Epidemiological Survey of Grapevine Leafroll-Associated Virus 1 and 3 in Sicily (Italy): Genetic Structure and Molecular Variability

Background: the most widely distributed and virulent Grapevine leafroll-associated viruses (GLRaV) that affect grapevine are GLRaV-1 and GLRaV-3, transmitted semi-persistently by different mealybugs and soft scales, mainly causing downward rolling of the leaf margins and interveinal reddening. Methods: the main objectives of this study were to investigate the genetic structure and molecular diversity of GLRaV-1 and GLRaV-3 in 617 samples from 11 autochthonous Sicilian grapevine cultivars, ascertaining their presence and spread. The detection was implemented by serological and molecular analyses and subsequently phylogenetic analyses on selected Sicilian isolates were conducted. Results: in total, 33 and 138 samples resulted positive to GLRaV-1 and GLRaV-3, with an incidence of 5.34% and 22.36%, respectively; 9 out of the 11 cultivars resulted positive, while the presence of both viruses was not found in ‘Grillo’ and ‘Moscato’ cultivars. Conclusions: phylogenetic analyses of the coat protein (CP) gene of 12 GLRaV-1 selected sequences showed a close relationship with European isolates; the discrete nucleotide differentiation and positive selection could demonstrate a current increase in population fitness. The phylogenetic analyses of the CP gene of 31 GLRaV-3 Sicilian CP sequences demonstrates a close relationship between Sicilian and different countries isolates; a certain stability of GLRaV-3 in the different cultivars analyzed is suggested by the discrete differentiation nucleotide and negative selection of the Sicilian isolates

Evolutionary Analysis of Grapevine Virus A: Insights into the Dispersion in Sicily (Italy)

Grapevine virus A (GVA) is a phloem-restricted virus (genus Vitivirus, family Betaflexiviridae) that cause crop losses of 5–22% in grapevine cultivars, transmitted by different species of pseudococcid mealybugs, the mealybug Heliococcus bohemicus, and by the scale insect Neopulvinaria innumerabilis. In this work, we studied the genetic structure and molecular variability of GVA, ascertaining its presence and spread in different commercial vineyards of four Sicilian provinces (Italy). In total, 11 autochthonous grapevine cultivars in 20 commercial Sicilian vineyards were investigated, for a total of 617 grapevine samples. Preliminary screening by serological (DAS-ELISA) analysis for GVA detection were conducted and subsequently confirmed by molecular (RT-PCR) analysis. Results showed that 10 out of the 11 cultivars analyzed were positive to GVA, for a total of 49 out of 617 samples (8%). A higher incidence of infection was detected on ‘Nerello Mascalese’, ‘Carricante’, ‘Perricone’ and ‘Nero d’Avola’ cultivars, followed by ‘Alicante’, ‘Grecanico’, ‘Catarratto’,‘Grillo’, ‘Nerello Cappuccio’ and ‘Zibibbo’, while in the ‘Moscato’ cultivar no infection was found. Phylogenetic analyses carried out on the coat protein (CP) gene of 16 GVA sequences selected in this study showed a low variability degree among the Sicilian isolates, closely related with other Italian isolates retrieved in GenBank, suggesting a common origin, probably due to the exchange of infected propagation material within the Italian territory

Development of a Real-Time Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Olea Europaea Geminivirus

A real-time loop-mediated isothermal amplification (LAMP) assay was developed for simple, rapid and efficient detection of the Olea europaea geminivirus (OEGV), a virus recently reported in different olive cultivation areas worldwide. A preliminary screening by end-point PCR for OEGV detection was conducted to ascertain the presence of OEGV in Sicily. A set of six real-time LAMP primers, targeting a 209-nucleotide sequence elapsing the region encoding the coat protein (AV1) gene of OEGV, was designed for specific OEGV detection. The specificity, sensitivity, and accuracy of the diagnostic assay were determined. The LAMP assay showed no cross-reactivity with other geminiviruses and was allowed to detect OEGV with a 10-fold higher sensitivity than conventional end-point PCR. To enhance the potential of the LAMP assay for field diagnosis, a simplified sample preparation procedure was set up and used to monitor OEGV spread in different olive cultivars in Sicily. As a result of this survey, we observed that 30 out of 70 cultivars analyzed were positive to OEGV, demonstrating a relatively high OEGV incidence. The real-time LAMP assay developed in this study is suitable for phytopathological laboratories with limited facilities and resources, as well as for direct OEGV detection in the field, representing a reliable method for rapid screening of olive plant material

