Chrysanthemum Stem Necrosis Virus Research Articles

Biological and Molecular Characterization of a Korean Isolate of Orthotospovirus chrysanthinecrocaulis (Formerly Chrysanthemum Stem Necrosis Virus) Isolated from Chrysanthemum morifolium

Biological and molecular characterization of a Korean isolate of <i>Orthotospovirus chrysanthinecrocaulis</i> (formerly known as chrysanthemum stem necrosis virus, CSNV) isolated from <i.Chrysanthemum morifolium</i> was determined using host range and sequence analysis in this study. Twenty-three species of indicator plants inoculated mechanically CSNV-Kr was investigated for determination of host range. CSNV-Kr induced various local and systemic symptoms in the inoculated plant species. CSNV-Kr could not infect three plant species and induced symptomless in systemic leaves in <i.Nicotiana tabacum</i> cultivars, though the plant samples reacted positively with the antiserum to CSNV by double-antibody sandwich–enzyme-linked immunosorbent assay. The complete genome sequence of CSNV-Kr was determined. The L RNA of CSNV-Kr consists of 8,959 nucleotides (nt) and encodes a putative RNA-dependent RNA polymerase. The M RNA of CSNV-Kr consists of 4,835 nt and encodes the movement protein (NSm) and the glycoprotein precursor (Gn/Gc protein). The S RNA of CNSV-Kr consists of 2,836 nt and encodes NSs protein and N protein. The Gn/Gc and N sequence of CSNV-Kr were compared with those of previously published CSNV isolates originating from different countries at nucleotide and amino acid levels. The Gn/GC sequence of CSNV-Kr shared 98.8−99.5% identity with CSNV isolated from other countries and the N sequence of CSNV-Kr shared 98.8−99.6% identity. No particular region of variability could be found in either grouping of viruses. All of the CSNV isolates did not show any relationship according to geographical origins and isolation hosts, suggesting no distinct segregation of the CSNV isolates.

First Report of Impatiens Necrotic Spot Virus Infecting Lettuce in Greece

HomePlant DiseaseVol. 104, No. 10First Report of Impatiens Necrotic Spot Virus Infecting Lettuce in Greece PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Impatiens Necrotic Spot Virus Infecting Lettuce in GreeceD. Beris, I. Malandraki, O. Kektsidou, N. Vassilakos, and C. VarveriD. Beris†Corresponding author: D. Beris; E-mail Address: d.mperi@bpi.grhttp://orcid.org/0000-0003-3308-8742Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, I. Malandrakihttp://orcid.org/0000-0001-5623-5261Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, O. KektsidouLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, N. VassilakosLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this author, and C. VarveriLaboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, GreeceSearch for more papers by this authorAffiliationsAuthors and Affiliations D. Beris † I. Malandraki O. Kektsidou N. Vassilakos C. Varveri Laboratory of Virology, Department of Phytopathology, Benaki Phytopathological Institute, Kifissia, Athens 14561, Greece Published Online:18 Aug 2020https://doi.org/10.1094/PDIS-11-19-2316-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat During the autumn of 2018 and the winter of 2019, virus-like disease symptoms were observed in four fields of lettuce (Lactuca sativa) in Kalamos, Marathonas, and Leonidio regions of central and south-eastern Greece. In all cases, the disease affected 30 to 40% of the plants. Symptoms consisted of brown ringspots, necrotic lesions, vein necrosis, and leaf distortion resembling those associated with tospovirus infections. Six symptomatic plants (four in 2018 and two in 2019) were collected from four different fields and were tested for the presence of tomato spotted wilt virus (TSWV), tomato chlorotic spot virus (TCSV), impatiens necrotic spot virus (INSV), groundnut ringspot virus (GRSV), and chrysanthemum stem necrosis virus (CSNV) with double antibody sandwich ELISA specific and generic polyclonal antisera, respectively (Loewe Biochemica, Germany). All samples were found negative for TSWV but positive for other Tospovirus infection with the generic ELISA test. Total RNA was purified from all six symptomatic samples and reverse transcription polymerase chain reaction (RT-PCR) was performed with the gM410/gM870c primer pair (Chen et al. 2012) targeting the conserved NSm genomic region of tospoviruses. Direct Sanger sequencing of the 500-bp PCR products obtained from all six samples revealed the presence of INSV Tospovirus, and showed 97.5 to 98.7% identity at nucleotide level with the respective region of the Chinese isolate HDL (GU112503). To further characterize our INSV isolate, total RNA isolated with NucleoZOL reagent (Macherey-Nagel, Germany) from one symptomatic lettuce was subjected to high-throughput sequencing (HTS) on a Next Seq platform (Illumina, U.S.A.) by using the TruSeq Stranded mRNA Library Prep kit (Illumina) for the construction of the poly-A selected HTS library. The analysis generated approximately 14,000,000 single-end reads of 75-bp length that were analyzed with Geneious bioinformatics software (Biomatters, New Zealand). After the de novo assembly of the unique reads (7,448,124 reads), contigs (in total 108,234 contigs ranging in size from 150 to 12,000 bp) were annotated with BLAST, and the presence of INSV was confirmed. Assembled nucleotide sequences of S (MN553562) and M (MN553561) segments exhibited 88 to 98% coverage and 99% identity at nucleotide level with an Italian isolate (DQ425096 and DQ425095, respectively). Nucleotide sequence of the L segment also showed 64% coverage with 88% identity with the Italian isolate (DQ425094). Furthermore, HTS analysis revealed the presence of lettuce big-vein associated virus (LBVaV, Rhabdoviridae, Varicosavirus genus). To isolate INSV, Nicotiana tabacum, an insusceptible host of LBVaV, was used. Infected N. tabacum tissues were used to reproduce symptoms in virus-tested-negative lettuce plants after mechanical inoculation. In the latter plants, INSV presence was confirmed with RT-PCR assays targeting specific regions of the three INSV segments by respective primer pairs designed based on the HTS analysis. Sequences of the three PCR products were 100% identical with the HTS-assembled INSV segments of the respective regions and confirmed the presence of all virus segments. INSV was first described in Impatiens spp. plants (Law and Moyer 1990) and is an important pathogen for several crops including ornamental plants, tomato, and lettuce (Kuo et al. 2014). The relatively high incidence of the virus in different regions of Greece suggests that INSV is an emerging pest for lettuce cultivation in Greece. To the best of our knowledge, this is the first report of INSV in Greece.The author(s) declare no conflict of interest.

