Academic literature on the topic 'Funghi'

The antimicrobial activity of dimethyl sulfoxide extract of moss Rhodobryum ontariense (Kindb.) Kindb. was evaluated by microdilution method against eight bacterial (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Enterobacter cloacae, Listeria monocytogens, Bacillus cereus, Micrococcus flavus and Staphylococcus aureus) and five fungal species (Aspergillus versicolor, Aspergillus fumigatus, Penicillium funiculosum, Penicillium ochrochloron and Trichoderma viride). The extract was proven to be active against all the bacteria and funghi tested but to varying degrees. It showed better inhibitory activity compared to the known antifungal drug against T. viride (MIC 100 and 200 ?g/ml, respectively). This finding implies that R. ontariense could be considered as a promising material for natural antifungal products.

Abstract The Tower of Beiern was colonized by various organisms, ranging from endolithic bacteria to higher plants. The paper focuses on the deterioration that the endolithic microorganisms: bacteria and cyanobacteria, algae and funghi, cause on the compact Lioz limestone. The presence of these microorganisms in the subparallel Assuring characteristic of this stone is illustrated as well as their association with the clay minerals present. Their biogeochemical action results in an increase in surface roughness, subsurface porosity and eventual flaking of the stone surface.

Le micotossine sono metaboliti secondari prodotti da alcuni funghi filamentosi, che possono contaminare piante coltivate o alimenti e mangimi immagazzinati; tra questi i più importanti funghi micotossigeni coinvolti nella contaminazione alimentare, appartengono a tre generi: Aspergillus, Fusarium e Penicillium. Più di 300 micotossine sono state identificate e questi metaboliti secondari possono essere dannosi per la salute umana ed animale quando ingeriti (Bennett and Klich, 2003). Le principali micotossine contaminati di alimenti e mangimi sono: aflatossine, ocratossina, fumonisine, tricoteceni e zearalenone. Le aflatossine rappresentano la classe più importante di micotossine, comunemente presenti nel mais e in altri cereali, i principali funghi responsabili della loro produzione sono Aspergillus flavus e A. parasiticus (Shepard, 2008). Invece i cereali, il caffè, il cacao, il vino, la birra e gli alimenti di origine animale sono spesso contaminati dall'ocratossina A, prodotta principalmente da A. ochraceus (Van der Merwe et al., 1965), A. carbonarius, Penicillium verrucosum e P. nordicum. Fusarium verticillioides e F. proliferatum producono fumonisine, che sono spesso rilevate nel mais e nei sottoprodotti (Dutton, 1996). I trichoteceni, che si trovano spesso nei cereali, in particolare nel frumento e nel mais, sono divisi in quattro gruppi, i due principali sono: il tipo A con le tossine T2 e HT-2, prodotte da F. langhsetiae e F. sporotrichioides (Van der Fels-Klerk e Stratakou, 2010); il tipo B con il deossinivalenolo (DON) e il nivalenolo (NIV) prodotti da F. graminearum e F. culmorum (Placinta et al., 1999; Turner, 2010). Inoltre, la contaminazione da DON si trova frequentemente in associazione con un'altra micotossina prodotta dagli stessi funghi, lo zearalenone (ZEA) (Logrieco et al., 2002a). Queste specie sono responsabili di infezioni che si verificano sia nel campo che durante la conservazione post-raccolta, in particolare quando i cereali sono immagazzinati in condizioni inadeguate (ad esempio alte temperature e alta umidità). Una grande varietà di effetti tossici negli animali e nell'uomo è stata osservata a causa dell'ingestione di cibo contaminato da micotossine, quali: immunosoppressione, effetti cancerogeni, genotossici, teratogeni o mutageni (Peraica et al., 1999; Richard, 2007). La contaminazione da micotossine è diventata una preoccupazione per la salute pubblica con gravi implicazioni economiche ed etiche. Dal momento che non è completamente possibile impedire la produzione di micotossine, le autorità nazionali e internazionali hanno adottato limiti regolatori e linee guida per monitorare i livelli di micotossine in vari prodotti alimentari e nei mangimi (EC 2006a e 2006b; Commision Recommendation 2013/165/UE). Diversi metodi fisici, chimici e biologici sono stati raccomandati per la detossificazione di alimenti e mangimi contaminati da micotossine. Tuttavia, solo alcuni di essi sono stati accettati per l'uso pratico. Molti specialisti pensano che l'approccio migliore per la decontaminazione da micotossine debba essere la degradazione biologica, dando la possibilità di rimuovere micotossine in blande condizioni, senza usare sostanze chimiche dannose e senza perdite significative nel valore nutritivo e nell’appetibilità di alimenti o mangimi detossificati. A seconda della loro modalità di azione, questi additivi per mangimi possono agire legando micotossine alla loro superficie (adsorbimento) o degradandoli o trasformandoli in metaboliti meno tossici (biotrasformazione). L'efficacia del legante di queste sostanze si basa sulle proprietà sia del legante che della micotossina. La biotrasformazione può essere ottenuta da enzimi che degradano le micotossine o da microrganismi (funghi e batteri) che producono tali enzimi. Vari adsorbenti inorganici, alluminosilicati e carboni attivi, sono stati testati e utilizzati come leganti micotossine (MB). Un'alternativa interessante agli adsorbenti inorganici per la detossificazione delle micotossine è l'uso di leganti organici, come i componenti della parete cellulare del lievito, i batteri dell'acido lattico, i conidi degli Aspergilli. Questi MB sono utilizzati l’alimentazione animale al fine di ridurre l'assorbimento delle micotossine dal tratto gastrointestinale e la loro distribuzione al sangue e agli organi bersaglio, prevenendo o riducendo le micotossicosi nel bestiame. Recentemente, l'uso di tali sostanze come additivi per mangimi tecnologici è stato ufficialmente autorizzato dall'Unione europea (Commission Regulation 2015/786). Enzimi ligninolitici, come le laccasi, dai funghi del marciume bianco, come Pleurotus spp. che catalizzano l'ossidazione di un ampio spettro di composti fenolici e di ammine aromatiche utilizzando l’ossigeno molecolare come accettore di elettroni, che viene quindi ridotto ad acqua (Reinhammar e Malstrom, 1981). Aggiungendo alla reazione l’appropriato mediatore redox può estendere l'attività degli enzimi laccasi a substrati non fenolici, come le micotossine. Questa tesi di dottorato è organizzata in sei capitoli e un allegato, in cui sono descritte le seguenti attività. Nel Capitolo 1 sono stati raccolti centosettantacinque campioni di grano durante le stagioni agricole: 2013-2014, 2014-2015 e 2015-2016 in diverse regioni italiane. I tricoteceni (DON, NIV, tossine T2 e HT-2) e i livelli di ZEA sono stati monitorati attraverso l'uso di metodi analitici validati, per fornire una visione d’insieme della distribuzione italiana delle micotossine nel grano. Le specie di Fusarium isolate dai semi sono state identificate in base alle loro caratteristiche morfologiche. Nel Capitolo 2, lo sviluppo di una tecnologia innovativa per la bioremedation del mais contaminato dall'AfB1 e della sua bioconversione in mangime ad un alto apporto nutrizionale, è stato realizzato attraverso lo sfruttamento della capacità degradativa del Pleurotus eryngii. A tale scopo, è stata studiata l'attività degradativa dei confronti dell’AfB1 da parte di un estratto enzimatico grezzo ottenuto da un substrato esausto e la capacità del fungo commestibile P. eryngii di degradare l'AfB1 sia in vitro che in una coltivazione dei funghi su scala di laboratorio. Nel Capitolo 3, è stata valutata la capacità del micelio non vitale del P. eryngii (ITEM 13681) di assorbire l’AfB1. Sono stati valutati l'influenza di diversi parametri, quali: pH (5, 7), concentrazioni dell’AfB1 (da 50 a 1000 ng/mL), tempo (da 30 a 120 min), temperatura (25, 37 ° C), massa fungina (da 50 a 1000 mg), sulla capacità di assorbimento del micelio di P. eryngii. La stabilità del legame AfB1-bioassorbente e gli studi di desorbimento sono stati effettuati variando, rispettivamente, il pH a 7 e 3, per 24 ore di incubazione a temperatura ambiente e al buio. Nel capitolo 4, l'attività di degradazione di due laccasi ottenute da due funghi commestibili (P. eryngii e P. pulmonarius) nei confronti di AfB1, AfM1, FB1, ZEA e tossina T2, sono state valutate singolarmente, aggiungendo alla reazione mediatori naturali ed artificiali. L'effetto dei sistemi laccasi-mediatore (LMSs) è stato analizzato mediante cromatografia liquida ad elevata prestazione accoppiata ad uno specifico rivelatore, scelto in base alle caratteristiche chimiche di ciascuna tossina. Nel capitolo 5, l'obiettivo perseguito era quello di investigare l'azione dei sistemi laccase-mediatore (LMSS) verso più tossine, simultaneamente. A tal fine, sono stati eseguiti diversi test di degradazione, esaminando l'effetto dei diversi mediatori, come acetosyringone (AS), syringaldehyde (SA) e del mediatore sintetico 2,2,6,6-tetrametil-piperidinil-ossil (TEMPO), sull'attività della laccasi da Trametes versicolor (CE 1.10.3.2) verso l'acido fusarico (FA) e micotossine, come: DON, T2, FB1, AfB1, OTA e ZEA. Un metodo multi micotossina, è stato sviluppato per determinare simultaneamente queste sette tossine, mediante cromatografia liquida con spettrometria di massa in tandem (LC-MS/MS). Nel capitolo 6, l'attività di degradazione dell’enzima laccasi verso lo ZEA è stata ulteriormente investigata. I prodotti della biodegradazione sono stati monitorati mediante cromatografia liquida con spettrometria di massa ad elevata risoluzione (LC-HRMS). I dati sono stati elaborati dal software MassHunter Workstation (Qualitative Analysis Navigator e Workflow di analisi qualitativa, versione B.08.00), Mass Profile Professional (versione 14.08) e MassHunter Molecular Structure Correlator (versione B.08.00)) di Agilent Technologies, per consentirne l'identificazione. Nell'Annesso A, la produzione di Enniatine (A, A1, B e B1) e Beauvericina da varie specie di Fusarium è stata valutata mediante cromatografia liquida ad alte prestazioni accoppiata con array di fotodiodi e spettrometro di massa a singolo quadrupolo (UPLC-PDA-QDa).
