【作者】 王红春；

【导师】 董立尧；

【作者基本信息】 南京农业大学 ， 农药学， 2012， 博士

【摘要】 本文以长江中下游地区小麦田、油菜田恶性杂草早熟禾(Poa annua L)为主要对象,研究其相关生物学生态学特性,特别就其对精嗯唑禾草灵的耐药性进行了较为系统的研究,初步明确了早熟禾发生日益严重的原因,丰富了早熟禾耐药性方面的有关理论,为早熟禾的防除提供了技术基础。本文具体研究了早熟禾籽实休眠及萌发出苗特性；调查了早熟禾的结实特性、田间发生动态、群体生物量以及不同前茬与不同耕作方式对早熟禾发生量的影响；测定了不同地区早熟禾生物型对乙酰辅酶A羧化酶(Acetyl coenzyme A carboxylase, ACCase)抑制剂类除草剂精噁唑禾草灵(fenoxaprop-P-ethyl)的敏感性；系统研究了早熟禾靶标酶ACCase活性、ACCase CT区片段氨基酸序列、ACCase基因相对表达量、谷胱甘肽硫转移酶(GST)活性、细胞色素P450氧化酶系主要组分含量及活性、抗氧化防御系统的相关指标与早熟禾耐药性的关系。1.早熟禾的生物学生态学特性对早熟禾籽实休眠、萌发及出苗特性的研究表明：成熟的早熟禾籽实具有接近20天的休眠期。0.5%次氯酸钠消毒后4℃清水浸泡、室外自然条件土壤层积、室外自然条件水下土壤层积均可快速解除早熟禾籽实的休眠,但低温干燥条件不利于其休眠解除；250mg·L-1赤霉素、31.25mg·L-1乙烯利或500mg·L-1硝酸钾室温浸泡早熟禾籽实24h均可有效的解除其休眠；早熟禾籽实萌发适宜温度为10～25℃,光照、酸碱度、盐胁迫均非其萌发的限制性因素,其萌发对水分胁迫非常敏感,0～2.0cm土壤深度时早熟禾的出苗率均可达到55%以上,土壤深度为3.5cm时,早熟禾籽实几乎不能出苗。田间调查早熟禾的结实特性、田间发生动态、群体生物量、不同前茬及不同耕作方式下早熟禾的发生量发现：2009年稻茬小麦田中早熟禾籽实成熟时,早熟禾平均株高为33.96±1.89cm,平均分蘖为6.7±1.2个,平均有效分蘖为5.6±1.2个,平均每穗粒数为1004±155粒,平均千粒重为0.2944±0.02g；稻茬小麦田早熟禾具有一个冬前出苗高峰；早熟禾的株高、鲜重均在小麦播后第15周进入快速增长期,比小麦推迟3周；前茬为玉米和水稻的试验田浅耕、免耕处理后撒播油菜和小麦,早熟禾的发生量具有显著的差异,以前茬为玉米,浅耕小麦田中早熟禾发生量最低。2.早熟禾对精噁唑禾草灵及其它除草剂的敏感性种子生测法和整株生测法测定了21个早熟禾生物型对精噁唑禾草灵的敏感性,结果表明早熟禾对精噁唑禾草灵具有天然抗药性,即耐药性。整株生测法测定了夏熟作物田禾本科杂草蔺草(Beckmannia syzigachne)、日本看麦娘(Alopecurus japonicus)、看麦娘(A. aequalis)、硬草(Sclerochloa kengiana)、棒头草(Polypogon fugax)对精噁唑禾草灵的敏感性,结果表明5种禾本科杂草中以看麦娘对精噁唑禾草灵最为敏感。整株生测法测定了早熟禾对其它ACCase抑制剂类除草剂的敏感性,结果表明早熟禾对高效氟吡甲禾灵(haloxyfop-P-methyl)、氰氟草酯(cyhalofop-butyl)、精吡氟禾草灵(fluazifop-P-butyl)、炔草酸(clodinafop-propargyl)、烯禾啶(sethoxydim)具有耐药性,而对吡喃草酮(tepraloxydim)、烯草酮(clethodim)敏感。土壤处理整株生测法测定早熟禾对19种非ACCase抑制剂类除草剂的敏感性,结果表明胺苯磺隆(ethametsulfuron-methyl)、磺酰磺隆(sulfosulfuron)、吡氟草胺(diflufenican)、氟噻草胺(flufenacet)、吡嘧磺隆(pyrazosulfuron)、叹草醚(bispyribac-sodium)、吡草胺(metazachlor)播后苗前处理对早熟禾均可达到比较理想的抑制效果。茎叶处理整株生测法测定早熟禾对15种非ACCase抑制剂类除草剂的敏感性,结果表明甲基二磺隆(mesosulfuron-methyl)、氟噻草胺(flufenacet)、胺苯磺隆(ethametsulfuron-methyl)、百草枯(paraquat)、草甘膦异丙胺盐(glyphosate)、草胺膦(glufosinate)茎叶处理均可有效抑制早熟禾的生长。3.早熟禾对精噁唑禾草灵的耐药性机理采用同位素标记检测法研究早熟禾及对照敏感杂草看麦娘ACCase对精噁唑禾草灵酸的敏感性,分子生物学技术研究早熟禾及对照敏感杂草看麦娘的ACCase氨基酸序列及其ACCase基因相对表达量的差异。结果表明早熟禾ACCase对精噁唑禾草灵酸敏感性极低,其ACCase氨基酸序列1781位点天然存在其它抗性禾本科杂草突变类型氨基酸亮氨酸(Leu),其ACCase基因表达量显著高于看麦娘。模式底物分光光度法测定施药前后早熟禾及看麦娘谷胱甘肽硫转移酶(GST)活性的变化动态。结果表明：施用精噁唑禾草灵初期早熟禾GST活性较未施药植株有所提升,后降至与未施药对照水平相当；而看麦娘GST活性则先稍有升高,后逐渐降至未施药水平之下。分光光度法测定施药前后早熟禾及对照敏感杂草看麦娘细胞色素P450氧化酶系主要组分细胞色素P450与细胞色素b5含量的变化动态。结果显示：施药后早熟禾细胞色素P450与细胞色素b5含量先升高,后逐渐降低,但其含量始终高于未施药植株；施药后看麦娘细胞色素P450含量先升高,后降至未施药水平之下,其细胞色素b5含量则先有所升高后降至未施药水平。荧光分光光度法测定施药前后早熟禾及看麦娘细胞色素P450氧化酶系介导的对硝基苯甲醚-O-脱甲基酶(PNOD)、乙氧基试卤灵-O-脱乙基酶(EROD)、乙氧基香豆素-O-脱甲基酶(ECOD)和NADPH-细胞色素P450还原酶活性的变化动态。施药后早熟禾细胞色素P450氧化酶系介导的PNOD、EROD、ECOD、NADPH-细胞色素P450还原酶活性均有所升高,尤以ECOD与NADPH-细胞色素P450还原酶活性升高相对较为显著。而看麦娘P450氧化酶系介导的PNOD、EROD、ECOD活性较未施药植株均有所下降,而NADPH-细胞色素P450还原酶活性则先升高,后降至未施药植株水平之下。整株生测法测定了细胞色素P450氧化酶系抑制剂胡椒基丁醚(PBO)、1-氨基苯并三唑(ABT)、马拉硫磷(malathion)寸精噁唑禾草灵的协同作用。研究表明细胞色素P450氧化酶系抑制剂PBO与ABT均可显著提高精噁唑禾草灵对早熟禾的抑制作用,而马拉硫磷则无增效作用。采用分光光度法或荧光分光光度法测定施药前后早熟禾体内活性氧伤害及抗氧化防御系统相关指标与其耐药性的关系。结果发现：施药后早熟禾超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性先升高,后又逐渐降低,过氧化物酶(POD)活性保持稳定,而看麦娘SOD、CAT活性保持稳定,POD活性先升高,后又逐渐降低。施药后早熟禾还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)含量先升高后降低,对照敏感杂草看麦娘则呈现逐渐下降的趋势。施药后早熟禾超氧阴离子(O2·-)、过氧化氢(H2O2)、抗坏血酸(AsA)、硫代巴比妥酸反应产物(TBARS)和可溶性蛋白含量保持稳定,而看麦娘则呈现逐渐升高的趋势。综上所述,早熟禾ACCase对精噁唑禾草灵酸极低的敏感性、氨基酸1781位点抗性突变类型氨基酸的天然存在及其相对较高的ACCase基因表达量可能是早熟禾对精噁唑禾草灵耐药性的主要机制之一。施药后早熟禾细胞色素P450与细胞色素b5含量及细胞色素P450氧化酶系活性得到了诱导性提升,表明细胞色素P450氧化酶系介导的代谢加强可能也是早熟禾对精噁唑禾草灵耐药性的主要机制之一。GST及抗氧化防御系统相关指标在早熟禾对精噁唑禾草灵的耐药性中可能也发挥了一定的作用。早熟禾对精噁唑禾草灵较高的耐药性水平可能是包含靶标酶、代谢酶及抗氧化防御系统等在内的多种机制共同作用的结果。更多还原