The NSm proteins of phylogenetically related tospoviruses trigger Sw-5b–mediated resistance dissociated of their cell-to-cell movement function

The cell-to-cell movement protein (NSM) of tomato spotted wilt virus (TSWV) has been recently identified as the effector of the single dominant Sw-5b resistance gene from tomato (Solanum lycopersicum L.). Although most TSWV isolates shows a resistance-inducing (RI) phenotype, regular reports have appeared on the emergence of resistance-breaking (RB) isolates in tomato fields, and suggested a strong association with two point mutations (C118Y and T120N) in the NSM protein. In this study the Sw-5b gene has been demonstrated to confer not only resistance against TSWV but to members of five additional, phylogenetically-related classified within the so-called “American” evolutionary clade, i.e., Alstroemeria necrotic streak virus (ANSV), chrysanthemum stem necrosis virus (CSNV), groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV) and tomato chlorotic spot virus (TCSV). Remarkably, bean necrotic mosaic virus (BeNMV), a recently discovered tospovirus classified in a distinct American subclade and circulating on the American continent, did not trigger a Sw-5b-mediated hypersensitive (HR) response. Introduction of point mutations C118Y and T120N into the NSM protein of TSWV, TCSV and CSNV abrogated the ability to trigger Sw-5b-mediated HR in both transgenic-N. benthamiana and tomato isolines harboring the Sw-5b gene whereas it had no effect on BeNMV NSM. Truncated versions of TSWV NSM lacking motifs associated with tubule formation, cell-to-cell or systemic viral movement were made and tested for triggering of resistance. HR was still observed with truncated NSM proteins lacking 50 amino acids (out of 301) from either the amino- or carboxy-terminal end. These data altogether indicate the importance of amino acid residues C118 and T120 in Sw-5b-mediated HR only for the NSM proteins from one cluster of tospoviruses within the American clade, and that the ability to support viral cell-to-cell movement is not required for effector functionality.

Development of a microarray for simultaneous detection and differentiation of different tospoviruses that are serologically related to Tomato spotted wilt virus

