El factor de transcripción bZIP Afap1 afecta al estrés oxidativo y la biosíntesis de aflatoxinas en Aspergillus flavus / The bZIP transcription factor Afap1 mediates the oxidative stress response and aflatoxin biosynthesis in Aspergillus flavus

Abstract Aflatoxin is a carcinogenic secondary metabolite produced mainly by Aspergillus flavus and Aspergillus parasiticus, which can seriously endanger the health of humans and animals. Oxidative stress is a common defense response, and it is known that reactive oxygen species (ROS) can induce the synthesis of a series of secondary metabolites, including aflatoxin. By using mutants lacking the afap 1 gene, the role of afap 1 gene in oxidative stress and aflatoxin synthesis was assessed. The growth of the mutant strains was significantly inhibited by the increase in the concentration of H2O2, inhibition was complete at 40mmol/l. However, in the quantitative analysis by HPLC, the concentration of AFB1 increased with the increased H 2O 2 until 10mmol/l. Following an analysis based on the information provided by the NCBI BLAST analysis, it was assumed that Afap1, a basic leucine zipper (bZIP) transcription factor, was associated with the oxidative stress in this fungus. Treatment with 5mmol/l H 2O 2 completely inhibited the growth of the mutant strains in afap 1 but did not affect the growth of the CA14PTs strain (non-mutant strain). In addition, the concentration of AFB 1 in the mutant strains was approximately V of that observed in the CA14PTs strain. These results suggested that Afap1 plays a key role in the regulation of oxidative stress and aflatoxin production in A. flavus. ©2018 Published by Elsevier España, S.L.U. on behalf of Asociación Argentina de Microbiología. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/ licenses/by-nc-nd/4.0/).

Resumen La aflatoxina es un metabolito secundario cancerígeno producido principalmente por Aspergillus flavus y Aspergillus parasiticus, que pone en riesgo grave a la salud de los humanos y los animales. El estrés oxidativo es una respuesta de defensa común, y es sabido que las especies reactivas de oxígeno (ROS) pueden inducir la síntesis de una serie de metabolitos secundarios, incluida la aflatoxina. Empleando mutantes carentes del gen afap1 se evaluó el papel de Afap1 en el estrés oxidativo y la síntesis de aflatoxinas. El crecimiento de las cepas mutadas se vio significativamente inhibido con el aumento de la concentración de H 2O 2, la inhibición fue completa a 40mmol/l. Sin embargo, en el análisis cuantitativo por HPLC, la concentración de la aflatoxina AFBi aumentó con el aumento de la concentración de H 2O 2 hasta 10mmol/l. Tras un análisis apoyado en la información provista por la herramienta NCBI BLAST, se supuso que Afap1, un factor de transcripción de la cremallera de leucina básica (bZIP), estaba asociado con el estrés oxidativo en este hongo. El tratamiento con 5mmol/l de H 2O 2 inhibió completamente el crecimiento de las cepas mutantes en afap1, pero no afectó el crecimiento de la cepa CA14PTs (cepa no mutada). Además, la concentración de AFB 1 en las cepas mutadas fue de aproximadamente 1/4 de la observada en CA14PTs. Estos resultados sugieren que Afap1 juega un papel clave en la regulación del estrés oxidativo y la producción de aflatoxinas en A. flavus.

The potential effects of Zataria multiflora Boiss essential oil on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes of toxigenic Aspergillus parasiticus

This study aims at evaluating the effects of Zataria multiflora (Z. multiflora) essential oil (EO) on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes. Total RNAs of Aspergillus parasiticus (A.parasiticus) ATCC56775 grown in yeast extract sucrose (YES) broth medium treated with Z. multiflora EO were subjected to reverse transcription-polymerase chain reaction (RT-PCR). Specific primers of nor-1, ver-1, omt-A and aflR genes were used. In parallel mycelial dry weight of samples were measured and all the media were assayed by high-pressure liquid chromatography (HPLC) for aflatoxinB1 (AFB1), aflatoxinB2 (AFB2), aflatoxinG1 (AFG1), aflatoxinG2 (AFG2) and aflatoxin total (AFTotal) production. The results showed that mycelial dry weight and aflatoxin production reduce in the presence of Z. multiflora EO (100 ppm) on day 5 of growth. It was found that the expression of nor-1, ver-1, omt-A and aflR genes was correlated with the ability of fungus to produce aflatoxins on day 5 in YES medium. RT-PCR showed that in the presence of Z.multiflora EO (100 ppm) nor-1, ver-1 and omtA genes expression was reduced. It seems that toxin production inhibitory effects of Z. multiflora EO on day 5 may be at the transcription level and this herb may cause reduction in aflatoxin biosynthesis pathway genes activity.

