Fusarium graminearum causa la fusariosi della spiga nei cereali a paglia. Il fungo produce sia conidi che ascospore sui residui della coltura precedente, le ascospore sono prodotte in periteci. La produzione e maturazione di periteci e ascospore in risposta a diverse condizioni di temperatura e umidità relativa sono state studiate. Dato che le condizioni atmosferiche influenzano anche l’umidità del substrato su cui l’inoculo è prodotto, la relazione tra i fattori atmosferici e l’umidità dei residui colturali di mais è stata esaminata. I fattori atmosferici influenzano anche il rilascio delle ascospore. L’effetto della temperatura è stato studiato in vitro. Mediante esperimenti in condizioni naturali, sono state definite regole per l’individuazione di condizioni favorevoli al rilascio di ascospore sulla base di pioggia e deficit di pressione di vapore. La distribuzione delle ascospore e dei conidi all’interno della vegetazione del frumento è quindi stata studiata mediante l’uso di captaspore passivi. Le ascospore possono essere rilasciate e depositarsi sulle spighe in condizioni non favorevoli per la germinazione. La germinazione di ascospore sottoposte a periodi asciutti di diversa durata, e a diverse condizioni di temperatura e umidità relativa durante il periodo asciutto, è stata studiata sia in vitro che in planta. / Fusarium graminearum causes Fusarium head blight of small-grain cereals. The fungus produces conidia and ascospores on the previous crop residues, ascospores are formed in perithecia. Production and maturation of perithecia and ascospores at several temperature and relative humidity conditions were studied. As environmental conditions also influence the moisture content of the substrate on which inoculum is produced, the relationship between environmental factors and moisture of maize residues was assessed. Environmental factors also influence ascospore discharge. The effect of temperature was studied in vitro. Experiments in natural condition allowed to define rules for conditions leading to ascospore discharge, based on rain and vapor pressure deficit. Once discharged, the distribution of ascospores and conidia in the wheat canopy was studied using passive spore traps. Ascospores can be discharged and deposit on wheat spikes also in conditions that are unfavorable for germination. Germination of ascospores incubated in dryness for periods of several length, in several condition of temperature and relative humidity during dryness, was studied both in vitro and in planta.

In questa tesi di dottorato sono stati indagati i punti critici del ciclo vitale del fungo Fusarium verticillioides, produttore di fumonisine in mais. Le fumonisine sono metaboliti secondari prodotte da funghi appartenenti tutti al genere fusarium e sono state classificate come sostanze cancerogene per l'uomo e per gli animali. Gli argomenti trattati nei vari capitoli sono stati: contaminazione da fumonisine nel mais coltivato nel nord Italia ed effetto delle diverse tecniche colturali, produzione di spore, dispersione di spore, infezione, parametri ecologici che condizionano la crescita e l'accumulo di fumonisina ed il controllo con mezzi chimici in campo su F. verticillioides e Ostrinia nubilalis, un lepidottero che aumenta la contaminazione. / Aim of this work was to collect missing information about critical point of the disease life cycle, to develop a conceptual model for the dynamic simulation of the life cycle of Fusarium verticillioides in maize and the consequent production of fumonisin B1 in kernels. Fumonisin is a secondary metabolite reported as toxigenic in humans and animals. Issues treated are: Fumonisin contamination in maize in Italy and effect of field managements, spore production, spore dispersal, variables influencing growth and toxin accumulation and chemical control of the disease in field.

