【作者】 李玮；

【导师】 操海群；

【作者基本信息】 安徽农业大学 ， 农药学， 2023， 硕士

【摘要】 多花黑麦草（Lolium multiflorum Lam.）是一种一年生禾本科草本植物,原产于地中海沿岸,于20世纪30年代作为一种冬季牧草引入我国并进行大面积种植。多花黑麦草具有较强的环境适应性,于近年来开始入侵麦田生态系统并且快速蔓延,目前已经成为我国北方小麦产区主要恶性杂草种类之一。乙酰辅酶A羧化酶（Acetyl-Co A carboxylase,ACCase）抑制剂炔草酯和乙酰乳酸合酶（Acetolactate synthase,ALS）抑制剂甲基二磺隆是两种小麦田常用除草剂,其除草活性高、杀草谱广、安全性好,自20世纪70年代以来广泛用于多种一年生杂草的防除。然而,上述药剂作用位点单一,随着长期高频超量使用,多种麦田杂草除草剂敏感性逐渐降低,其中某些杂草如多花黑麦草等已经对其产生严重抗性,对小麦安全生产构成严重威胁。明确我国主要小麦产区多花黑麦草对炔草酯和甲基二磺隆的抗性发生情况并探索其抗性发生机理,对于建立科学有效的抗性杂草防治策略、保障小麦稳产增收具有重要意义。因此,本研究在我国黄淮海部分区域小麦主产省份进行了多花黑麦草发生情况调查和种群资源采集,并进行了抗性鉴定和抗性机制初步研究,获得主要结果如下:1.多花黑麦草发生情况调查及除草剂敏感性测定。在我国陕西、河南、安徽和江苏等黄淮海区域省份进行麦田杂草发生情况调查和种群采集,其中针对多花黑麦草共采集得到36个种群。采用炔草酯和甲基二磺隆田间推荐剂量进行种群敏感性测定,结果表明,13个种群对炔草酯产生抗性,8个种群对甲基二磺隆产生抗性,其中7个种群同时对炔草酯和甲基二磺隆产生多抗性。抗性多花黑麦草在河南、安徽和江苏等省份广泛分布,表明目前黄淮海区域麦田多花黑麦草的抗药性发生较为普遍。通过整株剂量响应试验对多花黑麦草的抗性水平进行测定,结果表明,在13个炔草酯抗性种群中,10个种群产生高水平抗性,3个种群产生低水平抗性,GR₅₀值在4.22～1336.38 g a.i.hm^-2之间,相对抗性倍数在2.62～830.05之间;在8个甲基二磺隆抗性种群中,3个种群产生高水平抗性,2个种群产生中等水平抗性,3个种群产生低水平抗性,GR₅₀值在2.06～8.84 g a.i.hm^-2之间,相对抗性倍数在3.89～16.68之间。从种群抗性水平分布来看,安徽阜阳、滁州、蚌埠和宿州,河南驻马店以及江苏睢宁等地区小麦田多花黑麦草抗性水平相对较高,抗药性发生更为严重。2.多花黑麦草对炔草酯和甲基二磺隆的抗性机制。针对多花黑麦草抗性种群,分别探究其潜在的靶标及非靶标（代谢）抗性机理。首先对抗性种群靶标基因序列进行测序分析。结果表明,在13个炔草酯抗性种群中,10个种群ACCase基因第1781、2041和2078位点共发生3种突变形式（I1781L,I2041N和D2078G）,其余3个种群（AH-08、AH-12和HN-14）未检测到ACCase基因突变;在8个甲基二磺隆抗性种群中,3个种群ALS基因第197和574位点共检测到4种突变形式（P197T、P197S、P197A和W574L）,其中,HN-08种群ALS基因第197位发生脯氨酸到丝氨酸、丙氨酸或苏氨酸（P197S/A/T）的混合突变,其余5个种群未检测到ALS基因突变。在多抗性种群AH-05、AH-11和HN-08中,均同时检测到ACCase和ALS基因突变。选择3个炔草酯抗性种群AH-01（ACCase,I2041N）、AH-05（ACCase,I1781L）和HN-07（ACCase,D2078G）,2个甲基二磺隆抗性种群AH-05（ALS,P197T）和HN-08（ALS,P197S/A/T）,分别进行靶标基因ACCase和ALS相对表达水平分析。结果表明,与敏感种群AH-07相比,AH-05和AH-01种群在炔草酯处理后2 h和12 h ACCase基因表达分别上调3.00倍和3.85倍,而AH-05和HN-08种群在甲基二磺隆处理前后ALS基因表达与敏感种群没有显著差异。利用细胞色素P450单加氧酶（Cytochrome P450 monooxygenases,P450s）抑制剂胡椒基丁醚（Piperonyl butoxide,PBO）、1-氨基苯并三唑（1-aminobenzotriazole,ABT）、马拉硫磷（Malathion）和谷胱甘肽-S-转移酶（Glutathione S-transferases,GSTs）抑制剂4-氯-7-硝基苯并呋喃（4-chloro-7-nitro-benzofurazan,NBD-Cl）与除草剂结合处理的方法,研究除草剂解毒酶是否参与多花黑麦草的抗药性。结果显示,单独施用4种抑制剂对不同种群植株生长均无显著影响;PBO预处理后,炔草酯对抗性种群AH-01、AH-05、AH-08和AH-12防效增强,甲基二磺隆对抗性种群AH-11防效增强;ABT预处理后,炔草酯对抗性种群AH-05、AH-08、AH-10、AH-12和HN-03防效增强,甲基二磺隆对抗性种群AH-10防效增强;马拉硫磷预处理后,炔草酯对抗性种群AH-01、AH-08、AH-10和AH-12防效增强,甲基二磺隆对抗性种群AH-01和HN-08防效增强;NBD-Cl预处理后,炔草酯对抗性种群AH-01、AH-05、AH-08、AH-10和AH-12防效增强,甲基二磺隆对抗性种群AH-01、AH-11和HN-08防效增强。表明P450s和/或GSTs分别参与了部分多花黑麦草种群对炔草酯和甲基二磺隆的抗性。3.多花黑麦草除草剂抗性模式及其化学防治策略。针对AH-01（ACCase,I2041N）、AH-05（ACCase,I1781L;ALS,P197T）和HN-07（ACCase,D2078G）种群,分别研究其交互抗性和多抗性模式。结果表明,3个种群均对ACCase抑制剂高效氟吡甲禾灵、精喹禾灵、氰氟草酯、精噁唑禾草灵、噁唑酰草胺、烯草酮、烯禾啶和唑啉草酯表现出不同水平交互抗性;其中AH-01种群对ALS抑制剂双草醚表现多抗性,AH-05种群对ALS抑制剂双草醚、咪唑乙烟酸、啶磺草胺、甲氧咪草烟和氟唑磺隆表现交互抗性,HN-07种群对啶磺草胺、双草醚、甲氧咪草烟和咪唑乙烟酸表现多抗性;所有种群均对5-烯醇式丙酮酸-3-磷酸合酶（5-enolpyruvylshikimate-3-phosphate synthase,EPSPS）抑制剂草甘膦和光系统Ⅱ（Photosystem II,PSⅡ）抑制剂异丙隆敏感。采用苗后（Post-emergence,POST）除草剂对多花黑麦草的防治时期进行探索,结果表明,不同除草剂在多花黑麦草2～4叶期时防治效果较好（鲜重抑制率>70%）;随着杂草叶期增加,几乎所有除草剂防治效果均显著降低。在保证作物安全的前提下,应尽早使用苗后除草剂防治多花黑麦草。4.多花黑麦草抗性生物型种子生态学特征适应性变化。