A single amino acid in coat protein of Pepper mild mottle virus determines its subcellular localization and the chlorosis symptom on leaves of pepper.

Pepper mild mottle virus (PMMoV) causes serious economic losses in pepper production in China. In a survey for viral diseases on pepper, two PMMoV isolates (named PMMoV-ZJ1 and PMMoV-ZJ2) were identified with different symptoms in Zhejiang province. Sequence alignment analysis suggested there were only four amino acid differences between the isolates: Val262Gly, Ile629Met and Ala1164Thr in the replicase, and Asp20Asn in the coat protein. Infectious cDNA clones of both isolates were constructed and shown to cause distinctive symptoms. Chlorosis symptoms appeared only on PMMoV-ZJ2-infected plants and the Asp20Asn substitution in the CP was shown to be responsible. Confocal assays revealed that the subcellular localization pattern of the two CPs was different, CP20Asp was mainly located at the cell periphery, whereas most CP20Asn located in the chloroplast. Thus, a single amino acid in the CP determined the chlorosis symptom, accompanied by an altered subcellular localization.

Potato virus Y (PVY) Isolates from Physalis peruviana are Unable to Systemically Infect Potato or Pepper and Form a Distinct New Lineage Within the PVYC Strain Group.

Poha, or cape gooseberry (Physalis peruviana L.), is a plant species cultivated in Hawaii for fresh fruit production. In 2015, an outbreak of virus symptoms occurred on poha farms in the South Kohala District of the island of Hawaii. The plants displayed mosaic, stunting, and leaf deformation, and produced poor fruit. Initial testing found the problem associated with Potato virus Y (PVY) infection. Six individual PVY isolates, named Poha1 to Poha6, were collected from field-grown poha plants and subjected to biological and molecular characterization. All six isolates induced mosaic and vein clearing in tobacco, and three of them exhibited O-serotype while the other three reacted only with polyclonal antibodies and had no identifiable serotype. Until now, PVY isolates have been broadly divided into pepper or potato adapted; however, these six PVY isolates from poha were unable to establish systemic infection in pepper and in four tested potato cultivars. Whole-genome sequences for the six isolates were determined, and no evidence of recombination was found in any of them. Phylogenetic analysis placed poha PVY isolates in a distinct, monophyletic "Poha" clade within the PVYC lineage, suggesting that they represented a novel, biologically and evolutionarily unique group. The genetic diversity within this poha PVYC clade was unusually high, suggesting a long association of PVYC with this solanaceous host or a prolonged geographical separation of PVYC in poha in Hawaii.

Genome sequence variation in the constricta strain dramatically alters the protein interaction and localization map of Potato yellow dwarf virus.

The genome sequence of the constricta strain of Potato yellow dwarf virus (CYDV) was determined to be 12 792 nt long and organized into seven ORFs with the gene order 3'-N-X-P-Y-M-G-L-5', which encodes the nucleocapsid, phospho, movement, matrix, glyco, and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. Cloned ORFs for each gene, except L, were used to construct a protein interaction and localization map (PILM) for this virus, which shares greater than 80 % amino acid similarity in all ORFs except X and P with the sanguinolenta strain of this species (SYDV). Protein localization patterns and interactions unique to each viral strain were identified, resulting in strain-specific PILMs. Localization of CYDV and SYDV proteins in virus-infected cells mapped subcellular loci likely to be sites of replication, morphogenesis and movement.

Characterisation and diagnosis of frangipani mosaic virus from India.

Frangipani mosaic virus (FrMV) is known to infect frangipani tree (Plumeria rubra f. acutifolia) in India but the virus has not been characterized at genomic level and diagnosis is not available. In the present study, an isolate of FrMV (FrMV-Ind-1) showing greenish mosaic and vein-banding symptoms in P. rubra f. acutifolia in New Delhi was characterized based on host reactions, serology and genome sequence. The virus isolate induced local symptoms on several new experimental host species: Capsicum annuum (chilli), Nicotiana benthamiana, Solanum lycopersicum and S. melongena. N. benthamiana could be used as an efficient propagation host as it developed systemic mottle mosaic symptoms all round the year. The genome of FrMV-Ind-1 was 6643 (JN555602) nucleotides long with genome organization similar to tobamoviruses. The Indian isolate of FrMV shared a very close genome sequence identity (98.3 %) with the lone isolate of FrMV-P from Australia. FrMV-Ind-1 together with FrMV-P formed a new phylogenetic group i.e. Apocynaceae-infecting tobamovirus. The polyclonal antiserum generated through the purified virus preparation was successfully utilized to detect the virus in field samples of frangipani by ELISA. Of the eight different tobamoviruses tested, FrMV-Ind-1 shared distant serological relationships with only cucumber green mottle mosaic virus, tobacco mosaic virus, bell pepper mottle virus and kyuri green mottle mosaic virus. RT-PCR based on coat protein gene primer successfully detected the virus in frangipani plants. This study is the first comprehensive description of FrMV occurring in India.

