Resistance of dicot weeds to acetolactate synthase (ALS)-inhibiting herbicides in Australia

A biotype of Sonchus oleraceus L. and two bio types of Sisymbrium orientate Torn., SSO 3 and NSO 1, are the first dicot weeds in Australia to develop resistance to ALS-inhibiting herbicides. The resistant biotypes had been exposed to va rying periods of selection with sulfonylurea her bicides. All three biotypes are resistant to a range of sulfonylurea and imidazolinone herbicides. The S. orientale biotypes are also resistant to the triazolopyrimidine herbicide, flumetsulam. LD₅₀ ratios of resistant Sonchus oleraceus for sulfony lurea and imidazolinone herbicides are greater than 64-fold and 4.5-fold, respectively. GR₅₀ ratios are greater than 9 for sulfonylureas and 7.4 for imazapyr. The LD₅₀ ratios for both S. orien tale biotypes for chlorsulfuron, sulfometuron methyl, metsulfuron-methyl, flumetsulam and imazethapyr are greater than 110-, 15-, 7-, 24- and 29-fold, respectively. All resistant biotypes are susceptible to MCPA, diuron and diflufenican, herbicides which do not inhibit ALS.     

Resistance of Camelina microcarpa to acetolactate synthase inhibiting herbicides

An accession of Camelina microcarpa suspected to be resistant to sulfonylurea herbicides was identified in Oregon in 1998 field experiments. Greenhouse research confirmed that the putative resistant biotype was resistant to chlorsulfuron and metsulfuron on a whole plant level. Compared with the resistant (R) biotype, the susceptible (S) biotype was 1000 and 10 000‐fold more sensitive to metsulfuron and chlorsulfuron respectively. The R biotype was also resistant to other sulfonylurea, sulfonylaminocarbonyl‐triazolinone, imidazolinone and triazolopyrimidine herbicides. An in vivo enzyme assay indicated that acetolactate synthase (ALS) from the R plants required 111 times more chlorsulfuron to inhibit activity by 50% compared with the amount required to have a similar effect on ALS from S plants. Analysis of the nucleotide and amino acid sequences demonstrated that a single‐point mutation from G to T in the als1 gene conferred the change from the amino acid tryptophan to leucine at position 572 in the resistant biotype. This research confirmed that ALS inhibitor resistance in an Oregon accession of C. microcarpa is based on an altered target site conferred by a single‐point mutation.     

A mutation confers Monochoria vaginalis resistance to sulfonylureas that target acetolactate synthase

Acetolactate synthase (ALS) is the target enzyme for four distinct families of compounds: sulfonylureas (SUs), imidazolinones, triazolopyrimidine sulfonanilides, and pyrimidinyl oxybenzoates. We cloned and sequenced the fragments encoding ALS genes from biotypes of Monochoria vaginalis susceptible (S) and resistant (R) to SU-herbicides. The nucleotide sequences of the 39 bp Domain A region for R M. vaginalis biotype differed from that of the S biotype by a single nucleotide substitution at variable Pro codon of Domain A (CCT to TCT), predicting a Pro in the S but a Ser in the R biotype. No nucleotide differences between S and R M. vaginalis were observed in Domain D. We suggest that the amino acid substitution at Domain A region is responsible for resistance to SU-herbicides in M. vaginalis collected from Ushiku City, Ibaraki Prefecture, Japan.     

Sulfonylurea resistance in Stellaria media [L.] Vill.

A sulfonylurea resistant biotype of common chickweed (Stellaria media L. Vill.) was found in a field treated with chlorsulfuron or metsulfuron for eight consecutive years. In pot experiments the biotype was resistant to postemergence treatments with the following acetolactate synthase (ALS) inhibitors: chlorsulfuron, metsulfuron, tribenuron, triasulfuron, rimsulfuron, sulfometuron, flumetsulam and imazapyr. The level of resistance to chlorsulfuron and sulfometuron was higher than to the other sulfonylurea herbicides. Whereas the level of cross resistance to the triazolopyrimidine herbicide, flumetsulam was comparable to that of metsulfuron, that of imazapyr was significantly lower. In contrast to imazapyr the biotype was not resistant to imazethapyr, an other imidazolinone herbicide. ALS in vitro assays revealed that resistance was due to an ALS enzyme that was less sensitive to ALS inhibiting herbicides. Herbicides with different modes of action were equally effective on the susceptible and resistant biotypes.     

