Sabotage at the Powerhouse? Unraveling the Molecular Target of 2-Isopropylbenzaldehyde Thiosemicarbazone, a Specific Inhibitor of Aflatoxin Biosynthesis and Sclerotia Development in Aspergillus flavus, Using Yeast as a Model System

Amongst the various approaches to contain aflatoxin contamination of feed and food commodities, the use of inhibitors of fungal growth and/or toxin biosynthesis is showing great promise for the implementation or the replacement of conventional pesticide-based strategies. Several inhibition mechanisms were found taking place at different levels in the biology of the aflatoxin-producing fungal species such as Aspergillus flavus: compounds that influence aflatoxin production may block the biosynthetic pathway through the direct control of genes belonging to the aflatoxin gene cluster, or interfere with one or more of the several steps involved in the aflatoxin metabolism upstream. Recent findings pointed to mitochondrial functionality as one of the potential targets of some aflatoxin inhibitors. Additionally, we have recently reported that the effect of a compound belonging to the class of thiosemicarbazones might be related to the energy generation/carbon flow and redox homeostasis control by the fungal cell. Here, we report our investigation about a putative molecular target of the 3-isopropylbenzaldehyde thiosemicarbazone (mHtcum), using the yeast Saccharomyces cerevisiae as model system, to demonstrate how the compound can actually interfere with the mitochondrial respiratory chain.

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Inhibition of Aflatoxin Production by Paraquat and External Superoxide Dismutase in Aspergillus flavus

Toxins ◽

10.3390/toxins11020107 ◽

2019 ◽

Vol 11 (2) ◽

pp. 107 ◽

Cited By ~ 4

Author(s):

Tomohiro Furukawa ◽

Shohei Sakuda

Keyword(s):

Superoxide Dismutase ◽

Aspergillus Flavus ◽

Regulatory Protein ◽

Fungal Growth ◽

Aflatoxin Production ◽

Aflatoxin Contamination ◽

Sod Activity ◽

Fungal Cell ◽

Cell Extracts ◽

Ic50 Value

Aflatoxin contamination of crops is a worldwide problem, and elucidation of the regulatory mechanism of aflatoxin production, for example relative to the oxidative–antioxidative system, is needed. Studies have shown that oxidative stress induced by reactive oxygen species promotes aflatoxin production. However, superoxide has been suggested to have the opposite effect. Here, we investigated the effects of the superoxide generator, paraquat, and externally added superoxide dismutase (SOD) on aflatoxin production in Aspergillus flavus. Paraquat with an IC50 value of 54.9 µM inhibited aflatoxin production without affecting fungal growth. It increased cytosolic and mitochondrial superoxide levels and downregulated the transcription of aflatoxin biosynthetic cluster genes, including aflR, a key regulatory protein. The addition of bovine Cu/ZnSOD to the culture medium suppressed the paraquat-induced increase in superoxide levels, but it did not fully restore paraquat-inhibited aflatoxin production because bovine Cu/ZnSOD with an IC50 value of 17.9 µg/mL itself inhibited aflatoxin production. Externally added bovine Cu/ZnSOD increased the SOD activity in fungal cell extracts and upregulated the transcription of genes encoding Cu/ZnSOD and alcohol dehydrogenase. These results suggest that intracellular accumulation of superoxide impairs aflatoxin production by downregulating aflR expression, and that externally added Cu/ZnSOD also suppresses aflatoxin production by a mechanism other than canonical superoxide elimination activity.

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No Effect of Soybean Lipoxygenase on Aflatoxin Production in Aspergillus flavus–Inoculated Seeds

Journal of Food Protection ◽

10.4315/0362-028x-65.12.1984 ◽

2002 ◽

Vol 65 (12) ◽

pp. 1984-1987 ◽

Cited By ~ 6

Author(s):

J. E. MELLON ◽

P. J. COTTY

Keyword(s):

