Cytopathology of Tomato torrado virus Infection in Tomato and Nicotiana benthamiana

Electron microscopy studies were carried out to investigate the cytopathological changes induced in tomato leaves by Tomato torrado virus (ToTV) that infects tomato plants worldwide causing severe necrotic symptoms. Plants infected with one of the Polish isolates of ToTV were used for cytopathological research. The results revealed severe cellular alterations, especially in Solanum lycopersicum. Moreover, it was shown that crystalline aggregates of virions occurred not only within the phloem cells as it has been previously reported.     

Genome Characteristics,Phylogeny and Varying Host Specificity of Polish Kra and Ros Isolates of Tomato torrado virus

The genome sequences of two Polish Kra and Ros isolates of Tomato torrado virus (ToTV) were determined and compared with data of previously described ToTV isolates and other Torradovirus members. Whole‐genome sequence comparisons revealed 97.0–99.6% nucleotide sequence identities and close relatedness, with other known ToTV isolates. The high homology between Kra, Ros and Wal'03 ToTVs is likely responsible for the similar symptoms observed on infected plants. However, the symptoms differed in intensity and various host specificity. We report that Kra ToTV caused a milder expression of symptoms on Solanum tuberosum than Wal'03. We hypothesize this may be a result of the significant variability observed within the 3′‐UTR of RNA1 of Kra as well as of Ros ToTV isolates. In the light of this fact, potato may be considered an indicator plant for distinguishing Kra and Wal'03 ToTV isolates.     

Biological and Molecular Characterization of Three Isolates of Tomato aspermy virus Infecting Chrysanthemums in India

Severe mosaic, chlorotic ringspots and flower deformation were observed during the winter of November 2006–February 2007 on chrysanthemums ( Chrysanthemum morifolium ) at three locations in India: Lucknow (UP), Dhanbad (MP) and Kolkata (WB). Tomato aspermy virus (TAV) was detected in affected plants by ELISA and by RT-PCR using TAV specific primers. These TAV isolates were mechanically transmitted to test plant species and also by aphids ( Aphis gossypii ) to Lycopersicon esculentum . The complete RNA 3 of each TAV isolate was cloned and sequenced and determined to be 2386 nucleotides (nt) long, and to encode two open reading frames (ORFs): the movement protein (MP) of 741 nt and the coat protein (CP) of 657 nt translating in to 246 and 218 amino acid (aa), respectively. When RNA 3 sequences of the Indian isolates were multiple aligned with seven other strains of TAV occurring worldwide, Indian isolates shared 98–99% identities among themselves and with the KC, V, P, B, I and C strains of TAV. In phylogenetic analysis, the Lucknow and Kolkata isolates of TAV clustered together and showed a close relationship with the KC-TAV strain from South Korea, whereas the Dhanbad isolate formed an independent cluster and showed closeness with the V-TAV strains from Spain and Australia. Recombination events were also observed in the CP region of the Dhanbad isolate, supporting its diverse behaviour. This is the first report of the complete RNA 3 sequence of these three Indian TAV isolates.     

Characterization and Complete Nucleotide Sequence of Two Isolates of Tomato mosaic virus

Two virus isolates, designated S1 and TL, were obtained from tomato and camellia root in China, respectively, and their host ranges, symptomatology, serological reactions and complete nucleotide sequences were determined. Isolate TL systemically infected Chenopodium amaranticolor causing leaf chlorosis, but the isolate S1 induced only local necrotic lesions. The complete nucleotide sequences of S1 and TL were determined and consisted of 6384 and 6383 nucleotides (Genbank accessions AJ132845 and AJ417701 ), respectively. Sequence analysis revealed that both isolates have the highest nucleotide sequence identity (over 92%) with Tomato mosaic virus (ToMV), but less (80%) with other tobamoviruses. Phylogenetic analyses based on the amino acid sequences of 30‐kD and 17.5‐kD proteins also indicated that both the isolates form a cluster with the isolates of ToMV. These data suggest that S1 and TL are isolates of ToMV. The possible reasons that TL infected C. amaranticolor systemically but S1 induced only local necrotic lesions are discussed.     

New Necrotic Isolates of Pepino mosaic virus Representing the Ch2 Genotype

New necrotic isolates of Pepino mosaic virus (PepMV) were found in 2007 infecting greenhouse tomato plants in Poland. The isolates differ from previously identified PepMV isolates in host range and symptomatology. They induce severe necrosis on tomato plants ( Solanum lycopersicum ) and local necrotic lesions on Datura inoxia . Phylogenetic analysis, based on three distinct regions, triple gene block 1, the coat protein gene and a part of polymerase gene, revealed that the new necrotic isolates share high nucleotide sequence identity with isolates of the Ch2 genotype. This is the first report describing a necrotic type of PepMV of the Ch2 genotype.     

