摘要
盾壳霉(Coniothyrium minitans)是核盘菌(Sclerotinia sclerotiorum)的重寄生真菌,在世界范围内广泛分布,在菌核病的生物防治中有着非常重要的作用。本论文主要针对碳源,氮源和环境pH对盾壳霉产生抗真菌物质(AFS)的调控及其分子机理进行了研究。
研究发现盾壳霉菌株Chy-1在改良的查比培养基(MCD)中培养时能稳定产生AFS;而在马铃薯葡萄糖液体培养基(PDB)中培养时却不能产生AFS。为了进一步研究盾壳霉在MCD和PDB培养液中生长时产生AFS的差异,研究了营养和环境pH值对盾壳霉产生AFS的影响。结果显示:盾壳霉培养物滤液对核盘菌菌丝生长的抑制率与环境pH值相关,较适宜盾壳霉产生AFS的碳源或氮源,其培养液的环境pH值都较低。当盾壳霉在环境pH值缓冲为3的培养液中生长时,培养滤液对核盘菌菌丝生长的抑制率远远高于当环境pH值缓冲为6的培养液。
为了研究环境pH值调控盾壳霉产生抗真菌物质的分子机理,本文用简并引物和RACE技术从盾壳霉菌株Chy-1中克隆到了pH感应转录调控因子基因(cmpacC)和碳代谢抑制因子基因(cmcreA)。cmpacC的全长序列(GenBank Ace. No. FJ176399)含有一个开放阅读框和两个内含子。根据cmpacC的碱基序列推测cmpacC编码一个长度为596个氨基酸残基的蛋白质(GenBank Ace. No. ACN43303),其含有三个保守的锌指结构域。同时本文也用基因组步移的方法克隆到了长度为2740bp的cmpacC 5'端侧翼序列及长度为2730bp的cmpacC 3'端侧翼序列。其中,5’端侧翼序列含有5个PacC结合位点(5'-GCCAAG-3')和三个CreA结合位点(5'-SYGGRG-3')。确定了cmpacC转录时的加A位点。荧光定量RT-PCR的分析结果显示:当盾壳霉在不同环境pH条件下生长时,cmpacC、几丁质酶基因(cmchl)(?)口p-1,3-葡聚糖酶基因(cmgl)的表达量都随着环境pH值的上升而升高。当盾壳霉在以单糖为碳源的培养基中生长时,cmcreA基因的表达量低,而当碳源为微生物不易利用的多糖时,cmcreA的相对表达量高。利用农杆菌介导同源重组的方法对盾壳霉中cmpalF基因进行了敲除。结果显示:当cmpalF基因缺失后,cmpacC的表达量降低,在环境pH高于6的环境中生长速度显著低于野生型菌株。在缓冲环境pH值为6时,培养滤液对核盘菌菌丝生长的抑制率显著高于野生型菌株。
用数字化表达谱技术分析了盾壳霉菌株Chy-1在环境pH值为3和6条件下RNA转录水平的表达差异基因。共得到了1286个差异表达的基因,以盾壳霉RNA样品Cm3的基因表达量为参照时,RNA样品Cm6中有596个基因上调表达,690个基因下调表达。
上述结果为解释盾壳霉与核盘菌之间的互作以及进一步揭示盾壳霉产生AFS的分子机制奠定了基础。
Coniothyrium minitans (CM) is a promising biocontrol agent of Sclerotinia spp. including Sclerotinia sclerotiorum (SS). My research work mainly focused on carbon, nitrogen and ambient pH regulate the production of antifungal substances (AFS) by C. minitans and the molecular mechanism.
We found that the strain Chy-1 of C. minitans used in our lab could consistently produce AFS in modified Czapek-Dox (MCD) broth; however, production of AFS by this strain of C. minitans was hardly detected in potato dextrose broth (PDB). In order to elucidate the difference of antifungal activity (AA) of the mycoparasite C. minitans against S. sclerotiorum (AAcm-ss) of MCDcm and PDBcm, we studied the effects of ambient pH and nutritional factors on antifungal activity of C. minitans. The results showed that production of AACM-ss by C. minitans was closely related to ambient pH. The carbon or nitrogen suit for AFS production was closely related to low ambient pH of the medium. AACM-ss was much higher in C. minitans cultures buffered at pH 3 than that buffered at pH 6.
