苎麻疫霉抗甲霜灵菌株的生理生化研究
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摘要
本论文在苎麻疫霉抗甲霜灵诱变的基础上，就紫外光对苎麻疫霉抗甲霜灵诱变效应、紫外光对苎麻疫霉生物学性状的影响、苎麻疫霉抗甲霜灵突变株的生物学性状以及同工酶电泳分析等进行了系统的研究。
１.采用菌丝块紫外光诱变、游动孢子的紫外光诱变、紫外光+药物诱变、菌丝块药物诱变、游动孢子药物诱变等5种方法对苎麻疫霉野生型敏感菌株进行抗甲霜灵诱变。结果菌丝块药物诱变和菌丝块紫外光+药物诱变两种处理均能成功获得苎麻疫霉抗甲霜灵突变株，其它3种方法均未获成功。
2．研究了紫外光对苎麻疫霉生物学特性的影响，结果表明，30W的紫外灯管距培养皿50cm照射处理对苎麻疫霉的游动孢子具有明显杀灭作用，紫外光照射40sec可以抑制95%的游动孢子萌发；紫外光照射20min对菌丝块的菌丝生长的抑制率达最大，以后随着处理时间增加，抑制率却有降低的趋势，说明苎麻疫霉的菌丝块，经一定剂量紫外光照射后对紫外线产生了适应性。耐紫外光菌株的菌落形态较其亲本菌株及其它野生型菌株有较大的差异，但对甲霜灵的敏感性则无明显差异。此外，耐紫外光菌株的生长速率、卵孢子产生量、在不同pH值和温度下的生长情况明显小于其亲本野生菌株，说明紫外光降低苎麻疫霉对环境的适应能力，并且对其有性生殖能力也有显著影响 。
３.对抗甲霜灵（Mtr）苎麻疫霉菌株及其亲本的野生型菌株进行生物学研究，结果表明，突变苎麻疫霉菌株对甲霜灵的抗性水平均有极大的提高。抗性菌株的菌株形态较野生敏感性菌株有较大的变化，对于不同温度和pH值的适应范围没有改变，但抗性菌株在相同的温度和pH值情况下的生长情况明显低于野生型菌株及其亲本菌株。研究结果还表明，抗性菌株的卵孢子的产生量也明显低于其亲本菌株，抗性菌株在液体培养基中的菌丝较其亲本菌株生长量有所减少。研究结果说明抗性菌株在对甲霜灵产生抗药性的同时，对环境的适应能力有所降低，其有性生殖能力也明显下降。
４. 应用聚丙烯酰胺同工酶分析技术对苎麻疫霉Mtr突变株的酯酶（EST）、过氧化物酶（POD）、过氧化氢酶（CAT）、多酚氧化酶（PPO）的同工酶电泳图谱等进行了系统的研究，并利用光密度法对其POD和PPO活性进行了测定。结果表明，Mtr菌株的PPO活性较其亲本菌株都有不同程度的增加，并且其同工酶电泳具有１条与抗药性相关联的特征性谱带；可溶性蛋白的电泳图谱也发现了至少１条具有与抗药性相关联的特征性谱带。苎麻疫霉的EST、CAT、PPO的同工酶电泳图谱显示，供试菌株的同工酶有一定差异，但没有找到与抗甲霜灵特性相关联的酶带。
上述研究结果为阐明苎麻疫霉对甲霜灵抗性的产生机理、苎麻疫霉对甲霜灵抗药
性监测、风险评价及治理提供了必要的理论基础和实验依据，对其他疫霉菌的抗药性研究也有一定的参考价值。
The effects of ultraviolet rays on mutagenizing metalaxyl resistance, the effects of on biochemical characteristics of Phytophthora boehmeriae, biochemical characteristics of metalaxyl-resistant (Mtr) mutants of P.boehmeriae and isoenzyme electrophoreses were studied systemically in this paper based on mutagenizing resistence of P. boehmeriae to metalaxyl.
1. The metalaxyl-resistance mutagenesis was carried with ultraviolet rays to mycelium discs, ultraviolet rays to zoospore, ultraviolet rays + metalaxyl to mycelium discs, metalaxyl to mycelium discs, metalaxyl to zoospores methods. The result showed that the mutants could be obtained by applying the two methods, i.e. metalaxyl to mycelium discs and metalaxyl plus ultraviolet ray to mycelium discs, but not by the other methods.
