Creeping bentgrass: when herbicide resistance goes wrong Brigid Meints CROP /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 F 1 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, , January 5, 2004  Heck et al. (2005). Development and Characterization of a CP4 EPSPS-Based, Glyphosate-Tolerant Corn Event:Crop Sci. 45: (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 pollen- mediated gene flow from genetically modified creeping bentgrass using CP4 EPSPS as a marker. Proceedings of the National Academy of Sciences, USA, 101, 14533–  UC Davis. Statewide Integrated Pest Management: The UC Guide to Healthy Lawns, Creeping Bentgrass. creepbent.html  USDA. (2007). Release No USDA concludes genetically engineered creeping bentgrass investigation. 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.