K. Efthimiou

A novel grapevine badnavirus is associated with the Roditis leaf discoloration disease.

Roditis leaf discoloration (RLD), a graft-transmissible disease of grapevine, was first reported in Greece in the 1980s. Even though various native grapevine viruses were identified in the affected vines, the etiology of the disease remained unknown. In the present study, we used an NGS platform for sequencing siRNAs from a twenty-year old Roditis vine showing typical RLD symptoms. Analysis of the NGS data revealed the presence of various known grapevine viruses and viroids as well as a hitherto uncharacterized DNA virus. The circular genome of the new virus was fully reassembled. It is 6988 nts long and includes 4 open reading frames (ORFs). ORF1, ORF2 and ORF4 code for proteins with unknown functions while ORF3 encodes a polyprotein with motifs related to the replication, encapsidation and movement of the virus. Phylogenetic analysis classified the novel virus within the genus Badnavirus, with closest relationship to Fig badnavirus 1. Further studies showed that the new badnavirus is closely related with the RLD disease and the provisional name grapevine Roditis leaf discoloration-associated virus (GRLDaV) is proposed. Our findings extend the number of DNA viruses identified in grapevine, further drawing attention to the potential importance of this virus group on grapevine pathology.

A survey of tobacco viruses in tobacco crops and native flora in Greece

The most important tobacco producing areas in Greece were surveyed for virus presence, from 1997 to 2000. Tobacco seedlings or plants showing virus-like symptoms were randomly collected from seedbeds or fields, respectively, and tested by ELISA, and/or mechanical inoculation onto indicator plants. PotatovirusY (PVY), Cucumbermosaicvirus (CMV) and Tobaccomosaicvirus (TMV) were detected in all sampling areas, with TMV mainly found in oriental varieties. Tomatospottedwiltvirus (TSWV) consisted a serious endemic virus in Northern Greece (Thrace, Central and Eastern Macedonia), whereas Alfalfamosaicvirus (AMV) was mainly found in regions, where alfalfa was cultivated in the vicinity of tobacco crops. Eggplantmottleddwarfvirus (EMDV) was detected in several areas but always in very low incidence (<0.01%). Surveys were also conducted to assess the potential reservoir hosts of PVY, CMV and AMV among weeds collected from highly infected tobacco fields from 1998 to 2000. Among 3450 samples tested for PVY, plants from 17 species in 10 families were found infected. For CMV, 2891 weed samples were tested and 19 species in 12 families were positive. Assays for AMV infection were made on 961 samples and 12 species in 9 families were identified as hosts of this virus.

Transmission of Tomato chlorosis virus (ToCV) by Bemisia tabaci Biotype Q and Evaluation of Four Weed Species as Viral Sources

Tomato chlorosis virus (ToCV) is implicated in tomato yellows disease in many countries worldwide. It has a wide host range, including cultivated species as well as arable weeds, and it is transmitted in a semipersistent manner by at least five whitefly species or biotypes of the genera Trialeurodes and Bemisia. ToCV is not seed transmitted and more than 36 weed species have been recorded as natural reservoirs, acting as unique sources both for the virus and its vectors when susceptible crops are harvested. In this study, experiments were conducted to determine the transmission parameters of ToCV by biotype Q, the most abundant biotype of Bemisia tabaci in Greece. Results showed that biotype Q is an efficient vector of ToCV and it is able to retain the virus for at least 6 days. This vector was then used for the evaluation of four widespread weed species (Solanum nigrum, Sonchus oleraceus, Amaranthus retroflexus, and Chenopodium album) as ToCV sources through transmission experiments. Solanum nigrum was shown to be the most significant viral source among the tested weeds, followed by Sonchus oleraceus, A. retroflexus, and, lastly, C. album. Nevertheless, none of them was as efficient a ToCV source as tomato. This variation could be attributed to differences in virus concentration in each plant species or possible host preference by the whitefly vector.

A novel strategy for the determination of a rhabdovirus genome and its application to sequencing of Eggplant mottled dwarf virus

A novel strategy employing the rhabdovirus untranslated conserved intergenic regions was developed and applied successfully for the determination of the complete nucleotide sequence of Eggplant mottled dwarf virus (EMDV). The EMDV genome contains seven open reading frames with the same organization as Potato yellow dwarf virus (PYDV), the type species of the genus Nucleorhabdovirus. These two species encode five core genes [nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G), and the polymerase (L)] like other viruses of the genus and an additional one (X), located between N and P, giving rise to a protein with currently unknown function. Furthermore, both EMDV and PYDV contain a gene (Y), inserted between P and M, which probably encodes the virus movement protein, in concordance with the rest of the plant-infecting rhabdoviruses. Phylogenetic analysis of the polymerase gene confirmed the classification of EMDV within the genus Nucleorhabdovirus and showed a close evolutionary relationship to PYDV. The novel sequencing strategy developed is a useful tool for the genome determination of yet uncharacterized rhabdoviruses.

