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Cultivated plants belonging to the genus Prunus are globally widespread and for some countries, are economically important crops; and they play a key role in the composition of a landscape. Xylella fastidiosa is a key threat to plant health, and several Prunus species are heavily stressed by this pathogen, such as almond, peach, and plum; many strain types of different subspecies can cause severe diseases. This review highlights different approaches to managing epidemic events related to X. fastidiosa in stone fruit plants. In fact, in most new European and Asian outbreaks, almond is the main and very common host and peach, plum, apricot, and cherry are widespread and profitable crops for the involved areas. Various diseases associated with stone fruit plants show different degrees of severity in relation to cultivar, although investigations are still limited. The development and selection of tolerant and resistant cultivars and the study of resistance mechanisms activated by the plant against X. fastidiosa infections seem to be the best way to find long-term solutions aimed at making affected areas recover. In addition, observations in orchards severely affected by the disease can be essential for collecting tolerant or resistant materials within the local germplasm. In areas where the bacterium is not yet present, a qualitative-quantitative study on entomofauna is also important for the timely identification of potential vectors and for developing effective control strategies.

Agricultural activity replaces natural vegetation with cultivated land and it is a major cause of local and global climate change. Highly specialized agricultural production leads to extensive monoculture farming with a low biodiversity that may cause low landscape resilience. This is the case on the Salento peninsula, in the Apulia Region of Italy, where the Xylella fastidiosa bacterium has caused the mass destruction of olive trees, many of them in monumental groves. The historical land cover that characterized the landscape is currently in a transition phase and can strongly affect climate conditions. This study aims to analyze how the destruction of olive groves by X. fastidiosa affects local climate change. Land surface temperature (LST) data detected by Landsat 8 and MODIS satellites are used as a proxies for microclimate mitigation ecosystem services linked to the evolution of the land cover. Moreover, recurrence quantification analysis was applied to the study of LST evolution. The results showed that olive groves are the least capable forest type for mitigating LST, but they are more capable than farmland, above all in the summer when the air temperature is the highest. The differences in the average LST from 2014 to 2020 between olive groves and farmland ranges from 2.8 °C to 0.8 °C. Furthermore, the recurrence analysis showed that X. fastidiosa was rapidly changing the LST of the olive groves into values to those of farmland, with a difference in LST reduced to less than a third from the time when the bacterium was identified in Apulia six years ago. The change generated by X. fastidiosa started in 2009 and showed more or less constant behavior after 2010 without substantial variation, therefore, this can serve as the index of a static situation, which can indicate non-recovery or non-transformation of the dying olive groves. Failure to restore the initial environmental conditions can be connected with the slow progress of the uprooting and replacing infected plants, probably due to attempts to save the historic aspect of the landscape by looking for solutions that avoid uprooting the diseased plants. This suggests that social-ecological systems have to be more responsive to phytosanitary epidemics and adapt to ecological processes, which cannot always be easily controlled, to produce more resilient landscapes and avoid unwanted transformations.

The purpose of this research was to investigate phytochemical profiles and antioxidant activity in four Salvia species growing in Salento (Southern Italy). The hydrodistillation products obtained from the aerial parts of Salvia clandestina, Salvia fruticosa, Salvia officinalis and Salvia sclarea were characterized by GC–MS and 50 compounds were detected. With the exception of S. clandestina, that did not produce essential oils, the other species shared different amounts of monoterpenes oxygenated (camphor 2.13%-9.16%) and sesquiterpenes hydrocarbons (caryophyllene 4.65%-18.33%; humulene 1.87%-12.39%). The phenolic profiling, analyzed by HPLC ESI/MS-TOF, highlights that S. clandestina is a rich source of danshensu (4.76 mg/g DW) while S. sclarea of rosmarinic acid (15.57 mg/g DW). Mutivariate statistical analysis (PCA) of hitherto studied Salvia phenols have shown similarities in profiles between S. fruticosa and S. officinalis, while S. clandestina and S. sclarea showed distinctive profiles. Otherwise, essential oil profiles analysed by PCA are clearly different among the three productive species. The extracts from collected plants were found to be effective antioxidant in three different in vitro assays (DPPH, ABTS, FRAP and Superoxide anion scavenging activity). Thus, they can be proposed as natural ingredients in functional foods, herbal medicines or as sources of bioactive molecules.

