禾谷镰刀菌TEP1基因敲除及功能研究
|
摘要
禾谷镰刀菌(Fusarium graminearum)是引发我国小麦赤霉病的优势种之一。该病菌侵染植物的过程包括孢子的产生与寄主表面的附着、孢子萌发以及侵染菌丝的形成与扩展等关键阶段。这些阶段也是许多其它植物病原真菌所共有的,缺少其中任何一步将导致病菌不能侵染寄主。因此,研究禾谷镰刀菌这些发育阶段的分子基础不仅在理论上有助于阐明病原真菌致病性的分子机理,在应用上也有助于发现筛选和设计新型农药的靶标基因。
近年来全球对禾谷镰刀菌开展了大量的研究工作,并取得了一系列重大进展。但由于病菌在侵染过程中产生毒素,使得人们至今对禾谷镰刀菌的致病机理及寄主的抗病机制仍缺乏全面的了解与认识。目前,对禾谷镰刀菌的研究热点逐渐集中在弄清其致病机理和发现新的药物作用靶位点。本研究中,作者鉴定了一个控制禾谷镰刀菌分生孢子产生的新基因TEP1,并发现该基因与磷脂酰肌醇信号通路和致病性有关。具体结果如下:
首先,作者通过构建pMDa+b+G418敲除质粒,原生质体转化,转化子抗药性筛选和基因型鉴定,最终得到两个Fgtep1-t敲除体。与野生型PH-1菌株相比,发现Fgtep1-t缺失株对锂盐和3-磷酸磷脂酰肌醇(PI3)激酶抑制剂—涡曼青霉素敏感性显著增加,分生孢子产量下降约56%,对寄主小麦的致病力也下降了约38.5%。
为了进一步验证FgTEP1基因的生物学功能,作者构建了pUC-hph-FgTEP1回复质粒,并对Fgtep1 -t缺失株进行了回复实验。表型回复实验说明,包含该基因完整启动子和编码区的一个3.4kb的DNA片段能够使缺失株Fgtep1-t在锂盐和涡曼青霉素耐受性、分生孢子产量以及致病力等方面均恢复到野生型水平。这些结果证明:Fgtep1-t的突变表型是由于FgTEP1基因的缺失而引起的。
此外,本研究还运用了比较基因组学方法,探索了FgTEP1基因在模式真菌酿酒酵母中的互补功能。作者通过RT-PCR获得了不含内含子的FgTEP1 cDNA序列,并将其克隆到带有强启动子ADH的酵母表达载体YPB1-ADH1上,转化双倍体酿酒酵母Sctep1/tep1缺失株。互补实验结果表明,FgTEP1基因能够使双倍体酿酒酵母Sctep1/tep1缺失株对涡曼青霉素的耐受性恢复到野生型水平。这说明FgTEP1与ScTEP1都是磷脂酰肌醇信号通路调控中的功能蛋白,在生物学功能上存在相似性。
本研究对于进一步研究FgTEP1基因调控禾谷镰刀菌磷脂酰肌醇信号通路、分生孢子的产生以及致病机理奠定了基础,对于广谱新型杀菌剂的开发具有重要的指导意义。
Fusarium graminearum is the major causal agent of Fusarium head blast (FHB) disease on wheat, The infection process of F. graminearum in host plants includes conidia production and surface attachment,conidia germination,infectious hypha differentiation and spreading.All these stages are common in many other plant pathogenic fungi.Therefore,studying on the molecular basis of these developmental stages in F. graminearum will help to elucidate the molecular mechanism of fungal pathogenicity and to discover candidate target genes for screening and designing novel fungicides.
In recent years, F.graminearum was carried out a great deal of studies in the world and made a series of significant progress. However, due to toxins in the infection process, making it so far on the pathogenesis of F.graminearum and resistance mechanisms of host is still a lack of comprehensive understanding and awareness.At present,the studies on F. graminearum have been focused on the pathogenesis and the discovery of drug targets. In this study,a novel gene FgTEP1 required for conidiation was identified and linked to the phosphatidylinositol pathway and the pathogenicity of F. graminearum.The results in details are as the following:
Firstly,we constructed the knock-out plasmid pMDa+b+G418 for the FgTEP1 gene and successfully deleted the ORF of FgTPE1 gene by homologous recombination. Compared with wild-type strain PH-1, the knock-out strain Fgtep1-t are sensitive to both lithium ions and phosphatidylinositol-3-phosphate(PI3)kinase inhibitor wortmannin,the conidia production of Fgtep1-t is less than 56% and its virulence is less about 38.5%.To further validate the function of FgTPE1 gene,we constructed the complemental plasmid pUC-hph-FgTEP1 for gene complement to Fgtep1-t.The gene complement results showed that all the capacities of the resistance to wortmannin and to lithium ions,the conidia production and the pathogenicity in the complementary transforments were restored to the level of those in the wild-type strain PH-1 by 3.4kb DNA fragment,including functional promoter and the entire coding region of the FgTPE1.These results proved that the mutant phenotype was caused by the detection of FgTPE1.
