Molecular characterization of toxigenic and atoxigenic Aspergillus flavus isolates,collected from peanut fields in China

Aims: The objectives of this study were to assess the genetic relationships between toxigenic and atoxigenic isolates of Aspergillus flavus collected from peanut fields in China, and to analyse deletions within the aflatoxin biosynthetic gene cluster for the atoxigenic isolates. Methods and Results: Analysis of random‐amplified polymorphic DNA and microsatellite‐primed PCR data showed that the toxigenic and atoxigenic isolates of A. flavus were not clustered based on their regions and their ability of aflatoxin and sclerotial production. These results were further supported by DNA sequence of ITS, pksA and omtA genes. PCR assays showed that 24 of 35 isolates containing no detectable aflatoxins had the entire aflatoxin gene cluster. Eleven atoxigenic isolates had five different deletion patterns in the cluster. Conclusions: Toxigenic and atoxigenic isolates of A. flavus are genetically similar, but some atoxigenic isolates having deletions within the aflatoxin gene cluster can be identified readily by PCR assays. Significance and Impact of the Study: Because the extensive deletions within the aflatoxin gene cluster are not rare in the atoxigenic isolates, analysis of deletion within the cluster would be an effective method for the rapid screening of atoxigenic isolates for developing biocontrol agents.     

Selection of non-toxigenic strains of Aspergillus flavus for biocontrol of aflatoxins in maize in Thailand

Aflatoxin production in maize and peanuts remains a major public health problem, especially in developing countries. One promising method for combating aflatoxin formation is biocontrol using competitive exclusion, a management strategy currently being studied in maize crops in Thailand. It is important that the strains of Aspergillus flavus used in biocontrol be non-toxigenic and be incapable of reversion to toxigenicity. In the current study, 80 non-toxigenic strains of A. flavus, randomly selected from commercially produced dried maize samples from several sources in Thailand, were screened for their potential as biocontrol strains by examining the 24 aflatoxin biosynthesis genes, using a PCR assay. Assessment of the presence or absence of PCR products provides an indication of the function of pathway genes. Of the 80 strains, 78 showed no PCR products from one or more genes in the aflatoxin biosynthesis pathway. Twenty-three isolates showed only one failure, in the aflD (nor-1) gene, but most isolates failed to produce a PCR product for two or more genes. Nineteen isolates (24%) failed to show a PCR product in 10 or more genes. Altogether, 45 PCR product patterns were observed, usually common to only one or two isolates, indicating great diversity in the aflatoxin biosynthesis pathway in A. flavus isolates taken from dried Thai maize. Although the absence of a particular PCR product is not conclusive evidence that the particular gene is non-functional, the absence of several such PCR products provides reasonable evidence that the isolate in question will not revert to toxigenicity in the field.     

Evaluation of different genotypes of nontoxigenic Aspergillus flavus for their ability to reduce aflatoxin contamination in peanuts

Aflatoxins produced by the fungus Aspergillus flavus are potent carcinogens and account for large monetary losses worldwide in peanuts, maize, and cottonseed. Biological control in which a nontoxigenic strain of A. flavus is applied to crops at high concentrations effectively reduces aflatoxins through competition with native aflatoxigenic populations. In this study, eight nontoxigenic strains of A. flavus belonging to different vegetative compatibility groups and differing in deletion patterns within the aflatoxin gene cluster were evaluated for their ability to reduce aflatoxin B₁ when paired with eight aflatoxigenic strains on individual peanut seeds. Inoculation of wounded viable peanut seeds with conidia demonstrated that nontoxigenic strains differed in their ability to reduce aflatoxin B₁. Reductions in aflatoxin B₁ often exceeded expected reductions based on a 50:50 mixture of the two A. flavus strains, although one nontoxigenic strain significantly increased aflatoxin B₁ when paired with an aflatoxigenic strain. Therefore, nontoxigenicity alone is insufficient for selecting a biocontrol agent and it is also necessary to test the effectiveness of a nontoxigenic strain against a variety of aflatoxigenic strains.     

