Ruolo del deossinivalenolo nella competizione per la paglia da parte di funghi filamentosi

I funghi sono noti produttori di una vasta gamma di metaboliti secondari, tra i quali le micotossine sono attualmente oggetto di studi approfonditi per i risvolti tossicologici dovuti alla contaminazione di foraggi e derrate alimentari. Numerose specie fungine produttrici di micotossine sono patogeni dei cereali e tra questi, alcune specie appartenenti al genere Fusarium sono noti per la produzione di tricoteceni, una classe di micotossine di cui fa parte anche il deossinivalenolo (DON), comune contaminante del frumento in tutto il mondo. I suoi effetti tossici sui mammiferi sono ben noti e riconducibili a sindromi emetiche ed emorragiche. Sebbene si possa ipotizzare che la produzione di micotossine abbia lo scopo di danneggiare gli altri organismi o di fornire un “vantaggio per la sopravvivenza” dei produttori contro i predatori, attualmente il ruolo ecologico di queste tossine non è stato ancora determinato con precisione. Questo lavoro di tesi è stato condotto al fine di stabilire se la presenza di DON potesse svolgere un ruolo nella competizione per la paglia tra funghi filamentosi di origine tellurica. La prova è stata allestita utilizzando tre differenti terreni, tutti coltivati a frumento (di cui è stata preliminarmente caratterizzata la componente micologica residente) in cui sono stati interrati, come esche, pezzi di paglia trattati o non con DON. Dopo una settimana, la paglia è stata sterilizzata superficialmente e piastrata su mezzi selettivi; in seguito, sono stati isolati i ceppi fungini colonizzatori delle esche. Tutti gli isolati sono stati sottoposti ad indagine microscopica, previa induzione, laddove possibile, della sporulazione e della differenziazione di strutture riproduttive. I risultati delle identificazioni non hanno evidenziato differenze significative nella composizione del micobiota della paglia trattata o non con la micotossina, lasciando ipotizzare una mancanza di ruolo svolto dal DON nella competizione per il substrato da parte dei funghi presenti nel terreno. Alcuni isolamenti ottenuti sono stati assegnati ai generi Pythium e Trichoderma. Poiché è noto in letteratura che una delle possibili strategie di contenimento della produzione e accumulo di micotossine sul frumento prevede l’impiego di microrganismi antagonisti di Fusarium spp., gli isolati identificati come appartenenti al genere Pythium (all’interno del quale, ceppi che differenziano oogoni spinosi sono stati descritti come antagonisti e micoparassiti di diverse specie di Fusarium fitopatogene) e l’unico isolato di Trichoderma ottenuto dalla prova, sono stati sottoposti ad ulteriori indagini al fine di valutare le loro capacità antagonistiche e micoparassitarie nei confronti di vari ceppi di Fusarium anch’essi isolati dalle esche. Ceppi degli antagonisti e di Fusarium provenienti dalla stesse esche o dalle stesse tesi sono stati utilizzati per allestire culture duali volte a valutare l’eventuale inibizione della crescita del patogeno e la comparsa di evidenti indizi di micoparassitismo, quali produzione di avvolgimenti ifali (coilings), lacci o appressori da parte degli antagonisti nei confronti del patogeno. Inoltre gli isolati di Pythium sono stati valutati per la loro capacità di micoparassitizzare macroconidi del patogeno. I risultati ottenuti da questa prova preliminare aprono la strada ad ulteriori indagini che prevedono l‘impiego di antagonisti nella lotta biologica a Fusarium spp. patogeni del frumento quale strategia di prevenzione della produzione e accumulo di micotossine nelle cariossidi

DON on wheat crop residues: effects on mycobiota as a source of potential antagonists of Fusarium culmorum

Fusarium culmorum, a pathogenic fungal species associated with Fusarium Head Blight (FHB) of wheat, can produce the mycotoxin deoxynivalenol (DON) that is frequently found as contaminant in cereals. Wheat haulms, as decomposing plant material, are an important inoculum source of F. culmorum for subsequent crops. In the present work we exploited the mycobiota of haulms buried in natural soil as a source of potential antagonists of F. culmorum and evaluated the role played by DON in determining the composition of such mycobiota. DON was added to wheat haulm pieces that were incubated in three natural soils, namely sandy, clayey and mixed, all with a previous history of wheat cultivation. Composition of mycobiota associated with DON-treated cultural debris was evaluated and compared with mycobiota from control haulms, in order to evaluate the effect of soil type and of the presence of the mycotoxin. Our results showed that DON affected neither the number nor species profile of fungal isolates. Among fungi associated with cultural debris, thirty-nine Pythium spp. isolates were collected and five of them, both from treated and not treated haulms, were tested for their antagonistic ability against F. culmorum. The two strains showing the highest antagonistic activity were further tested against F. oxysporum, a species considered a major competitor of F. culmorum for wheat residues, and frequently recovered from haulms in our experiments. The two Pythium spp. isolates showed a positive antagonistic and mycoparasitic activity against F. culmorum, without affecting the growth of F. oxysporum. Mycobiota of haulms buried in soil represents an interesting source of potential antagonists and competitors for future exploitation in a multitrophic approach for biocontrol of FHB

