费氏中华根瘤菌的基因标记与竞争结瘤的研究
|
摘要
本研究以三亲本接合转移将含有发光酶基因luxAB的重组质粒pHN102导入费氏中华根瘤菌WWG18，得到标记菌株WWG18-lux。以同样的方法将含有绿荧光蛋白基因gfp的重组质粒pHN129导入费氏中华根瘤菌HNT29，得到另一标记菌株HN729-gfp。分别在自生和与大豆共尘条件下对标记菌株进行稳定性检测，结果表明：在两种条件下报告基因在标记菌株中遗传稳定性均为100％。
在砂培岔栽条件下，当供试菌株WWG18-lux与HNT29-gfp的体积比为100：1时，供试菌株从接种后第2天到第8天，WWG18-lux在根系的定殖密度高于HNT29-gfp，但是其接种后第11天到第32天，WWG18-lux在根系的定殖密度低于HNT29-gfp。当体积比为1：1时，供试菌株在接种后第2天，WWG18-lux在根系的定殖密度略高于HNT29-gfp，但是供试菌株从接种后第4天到第32天，WWG18-lux在根系的定殖密度始终低于HNT29-gfp。当体积比为1：100时，在接种后第2天到第32天，供试菌株WWG18-lux在根系的定殖密度始终低于HN729-gfp。占瘤率的检测结果表明：在三种不同体积比的条件下，HNT29-gfp的占瘤率始终明显高于WWG18-lux，表明HNT29-gfp的竞争结瘤能力高于WWG18-lux。砂培盆栽结瘤试验结果表明：占瘤率主要由菌株自身的竞争能力决定。根系定殖密度高的供试菌株的占瘤率高，占瘤率高的供试菌株的竞争结瘤能力强。接种量对占瘤率影响的结果表明：适当增大接种量能提高标记菌株的占瘤率。灭菌土培混合接种定殖密度测定结果表明：当体积比为100：1时，在接种后第2天到第16天，WWG18-lux在根系的定殖密度高于HNT29-gfp，在接种后第20天到第28天，其在根系的定殖密度低于HNT29-gfp。接种后第32天，其在根系的定殖密度高于HNT29-gfp。土培占瘤率的结果表明：HNT29-gfp的占瘤率高于WWG18-lux。灭菌土培混合接种时供试菌株的占瘤率高于非灭菌土培混合接种时的占瘤率。土培条件下结果表明：土壤中的其它因素(如：土著根瘤菌数量和土壤特性等)影响接种根瘤菌的竞争结瘤能力。供试菌株在土培和砂培时，其定殖密度变化不同。
用微根盒分根装置研究一侧根的结瘤对另一侧根结瘤影响的结果表明：用相同的菌株来接种根的左侧和右侧，在结瘤时期内，随着时间的延迟，一侧根的结瘤对另一侧根的结瘤有抑制作用。用竞争能力不同的标记菌株来接种根的左侧和右侧吋的结果表明：一侧根的结瘤对另一侧根的结瘤亦有抑制作用。本研究考察了(NH_4)_2SO_4对供试菌株结瘤的影响，结果表明：随着
费氏中华根瘤菌基因标记与竞争结瘤的研究
困H4)250。浓度的增加，总瘤数明显减少，1 omM倒H;)250;使供试菌株不结瘤。
在倒H月)2504存在情况下，一侧根的结瘤对另一侧根结瘤影响的结果表明:不
同浓度卿H4)2504存对根的两侧结瘤均有影响。随着倒H。)250;浓度的增加，
与不加(NH;)2504时相比，一侧根的结瘤对另一侧根的结瘤抑制作用加强。
10mM(NH刁2504使供试菌株两侧均不结瘤。
Two plasmids. pHN102 with luxAB and pHN129 with gfp were transferred into Sinorhizobium fredii WWG18 and HNT29 by the method of tri-parental mating. Transconjugants WWG18-/wx and HNT29-g/p were obtained. The results of stability experiments showed that both luxAB and gfp could stablely inherit in marked strains in either free-living or symbiosis conditions by means of light emission detection.
The results of competitive nodulation tests between the marked strains showed that the higher colonization density of the marked strain on the roots, the higher the ratio of nodule occupancy and the stronger competitive nodulation ability. The ratio of nodule occupancy of HNT29-g/p is higher than that of WWG18-lux and the ability of competitive nodulation of HNT29-gfp is stronger than that of WWG18-lux that proved the ratio of nodule occupancy was mainly determinated by the strain itself. The ratio of nodule occupancy could be increased according to the cell number of inoculated Sinorhizobium fredii.
