Fullerol C60(OH)24 Nanoparticles Affect Secondary Metabolite Profile of Important Foodborne Mycotoxigenic Fungi In Vitro.

Despite the efforts to control mycotoxin contamination worldwide, extensive contamination has been reported to occur in food and feed. The contamination is even more intense due to climate changes and different stressors. This study examined the impact of fullerol C60(OH)24 nanoparticles (FNP) (at 0, 1, 10, 100, and 1000 ng mL-1) on the secondary metabolite profile of the most relevant foodborne mycotoxigenic fungi from genera Aspergillus, Fusarium, Alternaria and Penicillium, during growth in vitro. Fungi were grown in liquid RPMI 1640 media for 72 h at 29 °C, and metabolites were investigated by the LC-MS/MS dilute and shoot multimycotoxin method. Exposure to FNP showed great potential in decreasing the concentrations of 35 secondary metabolites; the decreases were dependent on FNP concentration and fungal genus. These results are a relevant guide for future examination of fungi-FNP interactions in environmental conditions. The aim is to establish the exact mechanism of FNP action and determine the impact such interactions have on food and feed safety.

The application of natural food colorants as indicator substances in intelligent biodegradable packaging materials.

This paper presents innovative packaging materials made of environmentally friendly biodegradable polymers (polylactide and polyhydroxybutyrate) with the addition of natural colorants commonly used in the food industry. Colorants fulfilled the role of indicator, changing colour under the influence of external factors, and gave the materials the characteristics of intelligent packaging, where colour changes indicated the life time of the materials. The paper gives the mechanical and thermal properties of the materials obtained, and describes changes in the colour of the samples under the influence of thermooxidation, UV and weathering, as well as the biodegradability of the materials. The packaging materials presented are in line with current trends in the packaging market and legal requirements. The samples, in addition to the basic functions of packaging materials, are pro-ecological and fully biodegradable new generation materials.

Molecular identification and antibacterial properties of an ericoid associated mycorrhizal fungus.

BACKGROUND: The quest for novel sources of antibacterial compounds have necessitated the inclusion of ericoid mycorrhizal fungi (ERM) commonly found within the root of ericaceous plants. Agar-well diffusion method was used to detect antibacterial activity and was followed by the microbroth diffusion method [minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)]. RESULTS: The results of the phytochemical screening indicated that only alkaloids, flavonoids, phenols, saponins, cardiac glycosides and terpenoids were present, while steroids and tannins were absent. The MIC of the extracts ranged between 2 and 16 mg/mL, and the lowest MIC was obtained with Staphylococcus aureus. Also, the result of the MBC study indicated that the fungal extract was most active at concentrations of 2 and 4 mg/mL against Bacillus subtilis and S. aureus, respectively. CONCLUSIONS: This bioassay showed, for the first time, antibacterial activity of L. incrustata against some bacterial species. Subsequently, ERM fungi should be given attention when searching for antimicrobial agents because they could provide a solution to solve problems associated with conventional disease treatments (i.e. pathogenic microorganisms resistance).

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.

Symbiotic performance and induction of systemic resistance against Cercospora sojina in soybean plants co-inoculated with Bacillus sp. CHEP5 and Bradyrhizobium japonicum E109.

Soybean is an economically very important crop throughout the word and particularly in Argentina. Soybean yield may be affected by many factors such as the lack of some essential nutrients or pathogens attack. In this work we demonstrated that the co-inoculation of the native biocontrol bacterium Bacillus sp. CHEP5 which induces resistance against Cercospora sojina in soybean and the nitrogen fixing strain Bradyrhizobium japonicum E109, was more effective in reducing frog leaf spot severity than the inoculation of the biocontrol agent alone. Probably, this is related with the increase in the ability to form biofilm when both bacteria are growing together. Furthermore, Bacillus sp. CHEP5 inoculation did not affect Bradyrhizobium japonicum E109 symbiotic behavior and flavonoids composition of root exudates in pathogen challenged plants. These results suggest that co-inoculation of plants with rhizobia and biocontrol agents could be a strategy to improve soybean production in a sustainable system.

Quinone/hydroquinone meroterpenoids with antitubercular and cytotoxic activities produced by the sponge-derived fungus Gliomastix sp. ZSDS1-F7.

