Characterization of morphological changes within stromata during sexual reproduction in Aspergillus flavus.

Aspergillus flavus contaminates agricultural products worldwide with carcinogenic aflatoxins that pose a serious health risk to humans and animals. The fungus survives adverse environmental conditions through production of sclerotia. When fertilized by a compatible conidium of an opposite mating type, a sclerotium transforms into a stroma within which ascocarps, asci, and ascospores are formed. However, the transition from a sclerotium to a stroma during sexual reproduction in A. flavus is not well understood. Early events during the interaction between sexually compatible strains of A. flavus were visualized using conidia of a green fluorescent protein (GFP)-labeled MAT1-1 strain and sclerotia of an mCherry-labeled MAT1-2 strain. Both conidia and sclerotia of transformed strains germinated to produce hyphae within 24 h of incubation. Hyphal growth of these two strains produced what appeared to be a network of interlocking hyphal strands that were observed at the base of the mCherry-labeled sclerotia (i.e., region in contact with agar surface) after 72 h of incubation. At 5 wk following incubation, intracellular green-fluorescent hyphal strands were observed within the stromatal matrix of the mCherry-labeled strain. Scanning electron microscopy of stromata from a high- and low-fertility cross and unmated sclerotia was used to visualize the formation and development of sexual structures within the stromatal and sclerotial matrices, starting at the time of crossing and thereafter every 2 wk until 8 wk of incubation. Morphological differences between sclerotia and stromata became apparent at 4 wk of incubation. Internal hyphae and croziers were detected inside multiple ascocarps that developed within the stromatal matrix of the high-fertility cross but were not detected in the matrix of the low-fertility cross or the unmated sclerotia. At 6 to 8 wk of incubation, hyphal tips produced numerous asci, each containing one to eight ascospores that emerged out of an ascus following the breakdown of the ascus wall. These observations broaden our knowledge of early events during sexual reproduction and suggest that hyphae from the conidium-producing strain may be involved in the early stages of sexual reproduction in A. flavus. When combined with omics data, these findings could be useful in further exploration of the molecular and biochemical mechanisms underlying sexual reproduction in A. flavus.

Morphologic, molecular and metabolic characterization of Aspergillus section Flavi in spices marketed in Lebanon.

Spices are used extensively in Lebanon not only to flavour foods but also for their medicinal properties. To date, no data are available regarding the nature of the toxigenic fungal species that may contaminate these products at the marketing stage in this country. Eighty samples corresponding to 14 different types of spices were collected throughout Lebanon to characterize the Aspergillus section Flavi contaminating spices marketed in Lebanon and the toxigenic potential of these fungal species. Most fungal genera and species were identified as belonging to Aspergillus section Flavi. Aspergillus flavus was the most frequent species, representing almost 80% of the isolates. Although identified as A. flavus by molecular analysis, some strains displayed atypical morphological features. Seven strains of A. tamarii and one A. minisclerotigenes were also isolated. Analyses of toxigenic potential demonstrated that almost 80% of strains were able to produce mycotoxins, 47% produced aflatoxins, and 72% produced cyclopiazonic acid, alone or in combination with aflatoxins.

Thymol Induces Conidial Apoptosis in Aspergillus flavus via Stimulating K+ Eruption.

Aspergillus flavus is a notorious foodborne fungus, posing a significant risk to humans in the form of hepatocellular carcinoma or aspergillosis. Thymol, as a food preservative, could efficiently kill conidia of A. flavus. However, the underlying mechanisms by which thymol kills A. flavus are not completely understood. With specific fluorescent dyes, we detected several apoptotic hallmarks, including chromatin condensation, phosphatidylserine externalization, DNA damage, mitochondrial depolarization, and caspase 9 activation in conidia exposed to 200 µg/mL of thymol, indicating that thymol induced a caspase-dependent conidial apoptosis in A. flavus. Chemical-protein interactome (CPI) and autodock analyses showed that KCNAB, homologue to the ß-subunit of the voltage-gated potassium channel (Kv) and aldo-keto reductase, was the potential target of thymol. Following studies demonstrated that thymol could activate the aldo-keto reductase activity of KCNAB in vitro and stimulate a transient K+ efflux in conidia, as determined using a Port-a-Patch. Blocking K+ eruption by 4-aminopyridine (a universal inhibitor of Kv) could significantly alleviate thymol-mediated conidial apoptosis, indicating that activation of Kv was responsible for the apoptosis. Taken together, our results revealed a K+ efflux-mediated apoptotic pathway in A. flavus, which greatly contributed to the development of an alternative strategy to control this pathogen.

