Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and because they are seed borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tomato mottle mosaic virus, has recently gained attention due to reports in different continents and its common features with tomato brown rugose fruit virus. In this study, a new real-time RT-PCR detection system was developed for tomato brown rugose fruit virus and tomato mottle mosaic virus on tomato leaves and seeds using TaqMan chemistry. This test was designed to detect tomato mottle mosaic virus by amplifying the movement protein gene in a duplex assay with the tomato brown rugose fruit virus target on the CP-3’NTR region, which was previously validated as a single assay. The performance of this test was evaluated, displaying analytical sensitivity 10⁻⁵–10⁻⁶-fold dilution for seeds and leaves, respectively, and good analytical specificity, repeatability, and reproducibility. Using the newly developed and validated test, tomato brown rugose fruit virus detection was 100% concordant with previously performed analyses on 106 official samples collected in 2021 from different continents.

In 2020, a test performance study (TPS) for the specific detection of tomato brown rugose fruit virus (ToBRFV) was organized in the frame of the H2020 Valitest project. Since no validated tests were available, all the protocols reported in the literature were at first screened, performing preliminary studies in accordance with the EPPO standard PM 7/98 (4). Five molecular tests, two conventional RT-PCR and three real-time RT-PCR were found to be suitable and were included in the TPS. Thirty-four laboratories from 18 countries worldwide took part in TPS, receiving a panel of 22 blind samples. The panel consisted of sap belonging to symptomatic or asymptomatic leaves of Solanum lycopersicum and Capsicum annuum. The results returned by each laboratory were analyzed and diagnostic parameters were assessed for each test: reproducibility, repeatability, analytical sensitivity, diagnostic sensitivity and diagnostic specificity. All the evaluated tests resulted in being reliable in detecting ToBRFV and were included in an EPPO Standard PM 7/146—Diagnostics.

Among the cucurbit-infecting viruses, watermelon mosaic virus (WMV) and zucchini yellow mosaic virus (ZYMV) (Potyvirus: Potyviridae) are responsible for severe symptoms on cucumber, melon, watermelon, and zucchini cultivations worldwide. In this study, reverse transcription real-time PCR (real-time RT-PCR) and droplet-digital PCR (RT-ddPCR) assays targeting the coat protein (CP) genes of WMV and ZYMV were developed and validated according to the international standards of plant pest diagnosis (EPPO PM 7/98 (5)). First, the diagnostic performance of WMV-CP and ZYMV-CP real-time RT-PCRs was evaluated, and the assays displayed an analytical sensitivity of 10⁻⁵ and 10⁻³, respectively. The tests also showed an optimal repeatability, reproducibility and analytical specificity, and were reliable for the virus detection in naturally infected samples and across a wide range of cucurbit hosts. Based on these results, the real-time RT-PCR reactions were adapted to set up RT-ddPCR assays. These were the first RT-ddPCR assays aiming at the detection and quantification of WMV and ZYMV and showed a high sensitivity, being able to detect until 9 and 8 copies/µL of WMV or ZYMV, respectively. The RT-ddPCRs allowed the direct estimation of the virus concentrations and opened to a broad range of applications in disease management, such as the evaluation of partial resistance in breeding processes, identification of antagonistic/synergistic events, and studies on the implementation of natural compounds in the integrated management strategies.

The use of organic substances in integrated pest management can contribute to human- and environment-safe crop production. In the present work, a combination of organic biostimulants (Fullcrhum Alert and BioVeg 500) and an inorganic corroborant (Clinogold, zeolite) was tested for the effects on the plant response to the quarantine pest tomato leaf curl New Delhi virus (ToLCNDV). Biostimulants were applied to healthy and infected greenhouse-grown zucchini plants, and the vegetative parameters and viral titer were evaluated. Although no antiviral effects were observed in terms of both virus replication and symptom expression, these biostimulants were shown to influence plant fitness. A significant increase in biomass and in leaf, flower, and fruit production was induced in both healthy and infected plants. Biostimulants also enhanced the production of metabolites commonly involved in plant response to virus infection, such as carbohydrates, phenylpropanoids and free amino acids. These results encourage new field trials to evaluate the actual productivity of infected plants after treatments and the possible application of organic biostimulants in agriculture.

