枯草芽孢杆菌(Bacillus subtilis) Tpb55灌根与烟草根围细菌多样性变化的相关性

Correlation between root irrigation of Bacillus subtilis Tpb55 and variation of bacterial diversity in tobacco rhizosphere

摘要:

[目的] 利用454高通量测序技术分析生防菌株枯草芽孢杆菌(Bacillus subtilis) Tpb55对烟草根围土壤细菌群落的影响;同时测定施加Tpb55处理对烟草黑胫病的大田防效。[方法] 试验设置Tpb55菌剂10⁸ CFU/mL灌根和空白对照2个处理,分别在处理0、10、22 d采集烟草根围土壤,提取土壤细菌总DNA, 扩增细菌16S rDNA V1-V3区,对扩增产物进行454高通量测序,使用Qiime软件分析不同处理的细菌群落结构。[结果] 对照和处理样品测序后共获得了41207个优质序列,鉴定属于细菌的25个门。所有样品中优势菌群均为放线菌门(Actinobacteria)、变形菌门(Proteobacteria) 和酸杆菌门(Acidobacteria)。在病情发展过程中,放线菌门的细菌丰度逐渐下降,变形菌门含量呈上升趋势。施用Tpb55后,酸杆菌门含量明显上升并高于对照。对照中芽孢杆菌科(Bacillaceae)和多样性指示菌草酸杆菌科(Oxalobacteraceae) 的细菌丰度均明显下降,Tpb55处理的样品中芽孢杆菌科含量不断上升,草酸杆菌科含量相对稳定。Chao 1、ACE和Shannon指数分析表明,Tpb55处理的样品中细菌多样性和丰富度不断提高且高于对照。Tpb55处理后10 d和22 d, OTU序列数据库中与Tpb55 16S rDNA V1-V3区PCR产物高度同源OTU数目分别为31和45。Tpb55处理的烟草黑胫病病情指数(5.29)显著低于对照(38.52)。[结论] 施用Tpb55可以提高根围土壤细菌多样性和群落结构稳定性,这可能是其发挥良好生防作用的重要机制之一。

Abstract:

[Objective] The impact of inoculation with the biocontrol agent Bacillus subtilis on bacterial communities in rhizospheric soil of Nicotiana tabacum was assessed by using 454 pyrosequencing technology. The control effect of Tpb55 on tobacco black shank was also studied. [Methods] Two treatments were done as follows: irrigating root with Bacillus subtilis strain Tpb55 inoculants (10⁸ CFU/mL) and the control. Soil samples from tobacco rhizosphere were collected at 0d, 10d and 22 d after the treatment. Genomic DNA of soil samples was extracted and amplified for the 16S rDNA V1-V3 tags, and then the tags were sequenced by 454 sequencing. Qiime was used to analyze soil bacterial diversities. [Results] A total of 41207 high quality sequences were obtained from all samples, which were classified into 25 phyla. The dominant bacteriophyta were Actinobacteria, Proteobacteria and Acidobacteria in all samples. The content of Actinobacteria was decreased gradually in the development of disease, whereas Proteobacteria showed an opposite tendency. Acidobacteria revealed a marked increase andexceeded control in content after inoculation with Tpb55. The control showed a significant decline in Bacillaceae, as well as Oxalobacteraceae which was known as an indicator for bacterial diversity. However, Bacillaceae showed an increasing tendency and Oxalobacteraceae was relatively constant in Tpb55 treatment. The Chao 1, ACE and Shannon index of treatment showed a constant improvement of variety and richness. In 10 d and 22 d after Tpb55 inoculation, the number of sequences with high homology of V1-V3 regions of Tpb55 16S rDNA was 31 and 45, respectively. The disease index of tobacco black shank in inoculated tobacco (5.29) was significantly lower than the control (38.52). [Conclusion] Tpb55 could improve the diversity of soil bacterial community and ecosystem stability, which presented a possible reason for its biocontrol efficacy on tobacco black shank.