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

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