Chromium tolerance and accumulation in Aspergillus flavus isolated from tannery effluent.

Cr(VI) tolerance in Aspergillus flavus, strain SFL, isolated from tannery effluent was measured and compared with a reference strain of A. flavus, A1120. On solid medium, SFL had a high level of Cr(VI) tolerance (1,600 mg/L), which was 16 times that of A1120 and greater than most previously analyzed fungal strains. When in 100 mg/L of Cr(VI), SFL completely depleted Cr(VI) within 72 h while A1120 depleted 85% of Cr(VI). SFL was more effective in reducing extracellular Cr(VI) than A1120. While A1120 showed greater biosorption of Cr(VI) than SFL, intracellular accumulation was approximately 50% greater in SFL and was more energy-dependent than A1120. Cr(VI) modified the external surface of the hyphae. Cr speciation detected the presence of only Cr(III), corresponding to Cr(OH)3 , which precipitated on the hyphal surface. Cr(VI) bound to the functional groups carboxyl, amine, and hydroxyl in both SFL and A1120. Transmission electron microscopy energy-dispersive X-ray detected Cr on the fungal wall and within membrane-bound organelles of the cytoplasm. In conclusion, the greater tolerance of SFL to Cr(VI) relative to A1120 is due to more effective energy-dependant uptake of Cr(VI) into the cell and increased capacity of SFL to store Cr in intracellular vacuoles compared with A1120.

Morphological and Transcriptomic Analysis of the Inhibitory Effects of Lactobacillus plantarum on Aspergillus flavus Growth and Aflatoxin Production.

Lactobacillus plantarum, as a natural bio-preservative, has attracted a great deal of attention in recent years. In this study, 22 L. plantarum strains were tested against the aflatoxin-producing fungus, Aspergillus flavus; strain IAMU80070 showed the highest antifungal activity. At a concentration of 5 × 105 colony-forming units (CFU) mL-1, it completely inhibited A. flavus growth and decreased aflatoxin production by 93%. Furthermore, ultrastructural examination showed that IAMU80070 destroyed the cellular structure of hyphae and spores. To explore the inhibitory effect of IAMU80070 on A. flavus at the transcriptional level, transcriptome data were obtained and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The aflatoxin biosynthetic process was the most significantly downregulated functional category, while genes implicated in the synthesis and organization of cell wall polysaccharides were upregulated. Quantitative real-time PCR results verified the credibility and reliability of the RNA sequencing data. These results provided insight into the transcriptome of A. flavus in response to the antagonistic effects of L. plantarum IAMU80070.

Antifungal Activity of Essential Oil Compounds (Geraniol and Citral) and Inhibitory Mechanisms on Grain Pathogens (Aspergillus flavus and Aspergillus ochraceus).

The grain contamination by Aspergillus spp. has been a serious issue. This study exhibited the excellent antifungal effects of the essential oil compounds (EOCs) geraniol and citral against common grain pathogens (A. flavus and A. ochraceus) in vitro and in situ. The inhibitory mechanisms were also evaluated from the perspective of cell membrane permeability, reactive oxygen species (ROS) generation, and Aspergillus spp. growth-related gene expression. Meanwhile, the combined effects of EOCs in the vapor phase and modified atmosphere packaging (MAP) were examined to find an alternative preservation method for controlling Aspergillus spp. The results indicated that citral exhibited the antifungal activity mainly by downregulating the sporulation- and growth-related genes for both pathogens. Geraniol displayed inhibitory effectiveness against A. flavus predominantly by inducing the intracellular ROS accumulation and showed toxicity against A. ochraceus principally by changing cell membrane permeability. Furthermore, the synthetic effects of EOCs and MAP (75% CO2 and 25% N2) induced better grain quality than the current commercial fumigant AlP. These findings reveal that EOCs have potential to be a novel grain preservative for further application.

Nanoencapsulated Illicium verum Hook.f. essential oil as an effective novel plant-based preservative against aflatoxin B1 production and free radical generation.

