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.     

Molecular characterisation of resistance to ALS-inhibiting herbicides in Hordeum leporinum biotypes

BACKGROUND: The acetolactate synthase (ALS)-inhibiting herbicide sulfosulfuron is registered in Australia for the selective control of Hordeum leporinum Link. in wheat crops. This herbicide failed to control H. leporinum on two farms in Western Australia on its first use. This study aimed to determine the level of resistance of three H. leporinum biotypes, identify the biochemical and molecular basis and develop molecular markers for diagnostic analysis of the resistance. RESULTS: Dose-response studies revealed very high level (>340-fold) resistance to the sulfonylurea herbicides sulfosulfuron and sulfometuron. In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to herbicide inhibition. This altered ALS sensitivity in the resistant biotypes was found to be due to a mutation in the ALS gene resulting in amino acid proline to serine substitution at position 197. In addition, two- to threefold higher ALS activities were consistently found in the resistant biotypes, compared with the known susceptible biotype. Two cleaved amplified polymorphic sequence (CAPS) markers were developed for diagnostic testing of the resistant populations. CONCLUSION: This study established the first documented case of evolved ALS inhibitor resistance in H. leporinum and revealed that the molecular basis of resistance is due to a Pro to Ser mutation in the ALS gene.     

Selection for resistance to the herbicide imazethapyr in somaclones of soyabean

Cultivars of soyabean [ Glycine max (L.) Merr.] resistant to the herbicide imazethapyr were identified by suspending the roots of 5-day-old seedlings in nutrient culture containing 2.5 mg a.i. L^–1 imazethapyr and then comparing the inhibitory effect on root length and shoot dry weight. The four most resistant cultivars were subsequently screened as regenerating tissue cultures in a medium containing 2.0 mg a.i. L^–l imazethapyr to select somaclonal cells with increased resistance. Surviving portions of cultures were regenerated to give shoots, the plants isolated, allowed to flower and seed. These progeny were then used for further seed multiplication and seedlings from this latter generation were exposed to imazethapyr in vivo and callus and cell suspension cultures derived from these seedlings were exposed to imazethapyr in vitro . A reduction in the inhibitory effect of the imazethapyr was noted in the somaclone seedlings and tissue cultures. However, measurement of acetolactate synthase (ALS) activity showed no differences among the parent cultivars and in the selected somaclones in this trait.     

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.     

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.     

Varied occurrence of diverse sulfonylurea‐resistant biotypes of Schoenoplectus juncoides [Roxb.] Palla in Japan,as classified by an acetolactate synthase gene mutation

Suspected sulfonylurea (SU)‐resistant Schoenoplectus juncoides plants were collected from rice paddy fields at 24 sites in Japan in order to discover the occurrence pattern of target‐site substitutions on a nationwide scale and at a local field scale. A genetic analysis of the two acetolactate synthase (ALS) genes, ALS1 and ALS2, of the collected plants confirmed that a single‐nucleotide mutation at the Pro₁₉₇, Asp₃₇₆ or Trp₅₇₄ site of either ALS1 or ALS2 existed in each suspected SU‐resistant plant. On a nationwide scale, it was shown that the ALS1 mutations and the ALS2 mutations occurred at a similar frequency, that the P197S and the P197L substitutions were found most frequently among all the substitutions, and that the W574L substitutions (known as global resistance to any ALS‐inhibiting herbicide) were found at a relatively low frequency but in a geographically wide range. In the local field‐scale survey, which was conducted at two sites in Hyogo Prefecture, it was shown that the substitutions were less diverse, compared to on a nationwide scale, probably because the investigation involved a limited number of local fields, and that several substitutions and a susceptible biotype were found in single fields suggesting that a number of collections is required in order to understand the local SU‐resistant status of S. juncoides. In addition, this study reported new findings, that of the P197R, P197T and D376E substitutions in S. juncoides. This set of diverse substitutions in a weed species can be used for further research purposes.     

Sulfonylurea resistance in Lindernia micrantha, an annual paddy weed in Japan

Resistance to sulfonylurea herbicides, including bensulfuron-methyl, pyrazosulfuron-ethyl, imazosulfuron and ethoxysulfuron, was discovered in naturally occurring populations of Lindernia micrantha D. Don in rice fields that had been treated with sulfonylurea-based herbicides for 3–7 consecutive years. The resistant biotype was approximately 80≈300 times more resistant than the susceptible one to the above four sulfonylurea herbicides. This is the second confirmed occurrence of herbicide resistance resulting from the use of sulfonylurea herbicides in Japan. Several herbicides with different modes of action, including pretilachlor, cafenstrole, bifenox, naproanilide, thiobencarb + simetryn + MCPB, MCPA-thioethyl + simetryn and cyhalofop-butyl + bentazone, effectively controlled the resistant biotype in pot trials.     