Fifteen compounds, including six quinone/hydroquinone meroterpenoids, purpurogemutantin (1), macrophorin A (2), 4'-oxomacrophorin (3), 7-deacetoxyyanuthone A (4), 2,3-hydro-deacetoxyyanuthone A (5), 22-deacetylyanuthone A (6), anicequol (7), three roquefortine derivatives, roquefortine C (8), (16S)-hydroxyroquefortine C (9), (16R)-hydroxyroquefortine C (10), dihydroresorcylide (11), nectriapyrone (12), together with three fatty acid derivatives, methyl linoleate (13), phospholipase A2 (14), methyl elaidate (15), were isolated from the sponge-derived fungus Gliomastix sp. ZSDS1-F7 isolated from the sponge Phakellia fusca Thiele collected in the Yongxing island of Xisha. Their structures were elucidated mainly by extensive NMR spectroscopic and mass spectrometric analyses. Among these compounds, compounds 1-3 and 5-7 showed significant in vitro cytotoxicities against the K562, MCF-7, Hela, DU145, U937, H1975, SGC-7901, A549, MOLT-4 and HL60 cell lines, with IC50 values ranging from 0.19 to 35.4 µM. And compounds 2-4 exhibited antitubercular activity with IC50 values of 22.1, 2.44 and 17.5 µM, respectively. Furthermore, compound 7 had anti-enterovirus 71 activity with MIC value of 17.8 µM. To the best of our knowledge, this is the first report to product two quinone/hydroquinone meroterpenoids skeletons (linear skeleton and drimane skeleton) from the same fungal strain.

Antimicrobial activity of pomegranate peel extracts as affected by cultivar.

BACKGROUND: Some studies have reported that different parts of the pomegranate fruit, especially the peel, may act as potential antimicrobial agents and thus might be proposed as a safe natural alternative to synthetic antimicrobial agents. The high tannin content, especially punicalagin, found in pomegranate extracts, has been reported as the main compound responsible for such antimicrobial activity. Because the pomegranate peel chemical composition may vary with the type of cultivar (sweet, sour-sweet and sour), pomegranates may also differ with respect to their antimicrobial capacity. RESULTS: The extract from PTO8 pomegranate cultivar peel had the highest antimicrobial activity, as well as the highest punicalagins (α and ß) and ellagic acid concentrations. In the results obtained from both antibacterial and antifungal activity studies, the sour-sweet pomegranate cultivar PTO8 showed the best antimicrobial activity, and the highest ellagic acid concentrations. CONCLUSION: The results of the present study suggest that ellagic acid content has a significant influence on the antimicrobial activity of the pomegranate extracts investigated. The pomegranate peel of the PTO8 cultivar is a good source of antifungal and antibacterial compounds, and may represent an alternative to antimicrobial agents of synthetic origin. © 2016 Society of Chemical Industry.

Evaluation of Commercial Soybean Cultivars for Reaction to Phomopsis Seed Decay.

Phomopsis seed decay (PSD), caused by Phomopsis longicolla (syn. Diaporthe longicolla), is an economically important soybean disease causing poor seed quality. Planting resistant cultivars is one of the most effective means to control PSD. In this study, 16 commercially available maturity groups IV and V soybean cultivars, including two previously identified PSD-resistant and two PSD-susceptible checks, were evaluated for seed infection by P. longicolla in inoculated and noninoculated plots, and harvested promptly or with a 2-week delay in harvest. The test was conducted at Stoneville, Mississippi, in 2012 and 2013. Seed infection by P. longicolla ranged from 0.5 to 76%, and seed germination ranged from 18 to 97%. One MG IV cultivar (Morsoy R2 491) and five MG V cultivars (Progeny 5650, Progeny 5706, Asgrow 5606, Asgrow 5831, and Dyna-Gro33C59) had significantly (P ≤ 0.05) lower percent seed infected by P. longicolla than their respective susceptible checks and other cultivars in the same tests. Information obtained from this study will be useful for soybean growers and breeders for selection of cultivars for planting or breeding and future genetic studies in the development of cultivars with improved resistance to PSD.

Analytical Methods for Secondary Metabolite Detection.