This study also exploits the possible complementary function of the FgTEP1 gene in Saccharomyces cerevisiae. We cloned the cDNA sequence of the FgTEP1 gene into a yeast expression vector YPB1-ADH1 with a strong promoter of ADH1, the alcohol dehydrogenase gene. As a result, we have found that the FgTEP1 cDNA sequence could complement the function of ScTEP1 in wortmannin tolerance, which indicates that the FgTEP1 and ScTEP1 genes are functional homologues and linked to the phosphatidylinositol pathway.
This study provides a basis for further studies on the phosphatidylinositol pathway, conidia production and virulence of Fusarium graminearum. It also provides an important guiding significance for the development of a new type of broad-spectrum fungicide.
近年来全球对禾谷镰刀菌开展了大量的研究工作,并取得了一系列重大进展。但由于病菌在侵染过程中产生毒素,使得人们至今对禾谷镰刀菌的致病机理及寄主的抗病机制仍缺乏全面的了解与认识。目前,对禾谷镰刀菌的研究热点逐渐集中在弄清其致病机理和发现新的药物作用靶位点。本研究中,作者鉴定了一个控制禾谷镰刀菌分生孢子产生的新基因TEP1,并发现该基因与磷脂酰肌醇信号通路和致病性有关。具体结果如下:
首先,作者通过构建pMDa+b+G418敲除质粒,原生质体转化,转化子抗药性筛选和基因型鉴定,最终得到两个Fgtep1-t敲除体。与野生型PH-1菌株相比,发现Fgtep1-t缺失株对锂盐和3-磷酸磷脂酰肌醇(PI3)激酶抑制剂—涡曼青霉素敏感性显著增加,分生孢子产量下降约56%,对寄主小麦的致病力也下降了约38.5%。
为了进一步验证FgTEP1基因的生物学功能,作者构建了pUC-hph-FgTEP1回复质粒,并对Fgtep1 -t缺失株进行了回复实验。表型回复实验说明,包含该基因完整启动子和编码区的一个3.4kb的DNA片段能够使缺失株Fgtep1-t在锂盐和涡曼青霉素耐受性、分生孢子产量以及致病力等方面均恢复到野生型水平。这些结果证明:Fgtep1-t的突变表型是由于FgTEP1基因的缺失而引起的。
此外,本研究还运用了比较基因组学方法,探索了FgTEP1基因在模式真菌酿酒酵母中的互补功能。作者通过RT-PCR获得了不含内含子的FgTEP1 cDNA序列,并将其克隆到带有强启动子ADH的酵母表达载体YPB1-ADH1上,转化双倍体酿酒酵母Sctep1/tep1缺失株。互补实验结果表明,FgTEP1基因能够使双倍体酿酒酵母Sctep1/tep1缺失株对涡曼青霉素的耐受性恢复到野生型水平。这说明FgTEP1与ScTEP1都是磷脂酰肌醇信号通路调控中的功能蛋白,在生物学功能上存在相似性。
本研究对于进一步研究FgTEP1基因调控禾谷镰刀菌磷脂酰肌醇信号通路、分生孢子的产生以及致病机理奠定了基础,对于广谱新型杀菌剂的开发具有重要的指导意义。
Fusarium graminearum is the major causal agent of Fusarium head blast (FHB) disease on wheat, The infection process of F. graminearum in host plants includes conidia production and surface attachment,conidia germination,infectious hypha differentiation and spreading.All these stages are common in many other plant pathogenic fungi.Therefore,studying on the molecular basis of these developmental stages in F. graminearum will help to elucidate the molecular mechanism of fungal pathogenicity and to discover candidate target genes for screening and designing novel fungicides.
In recent years, F.graminearum was carried out a great deal of studies in the world and made a series of significant progress. However, due to toxins in the infection process, making it so far on the pathogenesis of F.graminearum and resistance mechanisms of host is still a lack of comprehensive understanding and awareness.At present,the studies on F. graminearum have been focused on the pathogenesis and the discovery of drug targets. In this study,a novel gene FgTEP1 required for conidiation was identified and linked to the phosphatidylinositol pathway and the pathogenicity of F. graminearum.The results in details are as the following:
Firstly,we constructed the knock-out plasmid pMDa+b+G418 for the FgTEP1 gene and successfully deleted the ORF of FgTPE1 gene by homologous recombination. Compared with wild-type strain PH-1, the knock-out strain Fgtep1-t are sensitive to both lithium ions and phosphatidylinositol-3-phosphate(PI3)kinase inhibitor wortmannin,the conidia production of Fgtep1-t is less than 56% and its virulence is less about 38.5%.To further validate the function of FgTPE1 gene,we constructed the complemental plasmid pUC-hph-FgTEP1 for gene complement to Fgtep1-t.The gene complement results showed that all the capacities of the resistance to wortmannin and to lithium ions,the conidia production and the pathogenicity in the complementary transforments were restored to the level of those in the wild-type strain PH-1 by 3.4kb DNA fragment,including functional promoter and the entire coding region of the FgTPE1.These results proved that the mutant phenotype was caused by the detection of FgTPE1.
