The thesis aims to expose the advances achieved in the practices of captive breeding of the European eel (Anguilla anguilla). Aspects investigated concern both approaches livestock (breeding selection, response to hormonal stimulation, reproductive performance, incubation of eggs) and physiological aspects (endocrine plasma profiles of players), as well as engineering aspects. Studies conducted on various populations of wild eel have shown that the main determining factor in the selection of wild females destined to captive breeding must be the Silver Index which may determine the stage of pubertal development. The hormonal induction protocol adopted, with increasing doses of carp pituitary extract, it has proven useful to ovarian development, with a synchronization effect that is positively reflected on egg production. The studies on the effects of photoperiod show how the condition of total darkness can positively influence practices of reproductions in captivity. The effects of photoperiod were also investigated at the physiological level, observing the plasma levels of steroids ( E2, T) and thyroid hormones (T3 and T4) and the expression in the liver of vitellogenin (vtg1 and vtg2) and estradiol membrane receptor (ESR1). From the comparison between spontaneous deposition and insemination techniques through the stripping is inferred as the first ports to a better qualitative and quantitative yield in the production of eggs capable of being fertilized, also the presence of a percentage of oocytes completely transparent can be used to obtain eggs at a good rate of fertility. Finally, the design and implementation of a system for recirculating aquaculture suited to meet the needs of species-specific eel showed how to improve the reproductive results, it would be preferable to adopt low-flow and low density incubation. / L’elaborato presentato si propone di esporre gli avanzamenti conseguiti nelle pratiche di riproduzione in cattività dell’anguilla europea (Anguilla anguilla). Gli aspetti indagati riguardano sia approcci zootecnici (selezione riproduttori, risposta a stimolazione ormonale, performance riproduttive, incubazione di uova) sia aspetti fisiologici (profili endocrini plasmatici dei riproduttori), nonché aspetti ingegneristici. Gli studi condotti sulle diverse popolazioni selvatiche di anguilla hanno evidenziato come il principale fattore discriminante nella selezione delle femmine selvatiche da destinare alla riproduzione in cattività deve essere il Silver Index che ne può determinare, a partire caratteri morfo-metrici e con un chiaro grado di precisione, lo stadio di sviluppo puberale. Il protocollo di induzione ormonale adottato, con dosi crescenti di estratto ipofisario di carpa, si è dimostrato utile allo sviluppo ovarico, con un effetto di sincronizzazione che si è positivamente riflesso sulla produzione di uova. Gli studi sugli effetti del fotoperiodo dimostrano come la condizione di totale buio, nelle vasche di mantenimento dei riproduttori, condizioni positivamente le pratiche di riproduzioni in cattività.. Gli effetti del fotoperiodo sono stati indagati anche a livello fisiologico, osservando i livelli plasmatici di steroidi (E2, T) e ormoni tiroidei (T3 e T4) nonché l’espressione a livello epatico dello vitellogenine (vtg1 e vtg2) e del recettore di membrana dell’estradiolo (esr1). Dalla comparazione fra deposizione spontanea e tecniche di inseminazione attraverso lo stripping si è dedotto come la prima porti ad una migliore resa qualitativa e quantitativa nella produzione di uova atte ad essere fecondate, inoltre la presenza di una percentuale di ovociti completamente trasparenti può essere utilizzato per ottenere le uova ad un buon tasso di fertilità. Infine, il design e la messa in opera di un impianto a ricircolo per l’acquacoltura adatto a soddisfare le necessità specie-specifiche dell’anguilla ha mostrato come per migliorare i risultati riproduttivi, sarebbe preferibile adottare basso flusso e bassa densità incubazione.

This PhD thesis is focused on cold atmospheric plasma treatments (GP) for microbial inactivation in food applications. In fact GP represents a promising emerging technology alternative to the traditional methods for the decontamination of foods. The objectives of this work were to evaluate: - the effects of GP treatments on microbial inactivation in model systems and in real foods; - the stress response in L. monocytogenes following exposure to different GP treatments. As far as the first aspect, inactivation curves were obtained for some target pathogens, i.e. Listeria monocytogenes and Escherichia coli, by exposing microbial cells to GP generated with two different DBD equipments and processing conditions (exposure time, material of the electrodes). Concerning food applications, the effects of different GP treatments on the inactivation of natural microflora and Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli on the surface of Fuji apples, soya sprouts and black pepper were evaluated. In particular the efficacy of the exposure to gas plasma was assessed immediately after treatments and during storage. Moreover, also possible changes in quality parameters such as colour, pH, Aw, moisture content, oxidation, polyphenol-oxidase activity, antioxidant activity were investigated. Since the lack of knowledge of cell targets of GP may limit its application, the possible mechanism of action of GP was studied against 2 strains of Listeria monocytogenes by evaluating modifications in the fatty acids of the cytoplasmic membrane (through GC/MS analysis) and metabolites detected by SPME-GC/MS and 1H-NMR analyses. Moreover, changes induced by different treatments on the expression of selected genes related to general stress response, virulence or to the metabolism were detected with Reverse Transcription-qPCR. In collaboration with the Scripps Research Institute (La Jolla, CA, USA) also proteomic profiles following gas plasma exposure were analysed through Multidimensional Protein Identification Technology (MudPIT) to evaluate possible changes in metabolic processes.