The study reports efficacy of Illicium verum essential oil (IvEO) against food borne moudls and its nanoencapsulation for enhancing antifungal and antiaflatoxigenic potency. Chemical characterization of the IvEO showed anethole (89.12%) as major compound followed by estragole (4.859%). The IvEO showed broad fungitoxic spectrum against common food borne moulds. It's minimum inhibitory concentration (MIC) and minimum aflatoxin B1 inhibitory concentration (MAIC) against aflatoxigenic strain Aspergillus flavus LHP-PV-1 were 0.7, and 0.5 µL/mL respectively. Morphological observations of treatment sets by SEM and TEM along with decrease in ergosterol content and enhanced leakage of Ca2+, K+ and Mg2+ ions denoted fungal cell membrane as site of action. The IvEO showed promising free radical scavenging activity and favourable safety profile with high LD50 value on mice. The IvEO also exhibited considerable protection of Pistacia vera from fungal contamination and complete protection from aflatoxin B1 contamination in storage containers. Nanoencapsulated IvEO in gel form and lyophilized form exhibited enhanced efficacy as fungal inhibitor and aflatoxin suppressor. The chemically characterised IvEO may be recommended as plant based preservative having favourable safety and its nanocapsules may be of industrial significance as shelf life enhancer of food items. This is the first report on in situ antiaflatoxigenic efficacy and nanoencapsulation of IvEO.

Farnesol induces apoptosis-like cell death in the pathogenic fungus Aspergillus flavus.

Farnesol (FOH) is known to induce apoptosis in some fungi and mammalian cells. We treated Aspergillus flavus, one of the leading causes of human invasive aspergillosis and a key producer of the most potent naturally occurring hepatocarcinogenic compounds, with FOH to assess its effect on the viability of the fungus. FOH strongly inhibited germination and growth of A. flavus and induced markers for apoptosis including nuclear condensation, phosphatidylserine (PS) externalization, DNA fragmentation and intracellular reactive oxygen species (ROS) generation, metacaspase activation and abnormal cellular ultrastructure. Moreover, FOH-induced apoptosis in A. flavus was inhibited by the broad-spectrum caspase inhibitor Z-VAD-fmk and partially inhibited by the ROS scavenger l-proline, which suggests that FOH induces apoptosis in A. flavus via a mechanism involving metacaspase activation and ROS production.

Antifungal activity of silver ion on ultrastructure and production of aflatoxin B1 and patulin by two mycotoxigenic strains, Aspergillus flavus OC1 and Penicillium vulpinum CM1.

OBJECTIVE: The antifungal activity of silver ion from silver nitrate solution was tested against two pathogenic and toxigenic fungal strains. The first was Aspergillus flavus OC1, a clinical aflatoxigenic strain that causes fungal keratitis and the second was Penicillium vulpinum CM1, a maize-pathogenic strain that is positive for patulin (PAT) producing ability. MATERIALS AND METHODS: Agar well diffusion assays on yeast sucrose (YES) agar were applied for determination of the antifungal activity of silver ions either filter- or autoclaved-sterilized. Transmission electron microscopy was used to analyze the cellular effects of silver ion. The mycotoxins AFB1 and PAT were analyzed in the fungal strains cultures treated with silver ion. RESULTS: Filter-sterilized ions have a greater potential for growth inhibition of both fungal strains than autoclaved-sterilized ions. The minimal inhibitory concentration of the filter-sterilized ions against A. flavus OC1 was 70 µg mL(-1) and against P. vulpinum CM1 was 60 µg mL(-1) and that the minimum fungicidal concentration was 120 µg mL(-1) against the first strain and 80 µg mL(-1) against the second strain. Hyphal cells treated with silver ion showed considerable changes in the nature of cell membranes and cytoplasmic organelles. Silver applied to YES broth inhibited mycelial growth and AFB1 and PAT formation of both strains. Growth and mycotoxin production appeared to be correlated processes. CONCLUSION: These findings indicate the future possibility to use silver ion as substitute for synthetic fungicides to control the growth of pathogenic fungi and their mycotoxin production.

