Dissertations / Theses on the topic 'Funghi'

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.

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.

L'opinione pubblica percepisce fortemente il rischio derivante da sostanze chimiche sintetiche, ma ignora spesso quello derivante dalla presenza di sostanze tossiche naturali. Tra queste sostanze vengono classificate le micotossine; prodotti del metabolismo secondario di alcuni funghi. La maggior parte degli alimenti di origine vegetale e in particolare i cereali, possono andare incontro a contaminazione da parte di questi funghi, produttori di micotossine, in qualunque stadio. In particolare nel frumento le micotossine che si riscontrano più frequentemente sono l'ocratossina A (OTA) e il deossinivalenolo (DON), mentre nel mais, si riscontrano le Aflatossine e le fumonisine. Queste micotossine hanno effetti epatotossici, nefrotossici, mutageni e cancerogeni, è necessario perciò, un continuo monitoraggio della materia prima (frumento e mais), ma anche dei prodotti destinati all'alimentazione umana a base di questi cereali.
People experience deeply, the risk of synthetics chemicals substances, but often don't know the risk from naturals toxics substances. Between these substances, they are mycotoxins; fungi secondary metabolism's products. Most of vegetable food, as cereals, can suffer a mycotoxins contamination, for effect of these fungi, during any step of alimentary chain. In particular, in wheat , the mycotoxins more frequents are ochratoxin A end deossinivalenol, while in the maize can find aflatoxins and fumonisins. These mycotoxins can have hepatotoxics, nefrotoxics, mutagens and cancerogens effects. This study has monitored contamination's levels of raw material (wheat and maize), but also of foodstuff containing cereals.

L'opinione pubblica percepisce fortemente il rischio derivante da sostanze chimiche sintetiche, ma ignora spesso quello derivante dalla presenza di sostanze tossiche naturali. Tra queste sostanze vengono classificate le micotossine; prodotti del metabolismo secondario di alcuni funghi. La maggior parte degli alimenti di origine vegetale e in particolare i cereali, possono andare incontro a contaminazione da parte di questi funghi, produttori di micotossine, in qualunque stadio. In particolare nel frumento le micotossine che si riscontrano più frequentemente sono l'ocratossina A (OTA) e il deossinivalenolo (DON), mentre nel mais, si riscontrano le Aflatossine e le fumonisine. Queste micotossine hanno effetti epatotossici, nefrotossici, mutageni e cancerogeni, è necessario perciò, un continuo monitoraggio della materia prima (frumento e mais), ma anche dei prodotti destinati all'alimentazione umana a base di questi cereali.
People experience deeply, the risk of synthetics chemicals substances, but often don't know the risk from naturals toxics substances. Between these substances, they are mycotoxins; fungi secondary metabolism's products. Most of vegetable food, as cereals, can suffer a mycotoxins contamination, for effect of these fungi, during any step of alimentary chain. In particular, in wheat , the mycotoxins more frequents are ochratoxin A end deossinivalenol, while in the maize can find aflatoxins and fumonisins. These mycotoxins can have hepatotoxics, nefrotoxics, mutagens and cancerogens effects. This study has monitored contamination's levels of raw material (wheat and maize), but also of foodstuff containing cereals.

Il presente progetto di dottorato di ricerca, in convenzione con Valpharma International S.p.A., è finalizzato all’ottenimento di estratti e biomolecole di elevata qualità, a partire da funghi medicinali ed erbe officinali marecchiesi, mediante l’utilizzo di processi ad alto livello tecnologico, elevata sostenibilità e ridotto impatto ambientale. Tra le piante che crescono spontaneamente in Valmarecchia (RN), Helichrysum italicum è stato selezionato come oggetto di studio, in quanto negli ultimi anni è emerso il potenziale delle specie appartenenti al genere Helichrysum. Inoltre, è stato scelto di indagare una seconda specie appartenente a questo genere, Helichrysum stoechas, in modo da avere un elemento di confronto rispetto ad H. italicum, ma anche per ampliare la conoscenza su questa specie, di cui vi è scarsa letteratura scientifica. Per quanto riguarda i funghi medicinali, sono state selezionate tre specie: Pleurotus eryngii, Pleurotus ostreatus e Ganoderma lucidum, delle quali è stato valutato il contenuto in polifenoli e in beta-glucani. Per ogni specie sono stati realizzati diversi estratti, utilizzando metodiche di estrazione con solvente. Il profilo fitochimico e l’attività antiossidante degli estratti ottenuti sono stati studiati in termini qualitativi e quantitativi mediante l’applicazione di varie metodiche, ovvero HPLC, PCL e i saggi di Folin-Ciocâlteau, FRAP e DPPH. Dai risultati è emerso che gli estratti con una maggiore attività antiossidante sono gli estratti glicerico, glicolico e con 1,3-propanediolo. Tali analisi sono state condotte anche su estratti commerciali di H. italicum e G. lucidum, con il fine di confrontarne le proprietà con i rispettivi estratti ottenuti in laboratorio; questi ultimi hanno dimostrato di avere un’attività antiossidante superiore ai rispettivi estratti commerciali. Per quanto riguarda il genere Helichrysum, gli estratti a base di H. italicum sono caratterizzati da un maggiore potere antiossidante rispetto a quelli di H. stoechas. Successivamente allo studio sistematico degli estratti, è stata caratterizzata la porzione volatile di H. italicum ed H. stoechas. Gli oli essenziali, ottenuti mediante un processo di idrodistillazione, sono stati iniettati in GC-MS per ottenere dati qualitativi e quantitativi sulla loro composizione fitochimica. La collaborazione con diversi gruppi di ricerca ha reso possibile la valorizzazione dell’elicriso marecchiese: sono state condotte numerose analisi (XRF, EA-IRMS, ICP-QQQ-MS), che consentissero di approfondire le conoscenze e le differenze tra l’elicriso autoctono e quello commerciale. In particolare, è stata condotta la caratterizzazione geochimica ed isotopica dei suoli e delle sommità fiorite di H. italicum proveniente da 7 località comprese nel territorio della Valmarecchia ed è stata effettuata la valutazione della variabilità genetica di 12 campioni di elicriso di diversa provenienza, mediante l’utilizzo di marcatori ISSR (Inter Simple Sequence Repeats). Infine, il progetto è stato finalizzato a valutare la possibile applicazione cosmetica di alcuni degli estratti botanici ottenuti a partire da H. italicum, H. stoechas e G. lucidum. Constatato il crescente interesse dei consumatori verso i prodotti cosmetici naturali ed ecosostenibili, è in atto un’intensa ricerca di materie prime di origine vegetale che permettano di ottenere ingredienti cosmetici eco-friendly da inserire all’interno di cosmetici naturali ed ecosostenibili. Per questo motivo, per fornire un esempio pratico di una possibile applicazione cosmetica degli estratti ottenuti, sono state formulate delle emulsioni O/A caratterizzate con gli estratti che hanno mostrato una maggiore attività antiossidante. Le emulsioni sono state poi sottoposte ad uno studio di stabilità della durata di tre mesi e all’analisi PCL per valutare la stabilità antiossidante degli estratti all’interno della formulazione cosmetica nel tempo.
This Ph.D. project, in agreement with Valpharma International S.p.A., is aimed at obtaining high-quality extracts and biomolecules from medicinal mushrooms and officinal herbs from the Marecchia Valley, through the use of high-tech, high sustainable and low environmental impact processes. Among the plants wild growing in the Marecchia Valley (RN), Helichrysum italicum was selected as the object of this study, as in recent years the potential of this species has emerged. In addition, a second species belonging to this genus, Helichrysum stoechas, was chosen to be investigated with the aim of getting a comparison with H. italicum, as well as for expanding knowledge about this species, for which there a limited scientific literature is available. As regards for medicinal mushrooms, three species were selected: Pleurotus eryngii, Pleurotus ostreatus and Ganoderma lucidum, and their polyphenol and beta-glucan contents evaluated. Several extracts were made for each species, using solvent extraction methods. The phytochemical profile and antioxidant activity of these extracts have been examined qualitatively and quantitatively by applying various methods, namely HPLC, PCL, and the Folin-Ciocâlteau, FRAP, and DPPH assays. The results showed that the extracts with higher antioxidant activity were the glyceric, glycolic, and 1,3-propanediol extracts. These analyses were also conducted on commercial extracts of H. italicum and G. lucidum in order to compare their featuring with the respective extracts obtained in the laboratory; the latter were shown to have higher antioxidant activity than the respective commercial extracts. As for the genus Helichrysum, extracts based on H. italicum have higher antioxidant power than those of H. stoechas. Following the systematic study of the extracts, the volatile portion of H. italicum and H. stoechas was characterized. The essential oils, obtained by a hydrodistillation process, were injected into GC-MS to obtain qualitative and quantitative data on their phytochemical composition. By collaborating with different research groups it has been possible to enhance the Marecchia's helichrysum: several analyses (XRF, EA-IRMS, ICP-QQQ-MS) were conducted, allowing to deepen the knowledge and differences between native and commercial helichrysum. In particular, the geochemical and isotopic characterization of soils and flowering tops of H. italicum from 7 localities within the Valmarecchia area was conducted, and the genetic variability of 12 helichrysum samples from different origins was evaluated using ISSR (Inter Simple Sequence Repeats) markers. Finally, the project was aimed at evaluating the possible cosmetic application of some of the botanical extracts obtained from H. italicum, H. stoechas and G. lucidum. Noting the growing consumer interest in natural and environmentally sustainable cosmetic products, there is an increasingly research for raw materials of plant origin that allow eco-friendly cosmetic ingredients to be included in natural and environmentally sustainable cosmetics. Therefore, to provide a practical example for a possible cosmetic application of the obtained extracts, O/W emulsions characterized with the extracts that showed higher antioxidant activity were formulated. The emulsions were then subjected to a three-month stability study and PCL analysis to assess the antioxidant stability of the extracts within the cosmetic formulation over time.

L'immissione intenzionale o accidentale nel suolo di sostanze inquinanti di natura organica o inorganica causa gravi conseguenze sia per l'ambiente che per la salute pubblica (tossicità e cancerogenicità). I metalli pesanti sono elementi non degradabili e quindi persistenti nell'ambiente e possono avere effetti teratogeni, mutageni e cancerogeni; gli idrocarburi policiclici aromatici (IPA) sono elementi difficili da trattare ed anch'essi possono causare cancerogenicità e tossicità nell'uomo. Diverse tecniche sono state utilizzate per la bonifica dei suoli contaminati, ma la fitorimediazione si propone come un'alternativa ecosostenibile e conveniente rispetto alle convenzionali tecniche chimico-fisiche. La fitorimediazione sfrutta le caratteristiche fisiologiche delle piante al fine di rimuovere o ridurre la presenza degli agenti inquinanti nel suolo. Per attuare tale tecnica è possibile impiegare diverse specie vegetali in grado di accumulare i contaminanti inorganici come i metalli pesanti o degradare, con l'aiuto dei microrganismi della rizosfera, diversi contaminanti organici; inoltre, la biomassa prodotta può essere utilizzata per altri scopi, quali la cogenerazione di energia e/o la produzione dei biocarburanti, ottenendo benefici per la salute, l'ambiente e la gestione dei costi. L'efficienza dei processi di fitorimediazione può essere incrementata grazie all'impiego di diversi microrganismi (funghi, lieviti, batteri) che popolano normalmente la rizosfera, instaurando rapporti simbiotici e facilitando così l'assorbimento e/o la degradazione dei contaminanti. In questo lavoro è stata effettuata la sperimentazione in vitro con tre microrganismi (Trichoderma harzianum, Saccharomyces cerevisiae, Wicherhamomyces anomalus) per testare la loro potenziale capacità bioaccumulatrice nei confronti di nove metalli pesanti (Ni, Cd, Cu, As, V, Pb, Zn, Cr e Hg) e di metabolizzazione dei sedici IPA definiti prioritari dalla U.S. EPA, al fine di valutare la loro possibile applicazione nei processi di fitorimediazione. Le prove di crescita controllata in serra hanno visto l'impiego della specie vegetale Arundo donax e di A. donax micorrizata con T. harzianum, al fine di valutare la loro efficacia nell'accumulare e degradare rispettivamente i metalli pesanti e gli IPA a cui sono stati esposti. T. harzianum è stato valutato come microrganismo più idoneo alla sperimentazione, in particolare per la sua attività anti patogena verso altri funghi infestanti le colture, per la dimostrata capacità di bioaccumulo verso un maggior numero di metalli pesanti e per aver mostrato buone capacità a degradare e metabolizzare diversi IPA come naphthalene, phenanthrene, chrysene, pyrene e benzo(a)pyrene. A. donax è una specie vegetale che ben si presta all'impiego nei processi di recupero dei suoli contaminati da metalli pesanti e IPA, in particolare perché produce una buona quantità di biomassa, non è appetibile per gli animali, resiste ai parassiti e agli stress idrici e, quindi, è in grado di adattarsi in ambienti inospitali. Da questo studio è emerso come A. donax e i microrganismi utilizzati si prestino molto bene ad un eventuale impiego nei processi di fitorimediazione. A. donax sia micorrizata che non, ha mostrato ottime capacità di sopravvivenza e di sviluppo nei confronti di un terreno contaminato sia con il mix di nove metalli pesanti, sia con il mix di sedici IPA. Ciò lascia intendere l'elevata resistenza ai contaminanti palesata da A. donax, e di conseguenza, potrebbero aprirsi nuove prospettive di studio e ricerca per un'applicazione di successo sul campo.

