Caractérisation et exploitation du microbiome de l'algue Haematococcus spp. : vers le biocontrôle du champignon pathogène Paraphysoderma sedebokerense

Haematococcus spp. is a freshwater green microalga cultivated worldwide to produce astaxanthin, a high value antioxidant and pigment used in nutraceutical and feed industries. The Haematococcus spp. large-scale production is threatened by the fungal pathogen Paraphysoderma sedebokerense (Blastocladiomycota). The complex life cycle of this fungus and its resistance to most disinfection methods result in a difficult control of the infection. Like for plants, there is increasing evidence that the algal microbiota plays a role for host health, including defence against pathogens. Initial lines of evidence - i.e. addition of antibiotics to Haematococcus spp. cultures - led us to formulate the hypothesis that, likewise, the bacterial microbiota of Haematococcus spp. interacts directly or indirectly with P. sedebokerense and thus might be engineered to prevent or limit infection. The aim of this work was to investigate the interactions in the tripartite "alga - microbiota - fungal pathogen" consortium, with the objective to identify bacteria that might be used for the biocontrol of the fungus. In an attempt to develop a quantitative and reproducible fungal inoculation protocol, we found that fungal propagules are heterogeneous morphologically, providing novel insights into the fungal infection capacity at different stages of its life cycle. The development of a high throughput cell classification method based on a digital slide scan combined with microscopy allowed us to qualitatively and quantitatively assess the outcome of the infection. Using this, we show that there is a heterogenous, non-random distribution of fungal cells on Haematococcus spp. Moreover, we describe a new infection pattern with P. sedebokerense cells located inside Haematococcus spp. cells, suggesting a novel endobiotic infection stage. In parallel, to understand the role of the bacteria during the infection, we analysed the bacterial communities associated to six laboratory-cultured Haematococcus spp. strains upon infection by P. sedebokerense, and isolated bacteria associated to a collection of 44 Haematococcus spp. strains. The metagenomic analysis revealed that the Haematococcus spp. microbiota clusters into reduced microbiota types, compared to “wild” microbiota, that remained remarkably stable both taxonomically and functionally upon infection. We show that P. sedebokerense is unable to infect axenic Haematococcus spp. strains, and that the addition of bacteria isolated from the algal microbiota, at least partially, restored the pathogenicity of the fungus. Overall, this work provides novel insights into the interaction of Haematococcus spp. and P. sedebokerense, showing heterogeneity in the type of fungal propagules and in the infection outcome. The combination of our methods optimised for reproductible and quantifiable infection assays, with the novel knowledge about the composition and functions of the Haematococcus spp. microbiota, provides bases to further investigate the interactions within the consortium and to design synthetic communities in a biocontrol perspective