Molecular characterization of Citrus tristeza virus isolates from Cyprus on the basis of the coat protein gene

Within the context of a program in Cyprus for the control of Citrus tristeza virus (CTV), the coat protein (CP) genes of 12 local isolates of the virus that induced different symptoms on host trees, were compared to those of known isolates. The CP genes were reverse-transcribed (RT) and amplified by polymerase chain reaction (PCR) and the resulting amplicons were cloned and sequenced. Nucleotide sequence analysis revealed no signs of geographic speciation. All the sequences obtained clustered close to those of previously known isolates of worldwide origin that are in five distinct groups. The nucleotide diversity was high compared to that found using a worldwide database of CP gene sequences. These data support the existence of different CTV introductions into Cyprus or an introduction from a location in which CTV is relatively diverse. Some of the isolates induced stem pitting on branches of grapefruit and sweet orange. Such isolates have not been noted often in the Mediterranean basin. They were close in CP sequence to isolate B249 from Venezuela, which induces stem pitting, and are of particular concern for the whole region.

Application of molecular tools for detection of plant viruses

Grapevine leafroll disease (GLRD) is one of the most important virus diseases of grapevines worldwide, causing major economical impact. The disease has a complex aetiology and currently eleven phloem-limited viruses, termed in general Grapevine leafroll-associated virus (GLRaVs), have been identified. Two of the GLRaVs, GLRaV-1 and GLRaV-3, are included in the European certification scheme of propagation material. However, the flawed notion that GLRaV-3 is more frequent than GLRaV-1 and that all other GLRaVs are possibly not as relevant for GLRD, has until now precluded the development of specific serological and molecular detection assays and limited the scope of molecular characterization of the viruses known to be associated with the disease. Hence, few studies have addressed the phylodynamics of GLRaVs or even characterized the genetic structure of their natural populations. This generalized lack of molecular information, in turn underlie the deficient capacity to detect the viruses. The phylogenetic analyses were conducted on the basis of the heat shock protein 70 homologue (HSP70h) and the coat protein (CP) genes for GLRaV-1 and the HSP70h, the heat shock protein 90 homologue (HSP90h) and the CP genes for GLRaV-5. The data obtained for GLRaV-1 contributed 83 new CP sequences. This information was combined with previous analysis by other authors and used for the production of new polyclonal IgG, capable of detecting CP variants from all the phylogroups observed. Successful testing of this new tool included tissue print immunoblotting (TPIB) and in situ immunoassay (ISIA). The data obtained for GLRaV-5, contributed 61 new CP and 28 new HSP90h gene sequences. Eight phylogenetic groups were identified on the basis of the CP. Characterization of the genetic structure of the isolates revealed a higher diversity than previously reported and allowed the identification of dominant virus variants. For both GLRaV-1 and GLRaV-5, the effect of vegetative propagation on the virus transmission dynamics was addressed.

First report of Citrus tristeza virus in the State Union of Serbia and Montenegro.

Citrus production in the State Union of Serbia and Montenegro has a strategic importance to the agricultural sector. Approximately 400,000 trees are now grown in the major citrus producing region, which is the Montenegrin Coastal Region. Satsuma mandarins and lemons grafted on Poncirus trifoliata are the most cultivated varieties. In December 2003, eight samples taken from the coastal region close to the towns of Bar and Ulcinj were analyzed using enzyme- linked immunosorbent assay (ELISA) with SP7 antibodies produced at Universidade do Algarve, Portugal (3). Further analysis was done using immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) targeting the entire coat protein (CP) gene (forward primer CTV1: 5(prime)- ATGGACGACGAAACAAAGAA-3(prime) and reverse primer CTV10: 5 (prime)-ATCAACGTGTGTTGAATTTCC-3(prime)). Using both techniques, seven of eight samples analyzed were found to be infected by Citrus tristeza virus (CTV), including samples from five trees that exhibited chlorosis, gummosis, and fruit deformation, and two trees that were symptomless.