三叶草根瘤中根瘤菌与土壤杆菌关系的初步研究
摘要
本论文采用来自中国西北地区白花三叶草的两株根瘤分离菌hbh54.2和hbh3.1作为研究对象,对二者的关系进行了初步的研究。经过对两株菌的16S rDNA全序列进行测定,构建了系统发育树状图,确定了根瘤分离菌hbh54.2位于土壤杆菌属Agrobaterium,hbh3.1位于快生根瘤菌属Rhizobium。在根瘤菌多样性研究领域,从根瘤中分离得到土壤杆菌的现象极为常见,但是对于这种现象还没有一个确切的解释,本研究希望通过设计一系列实验,能够初步揭示在三叶草根瘤中土壤杆菌与根瘤菌的关系问题,证明土壤杆菌存在于根瘤中的意义所在,为进一步的深入研究提供理论基础。
本研究通过对两株供试菌的功能基因进行PCR扩增,检验其是否具有共生性以及致病性,结果表明,土壤杆菌hbh54.2在常规条件下无法扩增产生相应的结瘤基因nodA和固氮基因nifH,与植物回接结瘤预实验中其不能与三叶草共生结瘤的结果一致,此外,二者均不能扩增出致病基因virC的目的条带,间接证明二者均不携带Ti质粒,不具有致病性。
本研究通过设计盆栽法植物回接结瘤实验,检验两株供试菌单独回接以及共同回接时对寄主植物白花三叶草各项生长指标的影响,结果表明,与不接菌对照相比,单独回接以及共同回接所导致的植株整体生长量的变化有一定的差异,此外,共同回接还能够促进根瘤菌hbh3.1与寄主植物的结瘤情况,体现在根瘤数量的增加,该结果从植物的角度证明了土壤杆菌hbh54.2与根瘤菌hbh3.1相互作用的意义可以体现在促进原寄主植物的生长发育上。
经过一系列促植物生长生理特性测定实验,结果发现,与根瘤菌hbh3.1相比,土壤杆菌hbh54.2具有较强的分泌铁载体和生长素吲哚乙酸IAA的能力,从生理的角度出发,可以推断土壤杆菌hbh54.2能够弥补根瘤菌hbh3.1的一些缺陷与不足,当前者与后者共存于根瘤中时,前者通过分泌IAA,为植物提供外源生长激素,控制植物体内IAA的浓度,调节植物的生长发育;通过分泌铁载体,螯合细胞周围有限的铁元素,除供自身所需之外,也有利于后者对铁元素的吸收,进而提高其固氮效率。
本研究还利用增强型绿色荧光蛋白EGFP基因标记土壤杆菌hbh54.2,将标记的土壤杆菌单独接入白花三叶草根部,并设计同步实验让其与未标记的根瘤菌hbh3.1共同侵染宿主植物,以及待植物与hbh3.1共生结瘤后再引入共生体系的根际附近,结果表明,只有当二者共同存在的情况下,标记土壤杆菌才能进入根瘤组织内部,单独侵染以及结瘤后再侵染,标记土壤杆菌均无法进入植物体内,说明土壤杆菌hbh54.2不具有单独入侵白花三叶草的能力,只有在相应根瘤菌hbh3.1的携带之下才有可能进入宿主植物体内并定殖于根瘤组织中。
本论文首次将促植物生长生理特性实验结果与标记菌回接实验结果结合起来,初步推断土壤杆菌能够在相应根瘤菌的携带下进入根瘤组织,在根瘤内,土壤杆菌通过分泌IAA调节植物生长,使植物为根瘤菌提供充足的碳源,同时土壤杆菌还能分泌铁载体,为维持根瘤菌固氮酶(钼铁蛋白)正常的生理活性提供铁元素。这个推断初步揭示了土壤杆菌与根瘤菌共存于根瘤中的意义所在,为今后进一步深入研究二者的关系开辟了一个新的方向。
Hbh54.2 and hbh3.1, two strains isolated from the same nodule of white clover in China’s northwest region , are used in this thesis for the preliminary study of the relationship of Rhizobium and Agrobacterium. A phylogenetic tree is constructed on the basis of the 16S rDNA sequences of the two strains, which locates hbh54.2 to the genus of Agrobaterium and hbh3.1 to the genus of Rhizobium. As we know, in the study of rhizobia diversity, it is very common to isolate Agrobacterium-related strains from root nodules. However, up to now, there is no precise explanation to account for this phenomenon. In our study, a series of experiments are designed under the aim of revealing the relationship of Rhizobium and Agrobacterium that can exist in the same nodules of the host plant, which can provide theoretical basis for the further research.
In our study, through the PCR amplification of the functional genes of the two tested strains, we can verify whether the tested strain is symbiotic or pathogenic. As is shown from the results, tested strain hbh54.2 habours no nodulation gene(nodA) and nitrogen-fixation gene(nifH), which is consistent with the results of the plant nodulation pre-experiment as is demonstrated that it cannot induce root nodules in the white clover. In addition, virulence gene(virC) can not be amplified when using the template of the two tested strains, indicating that both of the tested strains do not carry Ti plasmid, which is representative of pathogenecity.
Through pot plant-nodulation experiments, it can be seen from the consequence that there is difference on the mass growth of the host plant among different kinds of disposal condition, one is designed to inoculate with only one kind of strain, another is co-inoculating with two tested strains simutaneously or unsimutaneously. Additionally, it also shows that co-inoculation can promote the nodulation process. In a word, the results indicate that the significance of the interaction of hbh54.2 with hbh3.1 can be reflected in aspects of promoting the growth and development of the original host plant.
