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。
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.
引文
1 赖传雅.农业植物病理学(华南本)[M].北京:科学出版社,2004.
2 任春梅,高必达,何迎春.水稻抗纹枯病的研究进展[J].植物保护,2001,27(4):32-36
3 彭绍裘,曾昭瑞,张志光.水稻纹枯病及其防治[M].上海科学技术出版社,1986.
4 廖皓年,肖陵生,王华生.水稻纹枯病发生历史及演变原因简析[J].广西植保,1997,(3):35-38
5 彭绍裘,范坤成.水稻纹枯病研究论文选集[C].湖南省农业科学院植物保护研究所,1997.
6 Parmeter JR, Whitney HS. Affinities of Rhizoctonia species that resemble mycelium of Thanatephorus cucumeris[J]. Phytopathol, 1967, 57: 218-223.
7 Parmeter JR, sherwood RT, Platt WD. Anastomosis grouping among isolates of Thanatephorus cucumeris[J]. Phytopathol, 1969, 59:1270-1277.
8 Ogoshi A. Studies on the taxonomy of the genus Rhizoctonia. Annu Phytopathol Society of Japan[J], 1984, 50: 307-309.
9 Ogoshi, A.. Ecology and pathogenicity of anastomosis and intraspecific groups ofRhizoctonia solani[J] Kuhn. Annu. Rev. Phytopathol. 1987, 25: 125-143.
10 于金凤.中国冬小麦主产区及云南丝核菌属真菌的遗传多样性研究[D].山东农业大学博士学位论文,2003.
11 Oniki M, Ogoshi A, Araki T, et al. The perfect state of Rhizoctona oryzae and R. zeae and the anastomosis groups of Waitea circinata[J]. Transactions of the Mycological Society of Japan. 1985, 26: 189-198.
12 Martin RR, James D, Levesque CA. Impacts of molecular diagnostic technologies on plant disease management[J]. Annu Rev of Phytopathol, 2000, 38: 207-239
13 李华荣,颜思齐.四川省东南部水稻纹枯病菌系研究[J].真菌学报,1990:9(1):41-49.
14 杨根华,董文汉,李梅,等.云南省水稻纹枯病菌系研究[J].菌物系统,2002,21(2):274-279
15 Chien CC, Tong CC. Physiologic race of Pellicularia sasakii in Taiwan [J]. Agri. Res. Taiwan, 1963, 12(2): 1-6.
16 过崇俭,陈志谊,王法明.水稻纹枯病致病力分化及品种抗性鉴定技术的研究[J].中国农业科学,1985,18(5):50-57.
17 邓振山,张宝成,孙志宏,等.立枯丝核菌营养菌丝多型性观察[J].微生物学杂志,2005,25(6):56-58.
18 Suzuki H. Origin of variation in Pyricularia oryzae [A]. In: Ou S H, ed. The Rice Blast Disease [C]. Baltilmore, Maryland: The Johns Hopkins Press, 1963. 111-129.
19 Rosewich UL, Pettway RE, McDonald BA, et al. High levels ofgene flow and heterozygote excess characterize Rhizoctonia solani AG-1 IA (Thanatephorus (?)umeris ) from Texas [J]. Fungal Genet and Biol, 1999,28 : 148-159.
20 Flor HH. Host-parasite interaction in flax rust-its genetics and other implications [J]. Phytopathol, 1956, 45: 680-685.
21 易润华,染承邺,朱西儒,等.不同品种水稻纹枯病遗传多样性及致病力分化[J].华南农业大学学报(自然科学版),2002,23(2):22~-26.
22 袁筱萍,魏兴华,余汉勇,等.不同品种及有关外因对水稻纹枯病抗性的影响[J].作物学报,2004,30(8):739-744.
23 向珣朝,王世全,何立斌,等.一个高抗水稻纹枯病突变体的发现及其遗传特性的初步分析[J].作物学报,2005,31(9):1236-1248.
24 胡春锦,黄思良,岑贞陆,等.深水稻品种对稻纹枯病抗性鉴定初报[J].植物保护,2003,29(4):19-22.
25 唐定中,王金陵,李维明.水稻纹枯病体细胞突变体的离体筛选[J].福建农业大学学报,1997,26(1):8-12.