By using a self-designed split-root-system called Micro-rhizobox, it was demonstrated that the nodulation on one half-side root might suppress subsequent nodule formation on the opposite. Comparing with the results obtained from pot plant experiment using Fahraeus nitrogen-free plant grown solution, the total nodule numbers declined when chemical nitrogen was added. The suppression by different concentrations of (MH4)2SO4 on nodule formation was equal- in two sides, of roots. Suppression on nodule development from another side of a split-root system was enhanced with the increasement of (NH4)2SO4 concentrations When the (NH4)2SO4 concentration was increased to 10mM. the nodule could not be formed.
在砂培岔栽条件下，当供试菌株WWG18-lux与HNT29-gfp的体积比为100：1时，供试菌株从接种后第2天到第8天，WWG18-lux在根系的定殖密度高于HNT29-gfp，但是其接种后第11天到第32天，WWG18-lux在根系的定殖密度低于HNT29-gfp。当体积比为1：1时，供试菌株在接种后第2天，WWG18-lux在根系的定殖密度略高于HNT29-gfp，但是供试菌株从接种后第4天到第32天，WWG18-lux在根系的定殖密度始终低于HNT29-gfp。当体积比为1：100时，在接种后第2天到第32天，供试菌株WWG18-lux在根系的定殖密度始终低于HN729-gfp。占瘤率的检测结果表明：在三种不同体积比的条件下，HNT29-gfp的占瘤率始终明显高于WWG18-lux，表明HNT29-gfp的竞争结瘤能力高于WWG18-lux。砂培盆栽结瘤试验结果表明：占瘤率主要由菌株自身的竞争能力决定。根系定殖密度高的供试菌株的占瘤率高，占瘤率高的供试菌株的竞争结瘤能力强。接种量对占瘤率影响的结果表明：适当增大接种量能提高标记菌株的占瘤率。灭菌土培混合接种定殖密度测定结果表明：当体积比为100：1时，在接种后第2天到第16天，WWG18-lux在根系的定殖密度高于HNT29-gfp，在接种后第20天到第28天，其在根系的定殖密度低于HNT29-gfp。接种后第32天，其在根系的定殖密度高于HNT29-gfp。土培占瘤率的结果表明：HNT29-gfp的占瘤率高于WWG18-lux。灭菌土培混合接种时供试菌株的占瘤率高于非灭菌土培混合接种时的占瘤率。土培条件下结果表明：土壤中的其它因素(如：土著根瘤菌数量和土壤特性等)影响接种根瘤菌的竞争结瘤能力。供试菌株在土培和砂培时，其定殖密度变化不同。
用微根盒分根装置研究一侧根的结瘤对另一侧根结瘤影响的结果表明：用相同的菌株来接种根的左侧和右侧，在结瘤时期内，随着时间的延迟，一侧根的结瘤对另一侧根的结瘤有抑制作用。用竞争能力不同的标记菌株来接种根的左侧和右侧吋的结果表明：一侧根的结瘤对另一侧根的结瘤亦有抑制作用。本研究考察了(NH_4)_2SO_4对供试菌株结瘤的影响，结果表明：随着
费氏中华根瘤菌基因标记与竞争结瘤的研究
困H4)250。浓度的增加，总瘤数明显减少，1 omM倒H;)250;使供试菌株不结瘤。
在倒H月)2504存在情况下，一侧根的结瘤对另一侧根结瘤影响的结果表明:不
同浓度卿H4)2504存对根的两侧结瘤均有影响。随着倒H。)250;浓度的增加，
与不加(NH;)2504时相比，一侧根的结瘤对另一侧根的结瘤抑制作用加强。
10mM(NH刁2504使供试菌株两侧均不结瘤。
Two plasmids. pHN102 with luxAB and pHN129 with gfp were transferred into Sinorhizobium fredii WWG18 and HNT29 by the method of tri-parental mating. Transconjugants WWG18-/wx and HNT29-g/p were obtained. The results of stability experiments showed that both luxAB and gfp could stablely inherit in marked strains in either free-living or symbiosis conditions by means of light emission detection.
The results of competitive nodulation tests between the marked strains showed that the higher colonization density of the marked strain on the roots, the higher the ratio of nodule occupancy and the stronger competitive nodulation ability. The ratio of nodule occupancy of HNT29-g/p is higher than that of WWG18-lux and the ability of competitive nodulation of HNT29-gfp is stronger than that of WWG18-lux that proved the ratio of nodule occupancy was mainly determinated by the strain itself. The ratio of nodule occupancy could be increased according to the cell number of inoculated Sinorhizobium fredii.
