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Tomato (Solanum lycopersicum L.), family Solanaceae, has become in the past fifty years one of the most important and extensively grown horticultural crops in the Mediterranean region and throughout the world. In 2019, more than 180 million tonnes of tomato have been produced worldwide, out of which around 42 million tonnes in Mediterranean countries. Due to its genetic properties, tomato is afflicted by numerous plant diseases induced by fungal, bacterial, phytoplasma, virus, and viroid pathogens. Not only is its genetic inheritance of great importance to the management of the numerous tomato pathogens, but equally as important are also the present climate changes, the recently revised phytopathological control measures, and the globalization of the seed industry. Thus, the recognition of symptoms and the knowledge of the distribution and spread of the disease and of the methods for early detection of the pathogens are the major prerequisites for a successful management of the disease. In this review, we will describe the main tomato pathogens in the Mediterranean area that impact mostly the tomato yield and provide the current and perspective measures necessary for their successful management.

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed.

The increasing demand for hazelnut kernels is favoring an upsurge in hazelnut cultivation worldwide, but ongoing climate change threatens this crop, affecting yield decreases and subject to uncontrolled pathogen and parasite attacks. Technical advances in precision agriculture are expected to support farmers to more efficiently control the physio-pathological status of crops. Here, we report a straightforward approach to monitoring hazelnut trees in an open field, using aerial multispectral pictures taken by drones. A dataset of 4112 images, each having 2Mpixel resolution per tree and covering RGB, Red Edge, and near-infrared frequencies, was obtained from 185 hazelnut trees located in two different orchards of the Piedmont region (northern Italy). To increase accuracy, and especially to reduce false negatives, the image of each tree was divided into nine quadrants. For each quadrant, nine different vegetation indices (VIs) were computed, and in parallel, each tree quadrant was tagged as “healthy/unhealthy” by visual inspection. Three supervised binary classification algorithms were used to build models capable of predicting the status of the tree quadrant, using the VIs as predictors. Out of the nine VIs considered, only five (GNDVI, GCI, NDREI, NRI, and GI) were good predictors, while NDVI SAVI, RECI, and TCARI were not. Using them, a model accuracy of about 65%, with 13% false negatives was reached in a way that was rather independent of the algorithms, demonstrating that some VIs allow inferring the physio-pathological condition of these trees. These achievements support the use of drone-captured images for performing a rapid, non-destructive physiological characterization of hazelnut trees. This approach offers a sustainable strategy for supporting farmers in their decision-making process during agricultural practices.

Erysiphe corylacearum has recently been reported in northern Italy (Piedmont) and other European countries as the causal agent of a new emerging powdery mildew on hazelnut. This disease is much more dangerous than the common hazelnut powdery mildew caused by Phyllactinia guttata as it significantly reduces yield and quality of hazelnuts. This study aimed to perform morphological and molecular characterization of the fungal isolates from powdery mildew-infected plants in the Piedmont Italian region. Additionally, genetic diversity studies and pathogenicity tests were conducted. Thirty-six fungal isolates originating from symptomatic hazelnut plants exhibiting specific powdery mildew symptoms on the superior leaf side were identified morphologically as E . corylacearum . Single- and multilocus sequence typing of five loci (ITS, rpb2 , CaM , GAPDH and GS ) assigned all isolates as E . corylacearum . Multilocus and GAPDH phylogenetic studies resulted in the most efficient characterization of E . corylacearum . Studied fungal isolates were able to cause new emerging powdery mildew disease by fulfilling Koch’s postulates. The emergence of powdery mildew disease in Italy revealed the E . corylacearum subgrouping, population expansion, and high nucleotide similarity with other recently identified E . corylacearum hazelnut isolates. To contain this harmful disease and inhibit the fungus spread into new geographical zones, it will be necessary to implement more rigorous monitoring in neighboring hazelnut plantations near infected hazelnuts, use sustainable fungicides and search for new biocontrol agents.

Tomato spotted wilt virus (TSWV) is a devastating plant pathogen, causing huge crop losses worldwide. Unfortunately, due to its wide host range and emergence of resistance breaking strains, its management is challenging. Up to now, resistance to TSWV infection based on RNA interference (RNAi) has been achieved only in transgenic plants expressing parts of the viral genome or artificial microRNAs targeting it. Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants, namely RNAi-based vaccination, represents an attractive and promising alternative, already shown to be effective against different positive-sense RNA viruses and viroids. In the present study, the protection efficacy of exogenous application of dsRNAs targeting the nucleocapsid ( N ) or the movement protein ( NSm ) coding genes of the negative-sense RNA virus TSWV was evaluated in Nicotiana benthamiana as model plant and in tomato as economically important crop. Most of the plants treated with N -targeting dsRNAs, but not with NSm -targeting dsRNAs, remained asymptomatic until 40 ( N. benthamiana ) and 63 (tomato) dpi, while the remaining ones showed a significant delay in systemic symptoms appearance. The different efficacy of N - and NSm -targeting dsRNAs in protecting plants is discussed in the light of their processing, mobility and biological role. These results indicate that the RNAi-based vaccination is effective also against negative-sense RNA viruses but emphasize that the choice of the target viral sequence in designing RNAi-based vaccines is crucial for its success.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 37,000 people in Italy and has caused widespread socioeconomic disruption. Urgent measures are needed to contain and control the virus, particularly diagnostic kits for detection and surveillance, therapeutics to reduce mortality among the severely affected, and vaccines to protect the remaining population. Here we discuss the potential role of plant molecular farming in the rapid and scalable supply of protein antigens as reagents and vaccine candidates, antibodies for virus detection and passive immunotherapy, other therapeutic proteins, and virus-like particles as novel vaccine platforms. We calculate the amount of infrastructure and production capacity needed to deal with predictable subsequent waves of COVID-19 in Italy by pooling expertise in plant molecular farming, epidemiology and the Italian health system. We calculate the investment required in molecular farming infrastructure that would enable us to capitalize on this technology, and provide a roadmap for the development of diagnostic reagents and biopharmaceuticals using molecular farming in plants to complement production methods based on the cultivation of microbes and mammalian cells.