Mycotoxins are secondary metabolites produced by certain filamentous fungi, which can contaminate crop plants or stored food and feed; among them the most important mycotoxigenic fungi involved in food contamination, belong to three genera: Aspergillus, Fusarium and Penicillium. More than 300 mycotoxins have been identified and these secondary metabolites can be harmful to human and animal health when ingested (Bennett and Klich, 2003). Main mycotoxins contaminant in food and feed are: aflatoxins, ochratoxins, fumonisins, trichothecenes and zearalenone. Aflatoxins represent the most important class of mycotoxins, commonly found in maize and other cereals, the main fungi responsible for their production are Aspergillus flavus and A. parasiticus (Shepard, 2008). Instead grains, coffee, cocoa, wine, beer, and foods from animal origin are often contaminated by ochratoxin A, that is mainly produced by A. ochraceus (Van der Merwe et al., 1965), A. carbonarius, Penicillium verrucosum and P. nordicum. Fusarium verticillioides and F. proliferatum produce fumonisins, which are often detected in maize and by-products (Dutton, 1996). Trichothecenes, which are often found in cereal grains, in particular in wheat and maize, are divided in four groups, the principal two groups are: type-A with T2 and HT-2 toxin, produced by F. langhsetiae and F. sporotrichioides (Van der Fels-Klerk and Stratakou, 2010); Type-B with deoxynivalenol (DON) and nivalenol (NIV) produced by F. graminearum and F. culmorum (Placinta et al., 1999; Turner, 2010). Moreover, DON contamination is frequently found in association with another mycotoxin produced by the same fungi, zearalenone (ZEA) (Logrieco et al., 2002a). These species are responsible for infections occurring both in the field and during postharvest storage, particularly when cereals are stored under inappropriate conditions (e.g. high temperatures and high humidity). A large variety of toxic effects in animals and humans has been observed due to the ingestion of food contaminated with mycotoxins, such as: immunosuppression, carcinogenic, genotoxic, teratogenic or mutagenic effects (Peraica et al., 1999; Richard, 2007). Mycotoxin contamination became a public health concern with serious economical and ethical implications. Since it is not completely possible to prevent the synthesis of mycotoxins, national and international authorities have adopted regulatory limits and guidelines to monitor mycotoxin levels in various food and feed products (EC 2006a and 2006b; Commission Recommendation 2013/165/UE). Different physical, chemical and biological methods have been recommended for detoxification of food and feed contaminated by mycotoxins. Nevertheless, only a few of them have been accepted for practical use. A lot of specialists think that the best approach for mycotoxin decontamination should be the biological degradation, giving the possibility to remove mycotoxins under mild conditions, without using harmful chemicals and without significant losses in nutritive value and palatability of detoxified food or feed. Depending on their mode of action, these feed additives may act either by binding mycotoxins to their surface (adsorption), or by degrading or transforming them into less toxic metabolites (biotransformation). The binder efficacy of these substances is based on the properties of both the binder and the mycotoxin. Biotransformation can be achieved by mycotoxin-degrading enzymes or by microorganisms (fungi and bacteria) producing such enzymes. Various inorganic adsorbents, aluminosilicate and activated carbons, have been tested and used as mycotoxins binders (MB). An interesting alternative to inorganic adsorbents for the detoxification of mycotoxins is the use of organic binders, such as, cell wall components of yeast, lactic acid bacteria, conidia of Aspergilli. These MB are used to feed animal diet in order to reduce the absorption of mycotoxins from the gastrointestinal tract and their distribution to blood and target organs, thus preventing or reducing mycotoxicosis in livestock. Recently, the use of such substances as technological feed additives has been officially allowed in the European Union (Commission Regulation 2015/786). Ligninolytic enzymes, such as laccase, from white-rot fungi, as Pleurotus spp. catalyzed the oxidation of a broad number of phenolic compounds and aromatic amines by using molecular oxygen as the electron acceptor, which is then reduced to water (Reinhammar and Malstrom, 1981). Adding the appropriate redox mediator to the reaction can extend the activity of the laccase enzymes to nonphenolic substrates, such as mycotoxins. This PhD thesis is organized into six chapters and one annex, where the following tasks are described. In Chapter 1, one hundred and seventy-five wheat samples were collected during the growing seasons: 2013-2014, 2014-2015 and 2015-2016 in different Italian regions. Trichothecenes (DON, NIV, HT-2 and T2 toxins) and ZEA levels were monitored through the use of validated analytical methods, to provide an overview of the Italian distribution of mycotoxins in wheat. The Fusarium species isolated from the kernels were identified, based on their morphological characteristics. In Chapter 2, the development of an innovative technology for the bioremediation of AfB1-contaminated maize and its bioconversion into high nutritional feed, was realized through the exploitation of the degradative capability of Pleurotus eryngii. For this purpose, the AfB1–degradative activity of a crude enzymatic extract from a spent substrate and the ability of the white-rot and edible fungus P. eryngii to degrade AfB1 both in vitro and in a laboratory-scale mushroom cultivation, were investigated. In Chapter 3, the power of ground not-viable mycelium of P. eryngii (ITEM 13681) to absorb AfB1, was assessed. The influence of different parameters: pH (5, 7), AfB1 concentrations (50 and 1000 ng/mL), time (30 and 120 min), temperature (25 and 37°C), fungal mass (50 and 1000 mg), on the absorption capability of the mycelium of P. eryngii. were evaluated. Binding stability of AfB1-biosorbent and desorption studies were carried out varying, respectively, the pH to 7 and 3, for 24 hours of incubation at room temperature in the dark. In Chapter 4, the degradation activity of two laccases from two edible fungi (P. eryngii and P. pulmonarius) towards AfB1, AfM1, FB1, ZEA and T2 toxin, were evaluated separately, adding to the reaction natural and artificial mediators. The effect of laccase-mediator systems (LMSs) were analyzed by liquid chromatography with specific detector, based on the chemical feature of each single toxin. In Chapter 5, the aim pursued was to investigate the action of LMSs toward multiple toxins. For this purpose, several degradation assays were performed, screening the effect of different mediators, as acetosyringone (AS), syringaldehyde (SA), and synthetic mediator as 2,2,6,6-tetramethyl-piperidinyloxyl (TEMPO), on the activity of laccase from Trametes versicolor (EC 1.10.3.2) towards fusaric acid (FA) and mycotoxins, such as: DON, T2, FB1, AfB1, OTA and ZEA. A multi mycotoxin method, was set up to simultaneously screen these seven toxins, by liquid chromatography/tandem mass spectrometry (LCMS/ MS). In Chapter 6, the biodegrading activity of laccases enzymes towards ZEA has been further investigated. The degradation products were monitored by liquid chromatography–high resolution mass spectrometry (LC-HRMS). Data were processed by MassHunter Workstation Software (Qualitative Analysis Navigator and Qualitative Analysis Workflow, version B.08.00), Mass Profile Professional (version 14.08) and MassHunter Molecular Structure Correlator (version B.08.00)) from Agilent Technologies, to allow their identification. In Annex A, the Enniatins (A, A1, B and B1) and Beauvericin production from various Fusarium spp. were measured by ultra-performance liquid chromatography coupled with photodiode array and single quadrupole mass spectrometer (UPLC-PDA-QDa).

