Method for monitoring deletions in the aflatoxin biosynthesis gene cluster of Aspergillus flavus with multiplex PCR

The report presents a rapid, inexpensive and simple method for monitoring indels with influence on aflatoxin biosynthesis within Aspergillus flavus populations. PCR primers were developed for 32 markers spaced approximately every 5 kb from 20 kb proximal to the aflatoxin biosynthesis gene cluster to the telomere repeat. This region includes gene clusters required for biosynthesis of aflatoxins and cyclopiazonic acid; the resulting data were named cluster amplification patterns (CAPs). CAP markers are amplified in four multiplex PCRs, greatly reducing the cost and time to monitor indels within this region across populations. The method also provides a practical tool for characterizing intraspecific variability in A. flavus not captured with other methods.

Significance and Impact of the Study

Aflatoxins, potent naturally‐occurring carcinogens, cause significant agricultural problems. The most effective method for preventing contamination of crops with aflatoxins is through use of atoxigenic strains of Aspergillus flavus to alter the population structure of this species and reduce incidences of aflatoxin producers. Cluster amplification pattern (CAP) is a rapid multiplex PCR method for identifying and monitoring indels associated with atoxigenicity in A. flavus. Compared to previous techniques, the reported method allows for increased resolution, reduced cost, and greater speed in monitoring the stability of atoxigenic strains, incidences of indel mediated atoxigenicity and the structure of A. flavus populations.     

Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis gene cluster in Aspergillus flavus

Cyclopiazonic acid (CPA), an indole-tetramic acid mycotoxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins. Aflatoxin biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. flavus lacking aflatoxin production due to the loss of the entire aflatoxin gene cluster and portions of the subtelomeric region are often unable to produce CPA, which suggests a physical link of genes involved in CPA biosynthesis to the aflatoxin gene cluster. Examining the subtelomeric region in A. flavus isolates of different chemotypes revealed a region possibly associated with CPA production. Disruption of three of the four genes present in this region predicted to encode a monoamine oxidase, a dimethylallyl tryptophan synthase, and a hybrid polyketide non-ribosomal peptide synthase abolished CPA production in an aflatoxigenic A. flavus strain. Therefore, some of the CPA biosynthesis genes are organized in a mini-gene cluster that is next to the aflatoxin gene cluster in A. flavus.     

Aspergillus flavus expressed sequence tags and microarray as tools in understanding aflatoxin biosynthesis

Aflatoxins are the most toxic and carcinogenic naturally occurring mycotoxins. They are produced primarily byAspergillus flavus andA. parasiticus. In order to better understand the molecular mechanisms that control aflatoxin production, identification of genes usingA. flavus expressed sequence tags (ESTs) and microarrays is currently being performed. Sequencing and annotation ofA. flavus ESTs from a normalizedA. flavus cDNA library identified 7,218 unique EST sequences. Genes that are putatively involved in aflatoxin biosynthesis, regulation and signal transduction, fungal virulence or pathogenicity, stress response or antioxidation, and fungal development were identified from these ESTs. Microarrays containing over 5,000 uniqueA. flavus gene amplicons were constructed at The Institute for Genomic Research. Gene expression profiling under aflatoxin-producing and non-producing conditions using this microarray has identified hundreds of genes that are potentially involved in aflatoxin production. Further investigations on the functions of these genes by gene knockout experiments are underway. This research is expected to provide information for developing new strategies for controlling aflatoxin contamination of agricultural commodities.     

Enzyme reactions and genes in aflatoxin biosynthesis

Aflatoxins are highly toxic and carcinogenic substances mainly produced by Aspergillus flavus and Aspergillus parasiticus. Sterigmatocystin is a penultimate precursor of aflatoxins and also a toxic and carcinogenic substance produced by many species, including Aspergillus nidulans. Recently, the majority of the enzyme reactions involved in aflatoxin/sterigmatocystin biosynthesis have been clarified, and the genes encoding the enzymes have been isolated. Most of the genes constitute a large gene cluster in the fungal genome, and their expression is mostly regulated by a product of the regulatory gene aflR. This review will summarize the enzymatic steps and the genes in aflatoxin/sterigmatocystin biosynthesis.     