选择靶标基因ACCase发生I2041N突变频率100%的抗性种群JS-07以及2个敏感种群AH-06和AH-09作为研究对象,根据单株靶标基因测序结果,分别隔离培养具有抗性纯合突变的植株和各敏感种群植株,以获取其F1代种子材料,探究其在不同环境因子和不同土壤埋藏深度条件下的种子萌发和出苗特性。结果表明,不同种群对各处理的响应趋势基本一致。其中光照对种子萌发无显著影响,不同光照条件下所有种群萌发率均在85%以上;酸碱度变化也未对种子萌发产生显著影响,在p H水平为4～10范围内,各种群种子萌发率均在90%以上;多花黑麦草种子对温度具有广泛适应性,在5℃～35℃恒温和5/15℃～25/35℃变温条件下各种群均可萌发（萌发率在61%～99%之间）,但是恒定高温（35℃）会降低种子总萌发率,恒定低温（5℃）会推迟种子初始萌发时间,降低种子萌发速率;此外,盐胁迫、渗透势胁迫和土壤埋藏深度均会显著影响各种群种子萌发,但是相比2个敏感种群,抗性种群JS-07种子在这三方面表现出一定的生态适应性优势。采用土壤浅旋耕,将杂草种子埋藏在4 cm以下土层,是降低敏感和抗性多花黑麦草总体出苗率的有效手段。综上所述,本研究通过田间调查和抗性鉴定明确了黄淮海部分区域麦田多花黑麦草的发生情况及抗性分布;通过靶标基因测序、表达量分析和代谢酶抑制剂生物测定初步阐释了抗性种群的抗性分子机理;结合种群对不同除草剂抗性模式测定以及不同药剂化学防治时期筛选,探究了科学高效的抗性杂草化学防治策略;最后进一步研究多花黑麦草抗性种群相比敏感种群在种子萌发特性方面的变化,尝试探索可行的抗性杂草生态治理方法。本研究针对黄淮海部分区域麦田多花黑麦草的抗性研究结果,将为建立科学有效的抗性杂草防治策略,保障小麦的稳产增收提供科学依据。更多还原

【Abstract】 Italian ryegrass(Lolium multiflorum Lam.),an annual gramineous herb originated from the Mediterranean coast,was introduced as a winter forage in China in the 1930 s and widely planted.L.multiflorum has strong environmental adaptability.In recent years,it has gradually invaded the wheat ecosystem and spread rapidly,and has become one of the main malignant weeds in wheat fields in northern China.Acetyl-Co A carboxylase(ACCase)inhibitor clodinafop-propargyl and acetolactate synthase(ALS)inhibitor mesosulfuronmethyl are two herbicides commonly used in wheat fields.They have high herbicidal activity,broad herbicidal spectrum and good control effects.It has been widely used for weed control in farmland since the 1970 s.However,due to the single site of action of these herbicides,long-term high frequency and excessive use will make many weeds in wheat fields gradually reduce their herbicide sensitivity,such as L.multiflorum,have developed serious resistance to them,posing a serious threat to wheat production.Understanding the occurrence and mechanism of resistance of L.multiflorum to clodinafop-propargyl and mesosulfuronmethyl in the main wheat-producing areas of northern China is of great significance for establishing scientific and effective strategies for resistant weed control and ensuring stable and increasing wheat yield.Therefore,in this study,the occurrence investigation and population resource collection of L.multiflorum were carried out in the main wheatproducing provinces of Huang-Huai-Hai plain in China,and the resistance identification and resistance mechanism were preliminary studied.The main results are as follows:1.Occurrence investigation and herbicide susceptibility test of L.multiflorum.We investigated the occurrence of weeds in wheat fields and collected 36 L.multiflorum populations in five provinces,including Shaanxi,Henan,Anhui and Jiangsu,in the HuangHuai-Hai plain of China.The herbicide susceptibility was determined using the recommended field doses of clodinafop-propargyl and mesosulfuron-methyl.The results showed that 13 populations developed resistance to clodinafop-propargyl and 8 populations developed resistance to