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Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses |
HU Xin-xi, LEI Yan, WANG Pei, TANG Lin-fei, HE Chang-zheng, SONG Yong, XIONG Xing-yao, NIE Xian-zhou |
1、Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization, Hunan Provincial Engineering Research Center
for Potatoes, College of Horticulture and Landscape, Hunan Agricultural University, Changsha 410128, P.R.China
2、Potato Research Centre, Agriculture and Agri-Food Canada, Fredericton, E3B 4Z7, New Brunswick, Canada |
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摘要 A preliminary screening for garlic viruses in garlic plants in Hunan, China, using existing monoplex (simplex) reverse transcription- polymerase chain reaction (RT-PCR) procedures detected four viruses/virus groups. These viruses/virus groups were Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and allexiviruses (e.g., garlic viruses A, B, C, D, E, X). Sequence analysis of the projected allexivirus amplicons revealed the allexivirus in the infected garlic plants was Garlic virus D (GarV-D), which shared 92–97% sequence identities with various isolates from the world. A multiplex RT-PCR (mRT-PCR) was therefore developed to simultaneously detect and differentiate the four viruses/virus groups. To achieve this, four primer pairs targeting allexiviruses, OYDV, LYSV and SLV were designed. The anticipated amplicon sizes are 183 bp (allexiviruses), 265 bp (OYDV), 404 bp (LYSV) and 592 bp (SLV), respectively. All primer pairs produced virus-specific fragments in both simplex and multiplex formats, thus confirming the efficacy of the newly developed mRT-PCR for detection of these viruses. The mRT-PCR further was evaluated by applying it to garlic plant samples collected in two geographic locations in Hunan. Allexiviruses, OYDV, LYSV and SLV were detected in 50.9, 40.3, 28.3 and 58.5% of leaf samples, respectively; and mixed infections with two or more viruses accounted for 54% of the garlic samples. The results obtained by mRT-PCR were confirmed by simplex RT-PCR assays. In conclusion, this newly developed mRT-PCR provides a rapid, sensitive and reliable method for the detection and identification of major garlic viruses.
Abstract A preliminary screening for garlic viruses in garlic plants in Hunan, China, using existing monoplex (simplex) reverse transcription- polymerase chain reaction (RT-PCR) procedures detected four viruses/virus groups. These viruses/virus groups were Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and allexiviruses (e.g., garlic viruses A, B, C, D, E, X). Sequence analysis of the projected allexivirus amplicons revealed the allexivirus in the infected garlic plants was Garlic virus D (GarV-D), which shared 92–97% sequence identities with various isolates from the world. A multiplex RT-PCR (mRT-PCR) was therefore developed to simultaneously detect and differentiate the four viruses/virus groups. To achieve this, four primer pairs targeting allexiviruses, OYDV, LYSV and SLV were designed. The anticipated amplicon sizes are 183 bp (allexiviruses), 265 bp (OYDV), 404 bp (LYSV) and 592 bp (SLV), respectively. All primer pairs produced virus-specific fragments in both simplex and multiplex formats, thus confirming the efficacy of the newly developed mRT-PCR for detection of these viruses. The mRT-PCR further was evaluated by applying it to garlic plant samples collected in two geographic locations in Hunan. Allexiviruses, OYDV, LYSV and SLV were detected in 50.9, 40.3, 28.3 and 58.5% of leaf samples, respectively; and mixed infections with two or more viruses accounted for 54% of the garlic samples. The results obtained by mRT-PCR were confirmed by simplex RT-PCR assays. In conclusion, this newly developed mRT-PCR provides a rapid, sensitive and reliable method for the detection and identification of major garlic viruses.
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Received: 19 May 2014
Accepted: 13 May 2015
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Fund: This research was supported by the Non-Profit Industry Financial Program of Ministry of Agriculture of China (20130328) to Prof. Liu Yong and Prof. Dai Liangying, and the Natural Science Foundation of Hunan Province, China to Prof. Hu Xinxi (11JJ2018). |
Corresponding Authors:
XIONG Xing-yao, Tel/Fax: +86-731-84635295,E-mail: xiongxy@hunau.net; NIE Xian-zhou, Tel: +1-506-4604514,Fax: +1-506-4604377, E-mail: xianzhou.nie@agr.gc.ca
E-mail: xiongxy@hunau.net; xianzhou.nie@agr.gc.ca
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About author: HU Xin-xi, Tel: +86-731-84618171, E-mail: huxinxi163@163.com; LEI Yan, Tel: +86-731-84618171, E-mail: leiyan5181@sina.com; |
Cite this article:
HU Xin-xi, LEI Yan, WANG Pei, TANG Lin-fei, HE Chang-zheng, SONG Yong, XIONG Xing-yao, NIE Xian-zhou.