Filamentous fungal colonies show a remarkable diversity of different mycelial branching patterns. To date, the characterization of this biostructural complexity has been based on subjective descriptions. Here, computerized image analysis in conjunction with video microscopy has been used to quantify several aspects of fungal growth and differentiation. This was accomplished by applying the new branch of mathematics called Fractal Geometry to this biological system, to provide an objective description of morphological and biochemical complexity. The fractal dimension is useful for describing irregularity and shape complexity in systems that appear to display scaling correlations (between structural units) over several orders of length or size. The branching dynamics of Pycnoporus cinnabarinus have been evaluated using fractals in order to determine whether there was a correlation between branching complexity and the amount of extracellular phenol-oxidase that accumulated during growth. A non-linear branching response was observed when colonies were grown in the presence of the aminoanthraquinone dye, Remazol Brilliant Blue R. Branching complexity could be used to predict the generalized yield of phenol-oxidase that accumulated in submerged culture, or identify paramorphogens that could be used to improve yield. A method to optimize growth of discrete fungal colonies for microscopy and image analysis on microporous membranes revealed secretion sites of the phenoloxidase, laccase as well as the intracellular enzyme, acid phosphatase. This method was further improved using microwave-accelerated heating to detect tip and sheath bound enzyme. The spatial deposition of secreted laccase and acid phosphatase displayed antipersistent scaling in deposition and/or secretion pattern. To overcome inherent statistical limitations of existing methods, a new signal processing tool, called wavelets were applied to analyze both one and two-dimensional data to measure fractal scaling. Two-dimensional wavelet packet analysis (2-d WPA) measured the (i) mass fractal dimension of binary images, or the (ii) self-affine dimension of grey-scale images. Both 1- and 2-d WPA showed comparative accuracy with existing methods yet offered improvements in computational efficiency that were inherent with this multiresolution technique. The fractal dimension was shown to be a sensitive indicator of shape complexity. The discovery of power law scaling was a hallmark of fractal geometry and in many cases returned values that were indicative of a self-organized critical state. This meant that the dynamics of fungal colony branching equilibrium. Hence there was potential for biostructural changes of all sizes, which would allow the system to efficiently adapt to environmental change at both the macro and micro levels.

Per legno morto (Coarse Woody Debris; CWD) si intende il residuo di alberi vivi, inclusi alberi caduti a terra, rami caduti e frammenti legnosi presenti sul suolo forestale. Il CWD rappresenta una componente importante per il funzionamento degli ecosistemi forestali, influenzando il ciclo dei nutrienti, la formazione dell’humus e lo stoccaggio del carbonio. È considerato un elemento chiave per il mantenimento della biodiversità forestale, rappresentando un microhabitat per centinaia di specie di invertebrati, funghi, briofite, licheni, anfibi, piccoli mammiferi e uccelli. Negli ecosistemi forestali la quantità di CWD può variare in relazione alla tipologia forestale, alla gestione del popolamento ed ai disturbi naturali. In particolare, nelle foreste semi-naturali, la quantità di CWD è il risultato della mortalità arborea, mentre nelle foreste gestite, tale quantità è anche influenzata dalle attività di taglio e dalle pratiche di gestione (ad es. ceppaie e ramaglie rilasciate al suolo). Tuttavia, come conseguenza dei cambiamenti climatici in atto, che possono indurre un aumento dei tassi di decomposizione, è prevista una sua riduzione nel lungo periodo. Comprendere le fluttuazioni della quantità del CWD e dei relativi tassi di decomposizione è un aspetto molto importante per analizzare più in dettaglio le dinamiche legate al ciclo dei nutrienti e del carbonio negli ecosistemi forestali. Sulla base di tali considerazioni, obiettivo principale dell’elaborato di tesi è stato quello di indagare diversi fattori antropici (es gestione forestale) e biologici (es funghi) che influenzano l’abbondanza ed i processi di decomposizione del legno morto in due specie forestali quali il faggio (Fagus sylvatica L.) e l’abete bianco (Abies alba Mill.). Il lavoro è strutturato in tre capitoli, in cui nel primo vengono analizzati a scala di popolamento gli effetti a breve termine di pratiche selvicolturali innovative e tradizionali sulla presenza e distribuzione del legno morto e su altri indicatori di biodiversità (es. microhabitats e vegetazione del sottobosco) in tre faggete (Fagus sylvatica L.) montane dislocate lungo un transetto latitudinale in Italia. Nel secondo e nel terzo capitolo vengono invece analizzati i processi di decomposizione del legno morto, spostando quindi l’approccio sperimentale ad una scala di maggior dettaglio. In particolare, nel secondo capitolo viene analizzato il profilo degli zuccheri semplici nel legno vivo di abete bianco e nei campioni di CWD raccolti nelle cinque fasi di decadimento di Hunter in due periodi stagionali diversi, all’interno di due siti forestali alpini italiani (TN). Nel terzo capitolo viene invece effettuata un’analisi dei composti fenolici quali prodotti di degradazione della lignina nel legno di abete bianco prima e dopo l’azione enzimatica di nove funghi del marciume bianco, di cui sei appartenenti al genere Armillaria spp. (A. borealis, A. cepistipes, A. gallica, A.mellea, A. ostoyae, e A. tabescens) e tre al genere Heterobasidion spp. (H. abietinum, H. parviporum e H. annosum sensu stricto) in condizioni controllate in laboratorio. I risultati del primo lavoro hanno evidenziato diversi effetti dei trattamenti selvicolturali sugli indicatori analizzati (es. legno morto, microhabitats e vegetazione del sottobosco). In particolare, l’applicazione dei trattamenti innovativi (CTT), rispetto ai tradizionali (LT) ha comportato in tutte e tre le aree di studio indagate, un aumento significativo del volume di legno morto e degli altri indicatori esaminati, sottolineando l’importanza di tali trattamenti come valida alternativa gestionale per le faggete montane del Mediterraneo, in termini di conservazione della biodiversità. La seconda attività ha rappresentato un primo tentativo di analisi della produzione di carboidrati durante il processo di decomposizione del legno morto di abete bianco. Dai risultati ottenuti è stato possibile osservare differenze negli andamenti e nella variabilità del contenuto degli zuccheri semplici all’avanzare del processo di decomposizione del legno morto in campo, probabilmente legate alle diverse condizioni ambientali (es. temperatura e umidità) che caratterizzano i due siti indagati e all’azione degli organismi decompositori come i funghi. I risultati del terzo lavoro hanno infine evidenziato diverse strategie di accumulo e degradazione dei composti fenolici della lignina nel legno di abete bianco da parte di nove specie fungine dei generi Armillaria spp. e Heterobasidion spp., mediante la secrezione degli enzimi ligninolitici, sottolineando l’importanza di tali organismi nel processo di decomposizione. In definitiva, il presente elaborato può essere considerato un contributo utile ad incrementare le conoscenze scientifiche inerenti la complessità delle relazioni che intercorrono tra il legno morto e i diversi fattori che ne influenzano l’abbondanza ed i processi di decomposizione, con particolare riferimento a due specie forestali europee di ampia diffusione, quali il faggio e l’abete bianco.
Coarse woody debris (CWD) is the residue of living trees including fallen trees, fallen branches and lying woody debris (CWD). CWD is an important component of functioning of forest ecosystems, influencing the nutrient cycling, the humus formation and the overall forest carbon storage. It is a key element for forest biodiversity, representing a microhabitat for hundreds of species of invertebrates, fungi, bryophytes, lichens, amphibians, small mammals and birds. In forest ecosystems, the amount of CWD can largely varies in relation to forest types, stand management and natural disturbances. In particular, in semi-natural forests, the amount of CWD is the result of tree mortality while, in managed forests, logging and management practices also influence CWD (e.g. left-over stumps and branches). However, as a consequence of the climate warming, a decrease in CWD could be expected, due to the enhanced decomposition rates. Understanding the fluctuations in CWD abundance and the related decay rates is very important to better understand the nutrient cycling and carbon dynamics of forest ecosystems. Based on these considerations, the primary objectives of this thesis were to evaluate the effects of anthropic (e.g. forest management) and biological factors (e.g. fungi) on deadwood abundance, distribution and decomposition in two European tree species such as beech (Fagus sylvatica L.) and silver fir (Abies alba Mill.). The present work is structured in three chapters. In particular, in the first chapter, the short-term effects of innovative and traditional silvicultural treatments on the abundance and distribution of deadwood and other indicators of biodiversity (e.g. microhabitats and undergrowth vegetation) were analysed at a stand-scale in three mountain beech forests located along a latitudinal transect in Italy. In the second and third chapters, the decomposition processes of silver fir deadwood were analysed, moving the experimental approach to a more detailed scale. In particular, in the second chapter, the sugar profiles of living silver fir trees and CWD samples belonging to Hunter’s five decay stages was studied in two different seasonal periods and in two Alpine forests located in Northern Italy (TN). In the third chapter, a phenolic compounds analysis was carried out in silver fir wood before and after the enzymatic action of nine white rot fungi, belonging to Armillaria spp. (A. borealis, A. cepistipes, A. gallica, A. mellea, A. ostoyae, and A. tabescens) and Heterobasidion spp. (H. abietinum, H. parviporum and H. annosum sensu stricto) genera in controlled lab conditions. Results from the first research showed different effects of silvicultural treatments on the analysed indicators (e.g. deadwood, microhabitats and understory vegetation). In particular, the application of the innovative treatments (CTT), compared to the traditional ones, involved a significant increase of deadwood volume and other biodiversity indicators (e.g. microhabitats and understory vegetation), in all the three study areas, highlighting the importance of these treatments as a valid management option for Mediterranean beech forests in terms of biodiversity conservation. The second study was a preliminary attempt to investigate the production of carbohydrates during deadwood decay in field conditions. The results showed differences in trends and variability of simple sugar contents during deadwood decomposition in the field, probably related to the different environmental conditions (e.g. temperature and humidity) that characterize the two Alpine sites and by the action of decomposing organisms such as fungi. Finally, the results of the third research highlighted different strategies of phenolic compounds accumulation and consumption in lignin of silver fir wood after the action of nine fungal species belonging to Armillaria spp. and Heterobasidion spp. genera, through the secretion of ligninolytic enzymes, emphasizing the importance of these organisms during decomposition process. Ultimately, this thesis represents a useful contribution to increase the scientific knowledge on the complex relationships between deadwood and environmental factors influencing its abundance and decay processes, focusing on two widespread European tree species, such as beech and silver fir.

Forest decline, particularly for Quercus genus, is a well known phenomenon, spread in Europe as well as in the all world and with a complex aetiology, that involve interactions between host plant and several abiotic and biotic causes (i.e. tree genetic characteristics, environmental and climatic changes) Furthermore it has been hypothesized that some endophytic fungi can accelerate the decline of oaks, when trees are subjected to stress. In fact these fungi can become pathogenic, inflicting damage on trees, while often they produce no symptoms for most of their life cycle. The aim of the present study is to provide information concerning the fungal population present in the Q. robur acorns, considering all their growth phases, the influence of other soil fungi and the presence of external and internal seed-borne fungi.

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A tanase (EC 3.1.1.20) é uma enzima induzível que age sobre os taninos hidrolisando suas ligações éster e depsídicas obtendo-se como produtos a glicose e o ácido elágico ou ácido gálico, sendo este último, um importante substrato para as indústrias farmacêutica e química. Entre os diferentes organismos capazes de produzir tanases, os microorganismos, de modo especial os fungos filamentosos, vêm se destacando uma vez que são mais versáteis na degradação de diferentes tipos de taninos. Neste contexto, o objetivo deste trabalho foi estudar as tanases intra e extracelulares do fungo filamentoso Emericella nivea produzidas em Fermentação Submersa (FSbm) e em Fermentação em Substrato Sólido (FSS), purificando-as e caracterizando-as bioquimicamente, além de imobilizá-las em suportes de agarose. Em princípio, foi realizada a seleção da melhor cepa produtora de tanases, submetendo-se 42 linhagens fúngicas a FSbm em meio de cultura Khanna com 2% de ácido tânico como fonte de carbono, por 3 a 4 dias a 30ºC, tendo sido o fungo Emericella nivea selecionado para prosseguimento do trabalho. Para este microorganismo os maiores nívies enzimáticos extracelulares foram obtidos em 3 dias de cultivo em FSbm e 8 dias em FSS, sendo para esta última utilizados produtos agroindustriais e folhas de vegetais de diferentes espécies secas trituradas umedecidas com água de torneira (1:1; p/v). As tanases extra e intracelular foram purificadas 61 e 2,5 vezes com recuperação de 30% e 8,8%, respectivamente. Eletroforese em condições não desnaturantes (PAGE 7%) mostrou a presença de uma única banda protéica revelada por prata e para atividade tanásica com a mesma mobilidade relativa. A forma extracelular possui massa molecular nativa de aproximadamente 322kDa com 50% de conteúdo de carboidratos. Já a enzima intracelular apresentou massa molecular nativa de 258kDa e 17% de...
Tannases (EC 3.1.1.20) are inducible enzymes that catalyze the hydrolysis of ester and depside bonds in hydrolysable tannins releasing glucose and ellagic acid or gallic acid, which is an important compound used in pharmaceutical and chemical industries. Among different organisms able to produce these enzymes, the microorganisms, especially filamentous fungi deserve attention since they can act on different tannins degradation ways. In this context, the aim of this work was to study the intra and extracellular tannases from the filamentous fungus Emericella nivea produced in Submerged Fermentation (SbmF) and Solid Substrate Fermentation (SSF), purifying and characterizing them biochemically, as well to immobilize the extracellular enzyme in agarose supports. First of all, it was selected the best tannase producer among 42 strains, in Khanna culture medium with 2% tannic acid as carbon source for 3-4 days at 30°C, and the fungus Emericella nivea was selected. This fungus produced high levels of extracellular enzyme at 3 and 8 days when cultivated in SbmF and SSF at 30°C, respectivally. FSS was performed with agroindustrial products or crushed dried leaves of different plants umidified with tap water (1:1, w/v). The extra and intracellular tannases were purified 61 times and 2.5-times, with recovery of 30% and 8.8%, respectivally. Non-denaturing electrophoresis (PAGE 7%), showed a unique proteic band stained by silver and for activity, both with the same relative mobility. The extracellular enzyme, probably, is a hetero-dimeric protein with native molecular mass of 322 kDa with 50% of carbohydrate content and the intracellular with native molecular mass of 258 kDa and 17% of carbohydrate. The optimum temperature were 45ºC and 50°C for the extra and intracellular enzymes, respectively and the optimum pH for both enzymes was 5.0. The soluble tannases were thermostable with... (Complete abstract click electronic access below)

