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. 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Ecology of virus and virus-like diseases that affect olive trees in the Mediterranean basin

L’olivo (Olea europaea L.) riveste un’importanza storica e socio-economica notevole, in particolare nelle regioni del Bacino del Mediterraneo (Brunori et al., 2020). La crescente domanda globale di olive da tavola e di olio d’oliva ha portato all’adozione di pratiche agricole moderne, quali la creazione di uliveti ad elevata densità e l’implementazione di sistemi di irrigazione (Mili e Bouhaddane, 2021; Sobreiro et al., 2023). Tuttavia, tali cambiamenti comportano enormi sfide legate all’incremento dell’incidenza di parassiti e malattie che colpiscono tale coltura (Caselli e Petacchi, 2021), determinando consistenti perdite di raccolto e ingenti ripercussioni economiche. In risposta, la produzione olivicola nelle regioni mediterranee ha subito una significativa diminuzione a causa dell’emergere di agenti biotici che influenzano l’industria olivicola (Nuti et al., 2021). Inoltre, l’errato impiego di materiale infetto per la propagazione vegetativa dell’olivo ha favorito la diffusione di patogeni sistemici, quali i virus (Lambardi et al., 2023). Al fine di gestire tali minacce, sono state implementate misure preventive, come programmi di selezione e certificazione fitosanitaria (Montilon et al., 2023). In questo contesto, l’applicazione delle tecniche diagnostiche molecolari per la rilevazione di tali agenti patogeni ha assunto, nel recente passato, un’importanza critica per la valutazione dello stato sanitario (López et al., 2009), rappresentando la base per efficienti e validi programmi di certificazione. Pertanto, gli sforzi di ricerca affrontati nella presente tesi, condotta nel contesto di un programma di dottorato di ricerca, si sono concentrati particolarmente sull’identificazione, diagnosi e monitoraggio delle malattie virali che colpiscono l’olivo in ambiente mediterraneo. Con tale obiettivo, la presente tesi si propone di approfondire lo studio degli agenti virali e virus-simili che influenzano la coltivazione dell’olivo, sottolineando il ruolo indispensabile delle moderne tecniche diagnostiche nel preservare la salute e la sostenibilità degli uliveti. In particolare, gli studi di ricerca affrontati sono stati organizzati in otto capitoli. Il Capitolo I evidenzia l’importanza economica e culturale della coltivazione dell’olivo, ne sottolinea le potenziali minacce virali e descrive gli impatti negativi causati sia dai cambiamenti climatici, che dai moderni sistemi di coltivazione dell’olivo. Nel Capitolo II viene descritto lo scopo del lavoro di ricerca, enfatizzando l’importanza di affrontare le malattie virali come una nuova significativasfida globale per l’olivicoltura. Nel Capitolo III viene indagata la presenza di virus che colpiscono la coltura dell’olivo in Sicilia mediante analisi metagenomiche, tramite il dispositivo MinION dell’ Oxford Nanopore. Quest’ultimo ha fornito interessanti risultati, dimostrando il potenziale delle innovative tecniche diagnostiche nel rilevare patogeni virali. Infatti, il dispositivo MinION ha rilevato con successo tre virus dell’olivo: l’olea europaea geminivirus (OEGV), l’ olive leaf yellowing-associated virus (OLYaV) e l’olive latent virus 3 (OLV-3). Tali risultati sono successivamente stati confermati mediante diagnosi molecolare attraverso l’impiego della tecnica RT-PCR end-point, ad eccezione dell’OLV-3. Pertanto, i principali virus oggetto di studio sono stati l’OEGV e l’OLYaV. In particolare, il caso studio dell’OEGV è stato valutato nei Capitoli IV e V, mentre l’OLYaV è stato valutato nei Capitoli VI e VII. In particolare, lo sviluppo e la validazione di un protocollo di real-time Loop-Mediated Isothermal Amplification (LAMP) per la rilevazione e lo studio dell’incidenza dell’OEGV in Sicilia e una procedura rapida per la preparazione in loco del campione sono stati valutati e descritti nel Capitolo IV. Inoltre, con l’obiettivo di comprendere meglio la modalità di trasmissione di OEGV, nel Capitolo V sono riportati gli esperimenti condotti per indagare la trasmissione di tale virus mediante innesto. In particolare, il protocollo real-time LAMP descritto nel Capitolo IV si è rivelato un metodo rapido, semplice, specifico e sensibile per la rilevazione dell’OEGV, consentendo di processare un gran numero di campioni contemporaneamente, specialmente quando associato al metodo rapido di preparazione del campione. Inoltre, lo studio ha rivelato una presenza considerevole del virus all’interno degli uliveti