Spontaneous chlorate resistant mutants of Penicillium chrysogenum and Cephalosporium acremonium have been isolated. Putative genotypes of the P. chrysogenum mutants were identified using the phenotypic characterization of Cove (1979). This analysis was also attempted with C. acremonium, however it was found that the wild type organism could not grow on minimal media containing glucose and hypoxanthine and thus cnx and niaD mutants could not be differentiated using this method. Following the distinction of cnx and niaD mutants using cytochrome C reductase and purine hydroxylase I assays, a simple plate test using minimal media containing quinic acid as carbon source and inosine as nitrogen source was developed to analyse cnx and niaD mutants. Nonreverting (at less than 1 in 108) niaD mutants were isolated for later experiments. P. chrysogenum niaD mutant niaD19 was transformed to nitrate utilization at a frequency of up to 20 transformants/mug DNA using the Aspergillus nidulans niaD gene, up to nine/mug DNA with the A. niger niaD gene and up to three/mug DNA using the A. oryzae niaD gene. Vector constructs carrying the A. nidulans ans-1 sequence with the A niger niaD gene did not show increased transformation efficiency. Linearization of the A. niger niaD containing plasmid resulted in a two to three times increase in transformation frequency. Southern blot hybridization analysis demonstrated that vector sequences had integrated into the recipient genome. The control of heterologous niaD gene expression generally agreed with that found in the wild type strain, that is, induction by nitrate and repression in the presence of ammonium. The A. nidulans, A. niger and A. oryzae genes failed to transform C. acremonium niaD mutants. A DNA fragment containing the C. acremonium niaD gene was isolated by cross hybridization to the A. nidulans niaD gene. This fragment was sub-cloned into pUC18 (designated pSTA700), and showed back hybridization to C. acremonium wild type DNA exhibiting the expected hybridization pattern. When partially sequenced, it showed nucleotide and determined amino acid homology to the A. nidulans niaD gene and protein. The clone pSTA700 transformed a C. acremonium niaD mutant, CSG116 and P. chrysogenum STP19 to nitrate utilization at a frequency of up to 47 and six transformants/mug DNA, respectively. When heat shock was applied to C. acremonium protoplasts 10mins prior to the addition of DNA, transformation frequencies of up to 137/mug DNA were obtained. Southern analysis of transformants revealed multiple integration of vector sequences, with no detectable preference for the homologous niaD site. Some C. acremonium transformants showed a greatly increased nitrate reductase activity (up to 10 times wild type activity) when induced with nitrate. The niaD transformation system was successfully used to introduce unselected markers into C. acremonium such as hygromycin B and benomyl resistance. The co-transformation frequency was up to 25% when equal molar ratios of plasmids were used. Antibiotic biosynthetic genes isolated from C. acremonium (pcbC and cefEF) and A. nidulans (pcbC and penE) were introduced into C. acremonium CSG116 by co-transformation with the C. acremonium niaD gene or the construction of vectors containing both the C. acremonium niaD gene and antibiotic biosynthetic genes. Isolates transformed with the C. acremonium pcbC and cefEF genes exhibited an increase in antibacterial activity (greater than 20% compared to wild type) at frequencies of up to 10% and six percent of transformants, respectively. When A. nidulans constructs containing the pcbC and penE genes, but not the pcbC gene alone, were co-transformed into C. acremonium, up to 18% of transformants exhibited an increase in antibacterial activity. The presence of penicillin antibiotics could not be found in these transformants and thus the exact cause of this increase in antibacterial activity could not be determined.

I mitt examensarbete vill jag belysa vikten av kontakt mellan människa och natur samt uppmärksamma naturens läkande förmågor och ge utrymme för paus i våra hektiska liv. Projektet fokuserar kring hur inredningsarkitekter och möbelformgivare, genom design, kan främja ett bättre mående med association till naturen som inspiration och verktyg.  Genom trä som material utforskar jag olika hantverksmetoder och de självteurapeftiska effekter som genomföradeprocessen kan ha. Arbetet syftar till att förstå hur vi som människor genom inredningsarkitektur och möbeldesign påverkas fysiskt och psykologiskt av de strukturer, former och det material som omger oss.

