Identification And Biocontrol Efficacy Assessment Of Yeast Strain Against Colletotrichum Gloeosporioides Of Mango Fruit And Its Stress Resistance Improvement

Anthracnose, caused by Colletotrichum gloeosporioides(Penz.)Penz.&Sacc, is the major postharvest disease of mango in all mango producing areas of the world.The disease occurs as quiescent infections on immature fruit and the damage it incites is more important during transportation and storage.Currently,the main preventive approaches to reduce postharvest losses from anthracnose are chemical fungicides. Chemical fungicides not only cause health problems, but also result in pathogen resistance. Therefore, it is urgently needed to find alternative stategies for control of postharvest decay of fruit. The use of microbial antagonists to control postharvest diseases of fruits have been regarded as one of the potential safe alternatives to fungicides. Recently, minimal amounts of information about the application of antagonistic yeasts in controlling postharvest anthracnose of mango fruit have been reported,but these antagonism yeast mostly isolated from nontropical region fruit, the ability to adapt to the tropical climate and the control effect are not satisfactory.The purpose of this research is to screen effective antagonistic yeasts from surface and wound of various fruit in Hainan province by in vitro and in vivo methods, and identification were conducted on the selected antagonistic yeast against postharvest anthracnose decay of mango fruit, as well as possible antagonistic mechanisms of the yeast against were studied. In addition, the biocontrol efficacy of the yeast against combining with additives, and the method of stress resistance improvement to enhance the biocontrol ability of the yeast against were also investigated. The results of the experiments were drawn as following:1、130strains of yeasts which isolated from the surface and wound of various fruit in Hainan province were screened for antagonistic activity against postharvest anthracnose of mango fruit caused by C.gloeosporioides.4strains of yeasts screened through the in vitro and in vivo method showed that had stronger antagonistic action on postharvest anthracnose decay of mango fruit. Among them,the yeast strain (number LZ5)which had the best biocontrol efficacy against postharvest anthracnose of mango fruit was identified as Meyerozyma guilliermondii LZ5by morphology,physiology,and molecular biology (26S rRNA D1/D2) method.The inhibition zone reached13.2mm under in vitro condition and the disease incidence and lesion diameter of fruit in vivo only reached60%and4.27mm, respectively.2、Spore germination and germ tube elongation of C.gloeosporioides in PDB were significantly controlled by unwashed cell culture mixture and washed cell suspension of the yeast at1×108CFU/ml, while autoclaved culture and culture filtrate had no significantly inhibitory effect on C.gloeosporioides. Results of scanning electron microscopy showed that growth of the pathogens was significantly inhibited by M.guilliermondii LZ5in the wounds of fruit. The concentrations of M.guilliermondii LZ5had significant effects on biocontrol effectiveness:the higher the concentrations of M.guilliernondii LZ5, the lower the disease incidence and the smaller lesion diameter. Rapid colonization of M.guilliermondii LZ5on wound sites in the presence and absence of C.gloeosporioides were observed during the first48h, and then kept stable at a high level.In mango fruit, the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO) were significantly induced by M.guilliermondii LZ5treatment compared to those of the control fruit.3、 Biocontrol activity of M.guilliermondii LZ5against C.gloeosporioides in mango fruit was enhanced in combination with calcium chloride (CaCl2,4%)by directly inhibit the growth of pathogens and to promote the proliferation of antagonistic yeast, and its ability to significantly increased the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POD) and polyphenol oxidase (PPO) and the expressions of chitinase(CHI) and peroxidase (POD) genes.4、pFL61-neo-tpsl expression vector was constructed, and it was transformed into M.guilliermondii LZ5by lithium acetate method. This expression vector pFL61-neo-tpsl conferred M.guilliermondii LZ5drug resistance to250μg/mL G418and improved the stress tolerance of M.guilliermondii LZ5.The transformant could keep a high percentage of plasmid-containing of transformant with67.87%after50generations in non-selective medium. The results showed that yeast shuttle vector pFL61could be used to transform wild type antagonistic yeast and G418resistance can be used as a dominant selectable marker to select transformants.The trehalose-6-phosphate synthase overexpression in cells of M.guilliermondii LZ5transformed with pFL61-neo-tpsl expression vector correlated with increased tolerance to heat shock(48℃), osmostress(8%NaCI) and toxic levels of ethanol(15%), enabled the cells to increase viability.