2.1. Field Studies
Two non-crop studies were conducted at the Wiregrass Research and Extension Center in Henry County, AL (31°21′17.1″ N 85°19′35.3″ W) during summers of 2018 and 2019 in irrigated field with high Palmer amaranth pressure. The site included natural and augmented Palmer amaranth populations planted at 500 seeds m
−2 on June 6 and May 10 in 2018 and 2019, respectively. Prior to seeding, the study area was disked thoroughly to remove existing weeds, then field cultivated to ensure smooth soil surface. An artificial population combined with existing population was established by spreading Palmer amaranth seeds sourced from peanut and cotton fields in Alabama with known glyphosate and ALS-inhibitor resistance. Mature female seed heads were collected in late September before crop harvest and cleaned by hand on Auburn University campus for Palmer seeds. Other weed species presented in the non-crop plots included morning glory (
Ipomoea spp.), sicklepod (
Senna obtusifolia), Florida pusley (
Richardia scabra), Florida beggarweed (
Desmodium tortuosum), and southern crabgrass (
Digitaria ciliaris). The experimental design was a randomized complete block with four replications. Plot size was 3.3 m long by 3.3 m wide. Herbicide treatments and rates can be found in
Table 1 and consisted of combinations of 2,4-D choline (Enlist One
®; Dow AgroSciences LLC, Indianapolis, IN 46268, USA) or diglycolamine salt of dicamba with a built in pH buffer (Xtendimax
® with Vaporgrip
®; Monsanto Co. St. Louis, MO 63167, USA), glufosinate (Liberty
® 280 SL; BASF Corporation, Research Triangle Park, NC 27709, USA), and glyphosate (Roundup PowerMax
®; Monsanto Co. St. Louis, MO 63167, USA) at 1063, 559, 594, and 1540 g ai ha
−1 (
Table 1).
Glyphosate was used in tank mixture with dicamba and 2,4-D choline to provide control of annual grass weeds and broadleaf weeds that are not resistant to glyphosate, which is a common practice for row crop farmers in the southeast US. It does not provide any control of resistant Palmer amaranth in this field. Initial herbicide applications were performed on 6 July and 12 June in 2018. A blanket application of S-metolachlor (Dual Magnum®; Syngenta Crop Protection, LLC, Greensboro, NC 67419, USA) was applied once 7 days after initial herbicide application at 1469 g ai ha−1 on all treated plots to limit Palmer amaranth seed germination later in the study, thus confounding ratings and data collection. Herbicides were applied to Palmer amaranth with average height of 37 and 59 cm tall in 2018 and 2019, respectively. All herbicides were applied with a CO2-pressurized backpack sprayer equipped with four TeeJet nozzles (TeeJet Technologies, Wheaton, IL 60187, USA) delivering 187 L ha−1. Turbo TeeJet induction flat spray tips (TTI 110025, TeeJet Technologies, Wheaton, IL 60187, USA) producing ultra-coarse droplets were used for treatments that included synthetic auxins to reduce spray drift as label required, and Turbo TeeJet wide angle flat fan spray tips (TT 110025, TeeJet Technologies, Wheaton, IL 60187, USA) producing medium size droplets were used for glufosinate applications to increase spray coverage.
Palmer amaranth injury was visually estimated at 14 and 28 DAIT on a scale of 0 (no injury) to 100 (complete mortality). At 35 DAIT, ten Palmer amaranth heights were recorded randomly in each plot by measuring living individuals from the ground to the top of the plant. Palmer amaranth in each plot were cut at ground level 35 DAIT and immediately weighted to determine fresh biomass. Plants were only harvested from the middle area (2.4 × 2.4 m) in each plot to ensure adequate herbicide coverage was received.
Data were subjected to a mixed model analysis of variance through PROC GLIMMIX in SAS 9.4 (Statistical Analysis Systems®, version 9.4; SAS Institute Inc., Cary, NC 27513, USA). Treatment and year were considered fixed effects, while block was a random effect and all interactions were examined. If treatment by year interactions were observed, data was analyzed separately to show individual effects for each year. Means comparisons were generated using Tukey’s Honest Significant (HSD) different test with p = 0.05.
