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...     

苏门白酒草对草甘膦等除草剂的多抗性检测及防治药剂筛选

苏门白酒草Conyza sumatrensis是中国华南地区常见的阔叶杂草,在果园和非耕地常造成严重危害。本研究采用整株剂量反应法,明确了采自广东省广州市的苏门白酒草疑似抗性种群 (GZ-R) 对草甘膦、百草枯和敌草快的抗性水平,比对了GZ-R种群和采自广东省清远市的敏感对照种群 (QY-S) 的草甘膦靶标酶基因EPSPS2片段的差异,并测定了灭草松、氯氟吡氧乙酸等5种茎叶处理剂对不同叶龄苏门白酒草的室内防除效果。结果表明:GZ-R种群对草甘膦和百草枯分别产生了中等水平和高水平抗性,并已对敌草快产生交互抗性,3种药剂对GZ-R种群的LD₅₀值分别是对QY-S种群LD₅₀值的7.2、72.3和6.6倍;与QY-S种群相比,GZ-R种群的EPSPS2基因106位由脯氨酸突变为苏氨酸。在灭草松、氯氟吡氧乙酸或2甲4氯钠推荐剂量下,于4~5叶期施药,苏门白酒草死亡率均为100%,但于6~7叶期和10~12叶期施药,苏门白酒草死亡率显著下降至44.4%~91.7%;而在草铵膦或苯嘧磺草胺推荐剂量下,不同叶龄期施药苏门白酒草的死亡率均为100%,因此在植株生长早期可使用草铵膦和苯嘧磺草胺防除已对草甘膦和百草枯等除草剂产生抗性的苏门白酒草。     

玉米田四种禾本科杂草对草甘膦的敏感性测定

马唐Digitaria sanguinalis、牛筋草Eleusine indica、狗尾草Setaria viridis和虎尾草Chloris virgata是我国北方和黄淮海玉米田4种主要禾本科杂草,是影响玉米产量和品质的主要因素之一。为了明确禾本科杂草对草甘膦的敏感水平,本研究采用整株生物测定法测定上述4种杂草种群对草甘膦的敏感基线,为将来我国种植转基因耐草甘膦玉米后,玉米田杂草防除提供数据支撑。结果表明,马唐55个种群GR₅₀值范围在13.36～550.28 g a.i./ha,均值为120.02 g a.i./ha。狗尾草69个种群GR₅₀值范围在7.22～201.96g a.i./ha,均值为50.45 g a.i./ha。牛筋草50个种群GR₅₀范围在25.19～808.54 g a.i./ha,均值为218.78 g a.i./ha。测定的所有马唐、狗尾草和牛筋草种群GR₅₀值均低于900 g a.i./ha(田间推荐中剂量),表明这三种杂草对草甘膦敏感。虎尾草34个种群GR     

海南省香蕉园牛筋草对百草枯抗药性水平的初步研究

为明确海南省香蕉园杂草牛筋草的抗药性水平,利用温室生物测定法研究海南省儋州、白沙、昌江、东方、陵水、三亚、琼中等地区牛筋草种群对百草枯的抗药性水平。结果表明,采集的7个牛筋草种群对百草枯表现出不同程度的抗药性,其中采自陵水地区的牛筋草种群抗药性水平最高,ED_(50)为4 495. 702 2 g a. i./hm~2,相对抗药性倍数达50. 81倍,而昌江、琼中2个地区的牛筋草种群则表现出较低的抗药性,相对抗药性倍数在2～4倍之间。     

宁夏地区稗草种群对五氟磺草胺的抗性机制研究

为了明确宁夏稻区稗草对五氟磺草胺抗性水平及抗性机制。采用整株生物测定法测定了宁夏地区6个稗的原变种Echinochloa crus-galli var.crus-galli种群对五氟磺草胺的抗性水平,并测定了每个种群的乙酰乳酸合酶基因(ALS)序列和ALS酶离体活性,以及P450抑制剂马拉硫磷对稗草种群抗性水平的影响。结果显示,与敏感种群相比,5个疑似抗性种群对五氟磺草胺表现出不同程度的抗性(10.18倍～32.71倍),其中抗性种群N14,N22,N27和N51的ALS 574位色氨酸突变为亮氨酸,N53的197位脯氨酸突变为亮氨酸,敏感种群N43没有发现突变位点,五氟磺草胺对抗性种群ALS酶的IC₅₀均明显高于敏感种群,马拉硫磷对五氟磺草胺有增效作用,可提高稗草种群对五氟磺草胺的敏感性。综上所述,稗草种群对五氟磺草胺产生抗性是由于靶标基因ALS突变,同时稗草种群对五氟磺草胺的抗性也可能与细胞色素P450介导的代谢增强有关。     