Trypsin inhibitors from Capsicum baccatum var. pendulum leaves involved in Pepper yellow mosaic virus resistance.

Several plant organs contain proteinase inhibitors, which are produced during normal plant development or are induced upon pathogen attack to suppress the enzymatic activity of phytopathogenic microorganisms. In this study, we examined the presence of proteinase inhibitors, specifically trypsin inhibitors, in the leaf extract of Capsicum baccatum var. pendulum inoculated with PepYMV (Pepper yellow mosaic virus). Leaf extract from plants with the accession number UENF 1624, which is resistant to PepYMV, was collected at 7 different times (0, 24, 48, 72, 96, 120, and 144 h). Seedlings inoculated with PepYMV and control seedlings were grown in a growth chamber. Protein extract from leaf samples was partially purified by reversed-phase chromatography using a C2/C18 column. Residual trypsin activity was assayed to detect inhibitors followed by Tricine-SDS-PAGE analysis to determine the N-terminal peptide sequence. Based on trypsin inhibitor assays, trypsin inhibitors are likely constitutively synthesized in C. baccatum var. pendulum leaf tissue. These inhibitors are likely a defense mechanism for the C. baccatum var. pendulum- PepYMV pathosystem.

Grape seed extract for foodborne virus reduction on produce.

Grape seed extract (GSE) is reported to have antibacterial properties with few current studies on antiviral activity. Recently, we reported the effects of GSE against foodborne viral surrogates in vitro. This study evaluated the application of GSE (commercial Gravinol-S) against hepatitis A virus (HAV) and human norovirus surrogates, feline calicivirus (FCV-F9) and murine norovirus (MNV-1), on model produce. Washed and air-dried lettuce (3 × 3 cm(2)) and jalapeno peppers (25-30 g) were inoculated with FCV-F9, MNV-1, or HAV at high (∼7 log10 PFU/ml) or low (∼5 log10 PFU/ml) titers, and treated with 0.25, 0.5, 1 mg/ml GSE or water for 30 s to 5 min. Treatments were stopped/diluted with cell-culture media containing 10% heat-inactivated fetal bovine serum and evaluated using plaque assays. At high titers, FCV-F9 was reduced by 2.33, 2.58, and 2.71 log10 PFU on lettuce; and 2.20, 2.74, and 3.05 log10 PFU on peppers after 1 min using 0.25, 0.50, and 1 mg/ml GSE, respectively. Low FCV-F9 titers could not be detected after 1 min at all three GSE concentrations. Low titer MNV-1 was reduced by 0.2-0.3 log10 PFU on lettuce and 0.8 log10 PFU on peppers, without reduction of high titer. GSE at 0.25-1 mg/ml after 1 min caused 0.7-1.1 and 1-1.3 log10 PFU reduction for high and low HAV titers, respectively on both commodities. Instrumental color analysis showed no significant differences between treated and untreated produce. GSE shows potential for foodborne viral reduction on produce as part of hurdle technologies.

Helicase domain encoded by Cucumber mosaic virus RNA1 determines systemic infection of Cmr1 in pepper.

