When field tests of transgenic plants are precluded by practical containment concerns, manipulative experiments can detect potential consequences of crop-wild gene flow. Using topical sprays of bacterial
Commercialized transgenic, insect resistant (IR) crops currently grown in the United States have virtually no wild relatives near production sites, thus ensuring that novel crop traits are unlikely to move into local wild gene pools. However, an assessment of the consequences of gene flow will be necessary in future deregulation decisions because most of the major and minor crops in the world either exist in the wild themselves or hybridize with wild relatives somewhere in their range [
Identifying and quantifying environmental risks associated with gene flow from transgenic crops is subject to methodological tradeoffs because of containment restrictions, especially for plant fitness effects, which require pollen production. Field tests with pollen-producing transgenic plants must be contained physically in cages or greenhouses or established at sites where wild relatives do not occur. Conditions in regions that have no natural populations of wild relatives may differ from areas of concern for hybrid formation in ways that affect the results, and therefore the relevance, of such field tests. An alternative method, used in this study, is to conduct tests
We present herbivore protection experiments using Bt sprays on two very different species in the Brassicaceae, wild radish (
(a) Topographic map of California central coast, primarily Santa Cruz County, showing locations of four sites (Elkhorn Slough site was ~50 km south) used in year one (arrows) and of 90 1 m × 1 m plots established as 30 triplets of disked, field margin, and natural vegetation habitat plots in year two (GPS locations shown as white circles with black borders). (b) Example of disked field plot in year two, with T.R.F. Roubison spraying individual wild radish and wild mustard plants with Bt and denatured Bt amongst other forbs and grasses that emerged from soil seedbanks with the winter rains. Not shown are the associated field margin plot, which was within 5 m of the field, and the natural vegetation plot in uncultivated lands as shown in the distance.
Our experiments on wild radish and wild mustard were designed to detect plant population responses in complex habitats and determine (1) if protection from Bt susceptible herbivore damage would result in increased survivorship, longevity, and/or fecundity compared to plants incurring damage within the natural range occurring on local plants, (2) if tolerance to herbivory varies between wild plant species or among habitats that differ categorically in terms of plant resources and vegetational background (disked agricultural soil versus agricultural field margins versus natural vegetation), and (3) if seed limitation is a likely regulatory mechanism for either species in different habitats. The advantage of simulating plant protection conferred by transgenic traits in wild plants is the ability to include multispecies interactions that can alter fitness effects in ways that differ fundamentally from outcomes predicted by experiments with isolated plants, caged or greenhouse trials, or with artificial herbivory (see [
Drawbacks include the adequate matching of expression levels, persistence rates, and target insects with applications of insecticidal simulants, and any fundamental differences between actual transgenic hybrids and wild-type experimental plants. We compare the results of this simulation technique to our previous studies and to studies conducted with transgenic plants by other researchers to gauge the usefulness of a field simulation method in informing risk assessments and regulatory decision making more generally.