First report of Pepino mosaic virus infecting greenhouse cherry tomatoes in Greece.

Pepino mosaic virus (PepMV) (genus Potexvirus, family Flexiviridae) is a mechanically transmitted virus that has emerged as a significant problem of greenhouse tomato crops in Europe and around the world during the past 10 years (1). In spring of 2010, mosaic symptoms were observed on leaves of cherry tomato (Lycopersicon esculentum var. cerasiforme) greenhouse crops (hybrids Shiren, Tomito, and Rubino top) in the areas of Drymos and Vonitsa, located at Aitoloakarnania Prefecture, in Greece. A total of 63 tomato samples (55 from symptomatic and 8 from asymptomatic plants) were collected from 11 greenhouses where disease incidence ranged from 10 to 20%. All samples were tested by double-antibody sandwich (DAS)-ELISA using polyclonal antibodies from BIOREBA, AG (Reinach, Switzerland) for the presence of PepMV, Cucumber mosaic virus (CMV), and Tomato mosaic virus (ToMV). Leaf tissue from PepMV-, CMV-, and ToMV-infected samples and virus-free tomato plants were included in all tests as positive and negative controls, respectively. Results showed that 53 symptomatic samples collected from all greenhouses were infected with PepMV and two were co-infected with PepMV and CMV. Total RNA was extracted from all infected plants with a commercially available kit (Qiagen, Hilden, Germany) and amplified by conventional and real-time reverse transcription (RT)-PCR, using previously reported protocols (2). Positive and negative controls were also included in each assay. The 200-bp amplified PCR fragments of Triple Gene Block 3 (TGB3) obtained from five infected samples were purified and both strands were sequenced. Sequencing data were analyzed, deposited in the GenBank, and compared with other reported sequences. In addition, leaf tissue from five samples infected with only PepMV was used for mechanical inoculation of four plants of Nicotiana glutinosa, N. benthamiana, and tomato (L. esculentum FA 179 hybrid) plants. As negative controls, two plants from each species were used. Sequencing analysis showed that all five PepMV sequences were identical (GenBank Accession Nos. FR686904 to FR686908) and possessed 100% identity PepMVstrain CH2 (DQ000985). Inoculation results showed that the virus was successfully transmitted to N. benthamiana and tomato plants which developed mosaic symptoms, and tested positive by DAS-ELISA and RT-PCR. N. glutinosa plants did not develop any symptoms and were found to be free of PepMV when tested by DAS-ELISA and RT-PCR. To our knowledge, this is the first report of PepMV in Greece. Further studies on the disease prevalence and incidence and its economic impact on tomato production are required. PepMV is currently under quarantine status in the EU and therefore new protective measures should be recommended to prevent the spread of PepMV to other regions of Greece. References: (1) I. M. Hanssen and B. P. H. J. Thomma. Mol. Plant Pathol. 11:179, 2010. (2) K. S. Ling et al. J. Virol. Methods 144:65, 2007.

Development of one-tube real-time qRT-PCR and evaluation of RNA extraction methods for the detection of Eggplant mottled dwarf virus in different species

A one-tube real-time qRT-PCR assay was developed, for the detection and quantification of Eggplant mottled dwarf virus (EMDV), a pathogen affecting cultivated and ornamental plants. The amplification efficiency of the assay was 98% and the linear range of quantification was from 20 to 2×10(8) RNA transcripts. Total RNA extraction methods (three developed methods and one commercially available RNA extraction kit) were evaluated using tissues from seven different plant species and synthetic EMDV RNA transcripts of known concentration. The recovery rates of RNA and the effect of co-extracted inhibitors revealed that methods involving PVPP and phenol-chloroform extraction were the most efficient. These modifications were necessary for processing samples containing high phenolic and polysaccharide compounds such as woody plants. The developed EMDV detection protocol was successfully applied in forty naturally infected woody and herbaceous plants belonging to six different species. The protocol comprises a useful method for low-cost detection of ssRNA viruses in diverse plant tissues.

