接种根瘤菌HN01及其突变株GXHN100对大豆根系结瘤及微生物生态的影响
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摘要
与土著根瘤菌相比，接种根瘤菌竞争结瘤能力的强弱很大程度上决定了接种的效果。在提高接种效果的同时，根瘤菌的环境释放对土著微生物群落的生态效应也值得我们关注。早期的研究表明：(1)根系微生物群落因植物种类差异呈现波动变化；(2)根系微生物群落受季节变化的影响而波动；(3)一些根系微生物群落既不受植物也不受季节变化的影响。本试验将费氏中华根瘤菌HN01与其突变株GXHN100接种到大豆根系，以研究田间条件下HN01与GXHN100的结瘤及竞争结瘤能力，及接种后对大豆根系土著微生物微生态效应研究。
为了更好地了解接种根瘤菌对大豆根系微生物的群落的影响，本研究采用传统的平板分离培养计数、16SrDNA文库和变性梯度凝胶电泳技术相结合的方法。田间竞争结瘤试验混接种处理中，HN01的占瘤率为49.4％，而GXHN100的占瘤率仅为9.4％。平板分离培养计数试验显示种植大豆可以增加大豆根系微生物的数量。DGGE的图谱表明各处理大豆根系微生物群落是相当稳定的，接种根瘤菌处理与对照之间的差别微小。对比各处理的16SrDNA文库所构建的进化树发现，各处理大豆根系微生物群落结构相似性很高。试验结果表明接种根瘤菌对大豆根系微生物群落影响微小。
The success of a rhizobial inoculant in the soil depends to a large extent on itscapacity to compete against indigenous strains. With improving inoculantaffection, ecological effection of indigenous microbial communities also needto be attended by rhizobium release to soil. Previous researches show: (1)endorhizosphere microbial communities vary in various plants; (2)endorhizosphere microbial communities vary in seasons; (3) someendorhizosphere microbial communities are not affected by both plant andseason. With the wild type strain HN01 as the control, GXHN100, amutant(S. fredii, strain), was used as soil inoculants for soybean in a field releaseexperiment. The aim of this study is to investigate the differences of nodulationand indigenous microbial community in the root system soil after inoculatingHN01 and GXHN100.
Inorder to gain a better understanding dynamics of bacterial communities ofthe endorhizosphere of the soybean, traditional culture-dependent, 16S rDNAgene-based PCR followed by denaturing gradient gel electrophoresis (DGGE), and 16Sr DNA library were used to evaluated the effects of these two strainsinfluence the indigenous microbial community.
Nodulation competition test showed that the nodulation occupancy ofGXHN100 was 9.4% while that of the HN01 was 49.4%. Data showed soybeanplants increased the number of microbes. The DGGE patterns suggested that thebacterial communities as determined from direct endorhizosphere DNA extractswere largely stable between inoculanted treatment and noninoculanted treatment.Phylogenetic tree of each 16Sr DNA library showed that the structure ofendorhizosphere microbial community was very similar with each other. In ourstudy, no inoculation-dependent effects could be detected, while the soybeanplants appeared to have a much stronger influence on the microbial community.
为了更好地了解接种根瘤菌对大豆根系微生物的群落的影响，本研究采用传统的平板分离培养计数、16SrDNA文库和变性梯度凝胶电泳技术相结合的方法。田间竞争结瘤试验混接种处理中，HN01的占瘤率为49.4％，而GXHN100的占瘤率仅为9.4％。平板分离培养计数试验显示种植大豆可以增加大豆根系微生物的数量。DGGE的图谱表明各处理大豆根系微生物群落是相当稳定的，接种根瘤菌处理与对照之间的差别微小。对比各处理的16SrDNA文库所构建的进化树发现，各处理大豆根系微生物群落结构相似性很高。试验结果表明接种根瘤菌对大豆根系微生物群落影响微小。
The success of a rhizobial inoculant in the soil depends to a large extent on itscapacity to compete against indigenous strains. With improving inoculantaffection, ecological effection of indigenous microbial communities also needto be attended by rhizobium release to soil. Previous researches show: (1)endorhizosphere microbial communities vary in various plants; (2)endorhizosphere microbial communities vary in seasons; (3) someendorhizosphere microbial communities are not affected by both plant andseason. With the wild type strain HN01 as the control, GXHN100, amutant(S. fredii, strain), was used as soil inoculants for soybean in a field releaseexperiment. The aim of this study is to investigate the differences of nodulationand indigenous microbial community in the root system soil after inoculatingHN01 and GXHN100.
Inorder to gain a better understanding dynamics of bacterial communities ofthe endorhizosphere of the soybean, traditional culture-dependent, 16S rDNAgene-based PCR followed by denaturing gradient gel electrophoresis (DGGE), and 16Sr DNA library were used to evaluated the effects of these two strainsinfluence the indigenous microbial community.
Nodulation competition test showed that the nodulation occupancy ofGXHN100 was 9.4% while that of the HN01 was 49.4%. Data showed soybeanplants increased the number of microbes. The DGGE patterns suggested that thebacterial communities as determined from direct endorhizosphere DNA extractswere largely stable between inoculanted treatment and noninoculanted treatment.Phylogenetic tree of each 16Sr DNA library showed that the structure ofendorhizosphere microbial community was very similar with each other. In ourstudy, no inoculation-dependent effects could be detected, while the soybeanplants appeared to have a much stronger influence on the microbial community.
引文
1 李有国，周俊初，影响根瘤菌共生固氮效率的主要因素及遗传改造，微生物学通报，2002，29(6)：86-89．
2. Hurse LS, Date RA. Competitiveness of indigenous strains of Bradyrhizoium on Desmodium intortum cv Greenleaf om three soils of South East Queensland[J]. Soil Bilo Biochem, 1992, 24; 41-50.
3. George T, Bohlool BB, Singleton P W. Bradyrhizobium japonicum environment interactions: nodulation and interstrain competition in soils along an elevational transect [J]. Appl Environ Microbiol, 1987, 53: 1113-1117.
