小麦、水稻、玉米、白菜、芹菜内生固氮菌及其系统发育  

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

作　　者：孙建光[1] 罗琼[1] 高淼[1] 胡海燕[1] 徐晶[1] 周义清[1] 

机构地区：[1]中国农业科学院农业资源与农业区划研究所/农业部作物营养与施肥重点实验室,北京100081

出　　处：《中国农业科学》2012年第7期1303-1317,共15页Scientia Agricultura Sinica

基　　金：国家"863"计划项目(2009AA06Z316);国家行业科研专项(200803033-A002G);国家公益性行业(农业)科研专项(201203045)

摘　　要：【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。【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,

关 键 词：小麦 水稻 玉米 白菜 芹菜 内生固氮菌 系统发育 

分 类 号：S182[农业科学—农业基础科学]