibilité des limaces, leurs traits de vie dans les champs cultivés au cours des saisons, les effets des différentes pratiques agricoles, de la faune auxiliaire et des conditions météorologiques. Ce projet a contribué à la construction d’un outil d’évaluation multicritère du risque limace et d’un modèle conceptuel de dynamique des populations de limaces tenant compte des facteurs climatiques. Néanmoins leur développement informatique reste à faire afin de les rendre disponibles.

BGPI : équipe 6; International audience; The Wheat dwarf virus, the causal agent of the wheat dwarf disease, is transmitted by leafhoppers from the genus Psammotettix and currently the main protection strategy is based on the use of insecticide treatments. Sustainable management strategies for insect vectors should include methods that are targeted to disrupt reproductive behaviour and here we investigated the mating behaviour of Psammotettix alineus (Dahlbom 1850) in order to determine the role of vibrational signals in intra-specific communication and pair formation. Both genders spontaneously emit species- and sex-specific calling songs that consisted of regularly repeated pulse trains and differ primarily in pulse train duration and pulse repetition time. Females preferred the conspecific male calling song. After a coordinated exchange of pulse trains, the male approached the stationary female. During the close range courtship and also immediately prior to copulatory attempts distinct male vibrational signals associated with wing flapping and wing vibrations were recorded from the substrate. In the presence of a receptive female, competing males emitted vibrational signals most likely aimed to interfere with male-female interaction. Mated females regained sexual receptivity after they laid eggs. Although results suggest that the viruliferous status of insects may have an effect on vibrational songs, our current results did not reveal a significant effect of virus on leafhopper performance in mating behaviour. However, the present study also suggests, that detailed understanding of plant-vector-virus interactions relevant for vector mating behaviour is essential for trying new approaches in developing future control practices against plant viruses transmitted by insect vectors.

International audience; Bioinformatics can help scientists to develop hypotheses about proteins that may need to be tested further for risks of causing allergy. In silico methodologies and tools like databases and comparison software, play an important role in the assessment of protein allergenicity and allergenicity mechanisms. They can identify whether a novel protein is an existing allergen and/or has the potential to cross-react with an existing allergen. They cannot identify whether a novel protein will ‘become’ an allergen. AllergenOnline is the tool currently used for the safety assessment of novel proteins, but other tools are also available including the Structural Database of Allergenic Proteins (SDAP) and AllerTOP. Information concerning PeptideRanker, as well as the Hydrophobic Cluster Analysis (HCA) method used for identifying IgE-binding epitopes in food allergens is discussed.

Vection des bactéries par les insectes : la cuticule importe t’elle ?. 10. Réunion annuelle du Réseau de Biologie Adaptative des Pucerons et Organismes Associés : Bapoa

International audience; Wheat dwarf virus (WDV) infects plants of the Poaceae family. WDV species includes the WDV-wand the WDV-b strains. Contradictory data present in the literature led to a conclusion that WDV wand WDV-b preferentially infect wheat and barley, respectively. WDV is transmitted by the leaf hopper Psammotettix alienus. However, species identification in the genus Psammotettix, based on morphometric characterization of the aedeagus, is a challenging procedure. Indeed, due to high aedeagus variability, species identification can lead to the incorrect assignment of males.More over, it makes impossible the identification of larvae and females. Thus, in some published works on WDV, it is possible that leafhoppers were not accurately characterized before use in transmission experiments. To improve knowledge on WDV host range, our aim was to describe Psammotettix leafhoppers at the species level using multiple criteria and then to use P. alienus to inoculate WDV to wheat and barley. Leafhopper characterization included recordings of vibrational signals used during mating and morphometric analyses. In addition, sequences of the COI gene were obtained and compared to sequences available in databases. Field-collected P.alienus were characterized, reared and then used in WDV transmission experiments. Larvae, males and females were tested for their ability to transmit WDV strains. Moreover, the abilities of WDV strains to infect wheat and barley plants was tested using host-alternation procedures based on single and dual viral acquisitions/inoculations. Inoculation of WDV-w and WDV-b to barley and wheat, respectively, failed to produce infected plants while WDV-w/WDV-b dual inoculation made it possible to detect WDV-w in barley and WDV-b in wheat. These results suggest a helper effect between WDV strains during host infection. Such information is crucial for future work on the epidemiology of wheat dwarf disease.

