Chlorotic Ring Research Articles

First Report from Turkey of European Plum Line Pattern Caused by Apple mosaic virus.

European plum line pattern was first described in Bulgaria (1) and can now be found nearly worldwide. Characteristic symptoms in infected plum (Prunus domestica) include line and oak leaf patterns with chlorotic lines and rings in leaves. The causal agent of this economically damaging plum disease is Apple mosaic virus (ApMV). ApMV has been reported to naturally infect a number of hosts in the Rosaceae, including Rosa, Malus, and Rubus spp. as well as Humulus, Betula, and Corylus spp. in other plant families (3), but has not been reported to naturally infect plum in Turkey. In this study, disease symptoms were observed in only one local cultivar (Süt eriği) during the growing season of 2008-2009 in Amasya and Tokat provinces, situated between the Black Sea and inner Anatolia regions. Leaf samples were collected from 22 plum trees and tested by serological and molecular methods. In serological tests, double-antibody sandwich-ELISA was used with antisera to ApMV and Prunus necrotic ringspot virus (PNRSV) according to the manufacturer's (Agdia, Inc., Elkhart, IN) protocol. While none of the samples reacted positively to PNRSV antisera, 19 samples reacted positively to ApMV antisera. The presence of ApMV in symptomatic plum trees was confirmed by reverse transcription (RT)-PCR. RT-PCR was conducted with ApMV-specific primers (Forward 5'-ATCCGAGTGAACAGTCTATCCTCTAA-3'; reverse 5'-GTAACTCACTCGTTATCACGTACAA-3') as previously described (4) to specifically amplify a 262-bp product from viral sequences. Total RNA was extracted from plum leaf samples with a modified protocol based on silica-capture (2). Using serological and RT-PCR tests, ApMV was detected in all 19 samples that showed virus symptoms, but not from symptomless plants. To our knowledge, this is the first record of the presence of European plum line pattern in Turkey and provides a starting point for investigation of the incidence of ApMV in plum orchards of Turkey.

Identification of Peanut bud necrosis virus and Tomato spotted wilt virus in Indonesia for the first time

In surveys conducted in May 2008 in Warung Kondang, Cianjur, West Java, tomato plants ( Solanum lycopersicum ) growing in farmers’ fields showed stunting and leaf symptoms of either bronzing or general chlorosis with vein-banding. In addition, chilli peppers ( Capsicum spp.) were observed with chlorosis and vein-banding symptoms in Salabintana, Sukabumi, West Java. Finally, peanut plants ( Arachis hypogaea ) showing chlorotic rings and necrosis on leaves were observed in a field near to the Bogor Agricultural University Campus, Darmaga Bogor. All these symptoms suggested the possible involvement of tospoviruses. To identify the viruses involved, leaves with symptoms were pressed onto FTA classic cards [FTA ® Classic Card, Whatman International Ltd.] and shipped to Washington State University, with the appropriate permit from USDA-APHIS-PPQ. Nucleic acids were recovered from the cards using a sample extraction protocol (Alabi et al ., 2008) and tested by one-tube, single-step reverse transcriptionpolymerase chain reaction (RT-PCR) using degenerate ‘universal’ tospovirus primers [Upstream primer: 5 ′ -CCTTTAACAGT(A/T/G)GAAACAT-3 ′ and Downstream primer: 5 ′ -CAT(A/T/G)GC(A/G)CAAGA(A/G)TG(A/G)TA(A/ G)ACAGA-3 ′ ] conserved in the L RNA segment of tospoviruses (Chu et al ., 2001). A DNA fragment of approximately 800 base pairs was amplified from the tomato, pepper and peanut samples. Each was cloned separately into pCR 2.1-TOPO vector (Invitrogen). Three independent clones from each sample were sequenced in both directions. A comparison of the nucleotide sequences obtained from the tomato (GenBank Accession No. FJ 177301) and chilli pepper samples (FJ 177302) was made with corresponding sequences of tospovirus L-RNA deposited in GenBank. This analysis showed 96 and 97% nucleotide sequence identity with Tomato spotted wilt virus (TSWV) isolates from Hawaii, USA (AY070218) and Japan (AB198742), respectively. The sequences obtained from tomato and chilli shared only 93% identity between them, indicating that the Indonesian TSWV isolates from tomato and pepper may be distinct strains. An analysis of the nucleotide sequence obtained from the peanut sample (FJ177300) showed 94% sequence identity with corresponding L RNA sequence of a Peanut bud necrosis virus (PBNV) isolate from India (AF025538). These results confirm for the first time that two distinct tospoviruses, TSWV and PBNV, occur in Indonesia.

Detection of Sweet potato virus 2 in Sweet Potato in New Zealand.

