A review of mycotoxin biosynthetic pathways: associated genes and their expressions under the influence of climatic factors

Fungal contamination of agricultural commodities, particularly by mycotoxigenic fungi, represents an enormous concern for global food security in terms of feeding the world's growing population with sufficient and safe food. Not only do they reduce crop yield and quality, but they also produce substantial numbers of mycotoxins, which pose serious adverse health effects in human and animals. As the genome of most mycotoxigenic species have been sequenced, the gene clusters involved in the biosynthesis of agriculturally important mycotoxins including aflatoxins, fumonisins, ochratoxins, zearalenone and trichothecenes, have been largely identified and characterised, with their roles elucidated by researchers. This review provides a comprehensive overview of the current knowledge of genes involved in the biosynthetic pathways of mycotoxins. In addition, the influence of climatic factors including water, temperature and carbon dioxide on differential mycotoxin gene expressions have been highlighted. Overall, the relationship between the relative expression of key regulatory and structural genes under different environmental conditions is significantly correlated with mycotoxins production. This indicates that mycotoxin gene induction can be used as a reliable indicator or marker to monitor mycotoxin production pre-and-post harvest. Furthermore, current strategies to manage mycotoxin risks still require improvement. Thus, an accurate understanding of the molecular mechanisms of mycotoxin biosynthesis in mycotoxigenic species could help to develop an innovative, robust targeted control strategy. This could include the exploitation of novel compounds, which can inhibit biosynthetic genes, to minimise mycotoxin risks.     

Penicillic Acid Production by Blue-Eye Fungi on Various Agricultural Commodities

Of 10 Penicillium species reported to cause blue-eye disease of corn, four (P. martensii, P. palitans, P. cyclopium, P. puberulum) were found capable of producing the mycotoxin penicillic acid on various agricultural commodities. Commodities with high protein contents did not support toxin synthesis. The extent of toxin production varied with the strain of mold, the commodity, and the temperature; low temperatures (1 to 10 C) favored toxin accumulation.     

Mycotoxins in food systems in Sub Saharan Africa: A review

Mycotoxins, toxic secondary metabolites of fungi are now recognised as major cause of food intoxications in Sub Saharan Africa (SSA). Aflatoxins, the most important of the group have been implicated in acute aflatoxicoses, carcinogenicity, growth retardation, neonatal jaundice and immunological suppression in SSA. The hot and humid tropical climate provides ideal condition for growth of toxigenicAspergillus spp, making food contamination to be widespread in SSA, with maize and groundnuts being the most contaminated. The available data suggests that cassava products (the most important African food) are not prone to aflatoxin contamination. Recent data on ochratoxin A produced by species ofAspergillus on grains have indicated the necessity for it to be monitored in SSA. Fumonisins represent the most importantFusarium mycotoxins in SSA, and surveillance data indicate very high contamination rates of almost 100% in maize samples from West Africa. Limited information exists on the occurrence of trichothecenes, while the data currently available suggest that zearalenone contamination seems not to be a problem in SSA. The strategies under investigation to mitigate the mycotoxin problem in SSA include education of the people on the danger of consuming mouldy foods, pre and post harvest management strategies with emphasis on biological control, use of plant products to arrest fungal growth during storage, enterosorbent clay technology, and the search for traditional techniques that could reduce/detoxify mycotoxins during food processing.     

Impact of food processing and detoxification treatments on mycotoxin contamination

Mycotoxins are fungal metabolites commonly occurring in food, which pose a health risk to the consumer. Maximum levels for major mycotoxins allowed in food have been established worldwide. Good agricultural practices, plant disease management, and adequate storage conditions limit mycotoxin levels in the food chain yet do not eliminate mycotoxins completely. Food processing can further reduce mycotoxin levels by physical removal and decontamination by chemical or enzymatic transformation of mycotoxins into less toxic products. Physical removal of mycotoxins is very efficient: manual sorting of grains, nuts, and fruits by farmers as well as automatic sorting by the industry significantly lowers the mean mycotoxin content. Further processing such as milling, steeping, and extrusion can also reduce mycotoxin content. Mycotoxins can be detoxified chemically by reacting with food components and technical aids; these reactions are facilitated by high temperature and alkaline or acidic conditions. Detoxification of mycotoxins can also be achieved enzymatically. Some enzymes able to transform mycotoxins naturally occur in food commodities or are produced during fermentation but more efficient detoxification can be achieved by deliberate introduction of purified enzymes. We recommend integrating evaluation of processing technologies for their impact on mycotoxins into risk management. Processing steps proven to mitigate mycotoxin contamination should be used whenever necessary. Development of detoxification technologies for high-risk commodities should be a priority for research. While physical techniques currently offer the most efficient post-harvest reduction of mycotoxin content in food, biotechnology possesses the largest potential for future developments.     

