Endophytes and epiphytes from the grapevine leaf microbiome as potential biocontrol agents against phytopathogens

Plants harbor diverse microbial communities that colonize both below-ground and above-ground organs. Some bacterial members of these rhizosphere and phyllosphere microbial communities have been shown to contribute to plant defenses against pathogens. In this study, we characterize the pathogen-inhibiting potential of 78 bacterial isolates retrieved from endophytic and epiphytic communities living in the leaves of three grapevine cultivars. We selected two economically relevant pathogens, the fungus Botrytis cinerea causing gray mold and the oomycete Phytophthora infestans, which we used as a surrogate for Plasmopara viticola causing downy mildew. Our results showed that epiphytic isolates were phylogenetically more diverse than endophytic isolates, the latter mostly consisting of Bacillus and Staphylococcus strains, but that mycelial inhibition of both pathogens through bacterial diffusible metabolites was more widespread among endophytes than among epiphytes. Six closely related Bacillus strains induced strong inhibition (<60%) of Botrytis cinerea mycelial growth. Among these, five led to significant perturbation in spore germination, ranging from full inhibition to reduction in germination rate and germ tube length. Different types of spore developmental anomalies were observed for different strains, suggesting multiple active compounds with different modes of action on this pathogen. Compared with B. cinerea, the oomycete P. infestans was inhibited in its mycelial growth by a higher number and more diverse group of isolates, including many Bacillus but also Variovorax, Pantoea, Staphylococcus, Herbaspirillum, or Sphingomonas strains. Beyond mycelial growth, both zoospore and sporangia germination were strongly perturbed upon exposure to cells or cell-free filtrates of selected isolates. Moreover, three strains (all epiphytes) inhibited the pathogen’s growth via the emission of volatile compounds. The comparison of the volatile profiles of two of these active strains with those of two phylogenetically closely related, inactive strains led to the identification of molecules possibly involved in the observed volatile-mediated pathogen growth inhibition, including trimethylpyrazine, dihydrochalcone, and L-dihydroxanthurenic acid. This work demonstrates that grapevine leaves are a rich source of bacterial antagonists with strong inhibition potential against two pathogens of high economical relevance. It further suggests that combining diffusible metabolite-secreting endophytes with volatile-emitting epiphytes might be a promising multi-layer strategy for biological control of above-ground pathogens

Microbial life in the grapevine: what can we expect from the leaf microbiome?

Recent studies have shown that plants harbor complex bacterial communities, the so- called “microbiome”. We are only beginning to unravel the origin of these bacterial plant inhabitants, their community structure and their roles, which, in analogy to the gut microbiome, are likely to be of essential nature. The aim of this work was to analyze the abundance and diversity of the cultivable members of the bacterial microbiome living in and on the leaves of grapevine, and to identify microbiome members with putative plant-protective activities against phytopathogenic organisms.Methods and results: Grapevine leaves were sampled three times during the growing season at one location and used to quantify the abundance and isolate representative members of the epiphytic and endophytic bacterial communities. Results were compared for three grapevine cultivars: Pinot noir, Chasselas and Solaris. Greater bacterial abundance and diversity was observed among epiphytes than among endophytes. Leaf imprints revealed a higher colonization density for the upper than for the lower surface of leaves. A high relative frequency of strains belonging to the genera Staphylococcus and Bacillus was observed, especially in the endophytic communities.Conclusions: The first results of this ongoing study led to the conclusion that epiphytic bacteria of the cultivable grapevine microbiome were more abundant and diverse than endophytic bacteria. A tendency towards more abundant bacteria in the resistant variety Solaris than in the susceptible varieties Chasselas and Pinot noir contrasted with a higher diversity of epiphytic bacteria in the Pinot noir variety. A trend for higher frequency of strains showing antagonistic activity towards the grapevine pathogen Botrytis cinerea among isolates from the resistant variety Solaris could indicate a putative contribution of microbiome bacteria to this resistant phenotype, even though this remains to be confirmed.Significance and impact of the study: This study constitutes a first step in characterizing the endo- and epiphytic cultivable bacterial communities of three grapevine varieties grown at the same location. Further, presently ongoing studies shall reveal i) the true complexity of these leaf-associated communities through cultivation-independent, next generation sequencing techniques, and ii) their potential as a source of biofungicidal agents. In this last point, the contribution of the emission of volatile organic compounds (VOCs) as a newly discovered type of bacterial weapon against fungal and oomycetal pathogens shall be quantified