Aspergillus Flavus ◽

Fungal Pathogens ◽

Seed Viability ◽

Soybean Seed ◽

Fungal Growth ◽

Aflatoxin Production ◽

Aflatoxin Contamination ◽

Soybean Seeds ◽

Heat Treated ◽

Significant Difference

Soybean lines lacking lipoxygenase (LOX) activity were compared with soybean lines having LOX activity for the ability to support growth and aflatoxin B1 production by the fungal seed pathogen Aspergillus flavus. Whole seeds, broken seeds, and heat-treated (autoclaved) whole seeds were compared. Broken seeds, irrespective of LOX presence, supported excellent fungal growth and the highest aflatoxin levels. Autoclaved whole seeds, with or without LOX, produced good fungal growth and aflatoxin levels approaching those of broken seeds. Whole soybean seeds supported sparse fungal growth and relatively low aflatoxin levels. There was no significant difference in aflatoxin production between whole soybean seeds either with or without LOX, although there did seem to be differences among the cultivars tested. The heat treatment eliminated LOX activity (in LOX+ lines), yet aflatoxin levels did not change substantially from the broken seed treatment. Broken soybean seeds possessed LOX activity (in LOX+ lines) and yet yielded the highest aflatoxin levels. The presence of active LOX did not seem to play the determinant role in the susceptibility of soybean seeds to fungal pathogens. Seed coat integrity and seed viability seem to be more important characteristics in soybean seed resistance to aflatoxin contamination. Soybean seeds lacking LOX seem safe from the threat of increased seed pathogen susceptibility.

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Lack of aflatoxin production by Aspergillus flavus is associated with reduced fungal growth and delayed expression of aflatoxin pathway genes

World Mycotoxin Journal ◽

10.3920/wmj2014.1758 ◽

2015 ◽

Vol 8 (3) ◽

pp. 335-340 ◽

Cited By ~ 2

Author(s):

H. Zhang ◽

L.L. Scharfenstein ◽

C. Carter-Wientjes ◽

P.-K. Chang ◽

D. Zhang ◽

...

Keyword(s):

Aspergillus Flavus ◽

Animal Feed ◽

Regulatory Gene ◽

Fungal Growth ◽

Aflatoxin Production ◽

Aflatoxin Contamination ◽

Resistance Factors ◽

Crop Varieties ◽

Crop Resistance ◽

Underlying Mechanisms

Aflatoxins, produced by Aspergillus flavus and Aspergillus parasiticus, are the most toxic fungal secondary metabolites that contaminate agricultural commodities such as peanuts, cotton and maize. Understanding the underlying mechanisms of crop resistance to fungal infection is an important step for plant breeders to develop better and improved crop varieties for safe production of human food and animal feed. Infection studies have identified a resistant (R) peanut line, GT-C20, which is able to decrease aflatoxin contamination. The mycelial growth of A. flavus NRRL3357 on the R peanut line was much lower than that on the susceptible (S) peanut line, Tifrunner. Besides reducing fungal growth, the R line compared to the S line inhibited aflatoxin production completely. Real-time RT-PCR assays of both the R and S lines infected by A. flavus showed that expression of five aflatoxin biosynthetic pathway genes, the aflR regulatory gene and the aflD, aflM, aflP and aflQ structural genes, was not reduced but was significantly delayed on the R line. The results suggested that resistance factors of the R line acted negatively on A. flavus growth and also altered fungal development. The dysfunction in development changed the timing and the pattern of aflatoxin gene expression, which in part rendered A. flavus unable to produce aflatoxins.

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Comparison of Aflatoxin Production in Normal- and High-Oleic Backcross-Derived Peanut Lines

Plant Disease ◽

10.1094/pdis.2003.87.11.1360 ◽

2003 ◽

Vol 87 (11) ◽

pp. 1360-1365 ◽

Cited By ~ 25

Author(s):

H. Q. Xue ◽

T. G. Isleib ◽

G. A. Payne ◽

R. F. Wilson ◽

W. P. Novitzky ◽

...

