Diversity Of Root Endophytic Bacteria And Identification Of Promoting Growth Bacteria In Corn And Soybean

Endophytic bacteria are an important component of plant micro-ecosystem,and the distribution varies among different plant organs.The endophytic bacteria in plant roots are more abundant than that in other plant organs,and are characterized with the host diversities and species diversities.In this study,root samples of corn（Zea mays cv.Xinken-5）and soybean(Glycine max cv.Dongsheng^-1)under different soil amendments and soil erosion were collected from an experimental field in Guangrong Village,Hailun City,Heilongjiang Province of Northeast China established by Monitoring and Research Station for Soil and Water Conservation,Northeast Institute of Geography and Agroeology,Chinese Academy of Sciences.We combined the LNA–PCR clamping and 454 bar-coded pyrosequencing methods in investigating the diversity and composition of endophytic bacteria in the roots of soybean and corn.We aimed to compare the change of root endophytic bacterial communities under soil erosion condition,and to evaluate the response of root endophytic bacteria to manure fertilizer,and established a method for the diversity of endophytic bacteria based on the LNA–PCR clamping and 454 bar-coded pyrosequencing.Besides,we also used traditional cultural methods to screen plant growth promoting endophytic bacteria,which provides material for developing of plant growth promoting strains.The main results were as follows.（1）The LNA oligonucleotides were used to PCR amplified of endophytic bacteria,and the primers without using LNA oligonucleotides were used as control.The result of 454 pyrosequencing showed that bacterial genes were dominantly observed in the PCR products amplified with LNA oligonucleotides,on the other hand,the sequences belong to chloroplast and mitochondria was 97.4% and 67.8% in corn and soybean,respectively.Samples amplified with LNA oligonucleotides had higher OTUs numbers,ACE,Chao1,Shannon index but lower Simpson index than those corresponding samples without LNA oligonucleotides.The results showed that LNA-PCR and 454 pyrosequencing methods could provide a much more detailed description to the plant endophytic bacteria.（2）The LNA-PCR clamping and 454 bar-coded pyrosequencing methods was used to compare the diversity of root endophytic bacteria between soybean and corn.In soybean sample,Pseudomonas,Bradyrhizobium and Flavobacterium were the most abundant three genera,whiletop two predominant genera were Streptomyces and Niastella for corn roots.The rare phylotypes and uncultured endophytic bacteria members detected in this study revealed the soybean and corn root endophytic communities were very complex.（3）The LNA-PCR clamping and 454 bar-coded pyrosequencing methods was used to study the corn root endophytic bacterial communities at seedling stage under 30 cm soil erosion and manure fertilization conditions.No topsoil removal（0 cm）and only chemical fertilizer treatment were used as control.The results showed that 37820 valid sequences of 16 S r RNA genes were obtained,mainly distributed in 4 phyla,35 classes,214 genera and 782 OTUs.The dominant phyla were Proteobacteria、Firmicutes、Actinobacteria and Bacteroidetes,but the proportion varied in different samples.Alpha diversity indices of corn root endophytic bacteria were decreased in soil erosion condition.But in topsoil removal soil and no erosion soil,the diversity of corn root endophytic bacterial communities was increased by fertilizer application,and the effect was more obvious in 30 cm removal soil.（4）The density of root endophytic bacteria under different soil erosion,sampling dates and soil amendments of soybean and corn was investigated by plate counting method.The result showed that the population of root endophytic bacteria ranged from 0.79×104 to 19.95×104 CFU·g^-1·fresh weight irrespective of crops,sampling dates and soil amendments after incubation in 1/10 NA at 30 °C for 7 days.The population of endophytic bacteria on 10 July was greater than samples on 22 June and 24 August in soybean and corn roots,respectively,and the density of endophytic bacteria under soil erosion condition was greater than no topsoil removal condition.（5）119 and 277 bacterial isolates were isolated from soybean and corn roots,respectively,according to the culture-dependent method.39.6% of the total isolates showed IAA production in the range of 1²3 mg·L^-1 in culture medium supplemented with tryptophan.Fourteen isolates had the capacity of producing IAA over 10 mg·L^-1.Based on 16 S r RNA gene sequence analysis,the fourteen isolates were closely related to Psychrobacillus,Microbacterium,Lysinibacillus and Bacillus.Pot experiment indicated that the growth-promoting effects varied among these 14 bacterial strains and not all of the strains were able to promote growth of the tested soybean and wheat plants.Strains Microbacterium sp.C4 and Lysinibacillus sp.C7 showed a better performances in promoting soybean and wheat seedling growth.（6）Twelve antagonistic isolates were screened from 396 strains of endophytic bacteria obtained by the method of confrontation traing.All strains had broad spectrum antagonism against Rhizoctonia solani,and the radius of inhibition zone was from 1.4 cm to 3.5 cm.12 strains were also antagonism against Fusarium oxysporum f.sp.cucumerinum and Fusarium oxgsporum f.sp.lycopersici.Based on 16 S rRNA gene sequence analysis,the twelve isolates were closely related to Bacillus,Paenibacillus and Pseudomonas.Five isolates were selected for pot experiment,and the results showed that these isolates were beneficial for controlling soybean root rot disease and also promote the growth of soybean seedling.（7）IAA-producing strain C9^T was characterized using a polyphasic approach.The strain C9^T was determined to be most closely related to Lysinibacillus.chungkukjangi NBRC 108948^T（98.1 % 16 S r RNA gene sequence similarity）and Lysinibacillus sinduriensis DSM 27595^T（98.0 %）,and the 16 S r RNA gene sequence similarities between strain C9^T and other type strains of the genus Lysinibacillus were less than 98 %.Phylogenetic analysis based on 16 S rRNA gene sequences showed that strain C9^T formed a clade with its closely related strains in the neighbourjoining tree.The tree topology in this region is supported by the maximum-parsimony and maximum-likelihood methods.Morphological,physiological and biochemical characteristics,Chemotaxonomic characterisation and molecular analysis demonstrated that strain C9^T represents a novel species of the genus Lysinibacillus,for which the name Lysinibacillus endophyticus sp.nov.is proposed.The type strain is C9^T（=DSM 100506^T=CGMCC 1.15291^T）.