For the molecular mechanism of ambient pH regulate AFS production in C. minitans. We cloned a putative pH-responsive transcription factor gene (cmpacC) and a putative carbon repression gene (cmcreA) from strain Chy-1 of C. minitans by degenerate PCR and Rapid amplification of cDNA ends. The full-length DNA sequence of cmpacC (GenBank Ace. No. FJ176399) has one open reading frame (ORF) and two introns. cmpacC putatively encodes a polypeptide with 596 amino acid residues (GenBank Ace. No. ACN43303). The sequence of this polypeptide has three conserved zinc finger domains. Meanwhile, a 2,740 bp-long 5' flanking sequence of cmpacC and a 2,730bp-long 3'flanking sequence of cmpacC was obtained by genome walking. It contains five copies of PacC binding sites (5'-GCCAAG-3'). Real-time RT-PCR analysis indicated that with the increase of ambient pH from 3 to 8, expression of cmpacC, as well as the chitinase gene (cmchl) and theβ-1,3-glucanase gene (cmgl), increased consistently in C. minitans. When C.minitans grows in the media with the carbon source of Monosaccharide, the expression of cmcreA is relative lower than grow in the media with the carbon source of polysaccharide. We knocked out the cmpalF gene in C. minitans by Agrobacterium tumefacies-mediated homologous recombination. We found that when ambient pH of the media was higher than 6, cmpalF grew more slowly than the wild type strain Chy-1. AACM-ss of AcmpalF mutant was higher than Chy-1. The expression of cmpacC in cmpalF was significantly lower than that in strain Chy-1. We analysed the differentially expressed genes of C. minitans grew in the media of different ambient pH (pH 3 and pH 6). There were a total of 1286 genes of C. minitans differentially expressed,596 genes up-regulated and 690 genes down-regulated.
These results broadened our understanding of the interaction between C. minitans and S. sclerotiorum and laid a solid foundation for future studies to elucidate molecular mechanisms involved in AFS biosynthesis in C. minitans.
引文
1. 戴明丽.大豆菌核病发病规律及防治措施.中国农业推广,2003,1:53.
2. 傅廷栋.杂交油菜的育种与应用.武汉,湖北科技出版社,1995.
3. 高俊民,王双双,刘慧平,韩巨才.菌核重寄生菌盾壳霉生物学特性研究.山西农业大学学报,2002,71—75.
4. 高俊明,马丽娜,李欣,韩巨才.盾壳霉对核盘菌的拮抗作用研究.植物保护,2006,32(06):66-70
5. 黄剑华,陆瑞菊,张玉华.应用离体培养技术奠定不结球白菜耐热性及诱导耐热变异体.上海农业学报,1995,11:18-22
6. 姜道宏,李国庆,付艳平,易先宏,王道本.盾壳霉控制油菜菌核病再侵染及叶面存活的研究.植物病理学报2000,30(1):60—65.
7. 姜道宏,李国庆,易先宏,付艳平,王道本.盾壳霉所产抗细菌物质的特性.植物病理学报,1998,28(1):29-32
8. 姜道宏,李国庆,易先宏,王道本.影响盾壳霉寄生核盘菌菌核的几个生态因子的分析.植物病理学报,1997,27(1):47-52.
9. 姜道宏.核盘菌寄生菌盾壳霉(Coniothyrium minitans)的生物学及其寄生生态学研究.[硕士学位论文].武汉:华中农业大学图书馆,1995
10.李方球,官春云.油菜菌核病抗性鉴定,抗性机理及抗性遗传育种研究进展.作物学报,2001,3:85-92
11.李国庆,王道本,张顺和,但汉鸿.菌核寄生菌盾壳霉的研究Ⅰ生物学特性及在湖北省的自然分布.华中农业大学学报,1995,14-125-129
12.李国庆,王道本.拮抗细菌的筛选及其对油菜菌核病的防治效果.华中农业大学学报,1991,10:30-35
13.李金秀,陈文瑞,秦芸.油菜土壤中与核盘菌菌核存活有关的真菌.四川农业大学学报,1997,15:1-5
14.李荣禧,杨宝胜,严人沛等.小盾壳霉防治向日葵菌核病的初步研究.刘仪主编,植物病害研究与防治.中国农业科技出版社,1998,520-522
15.李子钦,曹丽霞、白全江,张庆平,徐丽敏.内蒙古向日葵主要病害及其防治对策。内蒙古农业科技,2004,S1:63—64。
16.罗宽,任新国,周必文等.油菜菌核病(Sclerotinia sclerotiorum)菌核上寄生真菌的研究,1987,3:40-45.
17.缪华军.盾壳霉抗杀菌剂农利灵突变菌株的获得、特性及其应用的研究.[硕士学位论文].武汉:华中农业大学图书馆,2004
18.潘以楼,汪智渊,吴汉章.油菜病害研究进展.江苏农业科学,1997,13:32-35
19.师俊玲,李寅,钱华丽,堵国成,陈坚.盾壳霉CCTCM203020的生长特性及其应用潜力.应用于环境生物学报,2004,10:798-802
20.石志琦,周明国,叶钟音,史建荣,陈怀谷,王裕中.油菜菌核病菌对多菌灵的抗药性监测。江苏农业学报,2000,16:226—229
21.王英超,李国庆,方媛,姜道宏.利用油菜秸秆发酵生产重寄生菌盾壳霉分生孢子的研究.中国生物防治,2006,22:308-312.
22.韦善君.盾壳霉在油菜花瓣上萌发的因子分析和防治核盘菌菌核病的研究.[硕士学位论文].武汉:华中农业大学图书馆,2000
23.薛根祥,李瑛,苟贤玉,钱爱林,林双喜.早春大棚瓜菜菌核病的发生与防治,2005,18(1):16-17.