To evaluate the effects on modulation of K+ channel in antiviral treatments, we report the application of potassium channel blockers (PCBs) tetraethylammonium chloride (TEA) and cesium chloride (Cs) in in vitro plantlets or callus cultures infected by Tobacco mosaic virus (TMV). The effect of PCBs were evaluated in: a) in vitro TMV-infection trials, where PCB-treatments were applied during infection process of healthy plantlet; b) chemotherapy trials, where PCB-treatments were applied in combination to antiviral drugs on TMV-infected plantlets; c) in antiviral trials using callus culture and sieving technique. Both PCBs were able to reduce relative concentration of virus [calculated as Fold Changes (FC) relative to control samples] during infection process, with major effect observed at 7 dpi and in medium or upper portions of plantlet. Positive effects of PCBs were also observed following chemotherapy trials, where TEA significantly improves antiviral effectiveness of the antiviral drugs, causing higher FC compared to that obtained with just chemotherapy treatment since 3th subculture. Nevertheless, plantlets were positive to TMV up to 6th subculture, thus virus elimination was not achieved). Finally, the callus culture and sieving technique lead to 11.1 ± 3.8% of TMV-negative plantlets, while TEA-treated mother-calli lead to 22.2 ± 3.8% of negative plantlets. Concluding, findings suggest that modulation of K+, previously indicated as essential in early stages of viral infection, could be also involved in antiviral techniques.

Heavy metal excess in soil represents a critical problem for crop productivity. Among these pollutants, cadmium (Cd) is one of the most dangerous in terms of food-chain contamination. Two durum wheat near-isogenic lines (NILs) and 12 commercial varieties (cultivars Arcangelo, Aureo, Aziziah, Cappelli, Cirillo, Creso, Iride, Maestrale, Parsifal, Russello, Strongfield, and Svevo) of durum wheat were exposed to a nontoxic level of Cd to evaluate its concentration in grains, roots, and shoots, as well as effects on biomass production. Cultivar Iride showed the most interesting behavior because it stored large amounts of Cd in the roots, preventing its translocation to grains. On the contrary, Cirillo and Svevo genotypes were characterized by a high Cd concentration in the grains. Furthermore, a molecular characterization employing the ScOPC20 marker associated with the Cd uptake locus has shown the absence of the expected fragment in the Iride variety and in other varieties characterized by low Cd concentration, as well as the presence of it in high Cd-accumulating cultivars.

Monitoring Xylella fastidiosa is critical for eradicating or at least containing this harmful pathogen. New low-cost and rapid methods for early detection capability are very much needed. Metabolomics may play a key role in diagnosis; in fact, mobile metabolites could avoid errors in sampling due to erratically distributed pathogens. Of the various different mobile signals, we studied dicarboxylic azelaic acid (AzA) which is a key molecule for biotic stress plant response but has not yet been associated with pathogens in olive trees. We found that infected Olea europaea L. plants of cultivars Cellina di Nardò (susceptible to X. fastidiosa) and Leccino (resistant to the pathogen) showed an increase in AzA accumulation in leaf petioles and in sprigs by approximately seven- and sixfold, respectively, compared with plants negative to X. fastidiosa or affected by other pathogens. No statistically significant variation was found between the X. fastidiosa population level and the amount of AzA in either of the plant tissues, suggesting that AzA accumulation was almost independent of the amount of pathogen in the sample. Furthermore, the association of AzA with X. fastidiosa seemed to be reliable for samples judged as potentially false-negative by quantitative polymerase chain reaction (cycle threshold [Ct] > 33), considering both the absolute value of AzA concentration and the values normalized on negative samples, which diverged significantly from control plants. The accumulation of AzA in infected plants was partially supported by the differential expression of two genes (named OeLTP1 and OeLTP2) encoding lipid transport proteins (LTPs), which shared a specific domain with the LTPs involved in AzA activity in systemic acquired resistance in other plant species. The expression level of OeLTP1 and OeLTP2 in petiole samples showed significant upregulation in samples positive to X. fastidiosa of both cultivars, with higher expression levels in positive samples of Cellina di Nardò compared with Leccino, whereas the two transcripts had a low expression level (Ct > 40) in negative samples of the susceptible cultivar. Although the results derived from the quantification of AzA cannot confirm the presence of the erratically distributed X. fastidiosa, which can be definitively assessed by traditional methods, we believe they represent a fast and cheap screening method for large-scale monitoring.

Background Among cereals, durum wheat (Triticum turgidum L. subsp. durum) accumulates cadmium (Cd) at higher concentration if grown in Cd-polluted soils. Since cadmium accumulation is a risk for human health, the international trade organizations have limited the acceptable concentration of Cd in edible crops. Therefore, durum wheat cultivars accumulating low cadmium in grains should be preferred by farmers and consumers. To identify the response of durum wheat to the presence of Cd, the transcriptomes of roots and shoots of Creso and Svevo cultivars were sequenced after a 50-day exposure to 0.5 μM Cd in hydroponic solution. Results No phytotoxic effects or biomass reduction was observed in Creso and Svevo plants at this Cd concentration. Despite this null effect, cadmium was accumulated in root tissues, in shoots and in grains suggesting a good cadmium translocation rate among tissues. The mRNA sequencing revealed a general transcriptome rearrangement after Cd treatment and more than 7000 genes were found differentially expressed in root and shoot tissues. Among these, the up-regulated genes in roots showed a clear correlation with cadmium uptake and detoxification. In particular, about three hundred genes were commonly up-regulated in Creso and Svevo roots suggesting a well defined molecular strategy characterized by the transcriptomic activation of several transcription factors mainly belonging to bHLH and WRKY families. bHLHs are probably the activators of the strong up-regulation of three NAS genes, responsible for the synthesis of the phytosiderophore nicotianamine (NA). Moreover, we found the overall up-regulation of the methionine salvage pathway that is tightly connected with NA synthesis and supply the S-adenosyl methionine necessary for NA biosynthesis. Finally, several vacuolar NA chelating heavy metal transporters were vigorously activated. Conclusions In conclusion, the exposure of durum wheat to cadmium activates in roots a complex gene network involved in cadmium translocation and detoxification from heavy metals. These findings are confident with a role of nicotianamine and methionine salvage pathway in the accumulation of cadmium in durum wheat. Electronic supplementary material The online version of this article (10.1186/s12870-018-1473-4) contains supplementary material, which is available to authorized users.

