Molecular identification of potato virus disease by RT-PCR

doi:10.6048/j.issn.1001-4330.2023.12.019

Abstract

Abstract:

【Objective】To study the types of virus diseases in potato in Xinjiang, China, in order to clarify the main virus pathogens of potato in this region and provide scientific basis for the scientific prevention China and control of potato virus diseases.【Methods】In 2021, 87 suspected susceptible potato virus samples were collected from Urumqi, Jimsar, Qitai, Balikun and Zepu counties in the main producing areas of Xinjiang, China,and RT-PCR was performed on the samples.CP genes of positive samples were cloned and sequenced, and homology, geographical origin and phylogenetic analysis were completed.【Results】Potato virus Y (PVY), Potato virus S (PVS) and Potato leaf roll virus (PLRV) were detected in 87 samples.The detection rates were 100%, 37.93% and 6.89%, respectively, with the highest detection rate of PVY, and no Potato virus X (PVX) and Potato virus M (PVM) were detected, indicating that PVY was the main virus pathogen harming Potato samples in this region.Cloning and sequence alignment of PVY positive disease samples were performed.The consistency of CP gene sequences of 5 samples with isolates from China(Fujian, Shandong, Jiangsu) and Poland reached 100%, and the consistency of CP gene sequences with isolates from other countries reached more than 99.04%.【Conclusion】Potatoes in Xinjiang,China main producing areas were mainly infected by one or more viruses of PVY, PVS and PLRV, among which PVY was the most serious pathogen.

Key words: potato; virus disease; molecular detection; phylogeny

摘要：

【目的】研究中国新疆马铃薯主产区的病毒病发病种类,分析该区域马铃薯主要病毒病原,为马铃薯病毒病科学防治提供科学依据。【方法】2021年在我国新疆马铃薯主产区乌鲁木齐县、吉木萨尔县、奇台县、巴里坤县、泽普县采集马铃薯病毒病疑似感病样本87份,并对样本进行RT-PCR检测,对于阳性样本的CP基因进行克隆及测序,完成同源性、地理来源和系统进化分析。【结果】从87份样本中检测到马铃薯Y病毒(Potato virus Y,PVY)、马铃薯S病毒(Potato virus S,PVS)、马铃薯卷叶病毒(Potato leaf roll virus,PLRV),检出率分别为100%、37.93%、6.89%,以PVY检出率最高,未检出马铃薯X病毒(Potato virus X,PVX)、马铃薯M病毒(Potato virus M,PVM),PVY是危害该区域马铃薯样本的主要病毒病原。试验对PVY阳性带病样本进行克隆与序列比对,其中5份样本CP基因序列与中国(福建、山东、江苏)及波兰等地分离物一致性达到100%,与其他国家的分离物的一致性达到99.04%以上。【结论】我国新疆马铃薯主产区主要由PVY、PVS、PLRV的一种或多种病毒所侵染,其中PVY病原较严重。

关键词: 马铃薯, 病毒病, 分子检测, 系统进化

CLC Number:

S435.32

YANG Ruwei, LIU Yi, LI Jiangtao, Gulimila Rehemutula, LUO Zhengqian, XU Linli, SHEN Hongfei. Molecular identification of potato virus disease by RT-PCR[J]. Xinjiang Agricultural Sciences, 2023, 60(12): 3032-3040.

杨茹薇, 刘易, 李江涛, 古丽米拉·热合木土拉, 罗正乾, 徐琳黎, 沈洪飞. 马铃薯病毒病病原RT-PCR分子鉴定[J]. 新疆农业科学, 2023, 60(12): 3032-3040.