4. Bushby HVA. Colonization of rhizaospheres by Bradyrhizobium sp. in relation to strain persistence and nodulation of some pasturelegumes[J]. Soil Biochem. 1993, 25: 597-605.
5．胡振宇，黄怀琼，刘世全．快生型花生根瘤菌株与土著性根瘤菌竞争结瘤能力的探讨[J]，四川农业大学学报，1994，12：12—181．
6 Hiltbold A E, Patterson RM, Reed R B. Soil populations of Rhizobium japonicum in a cotton-corn-soybean rotation. Soil Sci Soc Am J, 1985, 49: 343～348.
7 McDermott T R, Graham P H, Ferrey ML. Competitiveness of indigenous popultions of Bradyrhizobium japonicum serocluster 123 as determined using a root2tip marking procedure in growth pouches. Plant Soil, 1991, 135: 245～250.
8 A.K.A.va. D.G.Edwards, C.JAsher, et al. Effects of Acid Soil Infertility Factors on Growth and Nodulation of Soybean [J]. Agronomy Journal, 1987 (79): 302-306.
9 Lamrabet Y, Ramon A, Bellogin, et al. Mutation in GDP-Fucose synthesis genes of sinorhizobium fredii alters Nod factorsand significantly decrease competitiveness to nodulate soybeans[J]. Mol Plant-Microbe Interact, 1999, 12: 207～217.
10 杨江科．pH对土壤中土著快、慢生大豆根瘤菌结瘤的影响[J]．应用生态学报，2001，12(4)：639-640．
11 杨江科．占优势土著大豆根瘤菌的遗传多样性及pH对竞争结瘤的影响：[硕士学位论文]．武汉：华中农业大学，1999．
12 李新民，谷思玉，窦新田，等．不同土壤大豆接种根瘤菌剂反应的研究[J]．黑龙江农业科 学，1998，4：1-51．
13 陈因，陈永滨，唐锡华，等编著．《生物固氮》[M]．上海科技出版社．1985年8月1．
14 窦新田编著．《生物固氮》[M]．北京：农业出版社，1989年8月1．
15 Theis, J. E., Singleton, P. W., Bohlool, B. B. Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field grown legumes[J]. Applied and Environmental Microbiology. 1991, 57, 29～37.
16 Kapusta, G. D. L1Rowwenhost. Influence of inoculum size on Rhizobium japonicum sergroup distribution frequency in soybean nodules[J]. Agron. J. 1973, 65: 916～919.
17 Weaver, R. W., Frederick L.R., Dumeril, L.C. Effect of soybean cropping and soil properties on number of rhizobium Japonicum in Iowa Soils[J]. Soil Science, 1972 (114), 137～141.
18 丁武．影响根瘤菌竞争结瘤的生态学因素分析[J]．生态学杂志，1992，11(4)：50～54．
19 关桂兰，王卫卫，杨玉锁1 新疆干旱地区根瘤菌资源研究．根瘤菌种类及其共生固氮作用，微生物学报，1991，31(5)：356-404．
20 尤崇杓主编．生物固氮[M]．北京：科学出版社．1987年11月．
21 Cregan P B, er al. Host plant effects on nodulation and competitiveness of the Bradyrhizobium japonicum serotype strains constituting setocluster 123.Applied and Environmental Microbiology, 1989, 55(10): 2535～2536.
22 扬江科，刘墨青，周琴，周俊初。以luxAB为报告基因的大豆根瘤菌的竞争结瘤研究。中国农业科学，2002，35(1)：110～112
23 黄大昉，林敏主编，《农业微生物基因工程》，科学出版社，2001．
24 Araujo R S, Handeman J. Characteristics of exopolysaccharide-deficient mutants of Rhizobium spp. With altered nodulation competitiveness. Nitrogen Fixation: Achievements and Objecyives, 1990, New York: Champman and Hall Publisher
25 Zor R E, Pueppke S G. Nodulation competitiveness of Tn5-induced mutants of Rhizobium fredii 208 that are altered in mobility and extracellular polysaccharide production. Can. J. Microbiol., 1991, 37: 52～58
26 Ames P, Bergman K: Competitive advantage provided by bacterial motility in the formation of nodules by Rhizobium meliloti. J Bacteriol 1981, 148: 728～729
27 Triplett E W, Sadowsky MJ. Genetics for nodulation competition of legumes. Ann Rev Microbiol, 1992, 46: 399～428。
28 Sanjuan J, and Olivares J., 1989. Implication of nifA in regulation of genes located on a Rhizobium meliloti cryptic plasmid that affect nodulation efficiency. J. Bacteriol., 171: 4154～4161.
29 马庆生，武波，唐东阶等，外源nfeC基因导入快生型大豆根瘤菌菌株HN01的行为分析，复旦学报(自然科学版)，1998，37(4)：445～448。周刚林、武波、唐东阶等，慢生型大豆根瘤菌nfe基因的分子克隆，广西农业生物科学，1999，18(1)：29～32．
30 Osslak, RM and Bohlool, BB. Suppression of nodule development of one side of a split-root system of soybeans caused by prior inoculation of the other side. Plant Physiol. 1984, 75: 125～130
31 Eric W. Triplett. Isolation of Genes Involved in Nodulation Competitiveness from Rhizobium leguminosarum bv. trifolii T24, PNAS, Jun 1988; 85: 3810～3814.
32 Eduardo A. Robleto, Kenneth Kmiecik, Edward S. Oplinger. Trifolitoxin Production Increases Nodulation Competitiveness of Rhizobium etli CE3 under Agricultural Conditions. Appl. Envir. Microbiol., Jul 1998; 64: 2630～2633.
33 Alexandra J. Scupham, Yuemei Dong, and Eric W. Triplett. Role of tfxE, but not tfxG, in Trifolitoxin Resistance Appl. Envir. Microbiol., Sep 2002; 68: 4334～4340.
