Search

A real-time PCR technique was applied for the quantification of trichothecene-producing Fusariumspecies (TMTRI assay) as well as the highly toxigenic Fusarium graminearum(TMFg12 assay) present in barley grain and malt. PCR results were compared to the amounts of trichothecenes detected in the samples to find out if the PCR assays can be used for trichothecene screening instead of expensive and laborious chemical analyses. DNA was extracted from ground kernels using a commercial DNA extraction kit and analysed in a LightCycler® system using specific primers and fluorogenic TaqMan probes. Both naturally and artificially contaminated grains were analysed. The TMTRI assay and the TMFg12 assay enabled the quantification of trichothecene-producing FusariumDNA and F. graminearumDNA present in barley grain and malt samples, respectively. Both TaqMan assays were considered to be sensitive and reproducible. Linearity of the assays was 4–5 log units when pure FusariumDNAs were tested. The amount of FusariumDNA analysed with the TMTRI-trichothecene assay could be used for estimation of the deoxynivalenol (DON) content in barley grain. Furthermore, the TMFg12 assay for F. graminearumgave a good estimation of the DON content in north American barley and malt samples, whilst the correlation was poor among Finnish samples. DON content and the level of F. graminearumDNA were found to be naturally low in Finnish barleys.A real-time PCR technique was applied for the quantification of trichothecene-producing Fusariumspecies (TMTRI assay) as well as the highly toxigenic Fusarium graminearum(TMFg12 assay) present in barley grain and malt. PCR results were compared to the amounts of trichothecenes detected in the samples to find out if the PCR assays can be used for trichothecene screening instead of expensive and laborious chemical analyses. DNA was extracted from ground kernels using a commercial DNA extraction kit and analysed in a LightCycler® system using specific primers and fluorogenic TaqMan probes. Both naturally and artificially contaminated grains were analysed. The TMTRI assay and the TMFg12 assay enabled the quantification of trichothecene-producing FusariumDNA and F. graminearumDNA present in barley grain and malt samples, respectively. Both TaqMan assays were considered to be sensitive and reproducible. Linearity of the assays was 4–5 log units when pure FusariumDNAs were tested. The amount of FusariumDNA analysed with the TMTRI-trichothecene assay could be used for estimation of the deoxynivalenol (DON) content in barley grain. Furthermore, the TMFg12 assay for F. graminearumgave a good estimation of the DON content in north American barley and malt samples, whilst the correlation was poor among Finnish samples. DON content and the level of F. graminearumDNA were found to be naturally low in Finnish barleys.

Differences in morphology, ITS, IGS, mtSSU and β-tubulin sequences and UP–PCR hybridization were compared between morphologically identified F. avenaceum, F. arthrosporioides, F. anguioides, F. tricinctum, F. graminum and F. acuminatum strains. According to the combined β-tubulin, IGS and ITS tree, the strains of the Fusarium avenaceum/F. arthrosporioides/F. tricinctum species complex species can be divided into seven clusters supported by bootstrap values higher than 50%. The two main groups of European F. avenaceum, which cannot be distinguished by morphology, were separated in the tree based on β-tubulin sequences and less clearly in trees based on IGS and ITS sequences. MtSSU sequences were identical in all F. avenaceum and F. tricinctum strains studied. The European F. avenaceum strains of main group II had identical β-tubulin sequences with one American F. avenaceum strain and four European F. arthrosporioides strains, while F. avenaceum strains of main group I were closely related to two European F. arthrosporioides strains and to one Japanese F. anguioides strain. According to the combined β-tubulin/IGS/ITS sequence tree, European F. arthrosporioides strains were divided into four groups; F. tricinctum strains formed a well-supported cluster, in which two European clusters were separated from one African isolate. In the IGS sequence tree two European F. acuminatum strains together with one American F. acuminatum strain formed a cluster, which was separate from another American F. acuminatum strain. The F. acuminatum cluster was nested within the large F. tricinctum cluster together with one F. reticulatum strain in the combined IGS/β-tubulin tree. Several strains may be intermediates between the F. avenaceum/F. arthrosporioides/F. anguioides and F. tricinctum clusters and represent their own species. These results are partially supported by the results of UP–PCR hybridization analysis. Thus the molecular results may be helpful in future revision in the taxonomy of these species.

The randomly amplified polymorphic DNA (RAPD) technique was used to develop strain-specific primers for Gliocladium catenulatumstrain J1446, which is promising in biological control. One of the primer pairs developed proved to be strain-specific; strain J1446 was differentiated from 16 G. catenulatumstrains and six other strains of two Gliocladiumspecies, as well as from Trichoderma virens, and isolates of Nectriaspp. and Fusariumspp. Specific primers were also tested with DNA isolated from cucumber leaves, treated or untreated with a solution made from Gliocladiumpowder. The expected amplification product was produced only from treated leaves. DNA isolated from Gliocladium-treated potato tubers and fungi grown in peat was also used in amplification reactions. Strain-specific primers detected strain J1446 when the amount of DNA was 5 pg or more. Some variation between the Gliocladiumstrains was found by the random amplified microsatellites method (RAMS) and the universally primed polymerase chain reaction method (UP-PCR), but no clear fragments specific to strain J1446 were produced. Cross-blot hybridisation of UP-PCR products differentiated strain J1446 from T. virens, but not from the Gliocladiumisolates. The 28S rDNA sequences and β-tubulin sequences were identical or very similar in all Gliocladiumstrains. Thus, it is possible that the Gliocladiumstrains of the present study are conspecific, which means that a revision in the taxonomy of Gliocladiumspecies may be necessary.