Diversity and nitrogenase activity of endophytic diazotrophs colonized in the wild rice Oryza rufipogon Griff grown in Boluo, Huilai County in Guangdong Province and Lingshui County in Hainan Province were studied. Th...Diversity and nitrogenase activity of endophytic diazotrophs colonized in the wild rice Oryza rufipogon Griff grown in Boluo, Huilai County in Guangdong Province and Lingshui County in Hainan Province were studied. Thirty-seven isolates obtained from Oryza rufipogon were identified as putative endophytic nitrogen-fixing bacteria by ARA (acetylene reduction assay) test and further confirmed by PCR amplification of nifH gene fragments. All obtained strains have ARA activity and the same sized nifH gene fragments. Above the similarity level of 80%, the obtained isolates were assigned as Group Ⅰ to Ⅷ by the clustering of IS-PCR fingerprints. The SDS-PAGE whole-cell protein patterns were similar to those of IS-PCR fingerprints. Components and contents of fatty acid methyl esters (FAMEs) were used to differentiate the representative strains (Ls13, Ls8, BL1, BL12, HL6, Ls4) from Group Ⅰ to Group Ⅵ. The six representative strains showed significant difference in contents and components of cellular fatty acid methyl ester. 16S rDNA sequencing analysis showed that strains of Group Ⅰ to Ⅶ were located in Enterobacteraceae (γ-proteobacteria). Strains of Group Ⅰ and Group II were closely related to Klebsiella sp.; Strain Ls8 of Group Ⅱ was a little far away from the genus of Pantoea (homology level 96% with Pantoea agglomerans), which may represent a new species or genus in Enterobacteraceae; Strains of Groups IV and V belonged to different Enterobacter sp.; Strain Ls4 and Ls 9 representing Group Ⅵ were close to Citrobacter amalonaticus with 98% sequence similarity; Strain Ls15 of Group Ⅶ showed 98% sequence identity with Pantoea sp.; Strains of Group Ⅷ were assigned to the genus Ideonella (β-proteobacteria). Based on the above results, endophytic diazotrophs isolated from O. rufipogon showed great diversity and some diazotrophs showed high nitrogenase activity with 42.52 μmol/mL·h C2H4. Inoculation to rice tests indicated that the isolated endophytic diazotrophs significantly promoted the rice growth.展开更多
基金Supported by National Natural Science Foundation of China (Grant Nos. 30470002 & 30770001)Program for New Century Excellent Talents in University (Grant No. NCET-07-0315)Guangdong Province Key Research Project of Agriculture (Grant No. 2007B020711004)
文摘Diversity and nitrogenase activity of endophytic diazotrophs colonized in the wild rice Oryza rufipogon Griff grown in Boluo, Huilai County in Guangdong Province and Lingshui County in Hainan Province were studied. Thirty-seven isolates obtained from Oryza rufipogon were identified as putative endophytic nitrogen-fixing bacteria by ARA (acetylene reduction assay) test and further confirmed by PCR amplification of nifH gene fragments. All obtained strains have ARA activity and the same sized nifH gene fragments. Above the similarity level of 80%, the obtained isolates were assigned as Group Ⅰ to Ⅷ by the clustering of IS-PCR fingerprints. The SDS-PAGE whole-cell protein patterns were similar to those of IS-PCR fingerprints. Components and contents of fatty acid methyl esters (FAMEs) were used to differentiate the representative strains (Ls13, Ls8, BL1, BL12, HL6, Ls4) from Group Ⅰ to Group Ⅵ. The six representative strains showed significant difference in contents and components of cellular fatty acid methyl ester. 16S rDNA sequencing analysis showed that strains of Group Ⅰ to Ⅶ were located in Enterobacteraceae (γ-proteobacteria). Strains of Group Ⅰ and Group II were closely related to Klebsiella sp.; Strain Ls8 of Group Ⅱ was a little far away from the genus of Pantoea (homology level 96% with Pantoea agglomerans), which may represent a new species or genus in Enterobacteraceae; Strains of Groups IV and V belonged to different Enterobacter sp.; Strain Ls4 and Ls 9 representing Group Ⅵ were close to Citrobacter amalonaticus with 98% sequence similarity; Strain Ls15 of Group Ⅶ showed 98% sequence identity with Pantoea sp.; Strains of Group Ⅷ were assigned to the genus Ideonella (β-proteobacteria). Based on the above results, endophytic diazotrophs isolated from O. rufipogon showed great diversity and some diazotrophs showed high nitrogenase activity with 42.52 μmol/mL·h C2H4. Inoculation to rice tests indicated that the isolated endophytic diazotrophs significantly promoted the rice growth.
