Study Of Isolation And Diversity Of Rhizospheric And Endophytic Nintrogen-fixing Bacteria From Different Parts Of Maize

Clarifying the composition and function of the root-associated microbiomes is crucial to understand the interaction of plant and microbes,but the challenge is the complexity of root system and the limitation of technology.Here,We collected six maize samples to isolate and identify maize endophytic diazotrophs and find the advantages.we examined the phylogenetic of bacterial(16S rRNA gene)?fungus(ITS1gene)and functional gene(nifH)differences among different compartments,the endophytic(root?stem ?leaf),rhizosphere(soil close to the root surface)of mature maize using Illumina MiSeq? qPCR?DGGE technologies.Results revealed that:(1)Isolation?identification and construct phylogenetic tree of maize endophytic diazotrophs: There were 153 bacterias were isolated from maize root,stem,leaf and rhizosphere.After amplifying sequencing and blasting of 16 s rRNA gene,we found that these strains are from 24 generas.Enterobacter;Flavobacterium;Klebsiella;Pseudomonas;Kosakonia;Rhizobium are dominate generas.Biolog experiment shows that most of the tested strains could grow using starch as carbon source.There are 8 endophytic diazotrophs which can produce ACC deaminase,its activity are 0.04-5.388 umol/mg/h.(2)qPCR?RT-PCR?DGGE and high-throughput sequencing of nifH gene amplicons are used to analyze the diversity and activity of rhizospheric diazotrophs and endophytic diazotrophs.The diversity indices of rhizosphere soil was higher than endospher,Pseudacidovorax,Klebsiella,Enterobacter are the dominant genus.The diversity on DNA level is more higher than cDNA level.Enterobacter,Klebsiella,Bradyrhizobium,Burkholderia are the dominant genus on the cDNA level.(3)Based on 16 S rRNA gene high-throughput sequencing technique,sequencing produced 606833 high-quality reads by preprocessing and were assigned to individual operational taxonomic units(OTUs)at 3% dissimilarity.Subsequently,a total of 3222 OTUs were generated and belonged to 31 phyla,468 genera in these 24 samples.At the phyla level,Proteobacteria(46.2%)was the dominant phyla.At the genus level,Acinetobacter(33.4%)and Pseudomonas(26.1%)were the dominant genus,respectively.The rhizosphere soil has the highest diversity indices,significantly higher than endosphere.The potential function of the root-associated microbiomes were mainly involved in metabolism,cellular processes and environmental information processing.Redundancy analysis(RDA)revealed that the sample changes of microbial community structure in rhizosphere soil pond were closely relevant to their environmental factors.(4)Based on ITS high-throughput sequencing technique,sequencing produced 518064 high-quality reads by preprocessing and were assigned to individual operational taxonomic units(OTUs)at 3% dissimilarity.Subsequently,a total of 2295 OTUs were generated and belonged to 12 phyla,240 genera in these 24 samples.At the phyla level,Ascomycota(22%)was the dominant phyla.At the genus level,Fusariumwas was the dominant genus.The rhizosphere soil has the highest diversity indices,significantly higher than endosphere.RDA revealed that the sample changes of microbial community structure in rhizosphere soil pond were closely relevant to their environmental factors.