Article pubs.acs.org/JAFC
Understanding the Differential Response of Setaria viridis L. (green foxtail) and Setaria pumila Poir. (yellow foxtail) to Pyroxsulam Norbert M. Satchivi,* Gerrit J. deBoer,† and Jared L. Bell Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States S Supporting Information *
ABSTRACT: Green foxtail [Setaria viridis (L) Beauv.] and yellow foxtail [Setaria pumila (Poir.) Roem. & Schult.] are among the most abundant and troublesome annual grass weeds in cereal crops in the Northern Plains of the United States and the Prairie Provinces of Canada. Greenhouse and laboratory experiments were conducted to examine the differential responses of both weed species to foliar applications of the new triazolopyrimidine sulfonamide acetolactate synthase-inhibiting herbicide, pyroxsulam, and to determine the mechanism(s) of differential weed control. Foliar applications of pyroxsulam resulted in >90% control of yellow foxtail at rates between 7.5 and 15 g ai ha−1, whereas the same rates resulted in a reduced efficacy on green foxtail (≤81%). The absorption and translocation of [14C]pyroxsulam in green and yellow foxtail were similar and could not explain the differential whole-plant efficacy. Studies with [14C]pyroxsulam revealed a higher percentage of absorbed pyroxsulam was metabolized into an inactive metabolite in the treated leaf of green foxtail than in the treated leaf of yellow foxtail. Metabolism studies demonstrated that, 48 h after application, 50 and 35% of pyroxsulam in the treated leaf was converted to 5hydroxy-pyroxsulam in green and yellow foxtail, respectively. The acetolactate synthase (ALS) inhibition assay showed that ALS extracted from green foxtail was more tolerant to pyroxsulam than the enzyme extracted from yellow foxtail was. The in vitro ALS assay showed IC50 values of 8.39 and 0.26 μM pyroxsulam for green and yellow foxtail, respectively. The ALS genes from both green and yellow foxtail were sequenced and revealed amino acid differences; however, the changes are not associated with known resistance-inducing mutations. The differential control of green and yellow foxtail following foliar applications of pyroxsulam was attributed to differences in both metabolism and ALS sensitivity. KEYWORDS: pyroxsulam, Setaria viridis, Setaria pumila, acetolactate synthase, absorption, translocation, metabolism, ALS gene
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INTRODUCTION Green foxtail [Setaria viridis (L) Beauv.] and yellow foxtail [Setaria pumila (Poir.) Roem. & Schult.] are the most abundant summer annual grass weeds in the northern Great Plains of the United States1−3 and the Prairie Provinces of Canada.4−8 In surveys conducted between 1970 and 2000 in Western Canada, green foxtail was the most widespread annual grass weed with a frequency of >70% in Manitoba and >50% in Saskatchewan,6,9 whereas yellow foxtail was much less common with a frequency of 1500 plants m−2 for green foxtail16,17 and ≤600 plants m−2 for yellow foxtail.12 Apart from the economic impact of green and yellow foxtail, both weeds are known to produce prolific numbers of seeds, which perpetuates the problem.18 Although green and yellow foxtails often grow in mixed populations, both species appear to be morphologically distinct.19 Yellow foxtail possesses prominent silky, kinky hairs on the upper surfaces of the leaf blades just near the stem. On the other hand, green foxtail can be distinguished from yellow foxtail by the absence of long, kinky hair on the upper surface of the leaf blade near the stem and the presence of a fringe of hair on both margins of the leaf sheath. Phylogenetic and ploidy studies revealed differences between © XXXX American Chemical Society