Molecular basis of diverse responses to acetolactate synthase-inhibiting herbicides in sulfonylurea-resistant biotypes of Schoenoplectus juncoides

Sulfonylurea-resistant biotypes of Schoenoplectus juncoides were collected from Nakafurano, Shiwa, Matsuyama, and Yurihonjyo in Japan. All of the four biotypes showed resistance to bensulfuron-methyl and thifensulfuron-methyl in whole-plant experiments. The growth of the Nakafurano, Shiwa, and Matsuyama biotypes was inhibited by imazaquin-ammonium and bispyribac-sodium, whereas the Yurihonjyo biotype grew normally after treatment with these herbicides. The herbicide concentration required to inhibit the acetolactate synthase (ALS) enzyme by 50% (I₅₀), obtained using in vivo ALS assays, indicated that the four biotypes were > 10-fold more resistant to thifensulfuron-methyl than a susceptible biotype. The Nakafurano, Shiwa, and Matsuyama biotypes exhibited no or little resistance to imazaquin-ammonium, whereas the Yurihonjyo biotype exhibited 6700-fold resistance to the herbicide. The Nakafurano and Shiwa biotypes exhibited no resistance to bispyribac-sodium, but the Matsuyama biotype exhibited 21-fold resistance and the Yurihonjyo biotype exhibited 260-fold resistance to the herbicide. Two S. juncoides ALS genes (ALS1 and ALS2) were isolated and each was found to contain one intron and to encode an ALS protein of 645 amino acids. Sequencing of the ALS genes revealed an amino acid substitution at Pro₁₉₇ in either encoded protein (ALS1 or ALS2) in the biotypes from Nakafurano (Pro₁₉₇ → Ser₁₉₇), Shiwa (Pro₁₉₇ → His₁₉₇), and Matsuyama (Pro₁₉₇ → Leu₁₉₇). The ALS2 of the biotype from Yurihonjyo was found to contain a Trp₅₇₄ → Leu₅₇₄ substitution. The relationships between the responses to ALS-inhibiting herbicides and the amino acid substitutions, which are consistent with previous reports in other plants, indicate that the substitutions at Pro₁₉₇ and Trp₅₇₄ are the basis of the resistance to sulfonylureas in these S. juncoides biotypes.     

A European biotype of Amaranthus retroflexus cross-resistant to ALS inhibitors and response to alternative herbicides

An acetolactate synthase (ALS)‐resistant Amaranthus retroflexus biotype was collected in a soyabean crop after repeated exposure to imazethapyr and thifensulfuron‐methyl in north‐eastern Italy. Studies were conducted to characterise the resistance status and determine alternative post‐emergence herbicides for controlling this biotype. Whole‐plant bioassay revealed that the GR₅₀ values were 1898‐ and 293‐fold higher than those observed for the biotype susceptible to imazethapyr and imazamox respectively. The biotype also displayed high cross‐resistance to sulfonylureas. Molecular analysis demonstrated that a single nucleotide substitution had occurred in domain B (TGG to TTG at position 574), conferring a change from the amino acid tryptophan to leucine in the resistant biotype. However, herbicides with other modes of action (PSII, 4‐HPPD and PPO inhibitors) provided excellent control. The GR₅₀ ratios for metribuzin, terbuthylazine and mesotrione were close to 1 and treatments with fomesafen gave 100% control of both susceptible and resistant biotypes at the recommended field dose. This study documents the first case of an imidazolinone and ALS‐resistant biotype in European crops and identifies the post‐emergence herbicide options available for managing this troublesome weed in soyabean crops. Alternative management strategies are also discussed.     

