Simultaneous detection of the seven main tomato-infecting RNA viruses by two multiplex reverse transcription polymerase chain reactions

Cucumber mosaic virus, Tomato spotted wilt virus, Tomato mosaic virus, Tomato chlorosis virus, Pepino mosaic virus, Torrado tomato virus and Tomato infectious chlorosis virus cause serious damage and significant economic losses in tomato crops worldwide. The early detection of these pathogens is essential for preventing the viruses from spreading and improving their control. In this study, a procedure based on two multiplex RT-PCRs was developed for the sensitive and reliable detection of these seven viruses. Serial dilutions of positive controls were analysed by this methodology, and the results were compared with those obtained by ELISA and singleplex versions of RT-PCR. The multiplex and singleplex RT-PCR assays were able to detect specific targets at the same dilution and were 100 times more sensitive than ELISA. The multiplex versions were able to detect composite samples containing different concentrations of specific targets at ratios from 1:1 to 1:1000. In addition, 45 symptomatic tomato samples collected in different tomato-growing areas of Sicily (Italy) were analysed by multiplex RT-PCR, singleplex RT-PCR and commercially available ELISA tests. Similar results were obtained using the RT-PCR techniques, with a higher sensitivity than ELISA, revealing a common occurrence of mixed infections and confirming the presence of these seven virus species in ItalyPanno, S.; Davino, S.; Rubio, L.; Rangel, E.; Davino, M.; García Hernández, J.; Olmos Castelló, A. (2012). Simultaneous detection of the seven main tomato-infecting RNA viruses by two multiplex reverse transcription polymerase chain reactions. Journal of Virological Methods. 186(1-2):152-156. doi:10.1016/j.jviromet.2012.08.003S1521561861-

Genetic diversity and evolutionary analysis of Citrus Tristeza Virus p20 gene in Pakistan: insights into the spread and epidemiology

Background: Citrus tristeza virus (CTV) is a widespread disease and the most destruction causing agent of citrus. Pakistan is ranked amongst the top ten citrus producing countries around the globe and it contributes about 2% to its foreign exchange earnings. Based on this assumption it is very important to monitor and determine the evolutionary forces and the phylogeography of Pakistani CTV population.  Methods: A total of 49 sequences of p20 gene from Pakistan were phylogenetically compared with CTV sequences worldwide. These sequences were analyzed for their genetic diversity and evolution using a Bayesian Probability approach and predicted secondary structure.Results: Phylogenetic analysis using Bayesian probability inference and predicted secondary structures diversity of CTV indicated that Pakistani isolates were not diverse from global isolates. Lineage analysis showed that CTV was introduced in Pakistan in three individual events from various parts of the world.  After that CTV dispersed in Pakistan via vector transmission or by use of infected propagating material by local farmers.Conclusions: Our study confirmed multiple introductions of CTV in Pakistan and also confirmed the dissemination of CTV within Pakistan. This study also shows that the mutations are present in the predicted secondary structure of the p20 protein, however, it is not known if it affects the pathogenicity of the virus

First report of Tomato leaf curl New Delhi virus affecting zucchini squash in an important horticultural area of southern Italy

omato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) which infects species in the families Cucurbitaceae and Solanaceae (Padidam et al., 1995; Mizutani et al., 2011). Begomoviruses are transmitted by the whitefly Bemisia tabaci in a persistent manner (Rosen et al., 2015). In October 2015, severe symptoms not previously reported by growers in the horticultural area of the Province of Trapani (Sicily, Italy) were observed on zucchini squash (Cucurbita pepo) in open fields. The symptoms included yellow mosaic, severe leaf curling, swelling of veins of young leaves, shortening of internodes, roughness of the skin of fruit and reduced fruit size; the symptoms were reminiscent of those caused by begomoviruses. Total DNA was extracted from young leaves of 22 plants by phenol/chloroform extraction and ethanol precipitation. PCR was performed with the A1F/A1R primer pair (Mizutani et al., 2011) for the DNA-A component and the pair described by Ruiz et al. (2015) for the DNA-B component to amplify a ~1200-bp fragment of DNA-A and a ~890 bp fragment of DNA-B, respectively. All 10 samples were positive by PCR with both primer pairs. No amplification products were obtained using primers specific for the monopartite begomoviruses Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus (Davino et al., 2008). DAS-ELISA analysis for Cucumber mosaic virus, Papaya ring spot virus and Zucchini yellow mosaic virus (Loewe Phytodiagnostica, Germany) yielded negative results

