黄瓜根际土壤细菌种群及酮基合酶基因多样性与黄瓜枯萎病发病关系研究
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摘要
本文对黄瓜根际土壤中细菌种群以及酮基合酶基因(KS)多样性与枯萎病的发病关系进行了研究。试验利用传统的分离培养方法、PCR-DGGE技术和分子克隆技术,针对黄瓜不同根际土壤样品的微生物种群以及编码聚酮类化合物的酮基合酶(KS)’基因多样性,研究了二者与黄瓜枯萎病发病的相关性。
试验比较分析了发病程度不同的黄瓜根际土壤样品中微生物种群和KS基因的多样性。采用PCR-DGGE技术,分析了发病程度不同的黄瓜根际土壤样品中微生物种群的多样性,结果表明,罹病黄瓜植株根际土壤中细菌种群数量明显高于未发病黄瓜根际土壤,这与以往报道的未发病黄瓜根际土壤微生物种群数量更为丰富的结论并不一致。同时采用目的基因片段克隆及序列分析技术,分析了发病程度不同的黄瓜根际土壤中KS基因的多样性,结果表明,罹病黄瓜根际土壤中KS基因更为丰富。此结果与土壤中细菌种群多样性的分析结果趋于一致。
试验自行设计了不同土壤样品中生物基因组总DNA的提取方法——碱裂解法,并与其他3种DNA提取的方法进行了比较。结果表明,碱裂解法使用醋酸钾和氯化镁去除腐殖酸等杂质,可以获得高质量、大片段、高完整性的DNA,从而为后期实验提供了良好的’DNA模板,对于PCR扩增和后续的分子操作更为适用。试验证明,碱裂解法是自主创新的DNA提取的有效方法,该方法已经申请了专利(申请号：2010101032923)。
试验利用传统的常规分离方法,从发病程度不同的黄瓜根际土壤样品中分离培养获得放线菌。对各土壤样品中分离获得的拮抗放线菌不同种类的数量差异进行了比较,结果显示,感染黄瓜枯萎病的根际土壤中拮抗放线菌的种类多于未感染枯萎病的土壤。
试验采用PCR-DGGE方法,以特殊的带有"GC夹板”的引物,分析了发病程度不同的黄瓜根际土壤中细菌种群的变化,该方法大大提高了DGGE的检出率。通过对种植前、接菌前、罹病、未发病4种黄瓜根际土壤样品中细菌种群的变化分析,结果表明,罹病黄瓜根际土壤中细菌的种群更为丰富。
利用Ⅰ型聚酮合酶基因的简并引物,对提取的罹病和未发病根际土壤中的总DNA进行PCR扩增,并进行克隆测序,共获得KS基因205个,全部提交GenBank并获得登录号,供同类研究使用。对测序获得的KS基因数目统计分析结果表明,从罹病土壤样品中获得了更多的阳性克隆子,显示出了更为丰富的KS基因多样性,该结果与PCR-DGGE对土壤中细菌种群多样性的分析结果趋势一致。
The relationships between the diversity of bacterial community in cucumber rhizosphere soil, ketone synthase genes (KS) and the Fusarium wilt have been studied in this paper. This experiments have been finished by traditional isolation methods, PCR-DGGE molecular technology and molecular clone technology. According to the diversity of microbial populations and encoding polyketide ketone synthase (KS) genes from different cucumber rhizosphere soil samples, studied the relevance with cucumber wilt.
This experiments analyzed the differences of diversity about microbial and KS in cucumber rhizosphere soil. PCR-DGGE molecular technology was used to analyze the microbial populations diversity from different soil, the results showed that the species number of bacterial community in cucumber rhizosphere soil from infected cucumber by F. oxysporum f.sp. cucumerinum significantly higher than from healthy cucumber, it is inconsistency with the result that the health cucumber soil had richer microbials by previously literature. Meanwhile, the diversity of KS gene in different disease severity of rhizosphere soil of cucumber was construed using the target gene cloning technology and sequence analysis technology, the results were consistent with the diversity analysis of the species number of bacterial community, namely the KS gene in rhizosphere soil of diseased cucumber showed more abundant.
