Methods
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.
The experiments were conducted in research nurseries at Alice Holt (England), Wageningen (the Netherlands) and Ames (USA). The chief inoculation technique was the same as that used in the breeding programme in the Netherlands and employed in the earlier experiments of Gibbs et al. (1972) and Gibbs and Brasier (1973). A spore suspension was prepared for each isolate in mid-June 1973 by shaking pieces of mycelium in 10 ml of Tchernoff medium for 2 days (Tchernoff 1965). An incision was made in the main stem of each tree with a knife to expose the wood vessels, and 3 drops of the spore suspension were allowed to enter the cut. In part of the work at Ames an equal number of trees were also inoculated by the 'pinprick' technique. This involved placing a drop of spore suspension on a 2-year-old branch and pricking into the wood through the drop with a needle until the spore suspension was drawn in. The inoculum averaged 5 X 10^5 spores per tree. In all the experiments, the isolates were applied in randomized blocks with single-tree plots, the number of replications varying with the number of trees available. Because some of the isolates were exotic to the countries in which the experiments were being conducted, all inoculated trees received regular sprays of insecticide (methoxychilor in Ames, lindane at Alice Holt and Wageningen). Disease was assessed as the percentage of the crown showing leaf symptoms at 8 weeks after inoculation. Each accession was inoculated with either four or five isolates (aggressive and non-aggressive strains) separately and the data reported here are the average of all isolates.
The experiments were conducted in research nurseries at Alice Holt (England), Wageningen (the Netherlands) and Ames (USA). The chief inoculation technique was the same as that used in the breeding programme in the Netherlands and employed in the earlier experiments of Gibbs et al. (1972) and Gibbs and Brasier (1973). A spore suspension was prepared for each isolate in mid-June 1973 by shaking pieces of mycelium in 10 ml of Tchernoff medium for 2 days (Tchernoff 1965). An incision was made in the main stem of each tree with a knife to expose the wood vessels, and 3 drops of the spore suspension were allowed to enter the cut. In part of the work at Ames an equal number of trees were also inoculated by the 'pinprick' technique. This involved placing a drop of spore suspension on a 2-year-old branch and pricking into the wood through the drop with a needle until the spore suspension was drawn in. The inoculum averaged 5 X 10^5 spores per tree. In all the experiments, the isolates were applied in randomized blocks with single-tree plots, the number of replications varying with the number of trees available. Because some of the isolates were exotic to the countries in which the experiments were being conducted, all inoculated trees received regular sprays of insecticide (methoxychilor in Ames, lindane at Alice Holt and Wageningen). Disease was assessed as the percentage of the crown showing leaf symptoms at 8 weeks after inoculation. Each accession was inoculated with either four or five isolates (aggressive and non-aggressive strains) separately and the data reported here are the average of all isolates.
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 × 106 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). Disease severity ratings (DSR) were made at 90 days postinoculation (DPI) based on the percentage of the crown exhibiting permanent wilt using a 1–12 disease severity scale: 1 = 0%, 2 = 1%–9%, 3 = 10%–19%, 4 = 20%–29%, 5 = 30%–39%, 6 = 40%–49%, 7 = 50%–59%, 8 = 60%–69%, 9 = 70%–79%, 10 = 80%–89%, 11 = 90%–99% and 12 = 100%.
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).
One year old trees were planted into a field plot at Glenn Dale, MD, in 1989 and 1990, in a randomized-block, split-plot design with 7 blocks, and, depending on the number of trees available, four trees per clone in each whole plot within a block. Half the trees in each whole plot were inoculated May 18, 1992; the other half were inoculated on May 27, 1992 (sub-plot treatment). Inoculations on each date were made into a 2.4 mm (0.1 in.) hole in the bottom one-third of the main trunk of each tree with 0.11 mL (0.003 fl oz) of an aqueous spore suspension containing 3 × 10^6 spores/mL (1 × 10^8 spores/fl oz) of a mixture of two isolates each of O. novo-ulmi and O. ulmi. This inoculation was intentionally severe in order to induce symptom expression on even the most disease-tolerant clones. The percentage of the crown's branches showing a lack of foliage, or dieback, was estimated four weeks, one and two years after inoculation.
One year old trees were planted into a field plot at Glenn Dale, MD, in 1989 and 1990, in a randomized-block, split-plot design with 7 blocks, and, depending on the number of trees available, four trees per clone in each whole plot within a block. Half the trees in each whole plot were inoculated May 18, 1992; the other half were inoculated on May 27, 1992 (sub-plot treatment). Inoculations on each date were made into a 2.4 mm (0.1 in.) hole in the bottom one-third of the main trunk of each tree with 0.11 mL (0.003 fl oz) of an aqueous spore suspension containing 3 × 10^6 spores/mL (1 × 10^8 spores/fl oz) of a mixture of two isolates each of O. novo-ulmi and O. ulmi. This inoculation was intentionally severe in order to induce symptom expression on even the most disease-tolerant clones. The percentage of the crown's branches showing a lack of foliage, or dieback, was estimated one, two and seven years after inoculation. Percent survival was also assessed seven years after inoculation; trees that died back to below 1 m (1.1 yd) in height were considered dead, even if stump sprouts eventually arose from the base of the tree.
One year old trees were planted into a field plot at Glenn Dale, MD, in 1989 and 1990, in a randomized-block, split-plot design with 7 blocks, and, depending on the number of trees available, four trees per clone in each whole plot within a block. Half the trees in each whole plot were inoculated May 18, 1992; the other half were inoculated on May 27, 1992 (sub-plot treatment). Inoculations on each date were made into a 2.4 mm (0.1 in.) hole in the bottom one-third of the main trunk of each tree with 0.11 mL (0.003 fl oz) of an aqueous spore suspension containing 3 × 10^6 spores/mL (1 × 10^8 spores/fl oz) of a mixture of two isolates each of O. novo-ulmi and O. ulmi. This inoculation was intentionally severe in order to induce symptom expression on even the most disease-tolerant clones. The percentage of the crown's branches showing a lack of foliage, or dieback, was estimated one, two and seven years after inoculation. Percent survival was also assessed seven years after inoculation; trees that died back to below 1 m (1.1 yd) in height were considered dead, even if stump sprouts eventually arose from the base of the tree.
