抗精噁唑禾草灵和甲基二磺隆看麦娘ACCase和ALS基因突变

为明确看麦娘Alopecurus aequalis抗性种群YL的靶标抗性机制,采用基因克隆法对看麦娘抗性和敏感种群间乙酰辅酶A羧化酶(ACCase)和乙酰乳酸合成酶(ALS)基因序列进行扩增、克隆和测序,比对二者ACCase和ALS基因序列的差异,探寻其产生抗药性突变的基因位点,同时测定该突变型抗性种群YL对不同ACCase和ALS抑制剂类除草剂的交互抗性。结果显示,与看麦娘敏感种群TL相比,抗性种群YL的ACCase基因CT区域第2 041位氨基酸由异亮氨酸(ATT)突变为天冬酰胺酸(AAT),ALS基因Domain A区域第197位氨基酸由脯氨酸(CCC)突变为精氨酸(CGC)。看麦娘抗性种群YL对ACCase抑制剂炔草酯产生了高水平抗性,抗性倍数为43.96,对高效氟吡甲禾灵和精喹禾灵产生了中等水平抗性,抗性倍数分别为18.33和15.87,对唑啉草酯、烯草酮和烯禾啶较敏感;对ALS抑制剂氟唑磺隆产生了低水平抗性,抗性倍数为8.39,对啶磺草胺和咪唑乙烟酸较敏感。表明ACCase基因第2 041位和ALS基因第197位氨基酸突变是导致看麦娘抗性种群YL对精噁唑禾草灵和甲基二磺隆同时产生抗性的重要原因之一。     

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。     

抗精噁唑禾草灵的日本看麦娘ACCase基因突变

为明确日本看麦娘抗性种群对精噁唑禾草灵的抗性水平及抗性产生的分子机制,采用整株水平测定法测定了日本看麦娘对精噁唑禾草灵的抗性水平,扩增和比对了日本看麦娘抗性和敏感种群间乙酰辅酶A羧化酶(acetyl-Co A carboxylase,ACCase)基因的差异。结果显示,与敏感种群AH-7相比,抗性种群AH-25对精噁唑禾草灵的抗性倍数为33.82;AH-25种群ACCase基因CT区域2 078位氨基酸发生了突变,由天冬氨酸GAT突变为甘氨酸GGT;AH-25种群对炔草酯、烯草酮和烯禾啶产生了高水平的抗性,抗性倍数分别为35.66、38.64和29.14,对高效氟吡甲禾灵产生了低水平的抗性,抗性倍数为3.04,对精喹禾灵和唑啉草酯较敏感。表明ACCase基因2 078位氨基酸的突变可能是导致精噁唑禾草灵产生高水平抗性的重要原因。     

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...     

广东省稻菜轮作区中牛筋草对十种除草剂的抗性水平及靶基因序列分析

为明确广东省稻菜轮作区中牛筋草对10种常用除草剂的抗性水平及抗性分子机制,采用整株生物测定法测定广东省稻菜轮作区内8个牛筋草种群P1～P8对草甘膦、草铵膦和乙酰辅酶A羧化酶(acetyl-CoA carboxylase,ACCase)抑制剂类等10种除草剂的抗性水平,并进一步分析P1和P8种群相关靶标酶基因5-烯醇丙酮酰莽草酸-3-磷酸合酶(5-enolpyruvyl-shikimate-3-phosphate synthase,EPSPS)、谷氨酰胺合成酶(glutamine synthetase,GS)和ACCase的部分功能区序列特征。结果显示,牛筋草P1～P8种群对草甘膦抗性指数为敏感种群的5.9倍～17.7倍,其中P8种群对草甘膦的抗性水平最高;8个种群对草铵膦也产生了不同程度的抗性,抗性指数为敏感种群的2.3倍～14.2倍,其中P1种群抗性最高。牛筋草P1和P8种群均对ACCase抑制剂类除草剂精喹禾灵、氰氟草酯和噁唑酰草胺产生了交互抗性;P1种群ACCase基因在第2 041位氨基酸处发生突变,该突变在牛筋草种群中首次发现;而P8种群ACCase基因则在第2 027位氨基...     

