|本期目录/Table of Contents|

[1]谭志远,傅琴梅,彭桂香,等.青香茅和五节芒内生固氮菌的分离与生理生化鉴定[J].应用与环境生物学报,2013,19(04):643-649.[doi:10.3724/SP.J.1145.2013.00643]
 TAN Zhiyuan,FU Qinmei,PENG Guixiang,et al.Identification and Characterization of Endophytic Diazotrophs Isolated from Cymbopogon caesius and Miscanthus floridulus[J].Chinese Journal of Applied & Environmental Biology,2013,19(04):643-649.[doi:10.3724/SP.J.1145.2013.00643]
点击复制

青香茅和五节芒内生固氮菌的分离与生理生化鉴定()
分享到:

《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
19卷
期数:
2013年04期
页码:
643-649
栏目:
研究论文
出版日期:
2013-08-25

文章信息/Info

Title:
Identification and Characterization of Endophytic Diazotrophs Isolated from Cymbopogon caesius and Miscanthus floridulus
作者:
谭志远傅琴梅彭桂香原红娟程艳波江院
(1华南农业大学农学院 广州 510642)
(2华南农业大学资源环境学院 广州 510642)
Author(s):
TAN Zhiyuan FU Qinmei PENG Guixiang YUAN Hongjuan CHENG Yanbo JIANG Yuan
TAN Zhiyuan, FU Qinmei, PENG Guixiang, YUAN Hongjuan, CHENG Yanbo, JIANG Yuan
关键词:
乙炔还原法全细胞蛋白电泳IS-PCR内生固氮菌固氮酶活性微生物多样性青香茅五节芒
Keywords:
(1College of Agriculture South China Agricultural University Guangzhou 510642 China)(2College of Resources and Environment South China Agricultural University Guangzhou 510642 China)
分类号:
Q948.12+2.3 : Q939.11+3
DOI:
10.3724/SP.J.1145.2013.00643
文献标志码:
A
摘要:
为研究青香茅(Cymbopogon caesius)和五节芒(Miscanthus floridulus)内生固氮菌的多样性,基于两种无氮培养基结合乙炔还原法从这两种牧草中分别分离到30株和36株菌. 采用SDS-PAGE(Dodecyl sulfate sodium salt - polyacrylamide gel electrophoresis)全细胞蛋白电泳和IS-PCR(Insertion sequence-based PCR)指纹图谱将这66株内生固氮菌聚类为10个类群(I-X,每群3株以上,共61个聚群菌株)和3个未归类小组(共5个未聚群菌株). 从10个聚类群和3个未归类小组中各选1株菌作为代表进行16S rDNA序列分析,发现它们与洋葱伯克霍尔德菌(Burkholderia cepacia)、阴沟肠杆菌(Enterobacter cloacae)、固氮植物菌(Phytobacter diazotrophicus)、阿氏肠杆菌(Enterobacter asburiae)、织片草螺菌(Herbaspirillum seropedicae)、阪崎克洛诺菌(Cronobacter sakazakii)、水稻肠杆菌(Enterobacter oryzae)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、溶解肠杆菌(Enterobacter dissolvens)、无乳固氮螺螺(Azospirillum amazonense)、都柏林克罗诺杆菌(Cronobacter dublinensis)等的相似性分别达97%以上,表明所分离内生固氮菌多样性高. 进一步对它们进行生理生化测定,结果表明有些菌株具泌氨能力、产吲哚、解磷、解钾效果,在农业生产上具有一定的应用潜力. 图2 表3 参38
Abstract:
In order to investigate the diversity of endophytic diazotrophs in Cymbopogon caesius and Miscanthu floridulus, thirty and thirty-six isolates were obtained respectively from these two forage grasses by using two selective medium without nitrogen sources in combination with acetylene reduction assay. The sixty-six endophytic diazotrophs were assigned to 10 groups (61 strains, at least 3 for each group from I to X) and five ungrouped strains by analysis of SDS-PAGE (dodecyl sulfate sodium salt - polyacrylamide gel electrophoresis) patterns of whole-cell protein electrophoresis and IS-PCR (Insertion sequence-based PCR) DNA fingerprinting. 16S rDNA sequencing analysis of representative strains of each group showed that these strains were closely related to Burkholderia cepacia, Enterobacter cloacae, Phytobacter diazotrophicus, Enterobacter asburiae, Herbaspirillum seropedicae, Cronobacter sakazakii, Enterobacter oryzae, Klebsiella pneumoniae, Enterobacter dissolvens, Azospirillum amazonense, and Cronobacter dublinensis, indicating the high diversity of endophytic diazotrophs colonized in the host plants of C. caesius and M. floridulus. The results of physiological and biochemical tests showed that some isolates had the ability of ammonia secreting, indole producing, and phosphorus and potassium dissolving, suggesting the potential application in agricultural practices. Fig 2, Tab 3, Ref 38

