Establishment of Duplex RT-PCR Detection System for PlAMV and LMoV in Lily (Lilium spp.)
WANG Li-Hua1, YANG Xiu-Mei1,*, HE Xiao-Qin2, ZHANG Yi-Ping1, DUAN Qing1, YANG Shu-Qi1, SU Yan1,*
1 Flower Research Institute/Yunnan Key Laboratory for Flower Breeding/National Engineering Research Center for Ornamental Horticulture,Yunnan Academy of Agriculture Science, Kunming 650205, China; 2 Qingyun subdistrict office Panlong District Government, Kunming 650051, China
Abstract：The plantago asiatica mosaic virus (PlAMV) and the Lily mottle virus (LMoV) are currently the two most serious viral pathogens affecting the lily (Lilium spp.) industry in China. The aim of this study is to develop a rapid and efficient detection system for these 2 viruses. Firstly, total RNA was extracted from diseased lily plants, and cDNA was obtained through reverse transcription. Subsequently, a dual-synchronous detection system was optimised using cDNA as a template, and the specificity and sensitivity of the detection system were evaluated. The results demonstrated that the established dual-synchronous detection system amplified specific segments of 910 and 521 bp for PlAMV and LMoV, respectively, with no amplification for other common viruses, indicating strong detection specificity. The lowest detection limits for PlAMV and LMoV were 967.5 fg and 59.0 pg, respectively, which were 50 times and 5 times lower than the lowest detection limits of single-gene RT-PCR detection (PlAMV 19.35 fg, LMoV 11.8 pg). Sequence analysis of the amplified products with sequencing segments from 19 PlAMV isolates and 21 LMoV isolates from different sources revealed similarities of 86.71% to 99.40% for PlAMV and 94.75% to 99.49% for LMoV. The dual-synchronous detection system was employed to examine 19 field samples, and the results indicated that 3 samples were positive for both PlAMV and LMoV, 2 samples were positive for PlAMV, 4 samples were positive for LMoV, and the remaining 10 samples were not detected, which was consistent with the results of gene chip detection. In conclusion, the detection system this study established is capable of detecting both PlAMV and LMoV viruses simultaneously, with reliable results and broad application prospects.
王丽花, 杨秀梅, 何晓琴, 张艺萍, 段青, 杨舒淇, 苏艳. 百合PlAMV和LMoV双重RT-PCR检测体系的建立[J]. 农业生物技术学报, 2025, 33(3): 670-679.
WANG Li-Hua, YANG Xiu-Mei, HE Xiao-Qin, ZHANG Yi-Ping, DUAN Qing, YANG Shu-Qi, SU Yan. Establishment of Duplex RT-PCR Detection System for PlAMV and LMoV in Lily (Lilium spp.). 农业生物技术学报, 2025, 33(3): 670-679.
[1] 丁元明, 王继华, 刘忠善, 等. 2004. 应用特异引物和简并引物检测百合斑驳病毒[J]. 植物检疫, 18(3): 134-137. (Ding Y M, Wang J H, Liu Z S, et al.2004. Detection of Lily mottle virus by RT-PCR using special primers and degenerate primers[J]. Plant Quarantine, 18(3): 134-137.) [2] 洪玲艳, 聂江力, 乔雅琦, 等. 2024. 兰州百合和沂水百合在天津地区引种表现[J]. 分子植物育种, https://link.cnki.net/urlid/46.1068.s.20240206.1156.004.(Hong L Y, Nie J L, Qiao Y Q, et al. 2024. Introduction performance of Lilium davidii var. willmottiae and Yishui edible Lilium brownie var. viridulum in Tianjin area[J]. Molecular Plant Breeding, https://link.cnki.net/urlid/46.1068.s.20240206.1156.004.) [3] 李鑫, 陈雅诗, 朱连, 等. 2017. 进境荷兰百合中车前草花叶病毒的分子鉴定及序列分析[J]. 