Il cancro batterico dell’actinidia causato da Pseudomonas syringae pv.actinidiae (Psa) suscita grande interesse a livello globale a partire dal 2008. La malattia è comparsa in Giappone e in due anni ha avuto una diffusione epidemica in tutte le aree di coltivazione mondiale di actinidia. Gravi perdite economiche hanno attirato l’attenzione internazionale su questa problematica e grandi sforzi sono stati rivolti allo studio di questo patosistema ancora poco conosciuto. E’ emerso infatti che il patogeno può rimanere in fase latente per lunghi periodi senza causare sintomi caratteristici nelle piante infette, e che dalla comparsa dei sintomi la pianta muore nell’arco di un paio d’anni. Il monitoraggio ed il controllo della situazione è perciò di fondamentale importanza ed è ancora più importante prevenire la comparsa di nuovi focolai di infezione. A questo proposito sarebbe opportuno l’impiego di materiale vegetale di propagazione non infetto, ma in molti casi questo diventa difficile, dal momento che il materiale impiegato è generalmente quello asintomatico, non analizzato precedentemente per la presenza del patogeno. Negli ultimi anni sono state perciò messe a punto molte tecniche molecolari per l’identificazione di Psa direttamente da materiale vegetale. L’obiettivo di questo lavoro è stato quello di studiare l’epidemiologia di Psa in piante adulte infette e di verificare l’efficacia di metodi di diagnosi precoce per prevenire la malattia. A tale scopo il lavoro sperimentale è stato suddiviso in diverse fasi: i) studio della localizzazione, traslocazione e sopravvivenza di Psa nelle piante, a seguito di inoculazione in piante adulte di actinidia di ceppi marcati Psa::gfp; ii) studio della capacità di Psa di essere mantenuto in germogli di actinidia attraverso sette generazioni di micropropagazione dopo l’inoculazione delle piante madri con lo stesso ceppo marcato Psa::gfp; iii) studio ed applicazioni di un nuovo metodo di diagnosi precoce di Psa basato sull’analisi molecolare del “pianto”. / Bacterial canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) involved all global interest since 2008. The disease started from Japan and in two year it was causing epidemic outbreaks around the world, in every country that has an actinidia’s cultivations. Huge amount of economical losses brought the international attention on this problem and on strong efforts were devoted study this relatively unknown pathosystem. It appears in fact that pathogen can be maintained in latent form for long periods, without show any characteristic symptom on the affected plants, when it suddenly induce symptoms the plant die in one or two years. Monitor and control the real situation on symptoms are fundamental, but more important is to prevent the appearance of new infection events. This could be supported by the use of pathogen free propagation materials, but in several cases this is only theoretically achieved since materials employed are just asymptomatic but not tested to be pathogen-free. In the recent years a lot of molecular techniques were developed for Psa detection and diagnosis, directly from plant material. The objectives of this work were to clarify the spreading of Psa into the infected adult plant, and to verify the effectiveness of Psa early detection methods on disease prevention. Toward these aims the following experimental steps were carried out: i) study the localization, movement and survival ability of Psa into the plant after inoculation with a reference marked strains of Psa::gfp several actinidia adult plants; ii) study the Psa ability to be maintained in shoots during seven micropropagated generations after inoculation with the same marked strain of Psa::gfp mother plants; iii) study and application of a novel Psa detection method based on bleeding sap molecular testing.

