Study of the implication of exo-beta-1,3-glucanase encoding genes in the biocontrol activity of Pichia anomala (Hansen) Kurtzman (strain K) against Botrytis cinerea on post-harvest apples

The yeast Pichia anomala strain K protects post-harvest apples against Botrytis cinema. Two genes encoding exo-beta-1,3-glucanases, PaEXGl and PaEXG2 have been isolated from strain K genome thanks to conserved regions in fungal exo-beta-1,3-glucanases and to the sequencing of PaExg2, an exo-beta-1,3-glucanase from strain K culture fluids. PaEXGl potentially encodes a 498 amino acids long protein, with a calculated molecular weight of 58 kDa, an estimated pI of 4.79 and 8 potential sites for N-linked glycosylation. PaEXG2 has a coding capacity for an acidic protein of 427 amino acids with a predicted molecular weight of 49 kDa, a calculated pI of 4.70 and one potential N-glycosylation site. Haploid uracil auxotrophic mutants derived from strain K showed an inferior biocontrol effect and colonization of apple wounded sites compared to the prototrophic strain. Antagonism and colonization were recovered after restoration of the prototrophy by transformation with the URA3 gene encoding orotidine monophosphate decarboxylase. PaEXGl and PaEXG2 genes were separately disrupted by the insertion of the URA3 marker gene in their ORFs. Integrative transformation was shown to be mostly ectopic in strain K descendants (only 4/ of integration by homologous recombination). Disruption of PaEXGl or PaEXG2 had no visible effect on in vitro growth with B. cinerea cell walls preparation as the sole carbon source or in apple wounds. PaEXGl disruption had no effect on the in vitro or in situ extracellular exo-beta-l,3-glucanase activity production while PaEXG2 disruption abolished all detectable activity in vitro and in vivo. Disruption of PaEXGl or PaEXG2 did not affect the biocontrol towards B. cinerea on wounded apples, showing that the products of those genes are dispensable for protective activity (in singly disrupted mutants).