李正刚, 农媛, 汤亚飞, 佘小漫, 于琳, 蓝国兵, 邓铭光, 何自福^,广东省农业科学院植物保护研究所/广东省植物保护新技术重点实验室,广州510640

Molecular Characteristic and Pathogenicity Analyses of Cucumber green mottle mosaic virus (CGMMV) Infecting Bottle Gourd in Lianzhou, Guangdong

LI ZhengGang, NONG Yuan, TANG YaFei, SHE XiaoMan, YU Lin, LAN GuoBing, DENG MingGuang, HE ZiFu^,Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640

通讯作者: 何自福,Tel：020-87597476;E-mail： hezf@gdppri.com

责任编辑: 岳梅
收稿日期:2019-09-4接受日期:2019-11-19网络出版日期:2020-03-01

基金资助:

国家重点研发计划.2018YFD0201209 广东省现代农业产业共性关键技术研发创新团队建设项目.2019KJ134 广东省重点领域研发计划.2018B020202006 广东省重点领域研发计划.2018B020202007

Received:2019-09-4Accepted:2019-11-19Online:2020-03-01
作者简介 About authors
李正刚,E-mail：lzaagg@cau.edu.cn。










摘要
【目的】黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)是侵染瓜类作物的主要病毒之一,对瓜类产业造成巨大的危害。本研究旨在探明侵染广东省连州市葫芦的CGMMV分离物(CGMMV-GDLZ)分子特征及其在系统进化中的地位,并测定其对黄瓜、葫芦和西瓜的致病性,为CGMMV的防控提供理论依据。【方法】从广东省连州市葫芦种植基地采集2个疑似CGMMV侵染的病样以及1个无症状样品,提取总RNA,根据CGMMV参考序列（GenBank登录号：KX883801）设计引物进行RT-PCR检测,引物序列为F：CCACGAGTTGTTTCCTAATGCTG/R：TTTGCTAGGCGTGATCGGATTGT,退火温度53℃,扩增长度890 bp。将CGMMV全长序列分为前后两段,前半段1—3 511 nt,扩增引物序列为F：AAGTTCATTTCATTTGGAGAGGGTTTTAATTTTTATAA TTAAACAAA/R：AGTTCTGCATTAATTGCTATTTGGTAGGCACAGTGGTAG;后半段3 301—6 423 nt,扩增引物序列为F：GTGCGTGCTACCCCGACTCCAATAGGTTTGATTGCCCGTG/R：GGTGGAGATGCCATGCCGACCCTGGGCCCCTACCCGGGGAAAGG。将前后两段PCR产物通过同源重组的方法克隆到pCB301双元载体上,测序得到CGMMV-GDLZ分离物全长序列。利用CGMMV-GDLZ分离物全长序列在NCBI中进行Blast分析,然后通过MEGA7软件对CGMMV-GDLZ以及其他已经报道的CGMMV分离物进行系统进化树分析。将构建好的pCB301-CGMMV侵染性克隆注射接种本生烟验证其侵染性,然后再注射接种黄瓜、葫芦和西瓜的子叶,测定CGMMV-GDLZ分离物的致病性。【结果】RT-PCR结果证实,广东省连州市葫芦病样感染了CGMMV。CGMMV-GDLZ分离物全长序列为6 423 nt,编码4个蛋白,分别为129K复制酶（61—3 495 nt）、186K复制相关蛋白（61—5 007 nt）、运动蛋白MP（4 994—5 788 nt）和外壳蛋白CP（5 763—6 248 nt）。CGMMV-GDLZ核苷酸序列与CGMMV-eWT分离物（GenBank登录号：KY753928）同源性最高,为99.97%。系统进化树分析结果显示,CGMMV-GDLZ分离物与日本、韩国等东亚CGMMV分离物同属Group 1,在遗传距离上与山东、浙江和河南的CGMMV分离物最接近。pCB301-CGMMV侵染性克隆可以系统侵染本生烟,造成本生烟上部叶片出现皱缩、斑驳、凸起等症状,RT-PCR和Western blot进一步确认了侵染性克隆的侵染性。注射接种CGMMV-GDLZ分离物后15 dpi,葫芦和西瓜即可产生斑驳、花叶、突起、生长迟缓等症状,24 dpi时症状更明显。而15 dpi时,CGMMV-GDLZ分离物在黄瓜上的症状不明显,与未接种对照植株几乎没有区别;将植株从控温接种室移入网室中,30 dpi时,黄瓜植株上部叶片开始出现斑驳和花叶,40 dpi时,症状已经非常明显。RT-PCR和Western blot检测进一步确认了上述结果。【结论】侵染广东省连州市葫芦的CGMMV-GDLZ分离物与山东、浙江和河南的CGMMV分离物很可能具有相同的传染源;CGMMV-GDLZ分离物可以侵染本生烟、黄瓜、葫芦和西瓜等作物,但对这些作物的致病性存在差异。
关键词： 葫芦;黄瓜绿斑驳花叶病毒;分子特征;侵染性克隆;致病性