Kiwifruit (genus Actinidia) is an important horticultural crop grown in the temperate regions. The four world’s largest producers are China, Italy, New Zealand and Chile. More than 50 species are recognized in the genus but the principal species in cultivation are A. deliciosa and A. chinensis. In Italy, as well as in many other countries, the kiwifruit crop has been considered to be relatively disease free and then no certification system for this species has been developed to regulate importation of propagation plant material in the European Union. During the last years a number of fungal and bacterial diseases have been recorded such as Botrytis cinerea and Pseudomonas syringae pv. actinidiae. Since 2003, several viruses and virus-like diseases have been identified and more recent studies demonstrated that Actinidia spp can be infected by a wide range of viral agents. In collaboration with the University of Auckland we have been detected thirteen different viral species on kiwifruit plants. During the three years of my PhD I worked on the characterization of Cucumber mosaic virus (CMV) and Pelargonium zonate spot virus (PZSV). The determination of causal agents has been based on host range, symptom expression in the test plant species and morphological properties of the virus particles using transmission electron microscopy (TEM) and using specific oligonucleotide primers in reverse transcription-polymerase chain reaction (RT-PCR). Both viruses induced several symptoms on kiwifruit plants. Moreover with new technologies such as high-throughput sequencing we detected additional viruses, a new member of the family Closteroviridae and a new member of the family Totiviridae. Taking together all results of my studies it is clear that, in order to minimize the risk of serious viral disease in kiwifruit, it is vital to use virus-free propagation material in order to prevent the spread of these viruses.

Nanotechnology promises huge benefits for society and capital invested in this new technology is steadily increasing, therefore there is a growing number of nanotechnology products on the market and inevitably engineered nanomaterials will be released in the atmosphere with potential risks to humans and environment. This study set out to extend the comprehension of the impact of metal (Ag, Co, Ni) and metal oxide (CeO2, Fe3O4, SnO2, TiO2) nanoparticles (NPs) on one of the most important environmental compartments potentially contaminated by NPs, the soil system, through the use of chemical and biological tools. For this purpose experiments were carried out to simulate realistic environmental conditions of wet and dry deposition of NPs, considering ecologically relevant endpoints. In detail, this thesis involved the study of three model systems and the evaluation of related issues: (i) NPs and bare soil, to assess the influence of NPs on the functions of soil microbial communities; (ii) NPs and plants, to evaluate the chronic toxicity and accumulation of NPs in edible tissues; (iii) NPs and invertebrates, to verify the effects of NPs on earthworms and the damaging of their functionality. The study highlighted that NP toxicity is generally influenced by NP core elements and the impact of NPs on organisms is specie-specific; moreover experiments conducted in media closer to real conditions showed a decrease in toxicity with respect to in vitro test or hydroponic tests. However, only a multidisciplinary approach, involving physical, chemical and biological skills, together with the use of advanced techniques, such as X-ray absorption fine structure spectroscopy, could pave the way to draw the right conclusions and accomplish a deeper comprehension of the effects of NPs on soil and soil inhabitants.

Come molti altri settori produttivi, anche l'agricoltura deve affrontare una crescente dipendenza energetica da elettricità, petrolio e gas naturale. Tuttavia, l'agricoltura può rispondere direttamente a tali richieste ricorrendo alle fonti di energia rinnovabili (FER), come il solare fotovoltaico (PV) e gli impianti biogas/biometano (BP). Questi sistemi sono stati fortemente incentivati in passato. Gli attuali sistemi d’incentivazione prevedono invece sussidi commisurati alle caratteristiche aziendali come stalle, fienili, disponibilità di biomasse, ecc. Per questi motivi, per quanto riguarda il settore FV, è necessario utilizzare modelli analitici affidabili per valutare l’orientamento e la tecnologia migliore, in particolare per generatori integrati architettonicamente. Per la produzione di biometano, gli impianti di piccole dimensioni risultano quelli maggiormente incentivati previa alimentazione con sottoprodotti agro-industriali. Tuttavia, le loro performance dipendono fortemente dalla tecnologia d’upgrading. Pertanto, le prospettive economiche offerte da questi sistemi RES sono legate alla scelta della tecnologia da impiegare e non più esclusivamente al semplice dimensionamento. Su questa base, l'obiettivo principale di questo lavoro è stato lo sviluppo di strumenti di supporto decisionali (DSSS) per l'ottimizzazione energetica d’impianti FV e BP. Un primo studio si è focalizzato sull’analisi prestazionale per singolo componente e globale d’impianti FV installati in un’azienda agricola. Inoltre, una procedura analitica di ottimizzazione dei parametri d’impianto è stata definita per massimizzare il rendimento elettrico di un generatore integrato architettonicamente rispetto ad un’installazione a terra. Attraverso una serie d’informazioni tecnico-economiche di tecnologie di upgrading, un secondo studio è stato condotto con l'obiettivo di sviluppare un DSS per valutare la marginalità sul reddito aziendale data dall’installazione on-farm di un impianto BP collegato alla rete del gas naturale. I risultati dei due studi hanno dimostrato come questi DSS possono essere utili strumenti per valutare preventivamente le diverse potenzialità offerte da impianti FV e BP in base alle caratteristiche di un’azienda agricola. / Like many other productive sectors, even agriculture must tackle an increasing energy dependency on electricity, petroleum and natural gas. Nevertheless, agriculture can directly respond to such request thanks to renewable energy systems (RES) like solar photovoltaic (PV) and biogas/biomethane (BP) plants. These systems have been strongly incentivized in the past. On the contrary, current incentive schemes provide feed-in-tariffs proportionate to farming characteristics as stables, barns, biomass availability, etc. For this reason, as regards the PV sector, it is required to use reliable analytical models for assessing the best orientation and technology, in particular for architecturally integrated generators. For the biomethane production, small-medium plants have stronger incentives in particular using agro-food by-products as feed. However, their performances are strongly dependent on biomethane upgrading technology. Thus, the economic prospective offered by these RES systems are substantially tied to technology choice optimization and no longer solely in simple sizing. On this basis, the main goal of this work is to develop Decision Support Tools (DSSs) for energy optimization both for PV and BP plants. A first study was focused on the analysis of PV plants installed in the same farm. The aim is to examine the impact each plant component has on the PV generator global efficiency and define an analytical procedure for technical parameters optimization in order to maximize the electric yield of an architectonically integrated plant compared to a ground-mounted one. Based on the economic and efficiency features of a biomethane upgrading technology set, a second study was carried out with the aim to design a DSS to assess enterprise-wide profit margins resulting from the on-farm BP plant installation linked to the natural gas grid. The results of the two studies have shown how these DSSs can be useful tools for choosing PV and BP technologies based on farm characteristics.