After a series of experiments testing the physiological characteristics of plant-growth promoting bacteria, we find an interesting phenomenon. Compared with hbh3.1, hbh54.2 has a strong ability of producing siderophores and secreting plant-growth hormone (indole acetic acid, IAA). Therefore, it may be inferred that hbh54.2 can make up some of the defects and shortcomings of hbh3.1, when the two strains co-exist in the same nodules, the former can provide exogenous growth hormones to host plants through the secretion of IAA, controling the concentration of IAA of the plants in vivo and regulating plant growth condition, what’s more, through the secretion of siderophore, it can also chelate Fe element which is limited around the cells, not only for its own requirements, but also better for the latter in enhancing the efficiency of nitrogen fixation.
This study also used the EGFP(enhanced green fluorescent protein) gene as the marker gene of hbh54.2, expecting to find out how hbh54.2 could locate in the nodule. As the results demonstrate, only under the circumstance of inoculating with hbh54.2(marked with egfp) and hbh3.1 together, can hbh54.2 show its existence in the nodules, while there is no fluorescence detected in the nodules in other situation, which indicates that hbh54.2 do not have the ability to invade the white clover alone, only with the help of the appropriate rhizobia(hbh3.1) can it be carried into the host plants and colonize in the root nodule tissues.
This paper integrated the results of experiments testing the physiological characteristics of plant-growth promoting bacteria and plant nodulation tests with egfp marked strain for the first time. After analyzing the whole results, we conclude that Agrobaterium can enter the root nodules under the carrying of the appropriate Rhizobium; in the nodules, in order to make host-plant provide sufficient carbon source for Rhizobium, Agrobaterium can regulate plant-growth condition through the secretion of IAA, and also it can secrete siderophores to help Rhizobium absorb Fe for the maintenance of the normal physiological activity of nitrogenase (MoFe protein). The inferred conclusion revealed the significance of the coexistence of Agrobacterium with Rhizobium in the nodules, opening a new direction for further study of the relationship of Agrobacterium and Rhizobium in the future.
本研究通过对两株供试菌的功能基因进行PCR扩增,检验其是否具有共生性以及致病性,结果表明,土壤杆菌hbh54.2在常规条件下无法扩增产生相应的结瘤基因nodA和固氮基因nifH,与植物回接结瘤预实验中其不能与三叶草共生结瘤的结果一致,此外,二者均不能扩增出致病基因virC的目的条带,间接证明二者均不携带Ti质粒,不具有致病性。
本研究通过设计盆栽法植物回接结瘤实验,检验两株供试菌单独回接以及共同回接时对寄主植物白花三叶草各项生长指标的影响,结果表明,与不接菌对照相比,单独回接以及共同回接所导致的植株整体生长量的变化有一定的差异,此外,共同回接还能够促进根瘤菌hbh3.1与寄主植物的结瘤情况,体现在根瘤数量的增加,该结果从植物的角度证明了土壤杆菌hbh54.2与根瘤菌hbh3.1相互作用的意义可以体现在促进原寄主植物的生长发育上。
经过一系列促植物生长生理特性测定实验,结果发现,与根瘤菌hbh3.1相比,土壤杆菌hbh54.2具有较强的分泌铁载体和生长素吲哚乙酸IAA的能力,从生理的角度出发,可以推断土壤杆菌hbh54.2能够弥补根瘤菌hbh3.1的一些缺陷与不足,当前者与后者共存于根瘤中时,前者通过分泌IAA,为植物提供外源生长激素,控制植物体内IAA的浓度,调节植物的生长发育;通过分泌铁载体,螯合细胞周围有限的铁元素,除供自身所需之外,也有利于后者对铁元素的吸收,进而提高其固氮效率。
本研究还利用增强型绿色荧光蛋白EGFP基因标记土壤杆菌hbh54.2,将标记的土壤杆菌单独接入白花三叶草根部,并设计同步实验让其与未标记的根瘤菌hbh3.1共同侵染宿主植物,以及待植物与hbh3.1共生结瘤后再引入共生体系的根际附近,结果表明,只有当二者共同存在的情况下,标记土壤杆菌才能进入根瘤组织内部,单独侵染以及结瘤后再侵染,标记土壤杆菌均无法进入植物体内,说明土壤杆菌hbh54.2不具有单独入侵白花三叶草的能力,只有在相应根瘤菌hbh3.1的携带之下才有可能进入宿主植物体内并定殖于根瘤组织中。
本论文首次将促植物生长生理特性实验结果与标记菌回接实验结果结合起来,初步推断土壤杆菌能够在相应根瘤菌的携带下进入根瘤组织,在根瘤内,土壤杆菌通过分泌IAA调节植物生长,使植物为根瘤菌提供充足的碳源,同时土壤杆菌还能分泌铁载体,为维持根瘤菌固氮酶(钼铁蛋白)正常的生理活性提供铁元素。这个推断初步揭示了土壤杆菌与根瘤菌共存于根瘤中的意义所在,为今后进一步深入研究二者的关系开辟了一个新的方向。
Hbh54.2 and hbh3.1, two strains isolated from the same nodule of white clover in China’s northwest region , are used in this thesis for the preliminary study of the relationship of Rhizobium and Agrobacterium. A phylogenetic tree is constructed on the basis of the 16S rDNA sequences of the two strains, which locates hbh54.2 to the genus of Agrobaterium and hbh3.1 to the genus of Rhizobium. As we know, in the study of rhizobia diversity, it is very common to isolate Agrobacterium-related strains from root nodules. However, up to now, there is no precise explanation to account for this phenomenon. In our study, a series of experiments are designed under the aim of revealing the relationship of Rhizobium and Agrobacterium that can exist in the same nodules of the host plant, which can provide theoretical basis for the further research.
