The Diversity Of Endophyte Isolated From Oryza Meridionalis And Bryophyllum Pinnatum And Description Of Novel Species

There are rich diversity of microbial taxa in plants,which have an indispensable positive effect on the growth of plants.In order to investigate biodiversity and physiological function of endophytic bacteria,two kinds of host plant were chosen as experiment materials,including Oryza meridionalis and Bryophyllum pinnatum.The endophytic bacteria were isolated and identified,and the endophytes were tested for various functions for growth promoting and disease resistance.The pure culture of endophyte was obtained by spread plate and streak plate methods from surface disinfectant plants,utilizing nitrogen-free culture medium.Genetic diversity of isolates was studied by SDS-PAGE of whole cell protein and insertion sequence-based PCR(IS-PCR)finger-printing at protein and molecular levels.The 16S r RNA gene phylogenetic analysis and physiological and biochemical tests were used to identify the representative strains of all clusters.Microbiological polyphasic taxonomy method were tested for strains with uncertain taxonomic status.The novel species were identified and described by the methods of 16S r RNA gene complete sequence similarity comparison,phylogenetic analysis,phenotypic and physiological characterization,Chemotaxonomic characteristics,DNA base composition and DNA-DNA hybridization etc..In addition,functional diversity of identified endophytic bacteria was studied by nitrogenase activity test,secretion of IAA and ACC deaminase test,siderophore production test,dissolving phosphorus and potassium test,and antagonistic plant pathogenic fungi test.The main results were as follows:(1)175 strains were isolated from 2 kinds of herbaceous plants totally,among which the strains number isolated from Oryza meridionalis and Bryophyllum pinnatum were 159and 16 respectively.All isolates were divided into 33 clusters and 7 single strains.There were 14 large clusters with more than 6 isolates.According to the preliminary identification results,the representative strains belong to 40 species of 31 genera,showing rich microbial diversity.There were 4 clusters with uncertain taxonomic status may be novel species and need further taxonomy study.(2)There were physiological function diversity of the endophytic bacteria in this paper.In 40 representative strains,thirty four strains could secrete IAA and produce siderophore.There were 13 strains could fix nitrogen,18 strains could secrete ACC deaminase,17 strains could dissolve phosphorus,16 strains could dissolve potassium,15strains could against plant pathogenic fungi.Some functional bacteria with excellent characters were screened out.Strains L011,L021,L031,L081,L251,L411,L421,L461^T,L501 had multinomial and efficient functions to promote plant growth.Strains L101?L171?L271?L381?L451 not only showed variety of growth promoting function,but also had antagonistic some plant pathogens.Strains L161 and L371 had strong growth promoting ability and wide spectrum antibacterial activity.They could fix nitrogen,secrete IAA and ACC deaminase,produce siderophore,dissolve phosphorus and potassium,and resist 6kinds of plant pathogens(Rhizoctonia solani,Fusarium.oxysporum f.sp.cubense,Fusarium arysperum f.sp.cudense,Colletotrichum graminicola,Fusarium oxysporum,Fusarium oxysporum f.sp.momordicae).(3)Strain L201^T was isolated from Bryophyllum pinnatum,identified to be a novel specie belong to the genus of Paenibacillus.Cells are Gram-negative,aerobic,peritrichous flagella,motile rods(0.8-1.0×3.5-6.5?m)and occur singly or in short chains.Circular,cream coloured,translucent,smooth and moist colonies on LB medium.Acetylene is reduced to ethylene.The major fatty acid is anteiso-C_15:0.The predominant menaquinone is MK-7.The major polar lipids are diphosphatidylglycerol,phosphatidylethanolamine and phosphatidylglycerol.The G+C content of genomic DNA of the type strain is 43.92±1.47mol%.Strain L201^T(=KCTC 33951^T=GDMCC 1.1251^T)is designed as the type strain for the species and named as Paenibacillus bryophyllum sp.nov..(4)Strain L461^T was isolated from Bryophyllum pinnatum,identified to be a novel specie belong to the genus of Azotobacter.Cells are Gram-negative,aerobic,oval endospores,peritrichous flagella,motile short rods(0.6-0.8×1.0-2.0?m)and occur singly or in pairs.Circular,cream coloured,translucent,smooth and moist colonies on LB medium.Acetylene is reduced to ethylene.The major fatty acid are C_16:1?9c and C_16:0.The predominant menaquinone is Q-9.The major polar lipids are phosphatidylethanolamine,phosphatidylglycerol,aminophospholipid,phospholipid and one unknown lipid.The G+C content of genomic DNA of the type strain is 64.93±2.80 mol%.Strain L461^T(=KCTC62195 ^T=GDMCC 1.1250 ^T)is designed as the type strain for the species and named as Azotobacter bryophylli sp.nov..(5)The polyphasic taxonomy results indicated that strain L251,isolated from Oryza meridionalis,may represent a novel species belong to the genus of Azospirillum.The strain L251 showed high similarity with Azospirillum brasilense and Azospirillum formosense.This paper proved that L251 and Azospirillum brasilense ATCC 49958^T were not the same species.Closely related species of strain L361 belonged to 3 different genera.The 16S r RNA similarity values of them were more than 99%,while physiological and biochemical characteristics,whole cell fatty acid composition and DNA G+C mol%content were different from each other.L361 may be a novel species of actinomycetes Microbacteriaceae.