34 Okazaki, S., Sugawara, M., Minamisawa, K. Bradyrhizobium elkanii rtxC Gene Is Required for Expression of Symbiotic Phenotypes in the Final Step of Rhizobitoxine Biosynthesis. Appl. Environ. Microbiol. 2004. 70: 535～541
35 Ma, W., F. C. Guinel, and B. R. Glick. Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-carboxylate deaminase promotes nodulation of pea plants. Appl. Environ. Microbiol. 69: 4396～4402.
36 Ma W, Charles TC, Glick BR. Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in Sinorhizobium meliloti increases its ability to nodulate alfalfa. Appl Environ Microbiol. 2004 Oct; 70(10): 5891～7.
37 Parker, M. A., and N. K. Peters. 2001. Rhizobitoxine production and symbiotic compatibility of Bradyrhizobiurn from Asian and North American lineages of Amphicarpaea. Can. J. Microbiol. 47: 1-6.
38 Yuhashi, K., N. Ichikawa, H. Ezura. Rhizobitoxine production by Bradyrhizobium elkanii enhances nodulation and competitiveness on Macroptilium atropurpureum. Appl. Environ. Microbiol. 2000. 66: 2658～2663.
39 Hubber A, Vergunst AC, Sullivan JT, Hooykaas PJ, Ronson CW. Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium Ioti strain RTA VirB/D4 type Ⅳ secretion system. Mol Microbiol. 2004 Oct; 54(2) : 561～74.
40 武波．唐咸来．柏学亮等，慢生大豆根瘤菌与竞争结瘤相关的lrp基因的克隆，农业生物技术，2001，9(3)：286～288
41 Brock T D. The study of microorganisms in situ: progress and problem s. Symp. Soc. Gene Microbiol. icrobiol. , 1987, 41: 1～17.
42 Jonathan J, Alice L, et al. Cd (Ⅱ)-responsive and constitutive mutants implicate a novel domain in MerR. Journal of Bacteriology, 1999, 181(11) : 3462～3471.
43 Garland J L, Mills A L. Classification and characterization of heterotrophic microbial communities on the basis of patterns of community level sole—carbon source utilization. Appl Environ Microbiol 1991 2351～2359.
44 Bochner B R. Sleuthing out bacterial identities. Nature, 1989, 339: 157～158
45 Zak J C, Willig M R, Moorhead D L, et al. Functional diversity of microbial communities: a quantitative approach. Soil Biol. Biochem. , 1994, 26: 1101～1108
46 Bossio D D, Scow K M. Impact of carbon and flooding on the matablic diversity of microbial communities in soils. Appl. Environ. Microbiol. , 1995, 61: 4043～4050
47 Buyer J S, Drinkwater L E. Comparison of substrate utilization assay and fatty acid analysis of soil microbial communities. J. Microb. Methods, 1997, 30: 3～11
48 Grayston S J, Wang S, Campbell C D, et al. Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biol. Biochem. , 1998, 30: 369～378
49 FrostegardA, Tunlid A, Baath E. Journal of Microbiological Methods, 1991, 14: 151～163.
50 Yao H, He Z, Wilson M J, et al. M icro Ecol, 2000, 40: 223～237
51 Lei F, VanderGheynst J S. Process Biochemistry, 2000, 35: 923～929.
52 J 萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯．分子克隆实验指南，第二版
53 Van Dillewijn P, Villadas PJ, Toro N. Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa. Appl Environ Microbiol. 2002 Sep; 68 (9) : 4201～4208.
54 赵勇等。应用PCR-RFLP及PCR-TGGE技术监测农田土壤微生物短期动态变化，南京农业大学学报 2005，28(3)：53～57
55 Beat Frey, Michael Stemmer, Franco Eidmer, et al. Microbial activity and community structure of a soil after heavy metal contamination in a model forest ecosystem, Soil Biolgy &Biochemistry, 38(2006) : 1745-1756
56 Sangkyu Park, Youn Kyung KU Principal conponet analysis and discriminant analysis (PCA-DA) for discriminating profiles of terminal restriction fragment length polymorphism(T—RFLP) in soil bacterial communities Soil Biology & Biochemistry 38 (2006) 2344～2349
57 LuedersT, Friedrich M, Archael. Population dynamics during sequential reduction processed in rice. Appl. Environ Microbiol, 2000, 66: 2732～2742.
58 祖元刚等 喜树替代紫茎泽兰过程中根际微生物群落特征，《中国科学C辑》2006年第5期
59 陈强，陈文新，张小平等．分离自四川省葛藤属根瘤菌的遗传多样性研究．中国农业科学
60 Willems A, Doignon-Bourcier F, Coopman R. etal. 2000 AFLP fingerprint analysis of Bradrhizobiam strains isolated from Faidherbia albida and Aeschynomenes pecies, Syst Appl Microbiol. 23(1) : 137～47.
61 Head I M, Saunders J R, Pickup R W. Microbial evolution, diversity, and ecology: A decade of ribosomal RNA analysis of uncultivated microorganisms. Microbial Ecology. 1998. 35: 1～21.
62 Marilley L, Vogt G, Blanc M, et al. Bacterial diversity in the bulk soil and Rhizosphere fractions of Loliumperenne and Trifoliumrepensa srevealedb y PCR Restriction analysis. Plant and Soil. 1998. 198: 219～224.
63 Rank S, Christoph C T. A New Approach To Utilize PCR Single Strand Conformation Polymorphism for 16Sr RNA Gene-Based Microbial Community Analysis. Applied and Environmental Microbiology. 1998. 64 (12) : 4870～4876
64 FrankS, Christoph C T. Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicagosaliva) and a Non-Target Plant(Chenopodium album)Linking of 16SrRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the D iversity of Cultivated Bacteria. Applied and Environmental Microbiology. 2000. 66 (8) : 3556～3565
65 Sabine P, Stefanie K, FrankS, et al. Succession of microbial communities during hot composting as detected by PCR Single Strand Conformation Polymorphism Based genetic profiles of small-sub unit rRNA genes. Applied and EnvironmentalMicrobiology. 2000. 66 (3) : 930～936.
