枯草芽孢杆菌BSD-2对黄瓜叶际微生物数量及菌群结构的影响

Effects of Bacillus subtilis BSD-2 on cucumber phyllosphere microorganism community

摘要:

【目的】阐明枯草芽孢杆菌BSD-2菌株抗菌素、芽孢及发酵液不同施入形式对黄瓜叶际微生物数量及菌群多样性的影响，为该菌株的应用提供理论基础和依据。【方法】以黄瓜为实验材料于室内进行盆栽实验，设置于接种灰霉病原菌前后喷施抗菌素、芽孢、发酵液，单独接种灰霉病原菌和无菌水空白对照，共8个处理。采用传统微生物分离的方法，利用PB、高氏1号和PDA培养基分离并统计叶际可培养细菌、放线菌和真菌的数量；采用Bio-Rad公司的DcodeTM System进行样品的DGGE分析，对条带数量和灰度进行定量分析，采用UPGMA法对各条带进行聚类分析；采用Shannon-Wiener指数、pielou均匀度指数和丰富度分析可培养微生物和DGGE图谱中的群落多样性；并将条带回收测序。【结果】枯草芽孢杆菌BSD-2抗菌素、芽孢及发酵液以接种灰霉病原菌前喷施的效果最佳，均可提高可培养细菌的数量，降低可培养真菌的数量；可使叶际微生物的Shannon-Wiener多样性指数、Pielou均匀度指数和丰富度指数增加；其中抗菌素处理对叶际微生物种群结构的影响最小。【结论】枯草芽孢杆菌BSD-2的发酵液可有效抑制真菌的数量；芽孢可有效提高细菌及放线菌的数量；抗菌素则利于保持叶际微生物的丰富度及多样性指数。

Abstract:

[Objective] By analyzing the effects of Bacillus subtilis antibiotics, spores and fermentation on the microorganism quantity and microbial community in cucumber phyllosphere, providing theoretical basis for the effective application. [Methods] With cucumber as the experimental material, setting up eight treatments: applying the antibiotics, spores and fermentation before or after inoculating Botrytis cintrea, only inoculating Botrytis cintrea and spraying the sterile water. The experiment was conducted in the indoor. Using the method of traditional microbial separation to count the number, the cultured bacteria, actinomycetes and fungi of cucumber phyllosphere were respectively separated with PB, High’s No.1 and PDA medium. At the same time, analyzing the phyllosphere microorganism samples of different treatments by the Dcode Universal Mutation Detection System for DGGE, the quantitative analysis of strip number and gray level were carried on by Quantity One analysis software, the various belt was clustered by UPGMA method, the microorganism quantity and microbial community was analyzed by microbe richness index, Shannon-Wiener diversity index and Pielou index, the differential DNA sequencing results were analyzed by NCBI Blast program homology, and clustered by The Sequence match program. [Results] The results of different treatments on the microorganism quantity and microbial community in cucumber phyllosphere, showed that spraying before inoculation with the Botrytis cintrea is the best applying way of Bacillus subtilis antibiotics, spores and fermentation, can effectively reduce the number of culturable fungi, increase the number of cultured bacteria and actinomycetes, increase leaf microbe richness index, Shannon-Wiener diversity index and Pielou index. Antibiotic treatment on the occasion of leaf microbial population structure is minimized. [Conclusion] Bacillus subtilis BSD-2 fermentation can reduce the number of culturable fungi. Bacillus subtilis BSD-2 spores can increase the number of the cultured bacteria and actinomycetes. Bacillus subtilis BSD-2 antibiotics can keep phyllosphere microbial richness and diversity index.