臺灣博碩士論文加值系統

Trichoderma harzianum 為一具有顯著生物防治功能的真菌，常用於對抗引起植物病害的病原真菌，如Botrytis cinerea、Rhizoctonia solani 以及Fusarium oxysporum。因此，深入了解T. harzianum 對抗植物病原真菌的作用機制將有利於往後植物病害的防治。本研究將T. harzianum培養於四種不同碳源環境，分別是1% 葡萄糖、0.5% 葡萄糖混合0.5% 失去活性的B. cinerea 菌絲、1% 失去活性的B. cinerea 菌絲以及1% 失去活性的T. harzianum菌絲，以二維電泳配合LC MS/MS分析，分離並鑑定這些胞外蛋白質。從四種不同培養環境下取得的共100個蛋白質樣品進行LC MS/MS 分析，共91個樣品得到LC MS/MS的訊號，並成功鑑定出33個蛋白質。其中，專一於含有1% 失去活性的B. cinerea 菌絲的培養環境，鑑定出一個L-amino acid oxidase及兩個endochitinases。另外，本篇研究也在上述四種不同培養條件下，對T. harzianum所分泌的多種水解酵素進行活性分析，分別是chitinase、β-1,3-glucanase、β-1,6-glucanase、 xylanase、cellulase、mannanase 及 protease。結果顯示在B. cinerea的誘導下，β-1,3-glucanase、β-1,6-glucanase、chitinase、protease及xylanase的酵素活性明顯提升，顯然這些分泌到細胞外的水解酵素在T. harzianum對抗病原真菌時扮演著重要的角色。

Trichoderma harzianum is a notable biocontrol agent which has a natural ability to antagonize a wide range of phytopathogenic fungi such as Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Therefore, it is providing an alternative management of these pathogens to chemical fungicides. Understanding the biocontrol mechanism of T. harzianum responding to the pathogens will provide a direct benefit for the further applications in the plant diseases control. Two-dimensional electrophoresis was used to display secreted protein pattern of T. harzianum grown on media containing glucose, mixture of glucose and deactivated B. cinerea, deactivated B. cinerea, and deavtivated T. harzianum, respectively. Protein patterns were compared to identify T. harzianum proteins induced in the presence of B. cinerea. Protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ninety one out of 100 excised protein spots have been successfully analyzed by LC-MS/MS anaylysis. Thirty three out of these 91 proteins were identified. Among them, one L-amino acid oxidase, two endochitinases were uniquely induced in the medium contained deactivated B. cinerea as the sole carbon source. Seven secreted enzymes, including chitinase, β-1,3-glucanase, β-1,6-glucanase, xylanase, cellulase, mannanase and protease were also assayed for their activity. Enzyme activities of β-1,3-glucanases, β-1,6-glucanases, chitinases, proteases and xylanases were detected distinctly higher in medium with deactivated B. cinerea than others. Suggest that these enzymes play a critical role in biocontrol mechanism of T. harzianum in the mode of action pathogen cell wall degradation. To our knowledge, this is the first proteomic study conducted to identify the secreted proteins from T. harzianum against B. cinerea.

