小麦、水稻、玉米、白菜、芹菜内生固氮菌及其系统发育

【目的】了解小麦、水稻、玉米、白菜、芹菜内生固氮菌的主要类群，确定内生固氮菌的系统发育地位。【方法】样品表面灭菌后采用无氮培养基分离、培养内生固氮菌，乙炔还原法测定菌株固氮酶活性；PCR扩增得到菌株16S rDNA，通过序列测定和相似性分析研究菌株的系统发育。【结果】从大田小麦体内分离到内生固氮菌34株，固氮酶活性在0.30—30.24 nmol C2H4/h?mg蛋白，基于16S rDNA序列最大相似性，这些菌株分属于假单胞菌（Pseudomonas）、根瘤菌（Rhizobium）、芽孢杆菌（Bacillus）、黄杆菌（Flavobacterium）等13属21种，种群分布较为广泛；从大田水稻体内分离到内生固氮菌25株，固氮酶活性在3.12—254.12 nmol C2H4/h?mg蛋白，属于芽孢杆菌、伯克霍尔德氏菌（Burkholderia）、肠杆菌（Enterobacter）、克雷伯氏菌（Klebsiella）等9属16种，伯克霍尔德氏菌、肠杆菌和克雷伯氏菌是水稻内生固氮菌特有种群；从大田玉米体内分离到内生固氮菌9株，固氮酶活性在7.27—59.58 nmol C2H4/h?mg蛋白，属于根瘤菌、鞘氨醇单胞菌（Sphingomonas）等5属6种；从盆栽试验小白菜体内分离到内生固氮菌14株，固氮酶活性在2.33—205.21 nmol C2H4/h?mg蛋白，属于根瘤菌、节杆菌（Arthrobacter）等6属8种；从市售芹菜体内分离到内生固氮菌10株，固氮酶活性在1.23—46.70 nmol C2H4/h?mg蛋白，属于鞘氨醇单胞菌、假单胞菌等5属8种。【结论】在生长期小麦、水稻、玉米和部分蔬菜体内普遍存在内生固氮菌，菌株固氮酶活性差异较大，在0.30—254.12 nmol C2H4/h?mg蛋白，系统发育地位分属于假单胞菌、根瘤菌、芽孢杆菌等25个属的56个种，这些内生固氮菌对于农业生产有巨大潜能。

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.