BackgroundTospoviruses, the plant-infecting genus in the family Bunyaviridae, are thrips borne and cause severe agricultural losses worldwide. Based on the serological relationships of the structural nucleocapsid protein (NP), the current tospoviruses are divided into six serogroups. The use of NP-antisera is convenient for virus detection, but it is insufficient to identify virus species grouped in a serogroup due to the serological cross-reaction. Alternatively, virus species can be identified by the N gene amplification using specific primers. Tomato spotted wilt virus (TSWV) is the type species of the genus Tospovirus and one of the most destructive plant viruses. Eight known tospoviruses, Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Melon severe mosaic virus (MeSMV), Pepper necrotic spot virus (PNSV), Tomato chlorotic spot virus (TCSV) and Zucchini lethal chlorosis virus (ZLCV), sharing serological relatedness with TSWV in NP, are grouped in the TSWV serogroup. Most of the TSWV-serogroup viruses prevail in Europe and America. An efficient diagnostic method is necessary for inspecting these tospoviruses in Asia, including Taiwan.MethodsA microarray platform was developed for simultaneous detection and identification of TSWV-serogroup tospoviruses. Total RNAs extracted from Chenopodium quinoa leaves separately inoculated with ANSV, CSNV, GRSV, INSV, TCSV and TSWV were used for testing purposes. The 5’-biotinylated degenerate forward and reverse primers were designed from the consensus sequences of N genes of TSWV-serogroup tospoviruses for reverse transcription-polymerase chain reaction (RT-PCR) amplification. Virus-specific oligonucleotide probes were spotted on the surface of polyvinyl chloride (PVC) chips to hybridize with PCR products. The hybridization signals were visualized by hydrolysis of NBT/BCIP with streptavidine-conjugated alkaline phosphatase. The microarray was further applied to diagnose virus infection in field crop samples.ResultsAmplicons of approximately 0.46 kb were amplified from all tested TSWV-serogroup tospoviruses by RT-PCR using the degenerate primer pair Pr-dTS-f/Pr-dTS-r. Virus species were identified on chips by hybridization of PCR products with respective virus-specific probes. The microarray was successfully used to diagnose TSWV infection in field pepper samples.ConclusionsIn this study, a rapid, sensitive and precise microarray method has been developed to simultaneously detect and identify six TSWV-serogroup tospoviruses. The microarray platform provides a great potential to explore tospoviruses that can help researchers and quarantine staff to prevent invasions of tospoviruses.

The functional analysis of distinct tospovirus movement proteins (NSM) reveals different capabilities in tubule formation, cell-to-cell and systemic virus movement among the tospovirus species

The lack of infectious tospovirus clones to address reverse genetic experiments has compromised the functional analysis of viral proteins. In the present study we have performed a functional analysis of the movement proteins (NSM) of four tospovirus species Bean necrotic mosaic virus (BeNMV), Chrysanthemum stem necrosis virus (CSNV), Tomato chlorotic spot virus (TCSV) and Tomato spotted wilt virus (TSWV), which differ biologically and molecularly, by using the Alfalfa mosaic virus (AMV) model system. All NSM proteins were competent to: i) support the cell-to-cell and systemic transport of AMV, ii) generate tubular structures on infected protoplast and iii) transport only virus particles. However, the NSM of BeNMV (one of the most phylogenetically distant species) was very inefficient to support the systemic transport. Deletion assays revealed that the C-terminal region of the BeNMV NSM, but not that of the CSNV, TCSV and TSWV NSM proteins, was dispensable for cell-to-cell transport, and that all the non-functional C-terminal NSM mutants were unable to generate tubular structures. Bimolecular fluorescence complementation analysis revealed that the C-terminus of the BeNMV NSM was not required for the interaction with the cognate nucleocapsid protein, showing a different protein organization when compared with other movement proteins of the ‘30K family’. Overall, our results revealed clearly differences in functional aspects among movement proteins from divergent tospovirus species that have a distinct biological behavior.

Development of reverse transcription loop-mediated isothermal amplification assay as a simple detection method of Chrysanthemum stem necrosis virus in chrysanthemum and tomato

For a simple and rapid detection of Chrysanthemum stem necrosis virus (CSNV) from chrysanthemum and tomato, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed. A primer set designed to the genome sequences of CSNV worked most efficiently at 63°C and could detect CSNV RNA within 12min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. The result of a specificity test using seven other viruses and one viroid-infectable chrysanthemum or tomato showed that the assay could amplify CSNV specifically, and a sensitivity comparison showed that the RT-LAMP assay was as sensitive as the reverse transcriptase polymerase chain reaction. The RT-LAMP assay using crude RNA, extracted simply, could detect CSNV. Overall, the RT-LAMP assay was found to be a simple, specific, convenient, and time-saving method for CSNV detection.

Pest Identification Guide: Western Flower Thrips Frankliniella occidentalis (Pergande)

Western flower thrips transmit the carmovirus Pelargonium flower break virus (PFBV), the ilarvirus Tobacco streak virus (TSV), the tospoviruses Chrysanthemum stem necrosis virus (CSNV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Tomato chlorotic spot virus (TCSV), and Tomato spotted wilt virus (TSWV). This species is primarily a flower feeder, so most damage would be expected on the flower or fruit. Learn to identify the western flower thrips with this two-page illustrated guide written by Jeffrey D. Cluever and Hugh A. Smith, and published by the Department of Entomology and Nematology, May 2016. ENY-2034/IN1127: Pest Identification Guide: Western Flower Thrips Frankliniella occidentalis (Pergande) (ufl.edu)

The movement proteins (NSm) of distinct tospoviruses peripherally associate with cellular membranes and interact with homologous and heterologous NSm and nucleocapsid proteins

Tospovirus is the only genus containing virus species which infect plants in the Bunyaviridae family. The aims of this study were to understand the in vivo membrane association of the movement protein (NSm) of the tospovirus species Bean necrotic mosaic virus, Chrysanthemum stem necrosis virus, Tomato chlorotic spot virus and Tomato spotted wilt virus and the homologous and heterologous interactions among NSm and nucleocapsid protein (N). The results obtained by bimolecular fluorescence complementation (BiFC) assay and chemical treatments after membrane fractionation revealed that the four NSm proteins are associated with the biological membranes with the N- and C-termini oriented to the cytoplasm. BiFC analysis for protein–protein interactions showed: i) dimer formation for all NSm and N proteins; ii) interaction between NSm and the cognate N and iii) heterologous interactions between the NSm and N proteins. The implications of these interactions in the life cycle of tospoviruses are discussed.