Many oomycetes species are pathogens of plants, animals and humans; some of them are well studied because they cause significant economic losses in agriculture and acquaculture. The cell wall of oomycetes consists mainly of cellulose, β- (1,3) and β- (1,6)- glucan and in some species a small amount of chitin is also present. The biosynthesis of cellulose in oomycetes is still poorly studied and therefore misunderstood, although it’s the target of some fungicides that inhibit this process, such as carboxylic acid amides. Since 2010, single amino acid exchanges in CesA3 protein conferring CAA resistance in Plasmopara viticola have been identified. In this work, we initially provide the first evidence of the presence of mandipropamid resistant populations of Plasmopara viticola in commercial vineyards in Italy (paper I). We continue by studying the different activity of four CAAs fungicides (benthiavalicarb, dimethomorph, iprovalicarb, mandipropamid) toward P. viticola resistant strains (paper II). The results show that the G1105S mutation affects all four CAAs, but its impact is varied. These results confirm that they are cross resistant, although many gaps in the mode of action are still present. In order to confirm our previous findings, we performed a microscopical base method to assess the sensitivity of four CAAs, and preliminary microscopical data confirmed the different activity toward the CAA-resistant and CAA-sensitive populations (chapter ten). Furthermore, we present a study (chapter eleven) in which we randomly selected samples from paper I, in order to test them using a different approach, simulating a scenario more close to the field. With this test, we were able to confirm the data presented in the paper I.

Plant bacterial diseases are nowadays routinely managed with scheduled treatments based on heavy metal compounds or, in the worst cases, on antibiotics; to overcome the environmental consequences linked to the use of these chemical compounds, such as pollution or selection of antibiotic resistant pathogens, an integrated control management is required. The use of bacterial antagonists, biological agents, plant defence response elicitors or resistant host plant genotypes play an important role in the frame of sustainable agriculture. In this work, the activity of plasma activated water (PAW) and different bioagents aimed to the control of the two bacterial pathogens Xanthomonas vesicatoria and Pseudomonas syringae pv. actinidiae, causal agents of tomato bacterial leaf spot and kiwifruit bacterial canker, respectively, were studied. All these tools were assayed for their direct efficacy and for their ability, as elicitors, to trigger the plant immune system against these two bacterial pathogens. Moreover, a study on several Actinidia sp. accessions was carried out to evaluate their susceptibility against bacterial canker of kiwifruit. PAW resulted unable to direct inhibit X. vesicatoria growth in in vitro assays, however it showed the ability to trigger tomato plant immune system by reducing disease severity up to approx. 38% when tested in three experiments on two tomato genotypes conducted under greenhouse conditions. When tested in in vitro and in vivo experiments against X. vesicatoria and P. syringae pv. actidinidiae, the tested bioagents, based on natural extracted compounds or on different strains of Bacillus sp., showed a direct efficacy against both bacterial pathogens. Moreover, they were also able to elicit the plant defence response by significantly lowering the disease severity on tomato and kiwifruit leaves. In addition, the A. chinensis accession NPK3 resulted the less susceptible to the bacterial canker in comparison to more than 20 accessions tested.