玉米田反枝苋对烟嘧磺隆的抗性水平及靶标抗性分子机理

为明确玉米田主要杂草反枝苋对烟嘧磺隆的抗性水平及靶标抗性分子机理,采用整株水平测定法检测了黑龙江省玉米田反枝苋对烟嘧磺隆的抗性水平,通过靶标酶离体活性测定,分析了抗性和敏感种群反枝苋乙酰乳酸合成酶 (ALS) 对烟嘧磺隆的敏感性,并通过靶标ALS基因克隆测序进行了序列比对分析。结果显示:黑龙江省反枝苋疑似抗性种群 (HLJ-R) 对烟嘧磺隆已产生较高水平抗性,其抗性倍数达13.7;酶活性测定结果表明:烟嘧磺隆对HLJ-R种群ALS活性的抑制中浓度 (IC₅₀) 值是对敏感种群 (TA-S) IC₅₀值的43.9倍;与TA-S种群相比,HLJ-R种群ALS基因205位丙氨酸突变为缬氨酸,574位色氨酸突变为亮氨酸。研究表明,黑龙江省玉米田反枝苋对烟嘧磺隆已产生较高水平抗性,且靶标ALS基因的突变可能是其抗性产生的主要原因之一。     

玉米田主要杂草对烟嘧磺隆的抗性

为明确我国玉米田杂草对烟嘧磺隆的抗性水平及分布现状,于2010—2011年自山东、吉林、四川、河北4省采集连续多年施用烟嘧磺隆的玉米田杂草种子样本121个,在温室内采用盆栽法测定了其对烟嘧磺隆的抗性。结果显示:4个杂草样本对烟嘧磺隆产生了抗性,其中山东淄博张店区傅家镇高家村的牛筋草抗性种群的GR50为25.76g/hm2,是敏感种群(1.33g/hm2)的19.37倍,已产生明显的抗药性;四川彭山县谢家镇岳油村的稗草、河北大城县广安镇夏屯村的虎尾草和河北邯郸的狗尾草分别产生了6.14、5.43和5.65倍的低水平抗性;其余杂草样本均无明显抗性。同一杂草不同采集地点的敏感样本对烟嘧磺隆的敏感性存在差异。     

稗JS12种群对噁唑酰草胺的抗性水平及机理分析

稗Echinochloa crus-galli是中国水稻产区发生严重的恶性杂草之一，严重威胁水稻的产量和品质。为明确江苏省稻田稗JS12种群对噁唑酰草胺的抗性水平及抗性机理，本研究采用整株水平测定法测定了稗种群对噁唑酰草胺的敏感性；通过乙酰辅酶A羧化酶(acetyl-CoA carboxylase,ACCase)靶标基因测序和表达量测定，以及代谢酶抑制剂增效试验，阐明其产生抗性的靶标抗性机制和非靶标抗性机制；最后测定了稗JS12种群对ACCase抑制剂和其他不同作用机理除草剂的敏感性，以明确抗性种群的交互抗性和多抗性情况。结果表明：江苏省稻区稗JS12种群对噁唑酰草胺产生了13.71倍的高水平抗性；稗JS12种群ACCase基因的6个拷贝序列中均未发生氨基酸突变，药剂处理后其ACCase基因表达量显著低于敏感种群；细胞色素P450抑制剂马拉硫磷和谷胱甘肽-S-转移酶(glutathione-S-transferase,GST)抑制剂NBD-Cl均可显著提高JS12种群对噁唑酰草胺的敏感性，其鲜重抑制中量GR50由227.90 g/hm²分别降至77.51和137....     

