Complexity and Local Specificity of the Virome Associated with Tospovirus-Transmitting Thrips Species.

Frankliniella occidentalis (western flower thrips [WFT]) and Thrips tabaci (onion thrips [OT]) are insect species that greatly impact horticultural crops through direct damage and their efficient vectoring of tomato spotted wilt virus and iris yellow spot virus. In this study, we collected thrips of these species from 12 field populations in various regions in Italy. We also included one field population of Neohydatothrips variabilis (soybean thrips [ST]) from the United States. Total RNA data from high-throughput sequencing (HTS) were used to assemble the virome, and then we assigned putative viral contigs to each thrips sample by real-time reverse transcription-quantitative PCR (qRT-PCR). Excluding plant and fungal viruses, we were able to identify 61 viral segments, corresponding to 41 viruses: 14 were assigned to WFT, 17 to OT, and 1 to ST; 9 viruses could not be assigned to any species based on our stringent criteria. All these viruses are putative representative of new species (with only the exception of a sobemo-like virus that is 100% identical to a virus recently characterized in ST) and some belong to new higher-ranking taxa. These additions to the viral phylogeny suggest previously undescribed evolutionary niches. Most of Baltimore's classes of RNA viruses were present (positive- and minus-strand and double-stranded RNA viruses), but only one DNA virus was identified in our collection. Repeated sampling in a subset of locations in 2019 and 2020 and further virus characterization in a subset of four thrips populations maintained in the laboratory allowed us to provide evidence of a locally persistent thrips core virome that characterizes each population. IMPORTANCE Harnessing the insect microbiome can result in new approaches to contain their populations or the damage they cause vectoring viruses of medical, veterinary, or agricultural importance. Persistent insect viruses are a neglected component of their microbiota. In this study, for the first time, we characterize the virome associated with the two model systems for tospovirus-transmitting thrips species, of utmost importance for the direct and indirect damage they cause to a number of different crops. The thrips virome characterized includes several novel viruses, which in some cases reveal previously undescribed clades. More importantly, some of the viruses we describe are part of a core virome that is specific and consistently present in distinct geographical locations monitored over the years, hinting at a possible mutualistic symbiotic relationship with their host.

Biological and Molecular Characterization of Chenopodium quinoa Mitovirus 1 Reveals a Distinct Small RNA Response Compared to Those of Cytoplasmic RNA Viruses.

Indirect evidence of mitochondrial viruses in plants comes from discovery of genomic fragments integrated into the nuclear and mitochondrial DNA of a number of plant species. Here, we report the existence of replicating mitochondrial virus in plants: from transcriptome sequencing (RNA-seq) data of infected Chenopodium quinoa, a plant species commonly used as a test plant in virus host range experiments, among other virus contigs, we could assemble a 2.7-kb contig that had highest similarity to mitoviruses found in plant genomes. Northern blot analyses confirmed the existence of plus- and minus-strand RNA corresponding to the mitovirus genome. No DNA corresponding to the genomic RNA was detected, excluding the endogenization of such virus. We have tested a number of C. quinoa accessions, and the virus was present in a number of commercial varieties but absent from a large collection of Bolivian and Peruvian accessions. The virus could not be transmitted mechanically or by grafting, but it is transmitted vertically through seeds at a 100% rate. Small RNA analysis of a C. quinoa line carrying the mitovirus and infected by alfalfa mosaic virus showed that the typical antiviral silencing response active against cytoplasmic viruses (21- to 22-nucleotide [nt] vsRNA peaks) is not active against CqMV1, since in this specific case the longest accumulating vsRNA length is 16 nt, which is the same as that corresponding to RNA from mitochondrial genes. This is evidence of a distinct viral RNA degradation mechanism active inside mitochondria that also may have an antiviral effect.IMPORTANCE This paper reports the first biological characterization of a bona fide plant mitovirus in an important crop, Chenopodium quinoa, providing data supporting that mitoviruses have the typical features of cryptic (persistent) plant viruses. We, for the first time, demonstrate that plant mitoviruses are associated with mitochondria in plants. In contrast to fungal mitoviruses, plant mitoviruses are not substantially affected by the antiviral silencing pathway, and the most abundant mitovirus small RNA length is 16 nt.

