异源镰刀菌对小麦品种致病力分化的研究
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摘要
小麦赤霉病是我国长江中下游诸省麦类作物的重要病害。以穗腐危害最甚。引起小麦赤霉病的镰刀菌种类至少有20种，其中黄色镰刀菌、弯角镰刀菌、雪腐镰刀菌、禾谷镰刀茵、梨孢镰刀菌最为常见。近年来，许多地区对小麦赤霉病的病原鉴定、抗病品种选育开展了大量工作。本文对异源镰刀菌对小麦致病性测定、致病性测定方法及分子生物学进行了初步研究。本研究从全国各地及不同寄主收集到14种镰刀菌，共39个菌株，包括禾谷镰刀菌、串珠镰刀菌、尖孢镰刀菌、弯角镰刀菌、三线镰刀菌、半裸镰刀菌、锐顶镰刀菌、草燕麦镰刀菌、雪腐镰刀菌、梨孢镰刀菌、莞草镰刀菌、腹状镰刀菌、Fusarium annulatum、F. proliferatum。采用田间花期接种和室内芽期接种两种抗性鉴定方法将各镰刀菌分别接种于苏麦3号、扬麦158和安农8455三个小麦品种，测定小麦赤霉病镰刀菌和引起其它病害的镰刀菌对小麦的致病性，用SAS软件分析、比较各菌株致病力差异及相关性。观察各镰刀菌菌株的培养性状，将其与致病性测定相比较。选用15个随机引物，利用RAPD技术，对39个镰刀菌菌株的基因组DNA进行种群分析，评估RAPD分析区分不同种类镰刀菌的可行性。研究所取得的结果如下：
1、在所供试的非禾谷镰刀菌菌株中，串珠镰刀菌、尖孢镰刀菌、弯角镰刀菌等3种为“强一中”致病型，F. annulatum、雪腐镰刀菌、莞草镰刀菌、草燕麦镰刀菌、F. proliferatum、半裸镰刀菌等6种为“中一弱”致病型，三线镰刀菌、锐顶镰刀菌、梨孢镰刀菌、腹状镰刀菌等4种为致病力弱或为非致病型。禾谷镰刀菌仍属强致病型。各菌株之间和同一菌株在不同品种之间具有极显著的致病力分化现象，来源不同地区和不同寄主的菌株，以及地理来源相同、寄主植物相同的同一类型的镰刀菌菌株也存在极显著的致病力差异。室内芽期接种和田间花期接种结果一致，均可以鉴定菌株致病力，并可用于菌株与不同抗性小麦品种互作的研究。因此，室内芽期接种是一种方便、可靠的抗性鉴定方法。
2、不同镰刀菌菌株的培养性状差异明显，相同地理来源、相同寄主上分离的同一类型镰刀菌菌株的培养性状亦存在分化现象。培养性状与致病力的相关分析表明，菌落扩展速度与致病力之间线性相关性极高：菌落扩展速度快，菌株致病力最强，而产孢量和气生菌丝生长状况与致病力之间没有显著的线性关系。
3、RAPEI扩增图谱显示供试菌株在种间和种内均具有高度的多态性，综合所有谱
华中农业大学2003硕士论文
带系统聚类所得树状图将这39个菌株分为9组，组的划分能将非小麦寄主串珠镰刀
菌与小麦寄主串珠镰刀菌分成两组，将不同地理来源的尖抱镰刀菌分开，并将大多数
弱或非致病镰刀菌聚为一类。但对于所有供试镰刀菌株，由于遗传差异极大，组的划
分与菌株所属种、致病力及其寄主间没有明显的相关性。
Wheat head blight (wheat scab) caused by Gibberella zeae(Schw.)Petch is one of the most serious diseases of wheat in various parts of China. Fusarium head blight (FEB) of wheat has been linked to at least 20 causal organisms, most records of the diseases being associated with five species: Fusarium culmorum, F.avenaceum, F.graminearum, F.poue. F.nivale. In recent years, many works have been done in identification of wheat scab and screen of resistant wheat cultivars. In this experiment, 39 single-spore isolates of Fusarium spp. were collected from Hubei, Henan, Jiangsu provinces et al. in China. These isolates belong to 14 species including F.moniliforme, F.oxysporum, F.graminearum, Fcamptoceras, F.tricinctum, F.semitectum, F.annulatum, F.acuminatum, F.avenaceum, F.nivale, F.poae, F.proliferatum, F.scirpi, F.ventricosum. The variation of pathogenicity of these species was tested by artificial inoculation in budding and flowering stages using conidiospores and analyzed by SAS. The variation of cultural charac
teristics of Fusarium spp. was observed in PDA and compared with the differentiation of virulence. Randomly amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic diversity among 39 isolates of Fusarium spp.. The results include:
1. The test of pathogenicity of isolates which do not belong to F.graminearum showed that three species were highly virulent isolates, six species were intermediate virulent and the others were low virulent isolates. There was a significant difference in virulence of isolates and in resistance response of the wheat cultivars. Isolates of F.graminearum were also highly virulent. Isolates from different localites and different hosts were also different in virulence. Even isolates from the same regions and the same hosts varied significantly in their virulence. Both methods of inoculation in budding and flowering stage can idendicate the differentiation of the virulence of the causal oganisms and can be used to study the connection of isolates and wheat cultivars. Therefore, it is possible to identify disease ressistance of varieties in budding stage. It can be used as a reliable and advantageous method.
2. There was a marked difference in cultural characteristics. The isolates from the
same regions and the same hosts were also different in cultural characteristics, and the differentiation appeared to correlate the pathogenicity: the growth rate of mycelia correlated directly with pathogenicity: isolates which can grow fast in PDA have high virulent. The sporulation and mycelium growth did not correlate with pathogenicity. So maybe the cultural characteristics and virulence share common genetic background or the virulence of isolates correlates genetically with biologic characteristics.
3. Genetic relationships among the isolates were determined by hierarchical cluster method, and showed that there was high polymorphic among 39 isolates. Tree diagram divided these isolates into nine groups. Isolates of F.moniliforme which come from crops except wheat can be differentiated from those from wheat. And most of high and low virulent isolates can be differentiated here. Isolates of F.oxysporum can be differentiated by their orignation. But for all isolates RAPD analysis can not provide reliable molecular evidences of determining classification position to Fusarium spp. in this experiment.
1、在所供试的非禾谷镰刀菌菌株中，串珠镰刀菌、尖孢镰刀菌、弯角镰刀菌等3种为“强一中”致病型，F. annulatum、雪腐镰刀菌、莞草镰刀菌、草燕麦镰刀菌、F. proliferatum、半裸镰刀菌等6种为“中一弱”致病型，三线镰刀菌、锐顶镰刀菌、梨孢镰刀菌、腹状镰刀菌等4种为致病力弱或为非致病型。禾谷镰刀菌仍属强致病型。各菌株之间和同一菌株在不同品种之间具有极显著的致病力分化现象，来源不同地区和不同寄主的菌株，以及地理来源相同、寄主植物相同的同一类型的镰刀菌菌株也存在极显著的致病力差异。室内芽期接种和田间花期接种结果一致，均可以鉴定菌株致病力，并可用于菌株与不同抗性小麦品种互作的研究。因此，室内芽期接种是一种方便、可靠的抗性鉴定方法。
2、不同镰刀菌菌株的培养性状差异明显，相同地理来源、相同寄主上分离的同一类型镰刀菌菌株的培养性状亦存在分化现象。培养性状与致病力的相关分析表明，菌落扩展速度与致病力之间线性相关性极高：菌落扩展速度快，菌株致病力最强，而产孢量和气生菌丝生长状况与致病力之间没有显著的线性关系。
3、RAPEI扩增图谱显示供试菌株在种间和种内均具有高度的多态性，综合所有谱
华中农业大学2003硕士论文
带系统聚类所得树状图将这39个菌株分为9组，组的划分能将非小麦寄主串珠镰刀
菌与小麦寄主串珠镰刀菌分成两组，将不同地理来源的尖抱镰刀菌分开，并将大多数
弱或非致病镰刀菌聚为一类。但对于所有供试镰刀菌株，由于遗传差异极大，组的划
分与菌株所属种、致病力及其寄主间没有明显的相关性。
Wheat head blight (wheat scab) caused by Gibberella zeae(Schw.)Petch is one of the most serious diseases of wheat in various parts of China. Fusarium head blight (FEB) of wheat has been linked to at least 20 causal organisms, most records of the diseases being associated with five species: Fusarium culmorum, F.avenaceum, F.graminearum, F.poue. F.nivale. In recent years, many works have been done in identification of wheat scab and screen of resistant wheat cultivars. In this experiment, 