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.     

Characterisation and molecular basis of ALS inhibitor resistance in the grass weed Alopecurus myosuroides

Several UK populations of the grass weed Alopecurus myosuroides were identified where high proportions of individuals showed resistance to the acetolactate synthase (ALS)‐inhibiting herbicides, mesosulfuron‐methyl + iodosulfuron‐methyl sodium mixture and sulfometuron‐methyl. Screening with sulfometuron, followed by DNA sequencing of the ALS gene from resistant and susceptible individuals, led to the identification of eight populations where a single point mutation segregated with resistance to sulfometuron. All highly resistant individuals from seven of eight populations showed a single‐nucleotide polymorphism (SNP) in the first position of the Pro197 codon of an A. myosuroides ALS gene, conferring a predicted proline to threonine target‐site change. One population showed resistant individuals with single‐nucleotide polymorphism in the second position of the Trp574 codon, conferring a predicted tryptophan to leucine substitution. No other mutations segregating with resistance were found. Enzyme assays confirmed that resistance was due to an altered form of ALS enzyme, which was less susceptible to inhibition by sulfonylureas, making this one of the first fully characterised cases of ALS target‐site resistance in a European grass weed. Increased information regarding the nature and distribution of ALS target‐site mutation may help support sustainable management strategies, allowing continued use of mesosulfuron + iodosulfuron against this weed in the UK.     

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.     

Sulfometuron-resistant Amaranthus retroflexus: cross-resistance and molecular basis for resistance to acetolactate synthase-inhibiting herbicides

A biotype of Amaranthus retroflexus L. is the first weed in Israel to develop resistance to acetolactate synthase (ALS)-inhibiting herbicides. The resistant biotype (Su-R) was collected from Ganot, a site that had been treated for more than 3 consecutive years with sulfometuron-methyl + simazine. On the whole-plant basis, the resistance ratio ( ED₅₀ Su-R)/( ED₅₀ Su-S) was 6–127 for sulfonylureas, 4–63 for imidazolinones, 20–35 for triazolopyrimidines and 11 for pyrithiobac-sodium. Similar levels of resistance were found also when the herbicides were applied before emergence. Based on a root elongation bioassay, Su-R was 3240-fold more resistant to sulfometuron-methyl than Su-S. In vitro studies have shown that the Su-R biotype was resistant at the enzyme level to all ALS inhibitors tested. The nucleotide sequences of two amplified regions between the Su-S and the Su-R differed in only one nucleotide. One substitution has occurred in domain A, cytosine by thymine (C C C to C T C) at position 248, that confers an exchange of the amino acid proline in the susceptible to leucine in the Su-R. The proline to leucine change in domain A is the only difference in the amino acid primary structure of the regions sequenced, indicating that it is responsible for the ALS-inhibitor resistance observed.     

Mutations of the ALS gene endowing resistance to ALS-inhibiting herbicides in Lolium rigidum populations

BACKGROUND: In the important grass weed Lolium rigidum (Gaud.), resistance to ALS‐inhibiting herbicides has evolved widely in Australia. The authors have previously characterised the biochemical basis of ALS herbicide resistance in a number of L. rigidum biotypes and established that resistance can be due to a resistant ALS and/or enhanced herbicide metabolism. The purpose of this study was to identify specific resistance‐endowing ALS gene mutation(s) in four resistant populations and to develop PCR‐based molecular markers. RESULTS: Six resistance‐conferring ALS mutations were identified: Pro‐197‐Ala, Pro‐197‐Arg, Pro‐197‐Gln, Pro‐197‐Leu, Pro‐197‐Ser and Trp‐574‐Leu. All six mutations were found in one population (WLR1). Each Pro‐197 mutation conferred resistance to the sulfonylurea (SU) herbicide sulfometuron, whereas the Trp‐574‐Leu mutation conferred resistance to both sulfometuron and the imidazolinone (IMS) herbicide imazapyr. A derived cleaved amplified polymorphic sequences (dCAPS) marker was developed for detecting resistance mutations at Pro‐197. Furthermore, cleaved amplified polymorphic sequences (CAPS) markers were developed for detecting each of the six mutant resistant alleles. Using these markers, the authors revealed diverse ALS‐resistant alleles and genotypes in these populations and related them directly to phenotypic resistance to ALS‐inhibiting herbicides. CONCLUSION: This study established the existence of a diversity of ALS gene mutations endowing resistance in L. rigidum populations: 1–6 different mutations were found within single populations. At field herbicide rates, resistance profiles were determined more by the specific mutation than by whether plants were homo‐ or heterozygous for the mutation. Copyright © 2008 Society of Chemical Industry     

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.     