Characterization and Experimental Host Range of a Brazilian Tomato Isolate of Tomato severe rugose virus

We report the complete molecular characterization of the DNA‐A and DNA‐B of a Brazilian tomato isolate of Tomato severe rugose virus (ToSRV) and the experimental host range of the virus determined using whitefly transmission tests. Genome analysis showed that ToSRV has a close evolutionary relationship with Tomato rugose mosaic virus. Of 33 plants species inoculated with viruliferous Bemisia tabaci biotype B, 13 species were susceptible to ToSRV, nine asymptomatically. Therefore, ToSRV disease management strategy should include the control of infected weeds close to tomato fields.     

Expression of the Full‐length Coat Protein Gene of Tomato leaf curl Taiwan virus is Not Necessary for Recovery Phenotype in Transgenic Tomato

Transgenic tomato plants expressing full‐length (CPV1) and truncated coat protein (CP) gene (CPV2) of Tomato leaf curl Taiwan virus (ToLCTWV) were generated by Agrobacterium‐mediated transformation. Transgene integration and expression was confirmed by PCR and Southern blotting and Northern analysis, respectively. Resistance was evaluated both in plants of T0 and T1 progenies using viruliferous whiteflies under two different inoculum pressures (10–15 and 40–50 whiteflies/plant). Upon inoculation with ToLCTWV using viruliferous whiteflies, various levels of phenotypic reaction were observed. No complete resistance was observed in any of the plants tested. The reaction of the transgenic tomato lines carrying full‐length and truncated CP gene to ToLCTWV phenotype was (i) susceptible as non‐transgenic control, (ii) delayed symptom expression, (iii) complete susceptible (from delayed symptom expression phenotype) and (iv) recovered phenotype (either plants from symptom expression as non‐transgenic plants or delayed symptom expression phenotype). Dot blot quantification of the ToLCTWV using the replicase gene as a probe revealed that the recovered phenotypes accumulated a low level of ToLCTWV, and virus concentration was gradually reduced from 10 to 14 weeks postinoculation. The possible mechanisms of CP‐mediated resistance are discussed.     

Molecular and Biological Characterization of Potato virus Y Isolates from Vietnam

Eight isolates from different potato growing regions in Vietnam were characterized. All were highly pathogenic in some potato cultivars, but did not overcome the extreme resistance of Solanum stoloniferum and Solanum demissum. RT‐PCR analysis revealed that all of these isolates are recombinants. Sequence data for 4 isolates were obtained, and their reaction in potato cultivars harbouring specific N genes was determined. Different phylogenetic analyses of viral sequences confirmed previous results that the recombinant isolates evolved from different parental sequences. One of the Vietnamese isolates investigated had a specific structure. The need for a clear classification of PVY^NWi isolates is discussed.     

Molecular Structure and Properties of Lectin from Tomato Fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro₄ motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

Epidemics of Tomato torrado virus,Pepino mosaic virus and Tomato chlorosis virus in tomato crops: do mixed infections contribute to torrado disease epidemiology?

Torrado disease was first observed in protected tomato crops in the Murcia province of Spain in spring 2001, causing serious concern to regional tomato producers. The disease-causing agent was initially identified as a picorna-like bipartite plant RNA virus, now known as Tomato torrado virus (ToTV), but several additional torradoviruses inducing similar disease symptoms have been described more recently. We studied the incidence of torradoviruses between 2005 and 2008 in two parts of Murcia (Spain) where tomato crops are grown commercially. We also analysed the potential association among ToTV, Pepino mosaic virus (PepMV) and Tomato chlorosis virus (ToCV) in samples showing torrado symptoms of varying severity. ToTV was the only torradovirus found in the samples (predominantly as single infections), but double and triple infections comprising ToTV, PepMV and/or ToCV were also detected. There was no evidence of a specific association among the viruses as the frequencies of mixed infections did not deviate from those expected to occur by chance. Statistical analysis of the potential association between torrado symptoms and the type of infection (single or multiple) was inconclusive. To determine whether co-infections with ToTV and PepMV have any marked influence on the torrado disease, we analysed torrado symptom severity and virus accumulation in tomato plants experimentally infected with ToTV-CE, PepMV-Sp13 and PepMV-PS5 in single and mixed infections. The severity of the torrado symptoms was not affected by the presence of PepMV. In single infections, the ToTV titre remained very low, reaching its maximum in the early stages of infection and declining rapidly thereafter, whereas the disease symptoms became more severe over the same timescale. In mixed infections, the accumulation of both ToTV and PepMV was altered with respect to single infections, and the magnitude of this alteration appeared to be virus and strain specific. Therefore, ToTV and PepMV mixed infections may modulate the epidemiology of both viruses in a complex way by altering virus fitness. The impact of our studies on efforts to track and prevent the spread of torrado disease is discussed.     

