Partial Nucleotide Sequence and Genome Organization of a Canadian Isolate of Little cherry virus and Development of an Enzyme-Linked Immunosorbent Assay-Based Diagnostic Test

ABSTRACT Approximately 12.4 kb of the genome of a mealybug-transmissible, North American isolate of Little cherry virus (LChV-3, previously designated LChV-LC5) has been cloned and sequenced. The sequenced portion of the genome contains 10 open reading frames (ORFs) and, based on sequence comparisons, encodes a putative RNA helicase (HEL), RNA-dependent RNA polymerase (POL), two coat proteins (CPs), a homologue of HSP70, a 53K protein (p53) that is similar to an equivalent-size protein in other closteroviruses, and a 22K (p22) protein of unknown function. The genome also potentially encodes two small proteins (p5 and p6), one of which is similar to the small hydrophobic proteins of other closteroviruses. Phylogenetic analyses utilizing sequences of the HEL, POL, and HSP70 homologue suggest that LChV-3 is most similar to other mealybug-transmitted closteroviruses. Further comparisons between LChV-3 and a 4.7-kb region of the recently described Little cherry virus-2 (LChV-2) reveals 77% nucleotide sequence identity. Based on this low sequence identity, we propose that LChV-3 be considered a separate species, designated LChV-3. Unexpectedly, the LChV-3 CP duplicate ORF was found to lie upstream of the HSP70 ORF; therefore, the genome organization of LChV-3 is distinct from that of other closteroviruses. Polyclonal antiserum raised to bacterially expressed LChV-3 CP was useful for detection of LChV-diseased trees in the cherry-growing districts of British Columbia, Canada.     

Characterization and Detection of Several Filamentous Viruses of Cherry: Adaptation of an Alternative Cloning Method (DOP-PCR), and Modification of an RNA Extraction Protocol

For the identification and analysis of new RNA plant viruses infecting fruit trees, an initial step often involves the laborious procedure of isolation and cDNA synthesis and cloning from purified viral dsRNA. For subsequent RT-PCR detection of these and other viruses from tissue with high phenolic and polysaccharide concentrations, a simple and efficient extraction protocol for viral nucleic acid is also important. A method for rapid cDNA cloning from small amounts of purified dsRNA using a modification of degenerate oligo primed polymerase chain reaction mbox(DOP-PCR), and a modification of a protocol for effective extraction of viral RNA for use in RT-PCR are presented. Both methods were used to analyze a number of mottling diseases described in cherry. The causal agents for two of these diseases have been previously described, Cherry green ring mottle virus, a tentative member of the foveaviruses, and Cherry mottle leaf virus, a member of the trichoviruses. For the diseases cherry rusty mottle and cherry necrotic rusty mottle, data are presented identifying viruses associated with each disease. Viruses associated with cherry rusty mottle, cherry necrotic rusty mottle and European isolates of cherry mottle leaf diseases, are closely related to Cherry green ring mottle virus and can be tentatively included in the foveavirus genus. An additional virus, related to cherry green ring mottle virus, was discovered by RT-PCR cloning and appears to be a common latent virus of cherry. Finally, isolates of cherry necrotic mottle disease could be assayed positive by RT-PCR for a virus     

侵染肥城桃的病毒和类病毒的分子检测与鉴定

为明确山东肥城桃种植区桃树上主要存在的病毒和类病毒及其发生情况,采集具有花叶、斑驳和皱缩典型症状的肥城桃样品,提取叶片总RNA后,分别选用桃树上已报道的啤酒花矮化类病毒Hopstuntviroid(HSVd)、桃潜隐花叶类病毒Peach latent mosaic viroid(PLMVd)、苹果褪绿叶斑病毒Apple chlorotic leaf spot virus(ACLSV)、樱桃锉叶病毒Cherry rasp leaf virus(CRLV)、桃花叶病毒Peach mosaic virus(PMV)、李属坏死环斑病毒Prunus necrotic ringspot virus(PNRSV)、李痘病毒Plum pox virus(PPV)、李矮缩病毒Prunus dwarf virus(PDV)、樱桃绿环斑驳病毒Cherry green ring mottle virus(CGRMV)、杏假褪绿叶斑病毒Apricot pseudo-chlorotic leaf spot virus(APCLSV)、李树皮坏死茎纹孔伴随病毒Plum bark necrosis stem pitting-associated virus(PBNSPaV)和小樱桃病毒1号Little cherry virus 1(LchV1)的特异性引物进行RT-PCR检测。PCR结果显示仅HSVd、PLMVd、ACLSV、PNRSV和PBNSPaV的扩增产物中得到了预期大小的目的片段,将目的片段克隆测序后,经NCBI BLAST比对发现,山东肥城桃分离物HSVd、PLMVd、ACLSV、PNRSV和PBNSPaV与GenBank已报道分离物序列一致性均达90%以上。表明山东肥城桃已感染HSVd、PLMVd 2种类病毒和ACLSV、PNRSV、PBNSPaV 3种病毒。     

