Susceptibility of different plant species and tomato cultivars to two isolates of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis>

As Pepino mosaic virus has become a pathogen of major importance in worldwide tomato production, information is needed on possible differences between the sensitivity of cultivars towards infection. Furthermore, it is important what hosts other than Solanaceae may be virus reservoirs and are, therefore, threats for tomato cultivation. Two PepMV isolates (PepMV-Sav, E397, a European tomato isolate and PV-0554, a Peruvian pepino isolate) differing in their origin and virulence were used for several experiments to investigate these issues. The response to mechanical inoculation with PepMV was studied using 25 tomato cultivars, seven indicator plant species, and nine other possible horticultural host plants. Symptom development after infection with PepMV was monitored and the virus was detected by DAS-ELISA and IC-RT-PCR. Garlic and broad bean were shown to be additional hosts of PepMV depending on the virus isolate. Nicotiana benthamiana seems to be the most sensitive indicator among all tested indicator plants developing symptoms. Both PepMV isolates infected all tested tomato cultivars. Development of disease symptoms depended on the cultivar and the virus isolate but symptoms were not visible in all cases. None of the cultivars showed tolerance against the two isolates but two responded with a lower susceptibility at an absorbance level of 0.2 (healthy control 0.09). It was observed that some cultivars grown hydroponically showed also lower losses in biomass and yield. Data indicated a correlation between absorbance level in DAS-ELISA and reduction in total tomato growth.     

A transgenic RNAi approach for developing tomato plants immune to Pepino mosaic virus

RNA interference (RNAi) or gene silencing is a natural defence response of plants to invading viruses. Here, we applied this approach against pepino mosaic virus (PepMV) isolates in their natural host, tomato. PepMV isolates differ in their genetic sequences, the severity of the disease they induce, and their worldwide distribution. PepMV causes heavy crop losses, mainly due to impaired tomato fruit quality. Resistant varieties are not yet available, despite many years of resistance breeding efforts within the tomato seed industry. To generate broad resistance to PepMV strains, conserved sequences from three different strains of PepMV (US1, LP, and CH2) were synthesized as a single insert and cloned in a hairpin configuration into a binary vector, which was used to transform tomato plants. Transgenic tomato lines that expressed a high level of transgene-siRNA exhibited immunity to PepMV strains, including a new Israeli isolate. This immunity was maintained even after graft inoculation, in which a transgenic scion was grafted onto nontransgenic infected rootstocks. However, an immune transgenic rootstock was unable to induce resistance in a nontransformed scion. These results provide the first example of engineered immunity to diverse PepMV strains in transgenic tomato based on gene silencing.     

Genetic variation and evolutionary analysis of Pepino mosaic virus in Sicily: insights into the dispersion and epidemiology

Pepino mosaic virus (PepMV) is a highly infectious potexvirus that causes a severe disease in tomato (Solanum lycopersicum) crops worldwide. In Sicily, the first outbreak was detected in a single greenhouse in 2005 and it was promptly eradicated. However, in 2008, a large number of greenhouses were simultaneously affected, and it was impossible to eradicate or control the virus. This study addressed the dispersion and the genetic diversity of PepMV isolates obtained from the outbreak in Sicily, in comparison with worldwide PepMV isolates, to gain insight into the factors determining the evolution and epidemiology of the virus. A total of 1800 samples from plants with and without symptoms were collected in the Sicilian provinces of Agrigento, Caltanissetta, Palermo, Ragusa, Siracusa and Trapani. Three isolates collected at different times were biologically characterized. The incidence of the virus increased rapidly from 13% in 2011 to 63% in 2013, and phylogenetic analysis showed that all Sicilian isolates of PepMV belonged to the CH2 strain, one of the six strains previously described. Nucleotide diversity of the Sicilian isolates was low, thus suggesting rapid spread and genetic stability.     