La peronospora della vite è causata dall’Oomicete eterotallico Plasmopara viticola ed è ampiamente diffusa in tutte le aree caratterizzate da primavere miti e piovose. Gli organi svernanti di questo patogeno sono le oospore, che rappresentano l’unica fonte di inoculo per le infezioni primarie. Recenti studi molecolari hanno evidenziato l’importanza delle infezioni primarie e suggerito una revisione dell’epidemiologia di P. viticola. Questa tesi è costituita dagli studi epidemiologici realizzati per indagare il processo di maturazione e germinazione delle oospore mirati all’elaborazione di un modello dinamico per la simulazione delle infezioni primarie di P. viticola. Il modello è stato successivamente validato in diverse aree viticole italiane e, inoltre, utilizzato come base per un warning system in tempo reale per la gestione dei trattamenti in vigneto. / The heterothallic Oomycete Plasmopara viticola represents the causal agent of downy mildew of grapevine (Vitis spp.). The unique source of inoculum is represented by the overwintering sexual spores, originated by the fusion between anteridium and oogonium, so called oospores. Despite their critical impact on the epidemiology of this disease, knowledge about oospores presents some inconsistencies that are engaged in the present dissertation. Initially, the effect of water moistening the grape leaf litter holding overwintering P. viticola oospores was investigated. A close relationship was found between vapour pressure deficit (VPD in hPa) and aW (water activity) of the leaf litter, so that when VPD is lower than 2.13 hPa there is sufficient water for oospores to develop. Results showed that moisture of the leaf litter due to the water flow from the atmosphere makes the oospore development possible also during non rainy periods. Then, the effects of environmental conditions on the variability in germination dynamics of Plasmopara viticola oospores were studied over five years. The germination course was determined indirectly as the relative infection incidence (RII) occurring on grape leaf discs kept in contact with oospores sampled from a vineyard between March and July. The time elapsed between the 1st of January and the infection occurrence was expressed as physiological time, using sums of hourly rates from a temperature-dependent function only in hours when VPD was not a limiting factor (hydro-thermal time, HT). The Gompertz equation calculated over hydro-thermal time produced a consistent modelling of the general relationships between the germination dynamics of a population of P. viticola oospores and weather conditions. It represents the relative density of the seasonal oospores that should have produced sporangia when they have experienced favourable conditions for germination. Finally, a dynamic model for Plasmopara viticola primary infections on grapevine was elaborated according to a mechanistic approach. Development of the sexual stage of the pathogen was split into different state variables, in which changes from one state to another were regulated by rates depending on environmental conditions. The conceptual model was based on the definition of a primary inoculum season, a seasonal oospore (inoculum) dose, and its division into many coeval cohorts. Each cohort progresses along the primary infection cycle (production and survival of sporangia, release, survival and dispersal of zoospores, infection, appearance of disease symptoms) simultaneously, with a time step of one hour. The model was evaluated by comparing model predictions with disease onset in: i) 100 vineyards of Northern, Southern and Insular Italy (1995 to 2007); ii) 42 groups of potted grapevine plants exposed to inoculum (2006 to 2008). Most of the wrong positive predictions occurred in early season, when the host was in the earlier growth stages, or when the oospore germination was triggered by isolated weak rain events. Considering that neither calibration nor empirical adjustment of model parameters were necessary to obtain accurate simulation, it was concluded that this model produces a reasonable approximation of the primary infection processes underlying oospore development. A warning system based on such a model and on short-term weather forecasts was developed and its use was evaluated in experimental vineyards over a 3-year period in North Italy. An unsprayed control was compared with a “warning” treatment (fungicides were applied when the warning system predicted an infection), a “threshold” treatment (fungicides were applied as in the warning treatment, but only for the oospore cohorts higher than a fixed threshold), and the grower’s schedule. Average efficacy in decreasing disease incidence on leaves compared to the unsprayed control was > 90% for all treatments. On the average, 6.8 sprays were applied following the grower’s schedule; use of the warning system reduced applications by about one half (warning treatment) or two third (threshold treatment). The grower’s schedule was the most expensive control strategy, with average of 337 €/ha; the average saving was 174 and 224 €/ha for the warning and the threshold treatments, respectively. The value of this dissertation consists in two relevant and connected aspects. From one side the studies performed on the oospore maturation and germination allowed to better understand and clarify a key point of the downy mildew epidemics still wrapped by a lack of information. From the other side the model elaborated during this thesis represents a practical and efficient tool that leads to the reduction both of growers’ costs and chemical input in the environment.