BGPI : équipe 6; The most important cereal viruses are Barley/Cereal yellow dwarf virus (B/CYDV, genera Luteovirus/Polerovirus, family Luteoviridae) and Wheat dwarf virus (WDV, genus Mastrevirus, family Geminiviridae). B/CYDV is a complex of at least nine viral species and WDV consists of two main strains. B/CYDV and WDV are exclusively transmitted by aphids and leafhoppers, respectively. In spite of a wealth of data on both B/CYDV and WDV pathosystems, little information is available processes related to (i) the emergence, spread and maintenance of these viruses in a landscape including cultivated and wild compartments and (ii) the interactions between these viral species and between their vectors. An intensive survey was conducted to collect plants and insects in a 5 km² landscape (Annoix, Cher, France) where B/CYDV and WDV, and their vectors (i.e. cereal aphids and Psammotettix alienus), are usually highly prevalent. Previously characterized and putative host species of the Poaceae family present in the visited areas were sampled. Simultaneously, aphids and leafhoppers were caught using yellow water traps and sticky traps. The presence of BYDV-PAV, BYDV-MAV, CYDV-RPV and WDV in collected plants and insects was assessed using serological and molecular tools. A total of 37 wild gramineous species were identified. Serological analyses revealed that B/CYDV was present in 9 wild gramineous species.Surprisingly, none of the thousands collected leaves were infected by WDV. To complement the description of the gramineous species present in the studied landscape and their possible role as viral reservoirs (Abt et al., 2017), interactions between viruses and between vectors with overlapping host ranges will be analyzed in order to determine (i) how these pathogens (and their vectors) compete for host resources and (ii) the consequences of such interactions on the epidemiology of barley yellow dwarf and wheat dwarf diseases.

International audience; Wheat dwarf virus (WDV, family Geminiviridae, genus Mastrevirus) is the causal agent of dwarfing, mottling and yellowing symptoms in wheat and barley. WDV is exclusively transmitted by leafhoppers from the genus Psammotettix in a persistent manner and infects plants of the Poaceae family. Phylogenetic analyses carried out with WDV genomic sequences showed that this viral species comprises several strains including the originally described wheat (WDV-w) and barley (WDV-b) strains. Psammotettix leafhoppers collected in different French cereal-growing areas were characterized using recordings of vibrational signals used during mating, morphometric analyses and sequences of the COI gene. Characterized P. alienus individuals were used as WDV vectors to assess the host ranges of isolates from the WDV-w and WDV-b strains. Thirty-seven wild gramineous species were identified in a 5-km² cereal-growing area located in Annoix (Cher, France) where WDV and Psammotettix leafhoppers are usually highly prevalent (Gauthier et al., 2017). Whenever seeds were available, susceptibility of wild gramineous species to WDV was tested using P. alienus-mediated WDV inoculations. The characterization of field-collected Psammotettix showed that P. alienus is the prevalent Psammotettix species and sometimes it is found syntopically with P. confinis. Leafhopper mediated transmission experiments highlighted that (i) the two WDV strains have different but slightly overlapping host ranges, (ii) three gramineous species not yet reported to be WDV hosts (i.e. Alopecurus myosuroides, Cynosurus cristatus and Poa annua) could be infected by WDV, and (iii) Setaria viridis, described in the literature as non-host for WDV, was successfully infected by the WDV-w isolate.

National audience

BGPI : équipe 6; International audience; Wheat dwarf virus (WDV, Geminiviridae family, Mastrevirus genus) is the causal agent of dwarfing, mottling and yellowing symptoms in wheat and barley. WDV is exclusively transmitted by leafhoppers from the genus Psammotettix in a persistent manner. The circular single-stranded DNA genome of WDV, of about 2.75-kb long, encodes two replication-associated proteins, the movement protein and the coat protein. The WDV genome also contains two non-coding sequences that are important for viral replication and regulation of gene expression. Phylogenetic analyses carried out with WDV sequences showed that this viral species comprises several strains including the originally described wheat (WDV) and barley (WDV-Bar) strains. The WDV strains share 83-84% nucleotide identity. WDV isolates infect plants of the Poaceae family; however i) the host ranges of the WDV strains are not completely resolved, ii) contradictory results associated to virus-host interactions are present in databases, iii) WDV and WDV-Bar isolates have wheat and barley as their preferred host, respectively, and iv) each strain can infect, at least occasionally, the preferred host of the other strain.In this study, host ranges of isolates from the WDV and WDV-Bar strains were determined using well characterized isolates and Psammotettix alienus leafhoppers. Thirty-seven wild gramineous species have been reported in a 5-km² site located in Annoix (Cher, France) (Gauthier et al., 2017). Whenever seeds were available, susceptibility of these wild gramineous species to WDV was tested using leafhopper-mediated inoculations. The results show that the two WDV strains have different but slightly overlapping host ranges. Moreover, our results revealed variations for some parameters of viral infection (e.g. susceptibility and latent period in infected plant) according to host species/WDV strain combinations. To further characterize the viral molecular determinant(s) involved in WDV host range, WDV and WDV-Bar infectious clones were constructed. These tools will enable, through the production of artificial mutants/recombinants, to determine the genetic determinants of the specific biological properties of each WDV strain, including the ability to infect a given host species.

