RESUMO
Chlorpyrifos is one of the most used insecticides in agro-ecosystems and is repeatedly applied due to the increase in pest resistance, which leads to environmental accumulation. The aim of this work was to evaluate the effect of chlorpyrifos on growth and aflatoxin B1 (AFB1) production by four Aspergillus section Flavi strains, under different water conditions-aW (0.93, 0.95 and 0.98)-on maize-based medium (MMEA) and maize grains supplied with 0.06 to 1.4 mmol/L of chlorpyrifos. MMEA plates were incubated at 18, 28, and 37 °C and plates with maize grains at 25 °C during 21 days. Chlorpyrifos stimulated the growth and AFB1 production of non-target organisms, such as Aspergillus section Flavi strains, both at low (0.06 mmol/L) and at high concentrations (1.4 mmol/L) on MMEA and maize grains. Stimulation occurred over a wide range of temperature and aw conditions. The toxin concentration produced by the two strains on MMEA at 18 °C increased when the concentration of chlorpyrifos also increased, being most significant at 0.6 mmol/L. In conclusion, the presence of chlorpyrifos should be considered as a factor, together with environmental conditions, for the development of effective production practices of maize grains, in order to avoid fungal growth and AFB1 production, to prevent both economic losses and risks to human and animal health.
Assuntos
Aflatoxina B1/biossíntese , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/crescimento & desenvolvimento , Clorpirifos/farmacologia , Meios de Cultura/química , Inseticidas/farmacologia , Zea mays/microbiologia , Aflatoxina B1/análise , Aspergillus flavus/classificação , Aspergillus flavus/metabolismo , Meios de Cultura/farmacologia , Ecossistema , Temperatura , Água , Zea mays/metabolismoRESUMO
Aspergillus flavus is an opportunistic pathogen and may produce aflatoxins in maize, one of the most important crops in Argentina. A promising strategy to reduce aflatoxin accumulation is the biological control based on competitive exclusion. In order to select potential biocontrol agents among isolates from the maize growing region in Argentina, a total of 512 A. flavus strains were isolated from maize kernels and soil samples. Thirty-six per cent of the isolates from maize kernels did not produce detectable levels of aflatoxins, while 73% of the isolates from soil were characterized as non-aflatoxin producers. Forty percent and 49% of the isolates from maize kernels and soil samples, respectively, were not producers of cyclopiazonic acid (CPA). Sclerotia morphology was evaluated using Czapek Dox media. Eighty-six per cent of the isolates from maize kernels and 85% of the isolates from soil samples were L sclerotia morphotype (average diameterâ¯>â¯400⯵m). The remaining isolates did not produce sclerotia. All isolates had MAT 1-1 idiomorph. The competitive ability of 9 non aflatoxigenic strains, 4 CPA(+) and 5 CPA(-), was evaluated in co-inoculations of maize kernels with an aflatoxigenic strain. All evaluated strains significantly (pâ¯<â¯0.05) reduced aflatoxin contamination in maize kernels. The aflatoxin B1 (AFB1) reduction ranged from 6 to 60%. The strain A. flavus ARG5/30 isolated from maize kernels would be a good candidate as a potential biocontrol agent to be used in maize, since it was characterized as neither aflatoxin nor CPA producer, morphotype L, MAT 1-1 idiomorph, and reduced AFB1 content in maize kernels by 59%. This study showed the competitive ability of potential aflatoxin biocontrol agents to be evaluated under field trials in a maize agro-ecosystem in Argentina.
Assuntos
Antibiose/fisiologia , Aspergillus flavus/isolamento & purificação , Aspergillus flavus/metabolismo , Agentes de Controle Biológico/metabolismo , Zea mays/microbiologia , Aflatoxina B1/biossíntese , Argentina , Aspergillus flavus/classificação , Aspergillus flavus/patogenicidade , Produtos Agrícolas/microbiologia , Ecossistema , Indóis/metabolismo , Microbiologia do SoloRESUMO
Aflatoxin contamination of peanut, due to infection by Aspergillus flavus, is a major problem of rain-fed agriculture in India. In the present study, molecular characterisation of 187 Aspergillus flavus isolates, which were sampled from the peanut fields of Gujarat state in India, was performed using AFLP markers. On a pooled cluster analysis, the markers could successfully discriminate among the 'A', 'B' and 'G' group A. flavus isolates. PCoA analysis also showed equivalent results to the cluster analysis. Most of the isolates from one district could be clustered together, which indicated genetic similarity among the isolates. Further, a lot of genetic variability was observed within a district and within a group. The results of AMOVA test revealed that the variance within a population (84%) was more than that between two populations (16%). The isolates, when tested by indirect competitive ELISA, showed about 68.5% of them to be atoxigenic. Composite analysis between the aflatoxin production and AFLP data was found to be ineffective in separating the isolate types by aflatoxigenicity. Certain unique fragments, with respect to individual isolates, were also identified that may be used for development of SCAR marker to aid in rapid and precise identification of isolates.
