First Report of Hop stunt viroid Infecting Japanese Plum, Cherry Plum, and Peach in Greece.

Dapple plum and peach fruit is a widely distributed disorder of plum and peach resulting in significant economic losses (4). During a survey for the presence of Hop stunt viroid (HSVd) on stone fruit trees in Greece, samples from 30 European plums (Prunus domestica L., cvs. President, Tuleu Grass), 45 Japanese plums (Prunus salicina Lindl., cvs. Angeleno, Diamond, Santa Rosa), 12 cherry plums (Prunus domestica L. var. insititia (L.) Fiori & Paoletti of unknown cultivar), and 107 peaches (Prunus persica (L.) Batsch, cvs. Red Haven, Elberta, June Gold, Spring Crest, Lemonato) were collected in several orchards around Greece. Their fruit skin symptomatology indicated viroid infection (reddish dappling blotches and cracks in European and Japanese plum, green dappling in cherry plum, and light colored blotches and lines in peach). Samples were screened with tissue-print hybridization (TPH) for HSVd using a full length DIG-labelled riboprobe deriving from in vitro transcription of the positive control, a citrus isolate of HSVd (G. Vidalakis, CCPP, University of California, Riverside). In total, 44 out of the 194 trees surveyed were HSVd-positive with TPH. For a small number (40) of TPH-positive field samples, TNA phenol extraction from fruit skin, leaves, and bark and one-tube two-step reverse transcription (RT)-PCR assays followed, using a standardized protocol (3) with two different primer pairs, one new primer pair (this study) and a previously reported primer pair (2). RT-PCR analysis showed the presence of HSVd in peach and Japanese plum in prefectures Pella (Central Macedonia), Achaia, and Korinthia (Peloponnesus) and in cherry plum in Achaia (Peloponnesus). Six of 11 Japanese plums (cvs. Angeleno, Santa Rosa), 2 of 12 cherry plums, and 8 of 12 peaches (cvs. Spring Crest, Red Haven) examined were found HSVd-infected, but none of the five European plums were. Nucleotide sequence analyses of purified and cloned amplicons from peaches and Japanese and cherry plums revealed sizes of 297 to 308 nt and similarity to sequence variants of other HSVd isolates previously characterized: 95 to 97% identity with the Moroccan isolates apr.9, apr.10, apr.11, and apr.12 and the Spanish isolate apr.4 from apricot (1) (GenBank Accession Nos. AJ297825 to AJ297828 and Y09346, respectively). For confirmation of HSVd presence in field trees, 10 Japanese plums cv. Angeleno, 10 peaches cv. June Gold, and 10 peaches cv. Spring Crest, HSVd-negative (TPH), were bud- or chip-grafted from two of the aforementioned Japanese plums cv. Angeleno and two of the aforementioned peaches cv. Red Haven. Two years later, five Japanese plum trees (cv. Angeleno) and five peach trees (three cv. Spring Crest and two cv. June Gold) were found HSVd-positive with TPH; no fruits were observed to produce fruit symptoms as the grafted trees were kept in an insect-proof greenhouse (no bees for cross-pollination). To our knowledge, our investigation reports for the first time the occurrence of HSVd infecting Japanese plum, cherry plum, and peach in Greece, emphasizing the need for a certification program for the prevention of spreading stone fruit tree viroids in this country. References: (1) K. Amari et al. J. Gen. Virol. 82:953, 2001. (2) N. Astruc et al. Eur. J. Plant Pathol. 102:837, 1996. (3). F. Faggioli et al. Acta. Hort. 550:59, 2001. (4) T. Sano et al. J. Gen. Virol. 70:1311, 1989.

First Report of Olive mild mosaic virus and Sowbane mosaic virus in Spinach in Greece.