green and yellow foxtail, as well. Both species have a unique genome size range or ranges.19 Green foxtail is diploid,19,20 whereas yellow foxtail has levels of ploidy ranging from diploid to octoploid.19,21 Management strategies for green and yellow foxtail rely mainly on acetyl coenzyme A carboxylase (ACCase, EC 6.4.1.2) inhibitors and acetolactate synthase (ALS, EC 4.1.3.18) inhibitors. Pyroxsulam is the latest triazolopyrimidine sulfonamide ALSinhibiting herbicide developed by Dow AgroSciences (Indianapolis, IN). Pyroxsulam provides selective broad spectrum postemergence control of key annual grass weeds such as wild oat (Avena spp.), blackgrass (Alopecurus myosuroides Huds), brome (Bromus spp.), ryegrass (Lolium spp.), barnyardgrass [Echinochloa crus-galli (L.) Beauv.], windgrass [Apera spica-venti (L.) Beauv.], yellow foxtail [S. pumila (Poir.) Roem. & Schult.], and a wide range of annual broadleaf weeds like cleavers (Galium aparine L.), common chickweed [Stellaria media (L.) Vill.], flixweed [Descurainia sophia (L.) Webb ex Prantl], hempnettle (Galeopsis tetrahit L.), and many others.19 Pyroxsulam is also registered to provide suppression of green foxtail [S. viridis (L.) Beauv.] and quackgrass [Elymus repens (L.) Gould].22 Received: Revised: Accepted: Published: A
March 30, 2017 July 6, 2017 August 3, 2017 August 3, 2017 DOI: 10.1021/acs.jafc.7b01453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
leaf using an electronic pipet (Mettler-Toledo Rainin, Oakland, CA). Plants were harvested 0, 8, 24, and 48 h after treatment (HAT). The treated leaves (TL) were rinsed twice with 5 mL of an aqueous 20% (v/v) solution of ethanol for 20 s, followed by a third wash with 5 mL of 100% chloroform for an additional 10 s. The chloroform wash was performed to remove any [14C]pyroxsulam that may have been imbedded in the cuticle waxes and did not cause chlorophyll leakage. Each plant was dissected in TL, shoot above treated leaves (ATL), shoot below treated leaves (BTL), and roots. Each plant part was wrapped with Kimwipes EX-L paper tissues (Kimberly Clark Corp., Roswell, GA) and then dried at room temperature prior to being combusted. The non-absorbed radioactivity on the leaf surface was determined by adding 15 mL of Ultima Gold scintillation cocktail (PerkinElmer Inc., Waltham, MA) to each of the 5 mL ethanol rinsates and 15 mL of Hionic-Fluor scintillation liquid (Packard BioScience B.V., Groningen, The Netherlands) to the 5 mL chloroform rinsate. The non-absorbed [14C]pyroxsulam was counted via liquid scintillation spectrometry (LSS) using a Beckman LS6000 scintillation counter (Beckman Coulter Inc., Fullerton, CA). After the samples had been dried, the radioactivity of each plant part was determined by combustion of the samples to 14CO2 using a Packard Sample Oxidizer (Packard Instruments Co., Downers Grove, IL), and the radioactivity was quantified by LSS. The amount of 14C present in the leaf washes and plant sections was considered as total 14C recovered, which averaged >95% of the applied [14C]pyroxsulam. The total radioactivity present in all plant parts was considered the amount of [14C]pyroxsulam absorbed and expressed as a percentage of the recovered radioactivity in each plant. Translocation of radioactivity out of TL was assessed by counting the radioactivity present in all plant parts, except the TL. Data for the translocation of radioactivity were expressed as a percentage of 14C absorbed. At each harvest time, a separate single unwashed plant was taken and placed on a phosphor imager plate for 1 week, and then an image was developed using a Molecular Dynamics Storm 820 Phosphoimager (Molecular Dynamics, Sunnyvale, CA) to visualize movement of radiolabeled material in the plant (Figures S1 and S2). Metabolism of [14C]Pyroxsulam. All procedures for growing, treating, and harvesting plant parts were similar to those described in the absorption and translocation section. At each harvest time (0, 8, 24, and 48 HAT), the TL were excised and rinsed twice with 5 mL of an aqueous 20% (v/v) solution of ethanol, followed by a third wash with 5 mL of 100% chloroform. Extraction of [14C]pyroxsulam and 14 C-labeled metabolites was immediately performed upon harvest. Treated leaves (0.04 g) were individually placed in an Eppendorf tube with one small ball bearing (1/8 in. 440C s/s, GenMills Inc.), and 1 mL of an aqueous 80% (v/v) solution of methanol was added for extraction in a Spex CertiPrep Geno/Grinder (SPEX SamplePrep, Metuchen, NJ). Treated