【作者】 胡春锦；

【导师】 李杨瑞；

【作者基本信息】 广西大学 ， 作物栽培学与耕作学， 2007， 博士

【摘要】 水稻纹枯病是一种世界性病害，目前该病已成为限制水稻高产和稳产的主要障碍之一。病原菌的遗传多样性和变异性使得该病害的防治极为困难。为了解近年来广西水稻纹枯病病原菌的变化情况，本文对最近几年来从广西不同稻区收集到的160个水稻纹枯病菌分离物进行了系统的研究，明确了广西水稻纹枯病菌的菌系分类，分析了不同菌系间的遗传差异，建立了一些新菌系的分子检测技术手段，并且从营养体—蛋白质—核酸等3个不同层次分析“稻纹枯菌-寄主”互作以后病原菌的遗传分化，初步分析了病原菌的遗传变异与寄主的关系，为今后有针对性地抗病育种和品种合理布局提供了一定的理论依据。主要研究结果如下：1、采用了多种鉴定方法对160个水稻纹枯病的病原菌分离物进行了鉴定和分类，结果表明，与广西水稻纹枯病有关的病原菌主要有立枯丝核菌的AG1-IA(Rhizoctonia solani AG1-IA)和属于双核丝核菌的AG-Bb融合群的水稻丝核菌(Rhizoctonia oryzae-sativae)、水稻枯斑丝核菌(Rhizoctoniaoryzae)、双核丝核菌的AG-A融合群(Rhizoctonia sp AG-A)、嗜水小核菌(Sclerotium hydrophilue)等5个菌系，它们的出现频率依次为90.62％、2.50％；、1.25％；、1.25％和4.38％，R. solani AG1-IA是引起广西水稻纹枯病的主要的优势菌系。其中Rhizoctonia oryzae、Rhizoctonia sp AG-A、Sclerotium hydrophilum等3个菌系为广西的首次报道。除了Rhizoctonia spAG-A对水稻无致病力以外，其余4个菌系均能侵染水稻并引起相似的纹枯病症状，其中R. solani AG1-IA的致病力最强，但菌株间存在明显的致病力分化，其它几个菌系的致病力均比较弱，致病力强弱顺序为R. solani AG1-IA＞R. oryzae＞R. oryzae-sativae＞S. hydrophilum。此外，通过几种病原菌鉴定方法的比较，结果表明rDNA的PCR-RFLP是分析丝核菌不同菌系极为有用的技术手段，其结果与病原菌的形态学分类、菌丝融合测定、rDNA序列分析等的研究结果可以相互验证。2、从病原菌的生物学特性和rep-PCR指纹图谱等多方面比较了几种不同丝核菌的遗传差异，结果表明不同菌系在其菌落形态、生长适温、最适pH值以及对不同培养基营养元素的利用等生物学特性上均存在一定的差异；同一菌系内菌株间的rep-PCR指纹基本相似，而不同菌系间的差异非常明显，每种菌系均有多条各自的特征性谱带，可作为鉴别丝核菌不同菌系的特异性标记。3、分析和比较了5个不同菌系的ITS1-5.8S-ITS2间的核苷酸序列，并构建了它们之间的系统发育树。根据不同菌系rDNA ITS区序列的差异，设计了一对可专化性检测水稻枯斑丝核菌(R. oryzae)的特异性引物WP-1R／WP-2F，同时还设计了一条嗜水小核菌(S. hydrophilum)的专化性引物SP-1F，该引物与通用引物ITS1配对，可对S. hydrophilum进行特异性扩增。进一步的研究结果还表明引物WP-1R／WP-2F与SP-1F／ITS1能够分别准确地检测到罹病植株中的R. oryzae和S. hydrophilum,可以分别地用于对这两种病原菌引起的病害进行早期、快速诊断。4、R. solani AG1-IA菌系内的存在明显的致病力分化和一定的遗传变异潜能。致病力测定结果表明145个R. solani AG1-IA菌株对供试水稻品种的平均病情指数为37.3～78.3，大多数表现强致病性，少数菌株致病力比较弱。R. solani AG1-IA全部供试菌的28S扩增结果分别得到了3种不同的产物类型，扩增片段类型与菌株的地理来源有一定的相关性，初步推测这种相关性可能是因为一些菌株细胞中存在不同核型的细胞核所导致。该结果暗示了R. solani AG1-IA存在许多遗传变异的潜能，因为由于这种异核体引起的准性生殖会使菌株的遗传极不稳定，并最终引起菌株的遗传分化。5、根据rDNA ITS区以及28S的序列分析结果，构建了嗜水小核菌(S.hydrophilum)的系统发育树，结果表明该菌在rDNA的系统发育上与许多丝核菌属(Rhizoctonia)真菌有着非常高的同源性。结合该菌在形态上与Rhizoctonia比较相似的特点，初步推测该菌在分类上应属于丝核菌属(Rhizoctonia)真菌的一个种，而不是菌核属(Sclerotium)真菌。6、初步分析了“稻纹枯菌-寄主”互作后，再分离菌株在表型和致病性、生理生化等基础物质以及DNA水平上的遗传分化。结果表明，与起始菌株相比，再分离菌株在蛋白酶活性等基础物质上产生了一定的差异，少数再分离菌株还与起始菌株及其它菌株产生了菌体不亲合现象，即产生了自身不亲和突变体。进一步的AFLP分析结果表明，所有再分离菌均较起始菌株在DNA水平上发生了一定程度的改变，揭示了寄主的定向选择作用可能是稻纹枯菌发生遗传分化的主要原因，稻纹枯菌与不同水稻品种互作之后可产生寄主专化型不亲和VCGs。该研究结果可为采用多种抗性品种并进行合理时空布局来控制水稻纹枯病为害提供一定的理论依据。更多还原