直播稻田牛筋草对乙酰辅酶A羧化酶类除草剂抗性水平及其分子机制

为明确直播稻田牛筋草对乙酰辅酶A羧化酶 (ACCase) 类除草剂的抗药性水平及其抗性产生的分子机制,采用整株生物测定法测定了牛筋草对6种ACCase类除草剂的抗性水平,并分别对抗性种群和敏感种群的ACCase基因部分片段进行了扩增和测序。结果表明:疑似抗性种群SJ-1对唑酰草胺、氰氟草酯、精唑禾草灵、高效氟吡甲禾灵和烯禾啶产生了高水平抗性,其抗性倍数分别为56.6、62.5、128、52.0和16.3;对烯草酮产生了低水平抗性,相对抗性倍数为4.86。将抗性种群和敏感种群的ACCase基因片段序列进行比对分析发现,SJ-1种群ACCase基因2078位氨基酸由天冬氨酸 (GAT) 突变为甘氨酸 (GGT),该位点氨基酸突变可能是其对ACCase类除草剂产生抗药性的主要原因之一。     

嘉兴市稻田主要禾本科杂草发生规律及其抗性表现与防治策略研究

为了明确浙江省嘉兴市南湖区和秀洲区稻田主要禾本科杂草种群分布、发生和危害情况,对2地主要禾本科杂草进行了调查。采集2地稻田不同种群稗草(Echinochloa crusgalli)和千金子(Leptochloa chinensis),采用整株测定法测定了稗草的4个种群对二氯喹啉酸、五氟磺草胺、■唑酰草胺的敏感性以及千金子的4个种群对氰氟草酯、■唑酰草胺的敏感性,明确其抗性水平。结果表明,凤桥、古塘稗草种群对二氯喹啉酸的抗性指数分别达到60.0倍、10.7倍,属于高抗种群,其余种群对3种药剂的抗性指数均小于5倍,属于敏感种群。商务区、凤桥千金子种群对氰氟草酯的抗性指数分别达到9.4倍、6.9倍,对■唑酰草胺的抗性指数达到6.3倍、7.8倍,属于中抗种群,其余种群的抗性指数均小于5倍,属于敏感种群。将不同种群杂草采用不同药剂进行抗性鉴定,对其化学防除提供有效借鉴,以达到科学治理的目的。     

EPSPS gene amplification in glyphosate-resistant Italian ryegrass (Lolium perenne ssp. multiflorum) from Arkansas

BACKGROUND: Resistance to glyphosate in weed species is a major challenge for the sustainability of glyphosate use in crop and non‐crop systems. A glyphosate‐resistant Italian ryegrass population has been identified in Arkansas. This research was conducted to elucidate its resistance mechanism. RESULTS: The investigation was conducted on resistant and susceptible plants from a population in Desha County, Arkansas (Des03). The amounts of glyphosate that caused 50% overall visual injury were 7 to 13 times greater than those for susceptible plants from the same population. The EPSPS gene did not contain any point mutation that has previously been associated with resistance to glyphosate, nor were there any other mutations on the EPSPS gene unique to the Des03 resistant plants. The resistant plants had 6‐fold higher basal EPSPS enzyme activities than the susceptible plants, but their I₅₀ values in response to glyphosate were similar. The resistant plants contained up to 25 more copies of EPSPS gene than the susceptible plants. The level of resistance to glyphosate correlated with increases in EPSPS enzyme activity and EPSPS copy number. CONCLUSION: Increased EPSPS gene amplification and EPSPS enzyme activity confer resistance to glyphosate in the Des03 population. This is the first report of EPSPS gene amplification in glyphosate‐resistant Italian ryegrass. Other resistance mechanism(s) may also be involved. Copyright © 2012 Society of Chemical Industry     

