Real-time quantitative PCR assays for quantification of L1781 ACCase inhibitor resistance allele in leaf and seed pools of Lolium populations

The I1781L amino acid substitution in the target ACCase enzyme causes broad resistance to ACCase inhibitor herbicides in several monocotyledenous weeds of agronomic importance. This mutation results from a substitution of an adenine (A) residue by either a thymine (T) or cytosine (C) at position 5341 in Alopecurus myosuroides Huds and at an equivalent position in Lolium species, Avena fatua L. and Setaria viridis (L.) Beauv. Two different procedures, the PCR-based allele-specific assay (ASA) and the derived cleaved amplified polymorphic sequence (dCAPS) method, have previously been described for detecting this mutation. These methods are, however, only amenable to low sample throughput and are used in the analysis of single plants. Here, an alternative high-throughput ARMS/Scorpion real-time quantitative PCR (Q-PCR) method for measuring levels of the I1781L mutation in pools of leaf and seed samples of Lolium populations is presented. The limit of detection for C and T mutant alleles in a background of wild-type A is 0.02 and 0.0003% respectively. In this study, DNA from batches of 24 leaf segments measuring 0.5 cm from different plants or 1000 seeds could be conveniently extracted and accurately analysed. As part of assay validation, the comparative analysis of five geographically distinct Lolium populations with dCAPS and Q-PCR procedures demonstrated the accuracy of the latter method, and the three possible II1781, IL1781 and LL1781 ACCase genotypes being distributed as predicted by the Hardy-Weinberg principle. Given the dominance of the L1781 over the I1781 allele at recommended field rates for most ACCase inhibitors, the frequency of herbicide survivors in the field owing only to the presence of the I1781L mutation is thus predicted to be 2pq + q(2), where p and q are the frequencies of the I1781 and L1781 alleles as determined by Q-PCR. The Q-PCR assay established allows detection of very low levels of the L1781 ACCase mutation before resistance would normally be discernible in the field. Therefore, it offers the opportunity to tackle resistance at its very onset, potentially avoiding implementation of complicated and often costly weed management practices.     

An aspartate to glycine change in the carboxyl transferase domain of acetyl CoA carboxylase and non‐target‐site mechanism(s) confer resistance to ACCase inhibitor herbicides in a Lolium multiflorum population

BACKGROUND: The increasing use of ACCase‐inhibiting herbicides has resulted in evolved resistance in key grass weeds infesting cereal cropping systems worldwide. Here, a thorough and systematic approach is proposed to elucidate the basis of resistance to three ACCase herbicides in a Lolium multiflorum Lam. (Italian rye grass) population from the United Kingdom (UK24). RESULTS: Resistance to sethoxydim and pinoxaden was always associated with a dominant D2078G (Alopecurus myosuroides Huds. equivalent) target‐site mutation in UK24. Conversely, whole‐plant herbicide assays on predetermined ACCase genotypes showed very high levels of resistance to diclofop‐methyl for all three wild DD2078 and mutant DG2078 and GG2078 ACCase genotypes from the mixed resistant population UK24. This indicates the presence of other diclofop‐methyl‐specific resistance mechanism(s) yet to be determined in this population. The D2078G mutation could be detected using an unambiguous DNA‐based dCAPS procedure that proved very transferable to A. myosuroides, Avena fatua L., Setaria viridis (L.) Beauv. and Phalaris minor Retz. CONCLUSION: This study provides further understanding of the molecular basis of resistance to ACCase inhibitor herbicides in a Lolium population and a widely applicable PCR‐based method for monitoring the D2078G target‐site resistance mutation in five major grass weed species. Copyright © 2010 Society of Chemical Industry     

Cross-resistance patterns to ACCase-inhibiting herbicides conferred by mutant ACCase isoforms in Alopecurus myosuroides Huds. (black-grass), re-examined at the recommended herbicide field rate

