Mechanism of resistance to ACCase-inhibiting herbicides in wild oat (Avena fatua ) from Latin America

Whole-plant response of two suspected resistant Avena fatua biotypes from Chile and Mexico to ACCase-inhibiting herbicides [aryloxyphenoxypropionate (APP), cyclohexanedione (CHD), and pinoxaden (PPZ)] and the mechanism behind their resistance were studied. Both dose-response and ACCase enzyme activity assays revealed cross-resistance to the three herbicide families in the biotype from Chile. On the other hand, the wild oat biotype from Mexico exhibited resistance to the APP herbicides and cross-resistance to the CHD herbicides, but no resistance to PPZ. Differences in susceptibility between the two biotypes were unrelated to absorption, translocation, and metabolism of the herbicides. PCR generated fragments of the ACCase CT domain spanning the potential mutations sited in the resistant and susceptible biotypes were sequenced and compared. A point mutation was detected in the aspartic acid triplet at the amino acid position 2078 in the Chilean biotype and in isoleucine at the amino acid position 2041 in the Mexican wild oat biotype, which resulted in a glycine triplet and an asparagine triplet, respectively. On the basis of in vitro assays, the target enzyme (ACCase) in these resistant biotypes contains a herbicide-insensitive form. This is the first reported evidence of resistance to pinoxaden in A. fatua.     

Lolium rigidum, a pool of resistance mechanisms to ACCase inhibitor herbicides

Three diclofop-methyl (DM) resistant biotypes of Lolium rigidum (R1, R2, and R3) were found in different winter wheat fields in Spain, continuously treated with DM, DM + chlortoluron, or DM + isoproturon. Herbicide rates that inhibited shoot growth by 50% (ED50) were determined for DM. There were found that the different biotypes exhibited different ranges of resistance to this herbicide; the resistant factors were 7.2, 13, and 36.6, respectively. DM absorption, metabolism, and effects on ACCase isoforms were examined in these biotypes of L. rigidum. The most highly resistant, biotype R3, contained an altered isoform of ACCase. In biotype R2, which exhibited a medium level of resistance, there was an increased rate of oxidation of the aryl ring of diclofop, a reaction most likely catalyzed by a cytochrome P450 enzyme. In the other biotype, R1, DM penetration was significantly less than that observed in the resistant (R2 and R3) and susceptible (S) biotypes. Analysis of the leaf cuticle surface by scanning electron microscopy showed a greater epicuticular wax density in the leaf cuticles of biotype R1 than in the other biotypes.     

Inhibition of ACCase220 and ACCase240 isozymes from sethoxydim-resistant and -susceptible maize hybrids

Acetyl-coenzyme A carboxylase (ACCase) isozymes were separated from cyclohexanedione-resistant and -susceptible maize. ACCase240 from resistant maize was 3.7-, >77-, and 12.8-fold more resistant to inhibition by clethodim, sethoxydim, and tralkoxydim, respectively, than ACCase240 from susceptible maize. The resistant ACCase240 preparation had 3.0-fold more protein and 14.5-fold lower specific activity than susceptible ACCase240. Resistant ACCase240 has a V(max) 5.5-fold lower than that of susceptible ACCase240, whereas apparent K(m) values were similar. ACCase220 from resistant maize was >25- and 7.2-fold more resistant to inhibition by sethoxydim and tralkoxydim, respectively, than susceptible ACCase220 but was inhibited to the same extent by clethodim. In summary, sethoxydim-resistant corn has an altered herbicide-resistant ACCase220 isozyme and increased expression of a less efficient, herbicide-resistant ACCase240 isozyme. However, to what extent alteration of both isozymes contributes to sethoxydim resistance is not clear.     