One year old trees were planted into a field plot at Glenn Dale, MD, in 1989 and 1990, in a randomized-block, split-plot design with 7 blocks, and, depending on the number of trees available, four trees per clone in each whole plot within a block. Half the trees in each whole plot were inoculated May 18, 1992; the other half were inoculated on May 27, 1992 (sub-plot treatment). Inoculations on each date were made into a 2.4 mm (0.1 in.) hole in the bottom one-third of the main trunk of each tree with 0.11 mL (0.003 fl oz) of an aqueous spore suspension containing 3 × 10^6 spores/mL (1 × 10^8 spores/fl oz) of a mixture of two isolates each of O. novo-ulmi and O. ulmi. This inoculation was intentionally severe in order to induce symptom expression on even the most disease-tolerant clones. The percentage of the crown's branches showing a lack of foliage, or dieback, was estimated four weeks and one year after inoculation.
Trees were planted into a field plot at Glenn Dale, MD, in April 1993, in a randomized block design with 7 blocks and, when available, 4 trees per block per clone in each block. Inoculations were made on May 21, 2002, into a 2.4 mm (0.1 in) hole in the bottom one third of the main trunk of each tree with an aqueous spore suspension containing 3.72 × 10^6 spores/ml of a mixture of two strains of Ophiostoma novo-ulmi and two strains of Ophiostoma ulmi in a 2:1 ratio of O. novo-ulmi to O. ulmi spores. The percentage of the crown showing wilting or death of foliage was estimated on all trees 4 weeks after inoculation. The percentage of the crown showing dieback (lack of foliage) was estimated one and two years after inoculation. For all data collected on foliar symptoms and crown dieback, estimates were made by two observers examining a given tree at the same time, after which a consensus score was recorded.
The experiments were conducted in research nurseries at Alice Holt (England), Wageningen (the Netherlands) and Ames (USA). The chief inoculation technique was the same as that used in the breeding programme in the Netherlands and employed in the earlier experiments of Gibbs et al. (1972) and Gibbs and Brasier (1973). A spore suspension was prepared for each isolate in mid-June 1973 by shaking pieces of mycelium in 10 ml of Tchernoff medium for 2 days (Tchernoff 1965). An incision was made in the main stem of each tree with a knife to expose the wood vessels, and 3 drops of the spore suspension were allowed to enter the cut. In part of the work at Ames an equal number of trees were also inoculated by the 'pinprick' technique. This involved placing a drop of spore suspension on a 2-year-old branch and pricking into the wood through the drop with a needle until the spore suspension was drawn in. The inoculum averaged 5 X 10^5 spores per tree. In all the experiments, the isolates were applied in randomized blocks with single-tree plots, the number of replications varying with the number of trees available. Because some of the isolates were exotic to the countries in which the experiments were being conducted, all inoculated trees received regular sprays of insecticide (methoxychilor in Ames, lindane at Alice Holt and Wageningen). Disease was assessed as the percentage of the crown showing leaf symptoms at 8 weeks after inoculation. Each accession was inoculated with either four or five isolates (aggressive and non-aggressive strains) separately and the data reported here are the average of all isolates.
Two-year-old nursery grown callus cuttings of clone Belgica, which is susceptible to all strains of Ophiostoma ulmi, and of clone 390, which is resistant to the non-aggressive, but susceptible to the aggressive strains of O. ulmi (Elgersma, 1976), were inoculated with the aggressive strain H6 or with the non- aggressive strain E2 of O. ulmi with the techniques described earlier (Elgersma, 1969). The inoculum contained 10^6 conidia per ml. On the 3rd and 5th day after inoculation, 2 cm-stem pieces were cut 5 cm above the site of inoculation, using 10 plants in each combination at a time, which were fixed in formalin-ethanol (70%)-propionic acid (5:90:5) and sectioned transversely at 20 um thickness. The total number of vessels in the latest developed annual ring was counted and the proportion containing tyloses determined as described earlier (Elgersma, 1973).
Trees sampled were planted in Indian Mounds Park as part of the National Elm Trial. For all cultivars except for the ‘Valley Forge’ (only two trees available) at the Indian Mound site, three trees were randomly selected to be part of each evaluation. Ten leaves were
randomly selected from each tree. For the majority of the trees, these leaves were collected from
the lower branches which could be easily reached without assistance. For some of the taller
trees, a pole pruner was used. Leaves were selected evenly from all directions, as much as
possible, and were picked both near the trunk and near the tips of the branches to give a wide
breadth of coverage. Leaves were then placed in plastic, zipper-sealed bags labeled with the date
and tree number. After all samples were collected, they were brought back to the laboratory and
either were evaluated immediately or put into a refrigerator (40F) until they could be evaluated. The data from each sample of leaves were recorded within 48 hours of the leaves being picked. For each sample, the total number of adult feeding holes was recorded. The total number of larval
mines in each sample was also recorded. Finally, the length of each leaf was recorded. This measurement was used to estimate the area of each leaf by using the formula for the surface area of an oval (SA=0.5L*0.5(0.67L)*π) where L is the measured length and 0.67L
represents an estimate of the width. Although this estimate is not an exact measurement of the
surface area, this step was necessary normalize and compare the adult feeding damage per leaf
area. The total number of holes in the sample was then divided by the combined surface area of
the ten leaves. The final adult damage was then recorded as the number of holes per surface area
of leaf (holes/cm2)
We used newly emerged and unfed elm leaf beetle (Xanthogaleruca luteola) larvae hatching from eggs laid on the foliage of Ulmus pumila seedlings. These seedlings were growing in 7.6-liter pots and covered with light screen mesh to prevent beetles from escaping. Seedlings were held in the laboratory at 25C (77F) and under 16:8 hr (L:D) photoperiod. Adult beetles were reared from late instar larvae and pupae collected from U. pumila trees at North Platte, NE, and shipped overnight to The Morton Arboretum, Lisle, IL. On arrival, these larvae and pupae were held in clear Plexiglas cages in an incubator at 25C (77F) and 16:8 (L:D) hr photoperiod. As adults emerged, they were released onto U. pumila seedlings and allowed to feed, mate, and oviposit. For the host suitability test, we used 24-hr-old X. luteola larvae. Neonates of X. luteola initially cluster at or near the egg mass before initiation of feeding. These were randomly selected from an egg mass and transferred to a single leaf of the test elm that was placed in a plastic petri dish (0.6 × 10.0 mm). Ten such petri dishes were used for each single tree replicate and there were three trees per elm accession. Petri dishes were placed into a clear plastic bag to retain moisture. The petri dishes were checked daily for larval mortality, evidence of feeding, prepupation, pupation, and adult emergence. Candidate elm accessions growing at The Morton Arboretum were approximately 2 m (6.6 ft) high and growing in 8 liter (2.1 gal) pots. Leaves for the bioassays were randomly collected from the trees from all four cardinal directions. The leaf samples included the terminal 15 cm (5.9 in) of elm branches. Only fully expanded leaves were used. Leaf samples were taken in this way to compensate for variation in leaf quality within trees. Leaf samples were held in cold storage in plastic bags at 5C (41F) for a maximum of 2 days. Leaves collected from each test tree for each biotype were combined for the bioassays. Larval bioassays ceased when larvae died or when adults emerged. Larval suitability for each biotype was defined as the mean development time from larva to adult, mean proportion of larvae pupating, mean percent adult emergence, and mean pupal weight.