The colonization behavior of the Xylella fastidiosa strain CoDiRO, the causal agent of olive quick decline syndrome (OQDS), within the xylem of Olea europaea L. is still quite controversial. As previous literature suggests, even if xylem vessel occlusions in naturally infected olive plants were observed, cell aggregation in the formation of occlusions had a minimal role. This observation left some open questions about the whole behavior of the CoDiRO strain and its actual role in OQDS pathogenesis. In order to evaluate the extent of bacterial infection in olive trees and the role of bacterial aggregates in vessel occlusions, we tested a specific fluorescence in situ hybridization (FISH) probe (KO 210) for X. fastidiosa and quantified the level of infection and vessel occlusion in both petioles and branches of naturally infected and non-infected olive trees. All symptomatic petioles showed colonization by X. fastidiosa, especially in the larger innermost vessels. In several cases, the vessels appeared completely occluded by a biofilm containing bacterial cells and extracellular matrix and the frequent colonization of adjacent vessels suggested a horizontal movement of the bacteria. Infected symptomatic trees had 21.6 ± 10.7% of petiole vessels colonized by the pathogen, indicating an irregular distribution in olive tree xylem. Thus, our observations point out the primary role of the pathogen in olive vessel occlusions. Furthermore, our findings indicate that the KO 210 FISH probe is suitable for the specific detection of X. fastidiosa.

Due to its complex epidemiological cycle, including several polyphagous insect vectors and host plants, and the absence of efficient control strategies, Bois Noir (BN) disease of grapevine is encroaching wider territories in the main viticultural areas worldwide. Molecular approaches allowed to increase the knowledge about its etiological agent (Bois Noir phytoplasma, BNp; ‘Candidatus Phytoplasma solani’ species), revealing interesting features concerning BNp population structure and dynamics and transmission routes in vineyard agro‐ecosystems. In the present study, a multilocus sequence typing approach (vmp1 and stamp genes) was utilised for describing the genetic diversity among BNp strain populations in 17 vineyards localised in two distinct geographic areas in Tuscany (central Italy). The results confirmed that BNp ecology in Tuscan vineyards is mainly associated to the bindweed‐related host system, and allowed the identification of 14 BNp vmp1/stamp genotypes. Interestingly, the prevalent genotype (Vm43/St10) was never found in grapevines outside of Tuscany. Moreover, statistical analyses showed significant differences between the composition of BNp strain populations identified in grapevines from north‐western and central‐eastern Tuscany. These results reinforce the hypothesis that distinct geographic areas, probably associated with different ecological niches, can drive the selection of BNp strains, also favouring the entrance of unusual ‘Ca. Phytoplasma solani’ genotypes in vineyards.

Wheat, the main food source for a third of world population, appears strongly under threat because of predicted increasing temperatures coupled to drought. Plant complex molecular response to drought stress relies on the gene network controlling cell reactions to abiotic stress. In the natural environment, plants are subjected to the combination of abiotic and biotic stresses. Also the response of plants to biotic stress, to cope with pathogens, involves the activation of a molecular network. Investigations on combination of abiotic and biotic stresses indicate the existence of cross-talk between the two networks and a kind of overlapping can be hypothesized. In this work we describe the isolation and characterization of a drought-related durum wheat (Triticum durum Desf.) gene, identified in a previous study, coding for a protein combining features of NBS-LRR type resistance protein with a S/TPK domain, involved in drought stress response. This is one of the few examples reported where all three domains are present in a single protein and, to our knowledge, it is the first report on a gene specifically induced by drought stress and drought-related conditions, with this particular structure.

The trans-plasma membrane behavior in virus-infected grapevine leaves was investigated and the effects of six viruses included in European and Italian certification protocols of grapevine on trans-plasma membrane potential (t-PMEP) or electron transport (t-PMET) activity were evaluated. Electrophysiological tests were carried out on leaf samples of virus-infected Vitis vinifera cv. Sangiovese. Microelectrodes were placed in the central zone of the mesophyll for membrane potential measurement, while carbon fiber microelectrodes were used to estimate the membrane reductase activity of virus-infected resting cells. Viruses, the presence of which increased the NADH content, interfere differently with t-PMEP and t-PMET. Those that did not interfere negatively with membrane potential caused an increment in cell reductase activity, while virus-infected samples which showed a stressed status—as suggested by low energy availability and difficulty in the impalement procedure—were characterized by a lower t-PMET activity despite NADH content.

