树兰原球茎内生细菌群落多样性及功能分析

Diversity and functions of endophytic bacteria in protocorms of Epidendrum sp.

摘要:

【目的】通过分析树兰原球茎内生细菌群落组成、多样性特征和促生功能，探究树兰种子萌发相关的核心细菌类群及生物学功能。【方法】以树兰原球茎(树兰种子在树皮基质上共生萌发、在树叶基质上共生萌发、在MS1培养基上非共生萌发)和共生萌发基质(松树皮、腐熟树叶)共5个样本为研究材料，采用高通量测序技术分析不同萌发条件下原球茎内生细菌的16S rRNA基因多样性，比较分析细菌群落多样性和物种组成特征，通过传统的内生细菌分离方法获得共生萌发原球茎内生细菌菌株，并进行促生潜力评价。【结果】从5个研究样本中共获得2 735个可操作分类单元(operational taxonomic unit, OTU)，属于41门453科876属，其中变形菌门(Proteobacteria)和放线菌门(Actinobacteria)为优势门。主坐标分析(principal coordinates analysis, PCoA)结果表明，树兰原球茎与萌发基质细菌群落结构存在差异，非共生萌发原球茎与在树皮基质上共生萌发原球茎内生细菌群落结构最为接近。功能预测表明，在树叶基质上共生萌发的原球茎内生细菌固氮功能显著高于其他萌发条件。通过分离培养，共获得内生细菌19株，分属12属16种，其中鞭毛膨胀芽孢杆菌(Tumebacillus flagellatus)、Bradyrhizobium cenepequi和人参腐殖土魏茨曼氏菌(Weizmannia ginsengihumi)为共生萌发原球茎共有种；韩国假单胞菌(Pseudomonaskoreensis)和 W. ginsengihumi兼具有溶磷、产吲哚乙酸(indole-3-acetic acid, IAA)和铁载体的潜在能力。【结论】在不同环境中萌发的树兰原球茎均有丰富的内生细菌群落定殖；从共生萌发原球茎中分离的内生细菌具有固氮、溶磷、产IAA和铁载体等促生功能。本研究为兰科植物种子萌发相关微生物资源挖掘及兰科植物与微生物互作研究提供科学依据。

Abstract:

[Objective] To investigate the community structure, diversity, and plant-growth- promoting functions of endophytic bacteria in the protocorms of Epidendrum sp. and mine the core bacterial groups associated with the seed germination. [Methods] We collected five samples including three types of protocorms (germinated on MS1 medium, pine bark, and rotten oak leaves) and two types of substrates (pine bark and rotten oak leaves). High-throughput sequencing of the 16S rRNA gene was employed to compare the diversity and community composition of bacterial communities under different germination conditions. The conventional method was used to isolate the endophytic bacteria from the protocorms germinated on pine bark and rotten oak leaves (i.e., from symbiotic seed germination), and the growth-promoting potential of the isolates was evaluated. [Results] A total of 2 735 operational taxonomic units (OTUs) were obtained from the five samples, belonging to 876 genera, 453 families of 41 phyla, among which Proteobacteria and Actinobacteria were the dominant phyla. The results of principal coordinates analysis showed that there were differences in the bacterial community structure between the protocorms of Epidendrum sp. and the substrates, and the community structure of endophytic bacteria in the protocorms germinated on the MS1 medium was closest to that on pine bark. Functional prediction indicated that the endophytic bacteria in the protocorms germinated on rotten oak leaves had higher nitrogen-fixing ability than that in other types of protocorms. Nineteen isolates were obtained from protocorms geminated on pine bark and rotten oak leaves, belonging to 16 species of 12 genera. Tumebacillus flagellatus, Bradyrhizobium cenepequi, and Weizmannia ginsengihumi were the common species in the protocorms germinated on pine bark and rotten oak leaves. Pseudomonas koreensis and W. ginsengihumi had the potential of solubilizing phosphorus and producing indole-3-acetic acid (IAA) and siderophores. [Conclusion] The Epidendrum sp. protocorms germinated in different environments harbor rich endophytic bacteria. The endophytic bacteria isolated from the protocorms from symbiotic seed germination had plant growth-promoting functions, such as fixing nitrogen, solubilizing phosphorus, and producing IAA and siderophores. This study provides a scientific basis for mining the microbial resources related to seed germination of orchids and studying the interactions between orchids and microorganisms.