Composition,Diversity And Isolation Of Nitrogen Fixation Bacteria In Three Types Of Grasslands

Inner Mongolia grassland acrosses the arid,semi-arid,and semi-humid regions of northern China from east to west,which has widely geographical representation.However,the studies focus on the characteristics of nitrogen-fixing bacterial community composition and its driving environmental factors in this grassland is insufficient.In this study,the three types of grassland in Inner Mongolia,included meadow steppe(XWQ),typical steppe(MDMC),and desert steppe(SHTL)were taken as research objects.Soil physical and chemical properties of different types of steppes were determined.Fluorescence quantitative PCR(q PCR)and high-throughput sequencing approach with nifH gene as molecular were used to analyze the abundance of nifH genes,diversity and community composition of nitrogen-fixing bacterial community and their relationship with environmental factors.In addition,we try to isolate the nitrogen-fixing bacteria using traditional isolation approach.The research results obtained are as follows:(1)Qualitative PCR analysis of nifH gene showed that the nifH gene abundances in three types grassland was decreacing in the order of XWQ(5.78×10⁵-6.84×10⁵ copies·g^-1),MDMC(1.59×10⁵-2.94×10⁵copies·g^-1)and SHTL(1.38×10⁵-1.92×10⁵copies·g^-1).In addition,the nifH gene abundance of meadow steppe was significantly higher than that of the other two grasslands.(2)The results of high-throughput sequencing showed that Alphaproteobacteria(48.18%-77.12%)was the main group of nitrogen-fixing bacteria in the three type of steppe,and Azospirillum(34.10%-70.54%)was the most dominant genus.Other dominant nitrogen-fixing bacteria including Bacillus(8.30%-13.63%),Azohydromonas(0.25%-19.83%)and Bradyrhizobium(0.48%-1.95%).The Shannon index and Chao1 index of XWQ were significantly higher than those of MDMC and SHTL(P<0.05),indicating that the diversity of nitrogen-fixing bacterial community in meadow steppe was different from that in the other two steppes.The community composition of nitrogen-fixing microbes in three grassland types has regional characteristics,and there are significant differences in phylogenetic distance(P<0.05).(3)Pearson correlation analysis between gene abundance and community structure of nitrogen-fixing microorganisms and environmental factors showed that TN,nitrogen to phosphorus ratio(N/P),SOC and annual average precipitation were significantly positively with nifH gene abundance(P<0.01),with a linear correlation.The pH value and annual average temperature were negatively correlated with the abundance of nifH gene(P<0.01),while redundancy analysis(RDA)showed that SOC,N/P and pH value are the main physical and chemical environmental factors affecting nitrogen-fixing microbial community.(4)The nitrogen-deficient medium was used to isolate nitrogen-fixing bacteria from different types of grassland soils.A total of 63 bacteria were isolated.The results of 16S rRNA gene sequencing showed that the isolated strains belonged to 4 phyla,including Proteobacteria(42%),Firmicutes(18%),Actinobacteria(39%)and Bacteroidetes(1%).Among them,there were 14 strains with nifH gene,including Bradyrhizobium and Mesorhizobium,which detected in high-throughput sequencing.This study was chosen nifH gene as the marker of nitrogen-fixing microbial groups,and high-throughput sequencing technology and quantitative PCR technology,combined with microbial culture technology,were used to clarify the amount,community composition and diversity characteristics of nitrogen-fixing bacteria in three types of grassland in Inner Mongolia.The relationship between grassland type,environmental factors,microbial community composition and diversity was analyzed,which laid a scientific foundation and theoretical basis for further study on nitrogen input process and law of grassland ecosystem in Inner Mongolia,and also provided strain resources for grassland restoration and sustainable development in Inner Mongolia.