小麦田麦家公对苯磺隆的抗性机理

为明确麦田阔叶杂草麦家公Lithospermum arvense L.对苯磺隆的抗性机理，以苯磺隆抗性和敏感型麦家公为材料，比较分析这2个生物型麦家公靶标酶乙酰乳酸合成酶（acetolactate synthase，ALS）、解毒酶谷胱甘肽-S-转移酶（glutathione-S-transferase，GST）以及保护酶过氧化物酶（peroxidase，POD）、超氧化物歧化酶（superoxide dismutase，SOD）和过氧化氢酶（catalase，CAT）对苯磺隆的响应差异性。结果表明，抗性麦家公ALS对苯磺隆的敏感性较敏感型麦家公显著下降，苯磺隆的抑制中浓度分别为0.187、0.036 μmol/L。苯磺隆胁迫后，抗性和敏感型麦家公ALS活性都出现下降，但抗性麦家公ALS活性可恢复，而敏感型麦家公ALS活性则不能恢复；2个生物型麦家公GST活性都能被苯磺隆诱导，但抗性麦家公GST累计活性为29.31 U，高于敏感型麦家公（25.90 U）；抗性麦家公SOD累计活性为24.49 U，较敏感型麦家公（19.31 U）高，且具有较强的恢复能力；抗性麦家公POD和CAT累计活性分别为126.92~550.68 U和41.41~77.19 U，也高于敏感型麦家公的93.75~271.04 U、42.17~57.28 U。因此，靶标酶ALS对苯磺隆敏感性减弱是麦家公产生抗性的一个重要原因，解毒酶GST、SOD、POD和CAT活性升高可能与抗性有关。     

麦田抗性生物型猪殃殃对苯磺隆的抗性机制

为探讨猪殃殃Galium aparine抗药性生物型(R)对苯磺隆的抗性机制,测定了苯磺隆对猪殃殃抗性、敏感(S)生物型体内靶标酶 、代谢酶 及抗氧化酶 影响的差异。离体试验结果表明,苯磺隆对R、S生物型猪殃殃ALS的抑制中量(IC50)分别为0.682、0.718 μg/L(有效剂量),R、S生物型猪殃殃ALS对苯磺隆的敏感性不存在差异。活体试验结果表明,苯磺隆茎叶喷雾处理后,R、S生物型ALS活力均表现为先上升,但S生物型上升幅度小,且随后快速下降,第3 d即回落至对照之下,并维持在低于对照的水平,而R生物型ALS活力在第2 d可达对照的4.10倍,第5 d 基本回落至对照水平,之后基本维持在对照水平;R生物型GSTs活力在第1 d即开始上升,最高可达对照的 2.40 倍,而S生物型则表现为先下降,然后小幅回升,最高为对照的1.61倍,两者在10 d左右均回落至对照水平;R生物型SOD活力与对照基本相同,而S生物型虽略有下降,但R、S间不存在显著差异;两者POD活力虽均有大幅提高,但亦不存在显著差异。结果表明,低水平抗药性生物型猪殃殃对苯磺隆产生抗性的原因可能是ALS过量表达及GSTs对苯磺隆的代谢作用加强,而不是由于ALS的敏感性下降,同时POD、SOD在减轻药害中也具有一定作用。     

