【作者】 李红霞；

【导师】 周明国；

【作者基本信息】 南京农业大学 ， 植物病理学， 2003， 博士

【摘要】 苯并咪唑类杀菌剂作为一类高效、广谱内吸性杀菌剂在生产上应用，解决了保护性杀菌剂的环境毒性问题，同时也降低了对用药技术和用药时间的要求，提高了病害的防效。由于这类药剂的高度专化性，作用位点单一，加上施用频率高，使用2～3年后许多植物病原真菌很快会出现抗药性问题。苯并咪唑类杀菌剂是通过与病原菌β-微管蛋白结合，抑制微管的功能，阻止细胞的有丝分裂，抑制病原菌生长。病原菌抗药性的产生是因为细胞中β-微管蛋白由于控制该蛋白的基因发生突变，使蛋白三维构象改变，从而失去了与药剂分子的亲和性。近年来大量研究证明，除引起马铃薯储藏期干腐病的Gibberella pulicaris外，β-微管蛋白基因的198或200位氨基酸密码子的点突变是导致大多数病原菌产生抗药性的主要原因。 核盘菌（Sclerotinia sclerotiorum）、禾谷镰孢霉（Fusarium graminearum）、炭疽菌（Colletotrichum gloeosporiodies）和葡萄孢霉（Botrytis cinerea）引起的多种作物炭疽病和灰霉病是中国常见的重要农作物和蔬菜病害。长期以来对这4种病害的防治主要采用苯并咪唑类或与不同作用机制药剂复配的杀菌剂。1987、1992和1996年在田间分别检测到B．cinerea、F．graminearum和S．sclerotiorum对多菌灵的抗药性菌株。 通过室内菌落直径法测定这四种植物病原真菌对苯并咪唑类杀菌剂和乙霉威的敏感性。结果表明，小麦上F．graminearum的敏感菌株（MBC^S）对多菌灵（MBC）的EC₅₀值小于1μg／mL，无论是田间或诱导抗性菌株（MBC^R）的EC₅₀值均在10μg／mL左右，MBC^R菌株与乙霉威（NPC）间不存在负交互抗药性。2002年采集、分离获得江苏省通州市4乡镇油菜上的223个S．sclerotiorum菌株，对MBC和NPC的敏感性测定得到2种表型，其中143个菌株表现对多菌灵敏感而对乙霉威高抗(MBC^SNPC^HR)，其余80个菌株表现对多菌灵高抗而对乙霉威敏感(MBC^HRNPC^S)，且MBC^HRNPC^S菌株对7～28℃温度范围不敏感。2002年采集、分离获得江苏省江阴市番茄上的14个B．cinerea菌株，对MBC和NPC的敏感性测定得到2种表型，其中2个菌株表现MBC^SNPC^HR，其余12个菌株表现MBC^HRNPC^S。2001年从江苏省盐城地区采集分离到辣椒上的17个C．摘要gloeosrorio苗e:菌株，其中l个、MBeH勺护es菌株、l个MBeL物PeR菌株和15个MBesNPeHR菌株. 本研究通过根据其他相关真菌p一微管蛋白的保守序列合成的3对兼并性引物扩增获得F名7aminearum、s.:cleroriorum、丑.。inerea与抗多菌灵相关的卜微管蛋白全基因，通过其中的1对兼并性引物Bl和B3获得了c gloeosPoriodiesp-微管蛋白基因的部分序列。其中F graminearum的口一微管蛋白基因全长163lbp，包含3个内含子，编码447个氨基酸，与其他病原线状真菌除内元数目不同外，具有高度同源性一氮基酸同源性达95.120/to99.3o%.5:clerotiorum的卜微管蛋白基因全长1 685bp，包含4个内元，相应的编码447个氨基酸，氛基酸序列同源性达95.78协97.66%.B.einerea的p一微管蛋白基因全长1765bp，包含6个内元，相应的编码447个氛基酸，与s.:clerotio^和F graminearump一微管蛋白基因的氨基酸序列同源性分别为97.76%和%20%.c sloeosPoriodies已撒管蛋白基因的部分序列长586bp，包含1个内元（55bp），其位五与模式菌粗糙麦抱霉（Ne urosPora crassa）的内元6一致.与Hsl（MBCs黑色炭疽）相比，炭疽菌中的HGsZ（MBCs红色炭疽）、YCS（MBCLR）、YC41（MBCHR）:禾谷镰抱菌zF2054（MBeR）:核盘菌Tz25（MBeHR）:灰葡萄抱eoyool（MBeHR）的同源性分别为100%、97.18%、98.31%、98.87%、96.61%、98.87%. F graminearum的MBc”和MBc“菌株核普酸序列分析表明，MBC“菌株未发生任何位点的突变，说明F graminearum对多菌灵的抗药性机制并非像其他丝状真菌一样由口撒管蛋白198位氛基酸突变所致.5:clerotiorum、B.。inerea和C sloeosPortodies的p一微管蛋白基因编码的 198位氛基酸残基的GAG谷孰酸（Glu）突变为GCG丙氛酸（Ala）是导致上述病原菌产生对多菌灵高度杭药性的主要原因;突变位点和突变类型与其他杭多菌灵真菌一致，且与乙霉威之间存在明显负交互杭性.c sloeosPor勿die:的MBc咖Pc“菌株198位氛基酸未发生突变，尽管对四种病原真菌7个菌株的p撒管蛋白进行比较发现，除198位氛基酸外，其他位点也有所变化，那是属于菌株间的差异，但198位氮基酸的突变则是导致田间病原真菌产生高度抗药性的直接原因. 为了快速、准确检测和监测s.sclero如^的田间杭药频率，根据MBCHR菌株的点突变设计了2个快速检测方法:第一种方法别民据MBcHR菌株197和198位密码子（GACGAG一GACGCG）形成八al酶切位点（3’CGCGS，），将BI瓜3的扩增产物s74bp片段酶切成193bp和6slbP片段，而MBeS菌株的PeR产物不被酶切;第二种方法用198位突变密码子作为3’末端碱基设计2对等位基因特异性寡核普酸引物（ASO）用于“nested" PCR或直接从菌丝基因组DNA扩增.引物对s一T侧55一1只能从MBcHR菌株中扩增出373饰的条带，而引物对s一Ts/ss一1只能从MBcs菌株中扩增出373bp的条带.通过PcR扩增和T更多还原

