Bt Pollen Dispersal and Bt Kernel Mosaics: Integrity of Non-Bt Refugia ...

INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT

Bt Pollen Dispersal and Bt Kernel Mosaics: Integrity of Non-Bt Refugia for Lepidopteran Resistance Management in Maize ERIC C. BURKNESS

AND

W. D. HUTCHISON1

University of Minnesota, Department of Entomology, 1980 Folwell Avenue, St. Paul, MN 55108-6125

J. Econ. Entomol. 105(5): 1773Ð1780 (2012); DOI: http://dx.doi.org/10.1603/EC12128

ABSTRACT Field trials were conducted at Rosemount, MN in 2009 and 2010, to measure pollen movement from Bt corn to adjacent blocks of non-Bt refuge corn. As the use of Bt corn hybrids continues to increase in the United States, and new insect resistance management (IRM) plans are implemented, it is necessary to measure the efÞcacy of these IRM plans. In Minnesota, the primary lepidopteran pests of corn include the European corn borer, Ostrinia nubilalis (Hu¨ bner) and corn earworm, Helicoverpa zea (Boddie). The primary IRM plan in transgenic corn is the use of hybrids expressing a high dose of insecticidal proteins and an insect refuge containing hybrids not expressing insecticidal proteins that produce susceptible insects. Wind-assisted pollen movement in corn occurs readily, and is the primary method of pollination for corn. The combination of pollen movement and viability determines the potential for cross pollination of refuge corn. In 2009 and 2010, cross pollination occurred with the highest frequency on the north and east sides of Bt corn Þelds, but was found at some level in all directions. Highest levels of cross pollination (75%) were found within the Þrst four rows (3 m) of non-Bt corn adjacent to Bt corn, and in general decreasing levels of cross pollination were found the further the non-Bt corn was planted from the Bt corn. A mosaic of Bt cross-pollinated kernels was found throughout the ear, but in both years the ear tip had the highest percentage of cross-pollinated kernels; this pattern may be linked to the synchrony of pollen shed and silking between Bt and non-Bt corn hybrids. The dominant wind direction in both years was from WNW. However, in both years, there were also prevailing winds from SSW and WSW. Further studies are needed to quantify Bt levels in cross-pollinated kernels, measure the Bt dose of such kernels and associated lepidopteran pest survival, and measure the impact of Bt pollen on lepidopteran pests, particularly when considering the seed mixture refuge conÞguration. KEY WORDS refuge, Bacillus thuringiensis, Ostrinia nubilalis, Helicoverpa zea

The primary lepidopteran pests of Þeld and sweet corn in Minnesota include the European corn borer, Ostrinia nubilalis (Hu¨ bner) and corn earworm, Helicoverpa zea (Boddie). Throughout the major corn growing regions of the United States, including Minnesota, the use of transgenic corn expressing insecticidal proteins for managing lepidopteran pests has been widely adopted (Hutchison et al. 2010). An important management consideration, and challenge to long-term sustainability of the technology, is how best to minimize the risk of insect resistance to insecticidal proteins expressed in transgenic hybrids (Onstad 2008, Huang et al. 2011). The primary tactic employed for insect resistance management (IRM) in transgenic crops, expressing insecticidal proteins, is the high-dose, refuge (HDR) system where a structured insect refuge, consisting of nontransgenic hybrids are planted adjacent to, or 1 Corresponding author: William D. Hutchison, University of Minnesota, Department of Entomology, 219 Hodson Hall, 1980 Folwell Ave., St. Paul, MN 55108-6125 (e-mail: [email protected]).

within the high dose expressing transgenic Bt maize (Gould 1998, Tabashnik et al. 2008, Huang et al. 2011, Onstad et al. 2011). The refuge provides an area for susceptible individuals to develop without selection for resistance to one or more Bt toxins, and thus be available to interbreed with potentially resistant individuals that may emerge from the transgenic hybrids. Tabashnik et al. (2009) found that based on past instances of Þeld-developed insect resistance to Bt that abundant refuges of non-Bt crops, recessive inheritance of resistance, and the use of two-toxin Bt crops separately from one-toxin Bt crops, have likely delayed the development of resistance to Bt crops. Similarly, Huang et al. (2011) found that in three instances of documented Þeld resistance, Spodoptera frugiperda (J.E. Smith) in Puerto Rico, Busseola fusca (Fuller) in South Africa, and Pectinophora gossypiella (Saunders) in India, that at least two factors were associated with resistance developmentÑnot using high-dose Bt cultivars and insufÞcient plantings of non-Bt refugia. Complicating the logistical and ecological aspects of effective IRM, is the optimum spatial placement and