An experimental elm plantation was established near the Navajo County Fairground in the City of Holbrook, east central Arizona. The city landscape is dominated by Siberian elm trees, which sustain heavy defoliation by elm leaf beetles annually. Elm seedlings 2-4 yr old and between 30 and 45 cm in height were obtained from a variety of commercial and university sources for planting. Newly acquired trees were initially kept under shade near the Northern Arizona University Greenhouse Complex, and they were irrigated as needed until they were planted in the field. The plantation was established from 1995-1998, using a completely randomized design with 10 single-tree replicates per elm accession. Trees were planted on a 21-row by 14-column grid at 3.6- by 3.6-m spacing. In spring 1995, each accession, replicated 10 times, were randomly planted. The soil around each plant was mulched using a 1-sq m piece of black shade cloth covered with pine wood chips. Plants were watered regularly with a drip irrigation system that supplied water to the base of individual plants. Defoliation was estimated every third week of July, from 1999 to 2001. A 15-cm-long shoot was randomly selected from the north, south, east, and west quadrants of each plant and the number of elm leaf beetle eggs on the selected 15-cm shoots was recorded.
We used newly emerged and unfed elm leaf beetle (Xanthogaleruca luteola) larvae hatching from eggs laid on the foliage of Ulmus pumila seedlings. These seedlings were growing in 7.6-liter pots and covered with light screen mesh to prevent beetles from escaping. Seedlings were held in the laboratory at 25C (77F) and under 16:8 hr (L:D) photoperiod. Adult beetles were reared from late instar larvae and pupae collected from U. pumila trees at North Platte, NE, and shipped overnight to The Morton Arboretum, Lisle, IL. On arrival, these larvae and pupae were held in clear Plexiglas cages in an incubator at 25C (77F) and 16:8 (L:D) hr photoperiod. As adults emerged, they were released onto U. pumila seedlings and allowed to feed, mate, and oviposit. For the host suitability test, we used 24-hr-old X. luteola larvae. Neonates of X. luteola initially cluster at or near the egg mass before initiation of feeding. These were randomly selected from an egg mass and transferred to a single leaf of the test elm that was placed in a plastic petri dish (0.6 × 10.0 mm). Ten such petri dishes were used for each single tree replicate and there were three trees per elm accession. Petri dishes were placed into a clear plastic bag to retain moisture. The petri dishes were checked daily for larval mortality, evidence of feeding, prepupation, pupation, and adult emergence. Candidate elm accessions growing at The Morton Arboretum were approximately 2 m (6.6 ft) high and growing in 8 liter (2.1 gal) pots. Leaves for the bioassays were randomly collected from the trees from all four cardinal directions. The leaf samples included the terminal 15 cm (5.9 in) of elm branches. Only fully expanded leaves were used. Leaf samples were taken in this way to compensate for variation in leaf quality within trees. Leaf samples were held in cold storage in plastic bags at 5C (41F) for a maximum of 2 days. Leaves collected from each test tree for each biotype were combined for the bioassays. Larval bioassays ceased when larvae died or when adults emerged. Larval suitability for each biotype was defined as the mean development time from larva to adult, mean proportion of larvae pupating, mean percent adult emergence, and mean pupal weight.
An experimental elm plantation was established near the Navajo County Fairground in the City of Holbrook, east central Arizona. The city landscape is dominated by Siberian elm trees, which sustain heavy defoliation by elm leaf beetles annually. Elm seedlings 2-4 yr old and between 30 and 45 cm in height were obtained from a variety of commercial and university sources for planting. Newly acquired trees were initially kept under shade near the Northern Arizona University Greenhouse Complex, and they were irrigated as needed until they were planted in the field. The plantation was established from 1995-1998, using a completely randomized design with 10 single-tree replicates per elm accession. Trees were planted on a 21-row by 14-column grid at 3.6- by 3.6-m spacing. In spring 1995, each accession, replicated 10 times, were randomly planted. The soil around each plant was mulched using a 1-sq m piece of black shade cloth covered with pine wood chips. Plants were watered regularly with a drip irrigation system that supplied water to the base of individual plants. Defoliation was estimated every third week of July, from 1996 to 2001. A 15-cm-long shoot was randomly selected from the north, south, east, and west quadrants of each plant and evaluated for percentage of defoliation. Defoliation was independently visually estimated to the nearest 5% by two trained technicians and their estimates were averaged.
An experimental elm plantation was established near the Navajo County Fairground in the City of Holbrook, east central Arizona. The city landscape is dominated by Siberian elm trees, which sustain heavy defoliation by elm leaf beetles annually. Elm seedlings 2-4 yr old and between 30 and 45 cm in height were obtained from a variety of commercial and university sources for planting. Newly acquired trees were initially kept under shade near the Northern Arizona University Greenhouse Complex, and they were irrigated as needed until they were planted in the field. The plantation was established from 1995-1998, using a completely randomized design with 10 single-tree replicates per elm accession. Trees were planted on a 21-row by 14-column grid at 3.6- by 3.6-m spacing. In spring 1995, each accession, replicated 10 times, were randomly planted. The soil around each plant was mulched using a 1-sq m piece of black shade cloth covered with pine wood chips. Plants were watered regularly with a drip irrigation system that supplied water to the base of individual plants. Defoliation was estimated every third week of July, from 1999 to 2001. A 15-cm-long shoot was randomly selected from the north, south, east, and west quadrants of each plant and the number of elm leaf beetle larvae on the selected 15-cm shoots was recorded.
