JIA-2022-0065 一种同时检测6种侵染番茄的类病毒的通用探针的制备及马铃薯纺锤形块茎类病毒（PSTVd）在中国番茄上的首次报道

doi:10.1016/j.jia.2022.08.119

Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (3): 790-798.DOI: 10.1016/j.jia.2022.08.119

JIA-2022-0065 一种同时检测6种侵染番茄的类病毒的通用探针的制备及马铃薯纺锤形块茎类病毒（PSTVd）在中国番茄上的首次报道

收稿日期:2022-01-18 接受日期:2022-06-01 出版日期:2023-03-20 发布日期:2022-06-01

A universal probe for simultaneous detection of six pospiviroids and natural infection of potato spindle tuber viroid (PSTVd) in tomato in China

ZHANG Yu-hong^1*, LI Zhi-xin^2*, DU Ya-jie^{1, 3}, LI Shi-fang^1#, ZHANG Zhi-xiang^1#

¹State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China
²Keshan Branch of Heilongjiang Academy of Agricultural Sciences, Qiqihar 161005, P.R.China
³College of Agronomy and Biotechnology, Hebei Normal University of Science & Technology, Qinhuangdao 066004, P.R.China

Received:2022-01-18 Accepted:2022-06-01 Online:2023-03-20 Published:2022-06-01
About author:ZHANG Yu-hong, E-mail: yuhongzhang0105@139.com; #Correspondence LI Shi-fang, Tel: +86-10-62890875, E-mail: sfli@ippcaas.cn; ZHANG Zhi-xiang, Tel: +86-10-62815615, E-mail: zhangzhixiang02@caas.cn * These authors contributed equally to this study.
Supported by:
This research was funded by the National Natural Science Foundation of China (31670149).

摘要/Abstract

摘要：

【目的】马铃薯纺锤形块茎类病毒属的多种类病毒可侵染番茄，造成严重病害，威胁产业发展。研发番茄类病毒检测技术可为病害防控提供技术支撑。此外，虽然国外有报道说我国出口的番茄种子上携带类病毒，但仍未有定论，尚需确认，并且有必要弄清楚我国番茄上类病毒的发生情况。【方法】使用序列比对的方法，比较能够侵染番茄的不同类病毒的基因组序列，找到保守序列，以此为模板，通过体外转录的方法制备RNA探针。通过斑点杂交的方法，测试该探针的检测灵敏度和特异性。另外，利用该通用探针，使用斑点杂交的方法，普查我国番茄上的类病毒发生情况。【结果】能够侵染番茄的6种类病毒：金鱼花潜隐类病毒（CLVd）、辣椒小果类病毒（PCFVd）、马铃薯纺锤块茎类病毒（PSTVd）、番茄顶缩类病毒（TASVd）、番茄褪绿矮缩类病毒（TCDVd）和番茄雄性株类病毒（TPMVd）的基因组序列含有一段长度为61 bp的保守序列。利用该序列制备的RNA探针能够同时检测这6种类病毒，其杂交检测的适宜反应温度为55℃~60℃。虽然该探针的检测灵敏度略低于每种类病毒的特异性探针，但可以满足田间大量样品的快速检测的需求。利用该探针，从我国番茄温室栽培的番茄植株上检测到了PSTVd，这是PSTVd在我国番茄上的首次报道。基因组序列比较分析发现，我国PSTVd番茄分离物的序列与国外茄科作物上PSTVd的序列最为接近，并且感染PSTVd的番茄的种子是从国外进口的。这表明我国番茄上的PSTVd应该是通过进口的番茄种子从国外传入的。【结论】研发出了一种能够同时检测侵染番茄的6种类病毒的通用探针，该探针具有快速灵敏的特点，适用于大量样品的快速检测，为番茄类病毒的监测及防控提供了技术支撑；此外，证实我国温室栽培的番茄上的确有PSTVd的发生，应该是通过种子由国外传入的，因此，需要加强进口种子及繁殖材料的检验和检疫，也要加强田间种植番茄的监测。

Abstract:

Several viroids in the genus Pospiviroid can infect tomato (Solanum lycopersicum) and cause severe diseases, posing a serious threat to tomato production. For simultaneous detection of six tomato-infecting pospiviroids - columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd), and tomato planta macho viroid (TPMVd), we developed a universal probe based on a highly conserved 61 nt long sequence shared among them. Compared with their specific probes, the universal probe has a similar, though slightly reduced, detection sensitivity and has the advantages of simple and cost-effective preparation and simultaneous detection of the six pospiviroids. In addition, the universal probe was used in dot-blot hybridization assays for a large-scale survey of viroid(s) in tomato plantings in China. Only PSTVd was detected in a few greenhouse-planted tomato plants. Sequence analysis revealed that these tomato PSTVd isolates may have been introduced from tomato seeds imported from abroad.

Key words: tomato , viroid , simultaneous detection , molecular hybridization , pospiviroids

. JIA-2022-0065 一种同时检测6种侵染番茄的类病毒的通用探针的制备及马铃薯纺锤形块茎类病毒（PSTVd）在中国番茄上的首次报道[J]. Journal of Integrative Agriculture, 2023, 22(3): 790-798.

ZHANG Yu-hong, LI Zhi-xin, DU Ya-jie, LI Shi-fang, ZHANG Zhi-xiang. A universal probe for simultaneous detection of six pospiviroids and natural infection of potato spindle tuber viroid (PSTVd) in tomato in China[J]. Journal of Integrative Agriculture, 2023, 22(3): 790-798.

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JIA-2022-0065 一种同时检测6种侵染番茄的类病毒的通用探针的制备及马铃薯纺锤形块茎类病毒（PSTVd）在中国番茄上的首次报道

A universal probe for simultaneous detection of six pospiviroids and natural infection of potato spindle tuber viroid (PSTVd) in tomato in China

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