24.杨蕊.生防菌盾壳霉产生抗真菌物质及其特性和防病潜力研究.[硕士学位论文].武汉:华中农业大学图书馆,2006.
25.杨新美.油菜菌核病Sclerotinia sclerotiorum在我国的寄主范围及生态特性的调查研究.植物病理学报,1959,15(2):111-112.
26.张硎.安徽省2003年油菜菌核病重发原因分析及防治技术.安徽农业科学,2003,31(5):746-747.
27.郑在武.菌核病在武穴油菜苗期发生严重.湖北植保,2005,(1):7
28.周而勋,王克荣和陆家云.栗疫病菌不同毒力菌株胞外酶活性和草酸产量的比较,真菌学报1996,15(3)182-187
29. Adams P B and Ayers W A. Ecology of Sclerotinia species. Phytopathology,1979,69:896-899
30. Amaike S, Ozga J A, Basu U and Strelkov S E. Quantification of ToxB gene expression and formation of appressoria by isolates of Pyrenophora tritici-repentis differing in pathogenicity Plant Pathol,2008, 57 (4):623-633
31. Arst H N Jr, Bignell E and Tilburn J. Two new genes involved in signalling ambient pH in Aspergillus nidulans. Mol Gen Genet,1994,245:787-790
32. Arst H N Jr, E Bignell and J Tilburn. Two new genes involved in signalling ambient pH in Aspergillus nidulans. Mol Gen Genet,1994,245:787-790.
33. Audic S and Claverie J M. The significance of digital gene expression profiles. Genome Res,1997, 7(10):986-95.
34. Bailey C and Arst H N Jr. Carbon catabolite repression in Aspergillus nidulans. Eur JBiochem,1975, 51:573-577.
35. Bailey C R, Penfold H A and Arst H N Jr. cis-dominant regulatory mutations affecting the expression of GAB A permease in Aspergillus nidulans. Mol Gen Genet,1979,169:79-83.
36. Benjamini Y and D Yekutieli. The control of the false discovery rate in multiple testing under dependency. The Annals of Statistics,2001,29:1165-1188.
37. Bennett A J, Leifert C and Whipps J M. Survival of Coniothyrium minitans associated with sclerotia of Sclerotinia sclerotiorum in soil. Soil Biology and Biochemistry,2006,38:164-172.
38. Bert P F, Jouan I, Tourvieille de Labrouhe D, Serre F, Philippon J, Nicolas P and Vear F. Comparative genetic analysis of quantitative traits in sunflower (Helianthus annuus L.).2. Characterization of QTL involved in developmental and agronomic traits. TheorAppl Genet,2003,107:181-189.
39. Boland G J and Hall R. An Index of plant hosts of Sclerotinia sclerotiorum. Can J Plant Pathol,1994, 16:93-108.
40. Boland G J and Hall R. Index of plant hosts of Sclerotinia sclerotirum. Can J of Plant Pathol,1990, 16:93-108.
41. Budge S P and Whipps J M.Glasshouse trials of Coniothyrium minitans and Trichoderma species for the biological control of Sclerotinia sclerotiorum in celery and lettuce. Plant Pathol,1991,40:59-66.
42. Caddick M X and Arst H N Jr. Structural genes for phosphatases in Aspergillus nidulans. Genet Res 1986,47:83-91.
43. Caddick M X, Brownlee A G and Arst H N Jr. Regulation of gene expression by pH of the growth medium in Aspergillus nidulans. Mol Gen Genet,1986,203:346-353.
44. Caddick M X, Brownlee A G, and Arst H N Jr. Phosphatase regulation in Aspergillus nidulans: responses to nutritional starvation. Genet Res,1986,47:93-102.
45. Campbell W A. A new species of Coniothyrium parasitic on sclerotia. Mycologia,1947,39:90-195.
46. Carpita N C, Gibeaut D M, Structural models of primary cell-walls in flowering plants:consistency of molecular structure with the physical properties of the walls during growth. Plant J,1993,3:1-30
47. Cheng J S, Jiang D H, Fu Y P, Li G Q, Peng Y L and Ghabrial S A. Molecular characterization of a dsRNA totivirus infecting the sclerotial parasite Coniothyrium minitans. Virus Res,2003,93 (1):41-50
48. Cober E R, Rioux S, Rajcan I, Donaldson P A and Simmonds D H. Partial resistance to white mold in a transgenic soybean line. Crop Sci,2003,43,92-95.
49. Cohen B L. Transport and utilization of proteins by fungi, p.411-430. In J. W. Payne (ed.), Microorganisms and nitrogen sources. John Wiley and Sons, Inc., New York, N.Y.1980
50. Cubero B and Scazzocchio C. Two different, adjacent and divergent zinc finger binding sites are necessary for CREA-mediated carbon catabolite repression in the proline gene cluster of Aspergillus nidulans.EMBO J,1994,13:407-415.