Le micotossine sono metaboliti tossici prodotti da funghi in grado di svilupparsi sulle derrate alimentari. Diverse strategie sono state considerate per risolvere questo problema studiando la crescita di funghi/produzione di micotossine. Questa tesi è focalizzata sullo sviluppo/validazione di modelli matematici per prevedere la contaminazione di micotossine (deossinivalenolo, fumonisine e aflatossine) in cereali (mais/grano) sulla base di dati meteorologici. Il primo capitolo fornisce un’introduzione sulla teoria dei modelli e sui pat-sistemi modelizzati. Il secondo si concentra sulla presenza di tricoteceni e zearalenone nel frumento coltivato in Italia. Nel cap.3 sono stati confrontate le differenze predittive di modelli empirici/meccanicistici per la contaminazione di deossinivalenolo nel grano. Nel cap.4 è stata descritta la contaminazione da fumonisine e aflatossine in mais coltivato in Italia. I capitoli 5 e 6 analizzano il pato-sistema mais-Aspergillus flavus, il primo si concentra sulla sporulazione di A. flavus , il secondo sullo sviluppo di un modello per prevedere la contaminazione da aflatossina. Un altro modello meccanicistico per prevedere la presenza di fumonisina nel mais è descritto nel cap.7. L'ultimo capitolo riassume l'attività svolta nel progetto europeo MYCORED in cui sono stati coinvolti diversi paesi in tutto il mondo che hanno fornito i dati necessari per la validazione dei modelli. / Mycotoxin are toxic secondary metabolite produced by fungi able to colonize crops and thus posing a potential menace to human/animal health. Several strategies have been considered to mitigate the problem studying the variables related to mould growth and mycotoxin production. This thesis focuses on the development and validation of mechanistic models to predict mycotoxins (deoxinivalenol, fumonisins and aflatoxins) contamination in cereals (maize/ wheat) based on meteorological data. The first chapter introduce modelling theory, and patho-systems analysed. Chapter 2 focuses on trichothecenes and zearalenone occurrence in wheat produced in Italy. Predictive performance of empirical and mechanistic models for deoxnivalenol contamination in wheat were discussed in chapter 3. Chapter 4 described fumonisins and aflatoxins occurrence in maize grown in Italy. Chapters 5 and 6 analised the patho-system maize-Aspergillus flavus; the former focuses on the dynamics of A. flavus sporulation the lalatter on the development of a mechanistic model to predict aflatoxin produced by A. falvus. Another mechanistic model for Fusarium ear rot and fumosin production in maize (chapter 7). The last chapter summarised the activity done in the European project MYCORED in which several countries worldwide were involved and wheat and/or maize samples collected with data necessary for model validation.

In this work we investigated the in vivo and in vitro ecological conditions that can favour the fumonisin production, both free and hidden forms, in the maize-Fusarium verticillioides pathosystem. Samples of different maize hybrids have been collected from dough to the harvest maturity to follow the trend of fungal incidence and both fumonisin forms contamination, but also the changes in chemical composition. Differences in the level of contamination have been found among hybrids during the growing season. Furthermore, the production of fumonisins has been found correlated to the total lipids content, another parameter that changed during the growing season. This finding underlined the existence of a relationship between toxin contamination and fatty acids composition of the hybrids. Recently the existence of a cross talk between plant and pathogen has been demonstrated, based on some oxidized signal molecules (oxylipins) produced from fatty acid precursors. This result was also confirmed by the molecular analysis on the in vitro pathosystem that showed differences in the activation of the genes involved in plant and fungal oxylipins production during the incubation time. Also post-harvest contamination of maize was investigated in this study, with particular attention to the effects of the drying treatment, a common post-harvest practice aimed at decreasing the water availability, and to the storage capacity of a new low cost storage system, silo bag. The drying treatment was showed to affect fumonisins content, in particular an increased fumonisins contamination was detected after heat treatments. This increment seemed to be produced by chemical changes of matrix components, caused by high temperature, that produced the release of hidden fumonisin in free form. Silo bags were shown to be an effective system to store cereals because no significant change occurred in fungi or toxins contamination during a 9-month storage. Therefore, being more flexible and less expensive than traditional store houses, they should be very useful for farmers.