Distinct transcriptome responses to water limitation in isohydric and anisohydric grapevine cultivars

Background: Grapevine (Vitis vinifera L.) is an economically important crop with a wide geographical distribution, reflecting its ability to grow successfully in a range of climates. However, many vineyards are located in regions with seasonal drought, and these are often predicted to be global climate change hotspots. Climate change affects the entire physiology of grapevine, with strong effects on yield, wine quality and typicity, making it difficult to produce berries of optimal enological quality and consistent stability over the forthcoming decades. Results: Here we investigated the reactions of two grapevine cultivars to water stress, the isohydric variety Montepulciano and the anisohydric variety Sangiovese, by examining physiological and molecular perturbations in the leaf and berry. A multidisciplinary approach was used to characterize the distinct stomatal behavior of the two cultivars and its impact on leaf and berry gene expression. Positive associations were found among the photosynthetic, physiological and transcriptional modifications, and candidate genes encoding master regulators of the water stress response were identified using an integrated approach based on the analysis of topological co-expression network properties. In particular, the genome-wide transcriptional study indicated that the isohydric behavior relies upon the following responses: i) faster transcriptome response after stress imposition; ii) faster abscisic acid-related gene modulation; iii) more rapid expression of heat shock protein (HSP) genes and iv) reversion of gene-expression profile at rewatering. Conversely, that reactive oxygen species (ROS)-scavenging enzymes, molecular chaperones and abiotic stress-related genes were induced earlier and more strongly in the anisohydric cultivar. Conclusions: Overall, the present work found original evidence of a molecular basis for the proposed classification between isohydric and anisohydric grapevine genotypes

BIOCONTROL OF FUSARIUM HEAD BLIGHT: MOLECULAR INTERACTION BETWEEN TRICHODERMA AND MYCOTOXIGENIC FUSARIUM

Fusarium Head Blight (FHB) is a re-emerging disease of wheat that causes extensive agricultural damage through direct losses in yield and quality due to the presence of Fusarium damaged kernels and their associated mycotoxins such as the trichothecene deoxynivalenol (DON). Biological control, including the treatment of crop residues with antagonists such as mycoparasitic Trichoderma species to reduce pathogen inoculum of FHB, holds considerable promise. The main purpose of this PhD thesis is to select fungal antagonists able to compete with FHB causal agents, particularly Fusarium graminearum and Fusarium culmorum, in order to prevent the presence of DON on wheat. Potential antagonists were screened on the basis of their growth in presence of DON in liquid cultures in multi-wells microplates. For each isolate, growth curves in presence/absence of DON were submitted to linear regression and statistically compared. 10 out of 100 Trichoderma spp., 56 out of 93 F. oxysporum and 2 out of 40 Pythium spp. isolates showed a statistically comparable growth rate in presence/absence of DON. At the end of growth period, cultural filtrates were submitted to HPLC analysis in order to investigate the fate of the mycotoxin. No one of cultural substrates showed a DON content lower than the initial one, enabling us to reject the hypothesis of mycotoxin degradation. The ten Trichoderma isolates and two Pythium spp., selected for their ability to grow in presence of DON, were investigated as potential antagonists against Fusarium culmorum and F. graminearum mycotoxigenic isolates in plate confrontation assays. The results showed that three Trichoderma isolates were successful antagonists and exhibited antibiosis and mycoparasitism. In addition, we assessed the transcription of some chitinase-encoding genes, known for their involvement during mycoparasitic growth conditions, in various stages of plate confrontation assays with F. culmorum and F. graminearum by RT-PCR. The results showed that almost all investigated genes encoding chitinases from subgroups A, B and C responded to mycoparasitic conditions and were up-regulated before contact or/and at contact with the host. In order to evaluate the ability to prevent DON production by F. graminearum, competition tests on natural substrates of these three Trichoderma isolates against mycotoxigenic F. graminearum confirmed the positive results achieved on agar plates. Among these 3 isolates, one showed the best capacity of inhibiting DON production and was chosen for the following analysis. An in vitro competition test on natural substrates (wheat straw and rice) was performed using qPCR as a tool to estimate the effect of the selected T. gamsii isolate on pathogen’s growth and DON accumulation. Results achieved on rice, confirmed the ability of T. gamsii 6085 to antagonize F. graminearum and F. culmorum isolates whose biomass, in presence of Trichoderma, was lower then the control. On wheat straw, a substrate very poor in nutrients, T. gamsii 6085 seemed to develop very badly and growth of both Fusarium was unaffected by the presence of the antagonist. In the last part of the work we investigated a possible mechanism possibly involved in DON resistance in Trichoderma. We evaluated the gene expression of some PDR-ABC transporters in confrontation plate assay and in the above mentioned competition test on rice