2015.
Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses. Journal of Integrative Agriculture, 14(5): 900-908.
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Arya M, Baranwal V K, Ahlawat Y S, Singh L. 2006. RT-PCRdetection and molecular characterization of Onion yellowdwarf virus associated with garlic and onion. CurrentScience, 91, 1230-1234Canning E S, Penrose M J, Barker I, Coates D 1996. Improveddetection of Barley yellow dwarf virus in single aphids usingRT-PCR. Journal of Virological Methods, 56, 191-197Chen J, Chen J. 2001. Sequencing and phylogenetic analysisof Garlic virus E-A new member of allexivirus. ChineseScience Bulletin, 46, 1463-1468 (in Chinese)Chen J, Chen J, Adams M J. 2001. Molecular characterization ofa complex mixture of viruses in garlic with mosaic symptomsin China. Archives of Virology, 146, 1841-1853Cohen J, Zeidan M, Rosner A, Gera A. 2000. Biological andmolecular characterization of a new carlavirus isolated froman Aconitum sp. Phytopathology, 90, 340-344Conci V C, Canavelli A, Lunello P. 2003. Yield losses associatedwith virus-infected garlic plants during five successive years.Plant Disease, 87, 1411-1415Dai J, Cheng J, Huang T, Zheng X, Wu Y. 2012. A multiplexreverse transcription PCR assay for simultaneous detectionof five tobacco viruses in tobacco plants. Journal ofVirological Methods, 183, 57-62van Dijk P. 1993. Carlavirus isolates from cultivatedAllium species represent three viruses. NetherlandsJournal of Plant Pathology, 99, 233-257van Dijk P. 1994. Virus disease of Allium species and productsfor their control. Acta Horticulturae, 358, 299-305Dovas C I, Hatzibukas E, Salomon R, Barg E, Shiboleth Y M,Katis N I. 2001. Comparison of methods for virus detectionin Allium species. Journal of Phytopathology, 149, 731-737Dovas C I, Katis N I. 2003. A spot nested RT-PCR methodfor the simultaneous detection of members of the Vitivirusand Foveavirus genera in grapevine. Journal of VirologicalMethods, 107, 99-106Figueira A R, Domier L L, D’Arcy C J. 1997. Comparison oftechniques for detection of Barley yellow dwarf virus-PAVIL.Plant Disease, 81, 1236-1240Hadidi A, Montasser M S, Levy L, Goth R W, Converse R H,Madkour M A, Ckrzeckowski L J. 1993. Detection of potatoleafroll and strawberry mild yellow-edge luteovirusesby reverse transcription-polymerase chain reactionamplification. Plant Disease, 77, 595-601Ito T, Ieki H, Ozaki K. 2002. Simultaneous detection of six citrusviroids and Apple stem grooving virus from citrus plants bymultiplex reverse transcription polymerase chain reaction.Journal of Virological Methods, 106, 235-239Kanyuka K V, Vishnichenko V K, Levay K E. 1992.Nucleotide sequence of Shallot virus X RNA reveals a5´-proximal cistron closely related to those of potexvirusesand a unique arrangement of the 3´-proximal cistrons.Journal of General Virology, 73, 2553-2560Kobayashi K, Rabinowicz P, Bravo-Almonacid F, HelgueraM, Conci V, Lot H, Mentaberry A. 1996. Coat proteingene sequences of garlic and onion isolates of the Onionyellow dwarf potyvirus (OYDV). Archives of Virology, 141,2277-2287Liu X, Cheng Z. 2013. Research progress on cryopreservationof garlic germplasm and its virus eradication technique.Chinese Vegetables, 2, 12-19 (in Chinese)Majumder S, Baranwal V K, Joshi S. 2008. Simultaneousdetection of Onion yellow dwarf virus and Shallot latent virusin infected leaves and cloves of garlic bu duplex RT-PCR.Journal of Plant Pathology, 90, 371-374Marys E, Carballo O, Izaguirre-Mayoral M L. 1994. Isolation andcharacterization of viruses present in four clones of garlic(Allium sativum) in Venezuela. Journal of Phytopathology,142, 227-234Nagakubo T, Kubo M, Oeda K.1994. Nucleotide sequences ofthe 3´ regions of two major viruses from mosaic-diseasedgarlic: Molecular evidence of mixed infection by a potyvirusand a carlavirus. Phytopathology, 84, 640-645Nie X, Singh R P2001. A novel usage of random primers formultiplex RT-PCR detection of virus and viroid in aphids,leaves, and tubers. Journal of Virological Methods, 91,37-49Pan Y. 2012. Analysis and prospects of Chinese garlic industrydevelopment. Food and Nutrition in China, 18, 22-26 (inChinese)Park K S, Bae Y J, Jung E J, Kang S J. 2005. RT-PCR-baseddetection of six garlic viruses and their phylogeneticrelationships. Journal of Microbiology and Biotechnology,15, 1110-1114Pramesh D, Baranwal V K. 2013. Molecular characterization ofcoat protein gene of Garlic common latent virus isolates fromIndia: an evidence for distinct phylogeny and recombination.Virus Genes, 47, 189-193Shiboleth Y M, Gal-On A, Koch M, Rabinowitch H D, SalomonR. 