L'étude de l'impact des champignons sur les processus géologiques qui altèrent les mineraux s'appelle la “géo-mycologie”. Les serpentinites sont des roches de la famille des ophiolites et peuvent contenir du chrysotile (amiante serpentine). La réactivité des fibres d'amiante est due à la composition chimique de surface et principalement à la présence d'ions métalliques qui peuvent catalyser des réactions chimiques dangereuses. L'amiante est un problème ambiantale à cause de la présence de roches contenant ce minéral mais aussi à cause des anciennes mines d'amiante. La revalorisation (remediation) de ces sites naturellement contaminés passe par la modification de la toxicité des fibres. Les champignons sont des bons candidats pour la bioremediation de l'amiante. L'objectif de cette thèse est l'isolement de souches fongiques à partir de sols serpentiniques afin de savoir quelles sont les espèces fongiques les plus abondantes dans ces sols et de sélectionner les souches les plus efficaces pour leur interaction avec les fibres d'amiantes. Les modifications des fibres et l'altération du métabolisme fongique ont été considérés. Verticillium leptobactrum semble être l'espèce fongique dominante dans tous les sols serpentiniques examinés : cette espèce n'avait jusque là été que rarement isolée, ce qui rend ces résultats intéressants. Trois espèces fongique peuvent extraire Fe et Mg des fibres de amiante (chrysotile et crocidolite), en modifiant la composition et la réactivité chimique de la surface de fibres et la génotoxicité mesuré dans un système acellulaire. Les champignons expriment, en présence des fibres, des enzymes liés à la réponse aux stress oxydants
The interaction of soil fungi with rocks and minerals is called geomycology. Serpentine rocks belong to the ophiolites family and can contain chrysotile (serpentine asbestos). Asbestos fibres reactivity is related to their surface chemical composition, i. G. The presence of iron, catalysing free radicals release, which is harmful for cells and tissues. Asbestos represents an environmental issue, related not only to serpentine rocks naturally exposed and weathered, but also (and mainly) to asbestos mines and wastes. Soil fungi are good candidates for the bioremediation of asbestos rich soils. The main goal of this thesis is the isolation of soil fungi from asbestos rich soils and the selection of the more suitable to interact and modify asbestos fibres in vitro. The metabolic responses of fungi are also investigated. Verticillium leptobactrum is the most abundant specie in all the serpentinic soils considered. This and interesting result, since this specie has bees previously seldom isolated. V. Leptobactrum and other fungal species are able to extract iron and magnesium from chrysotile and crocidolite fibres, modifying their surface chemical composition and reactivity, and their génotoxicity (in acellular experiments). The fungi express anti-oxydant enzymes

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CAPEs
A Mata Atlântica destaca-se como um bioma recordista em termos de biodiversidade e endemismo. Entre os seres vivos presentes neste bioma, os fungos exercem a ciclagem de nutrientes, atividade de extrema importância para a manutenção e equilíbrio ambiental. Apesar disso, os estudos com fungos na Mata Atlântica ainda são escassos. Diante deste cenário, o presente estudo teve como objetivos: i) identificar e catalogar ascomicetos (anamórficos e teleomórficos) presentes no folhedo de Inga thibaudiana, Myrcia splendens e Pera glabrata (plantas representativas da Mata Atlântica) na Reserva Biológica de Una (REBIO-Una), município de Una, estado da Bahia, Brasil; e ii) avaliar a frequência de ocorrência, a constância e a similaridade de fungos conidiais presentes no folhedo das espécies estudadas. Quatro coletas semestrais (setembro/2011, abril e agosto/2012, janeiro/2013) foram realizadas na REBIO-Una. Cinco espécimes de cada uma das seguintes espécies: I. thibaudiana, M. splendens e P. glabrata foram identificados e marcados. Na serapilheira destas árvores coletou-se dez folhas (uma amostra composta) em diferentes estágios de decomposição de cada espécime selecionado, por coleta. Em laboratório, as amostras foram lavadas e incubadas em câmara úmida. O folhedo foi observado com um estereomicroscópio após 72 horas de incubação, por 30 dias. Lâminas dos espécimes fúngicos foram elaboradas com resina de álcool polivinílico, ácido lático e glicerol (PVLG) ou azul de algodão, e observadas em microscópio ótico para a identificação dos táxons, por meio de literatura específica. Após esse processo, os espécimes foram depositados na Coleção Micológica do Centro de Pesquisas do Cacau, Ilhéus-BA. Após a identificação das espécies fúngicas, os índices de frequência de ocorrência, constância e similaridade de fungos conidiais foram avaliados. Foram encontrados 58 táxons sobre folhedo das espécies avaliadas: três ascomicetos pertencentes a ordem Rhytismatales (Coccomyces leptosporus, Marthamyces quadrifidus e Terriera javanica) e 55 espécies de fungos conidiais. O presente estudo proporcionou o primeiro relato de Beltraniella botryospora e de T. javanica para as Américas, e de Beltraniopsis rhombispora para o Brasil. Todos os táxons encontrados estão sendo relatados pela primeira vez para as plantas e para a Reserva Biológica de Una. Com relação aos índices ecológicos, a maioria dos táxons apresentou frequência esporádica. Mais de 40% dos táxons foram classificados como constantes ou acessórios nas três plantas, porém, 53,8% dos táxons em P. glabrata apresentaram categoria acidental. O índice de similaridade demonstrou que há maior semelhança entre a composição da micota do folhedo de duas espécies (49 e 50%) do que entre o folhedo de três espécies vegetais (38%). Considerável diversidade de ascomicetos e de fungos conidiais compõem a micota decompositora do folhedo de I. thibaudiana, M. splendens e P. glabrata na Reserva Biológica de Una.
The Atlantic Forest stands out as a recordist biome in terms of biodiversity and endemism. Among the living organisms present in this biome, the fungi exert nutrient cycling, activity of extreme importance for the environmental maintenance and balance. Nevertheless, the studies with fungi in the Atlantic Forest are still scarce. Therefore, the present study aimed to: i) identify and catalog ascomycetes (anamorphic and teleomorphic) present on the leaf litter of Inga thibaudiana, Myrcia splendens and Pera glabrata (representative plants of the Atlantic Forest) in the Reserva Biológica de Una (REBIO-Una), municipality of Una, Bahia state, Brazil; and ii) evaluate the frequency of occurrence, constancy and similarity indexes of conidial fungi present on the leaf litter of the species studied. Four half-yearly surveys (september/2011, april and august/2012, january/2013) were done in the REBIO-Una. Five specimens of each one of the following species: I. thibaudiana, M. splendens and P. glabrata were identified and marked. In the litterfall of these trees were collected ten leaves (a compound sample) in different stages of decomposition of each specimen selected, for survey. In the laboratory, the samples were washed and incubated in moist chamber. The leaf litter was observed with a stereomicroscope after 72 hours of incubation, for 30 days. Slides of fungal specimens were prepared with polyvinyl alcohol, lactic acid and glycerol (PVLG) resin or cotton blue, and observed using a light microscope for the identification of the taxa, through specific literature. After this process, the specimens were deposited in the Mycological Collection of the Centro de Pesquisas do Cacau, municipality of Ilhéus, Bahia state. After the identification of fungal species, the frequency of occurrence, constancy and similarity of conidial fungi indexes were evaluated. Fifty-eight taxa were found on leaf litter of the plants evaluated: three ascomycetes belonging to the order Rhytismatales (Coccomyces leptosporus, Marthamyces quadrifidus and Terriera javanica) and 55 species of conidial fungi. The present study provided the first report of Beltraniella botryospora and of T. javanica for the Americas, and of Beltraniopsis rhombispora for the Brazil. All the taxa found are reported for the first time for the plants and for the Reserva Biológica de Una. Regarding to ecological indexes, the majority of the taxa was of sporadical frequency. More than 40% of the taxa were classified as accessory or constant in the three plants, however, 53.8% of the taxa in P. glabrata showed accidental category. The similarity index demonstrated that there is a greater resemblance between the leaf litter mycota composition of two species (49 and 50%) than among the leaf litter of three vegetable species (38%). Considerable diversity of ascomycetes and conidial fungi compose the decomposer mycota of I. thibaudiana, M. splendens and P. glabrata leaf litter in the Reserva Biológica de Una.

Thesis (MSc)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: It is known that many hyphomycetous fungi are dispersed by wind, water and insects. However, very little is known about how these fungi may differ from each other regarding their ability to be disseminated by different environmental vectors. Consequently, to obtain an indication of the primary means of spore dispersal employed by representatives of the genera Acremonium, Aspergillus and Penicillium, isolated from soil and indoor environments, we monitored spore liberation of cultures representing these genera in an airflow cell. The experimental data obtained, of plate counts conducted of the air at the outlet of the airflow cell, were subjected to an appropriate analysis of variance (ANOVA), using SAS statistical software. Intraspecific differences occurred regarding aerial spore release. Under humid conditions, however, Penicillium species were more successful in releasing their spores than Aspergillus and the Acremonium strain. Under desiccated conditions the Aspergillus took longer to release their spores than representatives of Acremonium and Penicillium. The taxa that were investigated did not differ from each other regarding the release of spores in physiological salt solution (PSS). Although not proven, indications are that water may act as an important dispersion agent for these fungi, because washing of cultures with PSS resulted in all cases in an immediate massive release of colony forming units. Subsequently, using standard plate count techniques, conidial adhesion of the fungi mentioned above to synthetic membranes, leaf cuttings and insect exoskeletons differing in hydrophobicity and electrostatic charge were investigated. We found that the different genera showed different adhesion profiles for the series of test surfaces, indicating differences in physico-chemical characteristics of the fungal spore surfaces. In general, the Penicillium strains showed a greater ability to adhere to the test surfaces, than the aspergilli, while the representative of Acremonium showed the least adherence. No significant difference in the percentage spore adhesion was found between hydrophobic and hydrophilic materials. Furthermore, evidence was uncovered supporting the contention that, under dry conditions, electrostatic surface charges play a role in the adherence of fungal spores to surfaces, because adherence was positively correlated (Correlation coefficient = 0.70898, p = 0.001) to positive electrostatic charges on the lamellar surfaces. In the next part of the study, standard plate count methods were used to determine the relative adhesion of the above mentioned hyphomycetous fungi, as well as a polyphyletic group of yeasts, to the test surfaces submerged in 10 mM sodium phosphate buffer (pH 7.0). As was found with the experiments with the dry surfaces, both intraspecific and intergenus differences were uncovered. Overall, the fungi adhered better to hydrophilic surfaces than to hydrophobic surfaces. This indicated that the fungal surfaces were covered with relatively hydrophilic compounds such as carbohydrates. Subsequently, it was demonstrated that all the fungi adhered to plasma membrane glycoprotein coated polystyrene and the presence of fungal carbohydrates on the surfaces of the fungal propagules was confirmed using epi-fluorescence microscopy. Differences in the strategy of the fungal genera to release their airborne spores, as well as differences in their adhesion profiles for the series of test materials, may be indicative of a unique environmental niche for each genus. In future, this phenomenon should be investigated further.
AFRIKAANSE OPSOMMING: Hifomisete fungi is daarvoor bekend om te versprei deur middel van wind, water, en insek vektore. Maar nietemin, daar is bykans geen kennis m.b.t. hoe hierdie fungi van mekaar verskil t.o.v. hul vermoë om versprei te word deur omgewings vektore nie. Gevolglik was spoorvrystelling van kulture, verteenwoordigend van die genera Acremonium, Aspergillus en Penicillium gemoniteer om ‘n aanduiding te kry van primêre wyse van spoorverspreiding waardeur verteenwoordigers van die onderskeie genera ingespan word. Eksperimentele data ingewin, vanaf plaat tellings wat uitgevoer was op lug afkomstig vanuit die uitlaat-klep van die lugvloei kapsule, was onderwerp aan ‘n toepaslike analise van afwyking (ANOVA), deur gebruik te maak van ‘n SAS statistiese pakket. Intraspesie verskille is waargeneem t.o.v. lug spoorvrystelling. Desnieteenstaande was Penicillium meer suksesvol onder vogtige kondisies t.o.v. spoorvrystelling in vergelyking met Aspergillus en die Acremonium stam. Onder droë kondisies het verteenwoordigers van Aspergillus langer geneem om hul spore vry te stel as verteenwoordigers van onderskeidelik, Penicillium en Acremonium. Geen verskille was waargeneem m.b.t. spoorvrystelling in fisiologiese soutoplossing (FSO) tussen die verskillende filogenetiese stamme nie. Alhoewel dit nie bewys is nie, wil dit voorkom asof water as belangrike verspreidingsagent van die betrokke fungi dien, aangesien die spoel van kulture met FSO tot ‘n oombliklike enorme vrystelling van kolonie-vormende eenhede gelei het. Gevolglik, deur gebruik te maak van standaard plaattellings tegnieke, was spoor aanhegting van bogenoemde fungi aan sintetiese membrane, blaar snitte en insek eksoskelette wat verskil in terme van hidrofobisiteit en elektriese lading, ondersoek. Daar was gevind dat die aanhegtingsprofiele m.b.t. hierdie reeks toetsoppervlaktes van die verskillende genera verskil, wat op sigself ‘n aanduiding was van verskille in fisieschemiese eienskappe van die swamspoor oppervlaktes. Penicillium stamme het ‘n hoër aanhegtings vermoë aan die toetsoppervlaktes getoon as die aspergilli, terwyl die verteenwoordiger van Acremonium die laagste aanhegting getoon het. Geen betekenisvolle verskille i.t.v. persentasie spoor aanhegting was gevind tussen hidrofobiese en hidrofiliese oppervlakte nie. Daarbenewens was die argument dat spoorvrystelling onder droë kondisies beïnvloed word deur elektrostatiese oppervlak ladings, bevestig deur ons bevindinge, want aanhegting het positief gekoreleer (Korrelasie koëffisient = 0.70898, p = 0.001) met positiewe ladings op die oppervlaktes. ‘n Standaard plaattellingstegniek was aangewend in die volgende fasset van die studie om die relatiewe aanhegting van bogenoemde hifomisete fungi, sowel as ‘n polifilitiese groep giste aan die toetsoppervlaktes, gedompel in 10 mM natrium fosfaat buffer (pH 7.0) vas te stel. Intraspesie en intragenus verskille was weereens waargeneem, net soos in die geval van die eksperimente met die droë oppervlakte. In die algemeen het die swamme baie beter geheg aan hidrofiliese oppervlaktes in vergelyking met hidrofobiese oppervlakte. Dit was ‘n aanduiding dat die swamspoor oppervlaktes bedek was met relatiewe hidrofiliese verbindings bv. koolhidrate. Verder was daar bewys dat alle swamme ingesluit in hierdie studie die vermoë het om plasmamembraan glikoproteïn bedekte polistireen te bind, en gevolglik was die teenwoordigheid van van koolhidrate op die swamspore bevestig m.b.v epi-fluoresensie mikroskopie. Verskille in die strategie van swamme om spore in die lug vry te stel, sowel as verskille in die aanhegtingsprofiele vir ‘n reeks toetsmateriale, mag net ‘n aanduiding wees van ‘n unieke omgewings nis vir elke genus wat in hierdie studie ondersoek is. Hierdie verskynsel moet dus in die nabye toekoms nagevors word.

