Spinach (Spinacia oleracea) is a New Natural Host of Impatiens necrotic spot virus in California.

Spinach (Spinacia oleracea) plants exhibiting severe stunting and leaves that showed interveinal yellowing, thickening, and deformation were found in an experimental trial adjacent to an artichoke field in Monterey County, CA in October of 2008. Percent incidence of symptomatic plants ranged from 20 to 39% in cvs. Bordeaux, Lazio, and Tigercat. Symptomatic plants were positive for Impatiens necrotic spot virus (INSV; family Bunyaviridae, genus Tospovirus) and were negative for Tomato spotted wilt virus, Cucumber mosaic virus, and Tobacco mosaic virus when tested with immunostrips (Agdia Inc., Elkhart, IN). The INSV-positive spinach was used for mechanical transmission to Nicotiana benthamiana, Chenopodium quinoa, and spinach. All inoculated plants were positive for INSV with immunostrips. To further confirm the presence of INSV, reverse transcription (RT)-PCR was conducted. Total RNA was extracted from the symptomatic spinach plants using a RNeasy Plant Kit (Qiagen Inc., Valencia, CA) and used as a template in RT-PCR using forward (5'-GGATGTAAGCCCTTCTTTGTAGTGG-3') and reverse (5'-CCTTCCAAGTCACCCTCTGATTG-3') primers specific to the INSV nucleoprotein (N) gene (GenBank Accession No. DQ425096). Amplicons of the expected size (approximately 364 bp) were obtained from both field-infected and mechanically inoculated spinach plants. Four amplicons were sequenced and compared with INSV N gene sequence in GenBank to confirm the identity of the products. Sequences obtained had 99% nucleotide identity with INSV sequences available under the GenBank Accession Nos. L20885, DQ523597, DQ523598, X66872, L20886, D00914, AB109100, and DQ425096. INSV can be one of the most serious viral pathogens of ornamental plants in North America and Europe. The host range of INSV is expanding and recent reports of INSV infection of vegetables include lettuce, peppers, peanut, and potato (1-4). To our knowledge, this is the first report of natural occurrence of INSV in spinach in California. Since INSV is vectored by thrips, its expanding natural host could make it an economically important problem in California and the United States. References: (1) S. T. Koike et al. Plant Dis. 92:1248, 2008. (2) R. A. Naidu et al. Online publication. doi:10.1094/PHP-2005-0727-01-HN, Plant Health Progress, 2005. (3) S. S Pappu et al. Plant Dis. 83:966, 1999. (4) K. L. Perry et al. Plant Dis. 89:340, 2005.

First Report of Pelargonium zonate spot virus from Tomato in the United States.

Pelargonium zonate spot virus (PZSV) was first isolated from tomato in southern Italy in 1982 (1) and later was also reported from Spain (3) and France (2). Infected tomato plants showed stunting, malformation, yellow rings and line patterns on the leaves, and concentric chlorotic ringspots on the stems. In June of 2006, more than 100 tomato (Lycopersicon esculentum Mill.) plants exhibiting symptoms similar to PZSV were observed in seven acres of tomato fields in Yolo County, California. The causal agent was mechanically transmitted to several indicator species. Symptoms on infected plants included local lesions on Beta macrocarpa, Chenopodium amaranticolor, C. capitatum, C. quinoa, Cucumis melo, Cucurbita pepo, and Tetragonia expansa, and systemic infection on Capsicum annuum, Chenopodium murale, L. esculentum, Nicotiana benthamiana, N. clevelandii, N. glutinosa, N. tabacum, Physalis floridana, and P. wrightii. Two field-infected tomato plants and one each of the mechanically inoculated host plant were positive with double-antibody sandwich (DAS)-ELISA using a commercial PZSV IdentiKit (Neogen Europe Ltd., Ayr, Scotland, UK). Partially purified virions stained with 2% uranyl acetate contained spherical to ovate particles. The particle diameters ranged between 25 and 35 nm. Published sequences of PZSV (GenBank Accession Nos. NC_003649 for RNA1, NC_003650 for RNA2, and NC_003651 for RNA3) were used to design three sets of primer pairs specific for PZSV RNA1 (R1-F: 5' TGGCTGGCTTTTTCCGAACG 3' and R1-R: 5' CCTAATCTGTTGGTCCGAACTGTC 3'), RNA2 (R2-F: 5' GCGTGCGTATCATCAGAAATGG 3' and R2-R: 5' ATCGGGAGCAG AGAAACACCTTCC 3'), and RNA3 (R3-F: 5' CTCACCAACTGAAT GCTCTGGAC 3' and R3-R: 5' TGGATGCGTCTTTCCGAACC 3') for reverse transcription (RT)-PCR tests. Total nucleic acids were extracted from field-infected tomato plants and partially purified virions for RT-PCR. RT-PCR gave DNA amplicons of the expected sizes. The DNA amplicons were gel purified and sequenced. The sequenced amplicons had 92, 94, and 96% nt sequence identity to PZSV RNA1, RNA2, and RNA3, respectively. The symptomatology, serology, particle morphology, and nucleotide sequences confirm the presence of PZSV in a tomato field in California. To our knowledge, this is the first report of the occurrence of PZSV in the United States. References: (1) D. Gallitelli. Ann. Appl. Biol. 100:457, 1982. (2) K. Gebre-Selassie et al. Plant Dis. 86:1052, 2002. (3) M. Luis-Arteaga et al. Plant Dis. 84:807, 2000.