In our study, through the PCR amplification of the functional genes of the two tested strains, we can verify whether the tested strain is symbiotic or pathogenic. As is shown from the results, tested strain hbh54.2 habours no nodulation gene(nodA) and nitrogen-fixation gene(nifH), which is consistent with the results of the plant nodulation pre-experiment as is demonstrated that it cannot induce root nodules in the white clover. In addition, virulence gene(virC) can not be amplified when using the template of the two tested strains, indicating that both of the tested strains do not carry Ti plasmid, which is representative of pathogenecity.
Through pot plant-nodulation experiments, it can be seen from the consequence that there is difference on the mass growth of the host plant among different kinds of disposal condition, one is designed to inoculate with only one kind of strain, another is co-inoculating with two tested strains simutaneously or unsimutaneously. Additionally, it also shows that co-inoculation can promote the nodulation process. In a word, the results indicate that the significance of the interaction of hbh54.2 with hbh3.1 can be reflected in aspects of promoting the growth and development of the original host plant.
After a series of experiments testing the physiological characteristics of plant-growth promoting bacteria, we find an interesting phenomenon. Compared with hbh3.1, hbh54.2 has a strong ability of producing siderophores and secreting plant-growth hormone (indole acetic acid, IAA). Therefore, it may be inferred that hbh54.2 can make up some of the defects and shortcomings of hbh3.1, when the two strains co-exist in the same nodules, the former can provide exogenous growth hormones to host plants through the secretion of IAA, controling the concentration of IAA of the plants in vivo and regulating plant growth condition, what’s more, through the secretion of siderophore, it can also chelate Fe element which is limited around the cells, not only for its own requirements, but also better for the latter in enhancing the efficiency of nitrogen fixation.
This study also used the EGFP(enhanced green fluorescent protein) gene as the marker gene of hbh54.2, expecting to find out how hbh54.2 could locate in the nodule. As the results demonstrate, only under the circumstance of inoculating with hbh54.2(marked with egfp) and hbh3.1 together, can hbh54.2 show its existence in the nodules, while there is no fluorescence detected in the nodules in other situation, which indicates that hbh54.2 do not have the ability to invade the white clover alone, only with the help of the appropriate rhizobia(hbh3.1) can it be carried into the host plants and colonize in the root nodule tissues.
This paper integrated the results of experiments testing the physiological characteristics of plant-growth promoting bacteria and plant nodulation tests with egfp marked strain for the first time. After analyzing the whole results, we conclude that Agrobaterium can enter the root nodules under the carrying of the appropriate Rhizobium; in the nodules, in order to make host-plant provide sufficient carbon source for Rhizobium, Agrobaterium can regulate plant-growth condition through the secretion of IAA, and also it can secrete siderophores to help Rhizobium absorb Fe for the maintenance of the normal physiological activity of nitrogenase (MoFe protein). The inferred conclusion revealed the significance of the coexistence of Agrobacterium with Rhizobium in the nodules, opening a new direction for further study of the relationship of Agrobacterium and Rhizobium in the future.
引文
安千里,杨学健,董越梅,冯丽洁,匡柏健,李久蒂. 2001.用共聚焦激光扫描显微镜观测GFP标记的内生固氮菌Klebsiella oxytoca SA2侵染水稻根.植物学报, 6:23~26
陈绍兴,赵翔,沈萍. 2006.高灵敏假单胞菌铁载体的平板检测方法.微生物学通报, 33(3):122~127
陈文新,李阜棣,闫章才.2007.我国土壤微生物学和生物固氮研究的回顾与展望.世界科技研究与发展,24(4):6~12
陈文新,李季伦,朱兆良.2006.发挥豆科植物-根瘤菌共生固氮作用从源头控制滥施氮肥造成的面源污染.科学新闻, 19:4~5
吕泽勋,李久蒂,朱至清. 2001.用绿色荧光蛋白基因(gfp)标记产酸克雷伯氏菌SG-11研究其在水稻苗期根部的定殖.农业生物技术学报, 1:13~17
史巧娟. 2000.绿色荧光蛋白基因gfp在华癸中生根瘤菌与紫云英共生固氮体系研究中的应用[博士学位论文].武汉:华中农业大学
田涛,王琦. 2004.绿色荧光蛋白作为分子标记物在微生物学中的应用.微生物学杂志, 25(1):68～72
王浩,绳志雅,隋新华,陈文新. 2006.用gfp基因标记法研究大豆根瘤菌在大豆根部定殖结瘤情况.微生物学杂志,26(2):2~4
Bai Y, D’Aous F, Smith D L, Driscoll B T. 2002. Isolation of plant-growth-promoting Bacillus strains from soybean root nodules.Canadian Journal of Microbiology 48:230~238.
