Role of hydrolases in the biocontrol of postharvest fungal pathogens by yeast antagonists

Most phytopathogenic fungi have cell walls composed of complex polymers of β-1,3- and β-1,6-glucans, mannoproteins, as well as some chitin. Different yeast – including strains of Pichia guilliermondii, Metschnikowia pulcherrima, M. fructicola and Aureobasidium pullulans – have been isolated from the carposphere of fruit, selected for their antagonistic properties against different postharvest pathogens of apple and peaches, and studied for their biocontrol mechanism. Production of hydrolases is one of the components of the mechanism of action: enzymatic assays permitted to evidence exo-1,3-beta-glucanase, chitinase and alkaline protease activities. The extracted crude enzymes produced by different antagonists showed a high activity in inhibiting the growth of the pathogens in vitro. The exo-1,3-beta-glucanase gene PgExg1 was amplified from the genomic DNA of P. guilliermondii M8. Similarly, two chitinase genes, MpChi1 and MfChi1, were amplified from the genomic DNA of M. pulcherrima strain MACH1 and M. fructicola strain AP47. An alkaline protease gene ALP5 was amplified respectively from the genomic DNA of A. pullulans PL5. The expression of ALP5 in Escherichia coli and Pichia pastoris, followed by analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolased the substrate casein, inhibited the mycelial growth of the pathogens and the development of postharvest rots on apples. Our results showed that chitinase, glucanase and protease are involved in the biocontrol activities of yeast antagonists.