Effect of curcumin on aspergillus parasiticus growth and expression of major genes involved in the early and late stages of aflatoxin biosynthesis

The effect of curcumin as a natural safe compound with different biological activities was examined on fungal growth and aflatoxin production in Aspergillus parasiticus NRRL 2999. The fungus was cultured in presence of serial two-fold concentrations of curcumin [125-2000 micro g/ml] in yeast extract sucrose broth for 3 days at 28°C. Mycelia dry weight was determined as an index of fungal growth, while aflatoxin production was assessed by high performance liquid chromatography [HPLC]. The expression of ver-1, nor-1, pksA, omtA and aflR genes in aflatoxin biosynthetic pathway was evaluated by real time PCR. Curcumin strongly inhibited aflatoxin B1 production in the range of 26.6 to 94.9% by serial two-fold concentrations from 125 to 2000 micro g/ml. Fungal growth was also inhibited by the compound in the range of 34.0 to 60.8%. Analysis of the expression of aflatoxin pathway genes by real time PCR showed that curcumin inhibited the expression of ver-1, nor-1, pksA, omtA and aflR genes at concentrations of 250 and 1000 micro g/ml. In concentration of 1000 micro g/ml, gene expression was reduced by 31.3%, 44.6%, 57.1% 110.9% and 286.7% accordingly. Reduction in the expression of aflatoxin biosynthesis genes was significant only for aflR. In ferric reducing ability of plasma [FRAP] assay, curcumin showed strong antioxidant activity at all concentrations tested. Curcumin may be employed successfully as a good candidate in controlling of toxigenic fungal growth on food and feed and subsequent contamination with aflatoxins in practice

Efficacy of Euphorbia splendens and Leonotis nepetaefolia on aflatoxin producing fungi Aspergillus flavus and Aspergillus parasiticus.

Efficacy of three different concentrations (5, 10 and 15 mg/ml) of dry flower powder of E. splendens and L. nepetaefolia was tested on the growth of aflatoxin-producing toxigenic strains of fungi A. flavus (NCBT 101) and A. parasiticus (NCBT 128) in Sabouraud dextrose agar medium (SDA). Maximum (75%) inhibition of growth of A. flavus was seen at 15 mg/ml concentration of E. splendens flower dry powder, while A. parasiticus showed 50% inhibition of growth at 10 and 15 mg/ml concentrations. Total inhibition (100%) of growth of A. flavus was seen at 10 and 15 mg/ml for L. nepetaefolia and maximum (75%) inhibition of growth was seen for A. parasiticus at 15 mg/ml concentration. Bioassay with groundnut seeds soaked with different concentrations of flower extract proved that both fungi were incapable of infecting the seeds at 10 and 15 mg/ml of L. nepetaefolia flower extracts.

Natural occurrence of mycotoxins in cereals from Mazandaran and Golestan provinces

Bakground-Aflatoxins [AF] and ochratoxin A [OA] are two toxic substances produced by specific types of fungi. Many reports consider them as carcinogens and nephropathogens, respectively. Objective-The aim of this study was to present the natural occurrence of AF and OA in some important foodstuffs in the southern Caspian littoral. Methods-Fourteen barley and nine corn samples, intended for animal feed, were collected from Golestan and Mazandaran provinces, northern Iran, and were analyzed for AF and OA by high performance liquid chromatography [HPLC]. Results-In corn samples, aflatoxin B1 [AFB1] and aflatoxin B2 [AFB2] were detected in 8 [88.8%] and 6 [66.6%] samples at a mean level of 15.83 and 2.99 ppb [median 1.72 and 1 ppb] respectively. Only one of the AF-contaminated corn samples was co-contaminated with OA at a concentration of 0.35 ppb. Neither AF nor OA were found in any of the barley samples. Conclusion-This study was the first report on natural occurrence of OA and its co-occurrence with AF in the corn samples of northern Iran. Although our findings showed a low level of AF and OA, we recommend a periodical survey to be conducted on the natural occurrence of mycotoxins in cereals from the Caspian littoral

Effect of propionic acid on fungal growth and aflatoxin production in moist inshell groudnuts

O efeito do ácido propiônico nas concentraçöes de 3,0 g/kg (AP1) e 5,0 g./kg (AP2) foi avaliado em laboratório para verificar sua eficiência sobre o crescimento fúngico e produçäo de aflatoxinas quando aplicado sobre amendoim em casca úmido. As avaliaçöes, crescimento fúngico sobre as vagens e seus gräos e as análises de aflatoxinas, foram realizadas antes da incubaçäo e aos 7, 14, 21 e 28 dias de incubaçäo. O tratamentoAP1 foi ineficiente no controle do crescimento fúngico a partir de 14§ dia