De nombreuses cultures subissent chaque année des attaques de larves de taupins qui occasionnent des pertes de rendement ou une dégradation de la qualité des produits. La protection des cultures est réalisée préventivement grâce à l’application de produits phytopharmaceutiques. En absence de références récentes concernant la répartition des principales espèces présentes en France et le manque de connaissance concernant leur biologie, il n’est pas possible de prévoir les risques d’attaques de taupins. Plus de 700 enquêtes parcellaires ont été réalisées dans le but de cartographier les espèces de taupins puis d’identifier et hiérarchiser les facteurs susceptibles d’influencer l’abondance de larves de taupins et l’intensité des attaques occasionnées aux plantes cultivées. L’analyse des données a été réalisée sur des parcelles de maïs. Les caractéristiques de sol (pH, taux de matière organique, texture du sol) et la pluviométrie estivale sont des facteurs qui influencent l’abondance de larves de taupins dans le sol. Les conditions météorologiques subies avant et après le semis, le type de rotation – avec notamment la présence d’une prairie dans la parcelle ou dans son environnement proche – sont des facteurs qui modifient le risque d’attaques sur la culture. Ces enseignements permettent d’appréhender les risques mais il demeure difficile de caractériser un risque à l’échelle de la parcelle avec précision. En parallèle, des expérimentations réalisées au champ ont permis d’évaluer l’intérêt de champignons entomopathogènes Metarhizium anisopliae pour la protection de différentes cultures. D’autres essais ont démontré l’efficacité de glucosinolates avec une diminution des attaques de taupins de l’ordre de 50%. Des travaux en conditions contrôlées ont également mis en évidence la pertinence de stratégies innovantes basées sur l’usage de macroorganismes (nématodes entomopathogènes) ou de semiochimiques., Many crops are annually attacked by wireworms which cause yield losses or degradation of product quality. Crop protection is carried out preventively through the application of plant protection products. In the absence of recent references concerning the distribution of the main species present in France and the lack of knowledge concerning their biology, it is not possible to predict the risks of attacks by wireworms. Over 700 fields were investigated in order to map wireworm species in France and to identify the main factors involved in the abundance of wireworms and intensity of damage on crops. Data analysis was carried out on maize fields. Soil characteristics (pH, organic matter content, soil texture) and the summer rainfall are factors that influence abundance of wireworms in the soil. Weather conditions encountered before and after planting, rotation type - including the presence of a meadow in the plot or in its immediate environment - are factors that influence the level of damage on maize. These knowledges allow comprehend the risks, but it remains difficult to characterize it precisely for each field. Experiments led in the fields have assessed the pathogenic fungus Metarhizium anisopliae interest for the protection of several crops. Other trials demonstrated effectiveness of glucosinolates provoking a 50% reduction of wireworms attack. Works under controlled conditions highlighted relevance of innovative strategies using macroorganisms (entomopathogenic nematode) or semiochemicals.

Six bacterial strains were isolated from the oak-Scleroderma citrinum ectomycorrhizosphere in acidic and nutrient-poor forest soil for their high efficacy to weather minerals. Four of the six isolates, PML1(12)T and PML1(4), PML1(14) and PML1(16), were further characterized extensively. They were Gram negative, obligate aerobic, motile, non spore forming and rod-shaped. The major fatty acids of strain PML1(12)T were cyclo-C17:0, cyclo-C19:0-ω8c, C16:0 and C18:1-ω7c. The GC content of the DNA was 60.8%. The 16S rRNA and GyrB analyses showed that the four PML strains formed a distinct phylogenetic lineage within the genus Caballeronia, most closely related to Caballeronia udeis. This result was confirmed by whole-genome phylogeny analyses done on strain PML1(12)T. The results of digital DNA–DNA relatedness further supported the separation of the new isolates from closely related species. Morphological, chemotaxonomic properties were also consistent with the description of the genus Caballeronia. It is therefore proposed that strains PML1(12)T and PML1(4), PML1(14) and PML1(16) be recognized as a novel species, for which the name Caballeronia mineralivorans sp. nov. is proposed. The type strain is PML1(12)T (= DSM 104028 and LMG 2991).

Vegetatively propagated crop plants often suffer from infections with persistent RNA and DNA viruses. Such viruses appear to evade the plant defenses that normally restrict viral replication and spread. The major antiviral defense mechanism is based on RNA silencing generating viral short interfering RNAs (siRNAs) that can potentially repress viral genes posttranscriptionally through RNA cleavage and transcriptionally through DNA cytosine methylation. Here we examined the RNA silencing machinery of banana plants persistently infected with six pararetroviruses after many years of vegetative propagation. Using deep sequencing, we reconstructed consensus master genomes of the viruses and characterized virus-derived and endogenous small RNAs. Consistent with the presence of endogenous siRNAs that can potentially establish and maintain DNA methylation, the banana genomic DNA was extensively methylated in both healthy and virus-infected plants. A novel class of abundant 20-nucleotide (nt) endogenous small RNAs with 5′-terminal guanosine was identified. In all virus-infected plants, 21- to 24-nt viral siRNAs accumulated at relatively high levels (up to 22% of the total small RNA population) and covered the entire circular viral DNA genomes in both orientations. The hotspots of 21-nt and 22-nt siRNAs occurred within open reading frame (ORF) I and II and the 5′ portion of ORF III, while 24-nt siRNAs were more evenly distributed along the viral genome. Despite the presence of abundant viral siRNAs of different size classes, the viral DNA was largely free of cytosine methylation. Thus, the virus is able to evade siRNA-directed DNA methylation and thereby avoid transcriptional silencing. This evasion of silencing likely contributes to the persistence of pararetroviruses in banana plants. IMPORTANCE We report that DNA pararetroviruses in Musa acuminata banana plants are able to evade DNA cytosine methylation and transcriptional gene silencing, despite being targeted by the host silencing machinery generating abundant 21- to 24-nucleotide short interfering RNAs. At the same time, the banana genomic DNA is extensively methylated in both healthy and virus-infected plants. Our findings shed light on the siRNA-generating gene silencing machinery of banana and provide a possible explanation why episomal pararetroviruses can persist in plants whereas true retroviruses with an obligatory genome-integration step in their replication cycle do not exist in plants.