麦田不同杂草对苯磺隆敏感性差异的分子机制

为了探讨麦田常见杂草对苯磺隆敏感性差异的分子机制,测定了苯磺隆对4种杂草活性及对杂草体内靶标酶(ALS)和代谢酶(GSTs)的影响差异。结果表明,泽漆对苯磺隆的敏感性最低,IC50为123.16gai/hm2;猪殃殃和荠菜次之,分别为8.47、1.07gai/hm2;播娘蒿最敏感,为0.29gai/hm2。离体条件下,苯磺隆对播娘蒿、泽漆ALS的IC50值分别为6.40、54.90mgai/L。活体条件下,苯磺隆处理后,播娘蒿体内ALS活力低于对照,第9天降为对照的31.20%,而敏感性低的泽漆处理后1～2天ALS活力虽略有下降,但第3天明显提高,第4天达到峰值,为对照的2.90倍,此后逐渐恢复至对照水平。4种杂草本身所含GSTs活性存在较大差异,泽漆GSTs活性最高。经苯磺隆处理后,泽漆与猪殃殃GSTs活性明显提高,第4天时达到峰值,相对活性分别为1.23和1.25,并持续在较高水平;而荠菜、播娘蒿GSTs活性虽分别在第3、5天达到峰值,但多数时间相对活性低于1。结果表明,麦田不同杂草体内ALS的敏感性差异和GSTs的代谢差异是对苯磺隆敏感性不同的两个重要原因。     

中国北方部分地区麦田荠菜对苯磺隆的抗性水平

为明确北方地区冬小麦田荠菜对苯磺隆的抗性水平,运用培养皿法和温室盆栽法分别测定了山东、山西、河南、河北、陕西5省13个地区采集点麦田潜在抗药性生物型对苯磺隆的抗性水平,并分别测定了驻马店梁祝镇采集点荠菜潜在抗药性生物型和敏感生物型乙酰乳酸合成酶(ALS)对苯磺隆的敏感性。培养皿法测定结果表明:驻马店梁祝镇采集点荠菜抗药性生物型对苯磺隆的抗性水平最高,抗性倍数为6.17倍,其他采集点荠菜抗性倍数在0.94～2.04倍之间,仍处于较为敏感状态。温室盆栽法测定结果表明:驻马店梁祝镇采集点荠菜抗性倍数仍为最高,达到233倍, 其他地区采集点荠菜抗性倍数在1.23～3.73倍之间,尚未产生明显的抗药性。离体条件下,苯磺隆对荠菜抗药性和敏感生物型ALS的抑制中浓度(IC50)分别为0.664 μmol/L和0.053 3 μmol/L,抗药性生物型的抗性倍数达12.5倍。结果表明,驻马店梁祝镇采集点荠菜已对苯磺隆产生了较高水平的抗药性,而其体内ALS敏感性降低可能是抗药性产生的原因之一。     

山东省部分市县麦田杂草麦家公Lithospermum arvense对苯磺隆的抗药性

采用温室盆栽法和培养皿法测定了山东省部分市县冬小麦田杂草麦家公Lithospermum arvense L.对苯磺隆的抗药性水平,以及其抗药性生物型乙酰乳酸合成酶(ALS)对苯磺隆的敏感性。温室盆栽结果显示,供试杂草对苯磺隆产生了不同程度的抗药性,其中胶州麦家公生物型抗性水平最高,抗性倍数为12.8倍;培养皿法测定结果也显示胶州麦家公生物型抗性水平最高,但抗性倍数为3.89倍。交互抗性测定结果表明,胶州抗性麦家公生物型对其他ALS抑制剂噻吩磺隆和苄嘧磺隆已产生不同程度的交互抗性,其中对噻吩磺隆的抗性倍数达到3.11倍。离体条件下,与敏感生物型ALS活力的抑制中浓度(IC50)相比较,胶州抗性麦家公生物型的IC50值是敏感麦家公的 2.65倍。表明ALS敏感性降低可能是山东部分市县麦家公对苯磺隆产生抗药性的重要原因之一。     

