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1.
Int J Food Microbiol ; 322: 108576, 2020 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-32240921

RESUMO

Aflatoxin contamination in food and feed products has been brought into sharp focus over the last few decades in the world. However, there is no effective strategy for solving the problem thus far. Therefore, basic research on the aflatoxin-producer Aspergillus flavus is an urgent need. The vital role of mitogen-activated protein kinases (MAPKs) in signal transduction has been documented in various pathogenic fungi, but their functions in A. flavus have rarely been investigated. Herein, we characterized the detailed function of one of these MAPKs, AflSlt2. Targeted deletion of AflSlt2 gene indicates that this kinase is required for vegetative growth, conidia generation, and sclerotium formation. The analysis of AflSlt2 deletion mutant revealed hypersensitivity to cell wall-damaging chemicals and resistance against hydrogen peroxide. Interestingly, the ability of the ΔAflSlt2 mutant to generate aflatoxins in medium was significantly increased compared to wild type. However, a pathogenicity assay indicated that the ΔAflSlt2 mutant was deficient in peanut infection. Site-directed mutation study uncovered that the function of AflSlt2 was dependent on the phosphorylated residues (Thr-186 and Tyr-188) within the activation loop and the phosphotransfer residue (Lys-52) within the subdomain II. Interestingly, an autophosphorylation mutant of AflSlt2 (AflSlt2R66S) displayed wild type-like phenotypes. Bringing these observations together, we propose that Slt2-MAPK pathway is involved in development, stress response, aflatoxin biosynthesis, and pathogenicity in A. flavus. This study may be useful to unveil the regulation mechanism of aflatoxin biosynthesis and provide strategy to control A. flavus contamination.


Assuntos
Aflatoxinas/biossíntese , Arachis/microbiologia , Aspergillus flavus/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Aspergillus flavus/crescimento & desenvolvimento , Aspergillus flavus/patogenicidade , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Transdução de Sinais , Estresse Fisiológico
2.
J Appl Microbiol ; 129(3): 652-664, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32196866

RESUMO

AIMS: This study aimed to apply the volatile organic compounds from Streptomyces philanthi RL-1-178 (VOCs RL-1-178) as a fumigant to protect soybean seeds against the two aflatoxin-producing fungi in stored soybean seeds. METHODS AND RESULTS: The antifungal bioassay tests on potato dextrose agar (PDA) dishes showed that 30 g l-1 wheat seed inoculum of S. philanthi RL-1-178 exhibited total (100%) inhibition on Aspergillus parasiticus TISTR 3276 and Aspergillus flavus PSRDC-4. Identification of the VOCs RL-1-178 using GC-MS revealed 39 compounds with the most abundant substances being geosmin (13·75%) followed by l-linalool (13·55%), 2-mercaptoethanol (9·71%) and heneicosane (5·96%). Comparison on the efficacy of the VOCs RL-1-178 (at 30 g l-1 wheat seed culture) and their four major components (100 µl l-1 each) on the suppression of the two aflatoxin-producing fungi on PDA plates revealed that the VOCs RL-1-178 as well as geosmin, l-linalool and 2-mercaptoethanol completely inhibited (100%) mycelial growth while heneicosane showed only 70·7% inhibition. Use of the VOCs RL-1-178 (30 g l-1 ) as a biofumigant on stored soybean seeds resulted in complete protection (100%) against the infection as well as complete inhibition on production of aflatoxin (B1 , B2 and G2 ) (analysed by HPLC) by the two aflatoxin-producing fungi. CONCLUSIONS: The VOCs RL-1-178 displayed strong inhibitory effects on A. parasiticus TISTR 3276 and A. flavus PSRDC-4 as well as inhibited aflatoxin (B1 , B2 and G2 ) production. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the VOCs RL-1-178 can be applied as a biofumigant to control the two aflatoxin-producing fungi on stored seeds products.


