Creeping bentgrass: when herbicide resistance goes wrong Brigid Meints CROP 540 11/19/2012 Herbicide resistant weeds Weeds become resistant to herbicides after repeated use; normal rates of the herbicide can no longer control the weed Transgenic plants engineered to be herbicide resistant that become weedy through gene flow Creeping Bentgrass (Agrostis stolonifera) Specialty grass used for golf course greens, lawn bowling greens, and lawn tennis High maintenance grass: requires high levels of fertilizer, frequent mowing, watering, aerating, and dethatching Identification: bright green, fine textured, no auricles, long and tapered ligule Perennial growth habit; spreads by stolons to form a mat or thatch layer above the soil line Low-growing with a shallow root system Allotetraploid Obligate outcrosser: has very small seeds, but can also propagate asexually Background Glyphosate-resistant creeping bentgrass created by Monsanto and Scotts: Event ASR368 In January 2004, Monsanto and Scotts petitioned the Animal and Plant Health Inspection Service (APHIS) seeking a determination of nonregulated status for creeping bentgrass APHIS began taking testimony to form an Environmental Impact Statement 14 species of Agrostis native to Oregon Found in agronomic and nonagronomic habitats Small seeds (6 million per pound) can be dispersed by wind, water, animals, or mechanical means Naturally forms interspecific F1 hybrids, low in fertility or sterile but under certain conditions have been shown to out-compete both parents Interspecific hybrid: mating of two species, generally from the same genus. The offspring show a combination of traits and characteristics from the two parents. Offspring are often sterile. Can hybridize with at least 12 other grass species from Agrostis and Polypogon extensive clonal propagation is still possible Genetics of glyphosate resistance Two components necessary: resistant target enzyme and sufficient expression of that enzyme within the transgenic plant 5-Enol-pyruvylshikimate-3-phosphate synthase (EPSPS) is the target enzyme for the inhibition of glyphosate in the aromatic amino acid biosynthetic pathway Agrobacterium sp. strain CP4 EPSPS was found to be an exceptional candidate. Does not have a negative impact on yield, quality, or nutritional value of the harvested crop Inherited as a single Mendelian locus History 2003: 162 ha of glyphosate-resistant transgenic creeping bentgrass tested under APHIS permit in central Oregon Conducted within a 4453 ha control area established by the ODA Located 144 km east of non-transgenic, commercial bentgrass production Watrud et al. (2004) Majority of gene flow observed within 2 km (in direction of prominent winds) Up to 21 km (sentinel plants) and 14 km (resident plants) Found evidence of gene flow in 75 of 138 (sentinel) and 29 of 69 (resident) A. stolonifera plants based on seedling progeny survival after spraying in the greenhouse Sampling grid designed to test viable pollen flow from transgenic plants Sentinel A. stolonifera plants cultivated in the Willamette Valley, transplanted in central Oregon (first tested for trait), transported very carefully PCR primers designed to test for the 1050-bp segment of the A. stolonifera CP4 EPSPS coding region. Matched GenBank accession for a CP4 EPSPS construct in glyphosate-resistant soybean Maximal distances of observed gene flow. Locations of sentinel A. stolonifera, resident A. stolonifera, and resident A. gigantea (A, B, and C respectively) Percent positive seedling progeny per location for sentinel A. stolonifera, resident A. stolonifera, and resident A. gigantea (A, B, and C respectively) Prevalence and incidence of plants positive for the transgene and seedling progeny Reichman et al. (2006) Resident populations of Agrostis species sampled in nonagronomic habitats outside of the ODA control zone Attempted to discover parentage of plants positive for CP4 EPSPS, but Monsanto and Scotts had proprietary constraints Used species-level molecular phylogenetic analyses Of 20,400 plant tissue samples taken, there were 9 positive plants (0.04%) Distributed over 6 of the surveyed population areas, including the Crooked River National Grassland Not possible to determine which field they came from, so distances are range All near waterways or roadside Gene trees showed that all wild transgenic plants had maternal and paternal A. stolonifera parents Why is this crop so problematic? Size, density, and viability of the pollen Wild, sexually compatible species Floral synchrony between crop and wild species Large source fields Small seed size History 2007: APHIS completed their investigation into alleged compliance infractions by The Scotts Company. Scotts agreed to pay a civil penalty of $500,000, which is the maximum penalty allowed by the 2000 Plant Protection Act The saga continues… 2012: Reports of intergeneric hybridization with rabbitfoot grass, (Polypogon monspeliensis) Transgenes confirmed using PCR primers Produced viable seed, had perennial growth habit and stolon production capability Intergeneric hybrid: hybrid between different genera Final thoughts Careful consideration of potential gene flow when introducing transgenic crops Consequences associated with that gene flow References APHIS (2004) Monsanto Co. and The Scotts Co.; Availability of petition for determination of nonregulated status for genetically engineered glyphosate-tolerant creeping bentgrass. Federal Register 69, 315-317, January 5, 2004 Heck et al. (2005). Development and Characterization of a CP4 EPSPS-Based, Glyphosate-Tolerant Corn Event:Crop Sci. 45:329-339 (2005). Reichman, J. R., Watrud, l. S., Lee, E. H., Burdick, C. A., Bollman, M. A., Storm, M. J., King, G. A. And Mallory-Smith, C. (2006), Establishment of transgenic herbicide-resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats. Molecular Ecology, 15: 4243–4255. Watrud LS, Lee EH, Fairbrother A et al. (2004) Evidence for landscape level pollenmediated gene flow from genetically modified creeping bentgrass using CP4 EPSPS as a marker. Proceedings of the National Academy of Sciences, USA, 101, 14533–14538. UC Davis. Statewide Integrated Pest Management: The UC Guide to Healthy Lawns, Creeping Bentgrass. http://www.ipm.ucdavis.edu/TOOLS/TURF/ TURFSPECIES creepbent.html USDA. (2007). Release No. 0350.07. USDA concludes genetically engineered creeping bentgrass investigation. http://www.usda.gov/wps/portal/usda/ usdahome?contentidonly=true&contentid=2007/11/0350.xml Zapiola, M. L. And Mallory-Smith, C. A. (2012), Crossing the divide: gene flow produces intergeneric hybrid in feral transgenic creeping bentgrass population. Molecular Ecology, 21: 4672–4680.