| Soybean is one of the most important crops in world agricultural productivity. Rhizobia can form nitrogen-fixing nodules with the soybean. The nodules plays a significant role to improve the N nutrition for plants and help preserve fertility in the soil. Many studies had suggested that the rhizobacterium could play an important role in the legume-rhizobia symbiosis. But there were some defects in the previous studies:the first, the rhizosphere was always looked as a whole though it actually could be separated into some small areas which had different environmental conditions according to the longitudinal and radial gradients; the second, most studies only described the phenomenon of rhizobacteria promoting plant growth and nodulation, but not tried to explore the mechanisms. In this study, we tried to isolate and identify some epiphytic bacteria from the surface of soybean nodules which was an important layer in rhizosphere and evaluate the impacts of these strains on the legume-rhizobia symbiosis. The aim of this study was to find out the mechanisms of these bacteria promoting plant growth and increasing nodulation and try to screen for related functional genes.A total of ninety-two bacterial strains were isolated from root nodule surface of soybean [Glycine max (L.) Merrill] cultivar Zhonghuang 13. These isolates were identified with phylogenetic analysis of 16S rRNA gene and screened in vitro for their plant growth promoting (PGP) traits. The results showed that these stains belonged to fourteen genera within the phyla Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes. More than 50% isolates belonged to Proteobacteria, just like the other layers of rhizosphere according the previously reported. Most genera (8/14) of the nodule epiphytic bacteria, such as Pseudochrobactrum (17.39%), Alcaligenes (18.48%) and Lysinibacillus (9.78%) were not reported for root epiphytic or for root and nodule endophytic bacteria of soybean, demonstrating that the nodule surface was a unique habitat for bacteria and had some unique mechanisms to select its epiphytic bacteria. According to the test of PGP traits, the production of IAA was the common feature (71%), just like previously reported for rhizosphere and rhizoplane bacteria. But the proportions of other three traits were different from the previous studies:chitinase production (30%) and siderophore production (42%) were more common and phosphate solubilization (8%) was apparently lower in the soybean nodule epiphytic bacterial communities. The results might be associated with the unique environmental conditions of nodule surface.The twenty-two nodule epiphytic isolates were chosen to do the inoculation tests according to their identification and presence of plant growth-promoting (PGP) traits. In general, sixteen of them showed the ability of enhancing the plant biomass or nodulation or nitrogen supply. Five isolates, including CCNWSP78, CCNWSP60, CCNWSP46, CCNWSP15 and CCNWSP13-4, could enhance plant growth and nodulation significantly. It indicated that many strains which could promote the soybean growth and nodulation colonized on the nodule surface, they were very clost to the symbionts and might have more interaction with the symbionts than the other rhizobacteria. So, the nodule surface was a good area to screen for plant growth-promoting rhizobacteria (PGPR). Furthermore, three possible modes of epiphytic bacteria to impact the symbiosis were proposed:the first might mainly increase the nodules number and then affected root growth; the second might mainly promote the root growth and then led to more nodules; the third might mainly influence the nodules size. For the physiological and biochemical traits tested in this study, we suggested that there was a positive correlation between the production of siderophore and the increase of root dry weight, nodules number and N accumulation, and there was also a positive correlation between the production of chitinase and the increase of nodules number and N accumulation.Two isolates which had been checked in inoculation tests, CCNWSP13-4 and CCNWSP30, were very similar in 16S rRNA gene sequence, but very different in the ability of promoting the soybean-rhizobia symbioses. So, we used the suppression subtractive hybridization (SSH) to find the differential gene of the two isolates, which might be the important functional gene for promoting the soybean-rhizobia symbioses. We constructed a subtractive library with more than two hundreds clones. Eleven subtracted fragments were sequenced and searched homologically in Genbank. Three of them might be associated with the modification of DNA and transcription, one with the glucose metabolism, seven were unknown genes.A polyphasic study was carried out to confirm the taxonomic position of the two nodule epiphytic isolates, CCNWSP36-1 and CCNWSP60. The result showed that the two strains were remarkable different from the type strains of their closest phylogenetic neighbors. So, we proposed two novel specieses:Sphingobacterium yanglingense, the type strain is CCNWSP36-1T; Microbacterium shaanxiense, the type strain is CCNWSP60T.As the first study on nodule epiphytic bacteria, our results revealed that Proteobacteria was the dominator on the nodule surface of soybean cultivar Zhonghuang 13. We proposed two novel specieses:Sphingobacterium yanglingense and Microbacterium shaanxiense. We got some isolates with strong application potentials which had significant capabilities to promote plant growth and nodulation. We also analyzed the possible models of these isolates to promote plant growth and nodulation. In the end, we constructed a subtractive library using the suppression subtractive hybridization (SSH) to find the function genes which might be relate with promoting the soybean-rhizobia symbioses. This study might help us to analyze the nodule epiphytic bacteria and understand the ternary interactions system of legumes-rhizobia-PGPR and provided the theoretic foundation for the development of new microbial inoculants. |
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