Assuntos
Aflatoxinas/metabolismo , Arachis/microbiologia , Aspergillus flavus , Agricultura , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Aspergillus flavus/classificação , Aspergillus flavus/genética , Aspergillus flavus/isolamento & purificação , DNA Fúngico/genética , Ensaio de Imunoadsorção Enzimática , Genes Fúngicos , Variação Genética/genética , Índia , Tipagem Molecular , Técnicas de Tipagem Micológica , Análise de Componente PrincipalRESUMO
Aflatoxin contamination of peanut, due to infection by
Assuntos
Aspergillus flavus , Aflatoxinas/metabolismo , Arachis/microbiologia , Agricultura , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Aspergillus flavus/classificação , Aspergillus flavus/genética , Aspergillus flavus/isolamento & purificação , DNA Fúngico/genética , Ensaio de Imunoadsorção Enzimática , Genes Fúngicos , Variação Genética/genética , Índia , Tipagem Molecular , Técnicas de Tipagem Micológica , Análise de Componente PrincipalRESUMO
The aim of this study was to use a polyphasic approach to identify Aspergillus section Flavi isolated from Brazil nuts collected in the Amazon forest: investigation of macro- and microscopic morphology, production of extrolites, heat-resistance fungi, and sequencing of DNA regions. The following Aspergillus section Flavi species were identified: Aspergillus flavus (75.5%), Aspergillus nomius (22.3%), and Aspergillus parasiticus (2.2%). All A. nomius and A. parasiticus isolates produced aflatoxins B and G, but not cyclopiazonic acid (CPA). A. flavus isolates were more diversified and a high frequency of mycotoxigenic strains was observed. The polyphasic approach permitted the reliable identification of section Flavi species. The rate of mycotoxigenic strains was high (92.7%) and mainly included A. flavus strains producing elevated levels of aflatoxins and CPA. These results highlight the possibility of co-occurrence of both toxins, increasing their potential toxic effect in this commodity.
Assuntos
Aspergillus flavus/isolamento & purificação , Bertholletia/microbiologia , Técnicas de Tipagem Micológica/métodos , Aspergillus flavus/classificação , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Contaminação de Alimentos/análise , Micotoxinas/metabolismo , Sementes/microbiologiaRESUMO
BACKGROUND: During 4 months, and while conducting an environmental sampling of air, 2 cases of aspergillosis by Aspergillus flavus (A. flavus) were diagnosed at an oncohematological center in Buenos Aires, Argentina. AIMS: The aim of this study was to know the variability and the genetic relationship between the clinical and environmental isolates, obtained in the oncohematological center. METHODS: Two genotyping techniques of different discriminatory power (RAPD and AFLP) were used. A genetic similarity matrix was calculated using Jaccard method and was the basis for the construction of a dendrogram by UPGMA. The level of genetic variability was assessed by measuring the percentage of polymorphic loci, number of effective allele, expected heterocygozity and association index test (I(A)). RESULTS: The dendrogram reveals that the A. flavus isolates recovered from the patients were not genetically related to those gotten from the rooms occupied by the patients. The environmental isolates had higher values of genetic diversity than the clinical isolates. The I(A) estimated for all the isolates suggest that recombination events occurred. CONCLUSIONS: Patients 1 and 2 were not infected with isolates from the nosocomial environment. Clinical and environmental isolates of A. flavus showed high genetic variability among them.
Assuntos
Microbiologia do Ar , Poluição do Ar em Ambientes Fechados/estatística & dados numéricos , Aspergillus flavus/isolamento & purificação , Institutos de Câncer/estatística & dados numéricos , Infecção Hospitalar/microbiologia , Aspergilose Pulmonar/microbiologia , Alelos , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Argentina/epidemiologia , Aspergillus flavus/classificação , Aspergillus flavus/genética , Líquido da Lavagem Broncoalveolar/microbiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/transmissão , DNA Fúngico , Contaminação de Equipamentos , Variação Genética , Genótipo , Humanos , Pulmão/microbiologia , Sinusite Maxilar/microbiologia , México/epidemiologia , Cavidade Nasal/microbiologia , Especificidade de Órgãos , Quartos de Pacientes , Aspergilose Pulmonar/epidemiologia , Aspergilose Pulmonar/transmissão , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
Aspergillus flavus is the second most common cause of aspergillosis infection in immunocompromised patients and is responsible for the production of aflatoxins. Little is known about the population structure of A. flavus, although recent molecular and phenotypic data seem to demonstrate that different genetic lineages exist within this species. The aim of this study was to carry out a morphological, physiological, and molecular analysis of a set of clinical and environmental isolates to determine whether this variability is due to species divergence or intraspecific diversity, and to assess whether the clinical isolates form a separate group. The amdS and omtA genes were more phylogenetically informative than the other tested genes and their combined analysis inferred three main clades, with no clear distinction between clinical and environmental isolates. No important morphological and physiological differences were found between the members of the different clades, with the exception of the assimilation of d-glucosamine, which differentiates the members of the clade II from the others.
