【作者】 黄青春；

【导师】 叶钟音；

【作者基本信息】 南京农业大学 ， 植物病理学， 2001， 博士

【摘要】 本文以拌种灵、噻枯唑、萎锈灵、以及二硫氰基甲烷等杀菌剂，柑桔溃疡病菌、水稻白叶枯病菌、大豆斑疹病菌等植物病原细菌为材料，着重从拌种灵对柑桔溃疡病菌的毒力机制、柑桔溃疡病菌的药理学机制、柑桔溃疡病菌对拌种灵的抗药风险性及生理特性等方面深入系统地研究了拌种灵对柑桔溃疡病菌的作用机制，并对拌种灵在菌体细胞内的分子生理生化靶标进行了初步的探索。 在国内外首次系统地研究了拌种灵对植物病原细菌的作用。在离体条件下，拌种灵对植物病原菌的抑制作用具有强烈的选择性，对水稻白叶枯病菌、水稻细菌性条斑病菌、柑桔溃疡病菌及大豆斑疹病菌的有效抑制中浓度(EC₅₀)分别为0.5745、1.3026、2.6257和7.4970μg·mL¹，对丁香假单胞病菌、番茄青枯病菌和大白菜软腐病菌基本无效，预示拌种灵仅对黄单胞病菌引起的病害具有较高的活性。噻枯唑、萎锈灵、二硫氰基甲烷和链霉素等杀菌剂对柑桔溃疡病菌的有效抑制中浓度分别为3.6269、2.8547、0.1485和0.0409μg·mL¹，拌种灵对柑桔溃疡病菌的抑菌作用稍优于萎锈灵和噻枯唑，而低于二硫氰基甲烷和硫酸链霉素，但福美双不能抑制柑桔溃疡病菌的生长。拌种灵在偏酸或偏碱的环境下都具有抑菌作用，并且拌种灵能够抑制柑桔溃疡病菌对外源葡萄糖的吸收和利用，拌种灵对柑桔溃疡病菌的活性在不同的底物培养基中差异较大，以乙酸钠为底物的培养基中，活性约为在以葡萄糖为底物培养基中的5.3倍以上。 对拌种灵、噻枯唑和萎锈灵的毒力机制进行比较表明，拌种灵与噻枯唑、萎锈灵、及二硫氰基甲烷具有相似、而功能相异的毒性基因。噻枯唑对小麦纹枯病菌的生长没有影响，拌种灵和萎锈灵防治细菌病害都具有较好的作用效果，但柑桔溃疡病菌对二者的耐受程度存在较大的差异，最低抑制浓度（MIC）表现为100μg·mL¹和400μg·mL¹。拌种灵、噻枯唑和萎锈灵的活性者低于二硫氰基甲烷。在相同的有效浓度下，拌种灵对柑桔溃疡病菌生长速率的影响优于噻枯唑与萎锈灵的作用效果之和。拌种灵和萎锈灵对柑桔溃疡病菌的呼吸作用都具有抑制作用，低浓度隍枯哩对病原菌的呼吸作用具有一定的刺激作用，与同浓度拌种灵相比，噬枯哩的抑制作用较弱，表明拌种灵在防治细菌病害过程中，分子结构中唆哩结构和苯胺基甲酚结构均能发挥生物活性。 柑桔溃疡病菌的胞外产物在拌种灵与病原菌互作过程中具有一定的调控作用。胞外产物尤其是胞外粘多糖（EPS）对拌种灵具有很强的桔抗作用，胞外产物可以使拌种灵对病原菌生长的抑制率降低2倍左右。经拌种灵处理的病原菌，其胞外产物如：电解质、EPS、胞外蛋白、以及胞外水解酶等的产量和生物活性都呈现出一定规律的变化，在20Pg·mL拌种灵的棚下，菌体胞外蛋白的产量仅占对照的26．26％，f巳胞外蛋白酶的活性在拌种灵浓度低于10Pg·mL’条件下随着药剂浓度的升高而增强。研究表明，病原菌在药剂处理下可通过胞外产物的变化产生一定的抗逆反应。同时菌体细胞结构亦发生相应的变化，细胞质向中心浓缩，细胞两端出现空泡结构，细胞内含物与拌种灵接触减少，茵体细胞质表现出对外界不良环境抗逆性生理的适应性调节。 菌体细胞的药理学研究表明，拌种灵对柑桔溃疡病菌的抑制作用在病原菌不同生长时朋具有很大差异，在病原菌生长静止期加入药剂，即使50％·mL‘拌种灵对病原菌生长的抑制作用都较小。拌种灵对病原菌的作用方式为抑菌作用而不是杀菌作用。丙酮酸、软酸氨基葡萄糖、N－乙酚－D－氨基葡萄糖、细胞色素C等可以抵消拌种灵在菌体内的抑制作用。拌种灵不改变菌体细胞膜通透性，对菌体细胞膜的功能没有影响，对细胞膜N广K”ATP酶及其活化能、菌体蛋白合成、丙酮酸和柠檬酸代谢，及细胞呼吸过程等都具有不同程度的活性，拌种灵能够抑制病原菌细胞磕用酸脱氢酶的活性，50％·mL－‘拌种灵作用于菌体30分钟后对琉琅酸脱氢酶的抑制率高达98％。但拌种灵对菌体脂质合成、以及末端活性氧的产生等都几乎没有影响。拌种灵对细胞呼吸作用的抑制与丁烯戳胺类杀菌剂的作用机制相似。有证据表明，菌体细胞糖代谢途径和细胞呼吸电子传递链上掳琅酸脱氢酶系是拌种灵在柑桔馈疡病菌菌体细胞内的作用靶标。 经药剂诱导和紫外光照射双重作用首次获得柑桔腿病菌抗拌种灵突变体，经致病性研究表明，该突变体为由 突变体，抗药性的产生伴随着致病性的丧失， 二互 抗药突变体具有遗传的稳定性。但抗药突变体的生长速率、菌体蛋白和胞外蛋白 组分等与敏感菌株基本一致，对萎锈灵和噎枯咬具有潜在的正文互抗性，与二硫 氰基甲烷没有交互抗性。胞外水解酶活性一般高于敏感菌株，其中以淀粉酶活性 变化最为明显。胰琅酸脱氢酶活性显著降低，抗药突变体受拌种灵的抑制作用亦 降低。同时抗药突变体从蔗糖产生还原物质的能力显著降低。抗药突变体的性质更多还原