BACKGROUND: Target‐site‐based resistance to acetyl‐CoA carboxylase (ACCase) inhibitors in Alopecurus myosuroides Huds. is essentially due to five substitutions (Isoleucine‐1781‐Leucine, Tryptophan‐2027‐Cysteine, Isoleucine‐2041‐Asparagine, Aspartate‐2078‐Glycine, Glycine‐2096‐Alanine). Recent studies suggested that cross‐resistance patterns associated with each mutation using a seed‐based bioassay may not accurately reflect field resistance. The authors aimed to connect the presence of mutant ACCase isoform(s) in A. myosuroides with resistance to five ACCase inhibitors (fenoxaprop, clodinafop, haloxyfop, cycloxydim, clethodim) sprayed at the recommended field rate. RESULTS: Results from spraying experiments and from seed‐based bioassays were consistent for all mutant isoforms except the most widespread, Leucine‐1781. In spraying experiments, Leucine‐1781 ACCase conferred resistance to clodinafop and haloxyfop. Some plants containing Leucine‐1781 or Alanine‐2096 ACCase, but not all, were also resistant to clethodim. CONCLUSION: Leucine‐1781, Cysteine‐2027, Asparagine‐2041 and Alanine‐2096 ACCases confer resistance to fenoxaprop, clodinafop and haloxyfop at field rates. Leucine‐1781 ACCase also confers resistance to cycloxydim at field rate. Glycine‐2078 ACCase confers resistance to all five herbicides at field rates. Only Glycine‐2078 ACCase confers clethodim resistance under optimal application conditions. It may be that Leucine‐1781 and Alanine‐2096 ACCases may also confer resistance to clethodim in the field if the conditions are not optimal for herbicide efficacy, or at reduced clethodim field rates. Copyright © 2008 Society of Chemical Industry     

'Universal' primers for PCR-sequencing of grass chloroplastic acetyl-CoA carboxylase domains involved in resistance to herbicides

Primers were designed to amplify two regions involved in sensitivity to herbicides inhibiting the plastidic acetyl-CoA carboxylase (ACCase) from grasses (Poaceae). The first primer pair amplified a 551-bp amplicon containing a variable Ile/Leu codon at position 1781 in Alopecurus myosuroides sequence. The second primer pair amplified a 406-bp amplicon containing four variable codons (Trp/Cys, Ile/Asn, Asp/Gly, Gly/Ala) at positions 2027, 2041, 2078 and 2096, respectively, in A. myosuroides sequence. Both primer pairs amplified the targeted fragments from genes encoding plastidic ACCases, but not from the very similar genes encoding cytosolic ACCases. Clear DNA sequences were obtained from fresh or dried plant material from the field, and from 29 various grass species. Sequences revealed that the gene encoding plastidic ACCase in Poa annua and Festuca rubra contained a Leu₁₇₈₁ codon, in agreement with both species being inherently tolerant to herbicides inhibiting ACCase. Sequencing confirmed the hybrid origin of P. annua. Compared with ACCase enzyme assay, polymerase chain reaction is faster, can be performed from a single plant and suppresses the need for radioactive experiments. It can be completed with basic molecular biology laboratory equipment. It is the tool of choice for diagnosing resistance caused by alteration(s) of the plastidic ACCase.     

Allelic variation of the ACCase gene and response to ACCase-inhibiting herbicides in pinoxaden-resistant Lolium spp

