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The "Stubbs Collection", began in 1956 by the late Dutch plant pathologist R.W. Stubbs, refers to a unique historic collection of urediniospore samples of Puccinia striiformis that had been stored in liquid nitrogen for decades. Since 2010 the collection has been maintained by the Global Rust Reference Center (GRRC) in Denmark. Part of the collection is now being in a study of past pathogen diversity. A subset of samples collected between 1958 and 1991 from 35 countries was investigated to assess recovery rate, race identity, and previously undetected virulences. A new method for recovery using an airbrush sprayer and NovecTM 7100 fluid as dispersal agent in inoculating host plants was highly successful, resulting in a 96% recovery from 231 isolates. Phenotyping on the World and European differential host sets and additional wheat genotypes revealed 181 apparently uniform isolates, of which race identities were confirmed for 102. Race identities were updated for additional isolates based on improved resolution due to updated and more informative differential lines. Additional virulences corresponding to Yr17, Yr25, and Yr27 were added, as these were not assayed earlier. The past population structure was investigated by genotyping 212 isolates using 19 multilocus microsatellites. Seven distinct populations were detected, including clonal populations and recombinant populations. These results were compared with recent studies and demonstrated an overall consistent population subdivision at the global scale with clear migration events between populations. The outcome of the study facilitates conclusions about long-term temporal dynamics and overall migration patterns within and among world-wide populations of Pst.