【Abstract】 Toxicity mechanism, pl~rmacology, resistance risk, physiological and biochemical characters, and targets of attacking site et al. were studied in citrus bacterial canker disease (CBCD) pathogen Xunthomonas citri inhibited by carboxamides fungicide amicarthiazol. Action of amicarthiazol to plant pathogen bacteria was first charactered systemly in abroad and domestic researches. Intensely selection was indicated in inhibition of amicarthiazol to plant pathogen bacteria Xanthomonade such as X oryzae pv. oryzae, X o. pv. oryzicola, X citri, and X glycines et al. with high activity of efficient inhibitory medium concentration (EC50) 0.5745, 1.3026, 2.6257 and 7.4970kg 秐L1 in vitro, respectively. Little inhibition was showed to Pseudomonas syringae pv. syringae, Ralstonia .solanacearum and Erwinia carotovora pv. carotovora. However. EC50 of hismerthiazol. carboxin. diisothiocyanatomethane and streptotnycin sulfate were 3.6269, 2.8547, 0.1485, 0.040%.tg mU? It showed that inhibitory efficiency of aniicarthiazol was better than that of carboxin and bismerthiazol, yet less than diisothiocyanatomathane and streptomycin sulfate to the growth of bacterium X citri. Thiram and pH tendency had no influence on inhibition to the pathogen, glucose oxidation and utility by cell of X citri was 5.3 times in medium with sodium acetate solely hig~r than with glucose as sole carbonic resource. The functions of toxicity radicals with the similar structures were minor 112 difference through comparison of toxicity mechanism among amicarthiazol, bismerthiazol and carboxin. They all had well inhibitory efficiency to the growth of plant bacterial disease pathogen and Rhizocto,iia cerealis. But bisnierthiazol had little toxicity to Rhizoctonia cerealis. The median inhibitory concentrations (MIC) of X curl to amicarthiazol and carboxin were 10014g. mV and 40014g ml]?respectively, it reflected that some different tolerances were exjsted between amicarthiazol and carboxin. Under the same efficient concentration effect of amiearthiazol was better than total of the effect of bismerthiazol and carboxin to the -growth and the respiration of X carl. However. Bismerthiazol could stimulate the respiration of the bacterium on some distance in low concentration. The results indicated that thiazole and benamidemetide structures in the molecular structure of amicarthiazol all had bioactivity to X cUrl. Extracellular secretions of X cUrl had certain abilities to regulate the interaction course between amicarthiazol and the bacterial cell. The secretions, especially extracellular polysaccharide (BPS), could strongly antagonize the inhibition of amicarthiazol. Output of electrolyte, El扴, extracelkilar protein, and bioactivity of extracehlular hydrolytic enzymes were regularized to vary with the concentration of amicarthiazol. To be noted that 73.74% product of ex~acelluIar protein of the bacterium treated with 2014g. mU?amicarthiazol was inhli,ited. l~ut the activity of extracellular proteinase increased with concentrati(n of aznicarthiazol till up to 1014g. mU1. The results made known that the pathogenic bacteria possess an active echo to the adverse environment by changing the atti~butions of extracellular secretions enriching its cytoplasm when surrounded by fungicides to minor the probable touch to amicarthia~ol. Many researches were further to make clear diet the inhibitory rate was great different during different growth stage of更多还原