Assuntos
Aspergilose/microbiologia , Aspergillus flavus/genética , Amidoidrolases/química , Amidoidrolases/genética , Aspergillus flavus/classificação , Aspergillus flavus/metabolismo , Aspergillus flavus/ultraestrutura , Sequência de Bases , DNA Fúngico/química , DNA Fúngico/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Variação Genética , Metiltransferases/química , Metiltransferases/genética , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Microbiologia do SoloRESUMO
Aspergillus flavus is one of the most common fungal eukaryotes on the planet. It is notorious for production of aflatoxins, for causing aspergillosis in humans and animals, and as an opportunistic pathogen of animals and plants. Its role in marine habitats is unclear. Until now, little phylogeographic structure has been detected for the species, except at very local scales, and it appears to fit the classic dictum of microbial biogeography: Everything is everywhere. Here we use genetic relationships among isolates to determine phylogeographic structure, mating types, and differences in preferences for: marine vs. terrestrial habitats, various substrates, and clinical vs. nonclinical environments. Phylogenetic relationships among isolates were estimated using amplified fragment length polymorphisms (AFLPs) and mating types were determined for a worldwide sample of A. flavus isolates from diverse substrates and geographic locations. All isolates composed a single population, with no significant differentiation of marine vs. terrestrial isolates, clinical vs. environmental isolates, or association with substrate or geographic origin. There was evidence for local dominance of a single clade, probably clonal in origin and short-lived. The proportion of mating types was 1:1, supporting the hypothesis of recombination in natural populations. However, a high proportion of clinical isolates were MAT1-1 (85%), suggesting that a gene linked to the MAT1-1 idiomorph could play a role in pathogenicity. This study suggests that a more appropriate description of the phylogeography of A. flavus is 'everything is everywhere, but not all the time.'
Assuntos
Aspergilose/microbiologia , Aspergilose/veterinária , Aspergillus flavus/classificação , Aspergillus flavus/isolamento & purificação , Microbiologia Ambiental , Filogeografia , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Animais , Aspergillus flavus/genética , Aspergillus flavus/fisiologia , Análise por Conglomerados , DNA Fúngico/genética , Humanos , Plantas , Reação em Cadeia da Polimerase , Recombinação Genética , Análise de Sequência de DNARESUMO
The Aspergillus genus belongs to a filamentous fungal group characterized by wide dispersion in the environment. Some species are associated with diseases, especially in immunocompromised patients, while others are of economical importance due to aflatoxin production or biotechnological applications. Its species identification is nowadays performed by traditional techniques combined with molecular markers, resulting in a higher efficiency of isolate characterization. In the present study, internal transcribed spacer, inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of Aspergillus flavus and strains of other species of the A. flavus group. High genetic diversity was revealed by RAPD and by ISSR, in which the use of the (GACA)4 primer yielded a higher diversity than with the (GTG)5 primer, although the latter showed a characteristic banding profile for each species. These data were used to create a similarity matrix for the construction of dendrograms by means of the UPGMA method. The ISSR and RAPD profiles showed that among the strains previously identificated as A. flavus, one should be A. oryzae, one A. parasiticus and two A. tamarii. On the other hand, a strain previously identified as A. parasiticus should be A. flavus. All these strains were retested by traditional methods and their new species identification was confirmed. These results strongly support the need for using molecular markers as an auxiliary tool in differentiating fungal species and strains.
Assuntos
Aspergillus flavus/classificação , Aspergillus flavus/genética , Brasil , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Marcadores Genéticos , Técnicas de Tipagem Micológica , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
AIMS: The objectives of this study were: (i) to evaluate genetic relatedness among Aspergillus section Flavi strains isolated from soil and peanut seeds in Argentina; (ii) to determine if AFLP molecular markers could be useful to identify isolates up to species level, and to correlate these markers with the isolates' toxigenic potentials and/or vegetative compatibility group (VCG) affiliations. METHODS AND RESULTS: Amplified fragment length polymorphism (AFLPs) analysis was applied to compare 82 isolates of Aspergillus section Flavi. Cluster analysis showed a clear separation of A. flavus and A. parasiticus, and comparison of fingerprints revealed several specific markers for each group of isolates. AFLP analysis indicates that no genotypical differences can be established between aflatoxigenic and nonaflatoxigenic producers in both species analysed. In addition, candidate AFLP markers associated with a particular VCG were not found. CONCLUSIONS: There was a concordance between morphological identification and separation up to species level using molecular markers. The findings of specific bands for A. flavus and A. parasiticus may be useful for the design of specific PCR primers in order to differentiate these species and detect them in food. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides new data on molecular characterization of Aspergillus section Flavi in Argentina.