Occurrence of Resistance-Breaking Beet necrotic yellow vein virus of Sugar Beet.

Rhizomania is an important virus disease of sugar beet and is caused by Beet necrotic yellow vein virus (BNYVV). During 2002-03, several sugar beet fields with cultivars partially resistant to BNYVV grown in the Imperial Valley of California were observed with severe rhizomania symptoms, suggesting that resistance conditioned by Rz1 had been compromised. Soil testing with sugar beet baiting plants followed by enzyme-linked immunosorbent assay (ELISA) was used to diagnose virus infection. Resistant varieties grown in BNYVV-infested soil from Salinas, CA, were ELISA-negative. In contrast, when grown in BNYVV-infested soil collected from the Imperial Valley, CA, all resistant varieties became infected and tested positive by ELISA. Based on host reaction, eight distinct BNYVV isolates have been identified from Imperial Valley soil (IV-BNYVV) by single local lesion isolation. Reverse transcription-polymerase chain reaction (RT-PCR) assays showed that the eight IV-BNYVV isolates did not contain RNA-5. Singlestrand conformation polymorphism banding patterns for the IV-BNYVV isolates were identical to A-type and different from P-type. Sequence alignments of PCR products from BNYVV RNA-1 near the 3' end of IV-BNYVV isolates revealed that both IV-BNYVV and Salinas BNYVV isolates were similar to A-type and different from B-type. Our results suggest that the resistancebreaking BNYVV isolates from Imperial Valley likely evolved from existing A-type isolates.

First Report of Calibrachoa mottle virus Infecting Petunia.

Calibrachoa mottle virus (CbMV), a tentative carmovirus, was first isolated and reported by Liu et al. (1) from infected Calibrachoa plants. During the spring of 2003, petunia samples from Florida and California sent to testing services at Agdia, Inc (Elkhart IN) tested positive for CbMV by enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (ImmunoStrips). These samples also tested positive by carmovirus group-specific polymerase chain reaction (PCR) primers and by immunocapture PCR (2). RNA extracted from these samples with the RNeasy Plant Kit (Qiagen Inc., Valencia, CA) hybridized with a digoxigenin labeled probe derived from purified CbMV viral RNA. All plant samples that tested positive for CbMV were symptomless except one symptomatic sample that also tested positive for Tobacco mosaic virus. From samples that tested positive for CbMV only, mechanical inoculations were made to Chenopodium quinoa at a USDA-ARS greenhouse in Salinas, CA. Representative single, local lesions were used to inoculate additional C. quinoa plants. The resulting local lesions from these inoculations were freeze-dried and further used as virus inoculum (CbMV petunia). Similar inoculum was made with CbMV isolated from Calibrachoa plants (CbMV calibrachoa). Virus-free Petunia hybrida cultivars Surfinia 'Baby Pink' and Surfinia 'Violet' (Jackson and Perkins Inc., Somis, CA) were mechanically inoculated with CbMV petunia and CbMV calibrachoa. Four weeks postinoculation, all plants were tested using ELISA for the presence of CbMV. In greenhouse conditions, 14.3% of 'Baby Pink' plants were positive for CbMV petunia, whereas none were positive for CbMV calibrachoa. 'Violet' plants were 64.3 and 33.3% positive for CbMV petunia and CbMV calibrachoa, respectively. None of the positive plants expressed virus-like symptoms. Virus particles resembling those of CbMV were observed from infected petunia plants with transmission electron microscopy in leaf-dip preparations. To our knowledge, this is the first report of CbMV infecting petunia. Commercial reproduction of petunia plants and maintenance of genetic mother stock are usually by vegetative propagation. CbMV can be transmitted mechanically and is readily propagated along with its host. To produce healthy petunia plants, virus-free mother stock should be used, which requires regular screening of mother stock for CbMV. Reference: (1) H.-Y. Liu et al. Plant Dis. 87:167, 2003. (2) A. M. Harness et al. (Abstr.) Phytopathology 92:S34, 2002.