The inhibitory effect of Bacillus megaterium on aflatoxin and cyclopiazonic acid biosynthetic pathway gene expression in Aspergillus flavus

Aspergillus flavus is one of the major moulds that colonize peanut in the field and during storage. The impact to human and animal health, and to the economy in agriculture and commerce, is significant since this mold produces the most potent known natural toxins, aflatoxins, which are carcinogenic, mutagenic, immunosuppressive, and teratogenic. A strain of marine Bacillus megaterium isolated from the Yellow Sea of East China was evaluated for its effect in inhibiting aflatoxin formation in A. flavus through down-regulating aflatoxin pathway gene expression as demonstrated by gene chip analysis. Aflatoxin accumulation in potato dextrose broth liquid medium and liquid minimal medium was almost totally (more than 98 %) inhibited by co-cultivation with B. megaterium. Growth was also reduced. Using expression studies, we identified the fungal genes down-regulated by co-cultivation with B. megaterium across the entire fungal genome and specifically within the aflatoxin pathway gene cluster (aflF, aflT, aflS, aflJ, aflL, aflX). Modulating the expression of these genes could be used for controlling aflatoxin contamination in crops such as corn, cotton, and peanut. Importantly, the expression of the regulatory gene aflS was significantly down-regulated during co-cultivation. We present a model showing a hypothesis of the regulatory mechanism of aflatoxin production suppression by AflS and AflR through B. megaterium co-cultivation.     

Evolution of the aflatoxin gene cluster

WhyAspergillus species produce aflatoxin remains an unsolved question. In this report we suggest that evolution of the aflatoxin biosynthesis gene cluster has been a multistep process. More than 300 million years ago a primordial cluster of genes allowed production of anthraquinones that may have served as insect attractants to facilitate spore dispersal. Later adaptive evolutionary steps introduced genes into the cluster that encoded enzymes associated with fungal virulence. These genes may have allowed the otherwise saprophytic fungi to be better able to colonize living plants. Later, genes for production of aflatoxins B1 and G1 were added to the basal cluster. Loss of the ability to produce aflatoxin G1 occurred with the divergence ofA. flavus, a species that, perhaps, was more successful than its ancestors at colonizing plants. This logical progression in evolutionary development of the aflatoxin biosynthetic cluster fits the phylogenetic data as well as known chemical reactivity of the initially formed anthraquinone polyketide metabolites.     

<Emphasis Type="Italic">Aspergillus flavus</Emphasis> hydrolases: their roles in pathogenesis and substrate utilization

Aspergillus flavus is a fungus that principally obtains resources for growth in a saprophytic mode. Yet, it also possesses the characteristics of an opportunistic pathogen with a wide, non-specific host range (plants, animals, and insects). It has attained a high level of agricultural significance due to production of the carcinogen aflatoxin, which significantly reduces the value of contaminated crops. To access a large variety of nutrient substrates and penetrate host tissues, A. flavus possesses the capacity to produce numerous extracellular hydrolases. Most work on A. flavus hydrolases has focused on the serine and metalloproteinases, pectinase P2c, and amylase. Many hydrolases are presumed to function in polymer degradation and nutrient capture, but the regulation of hydrolase secretion is complex and substrate dependent. Proteinases are employed not only to help access protein substrates, such as elastin that is found in mammals and insects, but may also play roles in fungal defense and virulence. Secretion of the endopolygalacturonase P2c is strongly correlated with isolate virulence (against plants) and maceration of cotton boll tissues. In some hosts, secretion of α-amylase is critical for starch digestion and may play a critical role in induction of aflatoxin biosynthesis. Despite a significant body of work, much remains to be learned about hydrolase production and utilization by A. flavus. This information may be critical for the formulation of successful strategies to control aflatoxin contamination in affected commodities.     

