Leonidas Lotos

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.

New poleroviruses associated with yellowing symptoms in different vegetable crops 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 a “Candidatus Phytoplasma solani” related strain associated with a disease of Datura stramonium in Greece

Jimsonweed (Datura stramonium), family Solanaceae, a common weed in spring crops in Greece, includes two botanical varieties (var. stramonium and var. tatula). During a field trial at the Aristotle University Farm (40o32N’ 22o59’E, 6 m asl), in which four accessions of D. stramonium were tested for growth rate and alkaloid content, phytoplasma-like symptoms were observed. Initially, the affected plants showed interveinal chlorosis of the upper leaves, stunting and flower malformation, whereas at maturity they did not form normal fruits and developed leaf necrosis. To investigate the possibility of a phytoplasma infection, DNA was extracted from leaf samples of symptomatic and apparently healthy plants of both varieties according to Psifidi et al. (2010). A nested PCR was performed 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. 1,200 bp product, amplified esclusively from symptomatic plants of both varieties, was cloned and sequenced. BLAST analysis revealed 99% similarity with sequence AF248959 of stolbur phytoplasma (16SrXII group, Ca. Phytoplasma solani). Sequences from both varieties were identical, indicating infection by the same phytoplasma strain and were deposited in EMBL-EBI (accession Nos HE598778 and HE598779). Diseased plants of var. tatula and stramonium showed 49-69% and 38% reduction of the above-ground fresh weight, respectively, compared to healthy plants, hence making the phytoplasma an important pathogen of jimsonweed, which constitutes a reservoir plant in the field. This is, to our knowledge, the first report of a Ca. Phytoplasma solani-related disease in jimsonweed in Greece.

Variability Studies of Two Prunus-Infecting Fabaviruses with the Aid of High-Throughput Sequencing

During their lifetime, perennial woody plants are expected to face multiple infection events. Furthermore, multiple genotypes of individual virus species may co-infect the same host. This may eventually lead to a situation where plants harbor complex communities of viral species/strains. Using high-throughput sequencing, we describe co-infection of sweet and sour cherry trees with diverse genomic variants of two closely related viruses, namely prunus virus F (PrVF) and cherry virus F (CVF). Both viruses are most homologous to members of the Fabavirus genus (Secoviridae family). The comparison of CVF and PrVF RNA2 genomic sequences suggests that the two viruses may significantly differ in their expression strategy. Indeed, similar to comoviruses, the smaller genomic segment of PrVF, RNA2, may be translated in two collinear proteins while CVF likely expresses only the shorter of these two proteins. Linked with the observation that identity levels between the coat proteins of these two viruses are significantly below the family species demarcation cut-off, these findings support the idea that CVF and PrVF represent two separate Fabavirus species.

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.

High-Throughput Sequencing Reveals Further Diversity of Little Cherry Virus 1 with Implications for Diagnostics

Little cherry virus 1 (LChV1, Velarivirus, Closteroviridae) is a widespread pathogen of sweet or sour cherry and other Prunus species, which exhibits high genetic diversity and lacks a putative efficient transmission vector. Thus far, four distinct phylogenetic clusters of LChV1 have been described, including isolates from different Prunus species. The recent application of high throughput sequencing (HTS) technologies in fruit tree virology has facilitated the acquisition of new viral genomes and the study of virus diversity. In the present work, several new LChV1 isolates from different countries were fully sequenced using different HTS approaches. Our results reveal the presence of further genetic diversity within the LChV1 species. Interestingly, mixed infections of the same sweet cherry tree with different LChV1 variants were identified for the first time. Taken together, the high intra-host and intra-species diversities of LChV1 might affect its pathogenicity and have clear implications for its accurate diagnostics.

A novel specific duplex real-time RT-PCR method for absolute quantitation of Grapevine Pinot gris virus in plant material and single mites

Grapevine Pinot gris virus (GPGV) is a widely distributed grapevine pathogen that has been associated to the grapevine leaf mottling and deformation disease. With the aim of better understanding the disease epidemiology and providing efficient control strategies a specific and quantitative duplex TaqMan real-time RT-PCR assay has been developed. This method has allowed reliable quantitation of the GPGV titer ranging from 30 up to 3 x 108 transcript copies, with a detection limit of 70 viral copies in plant material. The assay targets a grapevine internal control that reduces the occurrence of false negative results, thus increasing the diagnostic sensitivity of the technique. Viral isolates both associated and non-associated to symptoms from Greece, Slovakia and Spain have been successfully detected. The method has also been applied to the absolute quantitation of GPGV in its putative transmission vector Colomerus vitis. Moreover, the viral titer present in single mites has been determined. In addition, in the current study a new polymorphism in the GPGV genome responsible for a shorter movement protein has been found. A phylogenetic study based on this genomic region has shown a high variability among Spanish isolates and points to a different evolutionary origin of this new polymorphism. The methodology here developed opens new possibilities for basic and epidemiological studies as well as for the establishment of efficient control strategies.