Background Chitin, the second most abundant biopolymer on earth after cellulose, is found in probably all fungi, many animals (mainly invertebrates), several protists and a few algae, playing an essential role in the development of many of them. This polysaccharide is produced by type 2 glycosyltransferases, called chitin synthases (CHS). There are several contradictory classifications of CHS isoenzymes and, as regards their evolutionary history, their origin and diversity is still a matter of debate. Results A genome-wide analysis resulted in the detection of more than eight hundred putative chitin synthases in proteomes associated with about 130 genomes. Phylogenetic analyses were performed with special care to avoid any pitfalls associated with the peculiarities of these sequences (e.g. highly variable regions, truncated or recombined sequences, long-branch attraction). This allowed us to revise and unify the fungal CHS classification and to study the evolutionary history of the CHS multigenic family. This update has the advantage of being user-friendly due to the development of a dedicated website (http://wwwabi.snv.jussieu.fr/public/CHSdb), and it includes any correspondences with previously published classifications and mutants. Concerning the evolutionary history of CHS, this family has mainly evolved via duplications and losses. However, it is likely that several horizontal gene transfers (HGT) also occurred in eukaryotic microorganisms and, even more surprisingly, in bacteria. Conclusions This comprehensive multi-species analysis contributes to the classification of fungal CHS, in particular by optimizing its robustness, consensuality and accessibility. It also highlights the importance of HGT in the evolutionary history of CHS and describes bacterial chs genes for the first time. Many of the bacteria that have acquired a chitin synthase are plant pathogens (e.g. Dickeya spp; Pectobacterium spp; Brenneria spp; Agrobacterium vitis and Pseudomonas cichorii). Whether they are able to produce a chitin exopolysaccharide or secrete chitooligosaccharides requires further investigation. Electronic supplementary material The online version of this article (doi:10.1186/s12862-016-0815-9) contains supplementary material, which is available to authorized users.

Rispail, Nicolas et al., Mitogen-activated protein kinase (MAPK) cascades and the calcium–calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium–calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches., This analysis was carried out by members of the SIGNALPATH Marie Curie training network (MRTN-CT-2005-019277), which provided financial support for N.R., C.A., R.C., A.G., R.H., E.P.N., A.P., E.S., V.V. and M.Y.

International audience; Our view of genes involved in rice disease resistance is far from complete. Here we used a gene-for-gene relationship corresponding to the interaction between atypical avirulence gene ACE1 from Magnaporthe grisea and rice resistance gene Pi33 to better characterize early rice defence responses induced during such interaction. Rice genes differentially expressed during early stages of Pi33/ACE1 interaction were identified using DNA chip-based differential hybridization and QRT-PCR survey of the expression of known and putative regulators of disease resistance. One hundred genes were identified as induced or repressed during rice defence response, 80% of which are novel, including resistance gene analogues. Pi33/ACE1 interaction also triggered the up-regulation of classical PR defence genes and a massive down-regulation of chlorophyll a/b binding genes. Most of these differentially expressed genes were induced or repressed earlier in Pi33/ACE1 interaction than in the gene-for-gene interaction involving Nipponbare resistant cultivar. Besides demonstrating that an ACE1/Pi33 interaction induced classical and specific expression patterns, this work provides a list of new genes likely to be involved in rice disease resistance.

BGPI : équipe 6; International audience; Cereals can be infected by different pathogens among which the Wheat dwarf virus (WDV, family Geminiviridae, genus Mastrevirus) is the causal agent of dwarfing, mottling and yellowing symptoms in wheat, barley and oat. WDV is exclusively transmitted from plant to plant by leafhoppers from the genus Psammotettix in a persistent manner. Due to the lack of genetic resistance sources, the main protection strategy used against WDV is based on the use of chemicals against vectors. While large-scale insecticide treatments benefit the crops by reducing the number of insects in the protected area, their intensive use generate environmental and financial costs. Thus, it is important to identify parameters involved in the prevalence and the spread of the disease in fields to avoid using chemicals when they are not needed. Psammotettix alienus and P. provincialis are are the only two Psammotettix species for which it was shown that they are able to transmit WDV. However, species descriptions in the genus Psammotettix, exclusively based on male’s genitalia, are associated with many inconsistencies. Actually, many groups of organisms, including leafhoppers, contain a high level of cryptic diversity and in such cases a more complex approach interpreting species status is necessary. In this work, we propose several sources of taxonomic information (e.g. morphological, molecular, behavioural and ecological data) for a reliable identification of species and populations within the leafhopper genus Psammotettix. Furthermore, the described species/groups were tested, under controlled conditions, for their capacity to transmit WDV. In addition to scientific knowledge resulting from such type of work, the gathered data will be invaluable for the estimation of WDV risk associated with the presence of leafhoppers in cereal-growing areas. Such information is clearly needed for the future management of wheat dwarf disease.