A new blunervirus infects tomato crops in Italy and Australia.

In this study, we determined the complete genome sequence of a new blunervirus isolated from tomato plants grown in an open field in Italy in the fall of 2018. Like other blunerviruses, the RNA genome of this virus is quadripartite, positive-sense, and single-stranded. Excluding the polyA tail present in each segment, the RNAs 1 and 2 are 5790 nucleotides (nt) and 3621 nt in size, respectively, and each contains a single open reading frame (ORF). The RNAs 3 and 4 are 2842 and 1924 nt long and encode five and two ORFs, respectively. BLASTp analysis of the predicted products of RNA1 and RNA2 ORF1 showed the highest sequence identity (31% and 42%) to tea plant necrotic ring blotch virus (TPNRBV), while the protein encoded by RNA 4 ORF2 had the highest sequence identity (38%) to blueberry necrotic ring blotch virus (BNRBV). These are the only two recognized members in the genus Blunervirus. When the RNA3 ORF3 and ORF5 products were compared with the blunerviruses-encoded proteins, they had the highest sequence identity (30% and 32%) to their TPNRBV-encoded homologs; however, general comparisons showed stronger matches to two different proteins from Acinetobacter baumannii. The proteins encoded by ORFs 1, 2 and 4 of RNA3 and ORF 1 of RNA4 showed no significant BLASTp hits to any known proteins in the databases. Given the limited genetic similarity of this virus to those currently available in the databases, we suggest that this is a new virus, for which we propose the name "tomato fruit blotch virus" (ToFBV). A distinct isolate of the same virus was also detected in Australia.

Mycoviruses mediate mycotoxin regulation in Aspergillus ochraceus.

To date, no demonstration of a direct correlation between the presence of mycoviruses and the quantitative or qualitative modulation of mycotoxins has been shown. In our study, we transfected a virus-free ochratoxin A (OTA)-producing isolate of Aspergillus ochraceus with purified mycoviruses from a different A. ochraceus isolate and from Penicillium aurantiogriseum. Among the mycoviruses tested, only Aspergillus ochraceus virus (AoV), a partitivirus widespread in A. ochraceus, caused a specific interaction that led to an overproduction of OTA, which is regulated by the European Commission and is the second most important contaminant of food and feed commodities. Gene expression analysis failed to reveal a specific viral upregulation of the mRNA of genes considered to play a role in the OTA biosynthetic pathway. Furthermore, AoOTApks1, a polyketide synthase gene considered essential for OTA production, is surprisingly absent in the genome of our OTA-producing isolate. The possible biological and evolutionary implications of the mycoviral regulation of mycotoxin production are discussed.

Full-length genome sequence of the tospovirus melon severe mosaic virus.

The complete genome sequence of melon severe mosaic virus (MSMV), genus Tospovirus, family Bunyaviridae, was determined. The small segment is 3283 nucleotide (nt) long and contains two open reading frames in an ambisense organization. The medium segment is 4873 nt long and also encodes two proteins in an ambisense organization. The large segment is 9811 nt long and contains a single, negative-sense ORF. Phylogenetic analysis of each of the five encoded proteins compared to those of tospoviruses present in the databases reveals the same topology for each tree, suggesting that the MSMV genome did not result from recombination or reassortment. Sequence variants present in the RNA population of an infected leaf are described.

A new tobamovirus infecting tomato crops in Jordan.

In this study, we completed the whole genome sequence of a new tobamovirus isolated from tomato plants grown in greenhouses in Jordan during the spring of 2015. The 6393-nt single-stranded RNA (ssRNA) genome encodes four proteins, as do other tobamoviruses: two replication-related proteins of 126 kDa and 183 kDa, a 30-kDa movement protein (MP) and a 17.5-kDa coat protein (CP). Phylogenetic analysis showed that this virus does not group with either the tomato mosaic virus (ToMV) or the tobacco mosaic virus (TMV) clades. Instead, it stems from a branch leading to the TMV clade. Analysis of possible recombination events between this virus and representative isolates of closely related tomato-infecting tobamoviruses showed that at least one region originated by recombination. We provide evidence that we have identified a new tobamovirus, for which we propose the name "tomato brown rugose fruit virus".

