First Report of Potato spindle tuber viroid Naturally Infecting Field Tomatoes in the Dominican Republic.

In recent years, viroid disease outbreaks have resulted in serious economic losses to a number of tomato growers in North America (1,2,3). At least three pospiviroids have been identified as the causal agents of tomato disease, including Potato spindle tuber viroid (PSTVd), Tomato chlorotic dwarf viroid (TCDVd), and Mexican papita viroid (MPVd). In the spring of 2013, a severe disease outbreak with virus-like symptoms (chlorosis and plant stunting) was observed in a tomato field located in the Dominican Republic, whose tomato production is generally exported to the United States in the winter months. The transplants were produced in house. The disease has reached an epidemic level with many diseased plants pulled and disposed of accordingly. Three samples collected in May of 2013 were screened by ELISA against 16 common tomato viruses (Alfalfa mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Pepino mosaic virus, Potato virus X, Potato virus Y, Tobacco etch virus, Tobacco mosaic virus, Tobacco ringspot virus, Tomato aspermy virus, Tomato bushy stunt virus, Tomato mosaic virus, Tomato ringspot virus, Tomato spotted wilt virus, Groundnut ringspot virus, and Tomato chlorotic spot virus), a virus group (Potyvirus group), three bacteria (Clavibacter michiganensis subsp. michiganensis, Pectobacterium atrosepticum, and Xanthomonas spp.), and Phytophthora spp. No positive result was observed, despite the presence of symptoms typical of a viral-like disease. Further analysis by RT-PCR using Agdia's proprietary pospiviroid group-specific primer resulted in positive reactions in all three samples. To determine which species of pospiviroid was present in these tomato samples, full-genomic products of the expected size (~360 bp) were amplified by RT-PCR using specific primers for PSTVd (4) and cloned using TOPO-TA cloning kit (Invitrogen, CA). A total of 8 to 10 clones from each isolate were selected for sequencing. Sequences from each clone were nearly identical and the predominant sequence DR13-01 was deposited in GenBank (Accession No. KF683200). BLASTn searches into the NCBI database demonstrated that isolate DR13-01 shared 97% sequence identity to PSTVd isolates identified in wild Solanum (U51895), cape gooseberry (EU862231), or pepper (AY532803), and 96% identity to the tomato-infecting PSTVd isolate from the United States (JX280944). The relatively lower genome sequence identity (96%) to the tomato-infecting PSTVd isolate in the United States (JX280944) suggests that PSTVd from the Dominican Republic was likely introduced from a different source, although the exact source that resulted in the current disease outbreak remains unknown. It may be the result of an inadvertent introduction of contaminated tomato seed lots or simply from local wild plants. Further investigation is necessary to determine the likely source and route of introduction of PSTVd identified in the current epidemic. Thus, proper control measures could be recommended for disease management. The detection of this viroid disease outbreak in the Dominican Republic represents further geographic expansion of the viroid disease in tomatoes beyond North America. References: (1). K.-S. Ling and M. Bledsoe. Plant Dis. 93:839, 2009. (2) K.-S. Ling and W. Zhang. Plant Dis. 93:1216, 2009. (3) K.-S. Ling et al. Plant Dis. 93:1075, 2009. (4) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997.

Natural Infection of Verbena and Phlox by a Recently Described Member of the Carmovirus Genus.

A novel carmovirus infecting angelonia (Angelonia angustifolia) was recently described independently by researchers in the United States, Israel, and Germany (1,2,4). Angelonia flower break virus (AnFBV) and Angelonia flower mottle virus were proposed as appropriate names for this carmovirus. The virus, causing stunting, mild leaf mottle, flower mottling, and flower breaking symptoms has been detected in naturally infected angelonia in the United States, Israel, and Germany (2,4). Here we report the first detection of natural infection of verbena (in the United States and Israel) and phlox (in the United States) by using a recently developed double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA; Agdia, Elkhart, IN). Prior to this report, verbena was considered insusceptible to carmovirus infection (3) and phlox was known as an experimental host for AnFBV (2). A comparative serological study including 27 virus species, demonstrated that DAS-ELISA did not cross-react with any viruses that commonly infect ornamentals or are related to carmoviruses, showing that the polyclonal antibodies are specific to AnFBV. Antibody specificity was confirmed by the carmovirus group PCR test (Agdia). Furthermore, reverse transcription-polymerase chain reaction with AnFBV specific primers (2) produced the expected 1172-bp band from all ELISA-positive samples tested. Between November 2005 and March 2006, AnFBV was detected in 181 of 567 verbena, 26 of 143 phlox, and 193 of 267 angelonia samples submitted to Agdia Testing Services by commercial ornamental propagators for virus testing. Most samples were asymptomatic, although a few exhibited mild leaf mottle. It should be noted that the number of AnFBV-infected samples might not accurately reflect the actual number of commercially produced plants infected with AnFBV because most of the samples analyzed originated from virus elimination programs. The detection of natural AnFBV infection of verbena, phlox, and angelonia suggests that AnFBV may be more widespread in the ornamental industry than previously thought. References: (1) S. Adkins et al. Phytopathology (Abstr.) 95(suppl.):S2, 2005. (2) S. Adkins et al. Phytopathology 96:460, 2006. (3) G. P. Martelli and M. Russo. Online publication. ICTVdB-The Universal Virus Database. 00.074.0.02, 2004. (4) S. Winter et al. New Disease Reports. Vol 12. Brit. Soc. Plant Pathol. Online publication, 2005.

