Traditional Approaches and Emerging Biotechnologies in Grapevine Virology

Environmental changes and global warming may promote the emergence of unknown viruses, whose spread is favored by the trade in plant products. Viruses represent a major threat to viticulture and the wine industry. Their management is challenging and mostly relies on prophylactic measures that are intended to prevent the introduction of viruses into vineyards. Besides the use of virus-free planting material, the employment of agrochemicals is a major strategy to prevent the spread of insect vectors in vineyards. According to the goal of the European Green Deal, a 50% decrease in the use of agrochemicals is expected before 2030. Thus, the development of alternative strategies that allow the sustainable control of viral diseases in vineyards is strongly needed. Here, we present a set of innovative biotechnological tools that have been developed to induce virus resistance in plants. From transgenesis to the still-debated genome editing technologies and RNAi-based strategies, this review discusses numerous illustrative studies that highlight the effectiveness of these promising tools for the management of viral infections in grapevine. Finally, the development of viral vectors from grapevine viruses is described, revealing their positive and unconventional roles, from targets to tools, in emerging biotechnologies

Molecular characterization of ‘Candidatus Phytoplasma prunorum’ in Cacopsilla pruni insect vector

Recent investigations on molecular characterization of the ‘Candidatus Phytoplasma prunorum’ (16SrX-B subgroup), causal agent of the European Stone Fruit Yellows (ESFY) syndrome, on the non ribosomal tuf gene resulted in the finding of two groups of isolates, named ‘type a’ and ‘type b’, both with a distinctive geographical distribution in Italian stone fruit growing areas (Ferretti et al., 2007 and 2008). Considering the role of Cacopsylla pruni (Scopoli) in the epidemiological cycle of the disease, the presence of the two groups of isolates has also been investigated in infected psyllid individuals from different Italian areas. Both types have been identified in C. pruni specimens collected on apricot, plum and wild Prunus species, confirming the geographical distribution and the percentages of spread of the two isolates.Keywords: ESFY, phytoplasma, characterization, tuf gene, insect vecto

Prediction of the kiwifruit decline syndrome in diseased orchards by remote sensing

Eight years after the first record in Italy, Kiwifruit Decline (KD), a destructive disease causing root rot, has already affected more than 25% of the area under kiwifruit cultivation in Italy. Diseased plants are characterised by severe decay of the fine roots and sudden wilting of the canopy, which is only visible after the season's first period of heat (July-August). The swiftness of symptom appearance prevents correct timing and positioning for sampling of the disease, and is therefore a barrier to aetiological studies. The aim of this study is to test the feasibility of thermal and multispectral imaging for the detection of KD using an unsupervised classifier. Thus, RGB, multispectral and thermal data from a kiwifruit orchard, with healthy and diseased plants, were acquired simultaneously during two consecutive growing seasons (2017-2018) using an Unmanned Aerial Vehicle (UAV) platform. Data reduction was applied to the clipped areas of the multispectral and thermal data from the 2017 survey. Reduced data were then classified with two unsupervised algorithms, a K-means and a hierarchical method. The plant vigour (canopy size and presence/absence of wilted leaves) and the health shifts exhibited by asymptomatic plants between 2017 and 2018 were evaluated from RGB data via expert assessment and used as the ground truth for cluster interpretation. Multispectral data showed a high correlation with plant vigour, while temperature data demonstrated a good potential use in predicting health shifts, especially in highly vigorous plants that were asymptomatic in 2017 and became symptomatic in 2018. The accuracy of plant vigour assessment was above 73% when using multispectral data, while clustering of the temperature data allowed the prediction of disease outbreak one year in advance, with an accuracy of 71%. Based on our results, the unsupervised clustering of remote sensing data could be a reliable tool for the identification of sampling areas, and can greatly improve aetiological studies of this new disease in kiwifruit

Hypo- and hyper-virulence in apricot trees infected by European stone fruit yellows