Gene targets for fungal and mycotoxin control

It was initially shown that gallic acid, from hydrolysable tannins in the pelliele of walnut kernels, dramatically inhibits biosynthesis of aflatoxin byAspergillus flavus. The mechanism of this inhibition was found to take place upstream from the gene cluster, including the regulatory gene,aflR, involved in aflatoxin biosynthesis. Additional research using other antioxidant phenolics showed similar antiaflatoxigenic activity to gallic acid. Treatment ofA. flavus withtert-butyl hydroperoxide resulted in an almost doubling of aflatoxin biosynthesis compared to untreated samples. Thus, antioxidative response systems are potentially useful molecular targets for control ofA. flavus. A high throughput screening system was developed using yeast,Saccharomyces cerevisiae, as a model fungus. This screening provided an avenue to quickly identify fungal genes that were vulnerable to treatment by phenolic compounds. The assay also provided a means to quickly assess effects of combinations of phenolics and certain fungicides affecting mitochondrial respiration. For example, theS. cerevisiae sod2† mutant was highly sensitive to treatment by certain phenolics and strobilurins/antimycin A, fungicides which inhibit complex III of the mitochondrial respiratory chain. Verification of stress to this system in the target fungus,A. flavus, was shown through complementation analysis, wherein the mitochondrial superoxide dismutase (Mn-SOD) gene (sodA) ofA. flavus in the ortholog mutant,sod2†, ofS. cerevisiae, relieved phenolic-induced stress. Mitochondrial antioxidative stress systems play an important role in fungal response to antifungals. Combined treatment of fungi with phenolics and inhibitors of mitochondrial respiration can effectively suppress growth ofA. flavus in a synergistic fashion.     

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> NRRL 3357 and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> SRRC 143

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B₁ and B₂ biosynthesis, while A. parasiticus cultures had significantly increased B₁ and G₁ biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.     

Molecular and physiological aspects of aflatoxin and sterigmatocystin biosynthesis by Aspergillus tamarii and A. ochraceoroseus

Until recently, only three species (Aspergillus flavus, A. parasiticus and A. nomius) have been widely recognized as producers of aflatoxin. In this study we examine aflatoxin production by two other species, A. tamarii and A. ochraceoroseus, the latter of which also produces sterigmatocystin. Toxin-producing strains of A. tamarii and A. ochraceoroseus were examined morphologically, and toxin production was assayed on different media at different pH levels using thin layer chromatography and a densitometer. Genomic DNA of these two species was probed with known aflatoxin and sterigmatocystin biosynthesis genes from A. flavus, A. parasiticus and A. nidulans. Under the high stringency conditions, A. tamarii DNA hybridized to all four of the A. flavus and A. parasiticus gene probes, indicating strong similarities in the biosynthetic pathway genes of these three species. The A. ochraceoroseus DNA hybridized weakly to the A. flavus and A. parasiticus verB gene probe, and to two of the three A. nidulans probes. These data indicate that, at the DNA level, the aflatoxin and sterigmatocystin biosynthetic pathway genes for A. ochraceoroseus are somewhat different from known pathway genes. Received: 21 May 1999 / Received revision: 17 November 1999 / Accepted: 3 December 1999     

Fungi associated with food and feed commodities from Ecuador

Freshly harvested soybean, rice and corn from farms and corn-based pelleted feeds were collected from ranches from the coastal and mountain regions in Ecuador during 1998, and assessed for fungal contamination. The most prevalent fungi on pelleted feed were Aspergillus flavus and Fusarium graminearum. The prevalent fungi recovered from soybean were F. verticillioides, F. semitectum, Aspergillus flavus and A. ochraceus. In rice, F. oxysporum was the most prevalent toxigenic fungal species recorded, followed by F. verticillioides and A. flavus. In corn, F. verticillioides was the most prevalent fungus isolated in both the coastal and mountain regions, with high isolation frequencies of A. flavus and A. parasiticus at the coast. Based on the toxigenic species recovered, ochratoxin A may pose a contamination risk for soybean. A higher probability of aflatoxin contamination of corn was found in the coastal samples compared to those of the mountain region, while a risk of fumonisin contamination of corn exists in both regions.This revised version was published online in October 2005 with corrections to the Cover Date.     

Prevention of preharvest aflatoxin contamination through genetic engineering of crops

Current practices on prevention of aflatoxin contamination of crop species include time consuming, expensive agronomic practices. Of all the methods available to-date, conventional breeding and/or genetic engineering to develop host plant-based resistance to aflatoxin-producing fungi appear to be valuable for several reasons. However, breeding for disease-resistant crops is very time consuming, especially in tree crops, and does not lend itself ready to combat the evolution of new virulent fungal races. Moreover, availability of known genotypes with natural resistance to mycotoxin-producing fungi is a prerequisite for the successful breeding program. While it is possible to identify a few genotypes of corn or peanuts that are naturally resistant toAspergillus we do not know whether these antifungal factors are specific toA. flavus. In crops like cotton, there are no known naturally resistant varieties toAspergillus. Availability of transgenic varieties with antifungal traits is extremely valuable as a breeding tool. Several antifungal proteins and peptides are available for genetic engineering of susceptible crop species, thanks to the availability of efficient modern tools to understand and evaluate protein interactions by proteomics of host, and genomics and field ecology of the fungus. Transgenic approaches are being undertaken in several industry and academic laboratories to prevent invasion byAspergillus fungi or to prevent biosynthesis of aflatoxin. Recent trends in reducing aflatoxin contamination through genetic engineering of cultivated crop species with antifungal proteins are summarized in this report. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005     