66 陈洪，杨靖，薛国雄等RAPD技术在异精激发方正银卿比较研究中的应用．科学通报1994．39：661-663．
67 Fischer S Q Lerman L S. DNA fragments differing by single basepair sub stitutions are separated in denaturing gradient gels: correspondence with melting theory. Proceedings of the National Academy of Science of USA. 19 83. 80: 1579～1583.
68 Myers RM, Fischer S G, Lerman L S, et al. Nearly all single base substitutions in DNA fragments joined to a GC2clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res, 1985, 13: 3131～31451
69 Muyzer, G. , Ellen, C. D. W, Andre, CU. Profiling-of complex microbial Populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction genes coding for 16SrRNA. Applied and Environmental Microbiology. 1993. 59: 695～700.
70 Ferris M J, Muyaer G, Ward D M. Denaturing gradient gel electrophoresis Profiles of 16SrRNA-defined population inhabiting a hot spring microbial mat community. Applied and Environmental Microbiology. 1996. 62: 340～346.
71 Casamayor E O, Achafer H, Baneras L, et al. Identification of and spatiotemporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis. Applied and Environmental Microbiology.
72 Bano N, Hollibaugh JT. Phylogenetic compostion of bacterioplankton Assemblage from the Arctic ocean. Applied and Environmental Microbiology 2002. 68: 505-518.
73 Duineveld B M, Rosado A, Elsas J D, et al. Analysis of the dynamics of bacterial communities in the rhizophere of the chrysanthemum via denaturing gradient gel electrophoresis and substrate utlization patterns. Applied and Environmental Microbiology. 1998. 64. 4950～4957.
74 Smit E, L eeflangP, Gommans S, et al. Diversity and seasonal fluctuations of the dominant members of the bacterial soil community in a wheat field as determined by cultivation and molecular methods. Applied and Environmental Microbiology. 2001. 67: 2284～2291.
75 罗海峰，齐鸿雁，薛凯，张洪勋．PCR-DGGE技术在农田土壤微生物多样性研究中的应用．生态学报，2003，23(8)：1570～1575
76 Salles J F, De Souza F A, Van Elsas J D. Molecular method to assess the diversity of Brkolderia species in environmental samples. Appl. Environ. Microbiol, 2002, 68(4) : 1595～1603.
77 Short SM, Suttle C A. Sequence analysis of marine virus communities reveals that groups of related algal viruses are widely distributed in nature. A ppl. Env iron. Microbiol. , 2002, 68(3) : 1290～1296.
78 Ferris MJ, Muyzer G, and Ward D M1Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial matcommunity[J]. Appl Environ Microbiol ,1996 ,62(2):340-346.
79 Duineveld B M,Rosado A,Elsas J D,et al. Analysis of the dynamics of bacterial communities in the rhizophere of the chrysanthemum via denaturing gradient gel electrophoresis and substrate utilization patterns[J].Appl Environ Microbiol 1998,64(12):4950-4957.
80 Lottmann J,Heuer H,De Vries i,et al. Establishment of introduced antagonistic bacteria in rhizosphere of transgenic potatoes and their effect on the bacterial community. FEMS Microbiol. Ecol, 2000, 33 (1):41-49.
81 Ibekwe A M, Kennedy A C, Frohne P S, et al. Microbial diversity along a transect of agronomiczones. FEMS Microbiol Ecol., 2002, 39 (3): 183 - 191.
82 Heuer H, Kroppenstedt R M , Lo ttmann J, et al. Effects of T4 lysozyme release from transgenic po tato roo ts onb bacterial rhizosphere communities is negligible relative to natural factors. Appl. Environ. Microbiol,2002,68(3):1325-1335.
83 Sandaa R A, Enger A, To rsvik V. A bundahce and diversity of Archaea in heavy—metal—contaminated soils. Appl.Environ. Microbiol., 1999,65(8):3293-3297.
84 Santegoeds C M,Nold S ,and Ward D M.Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat [J]. Appl Environ Microbiol, 1996,62(11):3922-3928.
85 Teske A ,Sigalevich P ,Cohen Y, et al.Molecular identification of bacteria from a coculture by denaturing gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in pure cultures[J].Appl Environ Microbiol, 1996,62(11):4210-4215.
86 Dahll F I, Baillie H and Kjelleberg S. rpoB—based microbial community analysis avoids limitations inherent in 16SrRNA gene intraspecies heterogeneity.Appl.Environ. Microbiol,2000,66(8):3376-3380.
87 Ferris M J , Ward D M. Seasonal distributions of dominant 16SrDNA—defined populations in a hot spring microbial mat examined by denaturing gradient gel electrophoresis. A ppl. Environ. Microbiol,1997,63(4):1375-1381.
88 Yang C H, Crowley D E. Rhizosphere microbial community structure in relation to root location and plant iron nutritional status. Appl. Environ. Microbiol,2000,66(1):345-351.
89 Hold G L,Smith E A, Rappé M S,et al. Characterization of bacterial communities associated with toxic and nontoxic dinoflagellates: A lex and riumspp. and Scripp siella trochoidea. FEMS Microbiol. Ecol., 2001, 37 (2) : 161～173.
90 Straub K L, Buchho lz—Cleven B E E. Enumeration and detection of anaerobic ferrous iron—oxidizing, nitrate reducing bacteria from diverse European sediments. Appl Environ. Microbiol,1998, 64(12) : 4846～4856.
91 Phillip s C J, Harris D, Dollhopf S L, et al. Effects of agronomic treatments on structure and function of Ammonia—oxidizing communities. Appl. Environ. Microbiol, 2000, 66(12) : 5410～5418.