The entomopathogenic fungi Metarhizium brunneum, Beauveria bassiana, and B. brongniartii are widely applied as biological pest control agent in OECD countries. Consequently, their use has to be flanked by a risk management approach, which includes the need to monitor the fate of their relevant toxic metabolites. There are still data gaps claimed by regulatory authorities pending on their identification and quantification of relevant toxins or secondary metabolites. In this chapter, analytical methods are presented allowing the qualitative and quantitative analysis of the relevant toxic B. brongniartii metabolite oosporein and the three M. brunneum relevant destruxin (dtx) derivatives dtx A, dtx B, and dtx E.

[Advances in the regulation of cephalosporin C biosynthesis - A review].

The beta-lactam antibiotic cephalosporin C is produced industrially by Acremonium chrysogenum. Its derivative 7-aminocephalosporanic acid (7-ACA) is the intermediate of most chemical modification cephalosporins that are the most frequently used antibiotics for the therapy of infectious diseases. Due to its importance, the biosynthetic pathway of cephalosporin C has been elucidated in Acremonium chrysogenum. To improve the yield of cephalosporin C and reduce the cost of production, recent studies have been focused on the sophisticated regulation of cephalosporin C biosynthesis. In this review, recent advances in cephalosporin C biosynthesis and regulation are summarized.

Copper and zinc affect the activity of plasma membrane H+-ATPase and thiol content in aquatic fungi.

Aquatic hyphomycetes are the major microbial decomposers of plant litter in streams. We selected three aquatic hyphomycete species with different abilities to tolerate, adsorb and accumulate copper and zinc, and we investigated the effects of these metals on H+-ATPase activity as well as on the levels of thiol (SH)-containing compounds. Before metal exposure, the species isolated from a metal-polluted stream (Heliscus submersus and Flagellospora curta) had higher levels of thiol compounds than the species isolated from a clean stream (Varicosporium elodeae). However, V. elodeae rapidly increased the levels of thiols after metal exposure, emphasizing the importance of these compounds in fungal survival under metal stress. The highest amounts of metals adsorbed to fungal mycelia were found in the most tolerant species to each metal, i.e. in H. submersus exposed to copper and in V. elodeae exposed to zinc. Short-term (10 min) exposure to copper completely inhibited the activity of H+-ATPase of H. submersus and V. elodeae, whilst zinc only led to a similar effect on H. submersus. However, at longer exposure times (8 days) the most metal-tolerant species exhibited increased H+-ATPase activities, suggesting that the plasma membrane proton pump may be involved in the acclimation of aquatic hyphomycetes to metals.

Seasonal and spatial variation of Deuteromycetes population in polluted cost of Kiaochow Bay.

Objective: To reveal the relationship between Deuteromycetes community and the environmental in Kiaochow Bay of the Yellow Sea. Methods: Using recorded pollution survey, we used molecular methods to study seasonal and spatial variation of Deuteromycetes community diversity in different polluted waters of Kiaochow Bay of the Yellow Sea, China. Results: Denaturing gradient gel electrophoresis fingerprints varied obviously among different sites of similar level of pollution. Moreover, sequence analysis of recovered dominant bands exhibited the existence of plenty of uncultivable fungi, among which Penicillium was the dominant genus. Furthermore, in heavily polluted estuary, there were abundant animal pathogens such as amoeba and Pythium as well as Deuteromycetes. These discoveries demonstrate that the Deuteromycetes community structure is closely related to marine environment, and are indicative of different level of marine contamination. Conclusion: The relationship between Deuteromycetes community and different level of pollution and seasons varied were closely related.

A Critical Review of the Properties of Fusidic Acid and Analytical Methods for Its Determination.

Fusidic acid, an antibiotic produced from the Fusidium coccineum fungus, belongs to the class of steroids, but has no corticosteroid effects. It is indicated for the treatment of infections caused by methicillin-resistant Staphylococcus aureus strains. The aim of this study was to search for the properties of fusidic acid published so far in the literature, as well as the methods developed for its determination in biological samples and pharmaceutical formulations. From the findings, we can conclude that fusidic acid has been used for decades and is indicated for the treatment of serious infections caused by Gram-positive microorganisms to this day. Furthermore, it is a hypoallergenic agent, has low toxicity, shows low resistance, and has no cross-resistance with other clinically used antibiotics. The analytical method of high-performance liquid chromatography has been widely used for determining fusidic acid, since it can reduce the cost and time of analysis, making it more viable for routine quality control in the pharmaceutical industry.