2. The effects of ultraviolet rays on the biochemical characteristics of P. boehmeriae were studied, and the results indicated that the zoospores of P. boehmeriae could be inactivated obviously by irradiating with 30W ultraviolet light 50cm away from the culture dishes tested and that 95% germination of zoospores could be restrained by 30W ultraviolet light treatment for 40sec.The mycelium growth from mycelium discs of P. boehmeriae were inhibited mostly by 30W ultraviolet light treatment for 20min. Afterwards, the inhibitive rates were decreased with the rise of the treatment time, which showed that the mycelium discs of P. boehmeriae had produced flexibility to ultraviolet rays after being irradiated by certain dosage of ultraviolet light. Between ultraviolet-patient isolates and parental isolates there was obvious difference in colony morphology but not in the sensitivity to metalaxyl. Moreover, the growth rates, oospore production, growth states of ultraviolet-patient isolates in different pH and temperature were obviously less than those of the wild parental isolates, suggesting that the adaptability of P. boehmeriae to the environment was depressed by the ultraviolet rays, and that the ability of sexual reproduction was affected notably by the ultraviolet rays.
3.Biological characteristics of Mtr mutants of P.boehmeriae were studied, and the results showed that resistance levels of Mtr mutants to metalaxyl were over 498 times of those of the parental isolates. There was greatly difference between Mtr isolates and their parental isolates in colony morphology, but no difference in the adaptation scope at different pH and temperature. The growth rates of Mtr isolates were obviously lower than those of the parental wild isolates at the same pH and temperature. It also indicated that the oospore production of Mtr isolates was obviously less than those of the parental isolates,
and that the mycelium production of Mtr isolates was a little reduced compared to those of the parental isolates in liquid culture. The results suggested that the adaptability of Mtr isolates to environment was reduced and so did the sexual reproduction of Mtr isolates while they produced metalaxyl-resistance.
4. The variations of isozyme spectra of esterase (EST), peroxidase (POD) polyphenol oxidase (PPO), catalase (CAT) and soluble protein of P. boehmeriae mutants were studied systemically by applying polyacrlamide gel electrophoresis (PAGE) and isoenzyme analytic technique, and the enzyme activity of POD and PPO of the Mtr isolates of P.boehmeriea by applying the spectrophotometry. The results showed that PPO activity of Mtr isolates had increased in some degree compared to parental isolates, and that one band of isozyme spectrum of POD was found related to the character of the metalaxyl-resistance in the mutants. And at least one band of isozyme spectora of souble protein was found in mutants related to the character of metalaxyl-resistance too. The results showed that isozyme spectra of EST, PPO and CAT were different in tested isolates, but that there were no bands observed relative to the metalaxyl-resistance.
The results mentioned above provided necessary theoretic foundation and experimental evidences for clarifying the mechanism of metalaxyl-r
１.采用菌丝块紫外光诱变、游动孢子的紫外光诱变、紫外光+药物诱变、菌丝块药物诱变、游动孢子药物诱变等5种方法对苎麻疫霉野生型敏感菌株进行抗甲霜灵诱变。结果菌丝块药物诱变和菌丝块紫外光+药物诱变两种处理均能成功获得苎麻疫霉抗甲霜灵突变株，其它3种方法均未获成功。
2．研究了紫外光对苎麻疫霉生物学特性的影响，结果表明，30W的紫外灯管距培养皿50cm照射处理对苎麻疫霉的游动孢子具有明显杀灭作用，紫外光照射40sec可以抑制95%的游动孢子萌发；紫外光照射20min对菌丝块的菌丝生长的抑制率达最大，以后随着处理时间增加，抑制率却有降低的趋势，说明苎麻疫霉的菌丝块，经一定剂量紫外光照射后对紫外线产生了适应性。耐紫外光菌株的菌落形态较其亲本菌株及其它野生型菌株有较大的差异，但对甲霜灵的敏感性则无明显差异。此外，耐紫外光菌株的生长速率、卵孢子产生量、在不同pH值和温度下的生长情况明显小于其亲本野生菌株，说明紫外光降低苎麻疫霉对环境的适应能力，并且对其有性生殖能力也有显著影响 。
３.对抗甲霜灵（Mtr）苎麻疫霉菌株及其亲本的野生型菌株进行生物学研究，结果表明，突变苎麻疫霉菌株对甲霜灵的抗性水平均有极大的提高。抗性菌株的菌株形态较野生敏感性菌株有较大的变化，对于不同温度和pH值的适应范围没有改变，但抗性菌株在相同的温度和pH值情况下的生长情况明显低于野生型菌株及其亲本菌株。研究结果还表明，抗性菌株的卵孢子的产生量也明显低于其亲本菌株，抗性菌株在液体培养基中的菌丝较其亲本菌株生长量有所减少。研究结果说明抗性菌株在对甲霜灵产生抗药性的同时，对环境的适应能力有所降低，其有性生殖能力也明显下降。
４. 应用聚丙烯酰胺同工酶分析技术对苎麻疫霉Mtr突变株的酯酶（EST）、过氧化物酶（POD）、过氧化氢酶（CAT）、多酚氧化酶（PPO）的同工酶电泳图谱等进行了系统的研究，并利用光密度法对其POD和PPO活性进行了测定。结果表明，Mtr菌株的PPO活性较其亲本菌株都有不同程度的增加，并且其同工酶电泳具有１条与抗药性相关联的特征性谱带；可溶性蛋白的电泳图谱也发现了至少１条具有与抗药性相关联的特征性谱带。苎麻疫霉的EST、CAT、PPO的同工酶电泳图谱显示，供试菌株的同工酶有一定差异，但没有找到与抗甲霜灵特性相关联的酶带。
上述研究结果为阐明苎麻疫霉对甲霜灵抗性的产生机理、苎麻疫霉对甲霜灵抗药
性监测、风险评价及治理提供了必要的理论基础和实验依据，对其他疫霉菌的抗药性研究也有一定的参考价值。
The effects of ultraviolet rays on mutagenizing metalaxyl resistance, the effects of on biochemical characteristics of Phytophthora boehmeriae, biochemical characteristics of metalaxyl-resistant (Mtr) mutants of P.boehmeriae and isoenzyme electrophoreses were studied systemically in this paper based on mutagenizing resistence of P. boehmeriae to metalaxyl.