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Extratos etanólicos do ritidoma, da casca do caule e do fruto e da semente de Swartzia argentea, S. laevicarpa, S. panacoco, S. polyphylla e de S. sericea foram testados para os fungos degradadores de madeira. O volume de 10 mL de cada extrato nas concentrações de 0, 1; 0,01 e 0,001 mg/ L foi adicionado a 90 mL de meio de cultura agar-malte e inoculado com Pycnoporus sanguineus (L.:Fr.) Murr., Trametes villosa (Fr.) Ryv. e Lenzites trabea Pers.:Fr. A área (cm2) da colônia do fungo e o índice antifúngico (IAF) dos extratos foram calculados. A diferença significativa entre as médias do crescimento dos fungos (cm2) foi observada nos extratos do ritidoma, da casca do fruto e da semente, independente da espécie de Swartzia, mas para os extratos da casca do caule não houve diferença significativa para S. laevicarpa e para S. sericea. Houve variação significativa entre as médias da área micelial (cm2) dos fungos para cada concentração do extrato. Os extratos dos ritidomas de S. argentea, S. polyphylla e de S. sericea e da casca do caule de S. panacoco apresentaram o índice antifúngico de 100% para as três espécies de fungos. Estes extratos foram submetidos à partição líquido-líquido com solventes de polaridade crescente, obtendo-se as frações: hexânica, diclorometano, acetato de etila e aquosa, visando a atividade antifúngica das frações em ensaio de bioautografia. As frações: diclorometano, acetato de etila e aquosa, foram aplicadas em concentrações diferentes para cada espécie de Swartzia em cromatofolha de gel de sílica GF254, sendo que a fração aquosa foi eluída com a fase móvel butanol:etanol:agua (4,0;1,1;1,9), a fração acetato de etila com o sistema de solvente diclorometano:acetato de etila:metanol (5:3:2), acrescido de 0,01 mL de ácido acético, e a fração diclorometano com os sistema de solventes diclorometano:acetato de etila (7:3)...
Ethanolic extracts from the ritidome, the barks of the stem and of the fruit, and the seed of Swartzia argentea, S. laevicarpa, S. panacoco, S. polyphylla and S. sericea were tested for decay fungi. Pycnoporus sanguineus (L.:Fr.) Murr., Trametes villosa (Fr.) Ryv. and Lenzites trabea Pers.:Fr. were inoculated in 90 mL of Malt Agar plus 10 mL of each extract, in the concentrations of 0,1, 0.01 and 0,001 mg/mL. Colony area (cm2) and antifungal index of the extracts were calculated. Significant difference among the fungal growth average (cm2) was observed in the extracts of the ritidome, the barks of the fruit and of the seed, despite the species studied. However, there was no difference in the extracts of the barks of the stem in S. laevicarpa and S. sericea. There was significant variation among the averages of the fungal mycelial area (cm2) in each extract concentration. The extract of the ritidome of S. argentea, S. polyphylla, and S. sericea, besides one from the barks of the stem of S. panacoco, resulted in 100% antifungal index for all fungal species. These extracts were submitted to liquid-liquid partition with solvents of increasing polarity resulting the following fractions: hexanic, dichloromethane, ethyl acetate and water, aiming the fraction antifungal activity in thin layer cromatography TLC. Dichloromethane, ethyl acetate and the watery fraction were applied in different concentrations on each Swartzia species studied, in TLC GF254. The watery fraction was eluted with butanol:ethanol:water (4,0; 1,1; 1,9), the fraction ethyl acetate with the solvent system dichloromethane:ethyl acetate: methanol (5:3:2) plus 0,01mL acetic acid, and the dichloromethane fraction with the solvent system dichloromethane: ethyl acetate (7:3). The chromatograms were inoculated with P. sanguineus, T. villosa and L. trabea in Malt Agar, and chromatographic strips with no fractions...(Complete abstract click electronic access below)

A manipulação seletiva de alvos epigenéticos usando pequenas moléculas inibidoras das enzimas histona-desacetilases (HDACs) e DNA metiltransferases (DNMTs) é uma estratégia para estimular a expressão das vias biossintéticas e a produção de novos metabólitos secundários em fungos. Neste trabalho, inibidores de histonadesacetilases (butirato de sódio, ácido hidroxâmico suberoilanilida e ácido valproico) e inibidores de DNA metiltransferases (5-azacitidina, hidralazina, procaína e procainamida) foram suplementados em culturas líquidas e sólidas dos fungos endofíticos Fusarium oxysporum SS46, Hyphodermella corrugata FLe8.2 e Chaetomium globosum VR10, das linhagens comerciais Fusarium oxysporum ATCC MYA 4623 e Chaetomium globosum ATCC 56726 e do fitopatógeno Botrytis cinerea B0510. O fungo endofítico H. corrugata FLe8.2, em meio PDB, produziu o composto 2-(2-metoxifenil)-4H-piran-4-ona, e sua cultura em meio Czapek suplementada com hidralazina levou ao isolamento de 3-metil-1,2,4-triazolo[3,4-a]-ftalazina. O tratamento de F. oxysporum SS46 e F. oxysporum ATCC MYA 4623 com hidralazina em meio Czapek levou ao isolamento de um novo composto, 2H-[1,2,4]triazino[3,4- a]-ftalazina. Nestas culturas houve biotransformação da hidralazina pelos fungos, provavelmente como mecanismo de desintoxicação. Ainda, a hidralazina teve um efeito inibidor sobre a biossíntese do ciclohexadepsipeptídeo beauvericina em F. oxysporum SS46. Nas culturas de C. globosum VR10 em meio Czapek os inibidores de HDACs e de DNMTs suprimiram a biossíntese de chaetoglobosinas. A adição de ácido valproico na cultura de C. globosum VR10 em meio PDB eliciou a produção da chaetoviridina B. A adição de 5-azacitidina nas culturas de C. globosum VR10 em meio sólido PDA não modificou a produção da chaetoglosina A. A produção de chaetoviridina B por C. globosum ATCC 56726 em meio PDA foi inibida na menor concentração de 5-azacitidina, 50 ?M, e retomada na concentração de 150 ?M. O tratamento de B. cinerea B0510 com ácido hidroxâmico suberoilanilida SAHA levou à biossíntese de um novo composto, ácido 5-benzil-2,3-di-hidroxi-3-isopropil-4- oxotetrahidrofuran-2-carboxílico, o qual também foi isolado da linhagem geneticamente modificada B. cinerea Bc ?STC2. No geral, considerando as linhagens fúngicas estudadas, os resultados mostram que a adição de moduladores químicos que atuam em mecanismos epigenéticos promove mudanças no perfil de metabólitos secundários.
The selective manipulation of epigenetic targets using small molecule inhibitors of histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activities is a strategy to elicit the expression of biosynthetic pathways and production of new secondary metabolites in fungi. In this work, HDAC inhibitors (sodium butyrate, suberohydroxamic acid and valproic acid) and DNMT inhibitors (5-azacitidine, hydralazine, procaine and procainamide) were supplemented in liquid and solid cultures of the endophytic fungi Fusarium oxysporum SS46, Hyphodermella corrugata FLe8.2 and Chaetomium globosum VR10, of the commercial fungal strains Fusarium oxysporum ATCC MYA 4623 and Chaetomium globosum ATCC 56726 and of the phytopathogenic fungus Botrytis cinerea B0510. The endophytic fungus H. corrugata FLe8.2 produced 2-(2-methoxyphenyl)-4H-pyran-4-one in PDB medium, while in the presence of hydralazine in Czapek medium the fungus produced 3- methyl-1,2,4-triazolo[3,4-a]-phthalazine. Treatment of F. oxysporum SS46 and F. oxysporum ATCC MYA 4623 with hydralazine in a Czapek medium led to the isolation of new compound, 2H-[1,2,4]triazino[3,4-a]-phthalazine. Hydralazine was biotransformed by these three fungi probably as a detoxification strategy. In addition, hydralazine also inhibited the biosynthesis of the cyclodepsipeptide beauvericin by F. oxysporum SS46. HDAC and DNMT inhibitors suppressed chaetoglobosins\' biosynthesis by C. globosum VR10 cultures in Czapek medium. The biosynthesis of chaetoviridin B by C. globosum VR10 was elicited by acid valproic in PDB medium. The production of chaetogosin A by C. globosum VR10 in PDA medium has not been affected by 5-azacitidine. The biosynthesis of chaetoviridin B by C. globosum ATCC 56726 in PDA medium was inhibited in the presence of lower concentration 5- azacitidine (50 ?M) and recovered in the higher concentration (150 ?M). Treatment of B. cinerea B0510 with suberohydroxamic acid led to the biosynthesis of the new compound 5-benzyl-2,3-dihydroxy-3-isopropyl-4-oxotetrahydrofuran-2-carboxylic acid, which was also isolated from the genetically modified strain B. cinerea Bc ?STC2. Results showed that chemical compounds that act in epigenetic mechanisms can induce changes in the secondary metabolite profiles in the fungal strains studied in this work.

Microrganismos formam associações com a maioria das espécies animais e um exemplo fascinante são as múltiplas interações nas colônias de formigas-cortadeiras. Os efeitos dessas interações (positivos e negativos) se manifestam na sanidade e desenvolvimento das colônias. Sendo assim, a compreensão das interações que ocorrem entre os microrganismos das colônias de formigas-cortadeiras A. sexdens é importante para fundamentar o controle biológico desta praga. Esse presente trabalho foi dividido em três capítulos. O primeiro capítulo objetivou comparar o desenvolvimento de colônias de Atta sexdens (Linnaeus) em contato com dois solos: (i) de área com ninhos e (ii) de área sem ninhos de formigas-cortadeiras. Foram conduzidos dois experimentos em laboratório. No experimento I, fêmeas recémfecundadas fundaram a colônia em pote com gesso e, após 106 dias, entraram em contato com solo. No experimento II, as fêmeas recém-fecundadas fundaram suas colônias diretamente no solo. A taxa de mortalidade de colônias, após 106 dias da revoada e se desenvolvendo em pote com gesso, foi de 28,6%. Quando se desenvolveram desde o início em contato com o solo, a taxa de mortalidade elevouse a 67,2 %. Os resultados confirmam que as colônias incipientes de A. sexdens sofrem forte pressão seletiva de microrganismos do solo no momento da fundação. No entanto, após o surgimento da força operária, mecanismos de defesa imune social, provavelmente, garantem o desenvolvimento da colônia a despeito da presença de microrganismos patogênicos no solo dos ninhos. O segundo capítulo objetivou isolar e identificar actinobactérias de solos de câmaras de jardim de fungos de A. sexdens e avaliar o efeito inibitório desses isolados sobre fungos associados às colônias de formigas-cortadeiras. Foram sequenciados o gene 16S rRNA de nove actinobactérias, sendo seis do gênero Streptomyces, duas do gênero Nocardia e uma do gênero Kitasatospora. Foi verificado que dois isolados de Streptomyces e um de Kitasatospora inibiram não só o fungo Escovopsis sp., como também o fungo entomopatogênico Metarhizium anisopliae e o fungo antagonista do cultivar simbionte de cortadeiras Trichoderma aff. strigosellum. Uma vez que não existem evidências de cultivo de actinobactérias na cutícula de operárias do gênero Atta, é possível hipotetizar que essas operárias estabeleçam simbiose temporária adaptativa com microrganismos do solo produtores de substâncias antifúngicas e antibióticas e que vivem em alguma parte de seu ninho ou mesmo no interior do seu corpo. Além disso, 10 os fungos patogênicos para colônias de formigas-cortadeiras presentes no solo adjacente ao ninho, apesar de constituírem um risco, podem ser controlados pelas secreções produzidas pelas operárias, bem como pelos metabólitos de algumas actinobactérias. O terceiro capítulo teve como objetivo verificar a aceitação e incorporação de iscas contendo micélio de Escovopsis sp. em colônias jovens de Atta sexdens. Verificou-se o transporte de iscas em todas as colônias do teste. Houve redução no peso do jardim de fungos das colônias que receberam iscas com Escovopsis sp., e aumento no peso do jardim de fungos de colônias que receberam tratamento controle. Conclui-se que a utilização de iscas com micélio de Escovopsis sp. foi satisfatória para introduzir o fungo parasita no jardim de fungos de colônias de Atta sexdens.
Microorganisms form associations with most animal species, and a fascinating example is the multiple interactions in the colonies of leaf-cutting ants. The effects of these interactions (positive and negative) are exhibited in the health and development of the colonies. Therefore, the understanding of the interactions that occur among the microorganisms into leaf-cutting ants colonies is important to support of biological control of this pest. This work was divided into three chapters. The first chapter aimed to compare the development of colonies of Atta sexdens (Linnaeus) in contact with two types of soil: (i) from an area used for nesting and (ii) from an area not used for nesting of leaf-cutting ants. Two experiments were conducted in the laboratory. In experiment I, newly fertilized females founded the colony in a plastic pot with gypsum and, after 106 days were transferred to a plastic pot with soil. In experiment II, newly fertilized females founded their colonies directly on the soil. Colony mortality rate 106 days after nuptial flight and founding in a plastic pot with gypsum was 28.6%. When they developed directly in contact with the soil, mortality rate increased to 67.2%. The results support that incipient colonies of A. sexdens undergo strong selective pressure from soil microorganisms at the time of foundation. However, after the emergence of the worker force, social immune defense mechanisms likely guarantee the development of the colony, despite the presence of pathogenic microorganisms in the soil of the nests. The second chapter aimed to isolate and identify actinobacteria from soils of fungi garden chambers of A. sexdens and to evaluate the inhibitory effect of these isolates on fungi associated with leaf-cutting colonies. To identify the isolates, the 16S rRNA gene was sequenced from nine actinobacteria: six of Streptomyces genus, two of Nocardia genus and one of Kitasatospora genus. Two Streptomyces and one Kitasatospora isolates inhibited not only the fungus Escovopsis sp., but also the entomopathogenic fungus Metarhizium anisopliae and the antagonistic fungus of the cultivar symbiont of leaf-cutting ant Trichoderma aff. strigosellum. Since there is no evidence of cultivation of actinobacteria on the Atta worker cuticle, it is possible that these workers establish temporary adaptive symbiosis with soil microorganisms producing antifungal and antibiotic substances and living in some part of their nest or even in the interior of their body. It can be hypothesized that pathogenic fungi present in the soil adjacent to the leaf-cutting ant nest, despite the risk they represent, are controlled by the secretions produced by the workers, as well as by the metabolites of some actinobacteria. The third chapter had the objective of verifying the acceptance and incorporation of baits containing mycelium of Escovopsis sp. by young colonies of A. sexdens. We verified the transport of baits in all tested colonies. There was a reduction in the weight of the fungus garden of the colonies that received baits with Escovopsis sp., and an increase in the weight of the fungus garden of colonies that received control treatment. It is concluded that the use of baits with mycelium of Escovopsis sp. was satisfactory to introduce the fungus parasite in the fungus garden of A. sexdens colonies.