Assuntos
Aspergillus/efeitos dos fármacos , Fumigação/métodos , Controle Biológico de Vetores/métodos , Soja/microbiologia , Streptomyces/metabolismo , Aflatoxinas/biossíntese , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Aspergillus/crescimento & desenvolvimento , Aspergillus/metabolismo , Sementes/microbiologia
3.
J Environ Sci Health B ; 55(3): 210-219, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31653182

RESUMO

In the present study, ethanolic extract from Heliopsis longipes roots and affinin/spilanthol against Aspergillus parasiticus growth and aflatoxins production were studied in relation to the expression of aflD and aflR, two key genes of aflatoxins biosynthetic pathway. Phytochemical analysis of the ethanolic extract by GC-EIMS identified affinin/spilanthol (7.84 ± 0.27 mg g-1) as the most abundant compounds in H. longipes roots. The antifungal and anti-aflatoxigenic assays showed that affinin/spilanthol at 300 µg mL-1 produced the higher inhibition of radial growth (95%), as well as, the higher aflatoxins production inhibition (61%) in comparison to H. longipes roots (87% and 48%, respectively). qRT-PCR revealed that the expression of aflD and aflR genes showed a higher downregulation in affinin/spilanthol at 300 µg mL-1. The expression ratio of alfD was suppressed by affinin/spilanthol in 79% and aflR in 84%, while, a lower expression ratio suppressed by H. longipes was obtained, alfD (55%) and aflR (59%). Affinin/spilanthol possesses higher antifungal and anti-aflatoxigenic activity against A. parasiticus rather than H. longipes roots, and this anti-aflaxotigenic activity occurring via downregulation of the aflD and aflR genes. Thus, H. longipes roots and affinin/spilanthol can be considered potent antifungal agents against aflatoxigenic fungus, especially, affinin/spilanthol.


Assuntos
Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Asteraceae/química , Extratos Vegetais/farmacologia , Alcamidas Poli-Insaturadas/farmacologia , Aflatoxinas/biossíntese , Aflatoxinas/genética , Antifúngicos/química , Aspergillus/genética , Aspergillus/metabolismo , Vias Biossintéticas , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Extratos Vegetais/química , Raízes de Plantas/química , Alcamidas Poli-Insaturadas/análise , Fatores de Transcrição/genética
4.
Toxins (Basel) ; 11(12)2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31847206

RESUMO

Various signaling pathways in filamentous fungi help cells receive and respond to environmental information. Previous studies have shown that the mitogen-activated protein kinase (MAPK) pathway is phosphorylation-dependent and activated by different kinase proteins. Serine/threonine kinase plays a very important role in the MAPK pathway. In this study, we selected the serine/threonine kinase AflSte20 in Aspergillus flavus for functional study. By constructing Aflste20 knockout mutants and complemented strains, it was proven that the Aflste20 knockout mutant (ΔAflste20) showed a significant decrease in growth, sporogenesis, sclerotinogenesis, virulence, and infection compared to the WT (wild type) and complemented strain (ΔAflste20C). Further research indicated that ΔAflste20 has more sensitivity characteristics than WT and ΔAflste20C under various stimuli such as osmotic stress and other types of environmental stresses. Above all, our study showed that the mitogen-activated kinase AflSte20 plays an important role in the growth, conidia production, stress response and sclerotia formation, as well as aflatoxin biosynthesis, in A. flavus.


Assuntos
Aspergillus flavus/fisiologia , Proteínas Fúngicas/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Aflatoxinas/biossíntese , Morfogênese , Pressão Osmótica , Estresse Fisiológico
5.
Environ Microbiol ; 21(12): 4792-4807, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31608565