The NSs protein of tomato spotted wilt virus is required for persistent infection and transmission by Frankliniella occidentalis.

UNLABELLED: Tomato spotted wilt virus (TSWV) is the type member of tospoviruses (genus Tospovirus), plant-infecting viruses that cause severe damage to ornamental and vegetable crops. Tospoviruses are transmitted by thrips in the circulative propagative mode. We generated a collection of NSs-defective TSWV isolates and showed that TSWV coding for truncated NSs protein could not be transmitted by Frankliniella occidentalis. Quantitative reverse transcription (RT)-PCR and immunostaining of individual insects detected the mutant virus in second-instar larvae and adult insects, demonstrating that insects could acquire and accumulate the NSs-defective virus. Nevertheless, adults carried a significantly lower viral load, resulting in the absence of transmission. Genome sequencing and analyses of reassortant isolates showed genetic evidence of the association between the loss of competence in transmission and the mutation in the NSs coding sequence. Our findings offer new insight into the TSWV-thrips interaction and Tospovirus pathogenesis and highlight, for the first time in the Bunyaviridae family, a major role for the S segment, and specifically for the NSs protein, in virulence and efficient infection in insect vector individuals. IMPORTANCE: Our work is the first to show a role for the NSs protein in virus accumulation in the insect vector in the Bunyaviridae family: demonstration was obtained for the system TSWV-F. occidentalis, arguably one of the most damaging combination for vegetable crops. Genetic evidence of the involvement of the NSs protein in vector transmission was provided with multiple approaches.

The first complete genome sequences of two distinct European tomato spotted wilt virus isolates.

Tomato spotted wilt virus (TSWV) represents a major constraint to the production of important vegetable and ornamental crops in several countries around the world, including those in Europe. In spite of their economic importance, European TSWV isolates have only been partially characterized, and a complete genome sequence has not been determined yet. In this study, we completed the whole genome sequence of two distinct TSWV isolates from Italy, p105 and p202/3WT. The sequences of the L and M segments of p105 and of the L segment of p202/3WT were determined using a combined approach of RT-PCR and small RNA (sRNAs) contig assembly. Phylogenetic analysis based on RNA-dependent RNA polymerase and GN/GC protein sequences grouped the two isolates in two different clades, showing that different evolutive lineages are present among Italian TSWV isolates. Analysis of possible recombination/reassortment events among our isolates and other available full-length genome TSWV sequences showed a likely reassortment event involving the L segment.

A new ilarvirus isolated from Viola × wittrockiana and its detection in pansy germoplasm by qRT-PCR.

An infectious agent was transmitted mechanically from samples of Viola spp. showing white mosaic and leaf deformation to Nicotiana benthamiana. dsRNA extracted from the N. benthamiana plants migrated as four specific bands that were absent in non-inoculated plants. Sequence analysis of cDNA clones generated from the second-smallest dsRNA showed the greatest similarity to the RNA3 of prune dwarf virus (PDV) (genus Ilarvirus, family Bromoviridae). However, because of differences in molecular, biological, and serological properties between this virus isolate and PDV, a new ilarvirus species, named "Viola white distortion associated virus" (VWDaV) is proposed. Specific oligonucleotides and a TaqMan(®) probe were designed for diagnostic purposes. The possible association between the virus and the original white distortion symptoms is discussed.

The complete genome sequence of polygonum ringspot virus.