Molecular analysis of a Spanish isolate of chili pepper mild mottle virus and evaluation of seed transmission and resistance genes

[EN] An isolate of chili pepper mild mottle virus (CPMMV-Sp; GenBank OQ920979) with a 99% identity to CPMMV (GenBank MN164455.1) was found in symptomatic pepper plants in Spain. RACE analysis, performed using a stem-loop primer developed in this study to prime at the end of the introduced poly(A)/(U) tail, revealed the presence of an extra 22 nt at the 5' end, starting with a cytosine, which were essential to generate infectious clones. However, the 5' terminal cytosine was dispensable for initiating the infection. The design of two specific digoxigenin riboprobes targeting the more divergent area of CPMMV-Sp, compared to the closely related bell pepper mottle virus (BPeMV) (identity percentage of 80.6% and 75.8%, respectively), showed that both probes specifically detected CPMMV-Sp when the hybridization was performed at 68oC and 60oC, respectively. However, the BPeMV probe, targeting a region with an 89.4% identity percentage to CPMMV-Sp, showed cross-hybridization at 60oC but not at 68oC. The comparison of the detection limits between molecular hybridization and RT-PCR techniques revealed that the former was 125 times less sensitive than RT-PCR. The analysis of the vertical transmission of CPMMV-Sp using seeds from naturally or mechanically infected pepper plants revealed a transmission percentage ranging from 0.9% to 8.5%. Finally, the analysis of the resistance of capsicum species carrying different alleles of the L gene (L1, L2, L3, and L4) revealed that varieties with the L1 gene were infected by CPMMV-Sp (20-40% of inoculated plants), while varieties with the L2, L3, and L4 genes were resistant.This work was supported by grants PID2020-115571RB-100 and TED2021-131949B-I00 from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). Project 20-00032-VIRUSPIM from Dept. of Environment, Territorial Planning, Agriculture and Fisheries (Basque Government). Mikel Ojinaga was the recipient of a PhD contract "Introduction of Resistance to Tobamovirus and other Viruses in Landraces of Gernika Pepper and Ibarra Chili Pepper" (Order of 24 October 2018 of the Minister of Economic Development and Competitiveness of the Basque Government). Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.Ontañon, C.; Ojinaga, M.; Larregla, S.; Zabala, JA.; Reva, A.; Losa, A.; Heribia, R.... (2023). Molecular analysis of a Spanish isolate of chili pepper mild mottle virus and evaluation of seed transmission and resistance genes. European Journal of Plant Pathology. 1-18. https://doi.org/10.1007/s10658-023-02765-1118Al-Tamimi, N., Kawas, H., & Mansour, A. (2010). Seed Transmission Viruses in Squash Seeds (Cucurbita pepo) in Southern Syria and Jordan Valley. Jordan Journal of Agricultural Sciences, 5(4), 497–506. https://journals.ju.edu.jo/JJAS/article/view/864.Bhat, A.I. & Rao, G.P. (2020). Transmission Through Seeds. In: Characterization of Plant Viruses. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0334-5_10Davino, S., Caruso, A. G., Bertacca, S., Barone, S., & Panno, S. (2020). Tomato Brown Rugose Fruit Virus: Seed Transmission Rate and Efficacy of Different Seed Disinfection Treatments. Plants, 9(11), 1–13. https://doi.org/10.3390/PLANTS9111615Demski, J. W. (1981). Tobacco Mosaic Virus Is Seedborne in Pimiento Peppers. Plant Disease, 65(9), 723. https://doi.org/10.1094/PD-65-723Di Dato, F., Parisi, M., Cardi, T., & Tripodi, P. (2015). Genetic diversity and assessment of markers linked to resistance and pungency genes in Capsicum germplasm. Euphytica, 204(1), 103–119.Dombrovsky, A., Smith, E., Dombrovsky, A., & Smith, E. (2017). Seed Transmission of Tobamoviruses: Aspects of Global Disease Distribution. Advances in Seed Biology. https://doi.org/10.5772/INTECHOPEN.70244Gallois, J. L., Moury, B., & German-Retana, S. (2018). Role of the genetic background in resistance to plant viruses. International Journal of Molecular Sciences, 19(10), 2856. https://doi.org/10.3390/ijms19102856Genda, Y., Kanda, A., Hamada, H., Sato, K., Ohnishi, J., & Tsuda, S. (2007). Two amino acid substitutions in the coat protein of Pepper mild mottle virus are responsible for overcoming the L4 gene-mediated resistance in Capsicum spp. Phytopathology, 97(7), 787–793.Genda, Y., Sato, K., Nunomura, O., Hirabayashi, T., & Tsuda, S. (2011). Immunolocalization of Pepper mild mottle virus in developing seeds and seedlings of Capsicum annuum. Journal of General Plant Pathology, 77(3), 201–208. https://doi.org/10.1007/s10327-011-0307-0Genda, Y., Sato, K., Nunomura, O., Hirabayashi, T., Ohnishi, J., & Tsuda, S. (2005). Immunolocalization of Pepper mild mottle virus in Capsicum annuum seeds. Journal of General Plant Pathology, 71(3), 238–242. https://doi.org/10.1007/S10327-005-0189-0Ghodoum, P. M. H., & Keshavarz-Tohid, V. (2020). Identification and phylogenetic analysis of a tobamovirus causing hibiscus (Hibiscus rosa-sinensis L.) mosaic disease in Iran. Journal of Plant Pathology, 102(3), 813–824. https://doi.org/10.1007/S42161-020-00510-9/TABLES/3Gniffke, P. A., Shieh, S. C., Lin, S. W., Sheu, Z. M., Chen, J. R., Ho, F. I., et al. (2013). Pepper research and breeding at AVRDC - The World Vegetable Center. Breakthroughs in the genetics and breeding of capsicum and eggplantProceedings of the XV EUCARPIA meeting. https://worldveg.tind.io/record/50155. Accessed 2-4 Sept 2013Groth-Helms, D., Juszczak, S., & Adkins, S. (2022). First report of Chili pepper mild mottle virus in calibrachoa in the United States. 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Tomato Brown Rugose Fruit Virus: Seed Transmission Rate and Efficacy of Different Seed Disinfection Treatments