Genetic and serological characterization of chrysanthemum stem necrosis virus, a member of the genus Tospovirus.

Chrysanthemum stem necrosis virus (CSNV) is a member of a tentative tospovirus species. In this study, the complete genomic sequence of the Japanese CSNV isolate TcCh07A was determined. The L RNA is 8960 nt long and encodes the 331.0-kDa RNA-dependent RNA polymerase. The M RNA is 4828nt long and encodes the 34.1-kDa movement protein (NSm) and the 127.7-kDa glycoprotein precursor (Gn/Gc). The S RNA is 2949nt long and encodes the 52.4-kDa silencing suppressor protein (NSs) and the 29.3-kDa nucleocapsid (N) protein. The N protein of CSNV-TcCh07A was purified from virus-infected plant tissues and used for production of a rabbit polyclonal antiserum (RAs) and a monoclonal antibody (MAb). Results of serological tests by indirect ELISA and western blotting using the prepared RAs and MAb and a previously produced RAs against the N protein of tomato spotted wilt virus (TSWV) indicated that CSNV-TcCh07A, TSWV, tomato chlorotic spot virus, groundnut ringspot virus, alstroemeria necrotic streak virus and impatiens necrotic spot virus are serologically related.

The complete nucleotide sequence of chrysanthemum stem necrosis virus.

The complete genome sequence of chrysanthemum stem necrosis virus (CSNV) was determined using Roche 454 next-generation sequencing. CSNV is a tentative member of the genus Tospovirus within the family Bunyaviridae, whose members are arthropod-borne. This is the first report of the entire RNA genome sequence of a CSNV isolate. The large RNA of CSNV is 8955 nucleotides (nt) in size and contains a single open reading frame of 8625 nt in the antisense arrangement, coding for the putative RNA-dependent RNA polymerase (L protein) of 2874 aa with a predicted Mr of 331kDa. Two untranslated regions of 397 and 33 nt are present at the 5' and 3' termini, respectively. The medium (M) and small (S) RNAs are 4830 and 2947 nt in size, respectively, and show 99% identity to the corresponding genomic segments of previously partially characterized CSNV genomes. Protein sequences for the precursor of the Gn/Gc proteins, N and NSs, are identical in length in all of the analysed CSNV isolates.

First Report of Chrysanthemum stem necrosis virus on Russell Prairie Gentian in Brazil.

In November 2012, plants of Russell prairie gentian (Eustoma grandiflorum, Lisianthus russellianus) were collected from a commercial greenhouse in Atibaia, SP, Brazil, displaying necrotic spots on leaves and necrosis on stems, followed by generalized systemic necrosis. Disease symptom incidence was estimated at 10%. Preliminary electron microscopy observations of negatively stained leaf extracts prepared from those lesions revealed the presence of a large number of spherical tospovirus-like, approximately 100 nm in diameter. Samples of infected leaves were ground in 0.01 M phosphate buffer containing 0.5% sodium sulphide and mechanically inoculated in six plants of each species of Nicotiana glutinosa, N. tabacum cv. White Burley, N. megalosiphon, N. debneyii, Datura stramonium, Chenopodium amaranticolor, C. quinoa, and E. grandiflorum. All inoculated plants displayed local lesions 4 to 5 days after inoculation, while N. debneyii and D. stramonium showed systemic symptoms, typical of Tospovirus infection. In addition, E. grandiflorum reproduced the original symptoms. Total RNA was extracted from infected E. grandiflorum and D. stramonium, and reverse transcription (RT)-PCR was performed using universal primers BR60 and BR65 (2) targeting conserved regions of the nucleocapsid gene (N). The amplification products of approximately 450 bp were purified, cloned, and sequenced. The unknown virus was identified as Chrysanthemum stem necrosis virus (CSNV-Lis) based on host range and nucleotide sequence (Genbank Accession No. KC894721) and showed 99% identity with a CSNV chrysanthemum isolate from Japan (AB600872). Maximum likelihood phylogenetic analysis using nine homologous CSNV sequences available in GenBank classified CSNV-Lis into a monophyletic group formed by chrysanthemum isolates from Japan and China while a Japanese lisianthus isolate was separately clustered. CSNV is a member of the genus Tospovirus (Bunyaviridae) and was first reported on chrysanthemum in Brazil (1) and later in the Netherlands, Slovenia, United Kingdom, and Japan (3). Despite scattered recent reports of CSNV, the simultaneous production of chrysanthemum and lisianthus crops along the year by Brazilian farmers has contributed to the virus maintenance in the field. The high identity between Brazilian and Japanese isolates of CSNV suggest a possible reintroduction of the virus through exchange of vegetative propagating material.