Figures/Tables 11

References 26

[1]	SALAZARL. F. 马铃薯病毒及其防治[M]. 阎文昭. 北京: 中国农业科学技术出版社, 2000.
	SALAZARL. F. Potato virus and its control[M]. YAN Wenzhao, Trans. Beijing: China Agricultural Science and Technology Press, 2000.
[2]	吴丽萍, 王蒂, 司怀军, 等. 马铃薯S病毒的RT-PCR检测[M]. 中国马铃薯, 2006, 20(4):200-203.
	WU Liping, Wang Di, SI Huaijun, et al. Detection of potato S virus by RT-PCR[M]. Chinese potato, 2006, 20(4):200-203.
[3]	肖雅, 何长征, 聂先舟, 等. 马铃薯病毒病防治策略[J]. 中国马铃薯, 2008,(2):106-110.
	XIAO Ya, HE Changzheng, NIE Xianzhou, et al. Control strategy of potato virus disease[J]. Chinese Potato, 2008,(2):106-110.
[4]	王子民, 时家宁, 宫海建. 马铃薯病毒病的发生及防治[J]. 现代农业科学, 2009,(7):118.
	WANG Zimin, SHI Jianing, GONG Haijian. Occurrence and control of potato virus disease[J]. Modern Agricultural Sciences, 2009,(7):118.
[5]	张华鹏, 张剑锋, 刘俊莹. 马铃薯上PVY、PVS、PLRV的三重RT-PCR检测[J]. 华北农学报, 2011, 26(5):40-45. DOI
	ZHANG Huapeng, ZHANG Jianfeng, LIU Junying. Triplex RT-PCR Detection of PVY,PVS and PLRV in Potato.[J]. North China Journal of Agriculture, 2011, 26(5):40-45.
[6]	ZANG F H, Konstantinos G, Fan X Z, et al. Real time monitoring of macromolecular biosensing probe selfassembly and on chip ELISA using impedimetric microsensors[J]. Biosensors and Bioelectronics, 2016, 81:401-407. DOI URL
[7]	FENG J, ZENG R, Chen J. Accurate and efficient data processing for quantitative real -time PCR using a tripartite plant virus as a model[J]. BioTechniques, 2008, 44(7) :901-912. DOI URL
[8]	聂峰杰, 詹红, 张丽, 等. RT-PCR技术对宁夏马铃薯脱毒种薯病毒检测的研究[J]. 植物保护, 2016, 42(5):188-193.
	NIE Fengjie, ZHAN Hong, ZHANG Li, et al. Study on detection of potato virus in Ningxia potato virus-free seed by RT-PCR[J]. Plant protection, 2016, 42(5):188-193.
[9]	沈林林, 邹文超, 高芳銮, 等. 采用多基因联合方法鉴定福建长乐和福清产区马铃薯Y病毒株系组成[J]. 中国农业科学, 2016, 49(20):3918-3926. DOI
	SHEN Linlin, ZOU Wenchao, GAO Fangluan, et al. Identification of potato virus Y strains by multi-gene combination method in Changle and Fuqing areas of Fujian Province[J]. Chinese Agricultural Sciences, 2016, 49(20):3918-3926.
[10]	杨小龙, 陈细红, 蔡伟, 等. 福建省福清地区马铃薯病毒病病原的分子检测[J]. 植物保护, 2019, 45(3):201-205.
	Yang Xiaolong, Chen Xihong, CAI Wei, et al. Molecular detection of potato virus pathogens in Fuqing area of Fujian province[J]. Plant protection, 2019, 45(3):201-205.
[11]	姜立慧. 四川省6种主要马铃薯病毒的发生及其多重RT-PCR检测体系的建立[D]. 雅安: 四川农业大学, 2016.
	JIANG Lihui. Occurrence of six major potato viruses in Sichuan Province and establishment of a multiplex RT-PCR detection system[D]. Ya'an: Sichuan Agricultural University, 2016.
[12]	陈兆贵, 叶新友, 候红因, 等. 马铃薯卷叶病毒实时荧光定量PCR检测技术研究[J]. 湖南农业科学, 2018,(9):9-12.
	CHEN Zhaogui, YE Xinyou, HOU Hongyin, et al. Detection of potato leaf curl virus by real-time PCR[J]. Hunan Agricultural Sciences, 2018, (9):9-12.
[13]	邱彩玲, 范国权, 申宇, 等. 马铃薯纺锤块茎病毒RT-qPCR检测技术体系的建立[J]. 作物杂志, 2020,(3):79-84.
	QIU Cailing, FAN Guoquan, SHEN Yu, et al. Establishment of RT-qPCR for detection of potato spindle tuber virus[J]. Crops, 2020,(3):79-84.
