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Andrea Luvisi
Ruolo
Ricercatore a tempo determinato - tipo B
Organizzazione
Università del Salento
Dipartimento
Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali
Area Scientifica
Area 07 - Scienze agrarie e veterinarie
Settore Scientifico Disciplinare
AGR/12 - Patologia Vegetale
Settore ERC 1° livello
Non Disponibile
Settore ERC 2° livello
Non Disponibile
Settore ERC 3° livello
Non Disponibile
Table olives are a common and well-known food in the whole Mediterranean area, produced and consumed in great quantities. Many deepgreen olives can be found on sale in the South of Italy. Sometimes a deep color could be the result of the fraudulent addition of a coloring agent (E141ii, copper chlorophyllins) during the pickling process, in spite of the European Union legislation that does not allow the addition of any colorant to fruits included table olives. The objectives of this study were to use a relatively simple method of detection of E141ii added to table olives, to verify the presence on the Italian market of artificially colored table olives, and to show that also CuSO4 can be employed for table olive re-greening. Compounds with chromatographic and spectral characteristics similar to the ones from the E141ii (Cu chlorin e6, Cu isochlorin e4, Cu pyropheophorbide a) were found in 8 samples out of 16. These results show that the fraudulent addition of colorant to table olives is a quite common practice. More pressing controls and analysis are required to ensure the complete food safety and the compliance with the current law.
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.
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[Fe(2+)]) 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.
Therapiddevelopmentofnewtechnologiesandthechanginglandscapeoftheonlineworld (e.g., Internet of Things (IoT), Internet of All, cloud-based solutions) provide a unique opportunity for developing automated and robotic systems for urban farming, agriculture, and forestry. Technological advances in machine vision, global positioning systems, laser technologies, actuators, and mechatronics have enabled the development and implementation of robotic systems and intelligent technologies for precision agriculture. Herein, we present and review robotic applications on plant pathology and management, and emerging agricultural technologies for intra-urban agriculture. Greenhouse advanced management systems and technologies have been greatly developed in the last years, integrating IoT and WSN (Wireless Sensor Network). Machine learning, machine vision, and AI (Artificial Intelligence) have been utilized and applied in agriculture for automated and robotic farming. Intelligence technologies, using machine vision/learning, have been developed not only for planting, irrigation, weeding (to some extent), pruning, and harvesting, but also for plant disease detection and identification. However, plant disease detection still represents an intriguing challenge, for both abiotic and biotic stress. Many recognition methods and technologies for identifying plant disease symptoms have been successfully developed; still, the majority of them require a controlled environment for data acquisition to avoid false positives. Machine learning methods (e.g., deep and transfer learning) present promising results for improving imageprocessingandplantsymptomidentification. Nevertheless,diagnosticspecificityisachallenge for microorganism control and should drive the development of mechatronics and robotic solutions for disease management.
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.
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.
Recently, Xylella fastidiosa was reported in Italy, associated with the "Olive Quick Decline Syndrome". The cv. Leccino exhibits an evident tolerance with a slow disease progression compared with the other cultivars. Between the mechanisms proposed to explain the putative tolerance of some hosts to X. fastidiosa diseases, lignin deposition plays an important role. Analysis of phenolic compounds in healthy and infected Leccino and Cellina di Nardò leaves showed, in the two cultivars, a reduction of hydroxytyrosol glucoside (usually associated with drought and cold stress) and, only in Leccino, an increase of quinic acid, precursor of lignin. To determine if lignin biosynthesis is involved in defence response, we investigated the expression of genes coding for entry-point enzymes in different branches of the phenylpropanoid pathway. In stems of Cellina di Nardò infected plants, Cinnamate-4-Hydroxylase (C4H) and 4-Coumarate:CoA Ligase (4CL) resulted strongly down-regulated, indicating a plant disease response since the inhibition of C4H is reported to promote the accumulation of benzoic acid and salicylic acid as defence signals. Instead, in the cv. Leccino, Cinnamoyl-CoA Reductase (CCR, reported to be strongly induced during the formation of lignin defence response associated) was up-regulated in the stem of infected plants; moreover, Polyphenol oxidase (PPO), coding for an enzyme involved in the hydroxytyrosol biosynthesis, was down-regulated. The quantification of lignin in healthy and infected branches of both cultivars, showed a significant increase of total lignin in infected Leccino compared with the sensitive cultivar; moreover, histochemical observations of stem sections exhibited a different lignin distribution in the sclerenchyma and in the xylem tissue of infected Leccino plants compared to sections of healthy ones. Results suggest a critical role for lignin in X. fastidiosa tolerance of cv. Leccino.