First outbreak of Chrysanthemum stem necrosis virus (CSNV) on potted Chrysanthemum in Belgium

In Belgium, potted Chrysanthemum x morifolium cultivation covers about 400 ha, of which 375 ha is in open fields and the remaining portion in glasshouses. This represents nearly half of total European production. Among the most important pests and diseases with a significant impact on chrysanthemum production are the western flower thrips (Frankliniella occidentalis) and spotted wilt (Tomato spotted wilt virus TSWV), for which the thrips acts as a vector. So far, Chrysanthemum stem necrosis virus (CSNV), a distinct member of the genus Tospovirus (Bezerra et al., 1), has only been described in a limited host range and with a restricted geographical distribution. However, even with limited data available on losses caused by CSNV in its major areas of distribution i.e. Brazil and Japan, the phytosanitary risk to chrysanthemum and tomato crops, in areas where its vector F. occidentalis is known to occur, is considered high. Moreover, while symptoms of CSNV and TSWV are comparable, CSNV-induced disease often develops faster and is more destructive (Bezerra et al., 1). In October 2012, plants suspected to be virus-infected in showing wilting and having numerous necrotic lesions surrounded by yellow areas, were received from a seasonal chrysanthemum grower, growing under glass in western Belgium. Initially TSWV infection was suspected but the necrotic zones quickly extended to stems (Fig. 1), ultimately killing most of the crop in the case of cvs. Ludo and Jorca Pink (Fig. 2). Losses for the cv. Mirage Yellow (Fig. 3) also grew to more than 50%. Cultivar Miral White and an assorted cultivar mix were also infected, although symptoms remained less severe. Cuttings from all of these cultivars originated from Brazil. After an initial screen for TSWV with TSWV reagent set (Art. No. 190165, BIOREBA, Switzerland), followed by a CSNV test (AS-0529; DSMZ, Germany), both by DAS-ELISA, further detection and identification of the virus by PCR was based on the nucleocapsid (N) gene sequence. Total RNA was extracted from leaf and stem tissue using a Spectrum Total Plant RNA kit (Sigma-Aldrich, Belgium). The specific CSNV-N5/N3 PCR primers developed by Takeshita et al. (5) were used to amplify the N gene ORF (approx. 950 bp) and these fragments were cloned (Clonejet PCR cloning kit, Thermo Scientific, Belgium) and sequenced. CSNV infection was confirmed on all sampled plants with symptoms from all five infected cultivars, and no variability was noticed in their respective N gene sequences. The consensus 937 bp sequence of the N gene is available from GenBank (Accession No. KC525102). Symptomless plants did not test positive for CSNV nor any other tospoviruses. Infected plant material from the cv. Ludo was mechanically inoculated on a series of indicator plants and resulted in symptoms corresponding to those described by Bezerra et al. (1). RT-PCR on symptomatic indicator plants again confirmed the presence of CSNV. This is the first report of a CSNV outbreak in Europe in 10 years. Previous outbreaks in the Netherlands (Verhoeven et al., 2003), Slovenia (Ravnikar et al., 4) and UK (Mumford et al., 3) were all declared eradicated (EPPO, 2). The infected production facility is now under eradication measures and the virus will remain subject to official control in 2013.

Competence of Frankliniella occidentalis and Frankliniella intonsa strains as vectors for Chrysanthemum stem necrosis virus

The vector competence of Frankliniella occidentalis for Chrysanthemum stem necrosis virus (CSNV) was evaluated. Three vector strains with distinct competences for Tomato spotted wilt virus (TSWV) transmission were investigated, including an artificially selected strain (TsH) that has a particularly high competence (>90 %). Newly hatched larvae of F. occidentalis were given an acquisition access period of 5 days on CSNV-infected D. stramonium leaves, and reared to maturity. Their transmission efficiencies were examined using a leaf disk assay using Petunia x hybrida leaves. Following the leaf disk assay, the virus accumulation in the vectors was examined via a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of their bodies. The results showed that the CSNV acquisition and transmission efficiency of the TsH strain did not differ from those of the others, indicating that the competence of F. occidentalis as a vector for CSNV is not related to that for TSWV. The CSNV transmission and acquisition efficiencies of two F. intonsa strains (Hiroshima and Fukuoka) were also evaluated. In Hiroshima strain, 35 % of adults were viruliferous, but only two transmitters (3 %) were observed. In Fukuoka strain, 6 % were viruliferous, and no transmitters were observed. These results indicate that F. intonsa cannot be a major vector for CSNV. The accumulation of CSNV in the adults of F. occidentalis and F. intonsa evaluated using DAS-ELISA showed a significant difference in ELISA values among transmitter, viruliferous non-transmitter, and non-viruliferous individuals. These results clearly demonstrated that only transmitters that accumulated a threshold quantity of virus can transmit CSNV to plants.

