Luteapyrone, a Novel ƴ-Pyrone Isolated from the Filamentous Fungus Metapochonia lutea.

In the process of screening for new bioactive microbial metabolites we found a novel ƴ-pyrone derivative for which we propose the trivial name luteapyrone, in a recently described microscopic filamentous fungus, Metapochonia lutea BiMM-F96/DF4. The compound was isolated from the culture extract of the fungus grown on modified yeast extract sucrose medium by means of flash chromatography followed by preparative HPLC. The chemical structure was elucidated by NMR and LC-MS. The new compound was found to be non-cytotoxic against three mammalian cell lines (HEK 263, KB-3.1 and Caco-2). Similarly, no antimicrobial activity was observed in tested microorganisms (gram positive and negative bacteria, yeast and fungi).

The Fungi-specific histone Acetyltransferase Rtt109 mediates morphogenesis, Aflatoxin synthesis and pathogenicity in Aspergillus flavus by acetylating H3K9

Aspergillus flavus is a common saprophytic filamentous fungus that produces the highly toxic natural compound aflatoxin during its growth process. Synthesis of the aflatoxins, which can contaminate food crops causing huge losses to the agricultural economy, is often regulated by epigenetic modification, such as the histone acetyltransferase. In this study, we used Aspergillus flavus as an experimental model to construct the acetyltransferase gene rtt109 knockout strain (△rtt109) and its complementary strain (△rtt109·com) by homologous recombination. The growth of △rtt109 was significantly suppressed compared to the wild type (WT) strain and the △rtt109·com strain. The sclerotium of △rtt109 grew smaller, and the amount of sclerotia generated by △rtt109 was significantly reduced. The number of conidiums of △rtt109 was significantly reduced, especially on the yeast extract sucrose (YES) solid medium. The amount of aflatoxins synthesized by △rtt109 in the PDB liquid medium was significantly decreased We also found that the △rtt109 strain was extremely sensitive to DNA damage stress. Through the maize seed infection experiment, we found that the growth of △rtt109 on the surface of affected corn was largely reduced, and the amount of aerial mycelium decreased significantly, which was consistent with the results on the artificial medium. We further found that H3K9 was the acetylated target of Rtt109 in A. flavus. In conclusion, Rtt109 participated in the growth, conidium formation, sclerotia generation, aflatoxin synthesis, environmental stress response, regulation of infection of A. flavus. The results from this study of rtt109 showed data for acetylation in the regulation of life processes and provided a new thought regarding the prevention and control of A. flavus hazards.

Postharvest decay of mandarin fruit in Serbia caused by Penicillium expansum

Mandarin fruits are one of the most popular among the Citrus genus. They are consumed because of their nutritional and health benefits, as well as pleasant taste and smell. This paper describes the identification and characterization of Penicillium expansum, isolated from molded mandarin fruits. The obtained isolates were cultivated on five media [Czapek Yeast Autolysate agar (CYA), MEA (Malt extract agar), Creatine sucrose agar (CREA), Yeast extract sucrose agar (YES), and Oatmeal agar (OA)] and at five different incubation temperatures (5, 15, 25, 30, and 37 ?C). Isolates were sequenced for two molecular loci: internal transcribed spacer and beta-tubulin. Based on the results from morphological, physiological, molecular, and phylogenetic analyses, the recovered isolates were identified as P. expansum. The isolated species was confirmed as pathogenic to mandarin fruits in a pathogenicity test. To the best of our knowledge, this is the first report of P. expansum as a postharvest pathogen of mandarin fruit in Serbia.

Carbon Catabolite Repression Gene AoCreA Regulates Morphological Development and Ochratoxin A Biosynthesis Responding to Carbon Sources in Aspergillus ochraceus

Carbon is one of the most important nutrients for the development and secondary metabolism in fungi. CreA is the major transcriptional factor mediating carbon catabolite repression, which is employed in the utilization of carbon sources. Aspergillus ochraceus contaminates various food and feed containing different carbon sources by producing ochratoxin A (OTA). However, little is known about the function of AoCreA in regulating the morphology and OTA production of A. ochraceus. To give an insight into the mechanism of the carbon sources regulating development of A. ochraceus and OTA production, we have identified AoCreA in A. ochraceus. The homologous recombination strategy was used to generate the AoCreA deletion mutant (ΔAoCreA). We have investigated the morphology and OTA production of the wild type (WT) and ΔAoCreA of A. ochraceus with media containing different carbon sources (glucose, fructose, maltose, D-xylose, D-mannose, acetate, D-galactose, D-mannitol and lactose). ΔAoCreA showed a significant growth and conidiation defect on all media as compared with WT. Glucose and maltose were the most inducing media for OTA production by A. ochraceus, followed by sucrose and the nutrient-rich Yeast Extract Sucrose (YES) and Potato Dextrose Agar (PDA). The deletion of AoCreA led to a drastic reduction of OTA production on all kinds of media except PDA, which was supported by the expression profile of OTA biosynthetic genes. Furthermore, infection studies of ΔAoCreA on oats and pears showed the involvement of AoCreA in the pathogenicity of A. ochraceus. Thus, these results suggest that AoCreA regulates morphological development and OTA biosynthesis in response to carbon sources in A. ochraceus.