BACKGROUND: The repeated use of acetyl‐coenzyme A carboxylase (ACCase) inhibiting herbicides to control grass weeds has selected for resistance in Lolium spp. populations in Italy. The efficacy of pinoxaden, a recently marketed phenylpyrazoline herbicide, is of concern where resistance to ACCase inhibitors has already been ascertained. ACCase mutations associated with pinoxaden resistance were investigated, and the cross‐resistance pattern to clodinafop, haloxyfop, sethoxydim, clethodim and pinoxaden was established on homo/heterozygous plants for four mutant ACCase alleles. RESULTS: Seven different mutant ACCase alleles (1781‐Leu, 1999‐Leu, 2041‐Asn, 2041‐Val, 2078‐Gly, 2088‐Arg and 2096‐Ala) and 13 combinations with two types of mutation were detected in the pinoxaden‐resistant plants. The 1781‐Leu allele appears to confer a dominant resistance to pinoxaden, clodinafop, haloxyfop, sethoxydim and clethodim at 60 g AI ha^?1. The 2041‐Asn and 2041‐Val alleles are associated with dominant or partially dominant resistance to FOPs, no substantial resistance to DIMs and a moderate resistance to pinoxaden. The 2088‐Arg allele endows a partially dominant resistance to clodinafop, sethoxydim and most likely to pinoxaden. In addition, non‐target‐site resistance mechanisms seem to be involved in pinoxaden resistance. CONCLUSION: Almost all the ACCase mutations selected in the field by other ACCase inhibitors are likely to confer resistance to pinoxaden. Although pinoxaden is sometimes able to control FOP‐resistant populations, it should not be considered as a sustainable ACCase resistance management tool. The presence of non‐ACCase‐based resistance mechanisms that could confer resistance to herbicides with different modes of action further complicates the resistance management strategies. Copyright © 2011 Society of Chemical Industry     

Differences in the molecular basis of resistance to the cyclohexanedione herbicide sethoxydim in Lolium multiflorum

Resistance to the cyclohexanedione (CHD) herbicide sethoxydim was investigated in two UK Lolium multiflorum populations, Yorks A2 and PYL. Resistance screening experiments demonstrated a qualitative difference in the responses of the two populations to sethoxydim, suggesting that the molecular basis of resistance between them was different. After treatment, Yorks A2 plants were either alive (78% of sample tested) or dead (22% tested) but plants of the PYL population showed two further intermediate categories of response. The level of acetyl-CoA carboxylase (ACCase) insensitivity directly correlated with the degree of resistance at the whole plant level, indicating that the molecular basis of resistance is associated with differences in ACCase sensitivity in each population. Direct sequencing of the carboxyl transferase domain of the ACCase gene showed that an Ile-418-Leu substitution in the L. multiflorum chloroplastic ACCase (GenBank accession number AY710293 ) confers resistance to sethoxydim in Yorks A2. This corresponds to amino acid residue 1781 in the Alopecurus myosuroides full ACCase sequence. This is the first report of this mutation in this L. multiflorum, which has also been reported in four other grass-weeds, including L. rigidum. However, no amino acid substitutions were found to be specifically associated with the resistant phenotypes in the PYL population and the molecular basis of resistance in this population remains to be resolved.     

Cross‐resistance patterns to acetyl coenzyme A carboxylase (ACCase) inhibitors associated with different ACCase mutations in Beckmannia syzigachne

