【作者】 朱杰华；

【导师】 刘大群；

【作者基本信息】 河北农业大学 ， 植物病理学， 2004， 博士

【摘要】 马铃薯晚疫病（Phytophthora infestans de Bary）是世界马铃薯产区的重要病害。病菌群体结构的组成与变异直接影响着该病害的发生与流行，当进入80代年晚疫病再度流行以后，人们首先把研究的焦点集中在病原菌的群体结构方面。本研究从表现型性状和DNA分子水平两方面研究了中国部分马铃薯主产区晚疫病菌群体的遗传多样性，旨在为抗晚疫病育种及制定合理的综合治理措施提供理论依据。 1．利用8个含有单显性抗性基因的鉴别寄主R1、R3、R4、R6、R7、R9、R10和R11测定了采自云南、四川、河北和黑龙江省等地的135个菌株的生理小种，发现上述地区的生理小种组成非常复杂，参加测试的8个抗性基因全部都能被克服，且85％以上的被测菌株均能克服R3、R10和R11。在135个菌株中存在28个生理小种，出现频率最高的为小种1.3.4.7.9.10.11，发生频率为28.1％，其次为小种1.3.4.6.7.9.10.11、3.4.7.9.10.11、3.4.7.9.11、1.3.6.7.9.10.11、1.3.4.7.9.10发生频率为6.7～8.9％。同时发现生理小种的分布没有明显的地区性。 2．在云南、四川、河北及黑龙江省马铃薯主产区均发现对甲霜灵具有抗性的菌株。在80个被测菌株中对甲霜灵具有高度抗性、中度抗性、高度敏感和敏感菌株的出现频率分别为62.5％、3.8％、28.7％和5.0％。不同地区、不同年份所采集的菌株对甲霜灵的抗性程度有明显差异，2001～2002年采自黑龙江省和河北省的菌株对甲霜灵均具有高度抗性或中度抗性，而1996～1998年采自四川、云南和河北的菌株均以敏感菌株为主，出现频率分别为80.0％、86.7％和100％。经测定高抗菌株和敏感菌株的EC₅₀值分别为1479.11μg／mL和0.07244μg／mL。 3．在黑龙江省发现晚疫病菌A2交配型。在45个被测菌株中只有1个A2交配型菌株，出现频率仅为2.2％。 4．在前人工作的基础上，通过对影响AFLP体系建立的关键因素进行探索，建立了适合构建晚疫病菌AFLP DNA指纹图谱的技术体系，为研究晚疫病菌的遗传、构建遗传图谱以及无毒基因的克隆奠定了坚实的基础。 5．利用25对引物扩增采自不同地区的50个晚疫病菌株，共扩增出1515条带，其中多态性带为855条，多态带率为56.4％。基于AFLPs多态性带的聚类结果表明晚疫病菌的DNA多态性与病菌的地理来源及病菌对甲霜灵的抗性有一定相关性，和生理小种及交配型无相关性。 6．发现了3个有性杂交效率极高的晚疫病菌株，这3个菌株能和供试的任何1个菌株（共210个）产生大量卵孢子，卵孢子的数量为2000～8000个／cm²；用41对E+2／M+2引物进行AFLP扩增，发现引物组合E19／M8（E+CG／M+CC）、E7／M8（E+AT／M+CC）和E5／M10（E+AC／M+GT）能将这3个菌株与其它菌株区别开。 7．利用8个鉴别寄主鉴定后发现亲本SW98-2及无性游动孢子后代的生理小种相同；利用4对引物扩增菌株SW98-2及其10个单游动孢子后代，发现亲本菌株和单游动孢子后代的AFLP DNA指纹几乎完全一致，说明晚疫病菌无性繁殖的遗传是稳定的。 8.部分Fl代菌系的生理小种和亲本不同，说明有性后代菌系的毒力已发生变化。获得了杂交组合DX98一2 X SW98一2的Fl代菌系48个，利用24对引物扩增2个亲本及48个Fl代菌系获得AFLP DNA指纹图谱，发现有些Fl代菌系的DNA指纹与亲本菌株不同，说明晚疫病菌有性杂交的后代会发生变异。事实证明有性重组可引起晚疫病菌群体结构发生变化。更多还原