Evaluation and management of acetyl-CoA carboxylase inhibitor resistant littleseed canarygrass (Phalaris minor) in Pakistan

A field survey was conducted for the sampling of Acetyl-CoA carboxylase (ACCase) inhibitor resistance littleseed canarygrass, a major weed of wheat, from Punjab, Pakistan in 2014 for confirmation of resistance. The surveyed regions encompassed four different cropping systems including rice–wheat, maize–wheat, cotton–wheat and mixed cropping. Dose–response assay was conducted for confirmation of resistance. Efficacy of herbicide mixtures including clodinafop–propargyl, metribuzin, pinoxaden and sulfosulfuron at a range of doses was investigated to manage littleseed canarygrass. Results revealed that all populations were resistant to fenoxaprop except PM-BWL-2. The higher level resistance (6.5) was found in populations collected from rice–wheat cropping system. The tested herbicide mixtures at 75% and 100% of the recommended dose of each mixture component provided the effective control of resistant littleseed canarygrass. Mixtures at 50% provided more than 80% control and reduced growth and seed production potential of surviving plants. The confirmation of ACCase inhibitor resistance as the first case of herbicide resistance in Pakistan, leads us to discourage use of ACCase inhibitor herbicides alone. However, herbicide mixtures at 75% and 100% of the recommended dose are suggested to manage this weed for sustainable wheat production in the surveyed cropping systems.     

Late watergrass (Echinochloa phyllopogon): mechanisms involved in the resistance to fenoxaprop-p-ethyl

Fenoxaprop-p-ethyl (FE), 2-[4-[(6-chloro-2-benzoxazolyl)oxy]phenoxy] propanoate, ethyl ester (R), is an aryloxyphenoxypropionate herbicide for postemergence control of annual and perennial grasses in paddy fields; its site of action is acetyl-coenzyme A carboxylase (ACCase), an enzyme in fatty acids biosynthesis. The possible mechanism(s) of resistance to FE in a resistant biotype of Echinochloa phyllopogon was examined, namely, absorption, translocation, and metabolism of FE and ACCase susceptibility to fenoxaprop acid (FA). Studies of the in vitro inhibition of ACCase discounted any differential active site sensitivity as the basis of resistance to FE. There were differences in absorption rates between biotypes from 3 to 48 h after application (HAA). Biotypes did not differ in either the amounts or the rates of FE translocated; 98% of applied [14C]FE remaining in the treated leaf. However, there was a good correlation between the rate of herbicide metabolism and the plant resistance. The R biotype produced 5-fold less FA and approximately 2-fold more nontoxic (polar) metabolites 48 HAA than the S biotype. Moreover, the higher rate of GSH conjugation in the resistant biotype as compared to the susceptible one indicates that GSH and cysteine conjugation is the major mechanism of resistance of the R biotype against FE toxicity.     

Response of selected horseweed (Conyza canadensis (L.) Cronq.) populations to glyphosate

Horseweed (Conyza canadensis (L.) Cronq.) seed was collected in Illinois, Indiana, Kentucky, Mississippi, Missouri, and Ohio to determine susceptibility of different horseweed biotypes to glyphosate. Horseweed resistant to glyphosate was found in Mississippi, Ohio, and western Tennessee. In a separate experiment examining Tennessee biotypes, a dose response curve demonstrated that four times as much glyphosate was needed to achieve a 50% fresh weight reduction (GR(50)) in resistant biotypes when compared to a susceptible biotype. Resistant biotypes from Tennessee displayed a GR(50) of 1.6 kg/ha as compared to a GR(50) of 0.4 kg/ha in a susceptible horseweed population. Although growth was reduced, the resistant plants did not completely die and could potentially produce seed. Variation in glyphosate resistance was found among the populations tested.     

Monoclonal-based ELISA for the identification of herbicidal cyclohexanedione analogues that inhibit graminaceous acetyl coenzyme-A carboxylase

Cyclohexanediones are one of four known structural classes of herbicides that inhibit graminaceous acetyl coenzyme-A carboxylase (ACCase; EC 6.4.1.2). Five monoclonal antibodies were raised against cyclohexanediones conjugated to bovine serum albumin. Cross-reactivity studies using a homologous competitive indirect enzyme-linked immunosorbent assay (ciELISA) against 24 cyclohexanedione analogues revealed that two monoclonal antibodies (mAb A and mAb B) could segregate the analogues into active and inactive ACCase inhibitors on the basis of the analogue concentration required to inhibit 50% of antibody binding to the coating conjugate (IC(50)). Both mAb A and mAb B were also found to cross-react with various members of the indolizidinedione structural class of ACCase inhibitors in ciELISA, suggesting that both cyclohexanediones and indolizidinediones possess features recognized by monoclonal antibodies important for the inhibition of ACCase activity. In conclusion, pharmacophore-specific antibodies may be potentially valuable screening tools for the identification of new lead chemistries in a pesticide discovery program.     