siciliani, probabilmente a causa del movimento involontario di materiale di propagazione infetto, ma asintomatico. I risultati preliminari, inoltre, hanno dimostrato che l’OEGV è stato trasmesso con successo mediante innesto e i sintomi sono stati osservati entro 3-7 mesi post-innesto. Questi risultati sottolineano l’importanza di rigorosi ed efficaci metodi di screening, al fine di mitigare i rischi di trasmissione virale. Nel Capitolo VI, invece, viene focalizzata l’attenzione sullo sviluppo e la valutazione di un protocollo real-time RT-LAMP per la rilevazione dell’OLYaV; quest’ultimo viene, inoltre, comparato con la tecnica di diagnosi RT-PCR end-point per valutarne sensibilità e specificità. Successivamente, il protocollo sviluppato è stato impiegato per valutare l’incidenza dell’OLYaV nelle principali regioni olivicole in Spagna e Italia. I risultati hanno evidenziato che il protocollo di real-time RT-LAMP per la rilevazione di OLYaV si è rivelato maggiormente sensibile e specifico rispetto alla tecnica RT-PCR end-point. Inoltre, l’indagine condotta nelle principali regioni olivicole spagnole e italiane rappresenta, ad oggi, la prima analisi completa dell’incidenza dell’OLYaV in tali territori. In particolare, l’analisi dell’incidenza del virus ha evidenziato una maggiore presenza nel territorio spagnolo rispetto a quello italiano. Tuttavia, per fornire un quadro completo sulla prevalenza e distribuzione del virus in Sicilia, nel Capitolo VII viene descritto lo studio condotto sulla diffusione dell’OLYaV in diverse provincie e siti di produzione siciliani, mediante l’utilizzo del protocollo real-time RT-LAMP precedentemente sviluppato. I risultati hanno mostrato una significativa prevalenza dell’OLYaV negli uliveti campionati, con un numero considerevole di campioni positivi identificati in piante asintomatiche, indicando una possibile diffusione silenziosa del virus attraverso il materiale di propagazione infetto o tramite insetti vettori. Infine, nel Capitolo VIII sono state esposte le conclusioni e le prospettive future, insieme a tutte le possibili implicazioni per lo sviluppo di strategie mirate a mitigare le minacce virali nella coltivazione dell’olivo. I risultati presentati nella presente tesi mirano a contribuire in modo significativo alla comprensione scientifica e all’applicazione pratica delle strategie di controllo e prevenzione per la gestione delle malattie virali dell’olivo. Infine, gli esiti degli studi condotti mettono in luce le attuali sfide che l’industria olivicola affronta e sottolineano il ruolo cruciale delle moderne tecniche diagnostiche nel proteggere la salute, la produttività e la sostenibilità degli uliveti.The cultivation of olive tree (Olea europaea L.) has significant historical and socio-economic importance, especially in Mediterranean regions (Brunori et al., 2020). Due to the increasing global demand for olives and olive oil, modern agricultural practices, such as establishing high-density orchards and implementing irrigation systems have been employed (Mili and Bouhaddane, 2021; Sobreiro et al. 2023). Conversely, these changes pose challenges related to the increasing incidence of olive tree pests and diseases (Caselli and Petacchi, 2021), resulting in significant yield losses and economic consequences. Olive production in the Mediterranean regions has decreased significantly due to the emergence of biotic agents, affecting both the olive tree and the oil industry (Nuti et al., 2021). The improper use of infected olive material for vegetative propagation has facilitated the spread of systemic pathogens in olive orchards, such as viruses (Lambardi et al., 2023). To manage these threats, preventive measures such as phytosanitary selection and certification programmes have been implemented (Montilon et al., 2023). Within this context, the application of molecular diagnostic techniques for viruses detection has assumed, in the recent past, critical importance for the assessment of the sanitary status (López et al., 2009). These reliable methods are required as they are the basis for efficient and valid certification programmes, therefore research efforts focused on particular attention to the identification, diagnosis and monitoring of viral diseases affecting the olive plants in Mediterranean environment. For this aim, the present thesis, conducted as part of a three-year PhD program, delves into the investigation on viral and virus-like agents affecting olive cultivation, emphasizing the indispensable role of advanced diagnostic techniques in safeguarding olive orchards’ health and sustainability worldwide. The research effort is