RESUMO: Determinadas espécies de fungos são responsáveis por diversas doenças no ser humano. Estudos epidemiológicos avaliam essas infecções tanto superficiais quanto profundas, e alguns destes avaliam em relação ao trabalho e condição de vida e saúde de trabalhadores de diversas áreas. Entretanto a contaminação por fungos filamentosos na região da cavidade oral com relação à atividade laboral rural não apresenta referência. OBJETIVOS: Avaliar a incidência de contaminação fúngica na região orofaringe normal de trabalhadores agrícolas das culturas de cana-de-açúcar, laranja e abacaxi do município de Frutal, Minas Gerais. MÉTODOS: Esse é um trabalho longitudinal, prospectivo, tipo coorte, não randomizado, em que os participantes eram migrantes vindos para trabalho temporário nas culturas referidas. Foram selecionados 60 participantes no momento das contratações após realizados os exames pré-admissionais. Avaliou-se as características sociodemográficas dos participantes. A coleta de material investigativo seguiu a ordem: região do sulco gengivo - labial, próximo à região do freio superior e freio inferior; material da mucosa jugal direita e esquerda, com um swab para cada região e dispostos na placa de Petri, respeitando a anatomia descrita. A coleta e análise do material dos trabalhadores e do meio ambiente seguiram os tempos de cada cultura, determinados de (t0), (t1), (t2). Também foram usados para caracterizar cada fase de coleta, cultura e análise das amostras. As amostras foram semeadas em meio Agar Sabouraud Dextrose. Após o crescimento dos isolados, as culturas filamentosas foram submetidas às técnicas de colônias gigantes e microcultivo. Os fungos foram identificados no microscópio com objetiva de 40 vezes. RESULTADOS: Não houve diferença entre idade e sexo nos três grupos analisados. Houve predomínio de homens afrodescendentes nas culturas de cana e laranja quando comparados ao abacaxi, que apresentou um predomínio de caucasianos. Trabalhadores de origem oriental foram detectados apenas na cultura de abacaxi. Quanto à renda, os trabalhadores de abacaxi recebem salários mais elevados do que os outros dois grupos de trabalhadores. Não houve diferença em relação ao tabagismo e a ingestão de álcool entre os três grupos analisados. Em relação à contaminação do meio ambiente, tanto na planta quanto no ar, encontrou-se um maior índice de placas de Petri contaminadas nas plantações de abacaxi e cana-de-açúcar, respectivamente. Observou-se um predominio de F. moniliforme na cultura de cana-de-açúcar e de F.subglutians nas culturas de laranja e de abacaxi nos tempos definidos dessa pesquisa. A contaminação dos trabalhadores ocorreu no segundo tempo da pesquisa. A respeito dos voluntários das plantações de abacaxi, 13,3% dos trabalhadores foram infectados entre os sessenta analisados; todos os trabalhadores foram contaminados com F.subglutinans. Na cultura de cana-de-açúcar, 8,3% deles foram encontrados infectados, sendo 5% por A. niger e 3,3% por F.moniliforme. Dois voluntários infectados pelo A. niger apresentaram infecção concomitante com C. albicans. Na lavoura de laranja 1,6% foram infectados por F.subglutians. CONCLUSÕES: Esse estudo demonstrou que trabalhar na cultura do abacaxi se mostrou um fator de risco para infecção fúngica na mucosa oral quando comparado às atividades na cultura da laranja. Houve também contaminação fúngica na cultura de cana-de-açúcar quando comparado ao grupo de referência. Outros fatores como idade, ingestão de álcool, tabagismo, renda familiar ou etnia não se mostraram estatisticamente significativos para incidência da infecção
ABSTRACT: Certain species of fungi are responsible for several diseases in humans. Epidemiologic studies rate those infections, both superficial and profound, and some of them rate regarding the occupation and life and health conditions of workers from several areas. However, the filamentous fungal infection in the oral cavity regarding laboral activity presents no reference. OBJETIVE: To rate the incidence of fungal contamination in the normal oropharynx of agricultural workers of sugar cane, orange and pineapple crops in the municipal district of Frutal, Minas Gerais. METHODS: This is a longitudinal, prospective and cohort work, in which the participants were migrants to temporary work in the aforesaid crops. Sixty participants were selected in the hiring moment after pre-admission examinations. The participants\' sociodemographic features were rated. The gathering of investigative materials followed the order: region of the gingival sulcus, close to the upper and lower lip curb region; material from the right and left jugal mucous with a swab for each region and arranged in a Petri dish, respecting the anatomy described. The gathering and analysis from workers and environment material followed the time of each culture, determined of (t0), (t1), (t2). They were also used to feature each stage of gathering, culture and analysis of samples. The samples were sown amid Sabouraud Dextrose Agar. After the growth of the isolates, the filamentous cultures were subjected to the techniques of giant colonies and microcultivation. The fungi were identified in the microscope with a 40x magnification. RESULTS: There was no difference between age and sex in the three groups studied. There was a predominance of African descent men in cane and orange crops compared to pineapple, which showed a predominance of Caucasians. Oriental workers were detected only in pineapple crop. As for income, the pineapple workers receive higher wages than the other two groups of workers. There was no difference regarding smoking and alcohol intake among the three groups analyzed. Regarding the environment contamination, both in the plant and in the air, a higher rate was found in Petri dishes contamined in pineapple and sugar cane crops, respectively. There was a predominance of F. moniliforme in the sugar cane crop and of F.subglutians in orange and pineapple crops in the times defined in this research. Contamination of workers occured in the second half of the research. Regarding the volunteers of pineapple crops, 13,3% workers were infected among the sixty analyzed; all workers were contamined with F. subglutinans. In the sugar cane crop, 8,3% of them were found infected, 5% by A. niger and 3,3% by F. moniliforme. Two volunteers infected by the A. niger presented concomitant infection with C. albicans. A worker of the orange crop was infected (1,6% ) by F. subglutians. CONCLUSIONS: This study demonstrated that working in the pineapple crop showed a risk factor for the fungal infection in the oral mucosa when compared to the activities in the orange crop. Also, there was a fungal contamination in the sugar cane crop when compared to the reference group. Other factors such as age, alcohol intake, smoking, family income or ethnicity were not statistically significant for the incidence of infection

The eastern hemlock is a late-successional conifer species that is valued for its ecological functions, recreational importance, aesthetic beauty, and economic value. The hemlock woolly adelgid (HWA, Adelges tsugae Annand, Homoptera: Adelgidae) is an invasive aphid-like insect from Asia that is causing serious damage to the eastern and Carolina hemlock. HWA was first introduced to the Eastern United States to Virginia in the 1950’s and has since moved along the east coast from Georgia to Maine. It can kill a healthy tree in three to seven years depending on many environmental factors. Native predators have not been successful in reducing damage to hemlock trees or limiting the spread of HWA. Chemical, cultural, and biological control efforts have been implemented in states with high populations of HWA. Chemical applications are costly and current formulations are difficult to apply in large stands and in forested areas. Salvage cutting is a method to control the spread of adelgid and recover some economic value of the wood. Predatory beetles native to Asia, where HWA is present but not a severe problem, have been researched, reared, and released into infested stands in the Eastern United States. Their success for management of HWA has been difficult to ascertain. The entomopathogenic fungus Lecanicillium muscarium ((Petch) Zares & Gams) is another biological control agent with potential to manage and suppress adelgid populations. L. muscarium (Mycotal™ technical powder, Koppert Biological Systems) plus the nutritive base sweet whey, may promote conidia production without contact with a host and this means of increasing conidia is called a whey-based fungal microfactory. The whey would act as a nutritive substrate for fungi sprayed into hemlock forests. Formulation droplets deposited on hemlock needles should support fungal growth and serve as tiny factories for conidia production. Microfactory production was characterized in different combinations of sweet whey (0, 5, 10, and 15%) and conidia concentration (1 x 106, 1 x 107, and 1 x 108 conidia/ml) applied to lids of Petri dishes. A dramatic 42- and 29-fold increase in conidia production occurred with the addition of 10% sweet whey to 1 x 106 and 1 x 107 conidia/ml, respectively. Increasing whey concentration increased the number of conidia that were recovered. Conidia production was also obtained on hemlock foliage, with similar trends in influence of conidia and whey concentration. The hemlock branches also contained HWA and their mortality was evaluated. Adelgid mortality was highest in formulations containing sweet whey, but whey had an independent effect on mortality. Several antimicrobials were evaluated for compatibility with L. muscarium in the microfactory formulation. Nisin did not inhibit conidia production. The addition of antimicrobials to the whey-based formulations may limit the competition between the fungus and other microbes present in the spray tank and on foliage. Whey-based fungal microfactory technology is intended to facilitate multiplication of fungi in the natural environment. Transferring a portion of fungal massproduction into the treatment environment could reduce application costs and increase the feasibility of using fungi for biological control of HWA and other pest species.

Resumo: Os fungos constituem um reino de seres vivos que vêm despertando crescentes interesses em virtude do elevado potencial em elaborar substâncias economicamente importantes, como alimentos, solventes, enzimas, vitaminas, antibióticos, ácidos graxos e outros produtos, justificando a sua aplicação biotecnológica. As atividades enzimáticas destacando-se neste trabalho a da carboximetilcelulase e da xilanase foram avaliadas em oitenta linhagens de fungos isolados do solo da Estação Ecológica de Juréia-Itatins, SP. Os fungos foram cultivados em meio de farelo de trigo a 25oC por 4 dias. As enzimas liberadas no meio de cultura foram extraídas com 30 ml de água gelada e as respectivas atividades enzimáticas foram avaliadas em unidades (U) de enzima necessária para liberar um mol de glicose ou xilose por mililitro por minuto, nas condições do ensaio. As melhores atividades da carboximetilcelulase (8,38 U) e da xilanase (496,72 U) foram obtidas, respectivamente, nos cultivos com Trichoderma longibrachiatum (n. 31) e T. inhamatum (n. 33) a 28oC por 4 dias. A produção de ácido -linolênico foi avaliada em 25 linhagens de fungos zigomicetos, cultivados em meio contendo 2% de glicose e 1% de extrato de levedura, por 4 dias a 25oC. As culturas foram realimentadas com 20% de meio e permaneceram por 3 dias a 12oC. Nestas condições Mucor ramosissimus foi selecionado por apresentar a maior produção de ácido -linolênico (47,70 mg.l-1). Analisando-se diferentes fontes de carbono e nitrogênio e a influência do pH inicial do meio de cultura, verificou-se que o uso de glicose e extrato de levedura em meio com pH inicial de 4,5, após 4 dias a 25oC sob agitação constante de 150 rpm, a produção de ácido -linolênico correspondeu a 106,44 mg.l-1.
Abstract: The fungi constitute a group of organisms that wake up crescents interests by virtue of their high potential in elaborating substances economically important, as enzymes and fatty acids justifying for the vast biotechnology application that they offer. The CMCase and xylanase activities were evaluated in eighty isolated fungi strains in the Ecological Station of Juréia-Itatins soil, in São Paulo State. These microorganisms were cultivated in wheat bran medium at 25oC for four days. The enzymes liberated in the culture medium were extracted with 30 ml of cold distilled water. Enzyme production is expressed as units of enzyme and one unit of enzyme activity is defined as 1 mol glucose or xylose released per minute per milliliter of culture extract. The best CMCase (8.38 U) and xylanase (496.72 U) activities were obtained, respectively, with Trichoderma longibrachiatum (n. 31) and Trichoderma inhamatum (n. 33) after cultivations at 28oC, for four days in wheat brain medium. The -linolenic acid production was evaluated in 25 strains of zygomycetous fungi in a culture medium containing 2% glucose and 1% yeast extract, after four days of incubation at 25oC. The cultures were added with 20% (v/v) of culture medium and they stayed at 12oC for three days. In these conditions Mucor ramosissimus was selected by presenting the best -linolenic acid production (47.70 mg.l-1). After analyzed different carbon and nitrogen sources and the influence of the initial culture medium pH, it was verified that the -linolenic acid production in the culture medium with 2% glucose and 1% yeast extract and initial pH 4.5, after four days at 25oC with a rotary shaker at 150 rpm, corresponded to 106.44 mg.l-1.
Orientadora: Sâmia Maria Tauk Tornisiello
Coorientadora: Gláucia Maria Pastore
Banca: Sandra Regina Ceccato Antonini
Banca: Siu Mui Tsai
Banca: Dejanira de Franceschi de Angelis
Doutor