By using a self-designed split-root-system called Micro-rhizobox, it was demonstrated that the nodulation on one half-side root might suppress subsequent nodule formation on the opposite. Comparing with the results obtained from pot plant experiment using Fahraeus nitrogen-free plant grown solution, the total nodule numbers declined when chemical nitrogen was added. The suppression by different concentrations of (MH4)2SO4 on nodule formation was equal- in two sides, of roots. Suppression on nodule development from another side of a split-root system was enhanced with the increasement of (NH4)2SO4 concentrations When the (NH4)2SO4 concentration was increased to 10mM. the nodule could not be formed.
引文
1 马庆生，武波，唐东阶等．外源nfe基因导入快生型大豆根瘤菌GN01的行为分析．复旦大学学报(自然科学版)，1998，37(4)：445～448
2 王平，李阜棣．标记基因在根圈微生物定殖研究中的应用．生物技术通报，1994，5：9～12
3 王平，李阜棣．微生物分子生态学研究进展．见：中国土壤学会第8届全国会员代表大会及1995学术年会论文摘要集
4 王平．发光酶标记基因在微生物分子生态学研究中的应用．生态学杂志，1996，15(3)：26～34
5 石小岩．快生型大豆根瘤菌血清型、生性及竞争性[硕士学位论文]．华中农业大学 图书馆：华中农业大学，1986
6 农广．紫云英根瘤菌(Rhizoabium huakuii Chen)共生质粒共生基因克隆和早期共生关系的研究[博士学位论文]．华中农业大学图书馆：华中农业大学，1994
7 刘莉．化合氮对大豆根瘤菌结瘤固氮的影响及耐化合态氮灾变株筛选[硕士学位论文]．华中农业大学图书馆：华中农业大学，1996
8 李阜棣．分子生物学对微生物生态学的渗透：中国土壤科学的展望．1991．89～95
9 金山喜则．硝酸离子对大豆根瘤固氮作用的阻碍和有关亚硝酸豆血红蛋白的形成．见：日本土壤肥料学会讲演要旨集第35集．1989．93
10 莫才清，周俊初．含发光酶基因的转座子质粒载体的改造及向根瘤菌HN01的转座．应用与环境生物学报，1997．3(3)：252～257
11 莫才清，周俊初．根瘤菌与豆科植物竞争结瘤的分子生态学研究．生物技术通报，1996．4：6～8
12 曾广勤．花生根瘤菌与氮素化肥配合施用开发研究．微生物学研究与应用，1992．4：12～16
13 葛诚、樊蕙．快生型大豆根瘤菌竞争结瘤研究进展及其在田间自然分布调查，大豆科学，1986．5(4)：327～333
14 Anarger,N. and Lobreau, J. P.. Quantitaive study of nodulation competitiveness in Rhizobium strain. Appl. Environ. Microbiol. 1982.44:583～558
15 Barak, Behari, J., Ohnedo, G., Guzman, P., et al. PI Structure and function of the Bacillus SpollE protein and its localization to sites of sporrlation septum assembly.Mol.Microbiol, 1996. 19:1047～1060
16 Boivin, c., Berger,J., Rhizobium meliloti genes encoding catabolism of trigeoneline are induced under symbiotic conditions.The Plant Cell. 1990.2: 1157-1170
17 Brockwell J,Gault RR, Morthorpe LJ, Peoples MB, Turneer GL, Bergersen FJ.Effects of soil nitrogen status and rate of inoculation on the establishment of populations of Braclyrhizobium japonicum and on the nodulations of soybeans.Aust J Agri Res, 1989,
40:753-762
18 Bottomley, P. J., Jenkins, M. B.. Some characteristics of Rhizobium meliloti isolatcs from alfalfa fields in Oregin. Soil Sci. Soc., 1983 47:1153～1157
19 Caetano-Anolles G., Wall L.G., Micheli A.T. Role of motility and chemotaxis in efficiency of nodulation by Rhizobium melitoti Plant Physiol., 1988.86:1228-1235
20 Caetano-Anolles, G. Chemotaxis of Rhizobium meliloti to the plant flavone Lutenolin requires functional nodulation genes.J.Bacteriol,1988. 170:3164～3169
21 Carmi, O. A.,Stewart, G. S. A. B., Ulitzur,S. et al. Use of bacterial luciferase to establish a promoter probe vehicle capable of nondestructive real-time analysis of gene expression in Bacillus 899. J. Bacteriol.,1987.169:2165～2170
22 Cebolla, A.. Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters, J, Biol. Chem. 1991.6:177～184
23 Cebolla, A.. Stable tagging of Rhizobium meliloti with the firefly luciferase gene for environmental monitoring. Appl. Environ. Microbiol., 1993.59:2511～2519
24 Chabot, R., Antoun, H.and Kloepper,J. W. ct al..Root colonization of maize and lettuce by bioluminescent R. leguminosarum bv. Phaseoli. Appl. Environ. Microbiol.,1996.62:2767～2772
25 Chalfie, M., Tu, Y. and Euskirchen, G. et al.. Green fluorescent protein as a marker For gene expression. Science, 1994.263:802～805