In New Zealand, sweet potato (Ipomoea batatas) is a crop of cultural importance and an important food source; it is grown mainly in the districts of Kaipara, Auckland, and the Bay of Plenty in the North Island. In January of 2008, virus symptoms that included chlorotic spots, ring spots, and mottling were observed on the leaves of commercial sweet potato crops (cvs. Beauregard, Owairaka Red, and Toka Toka Gold) growing in the three main production areas. A survey was done to determine the extent of virus infection in these crops. Fifty to one hundred leaves were collected randomly from each of 26 different fields. Leaves from each field were bulked into groups of 10, giving a total of 173 composite samples. All samples tested negative for Cucumber mosaic virus, C-6 virus, Sweet potato caulimo-like virus, Sweet potato chlorotic fleck virus, Sweet potato chlorotic stunt virus (SPCSV), Sweet potato latent virus, and Sweet potato mild specking virus by nitrocellulose membrane enzyme-linked immunosorbent assays (International Potato Center-CIP, Lima, Peru). Total nucleic acid was extracted from all 173 composite samples and used in real-time PCR assays specific for Sweet potato leaf curl virus (SPLCV) and real-time reverse transcription (RT)-PCR specific for SPCSV, Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG), and Sweet potato virus 2 (SPV2; synonym Sweet potato virus Y) (1). No samples were positive for SPLCV or SPCSV, but 107 and 138 samples tested positive for SPFMV and SPVG, respectively. SPFMV and SPVG have been reported previously in New Zealand (2,3). Sixty four samples from 16 different fields tested positive for SPV2. Of the 64 samples, 52 were also infected with SPVG and SPFMV, and 10 were co-infected with SPVG but not SPFMV; no samples were co-infected with SPV2 and SPFMV when SPVG was absent. From a representative SPV2 positive sample, the 70-bp amplicon obtained by the real-time RT-PCR primers was cloned and sequenced A BLAST search showed 100% nucleotide sequence identity with SPV2 (GenBank Accession Nos. AM050887 and AY178992). Subsequently, primers (V2-F1c: 5'-AGAACAGGACAAACTCAACC-3'; V2-R1: 5'-TAATCACCCTTCACACCTTC-3') were designed to amplify an approximately 434-bp fragment within the SPV2 coat protein gene. One-step RT-PCR was done on four of the SPV2 positive samples and amplicons of the expected size were sequenced directly (GenBank Accession No. FJ461774). Sequence comparison showed 99% nucleotide sequence identity with SPV2 (GenBank Accession Nos. AM050886, AM050887, AY178992, and EF577437). SPV2 is a member of the genus Potyvirus but the virus has not been fully characterized. It is known that single-potyvirus infections cause mild or no symptoms in sweet potato, and consequently, no significant yield reduction is observed generally. However, co-infection with other viruses such as SPCSV produces a synergistic effect and more severe disease symptoms (4). To our knowledge, this is the first report of SPV2 infecting sweet potato in New Zealand.

Molecular evidence for the presence of Apple chlorotic leaf spot virus in infected peach trees in India

During surveys of Himachal Pradesh (HP) and Punjab states in northern India, Apple chlorotic ring spot virus (ACLSV) was detected in infected peach trees using Double Antibody Sandwich-Enzyme Linked Immunosorbent Assay (DAS-ELISA). Identity of the virus was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) giving an amplification of ∼800 bp product using primer pair to amplify the complete coat protein (CP) gene. Multiple alignment of sequences of the CP from samples collected from Kullu and Solan in HP with sequences of other isolates of ACLSV originating from peach trees showed identity of 69–84% and 69–93% at the nucleotide and protein level, respectively. This variation is similar to that observed with other isolates of ACLSV. This is the first report of molecular characterization of ACLSV infecting peach trees in India.

Partial characterisation of a novel Tobamovirus infecting Actinidia chinensis and A. deliciosa (Actinidiaceae) from China

Actinidia chinensis and A. deliciosa plants from China, showing a range of symptoms, including vein clearing, interveinal mottling, mosaics and chlorotic ring spots, were found to contain ~300 nm rod-shaped virus particles. The virus was mechanically transmitted to several herbaceous indicators causing systemic infections in Nicotiana benthamiana, N. clevelandii, and N. occidentalis, and local lesions in Chenopodium quinoa. Systemically- infected leaves reacted with a Tobacco mosaic virus polyclonal antibody in indirect ELISA. PCR using generic and specific Tobamovirus primers produced a 1,526 bp sequence spanning the coat protein (CP), movement protein (MP), and partial RNA replicase genes which showed a maximum nucleotide identity (88%) with Turnip vein clearing virus and Penstemon ringspot virus. However, when the CP sequence alone was considered the highest CP sequence identity (96% nt and 98% aa) was to Ribgrass mosaic virus strain Kons 1105. The morphological, transmission, serological and molecular properties indicate that the virus is a member of subgroup 3 of the genus Tobamovirus.

Pathogen‐tested material of grapevine varieties and rootstocks

Pathogen‐tested material of grapevine varieties and rootstocks

Capsicum chlorosis virus (Genus Tospovirus) Infecting Chili Pepper (Capsicum annuum) in India.

