【作者】 赵辉；

【导师】 高增贵；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2009， 博士

【摘要】 本论文从寄主-病原菌互作角度,采用性状培养、Ht单基因鉴别寄主鉴定技术、同工酶电泳技术、扩增片段长度多态性分析技术(AFLP)和血清学技术,系统研究了我国玉米大斑病菌(Exerohilum turcicum)生理分化和遗传多态性。本研究从我国玉米大斑病菌生理小种组成和种群变化等方面,全面分析了近年来我国玉米大斑病危害加重的原因。采用两套Ht单基因(Ht1、Ht2、Ht3和HtN)鉴别寄主,对2005年和2006年采自黑龙江、吉林、辽宁、河北、山东、陕西、山西、河南、四川和北京-天津-唐山地区共10个省区28个地区的204株玉米大斑病菌菌株进行了生理小种鉴定,共鉴定出0、1、2、3、12、13、23、123和23N等9个生理小种,其中0号和1号生理小种为优势小种,分别占供试菌株的62.25%和19.12%。毒力频率分析结果表明,玉米大斑病菌小种组成对Ht1抗性基因的毒力频率最高,为33.33%;对HtN抗性基因的毒力频率最低,为1.47%。Ht1和Ht2抗性基因在我国玉米产区均有不同程度的抗性“丧失”。玉米大斑病菌不断有新小种出现,生理分化明显。生理小种组成较复杂的省份为东北三省、河北和四川等地区。本研究从病原物的环境适应性、同工酶和DNA多态性等方面,系统探讨了导致病原菌生理小种变异的生理和分子生物学机制。利用继代培养、连续接种和rDNA ITS序列比较,探明了在稳定的寄主选择压力条件下,玉米大斑病菌生物学特征、致病性和rDNA-ITS序列是相对稳定的,rDNA-ITS序列同源性达96.9%～100%。在培养性状方面,玉米大斑病菌不同生理小种间菌落形态、菌丝生长速度、产孢量、孢子萌发和菌落产色素能力均存在明显的多态性。在同工酶方面,玉米大斑病菌不同生理小种在6种同工酶水平上均表现出不同程度的多态性,其中酯酶(EST)、超氧化物歧化酶(SOD)的多态性尤为明显。这种同工酶水平上的多态性,反映了病原菌遗传表达的多态性。在DNA遗传多态性方面,采用扩增片断长度多态性分析技术(AFLP)分析了玉米大斑病菌的遗传变异,供试菌株间遗传相似系数在0.6～1.0之间,表现出明显的遗传多态性。聚类结果表明,该病菌的遗传多态性与其生理分化存在明显相关性。但不同地域的同一生理小种的菌株相似系数达到了1.00,说明其生理分化与地域无关。在玉米大斑病菌生理小种辅助鉴定方法的研究方面,本研究首次利用AFLP特异条带序列设计,筛选出玉米大斑病菌生理小种的特异引物,引物组合可以为0、13号等生理小种鉴定提供准确的分子生物学辅助鉴定。在病原菌遗传多态性的基础上,首次利用免疫学方法制备了玉米大斑病菌1、2、12、13号生理小种的抗血清,在提高抗血清稀释度的情况下,经效价、交叉反应、ELISA和Western blot试验,发现1、12号生理小种菌株的抗血清表现出小种特异性。本研究完成了辽宁省主栽玉米品种对玉米大斑病菌生理小种的抗性鉴定。各品种对不同生理小种的抗病性存在明显差异,其中高抗品种占17.86%,中抗至抗的品种占53.57%。抗病性鉴定结果与品种系谱相关性分析表明,品种对不同生理小种的抗性与品种血缘关系相关。另外,气候条件对抗病性鉴定结果亦有影响。综合上述研究结果:玉米大斑病菌存在明显的生理分化。新小种的出现及其种群数量的上升和某些垂直抗病品种抗性的“丧失”是玉米大斑病逐年加重的重要原因。寄主更换的选择压力变化和病原菌种群内的遗传多态性是玉米大斑病菌发生变异的重要原因。AFLP和免疫学技术可以作为玉米大斑病菌生理小种的辅助鉴定技术。辽宁省主栽品种对玉米大斑病菌主要优势小种表现出较好的抗性。更多还原