leaves were ground twice for 1.5 min at a rate of 1500 strokes min−1, with a rotation of the rack between each grinding to achieve uniform grinding of the plant material, and then the samples were centrifuged for 2.5 min at 13200 rpm using an Eppendorf Centrifuge 5415 instrument (Eppendorf AG, Hamburg, Germany). The dark green supernatant was removed from the pellet and designated extract 1. The pellets were resuspended in 1 mL of an aqueous 80% (v/v) solution of methanol and centrifuged, and the supernatant was removed (extract 2). The content of 14C in both extracts was determined by analyzing 10 μL aliquots by LSS. If the radioactivity in the second extract was more than 10% of the total 14C in the combined radioactivity of extracts 1 and 2, the pellet was extracted a third time or until no radioactivity was observed in the 10 μL aliquot of the supernatant. All extracts were pooled and concentrated to dryness using a SpeedVac concentrator (Thermo Fisher Scientific, Waltham, MA). The resulting pellet was resuspended in a 10% (v/v) solution of acetonitrile in water and loaded onto an Alltech Alltima C-18 analytical column (250 mm × 4.6 mm) for purification and analysis by high-performance liquid chromatography (HPLC). A linear gradient from 10 to 100% acetonitrile over 20 min at a flow rate of 1 mL min−1 using an aqueous phase of 1% acetic acid in water was utilized to elute the parent pyroxsulam and metabolites for
Commercial formulations of pyroxsulam with the herbicide safener cloquintocet allow for selective weed control in cereal crops such as winter and spring wheat (Triticum aestivum L.), durum wheat (Triticum durum Desf.), winter rye (Secale cereale L.), and winter triticale (Triticosecale spp.). The objectives of this study were (1) to evaluate absorption, translocation, and metabolism patterns of pyroxsulam in both weed species, (2) to examine in vitro inhibition from partially enriched fractions of ALS, and (3) to determine the ALS gene sequences from both weed species to understand the mechanisms whereby green foxtail is less susceptible to pyroxsulam than yellow foxtail.
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MATERIALS AND METHODS
Plant Material. Seeds of green foxtail (S. viridis, SETVI) and yellow foxtail (S. pumila, SETPU) were planted in 729 cm3 plastic pots filled with a soil-less potting mix (Sun Gro MetroMix 306 Growing Media, Sun Gro Horticulture, Bellevue, WA) with a pH of 6.0−6.8 and an organic matter content of ∼30%. After emergence, green and yellow foxtail seedlings were thinned to six plants per pot and grown under greenhouse conditions with day and night temperatures of 23 and 22 °C, respectively, with a 14 h/10 h photoperiod at a relative humidity of 50−70%. Supplemental lighting was provided by overhead metal halide lights set to deliver a minimum of 500 μmol of photosynthetic photon flux density m−2 s−1. The plants were periodically watered as needed and fertilized weekly with a commercial fertilizer (Jack’s Professional LX, J. R. Peters, Inc., Allentown, PA) solution containing 200 mg L−1 nitrogen, 29 mg L−1 phosphorus, and 170 mg L−1 potassium for adequate propagation under greenhouse conditions. Whole-Plant Dose−Response Experiments. Green foxtail and yellow foxtail were treated at the four-leaf growth stage (5 cm tall). Plants were treated with 0, 0.23, 0.47, 0.94, 1.88, 3.75, 7.5, and 15 g of pyroxsulam ai ha−1 formulated as Simplicity. Pyroxsulam was applied with a compressed air-propelled track sprayer (Mandel Scientific Corp., Guelph, ON) equipped with a single Tee Jet 8002E flat-fan nozzle (Spraying System Co., Wheaton, IL), mounted 43 cm above the top of the plant canopy, set to deliver 187 L ha−1 at a speed of 3.2 km h−1 under an air pressure of 276 kPa. Pyroxsulam treatments also contained the crop oil concentrate adjuvant Assist at 0.8% volume per volume (v/v). Non-herbicide-treated green foxtail and yellow foxtail were sprayed with an aqueous 0.8% (v/v) solution of Assist crop oil concentrate. Visual assessment of herbicide control of green foxtail and yellow foxtail was made 21 days after treatment (DAT) on a scale of 0−100%, where 0% represents no control and 100% represents plant death. Radiolabeled Experiments. [14C]Pyroxsulam (Figure 1) was prepared by the Dow AgroSciences Specialty Synthesis Group