High Similarity Between Tomato Isolates of Pepino mosaic Virus Suggests a Common Origin

The almost simultaneous outbreaks of Pepino mosaic virus in tomato crops in different European and non-European countries, was reason to have a closer look at the relationship between these isolates and the original isolate from pepino. Fifteen isolates from tomato from different locations and the original pepino isolate, were compared on the basis of their symptomatology on a series of plant species. In addition, PCR fragments derived from the viral polymerase gene were sequenced and aligned. Both studies showed that the isolates from tomato clearly differed from the pepino isolate. The different tomato isolates, however, exhibited only minor differences to each other, both in symptomatology and nucleotide sequence. These results support the conclusion that the tomato isolates should be considered as a distinct strain (Mumford and Metcalfe (2001) Archives of Virology 146: 2455–2460; Van der Vlugt et al. (2000) Plant Disease 84: 103; Van der Vlugt et al. (2002) Bulletin OEPP/EPPO Bulletin 32: 503–508). Moreover, the high similarity of the different tomato isolates suggests the existence of a common source of infection for the recent outbreaks.     

Genetic Structure of the Population of Pepino mosaic virus Infecting Tomato Crops in Spain

ABSTRACT The population structure of Pepino mosaic virus (PepMV), which has caused severe epidemics in tomato in Spain since 2000, was analyzed. Isolates were characterized by the nucleotide sequence of the triple gene block and coat protein gene and, for a subset of isolates, a part of the RNA-dependent RNA polymerase gene. The full-length sequence of the genomic RNA of a Solanum muricatum isolate from Peru also was determined. In spite of high symptom diversity, the Spanish population of PepMV mostly comprised highly similar isolates belonging to the strain reported in Europe (European tomato strain), which has been the most prevalent genotype in Spain. The Spanish PepMV population was not structured spatially or temporally. Also, isolates highly similar to those from nontomato hosts from Peru (Peruvian strain) or to isolate US2 from the United States (US2 strain) were detected at lower frequency relative to the European strain. These two strains were detected in peninsular Spain only in 2004, but the Peruvian strain has been detected in the Canary Islands since 2000. These results suggest that PepMV was introduced into Spain more than once. Isolates from the Peruvian and US2 strains always were found in mixed infections with the European tomato strain, and interstrain recombinants were detected. The presence of different strains of the virus, and of recombinant isolates, should be considered for the development of control strategies based on genetic resistance.     

The use of attenuated isolates of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> for cross-protection

Pepino mosaic virus (PepMV) has recently emerged as a highly infectious viral pathogen in tomato crops. Greenhouse trials were conducted under conditions similar to commercial tomato production. These trials examined whether tomato plants can be protected against PepMV by a preceding infection with an attenuated isolate of this virus. Two potential attenuated isolates that displayed mild leaf symptoms were selected from field isolates. Two PepMV isolates that displayed severe leaf symptoms were also selected from field isolates to challenge the attenuated isolates. The isolates with aggressive symptoms were found to reduce bulk yields by 8 and 24% in single infections, respectively. Yield losses were reduced to a 0–3% loss in plants that were treated with either one of the attenuated isolates, while no effects were observed on the quality of the fruits. After the challenge infection, virus accumulation levels and symptom severity of the isolates with aggressive symptoms were also reduced by cross-protection. Infection with the attenuated isolates alone did neither affect bulk yield, nor quality of the harvested tomato fruits.     

Pepino mosaic virus isolates and differential symptomatology in tomato

Based on a survey conducted in commercial tomato production in Belgium in 2006, four Pepino mosaic virus (PepMV) isolates that differed in symptom expression in the crop of origin were selected for greenhouse trials. The selected isolates were inoculated onto tomato plants grown in four separate plastic tunnels. PepMV symptom development was assessed regularly and extensive sampling followed by ELISA analyses, genotyping and sequencing was performed to study viral presence and variation in PepMV sequences throughout the trial period. Two isolates (EU-mild and CH2-mild) that were selected based on mild symptom expression in the crop of origin caused only mild symptoms in the trial, while two other isolates (CH2-aggressive and EU + CH2) that were selected for severe symptom display, caused considerably more severe symptoms. Sequence homology between CH2-mild and CH2-aggressive was as high as 99·4%. Results of this study show that differential symptom expression can, at least partially, be attributed to the PepMV isolate, which may be related to minor differences at the nucleotide level between isolates.     