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.     

Identification of gene clusters associated with fusaric acid, fusarin, and perithecial pigment production in Fusarium verticillioides

The genus Fusarium is of concern to agricultural production and food/feed safety because of its ability to cause crop disease and to produce mycotoxins. Understanding the genetic basis for production of mycotoxins and other secondary metabolites (SMs) has the potential to limit crop disease and mycotoxin contamination. In fungi, SM biosynthetic genes are typically located adjacent to one another in clusters of co-expressed genes. Such clusters typically include a core gene, responsible for synthesis of an initial chemical, and several genes responsible for chemical modifications, transport, and/or regulation. Fusarium verticillioides is one of the most common pathogens of maize and produces a variety of SMs of concern. Here, we employed whole genome expression analysis and utilized existing knowledge of polyketide synthase (PKS) genes, a common cluster core gene, to identify three novel clusters of co-expressed genes in F. verticillioides. Functional analysis of the PKS genes linked the clusters to production of three known Fusarium SMs, a violet pigment in sexual fruiting bodies (perithecia) and the mycotoxins fusarin C and fusaric acid. The results indicate that microarray analysis of RNA derived from culture conditions that induce differential gene expression can be an effective tool for identifying SM biosynthetic gene clusters.     

Ochratoxins: A global perspective

Ochratoxins have been overshadowed by better-known mycotoxins, but they are gaining importance. Here we consider ochratoxins in the context of aflatoxins, which are better understood than ochratoxins on many levels. We review recent work on taxonomic distribution, contamination of commodities, biosynthesis, toxicity and regulatory aspects of ochratoxins. We focus on ochratoxins in coffee, since coffee is becoming a key commodity in ochratoxin research and regulation.     

镰刀菌真菌毒素产生与调控机制研究进展

镰刀菌是一种重要的植物病原菌,给世界范围内农作物生产带来巨大破坏。除导致产量下降外,由其产生的镰刀菌真菌毒素能够污染农产品品质,给动物和人类食物安全造成严重隐患。单端孢霉烯族毒素（Trichothecenes）、伏马菌素（Fumonisin）和玉米赤霉烯酮（Zearalenone）是三种最重要的镰刀菌真菌毒素。镰刀菌真菌毒素的生物合成与生产受到体内一系列相关功能基因的调控;此外,pH值、碳氮比等环境条件也能影响真菌毒素的产量。本文简述了镰刀菌真菌毒素在产生机理、主要分类、致病性以及调控因素等方面的研究进展。     

Trichothecenes and yellow rain: possible biological warfare agents

‘Yellow Rain’, an alleged biological warfare agent thought to be utilized in parts of both South East Asia and Afghanistan, may be composed in part of the mycotoxins, trichothecenes. However, more recent analyses suggest that the ‘Rain’ was mainly honey bee excreta. The history of the controversy together with the biological effects, chemistry as well as the fungi producing these mycotoxins and agricultural commodities affected by trichothecenes are reviewed.     

Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus

Aspergillus flavus and A. parasiticus are the two most important aflatoxin‐producing fungi responsible for the contamination of agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O‐methylsterigmatocystin, but not CPA. Only four of forty‐five attempted interspecific crosses between opposite mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important fungi.     

Biological detoxification of mycotoxins: a review

Mycotoxins are secondary fungal metabolites and are reported to be carcinogenic, genotoxic, teratogenic, dermato-, nephro- and hepatotoxic. Several studies have shown that economic losses due to mycotoxins occur at all levels of food and feed production, including crop and animal production, processing and distribution. Therefore, there is a great demand for a novel approach to prevent both the formation of mycotoxins in food and feed and the impact of existing mycotoxin contamination. Recently, investigators have reported that many microorganisms including bacteria, yeast, moulds, actinomycetes and algae are able to remove or degrade mycotoxins in food and feed. We have reviewed various strategies for the detoxification of mycotoxins using microorganisms such as bacteria, yeast and fungi.     

