Tracking new insights into antifungal and anti-mycotoxigenic properties of a biofilm forming Pediococcus pentosaceus strain isolated from grain silage.

The present study offers detailed insights into the antifungal and anti-mycotoxigenic potential of a biofilm forming lactic acid bacterium (Pediococcus pentosaceus) against one atoxigenic (Aspergillus flavus) and two toxigenic (Aspergillus nomius and Fusarium verticillioides) fungal strains. The antifungal effect of P. pentosaceus LBM18 strain was initially investigated through comparative analysis of fungi physiology by macroscopic visual evaluations and scanning electron microscopy examinations. The effects over fungal growth rate and asexual sporulation were additionally accessed. Furthermore, analytical evaluations of mycotoxin production were carried out by HPLC-MS/MS to provide insights on the bacterial anti-mycotoxigenic activity over fungal production of the aflatoxins B1, B2, G1 and G2 as well as fumonisins B1 and B2. Finally, reverse transcription quantitative real-time PCR (RT-qPCR) analysis was employed at the most effective bacterial inoculant concentration to evaluate, at the molecular level, the down-regulation of genes aflR, aflQ and aflD, related to the biosynthesis of aflatoxins by the strain of Aspergillus nomius. The effects over mycotoxin contamination were thought to be result of a combination of several biotic and abiotic factors, such as interaction between living beings and physical-chemical aspects of the environment, respectively. Several possible mechanisms of action were addressed along with potentially deleterious effects ascribing from P. pentosaceus misuse as biopesticide, emphasizing the importance of evaluating lactic acid bacteria safety in new applications, concentrations, and exposure scenarios.

Prospects for fungal bioremediation of unburied waste packages from the Goiânia radiological accident.

Goiânia, the Goiás State capital, starred in 1987, where one of the largest radiological accidents in the world happened. A teletherapy machine was subtracted from a derelict radiotherapy clinic and disassembled by scavengers who distributed fragments of the 50 TBq 137CsCl source among relatives and acquaintances, enchanted by the blue shine of the substance. During the 15 days before the accident was acknowledged, contaminated recycling materials were delivered to recycling factories in four cities in the state of São Paulo, Brazil, in the form of recycling paper bales. The contaminated bales were spotted, collected, and stored in fifty 1.6 m3 steel boxes at the interim storage facility of the Nuclear and Energy Research Institute (IPEN). In 2017, a check of the content was performed in a few boxes and the presence of high moisture content was observed even though the bales were dry when conditioned and the packages were kept sealed since then. The main objective of this work was to report the fungi found in the radioactive waste after they evolved for 30 years in isolation inside the waste boxes and their role in the decay of the waste. Examination of the microbiome showed the presence of nematodes and fungal communities. The fungi species isolated were Aspergillus quadricinctus, Fusarium oxysporum, Lecanicillium coprophilumi, Scedosporium boydii, Scytalidium lignicola, Xenoacremonium recifei, and Pleurostoma richardsiae. These microorganisms showed a significant capacity to digest cellulose in our trials, which could be one of the ways they survive in such a harsh environment, reducing the volume of radioactive paper waste. These metabolic abilities give us a future perspective of using these fungi in biotechnology to remediate radioactively contaminated materials, particularly cellulose-based waste.

Evaluating the potential of Pediococcus pentosaceus as a biocontrol agent against tenuazonic acid-producing Alternaria alternata on livestock feeds.

AIMS: This study aims to demonstrate the potential of the lactic acid bacteria (LAB) Pediococcus pentosaceus LBM18 against the mycotoxin-producing Alternaria alternata TEF-1A and highlight its application as an effective grain silage inoculant to control mycotoxin contamination. METHODS AND RESULTS: The antifungal properties of Ped. pentosaceus lyophilized (PPL) were assessed by evaluating its effect on A. alternata TEF-1A grown in a corn silage-based medium, which included morphological changes by Scanning Electron Microscopy (SEM) observations, growth rate, conidia production assays, and inhibition of Tenuazonic acid (TeA) production by high-performance liquid chromatography (HPLC-MS/MS) analyses. Furthermore, TeA biosynthesis was monitored for changes at the molecular level by PKS gene expression. The growth and sporulation processes of A. alternata TEF-1A were affected by Ped. pentosaceus LBM18 in a concentration-dependent manner. Moreover, a significant inhibition of TeA production (74.3%) and the transcription level of the PKS gene (42.9%) was observed. CONCLUSIONS: Ped. pentosaceus is one of the promising LAB to be applied as an inoculant for corn silage preservation, aiming to inhibit mycotoxigenic fungi growth and their mycotoxin production. SIGNIFICANCE AND IMPACT OF THE STUDY: Ped. pentosaceus could be used as an inoculant to reduce fungal and mycotoxins contamination in grain silage production.