This paper designed genomic DNA extraction methods of different soil samples-alkaline lysis method, and compared with the other three methods. The results showed that potassium acetate and magnesium chloride were used in alkaline lysis method to remove the precipitated impurities, and high DNA yields were obtained. The DNA showed good quality and big molecular size and suitable for PCR amplification as well. And it provided a good template DNA for later experiments. It is proved that alkaline lysis method was innovation DNA extraction method, the method has been applied for a patent (application No. 2010101032923).
Actinomycetes from different soil samples were isolated by traditional isolation methods, and the stocks differentia of culturable actinomycetes from various samples were compared. The results showed that the types of actinomycetes were higher from rhizosphere soil of diseased cucumber than from rhizosphere soil of no diseased cucumber.
The PCR-DGGE method was optimized, and the primer with "GC clamp" was conducted to amplify the expected KS gene gragements, and changes of bacterial populations in different levels of diseased cucumber rhizosphere soil were analyzed. The results showed that the detection rate of DGGE was greatly improved by this method. Bacterial populations from four rhizosphere soil samples were analyzed. The four rhizosphere soil samples included pre-planting soil, pre-inoculation soil, rhizosphere soil from diseased cucumber and healthy cucumber. The result showed that the soil samples from diseased cucumber had richer bacterial populations.
The extracted DNA from the rhizosphere soil from diseased and healthy cucumber were as template for PCR amplification, expected KS gene fragments were amplified by degenerate primers which were designed basing on type I polyketide synthase gene. Expected KS gene fragments were cloned and sequenced,205 gene sequences were submitted to GenBank and obtained GenBank Accession number, used for similar studies. The number of the KS gene from four rhizosphere soil samples were statistical analysis. The results showed that it can obtained more positive clones from rhizosphere soil of diseased cucumber and more abundant KS gene diversity, the results were consistent with the diversity analysis of the species number of bacterial community by PCR-DGGE.
试验比较分析了发病程度不同的黄瓜根际土壤样品中微生物种群和KS基因的多样性。采用PCR-DGGE技术,分析了发病程度不同的黄瓜根际土壤样品中微生物种群的多样性,结果表明,罹病黄瓜植株根际土壤中细菌种群数量明显高于未发病黄瓜根际土壤,这与以往报道的未发病黄瓜根际土壤微生物种群数量更为丰富的结论并不一致。同时采用目的基因片段克隆及序列分析技术,分析了发病程度不同的黄瓜根际土壤中KS基因的多样性,结果表明,罹病黄瓜根际土壤中KS基因更为丰富。此结果与土壤中细菌种群多样性的分析结果趋于一致。
试验自行设计了不同土壤样品中生物基因组总DNA的提取方法——碱裂解法,并与其他3种DNA提取的方法进行了比较。结果表明,碱裂解法使用醋酸钾和氯化镁去除腐殖酸等杂质,可以获得高质量、大片段、高完整性的DNA,从而为后期实验提供了良好的’DNA模板,对于PCR扩增和后续的分子操作更为适用。试验证明,碱裂解法是自主创新的DNA提取的有效方法,该方法已经申请了专利(申请号：2010101032923)。
试验利用传统的常规分离方法,从发病程度不同的黄瓜根际土壤样品中分离培养获得放线菌。对各土壤样品中分离获得的拮抗放线菌不同种类的数量差异进行了比较,结果显示,感染黄瓜枯萎病的根际土壤中拮抗放线菌的种类多于未感染枯萎病的土壤。
试验采用PCR-DGGE方法,以特殊的带有"GC夹板”的引物,分析了发病程度不同的黄瓜根际土壤中细菌种群的变化,该方法大大提高了DGGE的检出率。通过对种植前、接菌前、罹病、未发病4种黄瓜根际土壤样品中细菌种群的变化分析,结果表明,罹病黄瓜根际土壤中细菌的种群更为丰富。
利用Ⅰ型聚酮合酶基因的简并引物,对提取的罹病和未发病根际土壤中的总DNA进行PCR扩增,并进行克隆测序,共获得KS基因205个,全部提交GenBank并获得登录号,供同类研究使用。对测序获得的KS基因数目统计分析结果表明,从罹病土壤样品中获得了更多的阳性克隆子,显示出了更为丰富的KS基因多样性,该结果与PCR-DGGE对土壤中细菌种群多样性的分析结果趋势一致。
The relationships between the diversity of bacterial community in cucumber rhizosphere soil, ketone synthase genes (KS) and the Fusarium wilt have been studied in this paper. This experiments have been finished by traditional isolation methods, PCR-DGGE molecular technology and molecular clone technology. According to the diversity of microbial populations and encoding polyketide ketone synthase (KS) genes from different cucumber rhizosphere soil samples, studied the relevance with cucumber wilt.
