Insights gained from metagenomic shotgun sequencing of apple fruit epiphytic microbiota

The epiphytic plant microbial communities living at the surface of fruit have been the source of most current biocontrol agents (BCAs) and can influence fruit quality during storage. Despite this interest, their taxonomical and functional composition has been poorly studied so far. This paper describes the use of high-throughput sequencing (HTS) technologies to characterise the microbial phytobiome residing on apple surface at the taxonomic and functional levels through shotgun metagenome sequencing. Apples from the Pinova cultivar bearing no symptom of disease development were sampled in an orchard at harvest, and their epiphytic microbiota was isolated. After DNA extraction, 14.1 Gbases of raw sequences were generated by HTS. These sequences were annotated following two pipelines in parallel: (i) they were individually analysed by the MG-RAST server, and (ii) they were de novo assembled into contigs and the contigs were annotated by the IMG server. Our results showed a very high fungal and bacterial diversity, with a higher proportion of fungal sequences (79.0%) than bacterial sequences (13.8%). Among fungi, the phylum Ascomycota prevailed, while Bacteroides were dominant in the bacterial population. Among them, 24 species corresponded to known apple pathogens like Aspergillus spp., Botrytis spp., Sclerotinia spp., and Penicillium spp. for fungi, and Erwinia spp. and Agrobacterium spp. for bacteria. Moreover, several contigs were assigned to species of known BCA strains belonging to the following genera: Filobasidiella spp., Talaromyces spp, Candida spp., Saccharomyces spp., Bacillus spp., and Enterobacter spp. The functional analysis showed similar patterns of abundance and function in all samples, identified genes potentially involved in biocontrol properties, but also underlined the complexity of datum interpretation and the incompleteness of current databases.