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus.

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 106 conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

Transient transmembrane secretion of H2O2: a mechanism for the citral-caused inhibition of aflatoxin production from Aspergillus flavus.

A polydopamine-Fe3O4 nanocomposite-based H2O2 electrochemical sensor is fabricated to real-time monitor the transmembrane release of reactive oxygen species from citral-treated Aspergillus flavus, revealing a mechanism involving transient transmembrane secretion of H2O2 for the citral-caused inhibition of aflatoxin production from a fungus for the first time.

Self-sufficient redox biotransformation of lignin-related benzoic acids with Aspergillus flavus.

Aromatic carboxylic acids are readily obtained from lignin in biomass processing facilities. However, efficient technologies for lignin valorization are missing. In this work, a microbial screening was conducted to find versatile biocatalysts capable of transforming several benzoic acids structurally related to lignin, employing vanillic acid as model substrate. The wild-type Aspergillus flavus growing cells exhibited exquisite selectivity towards the oxidative decarboxylation product, 2-methoxybenzene-1,4-diol. Interestingly, when assaying a set of structurally related substrates, the biocatalyst displayed the oxidative removal of the carboxyl moiety or its reduction to the primary alcohol whether electron withdrawing or donating groups were present in the aromatic ring, respectively. Additionally, A. flavus proved to be highly tolerant to vanillic acid increasing concentrations (up to 8 g/L), demonstrating its potential application in chemical synthesis. A. flavus growing cells were found to be efficient biotechnological tools to perform self-sufficient, structure-dependent redox reactions. To the best of our knowledge, this is the first report of a biocatalyst exhibiting opposite redox transformations of the carboxylic acid moiety in benzoic acid derivatives, namely oxidative decarboxylation and carboxyl reduction, in a structure-dependent fashion.

Utilization of waste fruit-peels to inhibit aflatoxins synthesis by Aspergillus flavus: a biotreatment of rice for safer storage.

Antifungal activity in lemon and pomegranate peels was considerable against Aspergillus flavus, higher in pomegranate (DIZ 37mm; MIC 135µg/mL). Powdered peels (5, 10, 20% w/w) were mixed in inoculated rice. The inhibitory effect on fungal-growth and production of aflatoxins by A. flavus was investigated at storage conditions - temperature (25, 30°C) and moisture (18%, 21%) for 9months. The maximum total aflatoxins accumulated at 30°C, 21% moisture and at 25°C, 18% moisture were 265.09 and 163.45ng/g, respectively in control. Addition of pomegranate-peels inhibited aflatoxins production to 100% during four month-storage of rice at 25°C and 18% moisture, while lemon-peels showed similar inhibitory effect for 3months at same conditions. However a linear correlation was observed in aflatoxins level with temperature and moisture. Studies showed that both fruit-wastes are potent preventer of aflatoxin production in rice, useful for a safer and longer storage of rice.

Role of oxidative stress in Sclerotial differentiation and aflatoxin B1 biosynthesis in Aspergillus flavus.

We show here that oxidative stress is involved in both sclerotial differentiation (SD) and aflatoxin B1 biosynthesis in Aspergillus flavus. Specifically, we observed that (i) oxidative stress regulates SD, as implied by its inhibition by antioxidant modulators of reactive oxygen species and thiol redox state, and that (ii) aflatoxin B1 biosynthesis and SD are comodulated by oxidative stress. However, aflatoxin B1 biosynthesis is inhibited by lower stress levels compared to SD, as shown by comparison to undifferentiated A. flavus. These same oxidative stress levels also characterize a mutant A. flavus strain, lacking the global regulatory gene veA. This mutant is unable to produce sclerotia and aflatoxin B1. (iii) Further, we show that hydrogen peroxide is the main modulator of A. flavus SD, as shown by its inhibition by both an irreversible inhibitor of catalase activity and a mimetic of superoxide dismutase activity. On the other hand, aflatoxin B1 biosynthesis is controlled by a wider array of oxidative stress factors, such as lipid hydroperoxide, superoxide, and hydroxyl and thiyl radicals.

Biotransformation of bavachinin by three fungal cell cultures.