26 詹庆才,车克鹏,王子平,等.茭白DNA导入水稻选育出抗纹枯病的新品种[J].中国学术期刊文摘,2001,(7):665.
27 毛碧增,李德葆,李群,等.转化双价防卫基因获得抗纹枯病水稻[J].植物生理与分子生物学报,2000,29(4):322-326.
28 冯道荣,许新萍,范钦,等.获得抗稻瘟病和纹枯病的转多基因水稻[J].作物学报,2001,27(3):293-300.
29 Mckenzie KS, Rush MC, Groth DE. Registration of two disease-resistant germplasm lines of rice [J]. Crop Sci, 1986, 26:839-840.
30 陈宗祥,邹军煌,徐敬友,等.对水稻纹枯病抗源的初步研究[J].中国水稻科学,2000,14(10):15-18.
31 潘学彪,Rush M C.美国的水稻纹枯病抗病遗传育种研究[J].江苏农学院学报,1997,18(1):57-63.
32 Masajo TM. Varietal resistance in rice to sheath blight caused by Thanatephorus cucumeris (Frank) Donk Rhizoctonia solani kuhn) [D]. Ph.D. diss. Louisiana State Univ.Baton Rouge, 1976.
33 Jentong F. Evaluation of a dwarf rice sccession from chinese as a source of sheath blight resistance[D]. Thesis for M.S., Louisiana State Univ., Supervisor: Rush MC, 1985: 76-81.
34 Che KR, Zhan QC, Xing QH, et al. Tagging and mapping of rice sheath blight resistance gene[J]. Theor Appl Genet, 2003, 106(2): 293-297.
35 Li ZK, Pinson S RM, Marchetti MA, et al. Characterization of quantitative trait loci (QTL) in cultivated rice contributing to field resistance to sheath blight (Rhizoctonia solani) [J]. Theor Appl Genet, 1995, 91: 382-388.
36 邹军煌,潘学彪,陈宗祥,等.水稻两个品种抗纹枯病数量性状基因定位[J].云南大学学报(自然科学版),1999,21(3):97-98.
37 陶家凤.立枯丝核菌对水稻侵染过程的研究[J].四川农业大学学报,1992,10(3):471-477
38 Matsuura K. Scanning electron microscopy of the infection process of Rhizoctonia solani in leaf sheath of rice plants [J]. Phytopathol, 1986, 76:811-814.
39 Xie QJ, Rush MC. In vitro selection of rice protoplast derived calli for resistance to picolinic acid and phenylacetic acid [C]. Proc 24th Rice Tech. Working Group. Little Rock, AK, 1992.
40 陈兵.水稻纹枯病菌致病性与酶活力的关系[J].沈阳农业大学学报,1992,23(1):19-22.
41 孟庆忠,刘志恒,王鹤影,等.水稻纹枯病研究进展[J].沈阳农业大学学报,2001,32(5):376-381.
42 陈捷,唐朝荣,邹庆道.玉米纹枯病菌致病因子的研究[J].沈阳农业大学学报,1999,30(3):189-194.
43 张红生,朱立宏,沙学延,等.水稻纹枯病抗性机理的初步研究[A].朱立宏.主要农作物抗病性遗传研究进展[C].南京:江苏科学技术出版社,1990:154-164.
44 陈志谊,王玉环,殷尚智.水稻纹枯病抗性机制的研究[J].中国农业科学,1992,25(4):41-46.
45 Jedidah D,Kikuo W, Akihiro N. Infection of rice plants with the sheath blight fungus causes an activation of pentose phosphate and glycolytic pathways [J]. European Journal of Plant Pathology, 2000, 106 (6): 555-561.
46 Velazhahan R, Samiyappan R, Vidhyasekaran P. Purification of an elicitor-inducible antifungal chitinase from suspension cultured rice cells [J], Phytoparasitica, 2000, 28 (2): 131-139.
47 赵长江,鲁国东,杜晓昱,等.水稻纹枯病发病过程PR1和PBZ1的表达动态[J].植物病理学报,2006,36(4):317-321.
48 张楷正,李平,李娜,等.水稻抗纹枯病种质资源、抗性遗传和育种研究进展[J].分子植物育种,2006,4(5):713-720.
49 Stakman EC, Christnsen JJ. Problem of Variability in Fungi In: Plant Disease [M]. Agric: The Yearbook of US Dep Agricul, 1954: 35-62.