参考文献/References:

1 Boddey RM, De Oliveira OC, Urquiaga S, Reis VM, De Olivares FL, Baldani VLD, Doebereiner J. Biological nitrogen fixation associated with sugar cane and rice: contributions and prospects for improvement [J]. Plant Soil, 1995, 174 (1-2): 195-209
2 张国霞, 茅庆, 何忠义, 谭志远. 陵水普通野生稻(Oryza rufipogon)内生菌的固氮及溶磷特性[J]. 应用与环境生物学报, 2006, 12 (4): 457-460 [Zhang GX, Mao Q, He ZY, Tan ZY. Detection of nitrogenase activity and phosphorus dissolving ability of endophytic isolates from Oryza rufipogon in Lingshui [J]. Chin J Appl Environ Biol, 2006, 12 (4): 457-460]
3 谢光辉, 苏宝林. 长江流域水稻根际芽杆菌属固氮菌株的分离与鉴定[J]. 微生物学报, 1998, 38 (6): 480-483 [Xie GH, Su BL. Identification and isolation of nitrogen-fixing Bacillus sp. in rhizosphere of rice grown in the Yangtze River Basin [J]. Acta Microbiol Sin, 1998, 38 (6): 480-483]
4 吕泽勋, 李久蒂, 朱至清. 玉米内生固氮菌的回接分离及限菌条件下在玉米根内的定殖[J]. 应用与环境生物学报, 2001, 7 (3): 207-212 [Lu ZX, Li JD, Zhu ZQ. Inoculation, isolation of endophytic diaztorophic bacteria and their colonization in maize root tissue under gnotobiotic condition [J]. Chin J Appl Environ Biol, 2001, 7(3): 207-212]
5 Roesch LFW, Olivares FL, Pereira Passaglia LM, Selbach PA, Sá ELS, Camargo FAO. Characterization of diazotrophic bacteria associated with maize: effect of plant genotype, ontogeny and nitrogen-supply [J]. World J Microbiol Biotechnol, 2006, 22 (9): 967-974
6 田宏, 张德罡, 姚拓, 席琳乔. 禾本科草坪草固氮菌株筛选及部分特性初步研究[J]. 中国草地, 2005, 27 (5): 47-52 [Tian H, Zhang DG, Yao T, Xi LQ. Isolation and characteristics of nitrogen fixation bacteria in rhizosphere of turfgrass [J]. Grassl China, 2005, 27 (5): 47-52]
7 赵现伟, 彭桂香, 张志英, 邱永雄, 张泽民, 张桂权, 谭志远. 细菌聚类新方法: ddT聚类技术分析香根草联合固氮菌多样性[J]. 科学通报, 2010, 55 (7): 562-571 [Zhao XW, Peng GX, Zhang ZY, Qiu YX, Zhang ZM, Zhang GQ. A novel grouping method: ddT clustering assessment of the diversity of associated nitrogen-fixing bacteria isolated from Vetiveria zizanioides [J]. Chin Sci Bull, 2010, 55 (7): 562-571]
8 新楠, 樊明寿, 王海凤. 羊草和冰草根际高效固氮菌的分离及鉴定[J]. 天津农学院学报, 2008, 15 (2): 40-42 [Xin N, Fan MS, Wang HF. Study on separation and identification of Azotobacteria from rhizosphere of Aneurolepidium chinensis and Agropyron mongolicum [J]. J Tianjin Agric Coll, 2008, 15 (2): 40-42]
9 齐安国, 周建, 林紫玉. 菊花?月季?香石竹联合固氮细菌的分离筛选[J]. 安徽农业科学, 2006, 34 (4): 655-656 [Qi AG, Zhou J, Lin ZY. Methods of isolation and selection of N2-fixation bacteria from Dendranthema morifolium, Rosa chinensis and Pianthas caryophyllus [J]. J Anhui Agric Sci, 2006, 34 (4): 655-656]
