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| Identification of Four Viral Diseases in Melon (Cucumis melo) and Establishment of Multiplex Detection System |
| HAN Song-Lin*, LI Yao*, YANG Yu, WANG De-Fu, DU Jiang**, NIU Yan-Bing** |
| College of Life Sciences, Shanxi Agricultural University, Taigu 030801, China |
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Abstract Melon (Cucumis melo) is an annual trailing herbaceous plant that plays a significant role in the agricultural industry of the Taigu district in Shanxi province. In recent years, viral diseases have severely impacted melon production in this region. To identify the pathogens responsible for these viral diseases and to establish rapid detection techniques, this study collected symptomatic melon samples from Taigu district that exhibited spots, mosaic patterns, shrinkage, and necrosis. These samples were analyzed comprehensively using RT-PCR combined with bioinformatics. The results indicated that the presence of quadruple viral pathogens in melo specimens, specifically for: Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV), Cucurbit aphid-borne yellows virus (CABYV), and Watermelon mosaic virus (WMV). Mixed infections were found to be prevalent. Based on homology comparisons and phylogenetic analyses of the coat protein (CP) sequences of the viruses mentioned above, the results indicated that the amplified isolates obtained in this study CABYV isolate Fenghuang, CMV isolate Fengyun, CGMMV isolate Longquan, and WMV isolate Zhouquan exhibited sequence identities of over 99.5%, 98.0%, 99.6%, and 97.0%, respectively, when compared with corresponding virus sequences deposited in the GenBank database. Each isolate had a relatively close genetic relationship with the corresponding isolate of the same virus. In response to the phenomenon of complex infection of sweet melon virus diseases in the local area, a multiplex RT-PCR detection system was established to simultaneously detect CMV, CGMMV, CABYV and WMV. Multiple RT-PCR amplification was performed on materials with complex infection of 4 viruses, resulting in 4 specific bands of 1 106, 690, 521, and 400 bp, respectively. The multiplex RT-PCR detection system developed in this study allows for rapid identification of melon viral diseases and provides technical support for the monitoring and prevention of melon virus diseases.
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Received: 14 March 2025
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Corresponding Authors:
** Corresponding authors, dujiang20080803@163.com;niuyanbingbest@163.com
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| About author:: * Thses authors contributed equally to this work |
