Alessandra Panattoni

Selective chemotherapy on Grapevine leafroll-associated virus-1 and -3

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.

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.

RFID temperature sensors for monitoring soil solarization with biodegradable films

RFID sensors are effective for real-time temperature monitoring during solarization.Lesions of biodegradable film can be highlighted by RFID temperature assessment.Easy-to-use monitoring tools help the farmer to understand the thermal effect. Soil-borne pathogen and weed control can be achieved by soil solarization even if estimation of time treatment is difficult to assess. Thus, due to dependence to environmental conditions and the need to minimize the time of treatments, the implementation of monitoring tools may help in solarization managements, especially when biodegradable films were applied or weather condition are subjected to significant variation. Digitalization of data relative to plants thanks to RFID applications has been used for health or treatment monitoring, sample collecting and retrieving sanitary information: this paper presents the testing of RFID sensor application for soil solarization purposes. Different matrices were selected to assess RFID temperature sensors performances. Sandy, loam and clay soils with different moisture-holding capacity were selected for sensor burial. Sensors were covered by 5 or 10cm of fresh matrix and read immediately. Reliability was found to be more than 90% in all tested conditions, while higher failure in tag reading was recorded in clay soil at 90% of moisture-holding capacity (-7% of tag reliability). Soil solarization treatment was carried out as case of study during a period characterized by changeable weather using a biodegradable film. Data, expressed as thermal addition and temperature classes, collected continuously by sensors permitted to design real-time graphs that help the farmer to understand the thermal effect caused by treatment. Throughout the second and third week of treatments, Tmax at 5cm depth is increased by 9-13?C or 11-14?C compared to environment, respectively. Otherwise, Tmax at 10cm depth is increased by 7-9?C compared to environment throughout the second and third week, showing as sensors are able to collect temperature during solarization. The soil microbial community of soils treated with solarization exhibited a slight reduction of cumulative carbon metabolic activity compared to control (8.8% of reduction), while among 31 preselected carbon sources, the soil microbial communities were capable of utilizing up to 23 carbon source without difference between treatments. Unified Modeling Language activity diagrams for solarization management via digital sensors were designed and effects of biodegradable film on microbial population were observed. The integration of information technology solutions with new-generation biodegradable films may offer an interesting revaluation of soil solarization in actual farm organization.

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.

Synthesis of PAMAM Dendrimers Loaded with Mycophenolic Acid to Be Studied as New Potential Immunosuppressants

The terminal N-Boc protected diamino PAMAM 7 was condensed (EDC-DMAP) with two units of mycophenolic acid (MPA) giving the N-Boc protected dendron 8 in a good yield (76%). The ammonium trifluoroacetate 9 was prepared from 8 by acid treatment (TFA-THF-H2O) and was split into two equal parts. The first half was treated with di-2-pyridyl thionocarbonate (DPT) in the presence of Et3N to give the corresponding isothiocyanate 10. This was reacted with the second half of 9 providing the symmetrical dendrimer 11 (68% yield), exposing four MPA units around the thioureido-PAMAM core.

FIRST REPORT OF ZANTEDESCHIA MILD MOSAIC VIRUS ON ZANTEDESCHIA AETHIOPICA (L) SPRENG IN ITALY

Calla lily [Zantedeschia aethiopica (L.) Spreng] has become one of the most popular cut flowers worldwide. It has been reported as the natural host of various plant viruses, including potyviruses such as Bean yellow mosaic virus (BYMV), Dasheen mosaic virus (DsMV), Turnip mosaic virus (TuMV) and Zantedeschia mosaic virus (ZaMV). In 2005 a new potyvirus named Zantedeschia mild mosaic virus (ZaMMV) was identified in Taiwan (Huang and Chang, 2005). In 2012, 15 plants of Calla lily cultivated in Tuscan farms showed leaves with yellow spots and stripes, green islands and an unusual mild mosaic. Seventeen samples (15 symptomatic and two symptomless) were collected and assayed by ELISA for BYMV, DsMV, TuMV, ZaMV and potyviruses using antisera produced by DSMZ (Braunschweig, Germany) and LOEWE Biochemica (Sauerlach, Germany). Plants were positive for an anti-potyvirus group monoclonal antibody. Positive samples were assayed by reverse transcription-polymerase chain reaction for ZaMMV using total RNA extracted from leaves and specific primers for the coat protein gene (Wen-Chi et al., 2010). Amplicons of the expected size (792 bp) were obtained for 15 samples that reacted positively to the potyvirus antibodies, while no amplification was observed in symptomless samples. The sequence obtained from one ZaMMV amplicon (accession No. KF156666) had 99% nucleotide identity with the corresponding fragment of a reference ZaMMV isolate (GenBank accession no. AY626825.4). To our knowledge this is the first report of ZaMMV on Zantedeschia aethiopica in Italy.

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.

On the effects of exposure to realistic sulfur dioxide levels on six host/pathogen combinations

Abstract The effects of long‐term exposure to a gradient of realistic concentrations of sulfur dioxide (up to 135 ppb) on six host/pathogen combinations were investigated under experimental conditions. No visible symptoms of injury were induced by the treatments on the host plants. The results obtained were quite conflicting and did not show an overall trend in the interactions. No significant effect of the pollutant gas was detected on Alternaria raphani on Raphanus sativus (radish) and Rhizoctonia solani on radish and Cucumis sativus (cucumber). A linear inhibition of parasitism was observed in Oidium tuckeri on Vitis vinifera (grape); a mixed effect (stimulation in the range 0–75 ppb, followed by an inversion of this effect in the 75–135 ppb range) was observed for the rust fungus Puccinia recondita on Triticum aestivum (wheat); a linear stimulation of Colletotrichum trifolii was assessed on Trifolium repens (clover). The mechanisms responsible for these interactions and the practical implications of t...

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.