哈茨木霉WF2厚垣孢子发酵条件优化及其对烟草根际土壤微生物群落的影响

Optimization of chlamydospore fermentation conditions of Trichoderma harzianum WF2 and its effect on the microbial community in tobacco rhizosphere soil

摘要:

背景 本实验室分离筛选出1株哈茨木霉(Trichoderma harzianum) WF2，其对多种烟草病害具有优良的防治效果，并且能诱导植物抗病性，具有较好的应用前景。目的 研究并优化哈茨木霉WF2的发酵技术，提高厚垣孢子的产量，从而为该菌株的工业化发酵生产提供技术参考。同时分析哈茨木霉WF2厚垣孢子发酵液施用后，烟草植株根际微生物群落结构的变化，阐明其对烟草植株根际微生物群落的影响。方法 发酵培养条件优化采用单因素优化方法，对发酵培养基采用响应面法进行优化，并对优化后的发酵产物进行盆栽生防效果验证。采集经WF2厚垣孢子发酵液处理的烟草根际土壤样品，采用基于16S rRNA基因和ITS区域的扩增子测序技术，分析根际微生物的群落多样性与组成差异，并利用PICRUSt2和FUNGuild数据库对微生物功能进行预测分析。结果 最佳发酵培养条件为：25 ℃，240 r/min，初始pH 4.0，接种量3%，装液量60 mL。最佳发酵培养基组分：葡萄糖15.00 g/L、高粱粉15.00 g/L、玉米浆19.61 g/L、硫酸铵1.25 g/L、硫酸镁1.00 g/L、碳酸钙1.00 g/L、硫酸锌1.00 g/L、磷酸二氢钾1.00 g/L。在此条件下，厚垣孢子产量最大，可达5.16×10⁷ CFU/mL。盆栽生防效果试验表明，厚垣孢子稀释液在1×10⁷ CFU/mL浓度下，对烟草黑胫病的防效为87.33%，对烟草根腐病的防效为81.00%。结果表明，施用WF2厚垣孢子发酵液后，显著提高了细菌群落的多样性和丰富度，同时降低了真菌群落的多样性和均匀度。在群落组成上，提高了有益细菌的相对丰度，降低了有害真菌的相对丰度。结论 通过发酵条件优化得到了高产厚垣孢子的发酵配方，并且对烟草根际施用发酵液后能显著降低烟草黑胫病及烟草根腐病的病情指数，同时也能改善烟草根际土壤中微生物的群落结构，提高植物益生菌的相对丰度，降低植物病原真菌的相对丰度。

Abstract:

Background Trichoderma harzianum WF2 was isolated in our laboratory. It has excellent biocontrol effects on many tobacco diseases and can induce plant disease resistance, demonstrating a promising application prospect.Objective To optimize the fermentation conditions of T. harzianum WF2 for improving the yield of chlamydospores, providing a technological basis for the industrial production of this strain. In addition, the changes of the microbial community structure in the rhizosphere soil of tobacco after the application of the fermentation liquid of T. harzianum WF2 chlamydospores were analyzed.Methods The fermentation conditions were optimized by single factor experiments and the fermentation medium was optimized by the response surface methodology. The optimized fermentation product was verified by a pot experiment for biocontrol effect. The tobacco rhizosphere soil samples treated with WF2 chlamydospore fermentation broth were collected, and the community diversity and composition of rhizosphere microorganisms were analyzed by amplicon sequencing based on 16S rRNA gene and ITS region. The PICRUSt2 and FUNGuild databases were used to predict and analyze microbial functions.Results The optimum fermentation conditions were as follows: 25 ℃, 240 r/min, initial pH 4.0, an inoculation amount of 3%, and a liquid volume of 60 mL. The optimum fermentation medium was composed of 15.00 g/L glucose, 15.00 g/L sorghum powder, 19.61 g/L corn steep liquor, 1.25 g/L ammonium sulfate, 1.00 g/L magnesium sulfate, 1.00 g/L calcium carbonate, 1.00 g/L zinc sulfate, and 1.00 g/L potassium dihydrogen phosphate. Under these conditions, the yield of chlamydospores was the highest, reaching 5.16×10⁷ CFU/mL. The pot experiment showed that the biocontrol effects of chlamydospore dilution at 1×10⁷ CFU/mL on tobacco black shank and tobacco root rot were 87.33% and 81.00%, respectively. The application of WF2 chlamydospore fermentation liquid significantly increased the bacterial diversity and richness, while reducing the fungal diversity and evenness. In addition, the fermentation liquid increased the relative abundance of beneficial bacteria while reducing the relative abundance of harmful fungi.Conclusion The fermentation formula for the high yielding of chlamydospores is obtained by fermentation condition optimization. The application of the fermentation liquid significantly reduces the disease indexes of tobacco black shank and tobacco root rot. At the same time, it improves the microbial community structure in tobacco rhizosphere soil by increasing the relative abundance of beneficial bacteria and reducing the relative abundance of plant pathogenic fungi.