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[1] 白静科, 牛龙龙, 吴彦辉, 等. 2023. TMV、CMV和PVY三重荧光定量PCR同步检测方法的建立[J]. 中国烟草科学, 44(05): 55-61. (Bai J K, Niu L L, Wu Y H, et al.2023. Establishment of a triplex fluorescent quantitative PCR for simultaneous detection of TMV, CMV and PVY[J]. Chinese Tobacco Science, 2023, 44(05): 55-61.) [2] 陈永萱, 薛宝娣, 刘风权. 1988. A蛋白间接酶联免疫吸附法检测二种西瓜花叶病毒病(WMV-2和CMV)的研究[J]. 病毒学杂志, (04): 357-363. (Chen Y X, Xue B T, Liu F Q.1988. Research on two Watermelon mosaic virus (WMV-2 and CMV) aprotein detection indirect en zyme-linked immunosorbent assay[J]. Virilogica Sinica, (04): 357-363.) [3] 杜江, 马振男, 王晨燕, 等. 2024a. 利用小RNA深度测序技术鉴定西瓜病毒病病原[J]. 植物病理学报, 54(01): 69-81. (Du J, Ma Z N, Wang C Y, et al.2024a. Identification of the Citrullus ianatus infecting viruses by small RNA deep sequencing[J]. Acta Phytopathologica Sinica, 54(01): 69-81.) [4] 杜江, 马振男, 王德富, 等. 2024b. 基于小RNA深度测序和RT-PCR鉴定鹅绒藤花叶病的病毒病原[J]. 草业科学, 41(10): 2229-2239. (Du J, Ma Z N, Wang D F, et al.2024b. Small RNA sequencing and RT-PCR detection of viral pathogens infecting Cynanchum chinense[J]. Pratacultural Science, 41(10): 2229-2239.) [5] 冯丽婷, 张剑峰, 迟胜起. 2022. 甘薯4种RNA病毒多重RT-PCR检测方法的建立[J]. 华北农学报, 37(04): 206-211. (Feng L T, Zhang J F, Chi S Q.2022. Establish ment of a multiplex RT-PCR assay for detection of four sweet potato RNA viruses[J]. Acta Agriculturae Boreali Sinica, 37(04): 206-211.) [6] 付晶晶, 杨彩霞, 韩彤, 等. 2018. 西瓜花叶病毒辽宁臭椿分离物的鉴定与全序列分[J]. 江西农业大学学报, 40(01): 96-102. (Fu J J, Yang C X, Han T, et al.2018. Detection and complete viral sequence analysis of Watermelon mosaic virus on Ailanthus altissima in Liaoning[J]. Acta Agriculturae Universitatis Jiangxiensis, 40(01): 96-102.) [7] 高鹏, 刘识, 崔浩楠, 等. 2020. 甜瓜基因组学、功能基因定位及基因工程育种研究进展[J]. 园艺学报, 47(09): 1827-1844. (Gao P, Liu S, Cui H N, et al.2020. Research progress of melon genomics, functional gene mapping and genetic engineering[J]. Acta Horticulturae Sinica, 47(09): 1827-1844.) [8] 韩盛, 韩成贵, 玉山江·麦麦提, 等. 2016. 新疆吐鲁番地区三种甜瓜病毒病的发生与分子鉴定[J]. 新疆农业科学, 53(10): 1829-1842. (Han S, Han C G, Yujiangshan M M T, et al.2016. Occurrence and molecular identification of three kinds of melon virus diseases in Turpan area, Xinjiang[J]. Xinjiang Agricultural Sciences, 53(10): 1829-1842.) [9] 韩晓玉, 陈思宇, 李刚, 等. 2020. 河南省草莓病毒病病原的鉴定及5种草莓病毒多重PCR体系的建立[J]. 植物保护学报, 47(01): 219-220. (Han X Y, Chen S Y, Li G, et al.2020. Identification of the pathogens causing strawberry virus disease in Henan province and establishment of a multiplex PCR assay for five strawberry viruses[J]. Journal of Plant Protection, 47(01): 219-220.) [10] 黄爱军, 王莹, 丁敏, 等2019. 柑橘4种病毒多重PCR检测技术的建立及应用[J]. 园艺学报, 46(08): 1616-1622. (Huang A J, Wang Y, Ding M, et al.2019. Establishment and application of multiplex PCR rapid detection of four citrus viruses[J]. Acta Horticulturae Sinica, 46(08): 1616-1622.) [11] 李继洋, 杨渡, 韩盛, 等. 2015. 基于多重RT-PCR技术检测新疆甜瓜主栽区3种病毒病及其分布[J]. 