Physiological and molecular basis for ALS inhibitor resistance in Bromus tectorum biotypes

Primisulfuron‐resistant (AR and MR) and ‐susceptible (AS and MS) Bromus tectorum biotypes were collected from a Poa pratensis field at Athena, Oregon, and in research plots at Madras, Oregon. Studies were conducted to characterize the resistance of the B. tectorum biotypes. Whole plant bioassay and acetolactate synthase (ALS) enzyme assay revealed that the AR biotype was highly resistant to the sulfonylurea (SU) herbicides, primisulfuron and sulfosulfuron and to a sulfonylaminocarbonyltriazolinone (SCT) herbicide, propoxycarbazone‐sodium. However, the AR biotype was not resistant to imazamox, an imidazolinone (IMI) herbicide. Results of the whole plant bioassay studies showed that the MR biotype was moderately resistant to all ALS inhibitors tested. However, there were no differences in ALS sensitivities between the MR and MS biotypes. The nucleotide and amino acid sequence analysis of the als gene demonstrated a single‐point mutation from C to T, conferring the exchange of the amino acid proline to serine at position 197 in the AR biotype. However, this mutation was not found in the MR biotype. Results of this research indicate that: the resistance of the AR biotype to SU and SCT herbicides is based on an altered target site due to a single‐point mutation; resistance in the MR biotype is not due to a target site mutation.     

Conyza albida: a new biotype with ALS inhibitor resistance

Summary A biotype of Conyza albida resistant to imazapyr was discovered on a farm in the province of Seville, Spain, on land that had been continuously treated with this herbicide. This is the first reported occurrence of target site resistance to acetolactate synthase (ALS)-inhibiting herbicides in C. albida . In order to characterize this resistant biotype, dose–response experiments, absorption and translocation assays, metabolism studies, ALS activity assays and control with alternative herbicides were performed. Dose–response experiments revealed a marked difference between resistant (R) and susceptible (S) biotypes with a resistance factor [ED₅₀(R)/ED₅₀(S)] of 300. Cross-resistance existed with amidosulfuron, imazethapyr and nicosulfuron. Control of both biotypes using alternative herbicides was good using chlorsulfuron, triasulfuron, diuron, simazine, glyphosate and glufosinate. The rest of the herbicides tested did not provide good control for either biotype. There were no differences in absorption and translocation between the two biotypes, the maximum absorption reached about 15%, and most of the radioactivity taken up remained in the treated leaf. The metabolism pattern was similar and revealed that both biotypes may form polar metabolites with similar retention time (R_f). The effect of several ALS inhibitors on ALS (target site) activity measured in leaf extracts from both biotypes was investigated. Only with imazapyr and imazethapyr did the R biotype show a higher level of resistance than the S biotype [I₅₀ (R)/I₅₀(S) value of 4.0 and 3.7 respectively]. These data suggest that the resistance to imazapyr found in the R biotype of C. albida results primarily from an altered target site.     

Molecular basis for resistance to tribenuron in shepherd’s purse (Capsella bursa-pastoris (L.) Medik.)

Capsella bursa-pastoris, a winter annual weed in the mustard family, can not be controlled by tribenuron after the herbicide has been continuously used for several years. The resistant biotype Lz-R was the generation of a population collected from Liangzhu, a place where tribenuron had been used for more than 15 consecutive years. To confirm and characterize the resistance of C. bursa-pastoris to tribenuron, whole-plant bioassays were conducted in the greenhouse. The results of whole-plant bioassays revealed that Lz-R was highly resistant to tribenuron with the resistance index (GR₅₀ Lz-R)/(GR₅₀ Lz-S) up to 236.6. To investigate the molecular basis of resistance in C. bursa-pastoris, the acetolactate synthase (ALS) genes were sequenced and compared between susceptible and resistant biotypes. Analysis of the nucleotide and deduced amino acid sequences between the biotypes indicated that one substitution had occurred in Domain A, cytosine by thymine (CCT to TCT) at position 197, that led to a change of the amino acid proline in the susceptible to serine in the Lz-R.     

Mutations in the acetolactate synthase genes of sulfonylurea-resistant biotypes of Lindernia spp.