2.2. Greenhouse Study
In order to better understand Palmer amaranth physiological response to synthetic auxins and glufosinate, an experiment was conducted in a greenhouse with natural daylight at Auburn University in Auburn, AL during June of 2019. Temperatures ranged between 20 and 31 °C throughout the course of the study. Glyphosate and ALS-inhibitor resistant Palmer amaranth seed collected from Headland, AL were planted in 4 L pots filled with commercial potting soil (Miracle-Gro® Moisture Control® Potting Mix, The Scotts Company LLC, Marysville, OH 43040, USA). Plants were thinned to two plants per pot and grown until reaching 30 to 45 cm in height with irrigation and fertilizer applied as needed. One leaf on each plant was tagged with plastic string at the petiole, representing a young fully developed leaf (3–4 node position from the tip), to ensure the same leaf was measured throughout the study.
The experimental design was a randomized complete block with three replications in two separate runs. In addition to a nontreated control, treatments included a tank mixture of dicamba + glufosinate + glyphosate, dicamba + glyphosate fb glufosinate 7 DAIT, and glufosinate fb dicamba + glyphosate 7 DAIT. Reduced herbicide rates relative to the field study were chosen to allow Palmer amaranth survival and enable for physiological measurements up to 35 d after initial treatments. Dicamba, glufosinate, and glyphosate were applied at 186, 198, and 514 g ai ha−1. Herbicides were applied using a CO2-pressurized backpack sprayer equipped with two flat fan 110,025 nozzles delivering 187 L ha−1. Dicamba was applied with Turbo TeeJet induction flat fan nozzles (TTI110025, TeeJet Technologies, Wheaton, IL 60187, USA) and glufosinate with Turbo TeeJet wide angle flat fan nozzles (TT110025, TeeJet Technologies, Wheaton, IL 60187, USA).
Physiological measurements were recorded at 1, 3, 6, 8, 11, 13, and 35 DAIT. Measurements included mid-day photosynthesis, leaf stomatal conductance, and respiration, collected with a LI-6400XT (LI-COR Biosciences, Lincoln, NE 68504, USA). Before each midday photosynthesis measurements, light intensity in the greenhouse was recorded by a photosynthetic photon flux density meter (LI-190; LICOR Biosciences, Lincoln, NE, USA), temperature was monitored by an onsite weather station, and relative humidity was maintained between 60% and 70%. Conditions in the leaf cuvette were then set to match ambient environmental conditions, with the [CO2] in the cuvette set to match ambient [CO2] (~410 ppm). The methodology for respiration measurements was the same but the light intensity was set to 0 μmol mol−1. Photosystem II (PSII) quantum yield was recorded with a portable fluorometer (FluorPen FP 100, Photon Systems Instruments, Albuquerque, NM, USA 87106). Photosynthesis and stomatal conductance measurements were recorded at solar noon (11:00–13:00) while respiration and PSII quantum yield measurements were collected from dark-adapted plants two hours after sunset. At the end of physiological measurements, all Palmer amaranth leaves were removed at the petiole base 14 DAIT, fresh weight was recorded, then leaves were processed through a LI-3100C area meter (LI-COR Biosciences, Lincoln, NE 68504, USA) to determine total leaf area. Palmer amaranth bare stalks were allowed to resume growth until 35 DAIT when photosynthesis, respiration and fluorescence measurements were repeated as indicated above, and leaves were removed at the petiole base once more and weighted.
Each physiological measurement was averaged across the two tagged leaves per pot and data was subjected to a mixed model analysis of variance through PROC GLIMMIX in SAS® 9.4 (Statistical Analysis Systems®, version 9.4; SAS Institute Inc., Cary, NC 27513, USA) with treatment considered as a fixed effect. Data were pooled across experiment repetitions which was considered a random variable. Scatter plots were generated in Sigmaplot 13.0 (Systat Software, San Jose, CA 95131, USA) and means comparisons were generated using Tukey’s HSD test with p = 0.05.