Acquisition of Tomato spotted wilt virus by Adults of Two Thrips Species.

ABSTRACT Only larval thrips that acquire Tomato spotted wilt virus (TSWV), or adults derived from such larvae, transmit the virus. Nonviruliferous adults can ingest virus particles while feeding on TSWV-infected plants, but such adult thrips have not been shown to transmit TSWV. Immunofluorescence microscopy was used to show that thrips 1, 5, 10, and 20 days after adult emergence (DAE) fed on TSWV-infected plants acquired TSWV with virus replication and accumulation occurring in both epithelial and muscle cells of Frankliniella fusca (tobacco thrips [TT]) and F. occidentalis (western flower thrips [WFT]), as indicated by immunodetection of the nonstructural (NSs) protein encoded by the small RNA and the nucleocapsid (N) protein, respectively. Adult WFT acquired TSWV more efficiently than TT. There was no significant effect of insect age on TSWV acquisition by TT. In contrast, acquisition by adult WFT at 1 and 5 DAE was higher than acquisition at 10 and 20 DAE. Subsequent transmission competence of adult cohorts was studied by vector transmission assays. All adult thrips tested that had an acquisition access period as an adult were unable to transmit the virus. These results indicate the susceptibility of adult TT and WFT to infection of midgut cells by TSWV and subsequent virus replication and confirm earlier studies that adult thrips that feed on virus-infected plants do not transmit the virus. The role of a tissue barrier in TSWV movement and infection from midgut muscle cells to the salivary glands is discussed.

Symptom induction by Cowpea chlorotic mottle virus on Vigna unguiculata is determined by amino acid residue 151 in the coat protein.

The type strain of Cowpea chlorotic mottle virus (CCMV-T) produces a bright chlorosis in cowpea (Vigna unguiculata cv. California Blackeye). The attenuated variant (CCMV-M) induces mild green mottle symptoms that were previously mapped to RNA 3. Restriction fragment exchanges between RNA 3 cDNA clones of CCMV-T and CCMV-M that generate infectious transcripts and site-directed mutagenesis indicated that the codon encoding amino acid residue 151 of the coat protein determines the symptom phenotypes of CCMV-T and CCMV-M. Amino acid 151 is within an alpha-helical structure required for calcium ion binding and virus particle stability. No differences in virion stability or accumulation were detected between CCMV-T and CCMV-M. Mutational analysis suggested that the amino acid at position 151 and not the nucleotide sequence induce the symptom phenotype. Thus, it is likely that subtle influences by amino acid residue 151 in coat protein-host interactions result in chlorotic and mild green mottle symptoms.

Dynamics of Tomato spotted wilt virus Replication in the Alimentary Canal of Two Thrips Species.

ABSTRACT Transmission of Tomato spotted wilt virus (TSWV) is dependent on virus uptake in the midgut prior to virus movement to the salivary glands. Replication of TSWV in the alimentary canal of tobacco thrips (TT, Frankliniella fusca) and western flower thrips (WFT, F. occidentalis) was investigated by immunolocalization of the nonstructural protein (NSs) encoded by the small RNA of TSWV and fluorescence microscopy. Analysis of cohorts during development from larva to adults following virus acquisition by first instar larva indicated that virus replication followed a specific time-course pattern in the foregut, regions of the midgut, salivary glands, and ligaments between the midgut and salivary glands. Initial virus replication occurred only in epithelial cells of midgut-1 but, upon infection of muscle cells, the virus moved to the midgut-2, foregut, midgut-3, and salivary glands. The ligaments between the midgut and salivary glands appeared to be a route for virus to invade the salivary glands. No virus replication was observed in the hindgut, Malpighian tubules, or tubular salivary glands. The dynamics of TSWV replication, as measured by NSs accumulation, were similar in both TT and WFT.

Evaluation of Seed Transmission of Turnip yellow mosaic virus and Tobacco mosaic virus in Arabidopsis thaliana.

ABSTRACT The mechanism of virus transmission through seed was studied in Arabidopsis thaliana infected with Turnip yellow mosaic virus (TYMV) and Tobacco mosaic virus (TMV). Serological and biological tests were conducted to identify the route by which the viruses reach the seed and subsequently are located in the seed. Both TYMV and TMV were detected in seed from infected plants, however only TYMV was seed-transmitted. This is the first report of transmission of TYMV in seed of A. thaliana. Estimating virus seed transmission by grow-out tests was more accurate than enzyme-linked immunosorbent assay due to the higher frequency of antigen in the seed coat than in the embryo. Virus in the seed coat did not lead to seedling infection. Thus, embryo invasion is necessary for seed transmission of TYMV in A. thaliana. Crosses between healthy and virus-infected plants indicated that TYMV from either the female or the male parent could invade the seed. Conversely, invasion from maternal tissue was the only route for TMV to invade the seed. Pollination of flowers on healthy A. thaliana with pollen from TYMV-infected plants did not result in systemic infection of healthy plants, despite TYMV being carried by pollen to the seed.