【Abstract】 Rice sheath blight is a destructive disease of rice worldwide, and the disease hasbecome one of the major constraints for rice productivity and yield stability of rice inrecent years. It is very difficult to control the disease because of the diversity andvariability of the pathogen. In order to clarify the genetic mutations and diversity of thepathogens causing rice sheath blight in Guangxi, 160 pathogenic isolates derived fromtypical leaf lesions were analyzed for classification and genetic differentiation, and a PCRassay for detection of some of the strains has been established in the dissertation. To learnthe relationship between the directional selection of rice and the evolution of the pathogen,the genetic variations in the pathogen strains after host-pathogen interaction were studiedin the levels of vegetative compatibility, protein and DNA, which provided basic evidencesfor developing new strategies against this disease through reasonable cultivating measures.The main results were summarized as follows.1. Based on several classification and identification methods of Rhizoctonia, 160pathogenic isolates were divided into 5 groups, which were Rhizoctonia solani AG1-IA,with a percentage of 90.62%; Rhizoctonia oryzae-sativae, 2.5%; Rhizoctonia oryzae, 1.25%;Rhizoctonia sp AG-A, 1.25%; and Sclerotium hydrophilum, 4.38%, respectively. Among the5 groups, R. oryzae and S. hydrophilum were the first records in Guangxi. R. solani AG1-IAwas the preponderant pathogenic group and showed the strongest virulence, andRhizoctonia sp AG-A showed no virulence on rice, while others did infect rice but showedlower virulence. The results suggested that PCR-RFLP of rDNA was one of the mostpowerful tools to study the genetic variation of these Rhizoctonia fungi throughcomparison of 3 identification methods based on morphological features, hyphal fusions and sequencing analysis of rDNA, respectively.2. Genetic differences among the strains were studied comparatively with theirbiological characteristics and rep-PCR fingerprinting. The results indicated thatdifferences were obvious among the strains in their biological characteristics, such asoptimal initial pH, optimum temperature for hyphal growth, and so on. The rep-PCRfingerprinting technique utilized in this study was effective for distinguishing strains, andeach strains showed its own rep-PCR fingerprinting bands, which would be used as thespecific DNA fingerprints for distinguishing these strains.3. A UPGMA phylogenetic tree based on the rDNA ITS sequences of the strains usedanti relative species has been developed. Based on the conserved sequence determined bythe alignment rDNA sequence of all the strains used in the study, one pair of specificprimers (WP-1R/WP-2F) designed from the sequence of ribosomal DNA-ITS forRhizoctonia oryzae, and the other one (SP-1F/ITS1) for Sclerotium hydrophilum. Thespecific bands from Rhizoctonia oryzae and Sclerotium hydrophilum could be amplifiedwith the primer pairs WP-1R/WP-2F and SP-1F/ITS1, respectively, but not from otherfungal species associated with rice sheath blight, and the primer pairs showed the sameresults in detecting Rhizoctonia oryzae and Sclerotium hydrophilum from the infected leafof rice, which indicated that they could be used to develop a rapid PCR-based diagnosistechnique for this disease at early stage, and could be applied for quarantine and forecast.4. All the 145 R. solani AGI-IA isolates could induce typical lesions in the four ricecultivars screened, and the pathogenicity parameters showed high variability of virulenceamong the isolates with the disease index from 37.3 to 78.3, and most of the isolates showedhigh virulence, while few of them show lower virulence. The PCR products of 28S rDNAshowed 3 patterns among R. solani AG1-IA isolates, and the patterns showed certaincorrelation with geographical origins and ecological habitats of the isolates, which wasproposed preliminarily that there were different karyotypes among R. solani AG1-IAisolates. These results suggested a high genetic variation potential among R. solani AG1-IAisolates.5. A UPGMA phylogenetic tree of Sclerotium hydrophilum was established based onthe sequence analysis of ITS and 28S rDNA. The results indicated that Sclerotium hydrophilum showed high homology with many Rhizoctonia isolates in the phylogeneticrDNA. Based on these results, it is suggested that Sclerotium hydrophilum should belong toRhizoctonia but not Sclerotium in its taxonomy.6. The genetic variation in R. solani AG1-IA strains after host-pathogen interactionwas studied in terms of vegetative compatibility, protein and DNA. Compared with theparental isolate, the adapted isolates showed differences in certain extent at physiologicaland biochemical levels, and some of the adapted isolates even showed incompatibility withthe parental isolate in the vegetative compatibility test, suggesting that some geneticdifferences had happened among these isolates, and this was further demonstrated byAFLP analysis. These results indicated that the directional selection of rice was theimportant cause to the evolution of the pathogen, and suggested that maybe host-typeincompatible VCGs among the isolates exist under field conditions. It should be useful forcontrolling the disease through reasonable cultivating measures.更多还原