92 Filion M, Hamelin R C, Benier L, et al. Molecular Profiling of Rhizosphere Microbial Communities Associated with Healthy and Diseased Black Spruce (Picea mariana) Seedlings Grown in a Nursery. Appl Environ Microbiol, 2004, 70(6) : 3541～3551
93 戴欣，王保军，黄燕，等．普通和稀释培养基研究太湖沉积物可培养细菌的多样性．微生物学报，2005，45(2)：161～165．
94 Lottmann, J. , H. Heuer, J. de Vries, et al. Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community. FEMS Microbiol. Ecol. 2000, 33: 41～49.
95 Smalla, K. , G. Wieland, A. Buchner, et al. Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed. Appl. Environ. Microbiol. 2001, 67: 4742～4751.
96 Di Cello, F, A. Bevivino, L. Chiarini, R. et al. Biodiversity of a Burkholderia cepaciaBurkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages. Appl. Environ. Microbiol. 1997, 63: 4485～4493
97 Seldin, L. , A. S. Rosado, D. W. da Cruz, et al. Comparison of Paenibacillus azotofixansPaenibacillus azotofixans strains isolated from rhizoplane, rhizosphere, and non-root-associated soil from maize planted in two different Brazilian soils. Appl. Environ. Microbiol. 1998, 64: 3860～3868.
98 Picard, C, F. DiCello, M. Ventura, et, al. Frequency and biodiversity of 2, 4-diacetylphloroglucinol-producing bacteria isolated from the maize rhizosphere at different stages of plant growth. Appl. Environ. . Microbiol. 2000, 66: 948～955
99 Gomes, N. C. M. , H. Heuer, J. Schonfeld, et al. Bacterial diversity of the rhizosphere of maize (Zea maysZea mays) grown in tropical soil studied by temperature gradient gel electrophoresis. Plant Soil 2001. 232: 167～180.
100 Hamlen, R., F. Lukezic, and J. Bloom. Influence of age and stage of development on the neutral carbohydrate components in root exudates from alfalfa plants grown in a gnotobiotic environment. Can. J. Plant Sci. 1972. 52: 633～642.
101 Duineveld, B. M., A. S. Rosado, J. D. Van Elsas, et al. 1998. Analysis of the dynamics of bacterial communities in the rhizosphere of the chrysanthemum via denaturing gradient gel electrophoresis and substrate utilization patterns. Appl. Environ. Microbiol. 1998. 64: 4950～4957..
102 Duineveld, B. M., G. A. Kowalchuk, A. Keijzer, J. D. et al. Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments Coding for 16S rRNA. Appl. Environ. Microbiol. 2001. 67: 172～178.
2. Hurse LS, Date RA. Competitiveness of indigenous strains of Bradyrhizoium on Desmodium intortum cv Greenleaf om three soils of South East Queensland[J]. Soil Bilo Biochem, 1992, 24; 41-50.
3. George T, Bohlool BB, Singleton P W. Bradyrhizobium japonicum environment interactions: nodulation and interstrain competition in soils along an elevational transect [J]. Appl Environ Microbiol, 1987, 53: 1113-1117.
4. Bushby HVA. Colonization of rhizaospheres by Bradyrhizobium sp. in relation to strain persistence and nodulation of some pasturelegumes[J]. Soil Biochem. 1993, 25: 597-605.
5．胡振宇，黄怀琼，刘世全．快生型花生根瘤菌株与土著性根瘤菌竞争结瘤能力的探讨[J]，四川农业大学学报，1994，12：12—181．
6 Hiltbold A E, Patterson RM, Reed R B. Soil populations of Rhizobium japonicum in a cotton-corn-soybean rotation. Soil Sci Soc Am J, 1985, 49: 343～348.
7 McDermott T R, Graham P H, Ferrey ML. Competitiveness of indigenous popultions of Bradyrhizobium japonicum serocluster 123 as determined using a root2tip marking procedure in growth pouches. Plant Soil, 1991, 135: 245～250.
8 A.K.A.va. D.G.Edwards, C.JAsher, et al. Effects of Acid Soil Infertility Factors on Growth and Nodulation of Soybean [J]. Agronomy Journal, 1987 (79): 302-306.
9 Lamrabet Y, Ramon A, Bellogin, et al. Mutation in GDP-Fucose synthesis genes of sinorhizobium fredii alters Nod factorsand significantly decrease competitiveness to nodulate soybeans[J]. Mol Plant-Microbe Interact, 1999, 12: 207～217.
10 杨江科．pH对土壤中土著快、慢生大豆根瘤菌结瘤的影响[J]．应用生态学报，2001，12(4)：639-640．
11 杨江科．占优势土著大豆根瘤菌的遗传多样性及pH对竞争结瘤的影响：[硕士学位论文]．武汉：华中农业大学，1999．
12 李新民，谷思玉，窦新田，等．不同土壤大豆接种根瘤菌剂反应的研究[J]．黑龙江农业科 学，1998，4：1-51．
13 陈因，陈永滨，唐锡华，等编著．《生物固氮》[M]．上海科技出版社．1985年8月1．
14 窦新田编著．《生物固氮》[M]．北京：农业出版社，1989年8月1．
15 Theis, J. E., Singleton, P. W., Bohlool, B. B. Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field grown legumes[J]. Applied and Environmental Microbiology. 1991, 57, 29～37.
16 Kapusta, G. D. L1Rowwenhost. Influence of inoculum size on Rhizobium japonicum sergroup distribution frequency in soybean nodules[J]. Agron. J. 1973, 65: 916～919.
17 Weaver, R. W., Frederick L.R., Dumeril, L.C. Effect of soybean cropping and soil properties on number of rhizobium Japonicum in Iowa Soils[J]. Soil Science, 1972 (114), 137～141.
18 丁武．影响根瘤菌竞争结瘤的生态学因素分析[J]．生态学杂志，1992，11(4)：50～54．
19 关桂兰，王卫卫，杨玉锁1 新疆干旱地区根瘤菌资源研究．根瘤菌种类及其共生固氮作用，微生物学报，1991，31(5)：356-404．
20 尤崇杓主编．生物固氮[M]．北京：科学出版社．1987年11月．
21 Cregan P B, er al. Host plant effects on nodulation and competitiveness of the Bradyrhizobium japonicum serotype strains constituting setocluster 123.Applied and Environmental Microbiology, 1989, 55(10): 2535～2536.