Chili pepper (Capsicum annuum L.) is one of the most important spice crops in India. In October of 2006, symptoms indicative of tospovirus infection were noticed in several commercial fields of chili pepper near Bangalore in Karnataka State. Chlorotic and necrotic spots and rings on leaves, apical necrosis, and leaf distortion were observed. Disease incidence was more than 20%. Mechanical inoculation with sap extracts from these symptomatic plants showed that the host range and symptomatology of the virus was similar to those described for Capsicum chlorosis virus (CaCV) (1,3). The virus reacted with antisera specific to Groundnut bud necrosis virus (GBNV) and Watermelon silver mottle virus of serogroup IV tospoviruses in antigen-coated plate ELISA. It did not react with antisera specific to Tomato spotted wilt virus, Impatiens necrotic spot virus, or Iris yellow spot virus in double-antibody sandwich-ELISA. Immunosorbent electron microscopy of infected sap using GBNV antiserum revealed the presence of strongly decorated quasi-spherical virus particles. Reverse transcription (RT)-PCR was conducted to further identify the virus. No amplification was observed from extracts of symptomatic plants (n = 10) by RT-PCR using GBNV-specific primers (4), indicating that the diseased chili was not infected with GBNV. However, a DNA fragment of approximately 850 bp was amplified by using primers specific to the nucleocapsid (N) gene of CaCV (CaCF 5'-CTATAGAWGTACTAGGCTTTGAGC-3' and CaCR 5'-CATGTCTAACGTCAGGCAACTTAC-3'). Direct sequencing of the amplicon (GenBank Accession No. EF625227) revealed a nucleotide sequence identity ranging from 85.5% with isolates from Thailand (GenBank Accession Nos. AY846366, AY647437, and AF134400) and China (GenBank Accession No. DQ355974) to 98.1% with isolates from Australia (GenBank Accession Nos. AY036057 and AY036058). Phylogenetic analysis of the N protein sequences showed that the chili pepper isolate from India formed a cluster with those from Australia. This cluster was distinct from the one formed by the peanut isolates from Thailand and a tomato isolate from India (2). To our knowledge, this is the first report of CaCV infection of chili pepper in India. The potential impact of CaCV on tomato and chili pepper production in India remains to be seen.

과일 괴저 병징의 단 고추에서 분류동정한 오이모자이크바이러스의 새로운 계통 CMV-NP 특성

경북 청도지역의 단 고추 과일에 나타난 큰 괴저 반점 및 과일 괴저와 잎에 엽맥 녹대 및 기형 병징을 일으키는 바이러스는 오이모자이크바이러스의 새로운 계통인 CMV-NP로 분류동정되었다. CMV-NP는 직경이 26 nm의 구형이었으며, VC/RT-PCR 유전자 진단결과 CMV로 확인되었다. CMV-NP는 오이(Cucumis sativus) 등 9개 지표식물에 전신감염을 일으켰으며, 명아주(Chenopodim amaranticolor; C. quinoa)에 국부감염을 일으켰다. CMV-NP는 담배(N. rustica)와 번행초(Tetragonia expansa)의 접종잎과 상엽에 특이한 괴저 원형 반점을 일으켰다. 특히 오이에서는 상엽에 큰 퇴록 원형반점과 엽맥퇴록 병징을 일으켰으며 독말풀(Datura stramonium)에서는 병원성이 없었다. A strain of Cucumber mosaic virus(CMV) was isolated newly from sweet pepper(Capsicum annuum var. angulosum) showing necrosis with large necrotic spots on fruits and vein banding with malformation on leaf at Cheongdo area in Gyeongsangbukdo. The new strain was designated as a CMV-NP and the shape of virus particles was isometric of 26 nm in diameter from the sweet pepper fruit by Dip method. The strain of CMV-NP was identified genetically by VC/RT-PCR. CMV-NP could infect systemically on the 9 indicator plants including Cucumis sativus, but it could infect locally on Chenopodim amaranticolor and C. quinoa. CMV-NP induced the specific symptoms of necrotic rings on the inoculated and the upper leaves of N. rustica and Tetragonia expansa. On Cucumis sativus, the large chlorotic ring and vein chlorosis were produced on the upper leaves. CMV-NP had no virulence on Datura stramonium.