Low frequency tags can be safely implanted in 4 grape hybrids.Tags can be applied to one-year-old grafted rootlings.Hot water treatments against phytoplasmas can be integrated with tagging procedures.Semi-internal implants of UHF tags did not cause concerns about plant health. In order to adapt tracking implants in grapevine to production practices, four rootstocks belonging to two common Vitis hybrids V. berlandieri×V. riparia (420 A, Kober 5BB, SO4) and V. berlandieri×V. rupestris (1103 Paulsen) were tagged with radio frequency tags using the available methods: direct drilling of the pith from the distal cut of the rootstock or a "U" cut performed laterally on the rootstock below the grafting point. Tests were also combined with hot water treatments against phytoplasmas or applied to one-year-old grafted rootlings ready for transplantation in the vineyard to reduce tagging costs. In addition, novel health-compliant methodologies for ultra-high frequency (UHF) tagging were evaluated. To assess the effects of tag implantation in rootstocks, plant viability, functional vascular tissue area and tag reliability were calculated, as well as the effects of phytopathogenic fungi on wounds produced by tagging. The tagging procedure did not cause significant effects on viability and functional vascular tissue area. Tag reliability was set at more than 96%. Fungal infections caused less than 1% of infected vascular tissue area and tagging methods could be integrated with hot water treatments against phytoplasmas. Tracking implants were applied successfully to one-year-old rootlings that were ready for transplantation, even if tag reliability decreased. Novel semi-internal implants of UHF tags did not cause concerns about plant health but tags were exposed to environmental stress or fortuitous damage during farming practices.

To shed light on trends about elimination of viruses from plants, a bibliographic research was conducted to identify thermotherapy, chemotherapy and tissue culture trials published from 1991 through 2010. Among woody plants, grapevine, apple and peach are the most frequent targets of sanitation protocols because their health status is strictly regulated. Even if thermotherapy represents the preferred method for the host, grapevine viruses can also be eliminated with chemotherapy and tissue culture; apple viruses respond to chemotherapy as well. Although a similar trend was reported among herbaceous plants, chemotherapy was the most frequently used technique in potato. With regard to virus, thermotherapy was successfully applied against viruses belonging to 13 families and an unassigned genus. Instead, chemotherapy and tissue culture techniques eradicated viruses belonging to fewer families (nine). An interpretation of thermotherapy effects considers the new metabolic “pathways” triggered by the natural antiviral response emitted by the infected plant, with particular reference to virus-induced gene silencing. With regard to chemotherapy, several groups of antiviral drugs belong to inosine monophosphate dehydrogenase inhibitors, S-adenosylhomocysteine hydrolase inhibitors, neuraminidase inhibitors. Tissue culture, usually adopted to regenerate plantlets in biotechnological breeding programs, represents the less used tool for eliminate viruses from plants. To shed light on trends about elimination of viruses from plants, a bibliographic research was conducted to identify thermotherapy, chemotherapy and tissue culture trials published from 1991 through 2010. Among woody plants, grapevine, apple and peach are the most frequent targets of sanitation protocols because their health status is strictly regulated. Even if thermotherapy represents the preferred method for the host, grapevine viruses can also be eliminated with chemotherapy and tissue culture; apple viruses respond to chemotherapy as well. Although a similar trend was reported among herbaceous plants, chemotherapy was the most frequently used technique in potato. With regard to virus, thermotherapy was successfully applied against viruses belonging to 13 families and an unassigned genus. Instead, chemotherapy and tissue culture techniques eradicated viruses belonging to fewer families (nine). An interpretation of thermotherapy effects considers the new metabolic “pathways” triggered by the natural antiviral response emitted by the infected plant, with particular reference to virus-induced gene silencing. With regard to chemotherapy, several groups of antiviral drugs belong to inosine monophosphate dehydrogenase inhibitors, S-adenosylhomocysteine hydrolase inhibitors, neuraminidase inhibitors. Tissue culture, usually adopted to regenerate plantlets in biotechnological breeding programs, represents the less used tool for eliminate viruses from plants.

