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

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

Development of an In-Field Real-Time LAMP Assay for Rapid Detection of Tomato Leaf Curl New Delhi Virus

Tomato leaf curl New Delhi virus (ToLCNDV) represents a threat to economically important horticultural crops. A real-time loop-mediated isothermal amplification (LAMP) assay for in-field ToLCNDV detection was developed, coupled to a rapid sample preparation method, and tested both in field and laboratory conditions on zucchini squash, tomato, and pepper samples. A set of six LAMP primers was designed for specific ToCLNDV detection, targeting a 218-nucleotide sequence within the AV1 gene. The sensitivity, specificity and accuracy of the real-time LAMP assay and comparison with canonical PCR were evaluated. The real-time LAMP assay developed was about one-thousand times more sensitive than the conventional PCR method, detecting a total of 4.41 × 102 genome copies as minimum target; no cross-reactivity was detected with the other geminiviruses used as the outgroup. The rapid sample preparation method allows for a reliable detection with a low reaction delay (≈2–3 min) compared to canonical DNA extraction, providing results in less than 45 min. Lastly, an increase in ToLCNDV-positive sample detection was observed compared to PCR, in particular for asymptomatic plants (85% and 71.6%, respectively). The real-time LAMP assay developed is a rapid, simple, specific, and sensitive technique for ToLCNDV detection, and it can be adopted as a routine test, for both in-field and laboratory conditions

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

Mid-term results of complex distal humeral fractures.

The aim of this study was to assess outcomes following open reduction and internal fixation in complex fractures of the distal humerus. Between 2000 and 2006, 34 patients were operated for complex fractures of the distal humerus. Bone fixation was obtained with a reverse Y-shaped reconstruction plate in 13 cases and with double plating in 21 cases. At final follow-up, all the patients were assessed with the Mayo Elbow Performance Score. Satisfactory results were observed in 71% of the cases despite a high rate of complications. Age over 65 years is correlated with increased risk for an inferior postoperative result. Complex distal humeral fractures are difficult to treat and are associated with a high incidence of complications. It is therefore mandatory to obtain good anatomical reduction and a stable fixation of lateral and medial columns of the distal humerus. The results observed in older patients suggest that an alternative treatment for these patients may be joint replacement