0022-0493/12/1773Ð1780$04.00/0 䉷 2012 Entomological Society of America

1774

JOURNAL OF ECONOMIC ENTOMOLOGY

conÞguration of refuge relative to transgenic hybrids. Several conÞgurations of either blocks or seed mixtures have been implemented, which includes the use of different percentages of non-Bt refuge. However, each scenario provides both positive and negative aspects for IRM (Onstad et al. 2011). In addition, given the fact that corn pollination depends on wind-assisted pollen movement, it can be challenging to maintain “pure” non-Bt maize plants in the refuge, and speciÞcally non-Bt ears that have not been cross-pollinated by nearby Bt hybrids. The presence of kernels on non-Bt ears that express insecticidal proteins is of primary concern for ear feeding lepidopteran pests such as H. zea, and potentially to a lesser degree, O. nubilalis. However, even foliar feeding lepidopteran pests in refuge areas may be at risk of exposure during pollen shed as insecticidal proteins in the pollen will still be active and pollen will be present on leaves and in leaf axils. Regardless of the scenario, the potential exists to create less than adequate refugia. Reported distances of corn pollen dispersal are quite variable (Jemison and Vayda 2001) with Bateman (1947) reporting that pollen movement, as measured by cross pollination, declined by 99% within 12Ð15 m, and Jones and Newell (1948) calculated a half-distance (distance required for pollen amount to drop by half) of 47 m. Both estimates have been conÞrmed by several studies over the last decade, with cross pollination occurring outside of the pollen donor Þeld at distances ranging from 10 to 100 m (Luna et al. 2001, Byrne and Fromherz 2003, Ma et al. 2004, Goggi et al. 2006, Bannert and Stamp 2007, Weber et al. 2007, Bannert et al. 2008). Variability in cross pollination rates can be attributed to several factors, including pollen viability, which can be relatively short-lived at ⬇24 Ð 48 h (Purseglove 1972, Luna et al. 2001); wind speed and direction; and synchrony of pollen shed from the donor plant and silk receptivity of the receptor plant (Brookes et al. 2004). The combination of pollen movement, viability, and synchrony determine the potential for cross pollination of refuge corn. Research on corn pollen movement has focused on the use of modeling, small plot trials, or both to determine the likelihood of corn pollen moving from transgenic hybrids to nontransgenic hybrids, and distances corn pollen may travel from the source Þeld (Aylor 2002, Aylor et al. 2003, Chilcutt and Tabashnik 2004, Kuparinen et al. 2007, Bannert et al. 2008). In light of recent data from Burkness et al. (2011), conÞrming that non-Bt sweet corn kernels cross pollinated by Bt sweet corn pollen resulted in only 40% mortality of O. nubilalis larvae, and from Ives et al. (2011) suggesting that seed mixtures can hasten the development of resistance if larvae move between plants, further studies are needed to better understand the impact of cross pollination on IRM. This is particularly true for a seed mixture or Ôrefuge in the bagÕ (RIB) approach (Burkness et al. 2011). With the continued popularity of non-Bt strips as refugia for Bt corn (Andow et al. 2010), as well as an increasing emphasis on individual non-Bt plants adjacent to Bt plants (i.e., RIB; Onstad et al. 2011), there is a growing need to

Vol. 105, no. 5

better understand Bt pollen movement under Midwest conditions during the period of maximum pollen shed. We therefore conducted studies to assess pollen movement and cross pollination within large scale Þelds in 2009 and 2010. The objectives of this study were to assess the degree to which non-Bt corn hybrids in refuge areas are cross pollinated by nearby Bt corn hybrids; determine the location of Bt kernels on cross pollinated ears of non-Bt refuge corn; and quantify wind direction, during pollen shed, that may explain cross pollination patterns in non-Bt refuge corn. Materials and Methods In 2009 and 2010, at the Rosemount Research and Outreach Center, Rosemount, MN, Þelds of Bt corn (Cry1Ab) were planted in a block within 4.0 Ð 8.1-ha Þelds with 12Ð36 rows of a non-Bt hybrid planted adjacent to the Bt hybrid on all four sides. Commercial corn hybrids were used in both years with the Bt hybrid DKC 50 Ð 48 (Cry1Ab, event MON810) with a relative maturity of 100 d and the non-Bt hybrid DKC 48 Ð 40, a near isogenic non-Bt hybrid, with a relative maturity of 98 d. Measurements of pollen movement and cross pollination were made by collecting non-Bt ears just before harvest and testing the kernels for the presence of Cry1Ab protein. We assumed that non-Bt seed was not contaminated with Bt seed or by previous cross pollination by Bt pollen when the hybrid seed was produced. In addition, green leaf tissue was also tested on each plant to verify it was true to type. Therefore, presence of Cry1Ab was viewed as a conÞrmation that Bt pollen movement and cross pollination had occurred in the non-Bt refuge area. Data were recorded for presence and absence of Cry1Ab from a composite sample from each non-Bt ear collected, and in subsamples from the top, middle, and bottom third of each ear. Data were obtained for wind speed and direction during pollen shed. In 2009, the Bt hybrid DKC 50 Ð 48 was planted on 1 May and the non-Bt hybrid DKC 48 Ð 40 (DeKalb Corn Seed, Monsanto Co., St. Louis, MO) was planted 29 April. Both were planted with a six row planter on 0.76-m centers at a seeding rate of 79,040 SD/ha. Emergence of both Bt and non-Bt hybrids occurred 15 May and silking began 23 July. The Þeld was 9.63 ha total with 1.94 ha non-Bt (20%) and 7.69 ha Bt (80%) to reßect EPA regulations for refuge (USEPA 2001). The Þeld was arranged with a ring of non-Bt that consisted of 12 rows on the east and west sides, with rows planted north to south, and 36 rows planted on the headlands on the north and south, with rows planted east to west. Standard agronomic practices were followed with regard to fertilizer and herbicide applications. In 2010, both the Bt hybrid DKC 50 Ð 48 and non-Bt hybrid DKC 48 Ð 40 were planted in 4, 4.0-ha Þelds; two Þelds 26 April; and two Þelds 28 April. All Þelds were planted with a six-row planter on 0.76-m centers at a seeding rate of 79,040 SD/ha. Emergence of all Þelds occurred on 17 May and silking began on 10 July. Each 4.04-ha Þeld consisted of 1.46 non-Bt (36%) and 2.59