10 吴若菁, 陈奋飞, 王逸群, 林子都, 杨敏. 香椿中内生固氮菌的分离筛选与鉴定[J]. 福建师范大学学报(自然科学版), 2007, 23 (4): 34-36 [Wu RJ, Chen FF, Wang YQ, Lin ZD, Yang M. Isolation and characterization of endophytic diazotrophs in Toona sinensis Roem [J]. J Fujian Norm Univ (Nat Sci Edi), 2007, 23 (4): 34-36]
11 席琳乔, 姚拓, 张德罡. 固氮菌对燕麦不同生育期促生作用的研究[J]. 草业学报, 2007, 16 (3): 38-42 [Xi LQ, Yao T, Zhang DG. Effect of associative nitrogen fixation bacteria on Avena sativa growth [J]. Acta Pratacult Sin, 2007, 16 (3): 38-42]
12 林丽, 张新成, 李杨瑞, 梁俊. 甘蔗器官固氮酶活性及其对接种固氮菌的响应[J]. 西北植物学报, 2008, 28 (12): 2472-2477 [Lin L, Zhang XC, Li YR, Liang J. Changes of nitrogenase activity in sugarcane (Saccharum officinarum L.) and its response to innoculate nitrogen fixation bacteria [J]. Acta Bot Bor-Occid Sin, 2008, 28 (12): 2472-2477]
13 彭桂香, 王华荣, 张国霞, 侯伟, 袁清华, 谭志远. 糖蜜草内生固氮菌IS-PCR和16SrRNA基因全序列分析[J]. 华南农业大学学报, 2005, 26 (4): 73-76 [Peng GX, Wang HR, Zhang GX, Hou W, Yuan QH, Tan ZY. Molecular study of endophytic nitrogen fixing bacteria isolated from Melinis minutiflora [J]. J South China Agric Univ, 2005, 26 (4): 73-76]
14 夏觅真, 马忠友, 曹媛媛, 赵良侠, 唐欣昀, 甘旭华. 棉花根际固氮菌、解磷菌及解钾菌的相互作用[J]. 中国微生态学杂志, 2010 (2): 102-105 [Xia MZ, Ma ZY, Cao YY, Zhao LX, Tang XY, Gan XH. Interactions between a N-fixation bacterium, a phosphobacterium and a K-releasing bacterium of cotton rhizosphere [J]. Chin J Microecol, 2010 (2): 102-105]
15 崔美华, 郭坤友, 刘庆巍. 生物固氮菌肥在水稻上应用效果试验[J]. 北方水稻, 2009, 39 (2): 66 [Cui MH, Guo KY, Liu QW. Application effect of biological nitrogen fixation in rice [J]. North Rice, 2009, 39 (2): 66]
16 赵记军, 徐培智, 解开治, 严超, 谭志远. 内生固氮菌在水稻上的应用效果研究初报[J]. 广东农业科学, 2008 (1): 35-38 [Zhao JJ, Xu PZ, Xie KZ, Yan C, Tan ZY. Preliminary invocation effects of endophytic diazotroph on rice [J]. Guangdong Agric Sci, 2008 (1): 35-38]
17 陈夕军, 朱凤, 童蕴慧, 纪兆林, 徐敬友. 水稻内生联合固氮细菌的分离、种类及对水稻的促生长作用[J]. 扬州大学学报(农业与生命科学版), 2007, 28 (2): 61-64 [Chen XJ, Zhu F, Tong YH, Ji ZL, Xu JY. Isolation, species of rice endophytic bacteria with associative nitrogenfixing activity and their promotion to the growth of rice plants [J]. J Yangzhou Univ (Agric Life Sci Ed), 2007, 28 (2): 61-64]
18 陈朝勋, 席琳乔, 姚拓, 张虎, 张德罡. 生物固氮测定方法研究进展[J]. 草原与草坪, 2005 (2): 24-26 [Chen CX, Xi LQ, Yao T, Zhang DG. Research progress in determination methods of biological nitrogen fixation [J]. Grassl Turf, 2005 (2): 24-26]
19 Terakado-Tonooka J, Ohwaki Y, Yamakawa H, Tanaka F, Yoneyama T, Fujihara S. Expressed nifH genes of endophytic bacteria detected in field-grown sweet potatoes (Ipomoea batatas L.) [J]. Microb Environ, 2008, 23 (1): 89-93