Beckmannia syzigachne (American sloughgrass) is a competitive grass weed found in China. Fenoxaprop‐P‐ethyl is widely used for control of this species in China. Resistance to fenoxaprop‐P‐ethyl in B. syzigachne has been reported to be conferred by an isoleucine(Ile)‐1781‐leucine(Leu) substitution in the gene encoding the herbicide target, acetyl‐CoA carboxylase (ACCase). In this study, three mutations were detected by derived cleaved amplified polymorphic sequence (dCAPS) method in fenoxaprop‐P‐ethyl‐resistant B. syzigachne populations: Ile‐1781‐Leu in population JCWL‐R, Ile‐2041‐Asn in JCJT‐R and Gly‐2096‐Ala in JYJD‐R. The data indicated they were genetically homogeneous (homozygous mutant) at the ACCase locus. The use of cytochrome P450 inhibitors was shown to slightly reduce the GR₅₀ value of fenoxaprop‐P‐ethyl‐resistant populations, from which we inferred a combination of target‐site resistance (TSR) and non‐target‐site resistance (NTSR) was involved in fenoxaprop‐P‐ethyl‐resistance. We characterised the cross‐resistance patterns to ACCase inhibitors in B. syzigachne. The plants in the JCWL‐R population were highly resistant to all tested APPs (aryloxyphen‐oxypropionates), sethoxydim and pinoxaden, and moderately resistant to clethodim. The plants in the JCJT‐R population were highly resistant to fluazifop‐P‐butyl, clodinafop‐propargyl, cyhalofop‐butyl, metamifop and pinoxaden; moderately resistant to haloxyfop‐R‐methyl, quizalofop‐P‐ethyl and sethoxydim; and sensitive to clethodim. The plants in the JYJD‐R population were highly resistant to clodinafop‐propargyl, metamifop and pinoxaden; moderately resistant to haloxyfop‐R‐methyl, cyhalofop‐butyl, quizalofop‐P‐ethyl, fluazifop‐P‐butyl and sethoxydim; and sensitive to clethodim. If resistance to ACCase inhibitors is present in B. syzigachne populations in the field, then our results indicate that clethodim should be used. While we demonstrated the cross‐resistance patterns of TSR resulting from three mutations in B. syzigachne, we also demonstrated that NTSR plays a role in resistance, which will complicate weed management.     

Prevalence of cross‐ or multiple resistance to the acetyl‐coenzyme A carboxylase inhibitors fenoxaprop,clodinafop and pinoxaden in black‐grass (Alopecurus myosuroides Huds.) in France

BACKGROUND: Repeated use of acetyl‐CoA carboxylase (ACCase) inhibitors, especially fenoxaprop and clodinafop, since the late 1980s has selected for resistance in Alopecurus myosuroides Huds. (black‐grass) in France. We investigated whether resistance to pinoxaden, a phenylpyrazoline ACCase inhibitor to be marketed in France, was present in French black‐grass populations. We investigated pinoxaden resistance conferred by five mutant ACCase isoforms. Using 84 French black‐grass field samples, we also compared the frequencies of other mechanisms endowing resistance to fenoxaprop, clodinafop or pinoxaden. RESULTS: ACCase mutant isoforms Leu‐1781, Gly‐2078 and, likely, Cys‐2027 conferred cross‐resistance to pinoxaden, while isoform Asn‐2041 possibly conferred moderate resistance. Other mechanisms of resistance to fenoxaprop, clodinafop and pinoxaden were detected in 99, 68 and 64% of the samples investigated, respectively. Cross‐ or multiple resistance to fenoxaprop or clodinafop and pinoxaden was not systematically observed, suggesting a diversity of mechanisms exist. CONCLUSION: Pinoxaden resistance was observed before pinoxaden release in France. Only a fraction of the mechanisms endowing fenoxaprop or clodinafop resistance also confer pinoxaden resistance. Pinoxaden resistance was likely mostly selected for by ACCase inhibitors, and, in some cases, possibly by herbicides with other modes of action. This illustrates the necessity to use metabolisable herbicides cautiously where black‐grass has evolved non‐target‐site‐based resistance. Copyright © 2009 Society of Chemical Industry     

杂草对芳氧苯氧丙酸类(APPs)除草剂的抗性分子机理研究进展

芳氧苯氧丙酸类（aryloxyphenoxypropionates,APPs）除草剂是一类广泛使用的乙酰辅酶A羧化酶抑制剂,可高效专一抑制禾本科杂草的乙酰辅酶A羧化酶（acetyl-coenzyme A carboxylase,ACCase）。目前已出现大量抗芳氧苯氧丙酸类除草剂的禾本科杂草,其抗性大多由叶绿体乙酰辅酶A羧化酶的羧基转移酶（carboxyltransferase,CT）功能域中的氨基酸突变引起。在所有已发现的氨基酸突变中,最引人关注的是第1 781位（对应大穗看麦娘Alopecurus myosuroides质体ACCase的氨基酸残基位置）异亮氨酸到亮氨酸的单点突变,该特定位置形成亮氨酸会导致某些杂草对APPs类除草剂产生抗性。综述了乙酰辅酶A羧化酶CT功能域的研究进展及杂草对APPs类除草剂的抗性分子机理,探讨了杂草对APPs类除草剂抗性分子机理研究中存在的问题,以期为进一步深入研究APPs类除草剂的抗性机制提供参考。     

