Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) identified for the first time in 1995 in Asia, from where it spread into several countries of the Mediterranean basin. ToLCNDV was found in Spain in 2012, and subsequently in Tunisia and Italy. The first outbreak in Italy occurred at the end of 2015 in Trapani province (Sicily) on zucchini squashes. Then in 2016, ToLCNDV was found in infected zucchini plants in Campania, Lazio and Sardinia regions, and in 2017 in Calabria. This study addressed the dispersion and genetic diversity of ToLCNDV isolates in Italy. A total of 1400 plants were analysed. Phylogenetic analysis showed low variability among the Italian isolates, probably as a consequence of the recent introduction and rapid spread of this virus in Italy. Two statistically significant clusters were reported: one grouping only Italian isolates and the other grouping Italian, Spanish, Tunisian and Moroccan isolates. Furthermore, the highest incidence of ToLCNDV was observed in Sicily, although the disease also appears to be critical in other Italian regions. In this work, a high efficiency of ToLCNDV mechanical transmission into Cucurbita pepo, Cucumis melo inodorus and Cucumis melo cantalupensis has been demonstrated. The rapid spread of ToLCNDV in the Mediterranean basin represents a threat for horticultural production, thus it is very important to develop suitable crop management practices, applying genetic resistance strategies and more restrictive phytosanitary measures.

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Peach latent mosaic viroid (PLMVd) is a damaging pathogen for peach, its detection being of critical importance to both sanitary and certification programs worldwide. Here we report a quantitative realtime reverse transcription polymerase chain reaction assay (qRT-PCR) based on TaqMan® chemistry to improve the diagnosis of this pathogen. Critical to this approach is the design of a specific set of primers and of a probe, and the use of a suitable extraction method proving a reliable, sensitive and specific PLMVd diagnosis. More specifically, the sensitivity was evaluated using 12 ten-fold dilution series of an in vitro transcript of the entire PLMVd genome as a standard for quantification in infected samples. The assay detected up to 1 fg of target RNA. The protocol was also evaluated for its specificity using healthy peach, apricot, plum and pear controls and non-target viroids (Apple scar skin viroid, Hop stunt viroid, Pear blister canker viroid, Apple dimple fruit viroid and Potato spinder tuber viroid). None of the non-target or healthy analysed samples reacted in qRT-PCR. The efficiency and accuracy of the method was evaluated using different PLMVd peach isolates as templates, including three 'calico' variants: a strong signal was obtained from all of them. Finally, five RNA extraction methods were compared and evaluated to choose the best for detection and quantification of PLMVd by the qRT-PCR protocol.

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In a survey during [2006][2007][2008][2009] 947 field samples (bark, fruit and leaves) of cultivated and wild pome and stone fruit trees showing various symptoms, including scar skin, dappling, fruit malformation, leaf mottle and stem canker ( Fig. 1) were collected in different regions of Greece (Attica, Crete, Macedonia, Peloponnesus and Thessaly). Samples were tested for the presence of Apple scar skin viroid (ASSVd), Hop stunt viroid (HSVd) and Pear blister canker viroid (PBCVd), using tissue-print molecular hybridisation, reverse transcription-polymerase chain reaction (RT-PCR), cloning and sequencing.Tissue-print molecular hybridisation showed that approximately 400 trees were infected with only one viroid whereas 63 trees were co-infected by two or three viroids. RT-PCR confirmed mixed infections in 26 trees but the results were not clear for samples from the other trees. Fourteen trees in all, seven pear (Pyrus communis), three wild pear (Pyrus amygdaliformis), three apple (Malus domestica) and one quince (Cydonia oblonga) were infected with ASSVd and PBCVd; five sweet cherry trees (Prunus avium) were infected with ΑSSVd and HSVd, and seven trees, four apple and three wild apple (Malus sylvestris) were infected with ASSVd, PBCVd and HSVd. All three viroids were sequenced in two trees, ASSVd and PBCVd in a further six trees, and ASSVd and HSVd (partially) in a wild apple tree (Table 1) (Hassan et al., 2004; Tessitori et al., 2002) and of PBCVd, PLMVd and HSVd in pear (Fekih-Hassen et al., 2004) have been reported previously. The presence of natural viroid infections in wild species away from human influence in Greece suggests that ASSVd, PBCVd and PLMVd originated in wild hosts and are probably native to Greece (Kyriakopoulou et al., 2001); our data for ASSVd, PBCVd and HSVd support this hypothesis (Kaponi 2009).Mixed infections favour viroid recombination and evolution, and wild species used as rootstocks for cultivated species may serve as viroid reservoirs, making phytosanitary control critical (Randles, 2003).

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