Il fenomeno della contaminazione chimica dei sedimenti marini costieri è molto diffuso e rappresenta una grande preoccupazione per il benessere della biodiversità e dell’ecosistema. Il biorisanamento è una strategia ecocompatibile che sta ottenendo sempre più attenzione grazie al suo potenziale di ripulire sedimenti marini contaminati. In questa tesi di dottorato, prima di tutto, ho fornito una panoramica delle attuali conoscenze e prospettive sul biorisanamento di sedimenti marini, presenti in letteratura. Quindi ho valutato la diversità degli assemblaggi microbici in diversi sedimenti cronicamente contaminati delle aree di Bagnoli-Coroglio, Mar Piccolo di Taranto e Falconara Marittima (tutti inclusi nell'elenco dei Siti di Interesse Nazionale, SIN) e le loro relazioni con il livello e la tipologia degli inquinanti chimici. Ho testato l'efficienza delle strategie di biostimolazione nella degradazione degli Idrocarburi Policiclici Aromatici (IPA) in sedimenti a diverso livello di contaminazione sulla base di approcci di aggiunta di nutrienti inorganici e bioaumento utilizzando i singoli consorzi batterici o fungini o entrambi, precedentemente isolati e identificati; ho inoltre studiato i cambiamenti nella ripartizione dei metalli e nella diversità microbica dovuti ai biotrattamenti. I risultati qui presentati suggeriscono che i contaminanti chimici possono avere un ruolo importante nel modellare la diversità procariotica, potenzialmente selezionando taxa microbici tolleranti/resistenti. I sedimenti di Falconara Marittima ospitano taxa microbici con un'elevata capacità di biobonifica verso gli IPA. Questi taxa microbici, batteri e funghi, una volta isolati e cresciuti su terreni specifici, possono essere efficaci per il biorisanamento dei sedimenti di Bagnoli altamente contaminati da IPA. Nonostante i risultati riportati in questo studio non consentano di distinguere tra l'importanza relativa dei taxa microbici alloctoni e autoctoni sulla biodegradazione degli IPA, forniscono nuove conoscenze sulle interazioni batterico-fungine che si verificano durante il biorisanamento dei sedimenti marini altamente contaminati. Complessivamente, questi risultati suggeriscono che i biotrattamenti basati su consorzi batterici e/o fungini selezionati o su una combinazione di entrambi potrebbero costituire una strategia efficace per ridurre significativamente, in tempi relativamente brevi, la contaminazione da IPA dei sedimenti marini, portando possibilmente a opzioni di gestione alternative rispetto al dragaggio e allo smaltimento in discarica.
Chemical contamination of coastal marine sediments is a widespread phenomenon and represents a major concern for biodiversity and ecosystem health. Bioremediation is an environmental-friendly strategy gaining increasing attention for its potential to clean-up contaminated marine sediments. In this PhD thesis, first of all, I provided an overview of the current knowledge and perspectives on the bioremediation of marine sediments, based on literature review. Then I assessed the diversity of microbial assemblages in different chronically contaminated sediments of the Bagnoli-Coroglio, Mar Piccolo of Taranto and Falconara Marittima areas (all of them included in the list of Sites of National Remediation Interest) and their relationships with the level and typology of chemical pollutants. I tested the efficiency of biostimulation strategies based on inorganic nutrient addition and bioaugmentation approaches using selected bacterial or fungal consortia or both, previously isolated and identified, on PAH degradation in sediments displaying different contamination level and I investigated changes in metal partitioning and microbial diversity due to biotreatments. Results presented here suggest that chemical contaminants can have an important role in shaping prokaryotic diversity, potentially by selecting tolerant/resistant microbial taxa. Sediments of Falconara Marittima host microbial taxa with a high bioremediation capacity toward PAHs. These microbial taxa, including both bacteria and fungi, once isolated and growth on selected media, can be effective for the bioremediation of Bagnoli sediments highly contaminated with PAHs. Despite findings reported in this study do not allow disentangling the relative importance of the allochthonous vs. autochthonous microbial taxa on the biodegradation of PAHs, they provide new insights on bacterial-fungal interactions occurring during bioremediation of highly contaminated marine sediments. Overall, these results suggest that biotreatments based on selected bacterial and/or fungal consortia or a combination of both could be an effective strategy to significantly reduce in a relatively short time PAH contamination of marine sediments, possibly leading to alternative management options compared to dredging and landfill disposal.

Management improvements have caused a decline in plant species diversity in traditionally managed haymeadows. The aim of this study was examine the effects and causes of management improvements on the soil microbialocmmunity with particular emphasis on the fungal component. A seasonal study of 3 sites showed that management improvements to haymeadows consistently reduced soil microbial biomass C, but had no effect on dehydrogenase activity and basal respiration. Management improvements to these sites also caused a significant reduction in VAM spore numbers, soil fungal biomass, measured as soil ergosterol content and the PLFA 18:w6, and a decrease in the fungal:bacteria PLFA ratio. VAM spore numbers were not correlated with the possibly mycorrhizal NLFA 16:w5. In the Welsh haymeadow, fungi of the genera Fusarium, Mucor, Absidia, Cladosporium, Trichodenna, Acremonium, Zygorhynchus and Paecilomyces were commonly isolated on litter and soil. Commonly isolated fungi had proteolytic and urease activity, and approximately half had cellulose and lignin decay abilities. Management improvements induced shifts in the isolation frequency of these fungi, resulting in an increase in more general resource fungi, capable of growth on both litter and soil. Management improvements to haymeadows, may also have reduced species diversity of litter fungi. Agar and microcosm experiments established that changes in fungal community structure observed in the field could be in response to changes in plant litter inputs and applications of NPK fertiliser. Pairings of fungi on PDA showed that there was a combative hierarchy amongst the fungi, but was not able to show if this hierarchy was affected by NPK. A field experiment found no response of the soil microbial community to short term applications (2 years) of fertiliser or fungicide. The findings of this study suggest that management improvements to grasslands will induce changes in microbial and fungal community structure, this will be discussed.

The weathering of silicate rocks and minerals plays a foundational role in soil formation, provides essential nutrient elements required by terrestrial and marine ecosystems, and regulates atmospheric CO2 concentrations over geological time through transfer of calcium from continental silicates into marine carbonate sediments. Increasing evidence from experimental and modelling studies implicate evolutionary advances in plants and their mycorrhizal fungal symbionts in enhancing weathering rates over the past 400 million years. The rise of angiosperms to ecological dominance over gymnosperms during the Cretaceous (145.5-65.5 Ma) coincided with the first appearance of ectomycorrhizal fungi, which are more aggressive weathering agents than the ancestral arbuscular mycorrhizal fungi. However, the relative importance of the plant and fungal components of biotic weathering to the long-term decline of C02 from the Cretaceous onwards has not been fully resolved. To address these issues, this thesis reports axenic microcosm experiments that are used to investigate for the first time the influences of angiosperm and gymnosperm host type and atmospheric CO2 concentration on ectomycorrhizal fungal colonisation of silicate rock grains. Using a representative gymnosperm species, Pinus sylvestris and angiosperm species, Betula pendula in symbiosis with the common generalist EM fungi Paxillus involutus, carbon fluxes from ectomycorrhizal seedlings to fungal hyphae colonising weathering arenas were quantified. 14C02 pulse labelling studies and biomass quantification techniques elucidate temporal and spatial carbon dynamics of fungal hyphae colonising grains of basalt, apatite, granite and quartz. Fungal alteration of silicate mineral surfaces was investigated by vertical scanning interferometry and the formation of new biogenic minerals studied by scanning electron microscopy. Additional experiments investigated the effects of the generalist fungus Thelephora terrestris and the suilloid group specialists of the pinaceae. EM fungal proliferation and carbon allocation is shown to respond to elevated CO2 growth conditions, and to seedling host type. Furthermore, preferential fungal colonisation of the Ca and Mg rich silicate rock basalt and evidence of accelerated Ca weathering indicates a need to reassess the current paradigm of plant weathering which overlooks the eo-evolution of roots with their mycorrhizal symbionts.

Il verificarsi della presenza di micotossine nella granella di mais è di interesse mondiale perché la loro presenza negli alimenti è spesso associata a malattie acute e croniche nel bestiame e nell'uomo. Le micotossine sono metaboliti fungini secondari tossici per i vertebrati. Molti composti naturali e sintetici con proprietà antiossidanti sembrano avere una potenziale efficacia nella protezione contro gli effetti tossici delle micotossine. Questo lavoro ha avuto come obiettivo lo studio degli effetti degli antiossidanti naturali di mais (carotenoidi e tocoli) sulla crescita di funghi (fusarium verticillioides) e sull'accumulo di micotossine in preparati alimentari a base di mais e su alcuni sottoprodotti della lavorazione del mais.
The occurrence of mycotoxins in cereal grains is of great concern worldwide, because their presence in feeds and foods is often associated with chronic or acute mycotoxicoses in livestock and also in human. Mycotoxins are fungal secondary metabolites that are toxic to vertebrates. Several natural and synthetic compounds with antioxidant properties seem to be potentially efficient in protecting against the toxic effects of micotoxins. This work was aimed to study the effects of natural maize antioxidant (carotenoids and tocols) on fungi (fusarium verticillioides) growth and on micotoxins accumulation in maize-based food products and in maize by-products.