October 2012

BURKNESS AND HUTCHISON: BT CORN POLLEN DISPERSAL

ha Bt (64%). The Þeld was laid out with a ring of non-Bt that consisted of 24 rows around a block of Bt. Standard agronomic practices were followed with regard to fertilizer and herbicide applications. For both years, directional wind data for Rosemount, MN were obtained from the Minnesota State Climatology OfÞce and USDA-ARS Biometeorology Unit at the University of Minnesota. Hourly wind data were recorded at 3 m above the ground and covered an approximate 3 wk window, totaling 573 h including 1 wk before peak pollen shed and 2 wk after peak pollen shed began. The dates included 15 July-7 August and 2 July-25 July for 2009 and 2010, respectively. In 2009 and 2010, plants were allowed to mature as normal and samples were collected across a transect of the non-Bt refuge starting with the Þrst row of Bt corn and working out to the edge of the Þeld. In 2009, samples were collected along three transects on each side of the Þeld for a total of 12 transects. Transects on each side of the Þeld were 15.24 m apart. Samples on the east and west sides of the Þeld consisted of the Bt row and 12 non-Bt rows for 13 samples per transect in total. On the north and south sides of the Þeld, a Bt plant was sampled followed by the Þrst 12 non-Bt rows and then additional samples were taken at row 16, 24, and 36 (Þeld edge) for 16 samples per transect in total. At each sample location, the primary ear and one green leaf (leaf tissue used to verify the plant was either Bt or non-Bt, i.e., not an off-type) from the plant were collected and placed in a zip lock bag. Samples were collected 5 October and 13, returned to the lab, and kept in refrigeration at ⫺2⬚C until testing was done. A portion of the 5 October samples were tested on 5 October and the remainder were tested on 15 October. Tissue samples were tested 5, 15, and 17 October, using Envirologix (Portland, ME) strip test kits (limit of detection ⫽ 5Ð10 ppb) for Cry1Ab toxin, both the leaf and kernel tissue were tested for every sample collected. The presence or absence of the toxin were recorded for transect and row number for each tissue type. Tissue testing procedures from Envirologix were followed for all tissue samples. Kernels were collected from each ear as a composite sample taking three kernels from the top 6.35 cm of the ear (often described as the tip of the ear), three kernels from the middle 6.35 cm of the ear, and three kernels from the bottom 6.35 cm of the ear (often described as the butt of the ear). The kernels were all placed in a plastic bag, crushed using a hammer, and then mixed together by hand (shaking the bag and squeezing the pulverized kernel tissue together), and a composite sample was taken from the bag and placed in a microcentrifuge tube for testing. For the north transects, in addition to the composite samples, three kernels were tested separately from the top, middle, and bottom sections of each ear. Kernel samples from the three ear locations were processed for rows 1, 2, 3, 4, 6, 8, and 10 of each transect on the north side of the Þeld. In 2010, similar sampling procedures were followed and ear and leaf samples were collected in a transect starting at the Þrst Bt row and continuing out toward

1775

the Þeld edge. Sampled rows included the Þrst Bt row, the next 12 non-Bt rows, and row 16 and 24 (Þeld edge), for 15 samples in total on each side of the Þeld. In 2010, only one transect was sampled on each side of the Þeld and the same procedures were followed for all four Þelds. All tissue samples were collected from all Þelds on 22 September placed in plastic bags, returned to the lab, and kept in refrigeration at ⫺2⬚C until testing was done. All tissue samples were tested for Cry1Ab by using the same procedures as in 2009, on 23, 24, and 25 September. Kernel samples for the top, middle, and bottom ear locations were taken as in 2009 from the north side of each Þeld from rows 1, 2, 3, 4, 6, 8, and 10. Wind speed and direction data were based on mean hourly values and were divided into eight categories deÞned by the area between the cardinal and intercardinal directions on a compass (e.g., north to northeast, northeast to east). For each year, data for the presence or absence of Cry1Ab toxin in kernels of each ear were analyzed using a one-way analysis of variance (ANOVA) and a protected least signiÞcant difference (LSD) test (LSD, P ⫽ 0.05) for mean separation (SAS Institute 2008). Data for percentage of positive kernel samples were converted to a proportion and arcsine transformed for analysis, untransformed means are presented. Results and Discussion In 2009 and 2010, all leaf tissue samples from the Bt rows of corn tested positive and non-Bt leaf tissue tested negative for the presence of Cry1Ab toxin (data not shown), conÞrming that all seed were true to type. One important source of adventitious pollen in a refuge area can occur from seed contamination either through planting errors or during seed production and packaging. In refugia for transgenic cotton, Heuberger et al. (2010) found that the introduction of Bt toxins through planting errors or the presence of transgenic seed in nontransgenic seed bags were more prevalent in refugia than pollen mediated gene ßow from outside the Þeld. Furthermore, Heuberger et al. (2008) found, based on simulation modeling, that refugia contamination observed in the Þeld had negligible effects on resistance evolution in pink bollworm, unless the contamination allowed for a selective advantage to individuals with heterozygous resistance. During both years, Cry1Ab protein was found in kernel samples taken from all sides of the refuge-Bt block (Table 1). The highest percentages of positive samples were found within the Þrst four rows (3 m) and generally declined as the distance from Bt corn increased; however, the percentages of cross pollinated ears among rows were variable. With levels of cross pollination reaching as high as 75% of the ears in the Þrst four rows of refuge adjacent to the Bt corn (Table 1), it is likely that larvae feeding in the ears of these rows would be exposed to Bt toxins at some level. This phenomenon may also provide a direct mechanism for the “halo effect,” at a local Þeld level, where, at the interface of the Bt and non-Bt plantings, in-

