Genetic Diversity Of Endophytic Bacteria And Their Potential For Promotion Of Plant Growth Within Nodules Of The Sphaerophysa Salsula Grown In Horthwestern China

Sixty-five endophytic or non symbiotic and 50 symbiotic strains were isolated from root nodules on four different media, which were collected from the spontaneous legume Sphaerophysa salsula grown at six sampling sites in two ecological regions (Gansu province and Ningxia Autonomous Region) of northwestern China. The genetic diversity and phylogeny of the isolates was revealed by 16S rRNA gene RFLP and sequencing, ERIC-PCR and nifH gene sequence analysis. A relationship between the bacterial genotypes and their geographical origin was detected by princial component analysis and correspondence analysis, demonstrating the biogeography of symbiotic and endophytic bacteria in root nodules. Characterization of the plant growth promoting properties of endophytic bacteria:their effect on the growth and nodulation of Sphaerophysa salsula.Twenty-five different RFLP patterns of 16S rRNA genes were obtained from the 115 isolates. Phylogenetically, the symbiotic bacteria belonged to the genera Rhizobium, Sinorhizobium and Mesorhizobium, while the endophytic bacteria were Paracoccus, Sphingomonas, Inquilinus, Pseudomonas, Serratia, Mycobacterium, Nocardia, Streptomyces, Paenibacillus, Brevibacillus, Staphylococcus, Lysinibacillus and Bacillus. These results demonstrated the universal existence of endophytic bacteria in the nodules.The alignments of their nearly full-length (1337-1491 nucleotides) 16S rDNA sequences indicated that the 65 endophytes had a high level of genetic diversity and belonged to three different phyla:the Proteobacteria (Gram-negative), the Actinobacteria and the Firmicutes (Gram-positive). Within the Proteobacteria,4 isolates belonged to the a-class(Paracoccus, Sphingomonas, Inquilinus) and 11 isolates to the y-class(Pseudomonas and Serratia). The 6 isolates grouped within the Actinobacteria were related to Mycobacterium, Nocardia and Streptomyces, all belonging to the order Actinomycetales. The 45 isolates grouped in the phylum Firmicutes were identified into Paenibacillus, Brevibacillus, Staphylococcus, Lysinibacillus and Bacillus. Bacillus was an epidemic nodule endophyte that was isolated from the six sampling sites, accounted for 58.5% of all the endophytic bacterial isolates within nodules of Sphaerophysa salsula and being predominant. Phylogenetic analysis based on 16S rRNA gene sequences showed that 38 of them belonged to 5 species of the genera Bacillus(B. simplex, B. safensis, B. cereus, B. pumilus, B. licheniformis). And the species Serratia plymuthica, Lysinibacillus fusiformis and Staphylococcus saprophyticus were never previously recorded as nodule endophytes. Mesorhizobium which accounted for 64% of all the rhizobia as the predominant genera associated with this wild legume, and they were phylogenetically related to the species M. amorphae, M. gobiense, M. tianshanense and M. mediterraneum.ERIC-PCR resulted in specific PCR bands, from 6 to 15 for each strain and with fragments sized from 0.2 to 2.0 kb. Among the 115 isolates 57 ERIC types were identified. The isolates were grouped into 17 groups at the similarity level of 83%. The 50 symbiotic isolates were divided into nine groups (1-9) and 3 single isolates, and the 65 endophytic isolates were divided into eight groups (10-17) and 4 single isolates. The ERIC-PCR grouping was consistent with the 16S rRNA gene RFLP, with some exceptions.The nifH genes were amplified from the Rhizobium, Mesorhizobium strains and Bacillus whose sequence most similar to Mesorhizobium albiziae. They showed close relationships despite their chromosomal background, implying that lateral gene transfer might have happened, these transfers have occurred between different genera and the host legume might have a strong selection on the symbiotic gene background of rhizobia.A study on the Principal Component Analysis (PCA) was performed. According to our results, most of the ERIC types were restricted in their isolation sites, the distribution of nodule endophytic bacterial communities depends on their adaptation to the ecosystem, and is mainly related to C/N, soil pH and altitude. C/N, soil pH and altitude were the main impacting factors on the composition and distribution of nodule endophytic and symbiotic bacterial communities. These results confirmed that the genomic backgrounds of nodule endophytic bacteria were related to the geographical origin.According to correspondence analysis, six sampling sites had the wide genotypic diversity. The communities of endophytic and symbiotic bacteria in Sphaerophysa salsula nodules can be grouped into three ecological clusters according to their geographic origins: two were from sampling sites of Gansu province and the last included strains from Ningxia Autonomous Region. The high correlation between the geographic regions and the bacterial genotypes may be attributed to the different environmental factors and soil characteristics of the sampling sites.In the present study, our data indicated that the 7 different endophytic bacteria genera could coexist with Rhizobium in Minqin, Qingtongxia and Zhangye,4 with Sinorhizobium in Qingtongxia and 13 with Mesorhizobium in six sampling sites. Furthermore, the high isolation frequency of Bacillus genus (0.833) demonstrated that the coexistence of endophytic and symbiotic bacteria were common in root nodules of Sphaerophysa salsula. Under pot culture conditions,13 out of the 17 representative endophytic isolates showed plant growth promoting activity with respect to increase in various plant parameters:fresh weight, shoot length, chlorophyll content, nodule number and average nodule fresh weight per plant when co-inoculated with strain Mesorhizobium gobiense Zw-19. Qtx-10 inoculation enhanced the seeding fresh weight by 87.5%, the isolate Gt-10 enhanced the shoot length by 89.4%, respectively. Production of IAA, siderophore and antifungal activity was simultaneously exhibited by isolates of Qtx-10 and Gzn-9-1. Out of 20 representative endophytic isolates, only did treatment with Qtx-10, Gzn-9-1 and Zw-13-3 inoculation can result in deformed root hairs with bulging at the root tip in Sphaerophysa salsula.Cells of strain Zy-3T are aerobic, brown-pigmented, non-spore-forming, non-motile, Gram-negative rods. Phylogenetic analysis of the 16S rRNA gene indicated that the closest relative of this organism is Paracoccus halophilus HN-182T (96.6% sequence similarity). Based on phenotypic and genotypic studies, it is concluded that this endophytic isolate represents a novel species within the genus Paracoccus.Cells of type strain CCNWQtx-14T are Gram-negative, aerobic, motile, non-spore forming. Sequence similarities with its closed species Rhizobium giardinii and R. daejeonense is 95.8% and 95.6%, respectively. CCNWQtx-14T is classified in the genus Rhizobuim, and represents a novel species within the genus Rhizobuim.