mesosulfuron-methyl,and seven populations developed multiple resistance to both herbicides.Resistant L.multiflorum was widely distributed in Henan,Anhui and Jiangsu provinces,indicating that resistance of L.multiflorum in the wheat fields of Huang-Huai-Hai Plain was common.The whole plant dose response test was used to determine the resistance level of L.multiflorum.The results showed that among the 13 clodinafop-propargyl-resistant populations,ten populations developed high-level resistance and three populations developed low-level resistance,with GR50 values ranging from 4.22 to 1336.38 g a.i.hm-2.The relative resistance index ranged from 2.62 to 830.05.Among the eight mesosulfuronmethyl-resistant populations,three populations showed high-level resistance,two populations showed medium-level resistance an three populations showed low-level resistance,with GR50 values ranging from 2.06 to 8.84 g a.i.hm-2,and the relative resistance index ranged from 3.89 to 16.68.From the point of resistance distribution,the resistance level of L.multiflorum in Fuyang,Chuzhou,Bengbu and Suzhou of Anhui Province,Zhumadian of Henan Province and Suining of Jiangsu Province was relatively high and the herbicide resistance was more serious.2.Herbicide resistance mechanisms of L.multiflorum.For the resistant populations of L.multiflorum,the potential mechanisms of target-site resistance(TSR)and non-target-site resistance(NTSR)(metabolic)were explored.Firstly,the target gene sequences of resistant populations were sequenced and analyzed.The results showed that there were three mutations(I1781L,I2041 N and D2078G)in the ACCase gene at positions 1781,2041 and 2078 in 10 of the 13 clodinafop-propargyl resistant populations.No ACCase gene mutation was detected in the other three populations(AH-08,AH-12 and HN-14).Among the eight mesosulfuron-methyl-resistant populations,four mutations(P197T,P197 S,P197A and W574L)were detected at positions 197 and 574 of the ALS gene in three populations.A mixed mutation of proline to serine,alanine or threonine(Pro-197-Ser/Ala/Thr,P197S/A/T)was detected in population HN-08,while no mutation was detected in the other five populations.Of note,mutations in both ACCase and ALS were detected in the AH-05,AH-11,and HN-08 populations.Secondly,three clodinafop-propargyl-resistant populations AH-01(ACCase,I2041N),AH-05(ACCase,I1781L)and HN-07(ACCase,D2078G),two mesosulfuron-methylresistant populations AH-05(ALS,P197T)and HN-08(ALS,P197S/A/T)were selected to analyze gene expression level of target genes.Compared with the sensitive population AH-07,ACCase gene expression in the AH-05 and AH-01 populations was up-regulated 3.00-fold and 3.85-fold at 2 h and 12 h after clodinafop-propargyl treatment,respectively,while ALS gene expression in the AH-05 and HN-08 populations was not significantly different from that in the sensitive population before and after mesosulfuron-methyl