Table of Contents

Acknowledgements……………………………………………………….I
中文摘要………………………………………………………………...II
Abstract………………………………………………………………….III
Table of Contents………………………………………………………V
1. Introduction…………………………………………………………1
2. Literature review
2.1 Plant diseases and reigning managements…………………………4
2.2 Introduction of Trichoderma……………………………………….6
2.3 Taxonomic classification of Trichoderma…………………………7
2.4 The Trichoderma chronology………………………………………7
2.5 Geographic distribution of Trichoderma…………………………10
2.6 Morphology and Microscopic features of Trichoderma………….10
2.7 Trichoderma species as biocontrol agents………………………11
2.8 Trichoderma harzianum…………………………………………15
2.9 Introduction of Botrytis cinerea…………………………………..17
2.10 Morphology of Botrytis cinerea………………………………..19
3. Materials and methods
3.1 Fungal strains……………………………………………………..20
3.2 Chemicals…………………………………………………………20
3.3 Materials, Equipments and Software…………………………......21
3.4 Deactivated B. cinerea preparation……………………….………22
3.5 Deactivated T. harzianum ETS 323 preparation……………….....22
3.6 T. harzianum cultivation…………………………………………..23
3.7 Secreted protein precipitation from the culture media……………24
3.8 Protein assay………………………………………………………25
3.9 One-dimensional SDS-PAGE……………………………………25
3.10 Isoelectric focusing and 2D-PAGE……………………………26
3.11 Protein patterns analysis and protein spots in-gel digestion…….27
3.12 LC/ESI-MS/MS analysis of tryptic peptides…………………….28
3.13 Enzyme activity assay…………………………………………...30
3.14 Enzyme activity stain……………………………………………32
4. Result
4.1 T. harzianum ETS 323 cultivation on PDA……………………....34
4.2 T. harzianum ETS 323 germination in media culture……………36
4.3 B. cinerea B136 cultivation……………………………………….37
4.4 The antagonism between T. harzianum ETS 323 and B. cinerea B136……………………………………………………………38
4.5 One-dimensional SDS-PAGE……………………………………41
4.6 Enzyme activity assay……………………………………………43
4.7 Enzyme activity stain……………………………………………46
4.8 Two-dimensional gel electrophoresis analysis…………………....51
4.9 Protein identification……………………………………………...56
5. Discussion…………………………………………………………….64
6. Conclusion……………………………………………………………75
7. References……………………………………………………………77
8. Appendix
8.1 MS/MS spectra of the identified proteins…………………….......88

References

1.Ghisalberti EL, Sivasithamparam K. Antifungal antibiotics produced by Trichoderma spp. . Soil Biology and Biochemistry 1991;23:1011-1020.
2.Agrios GN. Plant Pathology. 4 ed. San. Diego, California, USA: Academic Press, 1997.
3.Manczinger L, Antal Z, Kredics L. Ecophysiology and breeding of mycoparasitic Trichoderma strains (a review). Acta Microbiol Immunol Hung 2002;49(1):1-14.
4.Elad Y, Yunis H, Katan T. Multiple resistance to benzimidazoles, dicarboximides and diethofencarb in field isolates of Botrytis cinerea in Israel. Plant Pathol. 1992;41:41-46.
5.Monte E, Llobell A. Trichoderma in organic agriculture. Proceedings V World Avocado Congress 2003;725-733.
6.Monte E. Understanding Trichoderma: between biotechnology and microbial ecology. Int Microbiol 2001;4:1-4.
7.Persoon CH. Dispositio methodica fungorum. Romer's Neues Mag. Bot. 1794;1:81-128.
8.Papavizas GC. Trichoderma and Gliocladium: biology, ecology and potential for biocontrol. Annu Rev Phytopathol 1985;23:23-54.
9.Samuels GJ. Trichoderma: a review of biology and systematics of the genus. Mycol. Res. 1996;100:923-935.
10.Harman GE, Björkman T. Potential and existing uses of Trichoderma and Gliocladium for plant disease control and plant growth enhancement. In: Kubicek CP, Harman GE, eds. Trichoderma and Gliocladium Vol. 2. London: Taylor and Francis, 1998;229-265.
11.Harman GE, Kubicek PK. Trichoderma and Gliocladium. Enzymes, biological control and commercial applications. Vol. 2. London: Taylor and Francis, 1998.
12.Tulasne L-R, Tulasne C. Selecta fungorum carpologia. Paris: Jussu, 1865.
13.Bisby GR. Trichoderma viride Pers. Ex Fries, and notes on Hypocrea. Trans. Br. Mycol. Soc. 1939;23:149-168.
14.Dingley JM. Life history studies in the genus Hypocrea Fr. New Zealand: Trans. Proc. R. Soc, 1957.
