The incidence and severity of light leaf spot epidemics caused by the ascomycete fungus Pyrenopeziza brassicae on UK oilseed rape crops are increasing. The disease is currently controlled by a combination of host resistance, cultural practices and fungicide applications. We report decreases in sensitivity of modern UK P. brassicae isolates to the azole (imidazole and triazole) class of fungicides. By cloning and sequencing the P. brassicae CYP51 (PbCYP51) gene, encoding the azole target sterol 14α-demethylase, we identified two non-synonymous mutations encoding substitutions G460S and S508T associated with reduced azole sensitivity. We confirmed the impact of the encoded PbCYP51 changes on azole sensitivity and protein activity by heterologous expression in a Saccharomyces cerevisiae mutant YUG37:erg11 carrying a controllable promoter of native CYP51 expression. In addition, we identified insertions in the predicted regulatory regions of PbCYP51 in isolates with reduced azole sensitivity. The presence of these insertions was associated with enhanced transcription of PbCYP51 in response to subinhibitory concentrations of the azole fungicide tebuconazole. Genetic analysis of in vitro crosses of sensitive and resistant isolates confirmed the impact of PbCYP51 alterations in coding and regulatory sequences on a reduced sensitivity phenotype, as well as identifying a second major gene at another locus contributing to resistance in some isolates. The least sensitive field isolates carry combinations of upstream insertions and non-synonymous mutations, suggesting that PbCYP51 evolution is ongoing and the progressive decline in azole sensitivity of UK P. brassicae populations will continue. The implications for the future control of light leaf spot are discussed.

The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.

A procedure has been developed for the determination of uranium and thorium in geological samples using extraction chromatography. Following sample preparation, uranium and thorium are pre-concentrated by precipitation with iron(iii) hydroxide and then separated using UTEVA resin. The separated uranium and thorium are electrodeposited onto stainless-steel discs and then measured by alpha spectrometry. The procedure was evaluated using uraninite ore, coral and granite reference materials. The uranium and thorium concentrations and the 234 U/ 238 U and 230 Th/ 234 U activity ratio values determined for the reference materials were in good agreement with the certified values. The presence of plutonium was found to interfere with the separation, but the inclusion of a reduction step using iron(ii) sulfamate eliminated the problem. Chemical recoveries for the procedure are similar to those for an anion-exchange procedure, but the extraction-chromatography procedure provides a more rapid separation using less reagents.