Barrett C F, Parker M A. 2006. Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica. Applied and Environmental Microbiology ,72:1198~1206
Bhupendra N. Tiwary , Birendra Prasad, Anuradha Ghosh, Sanjay Kumar, Rakesh K. Jain. 2007. Characterization of Two Novel Biovar of Agrobacterium tumefaciens Isolated from Root Nodules of Vicia faba. Curr Microbiol ,55:328~333
Bo Normander, Niels B. Hendriksen, Ole Nybroe. 1999. Green Fluorescent Protein-Marked Pseudomonas fluorescens: Localization, Viability, and Activity in the Natural Barley Rhizosphere. Applied and Environmental Microbiology, 65(10):4646~4651
Bouzar H, Jones J B, Hodge N C. 1993. Differential characterization of Agrobacterium species using carbon source utilization pattern and fatty acid profiles. Phytopathology ,83:733~739
Bouzar H, Moore L W. 1987 Isolation of different Agrobacterium biovar from a natural oak savanna and tallgrass prairie. Appl Environ Microbiol ,53:717~721
Bowyer P , Mueller E , John L. 2000. Use of isocitrate lyase promoter GFP fusion to monitor carbon metabolism of the plant pathogen Tapesia yallundae during infection of Wheat. Mol Plant Pathol, 1(4) :253 ~262
Chalfie M, Tu Y, Euskirchen G, Ward W W, Prasher D C.1994. Green-fluorescent protein as a marker for gene expression.Science,263:802~805
Chen W X, Yan G H, Li J L. 1988. Numerical taxonomy study of fast-growing soybean rhizobia and a proposal that Rhizobium fredii be assigned to Sinorhizobium gen. nov. Int. J. Syst.Bacteriol, 38: 392~397
Crosa J H. 1989. Genetics and molecular biology of siderophore- mediated iron transport in bacteria. Microbiol Rev, 53(4):517~530
D′Haeze W. A. 2004. gfp reporter plasmid to visualize Azorhizobium cauli nodans during nodulation of Sesbania rost rata. Plasmid , 51:185~191
De Lajudie P, Laurent-Fulele E, Willems A, Torck U, Coopman R, Collins M D, Kersters K, Dreyfus B, Gillis M. 1998. Allorhizobium undicola gen. nov., sp. nov.,nitrogen-fixing bacteria that efficiently nodulate Neptunia natans in Senegal. Int. J. Syst. Bacteriol, 48 :1277~1290.
De Lajudie P, Willems A, Nick G, Mohamed T S, Torck U, Filali-Maltouf A, Kersters K, Dreyfus B, Lindstrom K, Gillis M. 1999. Agrobacterium bv. 1 strains isolated from nodules of tropical legumes. Systematic and Applied Microbiology, 22:119~132
De Lajudie P, Willems A, Pot B. 1994. Polyphasic taxonomy of rhizobia: emendation of the genus Sinorhizobim and description of Sinorhizobium meliloti comb. nov., S. saheli sp. nov.. Int. J. Syst. Bacteriol, 44: 751~753
Dessaux Y, Petit A, TempéJ. 1992. Opines in Agrobacterium biology. In Verma DPS (ed) Molecular signals in plant-microbe communication, CRC Press, Boca Raton, FL pp:109~136
Dreyfus, B. 1988. Characterization of Azorhizobium caulinodans gen-nov, sp-nov, a stem-nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata. Int. J. Syst. Bacteriol, 38: 89~98
Farrand S K, Van Berkum P, Oger P. 2003. Agrobacterium is a definable genus of the family Rhizobiaceae. Int J Syst Evol Microbiol, 53:1681~1687
Fernando Ibáňez, Jorge Angelini, Tania Taurian, María Laura Tonelli, Adriana Fabra. 2009. Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria. Systematic and Applied Microbiology, 32 :49~55
Frank B. 1889.über die pilzsymbiose der leguminosen. Ber. Dtsch. Bot. Ges,7: 332~346
Gage D J, Bobo T, Long S R. 1996. Use of green fluorescent protein to visualize the early events of symbiosis between Rhizobium meliloti and alfalfa,Medicago sativa. J Bacteriol,178:7159~7166
Gage D J. 2002. Analysis of infection thread development using Gfp- and DsRed-expressing Sinorhizobium meliloti. J. Bacteriol,184:7042~7046
Gao J, Terefework Z, Chen W, Lindstrom K.. 2001. Genetic diversity of rhizobia isolated from Astragalus adsurgens growing in different geographical regions of China. Journal of Biotechnology, 91:155~168
Gaunt M W, Turner S L, Rigottier-Gois L. 2001. Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. Int J Syst Evol Microbiol, 51:2037~2048
Gherbi H. 2008. SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankia bacteria. Proc. Natl. Acad. Sci. U. S. A. 105:4928~4932
Glickmann E, Dessaux Y. 1995. A critical examination of the specificity of the Salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Applied and Environmental Microbiology, 61: 793~796
Han Su-Zhen, Wang En-Tao, Chen Wen-Xin. 2005. Diverse bacteria isolated from root nodules of Phaseolus vulgaris and species within the genera Campylotropis and Cassia grown in China.Systematic and Applied Microbiology, 28 :265~276
Heim R, Tsien R Y. 1996. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol,6:178~182
Jarvis B D W, Van Berkum P, Chen W X. 1997. Transfer of Rhizobium loti, Rhizobium huakuii, Rhizobium ciceri, Rhizobium mediterraneum, and Rhizobium tianshanense to Mesorhizobium gen. nov. Int. J. Syst. Bacteriol, 47: 895~898
