Isoepoxydon Dehydrogenase Gene Research Articles

The sequence of the isoepoxydon dehydrogenase gene of the patulin biosynthetic pathway in Penicillium species

Interest in species of the genus Penicillium is related to their ability to produce the mycotoxin patulin and to cause spoilage of fruit products worldwide. The sequence of the isoepoxydon dehydrogenase (idh) gene, a gene in the patulin biosynthetic pathway, was determined for 28 strains representing 12 different Penicillium species known to produce the mycotoxin patulin. Isolates of Penicillium carneum, Penicillium clavigerum, Penicillium concentricum, Penicillium coprobium, Penicillium dipodomyicola, Penicillium expansum, Penicillium gladioli, Penicillium glandicola, Penicillium griseofulvum, Penicillium paneum, Penicillium sclerotigenum and Penicillium vulpinum were compared. Primer pairs for DNA amplification and sequencing were designed from the P. griseofulvum idh gene (GenBank AF006680). The two introns present were removed from the nucleotide sequences, which were translated to produce the IDH sequences of the 12 species for comparison. Phylogenetic relationships among the species were determined from rDNA (ITS1, 5.8 S, ITS2 and partial sequence of 28S rDNA) and from the idh nucleotide sequences minus the two introns. Maximum parsimony analysis showed trees based on rDNA and idh sequences to be congruent. It is anticipated that the genetic information obtained in the present study will aid in the design of probes, specific for patulin biosynthetic pathway genes, to identify the presence of these mycotoxigenic fungi.

Byssochlamys nivea with patulin-producing capability has an isoepoxydon dehydrogenase gene ( idh) with sequence homology to Penicillium expansum and P. griseofulvum

Nucleotide sequences of the isoepoxydon dehydrogenase gene ( idh) for eight strains of Byssochlamys nivea were determined by constructing GenomeWalker libraries. A striking finding was that all eight strains of B. nivea examined had identical nucleotide sequences, including those of the two introns present. The length of intron 2 was nearly three times the size of introns in strains of Penicillium expansum and P. griseofulvum, but intron 1 was comparable in size to the number of nucleotides present in introns 1 and 2 of P. expansum and P. griseofulvum. A high degree of amino acid homology (88 %) existed for the idh genes of the strains of B. nivea when compared with sequences of P. expansum and P. griseofulvum. There were many nucleotide differences present, but they did not affect the amino acid sequence because they were present in the third position. The identity of the B. nivea isolates was confirmed by sequencing the ITS/partial LSU (28 S) rDNA genes. Four B. nivea strains were analysed for production of patulin, a mycotoxin found primarily in apple juice and other fruit products. The B. nivea strains produced patulin in amounts comparable to P. expansum strains. Interest in the genus Byssochlamys is related to the ability of its ascospores to survive pasteurization and cause spoilage of heat-processed fruit products worldwide.

The isoepoxydon dehydrogenase gene of the patulin metabolic pathway differs for Penicillium griseofulvum and Penicillium expansum

Purified DNA from isolates of Penicillium griseofulvum and P. expansum was used as a template to amplify a 600-bp fragment of the isoepoxydon dehydrogenase (idh) gene of the patulin biosynthetic pathway. Primer pairs designed from the P. griseofulvum gene to amplify specific regions of the idh gene yielded similar-sized bands for all strains. Asymmetrical amplification produced DNA products for sequencing and DNA sequences were translated to produce the corresponding amino acid sequences. After removal of two introns present in the region sequenced, amino acid sequences were compared. There were 12 amino acid differences between P. expansum and P. griseofulvum in the coding region. The differences correlated with the amount of patulin previously produced in culture, with strains of P. griseofulvum producing the greatest amounts of patulin.

