A molecular method for rapid detection of a Metschnikowia pulcherrima strain during grapevine biocontrol in industrial withering conditions

Microbial biocontrol is a biological strategy receiving increasing interest as a sustainable innovation, effective for preventing and managing crop diseases and food spoilage arising from microbial contamination. The work hereby presented was carried out within a research project focusing on bio- protection against Botrytis cinerea, in the context of post-harvest biocontrol on fruits. Among biocontrol agents, non-Saccharomyces yeasts have recently gained attention in the wine industry for their ability to enhance organoleptic characteristics and, at the same time, for their bioprotective activity which can reduce the need for the use of chemical preservatives. A species with great potential as a bioprotective agent is Metschnikowia pulcherrima, which was proven to inhibit the growth of spoilage microorganisms through various mechanisms. In this work, a specific strain of M. pulcherrima, previously selected for its potential to inhibit B. cinerea, was tested in post-harvest conditions. Its ability to persist throughout the withering period of grapes for Passito wine production has been investigated for the inhibition of B. cinerea in real-winemaking conditions (industrial-scale withering). In order to rapidly assess persistence during withering via PCR, a pair of strain-specific primers was designed to differentiate the experimental strain from other strains of Metschnikowia, possibly present in the environment since the M. pulcherrima clade is common in the winemaking environment. The proposed molecular method, indeed, consists of a rapid technique based on an optimized protocol of colony-PCR with primers designed through in silico comparison of different nucleotide sequences of a specific region that allows differentiation with the type strain of the same species and other relevant strains of closely related species due to a SNP. The application of the strain-specific PCR showed that the strain applied persists on grapes throughout all the withering period, moreover its ability to inhibit B. cinerea was confirmed compared with the untreated control.