Enrico Rinaldelli

Effect of mycophenolic acid on trans-plasma membrane electron transport and electric potential in virus-infected plant tissue

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.

In vivo inhibition of trans-plasma membrane electron transport by antiviral drugs in grapevine.

Electrophysiological techniques were applied to investigate the action of antiviral drugs during trans-plasma events in in vivo grapevine cells infected by GLRaV-1 and GLRaV-3. Carbon fiber microelectrodes and redox-sensitive dyes were used to measure trans-plasma membrane electron transport (t-PMET) activity in healthy and infected samples treated with ribavirin, tiazofurin and oseltamivir. Each drug caused a reduction in oxidation current (expressed as Δ[Fe2+]) in healthy samples, indicating t-PMET inhibition. In almost all infected samples, the effect of drugs on t-PMET activity was significantly lower, suggesting that higher content of NADH in infected plants can interfere with t-PMET inhibition caused by drugs. Moreover, virus-infected samples exhibited elevated t-PMET activity compared to healthy samples. Analogous effects were observed by dye tests. Considering the effects of drugs on trans-plasma membrane potential, tests showed the activity of a proton pump during drug treatments with no significant difference with regard to health status.

Virus interference with trans-plasma membrane activity in infected grapevine leaves

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.

Propagative material of grapevine: RFID technology for supporting traceability of "basic" and "certified" material along the wine production chain

Four main categories of propagative material in the European Union (EU) have been indentified for grapevine: the primary source, pre-basic, basic and certified material. Each type of material has to be periodically assayed for patho- gens and each material stage is intrinsically characterized by increasing risks. Radiofrequency (RFID) can be considered an efficient and durable traceability system to provide retrieval of propagated material or check producer identity. RFID tagging of grapevines of different categories along the production line could establish a durable link between stakeholder and products. To evaluate this approach, histological observations and growth parameters of basic or certified RFID- tagged grapevine were performed, as well as requirement analysis for management of sanitary checks and for traceability of the wine production chain. Basic material can be safety tagged with RFID tags to establish mother plant vineyards; derived certified material can also safely be tagged. No detrimental performance in terms of vegetative growth and bud production were reported for mother plant vineyards from the first year of production life. Requirement analysis made it possible to individuate traceability objectives, materials and stakeholders involved, as well as the RFID-tagging steps and methods to collect sanitary and production data that are useful for traceability purposes.

Electronic identification systems for reducing diagnostic workloads after disease outbreak

Diagnostic tests for grapevine viruses subjected to phytosanitary rules involve a heavy workload for plant protection services and laboratories. Propagation schemes enable nurseries, where mother plants (MPs) are cultivated, to be linked to batches of certified plants (CPs). This approach entails post-production checks of MPs once infection occurs in CPs. However, this traceability system is not tight and follow ups are demanding. This study assessed radio frequency identification (RFID) tagging of plants in terms of its ability to reduce laboratory workloads for nursery health checks. RFID-tagged plants (RFID-CPs) were produced from individually tagged MPs (RFID-MPs) or row-tagged MPs (RFID-ROW, a less expensive approach). In a 10-year case study, the health status of CPs and RFID-CPs were assessed and the occurrence of infections then led to health checks in MPs, RFID-MPs or RFID-ROWs. Laboratory workloads were evaluated by considering two sampling methods (single or pool sampling). Using single sampling, the workload was reduced by 93–98% in RFID-ROW or RFID-MP checks compared to the conventional approach. Considerable reductions in workload due to the tagging system (93–96%) were also observed using pool sampling. Traceability of CPs and MPs using RFID reduces laboratory workloads, and supports emergency measures that can be taken to stop any unsafe sales of plants after a virus outbreak.

Insight on trans-plasma membrane behavior of virus-infected plant cells

Little is known about the ion fluxes generated during plant-virus interactions, despite significant losses caused by viruses to agricultural crops. Changes in average ion currents were identifying an early event in the signal transduction pathway related to virus/host interaction. While significant decrease in the average inward currents, mainly due to Ca2+ moving into the cell was observed, the role of potassium may be significant. Host specific K+ efflux with a concomitant decrease in the intracellular K+ was observed in tobacco plants during the early minutes after infection, suggesting many hypothesis about the role of potassium in host-virus interaction. In the last years, trans-plasma membrane potential was evaluated for some viruses, observing as effect on membrane was different in relation to virus infection and host. Conversely, settle virus infection generally lead to an increase of activity in trans-plasma membrane electron transport.

Biosecurity of kiwifruit plants: effects of internal microchip implants on vines for monitoring plant health status

Abstract The sanitary condition and traceability of kiwifruit cuttings can represent a key point for pest control in the production of planting material. In recent years there has been concern about the rise of bacterial canker and thus investigation of systems to improve plant biosecurity. Radio frequency identification (RFID) microchips were implanted in 1- or 2-year-old Actinidia deliciosa in order to develop a system for plant traceability able to record health status, certifications or sanitary assay. To assess the health of implanted plants, histological observations were performed, while functional vascular tissue area and mean relative growth rate were evaluated. A small calibre trunk (5–7 mm) did not allow microchip implantation. Conversely, larger trunk diameters seem to sustain microchip positioning. Data reading tests were performed to evaluate the access to sanitary data, and RFID accuracy and reliability were satisfactory. Requirement analysis for the kiwifruit plant nursery chain was performed to help develop a computer-based information system able to record and retrieve sanitary data from each plant. Requirement analysis made it possible to identify traceability objectives, materials and stakeholders involved, as well as RFID-tagging steps and methods to collect information and matching data.