Pelargonium chlorotic ring pattern virus: first report in Spain

A recent survey of symptomless ornamental geraniums ( Pelargonium zonale , P. peltatum and P. grandiflorum ) in Spain (2000–2003) revealed viral infections in 52·4% of the tested plants (Alonso & Borja, 2005). Most infected plants contained tombus-like viruses such as Pelargonium line pattern virus (PLPV), either alone or in combination with other viral pathogens. Several little-studied PLPV-related viruses have been reported in Pelargonium spp. but their distribution and importance to geranium growers is unknown since they are not usually included in phytosanitary programmes. One of these viruses, Pelargonium chlorotic ring pattern virus (PCRPV), was recently sequenced (GenBank Acc. No. AY038069) and found to be related to PLPV in nucleotide sequence and genome organization (Kinard & Jordan, 2002; Castano & Hernandez, 2005). To assess the occurrence of PCRPV in Spain 5 samples from the previous survey and 3 gathered in 2006 were tested. Total RNA was extracted using Plant RNA Reagent (Invitrogen) and reverse transcribed with Superscript II-RT (Invitrogen) and primer CH149 (5 ′ -CCGATTCTCGAGCTGCCAATC-3 ′ ), complementary to positions 1543–1523 of PCRPV. PCR used primers CH149 and CH148 (5 ′ -CGGTTTGAGCACTCTGCGTATC-3 ′ ), homologous to positions 1277–1298 of PCRPV. An amplicon of the expected size (267 bp comprising a region of the viral polymerase gene) was generated in five samples from different geographical origins (Zaragoza, Zamora, Aranjuez and Cordoba) and collecting times (2000, 2003 and 2006). The amplicons derived from two separate samples were sequenced (GenBank Acc. Nos. EF635383 and EF635384) and showed 95% nucleotide and 99–100% amino acid sequence identity with the PCRPV reference sequence. An Italian isolate of PCRPV was recovered from P. zonale with vein banding and leaf distortion (Lisa et al ., 1996; Kinard & Jordan, 2002); however from our results, PCRPV infection can also be asymptomatic. This is the first report of PCRPV in Spain.

First Report of the Natural Infection of Coreopsis auriculata 'Nana' with Lettuce mosaic virus in the United States.

Perennial cultivars of Coreopsis, a genus native to the United States, are widely grown for aesthetics in home gardens and roadsides and are increasingly used in conservation projects and native-plant gardens. During the spring and summer of 2006 and 2007, Coreopsis auriculata 'Nana' plants with foliar symptoms showing chlorotic spots and rings were observed in wholesale and retail nurseries in Washington. Nicotiana benthamiana plants inoculated with crude sap extracts from symptomatic leaves of C. auriculata 'Nana' obtained from two different sources showed systemic mosaic mottling symptoms, indicating the presence of a virus. Symptomatic leaf samples from C. auriculata 'Nana' and N. benthamiana tested positive in antigen-coated plate-ELISA with potyvirus group-specific monoclonal antibodies (Agdia Inc., Elkhart, IN). Additional analysis by ELISA was positive for Lettuce mosaic virus (LMV; Agdia Inc.). To confirm these results, total RNA extracted from symptomatic N. benthamiana leaves was subjected to reverse transcription (RT)-PCR using potyvirus degenerate primers (PNIbF5: 5'-GCCAGCCCTCCACCGTNGTNGAYAA-3' and PCPR1: 5'-GGGGAGGTGCCGTTCTCDATRCACCA-3') covering the 3' end of the NIb gene and the 5' end of the CP gene (1). A single DNA band of approximately 1,000 bp amplified from symptomatic leaves of two independent plants was cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences with corresponding nucleotide sequences of potyviruses in GenBank showed 93 to 99% identity in the NIb/CP region with LMV sequences from France (GenBank Accession Nos. X97704, X65652, and X97705), China (GenBank Accession Nos. AJ306288 and AJ488153), and Brazil (GenBank Accession No. AJ278854). These results confirmed the presence of LMV in symptomatic leaves of N. benthamiana and C. auriculata 'Nana'. The occurrence of LMV has been reported in ornamental plants that included freeway daisy (Osteospermum fruticosum), lisianthus (Eustoma grandiflorum), and gazanias (Gazania spp.) (2-4). To our knowledge, this is the first documented evidence for the occurrence of LMV in Coreopsis, an economically important perennial ornamental widely grown in the United States. Although the origin of LMV in C. auriculata 'Nana' is not known, distribution of cuttings from LMV-infected C. auriculata 'Nana' plants to wholesale and retailers within Washington and across the country by movement of plant material could pose a risk to other ornamentals and crops like lettuce because of the broad host range of LMV and its potential transmission by several species of polyphagous aphids. Seed transmission as a potential means of dissemination of LMV in Coreopsis has not been examined, although the virus is seedborne in other plants such as lettuce.

Detection of Tomato spotted wilt virus in Tomato in the Baja California Peninsula of Mexico