Studies on the biocontrol of aflatoxin in maize in Thailand

Aflatoxins in maize and peanuts remain a major cause of liver cancer and other human and animal health issues. The principal causal fungi are Aspergillus flavus and A. parasiticus. Relatively little attention has been paid to reducing aflatoxin formation before harvest. The most promising approach is biocontrol by competitive exclusion. This project aimed to demonstrate the efficacy of locally isolated strains of A. flavus for biocontrol of aflatoxin in maize in Thailand. After a rigorous process utilising molecular methods was used to select non-toxigenic A. flavus strains, field inoculum was produced by using hulled rice coated with A. flavus spores in molasses. Field experiments were conducted over two years in two districts, one of light sandy soil (Chokchai), the other a heavy, close textured, soil (Pakchong). Postharvest treatments representative of local practice were also undertaken. Crops 1 and 2 were not significantly contaminated with aflatoxin at the time of harvest, so any impact of biocontrol could not be assessed. However, wet shelling plus storage before drying resulted in increased aflatoxin contamination; biocontrol had no impact on this increase. In crops 3 and 4, biocontrol had a beneficial impact in some freshly harvested maize. Biocontrol treatments also significantly reduced aflatoxin contamination in samples from some treatments stored for two or four days after shelling, but had minimal effect in others. These experiments demonstrated that biocontrol can be highly effective in reducing aflatoxin contamination in maize in Thailand, both at harvest and during poor postharvest crop handling. However, results were inconsistent.     

Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

The regulatory role of the Aspergillus flavus core retromer complex in aflatoxin metabolism

Aflatoxins are a series of highly toxic and carcinogenic secondary metabolites that are synthesized by Aspergillus species. The degradation of aflatoxin enzymes is an important regulatory mechanism which modulates mycotoxin producing. The retromer complex is responsible for the retrograde transport of specific biomolecules and the vacuolar fusion in the intracellular transport. Late endosomal-associated GTPase (Rab7) has been shown to be a downstream effector protein of the retromer complex. A deficiency in the retromer complex or Rab7 results in several cellular trafficking problems in yeast and humans, like protein abnormal accumulation. However, whether retromer dysfunction is involved in aflatoxin synthesis remains unclear. Here, we report that the core retromer complex, which comprises three vacuolar protein sorting-associated proteins (AflVps26-AflVps29-AflVps35), is essential for the development of dormant and resistant fungal forms such as conidia (asexual reproductive spore) and sclerotia (hardened fungal mycelium), as well as aflatoxin production and pathogenicity, in Aspergillus flavus. In particular, we show the AflVps26-AflVps29-AflVps35 complex is negatively correlated with aflatoxin exportation. Structural simulation, site-specific mutagenesis, and coimmunoprecipitation experiments showed that interactions among AflVps26, AflVps29, and AflVps35 played crucial roles in the retromer complex executing its core functions. We further found an intrinsic connection between AflRab7 and the retromer involved in vesicle-vacuole fusion, which in turn affected the accumulation of aflatoxin synthesis-associated enzymes, suggesting that they work together to regulate the production of toxins. Overall, these results provide mechanistic insights that contribute to our understanding of the regulatory role of the core retromer complex in aflatoxin metabolism.     

Use of microsatellite markers to assess the competitive ability of nontoxigenic Aspergillus flavus strains in studies on biocontrol of aflatoxins in maize in Thailand

Many nontoxigenic strains of Aspergillus flavus have been used in studies on biocontrol by competitive exclusion, but assessing their competitive ability is difficult. This paper reports on the use of a microsatellite marker technique for assessing competitiveness. The chosen microsatellite markers were able to differentiate, at an individual level, between the four biocontrol strains used in a study on the biocontrol of aflatoxins in maize in Thailand. The microsatellite markers were then used to determine which of the four biocontrol strains used were identical with 86 nontoxigenic strains of A. flavus taken from dried maize samples produced in that study. Fifty-one of the 86 strains (59%) were identified as one of the four biocontrol strains, with another four likely to be so. Analysis of microsatellites in A. flavus strains taken from dried samples at the conclusion of a field trial was shown to be of value in understanding the competitive ability of the specific strains used for biocontrol. This method provides an objective assessment of the competitiveness of biocontrol strains.     