Abstract This chapter focuses on the role of fungi. Fungi are a vital part of the mixture of microorganisms found in healthy soil. Fungal associations between plant roots and beneficial fungi are known as mycorrhizae (meaning 'fungus' and 'root'), and form a beneficial or symbiotic relationship with plants growing in the soil. Mycorrhizal fungi also facilitate plant interactions with other soil microbes. These include pathogens, and bacteria that produce vitamins and protect against attack. The most common of the mycorrhizae are divided into the following: (1) ectomycorrhizae; (2) endomycorrhizae; (3) arbuscular mycorrhizae; (4) ericoid mycorrhizae; and (5) orchid mycorrhiza. The role of saprophytes, pathogens and actinomycetes are also discussed.

Abstract This chapter introduces the kingdom Fungi, and other organisms referred to as fungi, as background for discussion of issues around the identification of fungi. The process of identification is sketched out, particularly with reference to names, taxa and taxon concepts, which is important to recognize as potentially changing over time. Some challenges in the identification of many fungal species are also presented.

Abstract This book chapter describes the advantages and limitations of the ITS Region as a universal barcode for fungal identification. The ITS region offers several practical advantages as a universal fungal barcode region. The region encompasses segments that permit resolution at different taxonomic levels as it includes the highly conserved 5.8S rRNA gene, the moderately rapidly evolving ITS2 region and the rapidly evolving ITS1 region, flanked by the highly conserved SSU and LSU genes which permit design of PCR primers that are almost panfungal. Over the last two decades the sequence-based identification of fungi has certainly come of age. The ITS region is universally accepted as the primary fungal barcoding region owing to the high barcode gap with the locus for many groups of fungi. Since the species-resolution power of ITS is poor for certain groups of fungi, and higher-level taxonomic resolution is greater with proteincoding genes, the TEF1α locus has been proposed as the universal secondary barcode region. In addition, the historical problems surrounding the reliability of fungal DNA sequences in centralized repositories are slowly being resolved by the development of an increasing number of publicly accessible, curated databases.

Abstract Precise naming of a species is very important for phytopathogenic fungi because names may carry key information for the management of the fungal diseases. Naming fungal species based on morphological traits or biological properties is outdated and unreliable. This chapter provides the classification of some plant pathogenic fungi including Rhizoctonia solani, Colletotrichum, Fusarium oxysporum, and Verticillum based on morphological, pathogenicity, molecular and phylogenetic analysis. Debate on species identification is no longer a question of being in favour of 'splitters' rather than of 'lumpers', but defining phytopathogenic species is particularly complicated and requires further consideration of subspecific categorizations.

Abstract Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is now used as a routine technique for the fast and reliable identification of fungi at the species level and, currently, it represents an important phenotypic methodology based on proteomic profiles. The main limitations to MALDI-TOF MS for fungal identification are related to sample quality (e.g. quality of biological material such as rigidity or pigmentation of cell walls), sample preparation (e.g. the myriad of sample preparation methodologies that deliver different data sets to different MALDI-TOF MS databases) and the databases themselves (e.g. the 'black-box' commercial databases). This chapter presents an overview and discussion of the use of MALDI-TOF MS for fungal identification. The major known limitations of the technique for fungal taxonomy, and how to overcome these, are also discussed.

The aim of the work was the identification of the antibiotic properties of highly virulent strains of the fungus Lecanicillium. Most of the strains were more active against phytopathogenic fungi than bacteria. Strain F 14 showed high antibiotic properties against both fungal and bacterial pathogens. Strain Vl 79 (L. dimorphum) was more active against phytopathogenic fungi, and Vl 61 showed good results against bacteria.

Introdução: As plantas são suscetíveis a um grande número de patógenos, incluindo os fungos. Fitopatógenos fúngicos são responsáveis ​​por inúmeras doenças, como ferrugem, oídio, podridão, entre outras. Diferentes estratégias têm sido desenvolvidas para aumentar a resistência fúngica em espécies de plantas com base no conhecimento atual dos mecanismos moleculares envolvidos na interação planta-patógeno. As tecnologias de edição de genoma progrediram rapidamente e se tornaram as ferramentas genéticas mais utilizadas para o melhoramento de plantas. Entre essas, temos a aplicação do sistema formado por repetições palindrômicas curtas, interespaçadas e regularmente agrupadas (CRISPR), e sua proteína associada-9 (Cas9). Objetivo: Apresentar a tecnologia de edição de genoma CRISPR/Cas9 com foco na sua aplicação para o aumento da resistência de plantas á patógenos fúngicos. Metodologia: A pesquisa foi realizada nas bases de dados: PubMed e Scopus. Para alcançar o máximo de precisão na estratégia de busca, utilizou-se os descritores: “plant”, “pathogen”, “fungi or fungus”, “CRISPR”. Resultados: A maioria dos trabalhos envolvendo a resistência de plantas contra patógenos fúngicos estavam relacionados como a capacidade do sistema CRISPR/Cas9 em induzir mutagênese direcionada, com competência em silenciar genes implicados na interação planta-fungo. Foi possível observar vários estudos onde os genes de suscetibilidade da planta hospedeira foram inativados, pois eram necessários para o ciclo de vida do patógeno, demonstrando que a tecnologia é aplicável à resistência a doenças fúngicas em plantas, pois o silenciamento de um determinado gene na planta pode resultar em uma suscetibilidade no fungo. Conclusão: Os resultados demonstram a aplicação vantajosa do sistema CRISPR/Cas9 para o melhoramento de culturas no que diz respeito à resistência a patógenos. O aumento dessa resistência possui um papel importante, pois os fungos fitopatogênicos representam uma ameaça para a produção e o rendimento das safras agrícolas.

Introdução: Fungos são seres vivos ubíquos, heterotróficos uni ou multicelulares, morfologicamente diversificados, importantes para o ecossistema, reciclagem da matéria orgânica e equilíbrio ecológico. Além dessas funções biológicas, os fungos, de uma maneira geral, fazem parte do imaginário coletivo sociocultural, sendo encontrados em diferentes estampas, desenhos infantis, jogos eletrônicos, filmes, desenhos animados, etc. Diversidade cultural define-se como diversidade biológica, cultural e linguística, incluindo inter-relações, dentro do complexo sistema adaptativo socioecológico. Este trabalho propõe o termo “Funga Cultural” como estudo da presença fúngica nas diferentes manifestações culturais. Como exemplo de manifestação cultural, este trabalho trata de relacionar fungos com filatelia. Objetivos: Realizar um inventário dos selos postais e comemorativos brasileiros que apresentam imagens de fungos, além de analisar suas representações na Funga Cultural e citar a classificação taxonômica das espécies estampadas nos selos. Material e Métodos: Pesquisa utilizou o “Catálogo de Selos do Brasil RHM”, principal obra de referência filatélica do país, sendo utilizada pelos colecionadores e comerciantes de selos brasileiros. Foram analisados todos os selos postais comemorativos emitidos pela Empresa de Correios e Telégrafos do Brasil durante o período de 1900-2019. Resultados: Desde o ano de 1900 até 2019, o Brasil emitiu 3.877 selos comemorativos. Desses, apenas dez (0,26%) têm estampas de fungos, distribuídos em três diferentes séries: três selos da série “Fungos”; um selo da série “Estação Ecológica do Taim-RS”; e seis selos da série “Diversidade de Fungos”. Os nomes científicos das espécies estão estampados nos selos. Exceto um selo da série Estação Ecológica. Porém, de acordo com suas características fenotípicas, provavelmente pertence ao filo Basidiomycota. Essa série é formada por uma dupla de selos e o fungo aparece somente no primeiro deles. Os táxons estampados nos outros selos foram: Pycnoporus sanguineus; Calvatia sp.; Pleurotus sp.; Clathrus chrysomycelinus; Clathrus columnatus; Geastrum violaceum; Hydnopolyporus fimbriatus; Laetiporus gilbertsonii e Oudemansiella cubensis. Conclusão: Os fungos apresentam pouca representatividade nos selos comemorativos brasileiros. Os selos fazem parte da cultura humana e aqueles com estampas de fungos podem ser inseridos como representantes da Funga Cultural. Ademais, pode-se usar o selo como mais um recurso didático no ensino da funga e na divulgação científica.