Polyphasic Assessment of Aflatoxin Production Potential in Selected Aspergilli

This study investigated the aflatoxin production potentials of selected fungi using a polyphasic approach. Internally transcribed spacer region of the fungi was amplified using the polymerase chain reaction. Forty-five Aspergillus strains were further assessed for aflatoxin production using the conventional methods such as growth on yeast extract sucrose, β-cyclodextrin neutral red desiccated coconut agar (β-CNRDCA); expression of the aflatoxin regulatory genes and the use of both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A large proportion (82.22%) of the isolates harbored the Nor-1 gene while 55.56%, 68.89%, and 80% possessed the ver-1, omt-A, and aflR genes, respectively. All 100% the isolates harbored the aflJ gene. Twenty-three isolates were positive for aflatoxin production based on the yeast extract sucrose medium (YES) test; ammonium vapor test (51%), yellow pigment production (75.5%), and β-CNRDCA tests; and blue/green fluorescence (57.7%). Based on TLC detection 42.2% produced aflatoxins while in the HPLC, total aflatoxin (AFTOT) production concentrations ranged from 6.77–71,453 µg/g. Detectable aflatoxin B1 (AFB1) concentrations obtained from the HPLC ranged between 3.76 and 70,288 µg/g; 6.77 and 242.50 µg/g for aflatoxin B2 (AFB2); 1.87 and 745.30 µg/g for aflatoxin G1 (AFG1); and 1.67 and 768.52 µg/g for aflatoxin G2 (AFG2). AFTOT contamination levels were higher than European Union tolerable limits (4 µg/kg). The regression coefficient was one (R2 = 1) while significant differences exist in the aflatoxin concentrations of Aspergillus (p ≤ 0.05). This study reports the potentials of Aspergillus oryzae previously known as a non-aflatoxin producer to produce AFG1, AFG2, AFB1, and AFB2 toxins. Aspergillus species in feedlots of animals reared for food are capable of producing aflatoxins which could pose hazards to health.

Impact of Silver Nanoparticles on Gene Expression in Aspergillus Flavus Producer Aflatoxin B1

AIM: In this study, we evaluated the effect of silver nanoparticles (AgNPs) on the production of aflatoxin B1 (AFB1) through assessment the transcription activity of aflatoxin biosynthesis pathway genes in Aspergillus flavus ATCC28542.MATERIAL AND METHODS: The mRNAs were quantitative by Real Time-polymerase chain reaction (qRT-PCR) of A. flavus grown in yeast extract sucrose (YES) medium containing AgNPs. Specific primers that are involved in the AFB1 biosynthesis which highly specific to A. flavus, O-methyltransferase gene (omt-A), were designed and used to detect the fungus activity by quantitative PCR assay. The AFB1 production (from A. flavus growth) which effected by AgNPs were measured in YES medium by high-pressure liquid chromatography (HPLC).RESULTS: The AFB1 produced by A. flavus have the highest reduction with 1.5 mg -100 ml of AgNPs were added in media those records 88.2%, 67.7% and 83.5% reduction by using AgNP HA1N, AgNP HA2N and AgNP EH, respectively. While on mycelial growth give significantly inhibitory effect. These results have been confirmed by qRT-PCR which showed that culture of A. flavus with the presence of AgNPs reduced the expression levels of omt-A gene.CONCLUSION: Based on the results of the present study, AgNPs inhibit growth and AFB1 produced by Aspergillus flavus ATCC28542. This was confirmed through RT-PCR approach showing the effect of AgNPs on omt-A gene involved in aflatoxin biosynthesis.

Improving Conditions for Gliotoxin Production by Local Isolates of Aspergillus fumigatus

Thirty two isolates of Aspergillus fumigatus were obtained from a total of 44 samples of sputum, nose swab and tracheal aspirate from suspected patient with aspergillosis were also collected from February 2014 to June 2014. A morphological examination of A. fumigatus was first made with naked eye and at low magnification power of microscope after that detailed examination was done by measuring the dimensions of the microscopic structures, photographing the microscopic structures and using relevant literature. Results appeared conical-shaped terminal vesicles, uniseriate row of phialides on the upper two thirds of the vesicle, conidiophore stipes were short, phialides arrange uniseriate upper vesicle conidia and parallel to axis of conidiophore, produced in chains of spore basipetally from phialides, the chains of spore were borne directly in the absence of metulae and represented by septet and branching hyphae. The ability of A. fumigatus for GT production was investigated using thin layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) techniques and results showed that GT was produced by 81.25% of A. fumigatus isolates. Optimum conditions for GT production by A. fumigatus 16 (AF-16) isolate were determined by submerged fermentation using Yeast extract sucrose medium. Results indicated that AF-16 isolate was the highest GT producer on Yeast Exract Sucrose medium with inoculum size 2×107 conidium and incubation at 32ºC for 15 days and the concentration of gliotoxin was (4511µg mL-1).

