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| Comparative genomics provide a rapid detection of Fusarium oxysporum f. sp. conglutinans |
| LING Jian*, ZHANG Ji-xiang*, ZENG Feng, CAO Yue-xia, XIE Bing-yan, YANG Yu-hong |
| Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China |
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摘要 Fusarium oxysporum f. sp. conglutinans (Foc) is the causal agent of Fusarium wilt disease of Brassica oleracea. A rapid, accurate, and reliable method to detect and identify plant pathogens is vitally important to integrated disease management. In this study, using a comparative genome analysis among Fusarium oxysporum (Fo), we developed a Foc-specific primer set (Focs-1/Focs-2) and established a multiplex-PCR assay. In the assay, the Focs-1/Focs-2 and universal primers for Fusarium species (W106R/F106S) could be used to detect Foc isolates in a single PCR reaction. With the optimized PCR parameters, the multiplex-PCR assay showed a high specificity for detecting Foc and was very sensitive to detect as little as 100 pg of pure Foc genomic DNA or 1 000 spores in 1 g of twice-autoclaved soil. We also demonstrated that Foc isolates were easily detected from infected plant tissues, as well as from natural field soils, using the multiplex-PCR assay. To our knowledge, this is a first report on detection Fo by comparative genomic method.
Abstract Fusarium oxysporum f. sp. conglutinans (Foc) is the causal agent of Fusarium wilt disease of Brassica oleracea. A rapid, accurate, and reliable method to detect and identify plant pathogens is vitally important to integrated disease management. In this study, using a comparative genome analysis among Fusarium oxysporum (Fo), we developed a Foc-specific primer set (Focs-1/Focs-2) and established a multiplex-PCR assay. In the assay, the Focs-1/Focs-2 and universal primers for Fusarium species (W106R/F106S) could be used to detect Foc isolates in a single PCR reaction. With the optimized PCR parameters, the multiplex-PCR assay showed a high specificity for detecting Foc and was very sensitive to detect as little as 100 pg of pure Foc genomic DNA or 1 000 spores in 1 g of twice-autoclaved soil. We also demonstrated that Foc isolates were easily detected from infected plant tissues, as well as from natural field soils, using the multiplex-PCR assay. To our knowledge, this is a first report on detection Fo by comparative genomic method.
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Received: 05 June 2015
Accepted:
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| Fund: This research was supported in part by the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, P.R.China, the National Natural Science Foundation of China (31571962, 31272003), the National Key Technology R&D Program of China (2012BAD19B06), the Special Fund for Agro-Scientific Research in the Public Interest, China (200903049-04), the National Staple Vegetable Industry Technology System Construction Project, China (CARS-25-B-01), and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences. |
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Corresponding Authors:
YANG Yu-hong, Tel: +86-10-82109545, E-mail: yangyuhong@caas.cn
E-mail: yangyuhong@caas.cn
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| About author: LING Jian, E-mail: lingjian2005@126.com |
Cite this article:
LING Jian, ZHANG Ji-xiang, ZENG Feng, CAO Yue-xia, XIE Bing-yan, YANG Yu-hong.
2016.
Comparative genomics provide a rapid detection of Fusarium oxysporum f. sp. conglutinans. Journal of Integrative Agriculture, 15(4): 822-831.
