Mycotoxin Biosynthetic Genes As Targets for Detection of Fungi

Mycotoxins are a chemically diverse group of fungal secondary metabolites that are harmful to animals and humans. Several hundreds of different mycotoxins have been identified, but only about 20 of them are relevant to human health. Most of the mycotoxin-producing species are filamentous ascomycetes or deuteromycetes, Aspergillus, Fusarium and Penicillium are considered as being the most important mycotoxin-producing genera. Most mycotoxins are very resistant to physical or chemical treatments. Although well-documented cases of mycotoxicoses are rare, the constant uptake of small amounts of mycotoxins, especially those with carcinogenic activity, can have profound effects on human health. Apart from the use of specific rDNA gene-based or RAPD-based probes, mycotoxin biosynthetic genes can serve as ideal targets for the detection of the producing fungi. To date, a number of biosynthetic genes of mycotoxins have been isolated and characterized. These gene sequences could serve as targets for molecular detection of the producing fungi as detailed below.

3.2.1 Aflatoxins

Aflatoxins (ATs) are among the most carcinogenic naturally occurring compounds known. Aflatoxins are produced mainly by species of Aspergillus section Flavi, e.g., by A. flavus, A. parasiticus, A. nomius, and A. bombycis. Sterigmatocystin, an intermediate of AT biosynthesis is also produced by other fungal species including, e.g., Aspergillus nidulans and A. versicolor. A. flavus and A. parasiticus are closely related to the nonaflatoxigenic A. oryzae and A. sojae species, respectively, which are used in food industry for producing soy sauce and frequently applied as hosts for the expression of heterologous proteins. Since these species are both morphologically and physiologically very similar to the AT-producing species, molecular methods have been extensively surveyed for the differentiation of these species, and AT-producing and nonproducing A. flavus isolates.

The biosynthetic pathway leading to AT production is one of the best known secondary metabolite pathways in fungi (Brown et al. 1996; 1999). Criseo et al. (2001), Farber et al. (1997), Geisen (1996; 1998), and Shapira et al. (1996) applied multiplex PCR targeting 3-4 genes of the gene cluster to identify AT-producing fungi. Bagnara et al. (2000) applied the real-time PCR system for the detection of an AT producing A. flavus isolate in black pepper. Mayer et al. (2003) developed a Taqman probe based on sequences of the norsolorinic acid reductase (nor-1) gene for quantitative detection of AT-producing fungi in foods including black pepper, red pepper, corn, and cereals. All authors found that some nonaflatoxigenic isolates gave false positive results. Even the AT nonproducing A. sojae and A. oryzae isolates were found to carry AT biosynthetic genes (Kusumoto et al. 2000; Matsushima et al. 2001). However, mRNA of the aflR, the regulatory gene of AT biosynthesis was not observed in A. sojae (Matsushima et al. 2001). These results indicate that a (multiplex) RT-PCR technique developed by Sweeney et al. (2000) for monitoring AT production in A. parasiticus could be used more efficiently for detection of AT producing fungi. Mayer et al. (2001) used Taqman-based real-time PCR for monitoring the expression of the nor-1 gene providing a possible mRNA-based method for the detection of AT-producing fungi in foods and feeds.

3.2.2 Trichothecenes

Trichothecenes are sesquiterpenoid mycotoxins produced by several fungal genera including Fusarium, Trichothecium, Acremonium, Gliocladium, Myrothecium, Trichoderma and Stachybotrys. More than 30 structurally related tetracyclic trichothecenes are known, among which T-2 toxin, diacetoxyscirpenol, deoxynivalenol, and nivalenol are economically the most important mycotoxins. The main producers of these toxins are in the genus Fusarium (e.g., F. acuminatum, F. culmorum, F. graminearum, F. poae, F. solani, and F. sporotrichioides). Trichothecenes have dermatotoxic, cytotoxic, and phytotoxic properties, and have been suggested to play a role in plant pathogenesis as virulence factors. Trichothecene biosynthesis genes were found to comprise a 25 kbp gene cluster including at least 10 genes in F. sporotrichioides and F. graminearum (Brown et al. 2001; Hohn et al. 1993). Similar clustering of the trichothecene biosynthesis genes was found in Myrothecium roridum, which produces macrocyclic trichothecenes (Trapp et al. 1998).

Fekete et al. (1997) determined the sequence of the Tri5 gene of Fusarium poae, and used a 378 bp fragment of it as a hybridization probe to detect similar sequences by dot blot hybridization in other fungi. Strong hybridization was observed to trichothecene producing Fusarium, Myrothecium and Stachybotrys species, but not to Trichoderma and Trichothecium DNAs. Niessen and Vogel (1998) developed a PCR method based on the amplification of Tri5 sequences for the detection of trichothecene producing Fusarium species in wheat samples. The authors also applied the LightCycler system with Sybr Green I for quantitation of their results. For distinguishing nonspecific products such as primer dimers, melting point analysis was carried out (Schnerr et al. 2001). Tri5 gene sequences were chosen as targets too in a quantitative competitive PCR approach developed for the detection and quantification of trichothecene producing fusaria in cereals by Edwards et al. (2001), and by Birzele et al. (2000). Doohan et al. (1999) developed a reverse-transcription-based PCR assay to quantify the expression of the Tri5 gene in Fusarium species. Lee et al. (2001) developed primer pairs based on sequences of the Tri7

gene for distinguishing between deoxynivalenol and nivalenol producing isolates of Gibberella zeae (Fusarium graminearum).