Lab-on-Chip (LoC) devices for performing real-time PCR are advantageous compared to standard equipment since these systems allow to conduct in-field quick analysis. The development of LoCs, where the components for performing the nucleic acid amplification are all integrated, can be an issue. In this work, we present a LoC-PCR device where thermalization, temperature control and detection elements are all integrated on a single glass substrate named System-on-Glass (SoG) obtained using metal thin-film deposition. By using a microwell plate optically coupled with the SoG, real-time reverse transcriptase PCR of RNA extracted from both a plant and human virus has been carried out in the developed LoC-PCR device. The limit of detection and time of analysis for the detection of the two viruses by using the LoC-PCR were compared with those achieved by standard equipment. The results showed that the two systems can detect the same concentration of RNA; however, the LoC-PCR performs the analysis in half of the time compared to the standard thermocycler, with the advantage of the portability, leading to a point-of-care device for several diagnostic applications.

Italy is the second largest producer of hazelnuts in the world after Turkey. The Viterbo province (Latium region) is one out of the four main productive districts, accounting for approximately 30% of the total Italian production. Several pathogens can severely affect hazelnut trees (Corylus avellana), mainly fungi and bacteria that are commonly spread in all the growing areas. However, other pathogens such as phytoplasmas, are known to infect this crop worldwide. Among these, ‘Candidatus Phytoplasma fragariae’ (16SrXII-E) has been recently associated with the decline and death of hazelnut trees in UK where it was identified for the first time on this plant species (Hodgetts et al., 2015), and in Slovenia (Mehle et al., 2018; Mehle et al., 2019). In July 2020, a survey was carried out in two orchards located in the Viterbo province, where symptoms resembling those induced by phytoplasmas and consisting in yellowing and rolling of leaves and tree decline (Supplementary figure 1) were recorded by growers. To ascertain the possible presence of phytoplasma infections, leaf samples were collected from five symptomatic plants: one plant from orchard 1, cv. Tonda Gentile Romana and four plants from orchard 2, cv. Tonda di Giffoni. Samples from four non-symptomatic plants (two per each orchard) were also collected and used as negative controls in the detection assays. DNA was extracted from 0.6 g of leaf ribs homogenised in CTAB buffer according to Marzachì et al. (1999). DNA extracts were amplified by direct and nested PCR employing the universal primer pairs P1/P7 and R16F2n/R2 according to the EPPO Standard PM7/133(1) - Appendix 2 (EPPO, 2018). Nested amplicons of the expected size (1,250 bp) were obtained from the five symptomatic samples whereas no bands were observed for the symptomless ones. The R16F2n/R2 fragments were submitted to both Sanger sequencing and RFLP analysis for the phytoplasma identification. Nucleotide BLAST analysis (NCBI) of the obtained sequences showed an identity percentage ranging from 99.65% and 99.91% with numerous strains of ‘Ca. Phytoplasma fragariae’ (16SrXII-E). The RFLP analysis was performed with the MseI restriction enzyme, and the obtained profiles were compared in the Codon Code Aligner software to those virtually generated from reference sequences of phytoplasmas belonging to the 16SrXII subgroups identified so far (Kazeem et al., 2021). All the five samples showed a common RFLP profile which was ascribable to that virtually generated from the 16SrXII-E reference sequence DQ086423 (Valiunas et al., 2006) (Supplementary figure 2). To our knowledge, this is the first finding of ‘Ca. P. fragariae' on hazelnut in Italy since the previous records on woody plant hosts regarded Cornus sanguinea and Sambucus nigra (Filippin et al. 2008). Further surveys are ongoing to assess the spread of ‘Ca. P. fragariae' in the Italian hazelnut orchards and to identify the putative insect vectors. The nucleotide sequences obtained in this study were deposited in GenBank NCBI database (accession numbers: MW767127, MW767128, MW767129, MW767130, MW767131).