This study also exploits the possible complementary function of the FgTEP1 gene in Saccharomyces cerevisiae. We cloned the cDNA sequence of the FgTEP1 gene into a yeast expression vector YPB1-ADH1 with a strong promoter of ADH1, the alcohol dehydrogenase gene. As a result, we have found that the FgTEP1 cDNA sequence could complement the function of ScTEP1 in wortmannin tolerance, which indicates that the FgTEP1 and ScTEP1 genes are functional homologues and linked to the phosphatidylinositol pathway.
This study provides a basis for further studies on the phosphatidylinositol pathway, conidia production and virulence of Fusarium graminearum. It also provides an important guiding significance for the development of a new type of broad-spectrum fungicide.
引文
1.陈顺和,杨士敏,张伯桥,季开桢,赵步洪,高德荣.小麦对赤霉病的抗扩展性鉴定方法的研究.中国农业科学,1994,27(2):45～49.
2.方中达.植病研究方法[M ].第3版.北京:中国农业出版社,1998.
3.康振生,黄丽丽,H.BUCHENAUER,韩青梅,蒋选利.禾谷镰刀菌在小麦穗部侵染过程的细胞学研究.植物病理学报.2004,34(4):329～335.
4.李克昌.小麦赤霉病及其防治[M].第2版.上海:上海科技出版社,1982:120～123.
5.刘宗镇,陆仕华,黄晓敏,姚泉洪,魏春妹,薛伟龙.DON毒素的类生长激素活性与小麦赤霉病.上海农业学报,1993,9(2):92～96.
6.陆维忠,程顺和,王裕中.小麦赤霉病研究[M].北京:科学出版社,2001,2～39.
7.汪天虹,钟耀华,王晓利.丝状真菌基因组学研究进展.Science & Technology Review,2007,25(11):48～51.
8.武爱波,廖玉才.禾谷镰刀菌(Fusarium graminearum)致病力鉴定、毒素检测及其分子生物学研究[D].武汉:华中农业大学,2005.
9.武爱波,赵纯森,廖玉才,马新霞,瞿波,陆维忠.禾谷镰刀菌接种小麦幼芽及致病力鉴定的初步研究[J].植物病理学报,2001,31(4):371～372.
10.姚金宝,陆维忠.中国小麦抗赤霉病研究进展.江苏农业学报,2000,16(4):242～248.
11.于钦亮,李成云.禾谷镰刀菌基因组中含寄主靶向模体分泌蛋白功能的初步分析[J].生物技术通报,2008,1:160～165.
12.俞刚,陈利锋,姚红燕.柴一秋.脱氧雪腐镰刀菌烯醇在小麦赤霉病病程中的作用.植物病理学报,3003,33(1):40～43.
13.袁婷露,曹秀云.禾谷镰刀菌致病力和致病基因的研究进展.安徽农业科学,2008,36(14):5915～591.
14.周明国,叶钟音,刘经芬.南京市郊灰霉病菌对苯并咪唑类杀菌剂田间抗性检测.南京农业大学学报,1987,11(2):53～58.
15. AdamasT.H.,Wieser J.and YuJ.H.Asexual sporrulationin Aspringullas nidiulans.Microbio.Mol.Biol. Rev.,1998,62:35～54.
16. Aibo Wu,Yucai Liao,et al.Comparative pathogenicity of Fusarium graminearum isolates from China revealed by wheat coleoptile and floret inoculations[J]. Mycopathologia,2005,160 (1): 75~83.
17. AlexanderN J,McCormick S P,Hohn TM.Tri12,a trithothecene efflux pump from Fusarium sporotrichioides: gene isolation and expression in yeast [ J ].Mol Gen Genet,1999,261: 977~984.
18. Anderson J A,Stack R W,Liu S,Waldron B L,Fild A D,Coyne C,Moreno-Sevilla B,Mitchell F J,Song Q J,Cregan P B,Frohberg R C.DNA markers for Fusarium head blight resistance QTL in two wheat populations.Theor Appl Genet,2001,102:1164~1168.
19. Andrew G T,Gulnara F G,Andrew W P.A novel regulatory gene,Tri10, controls trichothecene toxin p roduction and gene exp ression[ J ].Appl EnvironMicrobiol,2001,67 (11):5294~5302.
20. Bennett, J.W. and Klich, M.Mycotoxins. Microbiol,2003,16:497~516.
21. Birzele B,Prange A.Deoxynivalenol and ochratoxin A in German wheat and changes of level in relation to storage parameters.Food Addit Contam,2000,17(12):1027~1035.
22. Brown DW,Cheung F, Proctor RH, Butchko RAE, Zheng L, Lee Y, Utterback T, Smith S, Feldblyum T,Glenn AE,Plattner RD,Kendra DF,Town CD,Whitelaw CA.Comparative analysis of 87000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides[J].Fungal Genet Biol,2005,42: 848~861.
23. Bushnell, W.R., Hazen, B.E. and Pritsch, C.. Histology and physi-ology of Fusarium head blight. In Fusarium Head Blight of Wheat and Barley (Leonard, K.J. and Bushnell, W.R., eds).St. Paul,MN: APS Press, 2003, pp. 44~83.