Biotransformation of bavachinin (1) was investigated using three fungal cell cultures of Aspergillus flavus ATCC 30899, Cunninghamella elegans CICC 40250 and Penicillium raistrickii ATCC 10490, respectively. Two major converted products were identified by LC/MS, (1)H NMR and (13)C NMR and X-ray diffraction. Two biocatalyst systems, A. flavus ATCC 30899 and C. elegans CICC 40250 cell cultures, showed a great capacity of hydroxylation and two hydroxyl groups were attached at C-2″ and C-3″ positions in the side chain of the bavachinin A-ring, resulting in the formation of the same compound with a name, (S)-6-((R)-2,3-dihydroxy-3-methylbutyl)-2-(4-hydroxyphenyl)-7-methoxychromen-4-one (2). On the other hand, P. raistrickii ATCC 10490 cell cultures possessed the ability to reduction at C-4 of the substrate C-ring, resulting in the production of (2S,4R)-2-(4-hydroxyphenyl)-7-methoxy-6-(3-methylbut-2-en-1-yl)chromen-4-ol (3). Furthermore, the in vitro anti-tumor activities of the above compounds were evaluated by MTT assay. Compared with the substrate (1), product 3 possessed stronger inhibition activity on the human breast cancer cell line (MCF-7) and slightly lower inhibition activities against Hep G2, HeLa, Hep-2 and A549 cells lines; while the hydroxyl product 2 possessed much lower inhibition activity on tumor cells lines, which might be related to the insertion of two hydroxyl groups. Compounds 2 and 3 were considered to be novel. It was also the first time to biotransform bavachinin (1) by these three fungi, which suggested the potential role of microbial enzymes to synthesize novel compounds from plant secondary metabolites.

Sexual reproduction in Aspergillus flavus sclerotia naturally produced in corn.

Aspergillus flavus is the major producer of carcinogenic aflatoxins worldwide in crops. Populations of A. flavus are characterized by high genetic variation and the source of this variation is likely sexual reproduction. The fungus is heterothallic and laboratory crosses produce ascospore-bearing ascocarps embedded within sclerotia. However, the capacity for sexual reproduction in sclerotia naturally formed in crops has not been examined. Corn was grown for 3 years under different levels of drought stress at Shellman, GA, and sclerotia were recovered from 146 ears (0.6% of ears). Sclerotia of A. flavus L strain were dominant in 2010 and 2011 and sclerotia of A. flavus S strain were dominant in 2012. The incidence of S strain sclerotia in corn ears increased with decreasing water availability. Ascocarps were not detected in sclerotia at harvest but incubation of sclerotia on the surface of nonsterile soil in the laboratory resulted in the formation of viable ascospores in A. flavus L and S strains and in homothallic A. alliaceus. Ascospores were produced by section Flavi species in 6.1% of the 6,022 sclerotia (18 of 84 ears) in 2010, 0.1% of the 2,846 sclerotia (3 of 36 ears) in 2011, and 0.5% of the 3,106 sclerotia (5 of 26 ears) in 2012. For sexual reproduction to occur under field conditions, sclerotia may require an additional incubation period on soil following dispersal at crop harvest.

The inhibitory effects of Curcuma longa L. essential oil and curcumin on Aspergillus flavus link growth and morphology.

The essential oil from Curcuma longa L. was analysed by GC/MS. The major components of the oil were ar-turmerone (33.2%), α -turmerone (23.5%) and ß -turmerone (22.7%). The antifungal activities of the oil were studied with regard to Aspergillus flavus growth inhibition and altered morphology, as preliminary studies indicated that the essential oil from C. longa inhibited Aspergillus flavus Link aflatoxin production. The concentration of essential oil in the culture media ranged from 0.01% to 5.0% v/v, and the concentration of curcumin was 0.01-0.5% v/v. The effects on sporulation, spore viability, and fungal morphology were determined. The essential oil exhibited stronger antifungal activity than curcumin on A. flavus. The essential oil reduced the fungal growth in a concentration-dependent manner. A. flavus growth rate was reduced by C. longa essential oil at 0.10%, and this inhibition effect was more efficient in concentrations above 0.50%. Germination and sporulation were 100% inhibited in 0.5% oil. Scanning electron microscopy (SEM) of A. flavus exposed to oil showed damage to hyphae membranes and conidiophores. Because the fungus is a plant pathogen and aflatoxin producer, C. longa essential oil may be used in the management of host plants.