[14]	韩磊. 马铃薯病毒快速检测体系的建立及马铃薯BRP1基因dsRNA的体外表达[D]. 2016.
	HAN Lei. Establishment of a rapid detection system for potato virus and expression of potato BRP1 gene dsRNA in vitro[D]. 2016.
[15]	程胜群, 吕文河, 高艳玲, 等. 马铃薯S病毒RT-qPCR通用检测体系的建立与应用[J]. 华北农学报, 2020, 35(6):187-194. DOI
	CHENG Shengqun, LYU Wenhe, GAO Yanling, et al. Establishment and application of RT-qPCR system for detection of potato S virus[J]. North China Journal of Agricultural Sciences, 2020, 35 (6):187-194.
[16]	刘娴. 莲藕中一种马铃薯Y病毒属病毒的鉴定与全序列分析[D]. 扬州: 扬州大学, 2018.
	LIU Xian. Identification and complete sequence analysis of a potato virus of genus Y in lotus root[D]. Yangzhou: Yangzhou University, 2018.
[17]	尚慧, 韩树鑫, 高艳玲, 等. 利用RT-qPCR技术对PVY重组株系的鉴定[J]. 西南农业学报, 2018, 31(2):238-244.
	SHANG Hui, HAN Shuxin, GAO Yanling, et al. Identification of PVY recombinant lines by RT-qPCR[J]. Southwest China journal of agricultural sciences, 2018, 31(2):238-244.
[18]	秦亚南. 呼和浩特周边地区马铃薯病毒病的田间调查及分子鉴定[D]. 2018.
	Qin Yanan. Field investigation and molecular identification of potato virus diseases in surrounding areas of Hohhot[D]. 2018.
[19]	孙晓军, 周婷婷, 玉山江·麦麦提, 等. 设施番茄病毒病病原鉴定[J]. 新疆农业科学, 2021, 58(1):99-106. DOI
	SUN Xiaojun, ZHOU Tingting, Yushanjiang Maimaiti, et al. Pathogen identification of tomato virus disease in facilities[J]. Xinjiang Agricultural Sciences, 2021, 58(1):99-106. DOI
[20]	刘卫平. 马铃薯类病毒与马铃薯Y病毒的互作及其对马铃薯产量的影响[J]. 黑龙江八一农垦大学学报, 2016, 19(1):40-43.
	LIU Weiping. Interaction between potato-like and potato Y viruses and its effect on potato yield[J]. Journal of Heilongjiang Bayi Agricultural Reclamation University, 2016, 19(1):40-43.
[21]	刘卫平. 黑龙江省马铃薯脱毒种薯繁育发展现状与对策研究[D]. 北京: 中国农业科学院, 2013.
	LIU Weiping. Research on the current situation and countermeasures for the development of potato detoxification seed potato breeding in Heilongjiang Province[D]. Beijing: Chinese Academy of Agricultural Sciences, 2013.
[22]	胡新喜, 雷艳, 何长征, 等. 湖南省马铃薯主产区马铃薯病毒种类及流行分析[J]. 中国马铃薯, 2012, 54(6):358-361.
	HU Xinxi, LEI Yan, HE Changzheng, et al. Analysis of potato virus types and epidemiology in main potato producing areas of Hunan province[J]. Chinese potato, 2012, 54(6):358-361.
[23]	范国权, 白艳菊, 高艳玲, 等. 我国马铃薯主产区病毒病发生情况调查[J]. 黑龙江农业科学, 2014, 11(3):68-72.
	FAN Guoquan, BAI Yanju, GAO Yanling, et al. Survey on the occurrence of virus diseases in main potato producing areas in China[J]. Heilongjiang Agricultural Science, 2014, 11(3):68-72.
[24]	Wang A, Zhou X,Ed. Diagnosis of plant viruses using next-generation sequencing and metagenomic analysis.[J]. In Current Research Topics in Plant Virology. 2016,(3):323-335.
[25]	Jones S., Baizan-Edge A., MacFarlane S., et al. Viral diagnostics in plants using next generation sequencing: Computational analysis in practice.[J]. Front. Plant Sci. 2017,(10):1770.
[26]	Bhat A.I., Rao G.P. Next-generation sequencing for diagnosis of virus[J]. In Characterization of Plant Viruses. 2020,(10):389-395.

