高寒草地珠芽蓼内生拮抗固氮菌Z19的鉴定及其固氮功能

Identification, pathogen inhibiting and nitrogen fixation of endophytic bacterium Z19 of Polygonum viviparum

摘要:

【目的】从东祁连山高寒草地珠芽蓼内生细菌中筛选和鉴定具有固氮能力和拮抗能力的内生细菌。【方法】采用16S rRNA基因序列同源性分析和生理生化指标测定方法对该菌株进行鉴定。【结果】从珠芽蓼中分离获得的21株内生细菌中6株内生菌具有固氮能力，76.2%具有抑菌能力，其中5株内生细菌对5种以上的病原菌有抑制作用；菌株Z19具有较强固氮能力和分解纤维素的能力，其纤维素溶解圈直径与菌落直径比达3.33，产生的纤维素酶活性为 0.31 U，且对辣椒立枯丝核病菌(Rhizoctonia solani)、油菜菌核病菌(Sclerotinia sclerotiorum)、番茄灰霉病菌(Botrytis cinerea)、小麦根腐病菌(Bipolaris sorokiniana)和番茄早疫病菌(Alternaria solani)具有较强拮抗能力；经PCR扩增和测序，获得了菌株Z19的固氮基因(nifH)序列和16S rRNA基因序列，在GenBank中的登录号分别为EU693340和EU236746；菌株Z19呈革兰氏阳性，杆状，产芽孢。【结论】结合生理生化特征及16S rRNA基因序列同源性比较，鉴定其为枯草芽孢杆菌(Bacillus subtilis)。

关键词:内生细菌，筛选，鉴定，固氮功能

Abstract:

[Objective] in order to screen and identify endophytic bacteria with nitrogen fixation and pathogen inhibition functions, 21 endophytic bacteria were isolated from Polygonum viviparum on the Eastern Qilian Mountains in the Qinghai-Tibetan Plateau, China. [Methods] Nitrogen fixation endophytic bacteria were identified by the 16S rRNA genes sequences analysis, physiological and biochemical characterization. [Results] The results show that 28.6% endophytic bacteria of Polygonum viviparum could fix nitrogen and 76.2% had strong antagonistic against plant pathogen, furthermore, 23.8% endophytic bacteria inhibited more than 5 plant pathogen. Strain Z19 had strong ability of nitrogen fixation and decomposing-cellulose. The cellulase activity was 0.31 U, and ratio of the diameter of lysis zone to colony diameter was up to 3.33. Furthermore, strain Z19 inhibited pathogenic fungi, such as Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, Bipolaris sorokiniana and Alternaria solani. Through PCR amplification and sequencing, the sequence of nifH gene and 16S rRNA gene were obtained and they were registered in GenBank (accession numbers were EU693340 and EU236746, respectively). Strain Z19 is rod, Gram-positive, and produce spore. Based on the physiological and biochemical characteristics and 16S rRNA gene sequence analysis, Z19 was identified as Bacillus subtilis. [Conclusion] The study provides new insights for the exploration and utilization of promoting growth and antagonistic endophytic bacteria in alpine grassland.

Key words:Endophytic bacteria, Screening, Identification, Nitrogen fixation