An apricot orchard, located in an area of north eastern Italy under serious pressure from European stone fruit yellows (ESFY) infection, has been monitored since the year it was planted (1990). During this time, most of the trees displayed symptoms or were shown by PCR analyses to be infected. Two groups of apricot trees were particularly interesting: some trees were asymptomatically infected while others recovered from the symptoms but not from the pathogen. In order to isolate those strains of the phytoplasma characterised by varying virulence, each of the two groups was used as mother plants and propagated. The new trees were used to constitute experimental orchards, where they were observed for the presence of symptoms and in part were tested by PCR, starting in 2003. The results obtained confirmed the presence of strains of the pathogen characterised by varying virulence. The strains originally present in infected apricot trees which recovered from the symptoms of ESFY were seen to be hypovirulent; none of the propagated infected trees ever showed symptoms of the disease. Surprisingly, the strains present in asymptomatic apricot mother plants were hypervirulent and the propagated trees always displayed severe symptoms. In the propagated trees, the transmission of the pathogen was higher in the hypervirulent strains than in the hypovirulent ones. A graft transmission trial carried out in the greenhouse using some of the identified hypo- and hypervirulent strains, confirmed the results obtained in open field. Real time PCR analyses showed that in the trees infected by hypovirulent strains the colonisation of the pathogen was lower than in those infected by the hypervirulent strains. It is possible to affirm that the hypovirulent strains were present in those mother plants which had originally recovered. The research will continue with the aim of verifying the possibility of cross protection among the identified hypo- and hypervirulent strains.Keywords: ‘Candidatus Phytoplasma prunorum’, real-time PCR, Prunu

Incidence and molecular characterization of flavescence dorée and stolbur phytoplasmas in grapevine cultivars from different viticultural areas of Serbia

The presence and distribution of grapevine phytoplasmas was investigated from 2003 to 2005 in some of the most important viticultural areas of Serbia, considering in particular the susceptibility and sensitiveness of both local and imported grapevine cultivars. Both flavescence dorée (FD) and bois noir (BN) phytoplasmas were detected using molecular techniques. The presence of FD phytoplasma at the moment seems limited, while BN phytoplasma appears to be present in the majority of grape growing regions in Serbia. Field surveys demonstrate that grapevine yellows (GY) epidemics in the vineyards inspected in Serbia spread very fast, indeed the incidence of symptomatic plants increased considerably year by year. In particular, the average rate of FD diffusion increased from 45.5 to 93.0 % in the Sićevačko region, while the spread of BN resulted lower. The local cultivar 'Plovdina' appeared to be extremely sensitive to FD phytoplasma showing a percentage of infected plants ranging from 91 to 100 %. PCR-RFLP and phylogenetic analyses based on ribosomal protein (rp) and secY gene sequences performed on Serbian FD grapevine strains demonstrated their close relationship with the Italian FD-C strain present in north-east Italy. Based on both phylogenetic markers, Serbian FD strains represent a new distinct lineage and together with the FD-C strain form a major phylogenetic group within the elm yellows group.

Rare and Insidious Toxicities from New Combination Therapies in Metastatic Renal Cell Cancer: Lessons Learned from Real-Practice

The advent of immune checkpoint inhibitors in combination with multitarget tyrosine kinase inhibitors has become a standard first-line treatment for metastatic renal cell cancer. Along with survival improvement, new toxicities have emerged. Such adverse events are still complex to be managed and some of them are rare and could be insidious or even fatal. Medical oncologists dispose of guidelines about the management of toxicities from immune checkpoint inhibitors but not for combinations. Therefore, it is still difficult to properly attribute and manage additive or overlapping adverse events. We report two clinical cases regarding rare treatment-related endocrine toxicities—hypophysitis and thyroiditis—with particular focus on their management. To this purpose, immune checkpoint-related toxicities guidelines represent the starting point. However, their implementation with additional measures is needed, considering the increasing complexity of current clinical scenarios. The goal is to correctly recognize adverse events and address side effects, so as not to discontinue effective treatments. We, therefore, aim at discussing the points of proper management of toxicities and individuating potential areas of improvement

Studies on the aetiology of kiwifruit decline: interaction between soil-borne pathogens and waterlogging