Figure 1. Chemical structure of pyroxsulam indicating the location of the 14C label. (Indianapolis, IN). A dimethyl sulfoxide (DMSO) concentrate of [14C]pyroxsulam with a specific activity of 1.35 MBq μmol−1 was solubilized in an aqueous 0.8% (v/v) solution containing Assist crop oil concentrate. Nonlabeled pyroxsulam was added to the radioactive solution to obtain 15 g ha−1 in a carrier volume of 187 L ha−1. Foliar Absorption and Translocation of [14C]Pyroxsulam. Green foxtail and yellow foxtail were treated at the four-leaf growth stage. Seven 1 μL droplets of the treatment solution containing 1.78 kBq of [14C]pyroxsulam were applied to the adaxial side of the third B
DOI: 10.1021/acs.jafc.7b01453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 2. Putative location of the primer annealing and amplicon size based on foxtail millet [Setaria italica (L.) P. Beauv.] reference CDS of the ALS gene (GenBank entry XP_004952560). The three primer pairs were derived from previous sequencing of resistant and susceptible barnyardgrass and were used to amplify the ALS gene in green and yellow foxtail. quantification on Waters Alliance 2690 Separation Modules (Waters Corp., Wilford, MA) coupled to a radioactive HPLC Flow Scintillation Analyzer (Packard, Meriden, CT). Pyroxsulam degradation as well as metabolite formation was monitored via HPLC and oxidation of the pellets to quantify non-extractable radioactivity. Acetolactate Synthase Extraction and Assay. Green foxtail and yellow foxtail were grown as described above. The leaves were harvested at the four-leaf growth stage for enzyme extraction. The procedure for ALS extraction, purification, and assay was slightly modified from that described by Singh et al.23 and Ray.24 All procedures were performed at 4 °C unless noted otherwise. The excised leaves (10 g) were frozen in liquid nitrogen and ground with 2.5 g of polyvinylpolypyrrolidone. The powdered tissue was homogenized in 15 mL of extraction buffer [100 mM potassium phosphate (pH 7.5), 10 mM sodium pyruvate, 5 mM magnesium chloride, 0.5 mM thiamine pyrophosphate (TPP), 10 μM flavine adenine dinucleotide (FAD), 5 mM dithiothreitol (DTT), 0.02% (w/ v) sodium azide, 5 mM ethylenediaminetetraacetic acid (EDTA), and 10% glycerol]. DTT was added at the time of homogenization. The homogenate was filtered through four layers of cheesecloth and centrifuged at 27000g for 20 min at 4 °C. Acetolactate synthase was precipitated with ammonium sulfate from the supernatant fluid collected after centrifugation. The supernatant was brought to 50% saturation with saturated ammonium sulfate, stirred for 20 min, and stored for 60−75 min at 4 °C. The ammonium sulfate suspension was then centrifuged at 27000g for 20 min at 4 °C, and the resulting pellet was suspended in 2 mL of elution buffer [100 mM potassium phosphate (pH 7.5), 20 mM sodium pyruvate, 5 mM magnesium chloride, 1 mM DTT, 0.02% (w/v) sodium azide, and 1 mM EDTA]. The suspension was desalted on a PD-10 Sephadex G-25 column equilibrated with elution buffer (GE Healthcare Bio-Sciences AB, Upsalla, Sweden). The partially purified proteins were quantified using the method of Bradford25 and further utilized to measure ALS activity. The eluted ALS preparation (100 μL) and herbicide solution (50 μL) were incubated in 350 μL of reaction buffer [20 mM potassium phosphate (pH 7.0), 20 mM sodium pyruvate, 5 mM magnesium chloride, 0.5 mM TPP, and 10 μM FAD] for 60 min at 37 °C. The enzyme was assayed in the presence of varying concentrations of pyroxsulam (0−400 μM, 2-fold serial dilutions). The herbicide stock solution was freshly prepared by dilution of an aliquot of formulated pyroxsulam (Simplicity) in reaction buffer. An assay with a blank formulation (no active ingredient) was used as a control to assess the enzyme activity in the absence of pyroxsulam. The reaction was stopped by addition of sulfuric acid (6 M, 50 μL). Acetolactate was assessed as described by Westerfield26 with the following modifications. After a 15 min incubation at 60 °C, 500 μL of creatine (5 g L−1 solution freshly prepared in water) and 500 μL of naphthol (50 g L−1 solution freshly prepared in 2.5 M sodium hydroxide) were added, and the solution was incubated for an additional 15 min at 60 °C. The amount of acetoin formed from acetolactate was determined colorimetrically at a wavelength of 530 nm using a spectrophotometer
