The Antifungal Mechanisms To Botrytis Cinerea And Mutagenesis Breeding Of Yeast Strain 8-249

At present,bacteria,fungi and yeast have been used in the biocontrol of fruits and vegetables postharvest diseases,among of them,yeasts have become one of the hotspots for its’effectiveness and safety in postharvest diseases inhibition.In this study,firstly,we detected the antifungal activity of yeast strain 8-249 by the in vitro and in vivo experiments,identified the yeast strain 8-249 by morphology,physiology and molecular sequence,and studied the antifungal mechanism of 8-249 by the interaction system between Botrytis cinerea and cherry tomatoes.Secondly,the mutant strain with high antifungal activity was screened by atmospheric and room temperature plasma（ARTP）mutagenesis methord.Thirdly,we optimized the culture medium of the mutant strain and 8-249 by the response surface optimization method.Finally,we detected the relative expression of resistance-related genes according to the transcriptome database,this result will be the foundation of the resistance-related genes research.The mainly research of this paper are as follow:1.The antifungal activity and strain identification of yeast strain 8-249.The antifungal activity of the yeast strain 8-249 was detected by plate antagonism experiments,the result showed that,this strain had broad-spectrum antifungal activity,and the antifungal rates against B.cinerea,Coniella granati and Verticillium dahliae were 39.6%,36.3%and 38.7%,respectively,the strongest antifungal activity was against B.cinerea,during the first 8 hours of co-culture,the concentration to inhibit the germination of fungal spores was 10⁸ cells/m L.Further detecting the antifungal activities and colonization dynamics of the strain 8-249 by the interaction system between B.cinerea and cherry tomatoes,the results showed that,the strain8-249 could effectively inhibit the infection of B.cinerea on cherry tomatoes,and the maximum biomass of 8-249 was observed on the 3rd day after inoculation,which showed that the strain 8-249 had the strong colonization ability.According to the results of morphology,physiological,and the ITS sequence,the antifungal yeast strain 8-249 was identified as Moesziomyces globuligerus.2.The antifungal mechanisms of yeast strain 8-249.The results of antifungal mechanism experiments show that there have the nutritional competition and the hyperparasitism between yeast strain 8-249 and B.cinerea.The yeast strain 8-249 dose not have the ability to produce volatile antifungal substances,and has the abilities to produce chitinase,protease andβ-1,3-glucanase.The results of the metabolic ability of strain 8-249 showed that the strain has the ability to dissolve phosphorus,dissolve zinc,polyammonium,can produce siderophore,IAA and ammonia,and also has the biofilm-forming ability strongly.The results of the host induced resistance experiment showed that the strain 8-249 could induce the host resistance by improving the activities of four defense-related enzymes POD,PAL,CHI and GLU,and reducing the activity of PPO on cherry tomato fruits.In the determination of the influence of the strain 8-249 can improve the quality of fruits by delaying the fruit softening,the fruit respiration and the degradation of carbohydrates on cherry tomato fruits.3.ARTP mutagenesis breeding.Mutagenesis breeding of the strain 8-249 was performed using ARTP mutagenesis technique.The optimum treatment conditions were as follows:the ARTP power 110 W,flow rate 10 slpm,treatment distance 3 mm,and treatment time 40 s,a mutant strain 40-3 with a 37.5%antifungal activities increase was screened by the in vitro and in vivo.Mutant 40-3 showed a stronger ability to colonize in the cherry tomato wounds.The ability of the mutant strain 40-3 to inhibit the germination of fungal spores was increased to77.57%,the has a 34.53%increment than that of the original strain 8-249,mutant strain 40-3showed the stronger antifungal ability.4.Response surface methodology was used to optimize the composition of culture medium of the mutant strain 40-3 by Box-Behnken response surface optimization method.The optimized medium combination were as follows:molasses concentration 67.94 g/L,initial p H5.36,yeast powder concentration 8.72 g/L,（NH₄）₂SO₄ 0.5 g/L,KNO₃ 0.5 g/L,KH₂PO₄ 0.5g/L.Using the optimized medium,the biomass of the original strain 8-249 and the mutant 40-3 could reach 20.6±1.33 g/L and 29.3±1.54 g/L,which are a 3.37 and 4.8 fold increase over the pevious medium respectively,and the biomass of mutant strain 40-3 increased by 1.42times compared with the strain 8-249.5.The relative expression of resistance-related genes of strain 8-249 were analyzed based on the transcriptome data by q RT-PCR.The samples including the strain 8-249（control group）alone and co-cultured with B.cinerea after 3 days（experimental group）,the differentially expressed genes were selected by the threshold of multiple of difference be equal or greater than 1 and padjust less than 0.05,the results showed that,there were 1948 differentially expressed genes,including 584 up-regulated genes and 1364 down-regulated genes,and 7genes with an up-regulated expression of more than 5 times were selected from the up-regulated genes,and they were have the prediction functions including secondary metabolites,metabolic pathway genes related to biosynthesis,transport and catabolism.The relative expression of the seven genes were detected by q RT-PCR,the results showed that,the expression of 7 genes including Hu＿G04563,Hu＿G00946,Hu＿G05881,Hu＿G00884,Hu＿G00914,Hu＿G05293 and Hu＿G02932 were up-regulated by 82.17,6.91,15.49,18.07,5.15,4.40 and 83.88 times compared with the control group in the co-cultured strain 8-249,we inferred that these seven genes were associated with the antifungal of the strain 8-249.This experiment provides an experimental reference for the screening and applicating of antagonistic yeast strains,and provides an effective source of antagonistic yeast.