RESUMEN
In July 2009, the occurrence of pale yellow, bottle-shaped greenhouse cucumber (Cucumis sativus L.) fruits was reported by a horticultural adviser from Kannus in western Finland. The grower had observed the first symptoms in greenhouse cucumber cv. Rapides in May. The most distinctive symptoms were found in fruits, but also flowers were crumpled. Symptoms had spread along plant rows. Estimated yield loss by the grower was 2 to 3%. Fruit and flower symptoms were typical of cucumber pale fruit disease (3) caused by a strain of Hop stunt viroid (HSVd) (2). The original samples were collected by a phytosanitary inspector and the farmer from approximately 10 symptomatic plants growing in the same greenhouse. Testing two samples, one a cucumber leaf and the other a cucumber fruit, by return-polyacrylamide gel electrophoresis gave clear electrophoretic bands at the same position. However, the position of the bands differed slightly from the positive control (Potato spindle tuber viroid) and two other pospiviroids tested on the same gel, indicating the presence of a different viroid with a shorter length than those from the genus Pospiviroid. This observation of size combined with symptoms on cucumber gave a strong indication of the presence of a viroid, likely to be HSVd. RNA was extracted from two subsamples of fruit and leaf samples with a RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Extracted RNA was examined with primer pair HSVdF1/R1 designed to detect HSVd from citrus (1). The PCR reactions were performed using a reverse transcription-PCR protocol developed for Pospiviroids (4). Both fruit and leaf samples gave a PCR amplicon of the same size. Sequencing of the amplicon of approximately 300 bp revealed 99% similarity with 11 GenBank citrus isolates of HSVd and 98% similarity with two cucumber isolates of HSVd (Accession Nos. X07405 and X00524) On the basis of these results, the viroid of these cucumber plants was identified as HSVd. To our knowledge, this is the first finding of this viroid in Finland. Although we could determine the causal agent, we could not find the origin of the infection. The seedling plants had been grown in the same greenhouse where the infection was detected. Even though HSVd is not known to be seed transmitted (3) the other production places that had used the same seed lot were inspected and found to be free of the viroid. Very strict phytosanitary measures were taken to eradicate the infection. Since the viroid is easily sap transmissible, there is a certain risk of spreading of HSVd via human action, e.g., visitors, staff, and the use of common packing facilities. References: (1) L. Bernard and N. Duran-Vila. Mol. Cell. Probes 20:105, 2006. (2) T. Sano et al. Nucleic Acids Res. 12:3427, 1984. (3) H. J. M. van Dorst and D. Peters. Neth. J. Plant Pathol. 80:85, 1974. (4) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.
RESUMEN
In November 2005, 13 accessions of Petunia hybrida from the United States of America entered the post-entry quarantine station of the Plant Protection Service in the Netherlands. The plants were inspected and tested for quarantine organisms according to Directives 95/44 and 97/46 of the European Union. No virus and viroid symptoms were observed in the imported plants or in mechanically inoculated plants of Chenopodium quinoa, Nicotiana benthamiana, and N. occidentalis-P1 (3). Testing for pospiviroids by return-polyacrylamide gel electrophoresis (1) and reverse transcriptase-PCR with universal pospiviroid primers Pospi1-RE/FW (2) indicated the presence of pospiviroids in 3 and 11 P. hybrida accessions, respectively. The 196-bp amplicons of six accessions were sequenced. Sequence analysis showed the highest identity for all amplicons to both isolates of Tomato chlorotic dwarf viroid (TCDVd) in NCBI GenBank, Accession Nos. AF162131and AY372399, from Canada and the United States, respectively. Additional RT-PCRs with the Pospi1-RE/FW primers in opposite order and the semi-universal pospiviroid primers Vid-RE/FW (2) for one isolate, followed by sequence analysis, confirmed the identity as TCDVd. The isolate consisted of 359 nucleotides (GenBank Accession No. DQ859013) and showed sequence identities of 98.6 and 96.1% to the Canadian and American tomato isolates of this viroid, respectively. The next highest sequence identity was 90.0% to two accessions of Potato spindle tuber viroid (GenBank Accession Nos. AJ593449 and AY962324). On the basis of these results, the viroid from P. hybrida was identified as TCDVd. To our knowledge, this is the first report of TCDVd in this plant species. Reference: (1) J. W. Roenhorst et al. EPPO Bull. 30:453, 2000. (2) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (3) J. Th. J. Verhoeven and J. W. Roenhorst. EPPO Bull. 33:305, 2003.