【Abstract】 Annual bluegrass (Poa annua L.), a kind of worst grass weed in wheat (Triticum aestivum L.) and oilseed rape (Brassica campestris L.) fields in the middle and lower reach of Yangtze valley in China, was chosen as a main research object in this paper. The biological and ecological characteristics of P. annua and its mechanisms of tolerance to herbicide fenoxaprop-P-ethyl were studied to elucidate why the occurrence of P. annua were increased gradually and to make theoretic complementarity for herbicide tolerance mechanism of P. annua. In general, this paper did some exploration in following aspects: the characterization of seed dormancy and germination of P. annua; the characteration of fructification of mature P. annua plants, the occurrence dynamics, the population biomass and the influence of different kind of preceding crops and cultivation on plant biomass; the susceptibility of ACCase (acetyl coenzyme A carboxylase) to herbicide fenoxaprop-P-ethyl from P. annua; the relations of herbicide tolerance of P. annua with ACCase activity, amino acid sequence of ACCase CT domain, relative expression level of ACCase genes, GST activity, contents and activity of cytochrome P450monooxygenase, and anti-oxidant indexes.1.Biological and Ecological Characteristics of P. annuaThe mature seeds have about20days dormancy after being collected from fields. The dormancy of seeds could be broken by being buried underground5cm, buried ungerground5cm with water, soaked in the water at4℃after being disinfected using0.5%sodium hypochlorite (NaCIO). The seed dormancy of P annua could be broken by treated with chemicals such as250mg·L-1GA3,500mg·L-1KNO3and31.25mg·L-1Ethopon for24h. The optimum temperature range for seed germination was10～25℃and light was not necessary. Seed germination was sensitive to osmotic potential. But it was quite tolerant to salinity. More than80%of seeds could be germinated at pH values ranged from4to10. Seedling emergence was higher (55%) when seeds were sown at the depth of0～2.0cm.Few seedlings emerged when seeds were planted at a depth of3.0cm. Information gained in this study will load to a better understanding of the requirements of P. annua gerimination and seedling emergence.Fruiting traits of P. annua were investigated before the seed exfoliated in wheat fields rotated with rice. Its average plant height was33.96±1.89cm, average tillering nodes were6.7±1.2, average productive tillering nodes were5.6±1.2, average grain number per spike was1004±155and average1000-grain weight was0.2944±0.02g. Ecology exploration of P. annua showed that a main emergence fastigium was observed in winter with majority seedlings. The zooming period of plant height and fresh weight of P. annua occurred at15WAP (weeks after