Keyword(s):

Aspergillus Flavus ◽

Latin Square ◽

Fungal Growth ◽

Aflatoxin Production ◽

Aflatoxin Contamination ◽

High Oleic ◽

Plastic Bags ◽

The Mean ◽

Petri Dishes ◽

The Difference

The effect of the high-oleate trait of peanut on aflatoxin production was tested by comparing normal oleic lines with high-oleic backcross-derived lines. Seeds were blanched, quartered, and inoculated with Aspergillus flavus conidia, placed on moistened filter paper in petri dishes, and incubated for 8 days. In one experiment, dishes were stacked in plastic bags in a Latin square design with bags and positions in stacks as blocking variables. High-oleic lines averaged nearly twice as much aflatoxin as normal lines. Background genotype had no significant effect on aflatoxin content, and interaction between background genotype and oleate level was not detected. In a second experiment, dishes were arranged on plastic trays enclosed in plastic bags and stacked with PVC spacers between trays. Fungal growth and aflatoxin production were greater than in the first experiment. Background genotype, oleate level, and their interaction were significant. The mean of high-oleic lines was almost twice that of normal lines, but the magnitude of the difference varied with background genotype. Special care should be taken with high-oleic lines to prevent growth of Aspergillus spp. and concomitant development of aflatoxin contamination.

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A Novel Niosome-Encapsulated Essential Oil Formulation to Prevent Aspergillus flavus Growth and Aflatoxin Contamination of Maize Grains During Storage

Toxins ◽

10.3390/toxins11110646 ◽

2019 ◽

Vol 11 (11) ◽

pp. 646 ◽

Cited By ~ 6

Author(s):

García-Díaz ◽

Patiño ◽

Vázquez ◽

Gil-Serna

Keyword(s):

Aspergillus Flavus ◽

Fungal Growth ◽

Storage Conditions ◽

Aflatoxin Contamination ◽

Small Scale ◽

Low Concentrations ◽

Oil Formulation ◽

Scale Test ◽

Encapsulation Method

Aflatoxin (AF) contamination of maize is a major concern for food safety. The use of chemical fungicides is controversial, and it is necessary to develop new effective methods to control Aspergillus flavus growth and, therefore, to avoid the presence of AFs in grains. In this work, we tested in vitro the effect of six essential oils (EOs) extracted from aromatic plants. We selected those from Satureja montana and Origanum virens because they show high levels of antifungal and antitoxigenic activity at low concentrations against A. flavus. EOs are highly volatile compounds and we have developed a new niosome-based encapsulation method to extend their shelf life and activity. These new formulations have been successfully applied to reduce fungal growth and AF accumulation in maize grains in a small-scale test, as well as placing the maize into polypropylene woven bags to simulate common storage conditions. In this latter case, the antifungal properties lasted up to 75 days after the first application.

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In Vitro Activity of Balanites aegyptiaca and Tamarindus indica Fruit Extracts on Growth and Aflatoxigenicity of Aspergillus flavus and A. parasiticus

Journal of Food Research ◽

10.5539/jfr.v2n4p68 ◽

2013 ◽

Vol 2 (4) ◽

pp. 68 ◽

Cited By ~ 1

Author(s):

Saifeldin Ahmed El-nagerabi ◽

Abdulkadir E. Elshafie ◽

Mohamed R. Elamin

Keyword(s):

Aspergillus Flavus ◽

Fungal Growth ◽

Aflatoxin Contamination ◽

Tamarindus Indica ◽

Balanites Aegyptiaca ◽

Total Aflatoxin ◽

Animal Products ◽

Fruit Extracts ◽

Aflatoxin B

Aflatoxin and especially aflatoxin B1 (AFB1) is a carcinogenic secondary metabolite synthesized by certain Aspergillus species. They contaminate natural and processed agricultural and animal products which render them unfit for consumption. The aim of this study was to evaluate the in vitro effects of Balanites aegyptiaca and Tamarindus indica fruit extracts on the growth and aflatoxin secretion of Aspergillus flavus (SQU21) and A. parasiticus (CBS921.7) strains. The two fruit extracts significantly (P < 0.05) reduced aflatoxin and did not inhibit mycelial dry weights of the two Aspergillus strains. At different concentrations of balanites (2.5-10%), the inhibition of total aflatoxin was 49.9-84.8% for A. flavus (SQU21) and 32.1-84.4% for A. parasiticus (CBS921.7), whereas the inhibition of aflatoxin Bwas 38.2-81.4% and 32.8-80.6% for the two strains. Tamarind fruit extract (2.5-7.5%) caused 28.8-84.2% and 40.7-85.5% reductions in total aflatoxin and 37.1-83.5% and 33.9-85.9% in aflatoxin B for the two strains, respectively. None of these extracts inhibited the fungal growth or detoxified synthetic aflatoxin B1. We have concluded that these fruits contain various inhibitors to aflatoxin biosynthesis and secretion. Therefore, they can be used in combination as safe green biopreservatives to combat aflatoxin contamination of food.