Bacteriocinogenic probiotic bacteria isolated from an aquatic environment inhibit the growth of food and fish pathogens.

The conditions of aquatic environments have a great influence on the microbiota of several animals, many of which are a potential source of microorganisms of biotechnological interest. In this study, bacterial strains isolated from aquatic environments were bioprospected to determine their probiotic profile and antimicrobial effect against fish and food pathogens. Two isolates, identified via 16S rRNA sequencing as Lactococcus lactis (L1 and L2) and one as Enterococcus faecium 135 (EF), produced a bacteriocin-like antimicrobial substance (BLIS), active against Listeria monocytogenes, Salmonella Choleraesuis and Salmonella Typhimurium. Antimicrobial activity of BLIS was reduced when exposed to high temperatures and proteolytic enzymes (trypsin, pepsin, papain and pancreatin). All strains were sensitive to 7 types of antibiotics (vancomycin, clindamycin, streptomycin, gentamicin, chloramphenicol, rifampicin and ampicillin), exhibited a high rate of adherence to Caco-2 cells and expressed no hemolysin and gelatinase virulence factors. EF showed some resistance at pH 2.5 and 3.0, and L2/EF showed higher resistance to the action of bile salts. Finally, the presence of bacteriocin genes encoding for proteins, including Nisin (L1 and L2), Enterocin A, B, P, and Mundticin KS (EF) was detected. The molecular and physiological evidence suggests that the bacterial isolates in this study could be used as natural antimicrobial agents and may be considered safe for probiotic application.

Talaromyces amestolkiae uses organic phosphate sources for the treatment of uranium-contaminated water.

Fungi have received particular attention in regards to alternatives for bioremediation of heavy metal contaminated locales. Enzymes produced by filamentous fungi, such as phosphatases, can precipitate heavy metal ions in contaminated environments, forming metal phosphates (insoluble). Thus, this research aimed to analyze fungi for uranium biomineralization capacity. For this, Gongronella butleri, Penicillium piscarium, Rhodotorula sinensis and Talaromyces amestolkiae were evaluated. Phytate and glycerol 2-phosphate were used as the phosphate sources in the culture media at pH 3.5 and 5.5, with and without uranium ions. After 4 weeks of fungal growth, evaluated fungi were able to produce high concentrations of phosphates in the media. T. amestolkiae was the best phosphate producer, using phytate as an organic source. During fungal growth, there was no change in pH level of the culture medium. After 3 weeks of T. amestolkiae growth in medium supplemented with phytate, there was a reduction between 20 and 30% of uranium concentrations, with high precipitation of uranium and phosphate on the fungal biomass. The fungi analyzed in this research can use the phytic acid present in the medium and produce high concentrations of phosphate; which, in the environment, can assist in the heavy metal biomineralization processes, even in acidic environments. Such metabolic capabilities of fungi can be useful in decontaminating uranium-contaminated environments.

Histone H3K27 Methylation Perturbs Transcriptional Robustness and Underpins Dispensability of Highly Conserved Genes in Fungi.