Acetolactate synthase (ALS) is a key enzyme in the biosynthetic pathway of branched-chain amino acids. A mutation of the ALS gene causing amino acid substitution at the position of proline in Domain A makes ALS less sensitive to sulfonylureas, which are ALS-inhibiting herbicides. We cloned partial ALS genes from four Lindernia plants, L . dubia var. dubia , L . dubia var. major , L . micrantha and L . procumbens , for which biotypes resistant to sulfonylureas have been found in paddy fields. The clones were classified into two groups in each Lindernia plant: Als1 and Als2 . Sequencing of the clones and alignment of deduced amino acid sequences with previously reported ALS of other species suggested that the cloned region contains an intron in both Als1 and Als2 . Comparison of Als1 between resistant and susceptible biotypes showed that the proline of Domain A was replaced by alanine, serine or glutamine in all resistant biotypes of Lindernia plants, while it was conserved in all susceptible biotypes. This amino acid substitution in ALS encoded by Als1 is involved in the resistant mechanism of ALS to sulfonylurea in the four Lindernia plants.     

Inhibition of acetolactate synthase in susceptible and resistant biotypes of Stellaria media

Acetolactate synthase (ALS) from one susceptible and two chlorsulfuronresistant biotypes of Stellaria media(L.) Vill. was assayed in the presence of eight known ALS inhibitors. As expected, ALS from the chlorsulfuronresistant biotypes (R1 and R2) showed reduced sensitivity to chlorsulfuron and other sulfonylurea herbicides. The patterns of cross-resistance varied, however, indicating that the alteration in ALS that confers chlorsulfuron resistance does not confer the same level of resistance to other sulfonylurea herbicides. The resistant biotypes were highly cross-resistant to sulfometuron-methyl and DPX-A7H81, but less cross-resistant to triasulfuron. Both R1 and R2 were highly cross-resistant to DTPS (N-[2,6-dichlorophenyl]-5,7-dimethyl-1,2,4-iriazolo[1,5a]pyrimidine-2-siilfoiiamide), but only slightly cross-resistant to imazamethahenz, an imidazolinone herbicide. The differences in the patterns of cross-resistance observed presumably reflect differences in the binding affinity of the herbicides for the altered ALS. The data presented suggest, but do not confirm, that R1 and R2 contain the same ALS mutation.     

Mechanism of resistance to bensulfuron-methyl in Alisma plantago-aquatica biotypes from Portuguese rice paddy fields

Two Alisma plantago‐aquatica biotypes resistant to bensulfuron‐methyl were detected in rice paddy fields in Portugal’s Mondego (biotype T) and Tagus and Sorraia (biotype Q) River valleys. The fields had been treated with bensulfuron‐methyl‐based herbicide mixtures for 4–6 years. In order to characterize the resistant (R) biotypes, dose–response experiments, absorption and translocation assays, metabolism studies and acetolactate synthase (ALS) activity assays were performed. There were marked differences between R and susceptible (S) biotypes, with a resistance index (ED₅₀R/S) of 500 and 6.25 for biotypes Q and T respectively. Cross‐resistance to azimsulfuron, cinosulfuron and ethoxysulfuron, but not to metsulfuron‐methyl, imazethapyr, bentazone, propanil and MCPA was demonstrated. No differences in the absorption and translocation of ¹⁴C‐bensulfuron‐methyl were found between the biotypes studied. Maximum absorption attained 1.12, 2.02 and 2.56 nmol g⁻¹ dry weight after 96 h incubation with herbicide, for S, Q and T biotypes respectively. Most of the radioactivity taken up by the roots was translocated to shoots. Bensulfuron‐methyl metabolism in shoots was similar in all biotypes. The R biotypes displayed a higher level of ALS activity than the S biotype, both in the presence and absence of herbicide and the resistance indices (IC₅₀R/S) were 20 197 and 10 for biotypes Q and T respectively. These data confirm for the first time that resistance to bensulfuron‐methyl in A. plantago‐aquatica is target‐site‐based. In practice, to control target site R biotypes, it would be preferable to use mixtures of ALS inhibitors with herbicides with other modes of action.     

