Biocontrol activity of a novel endochitinase from Metschnikowia fructicola expressed in Pichia pastoris against postharvest pathogens of fruits

Metschnikowia fructicola strain AP47 was isolated from the carposphere of temperate fruits and showed a high efficacy in controlling Monilinia spp. on stone fruits, however its mechanism against postharvest pathogens is still unclear. M. fructicola AP47 was able to produce high chitinase activity in the presence of Monilinia spp. cell wall. A novel endochitinase gene MfChi (GenBank n° HQ113461) was amplified from the DNA and cDNA of M. fructicola AP47. An open reading frame of 1,098 bp encoding a 365 amino acid protein with a calculated molecular weight of 40.9 kDa and a predicted pI of 5.27 was characterized. Mfchi gene expression was significantly elevated in response to Monilinia spp. cell wall, suggesting MfChi chitinase role in the antagonistic activity of the yeast. Therefore, MfChi gene was transferred into Pichia pastoris KM71 for high heterologous expression. The antifungal activity of the recombinant chitinase was investigated against Monilinia fructicola and Monilinia laxa in vitro and on peaches cv. Redhaven. The recombinant chitinase MfChi reduced significantly spore germination and germ tube length of the tested pathogens in PDB medium and successfully reduced rot diameter on peaches. However, the efficacy was dependent on the enzyme concentration and the length of fruit storage.This study provided the direct evidence that extracellular chitinase Mfchi secreted by Metschnikowia fructicola AP47 plays an important role in the biocontrol activity against some postharvest pathogens of peaches, therefore MfChi could be developed as an efficient postharvest treatment with antimicrobial activity for fruits undergoing a short shelf life.