上海市水稻田千金子对3种乙酰辅酶A羧化酶抑制剂的抗性现状及酶突变机制

为明确上海市水稻田千金子对乙酰辅酶A羧化酶 (ACCase) 抑制剂类除草剂的抗性发生情况及可能存在的抗性机制,在上海市千金子发生严重地区的水稻田共采集51个种群,采用单剂量抗性甄别法测定了不同千金子种群对3种ACCase抑制剂类除草剂的抗性水平,扩增和比对了靶标酶ACCase基因部分片段的差异。结果显示:在氰氟草酯105 g/hm2有效成分剂量选择压下,8个千金子种群标记为抗性种群,6个种群为发展中抗性种群;在噁唑酰草胺 120 g/hm2有效成分剂量选择压下,4个千金子种群标记为抗性种群,5个种群为发展中抗性种群;在精噁唑禾草灵62.1 g/hm2有效成分剂量选择压下,6个千金子种群标记为抗性种群,2个种群为发展中抗性种群。对15个抗性千金子种群靶标酶基因片段的测序发现,9个种群共发生了4种ACCase基因突变类型,分别为ACCase基因 1999 位点色氨酸 (TGG) 突变为丝氨酸 (TCG)、1999位点色氨酸 (TGG) 突变为半胱氨酸 (TGT)、2027位点色氨酸 (TGG) 突变为丝氨酸 (TCG) 以及2027位点色氨酸 (TGG) 突变为半胱氨酸 (TGT/TGC)。其中,5个抗性千金子种群ACCase 基因突变频率均大于60%。研究表明,ACCase抑制剂类除草剂抗性千金子在上海市部分地区发生已较为严重,ACCase 基因突变是导致不同千金子种群对该类除草剂产生抗性的重要原因之一。     

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。     

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。     

安徽省稻田千金子对氰氟草酯和噁唑酰草胺的抗性及其靶标分子机制

为明确安徽省稻田杂草千金子Leptochloa chinensis对氰氟草酯和噁唑酰草胺的抗性，从安徽省12个市38个县（市、区）稻区千金子发生较严重的田块中共采集72个千金子种群，采用整株生物测定法检测其对氰氟草酯和噁唑酰草胺的抗性，筛选同时对噁唑酰草胺和氰氟草酯有疑似抗性的千金子种群并测定其对这2种药剂的抗性水平，同时利用分子生物学技术检测千金子高抗种群的乙酰辅酶A羧化酶（acetyl-CoA carboxylase,ACCase）基因是否发生抗性位点氨基酸突变。结果显示，在72个千金子种群中，13个千金子种群已对氰氟草酯产生抗性，5个千金子种群已对噁唑酰草胺产生抗性；AH-1、AH-3、AH-7、AH-34和AH-72这5个千金子种群同时高抗氰氟草酯和噁唑酰草胺，其中对氰氟草酯的抗性指数介于21.98～65.52之间，对噁唑酰草胺的抗性指数介于11.76～27.19之间；这5个种群的ACCase基因片段均在2 027位点发生氨基酸突变，色氨酸突变为半胱氨酸；此外AH-3和AH-72这2个种群在1 999位点也由色氨酸突变为半胱氨酸，表明安徽省千金子已经对氰氟草酯和噁唑酰草胺产生交...     