Physiological and biochemical characterization of quinclorac resistance in a false cleavers (Galium spurium L.) biotype

The physiological and biochemical basis for quinclorac resistance in a false cleavers (Galium spurium L.) biotype was investigated. There was no difference between herbicide resistant (R) and susceptible (S) false cleavers biotypes in response to 2,4-D, clopyralid, glyphosate, glufosinate-ammonium, or bentazon. On the basis of GR(50) (growth reduction of 50%) or LD(50) (lethal dose to 50% of tested plants) values, the R biotype was highly resistant to the acetolactate synthase (ALS) inhibitor, thifensulfuron-methyl (GR(50) resistance ratio R/S = 57), and quinolinecarboxylic acids (quinclorac R/S = 46), resistant to MCPA (R/S = 12), and moderately resistant to the auxinic herbicides picloram (R/S = 3), dicamba (R/S = 3), fluroxypyr (R/S = 3), and triclopyr (R/S = 2). The mechanism of quinclorac resistance was not due to differences in [(14)C]quinclorac absorption, translocation, root exudation, or metabolism. Seventy-two hours after root application of quinclorac, ethylene increased ca. 3-fold in S but not R plants when compared to controls, while ABA increased ca. 14-fold in S as opposed to ca. 3-fold in R plants suggesting an alteration in the auxin signal transduction pathway, or altered target site causes resistance in false cleavers. The R false cleavers biotype may be an excellent model system to further examine the auxin signal transduction pathway and the mechanism of quinclorac and auxinic herbicide action.     

Phytotoxic effects of wheat extracts on a herbicide-resistant biotype of annual ryegrass (Lolium rigidum)

Thirty-nine wheat accessions were used to evaluate their extract phytotoxicity against annual ryegrass (Lolium rigidum Gaud.). Aqueous extracts of wheat shoot residues significantly inhibited the germination and root growth of a biotype of annual ryegrass resistant to herbicides of acetyl CoA carboxylase inhibitors (group A), acetolactate synthase inhibitors (B), photosystem II inhibitors (C), and tubulin formation inhibitors (D). The germination of the herbicide resistant (HR) biotype was inhibited by 3-100%, depending upon the wheat accession. The phytotoxic effects on ryegrass root growth ranged from 12% stimulation to 100% inhibition, compared to a control. The germination and root growth of a herbicide-susceptible (HS) biotype of annual ryegrass were also inhibited by the wheat extracts, with germination inhibited by 4-100%, and root growth by 19-100%. Bioassays with two known wheat allelochemicals showed that p-coumaric acid and propionic acid significantly inhibited the growth of both HR and HS biotypes of annual ryegrass. The two compounds completely inhibited the root growth of HR ryegrass at concentrations greater than 5.0 mM. In comparison with p-coumaric acid, propionic acid was more inhibitory to seed germination, shoot, and root growth of both ryegrass biotypes. The root growth of the HR biotype was more sensitive when exposed to wheat extracts, to p-coumaric acid, and to propionic acid. The results suggest that residues of certain wheat cultivars with strong allelopathic potential could provide a nonherbicidal alternative for the management of herbicide-resistant weed species.     

Pool of resistance mechanisms to glyphosate in Digitaria insularis

Digitaria insularis biotypes resistant to glyphosate have been detected in Brazil. Studies were carried out in controlled conditions to determine the role of absorption, translocation, metabolism, and gene mutation as mechanisms of glyphosate resistance in D. insularis. The susceptible biotype absorbed at least 12% more (14)C-glyphosate up to 48 h after treatment (HAT) than resistant biotypes. High differential (14)C-glyphosate translocation was observed at 12 HAT, so that >70% of the absorbed herbicide remained in the treated leaf in resistant biotypes, whereas 42% remained in the susceptible biotype at 96 HAT. Glyphosate was degraded to aminomethylphosphonic acid (AMPA), glyoxylate, and sarcosine by >90% in resistant biotypes, whereas a small amount of herbicide (up to 11%) was degraded by the susceptible biotype up to 168 HAT. Two amino acid changes were found at positions 182 and 310 in EPSPS, consisting of a proline to threonine and a tyrosine to cysteine substitution, respectively, in resistant biotypes. Therefore, absorption, translocation, metabolism, and gene mutation play an important role in the D. insularis glyphosate resistance.     

