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The effects of enhanced UVB radiation and drought stress on willow secondary phenolics were studied using the leaves of 8-week-old micropropagated plantlets from interspecific hybrids (Salix myrsinites L. Â S. myrsinifolia Salisb.) and pure species (S. myrsinifolia). The plantlets were subjected for 4 weeks to two levels of UVB radiation (ambient, enhanced) and two levels of watering (well-watered, drought-stressed) according to a 2 Â 2 factorial design. Enhanced UVB radiation increased the total concentration of flavonoids and phenolic acids in all plantlets, while the total concentration of salicylates remained unaffected. Drought stress reduced the total concentration of salicylates and phenolic acids in S. myrsinifolia plantlets, while in hybrids only phenolic acids were affected. The response of phenolic acids to enhanced UVB in drought-stressed plantlets was different from that in well-watered ones, indicating that drought stress limited the accumulation of phenolic acids under enhanced UVB radiation. Flavonoids increased in response to enhanced UVB radiation in drought-stressed plantlets, although drought caused serious physiological stress on growth. There were significant differences between hybrid and S. myrsinifolia plantlets with respect to the composition of phenolics and between families and clones with respect to their concentration. In addition, the response of salicylates, flavonoids and phenolic acids to enhanced UVB and drought stress was clone-specific, which may indicate that climatic changes will alter the genetic composition of northern forests.

Vole feeding amongst herbal willows that have a high concentration of salicylates in their bark and leaves, and may therefore be cultivated for use as raw material for herbal medicine was tested in the field and in laboratory conditions. Eight clones of dark-leaved willow (Salix myrsinifolia Salisb.) were cultivated for two years with six different methods combining three fertilisation levels (none, low and high), black plastic mulch applied for suppressing weed competition and unmulched control. Samples for the laboratory feeding trial were taken from the unfertilised plants during willow winter dormancy and twigs were fed to 16 voles as a multi-choice experiment. The bark area removed was calculated from image analysis of the material left by the voles. The diameter and the bark thickness of the twigs were measured. Concentrations of salicin, salicortin, HCH-salicortin, picein, triandrin, triandrin derivative, gallocatechin, (+)-catechin, luteolin-7-glucoside, hyperin, total condensed tannins and total nitrogen were measured from the twigs fed to voles in the laboratory. Browsing by a natural population of voles amongst winter-dormant willows was measured in the field. In the laboratory, voles browsed on 80% of the twigs and in the field voles browsed on 33% of the twigs. Vole feeding followed similar patterns in the field and in the laboratory experiment; feeding was clearly higher amongst the plants grown in unmulched control compared to those in plastic mulch. The same clones, 1, 2 and 6 were preferred in both experiments. Voles preferred thin twigs to thick ones. Feeding correlated negatively with concentrations of salicylates and tannins. As vole feeding seems to be highly affected by willow cultivation method and plant genotype, careful selection of cultivated clones and cultivation methods can enhance the reliability of herbal willow cultivation.

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Ten clones of dark-leaved willows ( Salix myrsinifolia Salisb.) were grown for two years on two different sites, Luikonlahti and Punkaharju, using various combinations of soil cultivation, fertilization and mulch treatments, with the objective of comparing the effect of different cultivation methods on the growth and total salicylate yield. In cultivated soil, the total salicylate yield ranged from 20 to 220 kg/ha, depending on the clone and cultivation method. The use of black polythene mulch and the addition of mineral fertilizer reduced the total salicylate concentrations of several clones. On the other hand, polythene mulch noticeably increased the hectare yield of total salicylates by promoting biomass accumulation.