Enza Torchetti

Si riferisce sui risultati di una indagine condotta su piante di gerbera coltivate nel Nord barese ed in Provincia di Lecce interessate, rispettivamente, da estese necrosi delle foglie, degli steli e dei fiori e da virescenza e fillodia a carico dei soli fiori. L'agente causale dei sintomi necrotici è stato identificato in un ceppo 'resistance breaking' (RB) di Tomato spotted wilt virus (TSWV) mentre nel caso della virescenza e fillodia dei fiori la malattia è stata associata alla presenza di 'Candidatus Phytoplasma asteris'. Entrambi i patogeni sono stati già segnalati sulle coltivazioni di gerbera presenti in altre regioni italiane ma questa è la prima segnalazione della presenza di un ceppo RB di TSWV e la prima segnalazione pugliese della presenza del fitoplasma sulla ornamentale. Viene nuovamente sottolineata l'importanza del ritrovamento dei ceppi RB di TSWV anche nei confronti di colture orticole e sono fornite indicazioni sulla eco-epidemiologia delle...

Grapevine yellow speckle viroid 1 (GYSVd-1), Grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Hop stunt viroid (HSVd) and Citrus exocortis viroid (CEVd) are the five viroids known to infect naturally grapevines. We developed a multiplex RT-PCR (mRT-PCR) method for the simultaneous detection of these five viroids and the amplification of the cDNA fragment of a host-derived mRNA (actin mRNA) as an internal positive control. Specific primers for each targeted viroid were designed by taking into account the sequence variability within and between the viroid species and tested in silico. The method was validated by testing 57 grapevine samples from Iran and showed reliability and high sensitivity. The RT-PCR-negative samples were further assayed by Northern-blot hybridization. For this, a method was developed for the simultaneous detection of three different grapevine viroids on a single hybridization membrane. In this survey, HSVd, GYSVd-1, AGVd, and GYSVd-2 were detected in 100, 95, 93, and 65% of the samples tested, respectively, confirming the wide distribution of these viroids in Iran. CEVd was not detected in any of the samples collected. Based on these results, HSVd is proposed as a positive internal control for mRT-PCR in the areas where this viroid is widespread, so as to reduce the time and costs of DNase treatment, which is required when a host-derived internal control is used. The mRT-PCR method has the potential to be used routinely for large-scale surveys and certification programs.

The spread of viroids belonging to the genus Pospiviroid (family Pospiviroidae), recorded recently in ornamentals and vegetables in several European countries, calls for fast, efficient and sensitive detection methods. Based on bioinformatics analyses of sequence identity among all pospiviroids, a digoxigenin-labeled polyprobe (POSPIprobe) was developed that, when tested by dot-blot and Northern-blot hybridization, detected Potato spindle tuber viroid, Citrus exocortis viroid, Columnea latent viroid, Mexican papita viroid, Tomato planta macho viroid, Tomato apical stunt viroid, Pepper chat fruit viroid and Chrysanthemum stunt viroid. The end-point detection limits of the POSPIprobe ranged from 5(-2) to 5(-4), and from 5(-1) to 5(-3) for nucleic acid preparations obtained by phenol extraction and silica-capture, respectively, similar to those of single probes. Based on sequence identity, the POSPIprobe is expected to detect also the two pospiviroid species not tested in this study (Tomato chlorotic dwarf viroid and Iresine viroid-1). Dot-blot assays with the POSPIprobe were validated by testing 68 samples from tomato, chrysanthemum and argyranthemum infected by different pospiviroids as revealed by RT-PCR, thus confirming the potential of this polyprobe for quarantine, certification and survey programs.