39 single-spore isolates of Fusarium spp. were collected from Hubei, Henan, Jiangsu provinces et al. in China. These isolates belong to 14 species including F.moniliforme, F.oxysporum, F.graminearum, Fcamptoceras, F.tricinctum, F.semitectum, F.annulatum, F.acuminatum, F.avenaceum, F.nivale, F.poae, F.proliferatum, F.scirpi, F.ventricosum. The variation of pathogenicity of these species was tested by artificial inoculation in budding and flowering stages using conidiospores and analyzed by SAS. The variation of cultural charac
teristics of Fusarium spp. was observed in PDA and compared with the differentiation of virulence. Randomly amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic diversity among 39 isolates of Fusarium spp.. The results include:
1. The test of pathogenicity of isolates which do not belong to F.graminearum showed that three species were highly virulent isolates, six species were intermediate virulent and the others were low virulent isolates. There was a significant difference in virulence of isolates and in resistance response of the wheat cultivars. Isolates of F.graminearum were also highly virulent. Isolates from different localites and different hosts were also different in virulence. Even isolates from the same regions and the same hosts varied significantly in their virulence. Both methods of inoculation in budding and flowering stage can idendicate the differentiation of the virulence of the causal oganisms and can be used to study the connection of isolates and wheat cultivars. Therefore, it is possible to identify disease ressistance of varieties in budding stage. It can be used as a reliable and advantageous method.
2. There was a marked difference in cultural characteristics. The isolates from the
same regions and the same hosts were also different in cultural characteristics, and the differentiation appeared to correlate the pathogenicity: the growth rate of mycelia correlated directly with pathogenicity: isolates which can grow fast in PDA have high virulent. The sporulation and mycelium growth did not correlate with pathogenicity. So maybe the cultural characteristics and virulence share common genetic background or the virulence of isolates correlates genetically with biologic characteristics.
3. Genetic relationships among the isolates were determined by hierarchical cluster method, and showed that there was high polymorphic among 39 isolates. Tree diagram divided these isolates into nine groups. Isolates of F.moniliforme which come from crops except wheat can be differentiated from those from wheat. And most of high and low virulent isolates can be differentiated here. Isolates of F.oxysporum can be differentiated by their orignation. But for all isolates RAPD analysis can not provide reliable molecular evidences of determining classification position to Fusarium spp. in this experiment.
