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Research was conducted using a functional malachite green colorimetric assay to evaluate acetyl-coenzyme A carboxylase (ACCase) activity previously identified as resistant to sethoxydim and select aryloxyphenoxypropionate (FOPs) herbicides, fenoxaprop, and fluazifop. Two resistant southern crabgrass [Digitaria ciliaris (Retz.) Koeler] biotypes, R1 and R2, containing an Ile-1781-Leu amino acid substitution and previously identified as resistant to sethoxydim, pinoxaden, and fluazifop but not clethodim was utilized as the resistant chloroplastic ACCase source compared with known susceptible (S) ACCase. Dose-response studies with sethoxydim, clethodim, fluazifop-p-butyl, and pinoxaden (0.6 to 40 µM) were conducted to compare the ACCase–herbicide interactions of R1, R2, and S using the malachite green functional assay. Assay results indicated that R biotypes required more ACCase-targeting herbicides to inhibit ACCase activity compared with S. IC50 values of all four herbicides for R biotypes were consistently an order of magnitude greater than those of S. No sequencing differences in the carboxyltransferase domain was observed for R1 and R2; however, R2 IC50 values were greater across all herbicides. These results indicate the malachite green functional assay is effective in evaluating ACCase activity of R and S biotypes in the presence of ACCase-targeting herbicides, which can be used as a replacement for the 14C-based radiometric functional assays.

One putative-resistant (R) johnsongrass (Sorghum halepense L. Moench) population, originating from a cotton monoculture field in northern Greece, was evaluated for the possible evolution of cross-resistance to acetyl-CoA carboxylase (ACCase)- and multiple resistance to acetolactate synthase (ALS)-inhibiting herbicides, and to elucidate the levels and underlying mechanisms of resistance. Whole-plant rate-response assays showed that the R population was highly cross-resistant to the post-emergence applied ACCase-inhibiting herbicides fluazifop-P-butyl, propaquizafop (aryloxyphenoxypropionates) and cycloxydim (cyclohexanedione), but susceptible to the ACCase-inhibitor clethodim (cyclohexanedione) and the ALS-inhibitor nicosulfuron. The analysis of the ACCase gene sequence revealed a point mutation (ATA to WTA/TTA) at 1781 residue in the CT domain of ACCase, resulting in an amino acid substitution from isoleucine (Ile) to leucine (Leu). However, all sequenced plants of the S johnsongrass population were found with the wild-type allele encoding Ile-1781. The R johnsongrass population, grown without competition, produced more fresh weight, rhizome biomass and number of panicles than the S population. These findings indicate clearly that the R johnsongrass population has evolved target-site cross-resistance to three ACCase-inhibitors that increased most of its growth traits as compared with the S population, suggesting a fitness advantage associated with the ACCase Leu-1781 mutation.

Mutations conferring evolved herbicide resistance in weeds are known in nine different herbicide sites of action. This review summarizes recently reported resistance-conferring mutations for each of these nine target sites. One emerging trend is an increase in reports of multiple mutations, including multiple amino acid changes at the glyphosate target site, as well as mutations involving two nucleotide changes at a single amino acid codon. Standard reference sequences are suggested for target sites for which standards do not already exist. We also discuss experimental approaches for investigating cross-resistance patterns and for investigating fitness costs of specific target-site mutations.

This research has been supported by the Spanish Government, through project AGL2017-83325-C4-2-R (AEI/FEDER/UE). JT acknowledges support from the Spanish Ministry of Science, Innovation and Universities (grant Ramon y Cajal RYC2018-023866-I).

Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is a noxious grass weed that infests rice fields and causes huge crop yield losses. In this study, we collected 12 E. crus-galli populations from rice fields of Ningxia Province in China and investigated the resistance levels to the acetolactate synthase (ALS) inhibitor penoxsulam and the acetyl-CoA carboxylase (ACCase) inhibitor cyhalofop-butyl. The results showed that eight populations exhibited resistance to penoxsulam and four populations evolved resistance to cyhalofop-butyl. Moreover, all four cyhalofop-butyl–resistant populations (NX3, NX4, NX6, and NX7) displayed multiple herbicide resistance to both penoxsulam and cyhalofop-butyl. The alternative herbicides bispyribac-sodium, metamifop, and fenoxaprop-p-ethyl cannot effectively control the multiple herbicide–resistant (MHR) plants. To characterize the molecular mechanisms of resistance, we amplified and sequenced the target site–encoding genes in resistant and susceptible populations. Partial sequences of three ALS genes and six ACCase genes were examined. A Trp-574-Leu mutation was detected in EcALS1 and EcALS3 in two high-level (65.84- and 59.30-fold) penoxsulam-resistant populations, NX2 and NX10, respectively. In addition, one copy (EcACC4) of ACCase genes encodes a truncated aberrant protein due to a frameshift mutation in E. crus-galli populations. None of the amino acid substitutions that are known to confer herbicide resistance were detected in ALS and ACCase genes of MHR populations. Our study reveals the wide spread of MHR E. crus-galli populations in Ningxia Province that exhibit resistance to several ALS and ACCase inhibitors. Non–target site based mechanisms are likely to be involved in E. crus-galli resistance to the herbicides, at least in four MHR populations.

The resistance to fenoxaprop-P-ethyl, a herbicide that inhibits acetyl-CoA carboxylase (ACCase), has emerged in shortawn foxtail (Alopecurus aequalis Sobol.) since the 1990s, presenting a considerable challenge to wheat (Triticum aestivum L.) production in China. One of the primary mechanisms responsible for this high-level resistance is the presence of mutations at codons 1781, 2041, and 2078 in the ACCase gene. However, the conventional methods used to detect these mutations, such as polymerase chain reaction (PCR) and gene sequencing, are time-consuming and labor-intensive. To address this issue and enable the prompt and effective detection of these common ACCase mutations in A. aequalis, a loop-mediated isothermal amplification (LAMP) strategy was developed. The LAMP assay specifically targets the Ile-1781-Leu and Asp-2078-Gly mutations within the ACCase gene of A. aequalis. Through the optimization of primers, systems, and conditions, the LAMP assay enables rapid differentiation between wild-type individuals and mutants of A. aequalis carrying either of these two mutations. Including SYBR Green I dye in the final reaction mixtures enables detection of the target mutation through a noticeable color change that can be observed with the naked eye. It is noteworthy that the sensitivity of the LAMP assay was approximately 104-fold greater than that of conventional PCR methods. Additionally, a derived cleaved amplified polymorphic sequence (dCAPS) assay was established for each mutation to distinguish between homozygous and heterozygous mutants. Overall, the developed LAMP assay could efficiently detect the Ile-1781-Leu and Asp-2078-Gly mutations in the ACCase gene of A. aequalis, offering significant advantages for the monitoring and management of fenoxaprop-P-ethyl resistance.

Asia minor bluegrass (Polypogon fugax) is a common and problematic weed throughout China. P. fugax that is often controlled by acetyl-CoA carboxylase (ACCase) inhibitors in canola fields. Herein, we confirmed a P. fugax population (R) showing resistance to all ACCase inhibitors tested with resistance indexes ranging from 5.4–18.4. We further investigated the resistance mechanisms of this R population. Molecular analyses revealed that an amino acid mutation (Asp-2078-Gly) was present in the R population by comparing ACCase gene sequences of the sensitive population (S). In addition, differences in susceptibility between the R and S population were unlikely to be related to herbicide metabolism. Furthermore, a new derived cleaved amplified polymorphic sequence (dCAPS) method was developed for detecting the Asp-2078-Gly mutation in P. fugax efficiently. We found that 93.75% of plants in the R population carried the Asp-2078-Gly mutation, and all the herbicide-resistant phenotype of this R population is inseparable from this mutation. This is the first report of cross resistance to ACCase inhibitors conferred by the Asp-2078-Gly target-site mutation in P. fugax. The research suggested the urgent need to improve the diversity of weed management practices to prevent the widespread evolution of herbicide resistance in P. fugax in China.