The complete genome sequence of polygonum ringspot virus (PolRSV), genus Tospovirus, family Bunyaviridae, was determined. This is the first report of the complete genome sequence for a European tospovirus isolate. The large RNA of PolRSV was 8893 nucleotides (nt) in size and contained a single open reading frame of 8628 nucleotides in the viral-complementary sense, coding for a predicted RNA-dependent RNA polymerase of 330.9 kDa. Two untranslated regions of 230 and 32 nucleotides were present at the 5' and 3' termini, respectively, which showed conserved terminal sequences, as commonly observed for tospovirus genomic RNAs. The medium and small (S) RNAs were 4710 and 2485 nucleotides in size, respectively, and showed 99 % homology to the corresponding genomic segment of a previously partially characterized PolRSV isolate, Plg3. Protein sequences for GN/GC, N and NSs were identical in length in the two PolRSV isolates, while an amino acid insertion was observed for the NSm protein of the newly characterized isolate. The noncoding intergenic region of the S RNA was very short (183 nt) and was not predicted to form a hairpin structure, confirming that this unique characteristic within tospoviruses, previously observed for Plg3, is not isolate specific.

Ranunculus latent virus: a strain of artichoke latent virus or a new macluravirus infecting artichoke?

An elongated virus was isolated from artichoke crops in Liguria, and a 700-bp fragment was amplified by RT-PCR using oligonucleotides to detect members of the family Potyviridae. Comparison of fragment sequences showed 98% identity at the nucleotide level with the ranunculus isolate of the macluravirus Ranunculus latent virus (RaLV). RaLV was then detected by DAS-ELISA in symptomatic and asymptomatic artichoke plants from Liguria, Sardinia and Latium. The sequence of a 5.5-kb region was assembled from a cDNA library, and a 500-bp NIa fragment showed 80% identity to Artichoke latent virus.

Molecular characterization of two distinct strains of blueberry scorch virus (BlScV) in northern Italy.

During blueberry scorch virus (BlScV) surveys of highbush blueberries in Italy between 2005 and 2010, we initially discovered infected orchards only in Piedmont. Since 2009, however, three infected orchards have also been found in Trentino, where a new host species, Vaccinium ashei, was found to be infected by BlScV. Molecular characterization of isolates during the study period suggests that two very distinct epidemics are now present in Italy: the Piedmont isolates belong to a new BlScV strain, whereas the Trentino isolates are almost identical to the Washington State strain.

First Report of Tomato spotted wilt virus on Pepper in Montenegro.

In April 2009, chlorotic and necrotic ring spots, chlorotic line patterns, and stunting were observed on greenhouse-grown pepper plants in the vicinity of Podgorica, Montenegro. Disease symptom incidence was estimated at 40%. Symptomatic leaves were tested for the presence of Tomato spotted wilt virus (TSWV) with a commercial double-antibody sandwich (DAS)-ELISA diagnostic kit (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls were included in each ELISA. TSWV was detected serologically in 33 of 75 pepper samples. The virus was mechanically transmitted from ELISA-positive pepper samples to Nicotiana tabacum cv. Samsun using chilled 0.05 M phosphate buffer (pH 7) containing 0.1% sodium sulfite (1). Inoculated test plants produced chlorotic and necrotic concentric rings and necrotic spots, consistent with symptoms caused by TSWV on N. tabacum. For further confirmation of TSWV infection, reverse transcription (RT)-PCR was performed with the One-Step RT-PCR Kit (Qiagen, Hilden, Germany) using three sets of primers: S70-for/S890-rev (2) and S574-for/S1433-rev (3), both specific to the nonstructural (NSs) gene; and S1983-for/S2767-rev (2), specific to the nucleocapsid protein (N) gene. Total RNAs from naturally infected pepper and symptomatic N. tabacum cv. Samsun plants were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Total RNAs obtained from the Italian isolate of TSWV (GenBank Accession No. DQ398945) and healthy tobacco plants were used as positive and negative controls, respectively. The expected sizes of the RT-PCR products (820, 859, and 784 bp) were amplified from symptomatic pepper samples but not from healthy tissues. The PCR product obtained from isolate Is-344 using primers specific to N gene was purified by a QIAquick PCR Purification Kit (Qiagen), cloned into the pGEM-T Easy Vector (Promega, Madison, WI) and sequenced in both directions using the same primer pair as in RT-PCR. The sequences amplified with the two primer pairs specific to the NSs gene were obtained by direct sequencing (Bio-Fab Research Srl, Pomezia, Italy) and joined using MEGA4 software. Sequence analysis of the complete N gene (777 bp; GenBank Accession No. GU369717) revealed that the TSWV isolate originating from Montenegro shared 98.2 to 99.7% nucleotide identity (98.1 to 100% amino acid identities) with corresponding TSWV sequences deposited in GenBank. The Montenegrin isolate Is-344 was most closely related to Italian isolates from tomato (GU369725) and eggplant (GU369720). The partial (1,257 bp) nucleotide sequence of NSs gene (GU369737) showed 96 to 99.8% nucleotide identity (96.9 to 100% amino acid identity) with previously reported TSWV sequences, and in this case the highest identity was with French isolates from tomato (FR692835) and lettuce (FR692831). To our knowledge, this is the first report on the occurrence of TSWV in Montenegro. Data of this study sheds light on the importance of further survey studies and inspections of TSWV-susceptible crops cultivated in Montenegro. References: (1) Anonymous. OEPP/EPPO Bull. 29:465, 1999. (2) W. P. Qiu et al. Virology 244:186, 1998. (3) M. Tsompana et al. Mol. Ecol. 14:53, 2005.