BGPI : équipe 6; Assessing the risk of a viral disease based on expert opinions. 15. Rencontres de Virologie Végétale (RVV)

Sucrose starvation of Arabidopsis (Arabidopsis thaliana) cell culture was used to identify translationally regulated genes by DNA microarray analysis. Cells were starved by subculture without sucrose, and total and polysomal RNA was extracted between 6 and 48 h. Probes were derived from both RNA populations and used to screen oligonucleotide microarrays. Out of 25,607 screened genes, 224 were found to be differentially accumulated in polysomal RNA following starvation and 21 were found to be invariant in polysomal RNA while their total RNA abundance was modified. Most of the mRNA appears to be translationally repressed (183/245 genes), which is consistent with a general decrease in metabolic activities during starvation. The parallel transcriptional analysis identifies 268 regulated genes. Comparison of transcriptional and translational gene lists highlights the importance of translational regulation (mostly repression) affecting genes involved in cell cycle and cell growth, these being overrepresented in translationally regulated genes, providing a molecular framework for the arrest of cell proliferation following starvation. Starvation-induced translational control also affects chromatin regulation genes, such as the HD1 histone deacetylase, and the level of histone H4 acetylation was found to increase during starvation. This suggests that regulation of the global nuclear transcriptional activity might be linked to cytoplasmic translational regulations.

International audience; Barley yellow dwarf (BYD) disease is one of the most severe viral diseases in autumn sown cereals. In Western Europe, crop losses are mainly due to the PAV species of BYD viruses transmitted by Rhopalosiphum padi, the most abundant aphid in autumn. The proportion of migrant winged aphids that carry viruses in autumn is considered a major epidemiological factor for determining the disease incidence. In the main French cereal areas, during a 6-week period in autumn 1999-2002, the proportion of viruliferous R. padi assessed using a TAS-ELISA technique was on average of 4.98% (range 2.01-9.91%). Variations according to trap location were correlated with land use at the regional scale, annual variations being correlated with the climate of the year. Implementations of these results to improve BYD disease management program are discussed.

Barley yellow dwarf virus (BYDV) damage to winter cereals and population dynamics of the aphid Rhopalosiphum padi during fall were monitored in fields during 10 years at various locations in the northern half of France. Logistic regression was used to examine whether a simple risk probability algorithm based only on the autumnal population dynamics of R. padi can accurately predict yield losses caused by BYDV and, therefore, the need for insecticide treatment. Results showed that the area under the curve of the percentage of plants infested by R. padi during autumn was highly significantly related to BYDV yield losses. Then, a cost/benefit analysis was performed to estimate the optimal decision threshold resulting in the lowest annual average costs of BYDV damage and control. A “model use” strategy allowed a reduction in the annual average costs of BYDV disease and control of up to 36% when compared with a “prophylactic spraying” strategy. The optimal decision threshold was highly sensitive to variation in disease prevalence. This property was used to propose an easy way to adapt the model to any production situation through the determination of the most accurate decision threshold.

We report 34 new nuclear single-nucleotide-polymorphism (SNP) markers that have been developed from an expressed sequence tag library of Plasmopara viticola , the causal agent of grapevine downy mildew. This newly developed battery of markers will provide useful additional genetic tools for population genetic studies of this important agronomic species.

International audience; Carboxamide fungicides target succinate dehydrogenase (SDH). Recent field monitoring studies have identified Botrytis cinerea isolates resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B subunit. We confirmed, by site-directed mutagenesis of the sdhB gene, that each of the mutations identified in field strains conferred resistance to boscalid in B. cinerea, and in some cases cross-resistance to other SDHIs (fluopyram, carboxin). Enzyme inhibition studies showed that the studied modifications (SdhB_P225T/L/F, N230I, H272Y/R/L) affected the inhibition of SDH activity by SDHIs, directly contributing to resistance. Our results confirm the importance of H272, P225 and N230 for carboxamide binding. Modifications of P225 and N230 conferred resistance to the four carboxamides tested (boscalid, fluopyram, carboxin, bixafen). Modifications of H272 had differential effects on the susceptibility of SDH to SDHIs. SdhB(H272L), affected susceptibility to all SDHIs, SdhB(H272R) conferred resistance to all SDHIs tested except fluopyram, and SdhB(H272Y) conferred fluopyram hypersensitivity. Affinity-binding studies with radio-labelled fluopyram revealed strong correlations among the affinity of SDHIs for SDH, SDH inhibition and in vivo growth inhibition in the wild type. The sdhB(H272Y) mutation did not affect SDH and respiration activities, whereas all the other mutations affected respiration by decreasing SDH activity.

Highlights • History of seven fungal species used as models for studying development and pathogenicity. • Outline of central stages of their life cycle and their infection processes. • Molecular toolkits used to study different aspects of pathogenicity. • Insight gained from genome sequencing projects. • Current research trends and future challenges., Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity.