L'obiettivo della proposta è l'individuazione di accessioni di O. europaea resistenti (o tolleranti) al patogeno X. fastidiosa, da individuarsi nell'ambito del germoplasma olivicolo del Salento. L'attività prevede cinque workpackage (WP) principali: WP1. Monitoraggio delle superfici olivetate delle Province di Lecce e Brindisi. Tale azione sarà svolta con particolare riferimento alle aree territoriali presumibilmente colpite dalla batteriosi da tempi più remoti (vedi Gallipoli e comuni limitrofi). L'attività è finalizzata alla ricerca di accessioni di olivo asintomatici e/o con ridotta presenza di sintomi di disseccamento che abbiano vissuto più stagioni vegetative in territori compromessi dalla presenza della malattia. In tali soggetti, perciò, il cui quadro sintomatologico non sarebbe potenzialmente imputabile allo sviluppo erratico del patogeno e/o ad una alternanza dei sintomi, tipica delle fasi iniziali dell'evento infettivo, ma potrebbe rappresentare un effettivo indizio di tolleranza o resistenza. Le attività di monitoraggio saranno condotte nella zona infetta e saranno attuate con la collaborazione di esperti agronomi e tecnici individuati dal Soggetto Promotore, interessando i seguenti comuni della Provincia di Lecce e Brindisi. Inoltre, sempre di concerto con esperti agronomi e tecnici con comprovata conoscenza del territorio, sarà predisposta una azione di contatto con gli olivicoltori al fine di raccogliere indicazioni relative ad accessioni di interesse, finalizzate ad ulteriori azioni di monitoraggio e campionamento mirate. A seguito dell'individuazione di soggetti di interesse, questi saranno oggetto di una attività di catalogazione finalizzata a registrarne: • posizione geografica (georeferenziazione) • scala patometrica • descrizione del soggetto, con particolare riferimento a eventuali altre fitopatologie • immagini Le accessioni saranno oggetto di campionamento di materiale fogliare e legnoso in osservanza delle orme vigenti, con successivo trasferimento presso il laboratorio di Patologia Vegetale del DiSTeBA. In tale sede, i campioni biologici saranno oggetto di analisi diagnostica mediante real time PCR al fine di accertarne lo stato sanitario e quantificare lo stato infettivo. Analogo processo interesserà almeno un soggetto limitrofo a quello indagato (fenotipicamente analogo o, se assente, rappresentativo dell'area di coltivazione), in modo tale da accertare le condizioni fitosanitarie del sito e procedere ad eventuali comparazioni. WP2. Studio delle alterazioni metaboliche e modifiche istologiche coinvolte nei meccanismi di resistenza. L'invasione ed il consolidamento di X. fastidiosa all'interno di olivo dipendono dalla capacità del batterio di muoversi efficacemente tra i vasi xilematici e di colonizzare sistemicamente l'ospite. Tale abilità è contrastata nella varietà resistenti, nelle quali è contenuta la diffusione e la proliferazione del batterio nei tessuti. Questa azione progettuale prevede l'indagine dei meccanismi che sottintendono tale azione di contrasto, indagando le alterazioni metaboliche a carico dei processi di formazione del legno. L'obiettivo è quello di determinare differenze istologiche ed anatomiche a carico del tessuto xilematico (siano esse costitutive od indotte da infezione) nelle accessioni selezionale a seguito del WPl, e comprendere come queste possano influenzare la suscettibilità a X. fastidiosa, con particolare riferimento alla produzione da parte della pianta di composti che modulano la morfologia dello xilema, creando barriere fisiche che limitano la fase esplorativa del batterio, come l'accumulo di pectine, lignine e gomme. Attraverso l'impiego di microscopi elettronici a scansione/fluorescenza saranno condotte indagini anatomiche (lunghezza, diametro, distribuzione e connettività dei vasi xilematici) e istochimiche (colorazioni specifiche per determinare la natura chimica dei componenti vascolari) su cultivar suscettibili/resistenti, definendone i profili metabolici (es. accumulo di composti coinvolti nell'alterazione dello xilema come precursori della lignina). WP3. Studio dei processi di colonizzaziorìe in accessioni resistenti. Le accessioni oggetto dello screening saranno inoltre processate mediante analisi FISH (Fluorescent In Situ Hybridization) che utilizza sonde marcate disegnate su specifiche regioni del DNA batterico. L'analisi, altamente innovativa, rappresenta una combinazione di approcci molecolari abbinati alla microscopia che ottimizza l'esplorazione degli habitat microbici e permette l'osservazione dell'interazione ospite/patogeno in situ. Grazie alla specificità di riconoscimento del batterio nei tessuti, la FISH può rappresentare anche una efficiente tecnica di microscopia diagnostica utile per azioni di early detection del patogeno. L'identificazione diretta mediante FISH consentirebbe la visualizzazione con riconoscimento diretto e specifico del patogeno, oltre ad una valutazione quantitativa del grado di colonizzazione del tessuto infetto, attraverso l'impiego di tecniche di imaging associate ad analisi statistica dedicata, particolarmente utile per comprendere i processi di colonizzazione in piante potenzialmente resistenti o tolleranti. WP4. Riconoscimento varietale del germoplasma e caratterizzazione di endofiti. Le accessioni mostranti resistenza o tolleranza verso X. fastidiosa saranno oggetto, insieme ad opportuni controlli suscettibili, di analisi con marcatori molecolari. In particolare, il fmgerprinting sarà condotto utilizzando marcatori SSR riportati in letteratura su olivo (es. DCA 05, OCA 09,DCA17, DCA 18, GAPU 71B, GAPU 101, EMO 90) e/o marcatori SNP basati su tecniche di sequenziamento di nuova generazione (es. GBS). 1 dati molecolari saranno integrati con descrittori morfologici che comprendono caratteri qualitativi (nel caso dell'olivo vi sono la forma della foglia, forma del frutto e dell'endocarpo, etc.), quantitativi (pezzatura della drupa) e biologici (la resistenza della pianta ad agenti patogeni). Infine, in soggetti selezionati, saranno effettuate analisi finalizzate a caratterizzare la presenza di endofiti fungini e batterici mediante procedure di metabarcoding. WP5. Analisi dei dati e relazione finale. I dati collezionati nell'ambito dei WP1-WP4 saranno oggetto di analisi al fine di verificarne l'affidabilità scientifica e saranno oggetto di una relazione finale. Le accessioni selezionate saranno incluse in successivi piani di monitoraggio e ricerca condotti da parte del Soggetto Proponente ed altri Enti interessati.
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