22 扬江科，刘墨青，周琴，周俊初。以luxAB为报告基因的大豆根瘤菌的竞争结瘤研究。中国农业科学，2002，35(1)：110～112
23 黄大昉，林敏主编，《农业微生物基因工程》，科学出版社，2001．
24 Araujo R S, Handeman J. Characteristics of exopolysaccharide-deficient mutants of Rhizobium spp. With altered nodulation competitiveness. Nitrogen Fixation: Achievements and Objecyives, 1990, New York: Champman and Hall Publisher
25 Zor R E, Pueppke S G. Nodulation competitiveness of Tn5-induced mutants of Rhizobium fredii 208 that are altered in mobility and extracellular polysaccharide production. Can. J. Microbiol., 1991, 37: 52～58
26 Ames P, Bergman K: Competitive advantage provided by bacterial motility in the formation of nodules by Rhizobium meliloti. J Bacteriol 1981, 148: 728～729
27 Triplett E W, Sadowsky MJ. Genetics for nodulation competition of legumes. Ann Rev Microbiol, 1992, 46: 399～428。
28 Sanjuan J, and Olivares J., 1989. Implication of nifA in regulation of genes located on a Rhizobium meliloti cryptic plasmid that affect nodulation efficiency. J. Bacteriol., 171: 4154～4161.
29 马庆生，武波，唐东阶等，外源nfeC基因导入快生型大豆根瘤菌菌株HN01的行为分析，复旦学报(自然科学版)，1998，37(4)：445～448。周刚林、武波、唐东阶等，慢生型大豆根瘤菌nfe基因的分子克隆，广西农业生物科学，1999，18(1)：29～32．
30 Osslak, RM and Bohlool, BB. Suppression of nodule development of one side of a split-root system of soybeans caused by prior inoculation of the other side. Plant Physiol. 1984, 75: 125～130
31 Eric W. Triplett. Isolation of Genes Involved in Nodulation Competitiveness from Rhizobium leguminosarum bv. trifolii T24, PNAS, Jun 1988; 85: 3810～3814.
32 Eduardo A. Robleto, Kenneth Kmiecik, Edward S. Oplinger. Trifolitoxin Production Increases Nodulation Competitiveness of Rhizobium etli CE3 under Agricultural Conditions. Appl. Envir. Microbiol., Jul 1998; 64: 2630～2633.
33 Alexandra J. Scupham, Yuemei Dong, and Eric W. Triplett. Role of tfxE, but not tfxG, in Trifolitoxin Resistance Appl. Envir. Microbiol., Sep 2002; 68: 4334～4340.
34 Okazaki, S., Sugawara, M., Minamisawa, K. Bradyrhizobium elkanii rtxC Gene Is Required for Expression of Symbiotic Phenotypes in the Final Step of Rhizobitoxine Biosynthesis. Appl. Environ. Microbiol. 2004. 70: 535～541
35 Ma, W., F. C. Guinel, and B. R. Glick. Rhizobium leguminosarum biovar viciae 1-aminocyclopropane-carboxylate deaminase promotes nodulation of pea plants. Appl. Environ. Microbiol. 69: 4396～4402.
36 Ma W, Charles TC, Glick BR. Expression of an exogenous 1-aminocyclopropane-1-carboxylate deaminase gene in Sinorhizobium meliloti increases its ability to nodulate alfalfa. Appl Environ Microbiol. 2004 Oct; 70(10): 5891～7.
37 Parker, M. A., and N. K. Peters. 2001. Rhizobitoxine production and symbiotic compatibility of Bradyrhizobiurn from Asian and North American lineages of Amphicarpaea. Can. J. Microbiol. 47: 1-6.
38 Yuhashi, K., N. Ichikawa, H. Ezura. Rhizobitoxine production by Bradyrhizobium elkanii enhances nodulation and competitiveness on Macroptilium atropurpureum. Appl. Environ. Microbiol. 2000. 66: 2658～2663.
39 Hubber A, Vergunst AC, Sullivan JT, Hooykaas PJ, Ronson CW. Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium Ioti strain RTA VirB/D4 type Ⅳ secretion system. Mol Microbiol. 2004 Oct; 54(2) : 561～74.
40 武波．唐咸来．柏学亮等，慢生大豆根瘤菌与竞争结瘤相关的lrp基因的克隆，农业生物技术，2001，9(3)：286～288
41 Brock T D. The study of microorganisms in situ: progress and problem s. Symp. Soc. Gene Microbiol. icrobiol. , 1987, 41: 1～17.
42 Jonathan J, Alice L, et al. Cd (Ⅱ)-responsive and constitutive mutants implicate a novel domain in MerR. Journal of Bacteriology, 1999, 181(11) : 3462～3471.
43 Garland J L, Mills A L. Classification and characterization of heterotrophic microbial communities on the basis of patterns of community level sole—carbon source utilization. Appl Environ Microbiol 1991 2351～2359.
44 Bochner B R. Sleuthing out bacterial identities. Nature, 1989, 339: 157～158
45 Zak J C, Willig M R, Moorhead D L, et al. Functional diversity of microbial communities: a quantitative approach. Soil Biol. Biochem. , 1994, 26: 1101～1108
46 Bossio D D, Scow K M. Impact of carbon and flooding on the matablic diversity of microbial communities in soils. Appl. Environ. Microbiol. , 1995, 61: 4043～4050
47 Buyer J S, Drinkwater L E. Comparison of substrate utilization assay and fatty acid analysis of soil microbial communities. J. Microb. Methods, 1997, 30: 3～11
48 Grayston S J, Wang S, Campbell C D, et al. Selective influence of plant species on microbial diversity in the rhizosphere. Soil Biol. Biochem. , 1998, 30: 369～378
49 FrostegardA, Tunlid A, Baath E. Journal of Microbiological Methods, 1991, 14: 151～163.
50 Yao H, He Z, Wilson M J, et al. M icro Ecol, 2000, 40: 223～237
51 Lei F, VanderGheynst J S. Process Biochemistry, 2000, 35: 923～929.
52 J 萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯．分子克隆实验指南，第二版
53 Van Dillewijn P, Villadas PJ, Toro N. Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa. Appl Environ Microbiol. 2002 Sep; 68 (9) : 4201～4208.