First Report of a Carlavirus in Fuchsia spp. in New Zealand.

The genus Fuchsia has 110 known species and numerous hybrids. These ornamental plants with brightly colored flowers originate from Central and South America, New Zealand, and Tahiti, but a wider variety are now grown all over the world. Few viruses have been reported in Fuchsia spp.: a carlavirus, Fuchsia latent virus (FLV) (1-3), a cucumovirus, Cucumber mosaic virus (CMV) (3), and two tospoviruses, Impatiens necrotic spot virus (INSV) and Tomato spotted wilt virus (TSWV) (4). In August 2009, five plants, each representing a different cultivar of Fuchsia hybrid, from home gardens in the Auckland and Southland regions of New Zealand, displayed variable symptoms including mild chlorosis, mild mottle, or purple spots on leaves. Plants tested negative for CMV, INSV, and TSWV using commercial ImmunoStrips (Agdia Inc., Elkhart, IN); however, flexuous particles of ~650 to 700 nm were found by electron microscopy in all samples. Local lesions were also observed on Chenopodium quinoa plants 4 weeks after sap inoculation. Total RNA was extracted from all plants with a RNeasy Plant Mini Kit (Qiagen Inc., Doncaster, Australia) and tested by reverse transcription (RT)-PCR using two generic sets of primers (R. van der Vlugt, personal communication) designed to amplify fragments of ~730 and 550 bp of the replicase and coat protein genes of carlaviruses, respectively. Amplicons of the expected size were obtained for all samples, cloned, and at least three clones per sample were sequenced. No differences within clones from the same samples were observed (GenBank Accession Nos. HQ197672 to HQ197681). A BLASTn search of the viral replicase fragment showed the highest nucleotide identity (76%) to Potato rough dwarf virus (PRDV) (EU020009), whereas the coat protein fragment had maximum nucleotide identity (70 to 72%) to PRDV (EU020009 and DQ640311) and Potato virus P (DQ516055). Sequences obtained were also pairwise aligned using the MegAlign program (DNASTAR, Inc., Madison, WI) and results showed that the isolates had 83 to 97% identity to each other within each genome region. Further sequences (HQ197925 and HQ197926) were obtained from a Fuchsia plant originating from Belgium, a BLASTn analysis showed high nucleotide identity (84 to 99%) to the New Zealand isolates. The low genetic identity to other Carlavirus members suggests that these isolates belong to a different species from those previously sequenced. On the basis of electron microscopy and herbaceous indexing, the isolates had similar characteristics to a carlavirus reported from Fuchsia in Italy (1) and FLV reported in Canada (2). The Italian carlavirus isolate was obtained and tested with the same primers by RT-PCR. Pairwise analysis of the Italian sequences (HQ197927 and HQ197928) with the New Zealand and Belgian sequences showed between 84 and 95% similarity within each genome region. These results suggest that the carlavirus infecting these plants is the same virus, possibly FLV. To our knowledge, this is the first report of this carlavirus infecting Fuchsia spp. in New Zealand, but the virus has probably been present for some time in this country and is likely to be distributed worldwide. References: (1) G. Dellavalle et al. Acta Hortic. 432:332, 1996. (2) L. J. John et al. Acta Hortic. 110:195, 1980. (3) P. Roggero et al. Plant Pathol. 49:802, 2000. (4) R. Wick and B. Dicklow. Diseases in Fuchsia. Common Names of Plant Diseases. Online publication. The American Phytopathological Society, St. Paul, MN, 1999.

