【作者】 陈琪；

【导师】 丁克坚；

【作者基本信息】 安徽农业大学 ， 植物病理学， 2004， 硕士

【摘要】 本文以不同寄主来源的灰葡萄孢为供试菌株，通过抗药性诱变获得了对速克灵具有高抗性的突变株。进而对抗性突变株的致病力、致病生理生化机理、寄主抗病性生理生化机制等方面进行了较为系统的研究，探讨了灰葡萄孢抗药性变异及其致病力差异的机理，主要结果如下：1．灰葡萄孢抗药性研究 实验表明，速克灵、多菌灵、乙霉威、代森锰锌四种农药中，速克灵与多菌灵对灰葡萄孢的防治效果较好，Ec50基本上均小于 0.5ug/ml。对于来自不同寄主的灰葡萄孢菌株，四种农药的防治效果各有高低，但整体而言，防效高低顺序为多菌灵>速克灵>乙霉威>代森锰锌。 本实验通过药剂诱导灰葡萄孢产生对速克灵的高抗菌株 CF1-1,CF1-2, 其 Ec50值分别为 197.546 μg· ml-1与 370.984 μg· ml-1，抗性水平与敏感型菌株相比分别增加了 409倍与 911 倍。对抗性菌株毒力测定发现，CF1-1与 CF1-2不仅对速克灵抗性程度很高，对多菌灵抗性程度同样较高。抗性菌株在含低剂量速克灵的培养基上生长旺盛，生长速率较敏感菌株高，在无药培养基上菌丝生长较弱，菌落颜色较深并且菌丝畸形，说明这类抗性菌只适于有药的环境条件。抗性突变株与亲本菌株相比产孢量明显减少，说明抗速克灵突变株在繁殖能力上较亲本要减弱。接种草莓叶片实验表明，速克灵抗性菌株较敏感菌株而言发病较重且病斑较大，病斑边缘颜色深，致病力较强。即不仅获得了抗速克灵的高抗菌，同时具有较强的致病力。 将抗性突变株继代培养可以发现，在一定时期内的无药培养，抗性菌对速克灵与多菌灵的抗性程度没有下降，只是随着转移代数的增加，菌丝生长速率有所降低，而对多菌灵和速克灵的抗性程度均无影响，但是第十代的菌丝生长速率明显下降，进一步对抗性菌在含药平板上的生长频率实验表明，前 5 代抗性菌株的生长频率均较高，生长状态较好。第 7 代抗性菌对多菌灵的存活率仍较高，但对速克灵的存活率下降至50%，第 10 代，菌株存活率均下降至 25%左右，证明经过长时间转接与离体培养后，抗药性在一定程度上降低或丧失。可以认为在一定期限内，灰葡萄孢对速克灵的抗性遗传是稳定的，但超出一定的期限就有可能减弱或丧失。2．灰葡萄孢致病机理的研究 通过不同寄主来源的灰葡萄孢对不同寄主交互接种进行致病力的测定，可以看出，不同寄主来源的菌株之间有一定的差异，表现为在同种寄主植物上的发病情况有明显的差异；同一寄主来源的灰葡萄孢在不同的寄主上的致病力也表现出明显的差异。并且每一种菌株对其自身寄主与其它寄主来源的菌株相比并不具备致病力上的优 i<WP=7>势。从这一意义上来说，灰葡萄孢对寄主植物的致病力的确存在明显分化。 本文从草莓发病叶片中获得了灰葡萄孢的致病酶纤维素酶（Cx 和 ?-葡萄糖苷酶）与果胶酶（PMG）。在不同灰葡萄孢侵入同种草莓叶片后，产生的 Cx 与 ?-葡萄糖苷酶酶活性均随着培养天数的增加而升高，而对照的健康叶片在培养期间并没有明显变化，但不同菌株产生的纤维素酶活力有明显差异，强致病力的菌株产生的酶活力高，而侵染速率慢的菌株产生的纤维素酶活力较低。培养 10-15d 的灰葡萄孢均可测出果胶酶活性，酶活性差异不显著。接种草莓叶片后，果胶酶活性迅速升高，在第四天左右达到最大值，随后下降，而对照的变化并不明显。通过以上分析，可以认为灰葡萄孢的致病作用与致病酶有一定的相关性。测定的致病酶中，以 PMG 活性为最高，其次是 ?-葡萄糖苷酶，Cx 最少。由此可知，在病原菌致病过程中，主要是 PMG 起作用。3 灰葡萄孢诱导草莓抗病性的生理生化机制 本实验结果表明灰葡萄孢侵染草莓后 PPO 和 POD 活性明显提高，活性的变化量与感病程度有显著的正相关。不同菌株侵染草莓叶片后诱导 PPO 和 POD 酶活性变化有显著差异，不同寄主来源的敏感菌株中 FX1诱导产生的酶活性最强，并且酶活力增长的速率与病斑扩展的速率正相关。寄主来源是草莓的 CF1侵染草莓叶片的发病速率反而比较慢，CF1 的抗速克灵诱导菌株 CF1-1 和 CF1-2 虽然病斑面积与扩展速率和亲本菌株差异不大，但诱导产生的防御酶活力却远远强于亲本菌株。草莓接种后 1~3天之间 PAL 活性显著高于对照，至第四天起活性逐渐下降。在灰葡萄孢感染番茄过程中，从感染部位到未感染区域病斑处和病斑周围植物组织的防御反应，导致 PAL酶活性的增强。草莓接种后 1~4 天之间 SOD 活性明显升高，在灰葡萄孢感染草莓过程中，从感染部位到未感染区域的边缘，SOD 活性有一个明显的下降梯度。 本实验测试了不同灰葡萄孢接种草莓叶片后过氧化物酶同工酶(POD),多酚氧化酶同工酶(PPO)，超氧化物岐化酶同工酶(SOD)酶谱，从酶谱中可以看出，接种灰葡萄孢后三种酶的同工酶都有明显变化，并且不同寄主来源的灰葡萄孢株接种草莓后产生的同工酶谱带在数目和颜色上都存在差异，其中以过氧化物酶同工酶(POD)的差异最为明显，而超氧化物岐化酶同工酶(SOD)的差异最小。对同一品种而言，强致病力的菌株诱导产生的酶带颜色较深，带?更多还原

