【作者】 贾晓华；

【导师】 周明国；

【作者基本信息】 南京农业大学 ， 植物病理学， 2004， 硕士

【摘要】 嘧霉胺（pyrimethanil）为苯胺基嘧啶类杀菌剂，对由灰葡萄孢霉（Botrytis cinerea）引起的灰霉病有良好的防效。这类杀菌剂可抑制灰葡萄孢霉菌丝内甲硫氨酸的生物合成，并可抑制病菌胞外蛋白酶（包括水解酶）的分泌，减少病菌侵入位点寄主细胞的死亡。中国于20世纪90年代末开始推广使用嘧霉胺，由于其作用位点单一，加之灰葡萄孢霉具有繁殖速度快，遗传变异大和适合度高的特点，所以对嘧霉胺的抗药性风险较高，因此研究嘧霉胺对中国植物病原真菌的生物活性以及抗药性有重要的现实意义。 室内离体条件下采用菌落生长抑制法，选用马铃薯葡萄糖培养基（PDA）、麦芽浸膏培养基（MEA）和L-天冬酰氨合成培养基（L-asp）三种培养基测定油菜菌核病菌（Sclerotinia sclerotiorum）A2和A5菌株对嘧霉胺的敏感性，结果表明合成培养基L-asp测定嘧霉胺活性灵敏度高，10μg·mL^-1对A2和A5的抑制率分别为78.43％和87.50％；而相同药剂浓度下在PDA上测定的对A2和A5的抑制率分别为和56.54％和61.00％，在MEA培养基上测定的对A2和A5的抑制率则仅为38.25％和47.67％。 嘧霉胺对不同种真菌的毒力差异显著，具有专化性。在L-asp培养基上测试的12种真菌中，嘧霉胺对灰葡萄孢霉、番茄早疫病菌（Alternaria solani）和苹果轮斑病菌（A.solani）菌丝生长有强烈的抑制作用，EC₅₀值分别0.177、0.274和0.278μg·mL^-1。对芦笋茎枯病菌（Phoma asparagi）、辣椒炭疽病菌（Colletotrichum gloeosporioides）、油菜菌核病菌（Sclerotinia sclerotiorum）和水稻纹枯病菌（Rhizoctonia solani）的菌丝生长也有较高活性，EC₅₀值分别为1.444、1.623、2.017和4.106μg·mL^-1。对梨腐烂病菌（Cytospora carphosperma）、辣椒疫霉病菌（Phytophthora capsici）、白绢病菌（Sclerotium rolfsii）、稻瘟病菌（Magnaporthe grisea）和小麦赤霉病菌（Gibberella zeae）活性较差。 嘧霉胺处理灰霉分生孢子，在12h内表现对孢子萌发有强烈的抑制作用(处理12h时对灰葡萄孢霉h18菌株的EC₅₀为0.248μg·mL^-1)，但随着培养时间延长，对孢子萌芽抑制率下降，而表现为抑制芽管的发育。因此，嘧霉胺的作用方式是抑制灰葡萄孢霉菌丝生长，而不是抑制孢子萌发。 2000年从未用过喀霉胺等苯胺基嚓吮类杀菌剂的江苏盐城地区番茄上采集、单袍分离得到灰葡萄袍霉菌22个;2001年从未用过啥霉胺等苯胺基啥吮类杀苗剂的江苏南通市郊区、通州市等地区采集、分离得到油菜菌核病菌50个。采用菌落生长抑制法在L一asp培养基上测定这两种病原菌对峪霉胺的敏感性，测定结果表明，22个灰葡萄袍霉菌株的ECSO值变化范围在0.038一0.418拼g·mL一，之间，平均ECS。值为0.174。g’ mL一，，95%置信区间为0.125一0.222JJg·mL一‘。50个油菜菌核病菌ECS。值变化范围在0.303一5986 09·mL一，之间，平均ECS。值为1 .779 09·mL一‘，95%置信区间为l，738一2296似g·mL一l。 2002年从已使用过嚓霉胺药剂的江苏淮阴地区日光温室或塑料大棚中采集、分离获得55个番茄灰葡萄袍霉病菌，在L一asp培养基上采用菌丝生长抑制法测定各菌株对喀霉胺的敏感性，结果表明该地区灰葡萄袍霉菌对喀霉胺的敏感性ECs。值变化范围在0.108一22.94。g·mL一’，平均ECS。值为3.217。g·mL一‘;淮阴地区的灰葡萄袍霉对嚓霉胺已产生杭药性，抗药比例为4364%。抗性群体中83.33%的菌株表现中杭水平，1667%表现高杭水平。 室内紫外诱导获得高杭喀霉胺的菌株Huv16，ECS。值为24.95 09·mL一，，比亲本菌株高162倍。对田间敏感菌株、中杭菌株及紫外诱导的高抗菌株离体条件下的测定结果表明，三种类型的菌株在生长速率、产袍量和袍子萌芽率方面，菌株之间存在很大的差异，这些不同生物学性状与对嚓霉胺的抗性水平之间无明显相关性。田间中杭菌株和室内诱导的高杭菌株Huvl6均具有较好的遗传稳定性。在番茄叶片上测定的敏感性与在L一asp培养基上测得的敏感性之间存在相关性。交互杭性结果表明，苯胺基嚓咤类杀菌剂间存在正交互杭性，而与其他药剂如多菌灵（苯并咪吐类）、乙霉威（N-苯胺基甲酸醋类）和速克灵（二甲酞亚胺类）等不存在交互抗性。 将喀霉胺与多菌灵、异菌脉和福美双分别按1:l，3:1，6:1比例复配，离体条件下采用菌落生长抑制法测定各复配剂对灰葡萄袍霉菌株YS和Hyl6的毒力，结果表明，各复配剂对灰葡萄抱霉均表现相加作用。其中，啥霉胺和多菌灵3个比例的复配剂对Hyl6菌株（对多菌灵高杭，EC50习oo仁g·mL一，）具有优异的抑制作用。嚓霉胺与异菌豚3:1，6:1的复配剂比例对苹果轮斑病菌和番茄早疫病菌也表现相加作用。更多还原

