Evaluation expérimentale de stratégies de déploiement de gènes de résistance pour la gestion durable des nématodes à galles

Dans le cadre de projets soutenus par l'ANR Systerra et le GIS PICLeg (projets "Sysbiotel" et "Neoleg") menés en collaboration entre l'INRA PACA, l'IRD, l'APREL, la Chambre d'Agriculture du 06 et des entreprises privées de sélection de semences, plusieurs stratégies de déploiement de gènes de résistance ont été évaluées pendant 3 ans sur le terrain en conditions agronomiques pour mettre au point une gestion raisonnée des cultivars résistants permettant de gérer de manière durable les problèmes de nématodes à galles des racines. L'alternance des gènes de résistance dans la rotation et le "pyramiding" de gènes dans un même cultivar se sont révélés extrêmement efficaces pour supprimer l'émergence de populations virulentes et réduire les taux d'infestation du sol de plus de 80% en 3 ans. Un nouveau projet INRA "Gedunem", mis en place dans le cadre du Métaprogramme INRA SMaCH (Sustainable Management of Crop Health), vise maintenant à associer ces innovations variétales aux autres méthodes de lutte disponibles (gestion de l'interculture, plantes non hôtes, prophylaxie) afin de maintenir une pression parasitaire faible, tout en évaluant ces nouveaux systèmes de culture du point de vue agronomique et socio-économique

Identification of plant-parasitism genes in nematodes in silico screening and in vivo validation in Meloidogyne incognita.

International audienceRoot-knot nematodes, Meloidogyne spp., are root parasites that annually cause ca. 100 billion Euros losses in crop production worldwide. An initial analysis of the genome of Meloidogyne incognita has allowed the identification of thousands of genes that apparently did not have homolog in model eukaryotes. A proportion of these M. incognita-restricted gene models may be involved in functions linked to plant-parasitism. Our objective was to identify drug target genes to control primarily root-knot nematodes with potential applications to other plant pathogenic and parasitic species. Our hypothesis was, if a gene is involved in a key parasitism process, it might be conserved in plant-parasitic or plant-pathogenic species and absent from non-parasitic species. Our strategy to identify druggable parasitism target genes was to perform a comparative analysis of M. incognita and M. hapla predicted protein coding genes against a selection of fully sequenced genomes and transcriptomes of various eukaryotes with different lifestyles. Our pipeline conserves all sequences shared with parasitic or plant-associated species and eliminates those conserved in species that could be negatively affected by newly developed drugs or control means. In parallel, we performed automatic functional annotation of the identified genes. Those annotations include detection of signal peptide, Pfam domains, assignment of gene ontology (G.O.) terms, and identification of specific motifs. All these features and annotations were stored in a relational database and it is thus possible to produce any combination of feature to filter out genes of interest. Comparison of the relative abundance of G.O. terms assigned to genes restricted to Meloidogyne species versus those assigned to the whole gene sets of M. incognita and M. hapla revealed a number of differences. A second objective was to develop a new RNA interference (RNAi) procedure using siRNA to artificially silence target genes and analyze their role in parasitism. SiRNA are smaller than long dsRNA usually used for silencing in M. incognita. SiRNA might be easier to introduce by soaking in M. incognita. SiRNA are also more specific to a target sequence as they are 21 base-long and their specificity towards target mRNAs can be assessed by BLAST searches in M. incognita genome. SiRNA targeted to various positions on the mRNA secondary structure were tested to optimize siRNA design. Several transfection conditions were tested and validated by quantitative PCR to optimize reproducibility and target mRNA expression inhibition efficiency. To test the effect of gene silencing on nematode infectivity, nematodes treated with optimized siRNA were used for inoculation assays on tomato plants (Solanum esculentum cv. St Pierre). Infectivity was measured by counting gall and egg masse numbers. With this new method we will test the effect of gene silencing for a series of the most promising targets identified during bioinformatics screens

Identification of plant-parasitism genes in nematodes in silico screening and in vivo validation in Meloidogyne incognita.

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Pyramiding, alternating or mixing : comparative performances of deployment strategies of nematode resistance genes to promote plant resistance efficiency and durability

Background: Resistant cultivars are key elements for pathogen control and pesticide reduction, but their repeated use may lead to the emergence of virulent pathogen populations, able to overcome the resistance. Increased research efforts, mainly based on theoretical studies, explore spatio-temporal deployment strategies of resistance genes in order to maximize their durability. We evaluated experimentally three of these strategies to control root-knot nematodes: cultivar mixtures, alternating and pyramiding resistance genes, under controlled and field conditions over a 3-years period, assessing the efficiency and the durability of resistance in a protected crop rotation system with pepper as summer crop and lettuce as winter crop. Results: The choice of the resistance gene and the genetic background in which it is introgressed, affected the frequency of resistance breakdown. The pyramiding of two different resistance genes in one genotype suppressed the emergence of virulent isolates. Alternating different resistance genes in rotation was also efficient to decrease virulent populations in fields due to the specificity of the virulence and the trapping effect of resistant plants. Mixing resistant cultivars together appeared as a less efficient strategy to control nematodes. Conclusions: This work provides experimental evidence that, in a cropping system with seasonal sequences of vegetable species, pyramiding or alternating resistance genes benefit yields in the long-term by increasing the durability of resistant cultivars and improving the long-term control of a soil-borne pest. To our knowledge, this result is the first one obtained for a plant-nematode interaction, which helps demonstrate the general applicability of such strategies for breeding and sustainable management of resistant cultivars against pathogens

