Growth of an Aspergillus flavus Transformant Expressing Escherichia coli β-Glucuronidase in Maize Kernels Resistant to Aflatoxin Production

1997 ◽  
Vol 60 (1) ◽  
pp. 84-87 ◽  
Author(s):  
ROBERT L. BROWN ◽  
THOMAS E. CLEVELAND ◽  
GARY A. PAYNE ◽  
CHARLES P. WOLOSHUK ◽  
DONALD G. WHITE
Keyword(s):  
Escherichia Coli ◽  
Aspergillus Flavus ◽  
Aflatoxin B1 ◽  
Gene Promoter ◽  
Fungal Growth ◽  
Aflatoxin Production ◽  
Glucuronidase Activity ◽  
Glucuronidase Reporter ◽  
Aflatoxin Accumulation ◽  
Maize Kernels

Kernels of a maize inbred that demonstrated resistance to aflatoxin production in previous studies were inoculated with an Aspergillus flavus strain containing the Escherichia coli β-d-glucuronidase reporter gene linked to a β-tubulin gene promoter and assessed for both fungal growth and aflatoxin accumulation. Prior to inoculation, kernels were pin-wounded through the pericarp to the endosperm, pin-wounded in the embryo region, or left unwounded. After 7 days incubation with the fungus, β-glucuronidase activity (fungal growth) in the kernels was quantified using a fluorogenic assay and aflatoxin B1 content of the same kernels was analyzed. Kernels of a susceptible inbred, similarly treated, served as controls. Results indicate a positive relationship between aflatoxin levels and the amount of fungal growth. However, resistant kernels wounded through the pericarp to the endosperm before inoculation supported an increase in aflatoxin B1 over levels observed in nonwounded kernels, without an increase in fungal growth. Wounding kernels of the resistant inbred through the embryo resulted in both the greatest fungal growth and the highest levels of aflatoxin B1 for this genotype. Maintenance of resistance to aflatoxin B1 in endosperm-wounded kernels may be due to the action of a mechanism which limits fungal access to the kernel embryo.

Fungal Growth and Aflatoxin Production on Apiarian Substrates

1977 ◽  
Vol 60 (1) ◽  
pp. 96-99
Author(s):  
Jo Ann L Hilldrup ◽  
Thomas Eadie ◽  
Gerald C Llewellyn
Keyword(s):  
Aspergillus Flavus ◽  
Aflatoxin B1 ◽  
Fungal Growth ◽  
Aflatoxin Production ◽  
Brood Comb

Abstract Unprocessed honey, Lilium longiflorium pollen, brood comb, whole larvae, and whole bees were inoculated with Aspergillus flavus NRRL 3251, A. flavus ATCC 15548, and A. parasiticus NRRL 2999. The fungi grew, sporulated, and produced various amounts of aflatoxin on all substrates except the unprocessed honey. The largest quantity of aflatoxin B1 was produced on whole larvae supporting A. flavus NRRL 3251 growth. A. parasiticus NRRL 2999 growing on whole larvae produced the most aflatoxin G1. Aflatoxins B2 and G2 were seldom detected. Apiarian substrates with the exception of honey seem capable of supporting fungal growth and resultant aflatoxin production.

scholarly journals Maize Milling and Modifying Atmospheric Conditions Limit Formation of Aflatoxin B1 by Aspergillus Flavus

2021 ◽  
Vol 12 (2) ◽  
pp. 16
Author(s):  
Benigni Alfred Temba ◽  
Gaymary George Bakari
Keyword(s):  
Aspergillus Flavus ◽  
Aflatoxin B1 ◽  
Fungal Growth ◽  
Health Concern ◽  
Atmospheric Conditions ◽  
Physical Status ◽  
The World ◽  
Baseline Information ◽  
Petri Dishes ◽  
Maize Kernels