15.Rifai MA, Webster J. Culture studies of Hypocrea and Trichoderma II. H. aureo-viridis and H. rufa f. nov. Trans. Br. Mycol. Soc. 1966;49:296.
16.Rifai MA, Webster J. Culture studies of Hypocrea and Trichoderma III. H. lactea (=H. citrina) and H. pulvinata. Trans. Br. Mycol. Soc. 1966;49:297-310.
17.Webster J, Rifai MA. Culture studies of Hypocrea and Trichoderma I. Comparision of perfect and imperfect stares of H. gelatinosa, H. rufa and Hypocrea sp. 1. Trans. Br. Mycol. Soc. 1968;47:75-96.
18.Webster J, Rifai MA. Culture studies of Hypocrea and Trichoderma IV. Hypocrea pilulifera sp. nov. Trans. Br. Mycol. Soc. 1968;51:511-514.
19.Rifai MA. A revision of the genus Trichoderma. Mycol. Pap. 1969;116:1-56.
20.Lieckfeldt E, Kuhls K, Muthumeenakshi M. Molecular taxonomy of Trichoderma and Gliocladium and their teleomorphs. In: Kubicek CP, Harman GE, eds. Trichoderma and Gliocladium. Vol. 1. . London: Taylor & Francis, 1998;35-56.
21.Kindermann J, El-Ayouti Y, Samuels GJ, Kubicek CP. Phylogeny of the genus Trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA cluster. Fungal Genet Biol 1998;24(3):298-309.
22.Kullnig-Gradinger CM, Szakacs G, Kubicek CP. Phylogeny and evolution of the genus Trichoderma: a multigene approach. Mycological Research 2002;106:757-767.
23.Hermosa MR, Grondona I, Diaz-Minguez JM, Iturriaga EA, Monte E. Development of a strain-specific SCAR marker for the detection of Trichoderma atroviride 11, a biological control agent against soilborne fungal plant pathogens. Curr Genet 2001;38(6):343-50.
24.Lübeck M, Poulsen SK, Lübeck PS, Jensen DF, Thrane U. Identification of Trichoderma strains from building materials by ITS1 ribotyping, UP-PCR fingerprinting and UP-PCR cross hybridization. FEMS Microbiol Lett 2000;185(2):129-34.
25.Lieckfeldt E, Seifert KA. An evaluation of the use of ITS sequences in the taxonomy of the Hypocreales. Stud Mycol 2000(45):35-44.
26.Lutzoni F, Kauff F, Cox CJ, McLaughlin D, Celio G, Dentinger B, Padamsee M, Hibbett D, James TY, Baloch E, Grube M, Reeb V, Hofstetter V, Schoch C, Arnold AE, Miadlikowska J, Spatafora J, Johnson D, Hambleton S, Crockett M, Shoemaker R, Hambleton S, Crockett M, Shoemaker R, Sung GH, Lucking R, Lumbsch T, O'Donnell K, Binder M, Diederich P, Ertz D, Gueidan C, Hansen K, Harris RC, Hosaka K, Lim YW, Matheny B, Nishida H, Pfister D, Rogers J, Rossman A, Schmitt I, Sipman H, Stone J, Sugiyama J, Yahr R, Vilgalys R. Assembling the fungal tree of life: Progress, classification and evolution of subcellular traits. American Journal of Botany 2004;91(10):1446-1480.
27.Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC. Phylogenetic species recognition and species concepts in fungi. Fungal Genet Biol 2000;31(1):21-32.
28.Bissett J, Szakacs G, Nolan CA, Druzhinina I, Gradinger C, Kubicek CP. New species of Trichoderma from Asia. Canadian Journal of Botany-Revue Canadienne De Botanique 2003;81(6):570-586.
29.Chaverri P, Castlebury LA, Overton BE, Samuels GJ. Hypocrea/Trichoderma: species with conidiophore elongations and green conidia. Mycologia 2003;95(6):1100-1140.
