• Physiological analysis
• Biochemical analysis
• Molecular analysis
• Wisteria vein mosaic virus
• Grapevine Pinot gris virus
• Fig mosaic virus
• Plum pox virus

Plant viruses are responsible nearly 50% of plant diseases worldwide and are one of the major constraints in agricultural production by decreasing both quality and quantity of food crops. No cure is available against viral diseases, which can be controlled only by prophylactic measures. Thus, imporove knowledge about virus epidemiology and genomics, as well as the response mechanisms of plants challenged by viruses, is crucial to properly manage these harmful diseases. The overall scope of this research work was to investigate some major fruit and ornamental plant viroses in terms of disease epidemiologies, virus genetics, and host-pathogen interactions.
Sharka, caused by Plum pox virus (PPV), which is by far the most harmful disease of stone fruits (genus Prunus), was firstly investigated. Although Italy is a major producer of stone fruits, and sharka has spread in most Italian regions since its first identification in the 1970s, information on the diffusion of the disease in Italy results still scattered and incomplete. To address this knowledge gap, the present work firstly aimed to firstly provide a historical overview of sharka in Italy, summarizing outcomes on the disease achieved since the first identification of its causal agent PPV. Data retrieved from a variety of sources (i.e., published literature, official reports and personal communications) showed that (i) sharka spread across almost the whole Italian peninsula as only Liguria, Umbria and Aosta Valley regions officially result disease-free; (ii) the regions with higher rates of infected districts are not always those more dedicated to the production of stone fruits; (iii) only the three major PPV strains causing sharka, i.e., PPV-Dideron (D), PPV-Marcus (M), and PPV-Recombinant (Rec), have been reported in Italy; and (iv) only apricot, peach and plum (i.e., widespread and economically relevant species) resulted hosts of PPV, with different scenarios among regions.
Then, as a broad investigation and genetic characterization of PPV isolates was lacking in most of the Italian region, including Tuscany (Central Italy), field surveys were carried out in 2020 and 2021 in 80 commercial Tuscany orchards, and a total of 690 leaf samples were collected from different Prunus species. PPV was identified in 25 and 12 peach and plum samples, respectively (more than 5% of tested samples), whereas no positive samples were reported in apricot. Eighteen of the 37 PPV positive samples showed mixed infections with other viruses and viroids, mostly Prunus necrotic ringspot virus in plum and Peach latent mosaic viroid in peach. Molecular typing of PPV infected samples generated Nib/CP amplicons corresponding to PPV-Marcus (M) or PPV-Recombinant (Rec) strains. Furthermore, starting from the identification of eight PPV nucleotide sequences (among which five and two new PPV-M and PPV-Rec isolates, respectively), this study firstly identified the PPV-Mb subgroup in Italy, which was even prevalent than PPV-Ma. Finally, PPV-Rec isolates resulting phylogenetically close to Italian and Turkish isolates previously detected.
Furthermore, autochthonous plum cv. Coscia di Monaca was investigated to fill virological knowledges gap about Tuscan germplasm. To address this lack, an orchard composed by the autochthonous plum was selected in Arezzo (Tuscany, Italy) district and presence of widespread, (i.e., Plum pox virus, PPV; Prune dwarf virus, PDV; Prunus necrotic ringspot virus, PNRSV) and not widespread (i.e., Apple chlorotic ringspot virus, ACLSV; Apple mosaic virus, ApMV; Myrobalan latent ringspot virus, MLRSV; Plum bark necrosis steam pitting associated-virus, PBNSPaV) viruses in stone fruits were assayed also detailing the rates of mixed infection. Furthermore, plant-viruses interactions considering physiological and biochemical features were conducted. The diagnostic analysis carried out on leaves revealed presence of PPV and PBNSPaV. Mixed infections were the most widespread following by PBNSPaV and by PPV. Molecular typing revealed the presence of PPV-M strain. Furthermore, the physiological and biochemical analysis revealed as mixed infection and single infections has different effects in leaves tissue which didn’t include variation in water content. Mixed infection and PPV affected photosynthesis due to putative alteration in VAZ and DEPS. Conversely, PBNSPaV reduced VAZ but seemed not affected overall photosynthesis. Presence of PPV in tissue increased α-tocopherols, sucrose and fructose whereas decreased quinic acid. Differently, PBNSPaV increased malic acid and quinic acid content. The outcomes obtained will offer a starting point for autochthonous Tuscan plum trees studies and they can be useful to supporting their recovery and selection in breeding programs.