The sanitary status of grapevines has not yet been considered sufficiently in vineyards throughout Bosnia and Herzegovina (BiH). An extensive survey of five major grapevine viruses in the country was carried out in 2019. A total of 630 samples from the two dominant autochthonous cultivars, named Žilavka and Blatina, were tested by DAS-ELISA for the presence of grapevine leafroll-associated viruses (GLRaV-1 and 3), grapevine fleck virus (GFkV), grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV). Eighty-eight % of the samples were positive for at least one virus, and all five viruses were detected, thought with different incidence, i.e. GLRaV-3 (84%), GFLV (43%), GLRaV-1 (14%), GFkV (10%) and ArMV (0.2%). The majority of infected plants (about 75%) were asymptomatic. Specific virus symptoms were observed in the remaining infected plants, together with the reported GLRaV vectors, Planococcus ficus and Parthenolecanium corni , while nematodes of the Xiphinema genus were not found in the GFLV- or ArMV-infected vineyards. The GLRaV-3 CP phylogenetic analyses showed 75–100% nucleotide identity between the BiH and reference isolates, and the BiH isolates clustered into the major group. The dNS/dS ratio indicated a negative selection of the virus population, and the lack of geographical structuring within the population was observed. In addition, putative GLRaV-3 recombinants with breakpoints in the 5’ of the CP gene were detected, while no recombinant strains were identified for the other four viruses. The obtained results indicate a deteriorated sanitary status of the cultivated grapevines, the prevalence and intraspecies genetic diversity of GLRaV-3 throughout the country. The establishment of certified grapevine material and adequate virus vector control is therefore of primary importance to prevent further spread of these viruses. This study presents the results of the first molecular characterisation of grapevine viruses in Bosnia and Herzegovina.

Powdery mildew (PM) is one of the most important diseases of greenhouse and field-grown tomatoes. Viruses can intervene beneficially on plant performance in coping with biotic and abiotic stresses. Tomato yellow leaf curl Sardinia virus (TYLCSV) has been reported recently to induce tolerance against drought stress in tomato, and its C4 protein acts as the main causal factor of tolerance. However, its role in response to biotic stresses is still unknown. In this study, transgenic tomato plants carrying the TYLCSV C4 protein were exposed to biotic stress following the inoculation with Oidium neolycopersici , the causal agent of tomato PM. Phytopathological, anatomic, molecular, and physiological parameters were evaluated in this plant pathosystem. Heterologous TYLCSV C4 expression increased the tolerance of transgenic tomato plants to PM, not only reducing symptom occurrence, but also counteracting conidia adhesion and secondary hyphae elongation. Pathogenesis-related gene expression and salicylic acid production were found to be higher in tomato transgenic plants able to cope with PM compared to infected wild-type tomato plants. Our study contributes to unraveling the mechanism leading to PM tolerance in TYLCSV C4-expressing tomato plants. In a larger context, the findings of TYLCSV C4 as a novel PM defense inducer could have important implications in deepening the mechanisms regulating the management of this kind of protein to both biotic and abiotic stresses.

Grapevine is one of the most cultivated fruit plant among economically relevant species in the world. It is vegetatively propagated and can be attacked by more than 80 viruses with possible detrimental effects on crop yield and wine quality. Preventive measures relying on extensive and robust diagnosis are fundamental to guarantee the use of virus-free grapevine plants and to manage its diseases. New phenotyping techniques for non-invasive identification of biochemical changes occurring during virus infection can be used for rapid diagnostic purposes. Here, we have investigated the potential of Raman spectroscopy (RS) to identify the presence of two different viruses, grapevine fan leaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in Vitis vinifera cv. Chardonnay. We showed that RS can discriminate healthy plants from those infected by each of the two viruses, even in the absence of visible symptoms, with accuracy up to 100% and 80% for GFLV and GRSPaV, respectively. Chemometric analyses of the Raman spectra followed by chemical measurements showed that RS could probe a decrease in the carotenoid content in infected leaves, more profoundly altered by GFLV infection. Transcriptional analysis of genes involved in the carotenoid pathway confirmed that this biosynthetic process is altered during infection. These results indicate that RS is a cutting-edge alternative for a real-time dynamic monitoring of pathogens in grapevine plants and can be useful for studying the metabolic changes ensuing from plant stresses.

The propensity of viruses to acquire genetic material from relatives and possibly from infected hosts makes them excellent candidates as vectors for horizontal gene transfer. However, virus-mediated acquisition of host genetic material, as deduced from historical events, appears to be rare. Here, we report spontaneous and surprisingly efficient generation of hybrid virus/host DNA molecules in the form of minicircles during infection of Beta vulgaris by Beet curly top Iran virus (BCTIV), a single-stranded DNA virus. The hybrid minicircles replicate, become encapsidated into viral particles, and spread systemically throughout infected plants in parallel with the viral infection. Importantly, when co-infected with BCTIV, B. vulgaris DNA captured in minicircles replicates and is transcribed in other plant species that are sensitive to BCTIV infection. Thus, we have likely documented in real time the initial steps of a possible path of virus-mediated horizontal transfer of chromosomal DNA between plant species. Viruses are potential vectors for horizontal gene transfer. Here, studying viral infection of sugar beet plants, the authors report the generation of virus-host circular DNA hybrids and provide a picture of the initial steps in virus-mediated horizontal transfer of chromosomal DNA between plant species.