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A survey on the incidence of onion yellow dwarf virus (OYDV) and iris yellow spot virus (IYSV) was carried out over three production cycles of onion ‘Rossa di Tropea’ in Calabria, Italy. OYDV was found to be the prevalent virus. ‘Rossa di Tropea’ seedbeds adjacent to OYDV-infected green onion field had seedlings with 1.76% infection rate determining 36.2% and 98.67% infected plants in the bulbs and in the subsequent seed harvesting times, respectively. When seedbeds were at least one km away from other onion crops seedlings and bulb cultivation had the infection rate close to zero. OYDV was detected in whole plants except the roots and outer desiccated bulb skins. Seed transmission was not detected in ‘Rossa di Tropea’. Early OYDV infection significantly reduced the number and weight of seeds/inflorescence compared to late season infection, while the weight of 100 seeds was not different in the two early and late OYDV infected plants. IYSV was never found in seedbeds. It was always detected first in seed crops (April) than in bulb crops (June), and the final infection rate was higher in seed (2.67%–3.33%) than in bulb crops (0%–0.87%), suggesting there was an internal source of viral inoculum in the field. IYSV was detected in 3/123 apex bulbs randomly collected from stored bulbs and in 12/12 apex fresh bulbs collected at harvest time from infected plants, suggesting the role of bulbs as IYSV inoculum source. On the contrary, randomly collected bulbs (N = 109) from warehouse and bulbs of infected plants (N = 22), transplanted after storage, did not result in IYSV-infected plants.

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.

The genome of a new carlavirus isolate from asymptomatic wild Capparis spinosa L. plants in Sicily was sequenced via high-throughput sequencing (HTS) and 5'/3' RACE experiments. The complete genomic sequence was found to be 8,280 nt in length, excluding the poly(A) tail, and contained five putative open reading frames (ORFs). Molecular characterization revealed a close relationship to caper latent virus (CapLV), with 87% and 90% nucleotide sequence identity to available partial sequences of the ORFs encoding the replicase and coat protein of that virus. According to the molecular criteria for species demarcation, which is based on the ORF-1- and ORF-5-encoded proteins, the virus characterized in this study could be considered a variant of CapLV, and we have thus designated it as CapLV-W.

In the last decades, the evolution of molecular diagnosis methods has generated different advanced tools, like loop-mediated isothermal amplification (LAMP). Currently, it is a well-established technique, applied in different fields, such as the medicine, agriculture, and food industries, owing to its simplicity, specificity, rapidity, and low-cost efforts. LAMP is a nucleic acid amplification under isothermal conditions, which is highly compatible with point-of-care (POC) analysis and has the potential to improve the diagnosis in plant protection. The great advantages of LAMP have led to several upgrades in order to implement the technique. In this review, the authors provide an overview reporting in detail the different LAMP steps, focusing on designing and main characteristics of the primer set, different methods of result visualization, evolution and different application fields, reporting in detail LAMP application in plant virology, and the main advantages of the use of this technique.

Background Plant viral infections induce changes in metabolic components in the host plant, with potential effects on compositional, organoleptic and storability features of agricultural products. Identification of modulated metabolites may provide clues concerning pathways implementing responses in plant-pathogen interactions. A time course study of metabolic fingerprinting of onion yellow dwarf virus (OYDV)-infected versus healthy 'Rossa di Tropea' onion bulbs was performed using proton high-resolution magic angle spinning nuclear magnetic resonance (1 H HR-MAS NMR) and ultra-performance liquid chromatography (UPLC), providing an overview of the metabolic state of the bulb in response to OYDV infection during storage. Results Metabolites accumulated/depleted upon infection were identified, belonging to flavonoid, saccharide, amino acid and organic acid classes. A decrease in quercetin glucosides content and antioxidant activity was observed in infected bulbs; some amino acids (Arg, Asn, Phe, Val) accumulated, while others were depleted (Leu); for some metabolites, a bimodal time-course was observed during storage (Glc, Lys). Virus interference on metabolic pathways, and the effects of the metabolic shift on edible product storability, organoleptic and nutritional quality were discussed. Conclusions OYDV infection induces a metabolic shift in 'Rossa di Tropea' onion during bulb storage, involving several pathways and affecting storability and organoleptic and nutritional quality of bulbs at marketable stage. © 2020 Society of Chemical Industry.

