First Report of Tomato Brown Rugose Fruit Virus on Tomato Crops in Italy

In October 2018, virus-like symptoms were observed in four different greenhouses of tomato (Solanum lycopersicum) in Ragusa province (Sicily, Italy). Symptoms consisted in mosaic, deformation, and necrosis on young leaves, and discoloration and deformations on young fruits. In total 40 symptomatic samples were collected (10 for each greenhouse). Samples were tested by reverse transcription polymerase chain reaction (RT-PCR) using specific primers for different viruses that incite similar symptoms on tomato plants: Groundnut ringspot virus (Camelo-García et al. 2014), Parietaria mottle virus (Galipienso et al. 2015), Pepino mosaic virus (Panno et al. 2012), Tobacco etch virus (Zhang et al. 2012), Tomato brown rugose fruit virus (Salem et al. 2015), Tomato chlorotic spot virus (Webster et al. 2013), Tomato mosaic virus (Panno et al. 2012), Tomato mottle mosaic virus (Sui et al. 2017), Tomato necrotic spot virus (Bratsch et al. 2018), Tomato necrotic streak virus (Badillo-Vargas et al. 2016), Tomato torrado virus (Panno et al. 2012), and Tomato yellow leaf curl virus (Davino et al. 2008). Thirty-seven out of the 40 samples analyzed yielded fragments of the expected size only for tomato brown rugose fruit virus (ToBRFV). This screening identified ToBRFV as a putative causal agent of this disease. To confirm the presence of this virus, two new primers named ToBRFV-F-5722, 5′-CACAATCGCAACTCCATCGC-3′ (coordinates: 5,722 to 5,742 nt referred to GenBank no. KT383474), and ToBRFV-R-6179, 5′-CAGAGGACCATTGTAAACCGG-3′ (coordinates: 6,179 to 6,200 nt referred to GenBank no. KT383474), based on the sequence of the coat protein gene, were designed. RT-PCR, in one-step format, was performed in 25 μl (final volume) containing 2 μl of total RNA, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 0.4 mM dNTPs, 1 mM of primers, 4U of RNaseOut, 20 U of superscript II reverse transcription-RNaseH, and 2U of Taq DNA polymerase (Thermo Fisher, U.S.A.). RT-PCR was carried out according to the following conditions: 42°C for 45 min; 95°C for 5 min; 40 cycles of 30 s at 95°C, 30 s at 55°C, and 30 s at 72°C; and a final elongation of 10 min at 72°C. The RT-PCR yielded the expected amplicons of 458 bp, confirming the previous results. The amplification products were purified using the UltraClean PCR Clean-Up kit (Mo-Bio, U.S.A.), and the nucleotide sequences were determined in both directions using an ABI PRISM 3100 DNA sequence analyzer (Applied Biosystems, U.S.A.). The sequences obtained from the 37 samples showed 99% identity. BLAST analysis showed an identity >99% with ToBRFV isolates Tom1-Jo (accession no. KT383474) and ToBRFV-IL (accession no. KX619418). Only one sequence was deposited in GenBank (accession no. MK313803). Sap extracts of four samples retrieved from the four different greenhouses were mechanically inoculated into tomato cultivar Marmande (three plants per isolate). Plants were grown in sterilized soil in an insect-proof glasshouse, with a photoperiod of 14 h light at 28/20°C day/night. Symptoms were recorded weekly, with all plants showing the symptoms described for ToBRFV at 30 days postinoculation. Tomato brown rugose fruit virus is a single-stranded positive RNA virus, belonging to the genus Tobamovirus, family Virgaviridae (Salem et al. 2015). Sicily is an important region for horticulture in Southern Europe. This virus represents a serious problem for tomato crops in Sicily and in all regions where tomato is grown, owing to its ability to be transmitted by plant-to-plant contact, by manipulations, and particularly by seeds. To our knowledge, this is the first report of ToBRFV in Italy and in Southern Europe

Real-time reverse transcription polymerase chain reaction development for rapid detection of Tomato brown rugose fruit virus and comparison with other techniques