50 Andrivon D, Limpert E. Origin and proportions of the components of composite populations of Erysiphe graminis f.sp. hoidel[J]. Phytopathol, 1992, 135:6-19.
51 Milgroom MG, Wang KR, Zhou Y, et al. Intercontinental population structure of the chestnut blight fungus, Cryphonectria parasitica [J]. Mycology, 1995, 88:179-190.
52 Burdon JJ, Marshall DR, Luig NH, et al. Isozyme studies on the origin and evolution ofpuccinia graminis f. sp. Tritici in Australia. Aust [J]. Biol. Sci., 1982, 35: 231-238.
53 Barrett JA. The dynamics of genes in populations, pp 39~53 in Wolfe MS and Caten CE eds: Populations of plant pathogens: their dynamics and genetics [M]. Blackwell Sci.Publ., 1987, Oxford.
54 Flor HH. Host-parasite interaction in flax rust-its genetics and other implications [J]. Ann. Rev. Phytopathol, 1971, 45:680-685.
55 Levy M, FJ Correa, RS Zeigler, et al. Phytopathology [M], 1993, 83:1427-1433.
56 Dobinson KF, Patterson NA, White G J, et al. DNA fingerprinting and vegetative compatibility analysis indicate multiple origins for Verticillium dahliae race 2 tomato isolates from Ontario, Canada[J]. Mycol. Res, 1998, 102(9): 1089-1095.
57 Cayley DM. The phenomenon of mutual aversion between monospore mycelia of the same fungus (Diaporthe perniciosa) with a discussion of sex-heterothallism in fungi [J]. Genetics, 1923, 13: 1353-1370.
58 Todd NK, Rayner ADM. Fungal individualism [M], Science Progress, 1980, 66:331-354.
59 Elias KS, Schnerder RW. Vegetative compatibility groups in Fusarium oxysporum f. sp. Lycopersici [J]. Phytopathol, 1991, 81: 159-162.
60 夏淑春,鄢洪海,李如升,等.分子标记技术在植物病原菌遗传分化方面的应用[J].特产研究,2004,(4):56-59.
61 鄢洪海.玉米弯孢叶斑病菌生理分化及分子生理学研究[D].沈阳农业大学博士论文,2001.
62 袁凤杰.大豆灰斑病生理小种同工酶分析[J].植物病理学报,1998,28(1):39-42.
63 Liu Z, Nickrent DL, Sinclair JB. Genetic relationships among isolates of Rhizoctonia aolani anastomosiss group-2 based on isozyme analysis[J]. Canadian Journal of Plant Pathology, 1990, 12 (1): 376-382.
64 王守正,王海燕,李洪连,等.用酯酶同工酶酶谱鉴定病原菌专化型研究[J].河南农业科学,2000,(10):15-17.
65 赵桂东,周海波,朱玉梅,等.大麦纹枯病菌与其轮作物丝核菌的生物学特性及致病力比较研 究[J].大麦科学,1999,(1):37-39.
66 Jin MS, Korpradiskul V. Isozyme analysis of genetic diversity among Rhizoctonia solani Anastomosis Group 1-IA (AG I-IA) [J]. Mycosystema, 1998, 17(4):331-338.
67 Clare B C, Zentmyer G A. Starch-gel electrophoresis of proteins from species of Phytophthora [J]. Phytopathol, 1967, (56): 1334-1335.
68 Reynolds M, WeinholdAR, Morris TJ. Phytopathol [M], 1983, 73: 903-906.
69 刘力,葛起新.立枯丝核菌蛋白质电泳图谱研究[J].真菌学报,1997,(3):175—182.
70 熊庆,刘作易.儿种分子生物学方法在真菌中的应用[J].贵州农业科学,2002,30(3):69-73.
71 F.奥斯伯,R.布伦特,R.E.金斯顿.精编分子生物学[M],北京:科学出版社,1998.
72 Williamas JGK. DNA polymorphisms amplified by arbitrary primers are useful as genetic marker[J]. Nuc Aci Res, 1990, 18: 6531~6535.
73 Welsh J. Finger printing genomes using PCR with abitrary primers[J]. Nuc Aci Res, 1990, 18: 7213~7218.
74 邹喻苹,葛颂,王晓东.系统与进化植物学中的分子标记[M].北京:科学出版社,2001.