As part of a plant-pathogen interaction study between Onion yellow dwarf virus (OYDV) and onion cultivar Rossa di Tropea, a ΔΔCt-based relative quantification of OYDV was investigated to relate OYDV titer to accumulation of secondary metabolites in onion bulbs. An appropriate reference gene (RG) was required to achieve data normalization. Since no single internal control gene is universally used as an RG, multiple stably expressed reference genes were investigated. In particular, elongation factor (Elf), protein phosphatase 2A (PP2A), helicase (Hel-1), 5.8S rRNA, ubiquitin (UBQ) and s-Actin (s-Act) were compared one to another in both leaf and bulb tissues, at different growth and development stages, and with different infection status (healthy/OYDV-infected). Preliminary gene screening was carried out using an RT-qPCR assay (SYBR chemical), assessing both Ct values and melting curves. Expression stability of the reference genes in the sample sets was independently determined by three different software packages: geNorm, NormFinder and Bestkeeper. In contrast to Elf, PP2A, Hel-1 and s-Act, 5.8S rRNA and UBQ proved to be the most stable RGs. An OYDV specific RT-qPCR TaqMan® assay was also developed and validated for relative quantification of OYDV titer. The assay was shown to be specific and sensitive, able to identify virus presence up to 10−6 dilution, representing a rapid and sensitive diagnostic tool for OYDV detection for application in field surveys. Finally, a ∆∆Ct method was developed, to be applied in future studies describing the molecular interaction between OYDV and onion cv. ‘Rossa di Tropea’. This approach was used to provide relative quantification of OYDV titer in samples obtained from different experimental trials.

Allium sativum L. grown in Latium (central Italy) was shown to be infected by several common garlic viruses of the genera Potyvirus, Carlavirus and Allexivirus. Multiple viral infection with members of all three genera was found in most samples. In particular, the allexiviruses Garlic virus B (GarV-B), Garlic virus D (GarV-D) and Garlic virus X (GarV-X) were detected by reverse transcription polymerase chain reaction (RT-PCR) assay using both degenerate genus-specific and species-specific primer sets. Moreover, molecular phylogenetic relationships among the Italian isolates of GarV-B, GarV-D and GarV-X and isolates of the same and other allexivirus species were analyzed in a region including partial coat protein and nucleic acid binding protein (CP-NABP) genes. The present study reports the molecular characterization of one isolate of GarV-D and one isolate of GarV-X distantly related to those previously identified in Italy. Moreover, this is the first report of GarV-B in Italy.

Plant viral infections induce changes in metabolic components in the host plant, with potential effects on compositional, organoleptic and storability features of agricultural products. Identification of modulated metabolites may provide clues concerning pathways implementing responses in plant-pathogen interactions. A time course study of metabolic fingerprinting of onion yellow dwarf virus (OYDV)-infected versus healthy 'Rossa di Tropea' onion bulbs was performed using proton high-resolution magic angle spinning nuclear magnetic resonance (Metabolites accumulated/depleted upon infection were identified, belonging to flavonoid, saccharide, amino acid and organic acid classes. A decrease in quercetin glucosides content and antioxidant activity was observed in infected bulbs; some amino acids (Arg, Asn, Phe, Val) accumulated, while others were depleted (Leu); for some metabolites, a bimodal time-course was observed during storage (Glc, Lys). Virus interference on metabolic pathways, and the effects of the metabolic shift on edible product storability, organoleptic and nutritional quality were discussed.OYDV infection induces a metabolic shift in 'Rossa di Tropea' onion during bulb storage, involving several pathways and affecting storability and organoleptic and nutritional quality of bulbs at marketable stage. © 2020 Society of Chemical Industry.