引文
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54. Booth, C..Physical and biochemical techniques in the identification of Fusarium. J. Gen.Microbiol, 1966,42:7-8
55. Booth, C..Fusarium laboratory, guid to the identification of the major species. Comonwealth Mycological Institute, 1977
56. Buerstmayr, H.,Lemmes, M., Hartle, L.,Doldi, L. Steiner, B., Stierschneider, M.,Ruckenbauer, P..Molecular mapping of QTLS for Fusarium head blight resistance in spring wheat. Theor Appl Genet, 2002, (104):84-92
57. Cakmak, I.,Marschner, H..Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase in bean leaves. Plant Physiol, 1992,1222-1227
58. Carter, J.P.,Rezanoor, H.N, Holden, D.,Dejardins, A.E.,Plattner, R.D.,Nichol son, P..Variation in pathogenicity associated with the genetic diversity of Fusarium graminearum. Plant Pathology, 2000,49:452-460
59. Doohan, F.M.,Parry, D.W.,Jenkinson, P.,Nicholson, P..The use of species specific PCR based assays to analyse Fusarium ear blight of wheat. Plant pathology,1998,47:197-205
60. Edel, V. Steinberg, N.,Gautheron, N.,Alabouvette, C..Evaluation of restriction analysis of polymerase chain reaction (PCR)-amplified ribosomal DNA for the identification of Fusarium species.Mycological Research, 101(2):179-187
61. Gorden, W. L..The Occurrence of Fusarium Species in Canada Ⅱ Prevalence and Taxonomy of Fusarium Species in Cereal seed. Canada Jour Bot, 1952,30:209-251
62. John, G. K.,Williams..DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research, 22(18): 6531-6535
63. Linnemannstons, P.,Voss, T.,Hedden, P.,Gaskin, P.,Tudzynski, B.. Deletions in the gibberellin biosynthesis gene cluster of Gibberella fujikuroi by restriction enzyme mediated integration and conventional transformation mediated mutagenesis. Appl-environ-microbiol, 1999, 65(6):2558-2564
64. Miller, J.D., Arnison, P. G..Deoxynivalenol and experimental in Fusarium graminearum infection of wheat.Cananda Journal of Plant Pathology, 1986,8:147-152
65. Miedaner, T.,Schilling, A. G..Genetic variation of aggressiveness in individual field populations of Fusarium graminearum and Fusarium culmorum tested on young plants of winter rye. European Journal of Plant Pathology. 1996,102:823-830
66. McMullen, M.P.,Schartz, B.,Stover, R. Studies of fungicide efficacy, application timing, and application technologies to reduce Fusarium head blight and deoxynivalenol. Cereal Research Communications, 1997,25:779-780
67. Mullins, E.D.,Chen, X.,Romaine, P.,Raina, R.,Geiser, D.M.,Kang, S..Agrobacte rium-mediated transformation of Fusarium oxysporum:an efficient tool for insertional mutagenesis and gene transfer. Phytopathology, 2001,91(2):173-180
68. Nirenberg. H.I..A simplifiied method for identifying Fusarium spp. occurring on wheat. Cananda Journal of BOT 1981, 59:1599-1609
69. Nicholson, P.,Simpson, D.R.,Weston, G.,Rezanoor, H.N.,Lees, A.K.,Parry, D.W. Joyce, D..Detection and quantification of Fusarium culmorum and Fusarium gaminearum in cereals using PCR assays. Physiological and Molecular Plant Pathology, 1998, 53:17—37
70. Nightingale, M.J.,Marchylo,B.A.,Clear, R.M.,Dexter, J.E.,Preston, K.R..Fus
arium Head Blight:Effect of fungal proteases on wheat storage proteins. Cereal Chemistry,1999, 76(1):150-158
71. Nelson, P.E..Fusarium disease, biology, and taxonomy. The Pennsylvania state university press, 1983, 1-193
72. Nelson, P.E.,Toussoun, T.A.,Marasas, W.F.O..Fusarium species:an illustrated manual for identification. The Penn. State Uni. Press:193
73. Namiki, F.,Matsunaga, M.,Okuda, M.,Inoue, I..Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxyporum f. sp. melonis.Molecular plant microbe interactions, 2001,14 (4): 580-584
74. Nicol, J.J.,Frank, J.M.,Anke, P.L.,Wilhelm, S..Mating, conidiation and pathogenicity of Fusarium graminearum, the main caual agent of the head blight disease of wheat, are regulated by the MAP kinase gpmkl. Curr. Genet, 2003,43:87-95
75. Oreste ,A.,Laura, D.G.,Franca, T.,Rosalla, L..Changes in the Ascorbate System during seed Development of Vicia faba L. Plant Physiol, 1992,99:235-238
76. Prashant, K.M.,Roland, T.V.,Alastair, C..Development of a PCR based assay for rapid and reliable identification of pathogenic Fusarium. Science direct-FEMS Microbiology Letters, 2003,218(2):329-332
77. Schroeder, H.W.,Christensen, J.J..Factors affecting resistance of wheat scab caused by Gibberella zeae. Phytopathology, 1963, 53:831-838
78. Schilling, A.G, Moller, E.M.,Geiger, H.H..Polymerase chain restriction based assays for species detection of Fusarium culmorum, F. graminearum, and F. avenaceum. Phytopathology, 1996,86(5):515-522
79. Schilling, A.G, Moller, E.M.,Geiger, H.H..Molecular variation and genetic structure in field populations of Fusarium species causing head blight in wheat. Cereal Research Communications, 25,549-554.