We used newly emerged and unfed elm leaf beetle (Xanthogaleruca luteola) larvae hatching from eggs laid on the foliage of Ulmus pumila seedlings. These seedlings were growing in 7.6-liter pots and covered with light screen mesh to prevent beetles from escaping. Seedlings were held in the laboratory at 25C (77F) and under 16:8 hr (L:D) photoperiod. Adult beetles were reared from late instar larvae and pupae collected from U. pumila trees at North Platte, NE, and shipped overnight to The Morton Arboretum, Lisle, IL. On arrival, these larvae and pupae were held in clear Plexiglas cages in an incubator at 25C (77F) and 16:8 (L:D) hr photoperiod. As adults emerged, they were released onto U. pumila seedlings and allowed to feed, mate, and oviposit. For the host suitability test, we used 24-hr-old X. luteola larvae. Neonates of X. luteola initially cluster at or near the egg mass before initiation of feeding. These were randomly selected from an egg mass and transferred to a single leaf of the test elm that was placed in a plastic petri dish (0.6 × 10.0 mm). Ten such petri dishes were used for each single tree replicate and there were three trees per elm accession. Petri dishes were placed into a clear plastic bag to retain moisture. The petri dishes were checked daily for larval mortality, evidence of feeding, prepupation, pupation, and adult emergence. Candidate elm accessions growing at The Morton Arboretum were approximately 2 m (6.6 ft) high and growing in 8 liter (2.1 gal) pots. Leaves for the bioassays were randomly collected from the trees from all four cardinal directions. The leaf samples included the terminal 15 cm (5.9 in) of elm branches. Only fully expanded leaves were used. Leaf samples were taken in this way to compensate for variation in leaf quality within trees. Leaf samples were held in cold storage in plastic bags at 5C (41F) for a maximum of 2 days. Leaves collected from each test tree for each biotype were combined for the bioassays. Larval bioassays ceased when larvae died or when adults emerged. Larval suitability for each biotype was defined as the mean development time from larva to adult, mean proportion of larvae pupating, mean percent adult emergence, and mean pupal weight.
A male/female pair of field-collected (adult beetles used were field-collected from plants at The Morton Arboretum, Lisle, IL and The Chicago Botanic Garden, Glencoe, IL.) adult Japanese beetles was placed into a 5.7 L (6 qt) capacity Clear-View™ plastic container (SteriliteR, Townend, MA) with approximately 5.0 cm. of finely sifted moist silt-loam soil, and foliage of the candidate Ulmus taxon to be tested. Foliage was kept turgid and fresh using floral water piks. The boxes containing the beetles and foliage were kept at room temperature in the laboratory under natural daylength. The boxes were examined every third day and elm foliage was replaced if wilted or defoliated. At 7, 14, and 21 days from the initiation of the study, the soil in each container was visually examined for eggs, and the total number of eggs per container and adult beetle mortality was recorded. After each egg counting, the original male/female beetle pair was returned to the container. Ten single containers (replicates) containing one male/female pair were used for each of three single trees per Ulmus taxon for a total of 30 beetle pairs per taxon. The study was terminated after 21 days.
In late August, after all adult beetle feeding had ceased, a field defoliation survey was conducted by visually examining the tree canopy from all four cardinal directions for each accession. Trees were rated by two independent estimators for evidence of feeding using a scale of 0-4 as follows: 0=no feeding, 1=very light feeding, 2=light feeding, 3=moderate feeding, and 4=heavy feeding. Depending on the availability of trees, three to five single tree replicates were evaluated per taxon.
Ulmus taxa leaves for the no-choice (NC) laboratory feeding bioassays were randomly collected from ground level from all four cardinal directions and held in cold storage in plastic bags at 5 C (41 F) for a maximum of 2 d. Leaves collected from each test tree were combined for the NC laboratory feeding bioassays. Depending on availability, one to three individual trees of each Ulmus taxon were evaluated. Adult beetles used in the NC study were field-collected from plants at The Morton Arboretum, Lisle, IL and The Chicago Botanic Garden, Glencoe, IL. Upon collection, the adult beetles were held in clear plexiglass cages under a photoperiod of 16:8 (L:D) h at 25 C (77 F). While being held in the cages (no longer than 12 hours), the beetles were allowed to feed on fresh crabapple (Malus spp.) foliage to ensure predisposition to feeding. Prior to the beginning of the feeding trials, the Japanese beetles were sexed, and one adult male/female Japanese beetle pair was placed into each of 10 clear plastic petri dishes (10.0 cm diam by 0.6 cm depth) along with one leaf of the specific Ulmus taxon to be tested. Each beetle pair was used only once. There were 10 dishes (sub-replicates) for each tree for each taxon evaluated for a total of 10 to 30 male/female beetle pairs per taxon. Petri dishes were examined daily for beetle mortality and evidence of feeding. Foliage was replaced every 2 d. At the time of leaf removal, the leaves were visually rated (nearest 5%) for the percent of leaf tissue removed, by two independent estimators using a defoliation template. Petri dishes were placed in clear plastic bags to prevent drying of the foliage and were held in a growth chamber under a 16:8 (L:D) h photoperiod at ∼ 25 C (77 F). Condensation of water on the lid of the petri dish indicated a high relative humidity. At the end of seven days, the remaining foliage and fecal pellets were removed from each petri dish and the fecal pellets were oven dried and weighed (nearest mg). The NC feeding bioassays were terminated after 7 days. The measure of the susceptibility for each taxon was defined by mean percent leaf tissue removed and mean dried fecal pellet weights.
The main study site was an open, grassy area on Maury silt loam soil on the University of Kentucky campus in Lexington, Kentucky, adjacent to the Arboretum and State Botanical Garden of Kentucky (N38°1’; W84°30’; elevation 302 m). Five replicates of each elm accession were planted in a randomized complete block in rows spaced 7.6 m apart, with 7.6 m between trees within rows and 50 m between replicates. Trees were planted in spring 2005 or 2006. The trees were obtained from nurseries as bare-root transplants and ranged from 1.5–2.4 m height at time of planting. They were staked, watered as needed, and mulched over grass that had been killed with glyphosate herbicide. Twenty fully expanded leaves were collected from each tree in early June 2009 and measured with an electronic area meter (Li-Cor, Lincoln, NE) as square centimeters.