First Report of Tomato yellow ring virus (Tospovirus, Bunyaviridae) Infecting Tomato in Kenya.

Tomato (Lycoperscion esculentum) is one of the most popular vegetables and a major source of nutrition and income for smallholders in Africa. Thrips-transmitted tospoviruses are among the economically important pathogens of tomatoes that cause significant crop losses worldwide (3). In surveys for Tomato spotted wilt virus (TSWV) in the major tomato production areas of Kenya between March 2010 and January 2012, tomato fruits with chlorotic ring spots on fruits with stem and leaf necrosis were observed frequently. The symptoms were more evident in the dry seasons and disease incidence ranged from 28 to 42%. The pathogen did not react with antiserum specific to TSWV (Agdia Biofords, Ervy, France) in double-antibody sandwich (DAS)-ELISA. Furthermore, the pathogen did not react with antiserum specific to Capsicum chlorosis virus (CaCV), Chrysanthemum stem necrosis virus (CSNV), Groundnut ring spot virus (GRSV), Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV), and Watermelon silver mottle virus (WSMoV) (Agdia Biofords and DSMZ, Germany) in DAS-ELISA, but reacted positively to antiserum specific to Tomato yellow fruit ring virus (TYFRV) (DSMZ, AS0526). The nucleocapsid (N) gene specific primers (TFfor: 5'-ACTCATTAAAATGCATCGTTCT-3' and TFrev: 5'-CTAAGTAAACACCATGGCTACC-3' as forward and reverse primers, respectively) were designed by choosing six conserved regions of the N gene sequences of known TYFRV and Tomato yellow ring virus (TYRV) sequences available from GenBank. Using these primers, TYRV infection of tomatoes collected from Loitokitok, Kenya (2.73°S, 37.51°E) was confirmed by reverse transcription (RT)-PCR. PCR products of approximately 912-bp were obtained from six out of 11 symptomatic tomato samples tested, but not from healthy and water controls. Amplicons were gel-purified using QuickClean II Gel Extraction Kit (GenScript, UK) and sequenced using TFfor and TFrev primers. A consensus sequence was generated using Geneious Pro 5.5.6 Software (Biomatters Ltd., Auckland, NZ). The BLAST revealed that the N-gene sequence of the Kenyan tomato isolate (GenBank Accession No. JQ955615) had sequence identity with the Cineraria isolate (98.5%) (Accession No. DQ788693.1) and the Anemone isolate (98.1%) (Accession No. DQ788694.1) of TYRV (4) from Fars Province, Iran; an Alstroemeria isolate (98.4%) (Accession No. HQ154130.1) and two tomato isolates (98.3%) (Accession Nos. HQ154131.1 and AY686718.1) of TYRV from northern Khorasan Province, Iran, and a tomato isolate (98.1%) (Accession No. AJ493270.1) of TYFRV from Varamin, Iran. The Kenyan tomato isolate differed from a TYFRV potato isolate (87.5%) from Iran (Accession No. EU126931.1) (1), a TYRV potato isolate (87.5%) from Iran (Accession No. JF836812.1); a soybean isolate of TYRV (87.4%) from Iran (Accession No. DQ462163.1) (2), and showed significant divergence from that of Polygonum ringspot virus from Italy (81%) (Accession No. EF445397.1). To our knowledge, this is the first report of TYRV infecting tomatoes in Kenya. Further surveys and monitoring of TYRV incidence and distribution in the region, vector competence of thrips species, and impact on the crop yield are in progress.

Scientific Opinion on the pest categorisation of the tospoviruses

The European Commission requested EFSA's Panel on Plant Health to perform the pest categorisation for the 24 viruses of the Tospovirus genus for the EU territory. The following tospoviruses were analysed: Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV), Polygonum ringspot virus (PolRSV), Groundnut ringspot virus (GRSV), Tomato chlorotic spot virus (TCSV), Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Melon severe mosaic virus (MSMV), Tomato yellow (fruit) ring virus (TYRV), Tomato zonate spot virus (TZSV), Groundnut yellow spot virus (GYSV), Groundnut chlorotic fan-spot virus (GCFSV), Groundnut bud necrosis virus (GBNV), Zucchini lethal chlorosis virus (ZLCV), Capsicum chlorosis virus (CaCV), Watermelon bud necrosis virus (WBNV), Watermelon silver mottle virus (WSMoV), Tomato necrotic ringspot virus (TNRV), Calla lily chlorotic spot virus (CCSV), Melon yellow spot virus (MYSV), Soybean vein necrosis associated virus (SVNaV), Bean necrotic mosaic virus (BeNMV) and Pepper necrotic spot virus (PNSV). In reaching its conclusions, the Panel considered four parameters to be of critical importance in the risk assessment area: (i) the presence of a tospovirus, (ii) the existence of host plants, (iii) the existence of thrips vector species and (iv) the potential for damage to crops grown in Europe. Based on its analysis, the Panel concluded that the 24 viruses analysed could be allocated to four different risk groups. Seven viruses (GRSV, TCSV, ANSV, CSNV, MSMV, TYRV, TZSV) for which both thrips species vectors and natural or experimental hosts crops are present in the EU territory were considered by the Panel to represent the highest risk to the EU territory. In contrast, three viruses (INSV, IYSV and PolRSV) already present in the risk assessment area were not considered by the Panel to pose a risk justifying the development of full risk assessments.