Design and syntheses of novel phthalazin-1(2H)-one derivatives as acetohydroxyacid synthase inhibitors

A series of 2-substituted-8-(4,6-dimethoxypyrimidin-2-yloxy)-4-methylphthalazin-1-one derivatives, 7a-7w, were designed via an ortho-substituent cyclization strategy to discover a new herbicidal lead structure. These compounds were synthesized by a seven-step route using 3-hydroxy-acetophenone as a starting material. Determination of the Ki values against wild-type A. thaliana acetohydroxyacid synthase (AHAS) (EC 4.1.3.18) indicated that some of the compounds displayed good enzyme inhibition activity comparable to that of KIH-6127. The further preliminary bioassay data on weeds showed that the synthesized compounds exhibited typical injury symptoms of AHAS-inhibiting herbicides, and some of them showed broad-spectrum and high herbicidal activities in postemergence treatments against Echinochloa crusgalli, Digitaria sanguinalis, Setaria viridis, Brassica juncea, Amaranthus retroflexus, and Chenopodium album at an application rate of 150 g ai/ha. To our knowledge, this is the first report of methylphthalazin-1-one derivatives as AHAS inhibitors.     

小麦田杂草种类及危害情况调查

调查掌握小麦田杂草种类、分布和危害程度,可为筛选高效除草药剂提供科学依据。对定西市不同区域的小麦田杂草发生情况开展了调查,结果表明,定西市共有麦田杂草15科38种,其中单子叶杂草6种,双子叶杂草32种。安定区、通渭县、陇西县、临洮县以及渭源县北部等干旱半干旱区分布较多的杂草有12种,渭源县南部以及漳县、岷县等二阴区分布较多的杂草有13种。有8个杂草优势种,其中苦苣菜、藜、狗尾草、灰绿藜、菊叶香藜等5种杂草危害较重,危害等级达到Ⅲ级;苦苣菜、藜、狗尾草为绝对优势种,危害最重。杂草优势种是防除的重点对象,建议结合杂草特性,筛选高效除草剂加以科学防除。     

Shikimate accumulates in both glyphosate-sensitive and glyphosate-resistant horseweed (Conyza canadensis L. Cronq.)

Horseweed (Conyza canadensis) is a cosmopolitan weed that commonly grows throughout North America. Horseweed that is not completely controlled by normal applications of glyphosate has been reported in western Tennessee. This research had three objectives: (1) to develop and validate an analytical procedure for the quantitative determination of shikimate, an important indicator of glyphosate activity in plants; (2) to confirm resistance to glyphosate in a horseweed population; and (3) to examine the accumulation of shikimate in both glyphosate-resistant and glyphosate-susceptible horseweed plants. The analytical procedure to determine shikimate used extraction with 1 M HCl for 24 h, followed by liquid chromatography using photodiode array detection, and shikimate recoveries were >or=82%. Glyphosate applications of both 0.84 kg ae/ha (the standard application rate) and 3.8 kg ae/ha to susceptible plants caused complete plant death. The same glyphosate applications to putative resistant populations caused less than 15% growth reduction as determined by visual evaluations, and fresh weights of these resistant plants 17 days after glyphosate treatment (DAT) were reduced an average of 45% in one population and were not affected in a different population. This direct comparison conclusively confirms that horseweed plants collected in western Tennessee in 2002 are resistant to 4 times the normal application dosage of glyphosate. The glyphosate-resistant horseweed biotypes still exhibited some herbicidal effects from the glyphosate, such as yellowing in the most actively growing, apical shoot meristems. The yellowing in the shoot apexes was transitory, and the plants recovered from this damage. Shikimate concentrations in all untreated horseweed plants were less than 100 microg/g, which was significantly less than that in all plants which had been treated with 0.84 kg ae/ha of glyphosate. Unexpectedly, shikimate accumulated (>1000 microg/g) in both resistant populations and the susceptible population. However, there were differences in shikimate accumulation patterns between resistant and susceptible horseweed biotypes. Shikimate concentrations in resistant populations declined about 40% from 2 to 4 DAT, while shikimate concentrations in the susceptible horseweed plants increased about 35% from 2 to 4 DAT. The confirmed resistance of a widespread weed implies that alternative control strategies for glyphosate-resistant horseweed will be needed in those no-tillage production systems where it commonly occurs.     