First report of Southern tomato virus in tomato crops in Italy

Twenty-five tomato plants (Solanum lycopersicum) showing symptoms of viral disease were sampled from different greenhouses in the Ragusa province (Southern Italy) in summer 2015. Plants showed chlorosis on leaves and fruits and deformation and depressed spots of dark colour which later evolved into necrosis (Fig. 1). These symptoms were observed on the entire cluster of fruit making the product unsaleable. Based on these symptoms, samples were analysed for Cucumber mosaic virus, Pepino mosaic virus (PepMV), Potato virus Y (PVY), Tomato mosaic virus and Tomato spotted wilt virus by DAS-ELISA with polyclonal antibodies (Loewe Phytodiagnostica, Germany), and for the emerging Southern tomato virus (STV) by RT-PCR (Candresse et al., 2013). Three of the 25 samples analysed were positive only for PepMV whereas the rest of the samples had mixed infections: fifteen plants were co-infected with PepMV and PVY, and seven with STV, PepMV and PVY. The amplification product (894 bp) obtained from one STV-infected plant was purified using the UltraClean® PCR Clean-Up kit (Mo-Bio, USA) and the consensus nucleotide sequences were determined in both senses using an ABI 3130XL Genetic Analyzer (Life Technologies, USA) and deposited in GenBank under accession number KT948068. The nucleotide identity of the Italian STV isolate was greater than 99% with STV isolates Mexico1 (EF442780), BD-13 (KT634055), CN-12 (KT438549), MS7 (EU413670) and FR (KC333078) from Mexico, Bangladesh, China, USA and France, respectively

OCCURRENCE OF TOMATO LEAF CURL NEW DELHI VIRUS INFECTING ZUCCHINI IN SARDINIA (ITALY)

Tomato leaf curl New Delhi virus (ToLCNDV, genus Begomovirus) is a bipartite, circular, ssDNA virus, able to infect species within the Cucurbitaceae and Solanaceae. In August 2016, field observations carried out in Sardinia (Italy) highlighted in one location (Decimoputzu, CA) some plants of zucchini squash (Cucurbita pepo L.) showing a systemic disease never observed before, even in a previous survey (end-June 2016) on cucurbit viruses

Evolutionary Analysis of Grapevine Virus A: Insights into the Dispersion in Sicily (Italy)

Grapevine virus A (GVA) is a phloem-restricted virus (genus Vitivirus, family Betaflexiviridae) that cause crop losses of 5–22% in grapevine cultivars, transmitted by different species of pseudococcid mealybugs, the mealybug Heliococcus bohemicus, and by the scale insect Neopulvinaria innumerabilis. In this work, we studied the genetic structure and molecular variability of GVA, ascertaining its presence and spread in different commercial vineyards of four Sicilian provinces (Italy). In total, 11 autochthonous grapevine cultivars in 20 commercial Sicilian vineyards were investigated, for a total of 617 grapevine samples. Preliminary screening by serological (DAS-ELISA) analysis for GVA detection were conducted and subsequently confirmed by molecular (RT-PCR) analysis. Results showed that 10 out of the 11 cultivars analyzed were positive to GVA, for a total of 49 out of 617 samples (8%). A higher incidence of infection was detected on ‘Nerello Mascalese’, ‘Carricante’, ‘Perricone’ and ‘Nero d’Avola’ cultivars, followed by ‘Alicante’, ‘Grecanico’, ‘Catarratto’,‘Grillo’, ‘Nerello Cappuccio’ and ‘Zibibbo’, while in the ‘Moscato’ cultivar no infection was found. Phylogenetic analyses carried out on the coat protein (CP) gene of 16 GVA sequences selected in this study showed a low variability degree among the Sicilian isolates, closely related with other Italian isolates retrieved in GenBank, suggesting a common origin, probably due to the exchange of infected propagation material within the Italian territory

Epidemiological Survey of Grapevine Leafroll-Associated Virus 1 and 3 in Sicily (Italy): Genetic Structure and Molecular Variability

