| Cotton is one of the important fiber crops with high economic value.Soil-borne diseases such as verticillium wilt,fusarium wilt,cotton damping off and cotton red rot of cotton seriously affect the high quality and yield of cotton.In recent years,screening of effective biocontrol microorganisms has become one of the important ways to control soil-borne diseases of cotton.In this study,the endophytic fungus Penicillium simplicissimum CEF-818 was used as a biocontrol fungus and the strong pathogenic strains Verticillium dahliae,Fusarium oxysporum,Rhizoctonia solani and Fusarium verticilliodesin were used as pathogens in cotton.The inhibition rate of CEF-818 against pathogen was detected by confront culture method,plate-toplate culture method and exudate filtering culture method.In the greenhouse and field trials,the effect of CEF-818 on the control of major soil-borne diseases of cotton was further examined mainly by substrate inoculation method,root inoculation method and seed dip inoculation method.The ability of strain CEF-818 to induce disease resistance was initially investigated by measuring the reactive oxygen species burst and callus deposition in cotton leaves,detecting the expression of defense-related genes in cotton leaves by RTPCR and detecting the quorum of V.dahliae in cotton seedling roots by q-PCR.The composition of CEF-818 solid fermentation medium and the fermentation conditions were optimized using single-factor test method and orthogonal test method,which mainly yielded the following results.(1)The significant effect of the overall metabolites of strain CEF-818 on the growth of mycelium of V.dahliae,F.oxysporum,R.solani,and F.Verticillioides was determined by confront culture method,with inhibition rates of 67.63%、78.87%、71.31% and 74.65%;The volatile metabolites of strain CEF-818 had a significant effect on the growth of mycelium of V.dahliae with 58.09% inhibition,and the inhibition of F.oxysporum,R.solani and F.verticillioides was not significant by plate-to-plate culture method;the exudate filtering culture method determined that the non-volatile metabolites of strain CEF-818 had a highly significant effect on the growth of mycelium of V.dahliae,F.oxysporum,R.solani and F.verticillioides with100% inhibition.(2)The results of the greenhouse test showed that the disease index of cotton Verticillium wilt treated with root irrigation and substrate inoculation methods were significantly reduced,and the greenhouse control effect of cotton Verticillium wilt was up to 69.77%;the greenhouse control effect of CEF-818 on cotton Fusarium wilt was tested by root irrigation and seed dip inoculation methods,and the results showed that the control effect was 33.75% and 13.44%;The results showed that the control effect of CEF-818 on cotton damping off was 54.82% and 25.29% in greenhouse using substrate inoculation and seed dip inoculation.(3)The field control effect of CEF-818 on cotton Verticillium wilt was tested by substrate inoculation and seed dip inoculation methods,and the results showed that the field control effect of CEF-818 on cotton Verticillium wilt was up to 63.73%.(4)Strain CEF-818 induced the burst of reactive oxygen species and callus deposition in cotton leaves.RT-PCR assay for the expression of defense-related genes in cotton leaves showed that strain CEF-818 induced the up-regulated expression of POD、PAL and PR10 in cotton leaves.(5)The q-PCR method was used to detect the colonization of V.dahliae in cotton seedling roots,and the test results showed that the expression of V.dahliae in the control group was 3.61 times higher than that in the treated group,indicating that the strain CEF-818 was able to inhibit the invasion of cotton seedlings by Verticillium dahliae.(6)The results showed that the optimum culture for solid fermentation of P.simplicissimum CEF-818 was wheat bran,and addition of 2.5% glucose,0.6% urea and 0.6% potassium chloride,with the initial p H was 7.0.The ratio of material to water was 1: 0.5,and the liquid inoculums level was 5%.The fermentation process of the material thickness of 2 cm was conducted at 28°C for 7 days.Under this optimal medium and fermentation process,the spore yield of 8.28×109 CFU/g for P.simplicissimum CEF-818 can be obtained.In conclusion,this study clarified the effect of strain CEF-818 on the control of cotton Verticillium wilt、cotton Fusarium wilt、cotton damping off and cotton Red rot,provided a reference basis for assessing the biological control potential of the bacterium CEF-818 and its application scope,and provided technical support for the large-scale production of its fungicide to help control cotton diseases. |
PDF Full Text Request