planting), which was three weeks later than wheat, and suggested that chemical control and fertilization should be performed within15WAP.The seedling emergences of P. annua in the wheat fields were varied when it occurred in the different previous crop and different tillage methods. The seedling emergence was lowest when it occurred in the wheat fields, which corn (Zea mays) was the previous crop and the wheat was planted after shallow tillage.2. The Sensitivity of P. annua to Herbicide Fenoxaprop-P-ethyl and Other HerbicidesThe susceptibility of P. annua to fenoxaprop-P-ethyl were identified by seed-bioassay and whole-plant test, which were collected from different fields, and similar results were obtained by both of methods. The biotypes, which had never been applied with fenoxaprop-P-ethyl, had very high ED50values which were not impossibily applied in the fields. This indicated that P. annua was tolerant to fenoxaprop-P-ethyl.The sensitivity level of Alopecurus aequalis, A. japonicus, Beckmannia syzigachne, Sclerochloa kengiana, and Polypogon fugax to fenoxaprop-P-ethyl were evaluated by whole-plant bioassay, which were collected from Zijin Mountain (Nanjing, Jiangsu), where fenoxaprop-P-ethyl had never been applied. The A. aequalis was the most sensitive to fenoxaprop-P-ethyl among the five grasses.The sensitivities of P. annua and susceptible reference weed A. aequalis to other ACCase inhibiting herbicides were evaluated by whole-plant bioassay. P. annua and susceptible weed A. aequalis were collected from Zijin Mountain (Nanjing, Jiangsu), where fenoxaprop-P-ethyl had never been applied. P. annua was found to be tolerant to fenoxaprop-P-ethyl as well as haloxyfop-P-methyl, clodinafop-propargyl, fluazifop-P-butyl, cyhalofop-butyl and sethoxydim, whereas it was sensitive to clethodim and tepraloxydim.The susceptibility of P. annua to19herbicides belong to different classes was determined by pre-emergence applied methods. Ethametsulfuron-methyl, sulfosulfuron, diflufenican, flufenacet, bispyribac-sodium, and metazachlor could be used to control P. annua through pre-emergence applied.The susceptibility of P. annua to15herbicides belong to different classes was determined by post-emergence spraying methods. Ethametsulfuron-methyl, mesosulfuron-methyl, flufenacet, paraquat, glufosinate, and glyphosate could be used to control P. annua through post-emergence spraying.3.The Mechanisms of Tolerance to Fenoxaprop-P-ethyl in P annuaThe IC50(the concentration of fenoxaprop-P-ethyl (free acid) that inhibited50%of acetyl coenzyme A carboxylase (ACCase) activity) values for P. annua was1.70-fold higher than that for A. aequalis, which was susceptible to fenoxaprop-P-ethyl in fields. The presence of the Leu-1781residue, which had been reported to be involved in the resistance of gramineous weeds to ACCase inhibitors, was subsequently identified in the plastidic ACCase of P. annua. Furthermore, the relative gene expression level of P. annua ACCase genes was found to be approximately2.4-fold higher than that in A. aequalis, possibly explaining the tolerance to fenoxaprop-P-ethyl of