RESUMO

Aspergillus flavus is a pathogenic fungus that produces carcinogenic aflatoxins, posing a great threat to crops, animals and humans. Lysine acetylation is one of the most important reversible post-translational modifications and plays a vital regulatory role in various cellular processes. However, current information on the extent and function of lysine acetylation and aflatoxin biosynthesis in A. flavus is limited. Here, a global acetylome analysis of A. flavus was performed by peptide pre-fractionation, pan-acetylation antibody enrichment and liquid chromatography-mass spectrometry. A total of 1313 high-confidence acetylation sites in 727 acetylated proteins were identified in A. flavus. These acetylation proteins are widely involved in glycolysis/gluconeogenesis, pentose phosphate pathway, citric acid cycle and aflatoxin biosynthesis. AflO (O-methyltransferase), a key enzyme in aflatoxin biosynthesis, was found to be acetylated at K241 and K384. Deletion of aflO not only impaired conidial and sclerotial developments, but also dramatically suppressed aflatoxin production and pathogenicity of A. flavus. Further site-specific mutations showed that lysine acetylation of AflO could also result in defects in development, aflatoxin production and pathogenicity, suggesting that acetylation plays a vital role in the regulation of the enzymatic activity of AflO in A. flavus. Our findings provide evidence for the involvement of lysine acetylation in various biological processes in A. flavus and facilitating in the elucidation of metabolic networks.


Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/enzimologia , Aspergillus flavus/patogenicidade , Proteínas Fúngicas/metabolismo , Lisina/metabolismo , Metiltransferases/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Acetilação , Arachis/microbiologia , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Ciclo do Ácido Cítrico , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Espectrometria de Massas , Redes e Vias Metabólicas , Metiltransferases/química , Metiltransferases/genética , Via de Pentose Fosfato , Doenças das Plantas/microbiologia , Processamento de Proteína Pós-Traducional , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Esporos Fúngicos/patogenicidade , Virulência
6.
Int J Food Microbiol ; 310: 108313, 2019 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-31476580

RESUMO

Aflatoxin production of Aspergillus flavus is affected by abiotic factors such as temperature, water activity, oxidative stress, etc. These factors likely affect different metabolic pathways and result in altered aflatoxin production. Aflatoxin was determined in liquid media at 28 °C, solid media at 28 °C and solid media at 37 °C. The proteomic method was used to elucidate the mechanism of aflatoxin production in A. flavus in liquid media at 28 °C, solid media at 28 °C and solid media at 37 °C. Potential factors affecting aflatoxin production were found by GO and KEGG analysis. A. flavus produces more aflatoxin at 28 °C compared to 37 °C. Our study also found that A. flavus cultured on solid media produced more aflatoxin than in liquid media. In this study, we identified 5029 proteins from A. flavus NRRL3357, in which 1547 differential proteins were identified between liquid media and solid-state media, while 546 differential proteins were identified between 28 °C and 37 °C. Biological informatics analysis showed that these differential proteins were widely involved in a variety of biological processes, molecular functions, and cellular components, and were associated with multiple metabolic pathways. Compared to the liquid media, extracellular hydrolase for nutrient uptake and proteins related to sclerotia development were differentially expressed on solid media (p < 0.05). Enzymes involved in oxidative stress showed significantly down-regulated in liquid media and up-regulated at 28 °C (p < 0.05). Furthermore, our research also revealed aflatoxin synthesis is a complex process that is affected by a variety of factors such as nutrient uptake, oxidative stress, sclerotia development, G protein signaling pathways and valine, leucine and isoleucine degradation, and a speculative model summarizing the regulation of aflatoxin biosynthesis in A. flavus is presented.


Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/metabolismo , Meios de Cultura/farmacologia , Proteoma , Temperatura , Aflatoxinas/genética , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/genética , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Estresse Oxidativo/genética , Proteômica , Água/metabolismo
7.
Molecules ; 24(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426298

RESUMO

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.


