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从广东博罗、惠来和海南陵水县普通野生稻中分离到37株内生固氮菌,并测定其固氮酶活性.经固氮酶基因(nifH)的PCR扩增检测,37株内生固氮菌均能扩增出固氮酶基因(nifH)片段.在80%相似性水平上,IS-PCR指纹图谱聚类分析得到8个类群(Ⅰ～Ⅷ).SDS-PAGE全细胞蛋白电泳聚类分析与IS-PCR的聚类结果相似.对各类群相应代表菌株(Ls13,Ls8,BL1,BL12,HL6,Ls4)进行脂肪酸含量和成分比较,结果表明,这些菌株的脂肪酸成分和含量具有较大的差异.16SrDNA序列分析表明,类群Ⅰ～Ⅶ的菌株均属于肠杆菌科(γ-变形菌纲),其中类群Ⅰ和Ⅱ的菌株属于克雷伯氏菌属不同种群;类群Ⅲ菌株Ls8与成团泛菌(Pantoea agglomerans)的相似性为96%,可能代表该属内的一个新种或一个新属;类群Ⅳ和类群Ⅴ的菌株代表肠杆菌属的不同种群;类群Ⅵ菌株Ls4和Ls9与无丙二酸柠檬酸杆菌(Citrobacter amalonaticus)有98%的相似性;类群Ⅶ菌株Ls15与成团泛菌关系最近,有97%相似性,属于泛菌属;类群Ⅷ属于Ideonella属(β-变形菌纲).这些表明普通野生稻内生固氮菌具有较大的多样性,且部分菌株具高固氮酶活性,达42.52μmol C2H4·mL-1·h-1.水稻接种实验表明内生固氮菌能明显促进水稻生长.
Thirty-seven strains of endophytic diazotrophs were isolated from Boluo, Huilai, Guangdong and Lingshui County of Hainan Province, and their nitrogenase activity was determined. By PCR amplification of nifH, 37 endogenous nitrogen fixation The nifH gene fragment was amplified by PCR, and 8 groups (Ⅰ ~ Ⅷ) were obtained by IS-PCR fingerprinting at 80% similarity level. SDS-PAGE whole cell protein electrophoresis clustering analysis Similar to the results of IS-PCR, the fatty acid composition and composition of the corresponding representative strains (Ls13, Ls8, BL1, BL12, HL6, Ls4) of all the groups were compared and the results showed that the fatty acid composition and content of these strains were relatively large The results of 16S rDNA sequence analysis showed that all the isolates from group Ⅰ to Ⅶ belonged to Enterobacteriaceae (γ-Proteobacteria), of which the strains belonging to group Ⅰ and Ⅱ belonged to different populations of Klebsiella; The similarity of Pantoea agglomerans is 96%, which may represent a new species or a new genus in this genus. The strains of group Ⅳ and group Ⅴ represent different groups of Enterobacter. The group Ⅵ strains Ls4 and Ls9 and 98% similar to Citrobacter amalonaticus; Group VII Strain Ls15 has the closest relationship with Pantoea ananatis and 97% similarity, which belongs to Pantoea, and Ⅷ belongs to the genus Ideonella (β-Proteobacteria) .This indicated that the endophytic Azotobacter had a great diversity in common wild rice, And some strains with high nitrogenase activity, reaching 42.52μmol C2H4 · mL-1 · h-1. Rice inoculation experiments showed that endogenous nitrogen-fixing bacteria can significantly promote rice growth.