Physico-chemical study for zinc removal and recovery onto native/chemically modified Aspergillus flavus NA9 from industrial effluent.

Zinc biosorption characteristic of locally isolated Aspergillus flavus NA9 were examined as a function of pH, temperature, pulp density, contact time and initial metal ion concentration. The maximum zinc uptake was found to be 287.8 ± 11.1 mg g(-1) with initial metal concentration 600 mg L(-1) at initial pH 5.0 and temperature 30 °C. The equilibrium data gave good fits to Freundlich and Florry models with correlation coefficient value of 0.98. The contribution of the functional groups and lipids to zinc biosorption as identified by chemical pretreatment was in the order: carboxylic acids > hydroxyl > amines > lipids. The mechanism of biosorption was also studied using Fourier transform infrared (FTIR) spectrometry, scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX). The biosorbent was regenerated using 0.01 M HCl with 83.3% elution efficiency and was reused for five sorption-desorption cycles with 23.5% loss in biosorption capacity. The order of co-cations showing increased inhibitions of zinc uptake by A. flavus NA9 was Pb > Cu > Mn > Ni. The biosorption assays conducted with actual paint industry effluents revealed efficiency of 88.7% for Zn (II) removal by candidate biomass.

Antifungal action of Pichia anomala against aflatoxigenic Aspergillus flavus and its application as a feed supplement.

BACKGROUND: The disastrous problem of animal feed contamination by mycotoxigenic fungi continues to challenge researchers and health overseers worldwide. With the aim of preventing Aspergillus flavus growth in vitro and in corn feed, the yeast Pichia anomala was examined as a biocontrol agent. RESULTS: The yeast strain could efficiently prohibit the growth of A. flavus. P. anomala was able to produce exo-chitinase and ß-1,3-glucanase which could be suggested as a mode of action for its antifungal activity. Scanning electron microscopy of cultured P. anomala with fungal hypha revealed that A. flavus was colonised by the biocontrol yeast which subsequently led to complete hyphal lysis and deterioration. The supplementation with of P. anomala cells, as a protein source, led to an obvious increase in animals' weight gain and protein content in feed grain. Moreover, after consumption of P. anomala-supplemented feed, there was a remarkable decrease in the mortality rate among fed animals. CONCLUSION: P. anomala could be strongly recommended as a biocontrol agent against A. flavus which contaminates animal feed. Furthermore, the application of yeast cells, as a feed additive, proved its efficiency for escalating protein content and enhancing animal performance.

Molecular phylogeny and phenotypic variability of clinical and environmental strains of Aspergillus flavus.

Aspergillus flavus is the second most common cause of aspergillosis infection in immunocompromised patients and is responsible for the production of aflatoxins. Little is known about the population structure of A. flavus, although recent molecular and phenotypic data seem to demonstrate that different genetic lineages exist within this species. The aim of this study was to carry out a morphological, physiological, and molecular analysis of a set of clinical and environmental isolates to determine whether this variability is due to species divergence or intraspecific diversity, and to assess whether the clinical isolates form a separate group. The amdS and omtA genes were more phylogenetically informative than the other tested genes and their combined analysis inferred three main clades, with no clear distinction between clinical and environmental isolates. No important morphological and physiological differences were found between the members of the different clades, with the exception of the assimilation of d-glucosamine, which differentiates the members of the clade II from the others.

Effect of protein binding on the activity of voriconazole alone or combined with anidulafungin against Aspergillus spp. using a time-kill methodology.