51. de Vrije T, Antoine N, Buitelaar R H, Bruckner S, Dissevelt M, Durand A, Gerlagh M, Jones E E, Liith P, Oostra J, Ravensberg W J, Renaud R, Rinzema A, Weber F J and Whipps J M. The fungal biocontrol agent Coniothyrium minitans:production by solid-state fermentation, application and marketing. Appl Microbiobiotechnol,2001,56:58-68
52. Denison S H, Negrete-Urtasun S, Mingot J M, Tilburn J, Mayer W A, Goel A, Espeso E A, Penalva M A, Arst H N Jr. Putative membrane components of signal transduction pathways for ambient pH regulation in Aspergillus and meiosis in Saccharomyces are homologous. Mol Microbiol,1998, 30:259-64
53. Denison S H, Orejas M and Arst H N Jr. Signaling of ambient pH in Aspergillus involves a cysteine protease. JBiol Chem,1995,270:28519-28522.
54. Denison S H, S Negrete-Urtasun, Mingot J M, Tilburn J, Mayer W A, Goel A, Espeso E A, Penalva M A and Arst H N Jr.. Putative membrane components of signal transduction pathways for ambient pH regulation in Aspergillus and meiosis in Saccharomyces are homologous. Mol Microbiol,1998, 30:259-264.
55. Diez E, Alvaro J, Espeso E A, Rainbow L, Suarez T, Tilburn J, Arst H N Jr and M A Penalva. Activation of the Aspergillus PacC zinc-finger transcription factor requires two proteolytic steps. EMBO J,2002,21:1350-1359.
56. Donaldson P A, Anderson T, Lane B G, Davidson A L and Simmonds D H. Soybean plants expressing an active oligomeric oxalate oxidase from the wheat gf-2.8 (germin) gene are resistant to the oxalate-secreting pathogen Sclerotinia sclerotiorum. Physiol Mole Plant Pathol,2001,59:297-307
57. Dowzer C E A and Kelly J M. Cloning of the creA gene from Aspergillus nidulans:a gene involved in carbon catabolite repression. Curr Genet,1989,15:457-459.
58. Dowzer C E A, and Kelly J M. Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans. Mol Cell Biol,1991,11:5701-5709.
59. Drysdale M R, Kolze S I, and Kelly J M. The Aspergillus niger carbon catabolite repressor encoding gene, creA. Gene,1993,130:241-245.
60. Espeso E A, and H N Arst Jr. On the mechanism by which alkaline pH prevents expression of an acid-expressed gene. MolCell Biol,2000,20:3355-3363.
61. Espeso E A, and M A Penalva. Three binding sites for the Aspergillus nidulans PacC zinc-finger transcription factor are necessary and sufficient for regulation by ambient pH of the isopenicillin N synthase gene promoter. JBiol Chem,1996,271:28825-28830.
62. Espeso E A, J Tilburn, H N Arst Jr, and M A Penalva. pH regulation is a major determinant in expression of a fungal penicillin biosynthetic gene. EMBO J,1993,12:3947-3956.
63. Espeso E A, J Tilburn, L Sanchez-Pulido, C V Brown, A Valencia, H N Arst Jr and M A Penalva. Specific DNA recognition by the Aspergillus nidulans three zinc finger transcription factor PacC.J Mol Biol,1997,274:466-480.
64. Espeso E A, T Roncal, E Diez, L Rainbow, E Bignell, J Alvaro, T Suarez, S H Denison, J Tilburn, H N Arst Jr, and M A Penalva. On how a transcription factor can avoid its proteolytic activation in the absence of signal transduction. EMBO J,2000,19:719-728.
65. Foster J W. Microbial responses to acid stress, p.99-115. In G. Storz and R. Hengge-Aronis (ed.), Bacterial stress responses. American Society for Microbiology, Washington, D.C.2000.
66. Foster J W. When protons attack:microbial strategies of acid adaptation. Cur Opin Microbiol,1999, 2:170-174.
67. Gabor Giczey, Zoltan Kerenyi, Laszlo Fulop and Laszlo Hornok. Expression of cmgl, an exo-β-1, 3-glucanase gene from Coniothyrium minitans, increases during sclerotial parasitism. App Environ Microbiol,2001,67:865-871.
68. Gale E F, and H M R Epps. The effect of the pH of the medium during growth on enzymic activities of bacteria (Escherichia coli and Micrococcus lysodeikticus) and the biological significance of the changes produced. Biochem J,1942,36:600-618.
69. Gale E F. The bacterial amino acid decarboxylases. Ad Enzymol,1946,6:1-32.
70. Gerlagh M H, Goossen-van de Geijn M and Fokkenma N J. Long-term biosanitation by application of Coniothyrium minitans on Sclerotinia sclerotiorum-infected crops. Phytopathology,1999,89:141-147
71. Gielkens M, L Gonzalez-Candelas, P Sanchez-Torres, P van de Vondervoort, L de Graaff, J Visser, and D Ramon. The abfB gene encoding the major alpha-L-arabinofuranosidase of Aspergillus nidulans: nucleotide sequence, regulation and construction of a disrupted strain. Microbiology,1999, 145:735-741.