This experiments analyzed the differences of diversity about microbial and KS in cucumber rhizosphere soil. PCR-DGGE molecular technology was used to analyze the microbial populations diversity from different soil, the results showed that the species number of bacterial community in cucumber rhizosphere soil from infected cucumber by F. oxysporum f.sp. cucumerinum significantly higher than from healthy cucumber, it is inconsistency with the result that the health cucumber soil had richer microbials by previously literature. Meanwhile, the diversity of KS gene in different disease severity of rhizosphere soil of cucumber was construed using the target gene cloning technology and sequence analysis technology, the results were consistent with the diversity analysis of the species number of bacterial community, namely the KS gene in rhizosphere soil of diseased cucumber showed more abundant.
This paper designed genomic DNA extraction methods of different soil samples-alkaline lysis method, and compared with the other three methods. The results showed that potassium acetate and magnesium chloride were used in alkaline lysis method to remove the precipitated impurities, and high DNA yields were obtained. The DNA showed good quality and big molecular size and suitable for PCR amplification as well. And it provided a good template DNA for later experiments. It is proved that alkaline lysis method was innovation DNA extraction method, the method has been applied for a patent (application No. 2010101032923).
Actinomycetes from different soil samples were isolated by traditional isolation methods, and the stocks differentia of culturable actinomycetes from various samples were compared. The results showed that the types of actinomycetes were higher from rhizosphere soil of diseased cucumber than from rhizosphere soil of no diseased cucumber.
The PCR-DGGE method was optimized, and the primer with "GC clamp" was conducted to amplify the expected KS gene gragements, and changes of bacterial populations in different levels of diseased cucumber rhizosphere soil were analyzed. The results showed that the detection rate of DGGE was greatly improved by this method. Bacterial populations from four rhizosphere soil samples were analyzed. The four rhizosphere soil samples included pre-planting soil, pre-inoculation soil, rhizosphere soil from diseased cucumber and healthy cucumber. The result showed that the soil samples from diseased cucumber had richer bacterial populations.
The extracted DNA from the rhizosphere soil from diseased and healthy cucumber were as template for PCR amplification, expected KS gene fragments were amplified by degenerate primers which were designed basing on type I polyketide synthase gene. Expected KS gene fragments were cloned and sequenced,205 gene sequences were submitted to GenBank and obtained GenBank Accession number, used for similar studies. The number of the KS gene from four rhizosphere soil samples were statistical analysis. The results showed that it can obtained more positive clones from rhizosphere soil of diseased cucumber and more abundant KS gene diversity, the results were consistent with the diversity analysis of the species number of bacterial community by PCR-DGGE.
引文
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67 Neal J.L., Can J.,1971. Of microbiology.17:1143-1145.
68 Nyman J.A.,1999. Effeet of crude oil and chemical additives on metabolic aetivity of mixed microbial populations in fresh marsh soils. Microbial Ecology,37(2):152-162.
69 Ogram A., Sayler G.S., Barkay T.,1987. The extraction and purification of microbial DNA from sediments. Journal of Microbiological. Methods.7:57-66.
70 Pfeifer B.A., Wang C.C., Walsh C.T., et al.2003. Biosynthesis of Yersiniabactin a complex polyketide-nonribosomal peptide using Escherichia coli as a heterologous host. Applied and Environmental Microbiology,69(11):6698-6702.
71 Pfeifer B.A., Khosla C.,2001. Biosynthesis of polyketides in heterologous hosts. Microbiology and Molecular Biology Reviews,65(1):106-118.
72 Robe P., Nalin R., Capellano C., et al.2003. Extraction of DNA from soil. European Journal of Soil Biology,39:183-190.
73 Roose-Amsaleg C.L., Garnier-Sillam E., Harry M.,2001. Extraction and purification of microbial DNA from soil and sediment samples. Applied Soil Ecology,18:47-60.