80. Sona, J.,Kouichi, A.,Takuya, K.,Tomizo, O.,Renkichi, T..The G protein subunit FGB1 regulates development and pathogenicity in Fusarium oxysporum. Curr Genet, 2003,43:79-86
81. Turner, A.S.,Lees, A.K.,Rezanoor, H.N.,Nicholson, Refinement of PCR detection of Fusarium avenaceum and evidence from DNA marker studies for phenetic relatedness to Fusarium tricinctum. Plant Pathology, 1998,47:278-288
82. Theresa, L.,Woongoh, D.,Seonkim, H.,Jungkwan, L..Identification of Deoxynivalenol and Nivalenol producing chemotypes of Gibberella Zeae by using PCR. Applied And Environmental. Microbiology. 2001:2966-2972
83. Yoshiyuki, N.,Kozi, A. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant & Cell Physfol, 22(5):867-880
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30．徐雍皋，姚成林，方中达．麦类赤霉病病麦毒素对小麦毒性作用的研究。南京农业大学学报，1990，13(2)：58—63
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53. Anja, I.P.,Berne, L.J.,Marja, L.P..Purification and properties of an alkaline proteinase of Fusarium culmorum. Eur.J.Biochem, 2002, 269:798-807
54. Booth, C..Physical and biochemical techniques in the identification of Fusarium. J. Gen.Microbiol, 1966,42:7-8
55. Booth, C..Fusarium laboratory, guid to the identification of the major species. Comonwealth Mycological Institute, 1977
56. Buerstmayr, H.,Lemmes, M., Hartle, L.,Doldi, L. Steiner, B., Stierschneider, M.,Ruckenbauer, P..Molecular mapping of QTLS for Fusarium head blight resistance in spring wheat. Theor Appl Genet, 2002, (104):84-92
57. Cakmak, I.,Marschner, H..Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase and glutathione reductase in bean leaves. Plant Physiol, 1992,1222-1227
58. Carter, J.P.,Rezanoor, H.N, Holden, D.,Dejardins, A.E.,Plattner, R.D.,Nichol son, P..Variation in pathogenicity associated with the genetic diversity of Fusarium graminearum. Plant Pathology, 2000,49:452-460
59. Doohan, F.M.,Parry, D.W.,Jenkinson, P.,Nicholson, P..The use of species specific PCR based assays to analyse Fusarium ear blight of wheat. Plant pathology,1998,47:197-205
60. Edel, V. Steinberg, N.,Gautheron, N.,Alabouvette, C..Evaluation of restriction analysis of polymerase chain reaction (PCR)-amplified ribosomal DNA for the identification of Fusarium species.Mycological Research, 101(2):179-187
61. Gorden, W. L..The Occurrence of Fusarium Species in Canada Ⅱ Prevalence and Taxonomy of Fusarium Species in Cereal seed. Canada Jour Bot, 1952,30:209-251
62. John, G. K.,Williams..DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research, 22(18): 6531-6535
63. Linnemannstons, P.,Voss, T.,Hedden, P.,Gaskin, P.,Tudzynski, B.. Deletions in the gibberellin biosynthesis gene cluster of Gibberella fujikuroi by restriction enzyme mediated integration and conventional transformation mediated mutagenesis. Appl-environ-microbiol, 1999, 65(6):2558-2564
64. Miller, J.D., Arnison, P. G..Deoxynivalenol and experimental in Fusarium graminearum infection of wheat.Cananda Journal of Plant Pathology, 1986,8:147-152
65. Miedaner, T.,Schilling, A. G..Genetic variation of aggressiveness in individual field populations of Fusarium graminearum and Fusarium culmorum tested on young plants of winter rye. European Journal of Plant Pathology. 1996,102:823-830