A pollen test to detect ACCase target‐site resistance within Alopecurus myosuroides populations

This study was conducted to determine a suitable medium for in vitro germination of Alopecurus myosuroides pollen and to develop a reliable test for the rapid screening of ACCase target site‐resistant plants within populations. The assay is based upon germination of pollen in a medium supplemented with ACCase inhibitors. A 0.25% agar medium, containing 200 mg L^–1 CaNO₃, 100 mg L^–1 H₃BO₃, 200 g L^–1 sucrose, was selected as a suitable medium for in vitro pollen germination. At 25 °C, this medium supported a mean germination rate of 85% within two hours. Plants highly resistant (Rh) to aryloxyphenoxypropionate (APP), owing to the expression of an insensitive ACCase, were found to express this resistance in their pollen. In contrast, plants moderately resistant (Rm) to APP herbicides, owing to an enhanced capacity to detoxify herbicides, did not exhibit this resistance in their pollen. Concentrations of 120 μM fenoxaprop and 1000 μM clodinafop were selected as the best for reliable discrimination of the target‐site‐resistant biotypes. At these concentrations there was more than 50% germination of the Rh pollen grains whereas less than 10% of the S and Rm pollen grains germinated. This test, using haploid material, may also permit distinction between homozygous‐ and heterozygous‐resistant individuals.     

看麦娘对甲基二磺隆靶标抗性的快速检测

甲基二磺隆是防除小麦田看麦娘Alopecurus aequalis等禾本科杂草的主要除草剂品种之一,但目前中国山东、江苏及安徽等地已有部分看麦娘种群对其产生了抗性。ALS基因197位点突变是看麦娘对甲基二磺隆产生抗性的重要机理,根据突变型和野生型看麦娘在197位点处碱基序列的不同,本研究设计出了一种衍生性酶切扩增多态性序列(dCAPS)分子标记方法,可用于197位点突变的快速检测。通过在引物D197F序列的3′ 端引入一个错配碱基,扩增所得不同种群看麦娘的ALS片段经限制性内切酶BamH I酶切后表现出多态性:野生敏感型分别产生了200和36 bp的2个条带;纯合突变型因无法被切开,只有236 bp的一个条带;而杂合突变型则同时产生了上述3个条带。该dCAPS检测结果准确、可靠,与经典的整株水平测定结果一致,并且可同时检测197位点上任一形式的突变。研究结果可为看麦娘等禾本科杂草对甲基二磺隆靶标抗性的快速检测提供理论依据。     

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     

Status of black grass (Alopecurus myosuroides) resistance to acetyl-coenzyme A carboxylase inhibitors in France

We assessed the contributions of target site‐ and non‐target site‐based resistance to herbicides inhibiting acetyl‐coenzyme A carboxylase (ACC) in Alopecurus myosuroides (black grass). A total of 243 A. myosuroides populations collected across France were analysed using herbicide sensitivity bioassay (24 300 seedlings analysed) and ACC genotyping (13 188 seedlings analysed). Seedlings resistant to at least one ACC‐inhibiting herbicide were detected in 99.2% of the populations. Mutant, resistant ACC allele(s) were detected in 56.8% of the populations. Among the five resistant ACC alleles known in A. myosuroides, alleles containing an isoleucine‐to‐leucine substitution at codon 1781 were predominant (59.5% of the plants containing resistant ACC alleles). Comparison of the results from herbicide sensitivity bioassays with genotyping indicated that more than 75% of the plants resistant to ACC‐inhibiting herbicides in France would be resistant via increased herbicide metabolism. Analysis of herbicide application records suggested that in 15.9% of the populations studied, metabolism‐based resistance to ACC‐inhibiting herbicides was mostly selected for by herbicides with other modes of action. Our study revealed the importance of non‐target site‐based resistance in A. myosuroides. Using herbicides with alternative modes of action to control populations resistant to ACC‐inhibiting herbicides, the recommended management approach, may thus be jeopardised by the widespread occurrence of metabolism‐based resistance mechanisms conferring broad‐spectrum cross‐resistance.     