30.Lu BS, Druzhinina IS, Fallah P, Chaverri P, Gradinger C, Kubicek CP, Samuels GJ. Hypocrea/Trichoderma species with pachybasium-like conidiophores: teleomorphs for T-minutisporum and T-polysporum and their newly discovered relatives. Mycologia 2004;96(2):310-342.
31.Samuels GJ. Trichoderma: Systematics, the sexual state, and ecology. Phytopathology 2006;96(2):195-206.
32.Widden P. Fungal populations from forest soils in southern Quebec. Can. J. Bot. 1979;57:1324-1331.
33.Widden P, Abitol J. Seasonality of Trichoderma species in a spruce-forest soil. Mycologia 1980;72:775-784.
34.Roiger DJ, Jeffers SN, Caldwell RW. Occurrence of Trichoderma species in apple orchard and woodland soils. Soil Biol. Biochem. 1991;23:353-359.
35.Hagn A, Pritsch K, Schloter M, Munch JC. Fungal diversity in agricultural soil under different farming management systems, with special reference to biocontrol strains of Trichoderma spp. Biology and Fertility of Soils 2003;38(4):236-244.
36.Wuczkowski M, Druzhinina I, Gherbawy Y, Klug B, Prillinger H, Kubicek CP. Species pattern and genetic diversity of Trichoderma in a mid-European, primeval floodplain-forest. Microbiol Res 2003;158(2):125-33.
37.Chaverri P, Castlebury LA, Samuels GJ, Geiser DM. Multilocus phylogenetic structure of Trichoderma harzianum/Hypocrea lixii complex. Mol. Phylogenet. Evol. 2003;27:302-313.
38.Samuels GJ, Pardo-Schultheiss R, Hebbar P, Lumsden RD, Bastos CN, Costa JC, Bezerra JL. Trichoderma stromaticum, sp. nov., a parasite of the cacao witches broom pathogen. Mycol. Res. 2000;104:760-764.
39.Gams W, Bisset J. Morphology and identification of Trichoderma. In: Harman GE, Kubicek CP, eds. Trichoderma & Gliocladium. Vol. 1. London: Taylor and Francis, 1998;3-34.
40.Larone DH. Medically Important Fungi - A Guide to Identification. 3rd ed. Washington, D.C.: ASM Press, 1995.
41.St-Germain G, Summerbell R. Identifying Filamentous Fungi - A Clinical Laboratory Handbook. 1st ed. Belmont, California: Star Publishing Company, 1996.
42.Sutton DA, Fothergill AW, Rinaldi MG. Guide to Clinically Significant Fungi. 1st ed. Baltimore: Williams & Wilkins, 1998.
43.Weindling R. Trichoderma lignorum as a parasite of other soil fungi. Phytopathology 1932;22:837-845.
44.Weindling R. Studies on a lethal principle effective in the parasitic action of Trichoderma lignorum on Rhizoctonia solani and other soil fungi. Phytopathology 1934;24:1153-1179.
45.Weindling R. Experimental consideration of the mold toxin of Gliocladium and Trichoderma. Phytopathology 1941;31:991-1003.
46.Webster J, Lomas N. Does Trichoderma viride produce gliotoxin and viridin? Trans. Br. Mycol. Soc. 1964;47:535-540.
47.Rehner SA, Samuels GJ. Taxonomy and phylogeny of Gliocladium analyzed by large subunit ribosomal DNA sequences. Mycol. Res. 1994;98:625-634.
48.Taylor A. Some aspects of the chemistry and biology of the genus Hypocrea and its anamorphs, Trichoderma and Gliocladium. Proceedings of the Nova Scotia Institute of Science 1986;36:27-58.
49.Bliss DE. The destruction of Armillaria mellea in citrus soils. Phytopathology 1951;41:665-683.
