Herbicide-Resistant Mutations in Acetolactate Synthase Can Reduce Feedback Inhibition and Lead to Accumulation of Branched-Chain Amino Acids

The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that are critical for animal growth and development. Animals need to obtain BCAAs from their diet because they cannot synthesize them. Plants are the ultimate source of these amino acids. Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of BCAAs. The metabolic control of BCAA biosynthesis involves allosteric regulation of ALS by the end-products of the pathway, i.e., valine, leucine and isoleucine. ALS holoenzyme seems to consist of two large catalytic subunits and two small regulatory subunits. In a previous study, using homologous recombination dependent gene targeting we created rice plants in which W548Land S627I mutations were induced into the endogenous gene encoding the ALS catalytic subunit. These two amino acid substitutions conferred hypertolerance to the ALS-inhibiting herbicide bispyripac-sodium. In this study, we revealed that feedback regulation by valine and leucine was reduced by these two amino acid substitutions. Furthermore, in leaves and seeds of ALS mutants with W548Land/or S627I substitution, a 2- to 3-fold increase in BCAAs was detected. Our results suggest that the ALS catalytic subunit is also involved in feedback regulation of ALS, and that judicious modification of the regulatory and catalytic subunits of ALS-coding genes by gene targeting can lead to the efficient accumulation of BCAA in plants.

Efficacy of 2,4-D Choline/Glyphosate Dimethylamine on Glyphosate Resistant Canada Fleabane (<i>Conyza canadensis</i>) at Different Sizes

Glyphosate resistant (GR) Canada fleabane has spread quickly across southwestern Ontario and new strategies for the control of this competitive weed must be developed especially in no-tillage crops. A premix of 2,4-D choline and glyphosate dimethylamine (DMA) has been developed for application on tolerant corn, soybean and cotton crops that provides an option for the control of this problematic GR weed. The objective of this research was to determine the required dose needed to effectively control GR Canada fleabane at different size categories in field and greenhouse experiments. In the field experiments, nine rates of 2,4-D choline/glyphosate DMA (53.8 to 13,760 g·ae·ha-1) were applied to GR Canada fleabane that were 10 cm in diameter/tall, 20 cm tall or 30 cm tall. Similarly, in the greenhouse, seven rates of 2,4-D choline/glyphosate DMA (0 to 3440 g·ae·ha-1) were applied to 10, 20 and 30 cm tall GR Canada fleabane plants. The three different size classes of GR Canada fleabane responded similarly to 2,4-D choline/glyphosate DMA in the field experiment. In the greenhouse there were some differences in control for the three size classes of GR Canada fleabane with 2,4-D choline/glyphosate DMA;the 20 and 30 cm tall plants required similar rates to provide equivalent control, but the 10 cm plants required a lower rate. In all situations, greater than 1720 g·ae·ha-1 of 2,4-D choline/glyphosate DMA was required to provide 95% control of 10, 20 and 30 cm tall Canada fleabane in greenhouse (35 DAA) and field experiments (8 WAA), respectively.

Control of Glyphosate and Acetolactate Synthase Resistant Common Ragweed (Ambrosia artemisiifolia L.) in Soybean (Glycine max L.) with Preplant Herbicides

A population of common ragweed in Ontario was confirmed to be resistant to glyphosate in 2011. Group 2 [acetolactate synthase (ALS) inhibitors] resistant common ragweed was first confirmed in Ontario in 2000. Previously, glyphosate provided excellent control of common ragweed in glyphosate resistant soybean but with the confirmation of glyphosate resistant (GR) common ragweed, alternative herbicides need to be evaluated. Eight field trials with preplant herbicides were completed over two years (2013 and 2014) in fields with confirmed GR common ragweed. Tank-mixes of glyphosate and linuron or metribuzin provided 88% - 99% and 86% - 98% control 4 weeks after application (WAA) and 80% - 92% and 80% - 95% control 8 WAA, respectively. However, these herbicides also had among the highest environmental impact of the herbicides tested. Based on the results of these studies, GR common ragweed can be controlled with residual herbicides when applied preemergence in soybean. Currently, there are no post emergence herbicides that provide adequate control of GR common ragweed, therefore, preemergence herbicides with residual are essential for full season control.