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Control of Aspergillus flavus and aflatoxin production with spices in culture medium and rice

World Mycotoxin Journal ◽

10.3920/wmj2012.1437 ◽

2013 ◽

Vol 6 (1) ◽

pp. 43-50 ◽

Cited By ~ 6

Author(s):

V. Aiko ◽

A. Mehta

Keyword(s):

Aspergillus Flavus ◽

High Performance ◽

Fungal Growth ◽

Aflatoxin Production ◽

Culture Broth ◽

Minimum Inhibitory Concentrations ◽

Food Grains ◽

Star Anise ◽

Aflatoxin B ◽

Layer Chromatography

Cinnamon, cardamom, star anise and clove were studied for their effect on growth of Aspergillus flavus and aflatoxin B1 (AFB1) synthesis. The experiments were carried out in yeast extract sucrose culture broth as well as in rice supplemented with spices. AFB1 produced was analysed qualitatively and quantitatively using thin layer chromatography and high performance liquid chromatography, respectively. At a concentration of 10 mg/ml, cardamom and star anise did not exhibit any antifungal or anti-aflatoxigenic activity in culture broth, whereas cinnamon and clove inhibited A. flavus growth completely. The minimum inhibitory concentrations of cinnamon and clove were 4 and 2 mg/ml, respectively. Concentrations of cinnamon and clove below their minimum inhibitory concentrations showed enhanced fungal growth, while AFB1 synthesis was reduced. Clove inhibited the synthesis of AFB1 significantly up to 99% at concentrations ≥1.0 mg/ml. The spices also inhibited AFB1 synthesis in rice at 5 mg/g, although fungal growth was not inhibited. Clove and cinnamon inhibited AFB1 synthesis significantly up to 99 and 92%, respectively, and star anise and cardamom by 41 and 23%, respectively. The results of this study suggest the use of whole spices rather than their essential oils for controlling fungal and mycotoxin contamination in food grains.

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Interactions of Saprophytic Yeasts with anor Mutant of Aspergillus flavus

Applied and Environmental Microbiology ◽

10.1128/aem.65.6.2738-2740.1999 ◽

1999 ◽

Vol 65 (6) ◽

pp. 2738-2740 ◽

Cited By ~ 47

Author(s):

Sui-Sheng T. Hua ◽

James L. Baker ◽

Melanie Flores-Espiritu

Keyword(s):

Aspergillus Flavus ◽

Agar Plate ◽

Aflatoxin Production ◽

Aflatoxin Contamination ◽

Orange Pigment ◽

Yeast Strains ◽

Norsolorinic Acid ◽

Aflatoxin Accumulation ◽

Food Commodities ◽

Pigment Accumulation

ABSTRACT The nor mutant of Aspergillus flavus has a defective norsolorinic acid reductase, and thus the aflatoxin biosynthetic pathway is blocked, resulting in the accumulation of norsolorinic acid, a bright red-orange pigment. We developed a visual agar plate assay to monitor yeast strains for their ability to inhibit aflatoxin production by visually scoring the accumulation of this pigment of the nor mutant. We identified yeast strains that reduced the red-orange pigment accumulation in the normutant. These yeasts also reduced aflatoxin accumulation by a toxigenic strain of A. flavus. These yeasts may be useful for reducing aflatoxin contamination of food commodities.

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The Frequency of Sex: Population Genomics Reveals Differences in Recombination and Population Structure of the Aflatoxin-Producing Fungus Aspergillus flavus

mBio ◽

10.1128/mbio.00963-20 ◽

2020 ◽

Vol 11 (4) ◽

Cited By ~ 3

Author(s):

Milton T. Drott ◽

Tatum R. Satterlee ◽

Jeffrey M. Skerker ◽

Brandon T. Pfannenstiel ◽

N. Louise Glass ◽

...