Detection,characterization and host range studies of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> in Cyprus

Pepino mosaic virus (PepMV, Genus Potexvirus, Family Flexiviridae) is a mechanically transmitted viral disease that has emerged as a significant problem of greenhouse tomato crops in Europe and around the world. Although previous studies in Cyprus suggested that the virus was not present on the island, in 2009 tomato fruits from two major tomato production areas exhibited symptoms of yellow mosaic and discolouration, similar to those induced by PepMV. Consequently, an extensive survey was conducted in all tomato producing areas of the country to identify the incidence and prevalence of PepMV in protected and open field tomato crops. Analysis of 3500 leaf samples from tomato plants and weeds with DAS-ELISA and real-time RT-PCR showed that PepMV was present in all tomato growing areas of the island. The virus was detected in both protected and open field tomato plants, as well as in 20 weed species in the families of Amaranthaceae, Chenopodiaceae, Compositae, Convolvulaceae, Malvaceae, Plantaginaceae and Solanaceae. All Cypriot isolates assayed belonged to the CH2 genotype. Biological assays with two Cypriot isolates showed that they could infect cultivated and weed species including Vigna unguiculata, Solanum melongena, Nicotiana tabacum, Malva parviflora, Sonchus oleraceus, Solanum nigrum, Convolvulus arvensis, Chrysanthemum segetum and Calendula arvensis. To our knowledge, this is the first study to report Chrysanthemum segetum and Calendula arvensis as hosts of PepMV.     

A pith necrosis caused by Xanthomonas perforans on tomato plants

Pith necrosis is a common disease of tomato in Europe, mainly caused by Pseudomonas corrugata and other soil-borne species of Pseudomonas. During 2011–2012 a survey was conducted in soil-grown tomato crops in southeastern Sicily (Italy). Plants showed pith necrosis, brown discolouration of the vascular tissues, leaf chlorosis and sometimes wilting of leaves. Thirty bacterial isolates from symptomatic tissues, forming colonies on NA and KB, were identified by morphological, biochemical and physiological tests. Among them, seven isolates were analyzed for their 16S rDNA and 16S–23S spacer region sequence that resulted in 99 % identity to that of the Xanthomonas perforans type strain (GenBank accession number GQ46173over 2.085 bp.). Additional sequences of fusA, gapA, gltA, gyrB, lacF, and lepA from one selected isolate were 100% identical to sequences of the Xanthomonas perforans type strain. X. perforans local isolates showed similar genomic patterns with REP-PCR and fAFLP, and were clearly distinguished from other Xanthomonas spp. type strains. In stem-inoculation assays, bacteria isolated from symptomatic tomato plants identified as P. fluorescens, P. putida, P. marginalis, P. citronellolis, P. straminea, and Pantoea agglomerans induced discolouration of vascular tissues, while Pectobacterium carotovorum subsp. atrosepticum isolates induced soft rot. Conversely, the isolates here identified as Xanthomonas perforans were able to induce pith necrosis, vascular discolouration, longitudinal splits and external lesions on stems. This report of X. perforans causing pith necrosis on tomato represents a potentially serious problem that may limit the productivity of tomato crops.     

Role of sponge gourd in apical leaf curl disease of potato in Northern India

Potato (Solanum tuberosum) is one of the important vegetable crops in the world and its production is seriously affected by apical leaf curl disease in northern India. This paper reveals the role of cucurbits in maintaining Tomato leaf curl New Delhi virus (ToLCNDV) and Potato apical leaf curl (PALCD) disease in that region. The affected plants showed severe leaf curling and stunted growth. The begomovirus causing leaf curling and mosaic disease in cucurbits could be easily transmitted by the whitefly to potato crops and develop apical leaf curl disease in northern India. The movement of the virus by whiteflies from cucurbits to potato and tomato is possible because of overlapping of planting and harvesting dates of these crops. The causal virus was identified as a begomovirus on the basis of whitefly transmission, PCR, dot blot hybridization, cloning and sequencing of the coat protein gene. The comparison of full length coat protein gene sequence homology revealed that 90% identity with the coat protein gene of ToLCNDV- [Luffa] isolate and the phylogenetic tree derived from these sequences with other selected begomoviruses formed a close cluster with ToLCNDV isolates. The findings proved that the virus causing disease in cucurbits could easily move to tomato and potato and cause leaf curl disease naturally. This is the first observation on the role of sponge gourd for maintenance of ToLCNDV and serving as a host for PALCD in northern India. The findings indicate that the causal organism is a strain of ToLCNDV.     