organised into cohesive chapters as follows. Chapter I highlighted the economic and cultural importance of olive cultivation and underlined the potential viral threats and impacts of both climate change and the implemented modern plantation systems for olive cultivation. In Chapter II, the purpose of the research work is outlined, emphasizing the importance of addressing viral diseases as a global agriculture challenge. Chapter III screened the presence of the main olive tree viruses in Sicily using the metagenomic approach based on the Oxford Nanopore MinION sequencer. The implemented diagnostic tool gave interesting preliminary results, demonstrating the potential of innovative diagnostic tools in detecting viral pathogens. Indeed, MinION technology successfully detected olea europaea geminivirus (OEGV), olive leaf yellowing-associated virus (OLYaV) and olive latent virus 3 (OLV-3) that were confirmed, subsequently, by the conventional molecular diagnostic tool end-point RT-PCR, excepting for OLV-3. For these reasons, the case study of OEGV was evaluated in Chapters IV and V, whereas OLYaV was assessed in Chapters VI and VII. In particular, the development and practical feasibility of a real-time loop-mediated isothermal amplification (LAMP) protocol for the detection and investigation of OEGV incidence in Sicily, as well as a rapid procedure for on-site preparation of olive sample were assessed and are described in Chapter IV. Moreover, with the aim of better understanding the virus transmissibility, Chapter V reported the trials that were conducted to investigate OEGV transmission via grafting using infected propagation material. As results, from one hand, the real-time LAMP assay described in this work revealed to be a potential, rapid, simple, specific and sensitive tool for the recently described OEGV detection, allowing to process of a great number of samples at the same time, especially when associated with the rapidly developed on-site sample preparation method. Moreover, the survey revealed a considerable presence of the virus within the Sicilian olive groves, probably due to the inadvertent movement of clonally propagated and asymptomatic infected germplasms. From the other hand, Preliminary results also demonstrated that OEGV was successfully transmitted through grafting, with symptoms observed within 3-7 months post-grafting. These findings emphasize the importance of rigorous screening methods and cultivar selection to mitigate virus transmission risks in olive propagation. Chapter VI focused on the development and evaluation of a real-time RT-LAMP protocol for OLYaV detection, comparing it with end-point RT-PCR assay. Moreover, the real-time RT-LAMP protocol developed was used to assess OLYaV incidence in the main olive-producing regions of Spain and Italy. Obtained results showed that the developed real-time RT-LAMP protocol for OLYaV detection was higher sensitive and specific compared to the RT-PCR end-point technique. Furthermore, the investigation conducted in the main Spanish and Italian olive-growing regions represents, to date, the first comprehensive analysis of OLYaV incidence in these territories. Actually, the virus incidence analysis showed to be highly present in the Spanish territory than in the Italian one. However, in order to provide comprehensive insights into the prevalence and distribution of the virus in Sicily, Chapter VII describes the study conducted on the dispersion of OLYaV in different Sicilian olive production sites, using the previously developed real-time RT-LAMP. The results revealed a significant prevalence of OLYaV in the sampled olive trees, with a significant number of positive samples identified in asymptomatic plants, suggesting a potential silent spread of the virus through infected propagation material or via vector insects. Ultimately, a summarized conclusive considerations and future perspectives were reported in Chapter VIII, together with all the thesis possible contributions to the advancement of knowledge in plant virology and the development of strategies that could mitigate viral threats in olive cultivation. The interesting findings in the current PhD thesis aim to significantly contribute to scientific understanding and practical application of control and prevention strategies for managing olive viral diseases. Lastly, the results of the study shed light on the current challenges facing the industry and underscore the critical role of advanced diagnostic techniques in safeguarding the health, productivity and sustainability of olive orchards

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