Oggigiorno i dibattiti riguardanti il significato di sostenibilità, la sua misurazione attraverso indicatori e le metodologie nei quali quest’ultimi dovrebbero essere utilizzati rimangono spesso inconcludenti. Questo elaborato si propone come un prototipo per superare questi limiti. L’approccio, applicato per la coltura frumento duro, è un esempio di come è possibile consolidare i paradigmi teorici della sostenibilità e trasformarli in utili raccomandazioni per attività agricole più sostenibili. Attraverso l’implementazione di un Decision Support System (DSS) chiamato granoduro.net® e la stesura di un decalogo per una coltivazione del grano duro più competitiva (10 regole agronomiche), il divario tra i principi teorici e pratici della sostenibilità viene ridotto. Grazie alla disponibilità di Barilla S.p.A., sono stati pianificati per diversi anni, a partire dal 2011, studi di campo e il progetto è stato considerato dagli operatori del settore un esempio di come i principi teorici della sostenibilità possono essere messi in pratica con facilità. Le differenti fasi della validazione (un iniziale studio teorico, il confronto tra situazioni reali e teoriche ottimali e due anni di utilizzo sul campo di granoduro.net® e del decalogo) hanno permesso di dimostrare agli agricoltori che gli impatti antropici possono essere monitorati e ridotti e che quindi la sostenibilità è fruibile e calcolabile. Inoltre, il decalogo e granoduro.net® hanno dimostrato di essere due esempi di come è possibile migliorare la qualità delle decisioni strategiche (scelta della rotazione, lavorazione del terreno, scelta delle varietà, tecnica di fertilizzazione e uso di seme certificato) e tattiche (in risposta agli eventi generati da attacchi biotici, infestanti e necessità nutrizionali) intraprese dagli imprenditori agricoli. / Current debates concerning the meaning of sustainability, its measurement by means of indicators, and the framework in which these should be applied, often remain inconclusive. This work is a prototype aimed to overcome such shortcoming. The method adopted in this work was applied to the crop of durum wheat and represents an example on how to put the theoretical paradigms of sustainability into effect by transforming them into practical recommendations for implementing more sustainable agricultural practices. The implementation of a Decision Support System (DSS) called granoduro.net® and the drafting of a handbook (ten agronomical rules for a more competitive cultivation of durum wheat) allowed to bridge the gap between the theoretical principles and the practical implementation of sustainability. Since 2011 in-field validation activity has been carried out thanks to the collaboration with Barilla S.p.A.; the project was considered by Barilla’s stakeholders as an example of how theoretical principles of sustainability can be put into practice. The several steps of the validation process (i.e., a theoretical study, a comparison between real and target values, and the in-field adoption of both the handbook and granoduro.net® for two years) demonstrated to farmers and experts that human impacts can be monitored and reduced. Therefore, it was proved that sustainability is actionable and calculable. Moreover, the handbook and granoduro.net® showed to be tools able to help farmers improving the quality of their decisions, both strategic (choice of rotation, tillage, varieties, fertilization techniques, and use of certified seeds) and tactical (in response to nutritional needs or the risk of pests, disease or weeds infestation).

