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.

Tomato is one of the most important crops worldwide, with a production of ≈190 million tons, but it is constantly threatened by several viral diseases. Tomato brown rugose fruit virus (ToBRFV), identified in 2014 on tomato plants and subsequently reported in many countries, represents one of the major threats to tomato crops, due to production losses, different transmission modes and its rapid spread. This work aimed to evaluate 37 local Sicilian tomato ecotypes against ToBRFV infection. After a preliminary screening by molecular analyses for tomato mosaic virus (ToMV) and pepino mosaic virus (PepMV), and ToBRFV detection, tomato plants were grown in a greenhouse for their morphological characterization and for evaluating resistance and tolerance to ToBRFV. Resistance and tolerance levels were estimated by mechanical inoculation with ToB SIC01/19 ToBRFV isolate in ten plants per ecotype and evaluating virus accumulation by RT-qPCR and visual observation of symptoms. All ecotypes were infected with ToBRFV, showing several symptoms with different disease severity. No tomato ecotype showed a high level of resistance, but two ecotypes, Pop27 and Pop35, showed very moderate symptoms and therefore a high tolerance. These Sicilian tomato ecotypes could be used in genetic breeding programs as parental ones to obtain cultivars tolerant to ToBRFV.

Olive trees (Olea europea L.) are constantly threatened by many viruses, such as the olive leaf yellowing-associated virus (OLYaV), that belong to the Olivavirus genus, family Closteroviridae. In this work, the OLYaV incidence in different regions of Italy and Spain, which represent the two most important European areas for olive production, was evaluated through the development of a real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for reliable and sensitive OLYaV detection. The specificity and accuracy of the developed real-time RT-LAMP assay were determined; the assay showed that potential cross-reactivity with other viruses belonging to the Closteroviridae family was excluded. The LAMP assay detected OLYaV with a higher sensitivity than conventional end-point RT-PCR, detecting a total of 1.34 × 10⁻² genome copies. A total of 80 and 120 plants of different olive cultivars from Spain (Comunitat Valenciana, Andalusia) and Italy (Sicily, Calabria, Apulia, Lazio, and Umbria) regions were tested, respectively. The percentage of infected plants was 46.25% and 30% for Spain and Italy, respectively, while the most susceptible cultivars were “Serrana Espadán” and “Villalonga” from Comunitat Valenciana and Andalusia regions (Spain) and “Ogliarola barese” from Apulia region (Italy). In addition, the survey demonstrated that the real-time RT-LAMP showed good sensitivity for OLYaV-positive sample detection, especially on asymptomatic olive trees. For this reason, the developed assay could be very suitable for phytopathological laboratories as a reliable and efficient method for a rapid and sensitive routine test on olive samples.