Endophytic Diazotrophs Diversity And Physiological Function From Seashore Paspalum

Endophytic bacteria of the present study were isolated from roots or stems or leaves of four varieties of seashore paspalum,including Platinum, Sea Isle 2000, Salam, and Supreme, then were purified by streak plate methods and microscope examination. 55 strains of endophytic diazotrophs were isolated from the four varieties by using non-nitrogenous medium and acetylene reduction assay(ARA). According to the results of SDS-PAGE of whole-cell protein and IS-PCR analysis. 55 strains were classified using clustering analysis. Then 16 S r DNA sequences of representative strains of isolated endophytic bacteria were examined and dissolved phosphorus and morphology of some representative strains with high nitrogenase activity were studied. The two selected representative strains were inoculated into perennial ryegrass, stylosanthes and rice to verify the growth promotion effects to plant. The results obtained were as follows:(1) 55 strains of endophytic diazotrophs were selected by further acetylene-reduction assay and 360 bp fragment of nif H of all the strains have been obtained through PCR amplification of nitrogenase nif H gene fragments. The nitrogen-fixing bacteria showed different nitrogenase activity ranged from 34.09 to 2948.08 nmol C2H2?m L-1?h-1.with maximum strain Tt2 of 2948.08 nmol?m L-1?h-1, minimum strain Tt1 of 34.09 nmol?m L-1?h-1. There were big differences in nitrogenase activity among different strains.(2) 55 strains were clustered into 10 categories and 11 categories according to the two clustering methods. Contrast to the method of SDS-PAGE, the Group 1 and Group 2 were clustered into one group in the method of IS-PCR, while the Group 3 and Group 4 were classified into two groups. The rest groups were the same. Therefore the two clustering methods showed higher consistency.(3) 11 representative strains were selected as the results of clustering and nitrogenase activity of nitrogen-fixing to conduct 16 S r DNA sequencing. The results showed they belonged to 8 genus, including Stenotrophomo, Rhizobium, Escherichia, Enterobacter, Stenotrophomonas, Citrobacter, Curtobacterium and Leclercia, indicating that endophytic diazotrophs from seashore paspalum had rich genetic diversity. The results were the same as the SDS-PAGE of whole-cell protein patterns analysis and the IS-PCR of patterns analysis.(4) 11 representative strains from different groups were inoculated with VM-Ethanol solid medium to observe their morphological characteristics, whereas phosphorus-solubilizing ability of 17 representative strains with Pikovaskaia’s solid medium was tested by qualitative and quantitative methods. 16 strains among them showed phosphorus-solubilizing ability, ranging from 9.06 to 29.04 ug?m L-1 with Bt6 the least of 9.06 ug?m L-1, Zr2 the highest of 29.04 ug?m L-1. Colony morphology of different strains demonstrated big obvious differences. The most of strains were Gram-negative and rod-shaped morphology by microscope.(5) The results of inoculation on perennial ryegrass, stylo and rice showed that strains Bt10 and Tr4 had obviously growth-prompting effects on the three plants.The plant height, root length and root weight of inoculated plants were significantly increased in contrast with the control(P<0.05). The growth-prompting effect of strain Tr4 was better than strain Bt10. Strain Tr4 increased more plant height, root length and underground biomass than aboveground biomass while strain Bt10 enhanced more aboveground biomass and underground biomass than the plant height and root length.