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作物学报 ›› 2012, Vol. 38 ›› Issue (05): 791-799.doi: 10.3724/SP.J.1006.2012.00791

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

应用荧光定量PCR 技术分析普通菜豆品种中尖镰孢菜豆专化型定殖量

薛仁风1,朱振东1,*,黄燕1,2,王晓鸣1,王兰芬1,王述民1,*   

  1. 1 中国农业科学院作物科学研究所 / 农作物基因资源与基因改良国家重大科学工程, 北京 100081; 2 河北科技师范学院生命科技学院,河北昌黎066600
  • 收稿日期:2011-11-14 修回日期:2012-01-19 出版日期:2012-05-12 网络出版日期:2012-03-05
  • 基金资助:

    本研究由国家现代农业产业技术体系建设专项(CARS-09)资助。

Quantification of Fusarium oxysporum f. sp. phaseoli Detected by Real-time Quantitative PCR in Different Common Beans Cultivars

XUE Ren-Feng1, ZHU Zhen-Dong1,*, HUANG Yan1,2,WANG Xiao-Ming1,WANG Lan-Fen1,WANG Shu-Min1,*   

  1. 1 Institute of Crop Science, Chinese Academy of Agricultural Sciences / National Key Facility for Crop Gene Resources and Genetic Improvement,Beijing 100081, China; 2 College of Life Science and Technology, Hebei Normal University of Science & Technology, Changli 066600, China
  • Received:2011-11-14 Revised:2012-01-19 Published:2012-05-12 Published online:2012-03-05

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被引次数

11

摘要： 尖镰孢菜豆专化型(Fusarium oxysporum f. sp. phaseoli)引起的菜豆枯萎病是菜豆生产中最严重的维管束类病害之一, 防治该病害有效方法是利用抗病品种。因此, 一种能够从菜豆受侵染组织中准确鉴定并定量检测枯萎病原菌含量的方法将有助于筛选抗性品种, 应用于普通菜豆枯萎病抗病育种。本研究基于荧光定量PCR技术开发出一种能够对定殖于菜豆组织中的枯萎病原菌准确定量的新方法。该技术对根、茎组织中病原菌DNA的最低检测量为1 pg, 能在接种病原菌6 d后明显区分抗病性不同的品种, 可在菜豆植株表现出明显发病症状前准确鉴定不同品种抗性水平的差异。经验证参试的感病品种BRB-130和A0640-1根、茎组织中定殖的病原菌DNA量显著高于抗病品种260205和黑芸豆, 与表型鉴定的结果完全符合。利用荧光定量PCR技术能够在病原菌侵染早期快速、准确、高效定量菜豆组织中定殖的病原菌, 这对指导菜豆抗病育种和植物病害传播的研究都具有非常重要价值。

关键词: 普通菜豆, 镰孢菌枯萎病, 荧光定量PCR, 定量分析

Abstract: Fusarium wilt, caused by Fusarium oxysporum f. sp. phaseoli, is one of the most devastating vascular diseases of common bean. A efficient way to prevent and control the disease is to use resistant cultivars, so it is necessary to develop a rapid accurate method for detecting the pathogens. In this study, we developed a real-time quantitative polymerase chain reaction (qRT-PCR) protocol that quantified F. oxysporum f. sp. phaseoli DNA to a minimum of 1 pg in the plant roots and stems. Moreover, the qRT-PCR protocol asymptomatically distinguished resistant level of different bean cultivars challenged by the wilt pathogen FOP-DM01 at 6 d post inoculation. The result indicated FOP-DM01 DNA quantifications in the roots and stems of susceptible BRB-130 and A0640-1 were significantly higher than those in resistant 260205 and Heiyundou, which absolutely matched with the phenotypic identification. The use of this protocol for fast, reliable, and cost-effective quantification of F. oxysporum f. sp. phaseoli in asymptomatic tissues at early stages of the infection process is of great value for common bean breeding and studies of phytopathology and epidemiology.