This working group made a survey and drew maps of herbicide resistances in cereal crops according to modes of action and weed species. These resistances concern mainly grass weeds but also one dicot weeds., Ce groupe de travail a réalisé sous forme de cartes un état des lieux des résistances aux herbicides dans les cultures céréalières en fonction des modes d’action et par adventices. Ces résistances concernent essentiellement des graminées mais aussi quelques dicotylédones.

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National audience; Fusarium Head Blight (FHB) is one of the most important disease altering wheat crops. A field experiment was conducted to better understand the saprotrophic development of Fusarium graminearum and its consequences on FHB, to characterize the relative importance of the different sources of FHB inoculum and the accumulation of mycotoxins in grains and subsequently, to determine early indicators of future disease development on ears and accumulation of mycotoxins in grains. The development of F. graminearum in the soil and crop residues was monitored in controlled conditions. The inoculum hosted by seeds and/or buried with crop residues in the topsoil had only an effect on the winter development of the disease. In contrast, the main source of inoculum causing FHB disease on ears and accumulation of mycotoxins in wheat kernels came from residues left on the soil surface. Monitoring of plant development from sowing to harvest, crop management and soil and weather conditions produced a large database. Unfortunately, the role of climate was decisive in the development of the Fusarium-host plant interaction, thus prevented the use of early indicators to accurately predict the risks of yield losses and accumulation of mycotoxins involved. F. graminearum was regulated by the soil microflora. However, crop residues provide the fungus spatial and trophic niches favourable to its development. The exploitation of these niches by F. graminearum depends on the nature (previous crop and C/N) of the residues. Maize stubbles provide a greater carrying capacity than wheat straw and rapeseed residues while mustard has a suppressive effect for the fungus. The management of crop residues is a key point to control the development of FHB. A strong emphasis should be placed on the biological decomposition of crop residues at the soil surface or/and on the use of suppressive intermediate crops such as mustard to limit the soil inoculum potential of saprotrophic F. graminearum.

International audience; At an accelerating pace, advances in technology are providing an increasing number of molecular markers to the cereal geneticist. Most of these newly produced markers first need to be mapped before they can be used in breeding programmes. Although classical bi-parental segregating populations can be used for marker mapping, they are limited in value when mapping high numbers of markers, both because of a low rate of polymorphism and due to the limited probability of recombination between increasingly tightly linked markers. New populations based on multi-parental crosses and higher levels of recombination have been developed recently, for example MAGIC populations (Multi-parent Advanced Generation Inter-Cross), which overcome these drawbacks. The MAGIC wheat population we are studying is composed of 1100 SSD (Single Seed Descent) lines resulting from 12 cycles of random crosses of 62 founders with a large genetic base. These random crosses were facilitated by the integration of a nuclear male sterility allele (ms1b, Probus donnor) in the population. This population has been genotyped with an Illumina array of 9k SNPs (Single Nucleotide Polymorphisms), using a set of markers spread across the genome (Chao et al. 2010). The MAGIC parents were also genotyped with 3000 SNPs developed on chromosome 3B (Paux et al. 2006). Polymorphism and quality checks resulted in the selection of 6480 SNP markers from the 9k set, and 2690 SNP markers on the 3B chromosome. Before mapping markers, the genetic structure and linkage disequilibrium (LD) present in SSD lines and parental lines was assessed, confirming the efficiency of the 12 cycles of random crossing. Following this, the markers were mapped by means of a new method based on LD analysis, using the 3B dataset on the MAGIC parental panel. The quality of the method was assessed using previously mapped markers, or using 9k data on the SSD. The validity of the population for both marker mapping and quantitative trait locus (QTL) detection is discussed with regard to these first results.

Global change, in particular climate change, will affect agriculture worldwide in many ways: increased drought or flooding amplitude and frequency, variable temperature increases, loss of natural depuration of waters, soil erosion, loss of soil carbon content, invasion by alien species, increased pest events, changes in plant phenology, increased sensitivity of crops to stress and diseases etc. (Fisher et al. 2005; Howden et al. 2007). These anticipated or even already occurring stresses raise concerns about the sustainability of production and the ability of agriculture to feed human populations. All these changes could lead to an increased use of pesticides (Kattwinkel et al. 2011). Moreover, demographic pressure continues to rise, in particular in tropical and sub-tropical regions, where greater threats to agriculture and food sustainability are anticipated by the Intergovernmental Panel on Climate Change (IPCC) (Easterling et al. 2007). These trends will certainly lead to mounting conflicts involving water uses (irrigation versus drinking water production or freshwater ecosystem maintenance, sanitation etc.) and food production. This appeals to an “ecologically intensive agriculture” (Griffon 2006), i.e. a sustainable agriculture providing ecosystem services more efficiently than today and causing fewer adverse impacts on the environment and water resources. With EU Directive 2009/128/EC (EC 2009a) enforcement, requesting Member States to adopt action plans aiming to reduce risks and impacts related to pesticide uses, there will be a focus in the public and political debates in Europe on achieving a more sustainable use of pesticides. This should consequently lead to a reduction of the risks or impacts of pesticides on the environment. In Europe, there is currently a strong focus on source (including dose) reduction. This approach may nevertheless be too restrictive if the goal is to reduce the agriculture footprint while maintaining or increasing yield. Depending on the chemical properties of pesticides as well as environmental factors, decreasing the amounts of pesticides applied to crops will not automatically produce a decrease in the risk to non-target species or water supply. How could society meet the challenge of the forthcoming climate change? What adaptations should be envisaged for agriculture/pesticide risk management (RM)? These changes will probably have a profound effect on agricultural systems (crop selection, farming practices etc.) and to a lesser extent influence the fate and effects of chemicals (Schiedek et al. 2007). These questions have been addressed by two European research networks, namely Euraqua (the European Network of Freshwater Research Organisations, http://www.euraqua.org/) and PEER (Partnership for European Environmental Research, http://www.peer.eu/), which organised a workshop aiming to identify research needs and strategies induced by these questions in October 2011 in Montpellier, France. The workshop's specific goals were to (1) discuss the pesticide risk assessment (RA) approach, its limitations (e.g. spatial scale and multi-stress situations), the connections between different policies (pesticide regulation and Water Framework Directive), the use of models, (2) review integrated practices and innovative technologies which could or are intended to reduce pesticides' environmental impacts and (3) contribute to the future research and development agenda. This review summarises the workshop discussions.

