Isolation and Phylogeny of Nitrogen-Fixing Endophytic Bacteria in Wheat,Rice,Maize,Chinese Cabbage and Celery

【Objective】 The objective of this study is to determine the main groups and phylogenetic position of nitrogen-fixing endophytic bacteria in wheat,rice,maize,Chinese cabbage and celery.【Method】 Surface sterilization and nitrogen-free medium were used to isolate nitrogen-fixing endophytic bacteria.Acetylene reduction assay was used to measure the nitrogenase activity.16S rDNA was amplified by PCR and 16S rDNA sequence was analysed with MEGA software.【Result】 Thirty four nitrogen-fixing endophytic bacteria with nitrogenase activity ranging from 0.30 to 30.24 nmol C2H4/h?mg protein were isolated from field wheat.Based on 16S rDNA sequence similarity,these strains belonged to 21 species of 13 genera,including Pseudomonas,Rhizobium,Bacillus,Flavobacterium,etc.Twenty five nitrogen-fixing endophytic bacteria with nitrogenase activity ranging from 3.12 to 254.12 nmol C2H4/h?mg protein were isolated from field rice.They belonged to 16 species of 9 genera,including Bacillus,Burkholderia,Enterobacter,Klebsiella,etc.Moreover,Burkholderia,Enterobacter and Klebsiella were dominent.Nine maize endophytic diazotrophs with nitrogenase activity ranging from 7.27 to 59.58 nmol C2H4/h?mg protein were identified as 6 species of genera Rhizobium,Sphingomonas,Arthrobacter,Brevibacterium and Microbacterium.Fourteen endophytic diazotrophs with nitrogenase activity ranging from 2.33 to 205.21 nmol C2H4/h?mg protein were isolated from pot trial pakchoi and identified as 8 species of 6 genera of Rhizobium,Arthrobacter,Pseudomonas,etc.Ten endophytic diazotrophs with nitrogenase activity ranging from 1.23 to 46.70 nmol C2H4/h?mg protein were isolated from marcket celery and identified as 8 species of 5 genera of Sphingomonas,Pseudomonas,Brevundimonas,etc.【Conclusion】 Nitrogen-fixing endophytic bacteria colonized widely in wheat,rice,maize and vegetables.Their nitrogenase activity under pure culture ranged from 0.30 to 254.12 nmol C2H4/h?mg protein.Ninety two endophytic diazotrophs belonged to 56 species of 25 genera,including Pseudomonas,Rhizobium,Bacillus,Burkholderia,Sphingomonas,Enterobacter and Klebsiella,etc.These endophytic diazotrophs have a great potential to agriculture.