Herbicide-resistant Weeds in Oregon

Creeping bentgrass: when herbicide resistance goes wrongBrigid MeintsCROP 54011/19/2012Herbicide resistant weedsWeeds become resistant to herbicides after repeated use; normal rates of the herbicide can no longer control the weedTransgenic plants engineered to be herbicide resistant that become weedy through gene flowCreeping Bentgrass (Agrostis stolonifera)Specialty grass used for golf course greens, lawn bowling greens, and lawn tennisHigh maintenance grass: requires high levels of fertilizer, frequent mowing, watering, aerating, and dethatchingIdentification: bright green, fine textured, no auricles, long and tapered ligulePerennial growth habit; spreads by stolons to form a mat or thatch layer above the soil line Low-growing with a shallow root systemAllotetraploidObligate outcrosser: has very small seeds, but can also propagate asexually

BackgroundGlyphosate-resistant creeping bentgrass created by Monsanto and Scotts: Event ASR368In January 2004, Monsanto and Scotts petitioned the Animal and Plant Health Inspection Service (APHIS) seeking a determination of nonregulated status for creeping bentgrassAPHIS began taking testimony to form an Environmental Impact Statement14 species of Agrostis native to OregonFound in agronomic and nonagronomic habitatsSmall seeds (6 million per pound) can be dispersed by wind, water, animals, or mechanical meansNaturally forms interspecific F1 hybrids,low in fertility or sterilebut under certain conditions have been shown to out-compete both parentsInterspecific 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 possibleGenetics of glyphosate resistanceTwo components necessary: resistant target enzyme and sufficient expression of that enzyme within the transgenic plant5-Enol-pyruvylshikimate-3-phosphate synthase (EPSPS) is the target enzyme for the inhibition of glyphosate in the aromatic amino acid biosynthetic pathwayAgrobacterium 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 cropInherited as a single Mendelian locus

History2003:162 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 productionWatrud 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 zoneAttempted to discover parentage of plants positive for CP4 EPSPS, but Monsanto and Scotts had proprietary constraintsUsed species-level molecular phylogenetic analysesOf 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 GrasslandNot possible to determine which field they came from, so distances are rangeAll near waterways or roadsideGene trees showed that all wild transgenic plants had maternal and paternal A. stolonifera parentsWhy is this crop so problematic?Size, density, and viability of the pollenWild, sexually compatible speciesFloral synchrony between crop and wild speciesLarge source fieldsSmall seed size

History2007: 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 ActThe saga continues2012:Reports of intergeneric hybridization with rabbitfoot grass, (Polypogon monspeliensis)Transgenes confirmed using PCR primersProduced viable seed, had perennial growth habit and stolon production capabilityIntergeneric hybrid: hybrid between different genera

Final thoughtsCareful consideration of potential gene flow when introducing transgenic crops Consequences associated with that gene flowReferencesAPHIS (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, 2004Heck 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, 15:42434255. Watrud LS, Lee EH, Fairbrother A et al. (2004) Evidence for landscape level pollen-mediated gene flow from genetically modified creeping bentgrass using CP4 EPSPS as a marker. Proceedings of the National Academy of Sciences, USA, 101, 1453314538.UC Davis. Statewide Integrated Pest Management: The UC Guide to Healthy Lawns, Creeping Bentgrass. http://www.ipm.ucdavis.edu/TOOLS/TURF/TURFSPECIES creepbent.htmlUSDA. (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.xmlZapiola, M. L. And Mallory-Smith, C. A. (2012), Crossing the divide: gene flow produces intergeneric hybrid in feral transgenic creeping bentgrass population. Molecular Ecology, 21:46724680.