First record of Rhagoletis cingulata (Loew) (Dipt., Tephritidae) in Austria

The Nearctic eastern cherry fruit fly species Rhagoletis cingulata (Loew) (Dipt., Tephritidae) has been detected several times in different European countries during the last decades. This species as well as Rhagoletis indifferens (Curran) are major pests of cultivated cherries in North America and are classified as quarantine pests in Europe. The introduction and establishment of both species could result in severe problems for Austrian cherry production due to additional infestation pressure caused by overlapping developmental cycles of American and native cherry fruit flies. A survey of both non-European cherry fruit fly species was carried out during the growing seasons of 2007 and 2008 at eleven sampling sites in the eastern part of Austria. Pherocon^® AM yellow sticky traps were installed in cherry trees and replaced at weekly or fortnightly intervals. Identification of the cherry fruit flies caught was based on morphological characteristics. Two specimens of R. cingulata were caught in 2007 in different weeks and at different locations while none were caught in 2008. R. indifferens was not detected at all. While it is possible that these specimens originate from established populations with low densities, it is more likely that the catches derived from accidental introductions.     

Apple necrotic mosaic virus,a novel ilarvirus from mosaic-diseased apple trees in Japan and China

The causal agent of apple mosaic disease has been previously thought to be solely caused by apple mosaic virus (ApMV). In this study, we report that a novel ilarvirus is also associated with apple mosaic disease. Next-generation sequencing analysis of an apple tree showing mosaic symptoms revealed that the tree was infected with three apple latent viruses (apple stem pitting virus, apple stem grooving virus, and apple chlorotic leaf spot virus) and a novel ilarvirus (given the name apple necrotic mosaic virus (ApNMV)) that is closely related to Prunus necrotic ringspot virus (PNRSV) and ApMV. The genome of ApNMV consists of RNA1 (3378 nt), RNA2 (2767 nt), and RNA3 (1956 nt). A phylogenetic analysis based on the coat protein amino acid sequences indicated that the novel virus belongs to the same subgroup 3 of the genus Ilarvirus as PNRSV and ApMV. The presence of mosaic leaves, which tend to be unevenly distributed in diseased apple trees, was correlated with the internal distribution of ApNMV. RT-PCR detection of mosaic-diseased apple trees in Japan indicated that ApNMV was detected in apple trees introduced from China, whereas ApMV was detected from cultivated apple trees in domestic orchards. Consistent with these findings, a survey of mosaic-diseased apple trees in major apple-producing provinces in China revealed that the majority of apple trees showing mosaic symptoms in China are infected with ApNMV.     

Characterization of Beet Yellows Closterovirus-Specific RNAs in Infected Plants and Protoplasts

ABSTRACT Tetragonia expansa plants infected with a California isolate of beet yellows virus (BYV-60) contained multiple BYV-specific RNAs identified by Northern blot hybridization. These RNAs were identified by cDNA probes specific to six open reading frames (ORFs). One genomic RNA and five subgenomic (sg) RNAs representing the p65/p6.4, p64, p24, p22, and p21 ORFs were identified. A probe derived from the 3'-terminal ORF (p21) hybridized to each of the sgRNAs, indicating the RNAs are 3' coterminal. Hybridization with 5'- and 3'-end probes indicated that preparations of BYV particles contained the genomic RNA as well as two additional RNA molecules corresponding in size to the coat protein (CP) sgRNA and an unidentified RNA. A Chenopodium quinoa protoplast system also was used to study BYV replication. The temporal accumulation of BYV-specific RNAs and CP was investigated in protoplasts transfected with purified virion RNA. Accumulation of genomic plus-strand RNA was evident as early as 15 h postinoculation. The development of this protoplast system is significant for studies of closterovirus replication.     

