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Chancre de l’abricotier : diversité bactérienne en verger

International audience; Many phytopathogenic fungi are disseminated as spores via the atmosphere from short to long distances. The distance of dissemination determines the extent to which plant diseases can spread and novel genotypes of pathogens can invade new territories. Predictive tools including models that forecast the arrival of spores in areas where susceptible crops are grown can help to manage sustainable crop health. However, such models are difficult to establish for fungi with broad host ranges because sources of inoculum cannot be readily identified. Sclerotinia sclerotiorum, the pandemic agent of white mould disease, can attack > 400 plant species including economically important crops. Monitoring airborne inoculum of S. sclerotiorum in several French cropping areas has shown that viable ascospores are present in the air almost all the time, even when no susceptible crops are nearby. This raises the hypothesis of a distant origin of airborne inoculum. The objective of the present study was to determine the interconnectivity of reservoirs of S. sclerotiorum from distant regions based on networks of air mass movement. Viable airborne inoculum of S. sclerotiorum was collected in 4 distinct regions of France and 498 strains were genotyped with 16 specific microsatellite markers and compared among the regions. Air mass movements were inferred using the HYSPLIT model and archived meteorological data from the global data assimilation system (GDAS). The results show that up to 700 km could separate collection sites that shared the same haplotypes. There was low or no genetic differentiation between strains collected from the four sites. The rate of aerial connectivity between two sites varied according to the direction considered. The results also show that the aerial connectivity between sites is a better indicator of the probability of the incoming component of inoculum at a given site from another one than is geographic distance. We identified the links between specific sites in the trajectories of air masses and we quantified the frequencies at which the directional links occurred as a proof-of-concept for an operational method to assess the arrival of airborne inoculum in a given area from distant origins.

Mycobacterium avium subsp. paratuberculosis is responsible for paratuberculosis in animals. This disease, leading to an inflammation of the gastrointestinal tract, has a high impact on animal health and an important economic burden. The environmental life cycle of M. avium subsp. paratuberculosis is poorly understood and several studies suggest that free-living amoebae (FLA) might be a potential environmental host. FLA are protozoa found in water and soil that are described as reservoirs of pathogenic and nonpathogenic bacteria in the environment. Indeed, bacteria able to survive within these amoebae would survive phagocytosis from immune cells. In this study, we assessed the in vitro interactions between several strains of M. avium subsp. paratuberculosis and Acanthamoeba castellanii. The results indicate that the bacteria were able to grow within the amoeba and that they can survive for several days within their host. To explore the presence of M. avium subsp. paratuberculosis in environmental amoebae, we sampled water from farms positive for paratuberculosis. A M. avium subsp. paratuberculosis strain was detected within an environmental amoeba identified as related to the poorly described Rosculus genus. The bacterial strain was genotyped, showing that it was similar to previous infectious strains isolated from cattle. In conclusion, we described that various M. avium subsp. paratuberculosis strains were able to grow within amoebae and that these bacteria could be found on farm within amoebae isolated from the cattle environment. It validates that infected amoebae might be a reservoir and vector for the transmission of M. avium subsp. paratuberculosis.

Investigations into life history of microorganisms that cause plant diseases have been limited mostly to contexts where they are in interaction with plants, and with cropped or otherwise managed vegetation. Therefore, knowledge about the diversity of plant pathogens, about potential reservoirs of inoculum and about the processes that contribute to their survival and adaptation is limited to these contexts. The agro-centric perspective of plant pathogen life histories is incoherent with respect to the capacity of many of them to persist as saprophytes on various substrates. In this context we have investigated the ubiquity of the broad host range necrotrophic fungus Botrytis cinerea, outside of agricultural settings and have determined if the populations in these natural habitats can be distinguished phenotypically and phylogenetically from populations isolated from diseased crops. Over a period of 5 years, we isolated B. cinerea from 235 samples of various substrates collected in France including rainfall, snowpack, river, and lake water, epilithic biofilms in mountain streams, leaf litter and plant debris, rock surfaces, bird feathers and healthy wild plants from outside of agricultural fields. All substrates except rock surfaces harbored B. cinerea leading us to establish a collection of purified strains that were compared to B. cinerea from diseased tomato, grapes and various other crops in France. Phylogenetic comparisons of 321 strains from crop plants and 100 strains from environmental substrates based on sequences of 9 microsatellite markers revealed that strains from crops and the environment could not be distinguished. Furthermore, the genetic diversity of strains outside of agriculture was just as broad as within agriculture. In tests to determine the aggressiveness of strains on tomato stems, the mean disease severity caused by strains from environmental substrates was statistically identical to the severity of disease caused by strains from tomato, but was significantly greater than the severity caused by strains from grape or other crops. Our results suggest that highly diverse populations of this plant pathogen persist outside of agriculture in association with substrates other than plants and that this part of their life history is compatible with its capacity to maintain its potential as plant pathogen.