75 Delye C, Bousset L, Corio-Costet M F. PCR cloning and detection of point mutations in the eburicol 14alpha-demethylase (CYP51) gene from Erysiphe graminis f. sp. hordei, a "recalcitrant" fungus[J]. Curr Genet, 199834: 399~403.
76 Schilling AG, Moiler EM, Geiger HH. RAPDs ofFrsarium culmorum and F. Graminearum: Application for genotyping and species identification. In modern assays for plant pathogenic fungi: identification, detection, and quantification (ed. A. Schots, FM, Dewey and R. Oliver) [C], 1994: 37-46. CAB international, Wallingford, UK.
77 Zebeau M,Voss P. Selective restriction fragment amplication: a general method for DNA fingerprinting [J]. European Pattern Application, Number 92402629, publication number 0534858A1, 1993.
78 陈洪等.致病性念珠菌DNA的AFLP指纹图谱[J].科学通报,1996,41(10):934~938.
79 Mafia C. Julian, Javier Acero, Oscar Salazar, et al. Mating type-correlated molecular markers and demonstration of heterokaryosis in the phytopathogenic fungus Thanatephorus cucumeris (Rhizoctonia solani)AG 1-IC by AFLP DNA fingerprinting analysis[J]. Biotech, 1999, 67: 49-56.
80 Zitkiewicz E, Rafalski A, Laubuda D. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification [J]. Genomics, 1994, 20:176-183.
81 魏臻武.利用SSR、ISSR和RAPD技术构建苜蓿基因组DNA指纹图谱[J].草业学报,2004,13(3): 62-67.
82 Bruns TD, Vilgalys R, bruns SM. Evolutionary relationships within the fungi: analysis of nuclear small subunit rRNA sequence[J]. Molecular Phylogenetics Evolution, 1992, 1: 231-241.
83 于金凤;伍恩宇;夏海波.核糖体RNA基因在丝核菌分子系统学研究中的应用[J].菏泽学院学报,2006,28(5):103-107.
84 Michelmore RW and Hulbert SH. Molecular markers for genetic analysis ofphytopathologic fungi [J] Annu Rev Phytopathol, 1987, 25: 383-404.
85 White TJ, Bruns T, Lee S, et al. PCR protocols: a guide to methods and applications[M]. San Diego: Academic Press, 1990:315-322.
86 Edel V, Steinberg C, Avelange I, et al. Comparision of three molecular methods for the characterization of Fusarium oxysporum strains[J]. Phytopathol, 1995, 85: 579-585.
87 Godoy-Lutz G, Steadman JR, Higgins B, et al. Genetic variation among isolates of the web blight pathogen of common bean based on PCR-RFLP of the ITS-rDNA region[J]. Plant Dis, 2003, 87(7): 766-771.
88 Kong H-H, Shin J-Y, Yu H-S, et al. Mitochondrial DNA Restriction Fragment Length Polymorphism (RFLP) and 18S Small-Subunit Ribosomal DNA PCR-RFLP Analyses of Acanthamoeba Isolated from Contact Lens Storage Cases of Residents in Southwestern Korea[J]. J Clinical Microb., 2002, 40(4): 1199-1206.
89 Ying B, Song GE. Identification of Oryza Species with the CD Genome Based on RFLP Analysis of Nuclear Ribosomal ITS Sequences[J]. Acta Botanica Sinica, 2003, 45(7):762-765.
90 Masaru Matsumoto, Naruto Furuya, Nobuaki Matsuyarna. PCR-RFLPAnalysis of Amplified 28S Ribosomal DNA for Identification of Rhizoctonia spp., the Causal Agents of Sheath Diseases of Rice Plants[J]. J. Fac. Agr, Kyushu Univ. 1996, 41(1-2): 39-44.
91 Marianne Boysen, Marise Borjs, Catalina del Moral, et al. Identification at strain level of Rhizoctonia solani AG4 isolates by direct sequence of asymmetric PCR products of the ITS regions[J]. Current Genet, 1996, 29: 174-181.
92 Kuninaga S, and Yokosawa R. DNA based sequence homology in Rhizoctonia solani Kuhn. Ⅲ. Genetic relatedness within AG-3, AG-5, AG-7, and AG-BI [J]. Ann Phytopathol. Soc.Jpn, 1983, 49: 648-652.