72. Godoy G, Steadman J R, Dickman M B and Dam R. Use of mutants to demonstrate the role of oxalic acid in pathogenicity of Sclerotinia sclerotiorum on Phaseolus vulgaris. Physiol Mol Plant Pathol, 1990,37:179-191
73. Goldstein A L, Carpenter M A, Crowhurst R N and Stewart A. Identification of Coniothyrium minitans isolates using PCR amplification of a dispersed repetitive element. Mycologia,2000,92 (1):46-53.
74. Gong X Y, Fu Y P, Jiang D H, Li G Q, Yi X H and Peng Y L. L-Arginine is essential for conidiation in the filamentous fungus Coniothyrium minitans. Fungal GenetBiol,2007,44:1368-1379.
75. Gonzalez R, Gavrias V, Gomez D, Scazzocchio C, and Cubero B. The integration of nitrogen and carbon catabolite repression in the Aspergillus nidulans requires the GATA factor AreA and an additional positive-acting element, ADA. EMBO J,1997,16:2937-2944.
76. Grendene A, Minardi P, Giacomini A, Squartini A, Marciano P. Characterization of the mycoparasite Coniothyrium minitans:comparison between morpho-physiological and molecular analyses. Mycological Research,2002,106:796-807
77. Hall H K, K L Karem, and J W Foster. Molecular responses of microbes to environmental pH stress. Adv Microb Physiol,1995,37:229-272.
78. Hamlet W M and Plowright C B. On the occurrence of oxalic acid in fungi. Chemical News,1877, 36:93-94
79. Harel A, Bercovich S, Yarden O.2006. Calcineurin is required for sclerotial development and pathogenicity of Sclerotinia sclerotiorum in an oxalic acid-independent manner. Mol Plant Microbe Interact,19(6):682-93.
80. Hu X, Bidney D L, Yalpani N, Duvick J P, Crasta O and Lu G. Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower. Plant Physiol, 2003,133(1):170-181.
81. Huang H C and Erickson R S. Ulocladium afrum as a biological control agent for white mold of bean caused by Sclerotinia sclerotiorum. Phytoparastica,2007,35 (1):15-22.
82. Huang H C, Kokko E G. Ultrastructure of hyperparasitism of Coniothyrium minitans on sclerotia of Sclerotinia sclerotiorum. Can J,1988, Bot65:2483-2489.
83. Huang H C. Importance of Coniothyrium minitans in survival of sclerotia of Sclerotinia sclerotiorum in wilted sunflower. Can J of Botany,1976,55:285-295.
84. Hutchings H, K P Stahmann, S Roels, E A Espeso, W E Timberlake, H N Arst Jr and J Tilburn. The multiply-regulated gabA gene encoding the GAB A permease of Aspergillus nidulans:a score of exons. Mol Microbiol,1999,32:557-568.
85. Ilmen M, Onnela M L, Klemsdal S, Keranen S, and Penttila M. Functional analysis of the cellobiohydrolase I promoter of the filamentous fungus Trichoderma reesei. Mol Gen Genet,1996b, 253:303-314.
86. Ilmen M, Thrane C, and Penttila M. The glucose repressor gene crel of Trichoderma:isolation and expression of a full-length and a truncated mutant form. Mol Gen Genet,1996a,251:451-460.
87. Jones D A, Gordon A H and Bacon J S D. Co-operative action by endo- and exo-(3-(1,3)-glucanases from parasitic fungi in the degradation of cell-wall glucans of Sclerotinia sclerotiorum. Biochem J 1974,140:47-55.
88. Jones D. Ultrastructure and composition of the cell walls of Sclerotinia sclerotium. Trans Br Mycol Soc,1970,54:351-360
89. Jones E, Carpenter M D, Fong A, Goldstein A, Thrush Crowhurst and Stewart A. Co-transformation of the sclerotial mycoparasite Coniothyrium minitans with hygromycin B resistance and b-glucuronidase markers. Mycol Res,1999,103:929-937.
90. Katz M E, R N Rice and B F Cheetham. Isolation and characterization of an Aspergillus nidulans gene encoding an alkaline protease. Gene,1994,150:287-292.
91. Keller N P, Nesbitt C, Sarr B, Phillips T D and Burow G B. pH regulation of sterigmatocystin and aflatoxin biosynthesis in Aspergillus spp. Phytopathology,1997,87:643-648.
92. Kesarwani M, Azam M, Natarajan K, Mehta A and Datta A Oxalate decarboxylase from Collybia velutipes. Molecular cloning and its overexpression to confer resistance to fungal infection in transgenic tobacco and tomato. JBiol Chem,2000,275:7230-7238.