抗苄嘧磺隆雨久花乙酰乳酸合成酶突变的研究

本研究采用cDNA末端快速扩增技术(RACE)结合RT-PCR方法克隆抗苄嘧磺隆雨久花生物型和敏感性雨久花生物型乙酰乳酸合成酶(ALS)基因cDNA序列,并对测序结果进行比对分析。结果表明:与敏感性的雨久花ALS相比,公主岭(GZL)抗性生物型中第197位脯氨酸突变为组氨酸,第556位亮氨酸突变为苯丙氨酸;柳河(LH)抗性生物型中第358位天冬酰胺突变为天冬氨酸;磐石市(PS)抗性生物型中第525位缬氨酸突变为异亮氨酸。分析表明,高度保守区Domain A的第197位氨基酸残基的突变可能是导致公主岭稻区雨久花产生抗药性的主要原因之一,而其他抗性生物型抗性产生的原因有待进一步研究。     

非转基因抗除草剂玉米对三种乙酰乳酸合成酶抑制剂类除草剂的田间抗性表现

为评估中国农业大学培育的非转基因抗除草剂玉米品系958R和335R在大田条件下对乙酰乳酸合成酶（acetolactate synthase,ALS）抑制剂类除草剂的抗性表现,利用甲咪唑烟酸、砜嘧磺隆、唑嘧磺草胺3种除草剂对郑单958、958R、先玉335、335R共4种玉米杂交种进行了播后苗前土壤处理,每种除草剂设3个处理剂量（1、3、9倍推荐剂量）,并于施药2周和4周后进行株高测定,于收获晾干后测产。结果表明,在甲咪唑烟酸216、648 g（a.i.）/hm²处理下,郑单958和先玉335均已绝产,而958R和335R产量均未受影响;在砜嘧磺隆或唑嘧磺草胺高剂量处理下,常规玉米品种郑单958和先玉335株高的最高降幅分别为25.7%和35.2%,田间药害反应显著,而958R和335R则抗性反应显著。研究表明,非转基因抗除草剂玉米杂交种具有良好的田间抗性,不仅能有效解决玉米田砜嘧磺隆和唑嘧磺草胺等ALS除草剂的药害问题,还能够通过引入甲咪唑烟酸等新的ALS除草剂更好地防除玉米田的杂草。     

Characterization of acetolactate synthase from sulfonylurea herbicide‐resistant Schoenoplectus juncoides

Ten accessions of sulfonylurea‐resistant Schoenoplectus juncoides were collected from paddy fields in Japan. In order to characterize acetolactate synthase from sulfonylurea‐resistant S. juncoides, acetolactate synthase amino acid substitutions, whole‐plant growth inhibition and acetolactate synthase enzyme inhibition were examined. Schoenoplectus juncoides has two acetolactate synthase genes (ALS1 and ALS2). The sulfonylurea‐resistant accessions harbored amino acid substitutions at Pro197 or Trp574 in either ALS1 or ALS2 (the amino acid number is standardized to the Arabidopsis thaliana sequence). The whole plants of all the sulfonylurea‐resistant accessions showed resistance to imazosulfuron. The resistance level depended on the altered amino acid residues in acetolactate synthase. The acetolactate synthase enzyme that was partially purified from all the sulfonylurea‐resistant accessions was less sensitive to imazosulfuron, compared to the susceptible accession, suggesting that the resistance is related to the altered acetolactate synthase enzyme. In addition, the concentration–response inhibition of acetolactate synthase activity by imazosulfuron in the sulfonylurea‐resistant accessions was remarkably different with the presence of an amino acid substitution in either ALS1 or ALS2. Furthermore, the concentration–response inhibition of acetolactate synthase activity in the sulfonylurea‐resistant accessions with a P197S, P197T or W574L mutation showed a double‐sigmoid curve. The regression analysis of enzyme inhibition suggested that the abundance ratio of ALS1 to ALS2 enzymes was approximately 70:30%, with a range of ±15%. Taken together, these results suggest that the resistance of sulfonylurea‐resistant accessions of S. juncoides is related to altered acetolactate synthase in either ALS1 or ALS2, although the abundance of the altered acetolactate synthase in the plants is different among the sulfonylurea‐resistant accessions.     