3.2.3 Patulin

Patulin is an unsaturated lactone produced by a number of Aspergillus, Penicillium, and Byssochlamys (Paecilomyces) species. Representatives of other fungal genera, such as Mucor, Mortierella, Alternaria, Chrysosporium, Fusarium, and Trichoderma were also found to produce this mycotoxin (Steiman et al. 1989). The economically most important producer of patulin is Penicillium expansum, the causative agent of soft rot of apples and other pomaceous fruits. Patulin is receiving worldwide attention because of its occurrence in unfermented apple juice. The biosynthesis of patulin is well known (Paterson et al. 2000). Two genes of the biosynthetic pathway, the polyketide synthase gene, and an iso-epoxydon dehydrogenase (IDH) gene have been cloned and characterized to date (Beck et al. 1990; Wang et al. 1991; Gaucher GM, and Fedeschko RW, unpublished results). The IDH gene product catalyzes the epoxydon-phyllostine oxidation step of patulin biosynthesis (Sekiguchi and Gaucher 1979). Recently, a primer pair specific for the IDH gene (GenBank accession number AF006680) has been developed and used successfully to detect patulin-producing abilities of penicillia (Paterson et al. 2000). Patulin production of Aspergillus species was also analyzed using analytical procedures (thin layer chromatography and HPLC), agar diffusion test, and a PCR-based approach using the primer pair developed for the detection of the IDH gene (Varga et al. 2003). The analytical-, biological-, and PCR-based approaches used for patulin detection gave highly similar results indicating that the primers developed for a Penicillium IDH gene could also be used to detect patulin producing aspergilli in natural substrates. A quantitative PCR approach is also being tested for the detection of patulin producing organisms in foods including apple products and malt.

3.2.4 Other Mycotoxin Biosynthetic Genes

The PR-toxin is a sesquiterpenoid mycotoxin produced by P. roqueforti strains isolated from cheese and silage (Geisen 1998). This mycotoxin has been implicated in incidences of mycotoxicoses resulting from the consumption of contaminated grains (Proctor and Hohn 1993). The gene sequence of aristolochene synthase (a sesquiterpene cyclase), the key enzyme of PR-toxin biosynthesis is known (Proctor and Hohn 1993). Geisen (1998) developed a primer pair based on the sequence of aristolochene synthase, which was used successfully for the detection of PR-toxin producing fungi in cereals and cheese.

The ergot alkaloids are produced mainly by Claviceps purpurea and some other Claviceps species (e.g., C. paspali, C. fusiformis), although other species, e.g., Aspergillus fumigatus, A. clavatus, Penicillium species, and plants have also been described as producers of these mycotoxins. Since

Claviceps strains parasitize not only cereals but also different kinds of grasses, ergot poisoning of grazing animals is still an economic problem. Sequences of some of the biosynthetic genes responsible for ergot alkaloid production are known (Tudzynski et al. 1999). Boichenko et al. (2001) developed a primer pair based on sequences of 4-(g,g-dimethylallyl)-tryptophane (DMAT) synthase, the key enzyme of the ergot alkaloid biosynthesis, and used successfully in PCR reactions to detect ergot alkaloid producing fungi.

Fumonisin B1 was discovered in 1988 in Fusarium moniliforme (F. verticillioides) isolates. Fumonisins are most frequently encountered from stored corn and other cereals, and are the causal agents of leucoencephalomalacia, a fatal brain disease of horses, pulmonary edema in pigs and suspected to be responsible for high incidences of esophageal cancer in South Africa and China. Fumonisins are synthesized through the polyketide biosynthetic route. Seo et al. (2001) identified four coregulated genes associated with fumonisin production in F. verticillioides. These data indicate that these gene sequences could be used for the development of gene probes for the identification of potential fumonisin producing species in foods and feed products.

Since biosynthetic genes of other mycotoxins, e.g., those of fumonisins (Proctor et al. 1999; Seo et al. 2001) and the tremorgenic mycotoxin paxilline (Young et al. 2001) have also been characterized, molecular detection methods can potentially be applied in these cases as well. Apart from the mycotoxins mentioned earlier, the occurrence of the nephrotoxic ochratoxins in cereals, coffee beans, and other agricultural commodities also poses serious hazard to human and animal health (Varga et al. 2001a,b). Experiments are in progress in some European laboratories to isolate and characterize genes responsible for ochratoxin biosynthesis in Penicillium and Aspergillus species, and to identify suitable molecular probes for the detection of ochratoxin-producing fungi in foods and feeds (Quality of life and management of living resources project number QLK1-CT-1999-00433: Prevention of ochratoxin A in cereals; QLK1-CT-1999-01380: Early detection of toxigenic Fusarium species and ochratoxigenic fungi in plant products).

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