Il marciume del grappolo, causato da Botrytis cinerea, è una delle principali malattie che colpiscono la vite. Botrytis cinerea si sviluppa e cresce attivamente come patogeno necrotrofo e saprofita su diversi substrati. In vigneto, B. cinerea può produrre un ampio numero di conidi in un’ampia gamma di condizioni ambientali. Botrytis cinerea ha diversi pathway d’infezione e le infezioni occorrono principalmente in due periodi: dalla fioritura allo sviluppo dei giovani grappoli, e dopo invaiatura alla raccolta. A causa della complessità del ciclo di vita di B. cinerea e all’esistenza di questi diversi pathway d’infezione, i fungicidi vengono normalmente applicati a fine della fioritura (A), pre-chiusura del grappolo (B), invaiatura (C), e prima della raccolta (D). Le applicazioni durante la prima parte della stagione (A e B) hanno come obiettivo: i) la riduzione della germinazione dei conidi e le infezioni fiorali, ii) la prevenzione delle infezioni latenti delle bacche, e iii) la disinfezione dei residui di fioritura. Le applicazioni successive all’invaiatura (C e D) hanno lo scopo non solo di prevenire le infezioni durante la maturazione ma anche di disinfestare i residui di fioritura rimasti all’interno del grappolo. Questa strategia, basata su quattro trattamenti, garantisce una buona protezione contro il marciume del grappolo in vigneto, tuttavia presenta alcuni limiti: i) si tratta di un approccio preventivo che non considera il reale rischio d’infezione, con il conseguente effetto di eseguire trattamenti non necessari; ii) aumenta la probabilità che si sviluppino popolazioni resistenti ai fungicidi; iii) implica potenziali risvolti negativi sulla salute umana e sull’ambiente. Negli ultimi anni, nuove tipologie di prodotti, come gli agenti di biocontrollo (BCAs) e i botanicals, sono state proposte per ridurre l’uso eccessivo di prodotti chimici e aumentare la sostenibilità del controllo del marciume del grappolo in vigneto. Lo scopo di questa tesi di Dottorato è stato: i) comparare diverse strategie di controllo e valutare l’interazione tra fungicidi con diversi timing di applicazione, ii) valutare l’effetto sulla colonizzazione e sporulazione di B. cinerea su residui fiorali di diversi prodotti (fungicidi, agenti di biocontrollo, e botanicals) applicati con diversi timing; iii) valutare un modello meccanicistico sviluppato da González-Domínguez et al. (2015); e iv) includere all’interno di un modello per il biocontrollo, sviluppato da Jeger et al. (2009), l’effetto delle condizioni ambientali sulle interazioni BCA-patogeno. / Botrytis bunch rot (BBR), caused by Botrytis cinerea, is one of the main diseases affecting grapevine. Botrytis cinerea develops and actively grows as necrotrophic pathogen and as saprophyte on different substrates. In grapevine, B. cinerea can produce a large number of conidia on bunch and leaf trash and rotted berries under a wide range of environmental conditions. Botrytis cinerea has multiple infection pathways and infection mainly occurs in two periods: from flowering to young cluster development, and after veraison to harvest. Due to the complexity of the B. cinerea life cycle and the existence of these different infection pathways, fungicides are usually applied at the end of flowering (A), pre-bunch closure (B), veraison (C), and before harvest (D). The early season applications (A and B) are aimed at: i) reducing conidial germination and infection of flowers, ii) preventing latent infections of berries, and iii) disinfesting the bunch trash. The later-season applications (i.e., the applications from veraison until harvest; C and D) are aimed not only at preventing berry infection during ripening but also at disinfesting bunch trash in order to reduce the inoculum load. This fungicide schedule may results in unnecessary sprays because the applications are preventive and do not take into account the real risk of BBR infections. In the last decades, new products, like biological control agents (BCAs) and botanicals, have been proposed for reducing the excessive use of chemicals in agriculture and increase the sustainable of BBR control in vineyards. The aim of this Doctoral work was to: i) compare different control strategies and evaluate the interactions among fungicides applied at different timings; ii) investigate the effects of different products (fungicides, biological control agents, and botanicals) applied at different timings on B. cinerea bunch trash colonization and sporulation; iii) evaluate a weather-driven mechanistic model developed by González-Domínguez et al. (2015); and iv) include into a model for biological control, developed by Jeger et al. (2009), the effect of environmental conditions on the pathogen-BCA interactions.