Uncommon, viruslike symptoms (yellowing, line patterns, leaf deformation, and necrosis), were observed in spinach fields in the Marathon area, Greece in 2004. Seedlings from the same seed lot, grown in the greenhouse, also developed the same viruslike symptoms, indicating that the causal agent(s) of the disorder is seed-transmissible. Spinach seedlings of the same variety but a different lot and herbaceous indicators (Chenopodium quinoa, C. amaranticolor, Sonchus oleraceus, and Nicotiana benthamiana) were mechanically inoculated with infected material. Spinach developed yellowing or necrotic spots whereas indicators showed variety of symptoms including mosaic, vein banding, and necrotic lesions. Virus purifications, double-stranded RNA extractions, cloning, and sequencing (2,3) followed by a combination of molecular (reverse transcription [RT]-PCR and immunocapture RT-PCR) and serological (ELISA) techniques with antisera provided by Dr. Avgelis were performed as described (4), verifying the presence of two viruses in the diseased seedlings: Sowbane mosaic virus (SoMV), a sobemovirus, was present in spinach and indicators with mottling and leaf deformation, whereas Olive mild mosaic virus (OMMV), a necrovirus, was present in plants with necrotic spots. All RT-PCR products amplified with primers SoMV-F (5'-CAAATGGTCTTGGTCAGCAGTC)/SoMV-R (5'-GCATACGCTCGACGATCTG) and OMMV-F (5'-CAAACCCAGCCTGTGTTCGATG)/OMMV-R (5'-CATCAGTTTGGTAATCCATTGA) were sequenced and found to confirm the other results. The SoMV-spinach isolate polyprotein gene sequence (GenBank Accession No. DQ450973) has 95% sequence identity with the type isolate from C. quinoa (GenBank Accession No. GQ845002), whereas the OMMV-spinach isolate (GenBank Accession No. JQ288895) has 92% sequence identity with the OMMV type isolate from olive (GenBank Accession No. AY616760). SoMV has been found to naturally infect spinach in the Netherlands (1) and, to our knowledge, this is the first report on spinach in Greece. The presence of OMMV in spinach is, to our knowledge, the first report worldwide. Its natural host range is limited to olive, tulip, and now spinach. OMMV might be transmitted by Olpidium spp. and may, according to data of its close relatives, persist in the soil for several decades. Pollen- and seedborne viruses (PSVs) like sobemoviruses and necroviruses are of particular importance for a crop like spinach where crop increase takes place in small, seed production-designated areas. If a PSV spreads in such an area it has the potential to become a major problem for the industry, especially when it remains undetected. Infected seed can be shipped worldwide with PSVs, causing diseases and becoming endemic in areas where they were absent. For this reason and the fact that field losses can exceed 50%, rigorous monitoring for the presence of SoMV and OMMV in seed fields is essential to minimize the possibility of the viruses moving to new areas. References: (1) L. Bos and N. Huijberts. Eur. J. Plant Pathol. 102:707, 1996. (2) S. M. Girgis et al., Eur. J. Plant Pathol. 125:203, 2009, (3) I. E. Tzanetakis et al. J. Virol. Methods 124:73, 2005. (4) I. E. Tzanetakis et al. Virus Res. 121:199, 2006.

Sensitive and rapid detection of peach latent mosaic viroid by the reverse transcription loop-mediated isothermal amplification.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of peach latent mosaic viroid (PLMVd) was developed. Four primer sets (OLD, OLD1, NEW, and Fukuta's) were designed originally. Based on initial experiments the set OLD1 was selected for further evaluation. Simple and accelerated RT-LAMP was preformed using degenerate and no degenerate forward-loop (F-loop) and backward-loop (B-Loop) primers. Degenerate primers were selected, and after determination of their best concentration (0.8microM), the reaction was preformed at different temperatures (60-67.5 degrees C) using three different betaine concentrations (0.8M, 0.4M, and 0.2M). Optimal conditions were found to be 62.5 degrees C and 0.8M betaine. Under these conditions, using tRNA as template, PLMVd was detected within only 32min, compared to 180min of RT-PCR, using the Real Time Turbimeter (LA200, Teramecs) which measures the turbidity caused by the production of insoluble magnesium pyrophosphate. In addition, RT-LAMP was more sensitive than RT-PCR. PLMVd was detected in peach, plum, apricot, pear, wild pear and quince samples.

First Report of the Stubby-Root Nematode Paratrichodorus teres Associated with Artichoke (Cynara scolymus) in Greece.

