The postharvest phase has been considered an environment very suitable for successful application of biological control agents (BCAs). However, the tri-interaction between fungal pathogen, host (fruit) and antagonist is influenced by several parameters such as temperature, oxidative stresses, oxygen composition, water activity, etc. that could be determining for the success of biocontrol. Knowledge of the modes of action of BCAs is essential in order to enhance their viability and increase their potentialities in disease control. The thesis focused on the possibility to explain the modes of action of a biological control agent (BCA): Aureobasidium pullulans, in particular the strains L1 and L8, control effective against fruit postharvest fungal pathogen. In particular in this work were studied the different modes of action of BCA, such as: i) the ability to produce volatile organic compounds (VOCs), identified by SPME- gas chromatography-mass spectrometry (GC-MS) and tested by in vitro and in vivo assays against Penicillium spp., Botrytis cinerea, Colletotrichum acutatum; ii) the ability to produce lytic enzymes (exo and endo chitinase and β-1,3-glucanase) tested against Monilinia laxa, causal agent of brown rot of stone fruits. L1 and L8 lytic enzymes were also evaluated through their relative genes by molecular tools; iii) the competition for space and nutrients, such as sugars (sucrose, glucose and fructose) and iron; the latter induced the production of siderophores, molecules with high affinity for iron chelation. A molecular investigation was carried out to better understand the gene regulation strictly correlated to the production of these chelating molucules. The competition for space against M. laxa was verified by electron microscopy techniques; iv) a depth bibliographical analysis on BCAs mechanisms of action and their possible combination with physical and chemical treatments was conducted.

La Fusariosi della spiga (FDS) è una fitopatia diffusa a livello mondiale che colpisce le colture cerealicole, tra cui il frumento duro, ed è in grado di causare gravi danni di tipo qualitativo ed economico. Le specie fungine responsabili appartengono al genere Fusarium, tra cui F. graminearum, F. culmorum e più recentemente F. poae. La conseguenza più rilevante riguarda la contaminazione della granella da micotossine, molecole prodotte dai miceti, considerate dalla comunità scientifica ad alto rischio per la salute dell’uomo e animali. L’eziologia è molto complessa, dal momento che su una stessa spiga di frumento possono coesistere più specie fungine che contribuiscono ad influenzare i quantitativi di micotossine prodotte. Lo scopo della ricerca è incentrato sulla caratterizzazione di ceppi di F. poae, in termini di potenziale patogeno e aggressività. Tramite l’allestimento di un saggio di inoculazione in vitro “Petri-dish” è stato possibile attribuire un indice di aggressività a ciascun isolato fungino, basato su parametri quali AUHPC e AUDPC standard, insieme ad altre variabili come la riduzione della lunghezza del coleottile e del tasso di germinazione. Il saggio è stato esteso anche a F. culmorum, per valutare la riproducibilità del test su altre specie fungine. Il test in vitro offre diversi vantaggi, tra cui affidabilità e rapidità di esecuzione ed è quindi adatto allo screening di ceppi patogeni da utilizzare in successive sperimentazioni. Gli stessi ceppi di F. poae, provenienti da una prova di inoculazione artificiale in serra su piante di frumento duro, sono stati caratterizzati dal punto di vista bio-molecolare. Poichè lo studio della fusariosi della spiga richiede la determinazione quantitativa della biomassa dei patogeni nei tessuti della pianta-ospite, anche in assenza di sintomi, il protocollo di Real-Time PCR con chimica SYBR® Green I qui sviluppato, ha dimostrato essere un buon compromesso tra attendibilità, rapidità e costi complessivi della metodica. / Fusarium Head Blight (FHB) is one of the major diseases affecting worldwide cereral crops. It causes yield losses and decreasing grain quality, including the accumulation of mycotoxins. The causal agents, F. graminearum, F. culmorum and recently F. poae, belong to Fusarium genus. Contamination by mycotoxins is a great concern, as they are capable of harming the health of humans and farm animals. The aetiology is very complex, as one or more species can occur on a spike at the same time and can contribute to the total amount of mycotoxins. The aim of the research has been the characterization of F. poae strains regarding their pathogenicity and aggressiveness. It has been possible, by an in vitro “Petri-dish” test, to confer an aggressiveness index to each isolate, based on AUHPC and AUDPC standard parameters, together with the variables on germination rate reduction and coleoptile length reduction. The same test was also performed on F. culmorum strains, to assess its reproducibility on other Fusarium species; the benefits arisen from the test, which are reliability and rapidity in execution, make it suitable for screening analyses of pathogen strains that can be used for other purposes. The same F. poae strains, from a greenhouse artificial inoculation experiment on durum wheat plants, were also biomolecularly characterized. Estimation of fungal biomass, even in asymptomatic spikes, is fundamental to assess the pathogenicity of strains and it was possible by the Real-time PCR molecular technique with SYBR® Green I chemistry developed in this work, which showed to be sensitive, fast and specific for quantifying F. poae biomass.