54 赵勇等。应用PCR-RFLP及PCR-TGGE技术监测农田土壤微生物短期动态变化，南京农业大学学报 2005，28(3)：53～57
55 Beat Frey, Michael Stemmer, Franco Eidmer, et al. Microbial activity and community structure of a soil after heavy metal contamination in a model forest ecosystem, Soil Biolgy &Biochemistry, 38(2006) : 1745-1756
56 Sangkyu Park, Youn Kyung KU Principal conponet analysis and discriminant analysis (PCA-DA) for discriminating profiles of terminal restriction fragment length polymorphism(T—RFLP) in soil bacterial communities Soil Biology & Biochemistry 38 (2006) 2344～2349
57 LuedersT, Friedrich M, Archael. Population dynamics during sequential reduction processed in rice. Appl. Environ Microbiol, 2000, 66: 2732～2742.
58 祖元刚等 喜树替代紫茎泽兰过程中根际微生物群落特征，《中国科学C辑》2006年第5期
59 陈强，陈文新，张小平等．分离自四川省葛藤属根瘤菌的遗传多样性研究．中国农业科学
60 Willems A, Doignon-Bourcier F, Coopman R. etal. 2000 AFLP fingerprint analysis of Bradrhizobiam strains isolated from Faidherbia albida and Aeschynomenes pecies, Syst Appl Microbiol. 23(1) : 137～47.
61 Head I M, Saunders J R, Pickup R W. Microbial evolution, diversity, and ecology: A decade of ribosomal RNA analysis of uncultivated microorganisms. Microbial Ecology. 1998. 35: 1～21.
62 Marilley L, Vogt G, Blanc M, et al. Bacterial diversity in the bulk soil and Rhizosphere fractions of Loliumperenne and Trifoliumrepensa srevealedb y PCR Restriction analysis. Plant and Soil. 1998. 198: 219～224.
63 Rank S, Christoph C T. A New Approach To Utilize PCR Single Strand Conformation Polymorphism for 16Sr RNA Gene-Based Microbial Community Analysis. Applied and Environmental Microbiology. 1998. 64 (12) : 4870～4876
64 FrankS, Christoph C T. Effect of Field Inoculation with Sinorhizobium meliloti L33 on the Composition of Bacterial Communities in Rhizospheres of a Target Plant (Medicagosaliva) and a Non-Target Plant(Chenopodium album)Linking of 16SrRNA Gene-Based Single-Strand Conformation Polymorphism Community Profiles to the D iversity of Cultivated Bacteria. Applied and Environmental Microbiology. 2000. 66 (8) : 3556～3565
65 Sabine P, Stefanie K, FrankS, et al. Succession of microbial communities during hot composting as detected by PCR Single Strand Conformation Polymorphism Based genetic profiles of small-sub unit rRNA genes. Applied and EnvironmentalMicrobiology. 2000. 66 (3) : 930～936.
66 陈洪，杨靖，薛国雄等RAPD技术在异精激发方正银卿比较研究中的应用．科学通报1994．39：661-663．
67 Fischer S Q Lerman L S. DNA fragments differing by single basepair sub stitutions are separated in denaturing gradient gels: correspondence with melting theory. Proceedings of the National Academy of Science of USA. 19 83. 80: 1579～1583.
68 Myers RM, Fischer S G, Lerman L S, et al. Nearly all single base substitutions in DNA fragments joined to a GC2clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res, 1985, 13: 3131～31451
69 Muyzer, G. , Ellen, C. D. W, Andre, CU. Profiling-of complex microbial Populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction genes coding for 16SrRNA. Applied and Environmental Microbiology. 1993. 59: 695～700.
70 Ferris M J, Muyaer G, Ward D M. Denaturing gradient gel electrophoresis Profiles of 16SrRNA-defined population inhabiting a hot spring microbial mat community. Applied and Environmental Microbiology. 1996. 62: 340～346.
71 Casamayor E O, Achafer H, Baneras L, et al. Identification of and spatiotemporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis. Applied and Environmental Microbiology.
72 Bano N, Hollibaugh JT. Phylogenetic compostion of bacterioplankton Assemblage from the Arctic ocean. Applied and Environmental Microbiology 2002. 68: 505-518.
73 Duineveld B M, Rosado A, Elsas J D, et al. Analysis of the dynamics of bacterial communities in the rhizophere of the chrysanthemum via denaturing gradient gel electrophoresis and substrate utlization patterns. Applied and Environmental Microbiology. 1998. 64. 4950～4957.
74 Smit E, L eeflangP, Gommans S, et al. Diversity and seasonal fluctuations of the dominant members of the bacterial soil community in a wheat field as determined by cultivation and molecular methods. Applied and Environmental Microbiology. 2001. 67: 2284～2291.
75 罗海峰，齐鸿雁，薛凯，张洪勋．PCR-DGGE技术在农田土壤微生物多样性研究中的应用．生态学报，2003，23(8)：1570～1575
76 Salles J F, De Souza F A, Van Elsas J D. Molecular method to assess the diversity of Brkolderia species in environmental samples. Appl. Environ. Microbiol, 2002, 68(4) : 1595～1603.
77 Short SM, Suttle C A. Sequence analysis of marine virus communities reveals that groups of related algal viruses are widely distributed in nature. A ppl. Env iron. Microbiol. , 2002, 68(3) : 1290～1296.
