N. I. Katis

Multiplex detection of criniviruses associated with epidemics of a yellowing disease of tomato in Greece.

Since 1997, a yellowing disease has been observed in greenhouse tomato (Lycopersicon escu-lentum). By 2001, the disease was widespread, including open field tomato crops, and in most cases its incidence was 80 to 90% or even 100%. Epidemics in glasshouses were mainly associated with high populations of the whitefly Trialeurodes vaporariorum and Bemisia tabaci, the major whitefly pests in vegetable crops in Greece. The main leaf symptoms were severe yellowing, rolling, and brittleness. Samples from symptomatic plants were analyzed by polymerase chain reaction (PCR) and shown to be infected with Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) (family Closteroviridae, genus Crinivirus). TICV was found in 164 of 183 symptomatic samples, while ToCV was less representative (25/183). Sequence comparisons of the amplified 229-bp and 466-bp products revealed 99 and 100% identity with the reported sequences of TICV and ToCV, respectively. A reverse transcription (RT) multiplex PCR assay using a simple sample preparation procedure was developed to allow rapid, specific, and simultaneous detection of both ToCV and TICV sequences in two steps. The method involves a one-tube RT-PCR step in which the combination of primers amplifies part of the heat shock protein to homologue gene of both ToCV and TICV, followed by a multiplex nested PCR amplification. This is the first report of TICV and ToCV in Greece and, as far as we know, the first report of TICV in Europe.

A spot nested RT-PCR method for the simultaneous detection of members of the Vitivirus and Foveavirus genera in grapevine.

A reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to allow rapid, and simultaneous detection of Vitivirus and Foveavirus sequences in two steps. The method involved a one-step RT-PCR, in which the combination of degenerate deoxyinosine-substituted primers amplified part of the polymerase region of both genera, followed by a nested PCR amplification that increased specificity and sensitivity of detection. The increase in sensitivity also permitted the use of a simple and rapid template preparation protocol, involving the spotting of plant sap extract on a nylon membrane. Consistent amplification with infected grapevine plants was possible after inclusion of additives for inhibiting polyphenolic compounds during template preparation. This spot nested RT-PCR method can reliably detect virus species of both genera in grapevine allowing simple, fast, and cost-effective analysis of a large number of samples in certification schemes.

A spot multiplex nested RT-PCR for the simultaneous and generic detection of viruses involved in the aetiology of grapevine leafroll and rugose wood of grapevine

A spot nested RT-PCR assay using degenerate deoxyinosine-containing primers was developed, allowing rapid and simultaneous detection of Closterovirus sequences. Nested PCR amplification increased the specificity and sensitivity of detection. The sensitivity was also increased by a factor of 10 by using in addition to the deoxyinosine (dI)-containing primers, respective homologous primers in which dI was substituted by dG in the region of sequence homology. These homologous primers are shorter, having lower degeneracy and higher amplification efficiency than the dI-containing primers. This method was coupled to a similar nested RT-PCR detection method for Vitivirus and Foveavirus sequences. This permitted multiplex RT-PCR amplification of sequences belonging to the three genera in the same reaction tube and the two subsequent nested PCR amplifications (one for closteroviruses and one for viti- and foveaviruses) to run in parallel. Different primers and amplification parameters (additives and thermocycling conditions) were evaluated and optimised, respectively, in order to amplify efficiently all different templates. These improvements permitted the multiplex detection of fovea- and closteroviruses in petiole and cortical scraping preparations from 23 infected field-grown grapevines throughout the year, with the exception of GLRaV-1 in petioles that was only possible from June onwards. Preliminary results show that this method can detect reliably virus species from three genera in grapevine allowing simple, fast and cost-effective testing of a large number of samples in certification schemes.

First report of Tomato yellow leaf curl virus on tomato crops in Greece.

In late summer 2000, tomato (Lycopersicon esculentum Mill.) grown in greenhouses in Ierapetra, Tympaki, and Chania (Crete) showed leaf curling, reduced leaf size, yellowing, shortened internodes, and a bushy appearance. More than 30 ha of tomato greenhouses were affected and the disease incidence ranged from 15 to 60% with estimated crop losses of over

First report of Cucurbit chlorotic yellows virus in cucumber, melon, and watermelon in Greece.