【Abstract】 In this paper, different isolates of Botrytis cinerea were the research objects. Thehigh-resistant mutants were obtained by inducing sensitive strains with Procymidone. Thedifference of pathogenicity, the physiological and biochemical mechanisms topathogenicity, the physiological and biochemical mechanisms of host resistance werestudied systematically, and the variation of fungicide resistance of B.cinerea was alsodiscussed. The results were summary as follows:1. Study on fungicide resistance to procymidone of B.cinerea. The results showed that the control effect of procymidone and carbendazim was thebest among the four kinds of fungicide including procymidone, carbendazim, mancozeband dithofencarb. The Ec50 value of procymidone and carbendazim was less than 0.5ug/ml.There were significant differences between the control effects of different fungicides todiverse strains isolated from different hosts. In the whole, the sequences of the number ofcontrol effects were carbendazim > procymidone > dithofencarb > mancozeb. The Ec50 value of two high-resistant mutants CF1-1 and CF1-2 belonged tomulti-resistant strains because the two strains had the high resistant level to procymidoneand carbendazim, respectively the resistant mutants grew prosperously on the medium inwhich contained low content of procymidone. While the mycelial growth rate of resistantmutants was higher than that of sensitive strains. The color of resistant mutants colonybecame darker and more abnormal than that of sensitive. These characteristics weredemonstrated that the resistant mutants were accustomed to the environment with lowcontent of fungicide. The conidia bearing quantities of resistant mutants were less than thatof parents strains, it showed that the reproductive capability of resistant mutants toprocymidone decreased compared to inoculating the leaves of strawberry, thepathogenicity of resistant mutants to procymidone was stronger than that of sensitivestrains, and the lesion area was larger than that of sensitive strains. Maybe the co-evolutioneffect resulted in the characteristic of CF1-1 and CF1-2, these were the two mutants ownedthe high resistant level to procymidone and strong pathogenicity. The results of sub-culturing the resistant mutants showed the resistance level ofresistant mutants did not declined when they grew on the medium in which did not containthe two fungicides. The mycelia growth rate declined with the increase of culturegeneration. Maybe it resulted from culture in vitro with long-term. While there were noany influence on the resistance level to procymidone and carbendazim, but the myceliagrowth rate declined significantly when they grew to the tenth generation. The results ofgrowth frequent on the medium with fungicides showed, the growth frequent of resistantmutants in the fifth generation was high and they grew prosperously. The percentage ofsurvived of resistant mutants to carbendazim in the stage of the seventh generation washigh, but that to procymidone decreased to 50%, the percentage of survival of resistantmutants in the stage of the tenth generations decreased to 25%. These characteristicssuggested that the resistance level decrease or lose to some extent when these resistantmutants sub-cultured for a long time. The resistant inheritance of B.cinerea to procymidone iv<WP=10>was stable.2. The mechanism of pathogenicity of Botrytis cinerea Different hosts were inoculated by different strains of B.cinerea isolated from differenthosts, and the intensity of pathogenicity was determined. The result showed that differentstrains had obvious diversities pathogenicity in the same plant, and the same strains haddifferent expression in different plants. Making use of the measure of spectrophotometric analysis to study strawberry leaveswere inoculated by several different B.cinerea strains the pathogenicity enzyme activity ofcarboxymethylcellulose(CMC), β-glucosi更多还原