93 Gonzalez D, Carling DE, Kuninaga S, et al. Ribosomal DNA systematics of Ceratobasidium and Thanatephorus with Rhizoctonia anamorphs [J]. Mycologia, 2001, 93 (6): 1138-1150.
94 Salazar O, Johannes H M, Maria C J, et al. Phylogenetic Subgrouping of Rhizoctonia solani AG 2 isolates based on ribosomal ITS sequences[J]. Mycologia, 1999, 92 (3): 459-467.
95 Salazar O, Julian M C. Primers based on specific rDNA-ITS sequences for PCR detection of R.solani AG2 subgroups and ecological types and binucleate Rhizoctonia[J]. Mycological Research, 2000, 104 (3): 281-285.
96 Carling D E, Kuniaga S, Brainard A, et al. Hyphal anastomosis reactions, rDNA-intrnal transcribed spacer sequences, and virulence levels among subsets ofRhizoctonia solani anastomosis group-2 (AG-2) and AG-BI [J]. Phytopathol, 2002, 92 (1): 43-50.
97 喻子牛,刘作易,熊庆.线粒体DNA的研究与应用[J].西南农业学报,2002,15(3):111-115.
98 孟紫强,耿红,张波.线粒体DNA及其表达的研究进展[J].生命的化学,2001,21(6):500-502.
99 Ferreira MASV. Isolation of a Species-Specific Mitochondrial DNA Sequence for Identification of Tilletia indica, the Karnal Bunt of Wheat Fungns[J]. Applied and Environmental Microbiol, 1996, 87-93.
100 邢来君,李明春.电泳核型分析在丝状真菌研究中的应用[J].微生物学报,1996,23(4):244-248.
101 熊庆,刘作易.几种分子生物学方法在真菌中的应用[J].贵州农业科学,2002,30(3):69-73.
102 Carle G F, Olson M V. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alteration gel electrophoresis [J]. Nucleic Acids Res, 1984(12): 5647-5664.
103 Magee B B, Koltin Y, Gorman J A, et al. Assignment of cloned genes to the seven electrophoretically separated Candida albicans chromosomes [J]. Mol cell Biol, 1988 (8): 4721-4726.
104 Schwartz D. C. and Cantor C. R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis [J]. Cell, 1984, 37: 67-75.
105 Corby-Kistler H, Benny U, E. Boehm WA, et al. Genetic duplication in Fusarium oxysporum [J]. Current Genetic, 1995, 28(2): 173-176.
106 Vilgalys R. Genetic relatedness among anastomosis groups in Rhizoctonia as measured by DNA/DNA hybridization [J]. Phytopathol, 1988 (78): 698-702.
107 Naito, S. Basidiospore dispersal and survival. In Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control (B. Sneh, S. Jabaji-Hare, S. Neate, and G. Dijst, Eds.) [A], 1996, pp. 197-203. Kluwer Academic, Dordrecht.
108 高强,吴品珊,戴良英,等.分子生物学技术在腥黑粉菌种间分类鉴定的应用[J].植物检疫,2004,18(6):355-358.
109 Borrome ES, Nelson RJ, Bonman JM, et al. Genetic differentiation among isolates of Pyricularia infecting rice and weed hosts[J]. Phytopathol, 1993, 83: 393-399.
110 Baayen RP, O'Donnell K, Breeuwsma S, et al. Molecular relationships of fungi within the Fusarium redolens-F, hostae clade[J]. Phytopathol, 2001, 91: 1037-1044.
111 赵杰.ITS序列分析及其在植物真菌病害分子检测中的应用[J].陕西农业科学,2004(4):35-37.
112 刘雯霞 雷国莲.DNA分子诊断技术在中药鉴定中应用[J].陕西中医学院学报,2005,28(1):30-32.
113 秦旭升,刘学敏,周艳玲,等.植物病原真菌中DNA分子鉴定技术[J].植物生理学通讯,2000,36(4):342-347.
114 邢楠,谢响明,何晓青.核酸技术在立枯丝核菌分类与生态学研究中的应用[J].微生物学杂志,2005,25(2):93-97.