【Abstract】 Research on the Population Genetic Structure of Potato Late Blight Pathogen (Phytophthora infestans) in ChinaDoctoral candidate: Jiehua Zhu Supervisor: Professor Daqun Liu Major: Plant PathologyPotato late blight is a most serious disease in the potato growing areas all over the world. The occurrence and epidemics of this disease are affected by the composition and variation of Phytophthora infestans de Bary population. When the potato late blight emerged again in 1980s, the research on the late blight was focused on the population structure of the pathogen. This paper focuses on the phenotypic and (DNA) genotypic diversity of P. infestans population in areas investigated in China. And the results derived from the paper are of significance to the resistant-breeding as well as to the management of late blight disease.1. One hundred and thirty-five isolates colleted from five provinces i.e. Hebei, Sichuan, Yunnan, Inner Mongolia, and Heilongjiang were tested with 8 potato clones of the international late blight differential hosts containing single dominant resistant gene Rl, R3, R4, R6, R7, R9, R10, and Rll. The result showed that the composition of the physiological races was complex in above five provinces. All tested resistant genes were compatible to the virulent genes of isolates of P. infestans assayed, and 85% of the tested isolates overcome the resistant genes R3, R5 and Rl 1, which indicated that the virulent genes were very common in the tested population of P. infestans. In the 135 isolates tested there were 28 different races. Race 1.3.4.7.9.10.11 showed the highest frequency of 28.1%, followed by 1.3.4.6.7.9.10.11, 3.4.7.9.10.11, 3.4.7.9.11, 1.3.6.7.9.10.11, 1.3.4.7.9.10 with frequencies of 6.7~8.9%. The racial distribution was no correlation with the regions where the isolates came from.2. The metalaxyl-resistant isolates of P. infestans were discovered in potato production areas in Hebei, Sichuan, Yunnan and Heilongjiang provinces. The frequencies of isolates with highly-resistant, resistant, highly-sensitive, and sensitive were 62.5%, 3.8%, 28.7%, and 5.0%, respectively. All of the isolates collected in 2001-2002 from Hebei and Heilongjiang provinces were either highly-resistant or resistant, but the most isolates collected in 1996-1998 from Sichuan, Yunnan and Hebei provinces were metalaxyl-sensitive with the frequencies of 80.0%, 86.7%, and 100%, respectively. The EC50 values of the highly-resistant and the highly-sensitive isolate were 1479.11ug/mL and 0.07244ug/mL, respectively.3. The A2 mating type isolate was discovered in Heilongjiang province. Only 1 from 45 isolates expresses as mating type. The ratio of A2 mating type isolate was only 2.2%.4. An efficient AFLP DNA fingerprinting protocol for P. infestans of potato was modified. The improvement of the protocol could facilitate the research on the genotypic diversity, constructing genetic maps and cloning avirulent genes of the pothogen.5. Fifty P. infestans isolates collected from different provinces were amplified with 25 primer combinations. One thousand five hundred and fifteen DNA bands (fingerprints) were observed. Eight hundreds and fifty-five out of the 1515 DNA bands were polymorphic ones. The rate of polymorphic bands was 56.4%. The result based on the cluster analyse indicated that DNA polymorphism was correlated with the regions where isolates from and metalaxyl-sesitivity, but not with the races and mating types.6. Three specific isolates of the pathogen were discovered in this study. Oospores were produced when these three isolates crossed with any other 210 isolates tested, the amounts of oospores ranged from 2000 to 8000/cm2. The AFLP DNA markers were detected when these 3 isolates were amplified with the primer combinations E19/M8(E+CG/M+CC), E7/M8 (E+AT/M+CC), and E5/M10(E+AC/M+GT).7. Ten single zoospore progenies derived from parent SW98-2 were identical to their parental physiological race through pathogenesis test with the resistant gene. T更多还原