24. Chen L F.McCormick S P,Hohn T M.Altered regulation of I5-Acetyldedeoxynivalenol production in Fusarium graminearum.Appl Environ Microbio,2000,May:2062~2065.
25. Christina A.Cuomo,Ulrich Güldener,H.Corby Kistler. The Fusarium graminearum Genome Reveals a Link Between Localized Polymorphism and Pathogen Specialization[J].Science, 2007,317: 1400~ 1402.
26. Daren W. Brown, Robert A. E. Butchko & Robert H. Proctor Fusarium genomic resources:Tools to limit crop diseases and mycotoxin Conta分钟ation[J]. Mycopathologia,2006,162: 191~199.
27. Donnell.0 K,Kistler H C,TACKE B K,et a1.Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum,the fungus causing wheat scab[J].Proc Natl Acad Sci USA 1997,7:7905~7910.
28. Edwars S G,Pirgozzliev S R,Hare M C.Quantification of trichothecene-producing Fusarium species in harvested grain by pompetitive PCR to determine efficacies of fungicides against Fusarium Head Blight of wheat winter.Appl Environ Microbiol,2001,Apr:1575~1580.
29. Gale. L.R. Population biology of Fusarium species causing head blight of grain crops. In Fusarium Head Blight of Wheat and Barley (Leonard, K.J. and Bushnell, W.R., eds).St. Paul, MN: APS Press, 2003.pp.120~143.
30. Goswami RS, Xu JR, Trail F, Hilburn K, Kistler HC. Genomic analysis of host-pathogen interaction between Fusarium graminearum and wheat during early stages of disease development. Microbiology.2006,152(Pt 6):1877~1890.
31. Jansen C, von Wettstein D, Schafer W, Kogel KH, Felk A, Maier FJ. Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum. Proc Natl Acad Sci U S A. 2005,102(46):16892~16897.
32. Jennifer Heymont, Ludmilla Berenfeld, Jennifer Collins, Alexandra Kaganovich, Bradford Maynes, Aaron Moulin,Irina Ratskovskaya, Pak P. Poon, Gerald C. Johnston, Margarita Kamenetsky, John DeSilva, Hong Sun,Gregory A. Petsko, and JoAnne Engebrecht.TEP1, the yeast homolog of the human tumor suppressor gene PTEN/MMAC1/TEP1, is linked to the phosphatidylinositol pathwayand plays a role in the developmental process of sporulation.PNAS,2000,97(23):12672~12677.
33. Jungkwan Lee,James E. Jurgenson, John F. Leslie,and Robert L. Bowden. Alignment of Genetic and Physical Maps of Gibberella zeae[J].Applied and Environmental Microbiology, 2008,8:2349~2359.
34. Jurgenson JE, Zeller KA, Leslie JF. Expanded genetic map of Gibberella moniliformis (Fusarium verticillioides)[J].Appl Environ Microbiol,2002,68:1972~1979.
35. Kimura, M., Tokai, T., O’Donnell, K., Ward, T.J., Fujimura, M., Hamamoto, H., Shibata, T. and Yamaguchi, I.The trichothecene biosynthesis gene cluster of Fusarium graminearum F15 contains a limited number of essential pathway genes and expressed non-essential genes. FEBS Lett.,2003, 539,105~110.
36. KimuraM, Kaneko I, KomiyamaM.Trichothecene 3-O-acetyltransferase p rotects both the producing organism and transformed yeast from related mycotoxins.Cloning and characterization of Tri101 [ J ]. Biol Chem,1998,273 (3) :1654~1661.
37. Lee T, Han Y2K, Kim K2H. Tri13 and Tri7 determine deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae [ J ]. Appl EnvironMicrobiol, 2002, 68 (5) : 2148~2154.
38. Liang P,Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J]. Science,1992,257(5072):967~971.
39. Markell, S.G.and Francl, L.J. Fusarium head blight inoculum: species prevalence and Gibberella zeae spore type. Plant Dis.2003.87,814~820.
40. Mcmullen M,Jones R,Gallenberg D.Scab of wheat and barly:A re-emerging disease of devastating impact[J].Plant Dis,1997,81:1340~1348.
41. McMullen, M., Jones, R. and Gallenberg, D. 1997. Scab of wheat and barley: a re-emerging disease of devastating impact.Plant Dis.81,1340~1348.
42. Nicole J,Jenczmionka,Frank J.Mating,conidiation and pathogenicity of Fusarium graminearum.the main causal agent of the head-blight disease of wheat,are regulated by the MAPkinase gpmkl[J]. Curr Genet,2003,43:87~95.
43. O’Donnell, K., Kistler, H.C., Tacke, B.K. and Casper, H.H. Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab. Proc. Natl Acad. Sci. USA, 2000, 97, 7905~7910.
44. Parry D W,Nicholson P,Mcleod L.Fusarium ear blight(scab)in small grain cereals review[J].Plant Pathology,1995,44:207~238.
45. Parry,D.W.,Jenkinson,P.and McLeod,L.1995.Fusarium ear blight (scab) in small grains review. Plant Pathol.44,207~238 .