20 Ueda T, Suga Y, Yahiro N, Matsuguchi T. Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences [J]. J Bacteriol, 1995, 177 (5): 1414-1417
21 Lovell CR, Piceno YM, Quattro JM, Bagwell CE. Molecular analysis of diazotroph diversity in the rhizosphere of the smooth cordgrass, Spartina alterniflora [J]. Appl Environ Microbiol, 2000, 66 (9): 3814-3822
22 Zehr JP, Mcreynolds LA. Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii [J]. Appl Environ Microbiol, 1989, 55 (10): 2522-2526
23 Ohkuma M, Noda S, Usami R, Horikoshi K, Kudo T. Diversity of nitrogen fixation genes in the symbiotic intestinal microflora of the termite reticulitermes speratus [J]. Appl Environ Microbiol, 1996, 62 (8): 2747-2752
24 Loiret FG, Ortega E, Kleiner D, Ortega-Rodes P, Rodes R, Dong Z. A putative new endophytic nitrogen-fixing bacterium Pantoea sp. from sugarcane [J]. J Appl Microbiol, 2004, 97 (3): 504-511
25 丘雁玉, 李飞飞, 邓超宏, 崔大方, 陈玉芬. 广东省3种野生香茅属植物精油的化学成分及含量分析[J]. 植物资源与环境学报, 2009, 18 (1): 48-51 [Qiu YY, Li FF, Deng CH, Cui DF, Chen YF. Chemical components and content analysis of essential oil from three wild species of Cymbopogon spreng in Guangdong Province [J]. J Plant Resour Environ, 2009, 18 (1): 48-51]
26 张崇邦, 王江, 柯世省, 金则新. 五节芒定居对尾矿砂重金属形态、微生物群落功能及多样性的影响[J]. 植物生态学报, 2009, 33 (4): 629-637 [Zhang CB, Wang J, Ke SX, Jin ZX. Effects of natural inhabitation of Miscanthus floridulus on soil microbial biomass, respiration rate and enzyme activity in a mine tailing dump [J]. Chin J Plant Ecol, 2009, 33 (4): 629-637]
27 谭志远, 彭桂香, 徐培智, 艾绍英, 唐拴虎, 张国霞, 曾凤云. 普通野生稻(Oryza rufipogon)内生固氮菌多样性及高固氮酶活性[J]. 科学通报, 2009 (13): 1885-1893 [Tan ZY, Peng GX, Xu PZ, Ai SY, Tang SH, Zhang GX, Zeng FY. Diversity and high nitrogenase activity of endophytic diazotrophs isolated from Oryza rufipogon Griff [J]. Chin Sci Bull, 2009 (13): 1885-1893]
28 王华荣, 彭桂香, 张国霞, 侯伟, 谭志远. 糖蜜草(Melinis minutiflora Beauv.)内生固氮菌分离鉴定[J]. 生态学报, 2006, 26 (8): 2566-2571 [Wang HR, Peng GX, Zhang GX, Hou W, Tan ZY. Characterization of endophytic diazotrophs isolated from molasses grass [J]. Acta Ecol Sin, 2006, 26 (8): 2566-2571]
29 王春连, 章琦, 周永力, 赵炳宇. 我国长江以南地区水稻白叶枯病原菌遗传多样性分析[J]. 中国水稻科学, 2001, 15 (2): 131-136 [Wang CL, Zhang Q, Zhou YL, Zhao BY. Genetic diversity of pathogen Xanthomonas oryzae pv. oryzae from southern regions of Yangtze River in China [J]. Chin J Rice Sci, 2001, 15 (2): 131-136]