Occurrence of mycotoxins in foods poses a serious health concern all over the world. Aflatoxin B1 (AFB1) is the most toxic, with widest occurrence in various foods, but mainly in cereals and nuts and its accumulation depends on substrate and environmental factors. This study investigated the how physical status (milling) of maize kernels and atmospheric conditions (aeration, moisture and temperature) affect production of aflatoxin B1 by Aspergillus flavus (ATCC 28862). Intact kernels and flour were incubated for up to 20 days in open and partially sealed petri dishes under controlled temperatures of 25 ºC, 30 ºC and 37 ºC and initial moisture contents of 27%, 22%, 18%, 15% and 12%. It was found that on average, significantly higher (p < 0.05) aflatoxin B1 level was accumulated in intact kernels (145.7 µg/kg) as compared to milled kernels (2.2 µg/kg). Also, none of the samples incubated under partially sealed conditions, compared to up to 100% of the samples incubated in open atmosphere had detectable levels of aflatoxin B1 after 20 days. Fungal growth was not affected by milling or aeration, but sporulation was low at 37 ºC and high at 25 ºC and 30 ºC. The findings of this study provide baseline information on how conditions can be modified to control postharvest accumulation of aflatoxin B1 in cereals.

scholarly journals Antifungal and Antiaflatoxigenic Activities of 1,8-Cineole and t-Cinnamaldehyde on Aspergillus flavus

2018 ◽  
Vol 8 (9) ◽  
pp. 1655 ◽  
Author(s):  
Hyeong-Mi Kim ◽  
Hyunwoo Kwon ◽  
Kyeongsoon Kim ◽  
Sung-Eun Lee
Keyword(s):  
Propionic Acid ◽  
Aspergillus Flavus ◽  
Growth Medium ◽  
Aflatoxin B1 ◽  
Mode Of Action ◽  
Fungal Growth ◽  
Aflatoxin Production ◽  
Inhibitory Effects ◽  
Aflatoxin B2

Aspergillus flavus and A. parsiticus produce aflatoxins that are highly toxic to mammals and birds. In this study, the inhibitory effects of 1,8-cineole and t-cinnamaldehyde were examined on the growth of Aspergillus flavus ATCC 22546 and aflatoxin production. 1,8-Cineole showed 50% inhibition of fungal growth at a concentration of 250 ppm, while t-cinnamaldehyde almost completely inhibited fungal growth at a concentration of 50 ppm. Furthermore, no fungal growth was observed when the growth medium was treated with 100 ppm t-cinnamaldehyde. 1,8-Cineole also exhibited 50% inhibition on the production of aflatoxin B1 and aflatoxin B2 at a concentration of 100 ppm, while the addition of 100 ppm t-cinnamaldehyde completely inhibited aflatoxin production. These antiaflatoxigenic activities were related to a dramatic downregulation of the expression of aflE and aflL by 1,8-cineole, but the mode of action for t-cinnamaldehyde was unclear. Collectively, our results suggest that both of the compounds are promising alternatives to the currently used disinfectant, propionic acid, for food and feedstuff preservation.

Construction and Preliminary Evaluation of an Aspergillus flavus Reporter Gene Construct as a Potential Tool for Screening Aflatoxin Resistance

2003 ◽  
Vol 66 (10) ◽  
pp. 1927-1931 ◽  
Author(s):  
ROBERT L. BROWN ◽  
CARMEN S. BROWN-JENCO ◽  
DEEPAK BHATNAGAR ◽  
GARY A. PAYNE
Keyword(s):  
Reporter Gene ◽  
Aspergillus Flavus ◽  
Fungal Growth ◽  
Gus Expression ◽  
Alternative Methods ◽  
Preliminary Evaluation ◽  
Gene Construct ◽  
Reporter Gene Construct ◽  
Aflatoxin Accumulation ◽  
Maize Kernels