Insights into the genetic diversity and evolution of Little cherry virus 1

Little cherry virus 1 (LChV‐1), a member of the recently proposed genus Velarivirus, is a sweet cherry pathogen that has been recently reported to infect other Prunus species and is associated with various plant disorders. In this work the incidence of the virus on its putative hosts and possible mechanisms driving its evolution were investigated. Due to problems encountered with LChV‐1 detection, a new nested RT‐PCR assay was developed and applied. The virus was found to be prevalent in cherry plantations in Greece and only occasionally detected in other Prunus species. Sequences corresponding to the partial RNA‐dependent RNA polymerase (RdRp), heat‐shock protein homologue (HSP70h) and coat protein (CP) genes were determined from Greek LChV‐1 isolates originating from different hosts; these were analysed, along with published homologous genomic regions from other isolates. Phylogenetic analysis of the three genes revealed the segregation of four evolutionary distinct groups showing no host or geography‐based clustering. Mean genetic distances among the four groups were high with the CP region showing the highest divergence, although intragroup variability levels were low. Nevertheless, estimations of the mean ratio of nonsynonymous substitutions per synonymous site to synonymous substitutions per synonymous site (dN/dS) for the partial RdRp, HSP70h and CP indicated that these genomic regions are under negative selection pressure. Interestingly, a recombination event was identified at the 3′ end of RdRp on a Greek virus isolate, thus highlighting the role of this mechanism in the evolutionary history of LChV‐1.     

On the presence and distribution of olive viruses in Lebanon

A survey for viruses was carried out in 2003 in the main olive-growing areas of Lebanon (South Lebanon, North Lebanon, Mount Lebanon and Bekaa). A total of 300 samples was collected in 31 different locations in 76 different commercial orchards and checked by RT-PCR for the presence of Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Strawberry latent ringspot virus (SLRV), Olive latent virus 1 (OLV-1) and Olive leaf yellowing-associated virus (OLYaV), using virus-specific primers reported in the literature. About one third (31%) of the trees were infected. In particular, the closterovirus OLYaV was the most widespread, as it was detected in 23.7% of the samples, followed by the necrovirus OLV-1 (8.3%), the two nepoviruses CLRV (2%) and ArMV (0.3%), and the sadwavirus SLRV (0.3%). A high variability was detected in the HSP70 gene of Lebanese and Italian OLYaV isolates, for at least nine different patterns were obtained when this genomic region was subjected to single-strand conformation polymorphism (SSCP) analysis.     

The occurrence of beet pseudo-yellows virus in England

Beet pseudo-yellows virus (BPYV) has been identified in England for the first titne, in lettuce, following transmission by the greenhouse whitefly ( Trialeurodes vaporariorum ) to healthy plants. Infected plants had leaves with severe interveinal chlorosis and slight downward curling, and were gerterally smaller than healthy plants. Indirect ELISAs with an antiserum raised against the Californian isolate of BPYV were positive with extracts produced from infected plants. Infected but not healthy plants contained a single species of double-stranded RNA of about 7.5 × lO⁶Da, a size similar to that found with beet yellows virus, the type member of the closterovirus group.     

Viruses and virus-like diseases of olive1

Partial paralysis, leaf malformation, sickle leaf and infective yellowing are diseases of possible virus etiology recorded in different areas where olive is cultivated. Several attempts to reproduce by grafting the symptomatology observed in the field did not give completely satisfactory results. Attempts to isolate and identify the causal agent of these diseases also failed. Only in 1976 was a virus isolated in Italy from an olive tree, cv. Correggiolo, which did not however show any symptoms. The virus was identified as a strain of strawberry latent ringspot nepovirus (SLRV), which is present in Italy on a few other fruit trees (peach, cherry). On olive, SLRV is found in central Italy and has only in one case been associated with a specific malformation of the leaves and fruits. Since this first report, several other viruses have been isolated from olive trees which did not, anyway, present any symptoms of virus disease.     