In 2005, serological screening by ELISA of 24 tomato (Solanum lycopersicon Mill.) plants with virus-like foliar symptoms (locally known as "marchitez manchada" [spotted wilt] disease) was done for a variety of RNA viruses: Tomato spotted wilt virus (TSWV; family Bunyaviridae, genus Tospovirus) was specifically detected. The symptomatic plants testing positive were from the most important tomato areas in San Quintin, in the north of Baja California. Symptoms characteristic of TSWV (4), including chlorosis, malformation of apical leaves, stunting, and ringspot lesions, were observed in this region and throughout the peninsula. In 2006, 42 symptomatic tomato plants from La Paz, in the south of Baja California, were analyzed for TSWV by double-antibody sandwich (DAS)-ELISA with a commercially available kit (TSWV ImmunoStrip Kit; Agdia Inc., Elkhart, IN). Total nucleic acids of the TSWV ELISA-positive samples (16 of 42 = 38%) were extracted and preserved on FTA cards (Whatman, Brentford, U.K.) and processed according to the manufacturer's protocol. The positive TSWV samples were verified by reverse transcription (RT)-PCR with primers specific to the TSWV nucleocapsid protein gene, 5'-ATGTCTAAGGTTAAGCTC-3' and 5'-TTAAGCAAGTTCTGTGAG-3' (2). Amplicons of the expected size (approximately 800 bp) were obtained from all 16 positive samples but not in the ELISA-negative samples. The spotted wilt disease was mechanically transmitted to tomato (cv. Rutgers) and Nicotiana glauca seedlings. Symptoms on leaves consisting of chlorotic ring patterns and necrotic lesions were observed in tomato, and slightly concentric chlorotic lesions were observed in N. glauca. All symptomatic plants from San Quintin and La Paz were positive for TSWV in the DAS-ELISA and RT-PCR tests and none were positive for the tobamoviruses, Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV). TSWV was not detected in symptomless tomato plants used as negative controls. TSWV was detected in Mexico in tomatillo (Physalis ixocarpa), tobacco (Nicotiana tabacum), jimsonweed (Datura stramonium) (3), and recently, in tomato and pepper in the Central Plateau of Mexico (1). Although spotted wilt disease has been previously observed in San Quintin tomato-producing areas, to our knowledge, this is the first confirmation of TSWV in the Baja Peninsula. The role of weed hosts as a natural reservoir and the role of species of thrips in the epidemiology of the disease are currently unknown, although the incidence of the virus in these regions has risen to destructive levels in tomato.

First Report of Plum pox virus on Plum in New York State.

Plum pox, also known as Sharka, is one of the more significant viral diseases of stone fruit trees such as plum, peach, and apricot. It was first reported in Europe in the early 1900s and more recently in Chile in 1992, the United States (Pennsylvania) in 1999, Canada (Ontario and Nova Scotia) in 2000, China in 2001, and Argentina in 2004. Plum pox virus (PPV) was recently detected in two plum (Prunus domestica) trees in an orchard in Niagara County, NY, located within 5 miles from a Canadian plum pox eradication zone. Typical symptoms of chlorotic rings and spots were observed on some of the leaves from these trees. No symptoms were reported prior to the survey collection in July 2006. Survey samples were screened for the presence of PPV by ELISA using the Agdia PPV (Agdia, Elkhart, IN) specific kit that recognizes all strains but C of PPV. Approximately 5% of the survey samples were additionally analyzed by a validated immunocapture reverse transcription (IC-RT)-PCR TaqMan assay in a Cepheid SmartCycler (Cepheid, Sunnyvale, CA). Both replicates of the two New York plum trees produced a positive ELISA reaction in two consecutive tests. The ELISA-positive samples also produced positive results when subjected to the real-time IC-RT-PCR test. The PPV-positive trees were sampled again and an additional 53 samples were collected from trees in the surrounding area. Suspect trees again tested positive, while all the trees in the surrounding area tested negative. The methods used for confirmation included two ELISA tests (Durviz [Valencia, Spain] DAS indirect monoclonal ELISA and Agdia DAS polyclonal ELISA). Confirmatory real-time IC-RT-PCR was performed using universal 3' nontranslated region (NTR) primers (2,3) in a SYBR Green assay format and a coat protein (CP) primers/probe TaqMan assay (3,4). Further, the New York PPV isolate was identified as PPV D group using a subgroup specific conventional IC-RT-PCR (1). A 1.4-kb sequence fragment from the 3' end of the New York PPV was sequenced (GenBank Accession No. DG 883816). Comparison of the sequence with the database confirmed this isolate as subgroup D and exhibited a high degree of identity with other PPV D accessions (PPV D Teycheney [Accession No. X16415]; Penn4 [Accession No. DQ465243] Cnd 123-1 [Accession No. AY9553267]; and Cnd 3 [Accession No. AY953262]). To our knowledge, this is the first report of PPV in New York.

Identification and Host Relations of Turnip ringspot virus, A Novel Comovirus from Ohio.

Viruslike chlorotic ring spot symptoms and line patterns of unknown origin were observed on a greenhouse-grown turnip plant. The suspected virus was mechanically transmissible to plants in the Brassicaceae. Electron microscopic analysis revealed icosahedral particles approximately 28 nm in diameter. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses suggested that the pathogen is a comovirus, an observation that was confirmed by analysis of portions of the genomic sequence. This virus was provisionally named Turnip ringspot virus (TuRSV). Based on the RNA 1 sequence, TuRSV is most similar to Radish mosaic virus, another pathogen that infects members of the Brassicaceae. Arabidopsis thaliana is susceptible to TuRSV, and 12 out of the 23 ecotypes studied showed symptoms when inoculated with the virus. TuRSV induced a variety of responses on ecotypes from death to no infection. Some ecotypes showed one or two rounds of symptom display followed by recovery when inoculated with TuRSV. About half of the ecotypes (11/23) analyzed showed no symptoms when inoculated with TuRSV. Col-0 plants showed no symptoms, and infectious virus was not recovered from systemic leaves, although it could be detected by RT-PCR. Col-0 plants harboring mutations impairing the ethylene, jasmonic acid, or salicylic acid signaling pathways did not show symptoms when inoculated with TuRSV.