The polyphagous stubby-root nematode species, Paratrichodorus teres (Hooper) Siddiqi, was first described from soil under lettuce near Norwich, UK and subsequently reported from South Africa and the United States, but predominantly from temperate regions within Europe (4). P. teres is one of 13 economically important trichodorid species known to be vectors of Tobacco rattle virus (TRV) (4). Artichokes planted during 2000 in a field located in the Kandia area of the Argolis Region, Greece (37°32'N, 22°56'E) exhibited symptoms of a virus infection. Sampling was done to ascertain the presence of Longidorus fasciatus, a vector of artichoke Italian latent nepovirus known to occur in the area (1,4). In addition to L. fasciatus, an unknown trichodorid species and Tylenchorhyncus sp. were recovered from the root zone of artichoke at a number of sites within the field. Measurements and morphological examination of the female (n = 13, body length = 741.7 ± 25.5 µm, onchiostyle = 43.7 ± 0.8 µm, and position of vulva from anterior region relative to total body length V% = 53.8 ± 0.4 µm) and male (n =1, body length = 720.5 µm, onchiostyle = 43.5 µm, spicule length = 51.7 µm, and number of ventromedian precloacal supplements = 3) trichodorids isolated from soil samples conformed to the original description of P. teres and the generic polytomous key (2). Furthermore, morphological identification was supported by molecular data. DNA was extracted from seven individual trichodorids, each of which were placed into separate 0.5-ml micro-centrifuge tubes containing 20 µl of 0.25 M NaOH and incubated at 25°C overnight. Thereafter, samples were incubated at 99°C for 3 min and 10 µl of 0.25 M HCl, 5 µl of 0.5 M Tris-HCl, (pH 8.0) and 5 µl of 2% Triton X-100 were added to each tube. Samples were incubated at 99°C for a further 3 min and stored at -20°C. Template DNA was amplified using polymerase chain reaction with primers specific for 18S rDNA and sequenced (3). The resultant consensus sequence had 99.8% homology to P. teres populations isolated from Portugal and good homology (95 to 98%) with five other Paratrichodorus spp. listed on public sequence databases, e.g., NCBI GenBank. This constitutes a new geographic record and a possible association of P. teres on artichoke. References: (1) D. J. F. Brown et al. Eur. J. Plant Pathol. 103:501, 1997. (2) W. Decraemer. Fundam. Appl. Nematol. 21:37, 1998. (3) C. M. G. Oliveira et al. J. Nematol. 36:153, 2004. (4) C. E. Taylor and D. J. F. Brown. Nematode Vectors of Plant Viruses, CAB International Mycological Institute, Wallingford, UK, 1997.

A New Ilarvirus Isolated from Grapevine in Greece.

In 1994, characteristic viruslike symptoms on grapevine were reported in the collection of the Grapevine Institute in Athens, Greece, on the hybrid Baresana × Baresana. The symptoms were sharp angular mosaic, leaf crinkle, and little leaf. The affected vines showed gradual decline and severe stunting or death. Such vines produced abortive flowers or very few berries with smaller, wrinkled, and nongerminating seeds. Serological testing, by enzyme-linked immunosorbent assay (ELISA), of the affected vines against the most common grapevine viruses Alfalfa mosaic, Arabis mosaic, Grapevine fanleaf, Grapevine fleck, Grapevine A, Rasberry ringspot, and grapevine leafroll-associated viruses gave negative results. A virus was isolated from affected grapevine young leaves by mechanical inoculation of Gomphrena globosa and single lesioned. The virus host range included G. globosa (local and systemic dark red or necrotic lesions), Chenopodium quinoa (necrotic local lesions and systemic mottle), and three tobacco cultivars (sharp necrotic local lesions, 1 to 3 mm in diameter). Pollination of C. quinoa with pollen from infected plant gave about 30% infected seedlings. The virus was purified from C. quinoa by differential centrifugation using 0.02 M phosphate buffer pH 8.0, containing 0.01 M DIECA and 0.01 M sodium thioglycolate as extraction buffers. In a purified preparation, quasisphaerical virus particles of about 29 nm were observed. Electrophoretic mobility of the viral coat protein showed a molecular weight of 30 kDa. Using purified preparations, an antiserum was obtained with a titer of 1:1024 in microprecipitin test and an optimum IgG dilution in ELISA of 1:10,000 for maximum absorption at OD405 nm Using degenerate primers designed from homologous regions in RNA-2 corresponding to a fragment of the polymerase gene of Ilarviruses, the expected 381-bp polymerase chain reaction product was obtained. This product was cloned and sequenced. Comparisons with sequence data from the homologous regions of RNA-2 of other known Ilarviruses, showed that the sequence of the above 381-bp amplicon shared 72% sequence similarity with Tobacco streak virus, 67% of Citrus variegation virus and Spinach latent virus, 66% of Asparagus virus 2 and Elm mottle virus, and 65% of Citrus leaf rugose virus. Based on the above data, it is concluded that the isolated virus is an Ilarvirus with closest similarity to Tobacco streak virus. From the relative bibliography (1-3) it appears that the virus reported here is different from Grapevine line pattern virus, a possible Ilarvirus, previously reported from Hungary. References: (1) J. Lehoczky et al. Kertgazdasag 19:61, 1987. (2) J. Lehoczky et al. Phytoparasitica 17:59, 1989. (3) J. Lehoczky et al. Phytopathol. Medit. 31:115, 1992.