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| Alves-Santos F, Cordeiro-Rodrigues, Sayagués J, Martín-Domínguez R, García-Benavides P, Crespo M, Díaz-Mínguez J, Eslava A. 2002. Pathogenicity and race characterization of Fusarium oxysporum f. sp. phaseoli isolates from Spain and Greece. Plant Pathology, 51, 605–611.Chiocchetti A S L, Durando F, Garibaldi M. 2001. PCR detection of Fusarium oxysporum f. sp. basilici on Basil. Plant Disease, 85, 607–611.Dita M A, Waalwijk C, Buddenhagen I W, Souza Jr M T, Kema G H J. 2010. A molecular diagnostic for tropical race 4 of the banana Fusarium wilt pathogen. Plant Pathology, 59, 348–357.van der Does H C, Rep M. 2007. Virulence genes and the evolution of host specificity in plant-pathogenic fungi. Molecular Plant-Microbe Interactions, 20, 1175–1182.Edel V, Steinberg C, Cautheron N, Alabouvette C. 1997. Populations of nonpathogenic Fusarium oxysporum associated with roots of four plant species compared to soilborne populations. Ecology and Population Biology, 87, 693–697.Edel V, Steinberg C, Cautheron N, Recorbet G, Alabouvette C. 2001. Genetic diversity of Fusarium oxysporum populations isolated from different soils in France. FEMS Microbiology Ecology, 36, 61–71.Forsyth L M, Smith L J, Aitken E A. 2006. Identification and characterization of non-pathogenic Fusarium oxysporum capable of increasing and decreasing Fusarium wilt severity. Mycological Research, 110, 929–935.Fravel D, Olivain C, Alabouvette C. 2003. Fusarium oxysporum and its biocontrol. New Phytologist, 157, 493–502.Gao Y Y, Wang N. 2010. Triplex PCR detection of Cladosporuium cucumberinum, Fusarium oxysporum f. sp. niveum and Mycosphaerella melonis in infected plant tissues. Acta Phytopathologica Sinica, 4, 343–350. (in Chinese)Haegi A, Catalano V, Luongo L, Vitale S, Scotton M, Ficcadenti, N, Belisario A. 2013. A newly developed real-time PCR assay for detection and quantification of Fusarium oxysporum and its use in compatible and incompatible interactions with grafted melon genotypes. Phytopathology, 103, 802–810.Huang S, Van Der Vossen E A, Kuang H H, Vleeshouwers V G, Zhang N, Borm T J, Van Eck H J, Baker B, Jacobsen E, Visser R G. 2005. Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato. The Plant Journal, 42, 251–261.Jurado M, Vázquez C, Marín S, Sanchis V, Teresa González-Jaén M. 2006. PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in maize. Systematic and Applied Microbiology, 29, 681–689.Kelly A, Bainbridge B, Heale J, Pérez-Artés E, Jiménez-Díaz R. 1998. In planta polymerase chain reaction detection of the wilt-inducing pathotype of Fusarium oxysporum f. sp. ciceris in chickpea (Cicer arietinum L.). Physiological and Molecular Plant Pathology, 52, 397–409.Komada H. 1975. Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soil. Review of Plant Protection Research, 8, 114–124.Li M H, Yu X T, Wang H F, Zhou J N, Xi P G, Jiang Z D. 2012. Rapid detection and identification of Fusarium oxysporum f. sp. cubense race 1 and race 4. Scientia Agricultura Sinica, 45, 3971–3979. (in Chinese)Li M, Zhang T, Li X, Yan H. 2003. Fusariums wilt on cruciferous vegetables and identification of their pathogens. Plant Protection, 29, 44–45.Li Y, Mao L G, Yan D D, Ma T T, Shen J, Guo M X, Wang Q X, Ouyang C B, Cao A C. 2014. Quantification of Fusarium oxysporumin fumigated soils by a newly developed real-time PCR assay to assess the efficacy of fumigants for Fusarium wilt disease in strawberry plants. Pest Management Science, 70, 1669–1675.Lin Y H, Chang J Y, Liu E T, Chao C P, Huang J W, Chang P F. 2008. Development of a molecular marker for specific detection of Fusarium oxysporum f. sp. cubense race 4. European Journal of Plant Pathology, 123, 353–365.Lin Y H, Chen K S, Chang J Y, Wan Y L, Hsu C C, Huang J W, Chang P F L. 2010. Development of the molecular methods for rapid detection and differentiation of Fusarium oxysporum and F. oxysporum f. sp. niveum in Taiwan. New Biotechnology, 27, 409–418.Lin Y H, Su C C, Chao C P, Chen C Y, Chang C J, Huang J W, Chang P F. 2013. A molecular diagnosis method using real-time PCR for quantification and detection of Fusarium oxysporum f. sp. cubense race 4. European Journal of Plant Pathology, 135, 395–405.Lin Z, Zhang Y, Zhang H, Zhou Y, Cao J, Zhou J. 2012. Comparison of loop-mediated isothermal amplification (LAMP) and real-time PCR method targeting a 529-bp repeat element