Interactions Between Beet necrotic yellow vein virus and Beet soilborne mosaic virus in Sugar Beet.

Soils naturally infested with cultures of aviruliferous Polymyxa betae and viruliferous P. betae carrying two sugar beet benyviruses, Beet necrotic yellow vein virus (BNYVV) and Beet soilborne mosaic virus (BSBMV), alone and in combination, were compared with noninfested soil for their effects on seedling emergence, plant fresh weight, and virus content as measured by enzyme-linked immunosorbent assay (ELISA). Studies examined sugar beet with and without resistance to the disease rhizomania, caused by BNYVV. The Rz gene, conferring resistance to BNYVV, did not confer resistance to BSBMV. BSBMV ELISA values were significantly higher in single infections than in mixed infections with BNYVV, in both the rhizomania-resistant and -susceptible cultivars. In contrast, ELISA values of BNYVV were high (8 to 14 times the healthy mean) in single and mixed infections in the rhizomania-susceptible cultivar, but were low (approximately three times the healthy mean) in the rhizomania-resistant cultivar. Results indicate BNYVV may suppress BSBMV in mixed infections, even in rhizomania-resistant cultivars in which ELISA values for BNYVV are extremely low. Soils infested with P. betae, and with one or both viruses, showed significantly reduced fresh weight of seedlings, and aviruliferous P. betae significantly decreased sugar beet growth in assays.

Isolation and Characterization of a Carmo-like Virus from Calibrachoa Plants.

Spherical virus particles approximately 29 to 31 nm in diameter were isolated from Calibrachoa plants showing leaf mottling and chlorotic blotch symptoms. The virus was mechanically transmitted to Chenopodium amaranticolor, C. capitatum, C. quinoa, Nicotiana benthamiana, and N. clevelandii plants, but was not transmitted by green peach aphid (Myzus persicae), sweet potato whitefly (Bemisia tabaci), silverleaf whitefly (B. argentifolii), greenhouse whitefly (Trialeurodes vaporarium), or banded-wing whitefly (T. abutilonea). Virions contained a single species of single-stranded RNA of approximately 4.0 kb and a single capsid protein of approximately 41 kDa. The double-stranded (ds)RNA pattern consistently revealed one major band of about 4.0 kbp, and three minor dsRNA of approximately 3.1, 1.6, and 1.3 kbp. The virus-infected plants reacted with a homologous polyclonal antiserum in indirect enzyme-linked immunosorbent assay. The genome contained a sequence of a highly conserved motif of the RNA-dependent RNA-polymerase associated with the genus Carmovirus, and shared 94% identity with Carnation mottle virus (CarMV). However, the Calibrachoa virus and CarMV were distinct serologically and in host range. Based on the host ranges, particle morphology, dsRNA profile, properties of particles in sap, and features of the genome and protein, we concluded that the recently observed Calibrachoa disease is caused by a previously undescribed carmovirus on Calibrachoa plants. We propose to name this virus Calibrachoa mottle virus (CbMV).

Characterization of Distinct Tombusviruses that Cause Diseases of Lettuce and Tomato in the Western United States.

A soilborne disease of lettuce, associated with necrosis and dieback, has been found with increasing frequency in California and Arizona over the last 10 years. An isometric virus, serologically related to Tomato bushy stunt virus (TBSV), was consistently isolated from lettuce plants with these disease symptoms. Back-inoculation to healthy lettuce plants and subsequent reisolation of the virus from symptomatic lettuce leaves suggested that this virus was the causal agent of this disease. A tombusvirus was also associated with a necrosis disease of greenhouse-grown tomatoes in Colorado and New Mexico. Complementary DNA representing the 3' end of viral genomic RNAs recovered from diseased lettuce and tomato plants had identical nucleotide sequences. However, these sequences were divergent (12.2 to 17.1%) from sequences of the previously described strains of TBSV, Petunia asteroid mosaic virus (PAMV), Artichoke mottled crinkle virus, and Carnation Italian ringspot virus. Additional tombusvirus isolates were recovered from diseased lettuce and tomato plants and these were most closely related to the TBSV-cherry strain (synonymous with PAMV) and to Cucumber necrosis virus based on comparison of 3'-end sequences (0.1 to 0.6% and 4.8 to 5.1% divergence, respectively). Western blot analysis revealed that the new tombusvirus isolated from diseased lettuce and tomato plants in the western United States is serologically distinct from previously described tombusvirus species and strains. Based on genomic and serological properties, we propose to classify this virus as a new tombusvirus species and name it Lettuce necrotic stunt virus.