1776


Vol. 105, no. 5

Table 1. Percentage of ears in cardinal directions with kernels testing positive for cross pollination with Bt pollen within a non-Bt refuge strip surrounding Bt field corn and the max distance within the non-Bt refuge area that a positive kernel sample was found relative to the Bt field corn, Rosemount, MN 2009 –2010

Refuge locationa

Rows 1Ð 4 (0.8 Ð3.0 m)b

Rows 5Ð 8 (3.8 Ð 6.1 m)

Rows 9 Ð12 (6.9 Ð9.1 m)

x percentage of positive samples



75.0 ⫾ 14.4a 58.3 ⫾ 8.3ab 16.7 ⫾ 8.3b 25.0 ⫾ 14.4b 0.0464

75.0 ⫾ 0.0a 50.0 ⫾ 14.4ab 33.3 ⫾ 8.3bc 8.3 ⫾ 8.3c 0.0192

25.0 ⫾ 0.0a 16.7 ⫾ 8.3a 8.3 ⫾ 8.3a 16.7 ⫾ 8.3a 0.5720

8.4 ⫾ 0.4a 6.9 ⫾ 0.8a 6.9 ⫾ 0.8a 5.6 ⫾ 2.4a 0.5609

37.5 ⫾ 7.2b 75.0 ⫾ 10.2a 43.8 ⫾ 6.3b 6.3 ⫾ 6.3b 0.0077

18.8 ⫾ 6.3a 31.3 ⫾ 12.0a 6.3 ⫾ 6.3a 6.3 ⫾ 6.3a 0.1507

31.3 ⫾ 6.3a 50.0 ⫾ 10.2a 0.0 ⫾ 0.0b 6.3 ⫾ 6.3b 0.0033

8.4 ⫾ 0.3a 8.6 ⫾ 0.6a 1.9 ⫾ 0.7b 2.7 ⫾ 1.7b 0.0003

2009 East North West South P 2010 East North West South P

x max distance (m) of positive samples from Bt within Þrst 12 rows of non-Bt

Means ⫾ SEM within columns for each year followed by the same letter are not signiÞcantly different (P ⬎ 0.05); Protected Least signiÞcant difference Test (LSD P ⫽ 0.05). Mean percentages of positive samples were converted to proportions and transformed using the arcsine transformation to obtain mean separations; untransformed means are presented. All tissue testing was done using Envirologix test strips for Cry1Ab (LOD ⫽ 5Ð10 ppb). a Refuge location refers to the placement of non-Bt Þeld corn relative to a block of Bt Þeld corn. b One ear was taken from each row on a transect across the 12 row non-Bt refuge area (i.e., 12 samples total). The distances to positive Bt kernel samples were measured from the Þrst row of Bt Þeld corn immediately adjacent to the non-Bt Þeld corn refuge area.

creased larval mortality may occur because of non-Bt ears being pollinated by Bt pollen (Alstad and Andow 1995, Burkness et al. 2011). In both years, each of the Þrst four rows of non-Bt refuge maize, in downwind directions (north and east) had ⱖ25% of the ears containing kernels that expressed Cry1Ab (Table 2). Several studies have found relatively high levels of cross pollinated kernels in the Þrst row (⬇0.8 m from Bt corn) of non-Bt corn planted adjacent to Bt corn, with levels ranging from 3 to 82% (Byrne and Fromherz 2003, Ma et al. 2004, Bannert et al. 2008). Despite a trend for more consistent cross pollination in the dominant downwind directions, when viewed by individual row, Table 2 demonstrates the highly variable

nature of pollen movement in corn Þelds during our 2-yr study. In addition to ear feeding, it is possible that Bt pollen itself could provide exposure to larvae feeding on vegetative tissues shortly after egg hatch (Pilcher et al. 2001). Levels of Bt toxins present in pollen vary by event (Table 3) and, for example, have been measured at ⬍0.08 ␮g/g of pollen for Cry1Ab in MON810 to 21.9 ␮g/g of pollen for Cry1 F in TC1507. However, levels of expression in the Þeld may vary based on growing conditions and/or hybrid (Li et al. 2008). Li et al. (2008) found that after collecting pollen from the Þeld and air drying for 24 h for an assay with Chrysoperla carnea (Stephens), that Bt concentration was not sig-

Table 2. Percentage of ears in each non-Bt transect (12 rows) in each cardinal direction with kernels testing positive for cross pollination with Bt pollen within a non-Bt refuge block surrounding Bt field corn, Rosemount, MN 2009 –2010 x percentage of positive samples Row no.a 1 2 3 4 5 6 7 8 9 10 11 12 P

2009

2010

East

North

West

South

East

North

West

South

67 ⫾ 33ab 67 ⫾ 33ab 67 ⫾ 33ab 100 ⫾ 0a 100 ⫾ 0a 100 ⫾ 0a 100 ⫾ 0a 0 ⫾ 0b 0 ⫾ 0b 33 ⫾ 33ab 33 ⫾ 33ab 33 ⫾ 33ab 0.0337

