Clonal expansion of the Belgian Phytophthora ramorum populations based on new microsatellite markers

Co-existence of both mating types A1 and A2 within the EU1 lineage of Phytophthora ramorum has only been observed in Belgium, which begs the question whether sexual reproduction is occurring. A collection of 411 Belgian P. ramorum isolates was established during a 7-year survey. Our main objectives were genetic characterization of this population to test for sexual reproduction, determination of population structure, evolution and spread, and evaluation of the effectiveness and impact of control measures. Novel, polymorphic simple sequence repeat (SSR) markers were developed after screening 149 candidate loci. Eighty isolates of P. ramorum, broadly representing the Belgian population, were analyzed using four previously described and three newly identified polymorphic microsatellite loci as well as amplified fragment length polymorphisms. SSR analysis was most informative and was used to screen the entire Belgian population. Thirty multilocus genotypes were identified, but 68% of the isolates belonged to the main genotype EU1MG1. Although accumulated mutation events were detected, the overall level of genetic diversity within the Belgian isolates of P. ramorum appears to be limited, indicating a relatively recent clonal expansion. Based on our SSR analysis there is no evidence of sexual recombination in the Belgian population of P. ramorum. Metalaxyl use decreased the genetic diversity of P. ramorum until 2005, when the majority of the isolates had become resistant. Most genotypes were site-specific and despite systematic removal of symptomatic and neighbouring plants, some genotypes were detected over a period of several years at a single site, sometimes discontinuously, indicating (latent) survival of the pathogen at those sites.