The main study site was an open, grassy area on Maury silt loam soil on the University of Kentucky campus in Lexington, Kentucky, adjacent to the Arboretum and State Botanical Garden of Kentucky (N38°1’; W84°30’; elevation 302 m). Five replicates of each elm accession were planted in a randomized complete block in rows spaced 7.6 m apart, with 7.6 m between trees within rows and 50 m between replicates. Trees were planted in spring 2005 or 2006. The trees were obtained from nurseries as bare-root transplants and ranged from 1.5–2.4 m height at time of planting. They were staked, watered as needed, and mulched over grass that had been killed with glyphosate herbicide. Leaf pubescence was characterized by examining lower (abaxial) and upper (adaxial) leaf surfaces of leaves from each tree with a binocular microscope. Trichome density was subjectively rated as 0, 1, 2, 3, or 4 corresponding to glabrous (few or none), light, moderate, dense, or very dense, respectively. Twenty leaves analyzed.
Ulmus taxa leaves were randomly collected from ground level from all four cardinal directions and held in cold storage in plastic bags at 5 C (41 F) for a maximum of 2 d. Ten leaves for each taxon were measured for leaf thickness, and inner and outer leaf toughness. Leaf thickness was determined by using a Vernier caliper to measure the thickness of each leaf (nearest mm) approximately one-half the distance from the leaf margin to the mid-rib. Inner and outer leaf toughness was determined to the nearest gram using a Chatillon™ digital force meter (pentrometer) (Greensboro, N.C.) applied to within 0.5 cm from the edge of the leaf for measuring outer toughness, and in the center of the leaf adjoining the mid-rib for inner toughness, respectively.
The experiment was conducted at the U.S. Department of Agriculture, Agricultural Research Service station near Glenn Dale, Md., using a research field plot that is part of the U.S. National Arboretum Tree Breeding Program. The field plot consisted of two blocks of trees arranged within rows by elm selection in a completely randomized block design. Blocks were separated by a 9.2 m wide east-west corridor. Each block contained 11 rows 3.7 m apart with 17 or 18 trees separated by 3.1 m within rows. The plot was surrounded by mixed vegetation including such trees as maples and alders, and mowed every other week or as needed. Trees were irrigated only during very hot and dry periods. No fertilizers or pesticides were applied during the course of the study. Two replicate trees were used for each accession (clones for cultivars and half-sib seedlings for wild collected accessions). Yellow sticky traps (97 sq. cm) were hung on the west side of trees on a branch in the lower periphery of each tree at about 4 m above the ground on 1 May 2001. Traps were changed weekly until 28 Aug. 2001. Final traps were taken down on 4 Sept. 2001. Collected traps were examined in the laboratory with a stereomicroscope. Leafhoppers were carefully removed using a dissecting probe, transferred into transparent cups, and counted.
The main study site was an open, grassy area on Maury silt loam soil on the University of Kentucky campus in Lexington, Kentucky, adjacent to the Arboretum and State Botanical Garden of Kentucky (N38°1’; W84°30’; elevation 302 m). Five replicates of each elm accession were planted in a randomized complete block in rows spaced 7.6 m apart, with 7.6 m between trees within rows and 50 m between replicates. Trees were planted in spring 2005 or 2006. The trees were obtained from nurseries as bare-root transplants and ranged from 1.5–2.4 m height at time of planting. They were staked, watered as needed, and mulched over grass that had been killed with glyphosate herbicide. The number of leaves were counted in spring 2009 on the terminal 30 cm of new growth; average of ten shoots.
Composite score of resistance to Japanese beetle (Popillia japonica), European elm flea weevil (Orchestes alni), European fruit lecanium (Parthenolecanium corni), European elm scale (Gossyparia spuria), Kaliofenusa ulmi, Agromyza aristata, pouch gall aphids (Tetraneura nigriabdominalis), cockscomb gall aphid (Colopha ulmicola), woolly elm aphid (Eriosoma americanum), woolly apple aphid (Eriosoma lanigerum), cottony maple scale (Pulvinaria innumerabilis), and Oedophrys hilleri.
The main study site was an open, grassy area on Maury silt loam soil adjacent to the Arboretum and State Botanical Garden of Kentucky (Lexington, Kentucky) (38°1’N, 84°30’W; elevation 302 m). Five replicates of each accession, were planted in a randomized complete block in rows spaced 7.6 m apart, with 7.6 m between trees within rows and about 50 m between replicates. Trees were planted in spring 2005 or 2006. The trees were obtained from nurseries as bare-root transplants and ranged from 1.5 to 2.4 m height at time of planting. They were staked, watered as needed, and mulched over grass that had been killed with glyphosate herbicide. Leaf characteristics (mean area and number per shoot) were previously summarized (Condra et al. 2010) to standardize insect feeding by leaf area.