Biological and molecular characterization of Tomato spotted wilt Virus in Israel

Received April 24, 1997; received in final form June 29, 1997. Symptoms resembling tomato spotted wilt virus (TSWV) infections were documented among ornamental and vegetable crops in commercial greenhouses and open fields in Israel. Plants exhibiting these symptoms were collected from January 1992 to December 1996. Among cultivated plants analyzed for TSWV by enzyme-linked immunosorbent assay (ELISA), 19 species representing five families were found to be infected; natural infection was also recorded in six plant species of weeds. Virus identity was characterized by host range, serology and electron microscopy. Serological reaction with the isolates, found in Israel, using antisera from different sources as well as the sequence analysis of the nucleocapsid gene, demonstrated that the Israeli isolates of TSWV are a member of tospovirus serogroup I, type I (BR-01 strain). No virus transmission was found in seeds collected from virus-infected vegetable and ornamental crops. A non-radioactive molecular probe derived from the cloned nucleocapsid isolate enables specific detection of the virus in crude sap from infected plants. The detection of TSWV in Israel constitutes a severe potential threat to the ornamental and vegetable industry.     

引起番茄植株坏死病的病毒研究

 2004~2005年,在上海郊区夏番茄上连续发生严重的植株坏死病。表现为番茄植株矮化、顶端坏死、果实畸形和整株枯萎症状,并在叶柄和茎干上出现不规则坏死条斑,具有典型的病毒病特征。通过采集典型病叶、病果,进行病毒分离、dsRNA分析、寄主生物学研究、病毒纯化、病毒蛋白分析、组织病理学与形态学观察和ELISA检测,初步确定是由番茄花叶病毒(Tomato mosaic virus,ToMV)和黄瓜花叶病毒(Cucumber mosaic virus,CMV)侵染。经5次心叶烟单斑分离,获得N5分离物,对CP基因克隆测序的结果确定该分离物为ToMV。寄主反应测定显示,N5的寄主反应与常规ToMV株系存在明显区别,主要表现为常规番茄品种上出现严重坏死。进一步接种GCR品系番茄鉴定N5与ToMV-1株系相似。因此,作者认为,在上海地区番茄上流行并引起坏死病的主要病原可能是ToMV-1株系的1个变异株。     

Pepper (Capsicum annuum) Is a Dead-End Host for Tomato yellow leaf curl virus

ABSTRACT Tomato yellow leaf curl (TYLC) is one of the most devastating pathogens affecting tomato (Lycopersicon esculentum) worldwide. The disease is caused by a complex of begomovirus species, two of which, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), are responsible for epidemics in Southern Spain. TYLCV also has been reported to cause severe damage to common bean (Phaseolus vulgaris) crops. Pepper (Capsicum annuum) plants collected from commercial crops were found to be infected by isolates of two TYLCV strains: TYLCV-Mld[ES01/99], an isolate of the mild strain similar to other TYLCVs isolated from tomato crops in Spain, and TYLCV-[Alm], an isolate of the more virulent TYLCV type strain, not previously reported in the Iberian Peninsula. In this work, pepper, Nicotiana benthamiana, common bean, and tomato were tested for susceptibility to TYLCV-Mld[ES01/99]and TYLCV-[Alm] by Agrobacterium tumefaciens infiltration, biolistic bombardment, or Bemisia tabaci inoculation. Results indicate that both strains are able to infect plants of these species, including pepper. This is the first time that infection of pepper plants with TYLCV clones has been shown. Implications of pepper infection for the epidemiology of TYLCV are discussed.     

Genetic characterization of <Emphasis Type="Italic">Pepino mosaic virus</Emphasis> isolates from Belgian greenhouse tomatoes reveals genetic recombination

Over a period of a few years, Pepino mosaic virus (PepMV) has become one of the most important viral diseases in tomato production worldwide. Infection by PepMV can cause a broad range of symptoms on tomato plants, often leading to significant financial losses. At present, five PepMV genotypes (EU, LP, CH2, US1 and US2) have been described, three of which (EU, LP and US2) have been reported in Europe. Thus far, no correlation has been found between different PepMV genotypes and the symptoms expressed in infected plants. In this paper, the genetic diversity of the PepMV population in Belgian greenhouses is studied and related to symptom development in tomato crops. A novel assay based on restriction fragment length polymorphism (RFLP) was developed to discriminate between the different PepMV genotypes. Both RFLP and sequence analysis revealed the occurrence of two genotypes, the EU genotype and the CH2 genotype, within tomato production in Belgium. Whereas no differences were observed in symptom expression between plants infected by one of the two genotypes, co-infection with both genotypes resulted in more severe PepMV symptoms. Furthermore, our study revealed that PepMV recombinants frequently occur in mixed infections under natural conditions. This may possibly result in the generation of viral variants with increased aggressiveness.     