Citrus tristeza closterovirus (CTV) has been found several times in the last decades in Italy, and plant protection services are involved in monitoring and surveillance. Although orchards linked to the citrus industry are well monitored, there is an underestimated risk of viruses or virus-like diseases in ornamental nurseries. Our aim was to modify a CTV monitoring program to include other viruses (Citrus variegation virus, CVV; Citrus psorosis virus, CPsV) and viroids (Citrus exocortis viroid, CEVd; Hop stunt viroid, HSVd; Citrus bent leaf viroid, CBLVd; Citrus dwarfing viroid, CDVd; Citrus bark cracking viroid, CBCVd). Ornamental mother plants were monitored for four years in 15 nurseries in two locations in central Italy using inexpensive multiplex RT-PCR protocols. CTV incidence was 1.6–13.5%, with an average distribution of 11.9%. The average incidence of CVV and CPsV was 6.3% and 2.7%, respectively. Higher CTV, CVV and CPsV incidences were observed in C. x paradisi, C. grandis and C. x clementina. The most widespread viroid identified was CEVd (32.9%), frequently observed in C. x limonia and C. limon. HSVd (10.5%), and CDVd (7.1%) were mostly found in C. x limonia. Lower infection rates were observed for CBLVd (2.0%) and CBCVd (1.4%). However, the nurseries’ response to the virus alert by the protection services was only partially effective. Although the CTV incidence was lower in nurseries re-checked after the initial detection, it was not eradicated from two nurseries out of three, and the occurrence of viroids was reduced in just one nursery. Given that dangerous viruses along with the concomitant spread of viroids have unfortunately become a fact of every day life, multiplex RT-PCR diagnoses are likely to play an increasing role in warning nursery managers of possible infections.

The disease outbreak of Xylella fastidiosa subsp. pauca strain CoDiRO (Complesso del Disseccamento Rapido dell’Olivo) in Salento (Apulia, South Italy) associated with severe cases of olive quick decline syndrome may represent not just a new disease paradigm, but a challenge for policy formulation and science communication in plant pathology. Plant health management can be achieved by applying a technocratic model, in which objective science is thought to directly inform policy-making, or via decisionistic or inclusive models, in which scientific considerations drive risk assessment. Each could be applied to X. fastidiosa and CoDiRO strain management, thanks to consistent literature related to pathogen/host interactions, hosts, vectors, and diagnostic tools, reviewed here. However, consensus among stakeholders seems to be necessary in order to avoid plant health management failures or gridlocks, due to environmental, economic, and social implications in the X. fastidiosa threat. Here we discuss the role of consensus in building scientific opinion, reporting different approaches of governance after severe disease outbreaks in Europe. These case studies, and the available risk analysis for Xylella strains, should drive policy formulations towards more cooperative networks.

We have developed a vision-based program to detect symptoms of Olive Quick Decline Syndrome (OQDS) on leaves of Olea europaea L. infected by Xylella fastidiosa, named X-FIDO (Xylella FastIdiosa Detector for O. europaea L.). Previous work predicted disease from leaf images with deep learning but required a vast amount of data which was obtained via crowd sourcing such as the PlantVillage project. This approach has limited applicability when samples need to be tested with traditional methods (i.e., PCR) to avoid incorrect training input or for quarantine pests which manipulation is restricted. In this paper, we demonstrate that transfer learning can be leveraged when it is not possible to collect thousands of new leaf images. Transfer learning is the re-application of an already trained deep learner to a new problem. We present a novel algorithm for fusing data at different levels of abstraction to improve performance of the system. The algorithm discovers low-level features from raw data to automatically detect veins and colors that lead to symptomatic leaves. The experiment included images of 100 healthy leaves, 99 X. fastidiosa-positive leaves and 100 X. fastidiosa-negative leaves with symptoms related to other stress factors (i.e., abiotic factors such as water stress or others diseases). The program detects OQDS with a true positive rate of 98.60 ± 1.47% in testing, showing great potential for image analysis for this disease. Results were obtained with a convolutional neural network trained with the stochastic gradient descent method, and ten trials with a 75/25 split of training and testing data. This work shows potential for massive screening of plants with reduced diagnosis time and cost.

Plant tagging using radiofrequency identification (RFID) microchips is attractive for ornamental shrubs, such as rose (Rosa spp.), due to their high market value, wide distribution, health certification system, and numerous uses. Differently from other woody species for which methods of microchip implantation have been tested, rose tagging requires the possibility of insertion within canes of less than 10 mm diameter, such as those typically found in the rose nursery setting. Moreover, the possibility of tagging after grafting without changing common plant production procedures and aesthetic value are important considerations. Toward this aim, a new method of microchip insertion was developed. To test its effects on roses, two cultivars were subjected to a tagging procedure, and histological observations of tissues around the microchip and growth analysis of plant canes were performed. Microchip implantation did not cause xylem necrosis in 8- to 9-mm-diameter canes, but in lower diameter canes wilt of the lateral shoot and detriments in growth were observed compared with control plants. The tagged roses were tracked by a database developed for rose information, field log, and botanical sheet retrieval. Our findings suggest that rose plants can be safely tagged with a RFID microchip following suitable selection of cane calliper as early as the nursery phase without negative effects on plant appearance.