OBJECTIVES: the aims of the study were to explore the activity of total and free (according to protein binding) maximal concentrations achieved in serum after multiple doses of voriconazole 400/200 mg and anidulafungin 200/100 mg against Aspergillus fumigatus and Aspergillus flavus and the human albumin or serum effects on antifungal activity. MATERIAL AND METHODS: Time-kill curves were performed with two A. fumigatus and two A. flavus strains at voriconazole and anidulafungin Cmax concentrations using different media: a) RPMI broth (Cmax-RPMI); b) RPMI with human serum (Cmax-HS), and c) RPMI with human albumin (Cmax-HAlb). In parallel, free-drug (fCmax) concentrations considering theoretical protein binding were performed in RPMI broth. Aspergillus metabolic activity was measured by the XTT reduction assay. RESULTS: Voriconazol or voriconazole plus anidulafungin reduced >88.4% the metabolic activity of Aspergillus sp. at Cmax-RPMI and fCmax after 48 h of exposition. Anidulafungin alone showed poor metabolic reductions (<80.1% at Cmax- RPMI and <15% at fCmax). Anidulafungin activity, but not voriconazole activity alone or combined decreased in presence of HS or HAlb (more pronounced in A. flavus strains and HAlb). However, anidulafungin Cmax-HS or Cmax-HAlb against A. fumigatus strains were significantly more active (p<0.05) than fCmax in RPMI. These species and culture medium-dependent impact of human protein binding in the activity of anidulafungin was related to macroscopic and microscopic differences among mycelial mat grown in RPMI, HS or HAlb in whose XTT retention was different. CONCLUSIONS: Synergism could not be demonstrated due to the high activity showed by voriconazole. Protein binding has not impact on voriconazole activity and this impact is considerably less than predicted by free concentration extrapolated from theoretical binding rate on anidulafungin. The XTT colorimetric assay needs to be standardized for use with Aspergillus spp. since without DMSO extraction the activity of echinocandins in a free-human protein RPMI medium could be overestimated.

Ageratum conyzoides essential oil as aflatoxin suppressor of Aspergillus flavus.

Aflatoxin B(1) (AFB(1)) is a highly toxic and carcinogenic metabolite produced by Aspergillus species on food and agricultural commodities. Inhibitory effects of essential oil of Ageratum conyzoides, on the mycelial growth and aflatoxin B(1) production by Aspergillus flavus were studied. Cultures were incubated in yeast extract-sucrose (YES) broth for days at 25 degrees C at the following different concentrations of the essential oil (from 0.0 to 30mug/mL). The essential oil inhibited fungal growth to different extents depending on the concentration, and completely inhibited aflatoxin production at concentrations above 0.10microg/mL. The analysis of the oil by GC/MS showed that its main components are precocene II (46.35%), precocene I (42.78%), cumarine (5.01%) and Trans-caryophyllene (3.02%). Comparison by transmission electron microscopy of the fungal cells, control and those incubated with different concentrations of essential oil, showed ultra-structural changes which were concentration dependent of the essential oil of A. conyzoides. Such ultra-structural changes were more evident in the endomembrane system, affecting mainly the mitochondria. Degradation was also observed in both surrounding fibrils. The ability to inhibit aflatoxin production as a new biological activity of A.conyzoides L. indicates that it may be considered as a useful tool for a better understanding of the complex pathway of aflatoxin biosynthesis.

Differences in fungicidal efficiency against Aspergillus flavus for neutralized and acidic electrolyzed oxidizing waters.

Neutralized electrolyzed oxidizing water (NEW) and acidic electrolyzed oxidizing water (AcEW) are electrolyzed oxidizing waters (EOW) that have significantly different fungicidal efficiencies against Aspergillus flavus (A. flavus) (The actuation durations of no survival population to NEW and AcEW were 90s and 120s, respectively.), even when used at the same available chlorine concentration (30ppm). It has been verified by our previous research. This study hypothesized that this difference did not originate from the structure of water but based on the OH radical (OH). It was proved by the UV spectroscopy, (17)O-NMR spectroscopy and electron spin resonance analysis. NEW contains more OH compared with AcEW in the same available chlorine concentration level. The OH that exists in NEW and AcEW was found to have an important fungicidal factor that destroys the cellular structures of the A. flavus conidia. It also damages the cellular normal function of A. flavus conidia that brought about K+ and Mg2+ leakages. The levels of OH that exist in NEW and AcEW could be the important reason that leads to significant fungicidal efficiencies against A. flavus.

Microscopic studies on the Aspergillus flavus infected kernels of commercial peanuts in Georgia.

