3种水稻土中7株固氮蓝细菌的分离与特征

【背景】蓝细菌是水生和陆地生态系统中生物固氮的主要贡献者。【目的】增加对稻田土壤固氮蓝细菌的了解,获得用于进一步研究的可培养固氮蓝细菌菌株。【方法】选择3种具有不同固氮能力的水稻土,采用BG11-N培养基分离培养固氮蓝细菌菌株,对新分离菌株进行形态特征观察,通过基因组DNA的nifH基因扩增明确其固氮潜力,进一步采用乙炔还原法和~(15)N_2示踪法定量测定其固氮能力,通过基因组DNA的16SrRNA基因序列比对进行鉴定。【结果】在光照培养条件下,采用BG11-N培养基共分离纯化得到自养菌株7株,细胞呈圆形或椭圆形、单列、无分枝、丝状和念珠状,在固体培养基上形成团垫状菌落。新分离菌株在BG11-N培养基中生长状况良好,以基因组DNA为模板可扩增出nifH基因,乙炔还原法和~(15)N_2示踪法测定结果显示具有较高固氮能力,同时具有铁载体生成能力。结合16S rRNA基因序列比对和形态特征,7株菌被初步鉴定隶属于念珠藻科(Nostocaceae)。【结论】从水稻土中分离到在稻田生物固氮中发挥重要作用的蓝细菌(念珠藻科)菌株,可培养固氮蓝细菌菌株固氮能力较高,兼具铁载体生成能力,可作为进一步深入研究的微生物资源,具有潜在的研究应用价值。

Isolation and characterization of seven nitrogen-fixing cyanobacteria from three paddy soils

Background] Cyanobacteria are key contributors to biological nitrogen fixation in aquatic and terrestrial ecosystems. Objective] To increase our knowledge of nitrogen-fixing cyanobacteria in paddy soil and obtain culturable nitrogen-fixing cyanobacterial strains for further studies. Methods] Three paddy soils with different nitrogen fixation capabilities were selected to isolate nitrogen-fixing cyanobacterial strains with BG11-N medium. The morphological characteristics of newly isolated strains were observed. The nifH gene amplification of genomic DNA was used to clarify their nitrogen fixation potential, acetylene reduction assay and ¹⁵N₂ tracer method were used to quantitatively determine their nitrogen fixation activity. Preliminary identification of the newly isolated strains were determined through 16S rRNA gene sequence comparison. Results] Seven autotrophic strains were isolated and purified with BG11-N medium under light conditions. All cells were round or elliptical, uniseriate, unbranched, filamentous and moniliform, and formed mat-like colonies on solid medium. The newly isolated strains grew well in BG11-N medium, were positive for nifH gene amplification of genomic DNA, had high nitrogen fixation activity measured by acetylene reduction assay and ¹⁵N₂ tracer method, and had capabilities of siderophore production. Through 16S rRNA gene sequence comparison and morphological features, all the seven strains could be preliminarily identified as belonging to Nostocaceae. Conclusion] The cyanobacterial (Nostocaceae) strains that plays important role in biological nitrogen fixation were isolated from paddy soil. The culturable nitrogen-fixing cyanobacterial strains have high nitrogen fixation and siderophore production ability, which could be used as microbial resources for further studies and have potential research and application value.