74 Rosello M.R., Aman R.,2001. The species concept for prokaryotes. FEMS Microbiology Review,25: 39-67.
75 Sabine P., Stefanie K., Frank S., et al.,2000. Succession of microbial communities during hot composting as detected by PCR-Single-trand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes. Applied and Environmental Microbiology,66(3):930-936.
76 Sambrook J., Fritsch E.F., Maniatis T.,1989. Molecular Cloning:A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
77 Schafer H., Muyzer G.,2001. Denaturing gradient gel electrophoresis and marine microbial ecology. In:Paul, J. (Ed.), Methods in Microbiology, London:Academic Press, pp.425-468.
78 Shen B.,2003. Polyketide biosynthesis beyond the type Ⅰ, Ⅱ and Ⅲ polyketide synthase paradigms. Curr Opin Chem Biol,7:285-295.
79 Smalla K., Wachtendorf U., Heuer H., et al.1998. Analysis of BIOLOG GN substract utilization partterns by microbial community. Applied and environmental microbiology,64:1220-1225.
80 Smit E., Leeflang P., Gommans S., et al.2001. 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,67(5):2284-2291.
81 Solbrig O.T.,1991. From genes to ecosystems:a research agenda for biodiversity. Report of a IUBS-SCOPEUNESCO workshop, the international union of biological sciences parisfrance,51 Boulevar dole montmorenny.
82 Srivastava A.K., Ara A., Bhargava P., et al.2007. A rapid and cost-effective method of genomic DNA isolation from cyanobacterial culture, mat and soil suitable for genomic fingerprinting and community analysis. Journal of Applied Phycology,19:373-382.
83 Staddon W.J., Duchesne L.C., Trevors J.T.,1997. Microbial Diversity and Community Structure of Postdisturbance Forest soils as Determined by Sole-Carbon-Source Utilization Patterns. Microbial Ecology,34:125-130.
84 Stamper D.M., Walch M., Jacobs R.N.,2003. Bacterial population changes in a membrane bioreactor for gray-water treatment monitored by denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments. Applied Environmental Microbiology,69:852-860.
85 Stevenson F.J.,1976. Stability constants of Cu.Pb and Cd complexes with humic acids. Soil Science Society of America jouranl,40:665-672.
86 Tang L., Fu H., Betlach M.C., et al.1999. Elucidating the mechanism of chain termination switching in the picromycin/methymycin polyketide synthase. Journal of Biological Chemistry,6(8):553-558.
87 Teske A., Wawer C., Muyzer G., et al.1996. Distribution of sulatfe-redueing bacteria in a stratified Fjodr (Marigaer Fjodr, Denmark) as evaluated b ymost-Porbable-number counts and denaturing gardient gel electrophoresis of PCR-amplified ribosomal DNA fragments:Applied Environmental Microbiology,162(4):405-1415.
88 Tiedje J.M., Cho J.C., Murray A., et al.2001. Soil teeming with life:new frontiers for soil seience. In Sustainable Management of Soil Organic Matter. Edited by Rees R.M., Ball B.C., Campbell C.D., et al. CAB International,393-412.
89 Tien C.C., Chao C.C., Chao W.L.,1999. Methods for DNA extraetion from various soil:a comparison. Journal of Microbiological Methods,86:937-943.
90 Tsai Y.L., Olson B.H.,1991. Rapid method for direct extraction of DNA from soil and sediments. Applied Environmental Microbiology,57:1070-1074.
91 Viggo T.M., Kristjansson J.K., Kristmannsdottir H., et al.2001. Discovery and description of giant submarine smectite cones on the seafloor in Eyjafjordur, Northern Iceland, and a Novel Thermal Microbial Habitat. Applied and Environmental Microbiology,67:827-833.
92 Volossiou K.T., Robb E.J., Nazar R.N.,1995. Direct DNA extraction for PCR-mediated assays of soil organisms. Applied Environmental Microbiology,61(11):3972-3976.
93 Watve M.G., Gangal R.M.,1996. Problems in measuring bacterial diversity and apossible solution. Applied Environmental Microbiology,62(11):4299-4301.
94 Wikstrom P., Wiklund A., Andersson A., et al.1996. DNA recovery and PCR quantification of catechol 2,3-dioxygenase genes from different soil types. Journal of Biotechnology,52:107-120.