46. Proctor B H,Hohn T M,Mccormick S P.Reduced virulence of Gibberella zeae caused by disruption of a tfichothecene toxin biosynthetic gene[J].MPMI,1995,8(4):593~601.
47. Proctor R H, Hohn TM, McCormick S P. Restoration of wild type virulence to Tri5 disrup tion mutants of Gibberella zeae via gene reversion and mutant comp lementation [J].Microbiology, 1997,143:2583~2591.
48. Proctor R H,Hohn R M,McCormick S P.Reduced virulence of Gibberella zeae caused by disruptionof a trichothecene toxin biosynthetic gene.Mol Plant-Microbe Int,1995a,8:593~601.
49. Proctor R H,Hohn T M,McCormick S P,Desjardins A E.TRI6 encides an unusual zinc finger protein involved in regulation of trichothecene biosynthesis in fusarium sportrichioides.Appl Environ Microbiol,1995b,61:1923~1930.
50. R.S.Goswami,H.C.Kistler.Heading for disaster:Fusarium graminearum on cereal crops[J]. Mol. Plant Pathol,2004,5(6):515~525.
51. Seong, K.-Y. et al.Conidial ger分钟ation in the filamentous fungus Fusarium graminearum[J]. Fungal Genet. Biol,2007,10(1016).
52. Shinchi Oide,Gillian T.B.NPS6 , Encoding a nonribosomal peptide synthetase involved in siderophore mediated iron metabolism,is a conserved rulence determinant of plant pathogenic ascomycetes[J].Plant Cel1,2006,18:2836~2853.
53. Shinichi Oide,Gillian T.B.NPS6,Encoding a nonribosomal peptide synthetase involved in siderophore mediated iron metabolism,is a conserved rulence determinant of plant pathogenic ascomycetes[J].Plant Cel1.2006,18:2836~2853.
54. Shun-wen Lu.A novel class of gene controlling virulence in plant pathogenic ascomycete fungi[J].PNAS,2003,100(10):5980~5985.
55. Sonja S.Klemsdal,Karen R.Bone,Rasmus J.N.Frandsen, Thomas Johansen,Ulf Thrane, and Henriette Giese.The PKS4 Gene of Fusarium graminearum Is Essential for Zearalenone Production[J]. Applied and environmental microbiology, June 2006, 72(6):3924~3932.
56. Stack, R.W. Return of an old problem: Fusarium head blight of small grains. 1999. APSnet feature
57. Suga, H., Gale, L.R. and Kistler, H.C. 2004. Development of VNTR mark-ers for two Fusarium graminearum clade species. Mol. Ecol. Notes, 4, 468~470.
58. Takeshi Tokai,Naoko Takahashi-Ando,Makoto Kimura.Fusarium Tri4 encodes a key mulfifuncfional cytochrome P450 monooxygenase for four consecutive oxygenztion steps in trichothecene biosynthesis[J].Biochemical and Biophysical Research Communications,2007,353:412~417.
59. Tanaka T.,A.Hasegawa,S.Yamamota, Y.Sugiura,et al.World-wide contamination of cereals by Fusarium mycotoxins nivalenol, deoxynivalenol, and zearalenone [J]. Agric. Food Chem,1988 ,36: 979~983.
60. Trail F, Xu JR, San Miguel P, Halgren RG, Kistler HC.Analysis of expressed sequence tags from Gibberella zeae(anamorph Fusarium graminearum)[J]. Fungal Genet Biol,2003,38: 187~197.
61. Trail, F., Urban, M., Gaffoor, I., Mott, E., Andries, C. and Hammond-Kosack, K. Isolation and characterization of Fusarium graminearum mutants compromised in mycotoxin production and virulence. Fungal Genet. Newsletter, 2003a. 50 (Suppl.), 127.
62. Trail, F., Xu, H., Loranger, R. and Gadoury, D. Physiological and environmental aspects of ascospore discharge in Gibberella zeae. Mycologia, 2002:94,181~189.
63. Urban M,Daniels S,Mott E,Hammond-kosack,K E.Arabidopsis is susceptible to the cereal ear blight fungal pathogens Fusarium graminearum and Fusarium culmorum.Plant J,2002,32:961~966.
64. Vellaisamy,Ramamoorthy,Dilip M S.Two mitogen-activated protein.kinase signaling cascadesmediate basal resistance to antifungal plant defensins in Fusarium. graminearum[J].Cellular Microbiology,2007,9(6):1491~1506.
65. Windels, C.E. Economic and social impacts of Fusarium head blight: changing farms and rural communities in the northern Great Plains. Phytopathology, 2000:90,17~21.
66. Won bo Shim,Uma Shankar Sagaram,Yoon Echoi.FSR1 is essential for virulence and female fertility in Fusarium verticillioides and F.graminearum[J].Phytopathology, 2006,19(7):725~733.
67. Yang Z J,Ren Z L.Chromosomal disruption and genetic expression of Lophopyrum elongatum(Host) A.Love genes for adult plant resistance to stripe rust in wheat background.Gnent Crop Ev,2001,48:183~187.
68. Yong Sung Park,Ha Chin Sung . Physical and functional interaction of FgFtrl/FgFetl and FgFtr2/FgFet2 is required for iron uptake in Fusarium grammearum[J]. Biochemical Society.2007,27:102~108.