Resumo: Extratos etanólicos do ritidoma, da casca do caule e do fruto e da semente de Swartzia argentea, S. laevicarpa, S. panacoco, S. polyphylla e de S. sericea foram testados para os fungos degradadores de madeira. O volume de 10 mL de cada extrato nas concentrações de 0, 1; 0,01 e 0,001 mg/ L foi adicionado a 90 mL de meio de cultura agar-malte e inoculado com Pycnoporus sanguineus (L.:Fr.) Murr., Trametes villosa (Fr.) Ryv. e Lenzites trabea Pers.:Fr. A área (cm2) da colônia do fungo e o índice antifúngico (IAF) dos extratos foram calculados. A diferença significativa entre as médias do crescimento dos fungos (cm2) foi observada nos extratos do ritidoma, da casca do fruto e da semente, independente da espécie de Swartzia, mas para os extratos da casca do caule não houve diferença significativa para S. laevicarpa e para S. sericea. Houve variação significativa entre as médias da área micelial (cm2) dos fungos para cada concentração do extrato. Os extratos dos ritidomas de S. argentea, S. polyphylla e de S. sericea e da casca do caule de S. panacoco apresentaram o índice antifúngico de 100% para as três espécies de fungos. Estes extratos foram submetidos à partição líquido-líquido com solventes de polaridade crescente, obtendo-se as frações: hexânica, diclorometano, acetato de etila e aquosa, visando a atividade antifúngica das frações em ensaio de bioautografia. As frações: diclorometano, acetato de etila e aquosa, foram aplicadas em concentrações diferentes para cada espécie de Swartzia em cromatofolha de gel de sílica GF254, sendo que a fração aquosa foi eluída com a fase móvel butanol:etanol:agua (4,0;1,1;1,9), a fração acetato de etila com o sistema de solvente diclorometano:acetato de etila:metanol (5:3:2), acrescido de 0,01 mL de ácido acético, e a fração diclorometano com os sistema de solventes diclorometano:acetato de etila (7:3)...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Ethanolic extracts from the ritidome, the barks of the stem and of the fruit, and the seed of Swartzia argentea, S. laevicarpa, S. panacoco, S. polyphylla and S. sericea were tested for decay fungi. Pycnoporus sanguineus (L.:Fr.) Murr., Trametes villosa (Fr.) Ryv. and Lenzites trabea Pers.:Fr. were inoculated in 90 mL of Malt Agar plus 10 mL of each extract, in the concentrations of 0,1, 0.01 and 0,001 mg/mL. Colony area (cm2) and antifungal index of the extracts were calculated. Significant difference among the fungal growth average (cm2) was observed in the extracts of the ritidome, the barks of the fruit and of the seed, despite the species studied. However, there was no difference in the extracts of the barks of the stem in S. laevicarpa and S. sericea. There was significant variation among the averages of the fungal mycelial area (cm2) in each extract concentration. The extract of the ritidome of S. argentea, S. polyphylla, and S. sericea, besides one from the barks of the stem of S. panacoco, resulted in 100% antifungal index for all fungal species. These extracts were submitted to liquid-liquid partition with solvents of increasing polarity resulting the following fractions: hexanic, dichloromethane, ethyl acetate and water, aiming the fraction antifungal activity in thin layer cromatography TLC. Dichloromethane, ethyl acetate and the watery fraction were applied in different concentrations on each Swartzia species studied, in TLC GF254. The watery fraction was eluted with butanol:ethanol:water (4,0; 1,1; 1,9), the fraction ethyl acetate with the solvent system dichloromethane:ethyl acetate: methanol (5:3:2) plus 0,01mL acetic acid, and the dichloromethane fraction with the solvent system dichloromethane: ethyl acetate (7:3). The chromatograms were inoculated with P. sanguineus, T. villosa and L. trabea in Malt Agar, and chromatographic strips with no fractions...(Complete abstract click electronic access below)
Orientador: Vera Lucia Ramos Bononi
Coorientador: Ana Cláudia Fernandes Amaral
Banca: Mauricio Bacci Junior
Banca: Dejanira de Franceschi de Angelis
Banca: Luiz Carlos Couto
Banca: Mario Tomazello
Doutor

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
As formigas da tribo Attini são conhecidas pela complexa simbiose que mantêm com fungos, os quais cultivam como alimento. É sabido que além desse fungo, outros microrganismos podem ser encontrados nos ninhos desses insetos e estudos prévios apontaram que alguns microfungos (i.e. leveduras e fungos filamentosos) podem ser importantes nessa simbiose. O objetivo do presente trabalho foi avaliar o papel desses microfungos associados aos jardins dessas formigas. Analisando várias espécies do gênero Acromyrmex do sul do Brasil, demonstrou-se que as formigas importam uma comunidade diversa de microfungos para seus ninhos, provavelmente provenientes do solo e do substrato vegetal que as formigas utilizam para cultivar seu fungo. Num segundo estudo, avaliando formigas Attini da América do Norte (Atta texana, Trachymyrmex septentrionalis e Cyphomyrmex wheeleri) observou-se que a estrutura das comunidades de microfungos nos jardins desses insetos não se correlaciona com a variação sazonal, sugerindo que não existam relações espécie-específicas entre as formigas e os microfungos. Apesar de tais microrganismos não serem especialistas dos jardins desses insetos, é sugerido que os microfungos atuem como antagonistas do fungo simbionte. Ainda, descobriu-se que o parasita especializado Escovopsis spp. parece ser menos freqüente nas populações de formigas da América do Sul em relação as Attini da América Central, porém estudos adicionais são necessários para estabelecer a epidemiologia desse parasita nos ninhos das Attini. Num terceiro estudo, demonstrou-se que leveduras presentes nos jardins de fungos da formiga cortadeira A. texana inibem o crescimento de Escovopsis spp., sugerindo que esses insetos utilizam outros microrganismos, além das bactérias presentes em suas cutículas (Pseudonocardia spp.), para inibir esse parasita. Esse achado traz importantes implicações para essa...
Ants in the tribe Attini are well-known social insects that maintain a symbiotic relationship with fungi which they cultivate as food. Besides of the cultivated fungi, fungus gardens contain several other microorganisms considered to be potential players in this symbiosis. The aim of the present study was to evaluate the possible roles of microfungi (i.e. yeasts and filamentous fungi) in attine gardens. Our microbial profiling of gardens from several species in the genus Acromyrmex from South Brazil revealed that ants can harbor a diverse community of microfungi that probably originated from the surrounding soil or from the substrate used to manure the cultivated fungus. In this sense, additional studies of North American attine species (Atta texana, Trachymyrmex septentrionalis and Cyphomyrmex wheeleri) demonstrated that the structure of microfungal communities in gardens of these ants did not correlate with seasonal changes over a one year period, again suggesting there are no species-specific relationships among ants and microfungi species. Although, the microfungi are not specialized parasites of the attine ant-fungus symbiosis we suggest they can be considered antagonists to the cultivated fungus. Moreover, we demonstrated that the specialized parasite Escovopsis spp. is probably less frequent in South America than in Central America and we reinforce that additional studies are necessary to unravel the epidemiology of this parasite in attine gardens. In another study, we showed that yeasts isolated from gardens of the leafcutter ant A. texana can significantly inhibit the growth of Escovopsis sp. This interesting finding suggests that attine ants may use additional microbes to protect their gardens against Escovopsis spp. and not only actinomycete bacteria (Pseudonocardia spp.) found in their cuticles. Finally, we studied microfungi relationships with female alates (gynes) in two... (Complete abstract click electronic access below)

Kyoto University (京都大学)
0048
新制・論文博士
博士(農学)
乙第11673号
論農博第2556号
新制||農||912(附属図書館)
学位論文||H17||N4056(農学部図書室)
23486
UT51-2005-D591
(主査)教授 加藤 暢夫, 教授 清水 昌, 教授 宮川 恒
学位規則第4条第2項該当

Foi pesquisada a habilidade de carunchos Sitophilus zeamais em veicular esporos de Aspergillus flavus e Fusarium verticillioides e a conseqüente produção de micotoxinas. Grãos de milho foram mantidos em frascos conectados por uma mangueira formando um sistema fechado (lados A e B) e divididos em seis grupos: G1 (milho + caruncho - Lado A); G2 (Milho + A. flavus - Lado A); G3 (milho + A. flavus + caruncho - Lado A); G4 (milho + F. verticillioides - Lado A), G5 (milho + F. verticillioides + caruncho - Lado A) e G6 (milho + A. flavus + F. verticillioides + caruncho - Lado A). O lado B continha grãos estéreis. Após 10, 20 e 30 dias de incubação foram realizadas: pesagem, atividade de água, microbiota fúngica, determinação de micotoxinas, análise nutricional, microscopia eletrônica de varredura e PCR-RT em tempo real. Frente aos resultados obtidos constata-se a importância do Sitophillus zeamais como vetor de fungos e a importância de boas práticas de manipulação e armazenamento de grãos, visando reduzir os riscos de contaminação e deterioração.
The weevils Sitophilus zeamais ability was examined to propagate spores of Aspergillus flavus and Fusarium verticillioides and the production of mycotoxins. Corn grains was conserved in flasks connected by a rubber to form a closed system (side A and B) and divided in six groups: G1 (corn + weevil - side A); G2 (corn + A. flavus - side A); G3 (corn + A. flavus + weevil - side A); G4 (corn + F. verticillioides - side A), G5 (corn + F. verticillioides + weevil - side A) e G6 (corn + A. flavus + F. verticillioides + weevil - side A). The side B contained sterile grains. After 10, 20 and 30 days of incubation were realized: weighing, activity water, mycoflora, determination of mycotoxins, nutritional analysis, scanning electron microscope and Real time PCR-RT. In front of the results was observed the importance of Sitophilus zeamais like a fungus vector and the importance of Good Manufacturing Practices and Stores of grains, to reduce the risks of contamination and deterioration.

Eleven fungal strains were received from the clinical microbiology laboratory collection of Queen Mary Hospital and Pamela Youde Nethersole Eastern Hospital in Hong Kong from 2010-2011. The collection comprised of ten ascomycetes and one zygomycete. They were identified down to the genus level based on the morphological criteria. Internal transcribed spacer (ITS), beta-tubulin, actin and 28S gene sequencing were used for genotypic characterization. The ITS sequences of four of the strains demonstrate <3%-base difference to a single fungal species. They were species of the genus, Acremonium, Aspergillus, Cladophialophora and Ochroconis respectively. Five strains belonging to the genus, Trichophyton, Monascus, Mucor, Arthrinium and Acremonium could not be identified to the species level due to low interspecies heterogeneity. The tubulin gene was used for two of the strains. The tubulin sequence of a strain of Phaeoacremonium was identified to the species level with 0%-base difference. The ITS, partial beta-actin and 28S rDNA genes were sequenced for a strain of Exophiala. They showed a distinct cluster, mostly closely related to, but distinct from, Exophiala xenobiotica, Exophiala jeanselmei and Exophiala oligosperma. The genotypic characteristics suggest the strain to be a novel species of Exophiala. More genotypic and phenotypic characterization are required to described this strain of Exophiala.
published_or_final_version
Microbiology
Master
Master of Research in Medicine

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Agaricus blazei é uma das espécies de fungo do grupo basidiomiceto nativo do Brasil, popularmente conhecido como cogumelo-do-sol®. O Brasil é considerado um grande fornecedor desse fungo para o mercado internacional cujos padrões de qualidade são bastante restritivos em relação à concentração de metais pesados, principalmente cádmio. Esse fungo possui propriedades medicinais e seus cogumelos são consumidos por grande número de pessoas que procuram melhorar a qualidade de vida. O trabalho objetivou estudar o acúmulo de cádmio, cromo e chumbo por A. blazei. Foram realizados 3 experimentos, utilizando 2 tipos de composto e 3 tipos de solo para cobertura. O fungo foi cultivado em sacos de plástico preto contendo no fundo o composto inoculado, tendo como cobertura diferentes tipos de solo. O solo e o composto foram analisados para quantificação de Cd, Cr, Pb por espectrometria de absorção atômica, propriedades físicas e químicas (Instituto Campineiro de Análise de Solo e Adubo ICASA). Além das quantias de metais encontradas no solo e no composto foram adicionados 66 mg de cloreto de cádmio, 132 mg de sulfato de cromo e 12 mg de nitrato de chumbo ao solo de cobertura. Após a frutificação, os basidiocarpos foram analisados para quantificar proteínas, aminoácidos, metais pesados e identificação de modificações protéicas por espectroscopia de infravermelho (FTIR). O resultado das análises demonstrou que havia acúmulo de metais pesados nos basidiocarpos, ocorrendo maior percentual nos contaminados com cádmio.Além disso, detectou-se nos basidiocarpos contaminados menor teor de proteínas. Através de FTIR não foi possível detectar alterações nas proteínas nos basidiocarpos analisados.
Agaricus blazei is a basidiomycete fungus native from Brazil and popularly known as Cogumelo-do-sol® the sun-mushroom. Brazil is a supplier of this edible mushroom to international markets to which quality standards are very restrictive regarding the concentration of heavy metals, especially cadmium. This fungus exhibits medicinal properties and its fruiting bodies are consumed by a large number of people that seek to improve life quality. The objective of this work was to study the accumulation of cadmium, chromium and lead by A. blazei. Three experiments were made using two composts and three types of covering soil. The fungus was cultivated in black plastic bags contend in the deep inoculated compost and as covering different types of soils. The soil and the compost were analyzed to determine the amount of heavy metals: Cd, Cr and Pb by atomic absorption spectrophotometer, and physical and chemical properties (ICASA). Besides the metal found in the soil and compost, 66 mg of cadmium chloride, 132 mg of chromium sulfate and 12 mg of lead nitrate were also added to the covering soil. After fructification, the fruiting bodies were analyzed to measure the amount of raw proteins, amino acids, heavy metals and proteinic modifications for Spectroscopy of Infra Red (FTIR). The analyses of results showed accumulation of heavy metals in fruiting bodies, occurring a higher percentage in fruiting bodies contaminated of cadmium. Furthermore detected in contaminated fruiting bodies a less drift of raw proteins. Through FTIR was not possible to detect alteration in proteins of fruiting bodies analyzed.