Molasses supplementation promotes conidiation but suppresses aflatoxin production by small sclerotial Aspergillus flavus

AIMS: To find a supplemental ingredient that can be added to routinely used growth media to increase conidial production and decrease aflatoxin biosynthesis in small sclerotial (S strain) isolates of Aspergillus flavus. METHODS AND RESULTS: Molasses was added to three commonly used culture media: coconut agar (CAM), potato dextrose agar (PDA), and vegetable juice agar (V8) and production of conidia, sclerotia, and aflatoxins by A. flavus isolate CA43 was determined. The effect of nitrogen sources in molasses medium (MM) on production of conidia, sclerotia and aflatoxins was examined. Water activity and medium pH were also measured. Conidia harvested from agar plates were counted using a haemocytometer. Sclerotia were weighed after drying at 45 degrees C for 5 days. Aflatoxins B(1) and B(2) were quantified by high-performance liquid chromatography. Addition of molasses to the media did not change water activity or the pH significantly. Supplementing CAM and PDA with molasses increased conidial production and decreased aflatoxins. Two-fold increased yield of conidia was found on MM, which, like V8, did not support aflatoxin production. Adding ammonium to MM significantly increased the production of sclerotia and aflatoxins, but slightly decreased conidial production. Adding urea to MM significantly increased the production of conidia, sclerotia and aflatoxins. CONCLUSIONS: Molasses stimulated conidial production and inhibited aflatoxin production. Its effect on sclerotial production was medium-dependent. Water activity and medium pH were not related to changes in conidial, sclerotial or aflatoxin production. Medium containing molasses alone or molasses plus V8 juice were ideal for conidial production by S strain A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: Insight into molecular events associated with the utilization of molasses may help to elucidate the mechanism(s) that decreases aflatoxin biosynthesis. Targeting genetic parameters in S strain A. flavus isolates may reduce aflatoxin contamination of crops by reducing the survival and toxigenicity of these strains.     

Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations

Peanuts and other seed and grain crops are commonly contaminated with carcinogenic aflatoxins, secondary metabolites produced by Aspergillus flavus and A. parasiticus. Aflatoxin contamination of peanuts in the field can be reduced by 77–98% with biological control through the application of nontoxigenic strains of these species, which competitively exclude native aflatoxin-producing strains from developing peanuts. In this study, viable peanut seeds were artificially wounded and inoculated with field soil containing natural fungal populations that were supplemented with conidia of nontoxigenic A. flavus NRRL 21882 (niaD nitrate-nonutilizing mutant) and A. parasiticus NRRL 21369 (conidial color mutant). Increasing soil densities of applied nontoxigenic strains generally resulted in an increase in the incidence of seed colonization by applied nontoxigenic strains, a decrease in seed colonization by native A. flavus and A. parasiticus, and a decrease in aflatoxin concentration in seeds. Reduction of aflatoxins in peanut seeds depended on both the density and the aflatoxin-producing potential of native populations and on the fungal strain used for biological control. Wild-type strain A. flavus NRRL 21882 and its niaD mutant were equally effective in reducing aflatoxins in peanuts, indicating that nitrate-nonutilizing mutants, which are easily monitored in the field, can be used for evaluating the efficacy of biocontrol strains.     