Abstract
【Objective】Cucumber green mottle mosaic virus (CGMMV) is one of the main viruses that infect melon crops, which causes great harm to the melon industry. The objective of this study is to identify the molecular characteristic and phylogenetic status of CGMMV-GDLZ isolate, analyze the pathogenicity of CGMMV-GDLZ isolate on cucumber, bottle gourd, and watermelon, and to provide a theoretical basis for the prevention and control of CGMMV. 【Method】Two symptomatic samples and one asymptomatic sample were collected from a farm located in Lianzhou City of Guangdong Province and were subjected to total RNA extraction. RT-PCR detection was performed using primer pair F: CCACGAGTTGTTTCCTAATGCTG/R: TTTGCTAGGCGTGATCGGATTGT, which was designed according to CGMMV reference genome (GenBank accession number KX883801). The annealing temperature is 53℃, and the product is 890 bp in length. To construct pCB301-CGMMV infectious cDNA clones, two primer pairs were designed to amplify the first half (1-3 511 nt) and the second half (3 301-6 423 nt) of the nucleotide sequence of CGMMV-GDLZ isolate. The primer pair used to amplify the first half is F: AAGTTCATTTCATTTGGA GAGGGTTTTAATTTTTATAATTAAACAAA/R: AGTTCTGCATTAATTGCTATTTGGTAGGCACAGTGGTAG, and the primer pair used to amplify the second half is F: GTGCGTGCTACCCCGACTCCAATAGGTTTGATTGCCCGTG/R: GGTGGAGATGC CATGCCGACCCTGGGCCCCTACCCGGGGAAAGG. Then the acquired products were cloned into pCB301 by homologous recombination, followed by Sanger DNA sequencing. Blast analysis was performed in NCBI using the full-length sequence of CGMMV-GDLZ isolate, then the phylogenetic tree was constructed by MEGA7 software using the sequences of CGMMV-GDLZ and other reported CGMMV isolates. Agrobacterium containing pCB301-CGMMV was infiltrated into Nicotiana benthamiana leaves to verify the infectivity of CGMMV-GDLZ isolate. Subsequently, pCB301-CGMMV was agroinfiltrated into the cotyledon of cucumber, bottle gourd, and watermelon to analyze the pathogenicity of CGMMV. 【Result】RT-PCR detection verified that the two symptomatic samples were infected by CGMMV. Sanger sequencing reveals that CGMMV-GDLZ isolate contains 6 423 nt and encodes four proteins, 129K replicase (61-3 495 nt), 186K replication-associated protein (61-5 007 nt), movement protein (4 994-5 788 nt), and coat protein (5 763-6 248 nt). CGMMV-GDLZ isolate has the highest nucleotide similarity (99.97%) with CGMMV-eWT isolate (GenBank accession number KY753928). Phylogenetic analysis showed that CGMMV-GDLZ and other CGMMV isolates from Japan and Korea were clustered into Group 1, and CGMMV-GDLZ was closest to isolates from Shandong, Zhejiang, and Henan. In addition, the pCB301-CGMMV infectious cDNA clones could successfully infect N. benthamiana, causing crinkle, mottle, and mosaic symptoms in the upper leaves. RT-PCR and Western blot detection further verified the infection of the infectious cDNA clones. Moreover, CGMMV-GDLZ isolate could also infect bottle gourd and watermelon, causing mottle, mosaic, and growth retardation at 15 days post-infiltration (dpi), and showed more severe symptoms at 24 dpi. However, the upper leaves of cucumber agro-infiltrated with pCB301-CGMMV did not show obvious symptoms compared with control plants. Further studies found the agro-infiltrated plants began to appear mottle and mosaic symptoms at 30 dpi after transferred from the greenhouse to artificial net-room without temperature control. The symptoms were more obvious at 40 dpi. RT-PCR and Western blot detection further verified the above results. 【Conclusion】CGMMV-GDLZ isolate from Lianzhou City of Guangdong Province may have the same infection source with CGMMV isolates from Shandong, Zhejiang, and Henan provinces. CGMMV-GDLZ isolate can infect N. benthamiana, cucumber, bottle gourd, and watermelon, but the pathogenicity varies in different crops.
Keywords：bottle gourd;Cucumber green mottle mosaic virus (CGMMV);molecular characteristic;infectious cDNA clones;pathogenicity


