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.

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.

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.

Tomato Brown Rugose Fruit Virus: Seed Transmission Rate and Efficacy of Different Seed Disinfection Treatments.

Tomato brown rugose fruit virus (ToBRFV) is a highly infectious virus, that is becoming a threat to tomato production worldwide. In this work we evaluated the localization of ToBRFV particles in tomato seeds, its seed transmission rate and efficacy of disinfection, and the effects of different thermal- and chemical-based treatments on ToBRFV-infected seeds' germination. Analyses demonstrated that ToBRFV was located in the seed coat, sometime in the endosperm, but never in the embryo; its transmission from infected seeds to plantlets occurs by micro-lesions during the germination. The ToBRFV seed transmission rate was 2.8% in cotyledons and 1.8% in the third true leaf. Regarding the different disinfection treatments, they returned 100% of germination at 14 days post-treatment (dpt), except for the treatment with 2% hydrochloric acid +1.5% sodium hypochlorite for 24 h, for which no seed germinated after 14 dpt. All treatments have the ability to inactivate ToBRFV, but in six out of seven treatments ToBRFV was still detectable by RT-qPCR. These results raise many questions about the correct way to carry out diagnosis at customs. To our knowledge, this is the first study on the effective localization of ToBRFV particles in seeds.