Study Of Biocontrol Mechanisms On Pichia Anomala SRF After Picking Apples By Penicillium Expansum

Blue mold caused by Penicillium expansum is the first major disease of pome fruits,which seriously affects market value of fruits. Application of microbial antagonists to controlpostharvest blue mold has become one of promising alternatives to chemical fungicide,because microbial antagonists are able to effectively control the rot of fruit, and it reduces theamount of chemical fungicides.What is more,it increases food security and reduces thepotential environmental hazards. However, the biocontrol mechanism of antagonistic yeastsagainst pathogens is still unclear, even if many research has been made on revealing thepossible modes on action of antagonists against pathogens. This is mainly hampered by thelack of some efficient tools to probe into the interaction of antagonists and pathogens.Understanding the modes of action of antagonistic yeast is an important prerequisite forscreening for new effective antagonists, enhancing the biocontrol activity, and developingcommercial biocontrol agents. At present, modes on action of antagonistic yeasts mainlyinclude competition, hyperparasitism and production of chitinase and glucanase. This researchis mainly focusing on1) testing the efficacy of P. anomala SRF in controlling postharvest P.expansum on apples;2) preparing the haploid of P. anomala SRF;3) Cloning and expressionof exo-glucanase gene from P. anomala SRF;4) constructing the knockout vector ofexo-glucanase gene of P. anomala. The main results obtained are as follows:(1) P. anomala SRF can effectively suppress the occurrence of posharvest blue moldcaused by P. expansum on apples; The efficacy of SRF is dependent on the applicationconcentration of the antagonist, and it is positively correlated with concentrations ofantagonistic yeast used.(2) According to reported glucanase gene of P. anomala, specific primers were designedand one exo-glucanase gene, named PaExg (GenBank: AJ222862.1), it was cloned from P.anomala SRF. Gene and amino acid sequence analysis showed that, PaExg has an openreading frame (ORF) with a length of1284bp, encoding427amino acids, molecular weightof49.469kD, and an isoelectric point of4.72. Sequence alignment and homology analysisshowed that the sequences of PaExg had a100%similarity to the exo-glucanase gene from P.anomala strain K. Conserved domain analysis showed that PaExg belongs GH15family. A PaExg prokaryotic expression vector was constructed and successfully expressed in E. coliwith IPTG induction. The transformants showed glucanase activity.(3) By optimizing the pre-incubation time, temperature, and salt concentration gradiention and culture conditions, the optimal condition for producing haploid spores by wild-typediploid cells of P. anomala SRF was determined: Grown in YPD medium24h, theantagonistic yeast was serially passaged two generations, and transferred on the best inductionmedium (1%glucose,1.4%sodium acetate,0.4%potassium2%chloride agar). Grown oninduction culture at28℃for7d, the yeast produced spores and its spore rate can reach80%.Ac-and K+played an key role in the production of spores by P. anomala SRF.(4) By restriction site analysis of the relationship between the multi-cloning sites ofPUG6and PaExg gene sequence, three pairs of primers were designed. Using PCR with thethree primers, the flanking sequences of PaExg were obtained and ligated into PUG6vector.One PaExg-knockout vector was successfully constructed.