The geographical structure of resistance to herbicides inhibiting acetyl-coenzyme A carboxylase (ACCase) was investigated in the weed Alopecurus myosuroides (black-grass) across its geographical range to gain insight into the process of plant adaptation in response to anthropogenic selective pressures occurring in agricultural ecosystems. We analysed 297 populations distributed across six countries in A. myosuroides' main area of occupancy. The frequencies of plants resistant to two broadly used ACCase inhibitors and of seven mutant, resistant ACCase alleles were assessed using bioassays and genotyping, respectively. Most of the resistance was not endowed by mutant ACCase alleles. Resistance and ACCase allele distribution patterns were characterized by mosaicism. The prevalence of resistance and of ACCase alleles differed among countries. Resistance clearly evolved by redundant evolution of a set of resistance alleles or genes, most of which remain unidentified. Resistance in A. myosuroides was shaped by variation in the herbicide selective pressure at both the individual field level and the national level.

Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is a dominant weed species occurring in rice (Oryza sativa L.) fields across China. Metamifop, a common herbicide, is frequently applied to control E. crus-galli and other grassy weeds in rice fields. Herein, HS01, an E. crus-galli population suspected to be resistant (R) to metamifop, was collected from Hanshan County in Anhui Province, China. Whole-plant dose–response testing revealed that, compared with the susceptible (S) population FD03, HS01 had developed high-level resistance to metamifop with a resistance index (RI) of 11.76 and showed cross-resistance to cyhalofop-butyl (RI = 9.33), fenoxaprop-P-ethyl (RI = 5.80) and clethodim (RI = 3.24). Gene sequencing revealed a Cys-2088-Arg mutation in the ACCase 1,5 allele of all the R plants, while ACCase gene overexpression was not involved in the resistance. Molecular docking indicated that the less-negative binding energies might be the main reason for the resistance of HS01 to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. A derived cleaved amplified polymorphic sequence (dCAPS) method was developed for the rapid identification of the Cys-to-Arg mutation in the ACCase gene at codon position 2088 in E. crus-galli. Additionally, pretreatment with the cytochrome P450 inhibitor piperonyl butoxide or the glutathione S-transferase inhibitor 4-chloro-7-nitrobenzoxadiazole had no significant effects (P > 0.05) on the resistance of HS01 to metamifop. To our knowledge, this is the first report of a Cys-2088-Arg mutation in E. crus-galli ACCase that confers cross-resistance to ACCase-inhibiting herbicides.

Chinese sprangletop [Leptochloa chinensis (L.) Nees] is one grass weed severely affecting rice (Oryza sativa L.) growth in paddies in China. Cyhalofop-butyl is the main herbicide used to control grass weeds in Chinese paddy fields, especially for controlling L. chinensis; however, L. chinensis has evolved resistance to cyhalofop-butyl due to continuous and extensive application. To investigate cyhalofop-butyl resistance levels and mechanisms in L. chinensis in some of the Chinese rice areas, 66 field populations were collected and treated with cyhalofop-butyl. Of these tested populations, 10 showed a high level of resistance to cyhalofop-butyl; the 50% effective dose ranged within 108.4 to 1,443.5 g ai ha–1 with resistance index values of 9.1 to 121.8 when compared with the susceptible population. Acetyl-coenzyme A carboxylase genes (ACCase) of susceptible and all 10 resistant populations were amplified and sequenced. Among them, Ile-1781-Leu, Trp-2027-Cys, Trp-2027-Ser, and Ile-2041-Asn mutations were found in five resistant populations. No known resistance-related mutations were found in the other five resistant populations, indicating that resistance to cyhalofop-butyl in these populations was likely to be endowed by non–target site resistance mechanisms. Notably, the Ile-1781-Leu and Trp-2027-Cys substitutions have previously been reported, but this is the first report of Trp-2027-Ser and Ile-2041-Asn mutations in L. chinensis. Furthermore, three derived cleaved amplified polymorphic sequence methods were developed to rapidly detect these mutations in L. chinensis.