The Cmr1 gene in peppers confers resistance to Cucumber mosaic virus isolate-P0 (CMV-P0). Cmr1 restricts the systemic spread of CMV strain-Fny (CMV-Fny), whereas this gene cannot block the spread of CMV isolate-P1 (CMV-P1) to the upper leaves, resulting in systemic infection. To identify the virulence determinant of CMV-P1, six reassortant viruses and six chimeric viruses derived from CMV-Fny and CMV-P1 cDNA clones were used. Our results demonstrate that the C-terminus of the helicase domain encoded by CMV-P1 RNA1 determines susceptibility to systemic infection, and that the helicase domain contains six different amino acid substitutions between CMV-Fny and CMV-P1(.) To identify the key amino acids of the helicase domain determining systemic infection with CMV-P1, we then constructed amino acid substitution mutants. Of the mutants tested, amino acid residues at positions 865, 896, 957, and 980 in the 1a protein sequence of CMV-P1 affected the systemic infection. Virus localization studies with GFP-tagged CMV clones and in situ localization of virus RNA revealed that these four amino acid residues together form the movement determinant for CMV-P1 movement from the epidermal cell layer to mesophyll cell layers. Quantitative real-time PCR revealed that CMV-P1 and a chimeric virus with four amino acid residues of CMV-P1 accumulated more genomic RNA in inoculated leaves than did CMV-Fny, indicating that those four amino acids are also involved in virus replication. These results demonstrate that the C-terminal region of the helicase domain is responsible for systemic infection by controlling virus replication and cell-to-cell movement. Whereas four amino acids are responsible for acquiring virulence in CMV-Fny, six amino acid (positions at 865, 896, 901, 957, 980 and 993) substitutions in CMV-P1 were required for complete loss of virulence in 'Bukang'.

Cytopathological potato virus Y structures during Solanaceous plants infection.

The ultrastructural analysis of tobacco, potato and pepper tissues during infection with necrotic strains and the ordinary Potato virus Y strain of revealed the presence of virus inclusions not only in the epidermis and mesophyll but also in the vascular tissues. For the first time cytoplasmic inclusions were documented in companion cells and phloem parenchyma as well as in xylem tracheary elements. The ultrastructural features studied in this work consisted of mostly laminated inclusions (in the traverse and longitudinal section), which were frequently connected with enlarged cisternae of endoplasmic reticulum (ER) located in the direct vicinity of the cell wall attached to virus particles opposite to plasmodesmata. It was noticed that ER participates in synthesis and condensation of the PVY inclusions. During compatible interaction of tobacco and potato plants with PVY, amorphous and nuclear inclusions were observed. Such forms were not found in pepper tissues and potato revealing the hypersensitivity reaction to the infection with PVY necrotic strains. It was stated that the forms of cytoplasmic inclusions cannot serve as a cytological criterion to distinguish the potato virus Y strains and do not depend on host resistance level. Only in compatible interaction in Solanaceous plants tissues cytoplasmic inclusions were observed from the moment the morphological symptoms appeared. In the reaction of hypersensitivity, the inclusions were found on the 24th day following the infection with the PVY necrotic strains, whereas the symptoms were observed 3 days after the PVY infection.

Molecular characterization of a new begomovirus that infects Euphorbia heterophylla and Solanum lycopersicum in Venezuela.

We report the complete nucleotide sequence of a begomovirus isolate infecting Euphorbia heterophylla and tomato (Solanum lycopersicum) in central Venezuela. Based on the current taxonomic criteria for the genus Begomovirus, the isolate was shown to represent a novel species, tentatively named Euphorbia mosaic Venezuela virus (EuMVV). Its DNA-A is most closely related to those of Euphorbia-infecting begomoviruses from the Caribbean and Central America. The DNA B component forms a phylogenetic cluster with Euphorbia and Sida-infecting begomoviruses from the squash leaf curl virus (SLCV) cluster. EuMVV is transmissible to S. lycopersicum and Capsicum annuum by biolistics of infectious cloned DNA-A and DNA-B components and induces characteristic leaf downward curling and yellowing in S. lycopersicum and and yellowing and leaf distortion in Capsicum annuum.

A viral satellite RNA induces yellow symptoms on tobacco by targeting a gene involved in chlorophyll biosynthesis using the RNA silencing machinery.

Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5'-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene.

Importance and genetic diversity of vegetable-infecting tospoviruses in India.

A survey for Peanut bud necrosis virus (PBNV), Watermelon bud necrosis virus (WBNV), Capsicum chlorosis virus (CaCV), and Iris yellow spot virus (IYSV) was conducted between 2002 and 2009 in the major vegetable-growing areas in India. PBNV was documented widely in tomato and chili peppers in 14 states representing southern, north-western, north-eastern, and central regions and WBNV was predominantly detected in watermelons and cucurbits in all except north-eastern regions. In addition, the expanded host range of PBNV to watermelons and other cucurbits and WBNV to tomato and chili peppers was observed leading to natural mixed infection of the two viruses. IYSV was found in onion in southern, central, and north-eastern regions and CaCV in tomato and chili peppers in northern and southern regions, respectively. Phylogenetic analysis of the nucleocapsid gene revealed segregation of field isolates of PBNV and WBNV into two distinct subclades, whereas isolates of CaCV and IYSV each clustered into a single clade. A proposal for establishing WBNV as a distinct tospovirus species is made based on the molecular characterization of small- (S) and medium- (M) RNA segments.