Vitis vinifera L.(Vv), the European cultivated grapevine is one of the most worldwide important crops but is highly susceptible to the necrotrophic fungus Botrytis cinerea (Bc), the causal agent of bunch rot (BR) disease. In grapevine, as well as in other fruit species, it has been described a primary infection by Bc at full bloom, followed by a quiescence period during the berry development, and the fungal egression after veraison reaching the maximum at harvest. Today, this important disease is mainly controlled by massive use of fungicides, which are applied at different developmental stages that happen to be critical during the grapevine-Bc interaction. During the contact, the fungus must overcome several barriers from the host, which protect it from the pathogen attack and might be also modulated or activated due to the presence of the pathogen itself. The cuticle and the cell wall (CW) represent the first barriers from the plant encountered by the pathogen. To successfully colonize the plant tissue, Bc possesses several virulence factors, and CW modifying enzymes (CWMEs) are part of them. On the other hand, the regulated activity of the CWMEs, expressed both by the host and the pathogen, could alter the plant CW composition and porosity, therefore facilitating, or limiting the penetration of the fungus. Among the CWMEs, Pectin methylesterases (PMEs) regulate the degree of methyl esterification of the pectin homogalacturonan, also modifying the epitopes for the activity of other CWMEs such as polygalacturonases (PGs) and pectate lyases (PLs) by whose action, pectin becomes more susceptible to degradation and the CW more accessible by the pathogen. Previous works both in Arabidopsis thaliana and in crop species identified several PME genes with an altered expression in response to pathogens. A previous report characterizing Atpme17 mutant lines has highlighted a role of AtPME17 in the resistance response to Bc in contrast with the opposite role of AtPME3, suggesting that PME genes could have a completely different action during Bc response depending on the isoform involved. In this context, the main objective of the project was to identify new strategies for Bc BR-control, and specifically i) to identify candidate genes involved in the response to Bc, whose inactivation/overexpression would lead to Bc resistant plants and ii) to set up a molecular method to monitor the Bc load in the field and therefore implement a more sustainable control of the pathogen. To further understand the effects of the Bc primary infection in grapevine flowers at CW level, two contrasting genotypes (Souvignier gris (SG) and Teroldego (TE)) in their resistance to the fungus were considered. An artificial inoculation of different biological replicates, in vase maintained, was performed at full bloom, in controlled conditions, and samples were collected at 24 hours post-inoculation (hpi) with the fungus and post-treatment with the respective control, for the following RNA-seq analysis and biochemical characterization of PME activity and CW composition in the two genotypes before and upon infection. The Bc load was estimated in the flowers using qPCR and as expected, a higher biomass of Bc was found in TE, the susceptible cultivar, than in SG, the resistant one. The analysis of CW composition, PME activity and degree of pectin methyl-esterification, both in treated and control flowers, showed significant differences between the two genotypes, in particular SG showed a significant induction of PME activity with respect to the control, evidence not present in the susceptible genotype. The RNAseq analysis on the same samples showed a total of 4800 genes modulated, out of which 3064 are only modulated in TE, 739 only in SG and a common group of 997 genes. Regardless of the cultivar, upon infection there was a total 2919 genes upregulated vs 1909 genes downregulated. A gene set enrichment analysis (GSEA) indicated several over-represented categories upon infection, including response to pathogens and biosynthesis of secondary metabolites, with a general down-regulation of those genes related to CW organization and pectin modification (CWMEs), mostly in the resistant genotype. Within the down-regulated CWMEs, Pectin methylesterase (PME) genes were found highly represented. Unlike, a larger gene set, in many cases with a higher fold-change of induction, was identified in TE respect to