In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we developed an excised leaf assay combined with a vegetative cloning strategy to normalize herbicide uptake and remove translocation as contributing factors in herbicide-resistant (R) and -sensitive (S) waterhemp populations. Biokinetic analyses of organic pesticides in plants typically include the determination of uptake, translocation (delivery to the target site), metabolic fate, and interactions with the target site. Herbicide metabolism is an important parameter to measure in herbicide-resistant weeds and herbicide-tolerant crops, and is typically accomplished with whole-plant tests using radiolabeled herbicides. However, one difficulty with interpreting biokinetic parameters derived from whole-plant methods is that translocation is often affected by rates of herbicide metabolism, since polar metabolites are usually not mobile within the plant following herbicide detoxification reactions. Advantages of the protocol described in this manuscript include reproducible, accurate, and rapid determination of herbicide degradation rates in R and S populations, a substantial decrease in the amount of radiolabeled herbicide consumed, a large reduction in radiolabeled plant materials requiring further handling and disposal, and the ability to perform radiolabeled herbicide experiments in the lab or growth chamber instead of a greenhouse. As herbicide resistance continues to develop and spread in dicot weed populations worldwide, the excised leaf assay method developed and described herein will provide an invaluable technique for investigating non-target site-based resistance due to enhanced rates of herbicide metabolism and detoxification.
The Enlist weed control system allows the use of 2,4-D in soybean but slight necrosis in treated leaves may be observed in the field. The objectives of this research were to measure and compare uptake, translocation, and metabolism of 2,4-D in Enlist (E, resistant) and non-AAD-12 transformed (NT, sensitive) soybeans. The adjuvant from the Enlist Duo herbicide formulation (ADJ) increased 2,4-D uptake (36%) and displayed the fastest rate of uptake (U= 0.2 h) among treatments. E soybean demonstrated a faster rate of 2,4-D metabolism (M= 0.2 h) compared to NT soybean, but glyphosate did not affect 2,4-D metabolism. Metabolites of 2,4-D in E soybean were qualitatively different than NT. Applying 2,4-D-ethylhexyl ester instead of 2,4-D choline (a quaternary ammonium salt) eliminated visual injury to E soybean, likely due to the time required for initial de-esterification and bioactivation. Excessive 2,4-D acid concentrations in E soybean resulting from ADJ-increased uptake may significantly contribute to foliar injury.
Waterhemp (Amaranthus tuberculatus) is a common and troublesome weed in cropping systems throughout the United States. With the potential for future periods of low rainfall or drought, the need for improved weed control under drought stress is necessary. Drought stress typically reduces herbicide efficacy by reducing the foliar uptake of herbicides and their translocation. The objectives of this research were to determine the efficacy of 2,4-dichlorophenoxyacetic acid (2,4-D) and glyphosate, applied alone or when tank-mixed, on waterhemp under varying levels of drought stress, the effect of the timing of drought stress in relation to herbicide application and the absorption and translocation of each herbicide in drought-stressed waterhemp. At reduced herbicide rates, 2,4-D had a greater level of control of waterhemp under drought stress, compared to glyphosate. The level of herbicide efficacy was lower when the amount of water that was applied to the plants was reduced. The level of waterhemp control was greatest when drought stress occurred before the herbicide application and when the plants were watered to saturation after the application, compared to when drought stress occurred after the herbicide application or restricted watering levels occurred throughout the entire study. Glyphosate absorption and translocation were reduced in the drought-stressed plants, but 2,4-D absorption and translocation were not altered. The absence of a reduction in 2,4-D translocation in the drought-stressed weeds has not been previously reported. Applying herbicides prior to a rainfall event could increase the weed control level, even if the weed is stressed. Determining how and why 2,4-D absorption and translocation levels, compared to those of glyphosate, are unaffected by drought stress in waterhemp can aid in improving the control of drought-stressed weeds with other postemergence herbicides.
The Enlist™ traits provide 2,4-D resistance in several crops. Though corn is naturally tolerant to 2,4-D, the engineered trait conferred by the aryloxyalkanoate dioxygenase-1 (AAD-1) enzyme provides enhanced 2,4-D tolerance and confers resistance to the graminicide herbicide family, the aryloxyphenoxypropionates. The objectives of this research were 2-fold: (1) measure and compare uptake, translocation, and metabolism of 2,4-D in Enlist™ (E, +AAD1) and non–AAD-1 transformed (NT, −AAD1) isogenic corn hybrids; and (2) and investigate the effect of glyphosate and/or the Enlist™ adjuvant system (ADJ) on these factors and corn injury. Uptake of radiolabeled 2,4-D acid applied alone in corn was not altered by the addition of ADJ when tank mixed at 24 h after application (HAA). By contrast, uptake of radiolabeled 2,4-D was significantly lower (69%) compared with 2,4-D plus ADJ (89%) at 24 HAA with a premixed formulation of 2,4-D choline plus glyphosate-dimethylamine (Enlist Duo™ herbicide [EDH]). Translocation of 2,4-D between the two corn hybrids was not different. E corn metabolized more 2,4-D (100% of absorbed) than NT corn (84%), and glyphosate did not alter 2,4-D metabolism. Furthermore, the metabolism of 2,4-D to nonphytotoxic dichlorophenol (DCP) and subsequent DCP-derived metabolites formed in E corn was examined. Injury to E corn is not typically observed in the field; however, injury symptoms were clearly evident in E corn (within 24 HAA) when formulated acetochlor was tank mixed with EDH, which correlated with an increase in 2,4-D uptake during this time period. In summary, the lack of injury in E corn following EDH applied alone may be attributed to a relatively low amount of 2,4-D uptake and the combination of natural and engineered 2,4-D metabolic pathways.
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