Corn poppy (Papaver rhoeas) cross-resistance to ALS-inhibiting herbicides

BACKGROUND: Papaver rhoeas (L.) has evolved resistance to tribenuron in winter wheat fields in northern Greece owing to multiple Pro₁₉₇ substitutions. Therefore, the cross‐resistance pattern to other sulfonylurea and non‐sulfonylurea ALS‐inhibiting herbicides of the tribenuron resistant (R) and susceptible (S) corn poppy populations was studied by using whole‐plant trials and in vitro ALS catalytic activity assays. RESULTS: The whole‐plant trials revealed that tribenuron R populations were also cross‐resistant to sulfonylureas mesosulfuron + iodosulfuron, chlorsulfuron and triasulfuron. The whole‐plant resistance factors (RFs) calculated for pyrithiobac, imazamox and florasulam ranged from 12.4 to > 88, from 1.5 to 28.3 and from 5.6 to 25.4, respectively, and were lower than the respective tribenuron RF values (137 to > 2400). The ALS activity assay showed higher resistance of the ALS enzyme to sulfonylurea herbicides (tribenuron > chlorsulfuron) and lower resistance to non‐sulfonylurea ALS‐inhibiting herbicides (pyrithiobac > florasulam ≈ imazamox). CONCLUSION: These findings indicate that Pro₁₉₇ substitution by Ala, Ser, Arg or Thr in corn poppy results in a less sensitive ALS enzyme to sulfonylurea herbicides than to other ALS‐inhibiting herbicides. The continued use of sulfonylurea herbicides led to cross‐resistance to all ALS‐inhibiting herbicides, making their use impossible in corn poppy resistance management programmes. Copyright © 2011 Society of Chemical Industry     

Diclofop‐resistant Lolium rigidum from northern Greece with cross‐resistance to ACCase inhibitors and multiple resistance to chlorsulfuron

Repeated use of ACCase‐ and ALS‐inhibiting herbicides in northern Greece has resulted in the evolution of a population of Lolium rigidum resistant to diclofop and chlorsulfuron. The biotype from Athos was highly resistant to diclofop and also exhibited differential cross‐resistance to clodinafop, fluazifop, tralkoxydim and sethoxydim. Assay of ACCase activity confirmed that the resistant biotype was tenfold more resistant to diclofop than the susceptible biotype, suggesting that the resistance mechanism could involve an altered target site. The diclofop‐resistant biotype has also exhibited multiple resistance to chlorsulfuron and the mechanism for this is unknown. Seed‐bioassay was found to be a rapid, cheap and reliable method to identify populations of L rigidum resistant to ACCase inhibitors and chlorsulfuron. Moreover, root elongation in the seed bioassay was more sensitive to ACCase inhibitors and chlorsulfuron than shoot elongation. © 2000 Society of Chemical Industry     

Sulfonylurea herbicide resistance in Sonchus asper biotypes in Alberta, Canada

Summary Two Sonchus asper (spiny annual sow-thistle) biotypes, suspected of being resistant to the sulfonylurea herbicide metsulfuron-methyl, were collected in 1996 from two barley ( Hordeum vulgare ) fields in central Alberta, Canada. Both fields had received at least six applications of acetolactate synthase (ALS)-inhibiting herbicide(s). The responses of the two resistant (R) biotypes and two susceptible (S) biotypes to several sulfonylurea herbicides, and to herbicides and herbicide mixtures with other mechanisms of action, were compared. Both R biotypes were highly resistant to all sulfonylurea herbicides, but their control with other herbicides and mixtures was effective and comparable to that of the S biotypes. ALS extracted from an R biotype was about 440 times more resistant to metsulfuron-methyl than that of an S biotype, indicating that resistance was conferred by an ALS enzyme that was less sensitive to inhibition by the herbicide. Competitiveness and seed production of S. asper varied among biotypes, but the differences were probably the result of ecotype differences rather than resistance or susceptibility to sulfonylurea herbicides. This is the first reported occurrence of target site-based S. asper resistance to ALS-inhibiting herbicides.     