Aspergillus flavus infection and aflatoxin contamination in sorghum seeds and their biological management

In order to establish the current scenario of aflatoxigenic fungal infection and aflatoxin contamination in sorghum seeds across India, 58 seed samples were collected from different agro-climatic regions. Among these, 67.2% samples were infected with Aspergillus spp. and 28% were found contaminated with aflatoxins ranging from 0.0 to 130?μg?kg^?1. Greenhouse studies revealed no correlation between incidence of Aspergillus flavus and aflatoxin content, and its effect on seed quality parameters. Among the 37 A. flavus strains isolated, six were non-aflatoxigenic when analysed through cultural, TLC and ic-ELISA. Seed treatment with biocontrol agents (antagonistic Rhizobacteria and Trichoderma) suppressed the growth of A. flavus under laboratory and significantly enhanced seed quality variables under greenhouse conditions to a various extent. Field trials with selected biocontrol agents showed that talcum powder formulations of Pseudomonas putida Has-1/c, Bacillus spp. 3/a, Trichoderma asperellum M5 and T. asperellum T2 improved seedling emergence, % nutrient accumulation in plants, increased plant biomass and 1000 seed weight. Seeds harvested from treated plants showed significant increase in seed quality variables under laboratory and greenhouse conditions in comparison with control, but there was no significant difference in A. flavus infection and aflatoxin was completely absent in all treatments.     

Inhibition of aflatoxin B production of Aspergillus flavus, isolated from soybean seeds by certain natural plant products

AIMS: The inhibitory effect of cowdung fumes, Captan, leaf powder of Withania somnifera, Hyptis suaveolens, Eucalyptus citriodora, peel powder of Citrus sinensis, Citrus medica and Punica granatum, neem cake and pongamia cake and spore suspension of Trichoderma harzianum and Aspergillus niger on aflatoxin B(1) production by toxigenic strain of Aspergillus flavus isolated from soybean seeds was investigated. METHODS AND RESULTS: Soybean seed was treated with different natural products and fungicide captan and was inoculated with toxigenic strain of A. flavus and incubated for different periods. The results showed that all the treatments were effective in controlling aflatoxin B(1) production. Captan, neem cake, spore suspension of T. harzianum, A. niger and combination of both reduced the level of aflatoxin B(1) to a great extent. Leaf powder of W. somnifera, H. suaveolens, peel powder of C. sinensis, C. medica and pongamia cake also controlled the aflatoxin B(1) production. CONCLUSIONS: All the natural product treatments applied were significantly effective in inhibiting aflatoxin B(1) production on soybean seeds by A. flavus. SIGNIFICANCE AND IMPACT OF THE STUDY: These natural plant products may successfully replace chemical fungicides and provide an alternative method to protect soybean and other agricultural commodities from aflatoxin B(1) production by A. flavus.     

Biological control of pre-harvest aflatoxin contamination in groundnut (Arachis hypogaea L.) with Bacillus subtilis G1

The antagonistic activity of Bacillus subtilis strain G1 was tested against various isolates of Aspergillus flavus in vitro. A talc-based powder formulation of B. subtilis strain G1 was prepared and evaluated to control A. flavus infection and aflatoxin B1 contamination in groundnut under greenhouse and field conditions. The results showed that B. subtilis strain G1 could inhibit the growth of all isolates of A. flavus tested in dual culture assay and the growth inhibition ranged from 93 to 100%. Results of greenhouse and field experiments indicated that B. subtilis strain G1 when applied to groundnut as seed treatment and soil application significantly suppressed A. flavus population in the soil, A. flavus infection and aflatoxin B1 content in kernels and increased the pod yield. These studies show that B. subtilis strain G1 has potential as a biocontrol agent for control of aflatoxin contamination in groundnut.     

