Modern Methods in Cereal Grain Mycology

Olsson, J. 2000. Modem methods in cereal grain mycology. Doctor's 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 (DON) in naturally contaminated barley samples was investigated. The main volatile compounds of grain with normal odour were 2-hexenal, benzaldehyde and nonanal, while 3-octanone, methylheptanone and trimethylbenzene were the main volatile compounds of grain with off-odours. Both CFU and ergosterol levels could be predicted from data from either GC-MS or electronic nose measurement. It was also possible to classify the ochratoxin A level as either <5 or >5 pgkg cereal grain, and estimate the DON level. Samples with ochratoxin A levels below 5 pgkg had higher concentration of aldehydes (nonanal, 2-hexenal) and alcohols (I-penten-3-01, 1 octanol). Samples with ochratoxin A levels above 5 pgkg had higher concentration of ketones (2-hexanone, 3-octanone). Pentane, methylpyrazine, 3-pentanone, 3-octene-2-01 and isooctylacetate were positively correlated with DON, while ethylhexanol, pentadecane, toluene, I-octanol, I-nonanol, and 1-heptanol were negatively correlated with DON.