The total EU cereal production, for example, is approximately 173 million tonnes per year and it has also been reported that about 20% of the cereal crops grown in Europe and used foods and animals feeds contain measurable amounts of mycotoxins. Also contaminated imports to the EU infested by these toxins are causing concerns. In 1996, the WHO/FAO-Joint Expert Committee on Food Additives (JECFA) has considered various secondary metabolites produced by several fungal genera as potent mycotoxins and has set a Provisional Tolerable Weekly Intake for some of them. That means there is an urgent need to develop validated reliable, rapid, robust and user-friendly detection methods as part of a cost effective and time efficient food safety control strategy for both toxigenic fungi that are widespread on various economically important plant products as well as for their toxigenic secondary metabolites which may be carried-over in the food chain causing human, animal and plant health implications.
The aim of this project is to prevent and reduce consumer's health risk derived from mycotoxin contamination in food and feed. This will be achieved through: (1) development of molecular diagnostic methods for an early detection of toxigenic Fusarium species and ochratoxigenic fungi in plant products, (2) new and more sensitive immunological tests for detection of mycotoxins in food, (3) biochemical and molecular studies to characterise genes responsible for ochratoxin A synthesis.
The objectives are:
- To clone mating-type genes from Gibberella;
- To obtain molecular detection methods (biochips, species-specific quantitative PCR, NASBA) for detection of fumonisin and beauvericin producing Fusarium species;
- To obtain molecular detection methods (biochips, species-specific quantitative PCR, NASBA) for enniatin and trichothecenes producing Fusarium species;
- To develop specific monoclonal antibodies to beauvericin and enniatin and relative producing Fusarium species;
- To develop robust quantitative PCR detection system for ochratoxin A producing fungi;
- To clone genes responsible of OTA biosynthesis.
A BAC library of Fusarium proliferatum strain ITEM 2287, and a clone corresponding with the mat1-2 gene was identified. This has been sequenced and contains the entire mat locus. Moreover, 10 kb and 15kb of flanking sequence on either side of the locus have been identified. Strains of Fusarium, Liseola section, were characterised using DNA based techniques to differentiate them and develop tools to detect the FUM/BEA strains in natural products. Unique composition of populations of F. proliferatum, that is isolated with host specific mtDNA RFLP patterns generally predominant on a given host, suggests that the observed heterogeneity is principally the result of a long host-pathogen and toxins production coevolution. Primer pairs have been identified which can be used to specifically amplify Tri4, Tri5 and Tri6 genes from several trichothecene-producing Fusarium species. A specific assay for F. sporotrichioides has been developed, subject to the screening of further isolates. A primer pair has been developed to specifically amplify F. sporotrichioides, to produce a product of approximately 200bp. The assay has been screened against isolates of F. sporotrichioides and other Fusarium species in the John Innes Centre collection. Further isolates of F. sporotrichioides from diverse geographic sources and plant hosts have been obtained and will be used to fully evaluate the specificity of this assay. A variety of DNA extraction methods have been evaluated to determine the best method for DNA extraction from fungal mycelium. The results of this showed a considerable improvement in reproducibility compared to the established competitive PCR system of quantification. The Tri5 assay has been made quantitative using a competitive PCR system. Monoclonal antibody production was carried out using standard methods established and developed at CSL for these esadepsipeptides mycotoxins. Several arbitrary primers have been tested for their ability to produce a differentiating pattern with the chromosomal DNA of P. verrucosum. By RAPD analysis two major clusters (group I and group II) and a minor cluster (group III) occurred. The capacity of the strains to produce OTA, as determined by TLC, was assigned to the strains in the different RAPD groups. It became obvious that a clear correlation exists between the differentiation of the strains according to the RAPD analysis and their capacity to produce OTA. To further analyse the phylogenetic relationship between the two OTA producing Penicillium groups, the ITS1-5.8S-ITS2 region of selected strains of each group was sequenced and compared. The results support the view that two genetic different groups of Penicillium are able to produce OTA instead of a currently single species It becomes clear, that OTA producing strains of Penicillium does not belong to a genetically homogenous species as is the current view, but obviously rather to different taxonomic groups. The AFLP protocol established for studies in Fusarium was adapted to give optimum results with the DNA samples prepared from Aspergillus spp. AFLP fingerprints of different A. ochraceus, A. niger and A. carbonarius isolates, OTA producers as well as non producers, were obtained. The raw data were processed to fit into a newly created database. In addition, the A. ochraceus isolates analysed could be attributed to two different groups based upon their AFLP patterns. Two fragments was isolated with heterologous primers which apparently originates from a pks gene resonsible for OTA production. The presence of this gene is correlated to the capacity to produce high amounts of OTA. A more general approach for the isolation of OTA biosynthetic genes was planned. The differential display PCR approach (DDRT-PCR) makes the isolation of all genes possible, which are differentially expressed during OTA synthesis. Growth conditions which are permissive respectively restrictive for OTA biosynthesis have been described. In parallel a transformation system for P. verrucosum has been established based on the dominant selectable marker hygromycin C.