1. The metalaxyl-resistance mutagenesis was carried with ultraviolet rays to mycelium discs, ultraviolet rays to zoospore, ultraviolet rays + metalaxyl to mycelium discs, metalaxyl to mycelium discs, metalaxyl to zoospores methods. The result showed that the mutants could be obtained by applying the two methods, i.e. metalaxyl to mycelium discs and metalaxyl plus ultraviolet ray to mycelium discs, but not by the other methods.
2. The effects of ultraviolet rays on the biochemical characteristics of P. boehmeriae were studied, and the results indicated that the zoospores of P. boehmeriae could be inactivated obviously by irradiating with 30W ultraviolet light 50cm away from the culture dishes tested and that 95% germination of zoospores could be restrained by 30W ultraviolet light treatment for 40sec.The mycelium growth from mycelium discs of P. boehmeriae were inhibited mostly by 30W ultraviolet light treatment for 20min. Afterwards, the inhibitive rates were decreased with the rise of the treatment time, which showed that the mycelium discs of P. boehmeriae had produced flexibility to ultraviolet rays after being irradiated by certain dosage of ultraviolet light. Between ultraviolet-patient isolates and parental isolates there was obvious difference in colony morphology but not in the sensitivity to metalaxyl. Moreover, the growth rates, oospore production, growth states of ultraviolet-patient isolates in different pH and temperature were obviously less than those of the wild parental isolates, suggesting that the adaptability of P. boehmeriae to the environment was depressed by the ultraviolet rays, and that the ability of sexual reproduction was affected notably by the ultraviolet rays.
3.Biological characteristics of Mtr mutants of P.boehmeriae were studied, and the results showed that resistance levels of Mtr mutants to metalaxyl were over 498 times of those of the parental isolates. There was greatly difference between Mtr isolates and their parental isolates in colony morphology, but no difference in the adaptation scope at different pH and temperature. The growth rates of Mtr isolates were obviously lower than those of the parental wild isolates at the same pH and temperature. It also indicated that the oospore production of Mtr isolates was obviously less than those of the parental isolates,
and that the mycelium production of Mtr isolates was a little reduced compared to those of the parental isolates in liquid culture. The results suggested that the adaptability of Mtr isolates to environment was reduced and so did the sexual reproduction of Mtr isolates while they produced metalaxyl-resistance.
4. The variations of isozyme spectra of esterase (EST), peroxidase (POD) polyphenol oxidase (PPO), catalase (CAT) and soluble protein of P. boehmeriae mutants were studied systemically by applying polyacrlamide gel electrophoresis (PAGE) and isoenzyme analytic technique, and the enzyme activity of POD and PPO of the Mtr isolates of P.boehmeriea by applying the spectrophotometry. The results showed that PPO activity of Mtr isolates had increased in some degree compared to parental isolates, and that one band of isozyme spectrum of POD was found related to the character of the metalaxyl-resistance in the mutants. And at least one band of isozyme spectora of souble protein was found in mutants related to the character of metalaxyl-resistance too. The results showed that isozyme spectra of EST, PPO and CAT were different in tested isolates, but that there were no bands observed relative to the metalaxyl-resistance.
The results mentioned above provided necessary theoretic foundation and experimental evidences for clarifying the mechanism of metalaxyl-r
引文
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