SG. This is the case of genes involved in the defense response and its regulation, and in the modification/reinforcement of the cell wall, therefore attesting for an initial tentative by the susceptible genotype to counteract the pathogen, although at the end without success. This was also the case of the seven VviPME genes previously highlighted by the in-silico co-expression analysis and therefore of VviPME10, the gene with the highest homology to AtPME17. Among the regulators, one WRKY factor (VviWRKY3), known to be related to defense response in grapevine mediated by stilbene synthesis, was also further characterized as putative regulator of VviPME10, whose promoter hosts more than one several predicted binding sites for VviWRKY3. Indeed, luciferase assay results indicate a significant activation of VviPME10 promoter by VviWRKY3 factor. Parallelly, the genome-wide analysis of the last structural annotation of the grapevine genome assembly allowed us to identify 62 VviPME gene members, 15 more than a previous report, and manually curate the gene structure for 39 of them. Then, to corroborate the idea of the role of the CW, and in particular of PME activity, in the grapevine response to the fungus, an in silico co-expression analysis of the 62 VviPME members, considering the publicly available RNA-seq experiments related to grapevine-Bc interactions and the RNAseq experiment conducted in this project, was performed. The analysis highlighted a group of seven genes (VviPME1, VvPME9, VviPME10, VviPME11, VviPME12, VviPME13 and VviPME54) with significant induction upon Bc infection, five of them (VviPME8, VviPME9, VviPME10, VviPME11, and VviPME54) located in the same chromosome (chr06). VviPME10 showed the highest homology and was found to be phylogenetically close to the Arabidopsis thaliana PME17 gene, suggesting being considered as its putative orthologue. Afterward, Therefore, considering the increased VviPME10 expression upon infection, and the reported effect of AtPME17 in A. thaliana, VviPME10 was selected as a potential candidate to study its role in grapevine. In this regard, two strategies were adopted, i. VviPME10 knock-out (KO) with CRISPR/Cas9 and ii. VviPME10 overexpression (OE, under CaMV35S promoter) through embryogenic callus transformation of the grapevine cultivar ‘Sugraone’ mediated by Agrobacterium tumefaciens. More than 100 embryos developed, and around 20 plantlets per transformation were analyzed to check the presence of the transgenic construct. Then, the mutation profile, in the case of KO lines, and the expression analysis of the transgene, in the case of OE lines, were carried out to select the appropriate lines to acclimatize. OE lines were also tested for VviPME10 activity. A total protein extract was obtained from the leaves of the lines, showing a higher protein activity compared to the control, and indicating the functionality of the enzyme. Unfortunately, grapevine OE lines couldn’t be analyzed for their response to Bc, while KO lines showed a significantly larger lesion area when compared to the control at 5 days post fungal inoculation (dpi). However, the effect of VviPME10 overexpression upon Bc infection was evaluated also in Nicotiana benthamiana VviPME10-OE lines, generated in parallel. At 3 dpi a significant reduction was observed in the lesion area compared to the control. These results suggest that pectin modification, mediated by VviPME10, plays an important role in the grapevine response to Bc, in particular it seems to behave more like a resistance gene than a susceptibility one. For this reason, it could be considered as a valuable target to improve resistance to Bc in susceptible grapevine varieties. Finally, a molecular method for Bc detection, based on quantitative RT-PCR assays, was set up and applied to estimate the Bc load in field conditions. Although the method allowed the successful detection of the presence of the fungus in samples at different developmental stages, from two V. vinifera cultivars, in different vineyards, the lack of environmental conditions for the development of the disease might have impaired the correlation between detection and the development of the disease. Nonetheless, the method represents a good alternative for monitoring the Bc load in the field at the early season, to predict the BcBR severity at harvest and eventually apply the disease management protocols based on the real need.