Orientador: André Rodrigues
Banca: Michele de Cássia Pereira e Silva
Banca: Marina Correa Cortes
Resumo: Insetos cultivadores de fungos (formigas, cupins e besouros) evoluíram de forma independente uma associação simbionte com fungos que, ao metabolizar biomassa vegetal recalcitrante, produzem nutrientes disponíveis para seu hospedeiro. Esses sistemas de fungicultura também abrigam microbiomas bacterianos que apresentam importantes impactos fisiológicos na biologia do inseto fungicultor. Nesse trabalho, foram explorados os padrões de convergência funcional da microbiota associada a sistemas de fungicultura. Com o intuito de expandir a distribuição geográfica de microbiomas associados a sistemas de fungicultura, metagenomas de comunidades bacterianas de Mycocepurus goeldii (Attini basal) e Atta sexdens rubropilosa (Attini derivada, também denominada formiga-cortadeira de folhas) foram sequenciados e anotados. Tais amostras constituem os primeiros microbiomas de formigas Attini da América do Sul. Os gêneros Pseudomonas, Pantoea, Rhizobium, Enterobacter, Achromobacter, Stenotrophomonas e Serratia foram os mais abundantes na comunidade bacteriana associada ao jardim de fungo de A. sexdens rubropilosa. Pseudomonas também foi o gênero encontrado em maior abundância na comunidade bacteriana de M. goeldii, seguido de Dysgonomonas, Bacteroides, Parabacteroides, Prevotella, Comamonas e Burkholderia. A fim de explorar o perfil funcional da comunidade bacteriana, foram realizadas comparações entre microbiomas de Attini basais e derivadas; de insetos fungicultores; de insetos fungicultores e trato intestinal de insetos cuja alimentação é baseada em lignocelulose. As análises comparativas revelaram que os microbiomas associados a insetos fungicultores apresentam uma evidente convergência funcional e taxonômica. É possível verificar a existência de similaridades funcionais entre microbiomas de insetos fungicultores e do trato intestinal de inseto ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Fungus-growing insects (ants, termites, and beetles) independently evolved a symbiotic association with fungi that metabolize recalcitrant plant biomass, producing nutrients available to the insect host. These fungicultural systems also harbor bacterial microbiota of important physiological impacts for the host life style. Here, we explore convergence patterns of the microbiota associated with fungiculture systems. For expanding the geographic distribution of microbiomes fungiculture systems available, we sequenced and annotated metagenomes of bacterial communities from Mycocepurus goeldii (lower Attini ant) and Atta sexdens rubropilosa (higher Attini, a leaf-cutter ant), the first attine ants' microbiomes from South America. Pseudomonas, Pantoea, Rhizobium, Enterobacter, Achromobacter, Stenotrophomonas and Serratia were the most abundant genera in the bacterial community of A. sexdens rubropilosa fungus garden. Similarly, Pseudomonas was also the most abundant genus in the bacterial community of M. goeldii fungus garden, followed by Dysgonomonas, Bacteroides, Parabacteroides, Prevotella, Comamonas and Burkholderia. For metabolic profiling, these microbiomes were included in comparisons of several levels: between lower and higher attines, among fungus-growing insects, and between fungus-growing and non-fungus-growing insects. Comparative analysis of fungus-growing insects associated microbiomes support remarkable functional and taxonomic similarities, pointing to convergence in bacterial communities. Metabolic parallels may be found among microbiomes from fungus-growing insects and other lignocellulose-feeding insects, particularly for pathways involved with the metabolism of carbohydrates, amino acids, aromatic compounds, cofactors and vitamins. However, there are substantial taxonomic differences between microbiomes from fungiculture systems and ... (Complete abstract click electronic access below)
Mestre

Resumo: Fungos do grupo das leveduras negras têm revelado grande potencial em degradar compostos aromáticos como únicas fontes de carbono, capacidade de sobreviver sob atmosferas contendo hidrocarbonetos e habitar nichos com baixa concentração de nutrientes. Apesar do potencial de aplicação destes nas áreas ambiental e industrial, sua ecologia é pouco conhecida e seu isolamento na natureza ainda é difícil. A técnica de flotação em óleo (IWATSU et al., 1981) é aplicada com sucesso para recuperação de leveduras negras provenientes da natureza. No entanto, os mecanismos envolvidos neste método são desconhecidos. Assim, neste trabalho, testaram-se três hipóteses de seleção da técnica: hidrofobicidade celular, assimilação de hidrocarbonetos e oligotrofismo. A metodologia foi aplicada em amostras de solo de "landfarming" da Refinaria do Planalto Paulista (REPLAN). Cepas isoladas e padrão foram submetidas aos testes para verificação das hipóteses formuladas. A hidrofobicidade celular das cepas foi verificada mediante quantificação da concentração celular da suspensão na fase oleosa após agitação com hidrocarboneto. Os testes de assimilação de hidrocarbonetos e oligotrofismo foram realizados mediante análises de curvas de crescimento obtidas por leituras de absorbância. Os resultados obtidos mostraram que o solo de "landfarming" é um local propício para o estudo de leveduras negras, uma vez que 107 cepas foram recuperadas e a análise molecular revelou uma espécie nova: Cladophialophora immunda. O teste de hidrofobicidade indicou que, a hidrofobicidade celular pode não ser o principal fator seletivo, pois espécies hidrofóbicas e hidrofílicas foram recuperadas pela técnica em estudo. A maioria das cepas de leveduras negras testadas apresentou crescimento em meio contendo hidrocarbonetos como fontes únicas de carbono,...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Black yeast fungi have shown great potential to degrade aromatic hydrocarbons as single carbon source, ability to survive under atmospheres with hydrocarbons and to live in niches poor in nutrient. Despite the application potential of these fungi in the environmental and industrial areas, the knowledge about their ecology is incomplete and their isolation from nature is still difficult. The oil flotation technique (IWATSU et al., 1981) has been succeed on the black yeast strains recovery from nature, however, mechanisms involved in this method are still unknown. The present study tests three hypothesis on its selective mechanisms: cell surface hydrophobicity, hydrocarbon assimilation and oligotrophism. The methodology was applied in landfarming soil samples from Planalto Paulísta Refinery (REPLAN). Six isolates and control strains were submitted to experiments to verify the hypothesis done. Hydrophobicity was studied through quantification of oil-phase cell concentration after shaking the cell suspension with hydrocarbon. Hydrocarbon assimilation and oligotrophism were verified by analyzing the growth curves obtained with absorbance data. The results showed that landfarming soil has great potential for black yeast isolation, as 107 strains were recovered and a new species - Cladophialophora immunda- was revealed by molecular analyzes. Hydrophobicity test showed that cell hydrophobicity could not be the main selective factor, since hydrophobic and hydrophillic strains were recovered. Growth in media with hydrocarbons as sole carbon source was observed in the majority of the tested black yeast strains, indicating that hydrocarbon assimilation or tolerance to these substances could be the selective factors of the method. Exophiala xenobiotica was able to grow in the poor media tested, what suggests that this substrate with the addition of hydrocarbons could be used for the isolation...(Complete abstract, click electronic access below)
Orientador: Derlene Attili de Angelis
Coorientador: Dejanira de Francechi de Angelis
Banca: Rosely Ana Piccolo Grandi
Banca: Fernando Carlos Pagnocca
Mestre

Orientador: Andre Rodrigues
Coorientador: Fernando Carlos Pagnocca
Banca: Derlene Attili de Angelis
Banca: Vania Aparecida Vicente
Resumo: Os fungos negros são conhecidos por apresentarem melanina na parede celular, a qual proporciona resistência a diversos estresses ambientais. Nos últimos anos, foram descobertas várias associações entre formigas e fungos negros da ordem Chaetothyriales. No caso das formigas cultivadoras de fungos (atíneas), tais associações permanecem pouco exploradas. Fungos próximos ao gênero Cyphellophora (ex-Phialophora), encontrados no tegumento de formigas do gênero Apterostigma (uma atínea basal), inibem as actinobactérias simbiontes presentes no mesmo local, sendo considerados nocivos para as colônias dessas formigas. Por outro lado, espécies de fungos negros da mesma ordem foram encontradas em baixa abundância no tegumento de alados de Atta (uma atínea derivada). Nesse contexto, pouco se sabe sobre a abundância, diversidade e relação ecológica desses fungos em outros gêneros de atíneas derivadas. Este trabalho teve como objetivo descrever a presença e relação ecológica dos fungos negros no tegumento desses insetos. Utilizando dois métodos dependentes de cultivo, avaliamos a diversidade de fungos negros do tegumento de Acromyrmex coronatus (cortadeira de folhas) e Trachymyrmex tucumanus (não-cortadeira de folhas). Após o isolamento, os fungos foram purificados e identificados com base no sequenciamento da região ITS e um fragmento do gene tef1. Em seguida, foram realizados testes de co-cultivo in vitro entre quatro actinobactérias frente aos fungos negros obtidos das mesmas espécies de... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Black fungi are known for the presence of melanin in their cell walls, which provides resistance to different types of environmental stresses. Recently, many associations between ants and black fungi in the order Chaetothyriales have been discovered. When it comes to fungus-growing ants (the "attines"), these associations remain underexplored. Fungi related to the genus Cyphellophora (ex-Phialophora), found on the integument of Apterostigma ants (lower attines), inhibit the symbiotic actinobacteria also present on the ant integument, and for that are considered detrimental for the ant colonies. However, black fungi species belonging to the same order were found in low abundance on the integument of gynes and drones of Atta (higher attines). In this context, little is known about the abundance, diversity and ecological relation of these fungi in other genera of derived attines. The aim of this study was to describe the presence and ecological relation of black fungi on the integument of these insects. By applying two different culture dependent methods, we evaluated the diversity of black fungi on the integument of Acromyrmex coronatus (leafcutter) and Trachymyrmex tucumanus (non-leafcutter). After isolation, fungi were purified and identified based on ITS region and partial tef1 gene sequences. Next, we put together in vitro co-culture assays between four actinobacteria and black fungi obtained from the same ant species. We found 111 black fungi in both species of ants, inclu... (Complete abstract click electronic access below)
Mestre

Orientadora: Valéria Marta Gomes do Nascimento
Banca: Jonas Contiero
Banca: Cintia Duarte de Freitas Milagre
Resumo: A riqueza nutricional da biomassa de Lasiosiplodia theobromae, resíduo do bioprocesso que tem como finalidade a produção de exopolissacarídeo (EPS), foi estudada neste trabalho, bem como a produção de EPS pelo mesmo fungo, em cultivos suplementados com esse biomaterial. Inicialmente, o microrganismo foi cultivado em condição padrão (sacarose 50 g/L, sais mínimos de Vogel (SMV) e nitrato de amônio 2 g/L, 28 °C, 72 h), e o EPS e a biomassa (Biomassa A), provenientes deste cultivo, foram separados e quantificados por gravimetria. Parte da biomassa foi lavada, por duas vezes, com solução de NaCl 0,15 mol/L, autoclavada, liofilizada, tamisada e analisada quanto à composição bromatológica (Weende e Kjeldahl) e de minerais (Absorção Atômica), e utilizada como suplemento aos meios de cultivo. A biomassa A apresentou 332 g/kg de C e 51 g/kg de N, além de sais minerais, como Zn, Fe, Cu, Mn, K, Mg, Ca, S e P. Para avaliar a adição de biomassa em meio de cultivo para produção de EPS, foram realizados 18 experimentos, com 6 diferentes composições (meios BSVN, BSV, BVN, BV, BN e B), identificados de acordo com a composição de nutrientes: Biomassa A 20, 40 ou 60 g/L (B), sacarose 50 g/L (S), SMV (V) e nitrato de amônio 2 g/L (N). Houve produção de EPS em 16 experimentos, sendo que em apenas três condições experimentais a produção de EPS foi inferior à condição padrão de cultivo. Destaque para os resultados obtidos em meios BV, compostos por biomassa 40 e 60 g/L e SMV, sendo 3,22 e 3,99 g/L de EPS, respectivamente, enquanto que na condição padrão de cultivo, houve produção de 1,3 g/L de EPS. A biomassa resultante do primeiro cultivo com adição de Biomassa A, identificada como Biomassa B (Biomassa A residual + biomassa de novo crescimento microbiano), também foi avaliada quanto à composição bromatológica e ... (Resumo completo clicar acesso eletrônico abaixo)
Abstract: The nutritional value of Lasiodiplodia theobromae' biomass, waste of the bioprocess used for the production of exopolysaccharide (EPS) was studied in this work, as well as the production of EPS by the same fungus when grown in cultures supplemented with this biomaterial. Initially the microorganism was cultured in standard conditions (Sucrose 50 g/L, Vogel minimal salts (SMV) and ammonium nitrate 2 g/L, 28 °C, 72 h) and the EPS and the biomass (A Biomass) from this culture were separated and quantified by gravimetry. Part of the biomass was rinsed twice with NaCl 0.15 mol/L solution, autoclaved, lyophilized, sieved and analyzed for chemical composition (Weende and Kjeldahl) and minerals (Atomic Absorption) and used as a supplement to culture medium. Biomass A presented 332 g/kg C and 51 g/kg of N, and minerals such as Zn, Fe, Cu, Mn, Mg, Ca, S and P. In order to evaluate the addition of biomass in the culture medium for EPS production, 18 experiments were conducted with six different compositions (BSVN, BSV, BVN, BV, BN and B growth mediums), identified accordingly to the nutritional components: A Biomass 20, 40 or 60 g/L (B), Sucrose 50 g/L (S), SMV (V) and ammonium nitrate 2 g/L (N). EPS production occurred in 16 experiments, and in only three experimental conditions EPS production was lower than on the standard cultivation condition. Emphasis to the results obtained in BV medium, composed by biomass 40 and 60 g / L and SMV, being 3.22 and 3.99 g/L EPS, respectively, while the standard conditions of cultivation, production occurred of 1.3 g/L EPS. The biomass resulting this first cultivation with addition of A Biomass, identified as B Biomass (A Biomass waste + the biomass new microbial growth) was also evaluated for their chemical and mineral composition. As result 414 g/kg C and 46 g/kg N. Cultivations on BV condition with ... (Complete abstract click electronic access below)
Mestre

Cavity-excavating birds, such as woodpeckers, are ecosystem engineers and are often assumed to rely upon wood decay fungi to assist in softening the wood of potential excavation sites. Endangered red-cockaded woodpeckers (Picoides borealis; RCWs) are the only birds known to solely excavate through the sapwood and into the heartwood of living pine trees and generally take many years to complete their excavations. These birds may have developed a partnership with wood-inhabiting fungi to facilitate the excavation process. Past attempts to understand the complex relationships between cavity excavators and fungi relied on visual surveys of fruiting bodies, or evidence of decay, resulting in a one bird, one fungus paradigm. Using molecular methods, I investigated the relationships between RCWs and fungi, and found that the relationships between cavity-excavators and fungi involve multiple fungal species and are far more complex than previously imagined. Through a field survey, I showed that RCW excavations contain distinct communities of fungi, and propose two hypotheses to explain this result, (1) RCWs select trees with distinct fungal communities (tree selection hypothesis), or (2) RCWs promote distinct fungal communities via their excavations (bird facilitation hypothesis). By swabbing the birds, I found that RCWs carry fungal communities similar to those found in their completed excavations, demonstrating that RCWs may directly facilitate fungal dispersal during the excavation process. Through a test of the bird introduction hypothesis which implemented human-made experimental drilled cavity starts (incomplete excavations), half of which were inaccessible to the birds, I showed that RCW accessibility influences fungal community development in excavations. This experimental evidence demonstrates that the relationship between RCWs and fungal communities is a multipartite symbiosis may be mutualistic. Finally, by tracking fungal community development in experimental cavity starts through time, I also demonstrated that the fungal communities found in RCW excavations undergo succession, and that this process is influenced by the birds. The relationships described in this body of work provide the basis for future studies on cavity excavators and fungi, and also have implications for a diverse community of secondary cavity nesters, wood-inhabiting fungi, forest ecology, and the conservation of biodiversity.
Ph. D.