Assuntos
Aflatoxinas/antagonistas & inibidores , Antifúngicos/farmacologia , Regulação Fúngica da Expressão Gênica , Mitocôndrias/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Tiossemicarbazonas/farmacologia , Aflatoxinas/biossíntese , Antifúngicos/química , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/enzimologia , Aspergillus flavus/genética , Sítios de Ligação , Transporte de Elétrons/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Simulação de Acoplamento Molecular , Família Multigênica , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Tiossemicarbazonas/química
8.
J Dairy Sci ; 102(9): 7765-7772, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31301828

RESUMO

The expression of genes associated with aflatoxin biosynthesis by different Aspergillus flavus strains growing on a cheese model system has not been studied. To control aflatoxin biosynthesis, it would be useful to understand the changes in gene expression during cheesemaking and relate those changes to toxin production. The objective of this study was to evaluate the effects of pH, water activity, and temperature on the expression of 2 regulatory genes (aflR and aflS) and 1 structural gene (aflP) involved in aflatoxin biosynthesis, using 3 aflatoxigenic A. flavus strains growing on a cheese-based medium and reverse-transcription real-time PCR. The gene expression patterns were influenced by A. flavus strain and environmental conditions. The structural gene aflP and the regulatory genes aflR and aflS showed similar expression patterns in each A. flavus strain, but we also observed inter-strain differences. We observed the highest expression levels at 6 and 9 d of incubation by A. flavus strains CQ8 and CQ103, and saw a decrease in the days following. Strain CQ7 showed the lowest expression of these genes. We observed the highest expression levels of these genes at pH 5.5, water activity 0.95, and 20 to 25°C; strain CQ103 showed a different pattern for the aflS gene, with maximum expression at pH 6.0 on d 6 of incubation. For the 3 strains, we found a strong correlation between the relative expression of the aflR and aflS genes and the concentration of aflatoxins under conditions that simulated cheese ripening. Control strategies to avoid aflatoxin contamination during cheesemaking could use the detection of regulatory gene expression.


Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/metabolismo , Queijo/microbiologia , Microbiologia de Alimentos , Regulação Fúngica da Expressão Gênica , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados
9.
Mycotoxin Res ; 35(4): 381-389, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31161589

RESUMO

Application of atoxigenic strains to compete against toxigenic strains of Aspergillus flavus strains has emerged as one of the practical strategies for reducing aflatoxin contamination in corn, peanut, and tree nuts. The actual mechanism that results in aflatoxin reduction is not fully understood. Real-time RT-PCR and relative quantification of gene expression protocol were applied to elucidate the molecular mechanism. Transcriptional analyses of aflatoxin biosynthetic gene cluster in dual culture of toxigenic and atoxigenic A. flavus strains were carried out. Six targeted genes, aflR, aflJ, omtA, ordA, pksA, and vbs, were downregulated to variable levels depending on paired strains of toxigenic and atoxigenic A. flavus. Consistent with the decreased gene expression levels, the aflatoxin concentrations in dual cultures were reduced significantly in comparison with toxigenic cultures. Fluorescent images showed fungal hyphae in dual culture displayed green fluorescent, and contacts of live hyphae were seen. A coconut agar plate assay was used to show that toxigenic A. flavus colony produced blue fluorescence under long UV exposure, suggesting that aflatoxin is exported outside fungal hyphae. Furthermore, the assay was applied to demonstrate the potential role of thigmo-regulation in fungal interaction.


Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Interações Microbianas , Família Multigênica , Ágar/química , Genes Fúngicos , Técnicas Microbiológicas
10.
Food Chem ; 293: 472-478, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151636

RESUMO

Water activity (aw) and temperature are two pivotal environmental factors affecting Aspergillus flavus growth and aflatoxin production. Here, we found that AFB1 production on polished rice can occur over a wider range of temperature × aw levels than that on paddies. For fungal growth on polished rice, the optimum conditions were aw 0.92-0.96 and 28-37 °C. The maximum amounts of AFB1 on polished rice was observed at 33 °C and aw 0.96. Compared to 33 °C, all tested genes of A. flavus on polished rice were significantly up-regulated at 25 °C under aw 0.96. The late structural genes of pathway were significantly down-regulated at 37 °C under aw 0.96, although aflR and aflS and most of early structural genes were up-regulated. Compared to aw 0.96, most of pathway genes were significantly down-regulated at aw 0.90 and 0.99 under 33 °C, although two regulatory genes were up-regulated at aw 0.90.


Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/metabolismo , Aflatoxinas/análise , Aspergillus flavus/genética , Aspergillus flavus/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Oryza/microbiologia , Temperatura , Água/química , Água/metabolismo
11.
J Agric Food Chem ; 67(22): 6212-6221, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31099566

RESUMO

Common soil fungi, Aspergillus flavus and Aspergillus parasiticus, are opportunistic pathogens that invade preharvest peanut seeds. These fungi often produce carcinogenic aflatoxins that pose a threat to human and animal health through food chains and cause significant economic losses worldwide. Detection of aflatoxins and further processing of crops are mandated to ensure that contaminated agricultural products do not enter food channels. Under favorable conditions, the fungus-challenged peanut seeds produce phytoalexins, structurally related stilbenoids, capable of retarding fungal development. The purpose of the present study was to evaluate the potential influence of peanut phytoalexins on fungal development and aflatoxin formation in the course of peanut-fungus interaction. The present research revealed that during such interaction, aflatoxin formation was completely suppressed in A. flavus and A. parasiticus strains tested, when low concentrations of spores were introduced to wounded preincubated peanuts. In most of the experiments, when fungal spore concentrations were 2 orders of magnitude higher, the spores germinated and produced aflatoxins. Of all experimental seeds that showed fungal growth, 57.7% were aflatoxin-free after 72 h of incubation. The research provided new knowledge on the aflatoxin/phytoalexin formation in the course of peanut-fungus interaction.


Assuntos
Aflatoxinas/biossíntese , Arachis/microbiologia , Aspergillus/metabolismo , Sementes/química , Estilbenos/farmacologia , Arachis/química , Arachis/metabolismo , Aspergillus/efeitos dos fármacos , Aspergillus/crescimento & desenvolvimento , Interações Hospedeiro-Parasita , Doenças das Plantas/microbiologia , Sementes/metabolismo , Sementes/microbiologia , Sesquiterpenos/metabolismo , Sesquiterpenos/farmacologia , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Estilbenos/metabolismo
12.
Mol Plant Microbe Interact ; 32(9): 1210-1228, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30986121

RESUMO

Histone deacetylases (HDACs) always function as corepressors and sometimes as coactivators in the regulation of fungal development and secondary metabolite production. However, the mechanism through which HDACs play positive roles in secondary metabolite production is still unknown. Here, classical HDAC enzymes were identified and analyzed in Aspergillus flavus, a fungus that produces one of the most carcinogenic secondary metabolites, aflatoxin B1 (AFB1). Characterization of the HDACs revealed that a class I family HDAC, HosA, played crucial roles in growth, reproduction, the oxidative stress response, AFB1 biosynthesis, and pathogenicity. To a lesser extent, a class II family HDAC, HdaA, was also involved in sclerotia formation and AFB1 biosynthesis. An in vitro analysis of HosA revealed that its HDAC activity was considerably diminished at nanomolar concentrations of trichostatin A. Notably, chromatin immunoprecipitation experiments indicated that HosA bound directly to AFB1 biosynthesis cluster genes to regulate their expression. Finally, we found that a transcriptional regulator, SinA, interacts with HosA to regulate fungal development and AFB1 biosynthesis. Overall, our results reveal a novel mechanism by which classical HDACs mediate the induction of secondary metabolite genes in fungi.


Assuntos
Aflatoxinas , Aspergillus flavus , Regulação Fúngica da Expressão Gênica , Histona Desacetilases , Aflatoxinas/biossíntese , Aflatoxinas/genética , Aspergillus flavus/enzimologia , Aspergillus flavus/genética , Aspergillus flavus/patogenicidade , Regulação Fúngica da Expressão Gênica/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Ligação Proteica , Virulência/genética
13.
Toxins (Basel) ; 11(3)2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30934573

