Journal of Plant Pathology (2006) 88, p. S58 (Sciancalepore et al.)

A. Sciancalepore, A. De Stradis, A. Minafra, A. Campanale and G.P. Martelli (2006)
Fluorescent in situ hybridization on grapevine and Nicotiana tissues infected by grapevine viruses A and B
Journal of Plant Pathology 88 (3, Special Issue), S58-S58
S.I.Pa.V XIII National Meeting - Foggia, 12-16 September 2006 - Poster
Abstract: In grapevine, multiple natural infections by vitiviruses are relatively common since mealybug transmission and grafting favours the spread of both Grapevine virus A (GVA) and Grapevine virus B (GVB). The two viruses share a relatively high sequence similarity (average of 50% nucleotide identity on the whole genome). Thus, close or identical replication sites in infected cells may increase the chance of homologous recombination, producing viable chimeric RNAs. Since potential recombination events between low titer virus RNAs in infected grapevines, studied by nested RT-PCR, are difficult to detect, an accurate analysis of virus replication sites is needed. Fluorescent in situ hybridization was designed with oligo-labelled DNA or PCR-amplified fragments of CP genes with tetramethyl-rhodamine or Alexa Fluor-488 dUTP (Invitrogen), for GVA and GVB, respectively. This system was chosen because post-hybridization processing like use of antibodies or RNAse A incubation was not needed. The method was first validated on single infections by GVA in Nicotiana benthamiana and GVB in N. occidentalis. Hybridization was successfully obtained at 65°C in 50% formammide, after a pretreatment of sectioned tissues with proteinase K. In infected tobacco, viral RNAs were clearly identified as fluorescent spots on the plasma membranes. Comparable results were obtained when in vitro-grown shoots from two double-infected grapevines, were hybridized. In this case, however, the intrinsic fluorescence of polyphenols interfered with a clear-cut detection of viral RNAs.
Database assignments for author(s): Angelo De Stradis

Research topic(s) for pests/diseases/weeds:
molecular biology - genes

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Pest and/or beneficial records:

Beneficial	Pest/Disease/Weed	Crop/Product	Country	Quarant.
	Grapevine virus A	Grapevine (Vitis)
	Grapevine virus B	Grapevine (Vitis)