Three species of sap-feeding scale insects colonized trees at the study site. Their numbers were assessed by inspecting each tree on multiple dates. For some of the taller trees, observers stood on a step ladder or truck tailgate to reach and sample sufficient numbers of representative branches. European fruit lecanium, Parthenolecanium corni (Hemiptera: Coccidae), was abundant enough to provide data from three growing seasons (2010–2012). Five twigs were randomly selected from throughout the canopy of each tree; then beginning at the previous year’s node the number of adult scales was counted on a 30-cm long section of each twig. Data were standardized to number of scales per 1.0 m of twig length. Counts were taken over 2–3 days in early July in 2010 and 2011, and in June 2012. European elm scale, Eriococcus spuria (Hemiptera: Eriococcidae), was abundant enough to census in 2011 and 2012. Adult scales were counted on five 30 cm-long twigs per tree as previously described. In addition, because E. spuria also infested bark of the trunk and scaffold limbs, two observers standing on opposite sides of each tree inspected bases of those branches and main trunk of each tree to 2 m height, and counted scales spotted in 30 seconds, taking care not to count particular scales more than once. Counts from the two methods were pooled for analysis. Sampling dates were the same as for P. corni. Cottony maple scale, Pulvinaria innumerabilis (Hemiptera: Coccidae), was first noticed on the trees in 2012. Two observers slowly circled each tree on June 7–8 and together counted all of the distinctive white scales through the canopy. Oedophrys hilleri (Coleoptera: Curculionidae), a mottled gray, leaf-notching weevil was found feeding on elms at the study site in 2010. Adult populations on each tree were sampled on July 11–12 and August 11 in 2011, and on August 8, 2012. The first sampling was with a gasoline powered leaf blower reversed for suction and fitted with a paint strainer in the intake tube. Each tree’s canopy was sampled for 60 seconds; samples then were transferred to bags and frozen before counting. For subsequent samples, researchers used a beating sheet (71 cm × 71 cm, BioQuip, Rancho Dominguez, California, U.S.), striking eight branches (four each in lower and upper canopy) with a stick and counting dislodged weevils. Two species of foliage-distorting woolly aphids were abundant enough to evaluate in 2012. Spring feeding by woolly elm aphid, Eriosoma americanum (Hemiptera: Aphididae), causes developing leaves to swell and their edges to curl downward. The aphids feed and reproduce within the leaf rolls. Spring feeding by woolly apple aphid, Eriosoma lanigerum (Hemiptera: Aphididae), results in unsightly rosette-like clusters of deformed leaves at the ends of shoots. Both aphids also cause damage by sucking sap from the host, and by producing honeydew. Incidence of each pest’s damage was assessed in early June by two observers who counted all individually-rolled leaves (E. americanum) and rosettes (E. lanigerum) on each tree. Elm cockscomb gall aphid, Colopha ulmicola (Hemiptera: Aphididae), is a relatively minor pest that induces elongated, raised, irregularly-toothed galls on the adaxial surface of leaves. The gall’s shape and reddish color at maturity account for the common name. The leaf galls harden and turn brown after the aphids depart. The number of galls on each tree (to 2.5 m height) were counted by two observers on July 11–12, 2011, and on June 7–8, 2012.
Averages for each insect on each accession were calculated and then ranked on a 1–5 scale based on pest density or extent of damage. The most susceptible accession(s) always received a 5 rating, and those that sustained no damage or infestation by a particular insect received a 0 rating for that pest. For Japanese beetle defoliation, 0–5 ratings corresponded to <10, 22–20, 21–30, 31–40, 41–55, and >55% cumulative leaf loss, respectively. Tree ratings for the leafminers O. alni, A. aristata, and K. ulmi, and for T. nigriabdominalis aphid pod galls were based on numbers of mines or galled leaves per 100 leaves (2006–2008) or per ten 30-cm shoots (2009–2012), with 1–5 ratings generally assigned to accessions ranked in successive quintiles of the frequency distribution. For those pests first reported on in this paper, cultivar ratings were based on number of European fruit lecanium and European elm scale per 1.5 m of twig length, or whole tree counts for O. hilleri, elm cockscomb gall aphid, wooly elm aphid, wooly apple aphid, and cottony maple scale, as described herein. For susceptibility/resistance rankings, the study authors considered four of the insect species, Japanese beetle, European elm flea weevil, European fruit lecanium, and European elm scale, as “major” pests because, at least in Kentucky, their impact on heavily infested trees and/or problems associated with their honeydew can be serious enough to warrant control. Ratings for those four pests were doubled and then added to the sum of ratings for the remaining eight pests to get overall scores upon which the elm accessions were ranked for relative susceptibility to insect pests.
Ploidy level determined either through flow cytometry or chromosome counting. See citations for additional details.
Thirty-six ramets were planted in a completely randomized block design, with two blocks and three trees per clone per block, in a field plot at the USDA, USNA, Agricultural Research Service (ARS), station near Glenn Dale, MD. A laboratory colony of the potato leafhopper (Empoasca fabae (Harris)) was started in the summer of 1995 and invigorized yearly with adults collected in a potato field at the Beltsville Agricultural Research Center, ARS-USDA, Beltsville, MD. Insects were reared on a mixture of Henderson's bush lima bean (Phaseolus lunatus L.) and fava bean (Vicia faba L.) kept in clear plastic cages (60 by 60 by 30 cm, or 60 by 60 by 60 cm). Cages were maintained in a walk-in chamber at 25.5°C, 60-80% RH, and a photoperiod of 16:8 (L:D) h. For seven consecutive weeks during the 1998 season, one branch on each of two trees per clone per block was randomly selected. Potato leafhopper females, about 1 wk old, were collected from the laboratory colony in groups of five, and released into organza sleeves (30.5 by 38.1 cm) tied around branches as previously described (Bentz and Townsend 1999). Females were allowed to oviposit for 6 d. At this time, sleeves enclosing the branches with leaves were cut off the trees, brought to the laboratory and stored in a freezer at -20°C to kill the ovipositing females. The leaves within sleeves were collected and wrapped. Staining and counting of leafhopper eggs within leaves was done as previously described (Bentz and Townsend 1999). At each of the seven weeks during the 1998 season, a shoot from each of two trees per block of the six clones was randomly chosen, inspected for the presence of leafhoppers, and tagged if it had no evidence of leafhopper presence. Each tagged shoot was covered with a white organza sleeve (30.5 by 38.1 cm) as described in Bentz and Townsend (1999). Second instars were collected from the laboratory colony and released in groups of two into each organza sleeve. Nymphs were left in the sleeves until becoming adults. At this time, sleeves enclosing the shoots were cut off, brought to the laboratory and stored in a freezer at -20°C. Later, leaves were removed from the sleeves and adults counted.
The leaf samples used in the studies were obtained from plants in an experimental elm plantation located in the city of Holbrook, AZ. The plantation was established during 1995-1998, and each accession was represented by at least five single-tree replicates at the time of sampling. Elm seedlings 2-4-yr-old and 30-45 cm in height at the time of planting were used in establishing the plantation using a completely randomized design with 10 single-tree replicates per taxa. Seedlings were planted on a 21 rows x 14 columns grid at 3.6- by 3.6-m spacing. The soil around each plant was mulched using a 1-sq m piece of black shade cloth covered with pine wood chips. Plants were watered regularly with a drip irrigation system that supplied water to the base of individual plants. Detailed description of the experimental plantation can be found in Bosu et al. (2007). Undamaged single leaves were removed from each of the plants in the field and immediately cut with dissection scissors into ~1-sq cm pieces that encompassed at least the leaf midrib. The cut leaf samples were then placed in labeled glass vials containing 4% glutaraldehyde fixative and 0.01M sodium cacodaylate buffer solution, pH 7.2, before processing for scanning electron microscopy observation (St-Laurent et al. 2000). The samples were dehydrated in a series of ethanol concentrations of 25, 50, 70, 95, and 100%. Samples were then critical point dried using carbon dioxide, mounted on aluminum stubs, and sputtered with gold under vacuum. Trichome type and density were assessed with a scanning electron microscope (15 kV; LEO 435 VP, Leica, Cambridge, United Kingdom) on the abaxial leaf surface.