Sulfonylurea herbicide-resistant Monochoria vaginalis in Korean rice culture

Nine Monochoria vaginalis Pres1 accessions from Chonnam province, Korea were tested for resistance to the sulfonylurea herbicide, imazosulfuron, in whole-plant response bioassay. All accessions were confirmed resistant (R) to imazosulfuron. The GR50 (imazosulfuron concentration that reduced shoot dry weight by 50%) values of R accessions were 1112-3172 (accession #9) times higher than that of the standard susceptible (S) accession. Accession #9 exhibited cross-resistance to other sulfonylurea herbicides, bensulfuron-methyl, cyclosulfamuron and pyrazosulfuron-ethyl, but not to the imidazolinone herbicides, imazapyr and imazaquin. The R biotype could be controlled by other herbicides with different modes of action, such as mefenacet and pyrazolate, applied to soil at recommended rates. Foliar-applied herbicides, 2,4-D and bentazone, also controlled both the R and S biotypes. Sulfonylurea-based mixtures, except ethoxysulfuron plus fentrazamide, did not control resistant M. vaginalis. Rice yield was reduced 70% by resistant M. vaginalis that escaped pyrazosulfuron-ethyl plus molinate, compared with hand weeding in direct-seeded rice culture. In contrast, rice yield was reduced 44% by resistant M. vaginalis that survived the pyrazosulfuron-ethyl plus molinate treatment, compared with pyrazolate plus butachlor in transplanted rice culture. In vitro acetolactate synthase (ALS) activity of the R biotype was 183, 35, 130 and 31 times more resistant to imazosulfuron, bensulfuron-methyl, cyclosulfamuron and pyrazosulfuron-ethyl, respectively, than the S biotype. Imidazolinone herbicides, imazapyr and imazaquin had similar effect on in vitro ALS activity of the R and S biotypes. The in vivo ALS activity of the R biotype was also less affected than the S biotype by the sulfonylurea herbicides imazosulfuron and pyrazosulfuron-ethyl. Results of in vitro and in vivo ALS assays indicate that the resistance mechanism of M. vaginalis to sulfonylurea herbicides may be due, in part, to an alteration in the target enzyme, ALS. Since the level of resistance in the enzyme assay was much lower than that in the whole-plant assay, other mechanisms of resistance, such as herbicide metabolism, may be involved.     

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.     

Cross‐resistance of Echinochloa species to acetolactate synthase inhibitor herbicides

This study was conducted to evaluate the cross‐resistance of acetolactate synthase (ALS) inhibitors with different chemistries, specifically azimsulfuron (sulfonylurea), penoxsulam (triazolopyrimidine sulfonanilide) and bispyribac‐sodium (pyrimidinyl thio benzoate), in Echinochloa oryzicola and Echinochloa crus‐galli that had been collected in South Korea and to investigate their herbicide resistance mechanism. Both Echinochloa spp. showed cross‐resistance to the ALS inhibitors belonging to the above three different chemistries. In a whole plant assay with herbicides alone, the resistant/susceptible ratios for azimsulfuron, penoxsulam and bispyribac‐sodium were 12.6, 28.1 and 1.9 in E. oryzicola and 21.1, 13.7 and 1.8 in E. crus‐galli, respectively. An in vitro ALS enzyme assay with herbicides showed that the I ₅₀‐values of the resistant accessions were approximately two‐to‐three times higher than the susceptible accessions, with no statistical difference, suggesting that the difference in ALS sensitivity cannot explain ALS inhibitor resistance in Echinochloa spp. for azimsulfuron, penoxsulam and bispyribac‐sodium. A whole plant assay with fenitrothion showed that the GR ₅₀‐values significantly decreased in both the resistant E. oryzicola and E. crus‐galli accessions when azimsulfuron, penoxsulam and bispyribac‐sodium were applied with the P450 inhibitor, while no significant decrease was observed in the susceptible accessions when the P450 inhibitor was used. Thus, these results suggest that ALS inhibitor cross‐resistance for azimsulfuron, penoxsulam and bispyribac‐sodium is related to enhanced herbicide metabolism.     

Identification of resistance to either paraquat or ALS-inhibiting herbicides in two Western Australian Hordeum leporinum biotypes

BACKGROUND: Hordeum populations are becoming increasingly difficult to control in cropping fields. Two herbicide‐resistant H. leporinum populations were identified during a random crop survey after herbicides were applied. The study aimed to determine the herbicide resistance profile of these H. leporinum biotypes to a range of herbicides used for their control. RESULTS: Based on dose–response studies, one H. leporinum population was very highly resistant to sulfosulfuron and sulfometuron (both sulfonylurea herbicides) and also displayed low‐level resistance to imazamox (an imidazolinone herbicide). Reduced sensitivity of the ALS enzyme was identified with in vitro activity assays. Gene sequence analysis revealed a proline‐to‐threonine substitution at amino acid position 197 of ALS, which is likely to be the molecular basis for resistance in this population. Herbicide screening also revealed a different H. leporinum population with resistance to the bipyridyl herbicide paraquat. CONCLUSION: This study established the first cases of (1) sulfonylurea‐to‐imidazolinone cross‐resistance and (2) field‐evolved paraquat resistance in a Hordeum species in Western Australia. Copyright © 2012 Society of Chemical Industry     

Difference in ultraweak photon emissions between sulfonylurea-resistant and sulfonylurea-susceptible biotypes of Scirpus juncoides following the application of a sulfonylurea herbicide

We examined the ultraweak photon emissions from a paddy weed, Scirpus juncoides , to assess the availability of photon emissions for the identification of weed biotypes resistant to sulfonylurea herbicides. The emission intensity from the plant organs increased when treated with a sulfonylurea herbicide in a concentration-dependent manner. The increment in emissions was higher in the sulfonylurea-resistant biotypes than in the sulfonylurea-susceptible biotypes. The difference between the biotypes was greater in the culms than in the roots and remained so through the vegetative growth stage to the flowering stage. This difference was independent of the seed source or mutations in the acetolactate synthase genes of the resistant biotypes. These results suggest that the determination of ultraweak photon emissions can be a useful method for identifying the sulfonylurea-resistant biotypes of S. juncoides.     

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.