Further Molecular Diagnosis Determines Lack of Evidence for Real Seed Transmission of Tomato Leaf Curl New Delhi Virus in Cucurbits.

Begomoviruses (family Geminiviridae) cause serious diseases in many crop families. Since 2013, the Spanish isolate of tomato leaf curl New Delhi virus (ToLCNDV) has been a limiting factor for cucurbits production in the Mediterranean basin, forcing farmers to adapt new management and control techniques. Although it is well-known that begomoviruses are naturally transmitted by the whitefly Bemisia tabaci, the capacity of these viruses to be vertically transmitted through seeds remains controversial. Clarifying the potential ToLCNDV seed transmission is essential to understand the epidemiology of this threating-for-cucurbits virus and to design appropriate control strategies. We assessed ToLCNDV distribution in the leaves, flowers and seeds of the infected plants of susceptible Cucumis melo accessions and toleration to the infected genotypes of Cucurbita moschata by conventional and quantitative PCR. We analyzed whether the viral particle was transmitted to offspring. We also evaluated ToLCNDV presence in commercial seeds of cucurbits (zucchini (Cucurbita pepo), melon (C. melo), cucumber (Cucumis sativus) and watermelon (Citrullus lanatus)) and in their progenies. As the assayed seedlings remained symptomless, we increased the reliability and accuracy of detection in these samples by searching for replicative forms of ToLCNDV by combining Southern blot hybridization and rolling-circle amplification (RCA). However, integral genomic DNA was not identified in the plants of offspring. Although the seedborne nature of ToLCNDV was confirmed, our results do not support the transmission of this virus from contaminated seeds to progeny.

Detection and absolute quantitation of watermelon mosaic virus by real-time RT-PCR with a TaqMan probe.

Watermelon mosaic virus (WMV) causes serious damage to several crops worldwide, mainly cucurbits. Disease control is based on preventing spread and search for natural resistances for plant breeding, which requires tools for sensitive detection and precise quantitation. We developed a procedure based on reverse transcription followed by real-time quantitative polymerase chain reaction (RT-qPCR) with a primer pair and a TaqMan® probe specific for WMV. The primers and probe were designed from conserved sequence stretches to target a wide range of WMV isolates. A standard curve performed with transcripts enabled estimation of WMV RNA copies per ng of total RNA, with a wide dynamic range and sensitivity (104 to 1011). This RT-qPCR was assayed with field samples from different cucurbits and used to evaluate the temporal accumulation in pumpkin plants.

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.