Identifier les réservoirs et sources d’inoculum des agents phytopathogènes est un enjeu majeur en pathologie végétale. Les systèmes agricoles pérennes, tels que les vergers, sont soumis aux attaques de nombreux ravageurs et agents microbiens pathogènes. P. syringae, agent phytopathogène responsable de l’émergence de maladies des arbres fruitiers, dont la récente épidémie du chancre bactérien du kiwi causée P. syringae pv. actinidiae (Psa), représente un enjeu économique important au niveau mondial. En France, les moyens de lutte sont constitués de traitements cupriques et de gestes préventifs visant à réduire la propagation de la bactérie au sein et entre les vergers. Avec la prise de conscience de la nécessité de la conservation de l’environnement par les consommateurs et producteurs, les méthodes de cultures actuelles ont tendances à s’orienter vers celles de l’agroécologie et l’usage de l’ingénierie écologique au service de la santé des plantes. En verger, la gestion des communautés de plantes des couvre-sols donne de bons résultats dans la lutte conte certaines espèces de ravageurs, tels que les arthropodes herbivores, mais les effets sur les communautés de microorganismes pathogènes restent inexplorés. Les plantes couvre-sol et adventices des vergers hébergent d’abondantes communautés de P. syringae, cependant le rôle de ces couverts végétaux dans l’émergence des maladies des arbres fruitiers reste incompris. Par conséquent, les travaux de recherche présentés ici focalisent sur l’étude simultanée des communautés de P. syringae associées aux plantes couvre-sol et aux arbres fruitiers de trois vergers d’abricotiers et de quatre vergers de kiwis du département de la Drôme, sud-est de la France, choisis pour leur état sanitaire (sain, malade ou émergence de la maladie), ainsi que des pratiques de gestion du couvre-sol différentes (sol nu, enherbement des inter-rangs, enherbement des inter rangs et rangs des arbres). En l’absence d’outils permettant une identification rapide et une affiliation à l’un des 13 phylogroupes actuellement décris pour l’espèce P. syringae, l’étude du génome complet d’une cinquantaine de souches de P. syringae a permis la mise au point de marqueurs moléculaires capables d’identifier 9 des 13 phylogroupes. L’étude des communautés de P. syringae hébergées par les couvre-sols végétaux montre un effet de la composition des communautés des plantes couvre-sol sur l’abondance et la structure des communautés de P. syringae. La présence de Prunella vulgaris, une plante de la famille des Lamiaceae, est corrélée avec une diminution de l’abondance des P. syringae. La reproductibilité de ce résultat est actuellement en cours d’investigation dans une parcelle expérimentale. Cependant, les résultats préliminaires montrent une absence d’effet de P. vulgaris sur l’abondance de P. syringae. L’étude simultanée des communautés de P. syringae des couvre-sols végétaux et des arbres fruitiers montre que des échanges se font entre les deux compartiments en raison de la présence de souches génétiquement proches. Chez le kiwi, lorsque Psa est présent il coexiste toujours avec d’autres P. syringae, soulevant la question des interactions entre ces souches et leur rôle dans l’émergence de la maladie. Enfin, les résultats mettent en avant un potentiel antagonisme entre les phylogroupes 1 et 2., Identification of reservoirs and inoculum sources of plant pathogenic microorganisms is a major issue in plant pathology. Perennial agricultural systems, such as orchards, are exposed to many pests and pathogenic microorganisms. P. syringae, a phytopathogenic bacterium responsible for the emergence of diseases of fruit trees, including the recent outbreak of bacterial canker of kiwifruit caused P. syringae pv. actinidiae (Psa), represent an important economic issue worldwide. In France, means of control of bacterial canker consist of copper treatments and preventive measures in order to reduce the spread of bacteria within and between orchards. With the awareness for environmental conservation by consumers and producers, current cultivation methods tend to be progressively replaced by more agroecological ones and the use of ecological engineering to improve plant health. Ecological engineering of orchard ground cover plant communities provides good results for the control of orchard pests, such as herbivorous arthropods, but the effects on pathogenic microbial communities remains unexplored. The ground cover plants and orchard weeds host abundant P. syringae communities, however the role of ground covers in the emergence of fruit tree diseases remains ignored. Therefore, the research presented here is focused on the simultaneous study of P. syringae communities associated with ground covers and fruit trees from three apricot and four kiwifruit orchards of Drôme county, southeastern France, chosen for their health status (healthy, diseased, or disease emergence), as well as different ground cover management practices (bare soil, ground cover in inter-rows, ground cover in inter-rows and tree rows). In the absence of tools for rapid identification and affiliation to one of 13 currently described phylogroups for the P. syringae species, the screening of whole genomes of more than fifty P. syringae strains has allowed the development of specific molecular markers able to identify 9 of the 13 phylogroups. Results show that ground cover P. syringae community abundances and structures are correlated to plant community composition. The presence of Prunella vulgaris, a plant of the Lamiaceae family, is correlated to a decrease in the P. syringae abundances. Reproducibility of this result is currently under investigation in an experimental field. However, preliminary results from the experimental field show that the presence of P. vulgaris in 1-year-old ground covers is not correlated to a decrease in P. syringae abundances. Simultaneous study of ground cover and fruit tree P. syringae communities highlight bacterial exchanges between these two compartments because of the presence of genetically correlated strains in both of them. When present, Psa coexist with other P. syringae, raising the question of the interaction between these strains and their role in the emergence of the disease. Finally, the results highlight a potential antagonism between phylogroups 1 and 2.