Fungus-mediated biosynthesis and characterization of TiO2 nanoparticles and their activity against pathogenic bacteria.

In the present study, the biosynthesis of TiO(2) nanoparticles (TiO(2) NPs) was achieved by a novel, biodegradable and convenient procedure using Aspergillus flavus as a reducing and capping agent. Research on new, simple, rapid, eco-friendly and cheaper methods has been initiated. TiO(2) NPs were characterized by FTIR, XRD, AFM, SEM and TEM studies. The X-ray diffraction showed the presence of increased amount of TiO(2) NPs which can state by the presence of peaks at rutile peaks at 100, 002, 100 and anatase forms at 101 respectively. SEM observations revealed that synthesized TiO(2) NPs were spherical, oval in shape; individual nanoparticles as well as a few aggregate having the size of 62-74 nm. AFM shows crystallization temperature was seen on the roughness of the surface of TiO(2). The Minimum inhibitory concentration value for the synthesized TiO(2) NPs was found to be 40 µg ml(-1) for Escherichia coli, which was corresponding to the value of well diffusion test. This is the first report on antimicrobial activity of fungus-mediated synthesized TiO(2) NPs, which was proved to be a good novel antibacterial material.

Effect of Zataria multiflora Boiss. essential oil on colony morphology and ultrastructure of Aspergillus flavus.

The mode of inhibitory action of Zataria multiflora Boiss. essential oil (EO) on the fungus, Aspergillus flavus, was studied by colony morphology examination, light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The EO at concentrations used in this study suppressed the size of the colony as well as sporulation. SEM of mycelia treated with given concentrations of EO showed morphological alterations ranging from loss of turgidity and uniformity of mycelia at low concentrations of EO to evident destruction of the hyphae at higher concentration of EO. Semi-thin sections of mycelia exposed to different concentrations of EO were analysed by light microscopy and revealed that the major change at level as low as 50 ppm of EO was limited to vacuolisation of cytoplasm resulting in cell swelling, while at higher concentrations, detachment of the cell membrane from the cell wall, deformation of mycelia and shedding the cytoplasm from the cell were the main alterations. These damages were well documented by TEM, which showed that the main sites of action of EO were the plasma membrane and cell wall. In conclusion, morphological and structural changes observed in this study may be one of the mechanisms involved in growth inhibition of the fungi and reducing aflatoxin production.

Atypical Aspergillus flavus isolates associated with chronic azole therapy.

A case of chronic sinus disease due to morphologically atypical Aspergillus flavus is described. Multiple fungal isolates sporulated poorly or not at all, displaying unusual color and microscopic morphology, including the absence of typical vesicles and phialides, which caused the isolates to resemble several other fungal genera superficially. The patient received multiple antifungal therapies over at least 10 years with various azole drugs, including voriconazole, itraconazole, and posaconazole. We speculate that this lengthy exposure to azole antifungal drugs may have caused or promoted the atypical morphology seen in these isolates.

Sexual reproduction in Aspergillus flavus.

Aspergillus flavus is the major producer of carcinogenic aflatoxins in crops worldwide and is also an important opportunistic human pathogen in aspergillosis. The sexual state of this heterothallic fungus is described from crosses between strains of the opposite mating type. Sexual reproduction occurred between sexually compatible strains belonging to different vegetative compatibility groups. Multiple, indehiscent ascocarps containing asci and ascospores formed within the pseudoparenchymatous matrix of stromata, which places the fungus in genus Petromyces. The teleomorph of P. flavus could not be distinguished from that of P. parasiticus (anamorph = A. parasiticus), another aflatoxin-producing species, based on morphology of the sexual structures. The two species can be separated by anamorph morphology, mycotoxin profile and molecular characters.

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.

Use of a granular bioplastic formulation for carrying conidia of a non-aflatoxigenic strain of Aspergillus flavus.

Previous research demonstrated that aflatoxin contamination in corn is reduced by field application of wheat grains pre-inoculated with the non-aflatoxigenic Aspergillus flavus strain NRRL 30797. To facilitate field applications of this biocontrol isolate, a series of laboratory studies were conducted on the reliability and efficiency of replacing wheat grains with the novel bioplastic formulation Mater-Bi to serve as a carrier matrix to formulate this fungus. Mater-Bi granules were inoculated with a conidial suspension of NRRL 30797 to achieve a final cell density of approximately log 7 conidia/granule. Incubation of 20-g soil samples receiving a single Mater-Bi granule for 60-days resulted in log 4.2-5.3 propagules of A. flavus/g soil in microbiologically active and sterilized soil, respectively. Increasing the number of granules had no effect on the degree of soil colonization by the biocontrol fungus. In addition to the maintenance of rapid vegetative growth and colonization of soil samples, the bioplastic formulation was highly stable, indicating that Mater-Bi is a suitable substitute for biocontrol applications of A. flavus NRRL 30797.