RESUMO

Aspergillus flavus colonisation of maize can produce mycotoxins that are detrimental to both human and animal health. Screening of maize lines, resistant to A. flavus infection, together with a biocontrol strategy, could help minimize subsequent aflatoxin contamination. We developed a qPCR assay to measure A. flavus biomass and showed that two African maize lines, GAF4 and KDV1, had different fungal loads for the aflatoxigenic isolate (KSM014), fourteen days after infection. The qPCR assay revealed no significant variation in A. flavus biomass between diseased and non-diseased maize tissues for GAF4, while KDV1 had a significantly higher A. flavus biomass (p < 0.05) in infected shoots and roots compared to the control. The biocontrol strategy using an atoxigenic isolate (KSM012) against the toxigenic isolate (KSM014), showed aflatoxin production inhibition at the co-infection ratio, 50:50 for both maize lines (KDV1 > 99.7% and GAF ≥ 69.4%), as confirmed by bioanalytical techniques. As far as we are aware, this is the first report in Kenya where the biomass of A. flavus from maize tissue was detected and quantified using a qPCR assay. Our results suggest that maize lines, which have adequate resistance to A. flavus, together with the appropriate biocontrol strategy, could limit outbreaks of aflatoxicoses.


Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/crescimento & desenvolvimento , Agentes de Controle Biológico , Zea mays/microbiologia , Bioensaio , Biomassa , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real
14.
Appl Microbiol Biotechnol ; 103(11): 4623-4632, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997552

RESUMO

Recent studies from our laboratory indicate that engineered silver nanoparticles can inhibit aflatoxin biosynthesis even at concentrations at which they do not demonstrate antifungal activities on the aflatoxin-producing fungus. Whether such inhibition can be modified by altering the nanoparticles' physical properties remains unclear. In this study, we demonstrate that three differently sized citrated-coated silver nanoparticles denoted here as NP1, NP2, and NP3 (where, sizes of NP1 < NP2 < NP3) inhibit aflatoxin biosynthesis at different effective doses in Aspergillus parasiticus, the plant pathogenic filamentous fungus. Recapping NP2 with polyvinylpyrrolidone coating (denoted here as NP2p) also altered its ability to inhibit aflatoxin production. Dose-response experiments with NP concentrations ranging from 10 to 100 ng mL-1 indicated a non-monotonic relationship between aflatoxin inhibition and NP concentration. The maximum inhibitory concentrations differed between the NP types. NP1 demonstrated maximum inhibition at 25 ng mL-1. Both NP2 and NP3 showed maximum inhibition at 50 ng mL-1, although NP2 resulted in a significantly higher inhibition than NP3. While both NP2 and NP2p demonstrated greater aflatoxin inhibition than NP1 and NP3, NP2p inhibited aflatoxin over a significantly wider concentration range as compared to NP2. Our results, therefore, suggest that nano-fungal interactions can be regulated by altering certain NP physical properties. This concept can be used to design NPs for mycotoxin prevention optimally.


Assuntos
Aflatoxinas/antagonistas & inibidores , Aflatoxinas/biossíntese , Antifúngicos/metabolismo , Aspergillus/efeitos dos fármacos , Metabolismo/efeitos dos fármacos , Nanopartículas Metálicas/química , Prata/metabolismo , Aspergillus/crescimento & desenvolvimento , Aspergillus/metabolismo , Nanopartículas Metálicas/ultraestrutura , Venenos
15.
Food Microbiol ; 82: 269-276, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31027783

RESUMO

Dry-cured meat products, such as dry-cured ham or dry-fermented sausages, are characterized by their particular ripening process, where a mould population grows on their surface. Some of these moulds are hazardous to the consumers because of their ability to produce mycotoxins including aflatoxins (AFs). The use of native yeasts could be considered a potential strategy for controlling the presence of AFs in dry-cured meat products. The aim of this work was to evaluate the antagonistic activity of two native Debaryomyces hansenii strains on the relative growth rate and the AFs production in Aspergillus parasiticus. Both D. hansenii strains significantly reduced the growth rates of A. parasiticus when grown in a meat-model system at different water activity (aw) conditions. The presence of D. hansenii strains caused a stimulation of AFs production by A. parasiticus at 0.99 aw. However, at 0.92 aw the yeasts significantly reduced the AFs concentration in the meat-model system. The relative expression levels of the aflR and aflS genes involved in the AFs biosynthetic pathway were also repressed at 0.92 aw in the presence of both D. hansenii strains. These satisfactory results were confirmed in dry-cured ham and dry-fermented sausage slices inoculated with A. parasiticus, since both D. hansenii strains significantly reduced AFs amounts in these matrices. Therefore, both tested D. hansenii strains could be proposed as biocontrol agents within a HACCP framework to minimize the hazard associated with the presence of AFs in dry-cured meat products.


