Toxigenic profile of fungi and multi mycotoxins analysis as supporting tools for a risk evaluation and mycotoxins minimization/degradation

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)

Aflatoxin B1 and M1 Degradation by Lac2 from Pleurotus pulmonarius and Redox Mediators

Laccases (LCs) are multicopper oxidases that find application as versatile biocatalysts for the green bioremediation of environmental pollutants and xenobiotics. In this study we elucidate the degrading activity of Lac2 pure enzyme form Pleurotus pulmonarius towards aflatoxin B1 (AFB1) and M1 (AFM1). LC enzyme was purified using three chromatographic steps and identified as Lac2 through zymogram and LC-MS/MS. The degradation assays were performed in vitro at 25 °C for 72 h in buffer solution. AFB1 degradation by Lac2 direct oxidation was 23%. Toxin degradation was also investigated in the presence of three redox mediators, (2,2′-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid]) (ABTS) and two naturally-occurring phenols, acetosyringone (AS) and syringaldehyde (SA). The direct effect of the enzyme and the mediated action of Lac2 with redox mediators univocally proved the correlation between Lac2 activity and aflatoxins degradation. The degradation of AFB1 was enhanced by the addition of all mediators at 10 mM, with AS being the most effective (90% of degradation). AFM1 was completely degraded by Lac2 with all mediators at 10 mM. The novelty of this study relies on the identification of a pure enzyme as capable of degrading AFB1 and, for the first time, AFM1, and on the evidence that the mechanism of an effective degradation occurs via the mediation of natural phenolic compounds. These results opened new perspective for Lac2 application in the food and feed supply chains as a biotransforming agent of AFB1 and AFM1

Bioremediation of aflatoxin B1-contaminated maize by king oyster mushroom (Pleurotus eryngii).

Aflatoxin B1 (AFB1) is the most harmful mycotoxin that occurs as natural contaminant of agricultural commodities, particularly maize. Practical solutions for detoxification of contaminated staples and reduction of agricultural wastes are scarce. We investigated the capability of the white-rot and edible fungus Plerotus eryngii (king oyster mushroom) to degrade AFB1 both in vitro and in a laboratory-scale mushroom cultivation, using a substrate similar to that routinely used in mushroom farms. In malt extract broth, degradation of AFB1 (500 ng/mL) by nine isolates of P. eryngii ranged from 81 to 99% after 10 days growth, and reached 100% for all isolates after 30 days. The growth of P. eryngii on solid medium (malt extract-agar, MEA) was significantly reduced at concentrations of AFB1 500 ng/mL or higher. However, the addition of 5% wheat straw to the culture medium increased the tolerance of P. eryngii to AFB1 and no inhibition was observed at a AFB1 content of 500 ng/mL; degradation of AFB1 in MEA supplemented with 5% wheat straw and 2.5% (w/v) maize flour was 71-94% after 30 days of growth. Further, AFB1 degradation by P. eryngii strain ITEM 13681 was tested in a laboratory-scale mushroom cultivation. The mushroom growth medium contained 25% (w/w) of maize spiked with AFB1 to the final content of 128 μg/kg. Pleurotus eryngii degraded up to 86% of the AFB1 in 28 days, with no significant reduction of either biological efficiency or mushroom yield. Neither the biomass produced on the mushroom substrate nor the mature basidiocarps contained detectable levels of AFB1 or its metabolite aflatoxicol, thus ruling out the translocation of these toxins through the fungal thallus. These findings make a contribution towards the development of a novel technology for remediation of AFB1- contaminated corn through the exploitation of the degradative capability of P. eryngii and its bioconversion into high nutritional value material intended for feed production

Obesity-Related Chronic Kidney Disease: Principal Mechanisms and New Approaches in Nutritional Management

Obesity is the epidemic of our era and its incidence is supposed to increase by more than 30% by 2030. It is commonly defined as a chronic and metabolic disease with an excessive accumulation of body fat in relation to fat-free mass, both in terms of quantity and distribution at specific points on the body. The effects of obesity have an important impact on different clinical areas, particularly endocrinology, cardiology, and nephrology. Indeed, increased rates of obesity have been associated with increased risk of cardiovascular disease (CVD), cancer, type 2 diabetes (T2D), dyslipidemia, hypertension, renal diseases, and neurocognitive impairment. Obesity-related chronic kidney disease (CKD) has been ascribed to intrarenal fat accumulation along the proximal tubule, glomeruli, renal sinus, and around the kidney capsule, and to hemodynamic changes with hyperfiltration, albuminuria, and impaired glomerular filtration rate. In addition, hypertension, dyslipidemia, and diabetes, which arise as a consequence of overweight, contribute to amplifying renal dysfunction in both the native and transplanted kidney. Overall, several mechanisms are closely related to the onset and progression of CKD in the general population, including changes in renal hemodynamics, neurohumoral pathways, renal adiposity, local and systemic inflammation, dysbiosis of microbiota, insulin resistance, and fibrotic process. Unfortunately, there are no clinical practice guidelines for the management of patients with obesity-related CKD. Therefore, dietary management is based on the clinical practice guidelines for the nutritional care of adults with CKD, developed and published by the National Kidney Foundation, Kidney Disease Outcome Quality Initiative and common recommendations for the healthy population. Optimal nutritional management of these patients should follow the guidelines of the Mediterranean diet, which is known to be associated with a lower incidence of CVD and beneficial effects on chronic diseases such as diabetes, obesity, and cognitive health. Mediterranean-style diets are often unsuccessful in promoting efficient weight loss, especially in patients with altered glucose metabolism. For this purpose, this review also discusses the use of non-classical weight loss approaches in CKD, including intermittent fasting and ketogenic diet to contrast the onset and progression of obesity-related CKD

