Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (1): 90-102.doi: 10.3864/j.issn.0578-1752.2016.01.008

• PLANT PROTECTION • Previous Articles     Next Articles

Development of Detection Method for Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) Through Fluorescence Quantitative RT-PCR

LU Hui-xiang, LÜ Chang-wen, WU Zheng-dan, LUO Kai, YIN Wang, YANG Hang, WANG Ji-chun, ZHANG Kai   

  1. College of Agronomy and Biotechnology, Southwest University, Chongqing 400716
  • Received:2015-07-15 Online:2016-01-01 Published:2016-01-01

Abstract: 【Objective】 Sweet potato virus disease (SPVD), which could cause more than 90% of yield loss, is one of the most harmful diseases of the sweet potato. The objective of this study is to establish a rapid detection method of SPVD, provide a useful tool for the early warning of SPVD and epidemiological investigation, based on assay of co-infection of Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV).【Method】 The leaves of sweet potato cultivars/lines with typical SPVD symptoms were collected, and the co-infection of SPFMV and SPCSV were tested by typical symptoms analysis and Nitrocellulose Membrane Enzyme-Linked Immunosorbent Assay (NCM-ELISA). The partial genes encoding coat protein (CP) of SPFMV and heat shock protein70 (HSP70) of SPCSV were cloned from the diseased leaves. Fluorescence quantitative PCR (qRT-PCR) was used to detect SPFMV CP and SPCSV HSP70 by using primers designed according to the conservative regions, and by using sweet potato ubiquitin (UBI) and histone (H2B) as the reference genes. The rapid and accurate protocol for SPFMV and SPCSV detection were established bycomparison with the symptoms and virus detection results by NCM-ELISA, and the detection results were also compared among leaves collected from different sweet potato cultivars/lines, among different plants collected from the same cultivar/line, and among top young leaves and mature leaves in the middle collected from the same plant. 【Result】 Symptoms analysis suggested that infected leaves from different cultivars/lines might exhibit different typical symptoms, and the symptoms of infected leaves from different plants of the same sweet potato cultivar/line were similar but exhibited differentiation. Cluster analysis showed that there were at least two types of SPFMV strains (EA and RC) and one type of SPCSV strain (WA) existing in the tested sweet potato leaves. Among the 15 tested cultivars/lines, 10 cultivars/lines were detected to be co-infected by SPFMV and SPCSV by using NCM-ELISA detection and qRT-PCR method. The results obtained from qRT-PCR detection corresponded to that from NCM-ELISA testing, indicating that the qRT-PCR detection method developed in this study could accurately reflect the co-infections degree of SPFMVand SPCSV in the sweet potato plants. Differential transcriptional levels of SPFMV CP andSPCSV HSP70 were detected in diseased leaves from different cultivars/lines or different plants of the same cultivar/line, and even in top young leaves and mature leaves in the middle from the same plant by using qRT-PCR. The detected transcriptional levels of SPFMV CP in the tested diseased leaves were higher than and were 3-556 times of that ofSPCSV HSP70. In addition, qRT-PCR showed a transcriptional level of SPCSV HSP70 in two of four cultivars/lines which were detected to be only infected by SPFMV but not infected by SPCSV by NCM-ELISA detection, indicating the present qRT-PCR detection method was more sensitive and accurate when compared to the NCM-ELISA method. 【Conclusion】 The appropriate PCR primers for qRT-PCR detection of SPVD were selected and a rapid method for SPVD detection was developed, which could provide a useful tool for the monitoring and early warning of SPVD. The characteristic of co-infection of two viruses and differentiation in typical symptoms among different cultivars/lines should be comprehensively analyzed and targeting approach should be concerned when a new monitoring or warning system of SPVD was established.

Key words: Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), sweet potato virus disease (SPVD), fluorescence quantitative RT-PCR, NCM-ELISA