treatment.Finally,cytochrome P450 monooxygenases(P450s)inhibitor piperonyl butoxide(PBO),1-aminobenzotriazole(ABT),malathion and glutathione S-transferases(GSTs)inhibitor 4-chloro-7-nitro-benzofurazan(NBD-Cl)were applied in combination with herbicides to investigate whether herbicide detoxification enzymes were involved in the resistance of L.multiflorum.The results showed that single application of the four inhibitors had no significant effect on plant growth of different populations.After PBO pretreatment,the control effect of clodinafop-propargyl against the resistant populations AH-01,AH-05,AH-08 and AH-12 were enhanced,and the control effect of mesosulfuron-methyl against the resistant population AH-11 was enhanced.After pretreatment with ABT,the control effect of clodinafop-propargyl against the resistant populations AH-05,AH-08,AH-10,AH-12 and HN-03 were enhanced,and the control effect of mesosulfuron-methyl against the resistant population AH-10 was enhanced.After malathion pretreatment,the control effect of clodinafop-propargyl against the resistant populations AH-01,AH-08,AH-10 and AH-12 were enhanced,and that of mesosulfuron-methyl against the resistant populations AH-01 and HN-08 were enhanced.After NBD-Cl pretreatment,the control effect of clodinafoppropargyl against the resistant populations AH-01,AH-05,AH-08,AH-10 and AH-12 were enhanced,and the control effect of mesosulfuron-methyl against the resistant populations AH-01,AH-11 and HN-08 were enhanced.These results suggest that P450 s and/or GSTs are involved in the resistance of some L.multiflorum populations to clodinafop-propargyl and mesosulfuron-methyl,respectively.3.Resistance pattern determination and chemical control strategy of L.multiflorum.The cross-and multiple-resistance patterns of the three populations AH-01(ACCase,I2041N),AH-05(ACCase,I1781L;ALS,P197T)and HN-07(ACCase,D2078G)were studied,respectively.The results showed that all three populations showed varying degrees of cross-resistance to the ACCase inhibitors tested,namely,haloxyfop-R-methyl,quizalofop-P-ethyl,cyhalofop-butyl,fenoxaprop-P-ethyl,metamifop,clethodim,Sethoxydim and pinoxaden.Among them,the population AH-01 showed multiple-resistance to the ALS inhibitors bispyribac-sodium,while the population AH-05 showed crossresistance to bispyribac-sodium,imazethapyr,pyroxsulam,imazamox and flucarbazone-Na.The population HN-07 showed multiple-resistance to pyroxsulam,bispyribac-sodium,imazethapyr and imazamox.All populations were susceptible to 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS)inhibitor glyphosate and Photosystem II(PSⅡ)inhibitor isoproturon.Post-emergence(POST)herbicides were used to explore the best control period of L.multiflorum.The results showed that all tested herbicides had better control effects at the 2-4 leaf stage(fresh weight inhibition rate>70%).With the increase of weed leaf stage,the control effects of almost all herbicides decreased