First Report on Tomato bushy stunt virus Infecting Tomato in Korea

A new virus-like disease of tomato showing chlorotic spots, malformation and necrosis on leaves, and chlorotic blotching, rings, and necrosis on fruits was observed around Sacheon, Gyeongsangnam-do, Korea in 2004. Host range analysis could not differentiate 4 field isolates collected from tomatoes showing different symptoms but identified them as Tomato bushy stunt virus (TBSV). TBSV-tsf2 isolate induced symptoms in indicator plants similar to those caused by the TBSV-C, -S and -Nf. As the isolate could not systemically infect Chenopodium quinoa, the isolate might belong to the previously described TBSV-S isolate. TBSV-tsf2 isolate caused similar cytological alterations that were similar to that generally caused by previously reported TBSV isolates. TBSV-tsf2 isolate, however, could be considered to belong to new strain of TBSV because masses of small electron-dense patches that were not observed from the previously described TBSV. The complete nucleotide sequences of the genomic RNA of 4739 nt excluding non-translated sequences at both termini have been determined and compared to sequences of other TBSV strains. The complete nucleotide sequence identity among TBSV isolates was 98.9% to 99.7%, and to the other tombusviruses ranged from 80.8% to 94.9%. Comparison of the amino acid sequences all five ORFs with those of other TBSV strains shows a similar genomic organization, and high percentage of amino acid sequence homology with TBSV-Nf than TBSV-S isolate. Since the TBSV symptoms were only observed in Sacheon fields where imported seeds from Japan were planted, the TBSV incidence probably caused by the planting contaminated tomato seeds and thus require more through quarantine procedure to prevent settlement of TBSV in Korea. Altogether, these results support that the Korean isolate of TBSV infecting tomato might be new strain.

Occurrence of Tomato spotted wilt virus in Stevia rebaudiana and Solanum tuberosum in Northern Greece.

Tomato spotted wilt virus (TSWV) (genus Tospovirus, family Bunyaviridae) was first reported in Greece during 1972 (3) and currently is widespread in the central and northern part of the country infecting several cultivated and wild plant species (1,2). In June 2006, virus-like symptoms similar to those caused by TSWV were observed on sweet honey leaf (Stevia rebaudiana (Bertoni) Bertoni, [synonym Eupatorium rebaudianum Bertoni], family Asteraceae) plants in an experimental field in the prefecture of Kilkis and on potato (Solanum tuberosum, family Solanaceae) plants growing close to tobacco in the prefecture of Kozani in northern Greece. Diseased S. rebaudiana plants expressed chlorotic and necrotic rings and line patterns on systemically infected leaves and occasionally a general chlorosis or dwarfing of the plant. Potato plants expressed only necrotic rings or lesions and a mild plant dwarfing. Seven percent of the S. rebaudiana plants, but only 0.1% of the potato plants, were showing symptoms. TSWV was detected by ELISA using polyclonal antisera in all symptomatic plants of both species, but those plants tested negative for Potato Y virus (PVY), Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), and Potato leafroll virus (PLRV); all viruses known to occur in these areas. Sap from symptomatic plants was used to mechanically inoculate Petunia × hybrida cv. Blue magic, Nicotiana tabacum cvs. Samsun NN and Basmas, N. benthamiana, N. glutinosa, N. rustica, Datura stramonium, and Physalis floridana. Sap from both S. rebaudiana and potato produced typical symptoms of TSWV on the diagnostic test plants; all Nicotiana species and cultivars, as well as D. stramonium and P. floridana, reacted with chlorotic and necrotic lesions on the inoculated leaves followed by systemic mosaic and mottling of the leaves. None of the isolates produced systemic infection on P. × hybrida. Adult thrips, identified as Thrips tabaci, were collected from infected plants of both hosts in the field and determined to be infected with TSWV by the petunia leaf disc method (4). Symptoms observed in the field on both hosts where reproduced in the laboratory after infestation of healthy plants with TSWV-infected individuals from a tobacco population of T. tabaci. To our knowledge, this is the first official report of a TSWV infection in S. rebaudiana. Potato is a known host for TSWV, however, to our knowledge, this is the first natural infection detected in Greece.