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本文引用格式
李正刚, 农媛, 汤亚飞, 佘小漫, 于琳, 蓝国兵, 邓铭光, 何自福. 侵染广东连州葫芦的黄瓜绿斑驳花叶病毒的分子特征 及致病性分析[J]. 中国农业科学, 2020, 53(5): 955-964 doi:10.3864/j.issn.0578-1752.2020.05.008
LI ZhengGang, NONG Yuan, TANG YaFei, SHE XiaoMan, YU Lin, LAN GuoBing, DENG MingGuang, HE ZiFu. Molecular Characteristic and Pathogenicity Analyses of Cucumber green mottle mosaic virus (CGMMV) Infecting Bottle Gourd in Lianzhou, Guangdong[J]. Scientia Acricultura Sinica, 2020, 53(5): 955-964 doi:10.3864/j.issn.0578-1752.2020.05.008

0 引言

【研究意义】黄瓜绿斑驳花叶病毒（Cucumber green mottle mosaic virus,CGMMV）隶属于帚状病毒科（Virgaviridae）烟草花叶病毒属（Tobamovirus）^[1],该属病毒的特点是极易通过机械摩擦进行传播。CGMMV可以侵染黄瓜、葫芦、甜瓜、西瓜等葫芦科作物,造成植株生长迟缓、叶片褪绿斑驳、果实变色和纤维化,导致严重损失^[2,3,4,5]。CGMMV是典型的种传病毒^[6,7],是我国植物检疫性有害生物^[4]。因此,对CGMMV进行监测及鉴定,有利于对该病毒进行预防,减轻该病毒造成的危害,对葫芦科作物的可持续发展具有重要意义。【前人研究进展】CGMMV是正义单链RNA病毒,全长约6 400 nt,编码4个蛋白,分别为129 kD的复制酶、186 kD的复制相关蛋白、29 kD运动蛋白和17.4 kD的外壳蛋白^[8]。CGMMV最早是在英格兰发现的^[9],随后在全世界范围内陆续被报道^{[10,11,12,13,14,15,16]}。CGMMV在世界范围的传播可以分为3个时期^[17]：1935—1985年,该时期CGMMV在全球的传播速度较慢,仅局限在欧洲、中东、南亚和东亚少数几个国家以及中国台湾等地区^{[17,18,19,20]};1986—2006年,该时期CGMMV在全球传播的速度有所加快,已传播至欧洲大部分国家^[17],在亚洲包括南亚的巴基斯坦,东亚的中国和韩国,东南亚的印度尼西亚和泰国,中东地区的以色列、沙特阿拉伯和叙利亚^{[11,17,21-27]};2007—2016年,该时期CGMMV传播速度更加迅速,传播至更多的国家和地区^[17],加拿大、美国、尼日利亚、澳大利亚这些国家也陆续发现了CGMMV^[17,28-30]。中国大陆于2005年在广西南瓜上最早报道CGMMV,目前已经在山东、河北、湖北、云南、广东、辽宁等多地普遍发生^[3,22,31-33],并对多地的瓜类作物造成了毁灭性的损失^[2,4]。【本研究切入点】CGMMV广东分离物虽然已经报道^[32],但是只包含了运动蛋白和外壳蛋白的部分序列,不是全长序列,而且没有对其侵染性进行分析。【拟解决的关键问题】克隆侵染葫芦的CGMMV广东分离物基因组全长,构建其全长侵染性克隆,并测定其对3种重要葫芦科植物（黄瓜、葫芦和西瓜）的致病性,为该病毒的预防提供依据。

1 材料与方法

试验于2018年9月至2019年7月在广东省农业科学院植物保护研究所完成。

1.1 样本来源

2018年9月在广东省连州市葫芦产区发现有植株疑似感染CGMMV,采集2个有症状病样以及1个无症状样品后带回实验室,并置于-80℃超低温冰箱冻存。

1.2 RNA提取

采用TRIzol方法提取样品总RNA,TRIzol购自TaKaRa公司,提取好的RNA样品冻存于-80℃超低温冰箱备用。

1.3 RT-PCR反应

反转录试剂盒购自TaKaRa公司。CGMMV检测引物参考CGMMV序列（GenBank登录号：KX883801）设计,引物序列为F：CCACGAGTTGTTTCCTAAT GCTG/R：TTTGCTAGGCGTGATCGGATTGT,扩增长度为890 bp,退火温度为53℃。