2001. Molecular characterisation of Onion yellow dwarfvirus (OYDV) infecting garlic (Allium sativum L.) in Israel:Thermotherapy inhibits virus elimination by meristem tipculture. Annals of Applied Biology, 138, 187-195Singh R P, Nie X, Singh M. 2000. Duplex RT-PCR: Reagentconcentrations at reverse transcription stage affect the PCRperformance. Journal of Virological Methods, 86, 121-129Song S I, Song J T, Kim C H, Lee J S, Choi Y D. 1998. Molecularcharacterization of the garlic virus X genome. Journal ofGeneral Virology, 79, 155-159Sumi S I, Tsuneyoshi T, Furutani H. 1993. Novel rod-shapedviruses isolated from garlic, Allium sativum, possessing aunique genome organization. Journal of General Virology,74, 1879-1885Sward R J, Brennan A P. 1994. Diagnosis and control Alliumvirus disease in Victoria, Australia. Acta Horticulturae, 358,295-298Takaichi M, Yamamoto M, Nagakubo T, Oeda K. 1998. Fourgarlic viruses identified by reverse transcription-polymerasechain reaction and their regional distribution in NorthernJapan. Plant Disease, 82, 694-698Takaichi M, Yamamoto M, Nagakubo T, Oeda K. 2001. Mixedvirus infections of garlic determined by a multivalentpolyclonal antiserum and virus effects on diseasesymptoms. Plant Disease, 85, 71-75Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S.2011. MEGA5: Molecular evolutionary genetics analysis usingmaximum likelihood, evolutionary distance, and maximumparsimony methods. Molecular Biology and Evolution, 28,2731-2739Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, HigginsD G. 1997. The CLUSTAL_X windows interface: Flexiblestrategies for multiple sequence alignment aided by qualityanalysis tools. Nucleic Acids Research, 25, 4876-4882Tsuneyoshi T, Sumi S. 1996. Differentiation among garlicviruses in mixed infections based on RT-PCR proceduresand direct tissue blotting immunoassays. Phytopathology, 86, 253-259Tsuneyoshi T, Matsumi T, Deng T C, Sako I, Sumi S. 1998a.Differentiation of Allium carlaviruses isolated from differentparts of the world based on the viral coat protein sequence.Archives of Virology, 143, 1093-1107Tsuneyoshi T, Matsumi T, Natsuaki K T, Sumi S. 1998b.Nucleotide sequence analysis of virus isolates indicatesthe presence of three potyvirus species in Allium plants.Archives of Virology, 143, 97-113Uga H, Tsuda S. 2005. A one-step reverse transcriptionpolymerasechain reaction system for the simultaneousdetection and identification of multiple topovirus infections.Phytopathology, 95, 166-171Van der Vlugt R A A, Steffens P, Cuperus C, Barg E, LesemannD E, Bos L, Vetten H J. 1999. Further evidence thatShallot yellow stripe virus (SYSV) is a distinct potyvirusand reidentification of Welsh Onion yellow stripe virus as aSYSV strain. Phytopathology, 89, 148-155Vunsh R, Rosner A E, Stein A. 1990. The use of the polymerasechain reaction (PCR) for the detection of Bean yellowmosaic virus in gladiolus. Annals of Applied Biology, 117,561-569Walkey D G A, Antil D N. 1989. Agronomic evaluation of virusfreeand virus infected garlic (Allium sativum L.). Journal ofHorticultural Science, 64, 53-60Wei Y, Wei C, Yang Y, Hua J. 2012. Cloning and phylogeneticanalysis of six allexivirus CP genes from China isolates.Chinese Journal of Bioinformatics, 10, 79-82 (in Chinese)Yamashita K, Sakai J, Hanada K. 1996. Characterization of anew virus from garlic (Allium sativum L.), Garlic mite-bornemosaic virus. Annals of the Phytopathologicial Society ofJapan, 62, 483-489Zhang W, Bai Y, Shen Y, Gao Y, Fan G, Geng H, Meng X.2010. Study on pathogen identification and virus diseaseinvestigation of garlic virus in Heilongjiang Province. Journalof Northeast Agricultural University, 41, 21-26 (in Chinese) |
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