100 ⫾ 0a 33 ⫾ 33a 33 ⫾ 33a 67 ⫾ 33a 67 ⫾ 33a 33 ⫾ 33a 67 ⫾ 33a 33 ⫾ 33a 0 ⫾ 0a 67 ⫾ 33a 0 ⫾ 0a 0 ⫾ 0a 0.8313

0 ⫾ 0a 33 ⫾ 33a 33 ⫾ 33a 0 ⫾ 0a 33 ⫾ 33a 0 ⫾ 0a 33 ⫾ 33a 67 ⫾ 33a 0 ⫾ 0a 0 ⫾ 0a 33 ⫾ 33a 0 ⫾ 0a 0.6128

33 ⫾ 33a 0 ⫾ 0a 33 ⫾ 33a 33 ⫾ 33a 0 ⫾ 0a 33 ⫾ 33a 0 ⫾ 0a 0 ⫾ 0a 0 ⫾ 0a 33 ⫾ 33a 33 ⫾ 33a 0 ⫾ 0a 0.2351

50 ⫾ 29a 25 ⫾ 25a 25 ⫾ 25a 50 ⫾ 29a 25 ⫾ 25a 25 ⫾ 25a 25 ⫾ 25a 0 ⫾ 0a 25 ⫾ 25a 25 ⫾ 25a 50 ⫾ 29a 25 ⫾ 25a 0.9790

100 ⫾ 0a 100 ⫾ 0a 75 ⫾ 25a 25 ⫾ 25a 50 ⫾ 29a 0 ⫾ 0a 50 ⫾ 29a 25 ⫾ 25a 50 ⫾ 29a 25 ⫾ 25a 50 ⫾ 29a 75 ⫾ 25a 0.1203

100 ⫾ 0a 75 ⫾ 25a 0 ⫾ 0b 0 ⫾ 0b 25 ⫾ 25b 0 ⫾ 0b 0 ⫾ 0b 0 ⫾ 0b 0 ⫾ 0b 0 ⫾ 0b 0 ⫾ 0b 0 ⫾ 0b 0.0001

0 ⫾ 0a 25 ⫾ 25a 0 ⫾ 0a 0 ⫾ 0a 25 ⫾ 25a 0 ⫾ 0a 0 ⫾ 0a 0 ⫾ 0a 25 ⫾ 25a 0 ⫾ 0a 0 ⫾ 0a 0 ⫾ 0a 0.6077

Means ⫾ SEM within columns for each year followed by the same letter are not signiÞcantly different (P ⬎ 0.05); Protected Least signiÞcant difference Test (LSD P ⫽ 0.05). Mean percentages of positive samples were converted to proportions and transformed using the arcsine transformation to obtain mean separations; untransformed means are presented. All tissue testing was done using Envirologix test strips for Cry1Ab (LOD ⫽ 5Ð10 ppb). a Row number 1 was the Þrst row of non-Bt corn adjacent to the Bt corn.

October 2012


Table 3. Mean expression levels of insecticidal proteins in field corn pollen

Event

Insecticidal protein

Mean expression level in pollen (␮g/g dry weight)

TC1507a DAS-06275-8a Bt11b MON 810c MON 89034d MON 89034d MIR162b

poCry1F moCry1F Cry1Ab Cry1Ab Cry1A.105 Cry2Ab2 Vip3Aa20

21.90 3.67 0.08 ⬍0.08 12.00 0.64 97.20

a

Data taken from USEPA 2005. Data taken from USEPA 2009. Data taken from USEPA 2010a. d Data taken from USEPA 2010b. b c

niÞcantly reduced in the pollen after 3 d of the feeding assay when pollen was held at 22⬚C and 75% RH; these results suggest that Bt in the pollen remains relatively stable over time. Pilcher et al. (2001) examined the impact of Bt pollen on O. nubilalis in non-Bt corn adjacent to Bt corn; their results suggested that Bt pollen did not negatively inßuence O. nubilalis populations. However, data were not collected to quantify the actual amount of Bt pollen the larvae were exposed to or what concentration of Bt was in the pollen. These issues warrant further investigation to determine if Bt pollen results in enhanced larval mortality and/or exposure (e.g., levels of Bt toxins and longevity of toxins in pollen after shedding) in refuge areas, especially those areas planted within the Þrst four rows adjacent to Bt corn. Wind direction data indicated that the minimum amount of wind from any given direction occurred for at least 26.9 h (4.7%) of the 573 h in the sampling period (Fig. 1). The dominant wind direction was from the west-northwest at ⬇297 and 293⬚ in 2009 and 2010, respectively. These wind directions corresponded with 151.8 (26.5%) and 111.2 (19.4%) h of the 573 h in the sampling period (Fig. 1). However, wind velocities from the west-northwest were not the highest recorded during pollen shed (Fig. 1). Cross pollination was most prevalent in the non-Bt corn that was planted to the north and east of the Bt block (Table 1) and coincided with the wind directions with the highest percentages over the sampling period. Cross pollination of ears within the non-Bt corn was variable (Tables 1 and 2) but tended to decline the further away the non-Bt was from the Bt source. The movement of pollen depends on the wind direction, wind speed, and whether the wind is moving horizontally or vertically. Ma et al. (2004) found that the large variation in pollen dispersal is likely because of vertical wind moving pollen upward rather than horizontal wind movement of pollen. All of these factors are typically considered when looking at the coexistence of transgenic and nontransgenic crops, including Bt corn near organic crops or wild relatives (e.g., Brookes et al. 2004, Chilcutt and Tabashnik 2004). However, IRM has not typically been examined with regard to these issues and even