序号 Number	采样地点 Sampling locations	采样时间 Sampling time	样本数(份) Number of samples (copies)
1	乌鲁木齐市化肥厂	7月23日	4
2	奇台县总场、二分场7队	6月29日	40
3	巴里坤县大河镇、二十里、南元子村	7月10~11日	20
4	吉木萨尔县新地乡	7月16日	15
5	泽普县波斯喀木乡13村	6月8日	8

序号 Number	采样地点 Sampling locations	采样时间 Sampling time	样本数(份) Number of samples (copies)
1	乌鲁木齐市化肥厂	7月23日	4
2	奇台县总场、二分场7队	6月29日	40
3	巴里坤县大河镇、二十里、南元子村	7月10~11日	20
4	吉木萨尔县新地乡	7月16日	15
5	泽普县波斯喀木乡13村	6月8日	8

病毒种类 Virus type	序列(5'-3') Sequence(5'-3')	片段大小 Fragment size (bp)	Tm (℃)
PVX	F:TTCGACTTCTTCAATGGAGTC	711	49
	R:TCCAGTGATACGACCTCG
PVY	F:TGAAAATGGAACCTCGCC	801	54
	R:AATGTGCCATGATTTGCC
PVS	F;TCGTTGGAATTACATGCTG	885	57
	R:ATCAAATGTGTCAAAGCGG
PVM	F:ACATCTGAGGACATGATGCGC	520	52
	R:TGAGCTCGGGACCATTCATAC
PLRV	F:AAGAAGGCAATCCCTTCG	627	57
	R:ATGTCTCGCTTGAGCCTC

病毒种类 Virus type	序列(5'-3') Sequence(5'-3')	片段大小 Fragment size (bp)	Tm (℃)
PVX	F:TTCGACTTCTTCAATGGAGTC	711	49
	R:TCCAGTGATACGACCTCG
PVY	F:TGAAAATGGAACCTCGCC	801	54
	R:AATGTGCCATGATTTGCC
PVS	F;TCGTTGGAATTACATGCTG	885	57
	R:ATCAAATGTGTCAAAGCGG
PVM	F:ACATCTGAGGACATGATGCGC	520	52
	R:TGAGCTCGGGACCATTCATAC
PLRV	F:AAGAAGGCAATCCCTTCG	627	57
	R:ATGTCTCGCTTGAGCCTC

样本 Sample	检测数量 Detection The number	PVY 检测率 PVY Detection rate (%)	PVS 检测率% PVS Detection rate (%)	PLRV 检测率% PLRV Detection rate (%)	阳性样本率 Positive sample rate (%)
中国新疆乌鲁木齐县 Urumqi county,Xinjiang,China	4	4/100	0	0	4/100
中国新疆奇台县 Qitai county,Xinjiang,China	40	40/100	18/45	6/15	40/100
中国新疆巴里坤县 Balikun county,Xinjiang,China	20	20/100	0	0	20/100
中国新疆吉木萨尔县 Jimsar county,Xinjiang,China	15	15/100	7/46.67	0	15/100
中国新疆泽普县 Zepu county,Xinjiang,China	8	8/100	8/100	0	8/100
总计 Total	87	87/100	33/37.93	6/6.89	87/100