Molecular characterization of a grapevine leafroll-associated virus 4 from Slovenian vineyards.

During a survey conducted in vineyards in Slovenia, variety of grapevine leafroll disease symptoms were observed. Mixed infection with grapevine leafroll-associated viruses 3 and 4 (GLRaV-3, -4) in two grapevines from a vineyard in south-western part of Slovenia was confirmed by DAS-ELISA in 2010. The 3'final 1769 nucleotides of the Slovenian GLRaV-4 isolate were assembled from amplicons obtained by IC RT-PCR. The complete coat protein (CP) and p23 gene sequences were compared with other GLRaV-4 sequences from GenBank. Results showed that CP and p23 amino acid sequences of Slovenian variant (055-SI) are 88% and 85%, respectively, identical to corresponding genes of reference sequence GLRaV-4 LR106 (GenBank Acc. No. FJ467503). Phylogenetic analyses show that Slovenian variant clusters together with other corresponding strains of GLRaV-4. The sequencing results show great variability of the N-terminal part of the CP sequence indicating that this part of the genome is not suitable for molecular detection of the virus. To our knowledge this is also the first report of GLRaV-4 in Slovenian vineyards.

First Report of Grapevine Pinot gris virus Infecting Grapevine in Slovenia.

Unusual virus-like symptoms were first observed in 2001 on grapevine cvs. Pinot gris and Sauvignonasse in vineyards from the western part of Slovenia. Symptomatic plants showed shortened internodes, poor leaf development, mottling, and deformations of leaves that resulted in poor growth of symptomatic plants. In 2003 and 2004, several samples were tested for Arabis mosaic virus, Cherry leafroll virus, Grapevine fanleaf virus, Raspberry bushy dwarf virus, Strawberry latent ringspot virus, Tomato black ring virus, Tomato ringspot virus, and Tobacco ringspot virus by DAS-ELISA, but none of them could be confirmed as the cause of the observed symptoms. During intensive visual inspections between 2002 and 2006, the symptoms were observed on most grapevine cultivars grown in the Primorska region but predominantly on the two previously mentioned cultivars. In Trentino, northern Italy, similar virus-like symptoms, i.e., chlorotic mottling, puckering and deformation of the leaves, reduced yield, and low quality of the berries were observed in grapevine plants cv. Pinot gris in 2003 and in cvs. Traminer and Pinot noir in 2009 (2). No common grapevine viruses could be associated with the disease. In 2012, a new trichovirus named Grapevine Pinot gris virus (GPGV) was found in Pinot gris plants using deep sequencing. The virus was also detected in symptomless plants (2). GPGV was later reported also from Korea causing inner necrosis of berries and poor fruit set in grapevine cv. Tamnara (1). In 2012, 42 leaf samples from mostly symptomatic grapevine plants of cvs. Pinot gris, Pinot noir, and Muscat blanc were collected at three locations in the Primorska region. Total RNA was extracted from leaves using the MagMAX Express magnetic particle processor with MagMAX-96 Total RNA Isolation Kit and Plant RNA Isolation Aid in Lysis Binding Solution Concentrate (all by Life Technologies, Grand Island, NY). DNA fragments of 1,049 bp corresponding to the movement protein gene were successfully amplified by RT-PCR from 40 samples using primers GPgV5619 and GPgV6668 (2). Amplification products from three plants were cloned into the pGEM-T Easy vector (Promega, Madison, WI) and sequenced. The sequences were deposited in the EBI database under the accession numbers HG738850 to 52. All the nucleotide sequences shared 97.4 to 97.6% identity with GPGV from Italy (sequence FR877530) and 97.1 to 98.2% amino acid identity within the translated region. To our knowledge, this is the first report of GPGV in Slovenia. The disease seems to be spreading extensively in the Primorska region, causing considerable economic losses, and in 2013 it was also observed in other regions of Slovenia. Since the virus could be found in symptomless plants in Italy and in Slovenia, its role in the development of the disease should be further investigated. References: (1) I. S. Cho et al. New Dis. Rep. 27:10, 2013. (2) A. Giampetruzzi et al. Virus Res. 163:262, 2012.

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.

First Report of Potato spindle tuber viroid in Cape Gooseberry in Slovenia.