植物病理学报, 47(2): 197-202. (Li X, Chen Y S, Zhu L, et al.2017. Molecular identification and sequence analysis of Plantago asiatica mosaic virus in lily from Netherlands[J]. Acta Phytopathologica Sinica, 47(2): 197-202.) [4] 李鑫, 张寅寅, 黑多尔, 等. 2016. 辽宁局全国首次在进境荷兰百合中检出亚洲车前草花叶病毒[J]. 植物检疫, 30(1): 8.(Li X, Zhang Y Y, Hei D E, et al.2016. Plantago asiatica mosaic virus was first detected in imported dutch lily by Liaoning Bureau in China[J]. Asiatic, Plant Quarantine, 30(1): 8.) [5] 李月月, 孙健, 谭冠林, 等. 2018. 百合斑驳病毒云南分离物全基因组序列分析及CP结构预测[J]. 植物病理学报, 48(2): 187-194. (Li Y Y, SUN J, TAN G L, et al.2018. Sequencing and analysis of the genome of Lily mottle virus Yunnan isolates and structure prediction of their coat proteins[J]. Acta Phytopathologica Sinica, 48(2): 187-194.) [6] 廖富荣, 林石明, 吴媛, 等. 2011.侵染百合的病毒种类及其检疫重要性[J]. 植物检疫, 25(1): 70-74. (Liao F R, Lin S M, Wu Y, et al.2011. The viruses infecting lily (Lilium spp.) and their phytosanitary significance[J]. Plant Quarantine, 25(1): 70-74.) [7] 刘博, 付海滨, 李鑫, 等. 2023. 车前草花叶病毒对我国百合产业的风险评估[J]. 中国植保导刊, 43(09): 102-105. (Liu B, Fu H B, Li X, et al.2023.Risk assessment of Plantago asiatica mosaic virus on lily industry in China[J]. China Plant Protection, 43(09): 102-105.) [8] 马骏, 冯黎霞, 赵立荣, 等. 2015. 植物检疫性病毒传毒介体的传毒特征及检疫防控策略[J]. 植物检疫, 29(1): 1-7. (Ma J, Feng L X, Zhao L R, et al.2015. Virus transmission characteristics and quarantine control strategies for plant quarantine virus vectors[J]. Plant Quarantine, 29(1): 1-7.) [9] Robe B L.2018. 荷兰进口百合中车前草花叶病毒的鉴定及分子特征研究[D]. 博士学位论文, 中国农业科学院, 导师: 李世访, pp. 1-87. (Robe B L.2018. Identification and molecular characterization of Plantago asiatica mosaic virus in lily bulbs imported from the Netherlands[D]. Thesis for Ph. D., Chinese Academy of Agricultural Sciences, Supervisor: Li S F, pp. 1-87.) [10] 沈淑琳. 1996. 百合病毒病及其检验[J]. 植物检疫, 10(4): 223-226. (Shen S L.1996. The virus disease of lily and its test[J]. Plant Quarantine, 10(4): 223-226.) [11] 宋蒙, 徐雷锋, 曹雨薇, 等. 2021. 百合中车前草花叶病毒的实时荧光定量PCR检测[J]. 园艺学报, 48(12): 2497-2505. (Song M, Xu L F, Cao Y W, et al.2021.Real-time fluorescence quantitative PCR detection of Plantago asiatica mosaic virus in lilies[J]. Acta Horticulturae Sinica,48(12): 2497-2505.) [12] 苏学思, 张玉宝, 王若愚, 等. 2021. 车前草花叶病毒衣壳蛋白的原核表达和多克隆抗体制备[J]. 浙江农业学报, 33(01): 104-111. (Su X S, Zhang Y B, Wang R Y, et al.2021. Prokaryotic expression of Plantago asiatica mosaic virus capsid protein and preparation of itspolyclonal antibody[J]. Acta Agriculturae Zhejiangensis, 33(01):104-111.) [13] 孙爱青, 王丽花, 张艺萍, 等. 2024. TaqMan探针双重实时荧光定量PCR法同步检测兰花中的建兰花叶病毒和齿兰环斑病毒[J]. 植物保护, 50(02): 219-227. (Sun A Q, Wang L H, Zhang Y P, et al.2024. Simultaneous detection of Cymbidium mosaic virus and Odontoglossum ringspot virus by TaqMan duplex real-time fluorescence quantitative PCR[J]. Plant Protection, 50(02): 219-227.) [14] 唐开学, 王继华, 王丽花, 等. 2007. 花卉植物病毒检测规程(NYT 1491-2007)[S]. 农业行业标准, 中国标准出版社. (Tang K X, Wang J H, Wang L H, et al. 2007. Ruler for viruses detection on floral plants (NY/T 1491-2007) [S]. Agricultural industry standard, Standards Press of China.) [15] 王继华, 王丽花, 丁元明, 等. 2005. 应用多重RT-PCR检测百合无症病毒和百合斑驳病毒[J]. 园艺学报, 32(02): 284-287. (Wang J H, Wang L H, Ding Y M, et al. 2005. Detection of Lily symptom less virus and Lily mottle virus by Multiplex RT-PCR[J]. Acta Horticulturae Sinica, (02): 284-287.) [16] 王丽花, 瞿素萍, 杨秀梅,等. 2007. 百合上黄瓜花叶病毒的一步RT-PCR检测[J]. 