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

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-

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

Psychological Predictors of Energy Saving Behavior: A Meta-Analytic Approach

Understanding how psychological processes drive human energy choices is an urgent, and yet relatively under-investigated, need for contemporary society. A knowledge gap still persists on the links between psychological factors identified in earlier studies and people’s behaviors in the energy domain. This research applies a meta-analytical procedure to assess the strength of the associations between five different classes of individual variables (i.e.,: attitudes, intentions, values, awareness, and emotions) and energy-saving behavioral intentions and behaviors (self-reported and actual). Based on a systematic review of studies published between 2007 and 2017, we estimate the average effect size of predictor-criterion relations, and we assess relevant moderators and publication bias, drawing on data obtained from 102 independent samples reported in 67 published studies (N = 59.948). Results from a series of five single meta-analyses reveal a pattern of significant positive associations between the selected psychological determinants and energy-saving indicators: associations between individual-level predictors and energy-saving outcomes are positive and moderate in size, ranging from large effects for emotions to small-moderate effects for pro-environmental values. Interestingly, moderation analysis reveals, among other things, that attitude-behavior links are not statistically significant when actual behavior is considered as an outcome. Implications for policy interventions are discussed

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

Next-generation methods for early disease detection in crops

: Plant pathogens are commonly identified in the field by the typical disease symptoms that they can cause. The efficient early detection and identification of pathogens are essential procedures to adopt effective management practices that reduce or prevent their spread in order to mitigate the negative impacts of the disease. In this review, the traditional and innovative methods for early detection of the plant pathogens highlighting their major advantages and limitations are presented and discussed. Traditional techniques of diagnosis used for plant pathogen identification are focused typically on the DNA, RNA (when molecular methods), and proteins or peptides (when serological methods) of the pathogens. Serological methods based on mainly enzyme-linked immunosorbent assay (ELISA) are the most common method used for pathogen detection due to their high-throughput potential and low cost. This technique is not particularly reliable and sufficiently sensitive for many pathogens detection during the asymptomatic stage of infection. For non-cultivable pathogens in the laboratory, nucleic acid-based technology is the best choice for consistent pathogen detection or identification. Lateral flow systems are innovative tools that allow fast and accurate results even in field conditions, but they have sensitivity issues to be overcome. PCR assays performed on last-generation portable thermocyclers may provide rapid detection results in situ. The advent of portable instruments can speed pathogen detection, reduce commercial costs, and potentially revolutionize plant pathology. This review provides information on current methodologies and procedures for the effective detection of different plant pathogens. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry

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

Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Olea Europaea Geminivirus

A real-time loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of the Olea europaea geminivirus (OEGV), a virus recently reported in different olive cultivation areas worldwide. A preliminary screening by end-point PCR for OEGV detection was conducted to ascertain the presence of OEGV in Sicily. A set of six real-time LAMP primers, targeting a 209-nucleotide sequence elapsing the region encoding the coat protein (AV1) gene of OEGV, was designed for specific OEGV detection. The specificity, sensitivity, and accuracy of the diagnostic assay were determined. The LAMP assay showed no cross-reactivity with other geminiviruses and was allowed to detect OEGV with a 10-fold higher sensitivity than conventional end-point PCR. To enhance the potential of the LAMP assay for field diagnosis, a simplified sample preparation procedure was set up and used to monitor OEGV spread in different olive cultivars in Sicily. As a result of this survey, we observed that 30 out of 70 cultivars analyzed were positive to OEGV, demonstrating a relatively high OEGV incidence. The real-time LAMP assay developed in this study is suitable for phytopathological laboratories with limited facilities and resources, as well as for direct OEGV detection in the field, representing a reliable method for rapid screening of olive plant material

Rapid detection of tomato spotted wilt virus by real-time RT-LAMP and in-field application

Tomato spotted wilt virus (TSWV) is considered one of the most threatening viruses worldwide for diferent economically important agricultural crops. In this scenario, it is important to perform an early detection by laboratory tests to prevent TSWV spread. A rapid and sensitive TSWV detection protocol based on real time reverse transcription loop-mediated isothermal amplifcation (RT-LAMP) assay was developed in this work, also using cost-efective and simplifed sample preparation procedure, to assess the suitability of the RT-LAMP assay in feld conditions on tomato and pepper samples. A set of six primers was designed within the nucleotide sequence region coding for the nucleocapsid protein (N) of segment S, targeting a 220-nucleotide sequence. Sensitivity, specifcity, accuracy, and in-feld application of the real-time RT-LAMP assay were evaluated. The developed real-time RT-LAMP assay proved to be one thousand and one hundred times more sensitive than end-point RT-PCR and real-time RT-PCR methods, respectively, detecting a total of 9.191 × 101 genome copies as minimum target, and no cross-reactivity were detected with other viruses belonging to Tospoviridae and Bromoviridae families used as outgroup. In addition, the in-feld application of the assay using the rapid sample preparation gave adequate and reliable results within 60 minutes, with an acceptable reaction delay when compared to canonical RNA extraction. The in-feld analyses showed an increase of TSWV-positive samples (37%) detection compared with end-point RT-PCR and real-time RT-PCR (32% and 29%, respectively), particularly on asymptomatic samples, confrming that the real-time RT-LAMP assay can be implemented as a routine test both in-feld and laboratory conditions as a rapid and sensitive technique for TSWV detection