To enable the reliable genetic analysis of complex disease resistance traits in B. napus, a core collection of isolates of the two major fungal pathogens causing economic losses in oilseed rape (OSR) crops, Pyrenopeziza brassicae and Leptosphaeria maculans, was established. The genetic diversity within the pathogen collection was analysed to ensure that the assembled collection represented the majority of the pathogen diversity. This was necessary in order to enable future reliable genetic analysis of specific pathogen-host interactions in combination with the host crop DFFS. Framework standard operating procedures were established so that they can be applied to other oilseed rape pathogens in future projects associated with OREGIN.
Establishment of a worldwide OSR fungal pathogen resource collection
In 2003/04, isolates of Leptosphaeria maculans and isolates of Pyrenopeziza brassicae were assembled at Rothamsted Research from sources representative of the worldwide distribution of stem canker and light leaf spot epiphytotics in OSR. Information on these isolates and their properties was maintained in spreadsheets and a prototype database of the culture collection was tested and then made available on the OREGIN website. In 2004/05, additional collections were made from sources in western Australia, France, Portugal, Sweden, Germany and Scotland and the culture collection of pathogen isolates was enlarged with isolates of Leptosphaeria maculans and isolates of Pyrenopeziza brassicae. Information on the isolates and their properties was maintained in MS Excel spread sheets. Isolates were sourced and annotated. By April 2006, the culture collection comprised of Leptosphaeria maculans isolates andyrenopeziza brassicae isolates from the UK and other sources worldwide . These isolates are maintained on agar media at 4oC , and at -80oC as conidial suspensions in 10 % glycerol; the latter are kept in duplicate at Rothamsted Research and University of Warwick.
New pathogen isolates were included in the collection in 2014/15. These included 32 Leptosphaeria maculans isolates obtained from OREGIN field experiments in the 2014/15 cropping season. Moreover pathogen isolates of Leptosphaeria biglobosa (co-existing pathogen causing phoma stem canker in the UK and other European countries and the pathogen responsible for the disease in China) were also added in the OREGIN collection. Thirty-three Leptosphaeria biglobosa isolates were added; 10 of them were obtained from OREGIN field experiments in the 2014/15 cropping season and 23 were obtained in the 2013/14 cropping season from several locations in the UK.
Secure storage of the “founder isolate” collection.
Stock cultures of all isolates are maintained in 10% glycerol at -80oC and as duplicate nutrient media slopes. DNA extracted from freeze-dried mycelia was isolated for analysis of genetic diversity using molecular markers. For L. maculans, six minisatellite markers were initially evaluated. Amplified fragment length polymorphism (AFLP) markers were adopted for the characterization of P. brassicae isolates.