Jones K.M. 2007. How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model. Nat. Rev. Microbiol. 5:619~633
Jordan D C. 1982. Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. nov., a genus of slow-growing, root nodule bacteria from leguminous plants. Int. J. Syst. Bacteriol, 32: 136~139
Kan F L, Chen Z Y, Wang E T, Tian C F, Sui X H, Chen W X. 2007. Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai–Tibet Plateau and in other zones of China. Archives of Microbiology, 188:103~115
Kersters K, De Ley J, Sneath P H A. 1973. Numerical taxonomic analysis of Agrobacterium. J Gen Micrbiol ,78:227~239
Kloepper J W, John L, Teintze M. 1980. Enhanced plant growth by siderophores produced by plant growth-promoting rhizobacter. Nature, 286: 885~886
Leong J. 1986. Siderophores: their biochemistry and possible role in the biocontrol of plant pathogens. Annu Rev Phytopathol, 24: 187~209
Li Ji Hong, Wang En Tao, Chen Wen Feng, Chen Wen Xin. 2008. Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China. Soil Biology & Biochemistry, 40:238~246
Lindow S E. 1995. The use of reptorer genes in the study of microbial ecology. Mol . Ecol, 4:555～566
Lindstr?m and Martínez-Romero. 2002. International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Agrobacterium and Rhizobium. Int J Syst Evol Microbiol,52:2337
Machuca A, Milagres A M F. 2003. Use of CAS-agar plate modified to study the effect of different variables on siderophore production by Aspergills. Lett Appl Microbial, 36: 177~181
Marmur J. 1961. A procedure for the isolation of DNA from microorganisms. J. Mol. Biol, 3:208~218
Martinez E. 1987. Nitrogen fixing nodules induced by Agrobacterium tumefaciens harboring Rhizobium phaseoli plasmids. J. Bacteriol. 169:2828~2834
Mhamdi R, Mrabet M, Laguerre G. 2005. Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains. Can J Microbiol, 51:105~111
Moore L W, Bouzar H, Burr T. 1988. Agrobacterium. In: SchaadNW (ed) Laboratory Guide for identification of plant pathogenic bacteria, 2nd ed. APS Press, St Paul, MN, pp 16~36
Mrabet M, Mnasri B, Romdhane S B, Laguerre G, Aouani M E, Mhamdi R. 2006. Agrobacterium strains isolated from root nodules of common bean specifically reduce nodulation by Rhizobium gallicum. FEMS Microbiology Ecology, 56:304~309
Nico Stuurman. 2000. Use of Green Fluorescent Protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants . The American Phytopathological Society, 13 (11) :1163~1169
Odee D W, Haukka K, McInroy S G, Sprent J I, Sutherland J M, Young J P W. 2002. Genetic and symbiotic characterization of rhizobia isolated from tree and herbaceous legumes grown in soils from ecologically diverse sites in Kenya.Soil Biology&Biochemistry,34:801~811
Oke V. and Long S R. 1999. Bacteroid formation in the Rhizobium–legume symbiosis. Curr. Opin. Microbiol, 2:641~646
Prasher D C, Eckenrode V K, Ward W W, Prendergast F C, Cormier M J. 1992. Primary structure of the Aequorea victoria green-fluorescent protein. Gene, 111:229~233
Rivas R, Velázquez E, Willems A, Vizcaíno N, Subba-Rao N S, Mateos P F, Gillis M, Dazzo F B, Martínez-Molina E. 2002. A new species of Devosia that forms a unique nitrogen-fixing root-nodule symbiosis with the aquatic legume Neptunia natans (L. f.) Druce. Appl.Environ. Microbiol, 68:5217~5222.
Saitou N, Masatoshi N. 1987. The neighbor-joining method:a new method for reconstructing phylogenetic tree. Mol Biol Evol, 4(4):406~425
Sawada H. 2003. Changing concepts in the systematics of bacterial nitrogen-fixing legume symbionts. J. Gen. Appl. Microbiol.49:155~179
Shimomura O, Johnson F H, Saiga Y. 1962. Extraction,purification and properties of a bioluminescent protein from the luminous hudrmedusan, Aequorea. J Cell Comp Physiol, 59:223~240
Shimomura O. 1979. Structure of the chromophore of Aequorea green fluorescent protein. FEBS Lett,104:220~222
Sohail H, Fathia M, Kauser A M. 2004. Nodule co-occupancy of Agrobacterium and Bradyrhizobium with potential benefit to legume host. 14th International Congress on Nitrogen Fixation
Sohail Hameed, Sumera Yasmin, Kauser A. Malik, Yusuf Zafar, Fauzia Y. Hafeez. 2004. Rhizobium, Bradyrhizobium and Agrobacterium strains isolated from cultivated legumes. Biol Fertil Soils, 39:179–185
Stephen P. Cummings, Prasad Gyaneshwar, Pablo Vinuesa, Frank T. Farruggia, Mitchell Andrews, David Humphry, Geoffrey N. Elliott, Andrew Nelson, Caroline Orr, Deborah Pettitt, Gopit R. Shah, Scott R. Santos, Hari B. Krishnan, David Odee, Fatima M. S. Moreira, Janet I. Sprent, J. Peter W. Young and Euan K. James. 2009. Nodulation of Sesbania species by Rhizobium(Agrobacterium) strain IRBG74 and other rhizobia. Environmental Microbiology
Tan Z Y, Wang E T, Peng G X, Zhu M E, Martíne-Romero E, Chen W X. 1999. Characterization of bacteria isolated from wild legumes in the north-western regions ofChina. Int. J. Syst. Bacteriol, 49:1457~1469.