This article describes the use of microscopy to prove the presence of the aflatoxin producing pathogen, Aspergillus flavus Link ex Fries in commercially available edible peanuts in Georgia. Light microscopy in combination with electron microscopy has been used to describe the infection course established by the fungus. The alkali maceration technique used in the study was successful and sufficient to detect the kernel infection of A. flavus and monitor the infection percentage in edible peanuts. Percentage of infected kernel varied from one commercial outlet to another in the region. Briefly, peanut seeds from Cartersville had the highest percentage of A. flavus infection. Electron microscopy confirmed the seed-borne infection of this mold. Mycelium established inside the host tissues both intercellularly and intracellularly aided by active, continuous branching of young hyphae. Establishment of mycelium was also detected in the xylem vessels of roots indicative of systemic infection. Thus, edible peanuts can form an important source of inoculum and facilitate the spread of the fungus from one peanut to another in commercial outlets and elsewhere. Present study provides strong evidence that A. flavus can escape detection at selling points and lands in commercial outlets via edible peanuts. That these contaminated peanuts could pose public health hazards is discussed.

[Mitochondria aberration of Aspergillus flavus under citral stress circumstance].

Observation of mitochondrion in Aspergillus flavus damaged by citral under uransmission electron microscope, it was found that mitochondria had changes on number increase and shape aberrance as hyperplasia or hypertrophy, which resulted from the DNA replication system in mitochondria was damaged by citral. From the data that free radical in A. flavus which were determined by MDA method, it was showed that citral damaged mitochondria via induced free radical, which affected oxidation-reduction system and energy metabolism.

Culture media and sources of nitrogen promoting the formation of stromata and ascocarps in Petromyces alliaceus (Aspergillus section Flavi).

Petromyces alliaceus Malloch and Cain is the only known sexually reproducing fungus classified in Aspergillus section Flavi. The goal of this research was to identify culture media and sources of nitrogen that best support the formation of stromata with ascocarps. Three cultures of P. alliaceus isolated from crop field soils were grown on selected agar media in Petri dishes for 7 months at 30 degrees C in darkness. The largest numbers of stromata were recorded for cultures grown on Czapek's agar (CZA) and a mixed cereal agar (MCA), while the percentage of stromata containing ascocarps was greatest (P

Surface ultrastructural studies on the germination, penetration and conidial development of Aspergillus flavus Link:Fries infecting silkworm, Bombyx mori Linn.

Aspergillosis is a common disease of the silkworm Bombyx mori Linn., caused by an insect mycopathogen Aspergillus flavus Link:Fries. The present study reveals the germination, penetration and conidial development of A. flavus on the larval integument of B. mori under SEM. Four different strains (NB18, KA, NB4D2 and NB7) of B. mori was surface inoculated with ca. of 1 x 10(6) conidia/ml. Each conidium germinated on the cuticle approximately 6 h after inoculation, forming a humpy or suctorial appressoria within 24 h. The hyphae which entered into haemocoel 2 day post-inoculation, grew and multiplied extensively, forming a mycelial complex, causing death of the host larva in about 4-5 days. This occurred with minimal breakdown of the internal tissues. Death of the host was followed by ramification of the fungus through the mesodermal and epidermal tissues, leading to larval mummification about 5-6 days after inoculation. Extensive fungal growths on the entire larval body followed, consisting of aerial hyphae, which developed branched conidiophores. The aerial hyphae with abundant conidiophores formed a confluent yellowish green fungal mat over the entire larval body in 6-7 days of post-inoculation. The tip of each emerging conidiophores gradually dilated and developed to become a bulbous head known as the vesicle. A large number of conidiogenous cells were produced over the entire surface of vesicle, which later developed into finger-like projections termed as sterigmata or phialides. The phialides matured within 2 days after the aerial hyphae emerged as evidenced by chains of conidia at their tips. The conidia were globose with externally roughened walls. The life cycle of the fungus on B. mori was completed in six to seven days.

Distribution of aflatoxin-producing Aspergillus flavus and Aspergillus parasiticus in sugarcane fields in the southernmost islands of Japan.