Genetic diversity in curtoviruses: a highly divergent strain of Beet mild curly top virus associated with an outbreak of curly top disease in pepper in Mexico.

A full-length curtovirus genome was PCR-amplified and cloned from peppers in Mexico with symptoms of curly top disease. The cloned DNA of this isolate, MX-P24, replicated in Nicotiana tabacum protoplasts and was infectious in N. benthamiana plants. Sequence analysis revealed that the MX-P24 isolate had a typical curtovirus genome organization and was most similar to beet mild curly top virus (BMCTV). However, sequence identities were at the threshold value for establishment of a new curtovirus species. To further investigate the biological properties of MX-P24, an agroinoculation system was generated. Agroinoculated shepherd's purse plants developed typical curly top symptoms, and virus from these plants was transmissible by the beet leafhopper (Circulifer tenellus). The host range of MX-P24 was similar to that of BMCTV, with curly top symptoms induced in common bean, pepper, pumpkin, shepherd's purse and tomato plants and mild or no symptoms induced in sugar beet plants. Together, these results indicate that MX-P24 is a highly divergent strain of BMCTV associated with an outbreak of curly top disease in peppers in Mexico.

Differentiation of Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus using real-time TaqMan PCR.

During the past four decades, Tomato yellow leaf curl disease has become one of the major constraints in tomato production worldwide. In the Mediterranean basin, several isolates from two major Begomovirus species are involved in outbreaks and persistent epidemics. A real-time TaqMan PCR assay was developed and evaluated for the rapid and multiplex detection and differentiation of two begomoviruses often found in mixed infections in the region, Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV). This assay was 1000-fold more sensitive than conventional PCR assays described previously, allowing the use of simple template preparation methods and eliminating the need for total nucleic acid purification. The viral DNA template was obtained by spotting sap extract derived from TYLCV or TYLCSV infected tissues on a nylon membrane or by directly using crude plant extracts in the real-time reaction cocktail. Preliminary results showed that this method can successfully detect and discriminate virus species from infected tomato, bean, pepper and different weed species obtained from the Mediterranean basin, the USA and Japan, allowing the simple, fast and cost-effective testing of a large number of samples in certification schemes. The assay can also be used for the detection of these two begomovirus species in their whitefly vector biotypes of the Bemisia tabaci (Gennadius) species group.

Pepper chat fruit viroid: biological and molecular properties of a proposed new species of the genus Pospiviroid.

In autumn 2006, a new disease was observed in a glasshouse-grown crop of sweet pepper (Capsicum annuum L.) in the Netherlands. Fruit size of the infected plants was reduced up to 50%, and plant growth was also slightly reduced. Here we show that the disease is caused by a previously non-described viroid. The pepper viroid is transmitted by both mechanical inoculation and pepper seeds and, when inoculated experimentally, it infects several solanaceous plant species inducing vein necrosis and reduced fruit and tuber size in tomato and potato, respectively. The viroid RNA genome consists of 348 nucleotides and, with minor modifications, it has the central conserved and the terminal conserved regions characteristic of members of the genus Pospiviroid. Classification of the pepper viroid within the genus Pospiviroid is further supported by the presence and structure of hairpins I and II, the presence of internal and external RY motifs, and phylogenetic analyses. The primary structure of the pepper viroid only showed a maximum of 66% nucleotide sequence identity with other viroids, which is far below the main species demarcation limit of 90%. According to its biological and molecular properties, we propose to assign the pepper viroid to a new species within the genus Pospiviroid, and to name this new species Pepper chat fruit viroid.

Molecular characterization of Korean Pepper mottle virus isolates and its relationship to symptom variations.