1777

when IRM is considered it has been in the context of block refuges (Pilcher et al. 2001, Burkness et al. 2011). When considering IRM in the context of seed mixtures (i.e., “refuge in the bag” or RIB), research results suggest that the likelihood of cross pollination of non-Bt refuge plants is extremely high. Most studies suggest a buffer of at least 20m to reduce cross pollination between Bt and non-Bt corn (e.g., Luna et al. 2001, Ma et al. 2004, Bannert and Stamp 2007). At current seeding rates for corn of 69,160 Ð 86,450 SD/ha in the United States, and seed mixtures with 5% non-Bt seed, a non-Bt plant would potentially be between 0.15 and 0.19 m from the nearest Bt plant. In addition to the likelihood of cross pollination occurring, the location of cross pollinated kernels on the non-Bt ears could also be of concern. Although cross pollinated kernels tended to be more concentrated on the tip of the ear (Table 4), differences among the three sections of the ears examined were not signiÞcantly different in 2009 (P ⬎ 0.61) or 2010 (P ⬎ 0.26). The location of cross pollinated kernels within an ear can depend on the synchrony between the donor plant shedding pollen and the receptor plant having viable silk emerged from the husk. Bannert et al. (2008) found that temporal isolation of 5Ð7 d could decrease cross pollination by 80% versus full synchronization. Brookes et al. (2004) found that a 7-d planting difference resulted in ⬇60% decrease in cross pollination of nontransgenic receptor corn, but even these maturity differences are not sufÞcient to avoid the risk of Bt pollen contamination in non-Bt refugia. Manipulations of reproductive synchrony can also be achieved through the selection of different relative maturities in the Bt and non-Bt corn hybrids, and could be considered as a method to minimize cross pollination. In our study, the Bt hybrid relative maturity was 100 d and the non-Bt 98 d. This small difference in maturity may explain the higher proportion of cross pollinated kernels in the top portion of the ear (Table 4). For example, assuming that silk emerges from the bottom of the ear Þrst and proceeds sequentially up the ear and concludes with silk from the kernels at the tip of the ear (Abendroth et al. 2011), if the non-Bt hybrid relative maturity is longer than the Bt hybrid, then Bt development and pollen shed should occur earlier than the non-Bt silking. Subsequently, silks that emerge Þrst from the non-Bt hybrid would coincide with pollen from the later portion of pollen shed from the Bt hybrid and move a majority of cross pollinated kernels toward the bottom of the ear. Conversely, if the non-Bt hybrid relative maturity is shorter than the Bt hybrid, the termination of non-Bt silking would likely be in synchrony with the beginning of Bt pollen shed and could lead to more cross pollinated kernels on the tip of the ear. Therefore, the synchrony of Bt and non-Bt hybrids is not only important with respect to cross pollination in general but may have important implications for IRM, depending on what portion of the Bt contaminated ear typically incurs the most feeding damage by one or more lepidopteran pests of concern.

1778


Vol. 105, no. 5

Fig. 1. Mean wind speed, direction and duration for 2009 and 2010 during a three wk period surrounding pollen shed, 15 JulyÐ7 August 2009 and 2 JulyÐ25 July 2010, Rosemount, Minn.. This time frame began ⬇1 wk before pollen shed and continued until ⬇2 wk after pollen shed began. For both years, during this time interval, the total hours of wind data recorded was 573 h. Wind directions were divided into eight categories deÞned by the area between the cardinal and intercardinal directions on a compass (e.g., north to northeast, northeast to east). Arrows indicate the mean wind direction for each category and the mean wind speed in meters per second (m/s) and duration in hours (hrs) for the mean wind direction is given for each category. Numbers in parentheses indicate the percentage of the total hours over the 3-wk period.

In summary, pollen movement becomes more variable with increasing distance from the pollen source. Within the Þrst 2 m from the pollen source, movement is much more reliable and in fact is a key assumption, and historical approach used for creating hybrid corn seed. Corn seed production typically relies on a ratio

of one pollen donor row to four adjacent rows of pollen receptor plants (Brookes et al. 2004). This conÞguration routinely relies on pollen traveling at least two rows to either side of the donor row, or ⬇1.5 m in either direction. Therefore, based on the nature of pollen dispersal and cross pollination in corn, there is

October 2012


Table 4. Location of cross pollinated kernels within non-Bt ears, Rosemount, MN 2009 –2010 Location on eara

% positive samples for Cry1Ab

2009 Top Middle Bottom P 2010 Top Middle Bottom P

29.0 ⫾ 0.0a 24.0 ⫾ 12.7a 19.0 ⫾ 5.0a 0.6195 32.3 ⫾ 6.9a 17.8 ⫾ 3.8a 21.5 ⫾ 4.3a 0.2638

Means ⫾ SEM within columns for each year followed by the same letter are not signiÞcantly different (P ⬎ 0.05); Protected Least signiÞcant difference Test (LSD P ⫽ 0.05). Mean percentages of positive samples were converted to proportions and transformed using the arcsine transformation to obtain mean separations; untransformed means are presented. All tissue testing was done using Envirologix test strips for Cry1Ab (LOD ⫽ 5Ð10 ppb). a The ear was separated into 3 categories, the top, middle, and bottom. Each section of the ear was deÞned as: top: 6.35 cm from the tip of the ear, middle: 6.35 cm in the center of the ear, bottom: 6.35 cm from the butt of the ear. Samples consisted of three kernels selected arbitrarily from the top, middle, and bottom one-third of each ear. Seven ears were sampled in each of three replicates. Samples were collected from the non-Bt area on the North side of each Þeld.

