Molecular Methods for the Simultaneous Detection of Tomato Fruit Blotch Virus and Identification of Tomato Russet Mite, a New Potential Virus-Vector System Threatening Solanaceous Crops Worldwide.

Tomato fruit blotch virus (ToFBV) (Blunervirus solani, family Kitaviridae) was firstly identified in Italy in 2018 in tomato plants that showed the uneven, blotchy ripening and dimpling of fruits. Subsequent High-Throughput Sequencing (HTS) analysis allowed ToFBV to be identified in samples collected in Australia, Brazil, and several European countries, and its presence in tomato crops was dated back to 2012. In 2023, the virus was found to be associated with two outbreaks in Italy and Belgium, and it was included in the EPPO Alert list as a potential new threat for tomato fruit production. Many epidemiologic features of ToFBV need to be still clarified, including transmission. Aculops lycopersici Massee (Acariformes: Eriophyoidea), the tomato russet mite (TRM), is a likely candidate vector, since high population densities were found in most of the ToFBV-infected tomato cultivations worldwide. Real-time RT-PCR tests for ToFBV detection and TRM identification were developed, also as a duplex assay. The optimized tests were then transferred to an RT-ddPCR assay and validated according to the EPPO Standard PM 7/98 (5). Such sensitive, reliable, and validated tests provide an important diagnostic tool in view of the probable threat posed by this virus-vector system to solanaceous crops worldwide and can contribute to epidemiological studies by simplifying the efficiency of research. To our knowledge, these are the first molecular methods developed for the simultaneous detection and identification of ToFBV and TRM.

Carlavirus Species Infecting Hop Plants in Italy: Molecular Identification and Phylogenetic Analyses of the Detected Isolates.

Hop (Humulus lupulus L.) is a minor ingredient in the beer production but has a strong influence on the beer quality due to the high chemical complexity of the cones used in brewing. One of the major factors that can severely affect the chemical composition of the hop cones and their marketability is the presence of viral infections in the plant. Amongst the five major hop viruses, three belong to the Carlavirus genus: hop mosaic virus (HpMV), hop latent virus (HpLV), and American hop latent virus (AHLV). The occurrence of carlaviruses on hop germplasm in Italy was firstly recorded in 2017 but, in that context, a generic detection was only performed and no information on the infecting Carlavirus species was provided. To fill this gap, 51 hop samples previously found infected by carlaviruses were analysed by RT-PCR employing primer pairs specific for the coat protein (CP) of HpMV, HpLV and AHLV, respectively. HpLV resulted largely prevalent as it was detected in 96.1% of tested samples whereas for HpMV and AHLV an infection rate of 5.9% and 3.9% was recorded, respectively. CP nucleotide sequences from 13 selected virus isolates were obtained and analysed; moreover, the complete genome sequence of 7 isolates was obtained by using high throughput sequencing (HTS). Phylogenetic analysis showed close relationships among isolates from different geographical origin, including European and non-European countries, according to the worldwide movement of hop germplasm due to global trade. This is the first report of HpMV, HpLV and AHLV on hop germplasm in Italy.

Development, Validation, and Application of Reverse Transcription Real-Time and Droplet Digital PCR Assays for the Detection of the Potyviruses Watermelon Mosaic Virus and Zucchini Yellow Mosaic Virus in Cucurbits.

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.

Development and Validation of a Duplex RT-qPCR for Detection of Peach Latent Mosaic Viroid and Comparison of Different Nucleic-Acid-Extraction Protocols.

Peach latent mosaic viroid (PLMVd) is an important pathogen that causes disease in peaches. Control of this viroid remains problematic because most PLMVd variants are symptomless, and although there are many detection tests in use, the reliability of PCR-based methods is compromised by the complex, branched secondary RNA structure of the viroid and its genetic diversity. In this study, a duplex RT-qPCR method was developed and validated against two previously published single RT-qPCRs, which were potentially able to detect all known PLMVd variants when used in tandem. In addition, in order to simplify the sample preparation, rapid-extraction protocols based on the use of crude sap or tissue printing were compared with commercially available RNA purification kits. The performance of the new procedure was evaluated in a test performance study involving five participant laboratories. The new method, in combination with rapid-sample-preparation approaches, was demonstrated to be feasible and reliable, with the advantage of detecting all different PLMVd isolates/variants assayed in a single reaction, reducing costs for routine diagnosis.

Interlaboratory Performance of a Real-Time PCR Method for Detection of Ceratocystisplatani, the Agent of Canker Stain of Platanus spp.