Effective preharvest strategies to eliminate aflatoxin accumulation in crops are not presently available. The molecular biology of aflatoxin biosynthesis has been extensively studied, and genetic and molecular tools such as reporter gene systems for the measurement of fungal growth have been developed. A reporter construct containing the Aspergillus flavus β-tubulin gene promoter fused to Escherichia coli β-glucuronidase (GUS) has been shown to be a reliable tool for the indirect measurement of fungal growth in maize kernels. Since cost-saving alternative methods for the direct measurement of aflatoxin levels are needed to facilitate more widespread field and laboratory screening of maize lines, a new reporter gene construct involving the promoter region of the omtA gene of the aflatoxin biosynthetic pathway was constructed and tested. Expression of GUS activity by this construct (omtA::GUS) was correlated with aflatoxin accumulation in culture. In the fungal transformant GAP26-1, which harbors this construct, aflatoxin production and GUS expression on sucrose-containing medium showed the same temporal pattern of toxin induction. Furthermore, GUS expression by GAP26-1 was shown to be associated with aflatoxin accumulation in maize kernels inoculated with this strain. Our results suggest that this and other reporter gene pathway promoter constructs may provide superior alternatives to direct aflatoxin quantification with respect to time, labor, and materials for the screening of maize lines for resistance to aflatoxin accumulation.

RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels

Planta ◽  
2018 ◽  
Vol 247 (6) ◽  
pp. 1465-1473 ◽  
Author(s):  
Matthew K. Gilbert ◽  
Rajtilak Majumdar ◽  
Kanniah Rajasekaran ◽  
Zhi-Yuan Chen ◽  
Qijian Wei ◽  
...  
Keyword(s):  
Rna Interference ◽  
Aspergillus Flavus ◽  
Fungal Growth ◽  
Aflatoxin Production ◽  
Alpha Amylase ◽  
Maize Kernels

Wax and Cutin Layers in Maize Kernels Associated with Resistance to Aflatoxin Production by Aspergillus flavus

1995 ◽  
Vol 58 (3) ◽  
pp. 296-300 ◽  
Author(s):  
BAO Z. GUO ◽  
JOHN S. RUSSIN ◽  
THOMAS E. CLEVELAND ◽  
ROBERT L. BROWN ◽  
NEIL W. WIDSTROM
Keyword(s):  
Aspergillus Flavus ◽  
Potassium Hydroxide ◽  
Aflatoxin Production ◽  
Maize Kernel ◽  
Maize Hybrids ◽  
Maize Population ◽  
Maize Genotypes ◽  
Aflatoxin Accumulation ◽  
Maize Kernels ◽  
Susceptible Group

Thirteen maize hybrids and one maize population, MAS:gk, were screened for susceptibility to aflatoxin production by Aspergillus flavus. Marked differences in aflatoxin B1 production were detected among the maize genotypes tested. Most commercial hybrids consistently supported high levels of aflatoxin accumulation. Aflatoxin levels did not differ between intact and wounded kernels of these genotypes. However, different results were obtained from 4 of the 13 hybrids and the maize population MAS:gk. Levels of aflatoxin accumulation in intact kernels of these genotypes were lower than in the previous susceptible group of genotypes. In addition, aflatoxin levels were higher in wounded than in intact kernels. MAS:gk not only supported the lowest levels of aflatoxin production in intact kernels, but aflatoxin levels in endosperm-wounded kernels also were significantly lower in MAS:gk than in wounded kernels of all tested hybrids. Treatment with KOH to remove cutin from intact kernels prior to inoculation with A. flavus effected substantial increases in aflatoxin accumulation in MAS:gk, but only marginal increases in the susceptible hybrid Pioneer 3154. Removing wax from the surface of MAS:gk kernels greatly increased the susceptibility of this genotype to aflatoxin accumulation. When wax removal was combined with treatment with potassium hydroxide (KOH) or purified cutinase, aflatoxin levels in kernels were equal to those in wounded control kernels in both genotypes. These results indicated that wax and cutin layers of maize kernel pericarps may play a role in resistance to aflatoxin accumulation in MAS:gk and some other genotypes. However, results suggest further that resistance in MAS:gk also may be due to other preformed compounds as well.