We compared individual fitness parameters of wild radish (Brassicaceae:
Wild radish and mustard host a variety of herbivores, including cabbage aphids (
We added a single 1st or 2nd instar
Experiments were conducted in the central coast region of California (36.974°N, −122.029°W), where 780 mm annual precipitation in this Mediterranean climate falls between October and May. In 2003-2004 (year one), sixty 1 m × 1 m field plots were established at each of five sites (Figure
Treatment design was hierarchical in year one, with herbivory treatments (two levels) nested in habitat type (three levels—except at two locations where seedlings were scarce in disked plots), nested in location (five levels). Ten naturally occurring wild radish seedlings were selected in each plot (2,600 seedlings in total) and marked initially with numbered stakes and then with a numbered spiral binding ring around the plant stem. Paired plots were randomly designated to receive either weekly Bt sprays (protected plants) or denatured Bt sprays and larval additions (exposed plants). A single early instar
In year two, experimental plots (one set of three habitat types at each of 30 sites, Figures
For an increase in seed output to result in increased spread of the population, any production of additional seed has to result in additional plants surviving to reproduce in the habitat. To assess the potential fitness advantage of an increase in seed output by wild radish and wild mustard, we tested for seed limitation in a microcosm experiment with two of the habitat types (disked field and natural vegetation). Before the first rains (August/September 2006), large soil cores (25.4 cm diameter), with the existing dry vegetation and seed bank completely intact, were transferred to pots with care taken to minimize disturbance to the plant cover or soil profile. Two pairs of these large soil cores were taken from each of the 10 sites along the central coast with recently tilled soil, with one of each pair of the soil cores taken from a disked farm field before crops were sown and one taken from nearby areas (same soil type) with natural vegetation intact. To test for any impact of increased seed yield, eight wild radish pods (lightly crushed) yielding 4–10 seeds/pod and 15 seeds of wild mustard were added to one of each pair of soil cores in three-gallon pots representing either disked agricultural fields or grassland. The number of seeds added approximated one-half the increase in number of seeds produced by a single wild radish plant (36.5 seeds) or wild mustard plant (26.5 seeds) when that mother plant has been protected from herbivory (average of years one and two from our field results). The resulting potted microcosms were used to ensure that runoff from heavy rains would not cause unaccountable seed losses. Pots were transported to the rooftop greenhouse at UCSC where they could be watered biweekly as needed and exposed to coastal weather for 10 weeks until wild mustard in the pots produced siliques and wild radish plants produced pods. A small scale field experiment at two of our sites (UCSC CASFS and the Homeless Garden Project) was used to further test these seed additions
All analyses used PC-SAS v. 9.1 (SAS Institute 1990). In year one, both the average herbivore damage at 12 weeks after the first rain and the lifetime seed output for wild radish seedlings (rank transformed) were compared with respect to (1) herbivory treatment (protected versus exposed), (2) habitat type (disked field, field margin, and natural vegetation), and (3) interactions between herbivory treatment and habitat type. We used a General Linear Models (GLM) nested ANOVA to test for any significant effects, designating the error term as the type III mean square value for plot nested in the interaction term for site by treatment by habitat type. In year two, we used a logistic regression to compare categorical estimates of herbivory after eight weeks (0%, 2.5%, 25%, 75%, 95%), plant mortality (dead or alive), and lifetime production of siliques/pods (reproductive or not). Independent variables were plant species (wild radish versus wild mustard), herbivory treatment (protected versus exposed), and habitat type (disked field, field margin, and natural vegetation). Additionally, we used a GLM repeated measures ANOVA to test for changes in herbivory with time and to test for seasonal effects of habitat type, plant species, and interactions among these factors. Mean seed output per plant per plot, which could not be transformed to meet the assumptions of normality, was compared using a GLM ANOVA on ranks. Similarly, percent cover of bare soil after community development for 12 weeks was compared among subplots with ANOVA on ranks. All subplots for wild mustard and wild radish, and both herbivory treatments, were pooled to test for differences among habitat types because there were no significant differences in bare soil cover due to plant species or herbivore treatment.
The number of reproductive wild radish and wild mustard plants in the potted seed addition experiment was analyzed separately by plant species, with
In year one, 12 weeks after the first rains that caused seeds to germinate in experimental plots, juvenile wild radish plants protected with activated Bt sprays had an average of
In year two, eight weeks after the first rains, wild radish and wild mustard seedlings protected with Bt sprays again received approximately one-half (