Phylodynamics of Pepino mosaic virus in Spain

Pepino mosaic virus (PepMV) is an emerging pathogen that causes severe economic losses in tomato crops in the Northern hemisphere. After its first identification, the new viral strain PepMV-CH2 has been isolated in several countries worldwide. In order to further understand the evolutionary dynamics of PepMV before and after PepMV-CH2 emergence, we analyzed a collection of PepMV isolates from southeastern Spain, estimating the rate of PepMV molecular evolution and the coalescence process for the effective number of PepMV infections using a Bayesian phylogenetic approach. Our results show that the rate of PepMV molecular evolution was 5.570?×?10^?3 substitutions/site/year, a value which is approximately an order of magnitude higher than the rates recently reported for other plant RNA viruses. Moreover, PepMV-CH2 was estimated to have originated in 2000, coincident with the onset of PepMV-CH2 infections in southeastern Spain, its population following now an expansion process. This further illustrates that genetic and ecological interactions among different viral strains can modulate the evolutionary dynamics of PepMV and determine its epidemiological profile.     

First report of cucumber mosaic cucumovirus in banana from Pakistan

During regular surveys of banana plantations in the Sindh province of Pakistan for banana bunchy top nanavirus (BBTV), sporadic plants showing mosaic, chlorotic spots intermingled with a dark green area were observed at a few places in the districts of Thatta, Hyderabad and Nawabshah. The infected young plants were uprooted and grown in pots for 2 years to ascertain the cause of the disease. Apart from studying the development of symptoms, the infected plants were tested for the presence of BBTV, cucumber mosaic cucumovirus (CMV), chilli veinal mottle potyvirus (CVMV), tomato mosaic tobamovirus (TMV), potato X potexvirus (PVX) and potato Y potyvirus (PVY) by DAS-ELISA. Virions were also partially purified. The infected plants developed typical symptoms of CMV. All tested plants were ELISA-positive only for CMV, and a partially purified preparation revealed virus particles measuring 25–28 nm in diameter. Based on the characteristic field symptoms, serology and particle morphology, the presence of CMV in banana in Pakistan was established.     

西藏昌都市卡若区蔬菜主要病毒检测及番茄斑萎病毒鉴定

为明确西藏昌都市卡若区高原条件下的蔬菜主要病毒种类, 对温室、大棚和露地栽培的主要蔬菜进行了病毒病调查, 采集典型病毒病症状样品进行ELISA检测, 明确病毒种类; 并利用电子显微镜观察, RT-PCR扩增克隆与测序分析对主要病毒进行鉴定分析。ELISA检测结果表明, 西藏昌都市卡若区温室及大棚栽培的番茄、辣椒和莴苣上的主要病毒有番茄斑萎病毒(tomato spotted wilt virus, TSWV)、马铃薯Y病毒(potato virus Y, PVY)及凤果花叶病毒(pepino mosaic virus, PepMV)。其中TSWV检出率最高, 为45%。进一步对检出TSWV的蔬菜样品进行电子显微镜观察, 发现其中含有典型的正番茄斑萎病毒属Orthotospovirus病毒粒体, 应用TSWV-N基因特异性引物进行RT-PCR扩增克隆和序列分析, 发现西藏昌都市卡若区蔬菜感染的TSWV与云南TSWV分离株亲缘关系最近。本研究结果明确了西藏昌都市卡若区蔬菜的主要病毒种类。综合抗体检测、病毒粒体形态观察与分类相关基因的克隆测序结果, 明确了西藏昌都市卡若区蔬菜感染的主要病毒为TSWV。这也是TSWV在西藏的首次报道, 为了解TSWV的发生分布及其防控提供了依据。