We have witnessed a dramatic increase in the number of weeds showing herbicide resistance during the past three decades. The potentially most dangerous mechanisms of resistance endows Non-Target-Site based Resistance (NTSR): NTSR is polygenic, unpredictable and can confer cross-resistance to herbicides with different modes of action. High throughput sequencing methods, like RNA sequencing (RNA-seq), are particularly effective to identify genes differentially expressed between two experimental modalities. Therefore, RNA sequencing was implemented to identify genes involved in NTSR to clodinafop (an ACCase inhibitor) in black-grass. Black-grass transcriptome was sequenced from 4 sensitive plants and 4 clodinafop-resistant plants from the same F1 population, collected at different time-points before and after herbicide treatment. A black-grass transcriptome database was established that contains qualitative data (sequence of each gene and associated protein) and quantitative data (gene expression level of every gene during the time-course, in sensitive and in resistant plants). The expression of candidate NTSR genes was subsequently verified by quantitative RT-PCR., Nous avons assisté à une augmentation spectaculaire du nombre d’adventices résistantes à un herbicide ces 30 dernières années. Les mécanismes de résistance potentiellement les plus dangereux sous tendent la résistance non liée à la cible (RNLC) : la RNLC est polygénique, imprévisible et elle peut conférer des profils de résistance croisée à des herbicides ayant des modes d’action pouvant être différents. Les techniques de séquençage haut débit, comme le RNA sequencing (RNA-seq), sont particulièrement efficaces pour identifier les gènes différentiellement exprimés entre deux modalités. La méthode RNA-seq a donc été utilisée pour identifier des gènes de RNLC au clodinafop (inhibiteur de l’ACCase) chez le vulpin. Le transcriptome du vulpin a été séquencé à partir de quatre plantes sensibles et de quatre plantes résistantes au clodinafop provenant de la même F1 (première génération d'un croisement). Les plantes ont été prélevées avant et à différents moments après le traitement herbicide. Une base de données du transcriptome du vulpin a été établie, contenant des données qualitatives (séquence de chaque gène, protéine associée) et quantitatives (niveau d’expression de chaque gène au cours de la cinétique de prélèvement, chez les sensibles et chez les résistants). L’expression de gènes-candidats de RNLC a ensuite été vérifiée par RT-PCR quantitative.

Antifungal compounds such as ergosterol biosynthesis inhibitors are widely used to control crop diseases. Among them, one of the most recent, the hydroxyanilide fenhexamid, is efficient principally against Botrytis cinerea, the major causal agent of grey mould. Fenhexamid is a new type of ergosterol biosynthesis inhibitor affecting the sterol C4 demethylation processes due to its specific interaction with one of the four proteins of the enzymatic complex, the 3-ketoreductase. Our regular monitoring conducted on French vineyards allowed the identification of the first isolates of B. cinerea with acquired resistance. Two types of resistant isolates named HydR3- and HydR3+ were distinguished by their resistance level. This acquired resistance is due to point mutations in the erg27 gene leading to target modifications. These modifications induce a reduced in affinity of fenhexamid towards its target, the 3-ketoreductase. Because of their high resistant level, the HydR3+ strains have to be considered relative to the risk of resistance phenomenom occurrence in vineyards. Fitness studies conducted in vitro on isogenic mutants showed altered "overwintering" capacities of HydR3+ mutants suggesting that they probably do not impact fenhexamid's field efficacy. While B. cinerea's acquired resistance could be explained only by target modifications, as in most cases of fungicide resistance, the situation is different for the related species Botrytis pseudocinerea naturally resistant to fenhexamid. We show that erg27 polymorphism only slightly contributes to resistance whereas fenhexamid detoxification by a cytochrome P450 named cyp68.4 is the major mechanism responsible for the resistance. This is the first case of a functional validation of fungicide detoxification involved in resistance.

