Diversity Of Bacteria In Soybean Root Nodules Collected From Three Eco-Regions Of Shaanxi And Relationship With Environment

This study collected more than 200 soybean plants from three eco-regions, 11 sampling sites and 42 field plots in total. There were 511 isolates obtained from surface-sterilized root nodules. 16 S r RNA RFLP-PCR analysis of the 231 isolates revealed that they were classified into 43 RFLP types. The representive of all the RFLP types were chosen to be sequenced and used to construct phylogenetic tree, which revealed that Bacillus genus and Sinorhizobium americanus were predominant genus of non-symbiotic bacteria and domainated symbiotic bacteria.RAPD-PCR method was used to study the community genetic variation of the predominant non-symbiotic bacteria(Bacillus spp.) and the result showed the isolates that shared the same genotypes or from the same sampling site tend to clustered together. So RAPD-PCR is an effective way to discriminate the genotype and sampling site of Bacillus spp.. The RAPD-PCR fingerprints were combined and transferred into “1”, “0” matrix to make CCA analysis with 11 environment factors including longitude, latitude, altitude and common soil components. The CCA result revealed that the genetic variation in Bacillus community was influenced mostly by p H and organic matter at different level. The result may be related to the restrictive factors varied in different eco-regions. The detection of nif H gene result showed that Pol F/Pol R is a pair of primers more effective for amplification than the other two nif H primers. Nif H gene sequence in Bacillus spp. varied greatly from that of Rhizobia and showed great similarity to that of cyanobacteria.The predominant symbiotic bacteria in this study showed 100% sequence similarity of 16 S r RNA gene sequence to that of Sinorhizobium americanus, which was isolated from native Acacia spp. in Mexico originally and reported failed to inoculate soybean. We detected nif H and nod C gene showing 100% similarity to that of Sinorhizobium fredii in these Sinorhizobium spp., and they can form effective nodules with soybean(Brand: zhonghuang 13). This result enlarged the host range of Sinorhizobium americanus. Except for predominant Sinorhizobium americanus and Bacillus spp., we also detected Rhizobium spp., Bradyrhizobium spp., Ochrobactrum spp., Pseudochrobactrum spp., Morganella spp., Paenibacillus spp., Lysinibacillus spp., Exiguobacterium spp., Providencia spp., Diaphorobacter spp., Variovorax spp., Brevibacterium spp., Brevundimonas spp. and Alcaligenes spp. in soybean root nodules, which demonstrated as a rich niche for endophytic bacteria.In the detection of Plant-Growth-Promotion factors, there were more than 70% isolates can produce IAA and 14 isolates standing out as the most excellent ones according to the degree of red color. There were 14 isolats can produce siderophore effectively, 8 isolates with diameter ratio of solubilize phosphorus to colony between 1.85 and 3.45. Isolates that can produce siderophore or solubilize phosphorus are mostly belonging to Bacillus genus, while those that can produce IAA are mainly belonging to Alcaligenes genus. In the plant test, there was no signifcant difference existed among control and all treatments. All plants inoculated by rhizobia or together with endophytica bacteria can produce root nodules, but no significant different among treatments. The height of plants inoculated by endophytic bacteria without Plant-Growth-Promotion factors were longer significantly than that inoculated together by these endophytic bacteria and one rhizobium isolates(P<0.041); while when the plants were inoculated by 5 rhizobium isolates, the height were longer significantly than that inoculated together with non-PGP endophytic bacteria(P<0.043) or only inoculated by only one rhizobium isolate(P<0.004); the height of plants inoculated by 5 rhizobia and PGP endophytic bacteria together were longer significantly than that only inoculated by only PGP endophytic bacteria(P<0.009). According to the present results, it seems that the plants inoculated by more than one rhizobium can possess longer height, but non with only one rhizobium. If the so called PGP endophytic bacteria can promote plants growth is not clear, at least in this study. Not all endophytic bacteria which can produce PGP factors can lead to the increase of plant height or fresh weight or number of root nodules. From the plant test, we can see that the relationships between endophytic bacteria and plant are complicated, and we should be very careful and more experiments in studying their relationship and find the most efficient combination.The CCA analysis between the genotypes of 231 symbiotic or non-symbiotic and 11 kinds of environment factors revealed that: the genotypes distributed in three eco-regions differentiate significantly, and soil p H value is the most significant influenced factor.BN30T, is a gram-negative bacterium with well-distributed wrinkles in cell surface. Through DNA-DNA relationship analysis, 16 Sr RNA sequence alignment and phylogenetic tree constructing with closest neighbors, fatty acid component analysis, polar lipids, quinone and phenotypic character analysis, we proposed that BN30 T represent a new member in Diaphorobacterand named as Diaphorobacter ruginosibacter, which means a rod-shaped bacterium with wrinkled cell surface.