Fermentation Optimization Of Bacillus Methylotrophicus TA-1 And Research On Biocontrol Effects Of Its Bio-organic Fertilizer Against Fusarium Wilt Of Tomato

In China,production and plantation areas of tomatoes（Solanum lycopersicum Mill.）are both rank first in the world.In recent years,soil-borne diseases have become the most devastating threats in sustainable tomato production.Fusarium wilt of tomato,caused by Fusarium oxysporum f.sp.lycopersici（FOL）,is one of the main soil-borne diseases that limiting tomato yield and quality.Bio-organic fertilizers contain specific functional microorganisms,the combination of microorganisms with organic substances could prevent soil-borne diseases and promot plant growth,which is of great importance to the reduction of chemical pesticides and chemical fertilizer.Bacillus methylotrophic TA-1,showing broad-spectrum inhibition activity against a lot of phytopathogenic fungi,was isolated in our previous work.In this study,the laboratory antagonistic activity of TA-1 against FOL was determined.Meanwhile,the liquid fermentation conditions of TA-1 were optimized through response surface methodology,and the fermentation broth of TA-1 was mixed into matrixes with humic acid to prepare bio-organic fertilizer.Subsequently,the TA-1 bio-organic fertilizer was used to manage Fusarium wilt of tomato under greenhouse conditions to explore its biocontrol and promoting mechanism.In addition,a real-time fluorescent PCR detection system was established for quantitatively detecting FOL in order to provide scientific basis for the prediction and early prevention of Fusarium wilt of tomato.The results of the study were summarized as follows:1.Results of antagonistic examination against FOL revealed that TA-1 and its cell-free fermentation broth showed strong antagonistic activity,and the value of EC₅₀was 106.14 mg·L^-1 of the TA-1 crude lipopeptide against FOL.2.Response surface methodology found that the best carbon source for TA-1growth was glucose,nitrogen source was beef extract,and inorganic salt was Ca Cl₂.When the culture conditions were glucose 30 g·L^-1,beef extract 20 g·L^-1,Ca Cl₂ 5g·L^-1,shaking speed 211.55 rpm,temperature 30.54℃,fermentation time 48 h,loaded liquid 90 m L/250 m L,inoculation does 5.03%,the number of alive TA-1 in fermentation broth was estimated to be 9.13×10⁸ CFU·m L^-1.The optimized culture conditions were verified by plate counting method and the number of alive TA-1 was9.80×10⁸ CFU·m L^-1,which was 2.94 times higher than that before.TA-1 fermentation broth was mixed with humic acid to be granulated and dried to prepare TA-1bio-organic fertilizer,in which the number of alive TA-1 was stablely more than3×10⁸ CFU·g^-1.3.The results of greenhouse experiments showed that the biological control efficacy of TA-1 bio-organic fertilizer(3×10⁸CFU·g^-1)applied by soil drench at 12 g per plant against Fusarium wilt of tomato was 66.67%,and the plant height,stem diameter,above-ground fresh weight and above-ground dry weight were significantly increased.The application of TA-1 bio-organic fertilizer could reduce the content of ammonium nitrogen,increase nitrate nitrogen content and improve the activities of catalase,urease and phosphatase in soil.The results of plate-count in tomato rhizosphere soil showed that the application of TA-1 bio-organic fertilizer could increase the number of bacteria and Bacillus,and reduce the number of fungi and F.oxysporum.The copy number detection of FOL in base of tomato stem showed that TA-1 bio-organic fertilizer could effectively inhibit the infection of FOL in vascular bundles of tomatoes.4.The results of real-time fluorescent quantitative PCR detection of FOL indicated that pathogen could be detected at the base of the tomato stems on the 7th day after artificial inoculation,which could provide scientific basis for the epidemic monitoring and early prevention of the Fusarium wilt of tomato...