115 程颖慧,章桂明,王颖,等.小麦印度腥黑穗病菌PCR检测[J].植物检疫,2001,15(6):321-325
116 Dewey FM. Detection of plant-invading fungi by monoclonal sntibodies[J]. See Ref, 1992, 43: 47-62.
117 Schesser K, Luder A, Heson JM. Use of polymerase chain reaction to detec the take-all funguns, Gaeumannomyces graminis, in infected wheat plants[J]. Appl. Environ Microbiol, 1991, 57: 553-556.
118 王美南.禾顶囊壳(Gaeumannomyces graminis(sacc.)v.Arx Arx&Oliver)遗传差异研究[D].西北农林科技大学博士学位论文,2002.
119 周雪平,施曼玲.植物病毒病的诊断技术[J].微生物学通报,2000,27(2):149-151.
120 朱延书,康宁.生物技术在植物检疫检测中的应用[J].江苏林业科技,2003,30(3):42-47.
121 刘景梅,陈霞,王璧生,等.香蕉枯萎病菌生理小种鉴定及其SCAR标记[J].植物病理学报,2006,36(1):28-34.
122 曹丽华,康振生,郑文明,等.小麦条锈菌条中31号生理小种SCAR检测标记的建立[J].菌物学报,2005,24(1):98~103.
123 韩新运.甘蔗抗黑穗病性检测SCAR标记的建立[D].福建农林大学硕士学位论文,2003.
124 Li KN, Rouse DI, Eyestone EJ, et al. The generation of specific DNA primers using random amplified polymorphic DNA and its application to Verticillium dahliae[J]. Mycol. Res, 1999, 103(11): 1361-1368.
125 Hamelin CR, Ouellette GB, Bernier L. Identification of Gremmeniella abietina races with random amplified polymorphic DNA markers [J]. Appl Enviro Microgiol. 1993, 59: 1752-1755.
126 L Lubeck M, Poulsen H. UP-PCR cross blot hybridization as a tool for identification of anastomosis groups in the Rhizoctonia solani complex[J]. FEMS Microbiology letters, 2002, 201: 83-89.
127 Julian MC, Dullemans AM, Silfhout C, et al. Nuclear behavior in homokaryotic and heterokaryotic fiuiting of Thanatephorus cucumeris (Rhizoctonia solani) anastomosis group 1, subgroup IC [J]. Mycologia, 1998, 89: 361-374.
128 杨佩文,李家瑞,杨勤忠,等.根肿病菌核糖体基因ITS区段的克隆测序及其在检测中的应用[J].云南农业大学学报,2003,18(3):227-233.
129 陈怀谷,方正,陈厚德,等.小麦纹枯病菌核糖体基因内转录区序列比较[J].植物病理学报,2005,35(1):431-436.
130 Hayden K J, Rizzo D, Tse J, et al. Detection and quantification ofphytophthora ramorum from california forests using real-time polymerase chain reaction assay[J]. The America phytopathological society. 2004, 92(10): 1075-1083.
131 Le Cam B, Devaux M, Parisi L. Specific polymerase chain reaction identification of Venturia nashicola using internally transcribed spacer region in the ribosomal DNA[J]. Phytopathol, 2001, 91: 900-904.
132 Johanson A, Hurner HC, McKay GJ, et al. A PCR- R. solani based method to distinguish fungi of the rice sheath-blight complex, Rhizoctonia solani, R. oryzae and R. oryzae-sativae[J]. FEMS Microbiol letters. 1998, 162: 289-294.
133 Yu L M. Elicitins from Phytophthora and basic resistance in tobacco[J]. Proc Natal Acad Sci USA, 1995, 92: 4088-4094.
134 梁元存,刘爱新,董汉松,等.寄生疫霉parA1基因的克隆及在大肠杆菌中的表达[J].微生物学报,2004,44(2):202-205.
135 Fouly HM, Willkinson HT. Detection of Gaeumannomyces graminis varieties using polymerase chain reaction with variety-specific primers[J]. Plant Dis, 2000, 84:947-951.
136 詹金彪,郑树.天然蛋白毒素的研究和临床应用展望[J].浙江大学学报(医学版),2005,34(3):197-200.
137 Viji G, Gnanamanickam SS, Levy M. DNA polymorphisms of isolates of Magnaporthe grisea from India that are pathogenic to finger millet and rice[J]. Mycol Res, 2000, 104(2): 161-167.
138 Tan MK, Wong PTM, Holley MP. Characterization of nuclear ribosomal DNA (rDNA) in Gaeumannomyces graminis and correlation of rDNA variation with G graminis varieties[J]. Mycol Res, 1994, 98: 553-561.