Epigenetic modifications are key regulators of gene expression and underpin genome integrity. Yet, how epigenetic changes affect the evolution and transcriptional robustness of genes remains largely unknown. Here, we show how the repressive histone mark H3K27me3 underpins the trajectory of highly conserved genes in fungi. We first performed transcriptomic profiling on closely related species of the plant pathogen Fusarium graminearum species complex. We determined transcriptional responsiveness of genes across environmental conditions to determine expression robustness. To infer evolutionary conservation, we used a framework of 23 species across the Fusarium genus including three species covered with histone methylation data. Gene expression variation is negatively correlated with gene conservation confirming that highly conserved genes show higher expression robustness. In contrast, genes marked by H3K27me3 do not show such associations. Furthermore, highly conserved genes marked by H3K27me3 encode smaller proteins, exhibit weaker codon usage bias, higher levels of hydrophobicity, show lower intrinsically disordered regions, and are enriched for functions related to regulation and membrane transport. The evolutionary age of conserved genes with H3K27me3 histone marks falls typically within the origins of the Fusarium genus. We show that highly conserved genes marked by H3K27me3 are more likely to be dispensable for survival during host infection. Lastly, we show that conserved genes exposed to repressive H3K27me3 marks across distantly related Fusarium fungi are associated with transcriptional perturbation at the microevolutionary scale. In conclusion, we show how repressive histone marks are entangled in the evolutionary fate of highly conserved genes across evolutionary timescales.

Survey of Freshly Harvested Oat Grains from Southern Brazil Reveals High Incidence of Type B Trichothecenes and Associated Fusarium Species.

The current study investigated the fungal diversity in freshly harvested oat samples from the two largest production regions in Brazil, Paraná (PR) and Rio Grande do Sul (RS), focusing primarily on the Fusarium genus and the presence of type B trichothecenes. The majority of the isolates belonged to the Fusarium sambucinum species complex, and were identified as F. graminearum sensu stricto (s.s.), F. meridionale, and F. poae. In the RS region, F. poae was the most frequent fungus, while F. graminearum s.s. was the most frequent in the PR region. The F. graminearum s.s. isolates were 15-ADON genotype, while F. meridionale and F. poae were NIV genotype. Mycotoxin analysis revealed that 92% and 100% of the samples from PR and RS were contaminated with type B trichothecenes, respectively. Oat grains from PR were predominantly contaminated with DON, whereas NIV was predominant in oats from RS. Twenty-four percent of the samples were contaminated with DON at levels higher than Brazilian regulations. Co-contamination of DON, its derivatives, and NIV was observed in 84% and 57.7% of the samples from PR and RS, respectively. The results provide new information on Fusarium contamination in Brazilian oats, highlighting the importance of further studies on mycotoxins.

Wild rice (O. latifolia) from natural ecosystems in the Pantanal region of Brazil: Host to Fusarium incarnatum-equiseti species complex and highly contaminated by zearalenone.

We assessed the mycobiota diversity and mycotoxin levels present in wild rice (Oryza latifolia) from the Pantanal region of Brazil; fundamental aspects of which are severely understudied as an edible plant from a natural ecosystem. We found multiple fungal species contaminating the rice samples; the most frequent genera being Fusarium, Nigrospora and Cladosporium (35.9%, 26.1% and 15%, respectively). Within the Fusarium genus, the wild rice samples were mostly contaminated by the Fusarium incarnatum-equiseti species complex (FIESC) (80%) along with Fusarium fujikuroi species complex (20%). Phylogenetic analysis supported multiple FIESC species and gave support to the presence of two putative new groups within the complex (LN1 and LN2). Deoxynivalenol (DON) and zearalenone (ZEN) chemical analysis showed that most of the isolates were DON/ZEN producers and some were defined as high ZEN producers, displaying abundant ZEN levels over DON (over 19 times more). Suggesting that ZEN likely has a key adaptive role for FIESC in wild rice (O. latifolia). Mycotoxin determination in the rice samples revealed high frequency of ZEN, and 85% of rice samples had levels >100 µg/kg; the recommended limit set by regulatory agencies. DON was only detected in 5.2% of the samples. Our data shows that FIESC species are the main source of ZEN contamination in wild rice and the excessive levels of ZEN found in the rice samples raises considerable safety concerns regarding wild rice consumption by humans and animals.

Bioremediation of water contaminated with uranium using Penicillium piscarium.

