Willows in the Flemish region are threatened by the watermark disease, a wood disease caused by the bacterium Brenneria salicis (Hauben et al. Syst Appl Microbiol 21:384-397, 1998). Affected trees are observed to grow at specific sites which seem more sensitive to periodical watermark symptom development than other sites. In this study we aim at revealing abiotic site factors that could play a role in triggering the watermark disease. During three consecutive years we analyzed stands of young isogenic willows (Salix alba L. cv. Lichtenvoorde), which were planted on four different sites in an agricultural environment. These test sites were chosen, based on the occurrence/absence of watermark disease in the old willows that grow on these sites. Two sites had old willow pollards with clear watermark symptoms, and on the two other sites the old willows were unaffected. Differences in abiotic characteristics between the sites were analysed and their effects on the nutrient status in willow were measured in the isogenic willows that were newly planted. Brenneria salicis-specific multiplex PCR revealed the presence of this pathogenic bacterium in both the old and the freshly planted young willows. At each site, the physical and chemical characteristics of soil and groundwater were investigated; the young tree growth was monitored and nutritional status of the willow plantings was assessed by foliar analysis. Physical soil properties including soil type and profile water availability, and chemical soil analysis were unable to explain differences between healthy and affected sites. Diseased sites, however, had significantly higher N concentrations and traces of herbicides in the groundwater. In young trees, foliar levels of N and S were always greater at the affected sites, while P contents were lower. Dry mass based N concentration in the leaves of the newly planted trees increased systematically over the years, but more at affected than at healthy sites. Also, chlorophyll content was significantly greater at the affected sites. Growth rate of the young isogenic trees was twice as fast at the diseased sites. The presented data suggest that application of fertilisers in agriculture causes an imbalance in the nutritional status of Salix alba L. Excess nitrogen makes this species fast-growing, reflected by an average foliar N:P ratio >10, and more susceptible for watermark disease, indicated by an average foliar N:Ca ratio exceeding 1.5.