1.4 CGMMV-GDLZ分离物全长序列扩增

将CGMMV全长序列分为两段,前半段1—3 511 nt,后半段3 301—6 423 nt,分别设计引物扩增。前半段扩增引物为F：AAGTTCATTTCATTTGGAGAG GGTTTTAATTTTTATAATTAAACAAA （下划线序列为同源重组序列）/R：AGTTCTGCATTAATTGCTA TTTGGTAGGCACAGTGGTAG;后半段扩增引物为F：GTGCGTGCTACCCCGACTCCAATAGGTTTGAT TGCCCGTG/R：GGTGGAGATGCCATGCCGACCCT GGGCCCCTACCCGGGGAAAGG（下划线序列为同源重组序列）。将扩增好的前半段和后半段序列通过同源重组的方法构建到pCB301载体上^[34]。将构建好的pCB301-CGMMV质粒送往上海生工生物工程股份有限公司进行测序。

1.5 系统进化分析

将测序所得CGMMV全长序列在NCBI中进行Blast分析。利用MEGA7软件^[35]构建系统进化树。

1.6 CGMMV-GDLZ侵染性克隆构建及农杆菌注射接种

将约为0.5 μg的pCB301-CGMMV质粒转入100 μL GV3101（pSoup）农杆菌感受态细胞（上海唯地生物技术有限公司）,菌落PCR验证。将含有pCB301-CGMMV质粒的农杆菌接种于含有卡那霉素（100 μg·mL^-1）和利福平（25 μg·mL^-1）的LB培养基内,28℃摇床过夜培养,4 000 r/min离心10 min收集沉淀,弃上清,沉淀用农杆菌悬浮缓冲液（10 mmol·L^-1 MES,10 mmol·L^-1 MgCl₂,150 μmol·L^-1 As）重悬,利用紫外分光光度计将菌液OD₆₀₀调至1.0。28℃培养箱静置4 h,用注射器注射植物。

1.7 Western blot检测

采集0.5 g发病及健康对照样品于2 mL离心管中,液氮速冻,通过组织研磨机充分破碎,趁冷加入0.3 mL蛋白提取缓冲液（50 mmol·L^-1 Tris-HCl pH 6.8,10%甘油,2% SDS,2.5%巯基乙醇）,充分振荡混匀,沸水浴10 min,12 000 r/min离心10 min,取20 μL上清进行Western blot检测。Western blot一抗为CGMMV CP多克隆抗体,羊抗兔二抗购自于Sigma Aldrich。

2 结果

2.1 样品采集及检测

2018年9月在广东省连州市一个葫芦种植区调查时发现,部分葫芦植株叶片呈现明显的褪绿、花叶、斑驳等症状（图1-A）,疑似感染CGMMV。采集了2株发病植株及1株无症状植株,RT-PCR检测结果显示,2个发病样品均可检测到大小为900 bp左右的特异条带（图1-B）,无症状CK样品则没有条带,说明2个发病样品确实感染了CGMMV。

图1

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图1疑似感染CGMMV的葫芦植株田间发病症状以及RT-PCR检测

A：发病植株Diseased plant。B：M：DL2000 DNA Marker;CK：无症状对照样品Control sample without symptom
Fig. 1Symptoms of bottle gourd infected by CGMMV and RT-PCR detection of CGMMV

2.2 CGMMV-GDLZ基因组全长序列及其系统进化

为了得到CGMMV广东连州分离物（CGMMV- GDLZ）基因组的全长序列,将CGMMV全长分为两段扩增,PCR结果显示,从毒源病样中成功扩增到预期大小的目的条带（未展示数据）,将胶回收的片段与pCB301载体^[34]进行重组反应,得到CGMMV- GDLZ分离物基因组全长序列,同时也构建了其侵染性克隆pCB301-CGMMV。CGMMV-GDLZ分离物全长为6 423 nt（GenBank登录号：MK933286）,编码4个蛋白,分别为129K复制酶（61—3 495 nt）、186K复制相关蛋白（61—5 007 nt）、运动蛋白MP（4 994—5 788 nt）和外壳蛋白CP（5 763—6 248 nt）。CGMMV-GDLZ分离物与CGMMV-eWT分离物（GenBank登录号：KY753928）核苷酸同源性最高,为99.97%,仅有第31位和4 143位核苷酸不同,而其编码的所有蛋白的同源性则为100%。

为了分析比较CGMMV-GDLZ分离物与其他分离物之间的关系,利用MEGA7软件^[35]构建了系统进化树。选取不同地点（中国大陆、中国台湾、日本、韩国、加拿大、以色列、西班牙、俄罗斯）、不同作物（黄瓜、葫芦、西瓜）上的CGMMV分离物进行系统进化树构建。结果显示CGMMV分离物根据地点分成了3个组：中国大陆、中国台湾、韩国和日本等亚洲国家和地区的CGMMV分离物为第1组;以色列和加拿大CGMMV分离物为第2组;西班牙和俄罗斯CGMMV分离物为第3组。CGMMV-GDLZ分离物与山东黄瓜CGMMV-SD分离物（KJ754195）、河南西瓜CGMMV-hn分离物（KC851866）、浙江香瓜CGMMV-JD8分离物（KM873784）、浙江西瓜CGMMV-DY9分离物（KM873786）距离最近（图2）。

图2

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图2基于CGMMV全长序列构建的系统进化树

从GenBank中下载CGMMV不同分离物的序列,用MEGA7软件以邻接法构建系统进化树The sequences of different isolates of CGMMV were downloaded from the GenBank, and the phylogenetic tree was constructed by neighbor-joining method using MEGA7 software
Cu：黄瓜 Cucumber;Wm：西瓜 Watermelon;Om：香瓜 Oriental melon;Bg：葫芦 Bottle gourd;Lu：丝瓜 Luffa;Me：瓜 Melon;Pu：南瓜Pumpkin;Mm：甜瓜 Musk melon
Fig. 2Phylogenetic tree based on full-length sequence of CGMMV

2.3 CGMMV-GDLZ分离物侵染性克隆接种验证

为了验证侵染性克隆pCB301-CGMMV的侵染性,首先利用农杆菌注射的方法接种了本生烟,结果显示第7天时,与Mock植株相比,接种的本生烟上部叶片开始出现明显的泡状凸起,随着时间的发展,症状愈加明显（图3-A）。采集发病叶片进行RT-PCR验证,结果显示发病样品可以检测到CGMMV特异条带,Mock样品没有检测到（图3-B）。进一步利用Western blot进行验证,结果显示发病样品可以检测到CGMMV CP条带,而Mock没有检测到（图3-C）。说明侵染性克隆pCB301-CGMMV具有侵染性。

图3

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图3利用pCB301-CGMMV侵染性克隆接种本生烟

A：本生烟发病症状Diseased symptoms of N. benthamiana。Mock：未接种对照植株 Control plant without inoculation。下图为白色方框放大图The below figures are magnification of the white frames。B：本生烟接种CGMMV-GDLZ 7 dpi,RT-PCR检测上部叶片发病情况RT-PCR detection of the upper leaves of N. benthamiana at 7 dpi。M：DL2000 DNA Marker。C：Western blot检测上部叶片发病情况。箭头为CGMMV CP所在位置Western blot detection of the upper leaves of N. benthamiana at 7 dpi. The arrow indicates the location of the CGMMV CP。M：Protein marker
Fig. 3Ago-infiltration into N. benthamiana with pCB301-CGMMV infectious cDNA clones

2.4 CGMMV-GDLZ分离物的致病性

利用含有CGMMV-GDLZ侵染性克隆（pCB301- CGMMV）的农杆菌注射接种黄瓜、葫芦和西瓜的子叶,15 dpi时,葫芦和西瓜上部叶片出现明显的斑驳、花叶、突起,植株生长迟缓,24 dpi时症状更明显;而15 dpi时,CGMMV-GDLZ分离物在黄瓜上的症状不明显,与未接种对照植株几乎没有区别;将植株从控温接种室移入网室中,30 dpi,黄瓜植株上部叶片开始出现斑驳和花叶,40 dpi时,症状已经非常明显（图4-A）。RT-PCR和Western blot结果验证了CGMMV的成功侵染（图4-B、4-C）。这些结果说明,CGMMV- GDLZ分离物可以侵染黄瓜、葫芦、西瓜等瓜类作物,显症时间有所差异。

图4

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图4CGMMV-GDLZ分离物在黄瓜、葫芦和西瓜上的致病性