The health status of the native grapevine Vitis vinifera subsp. sylvestris (Gmeli) Hegi in natural areas in Europe has received little attention. A survey was carried out on wild grapevines in Tuscany (Italy), where isolates of the Grapevine rupestris stem pitting virus (GRSPaV), Grapevine leafroll-associated virus 1 and 3 (GLRaV-1 and GLRaV-3) and Grapevine virus A (GVA) were detected. The complete coat protein (CP) region of these isolates was sequenced to investigate the relationship of the viral variants from Tuscan wild grapevines with isolates from different geographical origins. According to the phylogenetic analyses, GLRaV-1 and GLRaV-3 isolates from Tuscan wild grapevines clustered with isolates from cultivated grapevines with nucleotide sequence identities ranging from 66% to 87% and from 72.5% to 99% respectively, without any correlation between the distribution and geographical origin. Conversely, GRSPaV and GVA isolates clustered together with other Italian isolates from V. vinifera with nucleotide sequence identities ranging from 71.14% to 96.12% and from 73.5% to 92%, respectively. Our analysis of the whole amino acid sequences revealed a high conservation level for the studied proteins explained by a selective pressure on this genomic region, probably due to functional constraints imposed on CP, such as specific interactions with cellular receptors in the insect vectors necessary for successful transmission. In addition, analyses of genetic recombination suggest no significant point mutations that might play a significant role in genetic diversification. The dN/dS ratio also estimated a low number of non-silent mutations, highlighting the purifying selective pressure. The widespread distribution of the Rugose wood complex (GRSPaV and GVA associated disease) in comparison with the Grapevine Leafroll associated viruses (GLRaV-1 and -3) could explain the major geographical correlation found for the viral variants detected in Tuscany.

To evaluate the role of purines in antiviral treatments in plants, ribavirin (RB) and tiazofurin (TZ) were applied in combination with guanosine (GS) or adenosine (AS) in in vitro grapevine or tobacco explants infected by Grapevine leafroll associated virus 3 (GLRaV-3) and Cucumber mosaic virus (CMV), respectively. Using a microelectrochemical (trans-plasma membrane electron transport, t-PMET) technique, in vivo assay of free reduced nicotinamide adenine dinucleotide (NADH) was also carried out to estimate the inosine monophosphate dehydrogenase inhibition caused by drugs. Antiviral effectiveness of TZ, evaluated as virus-free explants or virus copies, was significantly hindered by GS in both species, while AS did not interfere with the drugs. GS, but not AS, slightly hindered the antiviral effectiveness of RB. With regard to NADH tests, t-PMET inhibition caused by RB and TZ was dose dependent and the interference of drugs with the NAD+/NADH conversion was confirmed by NADH content. Findings indicate that exogenous GS up to 0.50 mM replenished the GS pool depleted by drugs, contrasting antiviral action. At higher doses of GS, the TZ antiviral action was completely inhibited and exogenous GS caused a feedback that reduced t-PMET activity. The reversal was partially against RB, suggesting that the reduction of the GS pool contributed to the antiviral activity of RB, but it was not the only cause of antiviral effectiveness.

International audience; Food contamination by toxic pesticides has induced intense research for alternative methods to control pests and diseases. For instance, the phasing out of methyl bromide used for soil disinfection has led to a reconsideration of heat-based methods to control soil-borne pathogens. Techniques such as soil steaming and soil solarization have been applied successfully. However, traditional steaming and solarization are unable to control viruses such as the Tobacco mosaic virus. Thus, methods for sustainable control of virus in soil are not available. Here, we tested the effect of short-duration soil steaming enhanced by the addition of exothermically reacting chemicals—potassium hydroxide or calcium oxide—and soil solarization. These methods were tested against three viruses having different stability: the highly stable Tobacco mosaic virus and the medium stable Potato virus Y, and the Cucumber mosaic virus as non-persistent control. Steaming was done in laboratory and open-field conditions after incorporation of exothermically reacting chemicals. Soils were solarized for 20 days using transparent polyethylene, ethylene–vinylacetate and high-effectiveness infrared films. Our results show that steaming with exothermic chemicals decreased the infectivity of the Tobacco mosaic virus below 3.0 %. We also found that the Tobacco mosaic virus was not controlled by solarization. The Potato virus Y was totally controlled by steam treatments and by soil solarization with ethylene–vinylacetate or high-effectiveness infrared films. Overall, our findings evidence promising ways to control soil viruses.

virus sanitation; in vitro explants; antiviral mechanism; virus longevity.

Antiviral drugs have demonstrated significant therapeutic potential against phytoviruses, such as inosine monophosphate dehydrogenase inhibitors like mycophenolic acid or thiopurines. However, drug delivery across cellular barriers is a challenging task that calls for investigation. In this study, membrane transport of three antiviral drugs such as mycophenolic acid (MPA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) was investigated in order to determine the mechanism involved in transmembrane flux. Electrophysiological tests were carried out on leaves of in vitro grapevine explants naturally infected by Grapevine leafroll associated virus 1 as well as virus-free explants treated with antiviral drugs. Results indicate that membrane electrical responses of the tested antiviral dugs are supported by the metabolism, and virus infection did not cause differences in membrane potential tests. The extent of depolarization was slight for MPA and 6-TG and greater for 6-MP. Complete repolarization always occurred and, in many cases, the electrical potential was greater than the starting level (overshoot). Treatments at 5 °C or in presence of CCCP, an uncoupler of oxidative phosphorylation, led to inhibition of drug effects of the membrane potential, suggesting a metabolic dependence of the transport. However, treatments carried out under sodium orthovanadate, a specific inhibitor of H+-ATPase, prove, although only partially, the activity of a proton pump. The complex of electrophysiological tests highlights that drugs such as MPA, 6-MP and 6-TG, when applied to infected or healthy grapevine leaf segments, lead to clear, biphasic, electrical membrane response suggesting an operation of H+ co-transport through systems that link substrate translocation across the membrane to the free energy available in a proton electrochemical potential difference.