Key words: Common bean, Fusarium wilt, Real time-PCR, Quantitative analysis

[1]Broughton W J, Hernandez G, Blair M, Beebe S, Gepts P, Vanderleyden J. Beans (Phaseolus spp.)—model food legumes. Plant Soil, 2003, 252: 55?128

[2]Buruchara R A, Camacho L. Common bean reaction to Fusarium oxysporum f. sp. phaseoli, the cause of severe vascular wilt in Central Africa. J Phytopathol, 2000, 148: 39?45

[3]Brouwer M, Lievens B, Hemelrijck W, Ackerveken G, Cammue B, Thomma B P H J. Quantification of disease progression of several microbial pathogens on Arabidopsis thaliana using real-time fluorescence PCR. FEMS Microbiol Lett, 2003, 228: 241?248

[4]Thomma B P H J, Tadesse Y S H, Jacobs M, Broekaert W F. Disturbed correlation between fungal biomass and β-glucuro¬nidase activity in infections of Arabidopsis thaliana with transgenic Alternaria brassicicola. Plant Sci, 1999, 148: 31?36

[5]Hoffman T, Schmidt J S, Zheng X, Bent A F. Isolation of ethylene-insensitive soybean mutants that are altered in pathogen susceptibility and gene-for-gene disease resistance. Plant Physiol, 1999, 119: 935?950

[6]Dan H, Ali-Khan S T, Robb J. Use of quantitative PCR diagnostics to identify tolerance and resistance to Verticillium dahliae in potato. Plant Dis, 2001, 85: 700?705

[7]Pegg G F, Brady B L. Verticillium wilts. Wallingford: CABI International, 2002

[8]Schena L, Nigro F, Ippolito A, Gallitelli D. Real-time quantitative PCR: a new technology to detect and study phytopathogenic and antagonistic fungi. Eur J Plant Pathol, 2004, 110: 893?908

[9]Böhm J, Hahn A, Schubert R, Bahnweg G, Adler N, Nechwatal J, Oehlmann R, Obszwald W. Real-time Quantitative PCR: DNA determination in isolated spores of the mycorrhizal fungus Glomus mosseae and monitoring of Phytophthora infestans and Phytophthora citricola in their respective host plants. J Phytopathol, 1999, 147: 409?416

[10]Mccartney H A, Foster S J, Fraaije B A, Ward E. Molecular diagnostics for fungal plant pathogens. Pest Manag Sci, 2003, 59: 129?142

[11]Mumford R A, Walsh K, Barker I, Boonham N. Detection of potato mop top virus and tobacco rattle virus using a multiplex real-time fluorescent reverse-transcription polymerase chain reaction assay. Phytopathology, 2000, 90: 448?453

[12]Schaad N W, Frederick R D, Shaw J, Schneider W L, Hickson R, Petrillo M D, Luster D G. Advances in molecular-based diagnostics in meeting crop biosecurity and phytosanitary issues. Annu Rev Phytopathol, 2003, 41: 305?324

[13]Vandemark G J, Barker B M. Quantifying Phytophthora medicaginis in susceptible and resistant alfalfa with a real-time fluorescent PCR assay. J Phytopathol, 2003, 151: 577?583

[14]Morrison T B, Weis J J, Wittwer C T. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques, 1998, 24: 954?962

[15]Winton L M, Manter D K, Stone J K, Hansen E M. Comparison of biochemical, molecular, and visual methods to quantify Phaeocryptopus gaeumannii in Douglas-Fir foliage. Phytopathology, 2003, 93: 121?126

[16]Alves-Santos F M, Ramos B, García-Sánchez M A, Eslava A P, Díaz-Mínguez J M. A DNA-based procedure for in planta detection of Fusarium oxysporum f. sp. phaseoli. Phytopathology, 2002, 92: 237?244

[17]de Vega-Bartol J J, Martín-Dominguez R, Ramos B, García- ¬Sánchez M A, Díaz-Mínguez J M. New virulence groups in Fusarium oxysporum f. sp. phaseoli: the expression of the gene coding for the transcription factor ftf1 correlates with virulence. Phytopathology, 2011, 101: 470?479

[18]Ramos B, Alves-Santos F M, García-Sánchez M A, Martín- ¬Rodrigues N, Eslava A P, Díaz-Mínguez J M. The gene coding for a new transcription factor (ftf1) of Fusarium oxysporum is only expressed during infection of common bean. Fungal Genet Biol, 2007, 44: 864?876

[19]Xue R-F(薛仁风), Zhu Z-D(朱振东), Wang X-M(王晓鸣), Wang L-F(王兰芬), Wu X-F(武小菲), Wang S-M(王述民). Cloning and expression analysis of the Fusarium wilt resistance-related gene PvCaM1 in common bean (Phaseolus vulgaris L.). Acta Agron Sin (作物学报), 2011, 38(4): 606????