National audience; Blights and cankers of fruit trees caused by Pseudomonas syringae generate significant economic losses worldwide. Given the epiphytic lifestyle of P. syringae orchard ground covers represent a potential source of inoculum of P. syringae for fruit trees. They also represent a reservoir of bacterial diversity that could be beneficial for plant health via natural antagonistic and competitive interactions within the microbial flora. This microbial reservoir could be exploited for the development of ecological engineering practices, specifically the management of ground covers to reduce their negative impact on plant health and in particular as a source of pathogenic strains of P. syringae. Within the species P. syringae there is a very high genetic variability, and strains are distributed into 13 phylogroups (Berge et al., 2014) with very marked differences in pathogenicity among some of these phylogroups. Until now the only method for phylogenetic identification was the sequencing of specific and conserved genes. We have now replaced this with a PCR-based method allowing fast and accurate identification of 9 of the 13 current phylogroups (Borschinger et al., submitted). Here we will present the results of an exploratory study of P. syringae populations hosted by different compartments from 4 kiwifruit and 3 apricot orchards located in the southwest of France. The analysis of the phylogenetic structure of the P. syringae populations isolated from samples of ground cover, buds, leaves, twigs, irrigation water and soil, indicates a very large inter- and intra-orchard variability. Populations are largely constituted by an assembly of strains belonging to different phylogroups. Similarities between the phylogenetic structures of populations, and genetic similarities between strains, from different samples suggests a connection between these different compartments.