First report of a tospovirus in a commercial crop of Cape gooseberry in Brazil

Viruses from the genus Tospovirus (family Bunyaviridae) are widespread in the Americas, where the following species have been reported: Tomato chlorotic spot virus (TCSV), Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV), Chrysanthemum stem necrosis virus (CSNV), Zucchini lethal chlorosis virus (ZLCV), Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV) (Jones, 2005), Alstroemeria necrotic streak virus (ANSV), Melon severe mosaic virus (MeSMV) and Bean necrotic mosaic virus (BeNMV) (Ciuffo et al., 2009; Hassani-Mehraban et al., 2010; Oliveira et al., 2011). This work describes the identification of a virus isolated from a commercial crop of Cape gooseberry (Physalis peruviana) in October 2009 in Santa Maria, State of Rio Grande do Sul, Brazil. The disease was identified in a 5 ha cultivated area, where 100% of the plants were showing symptoms of stunting, mosaic, necrosis and foliar distortion (Fig. 1). Spherical particles measuring 80-100 nm in diameter were observed by electron microscopy, giving the initial evidence that a tospovirus might be involved in the disease. Sap from the infected leaves of five different Cape gooseberry plants were inoculated on healthy indicator plants: Petunia hybrida and Chenopodium amaranticolor reacted with local lesions; Datura stramonium, Solanum gilo, S. lycopersicum, S. nigrum, Physalis angulata, P. floridana and P. peruviana (Fig. 1) displayed systemic symptoms. Host reactions were typical for those caused by tospoviruses and symptoms on Physalis species reproduced those observed in the original samples. Testing by plate trapped-ELISA, a positive reaction was only obtained from all five samples using anti-TCSV antiserum (Embrapa, CNPH, Brazil). Negative ELISA results were obtained using antiserum against four other tospoviruses: Tomato spotted wilt virus, Groundnut ringspot virus, Impatiens necrotic spot virus and Chrysanthemum stem necrosis virus. RT-PCR was performed using primers designed to conserved regions of the tospoviral S RNA (Eiras et al., 2001). This generated 440 bp DNA fragments, whose sequence revealed a high degree of nucleotide identity with TCSV isolates from Argentina (98-99%) and Brazil (93-95%). Alignment of the partial coat protein nucleotide sequences showed that the Physalis TCSV isolate (GenBank Accession No. JQ034525) clustered with other TCSV isolates, sharing a clade with Argentinean isolates (Fig. 2). Cape gooseberry is a fruit crop of great nutritional and economic value, which has gradually gained acceptance in international markets, due to its flavour and medicinal properties. In Brazil, it was introduced as a commercial crop into small farms in southern Brazil (Rufato et al., 2008). To our knowledge, this is the first report of natural occurrence of TCSV in commercial crops of Cape gooseberry. To date the disease is restricted to Rio Grande do Sul State. Further epidemiological studies are necessary for understanding the spread of TCSV into this relatively new introduced crop in Brazil.

RT-PCR Detection of Five Quarantine Plant RNA Viruses Belonging to Potyand Tospoviruses

In order to detect quarantine plant viruses, we developed reverse transcription-polymerase chain reaction (RT-PCR) primer pairs for five single-stranded (ss) plant RNA viruses that are not currently reported in Korea but could be potential harmful plant viral pathogens. Three viruses such as Chilli veinal mottle virus (ChiVMV), Colombian datura virus (CDV), and Tobacco etch virus (TEV) belong to the genus Potyvirus while Chrysanthemum stem necrosis virus (CSNV) and Iris yellow spot virus (IYSV) are members of the genus Tospovirus. To design RT-PCR primers, we used reported gene sequences corresponding to the capsid protein and polyprotein for ChiVMV, CDV, and TEV while using nucleocapsid protein regions for CSNV and IYSV. At least two different primer pairs were designed for each virus. Fifteen out of 16 primer pairs were successfully applied in detection of individual quarantine virus with high specificity and efficiency. Taken together, this study provides a rapid and useful protocol for detection of five quarantine viruses.