【Abstract】 Pyrimethanil is a new fungicide within the group of anilinopyrimidines (APs) with high activity against grey mould mainly caused by Botrytis cinerea. The modes of action of APs include inhibiting methionine biosynthesis, secreting hydrolytic enzymes and reducing host cell death at the site of fungal infection. Pyrimethanil had been widely used sine the end of the 1990s in China. Due to pyrimethanil with a single site of action and B. cinerea with the capacity of reproduction and mutation with high fitness, the combination of grey mould and pyrimehtanil is high risk of resistance; therefore, it is essential to embark on biological activity and resistance of pytimethanil to pathogenic fungi in China.Sensitivities of isolates A2 and A5 of Sclerotinia sclerotiorum to pyrimethanil were determined on potato dextrose agar (PDA) medium, malt extract agar (MEA) medium and chemically defined L-asparagine agar (L-asp) medium with mycelial growth test. The results revealed that L-asp medium is more sensitive for testing activity of pyrimethanil inhibiting mycelium growth of isolates A2 and A5. Growth inhibition rates were 78.43% and 87.50% at 10 u g mL-1 respectively for the both isolates. While, inhibition of mycelium growth was measured with 56.54% and 61.00% on PDA, 38.25% and 47.67% on MEA, respectively for the isolates A2 and A5.Of total 12 plant pathogens species tested on L-asp medium, pyrimethanil strongly inhibited the mycelium growth of B. cinerea, Alternaria solani and A.solani with EC50 values of 0.177, 0.274 and 0.278 u g mL-1 respectively. It also had high inhibition effect on the mycelium growth of Phoma asparagi, Colletotrichum gloeosporioid.es, Sclerotinia sclerotiorum and Rhizoctonia solani with EC50 values of 1.444, 1.623, 2.017 4.106 u g mL-1 respectively. With no or little effect on Gibberella zeae, Magnaporthe grisea, Sclerotium rolfsii, Phytophthora capsici and Cytospora carphosperma.Pyrimethanil strongly inhibited the germination of conidia of B.cinera within 12h treatment (EC50 value was 0.248 u g mL-1 of isolate h18). However, inhibition effect of germination of conidia of B.cinera descended with the treatment time gone on, but the development of germ tube was strongly inhibited. Therefore, the mode of action ofpyrimethanil was inhibiting the mycelium growth of B.cinerea and not the germination of conidia of B.cinera.22 isolates of B.cinrea were isolated from tomatoes sampling from Yancheng, Jiangsu Province in 2000 and 50 isolates of S. sclerotiorum were collected from Nantong, Tongzhou etc in 2001 where pyrimethanil had never been used before. The sensitivities to pyrimethanil were determined by the method of mycelial growth test on L-asp agar medium. The results showed that the EC50 value of B.cinrea was ranged from 0.038 0.418 u g mL-1 with the mean EC50 value of 0.174 ug mL-1 and 95% confidence interval was ranged from 0.125-0.222 u g mL-1. The EC50 value of S. sclerotiorum was ranged from 0.303-5.986 ug mL-1 with the mean EC50 value of 1.779ug mL-1 and 95% confidence interval was ranged from 1.738-2.296 u g mL-1.55 isolates of B.cinrea were collected from greenhouses or plastic tunnels in Huaiyin, Jiangsu Province in 2002 where pyrimethanil had been used for about 3 years. The sensitivities of these isolates to pyrimethanil were determined by the method of mycelial growth test on L-asp agar medium. The results showed that the EC50 value were ranged from 0.108-22.94 u g mL- 1with the mean EC50 value of 3.217 u g mL-1. Some isolates of B.cinrea in Huaiyin showed resistance to pyrimethanil and the proportion of resistant isolates was 43.64%. Among this resistant sub-population, 83.33% was moderate resistance and 16.67% was high resistantce.High resistance mutant Huvl6 to pyrimethanil was obtained by ultraviolet irradiation. EC50 of pyrimethanil to Huvl6 was 24.95 u g mL-1,162 times as that of its original isolate H16. Significant differences in growth rate, sporulation and conidium germination rate were found among isolates of B.cinerea. However, no correlation co更多还原