The Biogeography Of Rhizobia And Comparative Metagenomic Analysis Of Microbes In Alfalfa Rhizosphere

Interactions between bacteria and roots are critical to the terrestrial ecosystem.Plants can release up to 20%of their photosynthate via rhizodeposition,which is critical in thriving and shaping the root and rhizosphere microbiome.This microbiome composed of commensals,symbionts and pathogens that will interact with the plant and soil,and determines much of biological and agricultural productivity.Among the complicated bacteria and roots interactions,the legumes-rhizobium nitrogen fixing system was one of the most studied field,symbiotic rhizobia colonize intracellularly in nodules and in which they obtain essential nutrients from the plant host and reduce atmospheric dinitrogen.The efficiency of rhizobium-legume symbiosis is modulated by abiotic factors and biotic factors like the specificity and effectiveness of indigenous populations of rhizobia in soil.However,very little is known about the structural and functional signatures of the rhizobial population and the whole microbial community in the complex environment of rhizosphere soil.Here we firstly investigated the diversity of rhizobia from both nodules and rhizosphere of alfalfa from farmlands and natural ecosystems along the Yarlung Zangbo River valley in the Tibetan Plateau.581 nodule isolates were characterized using BOX-AIR PCR fingerprinting,sequences of rpoB and nodulation gene nodC.They belong to four rpoB haplotypes of Sinorhizobium meliloti(rpoB-?,473 isolates;rpoB-?,95;rpoB-?,1;rpoB-?,10),Sinorhizobium medicae(1 isolate)and Rhizobium sp.(1 isolate).Re-inoculation experiments of the 32 representative strains suggested a great variation in their symbiotic performance on alfalfa.High-throughput sequencing of rpoB for 34 rhizosphere samples uncovered that the predominant rpoB-? in nodules also dominated among fourteen rpoB haplotypes of S.meliloti in rhizosphere soils.In addition to S.meliloti,more than 40 rhizobial species documented as microsymbionts of other legumes were also identified.Although a very low level of nucleotide diversity for S.meliloti was found among sampling sites,multivariate statistical analysis demonstrated a significant differentiation of total rhizobial community between farmlands and natural ecosystems.Moreover,edaphic conditions,especially pH and nitrogen content,were revealed as important deterministic factors shaping the observed beta-diversity of rhizobial community.To further analyze the effects of soil sources and former crops on the rhizosphere microbial communities,we designed the alfalfa-alfalfa continuous cropping and barley-alfalfa rotation cropping experiment in greenhouse,and by incorporating amplicon sequencing of 16S rRNA gene and rpoB,and metagenomic sequencing,we surveyed the structural and functional differentiation of rhizosphere microorganisms of alfalfa,and compared it to Tibetan highland barley(THB).The structural differentiation of rhizosphere microbial communities shown no significant differences in greenhouse seedlings,bacterial taxa like Comamonadaceae,Rhizobiaceae and Planctomycetaceae were enriched in the rhziosphere soil of both plants,while bacterial taxa of Gemmatimonadetes and Xanthomonadaceae shown the opposite pattern.Remarkably,as revealed by rpoB sequencing,lots of rhizobial species like R.tibeticum?R.giardinii and R.herbae were also significantly enriched in rhizosphere of non-leguminous plant THB,and a few rhizobial species like R.leguminosarum and alfalfa microsymbiont S.meliloti were significantly higher abundant in rhizosphere soils of alfalfa,independent of plant age and growing environment.Continuous cropping can largely weaken the niche differentiation of the total microorganisms as well rhizobium populations in rhizosphere of alfalfa,depending mainly on the abundance of the source bacteria in the soil,as well as the cropping sequence its self.In contrast to rotation crops,more metabolic pathways involved in the biosynthesis of vitamins,antibiotic,siderophore,and phytohormone like flavonoids,gibberellins,cytokinins and abscisic acid were enriched in rhziosphere soils of continuous alfalfa.Enzymes involved in the degradation of carbohydrates,fatty acids and amino acids were enriched in the rhizosphere of crop rotation alfalfa,just in the opposite direction,many enzymes related to biosynthesis of these substances are enriched in the rhizosphere of continuous alfalfa.These findings indicated that the rhizospheric pool of rhizobial germplasms was significantly affected by land tillage compared to that of natural ecosystems.The enrichment of rhizobial species in the rhizosphere is not specific to leguminous plants,while a strong selection of symbiotic partners by alfalfa,leading to a population bottleneck effect on the diversity of these facultative microsymbionts in soils.Metabolic pathways involved in metabolisms of carbohydrates,fatty acids and amino acids,and host-microbe,microbe-microbe interaction were enriched in rhizosphere associated bacteria taxa.Plant host and soil source are the main factors that affect the structural differentiation of microbial community in the rhizosphere of plants,while the functional differentiation of microbial community can be varied or even opposite between different cropping sequences.