Antifungal activity of puroindoline protein from soft wheat against grain molds and its potential as a biocontrol agent

Mold growth reduces the quality of stored grains, besides producing toxins that pose a potential threat to human health. Therefore, prevention of grain mold growth during storage is important to ensure a safe and high-quality product, preferably using an eco-friendly antifungal agent. The puroindoline (PIN) protein was extracted by Triton X-114 and identified by QE mass spectrometry. Aspergillus flavus has attracted much attention because of its toxic secondary metabolites, and PIN protein showed a significant inhibition on A. flavus growth. Scanning electron microscopy revealed altered spore morphology of A. flavus following PIN protein treatment, and propidium iodide staining showed incomplete spore cell membranes. The disruption and deformation of A. flavus spores suggest that the cell walls and cell membranes were compromised. Decreased mitochondrial membrane potential and increased levels of intracellular reactive oxygen specieswere detected using JC-1 and 2,7-dichlorodihydrofluorescein diacetate staining, respectively. PIN protein could effectively inhibit the growth and aflatoxins B1 production of A. flavus in stored grains, such as wheat and rice. PIN proteins can inhibit the growth of many common grain storage molds, including Penicillium, Aspergillus spp. (A. flavus, A. glaucus, A. kawachii, A. ochraceus and A. niger), Alternaria and Fusarium graminearum, in a dose-dependent manner. PIN protein has a significant inhibitory effect on the growth of grain molds, with a stronger inhibitory effect noted in wheat and rice. Our study provides a novel and simple theoretical basis for the selection and storage of mold resistance in grains and food during storage.     

蛋白激酶Cla4对黄曲霉生长发育、毒素合成和致病力的影响

黄曲霉Aspergillus flavus是机会性的动物和植物致病性丝状真菌,保守的PAK(p21-activated protein kinases)样蛋白激酶对信号传导、细胞周期进程和细胞形态发生具有重要作用.通过同源重组方法构建敲除突变株(ΔAflcla4),研究Aflcla4基因对黄曲霉营养生长、分生孢子产生、...     

Evaluation of bacteria and Trichoderma for biocontrol of pre-harvest seed infection by Aspergillus flavus in groundnut

Pre- and post harvest aflatoxin contamination of groundnut caused by Aspergillus flavus is a major problem in the semi-arid tropics. Fluorescent Pseudomonas, Bacillus and Trichoderma spp. potentially antagonistic to A. flavus were isolated from the geocarposphere (pod-zone) of groundnut and used successfully for the control of pre-harvest groundnut seed infection by A. flavus. In greenhouse and field experiments, inoculation of selected antagonistic strains on groundnut resulted in significant reduction of seed infection by A. flavus, and it also reduced >50% of the A. flavus populations (as cfu) in the geocarposphere of groundnut.     

黑曲霉对黄曲霉生长、产毒及黄曲霉毒素B1的影响

目的研究黑曲霉对黄曲霉生长、产毒的抑制作用及对AFB1的降解作用。方法将黑曲霉分别与黄曲霉、AFB1共同培养,定期测定培养液pH、菌丝体干重、黄曲霉孢子数、AFB1含量。结果黑曲霉与黄曲霉混合培养时,黄曲霉孢子数、AFB1含量均比单独培养的低,2组之间差异有统计学意义(P<0.05),抑制率达到68.06%~91.52%;加入黑曲霉后,AFB1含量降低,实验组与对照组之间差异有统计学意义(P<0.05),降解率为46.19%。结论黑曲霉既能抑制黄曲霉生长、产毒,又能降解AFB1。     

黄曲霉菌次级代谢的组学研究进展

黄曲霉菌Aspergillusflavus是一种好氧型腐生真菌,其次级代谢产物黄曲霉毒素主要由黄曲霉和寄生曲霉产生,对诸如玉米、花生等在内的农作物侵染已经严重危及食品安全以及人和动物的健康。近年来,不同组学研究发展迅速,生物学研究的基础数据平台逐步建立。从基因组到转录组、蛋白质组、代谢组等组学技术在对黄曲霉菌次级代谢相关的研究中已有较多应用。本文概述了基因组、转录组、蛋白质组以及代谢组等组学技术在黄曲霉次级代谢研究中所取得的重要进展,并提出了相关研究的发展趋势以及有待解决的问题。为深入了解黄曲霉的生物学功能提供了重要的线索,并为今后的研究奠定了坚实的基础。