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。     

黑龙江省大豆田反枝苋对氟磺胺草醚的抗药性机制研究

为明确反枝苋抗性种群对氟磺胺草醚的抗性机制,分别测定了氟磺胺草醚对反枝苋抗性和敏感种群体内原卟啉原氧化酶(PPO)、谷胱甘肽S-转移酶(GSTs)、超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性的影响。结果表明:氟磺胺草醚处理后,抗性和敏感反枝苋种群PPO活性均受到一定程度的抑制,但抗性种群活性受到抑制后能逐渐恢复,而敏感种群则不能恢复;施用氟磺胺草醚后,抗性和敏感反枝苋种群GSTs和SOD活性变化无明显差异,抗性和敏感反枝苋种群POD和CAT活性均受到一定程度的抑制,但抗性种群活性受到抑制后能逐渐恢复,而敏感种群则不能恢复。研究表明,反枝苋抗性种群体内PPO对氟磺胺草醚敏感性降低是其产生抗药性的原因之一,反枝苋POD和CAT对活性氧的抵御能力差异也可能与反枝苋对氟磺胺草醚的抗性有关。     

不同小麦品种对苯磺隆耐药性差异及其机理

室内用砂培法测定11种小麦品种对苯磺隆的相对敏感性,其敏感性有较大差异.碧玛4号、维麦8号对苯磺隆有很强的耐药性,IC50可分别达到502.6和409.3aiμg/kg,济1 3号为156.9aiμg/kg,鲁麦22号、黄县大粒对苯磺隆较为敏感,IC50低于45.0 aiμg/kg.以上5种小麦离体ALS对苯磺隆的敏感性没有明显差异,敏感品种黄县大粒IC50为154.2nmol/L,甚至较维麦8号149.3nmol/L略高.ALS活体试验表明,小麦可以很快恢复苯磺隆对ALS活性的抑制,且耐药型品种恢复能力较强,碧玛4号、维麦8号ALS恢复活性只需1.5天,敏感品种需3天.碧玛4号、维麦8号除草剂解毒酶系GSTs相对于敏感品种对苯磺隆有较大的反应幅度和较短的反应时间,第2天就分别达到1.46、1.42的峰值,而济13号、鲁麦22号、黄县大粒变化幅度较小,第3天才达到峰值,活性分别为1.39、1.33和1.32.研究表明,小麦各品种对苯磺隆的耐药能力产生差异的主要原因之一是小麦GSTs对苯磺隆的代谢所致,而与小麦ALS对苯磺隆的敏感程度无关.     

河南省部分地区麦田荠菜对苯磺隆的抗性水平及抗性靶标分子机制

为明确河南省部分地区麦田荠菜Capsella bursa-pastoris对苯磺隆的抗性水平及抗性靶标分子机制,采用整株生物法测定了12个荠菜种群的抗性水平,并对乙酰乳酸合成酶(acetolactate synthase,ALS)离体活性和ALS基因突变进行了测定分析。结果表明,商丘市民权县花园村(MQ)、周口市西华县小于楼村(XH)、平顶山市叶县穆寨村(YX)、许昌市长葛市董庄村(CG)采集的荠菜种群对苯磺隆产生了较高的抗性,GR_(50)分别为129.14、110.67、62.91和85.29 g/hm~2,抗性倍数分别为215.23、184.45、104.85和142.15倍;ALS离体活性测定所得I_(50)分别为5.85、4.87、1.38和3.83μmol/L,抗性倍数分别为83.57、69.57、19.71和54.71倍;其余8个种群的GR_(50)在0.60~2.86 g/hm~2之间,抗性倍数在1.00~4.77之间;I_(50)在0.07~0.37μmol/L之间,抗性倍数在1.00~5.29之间。荠菜种群MQ、XH的ALS基因Domain A区域第197位脯氨酸(CCT)均突变为丝氨酸(TCT),荠菜种群CG的第197位脯氨酸(CCT)突变为亮氨酸(CTT),表明靶标ALS基因突变是荠菜对苯磺隆产生抗性的重要原因之一,但荠菜种群YX的ALS基因保守区内暂未发现突变位点,其抗药性可能由其它原因造成。     

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。     

Herbicide resistance in Aster squamatus conferred by a less sensitive form of acetolactate synthase