耳叶水苋对乙酰乳酸合成酶抑制剂的抗性及其分子机制初探

近年来长江下游地区稻田耳叶水苋Ammannia arenaria H.B.K.危害十分严重。采用盆栽法首次测定了耳叶水苋对苄嘧磺隆等药剂的抗性水平,同时分析了其抗性和敏感种群间乙酰乳酸合成酶(ALS)基因的DNA序列及其RNA表达差异。结果表明:采自浙江嘉兴(JX110)、江苏苏州(JS039)、浙江宁波(NB0143-05)和安徽广德(AH014)的耳叶水苋生物型对苄嘧磺隆的抗性指数(RI)分别为67.90、17.59、44.63和8.37,对苄嘧磺隆表现出中高水平抗性的生物型对五氟磺草胺、双草醚及咪唑乙烟酸也产生了低水平的抗性。获得了耳叶水苋ALS基因全长核苷酸序列2235 bp,编码667个氨基酸,仅发现NB0143-05等3种抗性生物型ALS酶的氨基酸序列非保守区第93位的亮氨酸被脯氨酸取代。然而,NB0143-05的ALS酶对ALS抑制剂的敏感性大幅度降低(RI 37.04),且在苄嘧磺隆处理后4 d的ALS基因表达量是敏感生物型(HZ001)的1.86倍。这表明,ALS酶对药剂的敏感性降低以及被苄嘧磺隆诱导后ALS基因表达量显著增加,很可能是耳叶水苋生物型NB0143-05对ALS抑制剂产生抗性的原因。     

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     

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.     

Target-site basis for resistance to acetolactate synthase inhibitor in Water chickweed (Myosoton aquaticum L.)

Water chickweed is a widespread and competitive winter annual or biennial weed of wheat in China. One Water chickweed population (HN02) resistant to several acetolactate synthase (ALS) inhibitors was found in Henan province of China. Whole-plant bioassays showed that HN02 was high resistance to tribenuron (292.05-flod). In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The I₅₀ value for HN02 was 85.53 times greater respectively than that of susceptible population (SD05). This altered ALS sensitivity in the resistant population was due to a mutation in the ALS gene resulting in a Pro197 to Ser substitution. Cross-resistance experiments indicated that HN02 exhibited various resistance patterns to pyrithiobac-sodium, florasulam and pyroxsulam, without resistance to imazethapyr. This is the first report of tribenuron-resistant Water chickweed in Henan province of China, target-site based resistance was established as being due to an insensitive form of ALS, resulting from a Pro to Ser substitution at amino acid position 197 in the ALS gene.     

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.     

A molecular assay for the proactive detection of target site-based resistance to herbicides inhibiting acetolactate synthase in Alopecurus myosuroides

Acetolactate synthase (ALS) inhibitors are the most resistance‐prone herbicide group. Rapid resistance diagnosis is thus of importance for their optimal use. We formulate rules to use the derived cleaved amplified polymorphic sequence method to develop molecular tools detecting a change at a given codon, the nature of which is unknown. We applied them to Alopecurus myosuroides (black grass) to develop assays targeting ALS codons A122, P197, A205, W574 and S653 that are crucial for herbicide sensitivity. These assays detected W574L or P197T, or both substitutions, in most plants analysed from a field where ALS inhibitors failed after 3 years of use. Similar assays can easily be set up for any species. Given the rapidity of selection for resistance to ALS inhibitors, these assays should be very useful in proactive herbicide resistance diagnosis.     

荠菜对乙酰乳酸合成酶抑制剂类除草剂的抗性水平及其分子机制

为明确荠菜种群对苯磺隆的抗性水平及其靶标抗性产生的分子机制,采用整株水平测定法测定了荠菜对苯磺隆及其他5种乙酰乳酸合成酶（ALS）抑制剂类除草剂的抗性水平,同时扩增和比对了荠菜抗性和敏感种群之间ALS基因的差异。结果显示:与敏感种群15-ZMD-1相比,抗性种群15-ZMD-5对苯磺隆产生了高水平抗性,抗性倍数为219.6;15-ZMD-5种群不同单株中共存在3种突变方式,分别为ALS基因197位点脯氨酸（CCT）突变为亮氨酸（CTT）、574位点色氨酸（TGG）突变为亮氨酸（TTG）以及单株同时发生上述197和574位点的氨基酸突变。15-ZMD-5抗苯磺隆种群对嘧草硫醚、啶磺草胺和氟唑磺隆均产生了高水平的交互抗性,抗性倍数分别为41.2、79.3和87.8;对双氟磺草胺和咪唑乙烟酸产生了低水平的交互抗性,抗性倍数分别为8.5和5.6。分析表明,荠菜抗性种群ALS基因发生的氨基酸突变可能是导致其对ALS抑制剂类除草剂产生抗性的重要原因之一。     

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.