Nonylphenol (NP) is the most abundant environmental estrogen listed as one of the priority hazardous substances in the Water Framework Directive (EC 2000) and the priority pollutant of Baltic Sea (HELCOM 2010). The present study aims to compare the effects of technical nonylphenol (tNP) on the cellulase, amylase and protease activity of the terrestrial fungal strains played a significant role in aquatic ecosystems due to their high adaptive capacity and a large range of functional activity. The study also attempts to understand the mechanisms behind the varying sensitivity of the terrestrial fungi to tNP. The fungal strains were isolated from the bottom sediments of the coastal area of the eastern part of the Gulf of Finland. The terrestrial fungi were identified based on their morphological characteristics and nucleotide sequence analysis of internal transcribed space region. One reason for significant differences in sensitivity to the toxicant studied among the fungi is the change in the fungal cell permeability, in particular in cell membrane permeability, induced by NP. Environmentally relevant concentrations of tNP cause significant changes in activity of hydrolytic enzymes in the terrestrial fungi Aspergillus tubingensis, Penicillium expansum, Penicillium glabrum, and Cadophora fastigiata involved in organic matter degradation in bottom sediments. There can be increasing or decreasing trend, depending on both the type of enzyme and the tNP concentration. The revealed changes may disrupt the destructive processes in bottom sediments, as well as succession and stability of microbial communities functioning in the aquatic environment. It was found that tNP contributes to the activation of proteolytic enzymes, considered as potential fungal virulence factors. This may lead to emergence fungal strains with enhanced virulence in aquatic microbiocenoses. The investigations of the physiological responses of terrestrial fungi under nonylphenol will be important for biochemical processes dynamics and their environmental consequences evaluation.

Nonylphenol (NP) is the most abundant environmental estrogen listed as one of the priority hazardous substances in the Water Framework Directive (EC 2000) and the priority pollutant of Baltic Sea (HELCOM 2010). The present study aims to compare the effects of technical nonylphenol (tNP) on the cellulase, amylase and protease activity of the terrestrial fungal strains played a significant role in aquatic ecosystems due to their high adaptive capacity and a large range of functional activity. The study also attempts to understand the mechanisms behind the varying sensitivity of the terrestrial fungi to tNP. The fungal strains were isolated from the bottom sediments of the coastal area of the eastern part of the Gulf of Finland. The terrestrial fungi were identified based on their morphological characteristics and nucleotide sequence analysis of internal transcribed space region. One reason for significant differences in sensitivity to the toxicant studied among the fungi is the change in the fungal cell permeability, in particular in cell membrane permeability, induced by NP. Environmentally relevant concentrations of tNP cause significant changes in activity of hydrolytic enzymes in the terrestrial fungi Aspergillus tubingensis, Penicillium expansum, Penicillium glabrum, and Cadophora fastigiata involved in organic matter degradation in bottom sediments. There can be increasing or decreasing trend, depending on both the type of enzyme and the tNP concentration. The revealed changes may disrupt the destructive processes in bottom sediments, as well as succession and stability of microbial communities functioning in the aquatic environment. It was found that tNP contributes to the activation of proteolytic enzymes, considered as potential fungal virulence factors. This may lead to emergence fungal strains with enhanced virulence in aquatic microbiocenoses. The investigations of the physiological responses of terrestrial fungi under nonylphenol will be important for biochemical processes dynamics and their environmental consequences evaluation.

Much literature is available on fungal lipids and their capability as a renewable oil platform for alternate fuels, chemicals, and food products. Microbial oils will not displace all edible oils soon, given techno-economical hurdles in commercialization. However, continued research & development can flatten the curve of deforestation and land-use impacts associated with cultivating these crops. To better understand how oleaginous yeasts and fungi could alleviate the challenges related to the energy-environment-food nexus, it becomes critical to investigate their potential environmental impacts quantitively compared to other feedstocks. Life cycle analysis or assessment (LCA) is a standard tool used for this purpose. LCA studies are not being conducted on a broader scale for fungus-derived oils than their phototrophic algal counterparts. The different stages in the life cycle of fungal lipid production that can be analyzed for environmental implications include cultivation and fermentation, oil extraction; further downstream processing; and end-use. The LCA method for fungal lipid-derived biofuel production systems should cover the main sustainability concerns of biofuel production systems: energy efficiency, climate change, and land occupation. With the scope of microbial oil applications expanding beyond non-fuel encompassing food, supplements, and medicines, their subsequent environmental implications need to be investigated. Further work is required in this area. There are significant knowledge gaps in life cycle inventory and impact assessment information for non-fuel applications.

Introdução: Fungos são grupos de seres vivos conhecidos por sua importância na decomposição natural de matéria orgânica e, ao lado de bactérias, são fundamentais para que haja o ciclo de renovação de nutrientes e da matéria como um todo. Em virtude dessa característica, há cada vez mais uma busca na natureza de fungos que podem ser usados para degradar, parcialmente ou integralmente, também matéria inorgânica, sobretudo polímeros sintéticos, como o policloreto de vinila (PVC), uma vez que estes podem demorar centenas ou milhares de anos para se decompor na natureza. Objetivo: Com isso em vista, o objetivo do presente estudo foi de realizar uma revisão sistemática sobre o tema e analisar o que já foi feito e as próximas etapas em relação à decomposição desses polímeros por fungos. Material e métodos: Para tal, usou-se das ferramentas Science Direct, Google Academics e Scielo para busca contendo as palavras: “Fungal decomposing synthetic polymers”, “Fungal decomposing plastics”, considerando artigos a partir do ano de 2018. Resultados: Foram encontrados 10 artigos que satisfaziam os critérios de busca no título, no resumo ou no corpo do texto. Os estudos encontrados nos mostram que existe ampla quantidade de fungos com potencial para degradar polímeros sintéticos, a depender de fatores como o ambiente, o tempo de avaliação, a disponibilidade de carbono no local ou até a associação com plantas e outras espécies. Gêneros como Aspergillus, Mucor, Penicillium e Trichoderma possuem atividade decompositora em diversos polímeros sintéticos, como o polietileno (PET), utilizando-os como fonte de carbono in vitro e na natureza a partir de algumas semanas. Também se identificou o exemplo do fungo Laccaria laccata que, em associação com plantas, ocorre aceleração do processo de degradação do PET. Conclusão: A partir do que foi encontrado, espera-se cada vez mais estudos e que mais fungos sejam descobertos com capacidade decompositora de polímeros sintéticos, contribuindo com o problema do acúmulo desse material na natureza e com sua contaminação de seres vivos e do meio ambiente.