500,000. Similar symptoms were observed in tomato samples from Marathon (Attiki) and Southern Peloponnese. All greenhouses with infected plants were infested with high populations of Bemisia tabaci (Gennadius), which were also observed outside the greenhouses on several weeds. Tomato symptoms were similar to those caused by Tomato yellow leaf curl virus (TYLCV). The assumed virus could not be transmitted mechanically but successful transmission was obtained by grafting onto healthy tomato plants. Over 100 samples of symptomatic tomato plants collected from Crete and southern Peloponnese gave positive reactions when tested by ELISA using monoclonal antibodies to TYLCV-European (Adgen Ltd). The serological results were confirmed by PCR using two pairs of primers, universal degenerate (1) and MA 13 and MA 17 (2), amplifying different parts of the virus genome. The restriction fragment length polymorphism (RFLP) analysis (AluI, HaeIII, and TaqI) of the 541 bp amplicon obtained with the degenerate primers showed patterns similar to TYLCV-Is (Israeli species). The second pair of primers gave the expected 348 bp product, which was sequenced. Sequence comparisons revealed 99% identity with TYLCV-Is (EMBL no. X15656, X76319). The resulting sequence was at least 97.7% identical to sequences of TYLCV isolates from the Dominician Republic (EMBL no. AF024715), Cuba (EMBL no. AJ223505), Portugal (EMBL no. AF105975), Iran (EMBL no. AJ13271), and Spain (EMBL no. AF071228). The disease appeared for the first time in 1992 in Tymbaki, but was limited to very few plants in one glasshouse. However, the cause was not determined. To our knowledge, this is the first report of TYLCV of the Begomovirus genus in Greece. References: (1) D. Deng et al. Ann. Appl. Biol. 125:327, 1994. (2) J. Navas-Castillo et al. J. Virol. Methods 75:195, 1998.

Demarcation of ilarviruses based on the phylogeny of RNA2-encoded RdRp and a generic ramped annealing RT-PCR

In 2011, an outbreak of a yellowing disease causing chlorosis and Interveinal chlorotic spots on lower leaves was observed in cucumber (Cucumis sativus) and melon (C. melo) plants in two greenhouses on the island of Rhodes, Greece. Similar symptoms were observed in 2012 in open field watermelon (Citrullus lanatus) plants in Rhodes and in November 2013 in a cucumber greenhouse in Tympaki, Crete. Disease incidence ranged from 10 to 40%. The observed symptoms were similar to those caused by whitefly transmitted criniviruses (family Closteroviridae) Cucurbit yellow stunting disorder virus (CYSDV) and Beet pseudo-yellows virus (BPYV), as well as Cucurbit chlorotic yellows virus (CCYV), a recently described crinivirus that infects cucurbits in Japan (4) and by the aphid transmitted polerovirus (family Luteoviridae) Cucurbit aphid-borne yellows virus (CABYV). Dense populations of whiteflies were present in all the affected crops. Leaf samples from cucumber (10 from Rhodes and 10 from Crete), melon (10), and watermelon (10) were collected and tested for the presence of the above viruses. Total RNA was extracted from the samples (2) and detection of BPYV, CYSDV, and CABYV was done as previously described (1,3) whereas detection of CCYV was conducted by herein developed two-step RT-PCR assays. Two new pairs of primers, CC-HSP-up (5-GAAGAGATGGGTTGGTGTAGATAAA-3)/CC-HSP-do (5-CACACCGATTTCATAAACATCCTTT-3) and CC-RdRp-up (5-CCTAATATTGGAGCTTATGAGTACA-3)/CC-RdRp-do (5-CATACACTTTAAACACAACCCC-3) were designed based on GenBank deposited sequences of CCYV for the amplification of two regions partially covering the heat shock protein 70 homologue (HSP70h) (226 bp) and the RNA dependent RNA polymerase (RdRp) genes (709 bp). Interestingly, CCYV was detected in all samples tested, while CYSDV was detected in 18 cucumbers (10 from Rhodes and 8 from Crete), 1 melon, and 3 watermelon plants. Neither BPYV nor CABYV were detected. In order to verify the presence of CCYV, the partial HSP70h and RdRp regions of a cucumber isolate from Crete were directly sequenced using the primers CC-HSP-up/CC-HSP-do and CC-RdRp-up/CC-RdRp-do. BLAST analysis of the obtained sequences (HG939521 and 22) showed 99% and 100% identities with the HSP70h and RdRp of cucumber CCYV isolates from Lebanon, respectively (KC990511 and 22). Also, the partial HSP70h sequence of a watermelon CCYV isolate from Rhodes showed 99% identity with the cucumber isolate from Crete. Whitefly transmission of CCYV was also carried out by using an infected cucumber from Crete as virus source. Four groups of 30 whitefly adults of Bemisia tabaci biotype Q were given an acquisition and inoculation access time of 48 and 72 h, respectively. Each whitefly group was transferred to a healthy cucumber plant (hybrid Galeon). Two weeks post inoculation, the plants, which have already been showing mild interveinal chlorosis, were tested for virus presence by RT-PCR. CCYV was successfully transmitted in three of four inoculated cucumbers, which was further confirmed by sequencing. In Greece, cucurbit yellowing disease has occurred since the 1990s, with CYSDV, BPYV, and CABYV as causal agents. To our knowledge, this is the first report of CCYV infecting cucurbits in Greece; therefore, our finding supports the notion that the virus is spreading in the Mediterranean basin and is an important pathogen in cucurbit crops. References: (1) I. N. Boubourakas et al. Plant Pathol. 55:276, 2006. (2) E. Chatzinasiou et al. J. Virol. Methods 169:305, 2010. (3) L. Lotos et al. J. Virol. Methods 198:1, 2014. (4) M. Okuda et al. Phytopathology 100:560, 2010.