Glyphosate-Resistant Giant Ragweed(Ambrosia trifida L.)in Ontario:Dose Response and Control with Postemergence Herbicides

Giant ragweed (Ambrosia trifida L.) is competitive with agronomic crops and can cause significant yield losses. Rapid adoption of glyphosate-resistant (GR) crops and a concomitant increase in the reliance on glyphosate for weed management has led to the evolution of GR giant ragweed in Ontario, Canada. Field studies were conducted to evaluate the level of resistance in giant ragweed biotypes from Ontario, and to evaluate the effectiveness of various postemer-gence (POST) herbicides in soybean (Glycine max L.). The effective dose (ED) to provide 50%, 80% and 95% giant ragweed control was up to 1658, 9991 and >43200 g?a.e.?ha–1 4 weeks after application (WAA), respectively. For effective control, growers would need to apply glyphosate 18 times greater than the recommended field application dose. Glyphosate applied at the recommended field dose of 900 g?a.e.?ha–1 provided up to 57% control and resulted in soybean yield equivalent to the weedy check. Cloransulam-methyl applied POST provided up to 99% control, reduced giant ragweed density 98%, reduced giant ragweed shoot dry weight 99% and resulted in soybean yield equivalent to the weedfree check. Chlorimuron-ethyl, fomesafen, imazethapyr and imazethapyr plus bentazon applied alone or with glyphosate did not provide adequate control of GR giant ragweed. Based on these results, some GR giant ragweed biotypes from Ontario have evolved a high level of resistance to glyphosate. Cloransulam-methyl applied POST was the only herbicide that provided adequate control and suggests that additional weed management tactics will need to be implemented in order to effectively manage GR giant ragweed.

Control of glyphosate resistant giant ragweed in soybean with preplant herbicides

Giant ragweed was the first glyphosate resistant weed identified in Canada. It is a very competetive weed in row crop production and has been found to drastically reduce yields of soybean;therefore, control of this competitive weed is essential. The objective of this study was to determine effective control options for glyphosate resistant giant ragweed in soybean with herbicides applied preplant. Eighteen herbicide combinations were evaluated in field studies conducted in 2011 and 2012 at five locations with confirmed glyphosate resistant giant ragweed. Glyphosate plus 2,4-D ester or amitrole provided the best control of glyphosate resistant giant ragweed 4 WAA. Glyphosate plus 2,4-D ester provided 98 to 99% control and was equivalent to the weed free check at all locations. Glyphosate plus amitrole provided 90% to 93% control and was equivalent to the weed free check at 4 of 5 locations. Herbicides providing residual activity provided variable control across all locations. Of the herbicides with residual activity evaluated, glyphosate plus linuron provided the best control of glyphosate resistant giant ragweed;however, control was inconsistent across locations and years. Glyphosate plus linuron provided 23% to 99% controland was equal to the weed free check at one location 8 WAA.

Generation of a high-efficiency adenine base editor with TadA8e for developing wheat dinitroaniline-resistant germplasm

Base editing using CRISPR technologies is an invaluable tool for crop breeding. One of the major base editors, the adenine base editor(ABE), has been successfully used in both model plants and many crops.However, owing to limited editing efficiency, the ABE has been difficult to apply in polyploid crops such as allohexaploid bread wheat that often require simultaneous mutation of multiple alleles for fast breeding. We have designed a wheat high-efficiency ABE(Whie ABE), using the newly developed high-activity adenosine deaminase Tad A8 e. In vivo and in vitro analysis demonstrated the improved applicability of Tad A8 e over the commonly used Tad A7.10. Dinitroaniline is a widely used herbicide with high effectiveness and low toxicity to animals. However, wheat cultivars with tolerance to dinitroaniline are rare, limiting the application of dinitroaniline in wheat planting. Using A-to-G editing with Whie ABE, we found that a Met-to-Thr mutation in wheat tubulin alleles located on chromosomes 1 A, 1 B, 1 D, 4 A, and 4 D increased the resistance of wheat to dinitroaniline, revealing a dosage effect of edited tubulins in resistance. The Whie ABE promises to be a valuable editing tool for accelerating crop improvement and developing herbicide-resistant wheat germplasm.

Glyphosate-Resistant Giant Ragweed(Ambrosia trifida L.):2,4-D Dose Response and Control with Postemergence Herbicides in Soybean

Glyphosate resistant giant ragweed is an increasing problem in glyphosate resistant cropping systems in southwestern Ontario. The postemergence herbicides registered for use in soybean in Ontario do not provide consistent control of glyphosate resistant giant ragweed. There is limited research on the lowest effective rate of 2,4-D for the control of glyphosate resistant giant ragweed. Consequently, the objectives of this study were a) to determine the efficacy of herbicides applied postemergence for the control of glyphosate resistant giant ragweed in glyphosate resistant soybean, and b) to determine the lowest effective rate of 2,4-D for the control of glyphosate-resistant giant ragweed. Ten postemergence herbicide combinations and seven rates of 2,4-D were evaluated in field studies conducted in 2011 and 2012 at six locations confirmed with glyphosate-resistant giant ragweed. The post emergence herbicides evaluated did not provide acceptable/consistent control. Of the herbicides evaluated, glyphosate plus cloransulam-methyl provided 26% to 70% control 8 WAA of glyphosate resistant giant ragweed, which was the best of the herbicides combinations evaluated. The doses of 2,4-D required to reduce giant ragweed shoot dry weight by 50, 80 and 95% were 142, 310 and 1048 g a.e. ha-1, respectively.