P. annua.The1-chloro-2,4-dinitrobenzene (CDNB) were used as model substrates to detect the activities of glutathione s-transferases (GST) in P. annua and susceptible A. aeuqlis in order to clarify the relationship with GST metabolic tolerance. The activity of GST in P. annua was higher than that in A. aequalis without herbicide treatment. GST activity in P. annua increased slowly after treated with fenoxaprop-P-ethyl, then it decreased to being similar to the untreated plants after3days treatment. The GST activity in A. aequalis increased when1day after treatment, then decreased to being smaller than the untreated plants in all of the cases.The contents of cytochrome P450and cytochrome b5in P. annua and A. aequalis were determined by spectrophotometric determination. After treated with fenoxaprop-P-ethyl, the contents of cytochrome P450and cytochrome b5in P. annua were significantly increased, compared with untreated plants. However, the increasing was less in A. aequalis, which was susceptible to fenoxaprop-P-ethyl. The activities of cytochrome P450monooxygenases mediating p-nitroanisole O-demethylase (PNOD), ethoxyresorufin O-dethylase (EROD), ethoxycoumarin Oxidase (ECOD), and NADPH-dependent cytochrome P450reductase were determined by influorescence spectrophotometry. The activities of cytochrome P450monooxygenase mediating PNOD, EROD, ECOD and NADPH-cytochrome P450reductase had increased in P. annua after application of fenoxaprop-P-ethyl, especially the activities of ECOD and cytochrome P450reductase. The O-deetylylation and reduction catalyzed by cytochrome P450monooxygenases may be involved in the metabolism of fenoxaprop-P-ethyl. It was also suggested that the induction of cytochrome P450monooxygenase activities is the mechanism involved in the fenoxaprop-P-ethyl tolerance observed in P. annua.The effects of fenoxaprop-P-ethyl and cytochrome P450monooxygenases inhibitor piperonyl butoxide (PBO), malathion and1-aminobenzotriazole (ABT) on the weed growth were evaluated by whole-plant bioassay. It had been found that PBO and ABT synergized fenoxaprop-P-ethyl activity in P. annua, while malathion had little effects. But the ED50vulues of fenoxaprop-P-ethyl had been applicaed with PBO or ABT were still very higher than the recommended dose in fileds. The synergisms of PBO and ABT valideted that cytochrome P450monooxygense is one of the mechanisms involved in the fenoxaprop-P-ethyl tolerance observed in P annua.The activities of superoxide dismutase, catalase, peroxidase, and contents of superoxide anion radial (O2·-), reduced/oxidized glutathione (GSH/GSSG), hydrogen peroxide (H2O2), ascorbic acid (AsA), thiobarbituric acid reactive substances (TBARS), and soluable protein in P. annua and A. aequalis were detected. Significant differences of all the indexes mentioned above were observed in P. annua and A. aequalis after treated with fenoxaprop-P-ethyl. The lower oxygen damages and higher activities of SOD, POD and CAT in P. annua indicated that tolerance due to oxidative stress may be one of the mechanisms of tolerance to herbicide fenoxaprop-P-ethyl in P. annua.更多还原