Fumonisins production potential of Fusarium verticillioides isolated from Serbian maize and wheat kernels

The production of fumonisins by potentially toxigenic Fusarium verticillioides isolates originating from Serbian maize and wheat kernels was tested in vitro. A total of six F. verticillioides isolates were incubated on yeast extract sucrose medium (YESA) for 4 weeks at 25 ?C in the dark. Their toxin production potential was tested by applying a modified HPLC method for determination of fumonisins in cereals, since the TLC method gave no results. Analyses were performed on a HPLC-FLD system after sample extraction from YESA and extract cleanup on a SPE column. Although the isolates were tested for fumonisin B1, B2 and B3, only fumonisin B1 was detected. The results showed that all tested isolates had toxigenic potential for fumonisin B1 production. The average fumonisin B1 production of the isolates ranged from 7 to 289 ?g/kg, thus indicating a highly variable toxigenic potential among the isolates. Isolate 1282 expressed the highest toxigenic potential for fumonisin B1 production (289 ?g/kg), while isolate 2533/A showed a questionable potential for fumonisin production (7 ?g/kg).

Antifungal Activity and Aflatoxin Degradation of Bifidobacterium Bifidum and Lactobacillus Fermentum Against Toxigenic Aspergillus Parasiticus

Food and feedstuff contamination with aflatoxins (AFTs) is a serious health problem for humans and animals, especially in developing countries. The present study evaluated antifungal activities of two lactic acid bacteria (LAB) against growth and aflatoxin production of toxigenic Aspergillus parasiticus. The mycelial growth inhibition rate of A. parasiticus PTCC 5286 was investigated in the presence of Bifidobacterium bifidum PTCC 1644 and Lactobacillus fermentum PTCC 1744 by the pour plate method. After seven days incubation in yeast extract sucrose broth at 30°C, the mycelial mass was weighed after drying. The inhibitory activity of LAB metabolites against aflatoxin production by A. parasiticus was evaluated using HPLC method. B. bifidum and L. fermentum significantly reduced aflatoxin production and growth rate of A. parasiticus in comparison with the controls (p≤0.05). LAB reduced total aflatoxins and B1, B2, G1 and G2 fractions by more than 99%. Moreover, LAB metabolites reduced the level of standard AFB1, B2, G1 and G2 from 88.8% to 99.8% (p≤0.05). Based on these findings, B. bifidum and L. fermentum are recommended as suitable biocontrol agents against the growth and aflatoxin production by aflatoxigenic Aspergillus species.

Identification and Toxigenicity of Aspergillus spp. from Soils Planted to Peanuts in Eastern Zambia

ABSTRACT It is not known which aflatoxigenic species are present in Zambia. Therefore, soil samples were collected during May to June 2012, at the end of the growing season in Eastern Province, from 399 farmers' fields that had been planted to groundnut (Arachis hypogaea L.) in Nyimba, Petauke, Mambwe, and Chipata Districts. Population densities of Aspergillus spp. were estimated by plating 10−3 soil dilutions on modified dichloran rose Bengal (MDRB) media. To test for toxigenicity, colonies were randomly selected from MDRB dilution plates, single-spored, transferred to vials with yeast extract sucrose (YES) liquid media, and grown for a week at room temperature. Agra Strip ® lateral flow cards were then used to test the filtered extracts, from the YES cultures, for total aflatoxin at 4 and 20 parts per billion (ppb). We identified Aspergillus flavus (small and large sclerotia strains), A. parasiticus, A. niger, A. nomius, A. oryzae, A. tamarii, and A. terreus. 100% of the S-strain A. flavus isolates produced aflatoxin at 4 and 20 ppb, whereas 86% and 56% of the L-strain A. flavus isolates produced aflatoxins at 4 and 20 ppb, respectively. All the A. nomius isolates produced aflatoxins at 4 and 20 ppb. 79% and 64% the A. parasiticus isolates produced aflatoxins at 4 and 20 ppb. To our knowledge, this is the first peer reviewed report from Zambia documenting the population densities of A. flavus across different agroecologies. In addition, it is also the first report on the identification of different Aspergillus spp., such as A. nomius, A. terreus, A. oryzae, and A. tamarii from Zambia. This information, taken together with cropping practices, soil characteristics, agroecological and climatic data, can form a basis for developing holistic pre-harvest aflatoxin mitigation strategies.