青香茅和五节芒内生固氮菌的分离与生理生化鉴定

Identification and Characterization of Endophytic Diazotrophs Isolated from Cymbopogon caesius and Miscanthus floridulus

为研究青香茅(Cymbopogon caesius)和五节芒(Miscanthus floridulus)内生固氮菌的多样性,基于两种无氮培养基结合乙炔还原法从这两种牧草中分别分离到30株和36株菌.采用SDS-PAGE(Dodecyl sulfate sodium saltpolyacrylamide gel electrophoresis)全细胞蛋白电泳和IS-PCR(Insertion sequence-based PCR)指纹图谱将这66株内生固氮菌聚类为10个类群(I-X,每群3株以上,共61个聚群菌株)和3个未归类小组(共5个未聚群菌株).从10个聚类群和3个未归类小组中各选1株菌作为代表进行16S rDNA序列分析,发现它们与洋葱伯克霍尔德菌(Burkholderia cepacia)、阴沟肠杆菌(Enterobacter cloacae)、固氮植物菌(Phytobacter diazotrophicus)、阿氏肠杆菌(Enterobacter asburiae)、织片草螺菌(Herbaspirillum seropedicae)、阪崎克洛诺菌(Cronobacter sakazakii)、水稻肠杆菌(Enterobacter oryzae)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、溶解肠杆菌(Enterobacter dissolvens)、无乳固氮螺螺(Azospirillum amazonense)、都柏林克罗诺杆菌(Cronobacter dublinensis)等的相似性分别达97%以上,表明所分离内生固氮菌多样性高.进一步对它们进行生理生化测定,结果表明有些菌株具泌氨能力、产吲哚、解磷、解钾效果,在农业生产上具有一定的应用潜力.

In order to investigate the diversity of endophytic diazotrophs in Cymbopogon caesius and Miscanthufloridulus, thirty and thirty-six isolates were obtained respectively from these two forage grasses by using two selective medium without nitrogen sources in combination with acetylene reduction assay. The sixty-six endophytic diazotrophs were assigned to 10 groups （61 strains, at least 3 for each group from I to X） and five ungrouped strains by analysis of SDS-PAGE （dodeeyl sulfate sodium salt - polyacrylamide gel electrophoresis） patterns of whole-cell protein electrophoresis and IS-PCR （Insertion sequence-based PCR） DNA fingerprinting. 16S rDNA sequencing analysis of representative strains of each group showed that these strains were closely related to Burkholderia cepacia, Enterobacter cloacae, Phytobacter diazotrophicus, Enterobacter asburiae, Herbaspirillurn seropedicae, Cronobacter sakazakii, Enterobacter oryzae, Klebsiella pneumoniae, Enterobacter dissolvens, Azospirillum amazonense, and Cronobacter dublinensis, indicating the high diversity of endophytic diazotrophs colonized in the host plants of C. caesius and M. floridulus. The results of physiological and biochemical tests showed that some isolates had the ability of ammonia secreting, indole producing, and phosphorus and potassium dissolving, suggesting the potential application in agricultural practices. Fig 2, Tab 3, Ref 38