Tomato brown rugose fruit virus (ToBRFV) is a highly infectious virus, that is becoming a threat to tomato production worldwide. In this work we evaluated the localization of ToBRFV particles in tomato seeds, its seed transmission rate and efficacy of disinfection, and the effects of different thermal- and chemical-based treatments on ToBRFV-infected seeds’ germination. Analyses demonstrated that ToBRFV was located in the seed coat, sometime in the endosperm, but never in the embryo; its transmission from infected seeds to plantlets occurs by micro-lesions during the germination. The ToBRFV seed transmission rate was 2.8% in cotyledons and 1.8% in the third true leaf. Regarding the different disinfection treatments, they returned 100% of germination at 14 days post-treatment (dpt), except for the treatment with 2% hydrochloric acid +1.5% sodium hypochlorite for 24 h, for which no seed germinated after 14 dpt. All treatments have the ability to inactivate ToBRFV, but in six out of seven treatments ToBRFV was still detectable by RT-qPCR. These results raise many questions about the correct way to carry out diagnosis at customs. To our knowledge, this is the first study on the effective localization of ToBRFV particles in seeds

Development of an In-Field Real-Time LAMP Assay for Rapid Detection of Tomato Leaf Curl New Delhi Virus

Tomato leaf curl New Delhi virus (ToLCNDV) represents a threat to economically important horticultural crops. A real-time loop-mediated isothermal amplification (LAMP) assay for in-field ToLCNDV detection was developed, coupled to a rapid sample preparation method, and tested both in field and laboratory conditions on zucchini squash, tomato, and pepper samples. A set of six LAMP primers was designed for specific ToCLNDV detection, targeting a 218-nucleotide sequence within the AV1 gene. The sensitivity, specificity and accuracy of the real-time LAMP assay and comparison with canonical PCR were evaluated. The real-time LAMP assay developed was about one-thousand times more sensitive than the conventional PCR method, detecting a total of 4.41 × 102 genome copies as minimum target; no cross-reactivity was detected with the other geminiviruses used as the outgroup. The rapid sample preparation method allows for a reliable detection with a low reaction delay (≈2–3 min) compared to canonical DNA extraction, providing results in less than 45 min. Lastly, an increase in ToLCNDV-positive sample detection was observed compared to PCR, in particular for asymptomatic plants (85% and 71.6%, respectively). The real-time LAMP assay developed is a rapid, simple, specific, and sensitive technique for ToLCNDV detection, and it can be adopted as a routine test, for both in-field and laboratory conditions

Tomato brown rugose fruit virus: A pathogen that is changing the tomato production worldwide

Tomato (Solanum lycopersicum L., family Solanaceae) represents one of the most cultivated horticultural crops worldwide, with over 5 million hectares of cultivated area and more than 182 million tons of tomato produced globally. Nevertheless, monoculture conditions, intensive selection, domestication throughout the last decades, international trade of infected propagating material and climate changes intensely favoured the establishment of many pathogens and the rapid spread of new diseases, allowing organisms to establish in new and unfavourable environments. Among different biotic agents, viruses are the most dangerous, because of their rapid diffusion and production losses. Here, we review an emerging viral threat to tomato production, tomato brown rugose fruit virus (ToBRFV), a new highly infectious tobamovirus that is currently causing great concern to tomato global production, especially in those areas where mitigation measures are absent or inadequate and which, in recent years, it has considerably increased its diffusion in new tomato cultivation areas. Through a review of all the existing literature, this article highlights the following aspects: (a) main characteristic of tomato species (origin, taxonomy and genome); (b) main diseases that undermine the tomato production, focusing on viral pathogens; (c) ToBRFV main characteristics (origin and spatiotemporal dispersal, taxonomy, genome organisation, host range and symptoms, transmission, spread and epidemiology, and genetic diversity); (d) detection methods developed and disease management; (e) breeding as a new weapon to control the ToBRFV diffusion. Moreover, future perspectives are highlighted, to understand the epidemiology key factors and the ToBRFV-tomato pathosystem management, in order to develop effective and appropriate control strategies

A Review of the Most Common and Economically Important Diseases That Undermine the Cultivation of Tomato Crop in the Mediterranean Basin

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

Genetic Structure and Molecular Variability of Grapevine Fanleaf Virus in Sicily

Grapevine fanleaf virus (GFLV) is one of the main causes of grapevine fanleaf degeneration disease (GFDD) and is present in almost all areas where grapevine is cultivated. In this work, we ascertained the presence and spread of GFLV in different commercial vineyards in four Sicilian provinces (Italy), and its genetic structure and molecular variability were studied. In detail, a total of 617 grapevine samples of 11 autochthonous grapevine cultivars were collected in 20 commercial vineyards. Preliminary screening by serological (DAS-ELISA) and molecular (RT-PCR) analyses for ArMV (arabis mosaic virus) and GFLV detection were conducted. Results obtained showed the absence of ArMV in all the samples analyzed, while 48 out of 617 samples gave positive results to GFLV, for a total of 9 out of 11 cultivars analyzed. Phylogenetic analyses carried out on the GFLV-CP gene of 18 Sicilian GFLV sequences selected in this study showed a certain degree of variability among the Sicilian isolates, suggesting a different origin, probably as a consequence of the continuous interchange of GFLV-infected propagating material with other Italian regions or viticultural areas located in other countries

Detection by Sensitive Real-Time Reverse Transcription Loop-Mediated Isothermal Amplification of Olive Leaf Yellowing Associated Virus and Its Incidence in Italy and Spain

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