Thomoson J D, Gibson T J, Plewniak F. 1997. The Clustal X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tool. Nucleic Acids Res, 24:4867~4882
Tighe S W, De Lajudie P, Dipietro K. 2000. Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobiumspecies using the Sherlock Microbial Identification System. Int J Syst Evol Microbiol, 50:787~801
Van de Peer Y, De Wachter R. 1994. Treecon for Windows:a software package for the construction and drawing of evolutionary trees for the Microsoft windows environment. Comput Appl Biosci, 10:569~570
Vincent J M. 1970. A Manual for the Practical Study of Root-Nodule Bacteria, Blackwell Scientific Publications, Oxford UK
Wang L L, Wang E T, Liu J, Li Y, Chen W X. 2006. Endophytic occupation of root nodules and roots of Melilotus dentatus by Agrobacterium tumefaciens. Microbial Ecology ,52:436~443
Weisburg W G, Barns S M, Pelletior D A, Lane D J. 1991. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173:697~703
Winans S C. 1992. Two-way chemical signaling in Agrobacterium–plant interactions. Microbiol Rev ,56:12~31
Woese C R, Stackebrandt E, Weisburg W G. 1984. The phylogeny of purple bacteria: the alpha subdivision. Syst Appl Microbiol, 5:315~326
Woese C R. 1987. Bacterial evolution. Microbiol Rev,51: 221~269
Wong L L, Wong E T, Liu J. 2006. Endophytic occupation of root nodules and root of Melilotus dentatus by Agrobacterium tumefaciens. Microbial Ecol ,52:436~443
Young J M, Bull C T, De Boer S H. 2001. Classification, nomenclature and plant pathogenic bacteria: a clarification.Phytopathology, 91:617~620
Young J M, Kuykendall L D, Martinez-Romero E. 2003. Classification and nomenclature of Agrobacterium and Rhizobium: a reply to Farrand et al(2003). Int J Syst Evol Microbiol, 53:1689~1695
Zimmer M. 2002. Green fluorescent protein ( GFP) : applications ,structure ,and related photophysical behavior. Chem Rev ,102(3) :759~781
陈绍兴,赵翔,沈萍. 2006.高灵敏假单胞菌铁载体的平板检测方法.微生物学通报, 33(3):122~127
陈文新,李阜棣,闫章才.2007.我国土壤微生物学和生物固氮研究的回顾与展望.世界科技研究与发展,24(4):6~12
陈文新,李季伦,朱兆良.2006.发挥豆科植物-根瘤菌共生固氮作用从源头控制滥施氮肥造成的面源污染.科学新闻, 19:4~5
吕泽勋,李久蒂,朱至清. 2001.用绿色荧光蛋白基因(gfp)标记产酸克雷伯氏菌SG-11研究其在水稻苗期根部的定殖.农业生物技术学报, 1:13~17
史巧娟. 2000.绿色荧光蛋白基因gfp在华癸中生根瘤菌与紫云英共生固氮体系研究中的应用[博士学位论文].武汉:华中农业大学
田涛,王琦. 2004.绿色荧光蛋白作为分子标记物在微生物学中的应用.微生物学杂志, 25(1):68～72
王浩,绳志雅,隋新华,陈文新. 2006.用gfp基因标记法研究大豆根瘤菌在大豆根部定殖结瘤情况.微生物学杂志,26(2):2~4
Bai Y, D’Aous F, Smith D L, Driscoll B T. 2002. Isolation of plant-growth-promoting Bacillus strains from soybean root nodules.Canadian Journal of Microbiology 48:230~238.
Barrett C F, Parker M A. 2006. Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. nodule bacteria on two Mimosa spp. in Costa Rica. Applied and Environmental Microbiology ,72:1198~1206
Bhupendra N. Tiwary , Birendra Prasad, Anuradha Ghosh, Sanjay Kumar, Rakesh K. Jain. 2007. Characterization of Two Novel Biovar of Agrobacterium tumefaciens Isolated from Root Nodules of Vicia faba. Curr Microbiol ,55:328~333
Bo Normander, Niels B. Hendriksen, Ole Nybroe. 1999. Green Fluorescent Protein-Marked Pseudomonas fluorescens: Localization, Viability, and Activity in the Natural Barley Rhizosphere. Applied and Environmental Microbiology, 65(10):4646~4651
Bouzar H, Jones J B, Hodge N C. 1993. Differential characterization of Agrobacterium species using carbon source utilization pattern and fatty acid profiles. Phytopathology ,83:733~739
Bouzar H, Moore L W. 1987 Isolation of different Agrobacterium biovar from a natural oak savanna and tallgrass prairie. Appl Environ Microbiol ,53:717~721
Bowyer P , Mueller E , John L. 2000. Use of isocitrate lyase promoter GFP fusion to monitor carbon metabolism of the plant pathogen Tapesia yallundae during infection of Wheat. Mol Plant Pathol, 1(4) :253 ~262
Chalfie M, Tu Y, Euskirchen G, Ward W W, Prasher D C.1994. Green-fluorescent protein as a marker for gene expression.Science,263:802~805
Chen W X, Yan G H, Li J L. 1988. Numerical taxonomy study of fast-growing soybean rhizobia and a proposal that Rhizobium fredii be assigned to Sinorhizobium gen. nov. Int. J. Syst.Bacteriol, 38: 392~397
Crosa J H. 1989. Genetics and molecular biology of siderophore- mediated iron transport in bacteria. Microbiol Rev, 53(4):517~530
D′Haeze W. A. 2004. gfp reporter plasmid to visualize Azorhizobium cauli nodans during nodulation of Sesbania rost rata. Plasmid , 51:185~191
De Lajudie P, Laurent-Fulele E, Willems A, Torck U, Coopman R, Collins M D, Kersters K, Dreyfus B, Gillis M. 1998. Allorhizobium undicola gen. nov., sp. nov.,nitrogen-fixing bacteria that efficiently nodulate Neptunia natans in Senegal. Int. J. Syst. Bacteriol, 48 :1277~1290.