Purified moniliformin does not affect the force or rate of contraction of isolated guinea pig atria

Aspergillus flavus Link ex Fries and A. parasiticus Speare can invade peanut kernels and under certain environmental conditions produce unacceptable levels of the mycotoxin aflatoxin. A concerted effort is underway to reduce aflatoxin contamination in peanut and peanut products. A potentially effective method of control in peanut is the discovery and use of genes for resistance to either fungal invasion or aflatoxin formation. The objective of the present experimental study was to develop an effective and efficient procedure for screening individual plants or pods of single plants for resistance to invasion by the aflatoxigenic fungi and subsequent aflatoxin production. Methods of obtaining adequate drought-stress and fungal infection were developed through this series of experiments. By completely isolating the pods from the root zone and imposing drought-stress only on pegs and pods, high levels of fungal infection were observed. High amounts of preharvest aflatoxin accumulation were also produced by completely isolating the pods from the root zone. Mid-bloom inoculation with A. parasiticus-contaminated cracked corn and drought-stress periods of 40 to 60 days were the most effective procedures. This technique was used to assess peanut genotypes previously identified as being partially resistant to A. parasiticus infection or aflatoxin contamination, and segregating populations from four crosses. Variability in aflatoxin contamination was found among the 11 genotypes evaluated, however, none were significantly lower than the standard cultivars. Broad-sense heritability of four crosses was estimated through evaluation of seed from individual plants in the F₂ generation. The heritability estimates of crosses GFA-2 × NC-V11 and Tifton-8 × NC-V11 were 0.46 and 0.29, respectively, but mean aflatoxin contamination levels were high (73,295 and 27,305 ppb). This greenhouse screening method could be an effective tool when genes for superior aflatoxin resistance are identified.Cooperative investigation of the USDA-ARS and the University of Georgia, College of Agriculture.     

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.     

Behaviour of Aspergillus flavus in presence of Aspergillus niger during biosynthesis of aflatoxin B1

Aspergillus niger or Aspergillus tamarii when grown as mixed cultures with toxigenic A. flavus inhibits biosynthesis of aflatoxin by A. flavus, owing primarily to its ability to produce inhibitors of aflatoxin biosynthesis and to their ability to degrade aflatoxin. Gluconic acid partly prevents aflatoxin production. The other factors such as changes in pH of the medium and the effect on the growth of A. flavus have no role in imparting capabilities to these cultures to inhibit aflatoxin production by A. flavus.     

Effect of sexual recombination on population diversity in aflatoxin production by Aspergillus flavus and evidence for cryptic heterokaryosis

Aspergillus flavus is the major producer of carcinogenic aflatoxins (AFs) in crops worldwide. Natural populations of A. flavus show tremendous variation in AF production, some of which can be attributed to environmental conditions, differential regulation of the AF biosynthetic pathway and deletions or loss‐of‐function mutations in the AF gene cluster. Understanding the evolutionary processes that generate genetic diversity in A. flavus may also explain quantitative differences in aflatoxigenicity. Several population studies using multilocus genealogical approaches provide indirect evidence of recombination in the genome and specifically in the AF gene cluster. More recently, A. flavus has been shown to be functionally heterothallic and capable of sexual reproduction in laboratory crosses. In the present study, we characterize the progeny from nine A. flavus crosses using toxin phenotype assays, DNA sequence‐based markers and array comparative genome hybridization. We show high AF heritability linked to genetic variation in the AF gene cluster, as well as recombination through the independent assortment of chromosomes and through crossing over within the AF cluster that coincides with inferred recombination blocks and hotspots in natural populations. Moreover, the vertical transmission of cryptic alleles indicates that while an A. flavus deletion strain is predominantly homokaryotic, it may harbour AF cluster genes at a low copy number. Results from experimental matings indicate that sexual recombination is driving genetic and functional hyperdiversity in A. flavus. The results of this study have significant implications for managing AF contamination of crops and for improving biocontrol strategies using nonaflatoxigenic strains of A. flavus.