Penicillium piscarium can be indicated as promising in the treatment of sites contaminated with uranium. Thus, this research aimed to analyze the P. piscarium dead biomass in uranium biosorption. This fungus was previously isolated from a highly contaminated uranium mine located in Brazil. Biosorption tests were carried out at pH 3.5 and 5.5 in solutions contaminated with concentrations of 1 to 100 mg/L of uranium nitrate. Our results showed that the dead biomass of P. piscarium was able to remove between 93.2 and 97.5% uranium from solutions at pH 3.5, at the end of the experiment, the pH of the solution increased to values above 5.6. Regarding the experiments carried out in solutions with pH 5.5, the dead biomass of the fungus was also able to remove between 38 and 92% uranium from the solution, at the end of the experiment, the pH of the solution increased to levels above 6.5. The analysis of electron microscopy, Energy-dispersive spectroscopy, and X-ray fluorescence demonstrated the high concentration of uranium precipitated on the surface of the fungal biomass. These results were impressive and demonstrate that the dead biomass of P. piscarium can be an important alternative to conventional processes for treating water contaminated with heavy metals, and we hope that these ecofriendly, inexpensive, and effective technologies be encouraged for the safe discharge of water from industrial activities.

Resistant fungi isolated from contaminated uranium mine in Brazil shows a high capacity to uptake uranium from water.

The Osamu Utsumi uranium mine occupies a 20 km2 area in the city of Caldas, which is located in the state of Minas Gerais, Brazil. Since mining activities ended at Osamu Utsumi 24 years ago, the surrounding area has become contaminated by acid effluents containing high concentrations of uranium. Thus, the aim of this study was to assess the uranium bioremediation capacity of 57 fungi isolated from the mine area. In tolerance tests, 38% (22) of the fungal isolates were considered tolerant to uranium, including 10 Penicillium species. At a uranium concentration of 2000 mg L-1 48 fungi did not exhibit mycelial growth index inhibition. Minimal inhibitory concentration (MIC) analysis showed growth of 25 fungi above a uranium concentration of 8000 mg L-1. At high uranium concentrations, some fungi (i.e., Talaromyces amestolkiae and Penicillium citrinum) showed morphological changes and pigment (melanin) production. Among the fungal isolates, those considered to be more tolerant to uranium were isolated from soil and sediment samples containing higher concentrations of heavy metal. When comparing the results of resistance/tolerance tests with those for uranium biosorption capacity, we concluded that the fungi isolated from the Osamu Utsumi mine with the best potential for uranium bioremediation were Gongronella butleri, Penicillium piscarium, Penicillium citrinum, Penicillium ludwigii, and Talaromyces amestolkiae. Biosorption tests with live fungal biomass showed that 11 species had a high potential for uranium uptake from contaminated water.

Cellular, physiological and molecular approaches to investigate the antifungal and anti-aflatoxigenic effects of thyme essential oil on Aspergillus flavus.

Several approaches, including the detection of apoptotic-like cell death, aflatoxin B1 (AFB1) production and gene expression analysis, were carried out to provide insights into the antifungal and anti-aflatoxigenic effects of thyme essential oil (EO) on Aspergillus flavus. At 0.5 µL mL-1, thyme EO completely inhibited A. flavus growth. Furthermore, this antifungal activity triggered significant apoptosis, via nuclear condensation (87.5% of nuclei analyzed) and plasma membrane damage (in 100% of treated hyphae). Further analysis of AFB1 production and gene expression related to secondary metabolism (laeA) and the mechanism of virulence (lipA and meT) of A. flavus in the presence of thyme EO indicated important physiological changes related to its anti-aflatoxigenic property. These results highlight the potent antifungal abilities of thyme EO in controlling A. flavus and AFB1 production, especially the abilities that operate by exerting changes at the molecular level and inducing significant apoptotic-like cell death.

Effect of temperature on growth, gene expression, and aflatoxin production by Aspergillus nomius isolated from Brazil nuts.

