Identification of Citrus tristeza virus (CTV) in Crete: standardization and optimization of diagnostic techinques
2010
Afifi, H.
Citrus tristeza virus (CTV) is the most important citrus virus causing serious economic problems for the citrus industry in the Mediterranean countries. There is currently a need for reliable and sensitive diagnostic routine methodology to identify CTV in Greece bearing in mind that is a quarantine virus and harsh virus elimination actions are taken once a positive sample is being identified in annual compulsory surveys. More than 2000 citrus trees in Crete were screened in 2009 for CTV infection using a commercial available immuno-print kit (Plant Print). The survey resulted in three infected trees (samples coded: TI, 342 and 802) all located in the north-western part of the island. The nucleotide sequences of the complete CP, p23, and HSP70 genes from trees TI, 342 and 802 were determined and compared with characterized CTV isolates (T385, Californian isolate, T30 and VT ). Nucleotide sequence analysis indicated that the obtained CTV infected trees samples were closer to the Spanish isolate T385 with nucleotide sequence identity around 99.7% for p23, CP and HSP70 genes which apparently has no effect on the amino acid level. Following several diagnostic assays, a PCR-amplified CTV dig-labeled probe convincingly detected the virus in hybridization experiments using stem prints, rolling-leaf prints, leaf and fruit crude sap (1:1 dilution) and RNA extracts. Total RNA extracts from CTV-infected plant tissues presented the template with the strongest reaction and minimum background. Furthermore, print-capture PCR and its variation immune-capture PCR protocols were also applied to detect immobilized viral p23 gene targets using the stem tissue prints or plant sap respectively.
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