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.

Citrus viroid V (CVd-V), a member of the species Apscaviroid epsiloncitri (family Pospiviroidae), is a graft-transmitted pathogen that spreads through infected propagation material and has been reported in several countries. Recently, it has been detected for the first time in Italy. Although CVd-V is generally considered asymptomatic, foliar and stem symptoms have been observed in Etrog citron. In several citrus species, mixed infections of CVd-V with other viroids give synergistic effects that cause more severe symptoms. To evaluate its spread, a specific and sensitive RT-qPCR assay was developed. The assay specificity, sensitivity, and performance were compared with a conventional end-point RT-PCR, showing a higher sensitivity and no cross-reactivity with other citrus viroids. Furthermore, the assay was evaluated using two RNA extraction procedures: total RNA extraction with commercial kits and a rapid crude extract preparation using a simple extraction buffer. Moreover, RNA extracts of 111 samples, collected from commercial citrus orchards across the main Sicilian citrus-producing areas and from old varieties maintained in germplasm collection, were analyzed. The RT-qPCR revealed an overall CVd-V incidence of 8.1%. Notably, the crude extract preparation showed a sensitivity comparable with conventional total RNA extraction, with substantial savings in cost and processing time. This finding paves the way for using the developed assay with portable qPCR instruments directly in the field, as well as for routine surveillance analyses in citrus production systems.

Citrus mal secco is a destructive tracheomycotic disease caused by the mitosporic fungus Plenodomus tracheiphilus (Petri) Gruyter, Aveskamp, and Verkley, severely affecting cultivated citrus species, particularly lemon trees in the Mediterranean region. A novel diagnostic assay based on real-time loop-mediated isothermal amplification (LAMP) was developed for the rapid detection of P. tracheiphilus. A set of six LAMP primers was designed to specifically target a 204-nucleotide sequence within the ITS1-5.8S-ITS2 region of nuclear rRNA genes. The assay’s sensitivity, specificity, exclusivity, and inclusivity were evaluated on a panel of 21 isolates and compared with conventional PCR and real-time quantitative PCR. Results showed that the developed real-time LAMP assay could rapidly detect the pathogen genomic DNA within only 15 min, with no registered cross-reactivity with nontarget fungal isolates from citrus plants or Citrus species. Moreover, reliable amplification was achieved using minimally processed samples. Specifically, two rapid extraction methods have been tested for an in-field plant diagnosis of P. tracheiphilus directly from the leaf surface. Overall, the developed real-time LAMP assay provides a potential tool for a quick, simple, specific, and sensitive detection of P. tracheiphilus, allowing an early disease prediction and reducing the risk of epidemics.

Field surveys conducted in citrus groves in Sicily, southern Italy, revealed sweet orange trees of different cultivars (‘Moro’, ‘Navelina’, and ‘Newhall’) grafted on ‘Forner Alcaide 5’ and citrange ‘C35’ showing typical symptoms of bud union crease in the canopy and at the grafting point. In order to better understand the possible etiology of this disorder, symptomatic samples were tested for the most common citrus viruses and viroids. Moreover, the identity of the surveyed rootstocks was confirmed with molecular analyses. In addition, to investigate the possible presence of viral and viroid agents not detected by targeted molecular assays, all citrus samples were subjected to high-throughput sequencing (HTS) analysis. Results showed that all samples resulted negative to all the tested viruses and viroids. Only one sample tested positive to Hop stunt viroid. Results from high-throughput sequencing generated sequence reads corresponding to Citrus Blight-associated Pararetrovirus (CBaPRV), representing the predominant “pathogen”-related sequences detected in the HTS dataset. The found association does not prove the causative effect with the bud union crease but highlights an important result that needs further investigations.

Tomato brown rugose fruit virus (ToBRFV) is one of the most aggressive viral pathogens affecting commercial tomato cultivars, causing severe economic losses worldwide. Since plant viral diseases cannot be effectively controlled with pesticides, alternative strategies aimed at reducing disease severity and enhancing plant tolerance are urgently needed but scarcely explored. In this study, L-citrulline, an amino acid known to improve plant tolerance to abiotic stress, was evaluated for its potential to increase tomato tolerance to ToBRFV infection. Healthy tomato seedlings were grown in a greenhouse and treated weekly with foliar applications of citrulline for six weeks. One week after the first treatment, half of the plants were mechanically inoculated with ToBRFV. Disease progression was evaluated by assessing disease severity and virus accumulation in plants. Tomato morphological and physiological traits, including plant height, biomass, leaf area, color and number, and chlorophyll content, were also assessed. Citrulline treatment significantly reduced the disease severity index by 64.5% and improved growth and physiological performance in ToBRFV-infected plants, despite having no significant effect on viral accumulation. Treated infected plants also exhibited partial recovery from typical ToBRFV leaf symptoms. These findings provide the first evidence that L-citrulline application can enhance tolerance to ToBRFV in tomato plants by reducing disease severity and ameliorating plant performance. This study highlights the potential of L-citruline as a sustainable strategy for management of ToBRFV infection in tomato plants.