93. Kim H S, Sneller C H and Diers B W. Evaluation of soybean cultivars for resistance to Sclerotinia stem rot in field environment. Crop Sci,1999,39:64-68.
94. Kim Y K, Xiao C L and Rogers J D. Influence of culture media and environmental factors on mycelial growth and pycnidial production of Sphaeropsis pyriputrescens. Mycol Res,2004,108:926-932.
95. Knudsen G R, Eschen D J, Dandurand L M and Bin L. Potential for biocontrol of Sclerotinia sclerotiorum though colonization of sclerotia by Trichoderma harzianum. Plant Disease,1991, 75:466-470.
96. Kobayashi H, H Sito and T Kakegawa. Bacterial strategies to inhabit acidic environments.J GenAppl Microbiol,2000,46:235-243.
97. Kulmburg P, Mathieu M, Dowzer C E A, Kelly J M, and Felenbok B. Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon catabolite repression in Aspergillus nidulans. Mol Microbiol,1993,7:847-857.
98. Levine M and Tjian R. Transcription regulation and animal diversity. Nature,2003,424 (6945):147-151.
99. Li B, Fu Y P, Jiang D H, Xie J T, Cheng J S, Li G Q, Hamid M I and Yi X H. Cyclic GMP as a Second Messenger in the Nitric Oxide-Mediated Conidiation of the Mycoparasite Coniothyrium minitans. Appl Environ Microbiol,2010,76:2830-2836
100. Li G Q, Huang H C and Acharya S N. Antagonism and biocontrol potential of Ulocladium atrum on Sclerotinia sclerotiorum. Biol Control,2003a,28:11-18
101. Li G Q, Huang H C and Acharya S N. Antagonism and biocontrol potential of Ulocladium atrum on Sclerotinia sclerotiorum. Biological Control,2003,28 (1):11-18.
102. Li G Q, Huang H C and Acharya S N. Importance of pollen and senescent petals in the suppression of Sclerotinia sclerotiorum by Coniothyrium minitans. Biocon Sci Technol,2003b,13:495-505.
103. Li G Q, Huang H C, and Acharya S N. Sensitivity of Ulocladium atrum, Coniothyrium minitans, and Sclerotinia sclerotiorum to benomyl and vinclozolin. Can J of Botany,2002,80:892-898.
104. Li G Q, Huang H C, Miao H J, Erickson R S, Jiang D H and Xiao Y N. Biological control of Sclerotinia diseases of rapeseed by aerial applications of the mycoparasite Coniothyrium minitans. Eur JPlant Pathol,2006,114:345-355.
105. Li M, Gong X, Zheng J, Jiang D, Fu Y, Hou M S Transformation of Coniothyrium minitans, a parasite of Sclerotinia sclerotiorum, with Agrobacterium tumefaciens. FEMS Microbiol Lett,2005, 243:323-329
106. Lindberg R A, Rhodes W G, Eirich L D, and Drucker H. Extracellular acid proteases from Neurospora crassa. JBacteriol,1982,150:1103-1108.
107. MacCabe A P, Orejas M, J A Perez-Gonzalez and D Ramon. Opposite patterns of expression of two Aspergillus nidulans xylanase genes with respect to ambient pH. J Bacteriol,1998,180:1331-1333.
108. Maccheroni W, May G S, Martinez-Rossi N M and Rossi A. The sequence of palF, an environmental pH response gene in Aspergillus nidulans. Gene,1997,194:163-67
109. Mach R L, Strauss J, Zeilinger S, Schindler M and Kubicek C P. Carbon catabolite repression of xylanase I (xynl) gene expression in Trichoderma reesei. Mol Microbiol,1996,6:1273-1281.
110. Mathieu M and Felenbok B. The Aspergillus nidulans CREA protein mediates glucose repression of the ethanol regulon at various levels through competition with the ALCR-specific transactivator. EMBO J,1994,13:4022-4027.
111. McLaren D L, Huang H C and Rimmer S R. Control of apothecial production of Sclerotinia sclerotiorum by Coniothyrium minitans and Talaromyces flavus. Plant Disease,1996,80 (12):1373-1378
112. McQuilken M P and Whipps J M. Production, survival and evaluation of solid-substrate inocula of Coniothyrium minitans against Sclerotinia sclerotiorum. Eur J Plant Pathol,1995,101:101-110.
113. McQuilken M P, Budge S P and Whipps J M. Biological control of Sclerotinia sclerotiorum by film-coating Coniothyrium minitans on to sunflower seed and sclerotia. Plant Pathol,1997,46 (6):919-929
114. McQuilken M P, Budge S P and Whipps J M. Effects of culture media and environmental factors on conidial germination, pycnidial production and hyphal extension of Coniothyrium minitans. Mycological Research,1997,101 (1):11-17.
115. McQuilken M P, Budge S P and Whipps J M. Effects of culture media and environmental factors on conidial germination, pycnidial production and hyphal extension of Coniothyrium minitans. Mycological Research,1997,101:11-17
116. McQuilken M P, Gemmell J, Hill R A and Whipps J M. Production of macrosphelide A by the mycoparasite Coniothyrium minitans. FEMS Microbiol Lett,2003,219(1):27-31.