Natural occurrence of mycotoxins in cereals

Besides peanuts and cottonseed, cereal grains are the most important feed and food source that occasionally are naturally contaminated with mycotoxins. The problem of mycotoxins occurring naturally in cereals, especially in corn, has become trouble-some because of changing agricultural technology. The mycotoxin problem in cereals is not restricted to any geographic or climatic region. Toxins are produced on cereals, both in the field and in storage; they involve both the grain and the whole plant. The genera of fungi most involved areAspergillus, Fusarium, Penicillium andClaviceps. Mycotoxins known to occur naturally in cereals include aflatoxins B₁, B₂, G₁ and G₂-as well as aflatoxins M₁ and M₂-ochratoxins A and B, penicillic acid, patulin, ergot, zearalenone, citrinin, T-2, tenuazonic acid, kojic acid and sterigmatocystin. Of these mycotoxins, aflatoxins, patulin, penicillic acid and sterigmatocystin are carcinogens.     

Understanding the genetics of regulation of aflatoxin production and Aspergillus flavus development

Aflatoxins are polyketide-derived, toxic, and carcinogenic secondary metabolites produced primarily by two fungal species, Aspergillus flavus and A. parasiticus, on crops such as corn, peanuts, cottonseed, and treenuts. Regulatory guidelines issued by the U.S. Food and Drug Administration (FDA) prevent sale of commodities if contamination by these toxins exceeds certain levels. The biosynthesis of these toxins has been extensively studied. About 15 stable precursors have been identified. The genes involved in encoding the proteins required for the oxidative and regulatory steps in the biosynthesis are clustered in a 70 kb portion of chromosome 3 in the A. flavus genome. With the characterization of the gene cluster, new insights into the cellular processes that govern the genes involved in aflatoxin biosynthesis have been revealed, but the signaling processes that turn on aflatoxin biosynthesis during fungal contamination of crops are still not well understood. New molecular technologies, such as gene microarray analyses, quantitative polymerase chain reaction (PCR), and chromatin immunoprecipitation are being used to understand how physiological stress, environmental and soil conditions, receptivity of the plant, and fungal virulence lead to episodic outbreaks of aflatoxin contamination in certain commercially important crops. With this fundamental understanding, we will be better able to design improved non-aflatoxigenic biocompetitive Aspergillus strains and develop inhibitors of aflatoxin production (native to affected crops or otherwise) amenable to agricultural application for enhancing host-resistance against fungal invasion or toxin production. Comparisons of aflatoxin-producing species with other fungal species that retain some of the genes required for aflatoxin formation is expected to provide insight into the evolution of the aflatoxin gene cluster, and its role in fungal physiology. Therefore, information on how and why the fungus makes the toxin will be valuable for developing an effective and lasting strategy for control of aflatoxin contamination.     

Trichothecene biosynthesis in Fusarium species: chemistry, genetics, and significance.

Several species of the genus Fusarium and related fungi produce trichothecenes which are sesquiterpenoid epoxides that act as potent inhibitors of eukaryotic protein synthesis. Interest in the trichothecenes is due primarily to their widespread contamination of agricultural commodities and their adverse effects on human and animal health. In this review, we describe the trichothecene biosynthetic pathway in Fusarium species and discuss genetic evidence that several trichothecene biosynthetic genes are organized in a gene cluster. Trichothecenes are highly toxic to a wide range of eukaryotes, but their specific function, if any, in the survival of the fungi that produce them is not obvious. Trichothecene gene disruption experiments indicate that production of trichothecenes can enhance the severity of disease caused by Fusarium species on some plant hosts. Understanding the regulation and function of trichothecene biosynthesis may aid in development of new strategies for controlling their production in food and feed products.     

Isolation of deoxynivalenol-transforming bacteria from the chicken intestines using the approach of PCR-DGGE guided microbial selection

Background  

Contamination of grains with trichothecene mycotoxins, especially deoxynivalenol (DON), has been an ongoing problem for Canada and many other countries. Mycotoxin contamination creates food safety risks, reduces grain market values, threatens livestock industries, and limits agricultural produce exports. DON is a secondary metabolite produced by some Fusarium species of fungi. To date, there is a lack of effective and economical methods to significantly reduce the levels of trichothecene mycotoxins in food and feed, including the efforts to breed Fusarium pathogen-resistant crops and chemical/physical treatments to remove the mycotoxins. Biological approaches, such as the use of microorganisms to convert the toxins to non- or less toxic compounds, have become a preferred choice recently due to their high specificity, efficacy, and environmental soundness. However, such approaches are often limited by the availability of microbial agents with the ability to detoxify the mycotoxins. In the present study, an approach with PCR-DGGE guided microbial selection was developed and used to isolate DON -transforming bacteria from chicken intestines, which resulted in the successful isolation of several bacterial isolates that demonstrated the function to transform DON to its de-epoxy form, deepoxy-4-deoxynivalenol (DOM-1), a product much less toxic than DON.     