66. McMullen, M.P.,Schartz, B.,Stover, R. Studies of fungicide efficacy, application timing, and application technologies to reduce Fusarium head blight and deoxynivalenol. Cereal Research Communications, 1997,25:779-780
67. Mullins, E.D.,Chen, X.,Romaine, P.,Raina, R.,Geiser, D.M.,Kang, S..Agrobacte rium-mediated transformation of Fusarium oxysporum:an efficient tool for insertional mutagenesis and gene transfer. Phytopathology, 2001,91(2):173-180
68. Nirenberg. H.I..A simplifiied method for identifying Fusarium spp. occurring on wheat. Cananda Journal of BOT 1981, 59:1599-1609
69. Nicholson, P.,Simpson, D.R.,Weston, G.,Rezanoor, H.N.,Lees, A.K.,Parry, D.W. Joyce, D..Detection and quantification of Fusarium culmorum and Fusarium gaminearum in cereals using PCR assays. Physiological and Molecular Plant Pathology, 1998, 53:17—37
70. Nightingale, M.J.,Marchylo,B.A.,Clear, R.M.,Dexter, J.E.,Preston, K.R..Fus
arium Head Blight:Effect of fungal proteases on wheat storage proteins. Cereal Chemistry,1999, 76(1):150-158
71. Nelson, P.E..Fusarium disease, biology, and taxonomy. The Pennsylvania state university press, 1983, 1-193
72. Nelson, P.E.,Toussoun, T.A.,Marasas, W.F.O..Fusarium species:an illustrated manual for identification. The Penn. State Uni. Press:193
73. Namiki, F.,Matsunaga, M.,Okuda, M.,Inoue, I..Mutation of an arginine biosynthesis gene causes reduced pathogenicity in Fusarium oxyporum f. sp. melonis.Molecular plant microbe interactions, 2001,14 (4): 580-584
74. Nicol, J.J.,Frank, J.M.,Anke, P.L.,Wilhelm, S..Mating, conidiation and pathogenicity of Fusarium graminearum, the main caual agent of the head blight disease of wheat, are regulated by the MAP kinase gpmkl. Curr. Genet, 2003,43:87-95
75. Oreste ,A.,Laura, D.G.,Franca, T.,Rosalla, L..Changes in the Ascorbate System during seed Development of Vicia faba L. Plant Physiol, 1992,99:235-238
76. Prashant, K.M.,Roland, T.V.,Alastair, C..Development of a PCR based assay for rapid and reliable identification of pathogenic Fusarium. Science direct-FEMS Microbiology Letters, 2003,218(2):329-332
77. Schroeder, H.W.,Christensen, J.J..Factors affecting resistance of wheat scab caused by Gibberella zeae. Phytopathology, 1963, 53:831-838
78. Schilling, A.G, Moller, E.M.,Geiger, H.H..Polymerase chain restriction based assays for species detection of Fusarium culmorum, F. graminearum, and F. avenaceum. Phytopathology, 1996,86(5):515-522
79. Schilling, A.G, Moller, E.M.,Geiger, H.H..Molecular variation and genetic structure in field populations of Fusarium species causing head blight in wheat. Cereal Research Communications, 25,549-554.
80. Sona, J.,Kouichi, A.,Takuya, K.,Tomizo, O.,Renkichi, T..The G protein subunit FGB1 regulates development and pathogenicity in Fusarium oxysporum. Curr Genet, 2003,43:79-86
81. Turner, A.S.,Lees, A.K.,Rezanoor, H.N.,Nicholson, Refinement of PCR detection of Fusarium avenaceum and evidence from DNA marker studies for phenetic relatedness to Fusarium tricinctum. Plant Pathology, 1998,47:278-288
82. Theresa, L.,Woongoh, D.,Seonkim, H.,Jungkwan, L..Identification of Deoxynivalenol and Nivalenol producing chemotypes of Gibberella Zeae by using PCR. Applied And Environmental. Microbiology. 2001:2966-2972
83. Yoshiyuki, N.,Kozi, A. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant & Cell Physfol, 22(5):867-880