Mycotoxins are secondary metabolites synthesized by mycotoxigenic fungi belonging mainly to three major fungal genera that are Aspergillus, Fusarium and Penicillium. The latter mycotoxigenic fungi contaminate plants and different food commodities and cause various health concerns (carcinogenicity, mutagenicity, nephrotoxicity, etc...) due to their corresponding mycotoxins. One of the most studied mycotoxins is ochratoxin A (OTA) owing to its toxicity level (classified in 2B group as possible human carcinogenic). To remediate the mycotoxins’ contamination, physical and chemical techniques can be proposed. However, the safest among all is the biological control approach. In this research, we have used the Qatari strain Burkholderia cepacia (QBC03) as a biological agent against mycotoxigenic fungi and the strain has possessed a wide antifungal spectrum against 21 species from different genera. Additionally, the antifungal activity of QBC03’s supernatant was explored on the fungal biomass and OTA synthesis of A. carbonarius in liquid media, and interestingly; both the biomass and OTA’s concentrations were massively reduced upon treatment. The effect of QBC03’s supernatant on the fungal spores’ germination was examined as well, and it was shown that the conidial germination was completely inhibited. Moreover, the supernatant of QBC03 has induced morphological alteration in the mycelia of the fungal strain. The thermal stability of the antifungal compounds in QBC03’s culture supernatant was investigated, and it was shown that metabolites of QBC03 were distinctively thermostable and they were still active even when heated at 100C. The findings of this research prove that Burkholderia cepacia strain QBC03 is an excellent candidate for the biological control of mycotoxigenic fungi and their mycotoxins particularly in local regions.

Given the vast expanse of Qatar’s dryland ecosystems, agricultural productivity and soil stability is highly dependent on the diversity of soil microbiota. The soil environment is a heterogeneous habitat shaped by various components like chemical (organic matter, salinity and nutrients) and biological (fungal diversity and vegetation) properties that form multitudes of different microhabitats. Soil microbial diversity changes along environmental gradients. It is hypothesized that a “stable” microhabitat is one that is inhabited by a large diversity of established microorganisms that are best adapted to the niche. Microorganisms like fungi serve as the underlying biological drivers for biochemical processes within the soil. The key objective of this study is to evaluate the fungal diversity and abundance present within the Qatari soil using molecular-based tools and evaluate potential relationships between the identified fungal communities with chemical properties of the habitat. We found that the composition of fungi and AMF varied between different habitats around Qatar. Despite the lack of significant differences in the measured soil chemical parameters between sampled sites, it is evident that AMF species are more abundant than compared to that of other fungal species in most of the study sites; thus, suggesting that other factors like land use may also be an essential component explaining the variation in fungal communities.

Spore germination is a common and fundamental event in fungal development and in many instances an essential phase of fungal infection and dissemination. Spore germination is also critical for hyperparasites to function as biocontrol agents as well as in fermentation proceses. Our common objective is to understand the mechanisms which regulated spore germination and identify factors involved in pathogenicity related prepenetration development. Our approach is to exploit the overall similarity among filamentous fungi using both a plant pathogen (Colletotricum trifolii) and a model system that is genetically sophisticated (Neurospora crassa). The simulataneous use of two organisms has the advantage of the available tools in Neurospora to rapidly advance the functional analysis of genes involved in spore germination and development of an economically important fungal phytopathogen. Towards this we have isolated a protein kinase gene from C. trifolii (TB3) that is maximally expressed during the first hour of conidial germination and prior to any visible gene tube formation. Based on sequence similarities with other organisms, this gene is likely to be involved in the proliferative response in the fungus. In addition, TB3 was able to functionally complement a N. crassa mutant (COT-1). Pharmacological studies indicated the importance of calmodulin in both germination and appressorium differentiation. Using an antisense vector from N. crassa, direct inhibition of calmodulin results in prevention of differentiation as well as pathogenicity. Both cAMP dependent protein kinase (PKA) and protein kinase C (PKC) like genes have been cloned from C. trifolii. Biochemical inhibition of PKA prevents germination; biochemical inhibitors of PKC prevents appressorium differentiation. In order to analyze reversible phosphorylation as a regulatory mechanism, some ser.thr dephosphorylative events have also been analyzed. Type 2A and Type 2B (calcineurin) phosphatases have been identified and structurally and functionally analyzed in N. crassa during this project. Both phosphatases are essential for hyphal growth and maintenance of proper hyphal architecture. In addition, a first novel-type (PPT/PP5-like) ser/thr phosphatase has been identified in a filamentous fungus. The highly collaborative project has improved our understanding of a fundamental process in fungi, and has identified targets which can be used to develop new approaches for control of fungal plant pathogens as well as improve the performance of beneficial fungi in the field and in industry. In addition, the feasibility of molecular technology transfer in comparative mycology has been demonstrated.

Postharvest decay of fruits and vegetables caused by pathogenic and saprophytic fungi significantly impairs the quality and quantity of fresh produce brought to market. Consequently, there is considerable interest in identifying factors that determine the susceptibility of these commodities to pathogen infection. Insidious postharvest decays remain quiescent during fruit growth and harvest, but activate during the postharvest period. A key response to the physiological changes occurring during fruit ripening is the initiation of ammonium secretion by the pathogen. Ammonium ions at the infection site (ammonification) have subsequent effects on both the pathogen and the host. An accompanying alkalinization process resulting from ammonia accumulation contributes to pathogenicity, since some important fungal virulence factors, (such as pectate lyase in Colletotrichum sp.), are significantly expressed only under alkaline conditions. In this proposal, investigated the mechanisms by which ammonification and alkalinization of infected tissues by the pathogen affect the host’s defense response to fungal attack, and instead increase compatibility during postharvest pathogen-host interactions. Our hypotheses were:1) that host signals, including ripening-related changes, induce secretion of ammonia by the pathogen; 2) that ammonia accumulation, and the resultant environmental alkalinization regulate the expression of fungal virulence genes that are essential for postharvest rot development; 3) that ammonification enhanced fungal colonization, by “suppression of host responses”, including production of reactive oxygen species, activation of superoxide, and polyphenol oxidase production. Our objectives were: to analyze: 1) factor(s) which activate the production and secretion of ammonia by the fungus; 2) fungal gene(s) that play role(s) in the ammonification process; 3) the relationship between ammonification and the activation of host defense response(s) during pathogen colonization; and 4) analyze hostgene expression in alkalinized regions of fruits attacked by hemibiotrophic fungi.

Sclerotinia sclerotiorum is a filamentous fungus (mold) that causes plant disease. It has an extremely wide range of hosts (>400 species) and causes considerable damage (annual multimillion dollar losses) in economically important crops. It has proven difficult to control (culturally or chemically) and host resistance to this fungus has generally been inadequate. It is believed that this fungus occurs in almost every country. Virulence of this aggressive pathogen is bolstered by a wide array of plant cell wall degrading enzymes and various compounds (secondary metabolites) produced by the fungus. It is well established that plant pathogenic fungi secrete proteins and small molecules that interact with host cells and play a critical role in disease development. Such secreted proteins have been collectively designated as “effectors”. Plant resistance against some pathogens can be mediated by recognition of such effectors. Alternatively, effectors can interfere with plant defense. Some such effectors are recognized by the host plant and can culminate in a programmed cell death (PCD) resistant response. During the course of this study, we analyzed an effector in Sclerotiniasclerotiorum. This specific effector, SsCM1 is the protein chorismatemutase, which is an enzyme involved in a pathway which is important in the production of important amino acids, such a Tryptophan. We have characterized the Sclerotiniaeffector, SsCM1, and have shown that inactivation of Sscm1 does not affect fungal vegetative growth, development or production of oxalic acid (one of this fungus’ secondary metabolites associated with disease) production. However, yhis does result in reduced fungal virulence. We show that, unexpectedly, the SsCM1 protein translocates to the host chloroplast, and demonstrated that this process is required for full fungal virulence. We have also determined that the fungal SsCM1 protein can interact with similar proteins produced by the host. In addition, we have shown that the fungal SsCM1 is able to suppress at least some of the effects imposed by reactive oxygen species which are produced as a defense mechanism by the host. Last, but not least, the results of our studies have provided evidence contradicting the current dogma on at least some of the mechanist aspects of how this pathogen infects the host. Contrary to previousons, indicating that this pathogen kills its host by use of metabolites and enzymes that degrade the host tissue (a process called necrotrophy), we now know that at least in the early phases of infection, the fungus interacts with live host tissue (a phenomenon known as biotrophy). Taken together, the results of our studies provide novel insights concerning the mechanistic aspects of Sclerotinia-host interactions. We hope this information will be used to interfere with the disease cycle in a manner that will protect plants from this devastating fungus.

