Field sites and treatments
Field plot experiments were repeated over 2 years at the Central Maryland Research and Education Center, Beltsville, MD (2013 and 2014), and at two Louisiana State University Agricultural Center research stations in St. Joseph (2013) and Winnsboro, LA (2014). At all sites, two simulated refuge systems were established each year. System 1 represented a structured refuge consisting of a planting of purple-seeded (PS) plants (hybrid ‘SL7571PG’; RM 113 days) flanked on both sides by stands of Bt yellow-seeded (YS) plants (Agrisure hybrid ‘NK1284-3000GT’; RM 112 days). System 2 represented a seed blend of Bt YS plants containing PS plants as refuges. Another seed blend planting (System 3) was evaluated only at the Maryland site in 2014 and consisted of PS plants containing its closely related non-Bt isoline YS plants (hybrid ‘NK1284-GT’; RM 112 days) as refuges.
Experimental plot design
Plots of each refuge system, consisting of 7.5 m long rows spaced 20 cm apart, were arranged side by side within in each of four (MD) or five (LA) randomized complete blocks. A buffer strip of 12 rows of male-sterile non-Bt corn running the opposite way was planted between blocks. For the structured refuge system 1, each plot consisted of four rows of PS plants flanked on both sides by four rows of Bt YS plants. For the blended seed systems, each plot consisted of 12 rows of either Bt YS (System 2) or PS plants (System 3) with either PS (System 2) or non-Bt YS (System 3) refuge plants, respectively. Refuge plants were hand planted after machine seeding in rows 3, 5, 7, and 9 and spaced 1.8 m apart, so that the eight refuge plants were not directly across from each other in the nearest adjacent rows. To ensure successful germination and seedling emergence, two seeds were planted at each refuge spot and marked with small stakes to identify refuge plants. At the two-leaf stage, the less vigorous seedling in a refuge spot was removed. Plots were seeded in early May at the LA sites and in late May at the MD site, using a no till corn planter to establish a stand of 64,200 plants per hectare. Each experiment consisted of either 8 (MD) or 10 (LA) plots, except for the Maryland experiment in 2014 which had 12 plots (three refuge systems replicated four times). For each experiment, standard fertility inputs were practiced according to site-specific nutrient management recommendations, and weeds were managed using pre- and post-emergent herbicide applications.
Measurements of gene flow
At the dough stage (R4), ear samples were collected from each refuge system to record the number of kernels expressing the purple color as a measure of cross-pollination. Any kernel showing partial or complete phenotypic expression of the purple trait was counted. In the structured refuge plots, ten randomly selected ears (LA) or all ears (MD) from the Bt YS plants on each row flanking the center four rows of PS plants were husked in situ and examined for purple kernels. Data were recorded on individual ears per row on each side of the center purple-seeded plants. For seed blend system 2, outcrossing of genes due to pollen drift from the PS refuge plants was estimated by the presence of purple kernels in ears of Bt YS neighboring plants around each refuge plant. Data on the number of purple kernels per ear were recorded on plants at 1, 2, 3, 4, and 5 positions away from the refuge plant on both sides within the same row. Ears were also examined for purple kernels on five or six consecutive plants (depending on the study site) on the adjacent row on each side of the refuge plants. For seed blend system 3 consisting of PS plants with YS refuge plants (MD, 2014), ears of all refuge plants in each plot were examined to record the number of purple kernels, indicative of the potential cross-pollination due to pollen drift from neighboring Bt plants. For each experiment, random samples of 10 ears in each plot were collected to record the number of kernel rows on each ear, length of kernel row (cm), and number of kernels per 10 cm.