significantly.Under the premise of ensuring crop safety,herbicides should be used as early as possible to control L.multiflorum.4.Adaptive changes of seed ecological characteristics of resistant biotype of L.multiflorum.The resistant population JS-07 and two sensitive populations AH-06 and AH-09 with 100% I2041N mutation frequency in the target gene ACCase were selected as the research objects.According to the sequencing results of individual target genes,the resistant homozygous mutant plants and the sensitive population plants were isolated and cultured to obtain their F1 generation seed materials.To explore the characteristics of seed germination and emergence under different environmental factors and different soil burial depths.The results showed that the response trends of different populations to each treatment were generally consistent.Under different light conditions,the total germination of all the populations was above 85%.Light had no significant effect on seed germination.p H did not affect seed germination.When the p H level was 4-10,seed total germination of all groups was above 90%.The seeds of L.multiflorum had a wide adaptability to temperature,which could germinate under the constant temperature of 5℃-35℃ and the variable temperature of 5/15℃-25/35℃(total germination between 61%-99%).The constant high temperature(35℃)would inhibit the germination,and the constant low temperature(5℃)would delay the initial germination time and reduce the total germination.In addition,salt stress,osmotic stress and soil burial depth could significantly affect seed germination.Compared with the sensitive population,the resistant population JS-07 showed obvious ecological adaptability advantages in these three aspects.Deep tillage with buried weed seeds below 4 cm was still an effective means to reduce the overall emergence rate of sensitive and resistant L.multiflorum.In summary,the occurrence and resistance distribution of L.multiflorum in wheat fields of Huang-Huai-Hai plain were clarified by field investigation and resistance identification.The molecular mechanism of the resistant population was preliminarily explained by target gene sequencing,expression analysis and metabolic enzyme inhibitor bioassay.To explore scientific and efficient chemical control strategies of resistant weeds,the resistance patterns of populations to different herbicides were measured and the chemical control periods of different herbicides were screened.Finally,we further studied the changes of seed germination characteristics between resistant and sensitive populations of L.multiflorum,and tried to explore feasible ecological management methods of resistant weeds.The results of resistance study on L.multiflorum in wheat fields of Huang-Huai-Hai plain would provide scientific basis for establishing scientific and effective resistant weed control strategies to ensure stable and increasing wheat yield.更多还原