Determination of mycotoxins in foods: current state of analytical methods and limitations

Mycotoxins are natural contaminants produced by a range of fungal species. Their common occurrence in food and feed poses a threat to the health of humans and animals. This threat is caused either by the direct contamination of agricultural commodities or by a “carry-over” of mycotoxins and their metabolites into animal tissues, milk, and eggs after feeding of contaminated hay or corn. As a consequence of their diverse chemical structures and varying physical properties, mycotoxins exhibit a wide range of biological effects. Individual mycotoxins can be genotoxic, mutagenic, carcinogenic, teratogenic, and oestrogenic. To protect consumer health and to reduce economic losses, surveillance and control of mycotoxins in food and feed has become a major objective for producers, regulatory authorities and researchers worldwide. However, the variety of chemical structures makes it impossible to use one single technique for mycotoxin analysis. Hence, a vast number of analytical methods has been developed and validated. The heterogeneity of food matrices combined with the demand for a fast, simultaneous and accurate determination of multiple mycotoxins creates enormous challenges for routine analysis. The most crucial issues will be discussed in this review. These are (1) the collection of representative samples, (2) the performance of classical and emerging analytical methods based on chromatographic or immunochemical techniques, (3) the validation of official methods for enforcement, and (4) the limitations and future prospects of the current methods.     

Oligonucleotide microarray for the identification of potential mycotoxigenic fungi

Background  

Mycotoxins are secondary metabolites which are produced by numerous fungi and pose a continuous challenge to the safety and quality of food commodities in South Africa. These toxins have toxicologically relevant effects on humans and animals that eat contaminated foods. In this study, a diagnostic DNA microarray was developed for the identification of the most common food-borne fungi, as well as the genes leading to toxin production.     

Nachweis von Fusarien und deren Mykotoxinen in Futterkonservaten aus Grünlandbeständen mit differenzierter Bewirtschaftungsintensität

Conservation forage (17 hay and 18 grass silage samples) from 15 farms with different intensities of grassland management in the Federal State of Brandenburg were examined for contamination with fusaria and their mycotoxins. The numbers of culturable filamentous fungi in hay were determined by plate counting andFusarium isolates were classified taxonomically. The mycotoxins Zearalenone (ZEA) and Deoxynivalenol (DON) were extracted from hay as well as silage by different procedures and detected chromatographically (HPLC). The numbers of filamentous fungi in the hay samples were 10² and 10⁶ CFU/g FM independently of intensive or extensive management. Only fourFusarium species were identified.Fusarium culmorum, a potential toxin producing species, was most frequently detected (52% of all isolates). ZEA was found in two hay and four silage samples (6-66 μg/kg), DON in three hay and seven silage samples (63–1290 μg/kg). There were no differences between forage samples of extensive and intensive cultivated grassland of the year 2003 regarding numbers of fusaria and the content of their mycotoxins.

Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004.     

Species-specific responses of dew fly larvae to mycotoxins

Abstract   The larval stages of saprophagous insects and filamentous fungi have been demonstrated to be serious competitors on decaying organic matter. When filamentous fungi appear to be competitively superior, fungal mycotoxins have frequently been suggested to constitute chemical weapons, causing high mortality among insect larvae. In this study, we tested whether typical fungal secondary compounds can indeed be considered as the underlying mechanism of interference competition between filamentous fungi and various saprophagous Drosophila species. In contrast to our expectation, we found no grand mycotoxin-specific effects, but insect survival appeared to be generally determined by complex interaction between toxin identity, toxin concentration and insect species. Three out of five drosophilids seemed to be equally affected by the mycotoxins used in this study, whereas two species showed toxin-specific changes in survival. Only two (Kojic acid and Ochratoxin A) out of seven mycotoxins caused insect-specific responses. Moreover, we discovered correlations between survival in toxin-free and spoiled substrates, which may indicate an interrelationship between intra-specific competitive ability and resistance to mycotoxins. We discuss the significance of mycotoxins as underlying mechanisms driving competitive insect–fungus interactions.