Evaluating sorghums as green manure against root-knot nematodes

Current restrictions on the use of chemical nematicides have led to an increase in root-knot nematode (RKN) damages in horticultural crops. The effects of two sorghums as summer cover crops, Sorghum sudanense sudangrass cv. 'Piper' or sudangrass hybrid [S. bicolor x S. sudanense] '270911', respectively with low and high dhurrin contents, were compared in their ability to suppress RKN in a vegetable production system. The use of both sorghums 'Piper' and '270911' as a green manure was found to be an effective strategy for decreasing RKN infestation in the soil, thereby protecting the subsequent planting of RKN susceptible crops (chard, lettuce or melon). Analytical experiments were further conducted in growth chamber and greenhouse pot experiments to investigate and compare the susceptibility of the sorghums and the factors affecting their efficacy for RKN management, in order to better explain the results obtained in the field trial. The two sorghums were poor hosts of RKN, acted as trap crops and as a biofumigant releasing hydrogen cyanide. Time of planting, time of biofumigation, and type of soil affected their efficacy for RKN management. For best RKN suppression, the sorghum cover crops need to be cultivated during one month or less and biofumigated for one month prior to crop planting. The trapping effect of both sorghums in clayey soil was less efficient than in sandy or sandy-loamy soils. Combining less than 30-days of sorghum culture and 10-days soil incorporation with solarization mulch was particularly efficient in suppressing nematodes. No effect relative to the sorghum type was detectable as long as they were used appropriately

Conception et évaluation de systèmes de culture maraîchers méditerranéens innovants pour gérer les nématodes à galles

Design and assessment of innovative Mediterranean vegetable cropping systems to manage root-knot nematodes. Description of the subject. A system approach based on co-design and experimental field evaluation of cropping systems (CSs), combining technical and varietal innovations, has been implemented for sustainable management of root-knot nematodes (RKN) in Mediterranean sheltered vegetable systems. Objectives. Cropping systems combining genetic resistance and cultural practices (crop rotations including susceptible, resistant, and non-host plants; intercropping management with nematicidal cover crops or soil solarization) were assessed over a period of 4 years (i) to reduce RKN populations and increase the durability of varietal resistances, (ii) to study the impact of these systems on soil ecology (plant-parasitic and free-living nematode communities), and (iii) to evaluate their acceptability by farmers. Method. Three CS prototypes, resulting from a co-design process with research and development stakeholders, were compared with CSs conventionally implemented in the Mediterranean region. The three prototypes were also evaluated using complementary methods: (i) system experiments in three commercial farms in Southern France; (ii) analytical experiments to decipher the mechanisms of action for some [agroecological??] levers; (iii) surveys to evaluate the acceptability of the prototypes by farmers. Results. All three CSs were found to be effective (90% RKN decrease, protection of partially resistant Solanaceae, no negative effect on non-phytoparasitic nematodes) and sustainable, when application conditions and soil biological equilibrium were favorable (global soil nematofauna diversified and abundant). The acceptability of the three systems depended on the type of farm where they were implemented and the attitude of the farmers towards innovation. Conclusions. These three CSs still need to be improved, in terms of their efficiency, in consultation with participating farmers, by introducing new agroecological levers, as well as innovation costs. Future research will also need to open up to a more comprehensive management of soil health

Varietal and technical innovations for the sustainable and integrated management of root-knot nematodes

International audienceThe current restrictions on the use of chemical nematicides have contributed to increased root-knot nematodes (Meloidogyne spp.) problems in horticultural crops. In this context, plant resistance (R) appears as the most effective method of control, but the restricted number of cultivated vegetable species with root-knot nematode R-genes available (tomato, pepper), and the possible occurrence of virulent nematodes able to reproduce on R-plants may constitute a severe threat to this control strategy. To increase the sustainability of the protection, a scientific challenge exists in building cropping strategies based on the combination of genetic resistance with cultivation practices. Our projects aimed at assessing such innovative strategies in a multi-site device in research stations and commercial farms. First, we looked at three components of crop protection, i.e., R-efficiency, R-durability, and sustainability of rotating cultivation. Overall, a constant hierarchy of management strategies was observed, with Pyramyding > Alternating > Mixture of R-genes > Sequential use of a single R-gene introgressed in a susceptible background. We are currently analysing (1) the impact of agronomic practices on the parasite pressure in the soil and on ecological diversity including other nematode species, (2) the linkage between reduction of Meloidogyne populations in the soil and increase of R-genes durability, and (3) the sustainability assessment of such varietal and technical innovations. Multidisciplinary approaches were combined to foster synergistic and long-term goals. The diversity of partners and associated forces brought all the complementary expertise needed for answering specific short-term questions as well as generic mid- to long-term expectations