A polyphasic approach for dereplication and characterization of wood endophytic bacteria with bio-protective potential

Microbial biocontrol is a biological strategy receiving increasing interest as a sustainable innovation, useful for managing diseases and spoilage arising from undesired microbial contaminations in foodstuff. The work hereby presented was carried out within a research project focusing on the exploration of the bio-protective potential of some bacterial isolates, mainly wood-endophytic Actinobacteria, against Botrytis cinerea, in the context of post- harvest biocontrol on fruits. For this purpose, the identification, characterization, and proper description of the genetic features of the recovered microbial species is also essential. The aim of this work was to evaluate the discriminatory power of a tailored method, combining 16S rDNA sequencing and Rep-PCR, to determine the isolates differentiation and their identification at genus level or below. More than fifty isolates were recovered from wood of different grapevine cultivars and cultured using appropriate growth media (CYA). The identification was firstly undertaken among complete and 16S-rDNA gene sequencing. The obtained sequences were compared against a proofread, robust and previously manually curated database through BLASTn algorithm. All the isolates were identified at the genus level, with more than 70% reaching the species level. As most taxa belonged to the Actinobacteria phylum, Gram-positive and GC rich, the Rep-PCR was chosen as molecular technique to dereplicate the isolates taking advantage from its well-known power to discriminate members of this taxon using the amplification of conserved sequences distributed throughout the bacterial genome, using specific primers such as GTG5, BOX-A1R and REP. In this work, we have shown how a polyphasic approach was useful for the correct characterization of our collection. In this way, we were able to obtain a good dereplication for endophytic bacteria, together with a reliable genetic identification, both essential preconditions to make a correct assessment of bio-protection.