Onion yellow dwarf virus (OYDV) is one of the most important viral pathogens of onion. In particular, on 'Rossa di Tropea' onion, granted with Protected Geographical Indication (PGI) trademarks, this pathogen represents the most limiting biotic stress in terms of spread, severity of symptoms and damage, and its detection is necessary to preserve high quality standards and avoid yield losses. A reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay was developed for detection of OYDV. The specificity, sensitivity, repeatability and reproducibility of the assay were validated according to EPPO standard PM7/98 (2). Diagnostic specificity, diagnostic sensitivity and diagnostic accuracy were determined in both leaf and bulb tissues. To enhance the feasibility of a LAMP-based method for field diagnosis, several nucleic acid extraction methods were compared to simplify sample preparation. The results showed the reliability of the method for OYDV detection, with a limit of detection (LOD) comparable to real time reverse transcription polymerase chain reaction (RT-qPCR). The ease of sample preparation, and the more than acceptable LOD, indicated that the RT-LAMP assay could be used in plant pathology laboratories with limited facilities and resources, as well as directly in the field. This work was carried out in the frame of "SI.ORTO" project.

Microarrays are an emerging methodology in viroid detection and identification. The principle is the hybridization of fluorescently labeled sequences (targets) to complementary probe sequences immobilized on a solid surface. The process includes probe development, array fabrication, target preparation, hybridization, washing, scanning, and interpretation of results. Probe design and labeling of the target are the key steps in detection and identification of viroids by microarrays. The main advantage of this methodology is the opportunity to detect many viroids simultaneously. Microarrays may be viroid-specific, or may be used for broad-spectrum screening with the use of genus-level probes, the latter being suitable for detecting newly emerging viroids, or established viroids occurring in new host or geographical regions.

Among the 15 virus species detected in olive trees, Olive leaf yellowing associated virus (OLYaV) is found with high incidence and frequency in Southern Italy in particular. Effects of OLYaV on virgin olive oil yield and quality of Leccino and Ottobratica cultivars in the Calabria region (Southern Italy) are analyzed. Oil yield, free acidity, number of peroxides, spectrophotometric indices, total content of chlorophylls and carotenoids, total phenol content, composition of the fatty acids, total tocopherols, and total sterols content are determined on oil obtained from olive fruits collected in healthy/virus‐free and OYLaV‐infected trees. Almost all analyzed oil parameters are not statistically different with some exceptions. Oils derived from ‘Ottobratica’ OLYaV‐infected plants have free acidity significantly lower than oil from healthy plants. K₂₃₂ of oil from virus‐free ‘Leccino’ trees is significantly lower than oil from infected trees. Even though some quality parameter differences between healthy and OLYaV‐infected oils are found, it is important to highlight that all oils can be considered in extra virgin olive oil category, within the UE maximum limit acceptance range. Results suggest a no negative interference by OYLaV in oil yield and quality, except for K₂₃₂ values, whereas surprisingly suggest a positive effect of virus infection on free acidity parameter. Practical Applications: Based on the evidence that OYLaV does not interfere negatively in oil yield and quality parameters, it seems appropriate that the European Union Council Directives (2014/96/EU, 2014/97/EU, 2014/98/EU) introduced in the last olive certification scheme only ArMV, CLRV, and SLRSV. The application of compulsory EU directive, in Italy as in other countries, will improve the facilities of olive plants commercialization, guaranteeing enough their sanitary status. The findings here reported support the suggestion that the Italian Ministry of Agriculture could be less restrictive in voluntary Italian regulation (D.M. 20/11/2006). Olive leaf yellowing associated virus (OLYaV) is found with high incidence and frequency especially in Southern Italy. Effects of OLYaV on virgin olive oil yield and quality of Leccino and Ottobratica cultivars in the Calabria region (Southern Italy) are analyzed, including free acidity, number of peroxides, spectrophotometric indexes, total content of chlorophylls, carotenoids and phenol, composition of the fatty acids, total tocopherols and total sterols.