【Abstract】 Benzimidazole fungicides, as a group of systemic fungicides with broad sprectrum of fungicidal activity, were widely used to control diseases caused by plant pathogenic fungi. The benzimidazoles envoid the harmless in the enviroment with protective fungicides, and reduce the requirment of application technique and time, further increase the control effect.Because benzimidazoles are characterized by high specificy, single action position, and high application frequency, many plant pathogenic fungi occurred resistance to these fungicides after 2-3 years’ application. Benzimidazole fungicides bind to β -tubulin, inhibiting microtubulin function or disassembling microtubulin, further prevent cell’s mitosis of fungi for growth. Mutations of pathogen’s β-tubulin gene make three-dimensional configuration of P -tubulin protein change and lose affinity with fungicides, leading to resistance to carbendazim.Many benzimidazole-resistant mutations in a variety of fungi have been mapped to the structural P -tubulin gene and the mutant genes were cloned and sequenced. Although single base-pair point mutations leading to the substitution of the amino acid by another occurred at least 10 different sites within the P -tubulin gene, these mutations are confined to amino acid codons 198 and 200 in resistant field strains of plant pathogens. Except for the pathogen Gibberella pulicaris causing potato dry rot, in recent years resistance to benzimidazoles in fungal pathogens has been attributed to single amino acid changes in the P -tubulin subunit. The majority of these changes in field benzimidazoles-resistant isolates were located in amino acids 198 or/and 200.Wheat head blight(caused by Fusarium graminearuni), Rape sclerotiniose (caused by Sclerotinia sclerotiorum), Tomato Grey(caused by Botrytis cinerea) and Pepper anthracnose(caused by Colletotrichum gloeosporiodies) are important crop and invegetable diseases in China. These diseases have been controlled by benzimidazole fungicides or its mixture with other fungicides with different mechnisms for 30 years.Carbendazim-resistant B. cinerea, F. graminearum and S.sclerotiorum isolates were detected out in the field populations in 1987,1992 and 1996, respectively.Sensitivity of the four plant pathogenic fungi to carbendazim(MBC) and diethofencarb(NPC) was tested with mycelial growth test. The result showed EC50 of carbendazim-sensitive isolates(MBCs) from F.graminearum was less than 1μg/mL. EC50 of field and induced carbendazim-resistant isolates(MBCR) was about 10μg/mL. There didn’t exist negative cross-resistance between carbendazim and diethofencarb in F. graminearum .223 S.sclerotiorum isolates were isolated from Tongzhou of Jiangsu Province in 2002. Two phenotypes were identified when these isolates were tested for sensitivity to carbendazim and diethofencarb. 143 of these isolates showed phenotype of carbendazim-sensitivity and diethofencarb-high-resistance (MBCsNPCHR and other 80 isolates showed carbendazim-high-resistance and diethofencarb-sensitivity (MBCHRNPCS). The MBCHRNPCS isolates were not sensitive to temperature Variation between 7℃ and 28℃. 14 isolates of Botrytis cinerea were isolated from Jiangyin of Jiangsu Province in 2002. Two phenotypes were identified when the isolates were tested for sensitivity to carbendazim and diethofencarb. 2 of the isolates showed phenotype of MBCSNPCHRand other 12 isolates showed MBCHRNPCS. 17 C.gloeosporiodies isolates were isolated from Yancheng of Jiangsu Province in 2001. Three phenotypes were identified when tested for sensitivity to carbendazim anddiethofencarb. One of these isolates showed MBC NPC , another showed carbendazim-low-resistance and diethofencarb-resistance (MBCLRNPCR) and other 80 isolates showed MBCSNPCHR.In this study, the whole β-tubulin genes were amplified from F.graminearum, S.sclerotiorum and B.cinerea using three pairs of chemic primers synthesized to conserved sequences of the P -tubulin gene derived from related fungi. Partical sequence of P -tubulin gene from C. gloeosporiod更多还原