scholarly journals Gene Expression Profile and Response to Maize Kernels by Aspergillus flavus

2011 ◽  
Vol 101 (7) ◽  
pp. 797-804 ◽  
Author(s):  
Brittiney N. Reese ◽  
Gary A. Payne ◽  
Dahlia M. Nielsen ◽  
Charles P. Woloshuk
Keyword(s):  
Aspergillus Flavus ◽  
Expression Profile ◽  
Developmental Stages ◽  
Fungal Growth ◽  
Aflatoxin Production ◽  
Sole Source ◽  
Ear Rot ◽  
Phytase Gene ◽  
Kernel Infection ◽  
Maize Kernels

Aspergillus flavus causes an ear rot of maize, often resulting in the production of aflatoxin, a potent liver toxin and carcinogen that impacts the health of humans and animals. Many aspects of kernel infection and aflatoxin biosynthesis have been studied but the precise effects of the kernel environment on A. flavus are poorly understood. The goal of this research was to study the fungal response to the kernel environment during colonization. Gene transcription in A. flavus was analyzed by microarrays after growth on kernels of the four developmental stages: blister (R2), milk (R3), dough (R4), and dent (R5). Five days after inoculation, total RNA was isolated from kernels and hybridized to Affymetrix Gene Chip arrays containing probes representing 12,834 A. flavus genes. Statistical comparisons of the expression profile data revealed significant differences that included unique sets of upregulated genes in each kernel stage and six patterns of expression over the four stages. Among the genes expressed in colonized dent kernels were a phytase gene and six putative genes involved in zinc acquisition. Disruption of the phytase gene phy1 resulted in reduced growth on medium containing phytate as the sole source of phosphate. Furthermore, growth of the mutant (Δphy1) was 20% of the wild-type strain when wound inoculated into maize ears. In contrast, no difference was detected in the amount of aflatoxin produced relative to fungal growth, indicating that phy1 does not affect aflatoxin production. The study revealed the genome-wide effects of immature maize kernels on A. flavus and suggest that phytase has a role in pathogenesis.

scholarly journals Mimosa tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus

Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 391
Author(s):  
Christopher Hernandez ◽  
Laura Cadenillas ◽  
Anwar El Maghubi ◽  
Isaura Caceres ◽  
Vanessa Durrieu ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Aqueous Extract ◽  
Aflatoxin B1 ◽  
Condensed Tannins ◽  
Fungal Growth ◽  
Oxidation Reactions ◽  
Expression Of Genes ◽  
Dependent Inhibition ◽  
Interesting Candidate ◽  
Mimosa Tenuiflora

Aflatoxin B1 (AFB1) is a potent carcinogenic mycotoxin that contaminates numerous crops pre- and post-harvest. To protect foods and feeds from such toxins without resorting to pesticides, the use of plant extracts has been increasingly studied. The most interesting candidate plants are those with strong antioxidative activity because oxidation reactions may interfere with AFB1 production. The present study investigates how an aqueous extract of Mimosa tenuiflora bark affects both the growth of Aspergillus flavus and AFB1 production. The results reveal a dose-dependent inhibition of toxin synthesis with no impact on fungal growth. AFB1 inhibition is related to a down-modulation of the cluster genes of the biosynthetic pathway and especially to the two internal regulators aflR and aflS. Its strong anti-oxidative activity also allows the aqueous extract to modulate the expression of genes involved in fungal oxidative-stress response, such as msnA, mtfA, atfA, or sod1. Finally, a bio-guided fractionation of the aqueous extract demonstrates that condensed tannins play a major role in the anti-aflatoxin activity of Mimosa tenuiflora bark.

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

2013 ◽  
Vol 6 (1) ◽  
pp. 43-50 ◽  
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.

scholarly journals Interactions of Saprophytic Yeasts with anor Mutant of Aspergillus flavus

1999 ◽  
Vol 65 (6) ◽  
pp. 2738-2740 ◽  
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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