De Lajudie P, Willems A, Nick G, Mohamed T S, Torck U, Filali-Maltouf A, Kersters K, Dreyfus B, Lindstrom K, Gillis M. 1999. Agrobacterium bv. 1 strains isolated from nodules of tropical legumes. Systematic and Applied Microbiology, 22:119~132
De Lajudie P, Willems A, Pot B. 1994. Polyphasic taxonomy of rhizobia: emendation of the genus Sinorhizobim and description of Sinorhizobium meliloti comb. nov., S. saheli sp. nov.. Int. J. Syst. Bacteriol, 44: 751~753
Dessaux Y, Petit A, TempéJ. 1992. Opines in Agrobacterium biology. In Verma DPS (ed) Molecular signals in plant-microbe communication, CRC Press, Boca Raton, FL pp:109~136
Dreyfus, B. 1988. Characterization of Azorhizobium caulinodans gen-nov, sp-nov, a stem-nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata. Int. J. Syst. Bacteriol, 38: 89~98
Farrand S K, Van Berkum P, Oger P. 2003. Agrobacterium is a definable genus of the family Rhizobiaceae. Int J Syst Evol Microbiol, 53:1681~1687
Fernando Ibáňez, Jorge Angelini, Tania Taurian, María Laura Tonelli, Adriana Fabra. 2009. Endophytic occupation of peanut root nodules by opportunistic Gammaproteobacteria. Systematic and Applied Microbiology, 32 :49~55
Frank B. 1889.über die pilzsymbiose der leguminosen. Ber. Dtsch. Bot. Ges,7: 332~346
Gage D J, Bobo T, Long S R. 1996. Use of green fluorescent protein to visualize the early events of symbiosis between Rhizobium meliloti and alfalfa,Medicago sativa. J Bacteriol,178:7159~7166
Gage D J. 2002. Analysis of infection thread development using Gfp- and DsRed-expressing Sinorhizobium meliloti. J. Bacteriol,184:7042~7046
Gao J, Terefework Z, Chen W, Lindstrom K.. 2001. Genetic diversity of rhizobia isolated from Astragalus adsurgens growing in different geographical regions of China. Journal of Biotechnology, 91:155~168
Gaunt M W, Turner S L, Rigottier-Gois L. 2001. Phylogenies of atpD and recA support the small subunit rRNA-based classification of rhizobia. Int J Syst Evol Microbiol, 51:2037~2048
Gherbi H. 2008. SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankia bacteria. Proc. Natl. Acad. Sci. U. S. A. 105:4928~4932
Glickmann E, Dessaux Y. 1995. A critical examination of the specificity of the Salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Applied and Environmental Microbiology, 61: 793~796
Han Su-Zhen, Wang En-Tao, Chen Wen-Xin. 2005. Diverse bacteria isolated from root nodules of Phaseolus vulgaris and species within the genera Campylotropis and Cassia grown in China.Systematic and Applied Microbiology, 28 :265~276
Heim R, Tsien R Y. 1996. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol,6:178~182
Jarvis B D W, Van Berkum P, Chen W X. 1997. Transfer of Rhizobium loti, Rhizobium huakuii, Rhizobium ciceri, Rhizobium mediterraneum, and Rhizobium tianshanense to Mesorhizobium gen. nov. Int. J. Syst. Bacteriol, 47: 895~898
Jones K.M. 2007. How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model. Nat. Rev. Microbiol. 5:619~633
Jordan D C. 1982. Transfer of Rhizobium japonicum Buchanan 1980 to Bradyrhizobium gen. nov., a genus of slow-growing, root nodule bacteria from leguminous plants. Int. J. Syst. Bacteriol, 32: 136~139
Kan F L, Chen Z Y, Wang E T, Tian C F, Sui X H, Chen W X. 2007. Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai–Tibet Plateau and in other zones of China. Archives of Microbiology, 188:103~115
Kersters K, De Ley J, Sneath P H A. 1973. Numerical taxonomic analysis of Agrobacterium. J Gen Micrbiol ,78:227~239
Kloepper J W, John L, Teintze M. 1980. Enhanced plant growth by siderophores produced by plant growth-promoting rhizobacter. Nature, 286: 885~886
Leong J. 1986. Siderophores: their biochemistry and possible role in the biocontrol of plant pathogens. Annu Rev Phytopathol, 24: 187~209
Li Ji Hong, Wang En Tao, Chen Wen Feng, Chen Wen Xin. 2008. Genetic diversity and potential for promotion of plant growth detected in nodule endophytic bacteria of soybean grown in Heilongjiang province of China. Soil Biology & Biochemistry, 40:238~246
Lindow S E. 1995. The use of reptorer genes in the study of microbial ecology. Mol . Ecol, 4:555～566
Lindstr?m and Martínez-Romero. 2002. International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Agrobacterium and Rhizobium. Int J Syst Evol Microbiol,52:2337
Machuca A, Milagres A M F. 2003. Use of CAS-agar plate modified to study the effect of different variables on siderophore production by Aspergills. Lett Appl Microbial, 36: 177~181
Marmur J. 1961. A procedure for the isolation of DNA from microorganisms. J. Mol. Biol, 3:208~218
Martinez E. 1987. Nitrogen fixing nodules induced by Agrobacterium tumefaciens harboring Rhizobium phaseoli plasmids. J. Bacteriol. 169:2828~2834
Mhamdi R, Mrabet M, Laguerre G. 2005. Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains. Can J Microbiol, 51:105~111
Moore L W, Bouzar H, Burr T. 1988. Agrobacterium. In: SchaadNW (ed) Laboratory Guide for identification of plant pathogenic bacteria, 2nd ed. APS Press, St Paul, MN, pp 16~36
Mrabet M, Mnasri B, Romdhane S B, Laguerre G, Aouani M E, Mhamdi R. 2006. Agrobacterium strains isolated from root nodules of common bean specifically reduce nodulation by Rhizobium gallicum. FEMS Microbiology Ecology, 56:304~309
Nico Stuurman. 2000. Use of Green Fluorescent Protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants . The American Phytopathological Society, 13 (11) :1163~1169
Odee D W, Haukka K, McInroy S G, Sprent J I, Sutherland J M, Young J P W. 2002. Genetic and symbiotic characterization of rhizobia isolated from tree and herbaceous legumes grown in soils from ecologically diverse sites in Kenya.Soil Biology&Biochemistry,34:801~811
Oke V. and Long S R. 1999. Bacteroid formation in the Rhizobium–legume symbiosis. Curr. Opin. Microbiol, 2:641~646
Prasher D C, Eckenrode V K, Ward W W, Prendergast F C, Cormier M J. 1992. Primary structure of the Aequorea victoria green-fluorescent protein. Gene, 111:229~233
Rivas R, Velázquez E, Willems A, Vizcaíno N, Subba-Rao N S, Mateos P F, Gillis M, Dazzo F B, Martínez-Molina E. 2002. A new species of Devosia that forms a unique nitrogen-fixing root-nodule symbiosis with the aquatic legume Neptunia natans (L. f.) Druce. Appl.Environ. Microbiol, 68:5217~5222.