河南省麦田荠菜对苯磺隆的抗性及其交互抗性

为明确河南省荠菜Capsella bursa-pastoris种群对苯磺隆的抗性水平及其可能存在的抗性机理,应用整株法测定了采自驻马店及南阳等6个荠菜发生严重市的10个荠菜种群对苯磺隆的抗性,扩增和比对了荠菜苯磺隆抗性种群及敏感种群之间靶标酶乙酰乳酸合成酶基因ALS的差异,并使用单剂量法测定了以上种群对双氟磺草胺、啶磺草胺及氟唑磺隆等ALS抑制剂类除草剂的交互抗性。结果表明,驻马店市的汝南县冯湾村(ZMD-1)及平舆县五里路村(ZMD-3)荠菜种群对苯磺隆的抗性倍数分别为3.1和2.5,表现出低水平抗性;驻马店市汝南县赖楼村(ZMD-2)和周口市川汇区文庄村(ZK-1)荠菜种群对苯磺隆的抗性倍数分别为21.7和57.8,表现出高水平抗性;南阳市唐河县上屯村(NY-2)荠菜种群对苯磺隆的抗性倍数为116.5,表现出极高水平抗性,其它种群对苯磺隆仍然较敏感。NY-2、ZMD-2和ZK-1种群的ALS基因第197位氨基酸由脯氨酸(CCT)分别突变为丝氨酸(TCT)、丙氨酸(GCT)和亮氨酸(CTT),其它种群中均未发现有突变产生;这3个种群在氟唑磺隆推荐剂量处理下,死亡率仅为18.9%、23.3%和11.1%,说明已对氟唑磺隆产生了较高水平的交互抗性,其中NY-2种群对双氟磺草胺和啶磺草胺产生了低水平交互抗性,推荐剂量下死亡率分别为82.2%和83.1%。表明ALS基因突变很可能是导致荠菜种群对苯磺隆等ALS抑制剂类除草剂产生抗性的重要原因。     

Multiple resistance across glufosinate,glyphosate, paraquat and ACCase‐inhibiting herbicides in an Eleusine indica population

An Eleusine indica population was previously reported as the first global case of field‐evolved glufosinate resistance. This study re‐examines glufosinate resistance and investigates multiple resistance to other herbicides in the population. Dose–response experiments with glufosinate showed that the resistant population is 5‐fold and 14‐fold resistant relative to the susceptible population, based on GR₅₀ and LD₅₀ R/S ratio respectively. The selected glufosinate‐resistant subpopulation also displayed a high‐level resistance to glyphosate, with the respective GR₅₀ and LD₅₀ R/S ratios being 12‐ and 144‐fold. In addition, the subpopulation also displayed a level of resistance to paraquat and ACCase‐inhibiting herbicides fluazifop‐P‐butyl, haloxyfop‐P‐methyl and butroxydim. ACCase gene sequencing revealed that the Trp‐2027‐Cys mutation is likely responsible for resistance to the ACCase inhibitors examined. Here, we confirm glufosinate resistance and importantly, we find very high‐level glyphosate resistance, as well as resistance to paraquat and ACCase‐inhibiting herbicides. This is the first confirmed report of a weed species that evolved multiple resistance across all the three non‐selective global herbicides, glufosinate, glyphosate and paraquat.     

Mechanisms of resistance to acetyl‐coenzyme A carboxylase‐inhibiting herbicides in a Hordeum leporinum population

A failure of acetyl‐coenzyme A carboxylase (ACCase)‐inhibiting herbicides to control a population of Hordeum leporinum Link (barleygrass) occurred following eight applications of these herbicides in both crops and pastures. This population was 7.6‐fold resistant to fluazifop‐P‐butyl compared with standard susceptible populations. The population was between 3.6‐ and 3.8‐fold resistant to other ACCase‐inhibiting herbicides, except butroxydim to which it was susceptible. ACCase extracted from resistant plants and assayed in the presence of herbicides in vitro was susceptible to fluazifop acid and other aryloxyphenoxypropanoate herbicides, but was 4‐fold less sensitive to sethoxydim compared with ACCase from susceptible plants. Resistant plants metabolised fluazifop acid about 1.3‐fold more rapidly compared with susceptible plants; however, sethoxydim was metabolised equally in both populations. Resistance to fluazifop‐P‐butyl and other aryloxyphenoxypropanoate herbicides may be the result of increased herbicide detoxification, whereas resistance to sethoxydim appears to be due to a modified target enzyme. Herbicide resistance in this population is unusual in that different mechanisms appear to confer resistance to the aryloxyphenoxypropanoate and cyclohexanedione herbicides. © 2000 Society of Chemical Industry     

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     

Characterisation of target-site resistance to ACCase-inhibiting herbicides in the weed Alopecurus myosuroides (black-grass)