Brachetto

Accessione Brachetto:Foto: Claudio D'Onofrio, Puntoni Piero Ampelografia: Claudio D'Onofrio, Belluomini Paolo, Calabrò Rolando, Rocco Fausta Ampelometria: Claudio D'Onofrio Fenologia: Claudio D'Onofrio, Ducci Eleonora, Matarese Fabiola, Cuzzola Angela SSR: Claudio D'Onofrio, Matarese Fabiol

Analysis of the expression of terpene synthase genes in relation to aroma content in two aromatic Vitis vinifera varieties.

Grape (Vitis vinifera L.) flavour management in the vineyard requires knowledge of the derivation of individual flavour and aroma characteristics. Some of the most prevalent wine grape aroma constituents are terpenoids and this study represents a wide report about grape terpene synthase (TPS) gene transcript profiling in different tissues of two aromatic grapevine varieties, particularly flowers and developing berries, correlated with the accumulation patterns of free aroma compounds. All investigated genes belonging to the TPS-a and TPS-b subfamilies reached the highest expression in accordance with the peak of accumulation of the respective compounds. In the TPS-g subfamily, only one of the genes characterised for linalool synthases showed major transcript abundance in ripening berries, whereas the only geraniol synthase had a peak of expression in green berries and at the beginning of ripening, when geraniol concentration started to increase and overcome the linalool concentration. The genes identified in this study as being mainly responsible for linalool and geraniol synthesis during berry development, and the phenological phases in which they are mostly expressed, should be of interest to viticulturists and wine makers to improve decision making along the chain of production

Effect of methyl jasmonate on the aroma of Sangiovese grapes and wines

Methyl jasmonate (MeJA) was applied in a vineyard on leaves and grape clusters of cv Sangiovese to test its ability to stimulate the production of aromas and identify the main genes involved in the biosynthetic pathways switched on by the elicitor. MeJA application led to a delay in grape technological maturity and a significant increase in the concentration of several berry aroma classes (about twice the total aroma: from around 3 to 6 μg/g of berry). Of these, monoterpenes showed the most significant increase. An analysis of the expression of terpenoid biosynthesis genes confirmed that the MeJA application activated the related biosynthetic pathway. The expression of all the TPS genes analyzedwas higher in samples treated with MeJA. Also the wines produced by microvinification of Sangiovese treated and untreated grapes showed a rise in the aroma concentration as in berries, with an important impact on longevity and sensorial characters of wines

Barghigiana

Per la prima volta è stata individuata in Lucchesia (molto probabilmente in Garfagnana) da Basso dell'Università di Pisa (1993) che l'ha introdotta nella collezione 'Vitiarium' dell'Agricola San Felice (Siena). Recentemente è stata individuata e caratterizzata in Garfagnana, dove al momento ha una presenza sporadica, confrontata con quella presente nella collezione 'Vitiarium' confermanodone l'identità (D'Onofrio et al., 2015) Al momento non corrisponde a nessuno dei genotipi presenti nei vari database viticoli, e inoltre presenta delle relazioni di parentela di primo grado con 'Canina bianca', 'Rossara tardiva' e 'Sillicana bianca', tutti vitigni strettamente autoctoni della Garfagnana. Pertanto, queste indicazioni, congiuntamente alle uniche citazioni storiche che la riportano in Lucchesia, si può supporre che sia effettivamente un vitigno autoctono della Garfagnana (D'Onofrio et al., 2015). Presenta una produzione tendenzialmente elevata, con grappoli medio-grandi e compatti. Fatica ad accumulare un adeguata grado zuccherino, e al contempo nel corso della maturazione l'acidità di riduce velocemente. Al momento non abbiamo dati sufficienti sulle caratteristiche tecnologiche

Moscato bianco

Moscato bianco (clone Rauscedo 2

Lombardesca

La prima citazione della 'Lombardesca nera' è di Basso (1992) che l'ha identificata in Garfagnana (Lucca), mentre il sinonimo 'Corvara nera' è stato accertato più recentemente sempre in garfagnana (D'Onofrio et a., 2015).La Corvara/Lombardesca nera non corrisponde a nessuno dei genotipi caricati nei vari Vitis database e appare essere presente esclusivamente in Garfagnana (D'Onofrio et al., 2015).L’uva è dolce e ha elevata acidità totale. Appare essere un vitigno di 3° epoca