Development of nitrocellulose membrane filters impregnated with different biosynthesized silver nanoparticles applied to water purification.

Bactericidal water filters were developed. For this purpose, nitrocellulose membrane filters were impregnated with different biosynthesized silver nanoparticles. Silver nanoparticles (AgNPs) from Aspergillus niger (AgNPs-Asp), Cryptococcus laurentii (AgNPs-Cry) and Rhodotorula glutinis (AgNPs-Rho) were used for impregnating nitrocellulose filters. The bactericidal properties of these nanoparticles against Escherichia coli, Enterococcus faecalis and Pseudomona aeruginosa were successfully demonstrated. The higher antimicrobial effect was observed for AgNPs-Rho. This fact would be related not only to the smallest particles, but also to polysaccharides groups that surrounding these particles. Moreover, in this study, complete inhibition of bacterial growth was observed on nitrocellulose membrane filters impregnated with 1 mg L(-1) of biosynthesized AgNPs. This concentration was able to reduce the bacteria colony count by over 5 orders of magnitude, doing suitable for a water purification device.

Molecular basis of the regulation of iron homeostasis in fission and filamentous yeasts.

When iron load exceeds that needed by fission and filamentous yeasts, iron-regulatory GATA-type transcription factors repress genes encoding iron acquisition systems. In contrast, under iron starvation, optimization of cellular iron utilization is coordinated by a specialized regulatory subunit of the CCAAT-binding factor that fosters repression of genes encoding iron-using proteins. Despite these findings, there is still limited knowledge concerning the mechanisms by which these iron-responsive regulators respond to high- or low-iron availability. To provide a framework for understanding common and distinct properties of iron-dependent transcriptional regulators, a repertoire of their functional domains in different fungal species is presented here. In addition, discovery of interacting partners of these iron-responsive factors contributes to provide additional insight into their properties.

[11beta-Hydroxylation of 6alpha-Fluoro-16alpha-Methyl-Deoxycorticosterone 21-Acetate by filamentous fungi].

Selected filamentous fungi--98 strains of 31 genera--were screened for the ability to catalyze 11beta-hydroxylation of 6alpha-fluoro-16alpha-methyl-deoxycorticosterone 21-acetate (FM-DCA). It was established that representatives of the genera Gongronella, Scopulariopsis, Epicoccum, and Curvularia have the ability to activate 11beta-hydroxylase steroids. The strains of Curvularia lunata VKM F-644 and Gongronella butleri VKM F-1033 expressed maximal activity and formed 6lpha-fluoro-16alpha-methyl-corticosterone as a major bioconversion product from FM-DCA. The structures of the major products and intermediates of the bioconversion were confirmed by TLC, H PLC, MS and 1H NMR analyses. Different pathways of 6alpha-fluoro-16alpha-methyl-corticosterone formation by C. lunata and G. butleri strains were proposed based on intermediate identification. The constitutive character and membrane-binding localization were evidence of a 11beta-hydroxylating system in G. butleri, while an inducible character and microsomal localization was confirmed for 11beta-hydroxylase of C. lunata. Under optimized conditions, the molar yield of 6alpha-fluoro-16alpha-methyl-corticosterone reached 65% at a FM-DCA substrate loading of 6 g/L.

Cadmium-induced formation of sulphide and cadmium sulphide particles in the aquatic hyphomycete Heliscus lugdunensis.

Freshwater fungi which can survive under metal exposure receive increasing scientific attention. Enhanced synthesis of sulphide and glutathione but no phytochelatin synthesis in response to cadmium (up to 80 µM Cd(2+) in the medium) was measured in the aquatic hyphomycete Heliscus lugdunensis. Up to 25 µmol g(-1) dry mass the fungus formed sulphide in an exponentially Cd(2+)-concentration-dependent manner. Using light microscopy, precipitates were observed outside of the hyphae which could be determined as amorphous particles by X-ray diffraction (XRD). Energy dispersive X-ray spectroscopy (EDS) analysis indicated that these particles were mainly composed of Cd and S with an atomic ratio of 1:1, but some elements of the culture medium such as P and Cl were also present. Fungal cells exposed to Cd(2+) accumulated 12-28 µmol metal g(-1) dry mass over a period of 7-28 days. The results may indicate that sulphide could sequester excess Cd(2+) under oxygen deprived conditions and thereby reduce its toxicity via an additional avoidance mechanism of this fungus.