Apple latent infection caused by Neofabraea alba: host-pathogen interaction and disease management Bull’s eye rot (BER) caused by Neofabraea alba is one of the most frequent and damaging latent infection occurring in stored pome fruits worldwide. Fruit infection occurs in the orchard, but disease symptoms appear only 3 months after harvest, during refrigerated storage. In Italy BER is particularly serious for late harvest apple cultivar as ‘Pink Lady™’. The purposes of this thesis were: i) Evaluate the influence of ‘Pink Lady™’ apple primary metabolites in N. alba quiescence ii) Evaluate the influence of pH in five different apple cultivars on BER susceptibility iii) To find out not chemical method to control N. alba infection iv) Identify some fungal volatile compounds in order to use them as N. alba infections markers. Results regarding the role of primary metabolites showed that chlorogenic, quinic and malic acid inhibit N. alba development. The study based on the evaluation of cultivar susceptibility, showed that Granny Smith was the most resistant apple cultivar among the varieties analyzed. Moreover, Granny Smith showed the lowest pH value from harvest until the end of storage, supporting the thesis that ambient pH could be involved in the interaction between N. alba and apple. In order to find out new technologies able to improve lenticel rot management, the application of a non-destructive device for the determination of chlorophyll content was applied. Results showed that fruit with higher chlorophyll content are less susceptible to BER, and molecular analyses comforted this result. Fruits with higher chlorophyll content showed up-regulation of PGIP and HCT, genes involved in plant defence. Through the application of PTR-MS and SPME GC-MS, 25 volatile organic compounds emitted by N. alba were identified. Among them, 16 molecules were identified as potential biomarkers.

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.

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.

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.

FHB dell’orzo è una malattia dall’eziologia complessa causata da specie fungine tossigene appartenenti al genere Fusarium in grado di compromettere interi raccolti. La predominanza di una specie di Fusarium rispetto ad un’altra dipende principalmente dal clima. Con i cambiamenti climatici in corso si sta assistendo ad una modificazione delle popolazioni fungine e per questo risulta fondamentale monitorare le specie fungine presenti in una determinata coltura, onde prevenire micotossicosi per l’uomo ed animali. Da analisi micologiche su granella di orzo proveniente da varie regioni italiane F. graminearum, F. poae e F. tricinctum sono risultate le specie più presenti. Prove in ambiente controllato hanno consentito di mettere a punto una scala fitopatometrica per la valutazione di FHB. Tale scala risulta pratica e facilmente applicabile ed è stata utilizzata per le prove di campo. In queste prove oltre a FHB sono stati valutati altri parametri quali produzione e contaminazione da micotossine. Il contenuto di micotossina Deossinivalenolo è correlata alla frequenza di F. graminearum su granella. I dati produttivi delle tesi inoculate non sono apparsi significativamente distinti dalle tesi non inoculate, facendo dedurre che granella contaminata da micotossina possa facilmente ritrovarsi nelle derrate alimentari, in quanto le cariossidi infette, solo lievemente più leggere di quelle sane. vengono ugualmente raccolte e commercializzate con serie conseguenze per l’industria mangimistica e alimentare. Con il presente lavoro si è dimostrato per la prima volta che per effetto della colonizzazione di culmi di piante di orzo da parte di tre specie Fusarium, responsabili sia della fusariosi del piede che della spiga, la micotossina DON può traslocare dalla base delle piante fino alla spiga, mentre la presenza dei funghi è rilevata da alte concentrazioni fino al secondo nodo. Da questo emerge che la presenza di Fusarium a livello basale contribuisce alla contaminazione della granella, oltre a quella dovuta ad FHB. / Fusarium head blight (FHB) is a barley disease with a complex etiology which is caused by different toxigenic Fusarium species able to compromise the whole crop yield. Climatic conditions establish the predominance of one Fusarium species respect to the others. With the climate change in progress we are attending to a modification of fungal populations that evidence the importance of monitoring the presence of fungal species in the crop, to prevent mycotoxicosis, dangerous for humans and animals. Mycological analysis in barley kernel from different Italian regions has shown that F. graminearum, F. poae and F. tricinctum are the main species. A phytopathometric scale to evaluate FHB was set up in controlled conditions. That scale, practical and easy to use, was used in field trials, where FHB, yield and mycotoxin contamination were evaluated. Mycotoxin DON content was correlated to F. graminearum frequency. Yield data of inoculated plots were not significant respect to the not inoculated ones, this implies that it is easy to find kernel contaminated by mycotoxins in food and feed, because infected grains are slightly lighter than those not infected and they are harvested and commercialized as well with serious consequences to public safety. For the first time on barley, with this study it was shown that after culm colonization of the plant by three toxigenic Fusarium spp., able to cause both FHB and FCR, DON mycotoxin can be transferred from the bottom to the head, whereas the presence of the fungi was with high level until the second node. This means that presence of Fusarium at base level contributes to the kernel contamination, additionally to FHB.