26 Choo Yeonsik, Sung Jun Kim and Swung Dal Song. Effects of nitrate gradient on nitrogen fixation, nitrate reduction and ureide content of soybean. Korean J. BOT, 1988.30(3): 205～214
27 Cregan, P. B., Keyser, H.H.. Soybean genotype restricting nodulation of a previously unrestricted serocluster 123 Bradyrhizobia. Crop. Sci.,1989.29:307～312
28 Cregan, P. B., Keyser, H.H.. Gene for gene interaction in the legume-Rhizobium Symbiosis. In Belstville Symposia in Agricalatural Research:The Rhizosphere and plant Growth (ⅩⅣ). 1990:163～171
29 De, Bruijn, F. J.. Use of the luciferase (Tn5-lux) reporter system to study R. meliloti genes responding to C/N/O2 limitation or plant factors and their role in rhizosphere competition. In Nitrogen Fixation: Fundamental and Applications: Tikhonovichia. 1995.195～200
30 Denarie, J. and Cullimore, J.. Lipo-oligosaccaride nodulation factors a minireviews: new class of signaling molecules mediating recognition and morphogenesis. Cell, 1993.74:951～954
31 Dowling, D. N. and Broghton, W. J.. Competition for nodulation of legumes. Ann. Rev.Microbiol., 1986.40:131～157
32 Doyle, T., and Bostein, D.. Movement of yeast cortical actin cytoskeleton visualized in-
vivo. Proc. Natl. Acad. Sci. USA, 1996, 93:3883～3891
33 Drahos, A.. Tracking recombinant organisms in the environment: β-galactosidase as a selective nonantibiotic marker for fluorescent pseudomonads. Bio-technology,1986.4:433～444
34 Firmin, J. L., Wilson K.E..Flavoniod activation of nodulation gene in Rhizobium reversed by other compounds present in plant. Nature,1986.324:90～92
35 Fuhrmann, J. J., Vasilas, B. L..Field response of the Glyeine-Bradyrhizobium Symbiosis to modified early-nodule occupancy. Soil biol. biochem.,1993.25(9):1203～1209
36 Hashem, F.M., Angle, J. S.. Rhizobiophage effects on Bradyrhizobium japonicum,nodulation and soybean growth. Soil Biol. Biochem., 1988.20:69～73
37 Hirsh, P. R., Spokes, J. D.. Survival and dispersion of genetically modified rhizobial in the field and genetic interaction with native strains. FEMS. Microbiol. Ecol..1994.15:147～159
38 Huang Chi-Ying, Zuei-leng Fong and Wan-Chium Fu. Bot Bull Acadsin (Taipei),1988.29(3):147～152
39 Jefferson. R. A., Burgess, S. M. and Hirsh, D., β-glucuronidase from Ecoli as a gene fusion marker. Proc. Natl. Acad. Sci. USA, 1986.86:8447～8451
40 Kjelleberg, S. Herrnansson, M. and Marden, P, et al.. The transient phase between growth and nongrowth of heterotrophic bacteria with emphasis on the marine environment. Ann. Rev. Microbiol.,1987.41:25～49
41 Klopper,J. W. A review of issues related to measuring colonization of plant roots by bacterial. Can. J. Microbiol., 1992.38:1219～1232
42 Kremer, L. Baulard, A. and Estaquier, J. et al.. Analysis of the Mycobacterium tuberculosis 85A antigen promotor region. J. Bacteriol., 1995. 177:642～653
43 Lowder, M. Unge, A. Maraha, N. and Jansson, J. K. et al.. Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506. Appl. Environ. Microbiol., 2000. 66(8):3160～3165
44 Manjanatho. M. G.,Loynachan. J. E.. Efficiency, competitiveness and persistency of Chinese Rhizobium fredii in lowa soil. Is. Agron.J., 1992.84 (4): 676～681
45 Mcloughiln, T. J., Bordelean, L.M.. Competition studies with Rhizobium trijolii in a field experiment. J. Appl. Bacteriol..1984.36:131～135
46 Michelmore. R. W.. Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating population. Proc. Natl. Acad. Sci. USA, 1991.88:9828～9832
47 Moawad. H. A.. Rhizospere response as a factor in competition among three serogroups
of indigenous Rhizobium japonicum for nodulation of field grown soybeans. Appl.Environ. Microbiol, 1984.:6:607～612