Deoxynivalenol and T-2 Toxin as Major Concerns in Durum Wheat from Italy

Fusarium Head Blight is a devastating disease of wheat caused by a complex of Fusarium species producing a wide range of mycotoxins. Fusarium species occurrence is variable in different geographical areas and subjected to a continuous evolution in their distribution. A total of 141 durum wheat field samples were collected in different regions of Italy in three years, and analyzed for Fusarium species and related mycotoxin occurrence. Mycotoxin contamination varied according to year and geographical origin. The highest mycotoxin contamination was detected in 2014. Deoxynivalenol was detected with an average of 240 µg/kg only in Central and Northern Italy; and T-2 and HT-2 toxins with an average of 150 µg/kg in Southern Italy. Approximately 80% of samples from Southern Italy in 2013/2014 showed T-2 and HT-2 levels over the EU recommended limits. Fusarium graminearum occurred mostly in Northern Italy, while F. langsethiae occurred in Southern Italy. These data showed that a real mycotoxin risk related to Fusarium exists on the whole in Italy, but varies according with geographical areas and environmental conditions. Consistent monitoring of Fusarium species and related mycotoxin distribution on a long period is worthwhile to generate more accurate knowledge on Fusarium species profile and mycotoxins associated and better establish the climatic change impact on wheat Fusarium epidemiology

Determination of AFOL in the biomass of <i>P</i>. <i>eryngii</i>.

<p>Overlay of HPLC/FLD chromatograms of a standard solution of aflatoxicol (AFOL, black line) and the extract of <i>P</i>. <i>eryngii</i> biomass developed on the contaminated mushroom substrate (red line). AFOL was not present in the extract.</p

Inhibitory effect of AFB₁ on growth of <i>P</i>. <i>eryngii</i>.

<p>The isolates ITEM 13681, ITEM 13688 and ITEM 13697 were grown for 15 days at 30 ± 1°C in the dark on malt extract agar (MEA) containing different concentrations (60, 120, 250, 500, 1000 ng/mL) of AFB₁. Data are the means ± SD (n = 5) of the percent reduction in colony diameters with respect to control. Statistically significant differences with control are indicated by asterisks (*** = <i>P</i> < 0.001, ** = <i>P</i><0.01; One-way Anova).</p

<i>Pleurotus eryngii</i> cultivated in a AFB₁-contaminated mushroom medium.

<p>Production of basidiocarps (fruit bodies) by <i>P</i>.<i>eryngi</i>i ITEM 13681 in a mushroom cultivation medium; A) medium contaminated with 128 μg/kg of AFB₁; B) non-contaminated control. In both the conditions <i>P</i>. <i>eryngii</i> ITEM 13681 produced well-developed basidiocarps, as well as growing immature fruit-bodies and fruit primordials in 42 days.</p

Degradation of AFB₁ by <i>P</i>.<i>eryngii</i> grown on malt extract-agar plus wheat straw and maize flour (MEASM) supplemented with 500 ng/mL of AFB₁, after 15 and 30 days of incubation at 30 ± 1°C in dark.

<p>Degradation of AFB₁ by <i>P</i>.<i>eryngii</i> grown on malt extract-agar plus wheat straw and maize flour (MEASM) supplemented with 500 ng/mL of AFB₁, after 15 and 30 days of incubation at 30 ± 1°C in dark.</p

Reduction of the inhibitory effect of AFB₁ on <i>P</i>. <i>eryngii</i> in the presence of wheat straw.

<p>The isolates ITEM 13681, ITEM 13688 and ITEM 13697 were grown on malt extract agar (MEA), MEA supplemented with 5% (w/v) wheat straw (MEAS) and MEA supplemented with 5% wheat straw and 2.5% (w/v) maize flour (MEASM) containing 500 and 1000 ng/mL of AFB₁. Data are the means ± SD (n = 5) of the percent reduction in colony diameters with respect to controls after 15 days of growth at at 30 ± 1°C in the dark. Statistically significant differences with control are indicated by asterisks (*** = <i>P</i> < 0.001, ** = <i>P</i><0.01; One-way Anova).</p