【Abstract】 The physiological differentiation and the genetic polymorphism of Exerohilum turcicum were studied systematically in terms of host-pathogen interaction with several techniques or methods,involving observation of pathogen culture traits,identification of pathotypes on differential hosts,electrophoresis of isozyme,analysis of amplified fragments length polymorphism(AFLP) and immunoassay techniques.In order to prove the factors of the increasing epidemic of northern corn leaf blight in China in some years,the composition and population variation of physiological races were investigated.204 isolates of the pathogen were isolated in 28 areas of Heilongjiang,Jilin,Liaoning,Hebei,Henan,Shandong,Shanxi, Shaanxi,Sichuan provinces and Beijing and Tianjin and Tangshan regions in 2005 and 2006 for identification of physiological races on differential hosts of corn inbred lines containing single resistance genes such as Ht1,Ht2,Ht3 and HtN.At last nine races were identified,including 0,1,2,3,12,13,23,123 and 23N.Among these physiological race 0 and 1 were predominant with occurrence frequency of 62.25% and 19.12%.The results about the analyzed based on virulence frequency showed that the virulence frequency for the race population against gene Ht1 and HtN were 33.33%and 1.47%.The resistance genes of Ht1 and Ht2 have to some extent lost their function against the pathogen in some areas of China.It was found that the physiological differentiation of the pathogen was complicated,and new races were frequently found.The variations of the physiological races of Exerohilum turcicum in Henan,Sichuan provinces and Northeastern China were higher than in other corn-planting regions.To explore the mechanism of the physiological and molecular biology about the physiological races variation of Exerohilum turcicum,the environmental adaptability of physiological races,the isozyme and DNA polymorphism of the pathogen were studied.By investigating the long-term sub-cultures of isolates, continuous inoculation,and ITS sequences analysis of physiological races of Exerohilum turcicum,the biological characteristics,the pathogenicity and rDNA-ITS sequences of Exerohilum turcicum were relatively stable.The homology of rDNA-ITS sequences of physiological races of Exerohilum turcicum was 96.9%～100%under the stable conditions of host selection pressure.The polymorphism of the biological characteristics and the isozyme of Exerohilum turcicum were studied.The results indicated that there were the significant difference among the different physiological races in the colony morphometries,the growth rate of mycelium,sporulation,spore germination and pigment-productivity of colony.The polymorphism of different physiological races could be well exhibited by the diversified changes of electrophoresis profile of six kinds isozyme including esterase(EST), peroxidase(PO),polyphenoloxidase(PPO),superoxide dismutase(SOD),catalase(CAT),and malate dehydrogenase(MDH).It was concluded that EST and SOD present the obvious polymorphism among the tested isolates.AFLP was conducted to analyze the genetic polymorphism of Exerohilum turcicum.The genetic similarity among the isolates was from 0.61 to 1.0.It was concluded that AFLP analysis could present the genetic polymorphism of Exerohilum turcicum.The clustering results of AFLP had correlation with physiological differentiation.But the genetic similarity of the same physiological races collected from different geographical regions reached 1.0.The physiological differentiation had no obvious correlation with their geological location.Assistant methods for physiological races classification were studied.The specific primers of physiological race of Exerohilum turcicum were screened by using AFLP bands specific.The primers being able to be used as an assistant method for No.0 and No.13 physiological race identification.Based on the genetic polymorphism of Exserohilurn turcicum,antiserum of 1,2,12 and 13 physiological race were prepared by immunological methods.The results showed that there was significant differences in antiserum titer among different physiological races through the titer,cross-reactivity,ELISA test and Western blot method.It had race-specific about the antiserum of the NO.1 and NO.12.Resistance identification of main cultivars in Liaoning province to physiological races of Exserohilum turcicum was completed.There was obvious difference in the physiological races to main cultivars.The highly resistant cultivars was 17.86%,and moderate resistant cultivars was 53.57%.Resistance identification to northern corn leaf blight and pedigree analysis showed that the resistance of cultivars to the different physiological races had correlation with the blood relations.In addition,climatic conditions impacted on the results of the resistance identification.The main conclusions about this paper were as follows:(1) the pathogenicity differentiation and biological polymorphism really existed in the population of the pathogen.(2)The increasing epidemic of northern corn leaf blight in some areas of China or in some years were ascribed to the continuing occurrence of new races of the pathogen,the increasing virulence frequencies of the minor races,and the resistance-losing of the cultivars.(3)The study could extrapolate that one of the important reason for genetic variation of Exerohilum turcicum was the variations of host selection pressure.4)AFLP analysis and immunological methods provided a new method for assistant identification of the physiological race of Exerohilum turcicum and improved the accuracy.(5)In recent years,common corn cultivars in Liaoning province had a strong resistnce agains the predominant races of Exerohilum turcicum.更多还原