A biotype of Aster squamatus (Sprengel) Hieronymus with suspected resistance to the ALS-inhibiting herbicide imazapyr was detected in a chicken farm in the province of Seville, Spain, which had been treated once a year with imazapyr for 10 years. Resistance to imazapyr in this biotype was studied using dose-response experiments, absorption and translocation assays, metabolism studies and ALS activity assays. The rate of imazapyr required to inhibit A squamatus growth by 50% (ED50) was 15 times higher for the R (resistant) than for the S (susceptible) biotype. Cross-resistance existed for the ALS-inhibitors imazamox, imazethapyr, amidosulfuron, nicosulfuron, rimsulfuron, triasulfuron and tribenuron, but not for bensulfuron. Control of A squamatus using alternative herbicides was poor with clopyralid, intermediate with quinclorac, amitrole and MCPA, and excellent with 2,4-D, glufosinate and glyphosate. Absorption of [14C]imazapyr increased over time for both the R and S biotypes, and translocation from the treated leaf to shoots and roots was similar in both biotypes, with most of the radioactivity remaining in the treated leaf. No metabolites of imazapyr were detected in either biotype. Sensitivity of the ALS enzyme (target site) to imazapyr was lower for the R biotype (I50(R) = 4.28 x I50(S)). The mechanism of imazapyr resistance in this R biotype appears to be an altered ALS conferring decreased sensitivity to imazapyr at the whole-plant level.     

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.     

Assessment of two biotypes of Solanum ptycanthum that differ in resistance levels to imazamox

Glasshouse and laboratory experiments were conducted on acetolactate synthase (ALS) homozygous resistant Solanum ptycanthum biotypes from Illinois (IL‐R) and Indiana (IN‐R), and homozygous susceptible biotypes from Illinois (IL‐S) and Indiana (IN‐S). Genetic similarity of biotypes was assessed by random amplified polymorphic DNA (RAPD) markers, which determined that the Illinois biotypes are more similar to each other than to the IN‐R biotype. ALS enzyme activity from the IL‐R and IN‐R biotypes had I₅₀ values of 362 and 352 μM imazamox respectively. Dose–response experiments using three‐ to four‐leaf‐stage plants of the IL‐R and IN‐R biotypes had GR₅₀ values of 242 and 69 g ae ha⁻¹ imazamox respectively. Whole‐plant and ALS enzyme results are different than previously reported values in the literature, which was attributed in the current study to the original IN‐R population having individuals that were segregating for ALS resistance. Metabolism studies showed no difference in percentage [¹⁴C]imazamox remaining between the IL‐R and IN‐R biotypes up to 72 h after treatment. The IL‐S biotype metabolised [¹⁴C]imazamox approximately two times faster than the IL‐R and IN‐R biotypes and this trait was heritable. Response of F₃ plants containing homozygous ALS‐resistant alleles from the IL‐R biotype in a genetic background of 50% Illinois and 50% Indiana biotypes suggests that genetic factors other than an altered target site or metabolism may also contribute to the magnitude of resistance at the whole‐plant level in resistant biotypes.     

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.     

湖南省部分地区棉田牛筋草(Eleusine indica)对精喹禾灵的抗性

采用整株法测定了湖南省部分地区棉田牛筋草(Eleusine indica)对精喹禾灵的抗性水平,还测定了精喹禾灵对牛筋草抗(R)、感(S)种群体内GSTs及SOD、POD、CAT影响的差异。整株法测定结果显示,相对于敏感的南县种群,其他地区棉田牛筋草对精喹禾灵的抗性指数在1.6~9.7之间,其中湘阴种群最高为9.7,澧县种群最低为1.6;精喹禾灵处理2d后,R种群GSTs活性急剧上升,在第3天达到最高,随后急剧下降,5d之后趋于平缓。S种群GSTs活性在施药后先上升后下降。药剂处理后,R种群的SOD和POD活性在中期显著低于S种群,R、S种群的CAT活性在药后第9天有明显差异。结果表明,R种群对精喹禾灵产生抗性的原因可能是GSTs对精喹禾灵的代谢作用加强,抗氧化酶系活性的变化可能与抗性相关。