Andrea Luvisi

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.

Electronic identification technology for agriculture, plant, and food. A review

Automation in agriculture should improve plant health, product quality, and production efficiency. However the actual use of electronic identification tools in agriculture is limited. Therefore, I review here electronic identification applications to support plant health and production and agricultural sustainability. The major points are as follows: (1) there is a tenfold increase of literature on the application of radio frequency identification in agriculture from 2000–2004 to 2005–2009. (2) Development of quick response code and radio frequency identification solutions are improving automated systems. (3) There is a major advancement in associating thermal sensors to electronic tags to preserve food quality and to manage temperature-controlled supply chain. Whereas tests with biosensors used for biological or chemical alerts are limited. (4) Agrochemical tagging, using radio frequency identification tags, improve plant health management and environmental monitoring. (5) While phytosanitary certifications are mandatory in high-cost cultivation systems and a need for risk management may promote radiofrequency identification (RFID) systems, the lack of specific economic analyses may discourage farmers and investors.

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.

Cadmium Concentration in Grains of Durum Wheat (Triticum turgidum L. subsp. durum)

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.

X-FIDO: An Effective Application for Detecting Olive Quick Decline Syndrome with Deep Learning and Data Fusion.

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.

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.

Control of Soil-Borne Diseases in Tomato by Use of Steam and an Exothermic Reaction

In 2005-2006 we tested pre-planting treatments applied using a self-propelled soil steaming machine designed for the release of steam after incorporation in the soil of a substance that causes an exothermic reaction able to heat it to a mild but effective temperature x time product. Experiments were conducted in open-field conditions by assessing the effectiveness of steam and potassium hydroxide (KOH) against Fusarium oxysporum f.sp. lycopersici and Sclerotium rolfsii on tomato plants. The combination of steam and exothermic reaction chemicals reduced the incidence of Fusarium wilt (93.4-94.8%) better than steam only (77.8-81.2%); control of Sclerotium rolfsii was likewise better with KOH (86.7-87.7%) than with steam only (65.0-71.0%). The effects of treatments on microbial density, plant growth and weed control were evaluated. Treatments caused significant reductions in the number of Fusarium oxysporum colonies compared to the untreated control, an increase in Trichoderma spp. and no significant effects on total fungus and actinomycete density. Significant reduction in weeds (above 82.5%) and drastic increase in plant growth was recorded with the use of steam/exothermic reaction. With a milder steam application, the exothermic reaction acts for a slight increase in Tmax and, most of all, on time in temperature x time product, obtaining a disease control similar to drastic steam treatments.

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.