Assuntos
Aflatoxinas/biossíntese , Aspergillus/metabolismo , Agentes de Controle Biológico , Debaromyces/fisiologia , Produtos da Carne/microbiologia , Aflatoxinas/genética , Antibiose , Aspergillus/crescimento & desenvolvimento , Regulação para Baixo , Microbiologia de Alimentos , Produtos da Carne/análise , Água/análise
16.
J Agric Food Chem ; 67(15): 4200-4213, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30916945

RESUMO

In Aspergillus, the cyclic adenosine monophosphate (cAMP) signaling modulates asexual development and mycotoxin biosynthesis. Here, we characterize the cyclase-associated protein Cap in the pathogenic fungus Aspergillus flauvs. The cap disruption mutant exhibited dramatic reduction in hyphal growth, conidiation, and spore germination, while an enhanced production of the sclerotia was observed in this mutant. Importantly, the cap gene was found to be important for mycotoxin biosynthesis and virulence. The domain deletion study demonstrated that each domain played an important role for the Cap protein in regulating cAMP/protein kinase A (PKA) signaling, while only P1 and CARP domains were essential for the full function of Cap. The phosphorylation of Cap at S35 was identified in A. flavus, which was found to play a negligible role for the function of Cap. Overall, our results indicated that Cap with multiple domains engages in mycotoxin production and fungal pathogenicity, which could be designed as potential control targets for preventing this fungal pathogen.


Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Aspergillus flavus/enzimologia , Aspergillus flavus/genética , Aspergillus flavus/patogenicidade , AMP Cíclico/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Doenças das Plantas/microbiologia , Domínios Proteicos , Esporos Fúngicos/enzimologia , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismo , Virulência , Zea mays/microbiologia
17.
Bioengineered ; 10(1): 13-22, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30836830

RESUMO

The deep-sea bacterium strain FA13 was isolated from the sediment of the South Atlantic Ocean and identified as Bacillus circulans based on 16S ribosomal DNA sequence. Through liquid fermentation with five media, the cell-free supernatant fermented with ISP2 showed the highest inhibition activities against mycelial growth of Aspergillus parasiticus mutant strain NFRI-95 and accumulation of norsolorinic acid, a precursor for aflatoxin production. Based on ISP2, uniform design was used to optimize medium formula and fermentation conditions. After optimization, the inhibition efficacy of the 20-time diluted supernatant against A. parasiticus NFRI-95 mycelial growth and aflatoxin production was increased from 0-23.1% to 100%. Moreover, compared to the original protocol, medium cost and fermentation temperature were significantly reduced, and dependence on seawater was completely relieved, thus preventing the fermentor from corrosion. This is the first report of a deep-sea microorganism which can inhibit A. parasiticus NFRI-95 mycelial growth and aflatoxin production.


Assuntos
Aflatoxinas/antagonistas & inibidores , Antraquinonas/antagonistas & inibidores , Antitoxinas/isolamento & purificação , Aspergillus/efeitos dos fármacos , Bacillus/metabolismo , Micélio/efeitos dos fármacos , Aflatoxinas/biossíntese , Antraquinonas/metabolismo , Antitoxinas/farmacologia , Organismos Aquáticos , Aspergillus/crescimento & desenvolvimento , Aspergillus/metabolismo , Aspergillus/patogenicidade , Oceano Atlântico , Bacillus/classificação , Bacillus/genética , Bacillus/isolamento & purificação , Reatores Biológicos , Meios de Cultura/química , Análise Fatorial , Fermentação , Sedimentos Geológicos/microbiologia , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Micélio/patogenicidade , Filogenia , RNA Ribossômico 16S/genética , Temperatura
18.
Rev Argent Microbiol ; 51(4): 292-301, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30905507

RESUMO

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 afap1 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 H2O2 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 H2O2 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 AFB1 in the mutant strains was approximately » 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.


Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Estresse Oxidativo/fisiologia , Aspergillus flavus/metabolismo
19.
J Basic Microbiol ; 59(6): 599-608, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30900741

RESUMO

Aflatoxins are part of fungal secondary metabolites which become serious health, environmental, and economic problems and can cause corruption of many crops and agricultural grains that used as food and feed for human and animal. Aflatoxins mainly produce by Aspergillus spp. especially Aspergillus flavus and Aspergillus parasiticus. The present work aimed to study the effect of nanoencapsulation of chitosan (CS) nanoparticles with two phenolic compounds 1-(2-ethyl,6-heptyl)phenol (EHP) extracted from Cuminum cyminum and 5-ethyl-2-(methoxymethyl)phenol (EMMP) extracted from black pepper on growth and aflatoxins production of A. flavus and A. parasiticus. A. flavus growth was completely inhibited by 0.6 mg/ml of EHP and EMMP as well as A. parasiticus which showed the same minimal inhibition concentration with the first compound and 0.8 mg/ml with the second one. CS nanoparticles inhibited the growth of the tested organisms more than CS especially with A. parasiticus and this potency became much better when nanoencapsulated with the two extracted phenolic compounds. In inhibition of aflatoxins production, EHP reduced the production of aflatoxin B1 and B2 of A. flavus by 68.6% and 69.7%, respectively. In the same manner EMMP reduce the production of the two toxins by 87.3% and 82.6%, respectively. The reduction effect of CS nanoparticles is much more than that of CS as it record in most cases about twofold increase. Nanoencapsulation of CS nanoparticles by the extracted phenolic compounds is much more effective with complete inhibition of aflatoxin B1 of both fungi and aflatoxin G1 of A. parasiticus.


Assuntos
Aflatoxinas/biossíntese , Aspergillus/crescimento & desenvolvimento , Aspergillus/metabolismo , Quitosana/química , Nanopartículas/química , Fenóis/química , Aspergillus/efeitos dos fármacos , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/crescimento & desenvolvimento , Aspergillus flavus/metabolismo , Quitosana/farmacologia , Cuminum/química , Estrutura Molecular , Nanopartículas/toxicidade , Fenóis/isolamento & purificação , Fenóis/farmacologia , Piper nigrum/química
20.
Toxins (Basel) ; 11(3)2019 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-30857280

RESUMO

In this work of quercetin's anti-proliferation action on A. flavus, we revealed that quercetin can effectively hamper the proliferation of A. flavus in dose-effect and time-effect relationships. We tested whether quercetin induced apoptosis in A. flavus via various detection methods, such as phosphatidylserine externalization and Hoechst 33342 staining. The results showed that quercetin had no effect on phosphatidylserine externalization and cell nucleus in A. flavus. Simultaneously, quercetin reduced the levels of reactive oxygen species (ROS). For a better understanding of the molecular mechanism of the A. flavus response to quercetin, the RNA-Seq was used to explore the transcriptomic profiles of A. flavus. According to transcriptome sequencing data, quercetin inhibits the proliferation and aflatoxin biosynthesis by regulating the expression of development-related genes and aflatoxin production-related genes. These results will provide some theoretical basis for quercetin as an anti-mildew agent resource.


Assuntos
Aflatoxinas/biossíntese , Antifúngicos/farmacologia , Aspergillus flavus/efeitos dos fármacos , Quercetina/farmacologia , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , RNA-Seq , Espécies Reativas de Oxigênio/metabolismo , Transcriptoma/efeitos dos fármacos
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