Biology and mechanisms of sulfonylurea resistance in Schoenoplectiella juncoides,a noxious sedge in the rice paddy fields of Japan

Schoenoplectiella juncoides is a noxious sedge weed in rice paddy fields that has evolved resistance to sulfonylurea (SU) herbicides. The molecular basis of resistance is amino acid substitutions at Pro₁₉₇, Trp₅₇₄ or Asp₃₇₆ in the acetolactate synthase (ALS) enzyme, which is the target of SUs. Schoenoplectiella juncoides has two ALS genes and resistant plants have point mutations that cause amino acid substitutions in either encoded protein. Single‐nucleotide substitutions at the codon for Pro₁₉₇ in the ALS genes can cause six types of amino acid substitutions and all of these substitutions have been found in both ALS genes among Japanese SU‐resistant biotypes. Whole‐plant herbicide responses differ among the amino acid substitution types. Furthermore, analyses of ALS activity in plant extracts show that the extracts’ responses to herbicides differ, depending on which ALS gene is mutated. The activity responses of the ALS extracts to the SU, imazosulfuron, showed double‐sigmoid curves with plateaus of ~30% inhibition for Pro₁₉₇ substitutions in ALS1 and ~70% for Pro₁₉₇ substitutions in ALS2. This indicates that ALS1 and ALS2 contribute to the responses with a proportion of 7:3. The double‐sigmoid curves can be reconstructed to show the responses of the resistant and susceptible enzymes separately by regression analysis. The resistance levels of the separate ALS1 or ALS2 mutated enzyme are highly correlated with the whole‐plant responses, with a relationship that the former is the square of the latter. This could provide a quantitative insight into the physiological basis of resistance.     

Assessment of two biotypes of Solanum ptycanthum that differ in resistance levels to imazamox

Glasshouse and laboratory experiments were conducted on acetolactate synthase (ALS) homozygous resistant Solanum ptycanthum biotypes from Illinois (IL‐R) and Indiana (IN‐R), and homozygous susceptible biotypes from Illinois (IL‐S) and Indiana (IN‐S). Genetic similarity of biotypes was assessed by random amplified polymorphic DNA (RAPD) markers, which determined that the Illinois biotypes are more similar to each other than to the IN‐R biotype. ALS enzyme activity from the IL‐R and IN‐R biotypes had I₅₀ values of 362 and 352 μM imazamox respectively. Dose–response experiments using three‐ to four‐leaf‐stage plants of the IL‐R and IN‐R biotypes had GR₅₀ values of 242 and 69 g ae ha⁻¹ imazamox respectively. Whole‐plant and ALS enzyme results are different than previously reported values in the literature, which was attributed in the current study to the original IN‐R population having individuals that were segregating for ALS resistance. Metabolism studies showed no difference in percentage [¹⁴C]imazamox remaining between the IL‐R and IN‐R biotypes up to 72 h after treatment. The IL‐S biotype metabolised [¹⁴C]imazamox approximately two times faster than the IL‐R and IN‐R biotypes and this trait was heritable. Response of F₃ plants containing homozygous ALS‐resistant alleles from the IL‐R biotype in a genetic background of 50% Illinois and 50% Indiana biotypes suggests that genetic factors other than an altered target site or metabolism may also contribute to the magnitude of resistance at the whole‐plant level in resistant biotypes.     

A novel amino acid substitution Ala-122-Tyr in ALS confers high-level and broad resistance across ALS-inhibiting herbicides

BACKGROUND: Wild radish, a problem weed worldwide, is a severe dicotyledonous weed in crops. In Australia, sustained reliance on ALS‐inhibiting herbicides to control this species has led to the evolution of many resistant populations endowed by any of several ALS mutations. The molecular basis of ALS‐inhibiting herbicide resistance in a novel resistant population was studied. RESULTS: ALS gene sequencing revealed a previously unreported substitution of Tyr for Ala at amino acid position 122 in resistant individuals of a wild radish population (WARR30). A purified subpopulation individually homozygous for the Ala‐122‐Tyr mutation was generated and characterised in terms of its response to the different chemical classes of ALS‐inhibiting herbicides. Whole‐plant dose‐response studies showed that the purified subpopulation was highly resistant to chlorsulfuron, metosulam and imazamox, with LD₅₀ or GR₅₀ R/S ratio of > 1024, > 512 and > 137 respectively. The resistance to imazypyr was found to be relatively moderate (but still substantial), with LD₅₀ and GR₅₀ R/S ratios of > 16 and > 7.8 respectively. In vitro ALS activity assays showed that Ala‐122‐Tyr ALS was highly resistant to all tested ALS‐inhibiting herbicides. CONCLUSION: The molecular basis of ALS‐inhibiting herbicide resistance in wild radish population WARR30 was identified to be due to an Ala‐122‐Tyr mutation in the ALS gene. This is the first report of an amino acid substitution at Ala‐122 in the plant ALS that confers high‐level and broad‐spectrum resistance to ALS‐inhibiting herbicides, a remarkable contrast to the known mutation Ala‐122‐Thr endowing resistance to imidazolinone herbicide. Copyright © 2012 Society of Chemical Industry     

