HYDRANGEA

Methods

FIELD WINTER DAMAGE - MN 2020

LEAF SPOT

In September 2019, leaves were sampled from 100 seedlings randomly selected from each population (two year old plants) or from 10 plants per cultivar. One leaf was sampled per seedling and three leaves were sampled per cultivar plant, all of which were growing in 7.57-L pots in Chanhassen, MN. Plants were exposed to naturally occurring Xanthomonas campestris inoculum under container nursery conditions including overhead irrigation. Populations were completely randomized within the nursery to ensure equivalent exposure to inoculum. The fourth fully expanded leaf was chosen for sampling based on the observation that the leaf spot symptoms were less severe on the top ofthe canopy, whereas leaves further down in the canopy were severely senesced or otherwise deteriorated. Leaves were immediately transported to the laboratory for imaging against a white background with uniform, cool white fluorescent lighting. Images were obtained with a Samsung SM-G950U1 camera (Samsung Electronics Co., Seoul, South Korea) with 4032- × 3024-pixel resolution, a 4.25-mm focal length and saved in the .jpg file format. Images were analyzed using Food Color Inspector software (v4.0; http://www.cofilab.com/portfolio/food-color-inspector/) to determine the percent leaf area affected by lesions and secondary symptoms. This software allows the user to define each of the categories in a training set; then, it assigns all pixelsin the image to one of the categories based on the color using a Bayesian algorithm. One leaf per population was used as the initial training set for the population, which was updated iteratively as needed for each leaf to ensure accurate classifications. Pixels were assigned to four categories: background; healthy leaf tissue; necrotic leaf tissue; and discolored leaf tissue. Discoloration was either chlorosis or other impacted tissue surrounding or immediately adjacent to necrotic tissue; it typically represented the leading edge of the lesion. The percent lea farea affected by leaf spot (necrotic tissue and discoloration) was calculated for each leaf by dividing the number of pixels classified as each of the diseased categories by the number of nonbackground pixels and then multiplying by 100. Population and cultivar means were assessed with an ANOVA and Bonferroni-corrected for multiple comparisons. Each leaf was considered a replicate; therefore, there were 100 replicates for each population and 30 replicates for each cultivar. Several representative infected leaves were submitted to the University of Minnesota Plant Disease Clinic to verify the identity of the pathogen. The pathogen was determined to be Xanthomonas campestris, which was consistent with the expectation. No additional pathogens were detected. See citation for additional details.

LT50

Seedlings, derived from wild collected seed which had germinated in Febraury 2018, were tested for cold hardiness in January 2019, with a controlled freezing test. These methods were based on those described previously for controlled hardiness screening (McNamara et al., 2002; McNamara and Hokanson, 2010). The container-grown seedlings were allowed to acclimate under ambient conditions in Chanhassen, MN until mid-November, when they were moved into a minimally heated greenhouse structure maintained at a minimum temperature of -9.5°C to avoid cold damage before sampling. The seedlings were sampled on four dates over the course of 2 weeks (6, 10, 13, and 17 Jan.) in a completely randomized experimental design. Each sample operation consisted of harvesting and preparing stem samples on the first day and freezing the stems the following day. On the first day of each sampling date, stems from seedlings of each population to be tested that day were taken to the laboratory and cut into 3.5-cm sections, color-coded, and then randomly assigned to one of six temperature treatments or a nonfrozen control. Stem sections from each population at each temperature treatment were placed into bags with moist paper towels (three replicate bags per temperature per sample date). The bags were placed in a ScienTemp freezer (ScienTemp Corporation, Adrian, MI) controlled by a Watlow series 942 temperature controller (Watlow, St. Louis, MO). Each bag contained stem sections from all populations, with a total of 8 to 12 stem samples per population per test temperature. A thermocouple was inserted into the pith of at least one stem section per temperature treatment to monitor the stem temperature throughout the experiment. Temperatures were slowly decreased to the first test temperature (-10°C) overnight to allow stem temperatures to equilibrate. The following day, temperatures were decreased at a rate of 3°C per hour, and three replicate bags for each treatment were removed from the freezer at increments of -5 to -35°C. Samples were removed from the freezer when each test temperature was reached. Stem sections were slowly thawed at 3°C overnight and incubated at room temperature for 1 week to allow damage symptoms to appear. Stem sections were evaluated for cold damage by slicing the stem longitudinally with a scalpel and rating stems as live or dead by observing the damage (oxidative browning of vascular tissue) under a dissecting microscope (12× magnification). Most stems were unambiguously dead or alive; however, some stems were intermediate. In that case, stems were classified according to having more than or less than 50% damage. Cold hardiness was determined as the mean lethal temperature (LT₅₀) and calculated for each population using a binomial logit model to interpolate the temperature at which 50% of the stems would have died (Suojala and Lindén, 1997). The binomial logit model was implemented in the R packages MASS (v7.3–50) and stats (v3.5.1).The experiment was repeated during late Fall 2019 through early Spring 2020, with a subset of populations that were tested in January 2019 plus a younger cohort of seedling populations (germinated in February 2019) as well as a selection of cultivars. During this experiment, plants were tested once per month from October to April to characterize the timing of fall acclimation and spring deacclimation in addition to midwinter hardiness using the methods described. The tested temperatures (and interval between test temperatures) were varied each month to exceed the expected range of LT₅₀ values. During each month, the sample from a population or cultivar comprised one random stem collected from multiple individual plants; therefore, each plant was resampled every month. Cultivars were tested with four to six stem samples per cultivar per temperature per month while the populations were tested with the same sample sizes as those during January 2019. See citations for additional details.