Resistance to aryloxyphenoxypropionate (AOPP), cyclohexanedione (CHD) and phenylurea herbicides was determined in UK populations of Alopecurus myosuroides Huds. Two populations (Oxford AA1, Notts. A1) were highly resistant (Resistance indices 13-->1000) to the AOPP and CHD herbicides fenoxaprop, diclofop, fluazifop-P and sethoxydim, but only marginally resistant to the phenylurea, chlorotoluron. Analyses of acetyl coenzyme A carboxylase (ACCase) activity showed that an insensitive ACCase conferred resistance to all the AOPP/CHD herbicides investigated. Another population, Oxford S1, showed no resistance to sethoxydim at the population level, but contained a small proportion of plants (<10%) with an insensitive ACCase. Genetic studies on the Notts A1 and Oxford S1 populations demonstrated that target site resistance conferred by an insensitive ACCase is monogenic, nuclearly inherited with the resistant allele showing complete dominance. Investigations of the molecular basis of resistance in the Notts A1 population showed that sethoxydim resistance in A myosuroides was associated with the substitution of an isoleucine in susceptible with a leucine in resistant plants, which has also been found in three other resistant grass-weed species (Setaria viridis (L) Beauv, Avena fatua L, Lolium rigidum Gaud).     

Confirmed resistance to aryloxyphenoxypropionate herbicides in Phalaris minor populations in Iran

Phalaris minor (littleseed canary grass) is a major weed in wheat fields in some parts of Iran. Diclofop‐methyl, fenoxaprop‐P‐ethyl, and clodinafop‐propargyl are three acetyl coenzyme A carboxylase (ACCase)‐inhibiting herbicides that are commonly used to control this grass in wheat fields. Thirty‐four P. minor populations with suspected resistance to ACCase‐inhibiting herbicides were sampled from wheat fields in the provinces of Fars and Golestan in Iran. The dose–response assays that were conducted under controlled greenhouse conditions indicated that 14 populations were resistant to fenoxaprop‐P‐ethyl, seven populations were resistant to both fenoxaprop‐P‐ethyl and diclofop‐methyl, and three populations were resistant to fenoxaprop‐P‐ethyl, diclofop‐methyl, and clodinafop‐propargyl. These populations showed different levels of resistance to the applied herbicides, compared to the susceptible population. These results suggest that different mechanisms of resistance could be involved. The enzyme assay revealed that the existence of modified ACCase in the three most‐resistant populations (AR, MR4, and SR3) is responsible for the resistance of these populations.     

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     

Mechanism of resistance to fenoxaprop in Japanese foxtail (Alopecurus japonicus) from China

Japanese foxtail is one of the most common and troublesome weeds infesting cereal and oilseed rape fields in China. Repeated use during the last three decades of the ACCase-inhibiting herbicide fenoxaprop-P-ethyl to control this weed has resulted in the occurrence of resistance. Dose–response tests established that a population (AHFD-1) from eastern China had evolved high-level resistance to fenoxaprop-P-ethyl. Based on the resistance index, this resistant population of A. japonicus is 60.31-fold resistant to fenoxaprop-P-ethyl. Subsequently, only a tryptophan to cysteine substitution was identified to confer resistance to fenoxaprop-P-ethyl in this resistant population. ACCase activity tests further confirmed this substitution was linked to resistance. This is the first report of the occurrence of Trp-2027-Cys substitution of ACCase in A. japonicus. From whole-plant pot dose–response tests, we confirmed that this population conferred resistance to other APP herbicides, including clodinafop-propargyl, fluazifop-P-butyl, quizalofop-P-ethyl, haloxyfop-R-methyl, cyhalofop-butyl, metamifop, DEN herbicide pinoxaden, but not to CHD herbicides clethodim, sethoxydim. There was also no resistance observed to ALS-inhibiting herbicides sulfosulfuron, mesosulfuron-methyl, flucarbazone-sodium, pyroxsulam, Triazine herbicide prometryne and glyphosate. However, this resistant population was likely to confer slightly (or no) resistant to Urea herbicides chlortoluron and isoproturon.