for diagnosis of toxoplasmosis. Veterinary Parasitology, 185, 296–300.Ling J, Zeng F, Cao Y X, Zhang J X, Chen G H, Mao Z C, Yang Y H, Xie B Y. 2015. Identification of a class of CFEM proteins containing a new conserved motif in Fusarium oxysporum. Physiological and Molecular Plant Pathology, 89, 41–48.Lievens B, Claes L, Vakalounakis D J, Vanachter A C, Thomma B P. 2007. A robust identification and detection assay to discriminate the cucumber pathogens Fusarium oxysporum f. sp. cucumerinum and f. sp. radicis-cucumerinum. Environmental Microbiology, 9, 2145–2161.Lievens B, Rep M, Thomma B P. 2008. Recent developments in the molecular discrimination of formae speciales of Fusarium oxysporum. Pest Management Science, 64, 781–788.Liu J, Chen X, Wang B S, He Z F, Peng A T, Cai M S. 2006. Race identification and SCAR markers development for the pathogen of banana Panama disease Fusarium oxysporum f. sp. cubense. Acta Phytopathologica Sinica, 36, 28–34. (in Chinese)del Mar Jiménez-Gasco, Milgroom M G, Rafael M, Jiménez-Díaz. 2004. Stepwise evolution of races in Fusarium oxysporum f. sp. ciceris inferred from fingerprinting with repetitive DNA sequences. Phytopathology, 94, 228–235.Möller E, Che?kowski J ,Geiger H. 1999. Species-specific PCR Assays for the fungal pathogens Fusarium moniliforme and Fusarium subglutinans and their application to diagnose maize ear rot disease. Journal of Phytopathology, 147, 497–508.Nelson P E, Toussoun T A, Marasas W. 1983. Fusarium Species: An Illustrated Manual for Identification. The Penn State University Press, PHL, PA, USA.Parry D, Nicholson P. 1996. Development of a PCR assay to detect Fusarium poae in wheat. Plant Pathology, 45, 383–391.Pasquali M, Dematheis F, Gullino M L, Garibaldi A. 2007. Identification of race 1 of Fusarium oxysporum f. sp. lactucae on lettuce by inter-retrotransposon sequence - Characterized amplified region technique. Phytopathology, 97, 987–996.Peng J, Zhan Y F, Zeng F Y, Long H B, Pei Y L, Guo J R. 2013. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. niveumin soil. FEMS Microbiology Letters, 349, 127–134.Peng J Z, He C, Zhang F P, Xie X, Hou Y X, Li X Y, Pu J J. 2014. Rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 in soil by real-time fluorescence loop-mediated isothermal amplification. Journal of Applied Microbiology, 117, 1740–1749.Raju G, Avuthu N, Anindita S, Mamta S. 2015. Development of loop-mediated isothermal amplification (LAMP) assay for rapid detection of Fusarium oxysporum f. sp. ciceris - wilt pathogen of chickpea. BMC Research Notes, 8, 40.Ramirez-Villupadua J, Endo R, Bosland P, Williams P. 1985. A new race of Fusarium oxysporum f. sp. conglutinans that attacks cabbage with type A resistance. Plant Disease, 69, 612–613.Reddy J M, Raoof M, Ulaganathan K. 2012. Development of specific markers for identification of Indian isolates of Fusarium oxysporum f. sp. ricini. European Journal of Plant Pathology, 134, 713–719.Shi X Z, Yu D S, Warner E D, Pan X Z, Petersen G W, Gong Z G, Weindorf D C. 2004. Soil database of 1:1,000,000 digital soil survey and reference system of the chinese genetic soil classification system. Soil Survey Horizons, 45, 129–136.Wilson A, Simpson D, Chandler E, Jennings P, Nicholson P. 2004. Development of PCR assays for the detection and differentiation of Fusarium sporotrichioides and Fusarium langsethiae. FEMS Microbiology Letters, 233, 69–76.Yergeau E, Filion M, Vujanovic V, St-Arnaud M. 2005. A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus. Microbiol Methods, 60, 143–154.Yoder W T, Christianson L M. 1998. Species-specific primers resolve members of Fusarium section Fusarium - Taxonomic status of the edible “Quorn” fungus reevaluated. Fungal Genetics and Biology, 23, 68–80.Zhang L J, Li S D, Liao Z Q. 2013. Pathogenicity variation and genetic structure differentiation of Fusarium oxysporum f.sp. conglutinans in soil under successive cultivation of Brassica oleracea. Acta Phytopathologica Sinica, 43, 58–68. (in Chinese)Zhang X, Zhang H, Pu J J, Qi Y X, Yu Q F, Xie Y X, Peng J. 2013. Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil. PLoS One, 8, e82841.Zhang Z, Zhang J, Wang Y, Zheng X. 2005. Molecular detection of Fusarium oxysporum f. sp. niveum and mycosphaerella melonis in infected plant tissues and soil. FEMS Microbiology Letters, 249, 39–47. |
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