a high probability that non-Bt ears of corn will be cross pollinated by Bt pollen under an RIB scenario. Although a detailed IRM and population model for O. nubilalis recently was used to assess the risk of Bt resistance within the RIB context, several assumptions were necessarily included to complete the analysis (Kang et al. 2012). Among these, the mean percentage of non-Bt ears cross pollinated was unknown, and the mosaic of Bt kernels, and Bt toxin levels per kernel were unknown. Our current study suggests these additional factors should be studied in more detail. SpeciÞcally, with an IRM goal of minimizing the risk of resistance evolution among ear-feeding lepidopteran pests in Bt corn, additional data are needed to quantify the Bt concentration of cross pollinated kernels and the associated lepidopteran pest survival, and to fully measure the impact of Bt pollen on lepidopteran pests for sustainable IRM of Bt corn. Acknowledgments We thank Suzanne Wold-Burkness, University of Minnesota, for technical assistance. We also thank Rick Hellmich (USDA-ARS) for his review of an early draft of this manuscript and Greg Spoden and Pete Boulay from the Minnesota State Climatology OfÞce and Bill Breiter from the USDAARS Biometeorology Unit at the University of Minnesota for providing access to state collected wind data. This research was supported by the University of Minnesota Agricultural Experiment Station.

References Cited Abendroth, L. J., R. W. Elmore, M. J. Boyer, and S. K. Marlay. 2011. Corn growth and development. PMR 1009. Iowa State University Extension, Ames, IA.

1779

Alstad, D. N., and D. A. Andow. 1995. Managing the evolution of insect resistance to transgenic plants. Science 268: 1894 Ð1896. Andow, D. A., S. L. Farrell, and Y. Hu. 2010. Planting patterns of in-Þeld refuges observed for Bt maize in Minnesota. J. Econ. Entomol. 103: 1394 Ð1399. Aylor, D. E. 2002. Settling speed of corn (Zea mays) pollen. J. Aerosol Sci. 33: 1601Ð1607. Aylor, D., N. Schultes, and E. Shields. 2003. An aerobiological framework for assessing cross-pollination in maize. Agric. For. Meteorol. 119: 111Ð129. Bannert, M., and P. Stamp. 2007. Cross-pollination of maize at long distance. Eur. J. Agron. 27: 44 Ð51. Bannert, M., A. Vogler, and P. Stamp. 2008. Short-distance cross-pollination of maize in a small-Þeld landscape as monitored by grain color markers. Eur. J. Agron. 29: 29 Ð32. Bateman, A. J. 1947. Contamination of seed crops Ñ II. Wind pollination. Heredity 1: 235Ð246. Brookes, G., P. Barfoot, E. Mele´, J. Messeguer, F. Be´ne´trix, D. Bloc, X. Foueillassar, A. Fabie´, and C. Poeydomenge. 2004. Genetically modiÞed maize: pollen movement and crop coexistence. PG Economics Ltd., Dorchester, United Kingdom. (http://www.pgeconomics.co.uk). Burkness, E. C., P. K. O’Rourke, and W. D. Hutchison. 2011. Cross-pollination of nontransgenic corn ears with transgenic Bt corn: efÞcacy against Lepidopteran pests and implications for resistance management. J. Econ. Entomol. 104: 1476 Ð1479. Byrne, P. F., and S. Fromherz. 2003. Can GM and non-GM crops coexist? Setting a precedent in Boulder County, Colorado, USA. J. Food Agric. Environ. 1: 258 Ð261. Chilcutt, C. F., and B. E. Tabashnik. 2004. Contamination of refuges by Bacillus thuringiensis toxin genes from transgenic maize. Proc. Natl. Acad. Sci. U.S.A. 101: 7526 Ð7529. Goggi, A. S., P. Caragea, H. Lopez-Sanchez, M. Westgate, R. Arritt, and C. Clark. 2006. Statistical analysis of outcrossing between adjacent maize grain production Þelds. Field Crops Res. 99: 147Ð157. Gould, F. 1998. Sustainability of transgenic insecticidal cultivars: integrating pest genetics and ecology. Annu. Rev. Entomol. 43: 701Ð726. Heuberger, S., C. Ellers-Kirk, C. Yafuso, A. J. Gassmann, B. E. Tabashnik, T. J. Dennehy, and Y. Carrie`re. 2008. Effects of refuge contamination by transgenes on Bt resistance in pink bollworm (Lepidoptera: Gelechiidae). J. Econ. Entomol. 101: 504 Ð514. Heuberger, S., C. Ellers-Kirk, B. E. Tabashnik, and Y. Carrie`re. 2010. Pollen- and seed-mediated transgene ßow in commercial cotton seed production Þelds. PLoS ONE 5: e14128. (doi:10.1371/journal.pone.0014128). Huang, F., D. A. Andow, and L. L. Buschman. 2011. Success of the high-dose/refuge resistance management strategy after 15 years of Bt crop use in North America. Entomol. Exp. Appl. 140: 1Ð16. Hutchison, W. D., E. C. Burkness, P. D. Mitchell, R. D. Moon, T. W. Leslie, S. J. Fleischer, M. Abrahamson, K. L. Hamilton, K. L. Steffey, M. E. Gray, et al. 2010. Areawide suppression of European corn borer with Bt maize reaps savings to non-Bt maize growers. Science 330: 222Ð225. Ives, A. R., P. R. Glaum, N. L. Ziebarth, and D. A. Andow. 2011. The evolution of resistance to two-toxin pyramid transgenic crops. Ecol. Appl. 21: 503Ð515. Jemison, J. M., and M. E. Vayda. 2001. Cross pollination from genetically engineered corn: wind transport and seed source. AgBioForum 4: 87Ð92. Jones, M. D., and L. C. Newell. 1948. Longevity of pollen and stigmas of grasses: buffalograss, Buchloe dactyloedees