Characterization of glyphosate resistance in cloned Amaranthus palmeri plants

Glyphosate‐resistant Palmer amaranth from Georgia (GA), USA, possesses multiple copies of the gene that encodes 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS), the enzyme target site of this herbicide. The cloned plants of glyphosate‐resistant and glyphosate‐susceptible Palmer amaranth biotypes from Mississippi (MS), USA, and GA were evaluated for glyphosate injury (digital imaging) in leaf disc bioassays. Four groups (three resistant groups: two from MS [G and R] and one from GA [C7]; one susceptible group from GA [C3]) were chosen for cloning to facilitate long‐term studies. After exposure to glyphosate (1.0 mmol L^–1, 144 h), the level of injury (mean value) was low in the resistant groups, while a higher level of injury was found in the susceptible group. However, the individual injury values within all groups varied widely. The mean EPSPS gene copy number of these groups was G ≥ R > C7 >>> C3. However, a higher copy number did not always convey increased resistance in these bioassays. When the copy number was high (>20), 81.5% of the bioassayed plants exhibited little or no injury and only ～20% were significantly injured, while 50% of the plants with a low copy number (<20) remained healthy. Overall, no strong statistical correlation of the copy number versus injury occurred in these cloned plants and no statistical relationship of resistance and copy number with the sex of the MS plants was observed. The results suggest that although an elevated copy number of the EPSPS gene can instill resistance, other mechanisms might contribute to the overall glyphosate resistance of Palmer amaranth in these plants.     

抗草甘膦杂草及其抗性机制研究进展

介绍了迄今为止全球发现的13种抗草甘膦杂草的发生、发展,并从草甘膦的吸收、输导和分布,5-烯醇丙酮莽草酸-3-磷酸合成酶(EPSPS)的活性以及抗药性遗传等方面对其抗性机制进行了讨论,指出了中国在未来出现抗草甘膦杂草的潜在风险性,并提出了延缓杂草对草甘膦抗性发生的策略。     

A target-site mutation is present in a glyphosate-resistant Lolium rigidum population

Annual ryegrass (Lolium rigidum) is the only weed species to have evolved resistance to the broad‐spectrum herbicide glyphosate in Australia. A population that had failed to be controlled by glyphosate was collected from a vineyard in the Adelaide Hills region of South Australia. Dose–response experiments on this population (SLR 77) showed that it was glyphosate resistant, with an LD₅₀ that was 1.9–3.4 times higher than that of a susceptible population (VLR 1). The movement of radiolabelled glyphosate within SLR 77 plants showed that this population did not have the differential glyphosate translocation mechanism of resistance common to several other Australian glyphosate‐resistant populations. Subsequent analysis of shikimic acid accumulation within the plant after glyphosate treatment showed that this population accumulated significantly less shikimic acid than a susceptible population, but more than a glyphosate‐resistant population with the translocation mechanism, indicating the possible involvement of another mechanism of resistance. Sequencing of a portion of the SLR 77 5‐enolpyruvylshikimate‐3‐phosphate synthase gene was carried out and a mutation causing an amino acid change at position 106 from proline to threonine was identified. This mutation is likely to be responsible for glyphosate resistance in this population, as mutations in this position have been found to be responsible for glyphosate resistance in goosegrass (Eleusine indica) from Malaysia. This paper represents the first report of target‐site glyphosate resistance in L. rigidum and provides evidence that this species has at least two mechanisms of glyphosate resistance present in Australia.     

Non‐target‐site mechanism of glyphosate resistance in Italian ryegrass (Lolium multiflorum)

In Shizuoka Prefecture, Japan, glyphosate‐resistant Lolium multiflorum is a serious problem on the levees of rice paddies and in wheat fields. The mechanism of resistance of this biotype was analyzed. Based on LD₅₀, the resistant population was 2.8–5.0 times more resistant to glyphosate than the susceptible population. The 5‐enolpyruvyl‐shikimate‐3‐phosphate synthase (EPSPS) gene sequence of the resistant biotype did not show a non‐synonymous substitution at Pro106, and amplification of the gene was not observed in the resistant biotype. The metabolism and translocation of glyphosate were examined 4 days after application through the direct detection of glyphosate and its metabolite aminomethylphosphonic acid (AMPA) using liquid chromatograph‐tandem mass spectrometer (LC‐MS/MS). AMPA was not detected in either biotype in glyphosate‐treated leaves or the other plant parts. The respective absorption rates of the susceptible and resistant biotypes were 37.90 ± 3.63% and 41.09 ± 3.36%, respectively, which were not significantly different. The resistant biotype retained more glyphosate in a glyphosate‐treated leaf (91.36 ± 1.56% of absorbed glyphosate) and less in the untreated parts of shoots (5.90 ± 1.17%) and roots (2.76 ± 0.44%) compared with the susceptible biotype, 79.58 ± 3.73%, 15.77 ± 3.06% and 4.65 ± 0.89%, respectively. The results indicate that the resistance mechanism is neither the acquisition of a metabolic system nor limiting the absorption of glyphosate but limited translocation of the herbicide in the resistant biotype of L. multiflorum in Shizuoka Prefecture.     