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

SummaryIn this study, a generic ramped-annealing (RAN) nested RT-PCR was developed, allowing the simultaneous detection and fast characterization of ilarviruses. The method involves a one-step RT-PCR in which a pair of degenerate primers amplifies a 381-bp part of the polymerase gene (RNA2), followed by a nested PCR amplification that increases detection sensitivity. The sensitivity and detection range of the method were further increased by applying a ramped annealing thermocycling step both in the first RT-PCR and in the subsequent nested PCR. The 371-bp nested amplicons can be sequenced directly, without cloning, to obtain initial sequence information on ilarvirus genomes, or can undergo a restriction enzyme analysis for rapid identification of already known virus species. Phylogenetic relationships among different members of the family Bromoviridae were inferred with maximum likelihood and Bayesian analysis, using published homologous partial amino acid sequences corresponding to the nested amplicon and also to a longer residue data set (432–453 aa) comprising all possible positions of homology among the RNA2-encoded polymerases of members of the family Bromoviridae. The implications of these analyses on the taxonomy of ilarviruses are discussed. The specific partial polymerase sequence, corresponding to the polymerase core palm structure (motifs A–D), was verified as phylogenetically informative and can be used to separate ilarviruses from other members of the family Bromoviridae, providing initial information for ilarvirus species characterization. However, the phylogenetic signal of this region is not reliable for inferring relationships among distantly related ilarviruses.

New poleroviruses associated with yellowing symptoms in different vegetable crops in Greece

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.

Occurrence of barley yellow mosaic and barley mild mosaic bymoviruses in Greece.

Four poleroviral isolates from Greece, two from lettuce, one from spinach and one from watermelon showing yellowing symptoms, were molecularly characterized by analyzing the sequence of a large part of the genome spanning from the 3′-terminal part of the RdRp to the end of the CP gene. The sequences were analyzed for their similarity and phylogenetic relationships to other members of the genus Polerovirus as well as for evidence of recombination events. The results revealed the existence of two putatively new viruses: one from lettuce and one from spinach, provisionally named “lettuce yellows virus” and “spinach yellows virus”, respectively. Also, a new recombinant virus infecting lettuce, herein named “lettuce mild yellows virus”, and a watermelon isolate of pepo aphid-borne yellows virus (PABYV) were identified. Our study highlights the existence of high genetic diversity within the genus Polerovirus, which could be associated with the emergence of new viral diseases in various crops worldwide.

First Report of Moroccan watermelon mosaic virus in Zucchini Crops in Greece

In March 1991, large chlorotic patches appeared in an autumn-sown barley crop growing near Thessaloniki, Greece. Leaves had characteristic mosaic symptoms and immunosorbent electron microscopy and enzyme-linked immunosorbent assay confirmed the presence of both soil-borne mosaic viruses of barley, barley mild mosaic and barley yellow mosaic bymoviruses. In the following year, similar symptoms appeared in a crop at Souroti, 30 km east of Thessaloniki but the disease has not been found in other areas of Macedonia. This report is the first record of these viruses from Greece and is the most southerly European record.