La presente tesi intende esplorare gli effetti della resistenza parziale sulle epidemie di peronospora della vite (Plasmopara viticola). È stato sviluppato un modello di simulazione teorico che comprende lo sviluppo della pianta ospite e le fasi principali della malattia, dalla mobilizzazione dell’inoculo, alla moltiplicazione della malattia sulle foglie, all’infezione dei grappoli. Attraverso esperimenti (monociclici) di inoculazione è stata studiata la risposta alle infezioni di P. Viticola di 16 varietà parzialmente resistenti, analizzando le seguenti componenti: frequenza d’infezione, durata del periodo di latenza, dimensione delle lesioni, produzione di sporangi, durata del periodo infezioso e infettività degli sporangi prodotti sulle lesioni. Queste componenti di resistenza sono state incorporate nel modello, attraverso cui sono stati studiati i loro effetti sull’epidemia (policiclica) in diversi scenari. Le componenti di resistenza hanno mostrato diversi livelli di efficacia nel sopprimere l’epidemia: l’efficienza di infezione e la produzione di sporangi risultano avere un maggiore impatto nella resistenza espressa a livello di pieno campo. Questo approccio è utile per guidare lo studio fenotipico della resistenza dell’ospite e per anticipare le prestazioni di un genotipo a livello di pieno campo, che risulterebbe difficile e dispendioso considerando la natura perenne della vite. / The present dissertation aims to explore the effects of partial resistance on grapevine downy mildew (Plasmopara viticola) epidemics. A theoretical simulation model was developed including host dynamics and main phases of the disease, from inoculum mobilisation to disease multiplication on foliage, and to infection of clusters. The response to P. Viticola infection was studied for 16 grapevine varieties through (monocyclic) inoculation experiments, by measuring components of partial resistance: infection frequency, duration of latent period, size of lesions, production of sporangia, duration of infectious period, and infectivity of sporangia produced on lesion. Components of partial resistance were incorporated into the model and their effects on the (polycyclic) epidemic were investigated accross different scenarios. Components of partial resistance showed different effectiveness on the suppression of epidemics, infection efficiency and spore production having the strongest impact on the overall field resistance response. This approach is an useful tool for phenotyping studies on host plant resistance and for anticipating the performance of a genotype at the field scale, that otherwise is difficult and time requiring due to the perennial nature of grapevine.

Il tema principale di questa tesi di dottorato verte sul metabolismo secondario di F. verticillioides, con particolare interesse alla via biosintetica, produzione e mascheramento delle fumonisine B (FB), fumonisine A (FA), fumonisine C (FC) e bikaverina, studiati in relazione all’ecologia fungina. E’ stato osservato che l’acqua libera (aw) esercita un effetto significativo maggiore rispetto alla temperatura sul metabolismo secondario di F. verticillioides. Alti valori di aw favoriscono l’espressione dei geni FUM ed una maggiore sintesi delle FB, FA e FC. Inoltre la produzione di bikaverina e l’espressione di BIK1 sono influenzate dall’ aw nello stesso modo che la produzione di FB e l’espressione dei geni FUM. Anche il tempo di incubazione è un fattore critico per la produzione di FB in F. verticillioides, così come per F. proliferatum: la produzione di FB, FA e FC aumenta nel tempo fino a 30 giorni, periodo dopo il quale si notano differenze tra le due specie fungine. Riguardo le fumonisine nascoste, queste sono state ritrovate in colture sia di F. verticillioides che di F. proliferatum. Non è stata registrata sintesi alcuna in colture cresciute su substrato di crescita sintetico, ma al contrario solo su colture cresciute su substrato a base di mais. / The main topic of this PhD thesis is F. verticillioides secondary metabolism, with regard to fumonisin B (FB), fumonisin A (FA), fumonisin C (FC) and bikaverin biosynthetic pathways and production and masking, studied in relation to fungal ecology. What we found is that water activity (aw) has a more significant effect than temperature on F. verticillioides secondary metabolism. Moreover bikaverin production and BIK1 expression are influenced by aw in the same way as FB production and FUM gene expression respectively. High aw levels favour FUM gene expression and allow the highest synthesis of FB, FA and FC. Incubation time is also critical for FB production both for F. verticillioides and F. proliferatum: the general trend is that FB, FA and FC production increases with time, up to 30 days, period after which differences can be noticed between F. verticillioides and F. proliferatum. As regard masked fumonisins, they are recovered in F. verticillioides and also F. proliferatum cultures, furthermore no synthesis is observed on cultures grown on synthetic medium, but only in those grown on maize-based substrates.