After Sharka, the present research work focused on Fig mosaic disease (FMD), a major disease affecting fig trees, for which only Fig mosaic virus (FMV) has been identified as etiological agent. Specifically, trees of common fig cv. Dottato, belonging to the old Tuscan germplasm, were investigated to pioneering (i) evaluate the presence of FMV in Tuscany (Central Italy), as well as other FMD-associated viruses previously reported in Italy, (ii) type and phylogenetically characterize the reported FMV isolates, and (iii) elucidate some physiological and biochemical responses of fig trees challenged by FMV. Although many studies on FMD have been carried out in Southern Italy, the present study represents the first identification of the disease in other Italian regions. This work (i) showed that FMD is present also in Central Italy, also suggesting a wider diffusion than what has been reported so far; (ii) confirmed that the disease is caused by FMV, tested positive in both symptomatic and asymptomatic leaves (100 and 27% of tested samples, respectively); and also Fig fleck-associated virus (FFKaV) was reported; (iii) identified three new FMV isolates (i.e., Dot-1, Dot-2 and Dot-3, deposited in GenBank), which resulted not close to other FMV Italian isolates; and (iv) pioneering elucidated that FMV impaired photosynthesis and organic acid biosynthesis in symptomatic leaves, but negative effects occurred also in asymptomatic ones in terms of photosynthetic and accessory pigments. More research should be carried out to improve our knowledge on FMD diffusion and FMV (and FFKaV) genetic features, as well on the effects of this regulated non quarantine pest on fig trees, also investigating its fruits representing an ancient source of food and health.
Later, Grapevine Pinot gris virus (GPGV), an emergent virus associated with macroscopic symptoms of Grapevine Leaf Mottling and Deformation disease (GLMD) detected in the main European and worldwide viticultural regions, was studied. Investigation was carried out in economic relevant Tuscany vineyards of Chianti Rufina, Chianti Classico and Val d’Orcia never inspected before to evaluate GPGV infection rate and characterize the isolates on susceptible grapevine cvs. Pinot gris, Tempranillo and Traminer. The results of this study showed GPGV presence in Chianti Classico area in cv. Tempranillo vines and any infection in cvs. Pinot gris and Traminer. Five nucleotide sequence variants were obtained, within two new ones located in movement protein (MP; OK350068, OK350069) and in movement protein/coat protein (MP/CP, OK350070, OK350071) genomic regions, respectively. One sequence (OK350072) was identical to previously found in Bolgheri (Tuscany) on cv. Sangiovese berries. Furthermore, the sequences comparison revealed presence of non-synonymous single nucleotide polymorphysms (SNPs).
Finally, the work focused on Wisteria vein mosaic virus (WVMV), which is a Potyvirus infecting Wisteria spp. worldwide, making these largely used ornamental plants unattractive and even unsalable. In 2021, nine Wisteria sinensis plants in Sarzana (Liguria, Italy) showing WVMV symptoms like vein mosaic with irregular patterns, mottling, deformation and twisting margin on leaves were reported. This work describes research on symptomatic and asymptomatic leaves to confirm the identity of the virus infection. All plants tested resulted positive to WVMV. Sequencing of Nib/CP genomic region and comparison on GenBank revealed the presence of eight new genetic variants named Sar 5-12. The eight nucleotide sequences alignments revealed identity ranging between 87.13% to 99.85%. Negative selection (dN/dS <1) was detected suggesting well adaptation in the area here examined and stability in population. Through a phylogenetic tree, WVMV isolates were grouped in five clades with high bootstrap values, four of which included the eight Italian variants here identified. Since the late 1950s and 1960s, the present study represents the first additional report of WVMV in Italy, as well as its first molecular characterization ever. More research should be carried out to improve our knowledge on WVMV diffusion and to elucidate the risk of growing virus infected plants that could represent a source for virus transmission to other potential neighbouring susceptible species.
Overall, the results here presented represent an important step to fill knowledge gaps about some major fruit and ornamental plant viroses in Italy, and we believe it may encourage other similar research to achieve more accurate data on virus populations at both national and international levels.