Primers from the isoepoxydon dehydrogenase gene of the patulin biosynthetic pathway to indicate critical control points for patulin contamination of apples

The mycotoxin patulin is subjected to European Union statutory limits, and has action levels imposed in the United States of America in fruit products. Samples from soil and an orchard were analysed by primers of the isoepoxydon dehydrogenase gene of the patulin biosynthetic pathway as an HACCP procedure in an initial assessment. A non-orchard soil appeared to inhibit the reaction. However, the reaction was positive from orchard soils. Negative results from orchard samples appeared to be because of target DNA being absent. Bramley samples contained the gene frequently. The method may be useful in the determination of critical control points.

The isoepoxydon dehydrogenase gene of patulin biosynthesis in cultures and secondary metabolites as candidate PCR inhibitors

The European Union (EU) has introduced statutory limits for patulin in fruit products. Species definitions need to be unambiguous for this capability, especially to determine 'weak spots' in food, drink and feed production. Fungi were analysed for the isoepoxydon dehydrogenase (IDH) gene of the patulin metabolic pathway to indicate potential patulin production. In several cases inhibition of PCR product formation was indicated. Dilution as a means of overcoming PCR inhibition and to determine optimal concentrations was assessed and the ramifications for nucleic acid analysis in general are highlighted. The inhibitors may be secondary metabolites. However, inhibition was not involved obviously for Penicillium brevicompactum. The gene was detected frequently from Aspergillus section Clavati and Penicillium subgenus Penicillium species. Some strains within certain species were negative for the gene. Detection of the gene product in Byssochlamys is reported.

Novel use of the isoepoxydon dehydrogenase gene probe of the patulin metabolic pathway and chromatography to test penicillia isolated from apple production systems for the potential to contaminate apple juice with patulin

Penicillia isolated from apple production systems were analysed with a probe for the isoepoxydon dehydrogenase (IDH) gene of the patulin metabolic pathway. Patulin production was also determined. These were used to assess which fungi had the potential to contaminate apple juice with patulin. Penicillium expansum was the most frequently isolated species, and the isolates were consistent for patulin production and the IDH gene. P. roquefortii was also positive for the traits. The presence of the gene in some strains of P. brevicompactum was confirmed and evidence of patulin production is presented. Most other species were negative. Some isolates were positive for the IDH gene and negative for patulin production. All fungi with the potential to produce patulin should be considered for optimal control of apple product contamination.

Mycotoxin production and evolutionary relationships among species of Aspergillus section Clavati.

Aspergillus clavatus is a commonly encountered fungus in the environment, producing a number of mycotoxins including patulin, kojic acid, cytochalasins and tremorgenic mycotoxins. A. clavatus belongs to Aspergillus section Clavati together with six other species, all of which possess clavate-shaped vesicles. Patulin production was analysed by thin layer chromatography and high performance liquid chromatography, while a primer pair developed for the detection of an iso-epoxydon dehydrogenase gene involved in the biosynthesis of patulin in penicillia was used to detect the ability of patulin production in the isolates examined. A good correlation was observed between patulin producing properties, and the presence of an iso-epoxydon dehydrogenase gene fragment among the isolates tested. A. longivesica was found for the first time to produce patulin. Ribotoxin production was also examined using a PCR-based approach. Ribotoxins were detected for the first time in an A. pallidus and a Hemicarpenteles acanthosporus isolate. A phylogenetic analysis of intergenic transcribed spacer sequence data indicated that most isolates belong to two main clades that have also been identified earlier based on 26 S rDNA sequence data. A. pallidus isolates clustered together with A. clavatus strains. Although A. clavatus isolates produced highly homogeneous random amplified polymorphic DNA profiles, phylogenetic analysis of these data let us cluster A. clavatus isolates into distinct clades. Correlations were not observed between either patulin or ribotoxin production, and the taxonomic position of the isolates tested, indicating that patulin and ribotoxin producing abilities were lost several times during evolution of Aspergillus section Clavati. Although patulin was earlier found to inhibit mycovirus replication, one of the mycovirus carrying isolates also produced patulin, and both carried the iso-epoxydon dehydrogenase gene.