Study On Fermentation And Soil Colonization And Control Of Tobacco Soil-borne Diseases Of Two Biocontrol Agents

Trichoderma asperellum THGY-01 and Bacillus methylotrophicus YB-1 were biocontrol agents screened and preserved in the laboratory.The liquid fermentation conditions of B.methylotrophicus YB-1 have been screened and optimized.The colonization ability of T.asperellum THGY-01 and B.methylotrophicus YB-1 in soil was determined.The antagonistic effects of T.asperellum THGY-01 and B.methylotrophicus YB-1 against Ralstonia solanacearum,Fusarium solani and Rhizoctonia solani were tested in vitro by confrontation test.The control effects of T.asperellum THGY-01 and B.methylotrophicus YB-1 to tobacco bacterial wilt,tobacco Fusarium root rot and tobacco blight were determined by pot experiment.The main results were as following:1.The liquid fermentation conditions of B.methylotrophicus YB-1 were studied.The liquid fermentation medium and conditions of B.methylotrophicus YB-1 were screened by single-factor and multi-factor orthogonal design test.The optimal fermentation medium formula were as follows:soybean meal 0.5%,corn starch 1%,KH₂PO₄ 0.2%,K₂HPO₄0.2%,MnSO₄ 0.1%.The optimal condition for liquid fermentation of B.methylotrophicus were as follows:initial pH 6.5,temperature 30?,inoculate size 4%,shaker speed 160 r/min and liquid volume 120 mL/500mL.The highest number of viable bacteria could reach 2.03×10¹00 cfu/mL in 21 h.2.Colonization ability of the two biocontrol agents in soil were determined.The colonization ability of T.asperellum THGY-01 in soil was determined by four methods:solid fermentation of T.asperellum THGY-01 mixed with sterile soil,solid fermentation of T.asperellum THGY-01 was spread on the sterile soil surface,fermentation liquid of T.asperellum THGY-01 and solid substrate were mixed with sterile soil,fermentation liquid of T.asperellum THGY-01 mixed with solid substrate was spread on the sterile soil surface,the results indicated that T.asperellum THGY-01 could colonize successfully in soil,and the number of spores produced below 3 cm of the soil surface were significantly higher than other soil layers.The number of spores below 3 cm of the soil layer were 1.59×10⁷ per gram,0.67×10⁷ per gram,0.91×10⁷ per gram,1.69×10⁷ per gram in 9 days.By pouring method,B.methylotrophicus YB-1 could colonize in different soil layers,the largest amount of the viable bacteria have been found below 6 cm of the soil surface which could reach 2.07×10⁹ cfu/g in 6 days.The results showed that the two biocontrol agents have occupied different optimal niches in soil,which provides a theoretical basis for the subsequent study of the mixed control of plant diseases using the two biocontrol agents.3.Antagonistic activity of T.asperellum THGY-01 and B.methylotrophicus YB-1 on three soil-borne pathogens of tobacco were tested.The results of plate confrontation test showed that T.asperellum THGY-01 had significant inhibitory effects against Fusarium solani and Rhizoctonia solani,the inhibition rates in 6 days were 67.28%and 77.31%,respectively.Both volatile metabolites and non-volatile metabolites have significant inhibitory effects on the two pathogens,the inhibition rates were 55.46%and 50.46%on Fusarium solani,respectively,60.74%and 56.73%on Rhizoctonia solani respectively in 6days.The inhibition rates of B.methylotrophic YB-1 on Rhizoctonia solani and Fusarium solani were 54.05%and 68.32%,respectively.The fermentation filtrate of B.methylotrophic YB-1 has significant inhibitory effect on Ralstonia solanacearum.4.The control effect of the two biological agents on tobacco bacterial wilt,Fusarium root rot and blight have been tested using pot experiments.The relative control effects of T.oxysporum THGY-01 applied alone were 59.2%,33.3%and 69.9%,respectively.The relative control effects of B.methylotrophic YB-1 applied alone were79.7%,65.1%and 54.8%,respectively.The control effect of T.oxysporum THGY-01 mixed with B.methylotrophic YB-1 was 83.3%,82.6%and 77.4%,respectively.The results showed that significant control effects have been made using solid fermentation of T.oxysporum THGY-01 mixed with fermentation liquid of B.methylotrophic YB-1compared to T.oxysporum THGY-01 or B.methylotrophic YB-1 applied alone,the reason may be due to the two biocontrol agents have different colonization niches in the soil.