| Wild rice is the ancestor of cultivated rice and an important gerpmlasm resource.Wild rice is also a valuable functional microbial resource pool.In order to study the diversity of endophytic bacteria in Oryza meridionalis,rich microbial fertilizer reserve bank,provide the basis for the selection of bacterial strains.In this study,Oryza meridionalis was used as the separation material.The isolation and purification of endophytic bacteria from Oryza meridionalis were carried out by using DN and CCM two media without nitrogen source,and a plate method.Determination nitrogenase activity of strains by acetylene reduction method,nif H gene amplification of nitrogen fixing bacteria,nitrogen fixation activity was further determined by molecular techniques,in order to screen the endophytic nitrogen fixing bacteria.IS-PCR fingerprints and SDS-PAGE whole cell protein electrophoresis cluster analysis were used to isolate the endophytic bacteria.Based on the analysis of 16S rRNA gene sequences,the phylogenetic relationships of representative strains were determined.Detection of dissolved phosphorus and potassium in endophytic bacteria by transparent circle method,salkowski colorimetric assay was used to detect the growth ability of each strain.The activity of ACC deaminase was tested quantitatively by 2,4-dinitrophenylhydrazine colorimetry.The method was used to detect whether the strains had the characteristics of siderophore production by iron?CAS?.A pot experiment was carried out to study the effect of high efficiency nitroge n fixing bacteria on rice growth.The results are as follows:?1?The abundance of endophytic bacteria in wild rice plants.In this study,58endophytic bacteria were isolated from the Oryza meridionalis,IS-PCR fingerprinting and SDS-PAGE of 58 endophytic bacteria were clustered into 11 groups,The sequence analysis of 16S gene of representative strains r RNA,the homology of 16S r RNA gene was 99.17%between group I and Kosakonia sacchari SP1T,99.88%between group II and Sphingomonas trueperi LMG 2142T,100%between group III and Burkholderia contaminans LMG 23361T,100%between group IV and Bacillus aryabhattai B8W22T,100%between group V and Rhizobium pusense NRCPB 10T,99.76%between group VI and Klebsiella pneumoniae subsp.rhinoscle romatis ATCC13884T,99.88%between group VII and Brevundimonas aurantiaca DSM 4731T,99.32%between group VIII and Citrobacter bitternis SKKUI-TP7T,99.88%between group IX and Burkholderia vietnamiensis LMG 10929T,99.65%between group X and Herbaspirillum seropedicae DSM 6445T,99.88%between group XI and Rhizobium radiobacter ATCC 19358T.The results showed that the endophytic bacteria of Oryza meridionalis were diversity,the strain resources were collected for microbial fertilizer production.?2?The results showed that 58 strains of endophytic bacteria had nitrogenase activity by the method of acetylene reduction and the amplification of nif H gene of the 58 strains of endophytic bacteria,nitrogenase activity distribution between 14.82494.14 nmol·mL-1·h-1,the nitrogenase activity of JH40 and JH50 are the highest,respectively 360.61nmol·mL-1·h-1,494.14nmol·mL-1·h-1.?3?Isolates were obtained from 2 taxa representing the strains JH40 and JH50,which were Citrobacter bitternis,Herbaspirillum seropedicae,JH40 and JH50 have the activity of nitrogenase,which can dissolve phosphorus and potassium,and secrete indole acetic acid,which has the ability of producing iron carrier and producing ACC deaminase,JH40 and JH50 have IAA activity were 27.84mg·L-1,29.97 mg·L-1,ACC eaminase activity were24.76 mol·mg-1·h-1 and 22.94 mol·mg-1·h-1.?4?It was found that JH40 had the ability of fixing nitrogen,releasing potassium producing iron carrier and producing ACC deaminase.The Citrobacter bitternis was carried out to promote the growth of rice.?5?The results of JH40 and JH50 rice pot experiment showed that all of them could promote the growth of rice,and the fresh weight increased by 32.97%and 40.99%,respectively,and the dry weight increased by about 63.06%,plant height,ear length,tiller number were significantly different.The above results show that JH40 and JH50 have strong growth promoting ability,they have a strong potential to promote rice growth,can be used as a microbial fertilizer reserve bacteria,and is expected to play a catalytic role in the development of green agriculture. |
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