A complex virome including two distinct emaraviruses associated with virus-like symptoms in Camellia japonica.

Camellia japonica plants manifesting a complex and variable spectrum of viral symptoms like chlorotic ringspots, necrotic rings, yellowing with necrotic rings, yellow mottle, leaves and petals deformations, and flower color-breaking have been studied since 1940, mainly by electron microscopic analyses; however, a strong correlation between the symptoms and one or more well-characterized viruses was never verified. In this work, samples collected from symptomatic plants were analyzed using the next-generation sequencing technique, and a complex virome composed of members of the Betaflexiviridae and Fimoviridae families was identified. In particular, the genomic fragments typical of the emaravirus group were organized in the genomes of two new emaraviruses species, tentatively named Camellia japonica-associated emaravirus 1 and 2. They are the first emaraviruses described in camellia plants and found in symptomatic plants. At the same time, in both symptomatic and asymptomatic plants, five betaflexivirus isolates were detected that, based on amino acid sequence comparisons, can be considered two new isolates of the recently characterized camellia ringspot-associated virus 1 and 2 (CRSaV-1/2). These recently identified betaflexiviruses associated with C. japonica disease show an unusual hyper-conservation of the coat protein at the amino acid level. The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are MN385581, MN532567, MN532565, MN385582, MN532566, MN385573, MN385577, MN385574, MN385578, MN385575, MN385579, MN385576, MN385580, MN557024, MN557025, MN557026, MN557027, and MN557028.

A New Tospovirus sp. in Cucurbit Crops in Mexico.

During the 2007 growing season, melon (Cucumis melo) samples from the state of Guerrero in Mexico showing mosaic and other virus-like symptoms were collected for analysis. Electron microscopic examination of negatively stained leaf-dip extracts revealed the presence of abundant virus-like particles with features characteristic of the family Bunyaviridae. No other viral particles were observed in these preparations. However, enzyme-linked immunosorbent assays (ELISAs) specific for the most common Tospovirus spp. gave negative results. Antibodies raised against purified nucleocapsids reacted specifically with the infected leaf extracts in Western blots and double-antibody sandwich ELISA. The viral RNA was used as a template for a cDNA library, and nucleotide sequence analysis identified cloned cDNAs representing sequences corresponding to the three Tospovirus genome segments. Sequence comparisons showed that the new virus had the highest similarity to Chrysanthemum stem necrosis virus (CSNV). Phylogenetic analysis of two genome regions confirmed that this virus, provisionally named Melon severe mosaic virus (MeSMV), is a previously undescribed Tospovirus sp. belonging to the "new world" clade of Tospovirus spp. An initial survey of various cucurbit crops in various states of Mexico confirmed the widespread occurrence of this virus.

The mycovirome of a fungal collection from the sea cucumber Holothuria polii.

Holothuria polii is a marine animal with an important ecological and economic impact. In the present study we analysed the presence of mycoviruses associated to fungi that were isolated from different H. polii tissues. Among the 48 fungal isolates analysed we identified 10 viruses in 8 strains belonging to 7 fungal species. Five out of nine viruses have a dsRNA genome: three of them belong to the Partitiviridae family, one belongs to a still undefined clade of bipartite viruses and the last one belongs to the Chrysoviridae family. We also identified two viruses belonging to a recently proposed new mycovirus taxon named polymycovirus. Two viruses belong to the positive single stranded RNA clade: one falls into the new Botourmiaviridae family, specifically in the Magoulivirus genus, and the other one falls into a still undefined clade phylogenetically related to tombusviruses. Finally, we also identified a virus with a negative stranded RNA genome showing similarity to a group of viruses recently proposed as a new family of mycoviruses in the order Bunyavirales. A bioinformatics approach comparing two datasets of contigs containing two closely related mycobunyaviruses allowed us to identify putative nucleocapsids (Nc) and non-structural (Ns) associated proteins. The GenBank/eMBL/DDBJ accession numbers of the sequences reported in this paper are: PRJNA432529, MG913290, MG913291, MG887747, MG887748, MG887749, MG887750, MG887751, MG887752, MG887753, MG887754, MG887755, MG887756, MG887757, MG887758, MG887759, MG887760, MG887761, MG887762, MG887763, MG887764, MG887765, MG887766, MG887767, MH271211, MN163273, MN163274.