西北农业学报, 24(08): 123-130. (Li J Y, Yang D, Han S, et al.2015. Detection and distribution of three viral diseases in Xinjiang melon region based on multiplex RT-PCR technique[J]. Acta Agriculturae Boreali-occidentalis Sinica, 24(08): 123-130.). [12] 李明骏, 廖萌, 蒋开蓉, 等. 2021. 五种玉米病毒多重PCR检测体系的建立[J]. 植物保护学报, 48(02): 465-466. (Li M J, Liao M, Jiang K R, et al.2021. Establishment of a multiplex PCR assay for detection of five maize-infecting viruses[J]. Journal of Plant Protection, 48(02): 465-466.) [13] 刘莉铭, 彭斌, 康保珊, 等. 2023. 西瓜花叶病毒西葫芦分离物的基因组及其侵染性克隆[J]. 中国瓜菜, 36(10): 16-22. (Liu L M, Peng B, Kang B S, et al.2023. The genome and infectious clone of Watermelon mosaic virus zucchini isolate[J]. China Cucurbits and Vegetables, 36(10): 16-22.) [14] 刘美, 解昆仑, 刘茜, 等. 2019. 宁夏回族自治区嫁接西瓜发生黄瓜绿斑驳花叶病毒病[J]. 中国瓜菜, 32(02): 7-10. (Liu M, Xie K L, Liu X, et al.2019. Occurence of Cucumber green mottle mosaic virus in grafted watermelon in Ningxia Hui Autonomous Region of China[J]. China Cucurbits and Vegetables, 32(02): 7-10.) [15] 鲁婧童, 孔祥凤, 李介文, 等. 2024. 北京地区四种百合病毒的检测及病毒序列分析[J]. 农业生物技术学报, 32(10): 2424-2436. (Lu J T, Kong X F, Li J W, et al.2024. Detection and viral sequence analysis of four lily-infecting viruses in Beijing[J]. Journal of Agricultural Biotechnology, 32(10): 2424-2436.) [16] 邱艳红, 王超楠, 朱水芳. 2017. 黄瓜花叶病毒致病性研究进展[J]. 生物技术通报, 33(09): 10-16. (Qiu Y H, Wang C N, Zhu S F.2017. Research advances on the pathogenicity of Cucumber mosaic virus[J]. Biotechnology Bulletin, 33(09): 10-16.) [17] 任春梅, 罗桂火, 朱云, 等. 2023. 山东寿光地区黄瓜上CGMMV的分布与危害[J]. 中国农学通报, 39(16): 96-100. (Ren C M, Luo G H, Zhu Y, et al.2023 Distribution and damage of CGMMV on cucumber in Shouguang of Shandong province[J]. Chinese Agricultural Science Bulletin, 39(16): 96-100.) [18] 邵旭鹏. 2021. 不同产地‘纳斯蜜’甜瓜香气相关基因及成分差异比较[D]. 硕士学位论文, 新疆农业大学, 导师: 刘峰娟. pp. 33-35. (Shao X P.2021. Comparison of differences in aroma-related genes and aroma components of 'Nasmi' melons from different producing areas[D]. Thesis for M.S., Xinjiang Agricultural University. Supervisor: Liu F J, pp. 33-35.) [19] 孙立新, 王晓君, 吴敬学, 等. 2023. 中国西瓜甜瓜生产区域布局变迁及驱动因素研究[J]. 中国农业资源与区划, 44(08): 42-51. (Sun L X, Wang X J, Wu J X, et al.2023. Research on regional distribution changes and driving factors of watermelon and melon production in China[J]. Chinese Journal of Agricultural Resources and Regional Planning, 44(08): 42-51.) [20] 王东, 麦麦提艾则孜·穆合塔尔, 刘艳全, 等. 2023. 新疆伽师县甜瓜病毒病种类鉴定[J]. 园艺学报, 50(08): 1793-1802. (Wang D, Maimaitiaizezi M, Liu Y Q, et al.2023. Identification of virus disease types of melon planting in Jiashi County of Xinjiang[J]. Acta Horticulturae Sinica, 50(08): 1793-1802.) [21] 王敏, 邱艳红, 古勤生, 等. 2023. 海南甜瓜的主要病毒病及其防控措施[J]. 中国瓜菜, 36(03): 15-20. (Wang M, Qiu Y H, Gu Q S, et al.2023b. Main virus diseases and the control measures of melon in Hainan province[J]. China Cucurbits and Vegetables, 36(03): 15-20.) [22] 王少立. 2018. 山东省辣椒病毒病病原的检测鉴定及BWYV和CABYV的序列分析[D]. 