Aims: In 2012, Italian kiwifruit orchards were hit by a serious root disease of unknown aetiology (kiwifruit decline, KD) that still causes extensive damage to the sector. While waterlogging was soon observed to be associated with its outbreak, the putative role of soil microbiota remains unknown. This work investigates the role of these two factors in the onset of the disease. Methods: Historical rainfall data were analysed to identify changes that might explain KD outbreak and mimic the flooding conditions required to reproduce the disease in a controlled environment. A greenhouse experiment was thus designed, and vines were grown in either unsterilized (U) or sterilized (S) soil collected from KD-affected orchards, and subjected (F) or not (N) to artificial flooding. Treatments were compared in terms of mortality rate, growth, and tissue modifications. Results: KD symptoms were only displayed by FU-treated vines, with an incidence of 90%. Ultrastructural observations detected tyloses and fibrils in the xylem vessels of all plants, irrespective of the treatment. Phytopythium vexans and Phytopythium chamaehyphon, isolated from roots of FU plants, emerged as the associated microorganisms. Conclusions: We succeeded in reproducing KD under controlled conditions and confirmed its association with both waterlogging and soil-borne microorganism(s)

PCR/RFLP-based method for molecular characterization of ‘Candidatus Phytoplasma prunorum’ strains using the aceF gene.

New molecular typing tools for phytoplasmas belonging to the 16SrX phytoplasma group have recently been developed based on the non-ribosomal genes aceF, pnp, imp, and SecY. In the present work we chose to perform a PCR-RFLP method based on the aceF gene. This genetic marker had previously shown high variability among strains of the 16SrX group, moreover, it had allowed for the differentiation of French hypovirulent ‘Candidatus Phytoplasma prunorum’ strains from virulent ones.Most of the stone fruit samples were collected in north-east Italy, although a few samples from Bosnia and Herzegovina, and Turkey were also included in the work to explore variability. French hypovirulent and virulent strains, one Azerbaijan strain and ‘Ca. P. prunorum’ strains maintained in periwinkles were used as reference strains. Some of the Italian samples were not collected in the field and they became infected by Cacopsylla pruni under controlled conditions.Sequencing of the aceF gene was performed on some of the samples tested and based on the alignment, a few restriction enzymes were selected for ‘Ca. P. prunorum’ strain differentiation. Nested PCR was performed using previously developed primers on all samples and RFLP analyses were carried out with BpiI, HaeIII and Tsp509I enzymes. BpiI and HaeIII enzymes generated two different profiles, one profile was undigested and the second one constituted by two different fragments. The Tsp509I enzyme enabled three different pattern types to be distinguished. Combining the results obtained with the three restriction enzymes, it was possible to distinguish between the ‘Ca. P. prunorum’ strains investigated in this study: 6 different RFLP subgroups AceF-A, -B, -C, -D, -E and –F. We confirmed that strains belonging to 4 subgroups, AceF-A, -B, -C and -E were present in north-east Italy, where a large number of the samples were processed. The strains of AceF-A and -E subgroups were the predominant ones (21.6% and 17.0%, respectively) and mixed infections of AceF-A+E subgroups (17.0%), and AceF-B+E (14.8%) subgroups were quite common. Keywords: phytoplasma, European stone fruit yellows, molecular differentiation, sequencin

Macropsis mendax as a vector of elm yellows phytoplasma of Ulmus species.-

A 3-year study was carried out in north-east Italy, the site of recent elm yellows epidemics, to identify vectors for the elm yellows phytoplasma. Using PCR analysis, Ulmus minor and Ulmus pumila , each with and without symptoms, were positive for the elm yellows phytoplasma. Macropsis mendax , a univoltine and monophagous leafhopper, was shown to be the vector of the elm yellows-associated disease agent. PCR analyses demonstrated that the insect was infected both in natural conditions and in the screenhouse after acquisition-feeding on infected elm plants. Groups of M. mendax , collected from naturally infected elm trees, transmitted elm yellows phytoplasma to elm test plants. In nature, Alnus glutinosa trees affected by alder yellows were found in the surroundings of yellows-affected elm trees; the associated disease agent of alder yellows was transmitted under controlled conditions from alder to elm test plants by grafting