Session 1 : Interactions Moléculaires. Poster n°1.2; absent

BACKGROUND: Fenhexamid, a sterol biosynthesis inhibitor effective against Botrytis, inhibits the 3-ketoreductase (Erg27) involved in C-4 demethylation. Several fenhexamid-resistant phenotypes have been detected in Botrytis cinerea populations from French vineyards. The field isolates with the highest resistance levels display amino acid changes in Erg27 (F412S, F412I or F412V). RESULTS: Fenhexamid-resistant mutants were generated by site-directed mutagenesis of the erg27 gene in a sensitive recipient strain to overcome the impact of different genetic backgrounds. The wild-type erg27 allele was replaced by the three mutated alleles (erg27F412S/I/V) by homologous recombination. These isogenic strains were shown to be fenhexamid-resistant and were used to quantify the impact of F412 mutations on fungal fitness. Several parameters, including radial growth, the production of sclerotia and conidia, freezing resistance and aggressiveness, were quantified in laboratory conditions. Analysis of variance demonstrated significant differences between the mutant and parental strains for some characters. In particular, the mutants grew more slowly than the wild-type strain and displayed variations in the production of sclerotia and conidia with temperature and susceptibility to freezing. CONCLUSIONS: The results highlight a moderate but significant impact of F412 mutations on the survival capacity of B. cinerea strains displaying high levels of resistance to fenhexamid in laboratory conditions, potentially limiting their dispersal and persistence, particularly in terms of overwintering, in field conditions. Copyright © 2011 Society of Chemical Industry

BGPI : équipe 6; National audience

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National audience; Dans deux parcelles, l'emploi systématique d'herbicides inhibiteurs de l'ACCase, puis d'inhibiteurs de l'ALS, a sélectionné des plantes de vulpin résistantes à la plupart des substances ayant ces modes d'action (fénoxaprop, clodinafop, pinoxaden, quizalofop, iodosulfuron+mésosulfuron et pyroxsulame). Ces plantes ne contiennent pas de mutations de l'ACCase ou de l'ALS, et sont uniquement résistantes par des mécanismes de résistance non liée à la cible (RNLC). Les solutions chimiques restant potentiellement disponibles pour les contrôler sont limitées et impliquent un plus grand nombre d'applications. En l'absence de nouveaux modes d'action et de capacité à détecter rapidement la RNLC, il est impératif de ralentir la sélection de la RNLC. Ceci passe par l'emploi d'une diversité de techniques de désherbage, chimiques et non chimiques

Publication de travaux de thèse; National audience; Pour approfondir les connaissances sur les évènements associés à la germination et identifier des marqueurs de vigueur germinative, nous avons analysé le protéome des graines d'Arabidopsis. Le choix de cette approche est fondé sur l'importance, dans la germination, des néosynthèses de protéines à partir des ARNm stockés dans les graines. Parmi 1272 protéines séparées par électrophorèse bi-dimensionnelle, le niveau d'accumulation de 80 protéines varie au cours de la germination sensu stricto ou de la phase de percée radiculaire. Plus de cent protéines ont été identifiées. Notre approche a révélé des protéines associées à l'état imbibé ou à l'état déshydraté des graines et des protéines essentielles dans le métabolisme cellulaire, comme l'AdoMet synthétase et la méthionine synthase. L'utilisation de DL-propargylglycine a démontré qu'une biosynthèse de méthionine et/ou des produits dérivés est requise pour la germination. Par ailleurs, nous avons utilisé cette carte protéique pour analyser le rôle des gibbérellines dans la germination. Cette stratégie a révélé que cette hormone contrôle principalement les évènements associés à la percée radiculaire.

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National audience; Cell wall integrity is crucial for fungal growth, development and stress survival. In yeast, Slt2MAP kinase and calcineurin signaling pathways monitor cell wall repair during stress and development. MPS1, the M. grisea SLT2 orthologue, is essential for cell wall repair and for appressorium mediated penetration into host plants (Xu 1998 PNAS 95:12713). In yeast, Slt2 activates the transcription factors Rlm1, Swi4 and Swi6, while calcineurin activates Crz1. Genes orthologous to yeast CRZ1, MPS1, RLM1, SWI4, and SWI6 genes were identified in M. grisea genome. Swi4 and Swi6 interact with Mps1 in yeast two hybrid assays. Deletion mutants were constructed by targeted gene replacement in M. grisea. Delta-mps1 mutants displayed an abnormal mycelial growth (no aerial hyphae), did not sporulate, and were nonpathogenic on plants as reported. Delta-crz1, delta-rlm1, delta-swi6 mutants have a normal mycelial growth and sporulation rates similar to wild type. Of these three mutants, only delta- Rlm1 displays a highly reduced pathogenicity on barley and rice (-98%, lesion number). Delta-mps1 mutants are highly sensitive to nikkomycin Z (chitin synthase inhibitor), CFW (disorganization of cell wall) and aculeacine (glucan synthase inhibitor), while delta-crz1 and delta-rlm1 mutant are only mildly hypersensitive to Nikkomycin, and delta-swi6 mutant is only slightly hypersensitive to CFW. These studies suggest that the transcription factors controlled by Mps1 are either functionally redundant or specialized in the control of specific target genes.