Le groupe des Pseudomonas syringae est très divers, au niveau phénotypique, génétique et de ses habitats. Récemment, 13 phylogroupes et 23 clades qui prennent en compte la diversité génétique et celle des habitats de cette bactérie ubiquiste ont été décrits (Berge et al., 2014). Des outils moléculaires permettant d’identifier les P. syringae et leur phylogroupes ont été développés au laboratoire (Borschinger et al., soumis), et nous permettent de travailler sur de relativement grands effectifs d’isolats bactériens. Avec ces outils, en réalisant nous-même des prélèvements dans des vergers ciblés en 2014 et 2015, et en collaboration avec le CTIFL qui a réalisé deux importantes prospections en 2011-2012, nous avons exploré la diversité des P. syringae rencontrés dans 36 vergers d’abricotiers de la vallée du Rhône, où le chancre bactérien était présent, et ce dans différents compartiments : le sol, le couvre-sol, les débris de culture, la plante (greffon) à différents stades de son développement, les racines (porte-greffe), la plante avec ou sans symptômes déclarées. Les résultats obtenus montrent que, parmi les 2436 isolats collectés, les isolats potentiellement pathogènes proviennent de 4 phylogroupes différents qui montrent une grande diversité; par ailleurs, certains phylogroupes sont ubiquistes et d’autres spécialisés. Le sol, le couvre-sol, les débris de culture, la plante (avec ou sans symptômes visibles) sont des réservoirs potentiels de la maladie. Les conséquences en termes d’étiologie et détection du chancre bactérien de l’abricotier sont discutées.

Blights and cankers of fruit trees caused by Pseudomonas syringae generate significant economic losses worldwide. Given the epiphytic lifestyle of P. syringae orchard ground covers represent a potential source of inoculum of P. syringae for fruit trees. They also represent a reservoir of bacterial diversity that could be beneficial for plant health via natural antagonistic and competitive interactions within the microbial flora. This microbial reservoir could be exploited for the development of ecological engineering practices, specifically the management of ground covers to reduce their negative impact on plant health and in particular as a source of pathogenic strains of P. syringae. Within the species P. syringae there is a very high genetic variability, and strains are distributed into 13 phylogroups (Berge et al., 2014) with very marked differences in pathogenicity among some of these phylogroups. Until now the only method for phylogenetic identification was the sequencing of specific and conserved genes. We have now replaced this with a PCR-based method allowing fast and accurate identification of 9 of the 13 current phylogroups (Borschinger et al., submitted). Here we will present the results of an exploratory study of P. syringae populations hosted by different compartments from 4 kiwifruit and 3 apricot orchards located in the southwest of France. The analysis of the phylogenetic structure of the P. syringae populations isolated from samples of ground cover, buds, leaves, twigs, irrigation water and soil, indicates a very large inter- and intra-orchard variability. Populations are largely constituted by an assembly of strains belonging to different phylogroups. Similarities between the phylogenetic structures of populations, and genetic similarities between strains, from different samples suggests a connection between these different compartments.

The phyllosphere is known for hosting a great diversity of fungi, yeasts and bacteria. These microorganisms interact with each other and with the host plant in the form of symbiosis, mutualism, commensalism, parasitism, competition or simply neutralism. Pseudomonas syringae is a ubiquitous epiphytic bacterium commonly found in these microbial communities. The phylogeny complex of P. syringae comprises 13 phylogroups, containing strains that are well-known pathogens and strains that apparently have limited capacity as pathogens. Emblematic among the pathogens are P. syringae pv. syringae (Pss) and P. syringae pv. actinidiae (Psa) belonging respectively to phylogroups 2 and 1. Bacterial blights of fruit trees caused by P. syringae lead to significant economic losses worldwide. With the expansion of bacterial blight of kiwifruit caused by P. syringae pv. actinidiae and bacterial blight of apricot caused by P. syringae pv. syringae, the identification of reservoirs of these pathogenic strains and an understanding of the role of the accompanying diversity of P. syringae in disease epidemiology are needed. Here we describe the structure of P. syringae populations on orchard ground covers based on a series of PCR that specifically identify most of the phylogroups of this species. This efficient technique allows us to link population structure on ground covers to those associated with trees and to the botanical diversity and location of the ground cover reservoirs. The overall goal of these analyses are to identify microbiological contexts that are unfavorable for disease and to conceive management strategies of ground covers to foster these microbiological contexts.