(Beckman Instruments Inc., Fullerton, CA). The data were expressed as the percent of the control (enzyme reaction without herbicide). Acetolactate Synthase Gene Sequencing. For ALS gene sequencing, three biological replicates of both green and yellow foxtail were completed and compared. Genomic DNA from three individual plants was extracted from leaf tissue using the Invitrogen PureLink Genomic Plant DNA Purification Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Extracted DNA was dissolved in DNAase free water and quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). All samples were diluted with water to a concentration of 10 ng μL−1 and stored at −20 °C until they were used. Forward and reverse primer sequences for amplification of the ALS gene were ordered through Life Technologies (Thermo Fisher Scientific) and had the same sequence that was used for sequencing of the barnyardgrass ALS gene.27 Primer pairs (BYG_12, BYG_22, and BYG_31) covered three overlapping regions of the ALS gene and created amplicons approximately 756−875 bp in length (Figure 2). DNA Amplification and Sequencing. Polymerase chain reactions (PCR) were performed in 25 μL total volume mixtures each containing 21 μL of Invitrogen AccuPrime Pfx SuperMix (Thermo Fisher Scientific), 2 μL of 10 μM forward and reverse primers, and 2 μL of 10 ng μL−1 template DNA. Amplification reactions were conducted on an Eppendorf Mastercycler (Eppendorf, Hamburg, Germany) thermocycler with the following PCR profile: initial denaturation at 95 °C for 5 min followed by 30 cycles at 95 °C for 15 s, 64 °C (for BYG_12 and BYG_31 primer pairs) or 60 °C (for the BYG_22 primer pair) for 30 s, and 68 °C for 60 s, and a final extension at 72 °C for 5 min. The PCR products were resolved by electrophoresis on a 1% agarose gel stained with GelRed Nucleic Acid Gel Stain (Biotium, Fremont, CA), and bands were compared to a 100 bp reference DNA ladder (Norgen Biotek Corp., Thorold, ON). DNA Fragment Sequencing and Analysis. The PCR-amplified ALS gene fragments were viewed under a Dark Reader blue transilluminator (Clare Chemical Research, Dolores, CO) and cut from the gel. DNA was purified from the gel using a PureLink Quick Gel Extraction and PCR Purification Combo Kit (Thermo Fisher Scientific) following the manufacturer’s protocol. Purified fragments were sent for direct sequencing to MWG Eurofin (Huntsville, AL), where capillary electrophoresis Sanger sequencing methods were used. Strands were sequenced from both directions by mixing the purified amplicon with the respective forward or reverse primer. Sequences were analyzed using Sequencher version 5.1 (Gene Codes Corp., Ann Arbor, MI). Sequences were trimmed and aligned on the basis of overlapping regions from both forward and reverse sequences to give one contiguous gene sequence for each replicate. The replicated sequences were compared within species to validate reproducibility and identify polymorphisms. The green and yellow foxtail sequences were compared to known plant ALS gene sequences, namely, Arabidopsis thaliana [(L.) Heynh.] found in GenBank as entry NM_114714, Setaria italica [(L.) P. Beauv.] found in GenBank as C
DOI: 10.1021/acs.jafc.7b01453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Table 1. Percent Visual Control and GR50a Values for Green Foxtail and Yellow Foxtail 21 Days after Treatment with Pyroxsulam % visual injury weed species
0.23 g ai ha−1
0.47 g ai ha−1
0.94 g ai ha−1
1.88 g ai ha−1
3.75 g ai ha−1
7.5 g ai ha−1
15 g ai ha−1
GR50
green foxtail yellow foxtail LSD (p = 0.05)
12 b 47 a 10.4
20 b 63 a 9.0
28 b 75 a 12.2
49 b 80 a 10.0
75 b 86 a 4.6
77 b 90 a 2.8
