Efficacy Evaluation Of Bacillus Subtilis EBS03 On Control Of Cotton Verticillium Wilt And Optimization Of Culture Conditions

A strain EBS03,which has an inhibitory effect on cotton Verticillium wilt,was screened in our laboratory,and its morphological observation,physiological and biochemical characteristics,and molecular biological identification were carried out.Taking cotton virulent strain Vd080 as the target strain,the inhibitory effect of EBS03 metabolites on cotton Verticillium wilt was determined by the plate confrontation method and buckle method.The effects of metabolites on spore production and sclerotia germination of V.dahliae were determined by the co-culture method.Furthermore,the control effect of the strain on cotton Verticillium wilt under different treatments was determined by greenhouse and field experiments,and the expression the of cotton defense gene and the colonization of V.dahliae in cotton tissue were detected by real-time fluorescence quantitative PCR.The optimum culture medium components of strain EBS03 were screened by single factor experimental design and orthogonal experimental design,and the fermentation conditions were optimized.The main results are as follows:The inhibition rate of EBS03 metabolites on mycelial growth of Bacillus subtilis was determined at 10d and 15 d in plate standoff culture,and the inhibition rate of EBS03 at 10 d and 15 d was 72.39%,and the the the 67.66%,respectively.The inhibition rates of volatile metabolites of EBS03 on V.dahliae colonies were 62.79%and 55.21%at 10 and 15 d,respectively,and the inhibition rates of non-volatile metabolites of EBS03 on V.dahliae sporulation and micronuclei were 40.10%-54.01%and 60.19%,respectively,using a mixed culture.The inhibition of V.dahliae sporulation and micronuclei by the non-volatile metabolites of EBS03 was determined to be 40.10-54.01%and 60.19-78.64%,respectively.The results of the greenhouse and field trials showed that seed soaking treated cotton seedlings and a low concentration of EBS03CMC solution had a significant promotion effect on seed germination.In the greenhouse test using vermiculite sand bottom Less paper bowl quantitative dipping method,the prevention effect of fermentation solution seed soaking and root irrigation treatments were 76.33%and 87.11%,respectively;in the field test,fermentation solution seed soaking method,root irrigation method and spray method were used to treat cotton seedlings,and the spray method had better prevention effect up to 42.54%,and root irrigation method and spray method had different degrees of prevention effect on cotton fiber quality and yield-related traits.Root irrigation methods and spray methods can promote the fiber quality and yield-related characteristics of cotton to different degrees.RT-q PCR showed that EBS03 induced up-regulation of peroxidase（POD）,polyphenol oxidase（PPO）,phenylalanine aminolase（PAL）,and disease-stage related genes（PR10）in V.dahliae.This resulted in the suppression of V.dahlia infestation in cotton.q PCR assays for colonization in cotton tissues showed that treatment with EBS03 fermentation solution significantly reduced the colonization of V.dahlia Vd080 in cotton tissues.The results showed that the optimum medium composition was 1.8 g of mannitol,2.0 g of peptone,and1.9 g of magnesium sulfate,and the optimum fermentation conditions were 37℃,50 m L of loading volume,4%of initial inoculum,and 200 r﹒min^-1 and p H 7.The maximum bacterial count of EBS03 fermented under these conditions was 9.59×10⁷ cfu/m L.In summary,the endophytic bacterium Bacillus subtilis EBS03 has a good effect on cotton Verticillium wilt and is expected to be used as a biological agent for the control of cotton Verticillium wilt.