入侵植物欧洲千里光内生固氮菌和溶磷菌多样性

Diversity of endophytic nitrogen-fixing and phosphate-solubilizing bacteria in the invasive Senecio vulgaris

摘要:

【背景】菊科(Asteraceae)外来入侵植物欧洲千里光(Senecio vulgaris L.)来源于欧洲，广泛分布于我国西南和东北地区，在湖北高海拔山区也有分布。在入侵过程中，内生细菌可能在其获取氮磷营养方面起到了一些关键性作用。【目的】探究欧洲千里光内生固氮菌和溶磷菌的多样性和功能，为理解其入侵机制及防治提供参考。【方法】选择来自6个不同种群的种子，萌发后转移到花盆生长6-8周，并从每个种群中各挑选9株生长情况良好的植株，对其叶片和根组织表面进行消毒处理。使用基于nifH基因(固氮功能基因)的高通量测序方法对植物的固氮微生物群落结构和多样性进行研究。通过涂布平板法和平板划线法，在固体无氮培养基(Ashby)和无机磷培养基(inorganic phosphate, NBRIP)上对植物内生菌进行分离、纯化，对纯化的固氮菌株和溶磷菌株进行16S rRNA基因测序。采用钼锑抗比色法分析纯化溶磷菌株的溶磷能力。【结果】基于nifH基因的内生菌高通量测序结果表明，欧洲千里光叶样本中固氮菌多样性显著高于根样本；固氮菌群落中丰度最高的属是慢生根瘤菌属(Bradyrhizobium，30.9%-34.0%)，其次是伯克氏菌属(Burkholderia, 27.2%-27.4%)、Methyloversatilis (2.1%-7.1%)和固氮螺菌属(Azospirillum, 2.9%-3.9%)；共6个门，其中变形菌门(Proteobacteria)在所有样本内相对丰度均达90%以上。用Ashby培养基筛选得到238株纯菌，分布在4门7纲10目16科19属，其中丰度前5的优势菌属包括微杆菌属(Microbacterium, 31.0%)、芽孢杆菌属(Bacillus, 24.8%)、假单胞菌属(Pseudomonas, 22.1%)、寡养单胞菌属(Stenotrophomonas, 6.2%)和类芽孢杆菌属(Paenibacillus, 2.8%)。用NBRIP培养基筛选共得到318株菌株，鉴定这些内生菌覆盖到3门5纲7目15科16属，其中丰度前5的优势菌属包括芽孢杆菌属(48.4%)、假单胞菌属(19.2%)、微杆菌属(15.2%)、类芽孢杆菌属(3.6%)、不动杆菌属(Acinetobacter, 3.6%)。挑选了24株代表性菌株对溶磷能力进行了定性及定量检测，结果表明有17株具备显著的溶磷能力，而且细菌培养过程中培养基pH值下降。【结论】欧洲千里光内生固氮菌和内生溶磷菌具有丰富的多样性，并且溶磷菌具有一定的溶磷能力，在欧洲千里光的入侵过程中可能起着促进作用。

关键词:nifH基因;高通量测序;纯培养;16S rRNA基因序列;溶磷活性

Abstract:

[Background] Native to Europe, Senecio vulgaris L. (Asteraceae) is widely distributed in southwest and northeast China as well as the high-altitude mountain areas of Hubei Province. In the invasion process, the endophytic bacteria may play a key role in nitrogen and phosphorus. [Objective] To investigate the diversity and functions of endophytic nitrogen-fixing and phosphate-solubilizing bacteria in S. vulgaris and thus to serve as a reference for elucidating the invasion mechanism of this species and controlling its invasion. [Methods] Seeds from six different populations were selected and after germination, seedlings were transferred to pots for growth for 6-8 weeks. Nine healthy plants from each population were picked and the leaf surface and root surface were disinfected. High-throughput sequencing of nifH gene (nitrogen fixation-related gene) was performed to investigate the structure and diversity of nitrogen-fixing bacteria in S. vulgaris. Endophytes were isolated and cultured on Ashby (nitrogen-free) medium and NBRIP (inorganic phosphate) medium, and 16S rRNA gene of the purified nitrogen-fixing and phosphate-solubilizing strains was sequenced. The phosphate-solubilizing ability of the isolated phosphorus-solubilizing strains was determined with the molybdenum-antimony anti-colorimetry method. [Results] The diversity of nitrogen-fixing bacteria in S. vulgaris leaf sample was significantly higher than that in root sample. Among the nitrogen-fixing bacteria, Bradyrhizobium (30.9%−34.0%) was most abundant, followed by Burkholderia (27.2%−27.4%), Methyloversatilis (2.1%−7.1%), and Azospirillum (2.9%−3.9%). A total of 6 nitrogen-fixing phyla were identified and the relative abundance of Proteobacteria was >90% in all samples. A total of 238 isolates (19 genera, 16 families, 10 orders, 7 classes, 4 phyla) were screened out via the Ashby medium. The top genera were Microbacterium (31.0%), Bacillus (24.8%), Pseudomonas (22.1%), Stenotrophomonas (6.2%), and Paenibacillus (2.8%). A total of 318 isolates (16 genera, 15 families, 7 orders, 5 classes, 3 phyla) were cultured on the NBRIP medium, and the top genera were Bacillus (48.4%), Pseudomonas (19.2%), Microbacterium (15.2%), Paenibacillus (3.6%), and Acinetobacter (3.6%). We performed quantitative and qualitative analysis of the phosphate-solubilizing ability of 24 representative strains. The result showed that 17 strains had remarkable phosphate-solubilizing ability and the phosphate-solubilizing process of these bacteria decreased the pH value of the culture medium. [Conclusion] S. vulgaris boasts abundant and diverse endophytic nitrogen-fixing and phosphate-solubilizing bacteria, and the phosphate-solubilizing bacteria may facilitate the invasion of this species.

Key words:nifH gene;high-throughput sequencing;pure culture;16S rRNA gene sequence;phosphate-solubilizing activity