Year 2: mean (±1 SE) percent damage per plant per plot 8 weeks after rains on exposed versus protected [tmt] wild mustard and wild radish [species] in disked field, field margin, and natural vegetation plots [hab]. Wild mustard bars, labeled “Mus,” precede wild radish bars, labeled “Rad.” Logistic regression (using
In year two, seedling mortality of wild mustard and wild radish was significantly lower for protected compared to exposed plants (Figure
(a) Year 2: mean (±1 SE) percent seedling mortality per plot (
Background vegetation had established fully by the time wild mustard and wild radish were reproductive, owing to the germination and growth of winter annuals after the fall rains began. By 12-13 weeks after the first rain, the average plant cover in all subplots was over 90%, including the disked field subplots, which initially had zero plant cover. The average plant species richness overall was
A significantly greater percentage of protected wild radish and mustard plants produced siliques or pods (
In year one, lifetime seed output of an average wild radish seedling, taking into account that early mortality results in zero seed production, was relatively low overall (Figure
(a) Year 1: mean (±1 SE) number of seeds per plant per plot (
Similarly, in year two, protected wild radish and wild mustard seedlings produced significantly more seeds (100.6 seeds/seedling) on average than exposed plants with higher levels of herbivory (46.1 seeds/seedling) (Figure
Experimental addition of wild radish and wild mustard seeds resulted in higher recruitment, even when the number of seeds per microcosm or field plot was relatively low (estimated as 1/2 the additional seed produced by an average plant in the low herbivory treatment). Mean wild radish density was significantly greater when seeds were added to the soil compared to controls with no added seeds, whether in the disked soil microcosms (initially bare) or in the natural vegetation microcosms (with plant cover intact) (Table
Mean number of radish or mustard plants surviving to reproduce when the approximate half the mean seed output advantage per plant due to herbivore protection was added to microcosms (
Disked soil habitat | Grassland habitat | |||
Experiment | Seeds added | Control | Seeds added | Control |
Radish microcosms1 | ||||
Mustard microcosms2 | ||||
Radish field plots3 | ||||
Mustard field plots4 |
1ANOVA on rank number of flowering radish plants per microcosm, with
2ANOVA on rank number of flowering wild mustard plants per microcosm, with
3Nested ANOVA on ranks for the number of wild radish plants after six months, seed addition treatment (site),
4Nested ANOVA on ranks for the number of wild mustard plants after six months, seed addition treatment (site),
Seed additions carried out at two field sites showed that six months after the first rains, the number of wild radish plants established in field plots was (1) significantly greater when seeds were added than in control plots with no seeds added and (2) significantly greater in disked fields than in grasslands (Table
Although there is general agreement that herbivores can have pronounced negative effects on plant fitness, plants also exhibit resistance to herbivores and tolerance to herbivore damage [
Lepidopteran resistant Bt-canola (
Seed output of wild mustard that survived to maturity was not consistently affected by differential herbivory, with a significant increase in Bt-protected plants only in the last year of our study [
Researchers and regulators involved in risk assessment face data gaps in predicting the consequences of host plant IR transgenes in wild plant populations [
We consider decreased female fitness due to naturally occurring levels of herbivory to be realistic outcomes for California agricultural and grassland habitats. Whereas individual fitness should be closely linked to seed production, the ecological population consequences of producing more seeds depend critically on whether those seeds will establish. Based on our seed addition experiments, an increase in seed production can allow more rapid spread of wild radish in local habitats. Results of our microcosm experiments are consistent with our casual observations of wild radish persisting in experimental plots that previously had no radish plants. Wild mustard, on the other hand, is not likely to invade grasslands by producing more seed but can increase numbers in disturbed sites such as agricultural fields.
Tiered frameworks for risk assessment (e.g., [
This research was supported by USDA Biotechnology Risk Assessment Grant 2003-33120-13968, faculty research grants from the UCSC Academic Senate and Social Sciences Division, and graduate student fellowship and research grants from the National Science Foundation and UCSC Department of Environmental Studies. The authors thank the UCSC Center for Agroecology and Sustainable Food Systems, Elkhorn Slough Foundation, UCSC Natural Reserves system, California State Parks, J. Velzy, and local growers and land managers for greenhouse, field, and logistical assistance. T. Roubison assisted with all field and lab experiments. They also thank I. Parker for help with experimental design and logistical dilemmas. S. Bothwell, A. Zeilinger, R. Abarca, D. Barrantes, L. Barth, S. L. Bryan, C. Conlan, E. Encarnacion, A. Fintz, L. Funk, E. Hampson, E. Hariton, F. Hesse, C. Josephson, A. LeComte, J. Martin, S. Moskal, R. Muscutt, Y. Pellman, T. Rogers, A. H. Stroud, A. Warner, J. Wilson, M. B. Winston, and K. Wong assisted in conducting field and lab experiments. The paper was improved by anonymous reviewers, P. Barbosa, S. Bothwell, T. Cornellise, J. Jedlicka, T. Krupnik, C. Moreno, I. Parker, A. Racelis, and A. Zeilinger.