Saitou N, Masatoshi N. 1987. The neighbor-joining method:a new method for reconstructing phylogenetic tree. Mol Biol Evol, 4(4):406~425
Sawada H. 2003. Changing concepts in the systematics of bacterial nitrogen-fixing legume symbionts. J. Gen. Appl. Microbiol.49:155~179
Shimomura O, Johnson F H, Saiga Y. 1962. Extraction,purification and properties of a bioluminescent protein from the luminous hudrmedusan, Aequorea. J Cell Comp Physiol, 59:223~240
Shimomura O. 1979. Structure of the chromophore of Aequorea green fluorescent protein. FEBS Lett,104:220~222
Sohail H, Fathia M, Kauser A M. 2004. Nodule co-occupancy of Agrobacterium and Bradyrhizobium with potential benefit to legume host. 14th International Congress on Nitrogen Fixation
Sohail Hameed, Sumera Yasmin, Kauser A. Malik, Yusuf Zafar, Fauzia Y. Hafeez. 2004. Rhizobium, Bradyrhizobium and Agrobacterium strains isolated from cultivated legumes. Biol Fertil Soils, 39:179–185
Stephen P. Cummings, Prasad Gyaneshwar, Pablo Vinuesa, Frank T. Farruggia, Mitchell Andrews, David Humphry, Geoffrey N. Elliott, Andrew Nelson, Caroline Orr, Deborah Pettitt, Gopit R. Shah, Scott R. Santos, Hari B. Krishnan, David Odee, Fatima M. S. Moreira, Janet I. Sprent, J. Peter W. Young and Euan K. James. 2009. Nodulation of Sesbania species by Rhizobium(Agrobacterium) strain IRBG74 and other rhizobia. Environmental Microbiology
Tan Z Y, Wang E T, Peng G X, Zhu M E, Martíne-Romero E, Chen W X. 1999. Characterization of bacteria isolated from wild legumes in the north-western regions ofChina. Int. J. Syst. Bacteriol, 49:1457~1469.
Thomoson J D, Gibson T J, Plewniak F. 1997. The Clustal X windows interface:flexible strategies for multiple sequence alignment aided by quality analysis tool. Nucleic Acids Res, 24:4867~4882
Tighe S W, De Lajudie P, Dipietro K. 2000. Analysis of cellular fatty acids and phenotypic relationships of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobiumspecies using the Sherlock Microbial Identification System. Int J Syst Evol Microbiol, 50:787~801
Van de Peer Y, De Wachter R. 1994. Treecon for Windows:a software package for the construction and drawing of evolutionary trees for the Microsoft windows environment. Comput Appl Biosci, 10:569~570
Vincent J M. 1970. A Manual for the Practical Study of Root-Nodule Bacteria, Blackwell Scientific Publications, Oxford UK
Wang L L, Wang E T, Liu J, Li Y, Chen W X. 2006. Endophytic occupation of root nodules and roots of Melilotus dentatus by Agrobacterium tumefaciens. Microbial Ecology ,52:436~443
Weisburg W G, Barns S M, Pelletior D A, Lane D J. 1991. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173:697~703
Winans S C. 1992. Two-way chemical signaling in Agrobacterium–plant interactions. Microbiol Rev ,56:12~31
Woese C R, Stackebrandt E, Weisburg W G. 1984. The phylogeny of purple bacteria: the alpha subdivision. Syst Appl Microbiol, 5:315~326
Woese C R. 1987. Bacterial evolution. Microbiol Rev,51: 221~269
Wong L L, Wong E T, Liu J. 2006. Endophytic occupation of root nodules and root of Melilotus dentatus by Agrobacterium tumefaciens. Microbial Ecol ,52:436~443
Young J M, Bull C T, De Boer S H. 2001. Classification, nomenclature and plant pathogenic bacteria: a clarification.Phytopathology, 91:617~620
Young J M, Kuykendall L D, Martinez-Romero E. 2003. Classification and nomenclature of Agrobacterium and Rhizobium: a reply to Farrand et al(2003). Int J Syst Evol Microbiol, 53:1689~1695
Zimmer M. 2002. Green fluorescent protein ( GFP) : applications ,structure ,and related photophysical behavior. Chem Rev ,102(3) :759~781
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |