烟草花叶病毒属条形码鉴定方法的建立

烟草花叶病毒属病毒可侵染多种植物,给农业生产带来严重危害。为建立该病毒属的条形码鉴定技术,以该属22个确定种的标准序列为凭证标本序列,分析其保守区域,设计6对候选条形码引物（Tobamo1-Tobamo6）,其扩增片段长度为200~1 240 bp。以田间样本对引物进行筛选,通过比较各引物的扩增效率、测序成功率、获得序列的亲缘关系对引物的通用性和物种分辨率进行评价。结果表明,Tobamo6的通用性最高,扩增率和测序成功率均为100%;物种分辨率良好,获得序列的种内种间变异可有效鉴定实际样本中的辣椒轻斑驳病毒、烟草花叶病毒、番茄花叶病毒和黄瓜绿斑驳花叶病毒;可作为烟草花叶病毒属的首选条形码引物。

杭州地区辣椒病毒病病原分子鉴定

通过双链RNA提取分析和病毒提纯与外壳蛋白，研究分析了杭州地区辣椒病毒病发生的情况，接合寄主生物学鉴定，发现侵染杭州辣椒的主要病毒为烟草花叶病毒属（Tobamovimses）和黄瓜花叶病毒（CMV）；且CMV的发病率明显低于Tobamovirses；同时，杭州地区辣椒上还可能存在前人未发现的新病毒或病毒因子，其dsRNA基因组大小约为1700hp。

Biological and molecular characterization of tomato brown rugose fruit virus and development of quadruplex RT-PCR detection

Tomato brown rugose fruit virus(ToBRFV) is a novel tobamovirus firstly reported in 2015 and poses a severe threat to the tomato industry. So far, it has spread to 10 countries in America, Asia, and Europe. In 2019, ToBRFV was identified in Shandong Province(ToBRFV-SD), China. In this study, it was shown that ToBRFV-SD induced mild to severe mosaic and blistering on leaves, necrosis on sepals and pedicles, and deformation, yellow spots, and brown rugose necrotic lesions on fruits. ToBRFV-SD induced distinct symptoms on plants of tomato, Capsicum annumm, and Nicotiana benthamiana, and caused latent infection on plants of Solanum tuberosum, Solanum melongena, and N. tabacum cv. Zhongyan 102. All the 50 tomato cultivars tested were highly sensitive to ToBRFV-SD. The complete genomic sequence of ToBRFV-SD shared the highest nucleotide and amino acid identities with isolate IL from Israel. In the phylogenetic tree constructed with the complete genomic sequence, all the ToBRFV isolates were clustered together and formed a sister branch with tobacco mosaic virus(TMV). Furthermore, a quadruplex RT-PCR system was developed that could differentiate ToBRFV from other economically important viruses affecting tomatoes, such as TMV, tomato mosaic virus, and tomato spotted wilt virus. The findings of this study enhance our understanding of the biological and molecular characteristics of ToBRFV and provide an efficient and effective detection method for multiple infections, which is helpful in the management of ToBRFV.

杨树上发生的两种病毒

从河南、湖南和北京采集到的具明显系统性紫红色坏死变形[PV-1]和叶褪绿皱缩[PV-2]杨树病株,经病害症状观察、病毒提纯和电镜观察、病毒回接、生化性状等试验,证明两种分离物是球状病毒粒子和杆状病毒粒子,是杨树上发生的两种病毒的病原。用杨树毒汁接种能感染多种草本植物。从病毒粒子的形状大小、理化性质、生物测定和血清反应,认为[PV-1]和[PV-2]分别属CMV和TMV。抗病品种鉴定看出,国内乡土种和北方型的品种抗病力较强,南方型的美洲黑杨和欧美杨易感病。

Robust molecular detection of the new Tomato brown rugose fruit virus in infected tomato and pepper plants from Turkey

Tomato brown rugose fruit virus(ToBRFV)causes severe fruit loss in tomato(Solanum lycopersicum)and pepper(Capsicum annuum)plants.It is an emerging Tobamovirus that is spreading globally.The major challenge is to develop a reliable method for the detection of the virus,and to better characterize the symptoms it causes.The aims of this study,therefore,were to characterize the symptom development on tomato and pepper plants,and to establish a reliable detection method for the virus.Following infection of the tomato and pepper plants with ToBRFV,the leaves turned chlorotic,mosaic or mottled,while the fruit became rugose,necrotic and marbled,and showed discoloration with yellow or brown spots.Transmission electron microscopy(TEM)revealed single rod-like virus particles characteristic of the Tobamoviruses.Classical reverse transcription PCR(RT-PCR)and quantitative PCR(qPCR)with specific primers and probes confirmed that the virus is ToBRFV.We found that the resistance genes from tomato,Tm-2^(2),and pepper,L1,L2,L3 and L4,did not confer resistance to ToBRFV.Here,we present a PCR-based method as a diagnostic test for detecting ToBRFV in infected seeds.This method will help to prevent further spread of the virus in commercial seeds.

Small RNA deep sequencing reveals the presence of multiple viral infections in cucurbit crops in Guangdong,China

Viral diseases are among the most critical damaging factors that impose a global threat to the cucurbit industry.China is the world’s leading country for the production and consumption of cucurbits.Guangdong,a province in southern China dominated by the tropical and subtropical climate,favors the survival of different plant viruses and their vectors.Five main cucurbit crops showing various disease symptoms were surveyed and collected to identify viruses infecting cucurbits in Guangdong during 2018–2020.In the field,the incidence ranged from 5-30%,or even 60-100% in the case of severely infected cucurbits.A total of 357 symptomatic samples were collected and subsequently screened for cucurbit viruses by small RNA deep sequencing and assembly(sRSA).Seventeen virus species belonging to 10 genera were identified in the five main cucurbit crops.The most common viruses were papaya ringspot virus(PRSV;Potyvirus),zucchini tigre mosaic virus(ZTMV;Potyvirus),zucchini yellow mosaic virus(ZYMV;Potyvirus),and watermelon silver mottle virus(WSMoV;Orthotospovirus),with infection rates of 24.4,19.0,17.1,and 14.3%,respectively.Notably,the most prevalent viruses were melon yellow spot orthotospovirus(MYSV)in cucumber,PRSV in squash,cucumber green mottle mosaic virus(CGMMV;Tobamovirus)in bottle gourd,WSMoV in white gourd,and ZYMV in luffa.Mixed infections were prevalent,and the types of mixed infections varied substantially in different cucurbit crops.Moreover,the full-length nucleotide sequences of watermelon green mottle mosaic virus(WGMMV),CGMMV,and watermelon virus A(WVA;Wamavirus)identified in bottle gourd were cloned and analyzed.This study is the first reporting WGMMV infecting bottle gourd in China mainland.In summary,the results demonstrate that in Guangdong,the most prevalent viruses belong to potyviruses,orthotospoviruses,and tobamoviruses groups.The findings will facilitate agricultural researchers and farmers to plan and implement effective disease control strategies aiming at timely detection and management of cucurbit-infecting viral pathogens.

Detection and characterization of an isolate of Tomato mottle mosaic virus infecting tomato in China

Tomato（Solanum lycopersicum） plants exhibiting severe leaf distortion, mottle and systemic crinkling symptoms were identified in Hainan province in China in 2016. To survey and control the disease, it is necessary to identify and characterize the pathogen causing the disease. Dot enzyme-linked immunosorbent assay showed that the crude saps of the infected tomato samples reacted positively with the monoclonal antibody against Tobacco mosaic virus which indicated that one or more tobamoviruses are likely associated with the disease. RT-p CR and DNA sequence analysis results further elucidated that Tomato mottle mosaic virus（To MMV） in Tobamovirus was the pathogen causing the mottle disease in tomato. We amplified and sequenced the full-length sequence of the genome which showed the highest nucleotide identity with To MMV YYMLJ and To MMV Ti Lha LJ isolates. The putative virus isolate was named To MMV Hainan. Biological indexing studies showed that To MMV Hainan can infect Nicotiana benthamiana, Capsicum annuum and Solanum lycopersicum showing serious symptoms. This was the first identification and characterization of To MMV infecting tomato in Hainan of China.

Tomato mottle mosaic virus: Characterization, resistance gene effectiveness, and quintuplex RT-PCR detection system

Tomato mottle mosaic virus(ToMMV), an economically important species of the genus Tobamovirus, causes significant loss in yield and quality of tomato fruits. Here, we identified the Shandong isolate of ToMMV(ToMMV-SD) collected from symptomatic tomato fruits in Weifang, Shandong Province of China. ToMMV-SD caused symptoms such as severe mosaic, mottling, and necrosis of tomato leaves, yellow spot and necrotic lesions on tomato fruits. The obtained full genome of ToMMV-SD was 6 399 nucleotides(accession number MW373515) and had the highest identity of 99.5% with that of isolate SC13-051 from the United States of America at the genomic level. The infectious clone of ToMMV-SD was constructed and induced clear mosaic and necrotic symptoms onto Nicotiana benthamiana leaves. Several commercial tomato cultivars, harboring Tm-2~2 resistance gene, and pepper cultivars, containing L resistance gene, were susceptible to ToMMV-SD. Plants of Solanum melongena(eggplant) and Brassica pekinensis(napa cabbage) showed mottling symptoms, while N. tabacum cv. Zhongyan 100 displayed latent infection. ToMMV-SD did not infect plants of N. tabacum cv. Xanthi NN, Brassica rapa ssp. chinensis(bok choy), Raphanus sativus(radish), Vigna unguiculata cv. Yuanzhong 28-2(cowpea), or Tm-2~2 transgenic N. benthamiana. A quintuplex RT-PCR system differentiated ToMMV from tomato mosaic virus, tomato brown rugose fruit virus, tobacco mosaic virus, and tomato spotted wilt virus, with the threshold amount of 0.02 pg. These results highlight the threat posed by ToMMV to tomato and pepper cultivation and offer an efficient detection system for the simultaneous detection of four tobamoviruses and tomato spotted wilt virus infecting tomato plants in the field.

An Attenuated Strain of Cucumber Green Mottle Mosaic Virus as a Biological Control Agent against Pathogenic Viral Strains

Cucumber green mottle mosaic virus (CGMMV), a member of the Tobamovirus genus, causes a severe disease of cucurbits. In the Moscow region of Russian Federation, the incidence of infection on cucumber plants in greenhouses is high;however, the virus is poorly studied. In this work, the full-length genomes of two pathogenic MC-1 and MC-2 strains of CGMMV isolated from cucumber plants grown in greenhouses in the Moscow region and the attenuated VIROG-43M strain were sequenced. Comparison of VIROG-43M nucleotide sequence with those of the pathogenic strains revealed three missense mutations. Their role in attenuation is discussed. For the first time, in a number of trials conducted under laboratory conditions and in commercial greenhouses, the efficiency of the attenuated VIROG-43M strain as a biocontrol agent for cucumber plant protection resulting in significant yield gain was demonstrated. Phylogenetic analysis with 83 full-length CGMMV coat protein genes isolated in 16 different countries showed that Russian strains are related to isolates from Spain, Greece, USA and Israel.

番茄抗病毒基因Tm-2^2及其等位基因tm-2氨基酸差异分析

番茄抗病蛋白Tm-2^2与其等位感病蛋白tm-2在序列上相对保守但也有一定的差异性，研究这些差异对于了解Tm-2^2抗病蛋白的结构和功能起着重要作用。本研究构建了不同的Tm-2^2／tm-2置换突变体，在本生烟草上利用农杆菌注射法共表达突变体及其效应因子MP或TMV—GFP载体。通过对植物坏死表型以及病毒量变化的观察。发现Tm-2^2ARC结构域中RNBS—D保守基序中的第413位甘氨酸突变成tm-2中的丝氨酸后，Tm-2^2介导的对烟草花叶病毒属病毒的抗性减弱。这说明RNBS-D保守基序对于Tm-2^2行使其抗病功能是至关重要的。