Specificity of TAS-ELISA for Beet Necrotic Yellow Vein Virus and Its Application for Determining Rhizomania Resistance in Field-Grown Sugar Beets.

Levels of beet necrotic yellow vein virus (BNYVV), as measured by triple-antibody sandwich-enzyme-linked immunosorbent assay (TAS-ELISA), were compared with biological evaluations in representative commercial and experimental sugar beet cultivars developed for production in the United States and ranging in their reactions to rhizomania from uniformly susceptible to highly resistant. TAS-ELISA was specific for BNYVV and did not react with related soilborne sugar beet viruses. Differences in absorbance (A405nm) values measured in eight cultivars closely correlated with the dosage and frequency of the Rz allele, which conditions resistance to BNYVV. A diploid (Rzrz) hybrid had a significantly lower absorbance value (less virus) than a similar triploid (Rzrzrz) hybrid. Cultivars that segregated (Rzrz:rzrz) had higher absorbance values than uniformly resistant (Rzrz) hybrids, as was expected. For all cultivars, absorbance values decreased as the season progressed. Absorbance value was significantly positively correlated with rhizomania disease index score and negatively correlated with individual root weight, plot root weight, and sugar yield. This information should be useful in resistance-breeding and -evaluation programs and in the sugar industry when considering cultivar choice, inoculum production, and future crop rotations.

First Report of Tomato Bushy Stunt Virus Isolated from Lettuce.

In recent years a disease causing dieback and necrosis of Romaine and leaf lettuce has become increasingly important in California and incidence is becoming more widespread. This disease has been primarily found in areas where soil has been dredged from a river or in flooded land. Tomato bushy stunt virus (TBSV) isolates have been isolated from roots and leaves of symptomatic lettuce. The particles are isometric with a diameter of 30 nm. Double-stranded RNA (dsRNA) profiles are identical to the tomato and Prunus isolates of TBSV. However, spurs are formed in agar double diffusion tests when antisera to the tomato and Prunus isolates were used. A similar dieback disease of lettuce was observed in several counties of California during the mid-1980s. Symptoms of this disease are very similar to those described for the "brown blight" disease of lettuce reported in the 1920s (1), including severe stunting of plants and extensive chlorosis, mottling, and necrosis of older leaves. Plants infected early in their development may die. Although inoculation under greenhouse conditions has not reproduced the dieback disease in lettuce, TBSV has been consistently isolated from field-grown, symptomatic lettuce. The question of whether this new dieback disease of lettuce is caused only by lettuce isolates of TBSV or if some other viruses are also involved needs further studies. Reference: (1) I. C. Jagger. Phytopathology 30:53, 1940.

A New Report of Rhizomania and Other Furoviruses Infecting Sugar Beet in Minnesota.

Several fields planted in sugar beet (Beta vulgaris) in the Southern Minnesota Beet Sugar Cooperative growing area showed patches of pale greenish yellow foliage and upright leaves characteristic of rhizomania. Other symptoms included reduced root size and root proliferation. Samples taken from these areas during August of 1996 were evaluated for beet necrotic yellow vein virus (BNYVV), the cause of rhizomania, and for other sugar beet furoviruses. BNYVV was identified in 59 of 90 beet samples tested with enzyme-linked immunosorbent assay and Western blot (immunoblot) analyses (molecular mass approximately 22 kDa) with specific (polyclonal antisera donated by K. Richards; monoclonal antisera donated by G. Grassi) and broadly reactive antisera produced at the USDA in Salinas, CA. Recovery by mechanical inoculation of Chenopodium quinoa and Beta macrocarpa confirmed identity. Beet leaves showing symptoms of vein clearing, vein banding, mosaic, and vein necrosis were all identified as being infected with beet soilborne mosaic virus (BSBMV). No systemic leaf symptoms of BNYVV were found in any sample. The BSBMV isolates were identical to one another based on symptomatology of indicator plants and molecular masses in Western blots (approximately 24 kDa), but symptoms were distinct from those of other members of the BSBMV serogroup isolates previously studied from Texas, Idaho, Nebraska, and Colorado. The beet soilborne virus (BSBV) was also recovered by mechanical inoculation and Western blot analysis (antisera donated by R. Koenig) in three samples of field-collected beets. This is a new report of BNYVV, BSBMV, and BSBV in Minnesota. The distribution of rhizomania in infested fields was not isolated to any general area, which indicates that the virus has been present and multiplying in previous sugar beet crops and was not detected. Severity of infection ranged from mild root symptoms with near normal yields and sugar content, to moderate and severe root symptoms with low yields and low sugar content.