In order to assess the occurrence of Wheat spindle streak mosaic virus (WSSMV) in Belgium, a reverse-transcription polymerase chain reaction (RT-PCR) was developed, targeting WSSMV isolates from Canada, France, Germany, Italy, and the United States. The primers also were designed for virus quantification by real-time RT-PCR with SYBR-Green. No cross-reaction with soilborne cereal viruses such as Barley mild mosaic virus, Barley yellow mosaic virus, Soilborne cereal mosaic virus, and Soil-borne wheat mosaic virus was observed. The RT-PCR and real-time quantitative RT-PCR allowed a more sensitive detection of WSSMV than enzymelinked immunosorbent assay. The incidence of WSSMV in Belgium was evaluated using a bioassay with wheat cvs. Cezanne and Savannah and rye cv. Halo, grown in 104 Belgian soils. The presence of WSSMV was detected from plants grown in 32% of the soils. The RT-PCR methods developed here, combined with large sampling, allowed WSSMV to be detected for the first time in Belgium. The real-time quantitative RT-PCR was developed as a tool for evaluating the resistance to WSSMV by quantifying the virus concentration in wheat cultivars.
Recent identifications of Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd) in Solanum jasminoides (3,4) prompted the testing of this plant species for infections with other pospiviroids. From autumn of 2006 to spring of 2007, samples from symptomless plants of S. jasminoides were collected in Belgium (3 samples ranging from 75 to 150 plants), Germany (3 samples ranging from 1 to 200 plants), and the Netherlands (3 samples ranging from 2 to 200 plants). Samples were tested for pospiviroids by reverse transcription (RT)-PCR assays using the Pospi1-FW/RE and Vid-FW/RE (2) and PSTV-Nb-FW (5′-ggatccccggggaaacctgga-3′)/RE (5′-ggatccctgaagcgctcctcc-3′) primer sets. Each set amplifies several but not all pospiviroids. The first and last primer sets amplified PCR products from six samples. The full-length genomes of all six isolates were amplified using primer pairs CEVd-FW1/RE1 (1) and CEVd-FW2 (5′-gtgctcacctgaccctgcagg-3′)/RE2 (5′-accacaggaacctcaagaaag-3′), which are fully complementary to both Citrus exocortis viroid (CEVd) and Tomato apical stunt viroid (TASVd). Sequence analysis of the PCR products identified CEVd from two samples each from Germany and the Netherlands and TASVd from one sample each from Germany and Belgium (plants were imported from Israel). Although the sequences of the different CEVd isolates from S. jasminoides were not identical, all exhibited more than 95% identity with a CEVd isolate from Vicia faba (GenBank Accession No. EF494687). Both TASVd sequences were identical and showed 99.2% identity to a TASVd isolate from tomato (GenBank Accession No. AY 062121). Two nucleotide sequences of CEVd were submitted to the NCBI GenBank (Accession Nos. EU094207 and EU094208). The two other CEVd sequences and the TASVd sequence were submitted to the EMBL Nucleotide Sequence Database as Accession Nos. AM774356, AM774357, and AM777161. In addition to identification from S. jasminoides by sequence analysis, TASVd infection in the S. jasminoides sample from Germany and CEVd in one sample from the Netherlands was confirmed by mechanical inoculation to tomato followed by RT-PCR using the two CEVd-FW/RE primer pairs and analysis of the sequenced PCR product. Infection by CEVd and TASVd was also confirmed in the German samples by Northern hybridization and TASVd was confirmed in the Belgian sample by return-polyacrylamide gel electrophoresis. To our knowledge, these are the first reports of CEVd and TASVd in S. jasminoides. The viroids do not reduce the quality of S. jasminoides plants; however, the infected plants may act as infection sources for other crops. References: (1) N. Önelge. Turk. J. Agric. For. 21:419, 1997. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (3) J. Th. J. Verhoeven et al. Plant Dis. 90:1359, 2006. (4) J. Th. J. Verhoeven et al. Plant Pathol. 57:399, 2008.
During August of 2006, a sample of a tomato plant (Solanum lycopersicum, formerly Lycopersicum esculentum) from a greenhouse in Belgium was received for diagnosis. The plant showed severe growth reduction and the young leaves were chlorotic and distorted. In the greenhouse, the disease had been spreading slowly along the row. These observations suggested the presence of a viroid infection, and reverse transcriptase (RT)-PCR with two sets of universal pospiviroid primers (Pospi1-RE/FW and Vid-FW/RE; 3) yielded amplicons of the expected size (approximately 196 and 360 bp). Sequence analysis of the larger PCR product revealed that the genome was 358 nt and 100% identical to two isolates of Potato spindle tuber viroid (PSTVd) previously submitted to the NCBI GenBank (Accession Nos. AJ583449 from the United Kingdom and AY962324 from Australia). A pathogen associated with the symptomatic tomato plants was therefore identified as PSTVd. Tracing the origin of the infection revealed the following information: during November of 2005, 8-day-old tomato seedlings raised from seed by a Dutch nursery were transferred to a small part of the greenhouse of the Belgian grower; 7 to 8 weeks later, the plants were transplanted to their final destination; during May of 2006, the grower first observed growth reduction in a single plant; several weeks later, similar symptoms were observed in two more plants in the same row close to the first symptomatic plant; and by September, there were approximately 20 symptomatic tomato plants, all located in two adjacent rows. The viroid outbreak was fully eradicated by destroying all tomato plants in the affected rows as well as in two adjacent rows at both sides. The absence of further infections was confirmed by testing approximately 1,200 tomato plants in pooled samples for PSTVd by RT-PCR (2) and real-time RT-PCR (1). The origin and the method of introduction and spread of the viroid remain unclear. References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) R. A. Mumford et al. Plant Pathol. 53:242, 2004. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.
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