Background: Tomato brown rugose fruit virus (ToBRFV) is a highly infectious tobamovirus that causes severe disease in tomato (Solanum lycopersicum L.) crops. In Italy, the first ToBRFV outbreak occurred in 2018 in several provinces of the Sicily region. ToBRFV outbreak represents a serious threat for tomato crops in Italy and the Mediterranean Basin. Methods: Molecular and biological characterisation of the Sicilian ToBRFV ToB-SIC01/19 isolate was performed, and a sensitive and specific Real-time RT-PCR TaqMan minor groove binder probe method was developed to detect ToBRFV in infected plants and seeds. Moreover, four different sample preparation procedures (immunocapture, total RNA extraction, direct crude extract and leaf-disk crude extract) were evaluated. Results: The Sicilian isolate ToB-SIC01/19 (6,391 nt) showed a strong sequence identity with the isolates TBRFV-P12-3H and TBRFV-P12-3G from Germany, Tom1-Jo from Jordan and TBRFV-IL from Israel. The ToB-SIC01/19 isolate was successfully transmitted by mechanical inoculations in S. lycopersicum L. and Capsicum annuum L., but no transmission occurred in S. melongena L. The developed real-time RT-PCR, based on the use of a primer set designed on conserved sequences in the open reading frames3, enabled a reliable quantitative detection. This method allowed clear discrimination of ToBRFV from other viruses belonging to the genus Tobamovirus, minimising false-negative results. Using immunocapture and total RNA extraction procedures, the real-time RT-PCR and end-point RT-PCR gave the same comparable results. Using direct crude extracts and leaf-disk crude extracts, the end-point RT-PCR was unable to provide a reliable result. This developed highly specific and sensitive real-time RT-PCR assay will be a particularly valuable tool for early ToBRFV diagnosis, optimising procedures in terms of costs and time

First report of Tomato leaf curl Sinaloa virus infecting tomato crops in Panama

In April 2011 and September 2012, virus-like symptoms were observed in open field- and greenhouse-grown tomato crops (Solanum lycopersicum) in Chiriqu\ued, the westernmost province of Panama. Samples from symptom-bearing plants (127 in all) were collected and tested for the presence of begomoviruses by polymerase chain reaction (PCR) assays with sets of degenerated primers designed to amplify parts of the DNA-A and DNA-B components (Rojas et al., 1993; Table 1). Products of the expected sizes, obtained with both DNA-A- and DNA-B-specific primers for 49 samples, suggested infection with New World bipartite begomoviruses. This corresponds to an incidence of 26% (8 plants) in open field, and 43% (41 plants) in greenhouse crops. Primers specific for ten tomato-infecting begomoviruses found in Central America (Engel et al., 1998; Nakhla et al., 2005; Table 1) were used to typify the PCR-positive samples

The Use of Ozenoxacin in Pediatric Patients: Clinical Evidence, Efficacy and Safety

Impetigo is the most common childhood skin infection in the world. There are two patterns of impetigo: nonbullous (or impetigo contagiosa) and bullous. The nonbullous type is due to Staphylococcus aureus and group A beta-haemolytic Streptococcus and occurs in 70% of impetigo cases. Impetigo is often a self-limited disease, but complications can sometimes occur. Therapy depends on the extent and site of the lesions and on the presence of systemic symptoms. The increase in multidrug resistance pathogens, such as methicillin-resistant Staphylococcus aureus, mupirocin-resistant Staphylococcus aureus or quinolone-resistant Staphylococcus aureus, requires the development of new antibiotics against these agents. The aim of this review is to evaluate the efficacy and safety of ozenoxacin in children compared to those of other approved topical antimicrobial therapies. The bactericidal activity against both susceptible and resistant organisms is a relevant feature of ozenoxacin because the bacterial strain and potential for resistance are generally not known at the beginning of therapy. Additionally, its minimal dermal absorption and its capability to reach high concentrations in the upper layers of the epidermidis agrees with the recommended practice aimed at avoiding the emergence of bacterial resistance in presence of a good safety profile. Further studies with real-life analyses and pharmacoeconomic evaluation are needed to confirm its role as first-line and second-line therapy in children with impetigo