Gli stress abiotici determinando modificazioni a livello fisiologico, biochimico e molecolare delle piante, costituiscono una delle principali limitazioni per la produzione agricola mondiale. Nel 2007 la FAO ha stimato come solamente il 3,5% della superficie mondiale non sia sottoposta a stress abiotici. Il modello agro-industriale degli ultimi cinquant'anni, oltre ad avere contribuito allo sviluppo economico dell'Europa, è stato anche causa di inquinamento di acqua, aria e suolo, mediante uno sfruttamento indiscriminato delle risorse naturali. L'arsenico in particolare, naturalmente presente nell'ambiente e rilasciato dalle attività antropiche, desta particolare preoccupazione a causa dell'ampia distribuzione come contaminante ambientale e per gli effetti di fitotossicità provocati. In tale contesto, la diffusione di sistemi agricoli a basso impatto rappresenta una importante risorsa per rispondere all'emergenza del cambiamento climatico che negli anni a venire sottoporrà una superficie agricola sempre maggiore a stress di natura abiotica. Nello studio condotto è stato utilizzato uno stabile modello di crescita in vitro per valutare l'efficacia di preparati ultra diluiti (PUD), che non contenendo molecole chimiche di sintesi ben si adattano a sistemi agricoli sostenibili, su semi di frumento preventivamente sottoposti a stress sub-letale da arsenico. Sono state quindi condotte valutazioni sia a livello morfometrico (germinazione, lunghezza di germogli e radici) che molecolare (espressione genica valutata mediante analisi microarray, con validazione tramite Real-Time PCR) arricchendo la letteratura esistente di interessanti risultati. In particolare è stato osservato come lo stress da arsenico, determini una minore vigoria di coleptile e radici e a livello molecolare induca l'attivazione di pathways metabolici per proteggere e difendere le cellule vegetali dai danni derivanti dallo stress; mentre il PUD in esame (As 45x), nel sistema stressato ha indotto un recupero nella vigoria di germoglio e radici e livelli di espressione genica simili a quelli riscontrati nel controllo suggerendo un effetto "riequilibrante" del metabolismo vegetale. / The unquestionable importance of abiotic stresses to world agriculture is demonstrated by the fact that altogether abiotic factors provide the major limitation to crop production worldwide. A 2007 FAO report stated that only 3.5% of the global land area is not affected by environmental stresses and that climate change will increase areas characterized by abiotic stresses. The agro-industrial model that enriched European population in the last fifty years, however has resulted in irreversible contamination of water, air and soil. In particular arsenic is a toxic metalloid widely disseminated in the environmental as a consequence of natural processes and anthropic activities, that causes global concern for health and environmental hazards. Within this context, sustainable farming systems and the use of organic treatments such as ultrahigh diluted treatments (PUD) could be important tools of mitigating the effects of climate change. The objective of the present study was to test the biological effect of PUD As45x on wheat seedling poisoned with a sub-letal dose of arsenic, using an in vitro plant system. The evaluation of the arsenic toxicity and the effectiveness of As45x was conducted through biometric measurements and molecular analyses (microarray and Real-Time PCR). The results provided evidence for a strong gene modulating effect of arsenic, upregulating molecular pathways involved in cellular protection and defense from oxidative stress. It was also observed a reduction in the length of shoots and roots. Inversely in seedlings grown from poisoned seeds and treated with PUD, a recovery of coleoptiles and roots vigor was detected together with a massive reduction of gene expression levels to values comparable to those of the control group. A plausible hypothesis is that PUD induced a reequilibration of those genes that were upregulated during arsenic stress by bringing the expression levels closer to the basal levels normally occurring in control plants.