69. Zhanming HOU,Chaoyang XUE,Youliang PENG.A mitoge.activated protein kinase gene(MGV1)in fusarlum graminearum is required for female fertility.heterokaryon formation , and plant Infection[J].MPMI,2002,15(11):1119~1127.
2.方中达.植病研究方法[M ].第3版.北京:中国农业出版社,1998.
3.康振生,黄丽丽,H.BUCHENAUER,韩青梅,蒋选利.禾谷镰刀菌在小麦穗部侵染过程的细胞学研究.植物病理学报.2004,34(4):329～335.
4.李克昌.小麦赤霉病及其防治[M].第2版.上海:上海科技出版社,1982:120～123.
5.刘宗镇,陆仕华,黄晓敏,姚泉洪,魏春妹,薛伟龙.DON毒素的类生长激素活性与小麦赤霉病.上海农业学报,1993,9(2):92～96.
6.陆维忠,程顺和,王裕中.小麦赤霉病研究[M].北京:科学出版社,2001,2～39.
7.汪天虹,钟耀华,王晓利.丝状真菌基因组学研究进展.Science & Technology Review,2007,25(11):48～51.
8.武爱波,廖玉才.禾谷镰刀菌(Fusarium graminearum)致病力鉴定、毒素检测及其分子生物学研究[D].武汉:华中农业大学,2005.
9.武爱波,赵纯森,廖玉才,马新霞,瞿波,陆维忠.禾谷镰刀菌接种小麦幼芽及致病力鉴定的初步研究[J].植物病理学报,2001,31(4):371～372.
10.姚金宝,陆维忠.中国小麦抗赤霉病研究进展.江苏农业学报,2000,16(4):242～248.
11.于钦亮,李成云.禾谷镰刀菌基因组中含寄主靶向模体分泌蛋白功能的初步分析[J].生物技术通报,2008,1:160～165.
12.俞刚,陈利锋,姚红燕.柴一秋.脱氧雪腐镰刀菌烯醇在小麦赤霉病病程中的作用.植物病理学报,3003,33(1):40～43.
13.袁婷露,曹秀云.禾谷镰刀菌致病力和致病基因的研究进展.安徽农业科学,2008,36(14):5915～591.
14.周明国,叶钟音,刘经芬.南京市郊灰霉病菌对苯并咪唑类杀菌剂田间抗性检测.南京农业大学学报,1987,11(2):53～58.
15. AdamasT.H.,Wieser J.and YuJ.H.Asexual sporrulationin Aspringullas nidiulans.Microbio.Mol.Biol. Rev.,1998,62:35～54.
16. Aibo Wu,Yucai Liao,et al.Comparative pathogenicity of Fusarium graminearum isolates from China revealed by wheat coleoptile and floret inoculations[J]. Mycopathologia,2005,160 (1): 75~83.
17. AlexanderN J,McCormick S P,Hohn TM.Tri12,a trithothecene efflux pump from Fusarium sporotrichioides: gene isolation and expression in yeast [ J ].Mol Gen Genet,1999,261: 977~984.
18. Anderson J A,Stack R W,Liu S,Waldron B L,Fild A D,Coyne C,Moreno-Sevilla B,Mitchell F J,Song Q J,Cregan P B,Frohberg R C.DNA markers for Fusarium head blight resistance QTL in two wheat populations.Theor Appl Genet,2001,102:1164~1168.
19. Andrew G T,Gulnara F G,Andrew W P.A novel regulatory gene,Tri10, controls trichothecene toxin p roduction and gene exp ression[ J ].Appl EnvironMicrobiol,2001,67 (11):5294~5302.
20. Bennett, J.W. and Klich, M.Mycotoxins. Microbiol,2003,16:497~516.
21. Birzele B,Prange A.Deoxynivalenol and ochratoxin A in German wheat and changes of level in relation to storage parameters.Food Addit Contam,2000,17(12):1027~1035.
22. Brown DW,Cheung F, Proctor RH, Butchko RAE, Zheng L, Lee Y, Utterback T, Smith S, Feldblyum T,Glenn AE,Plattner RD,Kendra DF,Town CD,Whitelaw CA.Comparative analysis of 87000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides[J].Fungal Genet Biol,2005,42: 848~861.
23. Bushnell, W.R., Hazen, B.E. and Pritsch, C.. Histology and physi-ology of Fusarium head blight. In Fusarium Head Blight of Wheat and Barley (Leonard, K.J. and Bushnell, W.R., eds).St. Paul,MN: APS Press, 2003, pp. 44~83.
24. Chen L F.McCormick S P,Hohn T M.Altered regulation of I5-Acetyldedeoxynivalenol production in Fusarium graminearum.Appl Environ Microbio,2000,May:2062~2065.
25. Christina A.Cuomo,Ulrich Güldener,H.Corby Kistler. The Fusarium graminearum Genome Reveals a Link Between Localized Polymorphism and Pathogen Specialization[J].Science, 2007,317: 1400~ 1402.