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98 Zelles., Alef K.,1995. Biomarkers. In:ALEF K, NANNIPIERI P eds. Methods in applied soil microbiology and biochemistry, London:Academic Press,422-439.
99 Zhou J., Bruns M.A., Tiedje J.M.,1996. DNA recovery from soils of diverse composition. Applied Environmental Microbiology,62:316-322.
100 Zhou J., Davey M.E., Ficueras J.B., et al.1997. Phylogenetic diversity of a bacterial community determined from Siberian.tundra soil DNA. Microbiology,143:3913-3919.
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61 Marsh T.L.,1999. Terminal restriction fragment length polyofmorphism(T—RPLP):An emerging method for characterizing diversity among homologous populations of amplification products. Current Opinion Microbiol,2:323-327.
62 McDaniel R., Ebert-Khosla S., Fu H., et al.1994. Engineered biosynthesis of novel polyketide: Influence of a downstream enzyme on the catalytic specificity of a minimal aromatic polyketide synthase. Proceedings of the National Academy of Sciences of the USA,91:11542-11546.
63 Meroth C.B., Walter J., Hertel C., et al.2003. Monitoring the baeterial population dynamics in sourdough fermentation proeesses by using PCR-denaturing gradient gel eleetrophoresis. Applied and Environmental Microbiology,69:475-482.
64 Miller D.N., Bryant J.E., Madsen E.L., et al.1999. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Applied and Environmental Microbiology, 65:4715-4724.
65 Morten K., Erland B.,1988. Microbial community dynamics during composting of straw material studied using phospholipids fatty acid analysis. FEMS Microbiology Ecology,27:9-20.
66 Muyzer G., Ellen C.W., Andre G.U.,1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction genes coding forl6Sr RNA. Applied and Environmental Microbiology,59:695-700.
67 Neal J.L., Can J.,1971. Of microbiology.17:1143-1145.
68 Nyman J.A.,1999. Effeet of crude oil and chemical additives on metabolic aetivity of mixed microbial populations in fresh marsh soils. Microbial Ecology,37(2):152-162.
69 Ogram A., Sayler G.S., Barkay T.,1987. The extraction and purification of microbial DNA from sediments. Journal of Microbiological. Methods.7:57-66.
70 Pfeifer B.A., Wang C.C., Walsh C.T., et al.2003. Biosynthesis of Yersiniabactin a complex polyketide-nonribosomal peptide using Escherichia coli as a heterologous host. Applied and Environmental Microbiology,69(11):6698-6702.
71 Pfeifer B.A., Khosla C.,2001. Biosynthesis of polyketides in heterologous hosts. Microbiology and Molecular Biology Reviews,65(1):106-118.
72 Robe P., Nalin R., Capellano C., et al.2003. Extraction of DNA from soil. European Journal of Soil Biology,39:183-190.
73 Roose-Amsaleg C.L., Garnier-Sillam E., Harry M.,2001. Extraction and purification of microbial DNA from soil and sediment samples. Applied Soil Ecology,18:47-60.
74 Rosello M.R., Aman R.,2001. The species concept for prokaryotes. FEMS Microbiology Review,25: 39-67.
75 Sabine P., Stefanie K., Frank S., et al.,2000. Succession of microbial communities during hot composting as detected by PCR-Single-trand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes. Applied and Environmental Microbiology,66(3):930-936.
76 Sambrook J., Fritsch E.F., Maniatis T.,1989. Molecular Cloning:A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
77 Schafer H., Muyzer G.,2001. Denaturing gradient gel electrophoresis and marine microbial ecology. In:Paul, J. (Ed.), Methods in Microbiology, London:Academic Press, pp.425-468.
78 Shen B.,2003. Polyketide biosynthesis beyond the type Ⅰ, Ⅱ and Ⅲ polyketide synthase paradigms. Curr Opin Chem Biol,7:285-295.
79 Smalla K., Wachtendorf U., Heuer H., et al.1998. Analysis of BIOLOG GN substract utilization partterns by microbial community. Applied and environmental microbiology,64:1220-1225.