Tissue-incorporated feeding bioassay
Laboratory bioassays of kernel tissue were conducted in 2015 to provide evidence of Bt toxin expression in ears of refuge plants in the seed blend system. Field plots of a pyramided Bt hybrid (SPS1031), expressing Cry1Ab and Vip3Aa20 toxins for lepidopteran control, and its closely related non-Bt hybrid (SPS1030) were established at the Central Maryland Research and Education Center, Beltsville, MD. Plots of 100% Bt hybrid, 95:5 seed blend of the Bt hybrid and non-Bt refuge, and 100% non-Bt hybrid were planted side by side in each of four randomized blocks. Each plot measuring 4 rows 4.6 m long was planted by hand on July 17 with 20 seeds per row spaced 23 cm apart. Each row of the seed blend treatment contained one refuge plant marked with a stake and placed at least 0.5 m away from the row ends.
Two ears at the dent stage (R5) were randomly collected from the center rows in each of the 100% Bt and non-Bt plots, and from two refuge plants in each seed blend plot. Kernels of each type were removed from the ears and pooled by replicate block as a composite sample, and then, a random subsample was frozen and dried in a freeze dryer. The lyophilized kernel tissue was ground to a fine powder in a commercial grinder (IKA Works, Inc, Wilmington, DE) and kept at − 80 °C until used in bioassays.
A diet-incorporated feeding bioassay consisting of a single concentration of each kernel tissue was conducted to measure the body weight gain of second instar H. zea and O. nubilalis. Eggs of laboratory colonies of both species obtained from Benzon Research (http://www.benzonresearch.com/) were incubated in a growth chamber until hatch and then reared on a meridic diet (Southland Products, Lake Village, AR) until the 2nd instar for testing. Separate bioassays were conducted exposing individual larvae of each species to four replicates of each kernel type. For each replicate sample of kernel powder, 500 ml of modified meridic diet (without soybean flour and adjusted with more water to offset the added kernel powder) was prepared, cooled to 55 °C in a water bath, and then incorporated with 30.5 g of kernel powder. The dilution by adding kernel tissue resulted in a concentration of 60 mg of kernel tissue per ml of diet. For each treatment, ~ 1.5 mL of diet was dispensed into each of 64 wells of a 128-well bioassay tray (C-D International). After the diet cooled, one larva was placed in each well using a camel-hair brush. Wells were sealed with a perforated adhesive lid, and trays were held in a growth chamber at 25 °C. After 7 days, the weight of individual larvae was recorded, and the average weight gain per larva was calculated for each treatment by replicate group (16 larvae/replicate).
The mean number of kernels per ear was calculated as [number of kernel rows per ear x number of kernels per row], using metrics recorded from ear samples in each plot. The percentage of purple kernels was then calculated as [number of purple kernels/mean number of kernels per ear × 100] and analyzed as a surrogate estimate of the percentage of cross-pollination. Although the refuge systems were planted together in the same experimental plot layout, each system was analyzed separately using the Proc Mixed model of SAS Version 9.4 (SAS Institute 2013), with the focus on testing for differences among the nested factors. Furthermore, analyses were conducted by year and study site due to differences in sampling protocols and environmental conditions. For the structured refuge system 1, we tested for mean differences across individual rows and between means pooled over rows on each side of PS plants. The ANOVA model treated row as a fixed nested factor and corrected for repeated measures across rows that were spatially dependent. For seed blend system 2, we tested for differences among three plant position groups relative to the refuge plant, as a fixed nested factor. These groups included the pooled data from four ears surrounding the refuge plant at 1 and 2 plant positions away, six ears at 3, 4, and 5 plant positions away, and four ears for the nearest plants on adjacent rows. Data recorded on individual ears within a plot row or plant position were considered sub-sampling and thus averaged by replicate block before analysis. Data from seed blend system 3 were summarized as mean (± standard error) percentage of kernels showing expression of the purple color in the refuge ears. In all analyses, replicate block was treated as a random effect, percentage data were arcsine transformed before the analysis, and Tukey’s option was used to test for significance (P < 0.05) among multiple mean comparisons. For the bioassay data, differences in larval weight gain among the kernel tissue types were tested using a one-way ANOVA, after data transformations were made for lack of normality and unequal variances.