Ceratocystis platani (CP), an ascomycetous fungus, is the agent of canker stain, a lethal vascular disease of Platanus species. Ceratocystis platani has been listed as a quarantine pest (EPPO A2 list) due to extensive damage caused in Southern Europe and the Mediterranean region. As traditional diagnostic assays are ineffective, a Real-Time PCR detection method based on EvaGreen, SYBR Green, and Taqman assays was previously developed, validated in-house, and included in the official EPPO standard PM7/14 (2). Here, we describe the results of a test performance study performed by nine European laboratories for the purpose of an interlaboratory validation. Verification of the DNA extracted from biological samples guaranteed the high quality of preparations, and the stability and the homogeneity of the aliquots intended for the laboratories. All of the laboratories reproduced nearly identical standard curves with efficiencies close to 100%. Testing of blind-coded DNA extracted from wood samples revealed that all performance parameters-diagnostic sensitivity, diagnostic specificity, accuracy and reproducibility-were best fit in most cases both at the laboratory and at the assay level. The previously established limit of detection, 3 fg per PCR reaction, was also validated with similar excellent results. The high interlaboratory performance of this Real-Time PCR method confirms its value as a primary tool to safeguard C. platani-free countries by way of an accurate monitoring, and to investigate the resistance level of potentially canker stain-resistant Platanus genotypes.

Effects of Biochar on the Growth and Development of Tomato Seedlings and on the Response of Tomato Plants to the Infection of Systemic Viral Agents.

Biochar is a rich carbon product obtained by pyrolysis of biomass under a limited supply of oxygen. It is composed mainly of aromatic molecules, but its agronomic value is hard to evaluate and difficult to predict due to its great variable characteristics depending on the type of starting biomass and the conditions of pyrolysis. Anyway, it could be used as soil amendment because it increases the soil fertility of acidic soils, increases the agricultural productivity, and seems to provide protection against some foliar and soilborne diseases. In this study, the effects of biochar, obtained from olive pruning, have been evaluated on tomato seedlings growth and on their response to systemic agents' infection alone or added with beneficial microorganisms (Bacillus spp. and Trichoderma spp.). First, experimental data showed that biochar seems to promote the development of the tomato seedlings, especially at concentrations ranging from 1 to 20% (w/w with peat) without showing any antimicrobial effects on the beneficial soil bacteria at the tomato rhizosphere level and even improving their growth. Thus, those concentrations were used in growing tomato plants experimentally infected with tomato spotted wilt virus (TSWV) and potato spindle tuber viroid (PSTVd). The biochar effect was estimated by evaluating three parameters, namely, symptom expression, number of infected plants, and pathogen quantification, using RT-qPCR technique and -ΔΔCt analysis. Biochar at 10-15% and when added with Trichoderma spp. showed that it reduces the replication of PSTVd and the expression of symptoms even if it was not able to block the start of infection. The results obtained on TSWV-infected plants suggested that biochar could contribute to reducing both infection rate and virus replication. For systemic viral agents, such as PSTVd and TSWV, there are no curative control methods, and therefore, the use of prevention means, as can be assumed the use biochar, for example, in the nursery specialized in horticultural crops, can be of great help. These results can be an encouraging starting point to introduce complex biochar formulates among the sustainable managing strategies of plant systemic diseases.

Effects of Organic Biostimulants Added with Zeolite on Zucchini Squash Plants Infected by Tomato Leaf Curl New Delhi Virus.

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.

Development and Validation of a One-Step Reverse Transcription Real-Time PCR Assay for Simultaneous Detection and Identification of Tomato Mottle Mosaic Virus and Tomato Brown Rugose Fruit Virus.

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.

Inter-Laboratory Comparison of RT-PCR-Based Methods for the Detection of Tomato Brown Rugose Fruit Virus on Tomato.

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.

Multiplex RT-PCR.

Amplification of different nucleic acid targets in the same reaction (multiplex polymerase chain reaction) is challenging but an extremely useful tool especially for viroid diagnosis. In the amplification mixtures, several pairs of primers work together in the same conditions to detect different targets. Here, we describe the development and use of a multiplex reverse transcription polymerase chain reaction protocol highlighting the most crucial factors that can significantly affect the quality of the method. First, particular attention must be paid to primer design. Then, the amplification mixture and temperature conditions must be calibrated precisely to avoid cross reactivity or loss in sensitivity. Finally, the detection system of the amplification results must allow a specific identification of the amplified target(s).