Diversity And Phylogeny Of Endophytes Isolated From Noudules Of Caragana Jubata And Oxytropis Ochrocephala In Qilian Mountains

The association between legume plants and rhizobia is quite old and enables the plant tosurvive nutrient poor conditions because of the nitrogen fixation capacity of the rhizobia thatreside inside their root nodules. Legumes are a large family which occurs almost everywhereon earth and is divided into three subfamilies, the Caesalpinioideae, Mimosoideae andFaboideae. In China most legumes occur from this last subfamily. The aim of this study wasto make an in-depth analysis from the diversity of root nodule endosymbionts present in twoindigenous legumes (Caragana jubata and Oxytropis ochrocephala) in Qilian Mountains.A total of201endophytic bacteria associated to root noudules collected from twoindigenous legumes in different altitude of Qilian Mountains (Hexi Corridor) werecharacterized by16S rDNA polymerase chain reaction (PCR)-restriction fragment lengthpolymorphism,16S rRNA gene sequence analysis and enterobacterial repetitive intergenicconsensus-PCR clustering. Phylogenetically, these isolates belonged to35species in thegenera Phyllobacterium, Ensifer, Rhizobium, Microvirga, Sphingomonas, Paracoccus,Mycobacterium, Paenibacillus, Cohnella, Sporosarcina, Bacillus, Staphylococcus,Brevibacterium, Xenophilus, Erwinia, Leclercia, Acinetobacter and Pseudomonas. Thisrevealed the presence of on the one hand traditional rhizobia (50%), Phyllobacterium,Sinorhizobium and Rhizobium and on the other hand non-rhizobial endophytes belonging toseveral genera in the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria,Actinobacteria, Firmibacteria and Sphingobacteria. The non-rhizobial endophyte populationwas dominated by Bacillus, Xenophilus and Acinetobacter species. Besides, eight new groupswith sequence similarity below97%were found within the genera Rhizobium, Microvirga,Mycobacterium, Cohnella, Paenibacillus, Sporosarcina, Bacillus and Xenophilus.The symbiosis genes nodA and nifH were investigated for a selection of strains whichrevealed the presence of different symbiovars in traditional rhizobia including Sophoraalopecuroides, Medicago sativa, Phaseolus vulgaris, Pisum sativum, Vigna unguiculata,Trifolium repens, Glycyrrhiza max and Galega oficinalis. As a result, only thirty-four stainscould form nodules with the host plants. Additionally, Phylogenetic sequence analysis ofnodA showed higher similarity with those of Sinorhizobium meliloti within strains related tothe genera Rhizobium, Acinetobacter and Sinorhizobium. NifH sequences revealed strains belonged to Rhizobium, Acinetobacter, Phyllobacterium and Sinorhizobium had similar nifHgene with Sinorhizobium. Two representive strains belong to Xenophilus and Acinetobacterformed a novel nifH type, which distinct from any previously described symbiovar withinMesorhizobium. This indicate that horizontal gene transfer could have occurred betweenrhizobia and non-rhizobial endophytes. Moreover, the elevation and the host plant types didcontribute to the separation of the bacterial endosymbionts. PGP test showed most strainshave the ability of production of IAA, siderophore and antifungal activity.Four strains isolated from nodules of Oxytropis ochrocephala were classified in thegenus Rhizobium on the basis of their16S rRNA gene sequences. These strains have identical16S rRNA gene sequences, except for one strain with one base-pair different from the others.Their sequences showed a mean similarity of94.4%with the most closely related species,Rhizobium oryzae. Analysis of recA and glnA sequences showed that these strains have lessthan88.1%and88.7%similarity with the defined species of Rhizobium, respectively. Thegenetic diversity revealed by ERCI-PCR fingerprinting indicated that the isolates correspondto different strains. Strain CCNWQLS01Tcontained Q-10as the predominant ubiquinone. Themajor fatty acids were feature8(C18:1ω7c and/or C18:1ω6c;67.2%), Therefore, a novelspecies Rhizobium qilianshanense sp. nov. is proposed, and CCNWQLS01T(=ACCC05747~T=JCM18337~T) is designated as the type strain.This study provided valuable information about the interactions among the symbioticbacteria, nonsymbiotic bacteria and their habitats, thus increased the knowledge about theirgenetic diversity and ecology.