A：CGMMV-GDLZ分离物在黄瓜、葫芦和西瓜上的发病症状Symptoms of cucumber, bottle gourd and watermelon infected by CGMMV-GDLZ isolate。Mock：健康对照Healthy control。dpi：Days post-infiltration。B：RT-PCR检测CGMMV-GDLZ在黄瓜、葫芦和西瓜上的发病情况RT-PCR detection of CGMMV-GDLZ in cucumber, bottle gourd and watermelon。Cum：黄瓜;Bg：葫芦;Wme：西瓜。Mock：未接种健康对照Control plant without inoculation。M：DL2000 DNA Marker。C：Western blot检测CGMMV-GDLZ在黄瓜、葫芦和西瓜上的发病情况Western blot detection of CGMMV-GDLZ in cucumber, bottle gourd and watermelon。M：Protein marker
Fig. 4Pathogenicity of CGMMV-GDLZ isolate on cucumber, bottle gourd, and watermelon

3 讨论

中国是世界上瓜类种植面积最大的国家,而CGMMV则是危害瓜类作物生产的主要病原菌之一,给我国的瓜类产业造成了巨大的损失。CGMMV属于烟草花叶病毒属^[1],该属病毒可以通过汁液、花粉、农事操作以及嫁接的方式传播,因此CGMMV在田间非常容易扩散。CGMMV还是典型的种传病毒^[6,7],种子带毒也是CGMMV长距离传播的主要方式。

早在2002年和2004年,中国口岸检疫部门就已在从日本进口的瓜类种苗或种子中检测到了CGMMV^[36];2005年,秦碧霞等报道在广西观赏南瓜上检测到了CGMMV,该南瓜品种是从欧洲、印度、日本等国引种后选育出的^[22];2007年,研究人员在从日本引进的西瓜中检测到了CGMMV^[37]。对不同国家和地区的CGMMV分离物进行进化树分析,结果显示亚洲的中国、日本、韩国被分到了一组,加拿大和以色列分到了一组,西班牙和俄罗斯等欧洲国家被分到了一组,其中中国与日本的CGMMV分离物在遗传距离上更为接近。这些结果均表明中国大陆的CGMMV很有可能是从日本传播扩散而来。CGMMV-GDLZ分离物在遗传距离上与山东、河南、浙江等地的分离物最接近,说明广东、山东、河南和浙江的CGMMV分离物可能来自相同的传染源。

接种后15 d,CGMMV-GDLZ在葫芦以及西瓜上的症状已经非常明显,而在黄瓜上的症状却不明显;将黄瓜植株由温度恒定的控温室移入变温的开放网室,30 dpi时黄瓜植株开始出现典型的斑驳和花叶,说明CGMMV-GDLZ在不同作物上的发病时间及发病温度有所差异,这些差异可能与不同的作物和品种有关。不同的作物和品种对同种病毒的抗性有很大的区别,包含抗性基因的作物和品种表现出抗病,反之则表现出感病;另外温度对植物的抗性水平也起着重要的调控作用,在不同的温度下植物表现出不同的抗性水平,比如包含Ry_chc抗性基因的马铃薯在不同的温度下对马铃薯Y病毒（Potaoto Y virus,PVY）表现出不同的抗性水平,在22℃下马铃薯表现出极端抗性（extreme resistance,ER）水平,只在接种叶上有少量小的坏死斑,上部叶片没有任何症状,而当温度升至28℃时,接种叶上出现了明显的坏死斑,上部叶片也开始有少量发病,另外研究人员还发现包含有Ry_chc抗性基因的马铃薯栽培品种在夏天比春天更容易出现坏死斑^[38]。再如小麦品种Adl-cross在25℃以下对小麦条纹花叶病毒（Wheat streak mosaic virus,WSMV）表现为抗病,而在32℃时则不抗病^[39]。这些结果都表明高温可能会降低抗性基因赋予的抗性,使植物在高温下更容易感病。黄瓜以及其他瓜类对CGMMV有没有抗性基因、抗性基因是否受到温度调控还需进一步研究。

4 结论

侵染广东省连州市葫芦的CGMMV-GDLZ分离物全长6 423 nt,与CGMMV-eWT分离物同源性最高,在遗传距离上与山东、浙江和河南的CGMMV分离物最接近,很可能具有相同的传染源。CGMMV-GDLZ分离物可以侵染黄瓜、葫芦和西瓜等作物,但在不同作物上显症时间存在差异。

致谢：

感谢中国农业大学植物保护学院王颖副教授惠赠CGMMV CP多克隆抗体;感谢南京农业大学植物保护学院陶小荣教授惠赠pCB301载体。

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