117. McQuilken M P, Mitchell S J, Budge S P, Whipps J M, Fenlon J S and Archer S A. Effect of Coniothyrium minitans on sclerotial survival and apothecial production of Sclerotinia sclerotiorum in field-grown oilseed rape. Plant Pathol,1995,44:883-896.
118. Merriman P R. Survival of sclerotia of Sclerotinia sclerotiorum in soil. Soil Biol Biochem,1976, 8:385-389.
119. Mingot J M, J Tilburn, E Dlez, E Bignell, M Orejas, D A Widdick, S Sarkar, C V Brown, M. X. Caddick, E A Espeso, H N Arst Jr, and M A Penalva. Specificity determinants of proteolytic processing ofAspergillus PacC transcription factor are remote from the processing site, and processing occurs in yeast if pH signalling is bypassed. Mol Cell Biol,1999,19:1390-1400.
120. Muthumeenakshi S, Sreenivasaprasad S, Rogers C W, Challen MP and Whipps J M. Analysis of cDNA transcripts from Coniothyrium minitans reveals a diverse array of genes involved in key processes during sclerotial mycoparasitism. Fungal Genet Biol,2007 44(12):1262-1284.
121. Muthumeenakshi S., Goldstein Alan L, Stewart A and Whipps J M. Molecular studies on intraspecific diversity and phylogenetic position of Coniothyrium minitans. Mycological research,2001, 105(9):1065-1074.
122. Nahas E, H F Terenzi and A Rossi. Effect of carbon source and pH on the production and secretion of acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) in Neurospora crassa. J Gen Microbiol,1982,128:2017-2021.
123. Negrete-Urtasun S, Denison S H, Arst H N Jr. Characterization of the pH signal transduction pathway gene palA of Aspergillus nidulans and identification of possible homologs. J Bacteriol, 1997,179:1832-35
124. Negrete-Urtasun S, W Reiter, E Dlez, S H Denison, J Tilburn, E A Espeso, M A Penalva and H N Arst Jr. Ambient pH signal transduction in Aspergillus:completion of gene characterization. Mol Microbiol,1999,33:994-1003.
125. Nehlin J O and Ronne H. Yeast MIG1 repressor is related to the mammalian early growth response and Wilms' tumour finger proteins. EMBO J,1990,9:2891-2898.
126. Nehlin J O, Carlberg M and Ronne H. Control of yeast GAL genes by MIG1 repressor:a transcriptional cascade in the glucose response. EMBO J,1991,11:3373-3377.
127. Orejas M, A P MacCabe, J A Perez-Gonzalez, S Kumar, and D Ramon. Carbon catabolite repression of the Aspergillus nidulans xlnA gene. Mol Microbiol,1999,31:177-184.
128. Orejas M, E A Espeso, J Tilburn, S Sarkar, H N Arst Jr, and M A Penalva. Activation of the Aspergillus PacC transcription factor in response to alkaline ambient pH requires proteolysis of the carboxy-terminal moiety. Genes Dev,1995,9:1622-1632.
129. Perez-Gonzalez J A, L H de Graaff, J Visser and D Ramon. Molecular cloning and expression in Saccharomyces cerevisiae of two Aspergillus nidulans xylanase genes. Appl Environ Microbiol,1996, 62:2179-2182.
130. Poussereau N, Creton S, Billon-Grand G, Rascle C and Fevre M. Regulation of acp1, encoding a non-aspartyl acid protease expressed during pathogenesis ofSclerotinia sclerotiorum. Microbiology, 2001,147:717-726
131. Ren L, Li G Q and Jiang D H. Characterization of some cultural factors affecting degradation of oxalate by the mycoparasite Coniothyrium minitans. J Appl Microbiol,2010,108:173-180
132. Ren L, Li G Q, Han Y C, Jiang D H and Huang H C. Degradation of oxalic acid by Coniothyrium minitans and its effects on production and activity of (3-1,3-glucanase of this mycoparasite. Biol Cont, 2007,43:1-11.
133.Riou C, Freyssinet G and Fevre M. Production of Cell Wall-Degrading Enzymes by the Phytopathogenic Fungus Sclerotinia sclerotiorum. Appl Environ Microbiol,1991,57(5):1478-1484
134. Rogers C W, Challen M P, Green J R and Whipps J M. Use of REMI and Agrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus, Coniothyrium minitans, FEMS Microbiol Lett,2004,241:207-214
135. Sakurai Y and H Shiota. Two distinct types of acid phosphatase formation in Aspergillus sp.J Gen Appl Microbiol,1977,23:311-323.