81 b 94 a 4.3
2.03 0.23
GR50 [dose (g ai ha−1) that reduced plant growth by 50% as compared to the untreated control] values are calculated using JMP 9 (SAS Institute Inc., Cary, NC). Means within a column followed by the same letter are not significantly different according to the Student−Newman−Keuls LSD test (p = 0.05). a
entry XP_004952560, and Echinochloa crus-galli [(L.) Beauv.] found in GenBank as entry JX415271. Sequences were further analyzed using BioEdit28 to make DNA−protein translations and perform multisequence alignments. Comparisons were made to identify the in-frame codon locations for protein translation and to validate and compare the foxtail sequences to other plant ALS genes. Amino acid positions were based on the Arabidopsis ALS gene (NM_114714). Comparisons were made between species at the nucleic acid and protein levels. Experimental Design and Statistical Analysis. Data were analyzed with JMP Pro version 12.2.0 (SAS Institute Inc., Cary, NC) and subjected to analysis of variance (ANOVA). All experiments were repeated once, and the number of replicates was varied for each experiment. Means were separated by the Student−Newman−Keuls LSD test at the p = 0.05 level. The whole-plant dose−response experiments were arranged as randomized complete block design with seven replicates for each treatment, whereas the ALS assay experiments were conducted with three replicates per treatment. Nonlinear regression analysis was used to determine the dose of herbicide causing 50% visible growth reduction (GR50) for the dose−response experiments and 50% inhibition (IC50) of ALS activity. Equation 1 relates response y to herbicide dose x using a log−logistic procedure:29
y=C+
D−C 1 + exp{b[log(x) − log(X50)]}
yellow foxtail plants were severely damaged by pyroxsulam with an average of 94% visual injury at the rate of 15 g ai ha−1 (Table 1). In general, green foxtail was more tolerant to pyroxsulam than yellow foxtail was in this study. Regardless of the rate of pyroxsulam applied in this study, visual injury to green foxtail did not exceed 81% (Table 1). The GR50 values for the green and yellow foxtail were 2.0 and 0.23 g ai ha−1, respectively, indicating that green foxtail was 10 times more tolerant to pyroxsulam than yellow foxtail was. Differential responses between green and yellow foxtail were previously reported for other ALS-inhibiting herbicides, including foramsulfuron and primisulfuron.30 In their study, the authors noted that both ALS inhibitors exhibited better efficacy against yellow foxtail than against green foxtail. Previous work has shown that differences in herbicide absorption, translocation, and metabolism may lead to differential sensitivity between weeds within the same genus.31−33 Absorption and Translocation Experiments. The total recovery of applied [14C]pyroxsulam was greater than 95% in both green foxtail and yellow foxtail. Foliar absorption of [14C]pyroxsulam in green foxtail and yellow foxtail proceeded rapidly up to 24 HAT with little further absorption by 48 HAT. Maximal [14C]pyroxsulam absorption was 53% in green foxtail and 44% in yellow foxtail (Figure 3). Regardless of the harvest
(1)
where C is the lower limit of the response, D is the upper limit of the response, b is the slope, and X50 refers to the GR50 or IC50 dose causing 50% visible growth reduction or ALS inhibition. Absorption and translocation experiments were organized as a three-factor factorial: (1) foxtail species, (2) HAT, and (3) pyroxsulam dose, arranged in a randomized complete block design (RCBD). Each treatment had four replicates. Absorption data were expressed as a percentage of the total 14C recovered, whereas translocation data were expressed as a percentage of 14C absorbed by each plant. Blocks were pooled when block-related interactions were not significant. Metabolism experiments were organized in a factorial design with (1) foxtail species, (2) HAT, (3) pyroxsulam dose, and (4) metabolites as factors. Metabolism data for [14C]pyroxsulam and 14C-labeled metabolites were expressed as a percentage of the total 14C recovered in the treated leaves.