The distribution of Aspergillus flavus and Aspergillus parasiticus in sugarcane field soils and on harvested sugarcane stems was studied on seven islands of Okinawa and Kagoshima Prefectures, the southernmost prefectures in Japan. With the use of a combination of dilution plate and plant debris plate techniques, the fungi were detected on all seven islands studied and in 74% of 53 soil samples. The fungi were also found on the cut surfaces of sugarcane stems from one of the islands. A. parasiticus was the predominant fungus, although many atypical A. parasiticus isolates that produced metulated conidial heads were also obtained. The proportions of isolates testing positive for aflatoxin production were ca. 89% (146 of 164) of all isolates and ca. 69% of A. flavus isolates. More than 40% of A. flavus isolates also produced G aflatoxins. Scanning electron microscopic observation of conidial wall texture was useful in distinguishing A. parasiticus from A. flavus. Cyclopiazonic acid, an indole mycotoxin, was never synthesized by any of the A. parasiticus or G aflatoxin-producing A. flavus isolates tested.

Aflatoxin related occupational exposure to maize processing workers.

A study was undertaken on environmental mycoflora of a maize processing industry in Ahmedabad. The airborne fungal communities were isolated and identified both qualitatively by Petri-plate exposure method and quantitatively by using Andersen-6-stage viable sampler, Midget impinger and high volume samples (cone and Hexhlet for total and respirable dusts, respectively). Of all the isolates genus Aspergillus was the dominant environmental mycoflora and among all the species of Aspergillus A. flavus was the common isolates irrespective of the method applied for sample collection. Maximum number of isolates were recovered from Elevator department. From total and respirable dusts, about 56.6% and 44.4% of recovery accounted for genus Aspergillus alone. Total percentages of aflatoxin positive strains of A. flavus were 5.65% and 9.73% from total and respirable dusts, respectively. These toxigenic strains were identified on various media like CZ with 0.05% anisaldehyde, APA and CAM. Surface morphology of toxigenic strains and dust samples were carried out using SEM.

[Study on biochemical mechanism of citral damage to the A. flavasi's mitochondria].

Adopted biochemical method and combinded with Scanning Electron Microscope(SEM) to observe, study the inhibition-growth mechanism to A. flavus when citral was permeated to the cell and damaged mitochondria, the result suggested that the mitochondria changed in many kinds of unrule shape and the oxidation-reduction system was destroyed, when the citral reached at the level of anaphylactic consistency to A. flavus. Compared with the control group, activities of MDH and SDH in experimental group non-reversibly decreased 27.1% and 23.8% respectively, and grandully lost. When respectively used the succinate, pyruvate and alpha-ketoglutarate as substrates, the respiration speed of mitochondria separatelly decreased 24.1%, 36.1%, 14.3%. It suggested that the citral could inhibit the biosynthesis of the mycelial DNA, RNA, lipoid and protein, so promoted to dath.

New antibiotics miyakamides produced by a fungus.

New antibiotics, miyakamides A1, A2, B1, and B2, were isolated from the cultured broth of Aspergillus flavus Link var. columnaris FKI-0739 together with known compounds, parasiticolide A, hydroxyaspergillic acid, and kojic acid. The structure of miyakamide A1 is N-acetyl-L-phenylalanyl-N-methyl-L-phenylalanyl-(alphaZ)-alpha,beta-didehydrotryptamine, and miyakamide A2 is E isomer of A1 at didehydrotryptamine. The structure of miyakamide B1 is N-acetyl-L-tyrosyl-N-methyl-L-phenylalanyl-(alphaZ)-alpha,beta-didehydrotryptamine, and B2 is E isomer of B1. Both miyakamides A1 and B1 existed as equilibrium isomers in solvents, and this isomerism was associated with cis-trans rotation of the amide bond between two amino acids. Conformational isomerism between two amino acids of miyakamides A2 and B2 is cis-form. Miyakamides showed growth inhibitory activity against brine shrimp, Artemia salina.