Elderberry latent virus: its relationship to Pelargonium ringspot virus and its identification as a distinct member of the genus Carmovirus, family Tombusviridae

The properties of Elderberry latent virus (ELV) and Pelargonium ringspot virus (PelRSV) were compared. The viruses were largely indistinguishable in herbaceous host range and symptomatology, particle morphology, sedimentation coefficient and RNA profiles and size. They were also very closely related serologically with SDI differences in agarose gel double‐diffusion tests of 1 to 3. Purified virus particle preparations of each virus contained isometric particles c. 30 nm in diameter that sedimented as a major component with an s^O_20W of 112–115S. Purified virus particle preparations contained a major and a minor ssRNA species that in polyacrylamide gel electrophoresis (PAGE) had estimated sizes of c. 3.8 kb and c. 1.6 kb respectively. Plants of Chenopodium quinoa infected with ELV or PelRSV each contained three dsRNA species of c. 3.8, 2.6 and 1.8 kbp, although the smallest of these species was not evident in all preparations. Protein from purified virus particle preparations contained a major polypeptide that, in SDS‐PAGE, had an estimated M^r of 40 000 (40K). However, after storage of purified virus particles for 7–10 days, protein preparations from PelRSV particles also contained an additional major polypeptide of estimated M^r of 37 000 that is probably derived by degradation of the 40K protein; this additional component was not observed in freshly prepared preparations of ELV. Neither virus was found to be related serologically to 16 other viruses with isometric particles and similar properties. These data, together with the recent finding by other researchers that the smallest RNA species is a sub‐genomic RNA, suggests that both viruses are members of the genus Carmovirus, and that PelRSV is a minor variant of ELV. However, the taxonomic status of these two viruses is discussed in relation to recent brief reports comparing the nucleotide and amino acid sequences of these two viruses.     

The circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci— insights from studies with Tomato yellow leaf curl virus

Our current knowledge concerning the transmission of begomoviruses by the whitefly vector Bemisia tabaci is based mainly on research performed on the Tomato yellow leaf curl virus (TYLCV) complex and on a number of viruses originating from the Old World, such as Tomato leaf curl virus, and from the New World, including Abutilon mosaic virus, Tomato mottle virus, and Squash leaf curl virus. In this review we discuss the characteristics of acquisition, transmission and retention of begomoviruses by the whitefly vector, concentrating on the TYLCV complex, based on both published and recent unpublished data. We describe the cells and organs encountered by begomoviruses in B. tabaci. We show immunolocalisation of TYLCV to the B. tabaci stylet food canal and to the proximal part of the descending midgut, and TYLCV‐specific labelling was also associated with food in the lumen. The microvilli and electron‐dense material in the epithelial cells of the gut wall were also labelled by the anti TYLCV serum, pointing to a possible virus translocation route through the gut wall and to a putative site of long‐term virus storage. We describe the path of begomoviruses in their vector B. tabaci and in the non‐vector whitefly Trialeurodes vaporariorum, and we follow the rate of virus translocation in these insects. We discuss TYLCV transmission between B. tabaci during mating, probably by exchange of haemolymph. We show that following a short acquisition access to infected tomato plants, TYLCV remains associated with the B. tabaci vector for weeks, while the virus is undetectable after a few hours in the non‐vector T. vaporariorum. The implications of the long‐term association of TYLCV with B. tabaci in the light of interactions of the begomovirus with insect receptors are discussed.     

侵染番茄的番茄花叶病毒的研究

从种传番茄苗中获得一病毒分离物Ｔｏ－Ｓｌ,人工摩擦接种７科２４种植物,Ｔｏ～Ｓｌ能侵染４科１５种植物,在番茄上产生花叶,在白肋烟上为局部枯斑。Ｔｏ－Ｓｌ的钝化温度为８５～９０℃,稀释限点为１０￣（－６）～１０￣（－７）．体外保毒期在一个月以上。病毒粒体杆状,长度主要分布于２８１～３００ｎｍ之间,平均长度２８８ｎｍ。病毒衣壳蛋白亚基只有一条多肽链,分子量为２１ｋＤａ。ｄｓＲＮＡ分析测得其基因组长度为６．４ｋｂ。琼胎糖双扩散和胶内交叉吸附试验证明,Ｔｏ－Ｓｌ与ＴＭＶ有血清关系,但有一定的差异,病毒粒体电泳分析也表明Ｔｏ－Ｓｌ与ＴＭＶ粒体有差异。在交叉保护试验中,ＴＭＶ和Ｔｏ－Ｓｌ之间均无保护作用。根据以上试验结果Ｔｏ－Ｓｌ被鉴定为番茄花叶病毒。这是我国首次系统报道番茄上番茄花叶病毒的侵染。     

Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus Invade South-east Coast of China

An epidemic outbreak of severe yellow leaf curl disease was reported in field grown tomato within Zhejiang Province of China in the autumn–winter cropping season of 2006. A molecular diagnostic survey was carried out based on comparisons of partial and complete viral DNA sequences. Comparison of partial DNA‐A sequences amplified with degenerate primers specific for begomoviruses confirmed the presence of two types of begomoviruses. The complete DNA sequences of five isolates, corresponding to the two types, were determined. Sequence comparisons and phylogenetic analysis revealed that they correspond to two previously identified begomoviruses, Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus. The satellite DNAβ molecule was not detected in these samples by either PCR or Southern blot hybridization analysis. There has been no previous report of geminivirus disease incidence in Zhejiang Province, indicating that the introduction of these two tomato infecting geminiviruses into the agro‐ecological zone of South‐eastern China is a fairly recent event. The implications for disease control are discussed.     

Biological and Molecular Characterization of Taiwanese Isolates of Cucumber mosaic virus Associated with Banana Mosaic Disease

Banana mosaic disease (BMD) caused by Cucumber mosaic virus (CMV) has become an important threat to the banana industry. We collected and characterized 10 CMV isolates associated with BMD in Taiwan and compared their biological characteristics and coat protein sequences. The isolates fell into four pathotypes on the basis of the symptoms they induce on banana, Nicotiana glutinosa and Vigna unguiculata (cowpea). Double-stranded RNA analysis revealed that the different pathotypes are not related to the presence of CMV satellite RNA. Phylogenetic analysis of worldwide CMV coat protein sequences revealed that among the currently known CMV subgroups IA, IB and II, subgroup IB is phylogenetically unresolved. Our CMV isolates form a new subgroup, IT, within subgroup I. In addition, we resolved another new CMV subgroup, IS, within subgroup I. The analysis also revealed that isolates within different subgroups can infect the banana.     

Molecular Analysis of the Coat Protein of Potato virus Y Isolates in Greece Suggests Multiple Introduction from Different Genetic Pools

The phylogenetic relationships among Potato virus Y (PVY) isolates from northern and southern Greece were investigated. A large part of coat protein gene of 49 tobacco isolates and three from pepper was examined. The analysis showed that all 52 isolates consisted of 34 distinct haplotypes, with only one haplotype found in both northern and southern regions. The southern population was more diverse than that from the north. The phylogenetic analyses of the Greek haplotypes alone or in combination with isolates from other countries using the maximum likelihood method classified unambiguously almost all the haplotypes examined. Nine tobacco haplotypes from the south were classified as C‐like (particularly C1), whereas 22 haplotypes from tobacco and two from pepper from both north and south were classified as N‐like. One tobacco haplotype from the south was found recombinant between N‐like and C1 lineages. The pattern of molecular evolution was examined using the fixed‐effects likelihood and the single‐likelihood ancestor counting methods. The analysis indicated that the evolution of PVY isolates appeared to be conservative (purifying selection and neutral evolution). These findings are discussed in relation to the introduction of PVY in the tobacco crop in Greece and the between region dispersal. A scenario of multiple introductions of PVY isolates in north and south Greece from different genetic pools and low or nil between region spread of the virus isolates was proposed.     

First Report of Tomato infectious chlorosis virus on Tomato in Bulgaria

Virus‐like chlorotic symptoms were observed on tomato plants, cv. Velocity, grown in a greenhouse, region of Plovdiv. Samples collected from the leaves with interveinal yellowing and with initial interveinal chlorosis were tested for virus presence. Only the samples collected from the upper leaves with slight interveinal chlorosis were positive for Tomato infectious chlorosis virus (TICV) in indirect ELISA. Further, RT‐PCR analysis with specific primers for Tomato chlorosis virus (ToCV) heat shock protein 70, for TICV heat shock protein 70 and for TICV minor capsid protein was positive for TICV in all tested samples. No signals were obtained with primers for ToCV. Phylogenetic analysis showed that the Bulgarian sequence of Hsp70 and a sequence of Greek isolate clustered together having the highest resampling score. Regarding CPm, the Bulgarian isolate was more relevant to the French isolate. The obtained results from phylogenetic analysis supported the idea of a close relationship between the Bulgarian and Greek isolates.