동부(Vigna sinensis)의 국부병반을 이용한 Cucumber mosaic virus와 Broad bean wilt virus 2의 구별

Cucumber mosaic virus(CMV) and Broad bean wilt virus 2(BBWV2) were isolated from Gentiana scabra plants showing typical mosaic and yellowing symptoms. When the inoculum of mosaic symptom propagated in Nicotiana benthamiana was inoculated to primary leaves of Vigna sinensis, the local lesions of different types was developed. Type one produced a small necrotic spot(SNS) of pinpoint type, while the other one showed a large necrotic spot(LNS) of halo type. LNS on primary leaves of V. sinensis was also induced by inoculum from yellowing symptom on G scabra. Single lesion from SNS induced a typical mosaic symptom on N. Benthamiana. On the other hand, LNS produced a chlorotic ring symptom on inoculated leaves and mosaic plus necrotic ringspot on upper leaves of N. benthamiana. An isolate of CMV from SNS and BBWV2 from LNS were detected by using dsRNA analysis, RT-PCR and agar gel double-diffusion test. Thus, our results should provide a tool of a simple method for discrimination from mixed infected plants by CMV and BBWV2.

IDENTIFICATION OF TOMATO MOSAIC VIRUS INFECTION IN JASMINE

Virus-like symptoms were recently observed on leaves of landscape and nursery downy and star jasmine (Jasminum multiflorum) and wax jasmine (J. gracile) in southeast Florida. Foliar symptoms included mottling, chlorotic ring spots and chlorotic line patterns. An agent was mechanically transmitted with difficulty from symptomatic leaves of downy jasmine to Nicotiana debneyi and N. tabacum 'Xanthi' and subsequently from these hosts to Chenopodium quinoa and other herbaceous test plants. Virions were isolated from N. tabacum 'Xanthi.' Rod-shaped particles (297 x 18 nm) similar to tobamoviruses were observed in partially purified virus preparations, and in leaf dips from symptomatic star jasmine and indicator plants. Extraction of viral-associated double-stranded (ds) RNA revealed a profile consistent with that of a tobamovirus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the virus contained one polypeptide with an approximate molecular weight (Mr) of 18 kDa. The virus reacted specifically with IgG for Tobacco mosaic virus (TMV) and related tobamoviruses [including Tomato mosaic virus (ToMV)] in double antibody sandwich, enzyme-linked immunosorbent assay. No reaction was observed with TMV specific IgG. Reverse transcription-polymerase chain reaction with total RNA isolated from symptomatic jasmine leaves and infected N. tabacum 'Xanthi' using ToMV coat protein specific primers amplified the expected product from downy and star (but not wax) jasmine and N. tabacum 'Xanthi.' The nucleotide and amino acid sequence of the products were 100% identical to the corresponding fragment of a Brazilian isolate of ToMV from Impatiens. To our knowledge, this the first report of ToMV in jasmine in the USA.

First Report in Argentina of Plum pox virus Causing Sharka Disease in Prunus.

Sharka disease, caused by Plum pox virus (PPV), is probably the most important disease of stone fruits crops worldwide because of tremendous yield losses from infected trees (1). During November 2004, symptoms resembling sharka disease were observed in a plum and apricot orchard consisting of 5,000 trees in Pocito, San Juan Province, Argentina. Apricot leaves showed chlorotic spots while plum leaves showed chlorotic rings, spots, and irregular edges. Plum fruits were deformed and much smaller than those from symptomless trees. Samples collected from 70 symptomatic trees were tested using double-antibody sandwich enzyme-linked immunosorbent assays with a polyclonal antiserum anti-PPV from BIOREBA (Reinach BL1, Switzerland), and immunosorbent electron microscopy with a polyclonal antiserum from our laboratory made against a recombinant PPV capsid protein (CP). The samples were also tested using double-antibody sandwich indirect enzyme-linked immunosorbent assay using the REAL kit (Durviz, Valencia, Spain) with two different monoclonal antibodies including Mab 5b that recognizes all strains of PPV and Mab 4DG5 that is specific for PPV strain D. Samples were positive with both antibodies in 80% of the cases. Leaf extracts from symptomatic plum samples were also analyzed by immuno-capture reverse-transcription polymerase chain reaction. A 1,209-bp fragment was amplified with specific primers that anneal at the 5' end of the coat protein coding region and the viral 3' end poly A tail. The amplified fragment was cloned and the nucleotide sequence was determined for two of the resulting clones (Gen-Bank Accession Nos. DQ299537 and DQ299538). The sequences were 98% identical with the PPV-strain D from the United States (GenBank Accession No. AF360579) and Germany (GenBank Accession No. X81081). The restriction sites for AluI and RsaI, previously described (2) as typical for the PPV-D strain, were present in the expected positions. To our knowledge, this is the first report of PPV-D in Argentina. Reference: (1) M. Németh. Virus, Mycoplasma, and Rickettsia Disease of Fruit Trees. Martinus Nijhoff Publishers, Dordrecht, the Netherlands, 1986. (2) T. Wetzel et al. J. Virol. Methods 33:355, 1991.