Mycophenolic acid (MPA) is an inosine monophosphate dehydrogenase inhibitor whose antiviral mechanism of action is supposed to interfere with NAD + /NADH conversion. Its effects on trans-plasma membrane electron transport (t-PMET) and on trans-plasma membrane electric potential (t-PMEP), which are involved in the NAD + /NADH conversion, were investigated using microelectrochemical techniques in tobacco plants infected by Cucumber mosaic virus . In these tests, ferricyanide (Fe 3+ ) was used as electron acceptor in assays performed with intact cells; ferricyanide is converted to ferrocyanide (Fe 2+ ) by one-electron reduction, and the rate of this reduction can be monitored in order to investigate the effects on t-PMET or t-PMEP. Considering tests on t-PMEP, MPA treatment of samples induced membrane depolarization and this effect was greater in healthy samples compared to infected ones. In any case, complete repolarization was achieved, indicating no irreversible damage to the membrane due to MPA administration. Moreover, in samples pre-treated with MPA, the extent of depolarization caused by Fe 3+ administration was lower than in samples without pre-treatment but the MPA effect was not related to virus infection. With regard to tests on t-PMET, MPA caused a reduction in Fe 3+ /Fe 2+ conversion compared to untreated plants. However, infected samples were less sensitive to MPA treatment, which may be due to the concurrent entry of MPA within the symplast that, as indicated by t-PMEP tests, was lower in infected samples. In conclusion, MPA interferes with membrane activity linked to NAD + /NADH conversion, acting differently in infected or healthy samples during drug uptake by cells.

The Prunus genus represents one of the main targets for regulating the health status of plants. Individual plant tagging with an implanted microchip may support plant certification or protect property rights, in addition to having research applications. Production of radiofrequency identification (RFID) implants in Prunus spp. plants was performed following two tagging procedures designed to consider Prunus anatomy, histology and grafting techniques. To assay the impact of microchip insertion, measurements of vascular tissue area by image analysis and histological observation were made, confirming the good healing capability of the plant. Moreover, neither production losses nor reduction in growth were noted, suggesting Prunus spp. as a suitable host for microchip implanting. An online database was developed to match microchips with information datasheets that can be read and updated by users to manage the marked plants. The present work concludes that implanted RFID microchips can be efficiently used for identifying, storing and tracking plants to support certification.

Different chemotherapeutic strategies on closely correlated phytoviruses, such as Grapevine leafroll-associated virus-1 (GLRaV-1) and -3 (GLRaV-3), were tested in the same host in order to investigate selective chemotherapy of virus infections in plants. To eradicate these viruses from grapevine in vitro explants, antiviral treatments using heat or chemical drugs such as inosine monophosphate dehydrogenase (IMPDH) inhibitors (ribavirin, tiazofurin), purine biosynthesis inhibitors (6-thioguanine) and neuraminidase inhibitors (oseltamivir) were conducted. Phytotoxicity assay and thermal stress tests were performed before treatments; viruses were detected using ELISA and reverse transcriptase–polymerase chain reaction (RT-PCR). All chemicals were able to produce virus-free explants, but showed significantly different sanitation rates considering virus type. For GLRaV-1, higher sanitation rates were obtained using IMPDH inhibitors: 72.0% eradication was obtained upon administration of an exclusive inhibitor such as tiazofurin and 40.0% when using ribavirin. The most effective drugs against GLRaV-3 did not belong to IMPDH inhibitors: 78.0% virus-free explants were obtained using a neuraminidase inhibitor and 75.1% explants were sanitized using a purine biosynthesis inhibitor. Conversely, heat treatments showed similar efficacy against both viruses, achieving more than 55.0% virus-free explants, with a non-selective effect between GLRaV-1 and GLRaV-3. These findings suggest how highly correlated viruses could be differentiated in virus-specific events that are linked to selective chemotherapy of virus infections. However, virus-specific events that can influence chemical treatment generating selectivity did not seem to interfere with physical treatment.