硕士学位论文, 山东农业大学, 导师: 竺晓平, pp. 45-49. (Wang S L.Detection and identification of the pathogen of pepper virus disease in Shandong province and the sequences analysis of BWYV and CABYV[D]. Thesis for M.S., Shandong Agricultural University, Supervisor: Zhu X P, pp. 45-49.) [23] 王威麟, 张昊, 于祥泉, 等. 2010. 侵染西瓜的5种病毒ZYMV、WMV、TMV、SqMV和CMV的多重RT-PCR检测体系的建立与检测应用[J]. 植物病理学报, 40(01): 27-32. (Wang W L, Zhang H, Yu X Q, et al.2010. Establishment and application of multiplex RT-PCR for simultaneous detection of five watermelon viruses ZYMV, WMV, TMV, SqMV and CMV[J]. Acta Phytopathologica Sinica, 40(01): 27-32.) [24] 魏宁生, 张满良, 吴云峰, 等. 1991. 新疆、甘肃甜瓜病毒病的鉴定及防治[J]. 植物保护学报, 18(01): 81-85. (Wei N S, Zhang M L, Wu Y F, et al.1991. Identification and control of muskmelon virus diseases in Xinjang and Gansu province of China[J]. Journal of Plant Protection, 18(01): 81-85.) [25] 文朝慧, 南志标. 2013. 甘肃省河西地区瓜类作物病毒病的病原鉴定[J]. 西北农林科技大学学报(自然科学版), 41(12): 131-137. (Wen C H, Nan Z B.2013. Detection of cucurbit virus diseases in Hexi region of Gansu province[J]. Journal of Northwest A&F University: Natural Science Edition, 41(12): 131-137.) [26] 吴洋, 刘莉铭, 彭斌, 等. 2017. 新疆、甘肃和河南部分地区甜瓜瓜类蚜传黄化病毒分子变异初步分析[J]. 园艺学报, 44(04): 777-783. (Wu Y, Liu L M, Peng B, et al.2017. Preliminary studies on molecular variation of Cucurbit aphid-borne yellows virus from Xinjiang, Gansu and Henan[J]. Acta Horticulturae Sinica, 44(04): 777-783.) [27] 张磊, 任婷璐, 刘艳, 等. 2023. 甜瓜病毒研究进展[J]. 中国果树, (04): 24-30. (Zhang L, Ren T L, Liu Y, et al. 2023. Research progress of melon virus[J]. China Fruits, (04): 24-30.) [28] 张建新, 吴云锋, 王睿, 等. 2007. 西瓜花叶病毒中国分离株全基因组核苷酸序列测定[J]. 病毒学报, 23(02): 153-156. (Zhang J X, Wu Y F, Wang R, et al.2007. Complete nucleotide sequence of Watermelon mosaic virus China isolate[J]. Chinese Journal of Virology, 23(02): 153-156.) [29] Herrera‐Vásquez J A, Córdoba‐sellés M C, Cebrián M C, et al.2010. Genetic diversity of Melon necrotic spot virus and Olpidium isolates from different origins[J]. Plant Pathology, 59: 240-251. [30] Lecoq H, Bourdin D, Wipfscheibel C, et al.1992. A new yellowing disease of cucurbits caused by a luteovirus, Cucurbit aphid‐borne yellows virus[J]. Plant Pathology, 41(6): 749-761. [31] Palukaitis P, Roossinck M J, Dietzgen R G, et al.1992. Cucumber mosaic virus[J]. Advances in Virus Research, 41: 281-348. [32] Park D, Kim H, Hahn Y.2017. Genome sequence of a distinct Watermelon mosaic virus identified from ginseng (Panax ginseng) transcriptome[J]. Acta Virologica, 61: 479-482. [33] Sun X, Wang Z, Gu Q, et al.2017. Transcriptome analysis of Cucumis sativus infected by Cucurbit chlorotic yellows virus[J]. Virology Journal, 14: 1-8. [34] Ugaki M, Tomiyama M, Kakutani T, et al.1991. The complete nucleotide sequence of Cucumber green mottle mosaic virus (SH strain) genomic RNA[J]. Journal of General Virology, 72: 1487-1495. |
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