Iris yellow spot virus in onions: a new tospovirus record from India

During 2002–03, field-infected onion (Allium cepa) plants, exhibiting characteristic symptoms of chlorotic spindle- or diamond-shaped lesions on the leaves and scapes with twisting or bending flower-bearing stalks, were observed in the Jalna and Nasik regions of Maharashtra, India. In the advanced stages, single spindle-shaped chlorotic lesions coalesced, leading to withering of leaves and flower-bearing stalks. The disease was transmitted to a number of virus-indicator plants by mechanical inoculation using phosphate buffer (0·01 m sodium sulfate, pH 7·0, containing 0·1% sodium sulfite). Inoculated Nicotiana benthamiana plants produced systemic necrotic lesions, eventually resulting in dieback and wilting of the plants, while Nicotiana tabacum (varieties Xanthi NC, White Burley, Samsun and GT-4) and Nicotiana clevelandii produced local chlorotic ring spots 3–6 days after inoculation. In Vigna unguiculata, necrotic local lesions developed 3–4 days postinoculation, while Nicotiana rustica failed to produce any symptoms and did not become infected. Field-infected onion samples and glasshouse-inoculated plants were tested by ELISA using the following antisera: Tobacco streak virus (DSMZ AS-0615); Watermelon silver mottle virus (DSMZ AS-0118); Iris yellow spot virus (DSMZ AS-0528); Tomato spotted wilt virus (DSMZ AS-0105); Impatiens necrotic spot virus (DSMZ AS-0115); Chrysanthemum stem necrosis virus (DSMZ AS-0529); Peanut bud necrosis virus (ICRISAT); and Potato virus Y (DSMZ AS-0573), utilizing the respective positive control samples. Field-infected onion samples and the respective mechanically inoculated tobacco plants reacted strongly with Iris yellow spot virus (IYSV) antisera, but failed to react with any of the other antisera tested. RT–PCR using primers designed to the capsid gene and flanking sequences of IYSV (GenBank accession number AF001387) produced the expected 925-bp amplicon from infected but not healthy onions. The results of symptomatology, host range, ELISA and RT–PCR indicate that the causal agent is a strain of IYSV, which is a new report from an important onion-growing region of Maharashtra State, India. This virus has been reported as a potentially devastating pathogen of onion in Europe (Cortes et al., 1998), Israel (Gera et al., 1998) and the USA (Gent et al., 2004).

Evaluation of Arabidopsis thaliana response to infection by Tomato spotted wilt virusand Groundnut ringspot virus

In Brazil the occurrence of different tospovirus species such as Tomato spotted wilt virus (TSWV) and Groundnut ringspot virus (GRSV) has been reported (De Avila et al., J. Gen. Virol. 74:153. 1993; De Avila et al., Arch. Virol. 128:211. 1993). Arabidopsis thaliana is currently the most studied plant and has had its genome completely sequenced since the year 2000. The ecotype Columbia of A. thaliana has been shown to be a host to TSWV (German et al., Plant Mol. Biol. Rep. 13:110. 1995). Here we expand on this previous report by describing in detail disease symptoms on A. thaliana and evaluating the response of 20 different ecotypes to TSWV. We also describe, for the first time, the symptoms of GRSV infection on Arabidopsis plants. Arabidopsis thaliana ecotypes CS903, CS920, CS1020, CS1072, CS1084, CS1194, CS1308, CS1354, CS1438, CS1466, CS1492,CS1540, CS1566, CS1594, CS1640, CS1643, CS2223, CS3112, CS6100, CS6699 were obtained from ABRC (Ohio, U.S.A.). Three to four weeks-old plants grown under continuous fluorescent illumination (100 imol.m-2.s-1), 24 °C and 50% relative humidity were used in all experiments. Rosette leaves of all 20 ecotypes were mechanically inoculated with TSWV, isolate BR-01 (De Avila et al., J. Gen. Virol. 71:2801. 1990). Symptoms of infection appeared two weeks after inoculation and ranged from mild to strong, sometimes leading to plant death, depending on the ecotype. Common symptoms were the development of chlorotic and necrotic spots (Figure 1). Sometimes chlorotic rings and deformation of leaves with curling, as well as purpling of leaves, inhibition of plant growth and reduced fertility were observed. All ecotypes showed positive DAS-ELISA results in at least one of the timepoints tested 12, 20 or 35 days post inoculation (d.p.i.) (Clark & Adams, J. Gen. Virol. 34:475. 1977). For experiments with GRSV isolate A5, six ecotypes (CS1194, CS1298, CS1308, CS1540, CS1640 and CS6922) were selected and their rosette leaves were mechanically inoculated. Symptoms such as leaf necrosis and chlorotic and necrotic spots were noticeable two weeks after inoculation. The bolt of many plants became contorted and stunting and inhibition of silique (pod) growth were also observed (Figure 1). Wrinkling of cauline leaves indicates that GRSV infection is systemic on A. thaliana. Symptoms correlated with GRSV detection by DASELISA at 14 d.p.i. on all tested ecotypes. All ecotypes tested were susceptible to the tospoviruses studied. Arabidopsis thaliana is a great model system for performing genetic experiments. Future studies such as mutant screens for increased susceptibility or resistance to TSWV and GRSV will provide the opportunity for uncovering plant genes involved in the plant-tospovirus interaction.