78 Ferris MJ, Muyzer G, and Ward D M1Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial matcommunity[J]. Appl Environ Microbiol ,1996 ,62(2):340-346.
79 Duineveld B M,Rosado A,Elsas J D,et al. Analysis of the dynamics of bacterial communities in the rhizophere of the chrysanthemum via denaturing gradient gel electrophoresis and substrate utilization patterns[J].Appl Environ Microbiol 1998,64(12):4950-4957.
80 Lottmann J,Heuer H,De Vries i,et al. Establishment of introduced antagonistic bacteria in rhizosphere of transgenic potatoes and their effect on the bacterial community. FEMS Microbiol. Ecol, 2000, 33 (1):41-49.
81 Ibekwe A M, Kennedy A C, Frohne P S, et al. Microbial diversity along a transect of agronomiczones. FEMS Microbiol Ecol., 2002, 39 (3): 183 - 191.
82 Heuer H, Kroppenstedt R M , Lo ttmann J, et al. Effects of T4 lysozyme release from transgenic po tato roo ts onb bacterial rhizosphere communities is negligible relative to natural factors. Appl. Environ. Microbiol,2002,68(3):1325-1335.
83 Sandaa R A, Enger A, To rsvik V. A bundahce and diversity of Archaea in heavy—metal—contaminated soils. Appl.Environ. Microbiol., 1999,65(8):3293-3297.
84 Santegoeds C M,Nold S ,and Ward D M.Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat [J]. Appl Environ Microbiol, 1996,62(11):3922-3928.
85 Teske A ,Sigalevich P ,Cohen Y, et al.Molecular identification of bacteria from a coculture by denaturing gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in pure cultures[J].Appl Environ Microbiol, 1996,62(11):4210-4215.
86 Dahll F I, Baillie H and Kjelleberg S. rpoB—based microbial community analysis avoids limitations inherent in 16SrRNA gene intraspecies heterogeneity.Appl.Environ. Microbiol,2000,66(8):3376-3380.
87 Ferris M J , Ward D M. Seasonal distributions of dominant 16SrDNA—defined populations in a hot spring microbial mat examined by denaturing gradient gel electrophoresis. A ppl. Environ. Microbiol,1997,63(4):1375-1381.
88 Yang C H, Crowley D E. Rhizosphere microbial community structure in relation to root location and plant iron nutritional status. Appl. Environ. Microbiol,2000,66(1):345-351.
89 Hold G L,Smith E A, Rappé M S,et al. Characterization of bacterial communities associated with toxic and nontoxic dinoflagellates: A lex and riumspp. and Scripp siella trochoidea. FEMS Microbiol. Ecol., 2001, 37 (2) : 161～173.
90 Straub K L, Buchho lz—Cleven B E E. Enumeration and detection of anaerobic ferrous iron—oxidizing, nitrate reducing bacteria from diverse European sediments. Appl Environ. Microbiol,1998, 64(12) : 4846～4856.
91 Phillip s C J, Harris D, Dollhopf S L, et al. Effects of agronomic treatments on structure and function of Ammonia—oxidizing communities. Appl. Environ. Microbiol, 2000, 66(12) : 5410～5418.
92 Filion M, Hamelin R C, Benier L, et al. Molecular Profiling of Rhizosphere Microbial Communities Associated with Healthy and Diseased Black Spruce (Picea mariana) Seedlings Grown in a Nursery. Appl Environ Microbiol, 2004, 70(6) : 3541～3551
93 戴欣，王保军，黄燕，等．普通和稀释培养基研究太湖沉积物可培养细菌的多样性．微生物学报，2005，45(2)：161～165．
94 Lottmann, J. , H. Heuer, J. de Vries, et al. Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community. FEMS Microbiol. Ecol. 2000, 33: 41～49.
95 Smalla, K. , G. Wieland, A. Buchner, et al. Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel electrophoresis: plant-dependent enrichment and seasonal shifts revealed. Appl. Environ. Microbiol. 2001, 67: 4742～4751.
96 Di Cello, F, A. Bevivino, L. Chiarini, R. et al. Biodiversity of a Burkholderia cepaciaBurkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages. Appl. Environ. Microbiol. 1997, 63: 4485～4493
97 Seldin, L. , A. S. Rosado, D. W. da Cruz, et al. Comparison of Paenibacillus azotofixansPaenibacillus azotofixans strains isolated from rhizoplane, rhizosphere, and non-root-associated soil from maize planted in two different Brazilian soils. Appl. Environ. Microbiol. 1998, 64: 3860～3868.
98 Picard, C, F. DiCello, M. Ventura, et, al. Frequency and biodiversity of 2, 4-diacetylphloroglucinol-producing bacteria isolated from the maize rhizosphere at different stages of plant growth. Appl. Environ. . Microbiol. 2000, 66: 948～955
99 Gomes, N. C. M. , H. Heuer, J. Schonfeld, et al. Bacterial diversity of the rhizosphere of maize (Zea maysZea mays) grown in tropical soil studied by temperature gradient gel electrophoresis. Plant Soil 2001. 232: 167～180.
100 Hamlen, R., F. Lukezic, and J. Bloom. Influence of age and stage of development on the neutral carbohydrate components in root exudates from alfalfa plants grown in a gnotobiotic environment. Can. J. Plant Sci. 1972. 52: 633～642.
101 Duineveld, B. M., A. S. Rosado, J. D. Van Elsas, et al. 1998. Analysis of the dynamics of bacterial communities in the rhizosphere of the chrysanthemum via denaturing gradient gel electrophoresis and substrate utilization patterns. Appl. Environ. Microbiol. 1998. 64: 4950～4957..
102 Duineveld, B. M., G. A. Kowalchuk, A. Keijzer, J. D. et al. Analysis of bacterial communities in the rhizosphere of chrysanthemum via denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA as well as DNA fragments Coding for 16S rRNA. Appl. Environ. Microbiol. 2001. 67: 172～178.