First outbreak of Pepper vein yellows virus infecting sweet pepper in Italy

Sweet pepper (Capsicum annum) is an economically important crop worldwide, including Sicily where about 4,000 hectares are grown each year. In October 2015, severe symptoms not previously reported by growers in the horticultural area of the province of Trapani (Sicily, Italy) were observed on sweet pepper plants in eight different greenhouses. Symptoms included upward leaf curling, internodal shortening and interveinal yellowing. Symptoms were more evident in the upper part of the plants. These symptoms were reminiscent of those caused by poleroviruses. In the greenhouse, symptoms were evident in about 35% of the plants. Three samples per greenhouse (24 in total) were collected for analysis

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

Detection of Parietaria Mottle Virus by RT-qPCR: An Emerging Virus Native of Mediterranean Area That Undermine Tomato and Pepper Production in Southern Italy

Parietaria mottle virus (PMoV) is considered an emerging virus in many countries of the Mediterranean basin, especially on tomato and pepper crops. Symptoms on tomato leaves and fruits can be easily confused with those induced by cucumber mosaic virus (CMV) with necrogenic satellite RNA (CMV-satRNA), tomato spotted wilt virus (TSWV) or tomato mosaic virus (ToMV). Mixed infection of these viruses has been also reported in some tomato cultivars, with an increase in the complexity of the symptoms and severity of the disease. Although a specific serum and riboprobes have been produced, nowadays no sensitive diagnostic methods are available for the rapid PMoV detection. Here, we have developed a RT-qPCR assay with the aim to establish a more sensitive and specific method for PMoV detection. Specific primers and TaqMan probe were designed and in silico tested with all PMoV isolates available in GenBank. Moreover, this method was evaluated on tomato naturally infected samples from Sicily region (Italy). Results obtained showed that the RT-qPCR assay developed in this work is extremely sensitive, in fact, it is able to detect as few as 10 PMoV RNA copies in tomato total RNA; moreover, it will be a particularly valuable tool for early detection of PMoV. Furthermore, the analyzes on field samples show how this pathogen is increasingly present in tomato crops in the last years, helping to undermine the Italian horticultural sector

Spread of tomato brown rugose fruit virus in sicily and evaluation of the spatiotemporal dispersion in experimental conditions

Tomato brown rugose fruit virus (ToBRFV) is an emerging pathogen that causes severe disease in tomato (Solanum lycopersicum L.) crops. The first ToBRFV outbreak in Italy occurred in 2018 in several Sicilian provinces, representing a serious threat for tomato production. In the present work, the spatiotemporal displacement of ToBRFV in Sicily was evaluated, analyzing a total of 590 lots of tomato seed, 982 lots of plantlets from nurseries and 100 commercial greenhouses. Furthermore, we investigated the ToBRFV spreading dynamic in a greenhouse under experimental conditions. Results showed several aspects related to ToBRFV dispersion in protected tomato crops. In detail, an important decrease of the ToBRFV-infected seed and plantlet lots was detected. Regarding the examined commercial greenhouses, ToBRFV still appears to be present in Sicily, although there has been a decrease during monitoring. In experimental conditions, it was demonstrated that the presence of few infected plants are sufficient to damage the entire crop in a short time, reaching almost 100% of infection

First Report of Tomato Leaf Curl New Delhi Virus Causing Yellow Leaf Curl of Pepper in Europe