Sorbicillinoid analogs with cytotoxic and selective anti-Aspergillus activities from Scytalidium album.

As part of an ongoing project to explore filamentous fungi for anticancer and antibiotic leads, 11 compounds were isolated and identified from an organic extract of the fungus Scytalidium album (MSX51631) using bioactivity-directed fractionation against human cancer cell lines. Of these, eight compounds were a series of sorbicillinoid analogs (1-8), of which four were new (scalbucillin A (2), scalbucillin B (3), scalbucillin C (6) and scalbucillin D (8)), two were phthalides (9-10) and one was naphthalenone (11). Compounds (1-11) were tested in the MDA-MB-435 (melanoma) and SW-620 (colon) cancer cell lines. Compound 1 was the most potent with IC50 values of 1.5 and 0.5 µM, followed by compound 5 with IC50 values of 2.3 and 2.5 µM at 72 h. Compound 1 showed a 48-h IC50 value of 3.1 µM when tested against the lymphocytic leukemia cell line OSU-CLL, while the nearly identical compound 5 had almost no activity in this assay. Compounds 1 and 5 showed selective and equipotent activity against Aspergillus niger with minimum IC values of 0.05 and 0.04 µg ml(-1) (0.20 and 0.16 µM), respectively. The in vitro hemolytic activity against sheep erythrocytes of compounds 1 and 5 was investigated and were found to provoke 10% hemolysis at 52.5 and 45.0 µg ml(-1), respectively, indicative of a promising safety factor.

Responsiveness of entomopathogenic fungi to menadione-induced oxidative stress.

Entomopathogenic fungi are predisposed to ROS induced by heat and UV-A radiation when outside the insect host. When inside the host, they are subject to phagocytic cells that generate ROS to eliminate invading pathogens. The oxidative stress tolerance of the entomopathogenic fungi Aschersonia aleyrodis (ARSEF 430 and 10276), Aschersonia placenta (ARSEF 7637), Beauveria bassiana (ARSEF 252), Isaria fumosorosea (ARSEF 3889), Lecanicillium aphanocladii (ARSEF 6433), Metarhizium acridum (ARSEF 324), Metarhizium anisopliae (ARSEF 5749), Metarhizium brunneum (ARSEF 1187 and ARSEF 5626), Metarhizium robertsii (ARSEF 2575), Tolypocladium cylindrosporum (ARSEF 3392), Tolypocladium inflatum (ARSEF 4877), and Simplicillium lanosoniveum (ARSEF 6430 and ARSEF 6651) was studied based on conidial germination on a medium supplemented with menadione. Conidial germination was evaluated 24 h after inoculation on potato dextrose agar (PDA) (control) or PDA supplemented with menadione. The two Aschersonia species (ARSEF 430, 7637, and 10276) were the most susceptible fungi, followed by the two Tolypocladium species (ARSEF 3392 and 4877) and the M. acridum (ARSEF 324). Metarhizium brunneum (ARSEF 5626) and M. anisopliae (ARSEF 5749) were the most tolerant isolates with MIC 0.28 mM. All fungal isolates, except ARSEF 5626 and ARSEF 5749, were not able to germinate at 0.20 mM.

The impact of proteomics on the understanding of functions and biogenesis of fungal extracellular vesicles.

Several microbial molecules are released to the extracellular space in vesicle-like structures. In pathogenic fungi, these molecules include pigments, polysaccharides, lipids, and proteins, which traverse the cell wall in vesicles that accumulate in the extracellular space. The diverse composition of fungal extracellular vesicles (EV) is indicative of multiple mechanisms of cellular biogenesis, a hypothesis that was supported by EV proteomic studies in a set of Saccharomyces cerevisiae strains with defects in both conventional and unconventional secretory pathways. In the human pathogens Cryptococcus neoformans, Histoplasma capsulatum, and Paracoccidioides brasiliensis, extracellular vesicle proteomics revealed the presence of proteins with both immunological and pathogenic activities. In fact, fungal EV have been demonstrated to interfere with the activity of immune effector cells and to increase fungal pathogenesis. In this review, we discuss the impact of proteomics on the understanding of functions and biogenesis of fungal EV, as well as the potential role of these structures in fungal pathogenesis. This article is part of a Special Issue entitled: Trends in Microbial Proteomics.