Arbuscular mycorrhizal fungi (AMF) represent an ancient and critical symbiotic partner with the majority of land plants, understood to promote ecosystem productivity and biodiversity and are also important to ecologically sound land management practices. The fungus is thought to be over 400 million years old, and due to a lack of an observable sexual cycle, has been placed into a select group of eukaryotes called 'ancient asexuals', which seemingly defy evolutionary theory by persisting for an extended period of time in the absence of sexual reproduction. Recently however, molecular evidence has accumulated which may suggest AMF harbour a cryptic sexual cycle. In the first chapter of this thesis, entitled "Searching for clues of sexual reproduction in the genomes of arbuscular mycorrhizal fungi", I review evidence supporting this notion of a cryptic sexual cycle in AMF which includes: the presence of recombinational events, meiosis-specific genes as well as mating-specific transcription factors called SexM and SexP of the MATA_HMG protein family which are otherwise found only in the genomes of sexual fungi. In the second chapter, I present the main research of my MSc work where I used bioinformatic, population genetic, molecular and experimental approaches to build on this existing evidence of sexuality in AMF. These findings include the presence of a dramatically expanded family of MAT-HMG genes which are present in several isolates of the Rhizophagus irregularis and also harbour significant allele variation amongst these isolates, some of which resembles variation expected at MAT-genes in other fungi. Q-RT-PCR procedures revealed that at least some of these genes tend to increase in expression during crosses of R. irregularis isolates. We also uncovered the presence of a unique genomic region where at least three of these genes are located in tandem. Finally, several tests of recombination support the presence of intraisolate as well as interisolate recombination events occurring between these MAT-HMG genes.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A introdução de moléculas xenobióticas no ambiente muitas vezes ocorre sem que sejam conhecidos muitos de seus aspectos bioquímicos e toxicológicos fundamentais. A presença anéis aromáticos na estrutura molecular muitas vezes é o fator determinante da toxicidade, recalcitrância e propriedades mutagênicas associadas e muitos destes compostos. O papel dos insumos agrícolas neste processo é de particular relevância, devido ao seu caráter de liberação intencional no ambiente e dos crescentes volumes aplicados em todo o mundo. Neste trabalho, foram selecionadas linhagens de basidiomicetos com base na sua tolerância aos herbicidas atrazina e diurom, para estudo detalhado do potencial de degradação e do papel desempenhado pelas enzimas ligninolíticas no processo. A tolerância não se mostrou relacionada à degradação dos xenobióticos; esta foi muito eficiente para algumas linhagens estudadas, chegando a 38% da atrazina e 96% do diurom, por MCA 17 agaricales e SXS 320 P. cubensis, respectivamente, após 20 dias de cultivo. As linhagens mais tolerantes à atrazina, Pluteus cubensis SXS 320 e Polyporus tenuiculus MCA 11, e ao diurom, Pycnoporus sanguineus MCA 16 e Dacryopinax elegans SXS 323, foram empregadas em ensaios mais detalhados para a degradação e produção de enzimas. Os efeitos de diferentes concentrações do diurom e da atrazina como única fonte de carbono ou em presença de fontes alternativas como glicose ou bagaço de cana (1%), foram determinados para os microrganismos tolerantes. Apenas Pycnoporus sanguineus MCA 16 e Polyporus tenuiculus MCA 11 produziram lacases, única enzima do sistema ligninolítico detectada nas culturas. No caso de Pycnoporus sanguineus, as lacases foram constitutivamente produzidas em meio contendo glicose, atingindo 283 U.l-1. A lacase de P. tenuiculus, por sua vez, foi produzida apenas em presença de constituintes...
The introduction of xenobiotic molecules in the environment often occurs without the knowledge about many basic aspects related to biochemistry and toxicology. The aromatic ring presence in their molecular structure many times is the determinant feature for toxicity, recalcitrance and mutagenic properties associated with those compounds. The role of agricultural chemicals in this process has particular relevance, due to their intentional release on environment and the crescent volumes employed worldwide. In this work, basidiomycete strains were chosen based on their tolerance to herbicides atrazine and diuron, for detailed study of the fungi’s degradative potential and the role developed by ligninolytic enzymes in this process. The tolerance was not related to xenobiotic’s degradation, which was very efficient for many strains, reaching 38% for atrazine and 96% for diuron, by MCA 17 Agaricales and SXS 320 Pluteus cubensis, respectively, after 20 days in culture. Those ones more tolerant to atrazine, Pluteus cubensis SXS 320 and Polyporus tenuiculus MCA 11, and for diuron, Pycnoporus sanguineus MCA 16 and Dacryopinax elegans SXS 323, were employed in assays focusing on degradation and enzyme production. The effect of different concentrations of diuron and atrazine as sole carbon source or in presence of alternative sources, like glucose and sugarcane bagasse (1%), were determined for tolerant microorganisms. Only Pycnoporus sanguineus MCA 16 and Polyporus tenuiculus MCA 11 have produced laccases, the unique enzyme of ligninolytic system detected in cultures. For Pycnoporus sanguineus, the laccases were constitutively produced in medium containing glucose, reaching 238 U.l-1. The laccase from P. tenuiculus was released only with the presence of lignocellulosic constituents in culture medium, resulting in 1,219 U.l-1 in medium containing wheat bran. The biochemical properties... (Complete abstract click electronic access below)

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Universidade Estadual Paulista (UNESP)
Neste trabalho o crescimento micelial, os aspectos morfológicos e o potencial para catálise enzimática foram avaliados em relação ao fungo termofílico Thermomyces lanuginosus, quando inoculado em meios de cultura YpSs (extrato de amido + água + Agar), com fonte de carbono substituída por matérias-primas encontradas na indústria química, como intermediários clássicos, na síntese de diversos ésteres. O monitoramento do crescimento do microrganismo envolvendo parâmetros tais como aspecto das hifas, quantidade de conídios e conidióforos, produção de pigmentos, pH, além das condições de imobilização do fungo, serviram para a averiguação da capacidade do microrganismo em expressar uma eventual condição de transformação de reagentes (di-álcoois e ácidos graxos) em produtos de síntese (ésteres). Os pellets contendo micélio de Thermomyces lanuginosus em erlenmeyers com as matérias-primas acima citadas, permaneceram em estufa microbiológica durante 15 dias, sendo, então, averiguadas as transformações decorrentes dos diferentes meios, evidenciando o consumo dos reagentes. Para a caracterização do material, foram realizadas medidas de absorção no infravermelho com transformada de Fourier (FTIR) efetuadas com o próprio micélio e meios de cultura, microscopia ótica (MO) de micro cultivo do fungo nos diversos meios de cultura propostos, e avaliações com cromatografia gasosa (GC). Os resultados obtidos, acrescidos ao crescimento de Thermomyces lanuginosus em todos os meios propostos, bem como a manutenção da sua capacidade de reprodução, mostram ser esse fungo termofílico altamente promissor em futuros trabalhos de biocatálise com microrganismos vivos. A capacidade demonstrada de catalisar reações de esterificação e a adaptação deste fungo as diversas fontes de carbono a que foi exposto, sugerem...
In this work, the mycelial growth, the morphologic aspects and the potential for enzymatic catalysis were evaluated considering the thermophilic fungus Thermomyces lanuginosus, when inoculated in YpSs media (starch extract + water + Agar), with its carbon source substituted by different raw materials normally found in the chemical industry, as classics intermediates used in diverse ester synthesis. The continuous observation of the microorganism growth involving parameters as hyphae aspect, amount of conidia and conidiophores, pigment production, pH and the fungus immobilization conditions, avoid the ascertainment of the fungus capacity to show an eventual condition of transform reagents (dialcohol and fatty acids) in synthesis products (esters). Erlenmeyers with the mentioned raw-materials and pellets containing Thermomyces lanuginosus mycelium, were allocated in microbiological oven for 15 days and transformations due to the different media were studied and interpreted showing the reagents consumption. Measures of absorption in the transformed infra-red ray with transformed of Fourier (FTIR) of the fungus mycelium and the media, optical microscopy (MO) of the fungus micro-cultive in several media, and evaluations with gas chromatography (GC) were evaluated for the material characterization. The obtained results, added to the growth of Thermomyces lanuginosus in all the considered media, as well as the maintenance of its capacity of reproduction, aimed to understand and evaluate that Thermomyces lanuginosus is promising microorganism alternative for immobilized biocatalysts. The demonstrated capacity to catalyze esterifications reactions and the adaptation of this fungus to different carbon sources, suggest several future works aiming the optimization of ester production, or even though, the use of its capacity in promoting such reactions when sterical hindrances already are significant.

Orientador: André Rodrigues
Coorientador: Priscila Cintra Socolowski
Banca: Mauricio Bacci Junior
Banca: Dartanhã José Soares
Resumo: Em interações parasita-hospedeiro é esperado que o parasita obtenha a maior parte de sua nutrição a partir do hospedeiro. Colônias de formigas atíneas podem ser utilizadas como modelo para estudar diferentes mecanismos envolvidos em interações de parasitismo. Esses insetos cultivam um fungo basidiomiceto, o qual é alvo de micoparasitas do gênero Escovopsis. É sabido que o jardim de fungo, local onde as formigas praticam a fungicultura, apresenta uma alta concentração de fontes de carbono simples, de fácil assimilação. No presente estudo investigamos se o parasita se beneficia desses recursos nutricionais e se esses influenciam na infectividade frente ao hospedeiro. Para tanto, realizamos ensaios para avaliar o desenvolvimento de seis isolados de Escovopsis em diferentes fontes de carbono tanto na ausência, quanto na presença do fungo cultivado pelas formigas. Além disso, observamos a interação Escovopsis - fungo cultivado com microscopia de epifluorescência, a fim de verificar em que momento é iniciada a degradação do micélio do hospedeiro. No geral, o parasita apresentou esporulação abaixo de 59 % quando somente uma fonte simples de carbono estava disponível. Quando interagindo com seus hospedeiros em meio com apenas uma ou duas fontes de carbono, três dentre seis isolados do parasita foram menos infectivos do que o esperado. Observamos que a adição de uma fonte nutricional não aumenta a infectividade do parasita, mas influencia seu crescimento, mesmo na presença do hospedei... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: In parasite-host interactions it is expected from the former to rely mostly on nutrients derived from the host. Attine ant colonies may be considered as a model system for studying different mechanisms of parasitism. These insects cultivate a basidiomycetous fungus, which is threatened by mycoparasites in the genus Escovopsis. It is known that the fungus garden, the substrate where the ants cultivate the fungal cultivar, has a high concentration of simple carbon sources. Here we investigated whether the parasite benefits from these nutritional resources and whether these nutrients influence host infectivity. We performed bioassays to evaluate the development of six Escovopsis isolates in different carbon sources in the absence as well as in the presence of the ant fungal cultivar. In addition, we observed the interaction Escovopsis - fungal cultivar with epifluorescence microscopy, to assess when the degradation of the host mycelium begins. Overall, the parasite showed sporulation below 59 % when only one carbon source was available. When interacting with their hosts in culture medium with one or two carbon sources, three out of six Escovopsis isolates were less infective than expected, probably due to nutritional stress. We observed that the parasite's infectivity did not increase with the addition of one nutritional source, however, influenced its growth, even in the presence of the host (the expected primary source of nutrition). Our results suggest that in addition to the... (Complete abstract click electronic access below)
Mestre

A despolimerização enzimática de componentes de biomassa é um processo chave para indústrias de bioenergia. A busca por novas enzimas aumentou os estudos dos secretomas de microrganismos degradadores de biomassa. Os fungos filamentosos são importantes agentes na reciclagem de carbono na natureza, apresentando potenciais enzimas para a conversão de biomassa. Malbranchea pulchella é um ascomiceto termofílico bioprospectado na Floresta Atlântica (região de Minas Gerais-Brasil), cuja análise e caracterização do secretoma, juntamente com a capacidade de biodegradação, foram foco deste estudo. O cultivo semi-sólido deste fungo em substratos lignificado (madeira de eucalipto moída) e \"não lignificado\" (polpa Kraft branqueada) por 60 dias resultou em perdas de peso de cerca de 5% e 52%, respectivamente. Para o estudo da diversidade das enzimas produzidas por M. pulchella, o secretoma foi avaliado em culturas submersas de 3 dias, utilizando as fontes de carbono anteriormente mencionadas. Os resultados de LC-MS/MS mostraram que o fungo foi capaz de secretar uma série de enzimas lignocelulolíticas. As enzimas proeminentes foram as glicosil-hidrolases com um total de 29 e 37 CAZymes nos substratos lignificado e \"não-lignificado\", respectivamente, sendo as famílias GH3 e GH18 encontradas em maior número. Considerando as enzimas oxidativas, um total de 12 e 13 CAZymes nos substratos lignificado e \"não lignificado\", respectivamente, sendo as famílias AA7 e AA9 as mais predominantes. Também foram detectadas proteínas não-CAZymes apresentando domínios alergênicos, que estão relacionados com proteínas semelhantes a expansinas de plantas. O secretoma produzido com substrato de polpa branqueada foi utilizado em ensaio de hidrólise com polpa Kraft e também em contribuição com Celluclast 1,5 L para hidrólise de polpa de dissolução e de Avicel. Os resultados indicaram que M. pulchella produz uma maquinaria enzimática interessante, que pode degradar celulose amorfa e cristalina, sendo potencial aplicação em hidrólise da biomassa lignocelulósica.
Enzymatic depolymerization of biomass components is a key process for bioenergy industries. The search for new enzymes for this task has increased secretome studies in biomass-degrading microorganisms. Filamentous fungi are important players in carbon recycling in nature, presenting potential enzymes for biomass conversion. Malbranchea pulchella is a thermophilic ascomycete bio prospected in Brazilian Atlantic forest whose secretome analysis and characterization together with biodegradation ability were focus of this study. Semi-solid cultivation of this fungus on lignified (milled eucalyptus wood) and non-lignified (bleached eucalyptus pulp) substrates for 60 days resulted in almost 5% and 52% weight losses, respectively. To understand the diversity of enzymes produced by M. pulchella the secretome was evaluated in 3-day submerse cultures using the previously mentioned carbon sources. LC-MS/MS results showed the fungus was able to secrete an array of lignocellulolytic enzymes. The prominent enzymes were glycosyl hydrolases with a total of 29 and 37 CAZymes in lignified and non-lignified substrates, respectively, with GH3 and GH18 families found in greatest number. Considering the oxidative enzymes, a total of 12 and 13 CAZymes were found in lignified and non-lignified substrates, respectively, being AA7 and AA9 families most expressive. Non-CAZymes proteins showing allergen domains, which are related with expansin-like proteins from plants, were also detected. The secretome produced with bleached eucalyptus pulp substrate was assayed for hydrolysis of Kraft pulp and in contribution with Celluclast 1.5 L for dissolving pulp and Avicel hydrolysis as well. Results indicated that M. pulchella produce an interesting enzymatic array that can disrupt both amorphous and crystalline cellulose, demonstred as potencial secretome for biomass hydrolysis.