机构地区:[1]中国地质大学(武汉)环境学院,湖北武汉430078
出 处:《微生物学通报》2023年第2期454-470,共17页Microbiology China
基 金:国家自然科学基金(31570537)。
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[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
关 键 词:NIFH基因 高通量测序 纯培养 16S rRNA基因序列 溶磷活性
分 类 号:Q948.122.3[生物学—植物学]
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