耿氏硬草对乙酰辅酶A羧化酶类除草剂抗性水平及分子机制初探

为明确耿氏硬草Pseudosclerochloa kengiana(Ohwi)Tzvel潜在抗性种群对不同乙酰辅酶A羧化酶(ACCase)类除草剂的抗性水平及其靶标抗性的分子机制,采用剂量-反应曲线法测定了耿氏硬草对精鰁唑禾草灵、炔草酯、烯禾啶、烯草酮和唑啉草酯5种ACCase类除草剂的抗性水平,扩增并比对了耿氏硬草抗性和敏感种群间ACCase基因的差异。结果显示:与敏感种群SD-6相比,耿氏硬草种群SD-32对精鰁唑禾草灵、炔草酯、烯禾啶、烯草酮和唑啉草酯产生了不同水平的抗性,抗性倍数分别为16.5、7.5、15.0、4.4和5.7;SD-32种群ACCase基因CT区域的2078位氨基酸基因由GAT突变为GGT,导致天冬氨酸(Asp)被甘氨酸(Gly)取代。分析表明,ACCase基因2078位氨基酸的突变可能是导致耿氏硬草对ACCase类除草剂产生抗性的重要原因之一。     

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     

浙江稻区千金子对氰氟草酯和噁唑酰草胺的抗药性及其分子机制研究

千金子是中国直播稻田的优势禾本科杂草之一,严重威胁水稻的产量和品质。为了进一步明确浙江地区水稻田千金子对芳氧苯氧丙酸类除草剂的抗性发生情况,本研究从浙江部分稻区共采集了11个千金子种群 (其中1个为敏感种群),通过整株植物测定法检测了各种群对氰氟草酯和噁唑酰草胺的敏感性。结果显示:共有8个种群对氰氟草酯产生了抗性 (抗性指数为2.1~79.1),9个种群对噁唑酰草胺产生了抗性 (抗性指数为2.0~31.0),其中对氰氟草酯的抗性问题更为显著。在此基础上,通过基因扩增和克隆,对敏感种群和抗性种群的乙酰辅酶A羧化酶 (Acetyl-CoA carboxylase,ACCase) 基因部分序列进行比对,结果在3个抗性种群中发现突变,其中1个种群为Ile-1781-Val突变,另外2个种群则为Trp-2027-Cys突变。该研究结果表明,目前浙江部分稻区千金子种群已对氰氟草酯和噁唑酰草胺产生了抗药性,其中靶标酶基因突变是导致部分种群产生抗药性的原因之一。     

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.     