Viruses and virus diseases of grapevine in Lebanon

Grapevines were surveyed for the presence of virus and virus-like diseases in the main viticultural areas of Lebanon (Bekaa valley, Mount Lebanon, South and North Lebanon). Symptoms of rugose wood were observed in vines ofall cultivars and areas surveyed, whereas leafroll was observed only in some vineyards of the Bekaa valley and, to a lesser extent, in South Lebanon on cvs Tfaifihi, Cinsaut and Cardinal. Symptoms of fanleaf and of phytoplasma-induced yellows were also observed with low frequency in the Bekaa valley on wine-grape cultivars. ELISA tests showed that 53% of 1536 Vitis vinifera vines individually checked were infected by one or more viruses. Grapevine trichovirus A (GVA) was the prevailing virus (32.4%), followed by grapevine fleck virus (GFkV) (19.5%) and grapevine leafroll-associated closterovirus 3 (GLRaV-3) (12.4%). Grapevine leafroll-associated closterovirus 1 (GLRaV-l), grapevine trichovirus B (GVB) and grapevine fanleaf nepovirus (GFLV) were also detected to a lesser extent, their incidence ranging between 1.1 and 3.6%.     

Purification and some properties of Papaya meleira virus, a novel virus infecting papayas in Brazil

'Meleira', or 'sticky disease', is currently the most damaging papaya disease in the mid-eastern Brazilian growing regions. Consistent disease transmission via latex injection, presence of similar isometric particles in the laticiferous vessels of diseased plants, and detection of double-stranded DNA in naturally and experimentally infected papaya trees suggest that a virus is the causal agent. Conclusive evidence for viral aetiology was previously lacking, mostly because every attempt to purify the putative virus from infected papayas had failed. Following the successful purification and partial characterization of the meleira virus, healthy papaya seedlings injected with purified virus particles later developed typical symptoms of the disease. Negatively stained, isometric, full and 'empty' purified virus particles measured 42 and 38 nm, respectively. The viral genome was a single dsRNA molecule of about 12 kbp. Several capsid proteins, ranging in size from 14·4 to 45 kDa, were consistently revealed by PAGE. Papaya meleira virus (PMeV) appears to represent a novel group of viruses, with no known similar counterpart among known plant-, vertebrate-, invertebrate- or prokaryote-infecting viruses.     

Molecular and biological characterization of Potato mop‐top virus (PMTV,Pomovirus) isolates from the potato‐growing regions of Colombia

Potato mop‐top virus (PMTV) causes necrotic flecks inside and on tubers in temperate countries. In South America, these symptoms have not been observed, although the presence of the virus has been confirmed in the Andes and in Central America. To characterize PMTV isolates from the Andes, soil samples were taken from the main potato‐producing regions in Colombia and virus was recovered by planting Nicotiana benthamiana as bait plants. The complete genomes of five isolates were sequenced and three of the isolates were inoculated to four different indicator plants. Based on sequence comparisons, three types of RNA‐CP (RNA2) and RNA‐TGB (RNA3) were found. The isolates from the centre of the country (CO3 and CO4) were similar to isolates from Europe. The genomes of CO1, CO2 and CO5 differ from other PMTV isolates, placing them in a separate clade in phylogenetic trees. The three Colombian isolates (CO1, CO2 and CO5) only induced slightly different symptoms in the indicator plants. However, the isolate from the northwest of the country (CO1) induced stronger symptoms in N. benthamiana including severe stunting. A correlation between the genotype of the isolates and the symptoms they induced on indicator plants was not found.     

北美森林新病害——山毛榉叶线虫病

2012年,北美地区发生了一种由山毛榉李氏垫刃线虫麦肯恩亚种(Litylenchus crenatae mccannii)引起的森林新病害——山毛榉叶线虫病,病情蔓延迅速,已扩散至美国和加拿大30个县。病原为害山毛榉属植物,可造成病树成片死亡。由于山毛榉是北美温带阔叶林的主要构成树种和重要用材树种,新病害已引起美国农业部的高度重视并采取积极的应对措施。我国分布5种山毛榉属植物(均为特有种),是我国南方森林的主要组成树种。鉴于我国每年从北美进口大量山毛榉木材,病原线虫存在随进境木材传入国内的巨大风险。因此,检疫部门应开展风险评估,口岸应针对性开展山毛榉叶线虫病的检测。本文主要介绍了山毛榉叶线虫病的发生历史、分布范围、为害症状、病原线虫形态学特征、生活史、传播途径、分子检测方法等方面的信息,以期为口岸检疫工作提供参考。