Phytotoxins from the leaves of Laggera decurrens.

Upon biological screening of a series of African medicinal plants, substantial phytotoxic activity was found in the leaves of Laggera decurrens (Vahl.) Hepper & Wood (Asteraceae), using a Lemna minor bioassay. Bioassay-guided fractionation of the leaves led to the isolation of two physiologically active compounds: 3-hydroxythymoquinone and 5-acetoxy-2-hydroxythymol, causing death of Lemna minor in the 25-100 microM range. Symptoms were a rapidly developing chlorosis, followed by necrosis of fronds. The compounds also inhibited growth and germination of the grass weed Agrostis capillaris down to 250 microM. The mode of action of both compounds could not be elucidated, but they do not appear to be photosystem II inhibitors.     

Influence of Harvest Time and Stubble Height on Weed Seedling Recruitment in a Green Cereal

A long-term field experiment was conducted in southern Sweden to evaluate the effect of harvest before crop ripeness on weed infestation. In addition, two stubble heights, 10 cm and 30 cm, were compared. The aim was to determine the potential of the treatments in weed regulation. The harvests were performed on three occasions: milk-ripeness (I), which is the normal harvest time for a green cereal, yellow-ripeness (II) and binder-ripeness (III). In control plots, harvested at full ripeness, herbicides were used in all years. Weed density initially increased exponentially in all treatments except for the control, but had a tendency to level off after 4 years. In all years weed density was higher when harvest was delayed and the stubble was high. Harvest at milk ripeness (I) favoured short-season annuals such as Stellaria media (L) Vill and worked against late-maturing species such as Polygonum tomentosum (Schrank). Weed density differed between the treatment plots during the last year of the experiment. In the treatment harvested at stage I combined with low stubble height, weed density was only slightly higher than in the control. Thus, when harvesting a green cereal, herbicides can be avoided.     

Degradation of cyhalofop-butyl (CyB) by Pseudomonas azotoformans strain QDZ-1 and cloning of a novel gene encoding CyB-hydrolyzing esterase

Cyhalofop-butyl (CyB) is a widely used aryloxyphenoxy propanoate (AOPP) herbicide for control of grasses in rice fields. Five CyB-degrading strains were isolated from rice field soil and identified as Agromyces sp., Stenotrophomonas sp., Aquamicrobium sp., Microbacterium sp., and Pseudomonas azotoformans; the results revealed high biodiversity of CyB-degrading bacteria in rice soil. One strain, P. azotoformans QDZ-1, degraded 84.5% of 100 mg L(-1) CyB in 5 days of incubation in a flask and utilized CyB as carbon source for growth. Strain QDZ-1 could also degrade a wide range of other AOPP herbicides. An esterase gene, chbH, which hydrolyzes CyB to cyhalofop acid (CyA), was cloned from strain QDZ-1 and functionally expressed. A chbH-disrupted mutant dchbH was constructed by insertion mutation. Mutant dchbH could not degrade and utilize CyB, suggesting that chbH was the only esterase gene responsible for CyB degradation in strain QDZ-1. ChbH hydrolyzed all AOPP herbicides tested as well as permethrin. The catalytic efficiency of ChbH toward different AOPP herbicides followed the order quizalofop-P-ethyl ≈ fenoxaprop-P-ethyl > CyB ≈ fluazifop-P-butyl > diclofop-methyl ≈ haloxyfop-P-methyl; the results indicated that the chain length of the alcohol moiety strongly affected the biodegradability of the AOPP herbicides, whereas the substitutions in the aromatic ring had only a slight influence.     