The symptom variations among Korean Pepper mottle virus (PepMoV) isolates infecting pepper, tomato and potato were described and the cause of variations in relation to molecular variability were investigated. In addition, the entire genome of the 13 PepMoV isolates, collected from five provinces (Kyonggi, Chungnam, Gyeongnam, Jeonbuk and Jeonnam) in Korea, were determined and compared including the previously reported Korean-Vb isolate and 2 other PepMoV isolates isolated from America (CA and FL). Our results showed that the nucleotide sequence of all Korean isolates tested were nearly identical (98-99%) and only 94% similar to American isolates. In general, the complete nucleotide sequences and deduced polyprotein sequences indicated low genetic variation among isolates showing 0.1-3% nucleotide changes per site. However, based on ratio between nucleotide diversity values in nonsynonymous and synonymous position (dN/dS ratio) surprisingly, P1 and 6K2 genes showed relatively high nucleotide substitution ratio (0.8 and 1.0 nucleotide, respectively). When the 6K2 amino acid were aligned, there were 15 amino acid substitutions found in PepMoV-infected potato and only 1 amino acid change from two isolates of PepMoV-infected bell pepper. Interestingly, three isolates including isolate numbers 731, 205135 and 205136 that possessed different aa changes at 6K2 region also showed distinct symptom differentiation in indicator hosts and cosegregated in the phylogenetic analysis. These results further proved previous studies that P1 and 6K2 genes with other proteins might have some involvement on host specificity and pathogenicity.

Functional study of hot pepper 26S proteasome subunit RPN7 induced by Tobacco mosaic virus from nuclear proteome analysis.

Two-dimensional gel electrophoresis (2-DE) was applied for the screening of Tobacco mosaic virus (TMV)-induced hot pepper (Capsicum annuum cv. Bugang) nuclear proteins. From differentially expressed protein spots, we acquired the matched peptide mass fingerprint (PMF) data, analyzed by MALDI-TOF MS, from the non-redundant hot pepper EST protein FASTA database using the VEMS 2.0 software. Among six identified nuclear proteins, the hot pepper 26S proteasome subunit RPN7 (CaRPN7) was subjected to further study. The level of CaRPN7 mRNA was specifically increased during incompatible TMV-P(0) interaction, but not during compatible TMV-P(1.2) interaction. When CaRPN7::GFP fusion protein was targeted in onion cells, the nuclei had been broken into pieces. In the hot pepper leaves, cell death was exacerbated and genomic DNA laddering was induced by Agrobacterium-mediated transient overexpression of CaPRN7. Thus, this report presents that the TMV-induced CaRPN7 may be involved in programmed cell death (PCD) in the hot pepper plant.

Potato virus Y CFH, a putative recombinant isolate from Capsicum chinense cv. Habanero.

Ornamental plants of Chili pepper, Capsicum chinense cv. Habanero, with symptoms of leaf mosaic, necrotic rings on fruits and necrotic stems were observed in June 2003 in a private garden in the province of Naples (Italy). Preliminary serological characterisation allowed the association of these symptoms with infections by Potato virus Y (PVY). The virus was isolated on Nicotiana tabacum cv. Xanthi and characterised by mechanical inoculation on herbaceous hosts and molecular characterisation of the P1 and the coat protein (CP) genes. Symptoms produced on indicator plants were generally consistent with those described for PVY. The identity of PVY was further confirmed by reaction with PVYN, PVYC and PVYO specific monoclonal antibodies: the isolate reacted only with the PVYC specific Mab. Immuno capture reverse transcription polymerase chain reaction (IC-RT-PCR) was performed on extracts of PVY-CFH infected N. tabacum cv. Xanthi plants, using two couples of primers specifically designed out of the P1 and the CP coding regions of the so far fully sequenced PVY isolates. PCR products were then cloned into pCRII-TOPO vector using TOPO-TA cloning kit (Invitrogen) and sequenced. Sequence analysis suggests that PVY-CFH originated from a recombination event involving a virus of the PVYO type and another parental virus, maybe resembling the PVYNP isolates, given the reasonably high similarity shared by PVY-CFH and, respectively, non potato PVY isolates in the CP coding region, PVYO isolates in the P1 coding region. Evidence for the existence of such a recombination comes, apart from similarity analysis, by the different locations of CFH within phylogenetic trees constructed from P1 and CP genomic regions.

Characterisation of Potato virus Y nnp strain inducing veinal necrosis in pepper: a naturally occurring recombinant strain of PVY.