48 Rasooly, A.. Epistasis of rji nonnodulation of soybean to nodulation by Sinorhizobium fredii. Crop-Sci.,1993.33(2): 329～331
49 Renee. M. Kosslak and B. Ben Bohlool,. 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
50 Reubert, L. Regulation of Rhizobium meliloti exogenes in free-loving ceils and in planta examined by using Tn-phoA fusions.J. Bacteriol..1991. 173:426～434
51 Rossbach, S. R. Kulpa, D. A. and Rossabach. U. et al.. Molecular and genetic characterization of the rhizopine catabolism genes of Rhizobium meliloti L5～30. Mol.Gen. Genet.,1994.245:11～24
52 Sanjuan,J.,Olivares.J.,Multicopy plasmids carrying the nifA gene enhance Rhizobium meliloti nodulation competitiveness on alfafa. Mol.Plant. Microb. Interact. 1991.4: 365-369
53 Schwinghamer, E.A.,Pankhust, C.. A phage-like bacteriocin of Rhizobium trifoli.Can. J.Microbiol.,1973.19:359～368
54 Sessitsch, A. A., Wilson, K. J. and Akkermans, A. D. et al.. Simultaneous detection of different Rhizobium strains marked with either the E.coli gusA gene or the Pyrococcus furious celB gene. Appl. Environ. Microbiol.,1996.62(11): 4191～4194
55 Sharma, S. B. and Singner, E. R..Temporal and spatial regulation of the symbiotic genes of Rhizobium meliloti in plant revealed by transponson Tn5-gusA. Genes Dev.,1990.4:344～356
56 Shaw, J. J. and C. I. Kado. Development of Vibrio gene-set to monitor phytopathogenic bacteria during the ongoing disease process in a non-disruptive manner.Bio-Techology.1986.4:560-564
57 Srinivasan, M., Persen, D. J.. Nodulation of Phaseolus vulgaris by Rhizobium etil is enhanced by the presence of Bacillus. Can. J. Microbiol.,1997.43:1～8
58 Streit, W. R., Joseph, C. M. and Phillips, D. A. et al..Biotin and other water-soluble vitamins are key factors for alfalfa. Rhizosphre colonization by Rhizobium meliloti 1021.Mol. Plant-Microb. Interact.,1996.5:331～338
59 Streit, W., Nodulatoin competitiveness of Rhizobium leguminosarum by. phaseoli and R. tropici stains measured by gus gene fusions. Biol. Fert. Soils, 1992. 14: 140～144
60 Thies, J. E., Bohlool, B. B.. Environ mental effects on competition for nodule occupancy between introduced and indigenous strains.1992.38(6):493～500
61 Thomas, J., Development of Rhizobium and Azospirillum inoculants with enhanced potential for field appication, in: Nitrogen Fixation, Fundamentals and Application:
Tikhnovich et al.1995. 659～664
62 Tripeltt E. W. The molecular genetics of nodulation competitiveness in Rhizobium and Bradyrhizobium. Mol. plant. Microb. Inter. 1990.3(4):199-206
63 Tripeltt,E. W. and Sadowsky, M. J.. Genetics of competition for nodulation of legumes.Annu. Rev. Microbiol.,1992.46:399～428
64 Truchet, G. L. and Dazzo, F. B.. Morphogenesis of Lucerne root nodules incited by Rhizobium meliloti in the presence of combined nitrogen. Planta, 1982. 154:352～360
65 Turk, D. and Kerser, H. H.. Response of tree legumes to rhizobial inoculation to relation to the population density of indigenous rhizobia. Soil Biol. Biochem.,1993.25(1):75～81
66 Unge, A., Tombolin, R. and Mlbak, L. et al.. Simulanous monitoring of cell Number and Metabolic Activity of Speciefic Populations with a dual gfp-luxAB marker system. Appl.Environ. Microbiol..1999.65(2):813～821
67 Vlassak, K. and Vander, Legden, J,. Competition and persistence of Rhizobium tropic and Rhizobium etli in tropical soil during successive bean cultures. Biol. Fert. Soils, 1996.21(1): 61～68
68 Wang ping and Li budi. Luminescence measurement of luxAB genes modified Pseudomonas in liquid culture and in soil microcosm. Proceedings of 3rd international symposium on biosafety of field results for genetically modified plants and microorganism (USA), 1995.437～447.