A member of a new Tospovirus species isolated in Italy from wild buckwheat (Polygonum convolvulus).

Electron microscopy of extracts from diseased Polygonum convolvulus plants from Piedmont (Italy) revealed particles with the morphological features of a tospovirus. Sequencing of the full-length small (S) and medium (M) genome segments indicated that the virus is a member of a new Tospovirus species provisionally named Polygonum ringspot virus. A feature distinguishing it from members of other Tospovirus species was the presence of a very short intergenic region on the S segment lacking the potential for formation of the predicted hairpin structure involved in subgenomic expression. Antibodies made against purified nucleocapsids allowed serological comparison with other tospovirus isolates and revealed a relationship with tomato yellow ring virus, and to a lesser extent, to iris yellow spot virus. Serological tests detected the virus in various locations in northern and central Italy. The experimental host range was wide, although in nature the virus appeared restricted to two Polygonum species.

Evidence that the nonstructural protein of Tomato spotted wilt virus is the avirulence determinant in the interaction with resistant pepper carrying the TSW gene.

All known pepper cultivars resistant to Tomato spotted wilt virus (TSWV) possess a single dominant resistance gene, Tsw. Recently, naturally occurring resistance-breaking (RB) TSWV strains have been identified, causing major concerns. We used a collection of such strains to identify the specific genetic determinant that allows the virus to overcome the Tsw gene in Capsicum spp. A reverse genetic approach is still not feasible for TSWV; therefore, we analyzed reassortants between wild-type (WT) and RB strains. Our results confirmed that the S RNA, which encodes both the nucleocapsid protein (N) and a nonstructural protein (NSs), carries the genetic determinant responsible for Tsw resistance breakdown. We then used full-length S RNA segments or the proteins they encode to compare the sequences of WT and related RB strains, and obtained indirect evidence that the NSs protein is the avirulence factor in question. Transient expression of NSs protein from WT and RB strains showed that they both can equally suppress post-transcriptional gene silencing (PTGS). Moreover, biological characterization of two RB strains carrying deletions in the NSs protein showed that NSs is important in maintaining TSWV infection in newly emerging leaves over time, preventing recovery. Analysis of another RB strain phenotype allowed us to conclude that local necrotic response is not sufficient for resistance in Capsicum spp. carrying the Tsw gene.

Characterization of Four Viral Species Belonging to the Family Potyviridae Isolated from Ranunculus asiaticus.

ABSTRACT Four different viral species were isolated from diseased Ranunculus asiaticus plants growing in Imperia Province (Italian Riviera-Liguria Region). Infected plants exhibited mosaic symptoms and growth abnormalities. The viruses were mechanically inoculated to a range of herbaceous hosts and differentiated biologically. Long flexuous particles were present in leaf dip extracts observed by electron microscopy. A general protocol for the amplification of potyvirus genome fragments through reverse transcription-polymerase chain reaction generated products that were cloned and sequenced. Sequence and phylogenetic analysis suggested that three of these isolates could be considered new viral species belonging to the genus Potyvirus. The fourth isolate is a new member of the genus Macluravirus. Purified virus was used as antigen to produce a specific polyclonal antiserum in rabbit; serological features were established through double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), antigen coated plate (ACP)-ELISA, and western blot analysis. DAS-ELISA was highly specific for each virus isolate, whereas some cross-reactivity was shown in ACP-ELISA and western blot analysis. Aphid transmission by Myzus persicae was demonstrated in a controlled environment for each of the four viral isolates, whereas no transmission through seed was observed.