固态共培养木霉和芽孢杆菌抗烟草黑胫病及其活性成分研究

Study on Co-culture of Trichoderma asperellum and Bacillus subtilis by Solid-state Fermentation against Tobacco Black Shank Disease and Their Bioactive Secondary Metabolites

为提高棘孢木霉HG1和枯草芽孢杆菌tpb55共培养菌的生物量和抗烟草疫霉活性,构建了两株菌固态共培养体系,并探究了其活性成分。采用菌丝生长速率法评价发酵提取物的抑菌活性,结合发酵后菌量,确定固态共培养的条件;运用正/反硅胶柱层析对活性成分进行追踪分离,使用高效液相色谱(HPLC)和核磁共振(NMR)及ESI质谱,纯化和鉴定活性化合物;通过盆栽试验验证共培养发酵对烟草黑胫病的防治效果。结果表明,两株生防菌固态共培养发酵的初始条件为:枯草芽孢杆菌(10^(6)CFU/mL,接种量3%)接种12 h后再接种棘孢木霉(10^(6)CFU/mL,接种量1.5%);发酵14 d时,枯草芽孢杆菌菌量达到1.58×10^(10)CFU/g,棘孢木霉菌量达到6.75×10^(10)CFU/g,并且提取物的抗菌活性最强,抑制率达到73.25%;从活性组分中分离得到一个单体化合物,鉴定为harziandione。盆栽防病效果表明,采用固态培养基共培养发酵的产物对烟草黑胫病的防效优于单培养,并能够改善土壤理化性质。该研究为烟草黑胫病生防菌剂的研发提供了新的思路和方法。

In order to improve the biomass of Trichoderma asperellum HG1 and Bacillus subtilis tpb55 in the co-culture system,and increase their anti-oomycetes activities against Phytophthora nicotianae,and to explore the bioactive ingredients,the solid-state fermentation system was constructed.The anti-oomycetes activities were evaluated by the mycelium growth rate method.The solid-state fermentation conditions were determined by the anti-oomycetes activities and biomass of HG1 and tpb55.The bioactivity guided separation and isolation of fermentation extracts by silica gel and octadecylsilyl silica gel(ODS)column chromatography(CC),as well as high performance liquid chromatography(HPLC),together with NMR and ESI mass spectrometry were applied to clarify and identify the bioactive ingredients.Finally,pot experiments were applied to verify the control effect of solid-state co-culture of HG1 and tpb55 on tobacco black shank.The results showed that the optimal solid-state fermentation condition was as follows:the inoculation sequence was first B.subtilis(10^(6)CFU/mL,3%)12 h and then inoculated with T.asperellum(10^(6)CFU/mL,1.5%).After 14 d of fermentation,under the optimal condition,the bacterial amounts of tpb55 were 1.58×10^(10)CFU/g and the spore amounts of HG1 were 6.75×10^(10)CFU/g,with the strongest anti-oomycete activity towards P.nicotianae(inhibition rate 73.25%).A pure compound was isolated from the bioactive fraction and identified as harziandione.The results of pot experiments showed that the control effect of solid co-culture strains on tobacco black shank was significantly better than that of single culture,reached 61.94%.The co-cultured strains could also improve soil physical and chemical properties.This study provided a new idea and method for the research and development of co-culture biocontrol agents for tobacco black shank disease.