A DNA microarray chip was developed for screening 10 major economically important tomato viruses from infected plants using "Combimatrix" platform 40-mer oligonucleotide probes. A total of 279 oligonucleotide virus probes were specific for simultaneous multiple detection, identification, differentiation and/or genotyping of each of the following tomato RNA viruses and/or strains and a virus satellite: Cucumber mosaic virus, Cucumber mosaic virus satellite RNA, Tomatoinfectiouschlorosisvirus, Tomato chlorosisvirus, Tomato spotted wilt virus, Pepino mosaic virus, Potato virus Y, Tobacco mosaic virus and Tomato mosaic virus. This selection included both positive and negative single-stranded RNA viruses. The single-stranded DNA viruses, Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus were detected but were not differentiated using probes designed from their coat protein genes. A sectored oligonucleotide microarray chip containing four sets of 2000 features (4 x 2 K) was designed. In this way, four samples were tested simultaneously in a hybridization event and 16 samples were analyzed by re-using the chip four times. The hybrids had low background signals. Many of the 40-mer oligonucleotide probes were specific for the detection and identification of each RNA viral species, RNA viral satellite and genotyping strains of Cucumber mosaic virus, Pepino mosaic virus and Potato virus Y. Universal probes were developed for strains of the last three viruses and also for the genus Tobamovirus which includes both Tobacco mosaicvirus and Tomato mosaic virus.

Since 2006, winter melon plants (Cucumis melo L. var inodorus) showing symptoms of pin-point yellow spots were noticed in Sicily (Italy). Leaf samples were tested by enzyme-linked immunosorbent assay to the most important viruses-infecting cucurbits. Zucchini yellow fleck virus (ZYFV, genus Potyvirus) was the only virus detected. Surveys in 2007 and 2008 revealed an increasing number of sites in Sicily with ZYFV-infected winter melon plants. To confirm the identity of the virus as ZYFV, two isolates from different locations were sequenced and shown to be approximately 85% identical to the published sequences of isolates previously identified in Italy and France. This is the first report of ZYFV occurring on melon in Italy.

During 2014 and 2015, commercial crops and germplasm collections of chilli ( Capsicum spp.) in Italy were surveyed for the presence of viral diseases. In 2015, 2% of plants from two fields located in different provinces of the Lazio region…

The current knowledge on viruses infecting asparagus (Asparagus officinalis) is reviewed. Over half a century, nine virus species belonging to the genera Ilarvirus, Cucumovirus, Nepovirus, Tobamovirus, Potexvirus, and Potyvirus have been found in this crop. The potyvirus Asparagus virus 1 (AV1) and the ilarvirus Asparagus virus 2 (AV2) are widespread and negatively affect the economic life of asparagus crops reducing yield and increasing the susceptibility to biotic and abiotic stress. The main properties and epidemiology of AV1 and AV2 as well as diagnostic techniques for their detection and identification are described. Minor viruses and control are briefly outlined.

The current knowledge on viruses infecting asparagus (Asparagus officinalis) is reviewed. Over half a century, nine virus species belonging to the genera Ilarvirus, Cucumovirus, Nepovirus, Tobamovirus, Potexvirus, and Potyvirus have been found in this crop. The potyvirus Asparagus virus 1 (AV1) and the ilarvirus Asparagus virus 2 (AV2) are widespread and negatively affect the economic life of asparagus crops reducing yield and increasing the susceptibility to biotic and abiotic stress. The main properties and epidemiology of AV1 and AV2 as well as diagnostic techniques for their detection and identification are described. Minor viruses and control are briefly outlined.

Tomato ( Solanum lycopersicum L.) is a vegetable crop which is affected by many viruses and several viroids, causing significant economic loss. Their detection and identification is of critical importance for plant protection and quarantine and certification programs. The potential was examined of an array based on the Combimatrix platform for the detection of 37 viruses belonging to 13 families, one of which is unassigned, together with six pospiviroid species, genus Pospiviroid , family Pospiviroidae . More than 470 oligonucleotide probes (40-mer) were selected for the microarray diagnostic technique developed in this investigation. Most of the virus probes were highly specific and were able to identify tomato viruses. Most pospiviroid probes, however, were non-specific in terms of species, but were specific at the genus level as they hybridized to members of the genus Pospiviroid. Only one probe of the Tomato apical stem viroid was species specific. The repeatability and specificity of the Combimatrix method showed that it can be considered for routine diagnostic use in suspected tomato germplasm since it detected 37 viruses and one pospiviroid at the species level and 5–6 pospiviroids at the genus level. The estimated cost for testing of a single tomato virus is similar to or less than the cost of using ELISA.

Chilli pepper ( Capsicum annuum ) is an important cash crop in Pakistan which is cultivated on an area of 62,500 ha with an annual yield of 145,100 tonnes (Farooq, 2014). Pepper mild mottle virus (PMMoV) is a single-stranded RNA virus…