1780


(NUTT) Engelm, and corn (Zea mays, L.). Agron. J. 40: 195Ð204. Kang, J., D. W. Onstad, R. L. Hellmich, S. E. Moser, W. D. Hutchison, and J. R. Prasifka. 2012. Modeling the impact of cross-pollination and low toxin expression in corn kernels on adaptation of European corn borer (Lepidoptera: Crambidae) to transgenic insecticidal corn. Environ. Entomol. 41: 200 Ð211. Kuparinen, A., F. Schurr, O. Tackenberg, and R. B. O’Hara. 2007. Air-mediated pollen ßow from genetically modiÞed to conventional crops. Ecol. Appl. 17: 431Ð 440. Li, Y., M. Meissle, and J. Romeis. 2008. Consumption of Bt maize pollen expressing Cry1Ab or Cry3Bb1 does not harm adult green lacewings, Chrysoperla carnea (Neuroptera: Chrysopidae). PLoS ONE 3: e2909. (doi:10.1371/ journal.pone.0002909). Luna, V. S., M. J. Figueroa, M. B. Baltazar, L. R. Gomez, R. Townsend, and J. B. Schoper. 2001. Maize pollen longevity and distance isolation requirements for effective pollen control. Crop Sci. 41: 1551Ð1557. Ma, B. L., K. D. Subedi, and L. M. Reid. 2004. Extent of cross-fertilization in maize by pollen from neighbouring transgenic hybrids. Crop Sci. 44: 1273Ð1282. Onstad, D. W. 2008. Major issues in insect resistance management, pp. 1Ð16. In D. Onstad (ed.), Insect resistance management: biology, economics and prediction. Academic, London, United Kingdom. Onstad, D. W., P. D. Mitchell, T. M. Hurley, J. G. Lundgren, R. P. Porter, C. H. Krupke, J. L. Spencer, C. D. DiFonzo, T. S. Baute, R. L. Hellmich, et al. 2011. Seeds of change: corn seed mixtures for resistance management and IPM. J. Econ. Entomol. 104: 343Ð352. Pilcher, C. D., M. E. Rice, R. D. Higgins, and R. Bowling. 2001. Pollen drift from Bacillus thuringiensis corn: efÞcacy against European corn borer (Lepidoptera: Crambidae) in adjacent rows of non-Bt corn. Environ. Entomol. 30: 409 Ð 414. Purseglove, J. W. 1972. Tropical crops. Chapter 1: Monocotyledons. Longman Group, London, United Kingdom. SAS Institute. 2008. SAS/STAT 9.2 userÕs guide. SAS Institute, Cary, NC.

Vol. 105, no. 5

Tabashnik, B. E., A. J. Gassmann, D. W. Crowder, and Y. Carrie`re. 2008. Insect resistance to Bt crops: evidence versus theory. Nature Biotechnol. 26: 199 Ð202. Tabashnik, B. E., J.B.J. Van Rensburg, and Y. Carrie`re. 2009. Field-evolved insect resistance to Bt crops: deÞnition, theory, and data. J. Econ. Entomol. 102: 2011Ð2025. [USEPA] U.S. Environmental Protection Agency. 2001. Biopesticides registration action document: Bacillus thuringiensis plant-incorporated protectants. U.S. Environmental Protection Agency, Washington, DC. [USEPA] U.S. Environmental Protection Agency. 2005. Biopesticides registration action document: Bacillus thuringiensis Cry1F corn updated August 2005. U.S. Environmental Protection Agency, Washington, DC. [USEPA] U.S. Environmental Protection Agency. 2009. Biopesticides registration action document: Bacillus thuringiensis Vip3Aa20 insecticidal protein and the genetic material necessary for its production (via elements of vector pNOV1300) in event MIR162 maize (OECD Unique IdentiÞer: SYN-IR162Ð 4). U.S. Environmental Protection Agency, Washington, DC. [USEPA] U.S. Environmental Protection Agency. 2010a. Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary for its production (Vector PVZMIR13L) in MON 863 corn (OECD unique identiÞer: MON-ØØ863Ð5); Bacillus thuringiensis Cry3Bb1 protein and the genetic material necessary for its production (Vector PV-ZMIR39) in MON 88017 corn (OECD unique identiÞer: MON-88Ø17Ð3). U.S. Environmental Protection Agency, Washington, DC. [USEPA] U.S. Environmental Protection Agency. 2010b. Biopesticides registration action document: Bacillus thuringiensis Cry1A. 105 and Cry2Ab2 insecticidal proteins and the genetic material necessary for their production in corn. U.S. Environmental Protection Agency, Washington, DC. Weber, W. E., T. Bringezu, I. Broer, J. Eder, and F. Holz. 2007. Coexistence between GM and non-GM maize crops - Tested in 2004 at the Þeld scale level. J. Agron. Crop Sci. 193: 79 Ð92. Received 26 March 2012; accepted 25 June 2012.