Palmer amaranth (Amaranthus palmeri) is a major weed in soybean cultivation across the state. In recent years, these weeds become resistant to multiple herbicides (acetolactate synthase, photosystem II, and protoporphyrinogen oxidase etc.) including glyphosate (ESPS synthase inhibitor). Glyphosate is one of the most widely used herbicide in soybean cultivation (97%) in the USA due to its high efficacy, low toxicity, and low environmental impacts. However, an excessive reliance on glyphosate lead to faster evolution of herbicide resistance (HR) in Palmer amaranth. Other causes are poor management practices, inadequate doses, and wrong application timings. This man-made evolution poses a serious threat to food security in term of yield losses up to 79%. HR is inevitable, but can be slow down using multiple technologies. We are proposing to evaluate the induction of non-target site resistance in Palmer Amaranth due to current applications of herbicides in DE. This work will help to decide what combination of herbicides will slow the evolution of HR in soybean cultivation.

Objective 1. Characterization of Glutathione-S-transferase activity in weed.
Leaves of 3-inch tall Palmer amaranth (Amaranthus palmeri) will be collected from the field and leaf discs will be incubated for 5 to 15 min in recommended herbicides to estimate the activity of GST. Each experiment will be replicated three times and analyzed by using completely randomized design.

Glutathione S-transferase assay (GST): Leaf samples (0.1 g) will be macerated in 10 ml 0.1 M potassium phosphate buffer, pH 7.5 containing 0.5 mM EDTA and filtered through cheesecloth. The filtrate will be centrifuged at 15,000g for 15 min. GST activity will be measured using 2,4-Dinitrochlorobenzene (CDNB) in a 1-mL reaction mixture containing 20 µL of leaf extract, 1.0 mm GSH, 1.0 mm CDNB, and 0.1 m potassium phosphate buffer (pH 7.5). The protein extract will be added in last and absorption will be measured at A340 for 300 s using a spectrophotometer.

Expected Outcome: We are expecting the selection of herbicide treatments, which causes minimum increase in GST activity and thus, can slow down the evolution of non-target-resistance in Palmer Amaranth.

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Herbicide resistance (HR) in soybean cultivation is a major challenge in front of soybean farmers in North and South America. Over the years, weeds have developed resistance against 23 targeted 2 sites out of 26 in different crop cultivation systems. Moreover, there is no progress on the development of new herbicide chemistry across the globe in last three decades. With ever evolving herbicide resistance and lack of novel herbicide mode of action, it become extremely difficult and expensive to control weed damage at farm. An estimate by Weed Science Society of America showed that US Corn and Soybean farmers could loss half of their crop and $43 billion per year in the absence of herbicides management.

A USDA report showed HR soybean acreage increased from 17% to 94% during 1997-2014. With the advent of glyphosate resistant (GR) soybean cultivation throughout the states there is a continuous decline in use of alternative herbicides, which enhanced the development of GR in weeds. Moreover, combination of GR corn/GR cotton/GR soybean crop rotation and multiple application (2 or 3) of glyphosate further accelerated the GR resistance in weeds much earlier than
expected. Total dependence on glyphosate also caused multiple resistance in weed populations through induction of novel resistance mechanisms by pollen transfer and sequential selection as observed in GR Lolium spp. and GR cotton in Australia and southern United States respectively (Beckie, 2011). The GR Palmer amaranth was first observed in Georgia
in 2004 (Yu et al., 2021). Herbicide resistance is conferred by two mechanisms; I: target-site alteration and II: non-target-site. The first mechanism involves mutation and overexpression of protein while in second mechanism herbicide is excluded or detoxified and translocated to vacuoles in plant cell, thus reduced the effective concentration of herbicide at the site of active growth in weeds. Recently, non-target-site resistance was observed in Palmer Amaranth against three herbicides including photosystem II (PSII: atrazine), acetolactate synthase (ALS: chlorsulfuron), and EPSP synthase (EPSPS: glyphosate; Choudhari et al., 2020). The enzyme glutathione S-transferase (GST) is chiefly responsible for the detoxification of herbicides in plants (Katerova et al., 2010). There is a possibility of glyphosate induced activation of these enzymes in GR weeds with concomitant evolution of non-target-site HR.

Evolution of HR is inevitable, however, it could be slowed/delayed or easily managed by simple long-term cost-effective integrated approaches. Understanding of mechanism of herbicides detoxification (activity of glutathione-S-transferase) in plant cells will help select type of herbicide (mode of action) for effective control of weeds without inducing herbicide resistance.