Christel Leyronas

Compared efficiency of the isolation methods for Botrytis cinerea

The phenotypic and genotypic studies on species responsible for grey mould disease, Botrytis cinerea and Botrytis pseudocinerea, are usually carried out on purified strains. Purification is classically achieved with the isolation of a single spore. This method entails fastidious and time-consuming steps and requires a good level of experience of the manipulator. An alternative method is based on the isolation of hyphal fragments under the stereomicroscope. Both the methods are widely used, but their efficiency has never been formally quantified. The two methods were compared for the purification of mixes of strains with known genotypic profiles. To assess the efficiency of the purification, the resulting isolates were characterized with micro-satellite markers and verified that each represented a single genotype. Both the methods had equally high efficiencies, up to 93–95%. The advantage of the simplified method is that it is much less time consuming than the single spore isolation (2 vs. 19 days) and requires less technical experience.

Host phenology and geography as drivers of differentiation in generalist fungal mycoparasites

The question as to why parasites remain generalist or become specialist is a key unresolved question in evolutionary biology. Ampelomyces spp., intracellular mycoparasites of powdery mildew fungi, which are themselves plant pathogens, are a useful model for studies of this issue. Ampelomyces is used for the biological control of mildew. Differences in mycohost phenology promote temporal isolation between sympatric Ampelomyces mycoparasites. Apple powdery mildew (APM) causes spring epidemics, whereas other powdery mildew species on plants other than apple cause epidemics later in the season. This has resulted in genetic differentiation between APM and non-APM strains. It is unclear whether there is genetic differentiation between non-APM Ampelomyces lineages due to their specialization on different mycohosts. We used microsatellites to address this question and found no significant differentiation between non-APM Ampelomyces strains from different mycohosts or host plants, but strong differentiation between APM and non-APM strains. A geographical structure was revealed in both groups, with differences between European countries, demonstrating restricted dispersal at the continent scale and a high resolution for our markers. We found footprints of recombination in both groups, possibly more frequent in the APM cluster. Overall, Ampelomyces thus appears to be one of the rare genuine generalist pathogenic fungi able to parasitize multiple hosts in natural populations. It is therefore an excellent model for studying the evolution of pathogens towards a generalist rather than host-specific strategy, particularly in light of the tritrophic interaction between Ampelomyces mycoparasites, their powdery mildew fungal hosts and the mildew host plants.

First report of Sclerotinia subarctica in France detected with a rapid PCR-based test

Abstract White mould can affect the production of a wide range of economically important crops worldwide. The symptoms may be caused by several species, including Sclerotinia subarctica, a species mostly occurring in northern latitudes in sympatry with S. sclerotiorum. Although the two species are morphologically indistinguishable, S. subarctica was reported to have different climatic requirements for mycelial growth and production of apothecia. These differences may affect the precision of white mould risk prediction models that are based on the production of ascospores by S. sclerotiorum. To assess the presence of S. subarctica in France, we adapted a rapid PCR-based test to distinguish S. subarctica from other commonly found species of Sclerotinia. This test was used to characterize a collection of 969 Sclerotinia sp. isolates originating from various plants (bean, canola, carrot, lettuce, melon and witloof chicory), air and soil samples in different regions of France. One single isolate, collected from witloof chicory in northern France, was identified as S. subarctica. When genotyped with five microsatellite markers designed for S. sclerotiorum, this isolate had a haplotypic profile that was clearly distinct from the other isolates. The ITS sequence of this isolate was identical to those of isolates collected in northern Europe and Alaska. Koch’s postulates were verified. When inoculated on witloof chicory, the isolate identified as S. subarctica induced white mould symptoms. This study is the first to report the presence of S. subarctica south of the 51st parallel north and on witloof chicory.

Assessing the Aerial Interconnectivity of Distant Reservoirs of Sclerotinia sclerotiorum

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 more efficiently manage 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 mold 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 four 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 (PIC) 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.

COMPARED DYNAMICS OF GREY MOULD INCIDENCE AND GENETIC CHARACTERISTICS OF BOTRYTIS CINEREA IN NEIGHBOURING VEGETABLE GREENHOUSES

Production of vegetables in southern France often relies on groups of greenhouses located in close vicinity. These crops are commonly affected by grey mould caused by Botrytis cinerea, a fungus known for its ability to produce abundant air-borne inoculum. Possible exchange of inoculum could affect the epidemics developing in neighbouring greenhouses. To test this hypothesis, grey mould incidence was assessed in four successive crops in six unheated polyethylene tunnels located in the Avignon area. On lettuce, the incidence was similar for a given harvest date in two tunnels where this crop was grown four times consecutively. In the four other tunnels, lettuce was grown in alternation with tomato. No grey mould developed on tomato and disease incidence was low on lettuce. One hundred and seventy four strains collected from lettuce in two tunnels were investigated for their genetic diversity, genetic structure and their mating type. Both known mating types of B. cinerea were observed in the tunnels but MAT1-1 was prevalent. The gene diversity of B. cinerea strains was similar in both tunnels. However haplotypic diversity and linkage disequilibrium were substantially higher in one tunnel. We hypothesize that this situation is related to differences in microclimatic conditions in the tunnels. It highlights a possible interest in individualizing disease management in the different tunnels of a given farm.