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus (family Geminiviridae) with two circular ssDNA genome components (DNA-A and DNA-B), is transmitted in a circulative nonpropagative manner by the whitefly Bemisia tabaci (Gennadius). Although it was first reported in Asia on tomato and other solanaceous crops such as eggplant, potato, and chilli pepper in the Mediterranean basin, this virus was mainly detected on cucurbits and only sporadically on tomato and on two wild solanaceous species, Datura stramonium L. and Solanum nigrum L. (Juárez et al. 2019). In 2018, separate surveys were carried out in protected cultivations of sweet pepper (Capsicum annuum L.) in two Italian regions: Lazio and Campania. The greenhouses were in areas with high density of B. tabaci and where ToLCNDV outbreaks occurred on cucurbits since 2016 (Panno et al. 2019). Some plants showing symptoms of yellowing and leaf curling were found in both regions, whereas fruit symptoms were neither observed nor reported by farmers. This disease syndrome, known as yellow leaf curl disease (YLCD), can be caused in pepper by several begomoviruses, as reported recently in a review listing the viruses causing YLCD in peppers in Thailand (Chiemsombat et al. 2018). Symptomatic leaves were collected during late summer 2018 from different pepper plants as well as from the neighboring zucchini cultivations, showing the typical symptomatology induced by ToLCNDV. Total DNA was extracted (DNeasy Plant Mini kit, Qiagen, Germany), and the presence of ToLCNDV was ascertained by PCR with the specific primers ToLCNDV-CP1 and ToLCNDV-CP2 (Panno et al. 2019; Parrella et al. 2018). ToLCNDV infection was further ascertained in three symptomatic leaf samples from Campania by using specific ToLCNDV ImmunoStrips (Agdia, Elkhart, IN). Successively, one symptomatic pepper sample from each greenhouse was selected and amplified by rolling circle amplification technique (RCA; Inoue-Nagata et al. 2004). The amplicons were cloned, and the DNA-A and DNA-B were full-length sequenced. The sequences were deposited in GenBank NCBI database (MK732932 DNA-A and MK732933 DNA-B, pepper sample from Campania; MK756106 DNA-A and MK756107 DNA-B, pepper sample from Lazio). The RCA analysis was performed also on a ToLCNDV-infected zucchini sample collected in the same area in Lazio region (MK756108 DNA-A and MK756109 DNA-B). The analysis of the ToLCNDV sequences showed a low level of genetic variability between the two pepper isolates from Lazio and Campania regions (rate of substitutions: 0.016 for DNA-A and 0.023 for DNA-B). A high genetic similarity was recorded between the zucchini isolate and both the pepper isolates from Campania (0.019 for DNA-A and 0.023 for DNA-B) and Lazio (0.003 for both DNA-A and B). The three characterized isolates showed a high sequence homology also with both the DNA-A (MH577751 from a melon isolate) and DNA-B (MH577673 from a zucchini isolate) of the ToLCNDV-ES genotype (Fortes et al. 2016), which differed in 15 and 13 nucleotide substitutions from pepper sample from Lazio, 29 and 51 substitutions from Campania sample, and 10 and 5 substitutions from zucchini sample. High homology was also identified compared with the other Spanish isolates collected since the first appearance of the virus (2014) and to the Tunisian (2015) and Moroccan (2018) isolates, confirming the hypothesis that the Mediterranean population of ToLCNDV is highly conserved (Juárez et al. 2019). To our knowledge, this is the first report of ToLCNDV infection on pepper in Europe and indicates that sweet pepper could also act as a reservoir of the virus for further spread to other solanaceous plants and cucurbits

In-Field LAMP Detection of Flavescence Dorée Phytoplasma in Crude Extracts of the Scaphoideus titanus Vector

One of the most destructive diseases affecting grapevine in Europe is caused by Flavescence Dorée phytoplasma (FDp), which belongs to the 16Sr-V group and is a European Union quarantine pathogen. Although many molecular techniques such as loop-mediated isothermal amplification (LAMP) are widely used for the rapid detection of FDp in infected grapevine plants, there is no developed isothermal amplification assay for FDp detection in the insect vectors that are fundamental for the spread of the disease. For this reason, a simple in-field real-time LAMP protocol was optimized and developed for the specific detection of FDp in the insect vector Scaphoideus titanus. The LAMP assay was optimized to work with crude insect extracts obtained by manually shaking a single insect in a buffer for 5 min. Such a simple, sensitive, specific, economic, and user-friendly LAMP assay allowed the detection of FDp in S. titanus in less than half an hour, directly in the field. The developed insect tissue preparation procedure, combined with the LAMP protocol, promptly revealed the presence of FDp in infected S. titanus directly in the vineyards, allowing for monitoring of the spread of the pathogen in the field and to apply timely strategies required for the mandatory control of this pathogen