26. Daren W. Brown, Robert A. E. Butchko & Robert H. Proctor Fusarium genomic resources:Tools to limit crop diseases and mycotoxin Conta分钟ation[J]. Mycopathologia,2006,162: 191~199.
27. Donnell.0 K,Kistler H C,TACKE B K,et a1.Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum,the fungus causing wheat scab[J].Proc Natl Acad Sci USA 1997,7:7905~7910.
28. Edwars S G,Pirgozzliev S R,Hare M C.Quantification of trichothecene-producing Fusarium species in harvested grain by pompetitive PCR to determine efficacies of fungicides against Fusarium Head Blight of wheat winter.Appl Environ Microbiol,2001,Apr:1575~1580.
29. Gale. L.R. Population biology of Fusarium species causing head blight of grain crops. In Fusarium Head Blight of Wheat and Barley (Leonard, K.J. and Bushnell, W.R., eds).St. Paul, MN: APS Press, 2003.pp.120~143.
30. Goswami RS, Xu JR, Trail F, Hilburn K, Kistler HC. Genomic analysis of host-pathogen interaction between Fusarium graminearum and wheat during early stages of disease development. Microbiology.2006,152(Pt 6):1877~1890.
31. Jansen C, von Wettstein D, Schafer W, Kogel KH, Felk A, Maier FJ. Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum. Proc Natl Acad Sci U S A. 2005,102(46):16892~16897.
32. Jennifer Heymont, Ludmilla Berenfeld, Jennifer Collins, Alexandra Kaganovich, Bradford Maynes, Aaron Moulin,Irina Ratskovskaya, Pak P. Poon, Gerald C. Johnston, Margarita Kamenetsky, John DeSilva, Hong Sun,Gregory A. Petsko, and JoAnne Engebrecht.TEP1, the yeast homolog of the human tumor suppressor gene PTEN/MMAC1/TEP1, is linked to the phosphatidylinositol pathwayand plays a role in the developmental process of sporulation.PNAS,2000,97(23):12672~12677.
33. Jungkwan Lee,James E. Jurgenson, John F. Leslie,and Robert L. Bowden. Alignment of Genetic and Physical Maps of Gibberella zeae[J].Applied and Environmental Microbiology, 2008,8:2349~2359.
34. Jurgenson JE, Zeller KA, Leslie JF. Expanded genetic map of Gibberella moniliformis (Fusarium verticillioides)[J].Appl Environ Microbiol,2002,68:1972~1979.
35. Kimura, M., Tokai, T., O’Donnell, K., Ward, T.J., Fujimura, M., Hamamoto, H., Shibata, T. and Yamaguchi, I.The trichothecene biosynthesis gene cluster of Fusarium graminearum F15 contains a limited number of essential pathway genes and expressed non-essential genes. FEBS Lett.,2003, 539,105~110.
36. KimuraM, Kaneko I, KomiyamaM.Trichothecene 3-O-acetyltransferase p rotects both the producing organism and transformed yeast from related mycotoxins.Cloning and characterization of Tri101 [ J ]. Biol Chem,1998,273 (3) :1654~1661.
37. Lee T, Han Y2K, Kim K2H. Tri13 and Tri7 determine deoxynivalenol- and nivalenol-producing chemotypes of Gibberella zeae [ J ]. Appl EnvironMicrobiol, 2002, 68 (5) : 2148~2154.
38. Liang P,Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction[J]. Science,1992,257(5072):967~971.
39. Markell, S.G.and Francl, L.J. Fusarium head blight inoculum: species prevalence and Gibberella zeae spore type. Plant Dis.2003.87,814~820.
40. Mcmullen M,Jones R,Gallenberg D.Scab of wheat and barly:A re-emerging disease of devastating impact[J].Plant Dis,1997,81:1340~1348.
41. McMullen, M., Jones, R. and Gallenberg, D. 1997. Scab of wheat and barley: a re-emerging disease of devastating impact.Plant Dis.81,1340~1348.
42. Nicole J,Jenczmionka,Frank J.Mating,conidiation and pathogenicity of Fusarium graminearum.the main causal agent of the head-blight disease of wheat,are regulated by the MAPkinase gpmkl[J]. Curr Genet,2003,43:87~95.
43. O’Donnell, K., Kistler, H.C., Tacke, B.K. and Casper, H.H. Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab. Proc. Natl Acad. Sci. USA, 2000, 97, 7905~7910.
44. Parry D W,Nicholson P,Mcleod L.Fusarium ear blight(scab)in small grain cereals review[J].Plant Pathology,1995,44:207~238.
45. Parry,D.W.,Jenkinson,P.and McLeod,L.1995.Fusarium ear blight (scab) in small grains review. Plant Pathol.44,207~238 .
46. Proctor B H,Hohn T M,Mccormick S P.Reduced virulence of Gibberella zeae caused by disruption of a tfichothecene toxin biosynthetic gene[J].MPMI,1995,8(4):593~601.
47. Proctor R H, Hohn TM, McCormick S P. Restoration of wild type virulence to Tri5 disrup tion mutants of Gibberella zeae via gene reversion and mutant comp lementation [J].Microbiology, 1997,143:2583~2591.