Effects of Cymbopogon citratus L. essential oil on the growth, morphogenesis and aflatoxin production of Aspergillus flavus ML2-strain.

The mycelial growth of Aspergillus flavus Link was completely inhibited using 1.5 (microl/ml or 2.0 (microl/ml of Cymbopogon citratus essential oil applied by fumigation or contact method in Czapek's liquid medium, respectively. This oil was found also to be fungicidal at the same concentrations. The sublethal doses 1.0 and 1.5 (microl/ml inhibited about 65% of fungal growth after five days of incubation and delayed conidiation as compared with the control. Microscopic observations using Light Microscope (LM), Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) were carried out to determine the ultra structural modifications of A. flavus hyphae after treatment with C. citratus essential oil. The hyphal diameter decreased and hyphal wall appeared as precipitates and disappeared in some regions. This oil also caused plasma membrane disruption and mitochondrial structure disorganization. Moreover, Ca(+2), K(+) and Mg(+2) leakages increased from the fumigated mycelium and its total lipid content decreased, while the saturated and unsaturated fatty acids increased. One of the most important results obtained during this study was the ability of C. citratus essential oil at its sublethal dose to completely inhibit aflatoxin B(1) production from A. flavus. These findings increase the possibility of exploiting C. citratus essential oil as an effective inhibitor of biodegradation and storage contaminating fungi and also in fruit juice preservation.

Production of cyclopiazonic acid, aflatrem, and aflatoxin by Aspergillus flavus is regulated by veA, a gene necessary for sclerotial formation.

The plant pathogenic fungus Aspergillus flavus produces several types of mycotoxins. The most well known are the carcinogenic compounds called aflatoxins. In addition, A. flavus produces cyclopiazonic acid and aflatrem mycotoxins, contributing to the toxicity of A. flavus infected crops. Cyclopiazonic acid is a specific inhibitor of calcium-dependent ATPase in the sarcoplasmic reticulum that results in altered cellular Ca++ levels. Aflatrem is a potent tremorgenic mycotoxin known to lead to neurological disorders. Previously we showed that a gene called veA controls aflatoxin and sclerotial production in A. parasiticus. In this study in A. flavus, we show that the veA homolog in A. flavus not only is necessary for the production of aflatoxins B1 and B2 and sclerotia, but also regulates the synthesis of the mycotoxins cyclopiazonic acid and aflatrem. The A. flavus DeltaveA mutant was completely blocked in the production of aflatrem and showed greater than twofold decrease in cyclopiazonic acid production. The genes involved in the synthesis of cyclopiazonic acid are unknown; however, the aflatrem gene cluster has been characterized. Northern hybridization analysis showed that veA is required for expression of the A. flavus aflatrem genes atmC, atmG, and atmM. This is the first report of a regulatory gene governing the production of cyclopiazonic acid and aflatrem mycotoxins.

[Study on morphological change of aspergillus flavus damaged by citral with a multi-channel micro-spectrophotometer system].

Citral extracted from litsea cubeb oil was used as antibacterial drug; toxigenic and atoxigenic Aspergillus flavus cell (AFC) were used as its target. Multi-channel micro-spectrophotometer (MMSP) and micro-image analysis were applied. Under the pressure of citral, changes in AFC were measured, including items such as absorbance, area, perimeter, major axis length and minor axis length. We found that toxigenic AFC had two characteristic absorbent peaks at 410 nm and 665 nm, and that the waveforms of absorption spectrum of toxigenic and atoxigenic AFC migrated and their peak areas increased, and that four morphological parameters reduced with citral concentration increasing. The results suggest that citral damages selective permeability and structure of membrane to enter to cell, and acts on target molecule or organelle to bring changes in physiology and biochemistry. In real-time, in-situ and non-invasive situation, the dynamic changes in shape and intracellular macromolecule are fastly measured during the drugs damage a living cell, which not only provides these changes with necessary physical parameters, but also has an importance in theoretical research and experimental method.