136. Sandys-Winsch C, Whipps J M, Gerlagh M and Kruse M. World distribution of the sclerotial mycoparasite Coniothyrium minitans. My col Res,1993,97(10):1175-1178
137. Screen S, Bailey A, Charnley K, Cooper R, and Clarkson J. Carbon regulation of the cuticle-degrading enzyme PR1 from Metarhizium anisopliae may involve a trans-acting DNA-binding protein CRR1, a functional equivalent of the Aspergillus nidulans CreA protein. Curr Genet,1997,31:511-518.
138. Shah A J, Tilburn J, Adlard M W, and H N Arst Jr. pH regulation of penicillin production in Aspergillus nidulans. FEMSMicrobiolLett,1991,77:209-212.
139. Shwab E K and Keller N P. Regulation of secondary metabolite production in filamentous ascomycetes. Mycol Res,2008,112:225-230.
140. Silvia Herranz J M, Rodrlguez, Henk-Jan Bussink Juan, C Sanchez-Ferrero, H N Arst Jr, Miguel, A. Penalva and Olivier Vincent. Arrestin-related proteins mediate pH signaling in fungi. PNAS,2005, 34:2141-12146
141. Smale S T and Kadonaga J T. The RNA polymerase Ⅱ core promoter. Annu Rev Biochem,2003, 72:449-479
142. Strauss J, Mach R L, Zeilinger S, Hartler G, Stoeffer G, Wolschek M and Kubiceka C P. Crel, the carbon catabolite repressor protein from Trichoderma reesei. FEBS Lett,1995,376:103-107.
143. Then-Bergh K, and Axel A Brakhage. Regulation of the Aspergillus nidulans penicillin biosynthesis gene acvA (pcbAB) by amino acids:implication for involvement of transcription factor PACC. Appl Environ Microbiol,1998,64:843-849.
144. Tilburn J, Sarkar S, Widdick D A, Espeso E A, Orejas M, Mungroo J, Penalva M A, and H N Arst Jr. The Aspergillus PacC zinc finger transcription factor mediates regulation of both acid-and alkaline-expressed genes by ambient pH. EMBO J,1995,14:779-790.
145. Tinsley J H, Lee I H, Minke P F and Plamann M. Analysis of actin and actin-related protein 3 (ARP3) gene expression following induction of hyphal tip formation and apolar Mol Gen Genet,1998,259 (6), 601-609
146. Toh-e A, Kobayashi S and Oshima Y. Disturbance of the machinery for the gene expression by acidic pH in the repressible acid phosphatase system of Saccharomyces cerevisiae. Mol Gen Genet,1978, 162:139-149.
147. Tribe H T. On the parasitism of Sclerotinia trifollorum by Coniothyrium minitans. Trans Brit My col Soc,1957,40:489-499
148. Tu J C. Mycoparasitism by Coniothyrium minitans on Sclerotinia sclerotiorum and its effect on sclerotial germination. J of Phytopathology,1984,109:261-268
149. Turner G J and Tribe H T. Preliminary field plot trails on biological control of Sclerotinia trifoliorum by Coniothyrium minitans. Plant Pathol,1975,24:109-113
150. van Kuyk P A, Cheetham B F and Katz M E. Analysis of two Aspergillus nidulans genes encoding extracellular proteases. Fungal Genet Biol,2000,29:201-210.
151. Whipps J M and Gerlagh M. Biology of Coniothyrium minitans and its potential for use in disease biocontrol. Mycol Res,1992,96:897-907
152. Whipps J M and Gerlagh M. Biology of Coniothyrium minitans and its potential for use in disease biocontrol. Mycological Research,1992,96:897-907.
153. Whipps J M, Budge S P and Mitchell S J. Observations on sclerotial mycoparasites of Sclerotinia sclerotiorum. Mycological Research,1993,97:697-700.
154. Whipps J M, Grewal SK and van der Goes P. Interactions between Coniothyrium minitans and sclerotia. Mycological Research,1991,95:295-299.
155. Yang L, Miao H J, Li G Q, Y L M Huang H C. Survival of the mycoparasite Coniothyrium minitans on flower petals of oilseed rape under field conditions in central China. Biological Control,2007,40 (2):179-186.
156. Yang R, Han Y C, Li G Q, Jiang D H and Huang H C. Effects of ambient pH and nutritional factors on antifungal activity of the mycoparasite Coniothyrium minitans. Biological Control,2008,44:116-127.
157. Yang R, Han Y C, Li G Q, Jiang D H. and Huang H C. Suppression of Sclerotinia sclerotinrum by antifungal substances produced by the mycoparasite Coniothyrium minitans. Eur J Plant Pathol,2007, 119:411-420.
158. Yu J H and Keller N. Regulation of secondary metabolism in filamentous fungi. Annu Rev Phytopathol,2005,43:437-458.
159. Zantinge J L, Huang H C and Cheng K J. Screening and identification of Coniothyrium minitans mutants with enhanced β-glucanase activity. Enzyme and Microbial Techno,2003,32:224-230
160. Zhao S and Shamoun S F. The effects of culture media, solid substrates, and relative humidity on growth, sporulation and conidial discharge of Valdensinia heterodoxa. Mycol Res,2006, 110:1340-1346