After comparison of various homogenization buffers and clarification methods, tulip breaking virus (TBV) was purified on a large scale by homogenization in 0.1 M tris buffer (pH 9.0), clarification with Triton X-100, and differential centrifugation. The TBV suspensions were absorbed with an anti-host protein antiserum. The purity of the product was verified by mechanical inoculation of test plants, serology, electron microscopy, spectrophotometry, and analytical ultracentrifugation.The TBV antisera obtained from rabbits after intramuscular injections had titres of 1/1280 to 1/10240 in the microprecipitin test and were mainly used in conjugate dilutions of 1/1000 in ELISA. The absorbance values for healthy tulips and lilies were (very) low. The antisera prepared against TBV from tulip cv. Jack Laan reacted best with TBV in lilies, whereas the antisera prepared against TBV from tulip cv. Texas Flame reacted best with TBV in tulips.

A distinct caulimovirus, associated with dahlia mosaic, was cloned and sequenced. The caulimovirus, tentatively designated as dahlia common mosaic virus (DCMV), had a double-stranded DNA genome of ca. 8 kb. The genome organization of DCMV was found to be typical of members of the genus Caulimovirus and consisted of six major open reading frames (ORFs), ORFs I-VI, and one minor ORF, ORF VII. Sequence comparisons with the DNA genomes of two known caulimoviruses isolated from dahlia, Dahlia mosaic virus (DMV) and an endogenous caulimovirus, DMV-D10, showed that DCMV is a member of a distinct caulimovirus species, with sequence identities among various ORFs ranging from 25 to 80%.

Dahlia mosaic, caused by Dahlia mosaic virus (DMV), is one of the most important viral diseases of dahlia. Molecular characterization of DMV showed the association of two distinct caulimoviruses (DMV-D10, DMV-Portland) and a D10-like sequence variant (DMV-Holland) with the disease. Using primers specific to these two viruses and the sequence variant, a polymerase chain reaction–based assay was used to determine their relative incidence in several dahlia samples from the United States and the Netherlands. Testing was done on samples collected in 2005 and 2006 in the United States and in 2006 in the Netherlands. Results indicated the predominance of DMV-D10 over DMV-Portland and DMV-Holland in both the United States and the Netherlands. Using conserved regions of the viral genome, primers were designed and used to detect all three sequences. Results suggested that DMV-D10 is predominantly associated with dahlia mosaic, but diagnostics should also include testing for DMV-Portland and DMV-Holland.

No abstract

A survey to identify virus diseases affecting Crocus spp. in the Netherlands was conducted during April 2004. Crocus spp. (cvs. Flavus, Pick-wick, Remembrance, and Grand Maitre) with symptoms suggestive of virus infection (stunting, yellowing, necrosis, and flower color breaking) were collected from several fields in the Breezand and Lisse districts in northern and southern Netherlands, respectively. All samples were tested for the presence of six known crocus-infecting viruses (1,2) using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) assays. The ELISA assay was performed with the following polyclonal and monoclonal antibodies: Iris severe mosaic virus (ISMV); Tobacco rattle virus (TRV) isolates F, Y, and J obtained from the Applied Plant Research Institute, Lisse, Netherlands; Arabis mosaic virus; Cucumber mosaic virus from the Plant Research International Institute, Wageningen, Netherlands; Iris yellow spot virus (IYSV) from the Virology Department at Wageningen University, Netherlands; and the potyvirus group-specific monoclonal antiserum from the DSMZ, Braunschweig, Germany. All samples that tested positive with a potyvirus antiserum were further tested for the presence of Bean yellow mosaic virus (BYMV) using a BYMV-specific antiserum. Serological results obtained indicated that BYMV, detected with the potyvirus antiserum and BYMV-specific antiserum, and ISMV were the most commonly encountered viruses. Tobacco necrosis virus (TNV) and TRV were only found occasionally, whereas IYSV, was not detected in any of the samples tested. To study the presence of viruses not yet reported, total RNA was extracted and tested with a RT-PCR assay with carlavirus, potexvirus, necrovirus (R. Miglino, unpublished), and potyvirus (3) genus-specific oligonucleotides. In accordance with the ELISA results, PCR amplicons were obtained with the potyvirus, TNV, and TRV primer sets. Furthermore, a 280-bp amplicon corresponding to the expected size was amplified in a RT-PCR assay performed on total RNA with a potexvirus genus-specific primer set. The reverse primer (5′-AGC ATG GCG CCA TCT TGT GAC TG-3′) was located upstream in the conserved viral replicaseencoding region at position 4254-4231 of Narcissus mosaic virus (NMV) RNA genome (Genbank Accession No. D13747) and the forward primer (5′-CTG AAG TCA CAA TGG GTG AAG AA-3′) was located downstream at position 3969–3992. Sequence homology using BLAST analysis of the cloned and sequenced PCR product showed 98% identity with NMV. Although the virus has a very narrow host range, the results of this study may have a significant impact on the crocus industry in the Netherlands. To our knowledge, this is the first report of NMV infecting crocus. References: (1) M. G. Bellardi and A. Pisi. Inf. Fitopatol. 37:33, 1987. (2) A. F. L. M. Derks. Crocus spp. Pages 260–264 in: Virus and Virus-like Diseases of Bulbs and Flower Crops. G. Loebenstein et al., eds. Wiley publishers, West Sussex, UK, 1995. (3) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991.