The involvement of glycans in microbial adherence, recognition and signaling is often a critical determinant of pathogenesis. Although the major glycan components of fungal cell walls have been identified there is limited information available on its ‘minor sugar components’ and how these change during different stages of fungal development. Our aim was to define the role of Rhacontaining-glycans in the gray mold disease caused by the necrotrophic fungus B. cinerea. The research was built on the discovery of two genes, Bcdhand bcer, that are involved in formation of UDP-KDG and UDP-Rha, two UDP- sugars that may serve as donors for the synthesis of cell surface glycans. Objectives of the proposed research included: 1) To determine the function of B. cinereaBcDh and BcEr in glycan biosynthesis and in pathogenesis, 2) To determine the expression pattern of BcDH and BcERand cellular localization of their encoded proteins, 3) Characterize the structure and distribution of Rha- containing glycans, 4) Characterization of the UDP-sugar enzymes and potential of GTs involved in glycanrhamnosylation. To address these objectives we generated a series of B. cinereamutants with modifications in the bchdhand bcergenes and the phenotype and sugar metabolism in the resulting strains were characterized. Analysis of sugar metabolites showed that changes in the genes caused changes in primary and secondary sugars, including abolishment of rhamnose, however abolishment of rhamnose synthesis did not cause changes in the fungal phenotype. In contrast, we found that deletion of the second gene, bcer, leads to accumulation of the intermediate sugar – UDP- KDG, and that such mutants suffer from a range of defects including reduced virulence. Further analyses confirmed that UDP-KDG is toxic to the fungus. Studies on mode of action suggested that UDP-KDG might affect integrity of the fungal cell wall, possibly by inhibiting UDP-sugars metabolic enzymes. Our results confirm that bcdhand bcerrepresent a single pathway of rhamnose synthesis in B. cinerea, that rhamnose does not affect in vitro development or virulence of the fungus. We also concluded that UDP-KDG is toxic to B. cinereaand hence UDP-KDG or compounds that inhibit Er enzymes and lead to accumulation of UDP-KDG might have antifungal activity. This toxicity is likely the case with other fungi, this became apparent in a collaborative work with Prof. Bart Thomma of Wageningen University, NETHERLANDS . We have shown the deletion of ER mutant in Verticillium dahlia gave plants resistance to the fungal infection.

Vesicular-arbuscular mycorrhizal (VAM) fungi and other beneficial rhizosphere microorganisms, such as Rhizobium bacteria, must locate and infect a host plant before either symbiont profits. Although benefits of the VAM association for increased phosphorous uptake have been widely documented, attempts to improve the fungus and to produce agronomically useful amounts of inoculum have failed due to a lack of in vitro production methods. This project was designed to extend our prior observation that the alfalfa flavonoid quercetin promoted spore germination and hyphal growth of VAM fungi in the absence of a host plant. On the Israeli side of the project, a detailed examination of changes in flavonoids and flavonoid-biosynthetic enzymes during the early stages of VAM development in alfalfa found that VAM fungi elicited and then suppressed transcription of a plant gene coding for chalcone isomerase, which normally is associated with pathogenic infections. US workers collaborated in the identification of flavonoid compounds that appeared during VAM development. On the US side, an in vitro system for testing the effects of plant compounds on fungal spore germination and hyphal growth was developed for use, and intensive analyses of natural products released from alfalfa seedlings grown in the presence and absence of microorganisms were conducted. Two betaines, trigonelline and stachydrine, were identified as being released from alfalfa seeds in much higher concentrations than flavonoids, and these compounds functioned as transcriptional signals to another alfalfa microsymbiont, Rhizobium meliloti. However, these betaines had no effect on VAM spore germination or hyphal growth i vitro. Experiments showed that symbiotic bacteria elicited exudation of the isoflavonoids medicarpin and coumestrol from legume roots, but neither compound promoted growth or germination of VAM fungi in vitro. Attempts to look directly in alfalfa rhizosphere soil for microbiologically active plant products measured a gradient of nod-gene-inducing activity in R. meliloti, but no novel compounds were identified for testing in the VAM fungal system in vitro. Israeli field experiments on agricultural applications of VAM were very successful and developed methods for using VAM to overcome stunting in peanuts and garlic grown in Israel. In addition, deleterious effects of soil solarization on growth of onion, carrot and wheat were linked to effects on VAM fungi. A collaborative combination of basic and applied approaches toward enhancing the agronomic benefits of VAM asociations produced new knowledge on symbiotic biology and successful methods for using VAM inocula under field conditions

The objectives of the research in this proposal were to (A) identify synergy among proteins that provide enhanced activity over single proteins for control of plant pathogenic fungi, (B) clone and characterize genetic sequences coding for proteins with ability to control pathogenic fungi, (C) produce transgenic organisms with enhanced biocontrol ability using genes and gene combinations and determine their efficiency in protecting plants against plant pathogenic fungi. A related objective was to produce disease-resistant plants. Fungal cell wall degrading enzymes from any source are strongly synergistic with any membrane active compound and, further, different classes of cell wall degrading enzymes are also strongly synergistic. We have cloned and sequenced a number of genes from bacterial and fungal sources including five that are structurally unrelated. We have prepared transgenic fungi that are deficient in production of enzymes and useful in mechanistic studies. Others are hyperproducers of specific enzymes that permit us, for the first time, to produce enzymes from T. harzianum in sufficient quantity to conduct tests of their potential use in commercial agriculture. Finally, genes from these studies have been inserted into several species of crop plants were they produce a high level of resistance to several plant pathogenic fungi.

Melanins are pigmented biomacromolecules found throughout all domains of life. Of melanins’ many unique properties, their malleable electrically conductive properties and their ability to chelate could allow them to serve as material for bioelectronics. Studies have shown that sheets or pellets of melanin conduct low levels of electricity; however, electrical conductance of melanin within a cellular context has not been thoroughly investigated. In addition, given the chelating properties of melanin, it is possible that introducing traditionally con-ductive metal ions could improve the conductivity. Therefore, this study investigated the conductive properties of melanized cells and how metal ions change these. We measured the con-ductivity of pulverized Curvularia lunata, a melanized filamentous fungi, with and without the addition of copper ions. We then com-pared the conductivity measurements of the fungus to chemically synthesized, commercially bought melanin. Our data showed that the conductivity of the melanized fungal biomass was an order of magnitude higher when grown in the presence of copper. However, it was two orders of magnitude less than that of synthetic melanin. Interestingly, conductance was measurable despite additional constituents in the pellet that may inhibit conductivity. Therefore, these data show promising results for using melanized cells to carry electrical signals.