139 Borrome ES, Nelson RJ, Bonman JM, et al. Genetic differentiation among isolates of Pyricularia infecting rice and weed hosts[J]. Phytopathol, 1993, 83: 393-399.
140 董金皋,王江柱,阎淑娟,等.几种旋孢腔菌毒素产生的分子生物学进展等[J].微生物学通报,1996,23(6):356-359.
141 葛丹,陈明杰,曹文伟.地高辛标记核酸探针的标记方法及其在食用菌研究领域的应用[J].食用菌学报,2002,(9):50-55.
142 黄媛,文翠容,牟兆钦,等.巢式PCR结合微孔板杂交技术鉴定脆弱拟杆菌[J].中国实验诊断学,2002,6(2):75-77.
143 Luo GZ, Mitchell TG. Rapid identification of pathogenic fungi directly from cultures by using multiplex PCR [J]. Journal of Clinical Microbiology, 2002, 40(8): 2860-2865.
144 杨红,李颖,关国华,等.棉枯萎病菌异核体及其不同表型分离子在基因转录水平上的差异[J].遗传学报,2004,31(2):166-170.
145 王鲁平 张建中.分子遗传学技术在诊断病理学中的应用[J].诊断病理学杂志,2003,10(6):329-331.
146 戴富明;刘少华;任小杰,等.西瓜蔓枯病分子诊断技术研究[J].植物病理学报,2006,36(5):439-445.
147 何月秋.稻瘟病菌定向选择的分子证据和抗稻瘟病近等基因累加系的分子选育[D].中国农业大学博士学位论文,1999.
148 石鸿文,丁文侠.水稻纹枯病的配套防治技术[J].河南农业科学,2001,(5):23.
149 陆岗,梁耀懋,黎坤爱,等.深水稻种质资源耐淹性及抗稻纹枯病特性研究[J].西南农业学报,2004,17(6):701-704
150 雷财林,凌忠专,王久林,等.水稻抗病育种研究进展[J].生物学通报,2004,39(11):4-7.
151 蒙姣荣,张超冲,李界秋,等.广西水稻纹枯病菌菌丝融合群鉴定初报[J]_中国农学通报,2006,22(6):327-329.
152 方中达.植病研究方法[M].农业出版社,1998.
153 J.萨姆布鲁克,D.W.拉塞尔,著.黄培堂,等译.分子克隆实验指南[M].北京:科学出版社,2002.
154 Lee SB, and Taylor JW. Isolation of DNA from fungal mycelia and single spores. In: PCR Protocols: A guide to methods and applications[A]. M. A. Innis, D. H. Gelfand, J.J. Sninsky, and T.J. white, eds. Academic Press, New York. 1990: 282-287.
155 陈延熙,张敦华,段霞渝,等.关于Rhizoctonia solani菌丝融合分类和有性世代的研究[J].植物病理学报,1985,15(8):139-143.
156 周而勋,曹菊香,杨媚,等.我国南方六省(区)水稻纹枯病遗传多样性的研究[J].南京农业大学学报,2002,25(3):10-15.
157 Neeraja CN, Shenoy VV, Rrddy CS, et al. Isozyme polymorphism and vhulence of Indian isolates of the rice sheath blight fungus [J]. Mycopathol, 2002, 156: 101-108.
158 Linde CC, Zalal M, David Paulraj RS, et al. Population structure of the rice sheath blight pathogen Rhizoctonia solani AG-1 IA from India[J]. European Journal of Plant Pathology. 2005, 112:113-121.
159 方正.小麦纹枯病菌的多样性和分子检测[D].扬州大学硕士学位论文,2003.
160 叶斌,张建平,胡贤春,等.稻菌核病的发生与防治[J].安徽农业科学,2005,33(8):1385-1386.
161 陈天寿.微生物培养基的制造与应用[M].北京:中国农业出版社,1995.
162 Ogoshi, A, and Ui, T. 1979. Specificity in vitamin requirement among anastomosis groups of Rhizoctonia solani Kuhn[J]. Ann. Phytopathol. Soc. Jpn. 1979, 45:47-53.
