Epidemiology of Plum pox virus strain M in Greece

Plum pox virus has been endemic in Greece since 1967 causing important losses in apricot and to a lesser extent in peach crops. A survey undertaken in 1992 in public and private mother-tree plantations to estimate its incidence revealed that the virus was absent in isolated areas far from commercial stone-fruit crops. Virus titers decrease significantly during the hot months in the infected trees but re-increase in October–November permitting reliable detection. It is virulent M-type isolates which are effectively transmitted by aphids that are mostly recovered. Aphis gossypii and Hyalopterus pruni were the most abundant virus vectors captured during the small scale monitoring undertaken in apricot orchards in 1999 and 2000. Virus spread was monitored in two apricot orchards from 1996 to 2000 and analysed. Initial infections followed a completely random spatial pattern, while loose clusters appeared in succeeding years, to finally reach a uniform distribution representing high infection levels. The nearby ecological conditions greatly affected the rate of disease development.     

High‐throughput sequencing technologies for plant pest diagnosis: challenges and opportunities

High‐throughput sequencing (HTS) technologies have revolutionized plant pest research and are now raising interest for plant pest diagnostics, with plant virus diagnostics at the forefront of development. However, the application of HTS in plant pest diagnostics raises important challenges that plant health regulators will have to address. Adapted infrastructures, technical guidelines and training are pivotal for further use and adoption of the HTS technologies in the phytosanitary framework.     

Biological and molecular characterization of a distinct Citrus tristeza virus isolate originating from a lemon tree in Greece

A Citrus tristeza virus (CTV) isolate (L192GR) naturally occurring in lemon trees of more than 100 years old in Greece was fully characterized. Virus‐derived small interfering RNAs, induced by Dicer processing of dsRNAs formed during RNA virus replication, were isolated and used as targets for sequencing. Next‐generation high‐throughput sequencing using the Ion Torrent platform was performed. A total of 432 632 sequences, 94·05% of which corresponded to L192GR, were determined. Subsequent bioinformatics analysis enabled the determination of the full‐length 19 251 nt genome of the L192GR isolate (GenBank no. KC262793 ). Comparative analysis of complete genomes revealed molecular homology with CTV‐VT isolate FS2‐2 from Florida (GenBank no. EU937519 ) with 98·2% nucleotide sequence identity. Recombination events were detected in L192GR and they probably contribute to its unique characteristics. Specifically, although most isolates of the CTV‐VT group induce the seedling yellows syndrome and react positively with the monoclonal antibody MCA13, which is typically associated with severe CTV isolates, the MCA13‐positive L192GR gave very mild or even no symptoms in the seedling yellows indicator plants. Furthermore, experimental aphid transmissibility studies revealed a poor transmission efficiency of 20%. This is the first report of a CTV isolate originating from a lemon tree being fully characterized at biological, serological and molecular levels. The present study further confirms that, when the goal is the risk assessment associated with a new pathogen or isolate in a particular area, molecular data have to be combined with the biological properties of the pathogen.     

Comparison of direct-RT-PCR and dot-blot hybridization for the detection of Potato spindle tuber viroid in natural host plant species

Potato spindle tuber viroid (PSTVd) is an EPPO A2-listed quarantine pathogen and its detection in large scale surveys requires complex decision schemes. In this study, a simple and rapid application of direct-RT-PCR was evaluated together with dot blot hybridization for the detection of PSTVd in dormant potato tubers harvested from primary infected plants, as well as in tomato and solanaceous ornamental plants. In all infected dormant potato tubers tested, both direct-RT-PCR and dot blot hybridization detected two different PSTVd isolates, with direct-RT-PCR being ten times more sensitive than dot blot. Similarly, in infected tomato and Brugmansia spp., PSTVd was detected by direct-RT-PCR with higher sensitivity compared to that of dot blot hybridization. However, in Brugmansia spp., a ten-fold decrease of the typical working concentration of the sap was required for an unequivocal detection of the viroid by direct-RT-PCR. The potential to use direct-RT-PCR for routine PSTVd examination is discussed.     

Characterization of cucumber mosaic cucumovirus isolates in Greece

Since 1990, massive cucumber mosaic cucumovirus (CMV) outbreaks of various symptomatology, especially on tomato, have been observed all over Greece. To characterize local virus populations, 40 CMV isolates of different origin and symptomatology in the field and glasshouse were examined for subgroup identification and presence of satellite RNA (satRNA). The IC-PCR method was used to amplify part of the coat protein gene of the isolates; the products were submitted to RFLP analysis using Eco RI and Msp I restriction enzymes. Most isolates gave the characteristic pattern attributed to CMV subgroup I, confirming the results obtained by serotyping with monoclonal antibodies. Some Greek isolates, however, possessed additional Eco RI and Msp I sites unusual for subgroup I isolates, and the patterns obtained, especially for Msp I, could be confused with those of subgroup II. These data suggest that restriction enzyme analysis of amplified PCR products used for strain characterization needs to be treated with caution, especially for viruses such as CMV showing genomic heterogeneity. IC-PCR was also developed for satRNA amplification and the results agreed with those of molecular hybridization with a dig-DNA probe. A satRNA was carried by 77% of the isolates.