Interaction of cyclohexanediones with acetyl coenzyme-A carboxylase and an artificial target-site antibody mimic: a comparative molecular field analysis

Similarities and differences between steric and electrostatic potentials of a monoclonal-antibody-based surrogate of a herbicide target-site and its in vitro enzyme target were investigated using three-dimensional quantitative structure-activity relationship comparative molecular field analysis (3D-QSAR CoMFA). Two separate, five-component, partial least squares CoMFA models were developed to compare the interaction of cyclohexanedione herbicides with their target site, acetyl coenzyme-A carboxylase (ACCase; EC 6.4.1.2) and a cyclohexanedione pharmacophore-specific monoclonal antibody (mAb A). On the basis of CoMFA models, similarities in steric and electrostatic requirements around position 2 of the binding site for the oxime functional group of the cyclohexanedione molecule appear to be crucial for interaction of the herbicide with both ACCase and mAb A. These similarities explain the observed quantitative relationship between binding of cyclohexandedione herbicides to ACCase mAb A. Furthermore, these results support the production and use of mAb-based surrogates of pesticide targets as screening tools in pesticide discovery programs.     

Determination of antagonism between cyhalofop-butyl and other rice (Oryza sativa) herbicides in barnyardgrass (Echinochloa crus-galli)

Herbicide antagonism is defined as the reduction of control of certain weeds as the result of applying mixtures of two or more herbicides. Cyhalofop-butyl, a graminicide used for postemergence grass weed control in rice, is antagonized by some rice herbicides when applied simultaneously. The result of this type of antagonism usually results in decreased control of grass weeds. Research has shown that herbicide antagonism between graminicides and other herbicides may be caused by different mechanisms as the result of activity of the tank-mix partner. Using HPLC, the objective of this experiment was to analyze the fate of cyhalofop-butyl in barnyardgrass tissue when applied alone and in combination with halosulfuron, propanil, or triclopyr. Results indicated that absorption of cyhalofop-butyl and hydrolysis to its phytotoxic metabolite, cyhalofop-acid, was rapid and that halosulfuron and triclopyr had no effect. Because of a likely interaction of propanil with an apoplastic esterase enzyme, increased levels of cyhalofop-butyl and cyhalofop-acid were detected in barnyardgrass tissue, indicating that cyhalofop-butyl metabolism was hindered by propanil.     

Structure-activity relationship studies of benzoxazinones and related compounds. Phytotoxicity on Echinochloa crus-galli (L.) P. Beauv

Echinochloa crus-galli (E. crus-galli; barnyardgrass) is a weed widely distributed. It constitutes a serious weed problem in 42 countries and has been found in at least 27 more. It is the world's main weed of rice affecting up to 36 crops worldwide. Several biotypes of this plant, with resistance to herbicides with different modes of action have evolved. In our ongoing studies regarding the potential application of benzoxazinones and their soil degradation products for weed control, a complete structure-activity relationships (SARs) study was made by using barnyardgrass as the target plant. Compounds used in this study were previously tested on a wide variety of standard target species (STS), and they include natural allelochemicals 2-O-beta-D-glucopyranosyl-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA-Glc), 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA), and 2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA), together with some degradation derivatives found in wheat crop soil and some synthetic analogues. Their phytotoxicity on E. crus-galli is discussed and compared with the results obtained from previous screening. This work constitutes the next step in the search for natural herbicide models based on benzoxazinones and their degradation products. The most active compounds were the degradation product 2-aminophenol (APH) and the synthetic analogue 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA). Their activities confirm the direction proposed in our previous SAR study, which establishes D-DIBOA to be the best lead for natural herbicide model development with benzoxazinone structure.     

Origin of enantiomeric selectivity in the aryloxyphenoxypropionic acid class of herbicidal acetyl coenzyme A carboxylase (ACCase) inhibitors

Molecular modeling was used to propose an "active conformation" for the R-2-phenoxypropionic acid portion of the aryloxyphenoxypropionic acid series of herbicidal acetyl CoA carboxylase (ACCase) inhibitors. This candidate active conformation is a low-energy conformer with the R-methyl distal to the phenoxy fragment, stabilized by the generalized anomeric effect around the propionate ether bond; the inactive S-enantiomer has difficulty accessing this conformation due to steric interaction of the S-methyl with the o-hydrogen of the phenyl. This candidate conformation was challenged by preparation of a series of novel rigid analogues. ACCase inhibition data suggest that the systems which contain a fused five-membered, but not a six-membered, ring present the necessary pharmacophore to the active site of ACCase, confirming the active conformation hypothesis and demonstrating that the precise placement of the carboxylate relative to the phenyl group is more critical than the placement of the methyl.