Molecular and biological characterization of Chrysanthemum stem necrosis virus isolates from distinct regions in Japan

Three isolates of Chrysanthemum stem necrosis virus (CSNV) were obtained from chrysanthemum plants in distinct regions of Japan in 2006 and 2007. All the original host plants showed severe necrotic symptoms on the leaves and stems. Amino acid sequence data of the nucleocapsid protein genes of the three isolates (CbCh07A, TcCh07A, and GnCh07S) showed high identities with those of two other CSNV isolates, HiCh06A L1 from Japan and Chry1 from Brazil. Furthermore, for the first time the complete nucleotide sequence of the S RNA was determined for CSNV (isolate HiCh06A). In phylogenetic analysis based on the non-structural protein genes from the genus Tospovirus, HiCh06A L1 was placed in the same genetic group as Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus. Host range examination for isolates HiCh06A L1 and CbCh07A showed that green pepper (cv. ‘Kyoyutaka’, ‘Saitamawase’, ‘Tosakatsura’, ‘L3 sarara’ and ‘L3 miogi’) and tomato (cv. ‘Sekaiichitomato’) were systemically susceptible hosts, whereas TSWV-resistant Solanaceae species, Capsicum chinense, Lycopersicon peruvianum and a TSWV-resistant cultivar of green pepper (cv. TSR miogi), were resistant.

Stem necrosis of aster and Russell prairie gentian caused by Chrysanthemum stem necrosis virus

A new disease causing necrotic streaks on stems and necrosis on leaves of aster (Callistephus chinensis) and Russell prairie gentian (Eustoma grandiflorum) was observed in Toyama Prefecture, Japan, in September 2009. On the bases of host reactions, serological reactions, viral particle morphology, and the nucleotide sequence of the nucleocapsid protein gene, the causal pathogen was identified as Chrysanthemum stem necrosis virus (CSNV). This report is the first in the world of natural infection of aster and Russell prairie gentian by CSNV. We propose the name “stem necrosis of aster and Russell prairie gentian” for these new diseases.

First Report of Tomato spotted wilt virus on Gerbera hybrida in Serbia.

In May 2009, approximately 30% of plants within a greenhouse-grown Gerbera hybrida crop in Vranjska Banja (Pčinj District) in Serbia displayed chlorotic oak-leaf patterns followed by necrosis and distortion of leaves. Symptoms on naturally infected gerbera plants and local necrotic spots on Petunia × hybrida mechanically inoculated with infected gerbera sap using chilled 0.05 M phosphate buffer (pH 7) containing 1 mM Na-EDTA, 5 mM Na-DIECA, and 5 mM Na-thioglycolate (4) suggested the presence of a Tospovirus. Symptomatic leaves were tested for the presence of Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), and Chrysanthemum stem necrosis virus (CSNV) by commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Loewe Biochemica, Sauerlach, Germany). Commercial positive and negative controls and extract from healthy gerbera tissue were included in each ELISA. All 20 tested plants were negative for INSV and CSNV. TSWV was detected serologically in 18 of 20 gerbera samples. The presence of TSWV in ELISA-positive symptomatic gerbera plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was conducted with the OneStep RT-PCR Kit (Qiagen) using Serbian tobacco TSWV isolate (GQ279731) and RNA extract from healthy gerbera as positive and negative controls, respectively. Two different sets of TSWV-specific primers, L1 TSWVR/L2 TSWVF (2) and M962/M66 (3), for a 276-bp fragment of the RNA-dependent RNA polymerase (RdRp) gene and a 897-bp fragment of the NSm gene, respectively, were used for both amplification and sequencing. RT-PCR analyses of each tested plant detected the presence of amplification fragments of expected size. The amplified products corresponding to part of the RdRp and NSm genes derived from the isolate 158-Gerb were purified (QIAquick PCR Purification Kit, Qiagen) and sequenced in both directions (GenBank Accession Nos. HQ246452 and HQ246453, respectively). Sequence analysis of the partial RdRp gene, conducted using MEGA4 software, revealed 91.1 to 98% nt identity (95.1 to 98.8% amino acid [aa] identities) with corresponding sequences of TSWV L RNA deposited in GenBank. The highest identity was found with an isolate from globe artichoke (AM940436) in Greece, and isolates from tomato (GQ279732), impatiens (GQ132190), and tobacco isolates (GQ279731, FJ189392, and FJ189393) found within Serbia. Analysis of the NSm sequence of isolate 158-Gerb demonstrated nucleotide identities varying between 90.6 and 99.6% (80.9 and 99.6% aa identities) with those of previously reported TSWV isolates. The highest identity was with tobacco isolate GQ373174 from Serbia. Therefore, while gerbera is one of the principal ornamental hosts of TSWV in the EPPO region (1), to our knowledge, this is the first report infecting gerbera in Serbia, which may have a devastating influence on its production.