OMERNIK LEVEL III ECOREGION

Listing of Omernik Level III Ecoregion where germplasm was obtained. Ecoregions determined using Omernik, J.M., 1987, Ecoregions of the conterminous United States (map supplement): Annals of the Association of American Geographers, v. 77, no. 1, p. 118-125, scale 1:7,500,000.

PLANT ARCHITECTURE - 2018 YR 1 POTS

Wild collected seed from 17 populations were grown and represented the latitudinal range of the species from Tennessee, Mississippi, and Florida. In 2018, seed from each maternal parent was surface-sown onto a soilless germination mix (Sungrow Horticulture, Agawam, MA). In the greenhouse, flats were placed on heat mats (Hydrofarm Horticulture, Petaluma, CA) and covered with clear plastic domes to maintain high relative humidity. Greenhouse temperatures were maintained between 25 and 30°C with natural daylength. After germination, seedlings were transplanted into 7.62-cm square pots holding a soilless potting medium (Sungrow Horticulture, Agawam, MA) and grown in the greenhouse until May. Seedlings were transplanted into 186.7-cm³ square pots with peat:pine bark (1:1) potting mix and transferred to an outdoor container nursery with overhead irrigation. Plants were watered as needed throughout the growing season. A minimum of 100 random seedlings from each population were evaluated after terminal buds had set at the end of the growing season. Because plants were completely randomized within the container nursery, random sampling was achieved by measuring the first 100 seedlings located in each population. Plants were measured from the top of the soil to the top of the apical bud on the tallest shoot, and nodes were counted on the same shoots. Internode length was estimated by dividing the total height by the number of nodes. See citation for additional details.

PLANT ARCHITECTURE - 2019 YR 1 POTS

In 2019, seeds were grown from 38 populations in Louisiana, Georgia, and Alabama. Seed was germinated in a greenhouse in Chanhassen, Minnesota. Seed from each maternal parent was surface-sown onto a soilless germination mix (Sungrow Horticulture, Agawam, MA). In the greenhouse, flats were placed on heat mats (Hydrofarm Horticulture, Petaluma, CA) and covered with clear plastic domes to maintain high relative humidity. Greenhouse temperatures were maintained between 25 and 30°C with natural daylength. After germination, seedlings were transplanted into 7.62-cm square pots holding a soilless potting medium (Sungrow Horticulture, Agawam, MA) and grown in the greenhouse until June. Seedlings were transplanted into 186.7-cm³ square pots with peat:pine bark (1:1) potting mix and transferred to an outdoor container nursery with overhead irrigation. Plants were watered as needed throughout the growing season. Because plants were completely randomized within the container nursery, random sampling was achieved by measuring the first 100 seedlings located in each population. After terminal buds were set, plants were measured from the top of the soil to the top of the apical bud on the tallest shoot, and nodes were counted on the same shoots. Internode length was estimated by dividing the total height by the number of nodes. See citations for additional details.

PLANT ARCHITECTURE - 2019 YR 2 MN FIELD

In 2018, seed from 17 populations which represented the latitudinal range of the species from Tennessee, Mississippi, and Florida were germinated. Seed from each maternal parent was surface-sown onto a soilless germination mix (Sungrow Horticulture, Agawam, MA). In the greenhouse, flats were placed on heat mats (Hydrofarm Horticulture, Petaluma, CA) and covered with clear plastic domes to maintain high relative humidity. Greenhouse temperatures were maintained between 25 and 30°C with natural daylength. After germination, seedlings were transplanted into 7.62-cm square pots holding a soilless potting medium (Sungrow Horticulture, Agawam, MA) and grown in the greenhouse until May. Seedlings were transplanted into 186.7-cm³ square pots with peat:pine bark (1:1) potting mix and transferred to an outdoor container nursery with overhead irrigation. Plants were watered as needed throughout the growing season. During Spring 2019, plants were cut back to three nodes above the soil to induce branching in their second year of growth. Cutting the plants back also served to equalize plant height at the beginning of the second growing season. Then, they were randomly assigned to three treatments: transplanted into 7.57-L containers, planted in the field at the Horticulture Research Center in Chanhassen, MN (44.859, -93.634) or in the field at the Otis L. Floyd Nursery Research Center in McMinnville, TN (35.709, -85.744) using a completely randomized experimental design. Natural rainfall was supplemented with drip irrigation at both locations to prevent soil moisture deficits. A minimum of 100 random seedlings from each population were evaluated after terminal buds had set at the end of the growing season. Because plants were completely randomized within the container nursery, random sampling was achieved by measuring the first 100 seedlings located in each population. Plants were measured from the top of the soil to the top of the apical bud on the tallest shoot, and nodes were counted on the same shoots. Internode length was estimated by dividing the total height by the number of nodes. Number of primary branches was also measured; because of the lack of secondary branches, branch counts were unambiguous. Seedlings were measured in 7.57-L pots and in both field locations (Minnesota and Tennessee) to assess genotype × environment interactions. Additionally, the canopy width was measured in the field by averaging the width in the widest dimension and the width perpendicular to the widest dimension. See citations for additional details.

PLANT ARCHITECTURE - 2019 YR 2 POTS

PLANT ARCHITECTURE - 2019 YR 2 TN FIELD

USDA COLD HARDINESS ZONE

Listing of USDA Cold Hardiness Zone where germplasm was obtained. Cold Hardiness Zones determined using the USDA Plant Hardiness Zone Map, 2012. Agricultural Research Service, U.S. Department of Agriculture.