80 Smit E., Leeflang P., Gommans S., et al.2001. 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,67(5):2284-2291.
81 Solbrig O.T.,1991. From genes to ecosystems:a research agenda for biodiversity. Report of a IUBS-SCOPEUNESCO workshop, the international union of biological sciences parisfrance,51 Boulevar dole montmorenny.
82 Srivastava A.K., Ara A., Bhargava P., et al.2007. A rapid and cost-effective method of genomic DNA isolation from cyanobacterial culture, mat and soil suitable for genomic fingerprinting and community analysis. Journal of Applied Phycology,19:373-382.
83 Staddon W.J., Duchesne L.C., Trevors J.T.,1997. Microbial Diversity and Community Structure of Postdisturbance Forest soils as Determined by Sole-Carbon-Source Utilization Patterns. Microbial Ecology,34:125-130.
84 Stamper D.M., Walch M., Jacobs R.N.,2003. Bacterial population changes in a membrane bioreactor for gray-water treatment monitored by denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments. Applied Environmental Microbiology,69:852-860.
85 Stevenson F.J.,1976. Stability constants of Cu.Pb and Cd complexes with humic acids. Soil Science Society of America jouranl,40:665-672.
86 Tang L., Fu H., Betlach M.C., et al.1999. Elucidating the mechanism of chain termination switching in the picromycin/methymycin polyketide synthase. Journal of Biological Chemistry,6(8):553-558.
87 Teske A., Wawer C., Muyzer G., et al.1996. Distribution of sulatfe-redueing bacteria in a stratified Fjodr (Marigaer Fjodr, Denmark) as evaluated b ymost-Porbable-number counts and denaturing gardient gel electrophoresis of PCR-amplified ribosomal DNA fragments:Applied Environmental Microbiology,162(4):405-1415.
88 Tiedje J.M., Cho J.C., Murray A., et al.2001. Soil teeming with life:new frontiers for soil seience. In Sustainable Management of Soil Organic Matter. Edited by Rees R.M., Ball B.C., Campbell C.D., et al. CAB International,393-412.
89 Tien C.C., Chao C.C., Chao W.L.,1999. Methods for DNA extraetion from various soil:a comparison. Journal of Microbiological Methods,86:937-943.
90 Tsai Y.L., Olson B.H.,1991. Rapid method for direct extraction of DNA from soil and sediments. Applied Environmental Microbiology,57:1070-1074.
91 Viggo T.M., Kristjansson J.K., Kristmannsdottir H., et al.2001. Discovery and description of giant submarine smectite cones on the seafloor in Eyjafjordur, Northern Iceland, and a Novel Thermal Microbial Habitat. Applied and Environmental Microbiology,67:827-833.
92 Volossiou K.T., Robb E.J., Nazar R.N.,1995. Direct DNA extraction for PCR-mediated assays of soil organisms. Applied Environmental Microbiology,61(11):3972-3976.
93 Watve M.G., Gangal R.M.,1996. Problems in measuring bacterial diversity and apossible solution. Applied Environmental Microbiology,62(11):4299-4301.
94 Wikstrom P., Wiklund A., Andersson A., et al.1996. DNA recovery and PCR quantification of catechol 2,3-dioxygenase genes from different soil types. Journal of Biotechnology,52:107-120.
95 William R.S.,1997. Biotechnology of Antibiotics, in Drugs and the Pharmaceutical Sciences. (Second edition). New York:Marcel Dekker, Inc New York.
96 Wu X., Zarka A., Boussiba S.,2000. A simplified protocol for preparing DNA from filamentous cyanobacteria. Plant Molecular Biology Reporter,18:385-392.
97 Yeates C., Gillings M.R., Davison A.D., et al.1998. Methods for microbial DNA extraction from soil for PCR amplification. Biological Procedures Online,1:40-47.
98 Zelles., Alef K.,1995. Biomarkers. In:ALEF K, NANNIPIERI P eds. Methods in applied soil microbiology and biochemistry, London:Academic Press,422-439.
99 Zhou J., Bruns M.A., Tiedje J.M.,1996. DNA recovery from soils of diverse composition. Applied Environmental Microbiology,62:316-322.
100 Zhou J., Davey M.E., Ficueras J.B., et al.1997. Phylogenetic diversity of a bacterial community determined from Siberian.tundra soil DNA. Microbiology,143:3913-3919.