Evaluation location: Minnesota, United States
Trees were purchased from commercial nurseries and were planted in a nursery field at the University of Minnesota, St. Paul campus during the summer of 2014. Trees were spaced 0.9 m apart within rows and 3 m apart between rows. Trees were three and four years old at the time of inoculation. All trees received water as needed during the growing season. In addition, trees were fertilized every 3 months during the growing season with 4.9 ml of Osmocote® Plus (15-9-12) (Everris NA Inc., Dublin, OH). Methods of inoculum preparation and inoculation have been described previously (Beier et al., 2017). In brief, an isolate of Ophiostoma novo-ulmi with known pathogenicity, collected from Minnesota, was used for inoculations. A drill was used to make a single small hole (2.38 mm wide by 4 mm deep) in the tree 0.5 m above the ground. Tape was wrapped tightly around the drill bit to maintain a consistent depth. Immediately following drilling, 25 μl of an O. novo-ulmi spore suspension (1 × 10^6 spores/ml) was injected into the hole using a micropipette and the wound was subsequently wrapped with Parafilm M® (Bemis Co., Inc., Neenah, WI) to avoid desiccation. To more accurately determine disease susceptibility in the cultivars, additional samples, which were not used for histological assessments, were included in 2015. Trees in the 2015 trial were inoculated on May 28 (43 days after budbreak), while trees in the 2016 trial were inoculated on May 26 (40 days after budbreak).
Trees were destructively harvested at 20 and 90 DPI. At each time point, two to six inoculated trees were harvested for each cultivar. To avoid cavitation, the stems were placed into a shallow tub of water and subsequently cut. A 20-cm sample, centred on the inoculation site, was cut and immediately placed on ice and subsequently stored at −20°C. A 5-cm segment was collected 10 cm above the inoculation site and immediately placed on ice and stored at −20°C. Since the trees in 2016 were too large to be cut underwater, harvesting was performed predawn to reduce the likelihood of cavitation. Transverse sections from 11 to 12 cm above the inoculation site were used. Thick cross sections (approximately 2 cm) of the main stem, which had been stored at −20°C, were quartered and thin freehand transverse sections, approximately 50 μm thick, were made using a high-profile microtome blade so the entire circumference of the most recent annual ring could be assessed. Sections were mounted in water. Each sample was examined using a Nikon E600 microscope (Nikon Instruments Inc., Melville, NY) at 200× to determine whether barrier zone formation had occurred. Images of barrier zones were taken at 100× using a Nikon DS-Ri1 camera (Nikon Instruments Inc., Melville, NY) mounted on a Nikon Eclipse Ni-U microscope (Nikon Instruments Inc., Melville, NY). Barrier zones were identified by examining the shape of cells, which generally appear more flattened than typical fibre and axillary parenchyma cells (Shigo & Tippett, 1981). In addition, barrier zone cells are often darker in appearance due to the accumulation of various tree defence compounds (Rioux & Ouellette, 1991). As many of the annual rings were larger than the field of view for the digital documenting system, multiple images of the same section were merged using the photomerge feature in Photoshop™ (Adobe Systems Inc., San Jose, CA) or the scan large image feature in Nikon Elements Advanced Research (Nikon Instruments Inc., Melville, NY). Z-stacking was performed as necessary using Photoshop™ or Nikon Elements Advanced Research. If barrier zones were present, the mean thickness was determined by averaging the thickness of the barrier zone at both sides of the cropped image. In the event that the barrier zone was not continuous across the section, only one measurement was used.