69 Wilson, K. J..Sessitsch, A, and Corbo, J. C. et al.. Beta-Glucuronidase(GUS)transposons for ecological and genetic studies of Rhizobia and other gram-negative bacteria. Microbiol., 1995. 141(7):1691～705
70 Winstanley, C.,Morgan.J. A. and Pickup, R. W. et al..Use of a xyIE marker gene to monitor-survival of recombinant Pseudomonas populations in lake water by culture on nonselective media. Appl. Enviro. Microbial.,1991.57:1905～1913
71 Zdor, R. E. and Pueppke, S.G. Nodulation competitiveness of Tn5-induced mutants of Rhizobium fredii 208 that are altered in motility and extracellular polysaccharide production, Can .J. Microbiol. 1991.37:52-58
2 王平，李阜棣．标记基因在根圈微生物定殖研究中的应用．生物技术通报，1994，5：9～12
3 王平，李阜棣．微生物分子生态学研究进展．见：中国土壤学会第8届全国会员代表大会及1995学术年会论文摘要集
4 王平．发光酶标记基因在微生物分子生态学研究中的应用．生态学杂志，1996，15(3)：26～34
5 石小岩．快生型大豆根瘤菌血清型、生性及竞争性[硕士学位论文]．华中农业大学 图书馆：华中农业大学，1986
6 农广．紫云英根瘤菌(Rhizoabium huakuii Chen)共生质粒共生基因克隆和早期共生关系的研究[博士学位论文]．华中农业大学图书馆：华中农业大学，1994
7 刘莉．化合氮对大豆根瘤菌结瘤固氮的影响及耐化合态氮灾变株筛选[硕士学位论文]．华中农业大学图书馆：华中农业大学，1996
8 李阜棣．分子生物学对微生物生态学的渗透：中国土壤科学的展望．1991．89～95
9 金山喜则．硝酸离子对大豆根瘤固氮作用的阻碍和有关亚硝酸豆血红蛋白的形成．见：日本土壤肥料学会讲演要旨集第35集．1989．93
10 莫才清，周俊初．含发光酶基因的转座子质粒载体的改造及向根瘤菌HN01的转座．应用与环境生物学报，1997．3(3)：252～257
11 莫才清，周俊初．根瘤菌与豆科植物竞争结瘤的分子生态学研究．生物技术通报，1996．4：6～8
12 曾广勤．花生根瘤菌与氮素化肥配合施用开发研究．微生物学研究与应用，1992．4：12～16
13 葛诚、樊蕙．快生型大豆根瘤菌竞争结瘤研究进展及其在田间自然分布调查，大豆科学，1986．5(4)：327～333
14 Anarger,N. and Lobreau, J. P.. Quantitaive study of nodulation competitiveness in Rhizobium strain. Appl. Environ. Microbiol. 1982.44:583～558
15 Barak, Behari, J., Ohnedo, G., Guzman, P., et al. PI Structure and function of the Bacillus SpollE protein and its localization to sites of sporrlation septum assembly.Mol.Microbiol, 1996. 19:1047～1060
16 Boivin, c., Berger,J., Rhizobium meliloti genes encoding catabolism of trigeoneline are induced under symbiotic conditions.The Plant Cell. 1990.2: 1157-1170
17 Brockwell J,Gault RR, Morthorpe LJ, Peoples MB, Turneer GL, Bergersen FJ.Effects of soil nitrogen status and rate of inoculation on the establishment of populations of Braclyrhizobium japonicum and on the nodulations of soybeans.Aust J Agri Res, 1989,
40:753-762
18 Bottomley, P. J., Jenkins, M. B.. Some characteristics of Rhizobium meliloti isolatcs from alfalfa fields in Oregin. Soil Sci. Soc., 1983 47:1153～1157
19 Caetano-Anolles G., Wall L.G., Micheli A.T. Role of motility and chemotaxis in efficiency of nodulation by Rhizobium melitoti Plant Physiol., 1988.86:1228-1235
20 Caetano-Anolles, G. Chemotaxis of Rhizobium meliloti to the plant flavone Lutenolin requires functional nodulation genes.J.Bacteriol,1988. 170:3164～3169
21 Carmi, O. A.,Stewart, G. S. A. B., Ulitzur,S. et al. Use of bacterial luciferase to establish a promoter probe vehicle capable of nondestructive real-time analysis of gene expression in Bacillus 899. J. Bacteriol.,1987.169:2165～2170
22 Cebolla, A.. Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters, J, Biol. Chem. 1991.6:177～184
23 Cebolla, A.. Stable tagging of Rhizobium meliloti with the firefly luciferase gene for environmental monitoring. Appl. Environ. Microbiol., 1993.59:2511～2519
24 Chabot, R., Antoun, H.and Kloepper,J. W. ct al..Root colonization of maize and lettuce by bioluminescent R. leguminosarum bv. Phaseoli. Appl. Environ. Microbiol.,1996.62:2767～2772
25 Chalfie, M., Tu, Y. and Euskirchen, G. et al.. Green fluorescent protein as a marker For gene expression. Science, 1994.263:802～805
26 Choo Yeonsik, Sung Jun Kim and Swung Dal Song. Effects of nitrate gradient on nitrogen fixation, nitrate reduction and ureide content of soybean. Korean J. BOT, 1988.30(3): 205～214
27 Cregan, P. B., Keyser, H.H.. Soybean genotype restricting nodulation of a previously unrestricted serocluster 123 Bradyrhizobia. Crop. Sci.,1989.29:307～312