FIRST REPORT OF A CANDIDATUS PHYTOPLASMA ASTERIS RELATED STRAIN ASSOCIATED WITH CABBAGE STUNTING IN GREECE

Cabbage (Brassica oleracea var. capitata) is one of the most im- portant vegetable crops in Greece grown on an area of 7,006 ha with a production of 188,000 tons (FAOSTAT, 2009). During a survey conducted on winter 2009 in northern Greece for pathogens infecting plant species of the Brassicaceae family, two cabbage plants showing phytoplasma-like symptoms were collect- ed near the village of Vasilika-Thessaloniki (40o28’N 23o08’E). These plants exhibited reduction of leaf size (little leaf), opening of the head, proliferation and generalized stunting. In order to in- vestigate the possibility of a phytoplasma infection, DNA was ex- tracted from leaf samples of two symptomatic and two apparently healthy plants according to the in house protocol developed by Psifidi et al. (2010). A nested PCR was done using two universal primer sets specific to the phytoplasma 16S rRNA gene: P1/P7 (Schneider et al., 1995) followed by R16F2n/R16R2 (Gundersen and Lee, 1996). The expected ca. 1200 bp amplicon was obtained from both symptomatic cabbage plants but not from the symp- tomless ones. One of the two amplicons was purified and directly sequenced. BLAST comparisons of the partial 16S rRNA se- quence revealed 99% identity with the homologous sequence from Ca. Phytoplasma asteris reference strain (M30790). The ob- tained sequence was deposited in EMBL-EBI database (accession No. HE601634). In silico RFLP analysis (Zhao et al., 2009) classi- fied the isolate in the 16SrI-B subgroup. Ca. Phytoplama asteris is an important pathogen of many herbaceous and woody plants; it has a worldwide distribution and has been previously associated with various diseases of several Brassicaceae plant species. How- ever this is, to our knowledge, the first record of a cabbage dis- ease associated with Ca. Phytoplasma asteris in Greece.

FIRST REPORT OF EGGPLANT MOTTLED DWARF VIRUS IN TOBACCO CROPS IN ALBANIA

In summer 2008 and 2009, tobacco plants of cvs Lovina and Katerini (Samsun) displaying severe stunting, leaf crinkling and vein clearing (VC) (incidence of diseased plants 0.1%) were ob- served in areas of Koritsa and Elbasan (Albania). Leaf extract from five affected plants collected in 2009 was used for mechani- cal inoculations of Nicotiana benthamiana, N. rustica and Gom- phrena globosa. Twenty-thirty days post inoculation, N. benthami- ana developed a systemic VC, while N. rustica reacted with yel- low spots in the inoculated leaves followed by systemic VC. In G. globosa only red local lesions were observed. Leaves from 10 symptomatic and 10 symptomless tobacco plants were collected from different fields and tested serologically by DAS-ELISA us- ing polyclonal antisera to Eggplant mottled dwarf virus (EMDV) (As 0136 antiserum, DSMZ, Braunschweig, Germany). All sam- ples from symptomatic plants tested positive. To confirm the presence of EMDV a one-step RT-PCR as described by Katis et al. (2011) was carried out to the same samples and one of the PCR products was sequenced. BLAST analysis of the obtained sequence revealed 98% nucleotide similarity with the homolo- gous published sequences of the EMDV isolates from tobacco (AM922318), cucumber (AM922317), Pittosporum tobira (AM922320) and 88% with those of virus isolates from eggplant (AM922319) and caper (AM922321), respectively. The obtained sequence was deposited in EMBL-EBI under the accession No. HE601787. EMDV is known since 1969 and is generally consid- ered to be of minor importance. It has been reported to infect to- bacco in Italy and Greece (Chatzivassiliou et al., 2004). To our knowledge this is the first report of EMDV in tobacco in Alba- nia, even though its economic importance is limited.

OCCURENCE OF APPLE CHLOROTIC LEAF SPOT VIRUS IN APPLE AND QUINCE IN SOUTHERN IRAN

Apple chlorotic leaf spot virus (ACLSV, genus Trichovirus, family Betaflexiviridae) is a widespread virus that naturally affects pome and stone fruit as well as other Rosaceae species. Recently, the virus has been reported in apple trees in Northern Iran whereas quince and pear were found to be ACLSV-free (Keshavarz and Shams-Bakhsh, 2015). During a survey, conducted from April to September of 2013 in Shiraz area (Southern Iran), leaf samples from two quince trees exhibiting chlorotic leaf spots and fruit deformation and from fifteen symptomless apple trees, were collected. ACLSV presence was ascertained serologically by DAS-ELISA using specific polyclonal antibodies (Dr. T. Candresse, INRA, Bordeaux) and by an ACLSV-specific nested RT-PCR (Katsiani et al., 2014) using the primer pair CLSup and CLSdo (Mathioudakis et al., 2010) targeting close to the 3 end of the coat protein (CP) gene sequence. All 17 tested samples were positive with both methods and direct sequencing of two amplified products (374 bps), one from quince and one from apple, confirmed the identification of ACLSV (accession Nos KP172503 and KP172502, respectively). As no Iranian ACLSV sequences were available in the database, BLAST comparison with ACLSV sequences from other countries retrieved from GenBank disclosed that the herein obtained sequences from apple and quince isolates share the highest identity at the nucleotide level with isolates FN386786 from Greece (94.4%) and AB326228 from Japan (94%), respectively. Nucleotide sequence comparison among the Iranian isolates revealed a 99.4% similarity. ACLSV association with the observed symptoms in quince trees remains to be determined, as the diseased trees were not tested for the presence of other common viruses such as Apple stem pitting virus (ASPV). To our knowledge this is the first report of ACLSV in quince in Iran.