[1]    Untiveros M, Fuentes S, Kreuze J. Molecular variability of sweet potato feathery mottle virus and other polyviruses infecting sweet potato in Peru. Archives of Virology, 2008, 153: 473-483.
[2]    Yamasaki S, Sakai J, Fuji S, Kamisoyama S, Emoto K, Ohshima K, Hanada K. Comparisons among isolates of Sweet potato feathery mottle virus using complete genomic RNA sequences.Archives of Virology, 2010, 155: 795-800.
[3]    Njeru R W, Mburu M W K, Cheramgoi E, Gibson R W, Kiburi Z M, Obudho E, Yobera D. Studies on the physiological effects of viruses on sweet potato yield in Kenya. Annals of Applied Biology, 2004, 145: 71-76.
[4]    Gibson R W, Mpembe I, Alicai T,Carey E E, Mwanga R O M, Seal S E, Vetten H J. Symptoms, aetiology and serological analysis of sweet potato virus disease in Uganda. Plant Pathology, 1998, 47: 95-102.
[5]    Melissa M, Cohen J, Loebenstein J. Effects of Sweet potato feathery mottle virus and Sweet potato sunken vein virus on sweet potato yields and rates of reinfection of virus-free planting material in Israel. Phytoparasitica, 1996, 24(3): 189-193.
[6]    Gutiérrez D L, Fuentes S, Salazar L F. Sweet potato virus disease (SPVD): Distribution, incidence, and effect on sweet potato yield in Peru. Plant Disease, 2003, 87(3): 297-302.
[7]    Mukasa S B, Rubaihayo P R, Valkonen J P T. Incidence of viruses and viruslike diseases of sweet potato in Uganda. Plant Disease, 2003, 87(4): 329-335.
[8]    张振臣, 乔奇, 秦艳红, 张德胜, 田雨婷. 中国发现由甘薯褪绿矮化病毒和甘薯羽状斑驳病毒协生共侵染引起的甘薯病毒病害.植物病理学报, 2012, 42(3): 328-333.
Zhang Z C, Qiao Q, Qin Y H, Zhang D S, Tian Y T. First evidence for occurrence of sweet potato virus disease (SPVD) caused by dual infection of Sweet potato feathery mottle virus and Sweet potato chlorotic stunt virus in China. Acta Phytopathologica Sinica, 2012, 42(3): 328-333. (in Chinese)
[9]    张盼, 兰新芝, 乔奇, 张德胜, 秦艳红, 田雨婷, 王爽, 张振臣. 甘薯病毒病害(SPVD)的多重RT-PCR检测方法及其应用. 植物保护, 2013, 39(2): 86-90.
Zhang P, Lan X Z, Qiao Q, Zhang D S, Qin Y H, Tian Y T, Wang S, Zhang Z C. Development and application of a multiplex RT-PCR detection method for sweet potato virus disease (SPVD). Plant Protection, 2013, 39(2): 86-90. (in Chinese)
[10]   王丽, 王振东, 乔奇, 秦艳红, 张德胜, 田雨婷, 王爽, 张立军, 张振臣. 甘薯羽状斑驳病毒实时荧光定量PCR检测方法的建立. 沈阳农业大学学报, 2013, 44(2): 129-135.
Wang L, Wang Z D, Qiao Q, Qin Y H, Zhang D S, Tian Y T, Wang S, Zhang L J, Zhang Z C. Development of real-time fluorescent quantitative PCR assay for detection of Sweet potato feathery mottle virus. Journal of Shenyang Agricultural University, 2013, 44(2): 129-135. (in Chinese)
[11]   王丽, 王振东, 乔奇, 秦艳红, 张德胜, 田雨婷, 王爽, 张立军, 张振臣. 甘薯褪绿矮化病毒西非株系实时荧光定量PCR检测方法的建立及应用. 植物病理学报, 2014, 44(5): 461-468.
Wang L, Wang Z D, Qiao Q, Qin Y H, Zhang D S, Tian Y T, Wang S, Zhang L J, Zhang Z C. Development and application of a real-time PCR method for detection of west African strain of Sweet potato chlorotic stunt virus. Acta Phytopathologica Sinica, 2014, 44(5): 461-468. (in Chinese)
[12]   IsHak J A, Kreuze J F, Johansson A, Mukasa S B, Tairo F, Abo El-Abbas F M, Valkonen J P T. Some molecular characteristics of three viruses from SPVD-affected sweet potato plants in Egypt. Archives of Virology, 2003, 148: 2449-2460.
[13]   Untiveros M, Quispe D, Kreuze J. Analysis of complete genomic sequences of isolates of the Sweet potato feathery mottle virus strains C and EA: molecular evidence for two distinct potyvirus species and two P1 protein domains. Archives of Virology, 2010, 155: 2059-2063.
[14]   Gibson R W, Aritua V. The perspective of Sweetpotato chlorotic stunt virus in sweetpotato production in Africa: a review.African Crop Science Journal, 2002, 10(4): 281-310.
[15]   Tairo F, Mukasa S B, Jones R A, Kullaya A, Rubaihayo P R, Valkonen J P. Unravelling the genetic diversity of the three main viruses involved in Sweet potato virus disease (SPVD), and its practical implications. Molecular Plant Pathology, 2005, 6(2): 199-211.