Influência da temperatura e umidade relativa na produçäo de aflatoxinas em amostras de milhos armazenados e contamanados artificialmente com Aspergillus flavus / Influence of temperature and relative humidity in the production of aflatoxins in samples of stored maize, artificially contamined with aspergillus flavus

resumo:Foram utilizadas 90 amostras de gräos de milho provenientes de silos de várias regiöes do Brasil, para se verificar a influência da temperatura e da umidade relativa na produçäo de aflatoxinas neste substrato.As amostras foram inoculadas com Aspergillus flavus toxigênico e incubadas em ambiente com temperatura e umidade relativa controladas(15ºC e 61 por cento UR, 86.5 por cento UR, 99 por cento UR;25ºC e 64 por cento UR, 85 por cento UR, 98 por cento UR;40ºC e 61.5 por cento UR, 85.5 por cento UR, 96 por cento UR)por Períodos de 10, 20 e 30 dias de incubaçäo.Foram detectadas aflatoxinas nas amostras incubadas a 15ºC e 86.5 por cento UR;25ºC nas três condiçöes de umidades estudadas;40ºC e 61.5 por cento UR.Conclui-se que temperaturas mais baixas(15ºC) com umidade relativa de 61 por cento e conteúdo de umidade de 13 por cento parecem ser as melhores condiçöes para armazenamento de gräos de milho

Effect of antimicrobial compounds of some lactic acid bacteria on growth and aflatoxins production by Aspergillus flavus

Effect of antimicrobial compounds of Lc. Thermophilus, Cit+ Lc. lactis, Lact. bulgaricus and Lact. casei on aflatoxins production and growth of Aspergillus flavus beside pH changes in growth media during 8 days incubation were determined. Addition of the antimicrobial compounds of lactic acid bacteria strains inhibited aflatoxins M1 and M2 completely and the total aflatoxins decreased markedly in the presence of these antimicrobial compounds. The increase in the antimicrobial compounds was correlated with increase in total aflatoxins inhibition. The effect of antimicrobial compounds ratios and intervals of incubation time had significant differences on aflatoxins production

Mycological evaluation of filtrated juice of agrave sisalana perrine's leafe

Conforme observado por Salomäo e Purchio, 1982, o suco filtrado da folha de Agave sisalana apresenta substância inibitória do crescimento de Aspergillus flavus e Aspergillus parasiticus e em doses subnibitórias interfere na síntese de aflatoxinas. Seguindo esta linha de pesquisa, verificou-se esta atividade inibitória em amostras de milho contaminadas pelos fungos toxigênicos citados. Realizou-se testes demonstrando a eficiência dessa atividade em 3 amostras de A. parasiticus, de A. flavus e 3 de Aspergillus sp. Esterilizou-se as amostras de miho por óxido de etileno sendo posteriormente colocadas em erlenmeyer. As amostras foram umedecidas com suco e contaminadas por fungos toxigênicos. Evidenciou-se inibiçäo de crescimento nos experimentos realizados, demonstrando-se, assim, a possibilidade do emprego do suco filtrado de A. sisalana para tal finalidade. Foram realizadas avaliaçöes toxicológicas do suco para animais de laboratório

Production of aflatoxins by fungi isolated from some medicinal plants used as drinks in Egypt

Thirty-one different plant samples of tea, anise, fenugreek and liquorice were purchased from different places in Egypt and tested for their total mold count on Sabouraud's agar mediumFrom the plant samples, 106 molds were isolated and screened for their ability to produce aflatoxinsOnly three fungal isolates were found to produce aflatoxins B1 and B2Two isolates were identified as Aspergillus parasiticus and one isolated as Aspergillus flavusThe spores of the aflatoxin producing fungal isolates were studied for their resistance to gamma radiation

Efeito de extratos proteícos de milho sobre a produçäo de aflatoxina B / Effect of corn proteic extracts on aflatoxin B production

Testou-se o efeito de extratos proteícos de milho à base de NaC1 5% sobre a produçäo de aflatoxina B, em meio de cultura líquido contendo, 2% de extrato de levedura e 20% de sacarose. Dois métodos de extraçäo foram avaliados a partir do peso seco do micélico do fungo Aspergillus flavus e da quantidade de aflatoxina B produzida. Os extratos proteícos das três variedades testadas estimularam o crescimento micelial do fungo. Obteve-se uma maior concentraçäo proteíca nos extratos previamente tratados com acetona. Contudo, os dois métodos se mostraram igualmente viáveis como instrumento na diferenciaçäo varietal do milho, caracterizando a suscetibilidade de "Pipoca Americana" com altos níveis de aflatoxina produzida. "Centralmex N" e "Centralmex O2" apresentaram uma menor produçäo de aflatoxina que o controle. Os resultados sugerem que a composiçäo proteíca do milho pode apresentar elementos tanto eliciadores como inibidores à produçäo de aflatoxina B