The full-length genome of Potato virus Y (PVY) nnp strain, recovered from pepper showing veinal necrosis of leaves, was cloned and sequenced, finding an organisation typical for PVY species. It consists of 9699 nucleotides (nt) excluding the 3' terminal poly(A) tail and contains an open reading frame of 9186 nt, encoding the putative polyprotein of 3061 amino acids. In ELISA, the isolate reacted with a monoclonal antibody specific for PVY(C) but not with antibodies against PVY(N) or PVY(O). Sequence analysis strongly suggests that PVY-nnp originated from a recombination event involving a virus of the PVY(O) type and another parental virus, maybe resembling the PVY(NP) isolates, given the reasonably high similarity shared by PVY-nnp and Lye84.2 and Son41 isolates. The recombination event involved a breakpoint near the middle of the P1 gene, around position 603 of the viral genome. Proof for the existence of such a recombination comes from several lines of evidence, including similarity analysis, recombination analysis using six different methods and the different locations of nnp within phylogenetic trees constructed from genomic regions on either side of the identified recombination breakpoint.

Strains of Peru tomato virus infecting cocona (Solanum sessiliflorum), tomato and pepper in Peru with reference to genome evolution in genus Potyvirus.

Two isolates (SL1 and SL6) of Peru tomato virus (PTV, genus Potyvirus) were obtained from cocona plants (Solanum sessiliflorum) growing in Tingo María, the jungle of the Amazon basin in Peru. One PTV isolate (TM) was isolated from a tomato plant (Lycopersicon esculentum) growing in Huaral at the Peruvian coast. The three PTV isolates were readily transmissible by Myzus persicae. Isolate SL1, but not SL6, caused chlorotic lesions in inoculated leaves of Chenopodium amaranticolor and C. quinoa. Isolate TM differed from SL1 and SL6 in causing more severe mosaic symptoms in tomato, and vein necrosis in the leaves of cocona. Pepper cv. Avelar (Capsicum annuum) showed resistance to the PTV isolates SL1 and SL6 but not TM. The 5'- and 3'-proximal sequences of the three PTV isolates were cloned, sequenced and compared to the corresponding sequences of four PTV isolates from pepper, the only host from which PTV isolates have been previously characterised at the molecular level. Phylogenetic analyses on the P1 protein and coat protein amino acid sequences indicated, in accordance with the phenotypic data from indicator hosts, that the PTV isolates from cocona represented a distinguishable strain. In contrast, the PTV isolates from tomato and pepper were not grouped according to the host. Inclusion of the sequence data from the three PTV isolates of this study in a phylogenetic analysis with other PTV isolates and other potyviruses strengthen the membership of PTV in the so-called "PVY subgroup" of Potyvirus. This subgroup of closely related potyvirus species was also distinguishable from other potyviruses by their more uniform sizes of the protein-encoding regions within the polyprotein.

Molecular resolution of a complex of potyviruses infecting solanaceous crops at the centre of origin in Peru.

Peru is a centre of origin and domestication of the potato, pepper and tomato (family Solanaceae). Many potyviruses (genus Potyvirus) that infect these crops were described 20-30 years ago. However, definitive classification of these viruses as distinct species remains unresolved for several reasons, including their close serological relationships, similar symptomatology in test plants and lack of genomic sequence data. Using samples collected from Peru, we have determined the complete genomic sequence of two strains of Peru tomato virus (PTV) as well as near-complete sequences for two additional PTV strains. We also obtained partial sequences of four strains of Potato virus V (PVV). Comparisons with genomic sequences of Wild potato mosaic virus (WPMV), Potato virus Y (PVY), Pepper mottle virus (PepMoV), Potato virus A (PVA) and other potyviruses established that all these viruses constitute different taxa (species). Phylogenetic comparisons indicated that PTV, PVV and WPMV are the most closely related species which, together with PepMoV, PVY, Pepper yellow mosaic virus and Pepper severe mosaic virus, constitute a group that is distinguishable from other potyviruses. Therefore, the members of this group may share a common ancestor. PVA does not belong to this group. PVV and PTV were also closely related serologically. However, PTV did not cross-protect against PVV and WPMV in tobacco plants or complement systemic infection of PVV and WPMV in pepper plants. Two biologically and phylogenetically distinguishable strain groups were identified within PTV and PVV. In future studies, the sequence data and virus-specific primers and probes for PTV, PVV and WPMV described in this study will enable accurate indexing of plants with respect to either single or mixed infection with these viruses.