28 Cregan, P. B., Keyser, H.H.. Gene for gene interaction in the legume-Rhizobium Symbiosis. In Belstville Symposia in Agricalatural Research:The Rhizosphere and plant Growth (ⅩⅣ). 1990:163～171
29 De, Bruijn, F. J.. Use of the luciferase (Tn5-lux) reporter system to study R. meliloti genes responding to C/N/O2 limitation or plant factors and their role in rhizosphere competition. In Nitrogen Fixation: Fundamental and Applications: Tikhonovichia. 1995.195～200
30 Denarie, J. and Cullimore, J.. Lipo-oligosaccaride nodulation factors a minireviews: new class of signaling molecules mediating recognition and morphogenesis. Cell, 1993.74:951～954
31 Dowling, D. N. and Broghton, W. J.. Competition for nodulation of legumes. Ann. Rev.Microbiol., 1986.40:131～157
32 Doyle, T., and Bostein, D.. Movement of yeast cortical actin cytoskeleton visualized in-
vivo. Proc. Natl. Acad. Sci. USA, 1996, 93:3883～3891
33 Drahos, A.. Tracking recombinant organisms in the environment: β-galactosidase as a selective nonantibiotic marker for fluorescent pseudomonads. Bio-technology,1986.4:433～444
34 Firmin, J. L., Wilson K.E..Flavoniod activation of nodulation gene in Rhizobium reversed by other compounds present in plant. Nature,1986.324:90～92
35 Fuhrmann, J. J., Vasilas, B. L..Field response of the Glyeine-Bradyrhizobium Symbiosis to modified early-nodule occupancy. Soil biol. biochem.,1993.25(9):1203～1209
36 Hashem, F.M., Angle, J. S.. Rhizobiophage effects on Bradyrhizobium japonicum,nodulation and soybean growth. Soil Biol. Biochem., 1988.20:69～73
37 Hirsh, P. R., Spokes, J. D.. Survival and dispersion of genetically modified rhizobial in the field and genetic interaction with native strains. FEMS. Microbiol. Ecol..1994.15:147～159
38 Huang Chi-Ying, Zuei-leng Fong and Wan-Chium Fu. Bot Bull Acadsin (Taipei),1988.29(3):147～152
39 Jefferson. R. A., Burgess, S. M. and Hirsh, D., β-glucuronidase from Ecoli as a gene fusion marker. Proc. Natl. Acad. Sci. USA, 1986.86:8447～8451
40 Kjelleberg, S. Herrnansson, M. and Marden, P, et al.. The transient phase between growth and nongrowth of heterotrophic bacteria with emphasis on the marine environment. Ann. Rev. Microbiol.,1987.41:25～49
41 Klopper,J. W. A review of issues related to measuring colonization of plant roots by bacterial. Can. J. Microbiol., 1992.38:1219～1232
42 Kremer, L. Baulard, A. and Estaquier, J. et al.. Analysis of the Mycobacterium tuberculosis 85A antigen promotor region. J. Bacteriol., 1995. 177:642～653
43 Lowder, M. Unge, A. Maraha, N. and Jansson, J. K. et al.. Effect of starvation and the viable-but-nonculturable state on green fluorescent protein (GFP) fluorescence in GFP-tagged Pseudomonas fluorescens A506. Appl. Environ. Microbiol., 2000. 66(8):3160～3165
44 Manjanatho. M. G.,Loynachan. J. E.. Efficiency, competitiveness and persistency of Chinese Rhizobium fredii in lowa soil. Is. Agron.J., 1992.84 (4): 676～681
45 Mcloughiln, T. J., Bordelean, L.M.. Competition studies with Rhizobium trijolii in a field experiment. J. Appl. Bacteriol..1984.36:131～135
46 Michelmore. R. W.. Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating population. Proc. Natl. Acad. Sci. USA, 1991.88:9828～9832
47 Moawad. H. A.. Rhizospere response as a factor in competition among three serogroups
of indigenous Rhizobium japonicum for nodulation of field grown soybeans. Appl.Environ. Microbiol, 1984.:6:607～612
48 Rasooly, A.. Epistasis of rji nonnodulation of soybean to nodulation by Sinorhizobium fredii. Crop-Sci.,1993.33(2): 329～331
49 Renee. M. Kosslak and B. Ben Bohlool,. 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
50 Reubert, L. Regulation of Rhizobium meliloti exogenes in free-loving ceils and in planta examined by using Tn-phoA fusions.J. Bacteriol..1991. 173:426～434
51 Rossbach, S. R. Kulpa, D. A. and Rossabach. U. et al.. Molecular and genetic characterization of the rhizopine catabolism genes of Rhizobium meliloti L5～30. Mol.Gen. Genet.,1994.245:11～24
