枣树花叶病毒病的分子检测

doi:10.6048/j.issn.1001-4330.2022.03.015

新疆农业科学 ›› 2022, Vol. 59 ›› Issue (3): 657-662.DOI: 10.6048/j.issn.1001-4330.2022.03.015

• 植物保护·农产品分析检测 • 上一篇下一篇

枣树花叶病毒病的分子检测

白剑宇¹^,²(), 罗达¹^,², 刘正兴³, 李宏²()

1.新疆林业科学院经济林研究所,乌鲁木齐 830063
2.新疆林业科学院,乌鲁木齐 830063
3.阿克苏地区农业农村局,乌鲁木齐 834000

收稿日期:2021-07-09 出版日期:2022-03-20 发布日期:2022-03-28
通信作者: 李宏
作者简介:白剑宇(1977-),男,河北承德人,研究员,博士,研究方向为林业有害生物防控,(E-mail) bjyhao2010@sina.com
基金资助:
新疆维吾尔自治区自然科学基金“基于小RNA深度测序的新疆枣树病毒病病原精准鉴定及传毒介体初探”(2017D01A28)

Molecular Detection of Jujube Mosaic Virus

BAI Jianyu¹^,²(), LUO Da¹^,², LIU Zhengxing³, LI Hong²()

1. Research Institute of Economic Forestry,Xinjiang Academy of Forestry Sciences,Urumqi 830063, China
2. Xinjiang Academy of Forestry Sciences,Urumqi 830063, China
3. Aksu Agricultural and Rural Bureau, Aksu Xinjiang 83400, China

Received:2021-07-09 Online:2022-03-20 Published:2022-03-28
Correspondence author: LI Hong
Supported by:
Natural Science Foundation of Xinjiang Uygur Autonomous Region “Pathogen Accurate Identification and Primary Exploration of Transmission Routes of Jujube Virus Based on miRNA Deep Sequencing in Xinjiang”(2017D01A28)

摘要/Abstract

摘要：

【目的】建立枣树花叶病毒病的分子检测方法,追踪检测与分析枣树花叶病毒病田间侵染动态,研究病害防治的关键时间节点,为新疆枣树种植区枣树花叶病毒病的流行监测和早期防治,以及枣树花叶病毒病的高效防治奠定基础。【方法】在前期准确获取病毒全基因组序列的基础上,以病毒基因组序列RNA5为靶标设计序列特异性引物,通过RT-PCR技术进行引物特异性验证与灵敏度检测,优化反应体系,建立枣树花叶病毒病分子检测技术,追踪检测与分析枣树花叶病毒病田间侵染动态。【结果】该检测技术对枣树花叶病毒病检测的最低浓度为4.79 pg/μL,可在叶片显症之前检测到该病毒。5月下旬至6月上旬是预防枣树病毒病的关键时间节点。【结论】建立的枣树花叶病毒病检测技术能够快速准确的检测和鉴定病原,可应用于枣树花叶病田间侵染动态的追踪检测。

关键词: 枣树; 花叶病毒病; 分子检测; 侵染动态; 流行监测

Abstract:

【Objective】 This project aims to develop a molecular detection method for carrying out the detection and analysis of field infection dynamic of jujube mosaic virus, so that we could explore the optimal time, and lay the foundation for the efficient control of the virus. 【Methods】 On the basis of obtaining the complete genome sequence accurately, specific primers were designed with RNA5 genome sequence as template. The specificity and sensitivity of primers were verified byRT-PCR technology, and the molecular detection technology was established by optimizing the reaction system. And also, the technology was applied to detect the infection dynamics of jujube mosaic virus. 【Results】 Experimental results showed that the lowest concentration of the virus was 4.79 pg/μL, which could be detected before the disease. The key time node of preventing jujube virus disease from late May to early June was identified by tracking and analyzing the field infection dynamics. 【Conclusion】 The established technique can detect and identify the pathogen quickly and accurately, and can be used to track and detect the infection dynamics between fields of jujube mosaic disease. Its establishment provides technical support for epidemic monitoring and early prevention of jujube mosaic virus disease in Xinjiang jujube planting area.

Key words: jujube; mosaic virus; molecular detection; infection dynamics; epidemiological monitoring

中图分类号:

S436.65

白剑宇, 罗达, 刘正兴, 李宏. 枣树花叶病毒病的分子检测[J]. 新疆农业科学, 2022, 59(3): 657-662.

BAI Jianyu, LUO Da, LIU Zhengxing, LI Hong. Molecular Detection of Jujube Mosaic Virus[J]. Xinjiang Agricultural Sciences, 2022, 59(3): 657-662.

图/表 3

表1 样品材料及枣树花叶病毒病田间发生动态的检测结果

Table 1 Specimen materials and the detection results of dynamic condition of black spot of jujube in field

采集地点 Collectionlocation	采集时间 Collection time	叶片样本数量 Sample number of leaves	带毒叶片样本数量 Sample number of pathogen	病原检出率 Detection rate of pathogens
阿克苏市依杆旗乡	5月15日	25	5	0.20
	6月10日	25	11	0.44
	7月13日	25	19	0.76
	8月16日	25	23	0.92
温宿县万亩枣园	5月16日	25	7	0.28
	6月12日	25	13	0.52
	7月15日	25	21	0.84
	8月18日	25	24	0.96
巴楚县阿拉格尔乡	5月17日	25	6	0.24
	6月14日	25	14	0.56
	7月17日	25	20	0.80
	8月19日	25	25	1.00

图1 枣树花叶病毒病引物的特异性RT-PCR检测扩增结果注:M:100 bp Marker;1~3:枣树花叶病毒;4~5:枣疯病;6~7:苹果花叶病毒;8~9:无花果病毒;10~11:西甜瓜病毒;12~13:苜蓿花叶病毒;14~15:番茄黄化曲叶病毒;16~17:丁香环斑病毒;CK:阴性对照

Fig.1 The sensibility amplification results of black spot of jujube by PCR of specific primers Note: M: 100 bp Marker; 1-3: Jujube mosaic virus 4-5: Jujube witches' broom; 6-7: Apple mosaic virus; 8-9: Fig mosaic virus; 10-11:Melon mosaic virus; 12-13: Alfalfa.mosaic.virus; 14-15: Tomato yellow leaf curl virus; 16-17:Lilacringspotvirus; CK: Negative control

图2 注:M:DNA Marker;1 ~8:cDNA 依次稀释梯度为1 ~ 107倍液;CK:阴性对照枣树花叶病毒病RT- PCR引物灵敏性检测

Fig.2 Sensibility detection of RT- PCR primers of jujube mosaic virus Note: M: DNA Marker; 1 ~ 8: Serial dilution of cDNA 1 ~ 10 7倍液; CK: Negative control

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枣树花叶病毒病的分子检测

Molecular Detection of Jujube Mosaic Virus

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