Negli ultimi anni le continue esigenze di mercato hanno spinto gli agricoltori a richiedere trattrici sempre più evolute e altamente specializzate al fine di limitare i costi e massimizzare le rese produttive. Per questo motivo i progettisti hanno dovuto sviluppare delle metodologie per definire con precisione il profilo di utilizzo delle macchine, al fine di ridefinire i test per la validazione finale dei componenti delle trattrici. Uno dei componenti più costosi è la trasmissione, in quanto è costituita da diversi automatismi e pertanto, in questa tesi, è stato descritto un piano sperimentale atto all’acquisizione di dati da trattrici utilizzate da clienti, con l’obiettivo di monitorare l’utilizzo delle trasmissioni. In una prima analisi sono state identificate relazioni statistiche del diverso utilizzo delle trasmissioni delle trattrici in funzione dell’area geografica di vendita e della potenza massima del motore. Sono state poi stimate le distribuzioni ed i parametri del tempo di neutro, delle gamme e retromarce, al fine di integrare le limitazioni riscontrate dalle analisi di correlazione cliente sulle trattrici. E’ stato riscontrato che la vita lavorativa di una trattrice è caratterizzata dal utilizzo del neutro e della gamma media per oltre il 75%. Non sono state evidenziate differenze significative tra i valori del 5° percentile della distribuzione del Neutro per l’intero campione rispetto a quelli calcolati per i gruppi, i quali sono stati formati in funzione dei fattori area geografica e intervalli di potenza massima motore. La gamma media, oltre ad essere la gamma più utilizzata, risulta essere influenzata dai fattori presi in esame, in particolare per le trattrici di potenza pari a A kW. / In the lasts years, the continuous demands of agricultural market, have prompted farmers of having more advanced and highly specialized tractors. This in order to limit the costs and maximize the working yields. Designers have, for the previous reasons, developed methods on the definition of machine usage profile, in order to re-define the final validation tests of tractor components. Considering that the most expensive components is the transmission, because it is constituted by various automatic functions, in this thesis it has been described an experimental acquisition data plan from tractors used by customers, to monitor the transmissions. First of all, there were identified statistical relations of the different tractor’s transmissions usage considering sale geographical areas and the maximum engine power. Therefore, there have been estimated distributions and parameters for neutral time, ranges and reverse gears, to supplement the limitations of customers correlation analysis about tractors. It has been verified that the tractor’s life time is characterized by the usage of the neutral and intermediate range for more than 75%. No significant differences were found between the values of the 5° percentile of the neutral gear distribution for the entire sample, compared to those calculated for the groups. These groups were formed considering the geographical area and maximum engine power ranges factors. The intermediate range, besides being the most used, is influenced by the considered factors, in particular for tractors with A kW of maximum engine power.

Colletrotrichum acutatum and Botrytis cinerea are among the major fungal pathogens of Fragaria spp. Both pathogens could infect strawberry fruits during the fruit’s early developing stage and remain quiescent until ripening. In strawberry, a fruit ontogenic resistance to pathogen infection was described and correlated with fungal quiescence during the unripe stages of the fruit. Due to the period of fungal quiescence, the management of anthracnose and gray mould diseases becomes more complex as symptoms only manifest in ripe fruits. To identify the underlying component in the ontogenic resistance of strawberry fruits, transcriptomic and metabolomic approaches were used. White and red fruits of strawberry were artificially inoculated with C. acutatum and B. cinerea. Transcriptome profile of B. cinerea infected fruits exhibited a general up-regulation of defense-related genes in white fruits after 24 h of infection. Meanwhile, accumulation of phenolic compounds such as proanthocyanidins, catechins and the ellagitannin casuarictin was also observed in white fruits after 48 h of interacting with C. acutatum and B. cinerea. The acquisition of these findings could provide a benchmark to further investigate the interaction of strawberry against pathogens with latent infection. Hence, a strawberry transformation was performed to study the mechanism of a gene encoding for a mannose-binding lectin protein which was previously identified to be correlated with the resistance of white strawberry fruits to C. acutatum. The regeneration system utilized in the transformation is also discussed.