Background: the most widely distributed and virulent Grapevine leafroll-associated viruses (GLRaV) that affect grapevine are GLRaV-1 and GLRaV-3, transmitted semi-persistently by different mealybugs and soft scales, mainly causing downward rolling of the leaf margins and interveinal reddening. Methods: the main objectives of this study were to investigate the genetic structure and molecular diversity of GLRaV-1 and GLRaV-3 in 617 samples from 11 autochthonous Sicilian grapevine cultivars, ascertaining their presence and spread. The detection was implemented by serological and molecular analyses and subsequently phylogenetic analyses on selected Sicilian isolates were conducted. Results: in total, 33 and 138 samples resulted positive to GLRaV-1 and GLRaV-3, with an incidence of 5.34% and 22.36%, respectively; 9 out of the 11 cultivars resulted positive, while the presence of both viruses was not found in ‘Grillo’ and ‘Moscato’ cultivars. Conclusions: phylogenetic analyses of the coat protein (CP) gene of 12 GLRaV-1 selected sequences showed a close relationship with European isolates; the discrete nucleotide differentiation and positive selection could demonstrate a current increase in population fitness. The phylogenetic analyses of the CP gene of 31 GLRaV-3 Sicilian CP sequences demonstrates a close relationship between Sicilian and different countries isolates; a certain stability of GLRaV-3 in the different cultivars analyzed is suggested by the discrete differentiation nucleotide and negative selection of the Sicilian isolates

Actas de Horticultura

El cultivo de los cítricos comenzó en Extremo Oriente hace unos 4.000 años, en las regiones que ocupan actualmente China y Japón. Los grandes movimientos migratorios que ocasionaron las conquistas de Alejandro Magno, la expansión del Islam y el descubrimiento de América favorecieron la expansión de este cultivo por todo el mundo. Sin embargo, fue a partir del siglo XVIII cuando la citricultura adquirió una relevancia económica, tanto desde el punto de vista industrial como ornamental. El movimiento de plantas fue acompañado por la difusión de diversos patógenos, aunque afortunadamente, sólo una parte de los presentes en las regiones de origen han llegado en las nuevas áreas de cultivo. En Italia, la superficie cultivada con cítricos es de aproximadamente 160.000 Ha y de éstas, alrededor del 60% se encuentran en Sicilia. En los últimos años ha cobrado relevancia la producción de cítricos destinados a fines ornamentales, con una producción media anual en Sicilia de unos 4,5 millones de plantas, lo que la convierte en el máximo productor de cítricos ornamentales de Europa. Entre estos se encuentran los limones ornamentales, distintos kumquats, calamondín, naranjo amargo, cidro, naranjo dulce, mandarinos y pomelos. Desde el punto de vista sanitario, hay que tener en cuenta que las plantas ornamentales que se venden por todo el territorio Europeo pueden actuar como reservorios y facilitar el tráfico y emergencia de nuevas enfermedades. Entre las enfermedades más comunes en los cítricos ornamentales se encuentra la exocortis, las protuberancias nerviales (vein enation), las manchas anulares (ringspot), la psoriasis, la tristeza, la variegación, las concavidades gomosas (concave gum), la impietratura, el stubborn y el Huanglongbing. En este artículo se describen las principales enfermedades que afectan a los cítricos ornamentales y que representan un riesgo en la Comunidad Europea

Fungal contaminants in Sicilian livestock feeds and first studies on the enzymatic activity of Aspergillus isolates

The purposes of this study were 1) to determine the total fungal contamination in Sicilian raw materials and livestock, 2) to evaluate the occurrence of Aspergillus spp., Penicillium spp. and Fusarium spp., 3) to identify fungi belonging to the genus Aspergillus and 4) to determine their ability to produce cellulolytic enzymes. Fourteen feed samples were collected in a feed mill near to Palermo (Sicily, Italy). Analysis of the total mycobiota was performed on Sabourad Dextros Agar (SAB) and Potato Dextrose Agar (PDA) and total fungal counts were expressed as CFU/g. Aspergillus spp. isolates were selected on the basis of the frequency of isolation and identifed using micro and macro-morphological characteristics and ITS sequence analysis. The ability of the Aspergillus isolates to produce cellulolytic enzymes was tested qualitatively by in vitro assay at two temperature, 25 and 30 °C, and in static and shaking condition. Total fungal population ranged from 1.11x106 to 1.31x108 and from 1.11x103 to 1.58x106 CFU/g on PDA and SAB, respectively. All feed samples showed the recurrent presence of colonies belonging mostly to the ubiquitous genera Aspergillus, Fusarium and Penicillium. Eight isolates of Aspergillus spp. were obtained and identifed as A. amstelodami, A. awamori, A. flavus, A. niger, A. oryzae and A. tubingensis. Between them, A. awamori, A. niger and A. tubingensis showed the highest enzymatic activity. The presence of potential mycotoxigenic isolates of Aspergillus spp. in the analysed feeds represents a risk for animal health; moreover their ability to produce cellulolytic enzymes can seriously affect feed quality