Aspergillus nomius is a potent producer of aflatoxins B and G and is one of the most common species of fungi found in Brazil nuts. Temperature is considered a major abiotic factor that influences fungal colonization and aflatoxin production in nuts during pre- and post-harvest. Therefore, assessment of the response of aflatoxigenic species to different temperatures is important to add information about the understanding of aflatoxin production by Aspergillus nomius and may help in the development of new strategies to prevent aflatoxin contamination. The aim of this study was to evaluate the effect of temperature (25, 30, and 35 °C) on the radial growth, aflatoxin production (B and G), and aflatoxin gene expression of seven A. nomius strains isolated from Brazil nuts. The optimal temperature for growth was 30 °C and was also the best condition for the expression of the aflR, aflD, and aflQ genes. However, maximum production of aflatoxins B and G occurred at 25 °C. Interestingly, high expression of the structural gene aflQ was observed in the maximum aflatoxin production condition (25 °C). The present study demonstrates that temperature may influence aflatoxin production by A. nomius. The combination of molecular and physiological data aids the understanding of the aflatoxigenic species response to different temperatures and can assist in predicting the driving environmental factors that influence aflatoxin contamination of Brazil nuts.

Complex Evolutionary Origins of Specialized Metabolite Gene Cluster Diversity among the Plant Pathogenic Fungi of the Fusarium graminearum Species Complex.

Fungal genomes encode highly organized gene clusters that underlie the production of specialized (or secondary) metabolites. Gene clusters encode key functions to exploit plant hosts or environmental niches. Promiscuous exchange among species and frequent reconfigurations make gene clusters some of the most dynamic elements of fungal genomes. Despite evidence for high diversity in gene cluster content among closely related strains, the microevolutionary processes driving gene cluster gain, loss, and neofunctionalization are largely unknown. We analyzed the Fusarium graminearum species complex (FGSC) composed of plant pathogens producing potent mycotoxins and causing Fusarium head blight on cereals. We de novo assembled genomes of previously uncharacterized FGSC members (two strains of F. austroamericanum, F. cortaderiae, and F. meridionale). Our analyses of 8 species of the FGSC in addition to 15 other Fusarium species identified a pangenome of 54 gene clusters within FGSC. We found that multiple independent losses were a key factor generating extant cluster diversity within the FGSC and the Fusarium genus. We identified a modular gene cluster conserved among distantly related fungi, which was likely reconfigured to encode different functions. We also found strong evidence that a rare cluster in FGSC was gained through an ancient horizontal transfer between bacteria and fungi. Chromosomal rearrangements underlying cluster loss were often complex and were likely facilitated by an enrichment in specific transposable elements. Our findings identify important transitory stages in the birth and death process of specialized metabolism gene clusters among very closely related species.

Fusarium Mycotoxins Stability during the Malting and Brewing Processes.

Mycotoxins are widely studied by many research groups in all aspects, but the stability of these compounds needs further research for clarification. The objective of this study is to evaluate deoxynivalenol and zearalenone stability during all steps of the malting and brewing processes. The levels of these compounds decreased significantly during the production process (barley to beer). During the malting process, the DON levels decreased significantly in the steeping, germination, and malting steps (62%, 51.5%, and 68%, respectively). Considering ZEN, when the levels were compared between barley and the last step of the process, a significant decrease was observed. Most of the mycotoxins produced were transferred to the rootlets and spent grains, which is advantageous considering the final product. Furthermore, the mycotoxin dietary intake estimation was included in this study. The results proved that if the concentrations of target mycotoxins in raw material are under the limits established by the regulations, the levels decrease during the malting and brewing processes and make the beer secure for consumers. The quality of the five commodities involved in the beer process plays a decisive role in the creation of a safe final product.

Correction: Multi-Method Approach for Characterizing the Interaction between Fusarium verticillioides and Bacillus thuringiensis Subsp. Kurstaki.

[This corrects the article DOI: 10.1371/journal.pone.0092189.].

Copper biosorption from an aqueous solution by the dead biomass of Penicillium ochrochloron.

The present study investigated the effect of contact time, the initial concentration of metal ions, and the biomass dose on the Cu(II) biosorption from an aqueous solution using dead biomass of filamentous fungus Penicillium ochrochloron, which was isolated at the Sossego mine, a copper-contaminated site located in Canaã dos Carajás city, Brazil. The Cu(II) biosorption started rapidly and increased gradually until the equilibrium was reached at 20 min. The Cu(II) uptake decreased as the initial Cu(II) concentration increased, reaching the saturation at 200 mg/L. The Cu(II) biosorption was considerably higher using 0.2 g than 0.5 g of the biomass in 50 mL of solution. The average biosorption capacity of Cu(II) was 7.53 mg/g and the maximum Cu(II) removal 75.0%. The Freundlich and Langmuir isotherm models adequately described the adsorption data. Our results evidenced that the dead biomass of P. ochrochloron has a great potential as a biosorbent to remove copper from an aqueous solution. Therefore, it could be explored for the development of the environmental recovery process.