[20]Silvar C, Díaz J, Merino F. Real-time polymerase chain reaction quantification of Phytophthora capsici in different pepper genotypes. Phytopathology, 2005, 95: 1423?1429

[21]Adams P S. Data analysis and reporting. In: Tevfik Dorak M eds. Real time-PCR. New York: Taylor & Francis Group, 2006. pp 39?61

[22]Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Nat Biotechnol, 1993, 11: 1026?1030

[23]Wang S-M(王述民), Zhang Y-Z(张亚芝), Wei S-H(魏淑红). Descriptors and Data Standard for Common Bean (Phaseolus vulgaris L.)(普通菜豆种质资源描述规范和数据标准). Beijing: China Agricultural Press, 2006. p 64 (in Chinese)

[24]Institute S. SAS 9.1.3 intelligence platform: single-user installation guide. SAS Institute Cary, NC, 2005

[25]Pastor C, Abawi G S. Reactions of selected bean germplasm to infection by Fusarium oxysporum f. sp. phaseoli. Plant Dis, 1987, 71: 990?993

[26]Salgado M O, Schwartz H F, Brick M A. Inheritance of resistance to a Colorado race of Fusarium oxysporum f. sp. phaseoli in common beans. Plant Dis, 1995, 79: 279?281

[27]Du Y-G(杜永刚), Li C(李昶). The occurrence and comprehensive control of common bean wilt. Jilin Veg (吉林蔬菜), 2008, (4): 89 (in Chinese)

[28]Miklas P N, Kelly J D, Beebe S E, Blair M W. Common bean breeding for resistance against biotic and abiotic stresses: from classical to MAS breeding. Euphytica, 2006, 147: 105?131

[29]Bates J A, Taylor E, Kenyon D M, Thomas J E. The application of real-time PCR to the identification, detection and quantification of Pyrenophora species in barley seed. Mol Plant Pathol, 2001, 2: 49?57

[30]Filion M, St-Arnaud M, Jabaji-Hare S H. Quantification of Fusarium solani f. sp. phaseoli in mycorrhizal bean plants and surrounding mycorrhizosphere soil using real-time polymerase chain reaction and direct isolations on selective media. Phytopathology, 2003, 93: 229?235

[31]Pasquali M, Acquadro A, Balmas V, Migheli Q, Lodovica Gullino M, Garibaldi A. Development of PCR primers for a new Fusarium oxysporum pathogenic on Paris daisy (Argyranthemum frutescens L.). Eur J Plant Pathol, 2004, 110: 7?11

[32]Pasquali M, Marena L, Fiora E, Piatti P, Gullino M L, Garibaldi A. Real-time polymerase chain reaction for identification of a highly pathogenic group of Fusarium oxysporum f. sp. chrysanthemi on Argyranthemum frutescens L. J Plant Pathol, 2004, 86: 53?59

[33]Abd-Elsalam K A, Asran-Amal A, Schnieder F, Migheli Q, Verreet J. Molecular detection of Fusarium oxysporum f. sp. vasinfectum in cotton roots by PCR and real-time PCR assay. J Plant Dis Protect, 2006, 113: 14?19

[34]Zhang Z, Zhang J, Wang Y, Zheng X. Molecular detection of Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in infected plant tissues and soil. FEMS Microbiol Lett, 2005, 249: 39?47

[35]Pasquali M, Piatti P, Gullino M L, Garibaldi A. Development of a real-time polymerase chain reaction for the detection of Fusarium oxysporum f. sp. basilici from Basil seed and roots. J Phytopathol, 2006, 154: 632?636

[36]Pasquali M, Dematheis F, Gullino M L, Garibaldi A. Identification of race 1 of Fusarium oxysporum f. sp. lactucae on lettuce by inter-retrotransposon sequence-characterized amplified region technique. Phytopathology, 2007, 97: 987?996

[37]Jiménez-Fernández D, Montes-Borrego M, Jiménez-Díaz R M, Navas-Cortés J A, Landa B B. In planta and soil quantification of Fusarium oxysporum f. sp. ciceris and evaluation of Fusarium wilt resistance in chickpea with a newly developed quantitative polymerase chain reaction assay. Phytopathology, 2011, 101: 250?262

[38]Inami K, Yoshioka C, Hirano Y, Kawabe M, Tsushima S, Teraoka T, Arie T. Real-time PCR for differential determination of the tomato wilt fungus, Fusarium oxysporum f. sp. lycopersici, and its races. J Gen Plant Pathol, 2010, 76: 116?121

[39]Ririe K M, Rasmussen R P, Wittwer C T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem, 1997, 245: 154?160

[40]Brick M A B, Schwartz P F, Ogg H F, Otto J B, Fall K, Gilbert A L. Reaction to three races of Fusarium wilt in the core collection. Crop Sci, 2006, 46: 1245?1252

[41]Ribeiro R L D, Hagedorn D J. Screening for resistance to and pathogenic specialization of Fusarium oxysporum f. sp. phaseoli, the causal agent of bean yellows. Phytopathology, 1979, 69: 272?276
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