First Report of Citrus exocortis viroid in a Verbena sp. in Montenegro.

Numerous ornamental plants have been found to be symptomless hosts of various pospiviroids including Citrus exocortis viroid (CEVd), and hence, may serve as potential inoculum reservoirs for susceptible vegetable plants. Production of tomato, potato, and pepper, on which viroids from the genus Pospiviroid can cause severe damage, represents two-thirds of the vegetable production in Montenegro. We tested vegetable, ornamental, and weed host plants for the presence of pospiviroids in September 2011. Altogether 80 samples were taken. Samples of ornamental plants (15 of Petunia spp., 7 of Impatiens spp., 4 of Verbena spp., 3 of Dahlia spp., 3 of Pittosporum tobira, 3 of Vinca spp., 2 of Brugmansia spp., 2 of avocado, 2 of Portulaca spp., and 1 of Datura sp.) were taken from three places of production. One sample per species was collected from symptomless eggplants, tomatoes, sweet peppers, and avocados in the vicinity of one glasshouse with ornamental plants. Twenty-two samples from sweet pepper and seven samples from tomato, all grown under cover and all showing potential virus-like symptoms, were collected from three places of vegetable production. Two samples of Solanum nigrum and three samples of unidentified weed species belonging to genus Solanum were taken from two glasshouses. With the exception of weed plants, samples consisted of fully developed leaves collected from five plants. All sampled ornamental and weed plants were symptomless. RNA was extracted from approximately 15 mg of leaf tissue with the MagMAX-96 Total RNA Isolation Kit (Life Technologies, Foster City, CA) in accordance with the manufacturer's instructions for the MagMAX Express Magnetic Particle Processor. Samples were tested by reverse transcription (RT)-PCR using semi-universal pospiviroid primers (Pospi1-RE/FW and Vid-RE/FW [3]). None of the samples reacted with the Vid-RE/FW primer pair. An amplicon of an expected size (approximately 196 nt) was produced with the Pospi1-RE/FW primer pair from one Verbena sp. sample. Direct sequencing was performed by Macrogen (Amsterdam, The Netherlands). Sequence analysis indicated the presence of CEVd. This finding was confirmed by sequence analysis of the DNA product obtained by RT-PCR using Pospi1-FW/RE from a new extraction. Further analyses using primer pairs CEVd-AS/S (1) and CEVd-FW2/RE2 (4) were performed to obtain the full viroid sequence. The sequence of 372 nt was deposited in GenBank (Accession No. JN872140) and had 99% identity with two CEVd sequences from Verbena spp. (Accession Nos. EF192396 and DQ094297). To our knowledge, this is the first report of CEVd in a Verbena sp. in Montenegro and the second report in Europe (4). CEVd has been detected in Verbena spp. also in India and Canada and can be transmitted by seed (2). The infected Verbena sp. plants were not destroyed, since CEVd is not listed as a quarantine organism in Montenegro. The spread of CEVd infection to tomato could devastate the production of this crop in Montenegro. References: (1) A. Elleuch et al. Plant Protect. Sci. 39:139, 2003. (2) R. P. Singh et al. Eur. J. Plant Pathol. 124:691, 2009. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) J. Th. J. Verhoeven et al. Plant Dis. 92:973, 2008.