The occurrence of herbicide-resistant weeds worldwide

The 1995/6 International Survey of Herbicide-Resistant Weeds recorded 183 herbicide-resistant weed biotypes (124 different species) in 42 countries. The increase in the number of new herbicide-resistant weeds has remained relatively constant since 1978, at an average of nine new cases per year worldwide. Whilst 61 weed species have evolved resistance to triazine herbicides, this figure now only accounts for one-third of all documented herbicide-resistant biotypes. Triazine-resistant weeds have been controlled successfully in many countries by the use of alternative herbicides. Due to the economic importance of ALS and ACCase inhibitor herbicides worldwide, and the ease with which weeds have evolved resistance to them, it is likely that ALS and ACCase inhibitor-resistant weeds will present farmers with greater problems in the next five years than triazine-resistant weeds have caused in the past 25 years. Thirty-three weed species have evolved resistance to ALS-inhibitor herbicides in 11 countries. ALS-inhibitor-resistant weeds are most problematic in cereal, corn/soybean and rice production. Thirteen weed species have evolved resistance to ACCase inhibitors, also in 11 countries. ACCase inhibitor resistance in Lolium and Avena spp. threatens cereal production in Australia, Canada, Chile, France, South Africa, Spain, the United Kingdom and the USA. Fourteen weed species have evolved resistance to urea herbicides. Isoproturon-resistant Phalaris minor infesting wheat fields in North West India and chlorotoluron-resistant Alopecurus myosuroides in Europe are of significant economic importance. Although 27 weed species have evolved resistance to bipyridilium herbicides, and 14 weed species have evolved resistance to synthetic auxins, the area infested and the availability of alternative herbicides have kept their impact minimal. The lack of alternative herbicides to control weeds with multiple herbicide resistance, such as Lolium rigidum and Alopecurus myosuroides, makes these the most challenging resistance problems. The recent discovery of glyphosate-resistant Lolium rigidum in Australia is a timely reminder that sound herbicide-resistant management strategies will remain important after the widespread adoption of glyphosate-resistant crops. ©1997 SCI     

Multiple origins for black-grass (Alopecurus myosuroides Huds) target-site-based resistance to herbicides inhibiting acetyl-CoA carboxylase

We have investigated the process of evolution of target-site-based resistance to herbicides inhibiting acetyl-CoA carboxylase (ACCase) in nine French populations of black-grass (Alopecurus myosuroides Huds). To date, two different ACCase resistant alleles are known. One contains an isoleucine-to-leucine substitution at position 1781, the second contains an isoleucine-to-asparagine substitution at position 2041. Using phylogenetic analysis of ACCase sequences, we showed that 1781Leu ACCase alleles evolved from four independent origins in the nine black-grass populations studied, while 2041Asn ACCase alleles evolved from six independent origins. No geographical structure of black-grass populations was revealed. This implies that these populations, although geographically distant, are, or have until recently been, connected by gene flows. Comparison of biological data obtained from herbicide sensitivity bioassay and molecular data showed that distinct resistance mechanisms often exist in a single black-grass population. Accumulation of different resistance mechanisms in a single plant was also demonstrated. We conclude that large-scale evolution of resistance to herbicides in black-grass is a complex phenomenon, resulting from the independent selection of various resistance mechanisms in local black-grass populations undergoing contrasted herbicide and agronomical selection pressures, and connected by gene flows whose parameters remain to be determined.     

河南省多花黑麦草对ACCase和ALS抑制剂的抗性及其靶标基因突变分析

为明确河南省部分地区的多花黑麦草Lolium multiflorum种群对乙酰辅酶A羧化酶（acetylCoA carboxylase,ACCase）和乙酰乳酸合成酶（acetolactate synthase,ALS）抑制剂类除草剂的抗性水平和抗性机理,采用整株生物测定法测定采自新乡市和驻马店市的多花黑麦草种群对ACCase抑制剂类除草剂精噁唑禾草灵、炔草酯、唑啉草酯和ALS抑制剂类除草剂甲基二磺隆、氟唑磺隆、啶磺草胺的抗性水平,并对多花黑麦草ACCase和ALS靶标酶编码基因进行克隆及氨基酸序列比对,分析其靶标抗性机理。结果显示,与多花黑麦草敏感种群HNXX01相比,HNZMD04和HNXX05种群对6种除草剂均产生了抗性,HNZMD04种群对精噁唑禾草灵和啶磺草胺的相对抗性倍数分别为44.65和40.31,对炔草酯和氟唑磺隆的相对抗性倍数分别为11.91和11.93;HNXX05种群对精噁唑禾草灵和氟唑磺隆的相对抗性倍数分别为27.70和25.67。HNZMD04和HNXX05抗性种群的ACCase基因均发生了D2078G突变,2个种群的突变率分别为55%和70%;HNZMD04...