30 彭桂香, 陈文新, 谭志远. rRNA基因间隔区序列用于亲缘关系密切的根瘤菌种群鉴定及系统发育分析[J]. 华南农业大学学报, 2004, 25 (4): 58-62 [Peng GX, Chen WX, Tan ZY. Identification and phylogenetic analysis of closely related rhizobium species by rRNA gene intergenic spacer sequence [J]. J South China Agric Univ, 2004, 25 (4): 58-62]
31 Tan Z, Hurek T, Vinuesa P, Muller P, Ladha JK, Reinhold-Hurek B. Specific detection of Bradyrhizobium and Rhizobium strains colonizing rice (Oryza sativa) roots by 16S-23S ribosomal DNA intergenic spacer-targeted PCR [J]. Appl Environ Microbiol, 2001, 67 (8): 3655-3664
32 赵斌, 何绍江. 微生物学实验[M]. 北京: 科学出版社, 2002 [Zhao B, He SJ. Microbiology Experiment [M]. Beijing: Science Press, 2002 ]
33 田宏, 李凤霞, 张德罡, 姚拓. 草坪草溶磷菌筛选及溶磷能力的初步研究[J]. 草业科学, 2005, 22 (10): 92-96 [Tian H, Li FX, Zhang DG, Yao T. Primary research on isolation and ability of phosphorus-solubilizing of turf phosphorus-solubilizing bacteria [J]. Pratacult Sci, 2005, 22 (10): 92-96 ]
34 侯伟, 彭桂香, 许志钧, 陈仕贤, 谭志远. 广东省刺竹内生固氮菌多样性[J]. 农业生物技术学报, 2007, 15 (2): 290-294 [Hou W, Peng GX, Xu ZJ, Chen SX, Tan ZY. Diversity of endophytic diazotrophs isolated from Bambusa blumeana in Guangdong Province [J]. J Agric Biotechnol, 2007, 15 (2): 290-294 ]
35 许晓东, 陈文新. 天山根瘤菌(Rhizobium tianshanense)全细胞蛋白电泳和多位点酶电泳分析[J]. 微生物学通报, 1996, 23 (3): 131-134 [Xu XD, Chen WX. Analysis on Rhizobium tianshanense by whole-cell protein electrophoresis and multilocus enzyme electrophoresis [J]. Microbioloy, 1996, 23 (3): 131-134]
36 谭志远, 陈文新. 根瘤菌新类群的全细胞蛋白电泳及16S rDNA全序列分析[J]. 应用与环境生物学报, 1998, 4 (1): 65-69 [Tan ZY, Chen WX. SDS-Page of whole cell protein of new rhizobial groups and 16S rDNA sequencing of their representatives [J]. Chin J Appl Environ Biol, 1998, 4 (1): 65-69]
37 安千里, 李久蒂. 植物内生固氮菌[J]. 植物生理学通讯, 1999, 35 (4): 265-272 [An QL, Li JD. The endophytic diazotrophs [J]. Plant Physiol Commun, 1999, 35 (4): 265-272]
38 Sprent JI, James EK. N2-fixation by endophytic bacteria: questions of entry and operation [M]. In: Fendrik I, Del Gallo M, Vanderleyden J, De Zamaroczy M. Azospirillum VI and Related Microorganism: Genetics, Physiology, Ecology. Berlin: Springer-Verlag, 1995. 15-30

备注/Memo

备注/Memo:
国家高校博士点专项基金(20094404110006)、国家重点基础研究发展计划(2010CB126502)和广东省科技厅项目(2010B090400434,2012B010300018)资助 Supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20094404110006), the National Basic Research Program of China (“973” Program, No. 2010CB126502), and the Science and Technology Program of Guangdong Province (Nos. 2010B090400434, 2012B010300018)
更新日期/Last Update: 2013-08-22