The behaviour of the sporulating soil-dwellingBacillus cereus sensu lato(B. cereus sl) which includes foodborne pathogenic strains has been extensively studied in relation to its various animal hosts. The aim of this environmental study was to investigate the water compartments (rain and soil water, as well as groundwater) closely linked to the primaryB. cereus slreservoir, for which available data are limited.B. cereus slwas present, primarily as spores, in all of the tested compartments of an agricultural site, including water from rain to groundwater through soil. During rain events, leachates collected after transfer through the soil eventually reached the groundwater and were loaded withB. cereus sl. In groundwater samples, newly introduced spores of aB. cereusmodel strain were able to germinate, and vegetative cells arising from this event were detected for up to 50 days. This firstB. cereus slinvestigation in the various types of interrelated environments suggests that the consideration of the aquatic compartment linked to soil and to climatic events should provide a better understanding ofB. cereus slecology and thus be relevant for a more accurate risk assessment of food poisoning caused byB. cereus slpathogenic strains.

Bacterial cankers of perennial trees caused by Pseudomonas syringae provoke serious economic losses all around the world, and also affect tree species in the genus Prunus. When pedo-climatic conditions are favorable, the disease can severely affect apricot (Prunus armeniaca).The epidemiology of this complex disease is not completely understood. Based on recent work on the presence of P. syringae in very diverse non-agricultural habitats, the complexity of the phylogeny of P. syringae, and the existence of phylogroups containing more aggressive strains than others, the scope of this work was the identification of the potential reservoirs of pathogenic strains of P. syringae in Prunus orchards. We addressed the following questions: What is the structure of the P. syringae population in the ground-cover, in the soil, in the irrigation water, and in the plant (epiphytic and endogenous)? What are the relationships between these populations and the populations in non-agricultural habitats? What are the factors that trigger disease when pathogenic populations are present? To answer these questions, a specific molecular detection method, by PCR, allowing rapid and accurate identification of the different phylogroups of P. syringae was developed. With this method, the different potential reservoirs: plants (buds, leaves, twigs), cover-grass, soil, and water, were investigated in 2014 in 3 different French orchards of apricot, at 3 different periods of the plant’s vegetative development. Leaf litter was collected and analyzed during winter. Preliminary results showed different structuration of the populations according to their origin. For example, in March, the abundance of isolates of phylogroup 1 (including P. syringae morsprunorum race 2) and phylogroup 2 (including P. syringae pv syringae) was different in plant, soil and ground-cover populations.

National audience

International audience; The deployment of resistant crops often leads to the emergence of resistance-breaking pathogens that suppress the yield benefit provided by the resistance. Here, we theoretically explored how farmers' main leverages (resistant cultivar choice, resistance deployment strategy, landscape planning and cultural practices) can be best combined to achieve resistance durability while minimizing yield losses as a result of plant viruses. Assuming a gene-for-gene type of interaction, virus epidemics are modelled in a landscape composed of a mosaic of resistant and susceptible fields, subjected to seasonality, and a reservoir hosting viruses year-round. The model links the genetic and the epidemiological processes, shaping at nested scales the demogenetic dynamics of viruses. The choice of the resistance gene (characterized by the equilibrium frequency of the resistance-breaking virus at mutation-selection balance in a susceptible plant) is the most influential leverage of action. Our results showed that optimal strategies of resistance deployment range from 'mixture' (where susceptible and resistant cultivars coexist) to 'pure' strategies (with only resistant cultivar) depending on the resistance characteristics and the epidemiological context (epidemic incidence and landscape connectivity). We demonstrate and discuss gaps concerning virus epidemiology across the agro-ecological interface that must be filled to achieve sustainable disease management.

