A High Antibacterial Biopesticide Production From A Mutant Strain Of Trichoderma Harzianum Based On Moso Bamboo Substrate

T.harzianum is a highly efficient microbial pesticide that can be used to control various soil-borne fungal diseases.The aim of this study was to screen high-antibacterial biocontrol strains using ARTP mutagenesis technology,optimize the fermentation process of T.harzianum,optimize the pretreatment technology of moso bamboo,reduce the complexity of bamboo components,use moso bamboo as a fermentation substrate,and prepare T.harzianum microbial pesticides by solid-state fermentation.The effect was evaluated by antibacterial experiments.The effect of microwave-assisted hydrothermal pretreatment on the components and microstructure of moso bamboo was investigated.The results showed that:microwave-assisted hydrothermal pretreatment of moso bamboo removed 7.18%of lignin and 14.73%of cellulose in the material,respectively,and increased the cellulose recovery rate by 22.84%compared to the hydrothermal method,which maximized the retention of cellulose in the sample;compared to the hydrothermal method,microwave-assisted hydrothermal pretreatment of moso bamboo further promoted the breakage of intramolecular hydrogen bonds,intermolecular hydrogen bonds and methylene bonds in the material and increased the crystallinity of the material after microwave-assisted hydrothermal pretreatment by 14.84%.Utilizing atmospheric pressure room temperature plasma(ARTP)mutagenesis technology,high-yield xylanase andβ-1,3-glucanase T.harzianum mutant strains were selected by studying the mutagenic effect of ARTP on T.harzianum.The optimal mutagenesis time was determined to be 200s.Under this condition,after 5 rounds of mutagenesis screening,a mutant strain T57 with high enzyme activity was obtained.Compared with T334,the xylanase activity of T57 was increased by 459.29%and theβ-1,3-glucanase activity was increased by 817.42%.The liquid fermentation conditions of T57 were optimized.Under the conditions of 28℃culture temperature,8%inoculation volume,160r/min shaking speed and 48h fermentation,the xylanase activity of bamboo after microwave hydrothermal pretreatment was increased by 66.95%to 13.74U/m L when 1%of the material was added into the liquid medium.After fermentation for 96h,β-1,3-glucanase activity reached 1.71U/m L,which was increased by 17.93%compared with before optimization.The residue of microwave hydrothermal pretreatment of moso bamboo is used as a solid-state fermentation substrate.The optimal conditions for solid-state fermentation production of xylanase by T.harzianum(T57)are:culture temperature of 28°C,moso bamboo(moso bamboo raw material content93.75%,microwave hydrothermal pretreated moso bamboo content 6.25%)64g,bran 16g,fermentation time of 216h,inoculation amount of 10%,under which xylanase activity and spore number is 17.84U/g and 1.83×10~9 CFU/g.The optimal conditions for solid-state fermentation production ofβ-1,3-glucanase by T57 are:culture temperature of 28°C,moso bamboo(moso bamboo raw material content93.30%,microwave hydrothermal pretreated moso bamboo content 6.67%)60g,bran 20g,fermentation time of 120h,inoculation amount of 10%,under whichβ-1,3-glucanase activity and spore number is 5.64U/g and1.85×10~9 CFU/g.The antibacterial activity of spore preparations from high-enzyme-activity T.harzianum mutant strains was verified by combining confrontation experiments and plant cultivation experiments.T.harzianum T57 showed an obvious competitive advantage in confrontation culture experiments.In week confrontation experiments,the inhibition rate was 12%at 48 hours,37%at 72 hours,and 100%at 96 hours;in week confrontation experiments,the inhibition rate was 30%at 48 hours,63%at 72 hours,and 100%at 96 hours.The results of wheat planting experiments showed that T57’s intervention increased the germination rate of healthy wheat seeds by 5.2%and the germination rate of wheat seeds infected with Fusarium graminearum by 51.6%.This indicates that the spore preparations of high-enzyme-activity T.harzianum mutant strains can not only effectively inhibit Fusarium oxysporum activity but also promote wheat growth to a certain extent.