Cape gooseberry (Physalis peruviana) was first reported as a host of Potato spindle tuber viroid (PSTVd) in 2009 (4). In Slovenia, 10 young plants of cape gooseberry that were grown in a glasshouse were inspected in April 2011. Plants were multiplied from an adult plant of unknown origin. During sampling, the inspected plants showed no disease symptoms. Total RNA was extracted twice from leaves of five plants with an RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription (RT)-PCR assay employing two pairs of semi-universal pospiviroid primers (Pospi1-RE/FW and Vid-RE/FW [3]) yielded amplicons of the expected size (approximately 196 and 360 bp) from each total RNA preparation. All four DNA products were sequenced directly (Macrogen, Amsterdam, the Netherlands). Sequence analysis confirmed the identity of a viroid as PSTVd. Further RT-PCRs using primer pairs of Shamloul et al. (2) and Di Serio (1) were made to obtain a full viroid sequence. The sequence was deposited in the NCBI GenBank under Accession No. JN543964. Sequence analysis confirmed the identity of the viroid as PSTVd. The Slovenian isolate had 358 nucleotides and was 100% identical to the cape gooseberry isolate from Germany (GenBank Accession No. EU862231) and the tomato isolate from New Zealand (GenBank Accession No. AF369530). The analyzed sample was the only sample of cape gooseberry taken from the start of the survey for PSTVd in 2006 because P. peruviana is rarely grown in Slovenia. To our knowledge, this is the first report of PSTVd infection of P. peruviana in Slovenia and the fourth reported case after the detection of PSTVd infection in Germany, Turkey, and New Zealand. This emerging host for PSTVd could potentially serve as a source of infection for tomato and potato, where the viroid can cause severe losses. References: (1) F. Di Serio. J. Plant Pathol. 89:297, 2007. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) J. Th. J. Verhoeven et al. Plant Dis. 93:316, 2009.

First Report of Raspberry bushy dwarf virus Infecting Grapevine in Hungary.

Raspberry bushy dwarf virus (RBDV) is the sole member of genus Idaeovirus and naturally infects Rubus species worldwide. It can be experimentally transmitted to many dicotyledonous plant species from different families. In Slovenia it has been reported to naturally infect grapevine, the first known non-Rubus natural host (3). However, RBDV from red raspberry and grapevine were found to be different in biological, serological, and molecular characteristics (4). From 2007 to 2010, grapevine (Vitis vinifera L.) vineyards were sampled in different parts of Hungary and tested for RBDV infection by double antibody sandwich (DAS)-ELISA using commercial reagents (Bioreba, Reinach, Switzerland). Overall, 181 samples were collected from 10 vineyards around Csörnyeföld, Badacsony, Eger, Tolcsva (Orémus), and Nagyréde. Samples were taken randomly unless plants showing virus-like symptoms were present, which were preferentially included in the survey. Two samples collected in 2010, each consisting of five leaves from five individual plants, tested positive by DAS-ELISA. They originated from a small private vineyard of Italian Riesling, Pinot Gris, and Rhein Riesling in the southwestern part of Hungary near Csörnyeföld where 29 samples were collected. All leaves were asymptomatic. Total RNA was extracted from positive samples using a RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesized using primer RNA12 as described (4) and further amplified by PCR using primers RBDVUP1/RBDVLO4 that amplified an 872-bp fragment of RBDV coat protein and 3' non-translated region (2). Amplification products from both samples were directly sequenced (Macrogen, Seoul, Korea). The sequences showed 98.6% identity between each other and were deposited in GenBank (Accession Nos. JQ928628 and JQ928629). Sequences were also compared with RBDV sequences deposited in GenBank. They showed 97.7 to 99.3% identity with RBDV sequences from grapevine from Slovenia and 94.2 to 96.1% with RBDV sequences from Rubus sp. Natural infection of grapevine with RBDV was first reported from Slovenia in 2003 (3) and was recently reported also from Serbia (1). To our knowledge, this is the first report of RBDV infection of grapevine in Hungary and suggests a wider presence of the virus in the region. References: (1) D. Jevremovic and S. Paunovic. Pestic. Phytomed. (Belgrade) 26:55, 2011. (2) H. I. Kokko et al. BioTechniques 20:842, 1996. (3) I. Mavric Plesko et al. Plant Dis. 87:1148, 2003. (4) I. Mavric Plesko et al. Eur. J. Plant Pathol. 123:261, 2009.

First Report of Peach latent mosaic viroid in Peach Trees in Montenegro.

Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) are known to infect stone fruit species worldwide. The viroid infection can be latent or induce a variety of disease symptoms. Stone fruit samples were collected in Montenegro for a Plum pox virus (PPV) survey in 2007. Thirteen samples infected with PPV, taken from 12-year-old peach trees (Prunus persica (L.) Batsch, cv. Elegant Lady) in the area of Cemovsko field, were tested for the presence of PLMVd and HSVd by reverse transcription (RT)-PCR. Mild or severe mosaic, chlorotic rings, and fruit deformations were observed on some trees. Total RNA was extracted from all samples with a RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA) and RT-PCR was performed. Samples were tested for HSVd and PLMVd infection using primer pairs RF-43/RF-44 for PLMVd (1) and VP-19/VP-20 for HSVd (2). Amplification products of approximately 348 bp were obtained from nine samples with PLMVd primers. Amplification products from seven samples were successfully cloned into pGEM-T Easy Vector (Promega, Madison, WI) and used for transformation of Escherichia coli. At least four clones of each sample were sequenced. Obtained sequences were 337 and 338 nucleotides long and shared 90.3 to 100% identity. Consensus sequences of each sample were deposited in GenBank under Accession Nos. JF927892-JF927898. They showed 92.6 to 97.9% identity among each other, 94 to 98% identity with the PLMVd isolate G sequence (Accession No. EF591868) and 91.8 to 94.4% identity with PLMVd sequence M83545. HSVd was not detected in analyzed samples. PLMVd infections were found on peach trees in an area where approximately 40% of the peach production is located. Therefore, PLMVd infections can pose a threat to peach production in Montenegro. To our knowledge this is the first report of PLMVd infection of peach in Montenegro. References: (1) S. Ambrós et al. J. Virol. 72:7397, 1998. (2) S. A. Kofalvi et al. J. Gen. Virol. 78:3177, 1997.

Xiphinema rivesi from Slovania Transmit Tobacco ringspot virus and Tomato ringspot virus to Cucumber Bait Plants.

The dagger nematode, Xiphinema rivesi Dalmasso, a member of the X. americanum group, was detected in 2002 for the first time in Slovenia and for the fourth time in Europe (4). X. rivesi is a vector of at least four North American nepoviruses including Cherry rasp leaf virus (CRLV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Peach rosette mosaic virus (PRMV) (1,2). All of these viruses are included on the EPPO and EU lists of quarantine organisms, but none of the Xiphinema species found in Europe have been reported to transmit these nepoviruses. Three virus isolates, including TRSV (from Lobelia spp.; virus collection of the Plant Protection Service, Wageningen, The Netherlands), ToRSV (grapevine isolate PV-0381; DSMZ, Braunschweig, Germany), and Arabis mosaic virus (ArMV) (from Vinca spp.; virus collection of the Plant Protection Service), were used in transmission tests with a population of X. rivesi found in Slovenia. X. rivesi is not known to transmit ArMV and this virus was included as a check. The nematodes were extracted from peach orchard soil collected near the village of Dornberk, and transmission tests fulfilled the set of criteria proposed by Trudgill et al. (3). Cucumis sativus cv. Eva, grown in a growth chamber at 25°C, was used as acquisition hosts and transmission bait plants. The acquisition hosts were mechanically inoculated and showing systemic symptoms before the introduction of nematodes. Noninoculated acquisition plants were included as controls. After a 10-day acquisition feeding period, the nematodes were transferred to healthy bait plants and allowed a 14-day inoculation feeding period. X. rivesi transmitted TRSV and ToRSV but not ArMV. TRSV and ToRSV bait plants developed systemic symptoms 4 to 6 weeks after the nematodes were transferred. Transmission of TRSV and ToRSV was confirmed by testing leaf and root sap of bait plants in a double antibody sandwich (DAS)-ELISA. High virus concentrations were detected in the roots and leaves of TRSV and ToRSV symptomatic plants. DAS-ELISA on bait plants from nematodes that had been allowed to feed on ArMV-infected or the virus-free control acquisition plants gave negative results. No symptoms appeared on bait plants used for ArMV transmission or the control bait plants. To our knowledge, this is the first report of transmission of TRSV and ToRSV with a Xiphinema population from Europe. References: (1) D. J. F. Brown et al. Phytopathology 84:646, 1994. (2) L.W. Stobbs et al. Plant Dis. 80:105, 1996. (3) D. L. Trudgill et al. Rev. Nematol. 6:133, 1983. (4) G. Urek et al. Plant Dis. 87:100, 2002.