Resistance to glyphosate in Lolium rigidum

Annual ryegrass (Lolium rigidum) is a widespread and important weed of Australia and populations of this weed have developed resistance to most major herbicides, including glyphosate. The possible mechanisms of resistance have been examined in one glyphosate-resistant Lolium population. No major differences were observed between resistant and susceptible biotypes in respect of (i) the target enzyme (EPSP synthase), (ii) DAHP synthase, the first enzyme of the target (shikimate) pathway, (iii) absorption of glyphosate, or (iv) translocation. Following treatment with glyphosate, there was greater accumulation of shikimate (derived from shikimate-3-Pi) in susceptible than in resistant plants. In addition, the resistant population exhibited cross-resistance to 2-hydroxy-3-(1,2,4-triazol-1-yl)propyl phosphonate, a herbicide which, although structurally similar to glyphosate, acts at an unrelated target site. On the basis of these observations we speculate that movement of glyphosate to its site of action in the plastid is involved in the resistance mechanism. © 1999 Society of Chemical Industry     

转基因抗虫耐除草剂(Cry1Ac+EPSPS)玉米对草甘膦耐受性研究

将具有我国自主知识产权的抗虫、耐除草剂基因转化到玉米中获得兼具抗虫和耐除草剂的复合性状转基因玉米,实现了玉米复杂性状的有效改良,研究了转耐除草剂CC-2基因和可表达Bt毒素的cry1Ac基因的双抗玉米对除草剂的耐受性及目标蛋白表达量。通过调查喷施除草剂后玉米植株存活率及株高和生长发育情况,研究了转抗虫耐除草剂基因玉米‘CC-2×BT799’、抗虫玉米‘BT799’、耐除草剂玉米‘CC-2’,以及常规玉米‘郑58’在田间对除草剂的耐受性,并用ELISA测定了供试品种中CP4EPSPS和Cry1Ac蛋白含量。对草甘膦的耐受性试验表明,转复合基因玉米‘CC-2×BT799’与单抗草甘膦品种‘CC-2’对草甘膦均具有良好的耐受性。心叶期喷施草甘膦推荐施用剂量中量和2倍剂量对其株高和后期生长发育无不良影响。ELISA检测显示,CP4EPSPS蛋白在雄穗中的表达量较叶片稍高。且双抗品种‘CC-2×BT799’较‘CC-2’中的CP4EPSPS蛋白含量稍高。在雄穗中,‘BT799’的Cry1Ac蛋白含量与‘CC-2×BT799’相当。在叶片中,‘BT799’的Cry1Ac蛋白含量约为‘CC-2×BT799’的2倍。抗虫耐除草剂玉米能够有效抵御草甘膦的喷施,Cry1Ac蛋白含量在不同品种不同组织中存在差异。     

田旋花EPSPS基因表达特征及草甘膦对其表达的影响

采用实时荧光定量RT-PCR测定了田旋花不同组织、不同叶龄的EPSPS基因mRNA的相对表达量以及草甘膦对EPSPS基因相对表达量的影响。结果表明:田旋花EPSPS基因在不同组织表达量差异显著,在叶的表达量高于茎和根;该基因在9叶期的表达量最高,是3叶期的1.5倍;在草甘膦处理后,田旋花EPSPS基因的表达量先升高后降低,在处理后24h达最大值。随草甘膦剂量增加,该基因的表达量升高。研究结果可为深入解析田旋花对草甘膦耐药性机理提供参考。