Evaluation location: Gelderland, Netherlands
The plants used in this study were propagated by grafting on an U. glabra rootstock in 2006. In April 2007 these plants were planted in a field trial at a tree nursery in Ommeren, the Netherlands (51° 56’ N, 5° 29’ E) on a clay soil (river deposit) at a spacing of 1 m within and 1.35 m between rows. The plants were planted in a randomized block design with nine blocks an four ramets of each clone per block. After planting the plants were watered several times, nevertheless the very dry weather conditions in the spring of 2007 resulted in a substantial loss of plants in the first season. These plants were replaced by spare plants from another part of the field in April 2008. The soil was kept free from weeds by using a herbicide until the canopy closed. No regular pruning was done, only some low branches were removed to enable better access to the field and when needed to maintain stability of the trees. Four independent inoculations with Ophiostoma novo-ulmi were performed on different replicates of the same plant material, thus each tree was inoculated once. In both 2008 and 2009 two inoculations were carried out, the first in June and the second in July, coinciding with the period in which elms in the Netherlands are at their greatest susceptibility to the fungus. For each inoculation, 9 plants per clone were used (one in each block). Inoculations were performed with an aggressive well-known tester isolate H328 belonging to the subspecies O. novo-ulmi ssp. novo-ulmi, formerly known as the EAN race (kindly provided by C. M. Brasier, Forestry Commission, UK). The conidial suspension was prepared according to the method by Tchernoff (1965), modified according to Sutherland et al. (1995). The inoculum consisted of a conidial suspension of O. novo-ulmi (10^6 spores per ml). Inoculations were performed using the Dutch inoculation method (Heybroek 1993), by forcing a knife with a drop of 0.12 ml inoculum on it into the vascular xylem of the lower stem at 30-50 cm height. This severe method ensures that even clones with reasonable resistance may develop some disease symptoms and that maximum differentiation in resistance level between clones can be obtained. Inoculations were performed in the morning only on dry and sunny days to be sure that the inoculum was absorbed by the trees very easily. Knowing that symptoms are influenced by the vigor of the clone and ramet (Santini et al. 2005), care was taken to inoculate well established and vigorously growing plants only. Trees with low vigor (shorter < 1 m) or incomplete bud burst were excluded from inoculation and dropped from the experiment. Also replacement trees, planted in 2008, were not inoculated in 2008 but left for inoculation in 2009. The average stem diameter of the clones measured at 1m
height in April 2008 prior to inoculation varied from 4.5 cm to 17.5 cm. Typical symptoms for Dutch elm disease are wilting, yellowing of the leaves, leaf fall, formation of “shepherd’s crooks” and dieback of twigs. To evaluate symptom development in the trees, the percentage of defoliation 4 and 8 weeks after inoculation, and recovery ability of trees one year after inoculation were assessed. During the next growing season following inoculation of trees, the ability to recover was assessed based on the percentage of defoliation of the restored foliage and proportion of crown mortality. Mortality in the crown was assessed using 5 classes: 0 = healthy, no crown dieback; 1 = dieback in < 25 % of the crown; 2 = 25-50%; 3 = 51-75%; and 4 ≥ 75 % of the crown shows dieback. All symptom assessments were performed by two independent observers. For the statistical analysis only the maximum (index, percentage) value was used (bias to classify clones to be more susceptible).