Caractériser la dissémination des bio-agresseurs est un enjeu majeur pour la gestion et la prédiction des maladies en santé des plantes. Face aux limites des approches usuelles en pathologie végétale, une nouvelle vision a été proposée abordant les paradigmes d’histoire de vie des agents phytopathogènes en dehors des limites du système hôte-pathogène. Parmi ces agents phytopathogènes, les études sur P. syringae sont celles qui ont contribuées le plus à ce nouveau courant de pensée et dont on connaît le mieux l’histoire de vie en relation avec ses réservoirs « non hôtes ». L’espèce est détectée dans de nombreux compartiments du cycle de l’eau, des précipitations jusqu’aux rivières et eaux d’irrigation, en passant par les plantes sauvages et le manteau neigeux. L’ensemble de ces observations a soulevé de nouvelles questions sur la manière dont P. syringae se dissémine au travers de ces environnements et sur les processus impactant sur la dynamique des populations à l’échelle d’un bassin versant. Ces recherches se sont donc intéressées à ces processus des précipitations jusqu’aux cours d’eau alpins dans l’optique d’acquérir des données pour la modélisation des flux de P. syringae. Elles ont mis en évidence les populations résidentes de la litière et sa survie dans le sol, processus jamais identifiés à l’histoire de vie de P. syringae. Elles ont également caractérisé (i) les conditions propices à son transport via les précipitations, (ii) le rôle du manteau neigeux comme réservoir et protecteur des populations des prairies alpines et (iii) ont mis en évidence la chimie de l’eau comme indicateur témoin de la dynamique des populations des rivières. Ces observations suggérant un transport de P. syringae dans le sol, nous l’avons quantifiée à travers des études de terrain et des simulations en laboratoire. Enfin, l’ensemble des données de ces recherches couplées à des outils SIG et des modèles météorologiques et hydrologiques ont permis de proposé un modèle sur les flux de P. syringae des habitats naturels vers les agro-systèmes., The characterization of the spread of bio-agressors spread is a major issue for the management of plant health and the prediction of disease emergence. Given the limitations of conventional approaches in plant pathology, a new vision has been proposed addressing paradigms life history of plant pathogens outside the limits of the crop host-pathogen system. Among the plant pathogens, studies on P. syringae are those that have contributed the most to this new way of thought for which life history in relation to "non host" reservoirs has been highlighted. The species is found in many compartments of the water cycle, from precipitation to rivers and irrigation water, wild plants and snowpack. All these observations have raised new questions about how P. syringae spreads through these environments and on the processes impacting population dynamics at the scale of a watershed. This research was therefore interested in these processes with the objective to acquire data for modeling the tranfer of P. syringae through the watershed. They highlighted the resident populations of litter and their survival in the soil, processes never identified in association with the life history of P. syringae. They also revealed (i) the conditions for transport via precipitations, (ii) the role of snowpack as a reservoir and protector of the populations in alpine meadows and (iii) showed that water chemistry can be used as an indicator of the population dynamics in headwaters. These observations suggested a transport of P. syringae via the soil that we subsequently characterized through field studies and laboratory simulations. Finally, all data from this research combined with GIS tools and meteorological and hydrological models have permitted us to propose a model of the flux of P. syringae of natural habitats to agricultural systems.

Récemment, le laboratoire a mis en évidence l'ubiquité et la diversité de la bactérie phytopathogène Pseudomonas syringae dans les eaux de sources et des expériences préliminaires suggéraient son transport à travers le sol. Afin de mieux prédire la dynamique des populations dans les réseaux hydrologiques, nous avons estimé le transfert de plusieurs souches en conditions abiotiques dans des colonnes de sable, puis dans des colonnes de sol prélevées dans des prairies alpines. Le taux de transfert de P. syringae en milieu poreux dépendait de la souche et de la force ionique de la solution mais pas de ses propriétés physiques. Ce taux était comparable à ceux observés avec les colonnes de sol et supérieur à celui des bactéries totales. Enfin le sol constituait un réservoir de P. syringae dans lequel les populations ont pu survivre plusieurs mois et dont la taille des populations (102 à 105 cfu 1 L) correspondait à la gamme trouvée dans les eaux de sources. Nous avons donc pu formellement prouver le passage à travers le sol de cette espèce.