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RESULTS AND DISCUSSION Whole-Plant Dose−Response Experiments. Dose− response data indicated that visual injury to green and yellow foxtail increased as pyroxsulam rates increased (Table 1). Visual injury to both green and yellow foxtail was first observed 7 days after treatment (DAT), and final assessment of herbicide injury was performed 21 DAT. In general, the activity of pyroxsulam resulted in chlorosis of the foliage and stunting of the whole plant followed by foliar necrosis. Symptoms caused by this triazolopyrimidine sulfonamide were similar to injury caused by ALS herbicides observed on other grass weeds.27−29 The whole-plant experiments revealed a differential dose response to pyroxsulam between green and yellow foxtail. At 21 DAT,
Figure 3. Absorption of [14C]pyroxsulam in green foxtail (S. viridis, SETVI) and yellow foxtail (S. pumila, SETPU) during a 48 h treatment. Each point represents the mean and a 95% confidence interval for six replicates.
time, differences in the amount of [14C]pyroxsulam absorbed by green and yellow foxtail were negligible. The data suggest that mechanisms other than differential absorption may account for the difference in sensitivity observed between green and yellow foxtail. The amount of radioactivity translocated out of the TL into other plant parts increased over time and was the same in both D
DOI: 10.1021/acs.jafc.7b01453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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min, and metabolite 3 (Met 3) eluted at 7.1 min. Met 1 and 3 were observed in both species. However, metabolite 2 (Met 2) eluted at 10.3 min and was observed in only green foxtail (Figure 5). The major metabolite, Met 1, was previously identified as 5-hydroxy-pyroxsulam (Figure 6) and was inactive on wheat.34 The two minor metabolites (Met 2 and Met 3) have not been identified in the study presented here. Previous studies of pyroxsulam metabolism identified two other hydroxypyroxsulam metabolites; i.e., 2′-hydroxy-pyroxsulam and 7hydroxy-pyroxsulam were identified in wheat.35 Metabolites that corresponded to 5,7-dihydroxy-pyroxsulam, pyridinesulfonic acid, pyridine-sulfonamide, and amino-dimethoxytriazole pyrimidine were also detected in tissue extracts of wheat.35 These metabolites showed no herbicidal activity when they were tested on various weed and crop species (R. Gast and P. Schmitzer, Dow AgroSciences Internal Report, 2006). The retention times of Met 2 and Met 3 are very close to those of 2′-hydroxy-pyroxsulam and 7-hydroxy-pyroxsulam, respectively. Therefore, Met 2 could assumed to be the 2′-hydroxypyroxsulam metabolite, and Met 3 could be identified as the 7-hydrox-pyroxsulam metabolite (Figure 6). The treated leaf of green foxtail had less pyroxsulam remaining than did that of yellow foxtail 24 and 48 HAT, and the amount of pyroxsulam decreased over time for green foxtail (Figure 7). At 24 HAT, 76% of the radioactivity remaining in the treated leaf of yellow foxtail was pyroxsulam whereas only 59% of the radioactivity remaining in the treated leaf of green foxtail was pyroxsulam. Differential metabolism of pyroxsulam in the treated leaf became more apparent 48 HAT with 62 and 38% of the radioactivity remaining as pyroxsulam in the treated leaf of yellow and green foxtail, respectively (Figure 7). At 48 HAT, 50% of the radioactivity in the TL of green foxtail was identified as metabolite 1 whereas only 35% was Met 1 in yellow foxtail. Metabolite 2 detected in only green foxtail corresponded to 11% of the radioactivity counted, while a negligible amount (