Cultivars were acquired from commercial nurseries. Trees were planted during the summer of 2014 in the nursery fields at the University of Minnesota, St. Paul, MN, and were watered as needed. In 2015, trees were 3 years old and 1–3 cm diameter at 0.5 m above the ground. A portion of stem was collected approximately 0.60 m above the ground. Samples were then placed in a −20°C freezer until sectioned. Four pieces (approximately 2 mm wide) spaced 90° apart with a random starting point were cut from the stem segment using a high-profile microtome blade. Pieces were soaked in 100% TFM™ tissue freezing medium (Electron Microscopy Sciences, Hatfield, PA) for approximately 16 hr. An IEC Minotome® cryostat (International Equipment Co.) at −20°C was used to cut 15- to 30-μm-thick transverse sections. After sectioning, the sections were cleared with water and subsequently stained with 0.1% safranin O (dye content ≥85%) (Sigma-Aldrich®) (w/v) solution for 20 s. After removing excess safranin O with a paper towel, sections were mounted in water by placing a drop of DI water onto the surface of the sample and then subsequently covering the sample with a coverslip. Sections were allowed to air-dry at room conditions for 24 hr. Images were taken at 40× using a Nikon Eclipse E600 microscope (Nikon Instruments Inc.) with a Nikon Digital Camera DXM 1200F (Nikon Instruments Inc.). Many samples were larger than the field of view for the low magnification of the microscope objective and field of view for the digital documenting system, so multiple images of the same section were stitched together in Photoshop™ (Adobe Systems Inc.) using the photomerge feature. Focus stacking was performed as needed in Photoshop™. A 500-μm-wide area of the xylem following the same ray parenchymal cells of the annual ring of interest was analysed. This area included earlywood and latewood vessels. Vessel elements were manually traced or selected using the magic wand in Photoshop™. Images were subsequently analysed using the thresholding feature in ImageJ (Schneider et al., 2012) to generate a mask of vessel elements with a D (equivalent circle diameter) ≥15 μm. The masked images were then analysed using ROXAS 3.0 (von Arx et al., 2013). Jansen et al. (2009) found that the total intervessel cell wall thickness (mean ± SD) in Ulmus americana was 2.946 ± 0.665 μm; therefore, the double cell wall thickness was set to 4 μm to ensure most connected vessels would be included when analysed for vessel aggregation. Vessel density was measured as number of vessels per square milimeter.
Cultivars were acquired from commercial nurseries. Trees were planted during the summer of 2014 in the nursery fields at the University of Minnesota, St. Paul, MN, and were watered as needed. In 2015, trees were 3 years old and 1–3 cm diameter at 0.5 m above the ground. A portion of stem was collected approximately 0.60 m above the ground. Samples were then placed in a −20°C freezer until sectioned. Four pieces (approximately 2 mm wide) spaced 90° apart with a random starting point were cut from the stem segment using a high-profile microtome blade. Pieces were soaked in 100% TFM™ tissue freezing medium (Electron Microscopy Sciences, Hatfield, PA) for approximately 16 hr. An IEC Minotome® cryostat (International Equipment Co.) at −20°C was used to cut 15- to 30-μm-thick transverse sections. After sectioning, the sections were cleared with water and subsequently stained with 0.1% safranin O (dye content ≥85%) (Sigma-Aldrich®) (w/v) solution for 20 s. After removing excess safranin O with a paper towel, sections were mounted in water by placing a drop of DI water onto the surface of the sample and then subsequently covering the sample with a coverslip. Sections were allowed to air-dry at room conditions for 24 hr. Images were taken at 40× using a Nikon Eclipse E600 microscope (Nikon Instruments Inc.) with a Nikon Digital Camera DXM 1200F (Nikon Instruments Inc.). Many samples were larger than the field of view for the low magnification of the microscope objective and field of view for the digital documenting system, so multiple images of the same section were stitched together in Photoshop™ (Adobe Systems Inc.) using the photomerge feature. Focus stacking was performed as needed in Photoshop™. A 500-μm-wide area of the xylem following the same ray parenchymal cells of the annual ring of interest was analysed. This area included earlywood and latewood vessels. Vessel elements were manually traced or selected using the magic wand in Photoshop™. Images were subsequently analysed using the thresholding feature in ImageJ (Schneider et al., 2012) to generate a mask of vessel elements with a D (equivalent circle diameter) ≥15 μm. The masked images were then analysed using ROXAS 3.0 (von Arx et al., 2013). Jansen et al. (2009) found that the total intervessel cell wall thickness (mean ± SD) in Ulmus americana was 2.946 ± 0.665 μm; therefore, the double cell wall thickness was set to 4 μm to ensure most connected vessels would be included when analysed for vessel aggregation. Vessel diameter was measured as the equivalent circle diameter (D) in μm, which is the diameter of a circle having the same lumen area (cell walls excluded) as the measured vessel
Plants of each accession were cultivated in a field setting in Ames, IA or at Wageningen, Netherlands. All xylem material came from 2-year-old twigs growing on sapling-size or larger trees. The twigs were of comparable size. The material from The Netherlands was collected in early April and that from Ames, in mid-June. Lengths of twigs adjacent to twig crotches were killed and fixed in formalin-alcohol-acetic acid solution (FAA) (Sass, 1958). Isopropyl alcohol was used in The Netherlands and ethyl alcohol in Iowa. All twig material was stored in FAA for up to 2 years before crosssections were made. For sectioning, the fixed twigs were wrapped tightly in paper towelling and cut 15 to 20 ~m thick with a sliding microtome. The cross-sections were stored in distilled water at 4 ~ before anatomical observations were made. Three of the Ames selections were also embedded in paraffin, cut 10 ~m thick on a rotary microtome and stained with saffranin-fast green (Sass, 1958). The latter was done to check the accuracy of vessel determination in the thicker, unstained sections. Observations were made on images of twig cross-sections projected on white cardboard using a projection mirror mounted on a compound microscope. With the microscope placed at stand-up bench height, the image was projected to sit-down table height. An enlargement of x 248 was obtained thereby. Determination of vessels was aided by the appearance of bordered pits common to these cells. Vessel lumen diameter in the projected image, then converted to micrometers on the sample. The data represent the vessel characteristics of a constant area of xylem grown the year prior to collection (second-year growth). All earlywood and most, if not all, late wood were included in this area. For each cross-section observed, data were recorded for four different areas. Two cross-sections were checked from each of two branches collected from each elm selection. Only one tree for each selection was used. The data from the sixteen areas for each elm selection were averaged. Although variability within a selection was present, it did not affect statistically the final comparisons among the different selections.