Keyword(s):

Aspergillus Flavus ◽

Population Genomics ◽

Population Expansion ◽

Aflatoxin Production ◽

The United States ◽

Aflatoxin Contamination ◽

Deleterious Mutations ◽

Content Type ◽

Sexual And Asexual Reproduction ◽

Potent Carcinogen

ABSTRACT The apparent rarity of sex in many fungal species has raised questions about how much sex is needed to purge deleterious mutations and how differences in frequency of sex impact fungal evolution. We sought to determine how differences in the extent of recombination between populations of Aspergillus flavus impact the evolution of genes associated with the synthesis of aflatoxin, a notoriously potent carcinogen. We sequenced the genomes of, and quantified aflatoxin production in, 94 isolates of A. flavus sampled from seven states in eastern and central latitudinal transects of the United States. The overall population is subdivided into three genetically differentiated populations (A, B, and C) that differ greatly in their extent of recombination, diversity, and aflatoxin-producing ability. Estimates of the number of recombination events and linkage disequilibrium decay suggest relatively frequent sex only in population A. Population B is sympatric with population A but produces significantly less aflatoxin and is the only population where the inability of nonaflatoxigenic isolates to produce aflatoxin was explained by multiple gene deletions. Population expansion evident in population B suggests a recent introduction or range expansion. Population C is largely nonaflatoxigenic and restricted mainly to northern sampling locations through restricted migration and/or selection. Despite differences in the number and type of mutations in the aflatoxin gene cluster, codon optimization and site frequency differences in synonymous and nonsynonymous mutations suggest that low levels of recombination in some A. flavus populations are sufficient to purge deleterious mutations. IMPORTANCE Differences in the relative frequencies of sexual and asexual reproduction have profound implications for the accumulation of deleterious mutations (Muller’s ratchet), but little is known about how these differences impact the evolution of ecologically important phenotypes. Aspergillus flavus is the main producer of aflatoxin, a notoriously potent carcinogen that often contaminates food. We investigated if differences in the levels of production of aflatoxin by A. flavus could be explained by the accumulation of deleterious mutations due to a lack of recombination. Despite differences in the extent of recombination, variation in aflatoxin production is better explained by the demography and history of specific populations and may suggest important differences in the ecological roles of aflatoxin among populations. Furthermore, the association of aflatoxin production and populations provides a means of predicting the risk of aflatoxin contamination by determining the frequencies of isolates from low- and high-production populations.

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Chestnut Drying Is Critical in Determining Aspergillus flavus Growth and Aflatoxin Contamination

Toxins ◽

10.3390/toxins10120530 ◽

2018 ◽

Vol 10 (12) ◽

pp. 530 ◽

Cited By ~ 6

Author(s):

Simona Prencipe ◽

Ilenia Siciliano ◽

Carlotta Gatti ◽

Maria Gullino ◽

Angelo Garibaldi ◽

...

Keyword(s):

Antioxidant Activity ◽

Fungal Growth ◽

Aflatoxin Production ◽

Aflatoxin Contamination ◽

Total Phenol ◽

Fungal Colonization ◽

Total Phenol Content ◽

Phenol Content ◽

Drying Treatment ◽

Drying Conditions

Chestnut drying is used to prevent postharvest losses and microorganism contamination during storage. Several studies reported the contamination by aflatoxins (AFs) produced by Aspergillus spp. in chestnuts. The effect of drying temperatures (from 30 to 50 °C) was evaluated on the growth of A. flavus and the production of aflatoxins in chestnuts. The influence of the treatment on the proximate composition, the total phenol content and antioxidant activity of chestnuts was considered. Fungal colonization was observed on the nuts dried at 30, 35, and 40 °C; the incidence was lower at 40 °C. The highest concentrations of AFB1 and AFB2 were produced at 40 °C. No aflatoxins were detected at 45 or 50 °C. At 40 °C A. flavus was under suboptimal conditions for growth (aw 0.78), but the fungus was able to synthesize aflatoxins. As the temperatures applied increased, the total phenol content increased, while the antioxidant activity decreased. A drying treatment at 45 °C for seven days (aw 0.64) could be a promising method to effectively control both the growth of aflatoxigenic fungi and the production of aflatoxins. This study provides preliminary data useful to improve the current drying conditions used in chestnut mills, to reduce both fungal growth and aflatoxin production.

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