Molecular bases for resistance to acetyl-coenzyme A carboxylase inhibitor in Japanese foxtail (Alopecurus japonicus)

BACKGROUND: Haloxyfop‐R‐methyl is a widely used herbicide to control Poaceae weeds. Alopecurus japonicus, a widespread annual grass, can no longer be controlled by haloxyfop‐R‐methyl after continuous use of this herbicide for several years. RESULTS: Dose‐response experiments have established that the Js‐R biotype of A. japonicas has evolved resistance to aryloxyphenoxypropionates (APPs). Target‐site enzyme sensitivity experiments have established that the haloxyfop (free acid) rate causing 50% inhibition of acetyl‐CoA carboxylase (ACCase) activity (I₅₀) for the resistant (Js‐R) biotype is 11 times higher than that for the susceptible (Js‐S) biotype. In many cases, resistance to ACCase‐inhibiting herbicides is due to a resistant ACCase enzyme. Full‐length DNA and mRNA sequences of the plastidic ACCase gene were amplified. Eight single‐nucleotide differences were detected in this region. Four of the nucleotide changes were silent mutations. However, the other four nucleotide mutations caused four amino acid substitutions, replacing Arg‐1734 with Gly, Met‐1738 with Leu, Thr‐1739 with Ser and Ile‐2041 with Asn in the R biotype respectively; the substitution at position 2041 had been reported, while the other three had not. CONCLUSION: The ACCase in the Js‐R biotype was less susceptible to haloxyfop‐R‐methyl than that in the Js‐S biotype. Moreover, the amino acid substitution of Ile‐2041 with Asn might confer resistance to haloxyfop‐R‐methyl in A. japonicas. Copyright © 2012 Society of Chemical Industry     

Occurrence of sulfonylurea resistance in Sagittaria trifolia,a basal monocot species,based on target‐site and non‐target‐site resistance

Sagittaria trifolia L. is one of the most serious weeds in paddy fields in Japan. Since the late 1990s, severe infestations of S. trifolia have occurred following applications of sulfonylurea herbicides in Akita prefecture. In this study, two accessions of S. trifolia, R1 and R2, were collected from paddy fields with severe infestations and their resistance profiles were determined in comparison to a susceptible accession, S1. R1 and R2 were highly resistant to bensulfuron‐methyl. R1 was also highly resistant to pyrazosulfuron‐ethyl, but R2 was susceptible. Relative to S1, R1 had an amino acid substitution at the Pro₁₉₇ residue of acetolactate synthase (ALS), a well‐known mutation that confers sulfonylurea resistance, suggesting that R1 has a target‐site‐based resistance (TSR) mechanism. The sequence of the ALS gene in R2 was identical to that in S1. A Southern blot analysis indicated that there was only one copy of the ALS gene in S1 and R2. These results suggest that R2 has a non‐target‐site‐based resistance (NTSR) mechanism. R2 was moderately resistant to imazosulfuron but susceptible to thifensulfuron‐methyl. R2 and S1 were susceptible to pretilachlor, benfuresate, MCPA‐ethyl and bentazon. The results reveal the occurrence of two sulfonylurea‐resistant biotypes of S. trifolia that show different mechanisms of cross‐resistance to sulfonylureas related to TSR in R1 and NTSR in R2.