Plants of each accession were cultivated in a field setting in Ames, IA or at Wageningen, Netherlands. All xylem material came from 2-year-old twigs growing on sapling-size or larger trees. The twigs were of comparable size. The material from The Netherlands was collected in early April and that from Ames, in mid-June. Lengths of twigs adjacent to twig crotches were killed and fixed in formalin-alcohol-acetic acid solution (FAA) (Sass, 1958). Isopropyl alcohol was used in The Netherlands and ethyl alcohol in Iowa. All twig material was stored in FAA for up to 2 years before cross-sections were made. For sectioning, the fixed twigs were wrapped tightly in paper towelling and cut 15 to 20 um thick with a sliding microtome. The cross-sections were stored in distilled water at 4C before anatomical observations were made. Three of the Ames selections were also embedded in paraffin, cut 10 um thick on a rotary microtome and stained with saffranin-fast green (Sass, 1958). The latter was done to check the accuracy of vessel determination in the thicker, unstained sections. Observations were made on images of twig cross-sections projected on white cardboard using a projection mirror mounted on a compound microscope. With the microscope placed at stand-up bench height, the image was projected to sit-down table height. An enlargement of x 248 was obtained thereby. Determination of vessels was aided by the appearance of bordered pits common to these cells. Vessel lumen diameter was measured in the projected image, then converted to micrometers on the sample. The diameter of a vessel group was estimated as the product of the average vessel diameter and the average number of vessels per group. The data represent the vessel characteristics of a constant area of xylem grown the year prior to collection (second-year growth). All earlywood and most, if not all, late wood were included in this area. For each cross-section observed, data were recorded for four different areas. Two cross-sections were checked from each of two branches collected from each elm selection. Only one tree for each selection was used. The data from the sixteen areas for each elm selection were averaged. Although variability within a selection was present, it did not affect statistically the final comparisons among the different selections.
Cultivars were acquired from commercial nurseries. Trees were planted during the summer of 2014 in the nursery fields at the University of Minnesota, St. Paul, MN, and were watered as needed. In 2015, trees were 3 years old and 1–3 cm diameter at 0.5 m above the ground. A portion of stem was collected approximately 0.60 m above the ground. Samples were then placed in a −20°C freezer until sectioned. Four pieces (approximately 2 mm wide) spaced 90° apart with a random starting point were cut from the stem segment using a high-profile microtome blade. Pieces were soaked in 100% TFM™ tissue freezing medium (Electron Microscopy Sciences, Hatfield, PA) for approximately 16 hr. An IEC Minotome® cryostat (International Equipment Co.) at −20°C was used to cut 15- to 30-μm-thick transverse sections. After sectioning, the sections were cleared with water and subsequently stained with 0.1% safranin O (dye content ≥85%) (Sigma-Aldrich®) (w/v) solution for 20 s. After removing excess safranin O with a paper towel, sections were mounted in water by placing a drop of DI water onto the surface of the sample and then subsequently covering the sample with a coverslip. Sections were allowed to air-dry at room conditions for 24 hr. Images were taken at 40× using a Nikon Eclipse E600 microscope (Nikon Instruments Inc.) with a Nikon Digital Camera DXM 1200F (Nikon Instruments Inc.). Many samples were larger than the field of view for the low magnification of the microscope objective and field of view for the digital documenting system, so multiple images of the same section were stitched together in Photoshop™ (Adobe Systems Inc.) using the photomerge feature. Focus stacking was performed as needed in Photoshop™. A 500-μm-wide area of the xylem following the same ray parenchymal cells of the annual ring of interest was analysed. This area included earlywood and latewood vessels. Vessel elements were manually traced or selected using the magic wand in Photoshop™. Images were subsequently analysed using the thresholding feature in ImageJ (Schneider et al., 2012) to generate a mask of vessel elements with a D (equivalent circle diameter) ≥15 μm. The masked images were then analysed using ROXAS 3.0 (von Arx et al., 2013). Jansen et al. (2009) found that the total intervessel cell wall thickness (mean ± SD) in Ulmus americana was 2.946 ± 0.665 μm; therefore, the double cell wall thickness was set to 4 μm to ensure most connected vessels would be included when analysed for vessel aggregation. Mean number of vessels per group were counted, where solitary vessels are also considered a group (Carlquist, 2001)
Netherlands and ethyl alcohol in Iowa. All twig material was stored in FAA for up to 2 years before cross-sections were made. For sectioning, the fixed twigs were wrapped tightly in paper towelling and cut 15 to 20 um thick with a sliding microtome. The cross-sections were stored in distilled water at 4C before anatomical observations were made. Three of the Ames selections were also embedded in paraffin, cut 10 um thick on a rotary microtome and stained with saffranin-fast green (Sass, 1958). The latter was done to check the accuracy of vessel determination in the thicker, unstained sections. Observations were made on images of twig cross-sections projected on white cardboard using a projection mirror mounted on a compound microscope. With the microscope placed at stand-up bench height, the image was projected to sit-down table height. An enlargement of x 248 was obtained thereby. Determination of vessels was aided by the appearance of bordered pits common to these cells. The data represent the vessel characteristics of a constant area of xylem grown the year prior to collection (second-year growth). All earlywood and most, if not all, late wood were included in this area. For each cross-section observed, data were recorded for four different areas. Two cross-sections were checked from each of two branches collected from each elm selection. Only one tree for each selection was used. The data from the sixteen areas for each elm selection were averaged. Although variability within a selection was present, it did not affect statistically the final comparisons among the different selections.
Images captured with an Epson Expression 12,000 XLGA flatbed scanner set at 600 DPI. Silverfast used to capture and color calibrate. Random sample of seeds (samaras). Seed length and width measurements (millimeters) includes membranous wing.