48. Proctor R H,Hohn R M,McCormick S P.Reduced virulence of Gibberella zeae caused by disruptionof a trichothecene toxin biosynthetic gene.Mol Plant-Microbe Int,1995a,8:593~601.
49. Proctor R H,Hohn T M,McCormick S P,Desjardins A E.TRI6 encides an unusual zinc finger protein involved in regulation of trichothecene biosynthesis in fusarium sportrichioides.Appl Environ Microbiol,1995b,61:1923~1930.
50. R.S.Goswami,H.C.Kistler.Heading for disaster:Fusarium graminearum on cereal crops[J]. Mol. Plant Pathol,2004,5(6):515~525.
51. Seong, K.-Y. et al.Conidial ger分钟ation in the filamentous fungus Fusarium graminearum[J]. Fungal Genet. Biol,2007,10(1016).
52. Shinchi Oide,Gillian T.B.NPS6 , Encoding a nonribosomal peptide synthetase involved in siderophore mediated iron metabolism,is a conserved rulence determinant of plant pathogenic ascomycetes[J].Plant Cel1,2006,18:2836~2853.
53. Shinichi Oide,Gillian T.B.NPS6,Encoding a nonribosomal peptide synthetase involved in siderophore mediated iron metabolism,is a conserved rulence determinant of plant pathogenic ascomycetes[J].Plant Cel1.2006,18:2836~2853.
54. Shun-wen Lu.A novel class of gene controlling virulence in plant pathogenic ascomycete fungi[J].PNAS,2003,100(10):5980~5985.
55. Sonja S.Klemsdal,Karen R.Bone,Rasmus J.N.Frandsen, Thomas Johansen,Ulf Thrane, and Henriette Giese.The PKS4 Gene of Fusarium graminearum Is Essential for Zearalenone Production[J]. Applied and environmental microbiology, June 2006, 72(6):3924~3932.
56. Stack, R.W. Return of an old problem: Fusarium head blight of small grains. 1999. APSnet feature
57. Suga, H., Gale, L.R. and Kistler, H.C. 2004. Development of VNTR mark-ers for two Fusarium graminearum clade species. Mol. Ecol. Notes, 4, 468~470.
58. Takeshi Tokai,Naoko Takahashi-Ando,Makoto Kimura.Fusarium Tri4 encodes a key mulfifuncfional cytochrome P450 monooxygenase for four consecutive oxygenztion steps in trichothecene biosynthesis[J].Biochemical and Biophysical Research Communications,2007,353:412~417.
59. Tanaka T.,A.Hasegawa,S.Yamamota, Y.Sugiura,et al.World-wide contamination of cereals by Fusarium mycotoxins nivalenol, deoxynivalenol, and zearalenone [J]. Agric. Food Chem,1988 ,36: 979~983.
60. Trail F, Xu JR, San Miguel P, Halgren RG, Kistler HC.Analysis of expressed sequence tags from Gibberella zeae(anamorph Fusarium graminearum)[J]. Fungal Genet Biol,2003,38: 187~197.
61. Trail, F., Urban, M., Gaffoor, I., Mott, E., Andries, C. and Hammond-Kosack, K. Isolation and characterization of Fusarium graminearum mutants compromised in mycotoxin production and virulence. Fungal Genet. Newsletter, 2003a. 50 (Suppl.), 127.
62. Trail, F., Xu, H., Loranger, R. and Gadoury, D. Physiological and environmental aspects of ascospore discharge in Gibberella zeae. Mycologia, 2002:94,181~189.
63. Urban M,Daniels S,Mott E,Hammond-kosack,K E.Arabidopsis is susceptible to the cereal ear blight fungal pathogens Fusarium graminearum and Fusarium culmorum.Plant J,2002,32:961~966.
64. Vellaisamy,Ramamoorthy,Dilip M S.Two mitogen-activated protein.kinase signaling cascadesmediate basal resistance to antifungal plant defensins in Fusarium. graminearum[J].Cellular Microbiology,2007,9(6):1491~1506.
65. Windels, C.E. Economic and social impacts of Fusarium head blight: changing farms and rural communities in the northern Great Plains. Phytopathology, 2000:90,17~21.
66. Won bo Shim,Uma Shankar Sagaram,Yoon Echoi.FSR1 is essential for virulence and female fertility in Fusarium verticillioides and F.graminearum[J].Phytopathology, 2006,19(7):725~733.
67. Yang Z J,Ren Z L.Chromosomal disruption and genetic expression of Lophopyrum elongatum(Host) A.Love genes for adult plant resistance to stripe rust in wheat background.Gnent Crop Ev,2001,48:183~187.
68. Yong Sung Park,Ha Chin Sung . Physical and functional interaction of FgFtrl/FgFetl and FgFtr2/FgFet2 is required for iron uptake in Fusarium grammearum[J]. Biochemical Society.2007,27:102~108.
69. Zhanming HOU,Chaoyang XUE,Youliang PENG.A mitoge.activated protein kinase gene(MGV1)in fusarlum graminearum is required for female fertility.heterokaryon formation , and plant Infection[J].MPMI,2002,15(11):1119~1127.