A method to determine the pollen-mediated spread of target-site resistance to acetyl-coenzyme A carboxylase inhibitors in black grass (Alopecurus myosuroides Huds.)

In this study the spread of target-site resistance to ACCase inhibitors via pollen was characterized in a population of Alopecurus myosuroides. The spread of target-site resistance was assessed in a three-year model crop rotation experiment with 20 plots (round circles of 1 m diameter and enclosed by concrete walls of 50 cm height to avoid seed spread) in a total area of 90 m(2). In one plot (so called initial plot) an A. myosuroides population with a known target-site resistance (Leu1781) was sown. A herbicide sensitive biotype (Ile1781) was sown in all others plots. In a cereal crop rotation the second and third crop were treated with field rate of fenoxaprop and leaf samples of surviving plants were taken for DNA analysis. The Pyrosequencing (R) technology was applied to determine the genotype of sensitive (Ile1781) heterozygous (Ile/Leu1781) and homozygous (Leu1781) individuals. Plants carrying a heterozygous target-site resistance outside the initial plot were a first indication for outcrossing via pollen between the plots. Results showed an outcrossing frequency of 0.4 to 3.3% of Leu1781 into wild-type (Ile1781). The maximal distance for outcrossing from the initial plot was 4 m and 37% of the plots were infested with resistant plants. The frequency of Leu1781 increased in the second year from 7.9 to 26.6% and most plots were infested with herbicide-resistant plants. Outcrossing events decreased with increasing distance from the initial plot. After the first application, all plants identified as resistant outside the initial plot were heterozygous, while homozygous resistant plants were detected in the second year and indicated inbreeding between and within the plots. The results obtained over two years demonstrate the strong potential of the study to model the spread of resistance via pollen in allogamous species in narrow spaces. Hence, we provide a method for obtaining data under conditions close to field conditions.