[16]   Karyeija R F, Kreuze J F, Gibson R W, Valkonen J P. Synergistic interactions of a potyvirus and a phloem-limited crinivirus in sweet potato plants. Virology, 2000, 269: 26-36.
[17]   马丽, 张春庆, 周玉亮. 甘薯病毒病检测技术研究进展. 中国农学通报, 2005, 21(2): 88-91.
Ma L, Zhang C Q, Zhou Y L. Progress of study on sweet potato viruses detection technique. Chinese Agricultural Science Bulletin, 2005, 21(2): 88-91. (in Chinese)
[18]   Cali B B, Moyer J W. Purification, serology and particle morphology of two russet crack strains of Sweet potato feathery mottle virus. Phytopathology, 1981, 71(3): 302-305.
[19]   Park S C, Kim Y H, Ji C Y, Park S, Jeong J C, Lee H S, Kwak S S. Stable internal reference genes for the normalization of real-time PCR in different sweetpotato cultivars subjected to abiotic stress conditions. PLoS ONE, 2012, 7(12): e51502.
[20]   Bustin S A, Benes V, Garson J A, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl M W, Shipley G L, Vandesompele J, Wittwer C T. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 2009, 55(4): 611-622.
[21]   Taylor S, Wakem M, Dijkman G, Alsarraj M, Nguyen M. A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines. Methods, 2010, 50: S1-S5.
[22]   Kokkinos C D, Clark C A. Real-time PCR assays for detection and quantification of sweetpotato viruses. Plant Disease, 2006, 90(6): 783-788.
[23]   Mcgregor C, Miano D, Bonte D L, Hoy M, Clark C. The effect of the sequence of infection of the causal agents of sweet potato virus disease on symptom severity and individual virus titres in sweet potato cv. Beauregard. Journal of Phytopathology, 2009, 157: 514-517.
[24]   陈锴, 姚华伟, 王长江, 徐璐, 范学政, 赵启祖, 邹兴启, 朱元 源, 赵燕, 杨光友, 王琴. 荧光定量PCR作为猪瘟兔化弱毒疫苗效价检验替代方法的研究与应用. 中国农业科学, 2013, 46(1): 162-169.
Chen K, Yao H W, Wang C J, Xu L, Fan X Z, Zhao Q Z, Zou X Q, Zhu Y Y, Zhao Y, Yang G Y, Wang Q. Fluorescent quantitative PCR as an alternative method for efficacy testing of lapinized hog cholera virus. Scientia Agricultura Sinica, 2013, 46(1): 162-169. (in Chinese)
[25]   Eun A J, Seoh M, Wong S. Simultaneous quantitation of two orchid viruses by the TaqMan real-time RT-PCR. Journal of Virological Methods, 2000, 87: 151-160.
[26]   于春梅, 刁有祥, 唐熠, 崔京腾, 高绪慧, 张颖, 鞠小军, 武利利. 坦布苏病毒荧光定量RT-PCR 方法的建立. 中国农业科学, 2012, 45(21): 4492-4500.
Yu C M, Diao Y X, Tang Y, Cui J T, Gao X H, Zhang Y, Ju X J, Wu L L. Fluorescence quantitative RT-PCR assay for detection of Tembusu virus. Scientia Agricultura Sinica, 2012, 45(21): 4492-4500. (in Chinese)
[27]   李玲娣, 周常勇, 李中安, 田晓, 王永江, 唐科志, 周彦, 刘金香. 褐色橘蚜中柑橘衰退病毒实时荧光定量RT-PCR检测方法的建立与应用. 中国农业科学, 2013, 46(3): 525-533.
Li L D, Zhou C Y, Li Z A, Tian X, Wang Y J, Tang K Z, Zhou Y, Liu J X. Development and application of a real-time RT-PCR approach for quantification of CTV in Toxoptera citricida. Scientia Agricultura Sinica, 2013, 46(3): 525-533. (in Chinese)
[28]   Roberts C A, Dietzgen R G, Heelan L A, Maclean D J. Real-time RT-PCR fluorescent detection of Tomato spotted wilt virus. Journal of Virological Methods, 2000, 88: 1-8.
[29]   Valasek M A, Repa J J. The power of real-time PCR. Advances in Physiology Education, 2005, 29: 151-159.
[30]   Giulistta A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C. An overview of real-time quantitative PCR: application to quantify cytokine gene expression. Methods, 2001, 25: 386-401.
[31]   Sakai J, Mori M, Morishita T, Tanaka M, Hanada K, Usugi T, Nishiguchi M. Complete nucleotide sequence and genome organization of Sweet potato feathery mottle virus (S strain) genomic RNA: the large coding region of the P1 gene. Archives of Virology, 1997, 142: 1553-1562.
[32]   Kreuze J F, Savenkov E I, Valkonen J P. Complete genome sequence and analyses of the subgenomic RNAs of Sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus. Journal of Virology, 2002, 76(18): 9260-9270.
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