Interesting applications for traceability in agriculture have recently been developed using radiofrequency identification (RFID) technology. A preliminary report of survival and growth in grapevine suggested pith not only as optimal microchip localization within the plant, but only continuous monitoring of performances, supported by histological observation of tissues around microchips, can validate this approach as a long-term strategy for grapevine identification. In this study, histological assays of grapevine plants are reported, considering different strategies in RFID marking. Microchip insertion after direct drilling of pith from a distal cut on rootstocks did not show any differences in tissue status compared to control, and this can be adequately correlated to an absence of effect in plant growth. Conversely, a “U” cut performed laterally on the rootstock to insert the microchip, which involved tissues from bark to pith, caused development of callus tissues, restoring transversal continuity, but with a partial loss of functionality in terms of open vessels. This phenomenon can be considered permanent damage to plant vascular function, but with limited extension.

The results of the grapevine clonal selection carried out in Tuscany in the last seven years not only provided evidence of a high concentration of viral infections, but also showed that the sanitary status of the variety Aleatico is extremely worrying. The sanitary tests conducted on 172 plants, selected in the Elba Isle, on the hills around Lucca and in Maremma region showed a critical phytovirological condition. In fact, only 5 plants passed the first sanitary screening, being found free of all viruses investigated (GFLV, ArMV, GFkV, GLRaV-1, GLRaV-3, GLRaV-7, GVA and GVB). The candidate clones selected in Maremma showed remarkable differences in their morphological traits, both from one another ('ALESCA 59' and 'ALESCA 60' on one side, and 'ALCHI V on the other), and from those that had been identified on the Elba Isle ('ALE 102' and 'ALE 119'). The differences found among the 3 biotypes were statistically confirmed by cluster analysis procedures concerning the OIV traits of the shoots, leaves and bunches, and through the results of preliminary DNA analyses conducted in 6 micro-satellite loci. The general analysis of the available data showed the following decreasing scale of virus spreading values for the variety Aleatico (for the studied viruses): GLRaV-3>GLRaV-l>GVA>GFLV>GFkV. ArMV, GLRaV-7 and GVB have never been found. Differences in the infectious combinations ascertained in the 3 areas of selection are particularly interesting. The presence of mixed infections did not affect the vines that had been selected in the Maremma region and only slightly affected those from the Lucca hills, while 16 different combinations were found for the Elba Isle. The worrying sanitary status of Aleatico and the lack of registered clones of this variety seem to attach great importance to the results of this work, even if still quantitatively poor, for the improvement of the island's vine-growing activity along with the promotion of Aleatico wine, whose excellent organoleptic features and uniqueness are well-known.

SUMMARY The search for non-chemical methods to control soil-borne pathogens has recently intensified ahead of the forthcoming phase-out of the fumigant methyl bromide in 2005. Indeed, none of the previously available alternatives match methyl bromide wide-spectrum control. Here, we tested two pre-planting treatments: steaming with a soil-steaming machine and exothermic reaction by incorporation into the soil of potassium hydroxide (KOH), a substance that causes an exothermic reaction with water. Experiments were conducted from 2003 to 2005 in open-field conditions by assessing the effectiveness of steam and exothermic reaction against Fusarium oxysporum f.sp. basilici and Sclerotinia minor on basil. The results show that steam combined with exothermic reaction reduce the incidence of Fusarium wilt (77–96%) better than steam only (70–89%). The control of Sclerotinia minor was better with potassium hydroxide (94–92%) than with steam only (83–87%). Further, we evaluated the effects of the treatments on microbial population, yield and weed control. Here, the treatments induced the following major effects: (1) a reduction of more than 99% of the number of Fusarium oxysporum colonies in soil; (2) an increase of about 200% in Trichoderma spp. in soil; (3) a drastic increase in the plant yield, from 6.9–7.5 g for the control to 23.0–25.5 gr for steam treatments; (4) a high reduction of plants per square meter of 94–95% for Amaranthus retroflexus, of 96–99% for Portulaca oleracea, of 93–94% for Chenopodium spp. and of 86–90% for Hordeum vulgare, using both steam and exothermic reactions. Similar results were obtained with steam only. Therefore, our results evidence the potential of this approach to control various soil-borne pathogens. Steam and exothermic reaction are thus promising alternatives to chemical soil disinfestation for high-value crops.

Xylella fastidiosa subsp. pauca strain CoDiRO, a pathogen responsible for Olive Quick Decline Syndrome (OQDS), is strongly threatening the agricultural-based economy of South Italy and making its typical landscape collapse. The bacteria can also infect more than other twenty woody or shrub species and quarantine programs are carried out in Italy. Since symptoms of OQDS like leaf scorching and wilting of canopy may appear several months after infection and some hosts are asymptomatic, a tool for the rapid and early screening of plants is desirable, in order to plan a sudden control strategy and apply programs for pest management. X. fastidiosa detection is usually performed by ELISA and PCR methods. In this work, the two standard methods are compared with an innovative on-chip detection strategy for X. fastidiosa assay from leaves samples, based on an electrochemical transduction method. The realized lab-on-chip includes also a microfluidic module and its performances are competitive with conventional diagnostic methods in terms of reliability, but with further advantages of portability, low-costs and ease of use. Thus, the proposed technology has the potential to provide a useful assay method for large-scale monitoring programs.