163 Ogoshi, A. Studies on the grouping of Rhizoctonia solani Kuhn with hyphal anastomosis, and on the perfect stage of groups in Japan [J]. Natl. Inst. Agric. Sci. Bull. Ser. C, 1976, 30: 1-63.
164 Cubeta MA, Echandi E, Abernethy T, et al. Characterization anastomosis groups of bincleate Rhizoctonia species using restriction analysis of an amplified ribosomal RNA gene[J]. Phytopathol, 1991, 81: 1935-1400.
165 陈宗祥,邹军煌,韩月澎,等.抗水稻纹枯病遗传研究的改良方法及其验证[J].中国水稻科学,2002,16(1):74-76.
166 Gillings M, Holley M. Repetitive element PCR fingerprinting (rep-PCR) using enterobacterial Repetitive Intergenic Consensus (ERIC) primers is not necessarily directed at ERIC elements[J]. Letters in Applied Microbiol, 1997, 25: 17~21.
167 张星耀.基于培养性质模糊解析和28SrDNA-PCR-RFLP解析的外担子菌属真菌的分类学研究[J].林业科学,1999,34(4):59-70.
168 王成树,王四宝,李增智,等.球孢白僵菌种群野生菌株异核现象的分子验证[J].菌物系统,2001,20(4):475-481.
169 Neuveglise C, Brygoo Y, Riba G. 28S rDNA group-Ⅰ introns: a powerful tool for identifying strains of Beauveria brongniartii[J]. Mol Ecol, 1997, 6(4): 373-381.
170 Hellmich, S., and K. Schauz. Production of extracellular, alkaline and neutral proteases of Ustilago maydis[J]. Exp Mycol. 1988, 12: 223-232.
171 Movahedi, S., Heale, J. B. The roles of aspartic proteinase and endo-pectin lyase enzymes in the primary stages of infection and pathogenesis of the various host tissues by different isolates ofBotrytis cinerea Pers ex. Pers[J]. Physiol. Mol. Plant Pathol. 1990b, 36: 303-324.
172 Carlile AJ. Characterization of SNP1, a cell wall-degrading Trypsin, produced during infection by Stagonospora nodorum[J]. Mol. Plant-Microbe Interact. 2000, 13: 538-550.
173 Walton JD. Deconstructing the cell wall[J]. Plant Physiol. 1994, 104:1113-1118.
174 Murphy JM., Walton JD. Three extracellular proteinase from Cochliobolus carbonum: Cloning and targeted disruption of ALP1 [J]. Mol. Plant-Microbe Interact. 1996, 9: 290-297.
175 赵蕾.小麦纹枯病菌产生的一种胞外蛋白酶及其致病机理的研究[D].山东农业大学博士学位论文,2003.
176 Kimiharu I. Dispersal office sheath blight fungus Rhizoctonia solani AG-1 (IA), and subsequent disease development in paddy fields from survey of vegetative compatibility groups [J]. Mycoscience, 1998, 39: 391-397.
177 邓振山,张宝成,孙志宏,等.新疆北疆棉田立枯丝核菌不同菌丝融合群致病力的研究[J].植物保护,2006,32(4):36-39.
178 Ceresini PC, Shew HD, Rosewich L, et al. Genetic diversity ofRhizoctonia solani AG-3 from potato and tobacco in North Carolina [J]. Mycologia, 2002, 94(3): 437-449.
179 Elena K, Paplomatas EJ. Vegetative compatibility groups within Verticillium dahlliae isolates from different hosts in Greece [J]. Plant Path01, 1998, 47: 635-640.
180 Daayf F, Nicole M., Belanger RR, et al. Hyaline mutants from Verticillium dahlliae, and example of selection and characterization of strains for host-parasite interaction studies [J]. Plant Dis, 1998, 47: 523-529.
181 阮兴业,陈兴全.云南省稻球小菌核病的病原鉴定报告[J].云南农业大学学报,2002,17(2):126-129
182 肖平,易润华,陈小丽,等.水稻纹枯病菌性状多样性的研究[J].江西农业学报,2006,18(3):125-128
183 王宗华,单卫星.对植物病原真菌群体遗传研究范畴及其意义的认识[J].植物病理学报,1998,28(1):5-9.
184 Manici LM, and Bonora P. Molecular genetic variability of Italian binucleate Rhizoctonia spp. isolates from strawberry [J]. European Journal of Plant Pathology, 2007, 118: 31-42.