Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) play a key role in the suppressiveness of some soils to take-all of wheat and other diseases caused by soilborne pathogens. Soils from side-by-side fields on the campus of North Dakota State University, Fargo, USA, which have undergone continuous wheat, continuous flax or crop rotation for over 100 years, were assayed for the presence of 2,4-DAPG producers. Flax and wheat monoculture, but not crop rotation, enriched for 2,4-DAPG producers, and population sizes of log 5.0 CFU g root(-1) or higher were detected in the rhizospheres of wheat and flax grown in the two monoculture soils. The composition of the genotypes enriched by the two crops differed. Four BOX-PCR genotypes (D, F, G, and J) and a new genotype (T) were detected among the 2,4-DAPG producers in the continuous flax soil, with F- and J-genotype isolates dominating (41 and 39% of the total, respectively). In contrast, two genotypes (D and I) were detected in the soil with continuous wheat, with D-genotype isolates comprising 77% of the total. In the crop-rotation soil, populations of 2,4-DAPG producers generally were below the detection limit, and only one genotype (J) was detected. Under growth-chamber and field conditions, D and I genotypes (enriched by wheat monoculture) colonized the wheat rhizosphere significantly better than isolates of other genotypes, while a J-genotype isolate colonized wheat and flax rhizospheres to the same extent. This study suggests that, over many years of monoculture, the crop species grown in a field enriches for genotypes of 2,4-DAPG producers from the reservoir of genotypes naturally present in the soil that are especially adapted to colonizing the rhizosphere of the crop grown.