天津农学院学报, 14(04): 9-12. (Wang L H, Qu S P, Yang X M, et al.Detection of Cucumber mosaic virus on lily by one-step RT-PCR[J]. Journal of Tianjin Agricultural University, 14(04): 9-12.) [17] 吴青青, 胡小京, 崔嵬, 等. 2019. 百合车前草花叶病毒的脱毒组培技术[J]. 农技服务, 36(05): 41-43. (Wu Q Q, Hu X J, Cui W, et al.2019.Virus-free tissue culture technology for Plantago asiatica mosaic virus of lily[J]. Agricultural Technology Service, 36(05): 41-43.) [18] 徐品三, 宋炯, 张郑瑶. 2017. 百合斑驳病毒外壳蛋白基因原核表达、抗血清制备及其应用[J]. 植物保护学报, 44(4): 630-635. (Xu P S, Song J, Zhang Z Y.2017. Prokaryotic expression of coat protein gene of Lily mottle virus and preparation, application of its antiserum[J]. Journal of Plant Protection, 44(4): 630-635.) [19] 杨丹, 赵斌安, 许燕, 等. 2021. 百合病毒检测技术研究进展[J]. 生物学通报, 56(07): 1-3. (Yang D, Zhao B A, Xu Y, et al.2021. Research progress of lily virus detection technology[J]. Bulletin of Biology, 56(07): 1-3.) [20] Anonymous.2010. Plantago asiatica mosaic virus on Lilium spp.: Pest report-The Netherlands, Plant protection Service of the Netherlands[J/OL]. https://145.12.37.103/txmpub/files/?p_file_id=2001424. [21] Chin C C, Yu L J, Bi Y L.2013. Serological reagent preparation and improvement of serological method for thedetection of Plantago asiatica mosaic virus in lily[J]. Jorunal of Taiwan Agricultural Research, 62(3): 268-279. [22] Hammond J, Bampi D, Reinsel M D.2015. First report of Plantago Asiatica mosaic virus in imported Asiatic and oriental lilies (Lilium hybrids) in the United States[J]. Plant Disease, 99: 292. [23] Ki H R, Hye W P, Jang K C.2002. Characterization and sequence analysis of a lily isolate of Cucumber mosaic virus form lilium tsingtauense[J]. Plant Pathology Journal, 18(2): 85-92. [24] Kostin V D, Volkov Y G.1976. Some properties of the virus affecting Plantago asiatica L.[J]. Virusnye Bolezni Rastenij Dalnego Vostoka, 25: 205-210. [25] Lim S, Igori D, Zhao F, et al.2016. Molecular detection and characterization of a divergent isolate of Plantago Asiatica mosaic virus in Plantago asiatica[J]. Virus Disease, 27: 307-310. [26] Mauricio M A, Laura G, Elena V, et al.2017. Detection of Plantago asiatica mosaic virus in lily hybrid plants (Lilium spp.) in Costa Rica grown from imported bulbs[J]. Australasian Plant Disease, 12: 57. [27] Ozeki J, Takahashi S, Komatsu K, et al.2006. A single amino acidin the RNA-dependent RNA polymerase of Plantago asiatica mosaic virus contributes to systemic necrosis[J]. Archives of Virology, 151: 2067-2075. [28] Pájtli É, Eke S, Palkovics L.2015. First report of the Plantago asiaticamosaic virus (PlAMV) incidence on Lilium sp. in Hungary[J]. Plant Disease, 99: 1288. [29] Parrella G, Greco B, Pasqualini A, et al.2015. Plantago asiatica mosaic virus found in protected crops of lily hybrids in Southern Italy[J]. Plant Disease, 99: 1289. [30] Solovyev A G, Novikov V K, Merits A, et al.1994. Genome characterization and taxonomy of Plantago asiatica mosaic potexvirus[J]. Jouranl of General Virology, 75: 259-267. [31] Vidal A K, Camps R, Besoain X.2016. First report of necrotic streaking of Asiatic lilies caused by Plantago asiatica mosaic virus in Chile[J]. Plant Disease, 100: 1799. [32] Xu L F, Ming J, Yuan S X.2017. First report of Plantago asiatica mosaicvirus in lily hybrids in China[J]. Plant Disease, 101: 263.