52 Sanjuan,J.,Olivares.J.,Multicopy plasmids carrying the nifA gene enhance Rhizobium meliloti nodulation competitiveness on alfafa. Mol.Plant. Microb. Interact. 1991.4: 365-369
53 Schwinghamer, E.A.,Pankhust, C.. A phage-like bacteriocin of Rhizobium trifoli.Can. J.Microbiol.,1973.19:359～368
54 Sessitsch, A. A., Wilson, K. J. and Akkermans, A. D. et al.. Simultaneous detection of different Rhizobium strains marked with either the E.coli gusA gene or the Pyrococcus furious celB gene. Appl. Environ. Microbiol.,1996.62(11): 4191～4194
55 Sharma, S. B. and Singner, E. R..Temporal and spatial regulation of the symbiotic genes of Rhizobium meliloti in plant revealed by transponson Tn5-gusA. Genes Dev.,1990.4:344～356
56 Shaw, J. J. and C. I. Kado. Development of Vibrio gene-set to monitor phytopathogenic bacteria during the ongoing disease process in a non-disruptive manner.Bio-Techology.1986.4:560-564
57 Srinivasan, M., Persen, D. J.. Nodulation of Phaseolus vulgaris by Rhizobium etil is enhanced by the presence of Bacillus. Can. J. Microbiol.,1997.43:1～8
58 Streit, W. R., Joseph, C. M. and Phillips, D. A. et al..Biotin and other water-soluble vitamins are key factors for alfalfa. Rhizosphre colonization by Rhizobium meliloti 1021.Mol. Plant-Microb. Interact.,1996.5:331～338
59 Streit, W., Nodulatoin competitiveness of Rhizobium leguminosarum by. phaseoli and R. tropici stains measured by gus gene fusions. Biol. Fert. Soils, 1992. 14: 140～144
60 Thies, J. E., Bohlool, B. B.. Environ mental effects on competition for nodule occupancy between introduced and indigenous strains.1992.38(6):493～500
61 Thomas, J., Development of Rhizobium and Azospirillum inoculants with enhanced potential for field appication, in: Nitrogen Fixation, Fundamentals and Application:
Tikhnovich et al.1995. 659～664
62 Tripeltt E. W. The molecular genetics of nodulation competitiveness in Rhizobium and Bradyrhizobium. Mol. plant. Microb. Inter. 1990.3(4):199-206
63 Tripeltt,E. W. and Sadowsky, M. J.. Genetics of competition for nodulation of legumes.Annu. Rev. Microbiol.,1992.46:399～428
64 Truchet, G. L. and Dazzo, F. B.. Morphogenesis of Lucerne root nodules incited by Rhizobium meliloti in the presence of combined nitrogen. Planta, 1982. 154:352～360
65 Turk, D. and Kerser, H. H.. Response of tree legumes to rhizobial inoculation to relation to the population density of indigenous rhizobia. Soil Biol. Biochem.,1993.25(1):75～81
66 Unge, A., Tombolin, R. and Mlbak, L. et al.. Simulanous monitoring of cell Number and Metabolic Activity of Speciefic Populations with a dual gfp-luxAB marker system. Appl.Environ. Microbiol..1999.65(2):813～821
67 Vlassak, K. and Vander, Legden, J,. Competition and persistence of Rhizobium tropic and Rhizobium etli in tropical soil during successive bean cultures. Biol. Fert. Soils, 1996.21(1): 61～68
68 Wang ping and Li budi. Luminescence measurement of luxAB genes modified Pseudomonas in liquid culture and in soil microcosm. Proceedings of 3rd international symposium on biosafety of field results for genetically modified plants and microorganism (USA), 1995.437～447.
69 Wilson, K. J..Sessitsch, A, and Corbo, J. C. et al.. Beta-Glucuronidase(GUS)transposons for ecological and genetic studies of Rhizobia and other gram-negative bacteria. Microbiol., 1995. 141(7):1691～705
70 Winstanley, C.,Morgan.J. A. and Pickup, R. W. et al..Use of a xyIE marker gene to monitor-survival of recombinant Pseudomonas populations in lake water by culture on nonselective media. Appl. Enviro. Microbial.,1991.57:1905～1913
71 Zdor, R. E. and Pueppke, S.G. Nodulation competitiveness of Tn5-induced mutants of Rhizobium fredii 208 that are altered in motility and extracellular polysaccharide production, Can .J. Microbiol. 1991.37:52-58