Assessment of Toxigenic Fusarium Species and Their Mycotoxins in Brewing Barley Grains.

Fusarium species threaten yield and quality of cereals worldwide due to their ability to produce mycotoxins and cause plant diseases. Trichothecenes and zearalenone are the most economically significant mycotoxins and are of particular concern in barley, maize and wheat. For this reason, the aim of this study was to characterize the Fusarium isolates from brewing barley and to assess deoxynivalenol and zearalenone contamination in grains. Characterization of the Fusarium strains was carried out by the phylogeny based on two loci (EF-1α and RPB2). Mycotoxin detection and quantification were performed by LC-MS. The results show that Fusarium was the predominant genus. Phylogenetic study demonstrated that the majority of the strains clustered within the Fusarium sambucinum species complex followed by the Fusarium tricinctum species complex. The results revealed high incidence of deoxynivalenol (DON) and zearalenone (ZEA) contamination (90.6% and 87.5%, respectively). It was observed that 86% of the samples contaminated with ZEA were above the limits set by the EU and Brazilian regulations. These results may highlight the importance of controlling Fusarium toxins in barley, mainly because of its use in the brewing industry and the resistance of various mycotoxins to food processing treatments.

Polyphasic characterization of Epicoccum sorghinum: A tenuazonic acid producer isolated from sorghum grain.

Epicoccum sorghinum isolates from sorghum grain grown in Brazil were characterized using a polyphasic approach based on morphological, genetic and physiologic features. A considerable intraspecific variability was observed. The phylogenetic analysis showed that the 53 isolates studied formed four distinct clades, and their morphological features revealed a high variability in culture. The four clades could not be distinguished clearly by morphology or enzyme production patterns. The tenuazonic acid (TeA) production capability of 11 isolates was also determined and revealed a good agreement with the phylogeny results. All of the 11 isolates were revealed as TeA producers, indicating a potential toxicological risk to sorghum crops. This is the first study to provide a detailed morphological description of E. sorghinum isolates from sorghum grains in Brazil and it clearly confirms the wide genetic and phenotypic variability previously reported for this species in other countries.

Influence of environmental factors on tenuazonic acid production by Epicoccum sorghinum: An integrative approach of field and laboratory conditions.

Sorghum is the fifth most cultivated and consumed grain in the world. However, this grain is frequently contaminated with toxins from fungi. The present study evaluated the effects of environmental factors on tenuazonic acid (TeA) production by Epicoccum sorghinum in the field and in controlled laboratory conditions. In this study, 50 sorghum grain samples were collected from summer and autumn growing seasons and analyzed for TeA contamination using LC-MS/MS. To further understand the ecophysiology of this fungus, an isolated strain of E. sorghinum from the field was investigated for its development and TeA production under controlled environmental conditions in the laboratory. In the ecophysiological investigation, the effects of water activity (0.90, 0.95, 0.99) and temperature (18, 22, 26 and 30 °C) were evaluated on the radial growth, enzymatic production and expression of TAS1, which is the gene involved in TeA production. Results showed that in the field, the summer season presented the highest TeA average level in the grains (587.8 µg/kg) compared to level found in the autumn (440.5 µg/kg). The ecophysiological investigation confirmed that E. sorghinum produces more actively TeA under environmental conditions simulating the summer season. Optimum growth, maximum TAS1 gene expression, and higher extracellular enzymatic production were observed at 26 °C with a water activity of 0.99. Pearson correlation analyses showed that the production of TeA highly correlates with fungal growth. The present study demonstrates that abiotic factors in a combined approach of field and laboratory conditions will assist in predicting the driving environmental factors that could affect growth of E. sorghinum and TeA production in sorghum grains.