Modern Methods in Cereal Grain Mycology.pdfVIP

Modern Methods in Cereal Grain Mycology Johan Olsson Department of Microbiology Uppsala Doctoral thesis Swedish University of Agricultural Sciences Uppsala 2000 Acta Universitatis Agriculturae Sueciae Agraria 24 1 ISSN 1401-6249 ISBN 91-576-5792-0 02000 Johan Olson, Uppsala Tryck: SLU Service/Repro, Uppsala 2000 Abstract Olsson, J. 2000. Modem methods in cereal grain mycology. Doctors dissertation. ISSN 1401-6249, ISBN 91-576-5792-0. A simple rapid DNA extraction method, equally suitable for spores and mycelia is proposed. Heating samples in NaOH and SDS provides DNA of high purity, suitable for Polymerase Chain Reaction (PCR) analysis. For Penicillium roqueforti the detection limit was 6 x lo3 conidia and 1 mg (fresh weight) mycelium in the extraction liquid. The method proved efficient with Aspergillus jlavus, Fusarium graminearum, Rhizopus stolonifer, Eurotium herbariorum, and Cladosporium herbarum, as well. An optimised competitive PCR (cPCR) method for quantifying fungal growth in cereal grain was developed using experimental design. DNA extraction efficiency was quantified by cPCR using primers specific for the internal transcribed spacers (ITS) of the ribosomal DNA of P. roqueforti. The proposed method can detect P. roqueforti at levels as low as 10 CFU/g grain and at levels higher than lo2 CFU/g grain. Quantification is consistent (CV 8%) and highly correlated with results from traditional dilution plating. The possibilities of using an electronic nose or gas chromatography combined with mass spectrometry (GC-MS) to quantify ergosterol, colony forming units (CFU), ochratoxin A, and deoxynivalenol