50.Aluko MO, Hering TF. The mechanism associated with the antagonistic relationship between Corticium solani and Gliocladium virens. Trans. Br. Mycol. Soc. 1970;55:173-179.
51.Wells HD, Bell DK, Jaworski CA. Efficacy of Trichoderma harzianum as a biocontrol for Sclerotium rolfsii. Phytopathology 1972;62:442-447.
52.Howell CR. Effect of Gliocladium virens on Pythium ultimum, Rhizoctonia solani, and damping-off of cotton seedlings. Phytopathology 1982;72:496-498.
53.Chet I. Trichoderma: application, mode of action and potential as a biocontrol agent of soilborne plant pathogenic fungi. In: Chet I, ed. Innovative approaches to plant disease control. New York: John Wiley & Sons, 1987;137-160.
54.Howell CR, Stipanovic RD, Lumsden RD. Antibiotic production by strains of Gliocladium virens and its relation to the biocontrol of cotton seedling diseases. Biocontrol Sci. Technol. 1993;3:435-441.
55.Lo CT, Nelson EB, Hayes CK, Harman GE. Ecological studies of transformed Trichoderma harzianum strain 1295-22 in the rhizosphere and on the phylloplane of creeping bentgrass. Phytopathology 1998;88:129-136.
56.Griffin DH. Fungal physiology. New York: Wiley-Liss, 1994.
57.Vey A, Hoagland RE, Butt TM. Toxic metabolites of fungal biocontrol agents. In: Butt TM, Jackson C, Magan N, eds. Fungi as biocontrol agents: Progress, problems and potential. Bristol: CAB International, 2001;311-346.
58.Sawa R, Mori Y, Iinuma H, Naganawa H, Hamada M, Yoshida S, Furutani H, Kajimura Y, Fuwa T, Takeuchi T. Harzianic acid, a new antimicrobial antibiotic from a fungus. J Antibiot (Tokyo) 1994;47(6):731-2.
59.Ghisalberti EL, Rowland CY. Antifungal metabolites from Trichoderma harzianum. J Nat Prod 1993;56(10):1799-804.
60.Graeme-Cook KA, Faull JL. Effect of ultraviolet-induced mutants of Trichoderma harzianum with altered antibiotic production on selected pathogens in vitro. Can J Microbiol 1991;37(9):659-64.
61.Corley DG, Miller-Wideman M, Durley RC. Isolation and structure of harzianum A: a new trichothecene from Trichoderma harzianum. J Nat Prod 1994;57(3):422-5.
62.Itoh Y, Kodama K, Furuya K, Takahashi S, Haneishi T, Takiguchi Y, Arai M. A new sesquiterpene antibiotic, heptelidic acid producing organisms, fermentation, isolation and characterization. J Antibiot (Tokyo) 1980;33(5):468-73.
63.Mandels M, Reese ET. Induction of cellulase in Trichoderma viride as influenced by carbon sources and metals. J. Bacteriol. 1957;73:269-278.
64.Mandels M. Microbial sources of cellulase. Biotechnol. Bioeng. Sym. 1975;5:81-105.
65.Chet I, Benhamou N, Haran S. Mycoparasitism and lytic enymes. In: Harman GE, Kubicek CP, eds. Trichoderma and Gliocladium. Enzymes, biological control and commercial application. Vol. 2. London: Taylor and Francis, 1998;153-171.
66.Elad Y, Chet I, Henis Y. Degradation of plant pathogenic fungi by Trichoderma harzianum. Can. J. Microbiol. 1982;28:719-725.
67.Kubicek CP, Mach RL, K. PC, Lorito M. Trichoderma: from genes to biocontrol. J. Plant Path. 2001; 83:11-23.
68.Benitez T, Limon C, Delgado-Jarana J, Rey M. Glucanolytic and other enzymes and their control. In: Harman GE, Kubicek CP, eds. Trichoderma and Gliocladium. Vol. 2. London: Taylor & Francis, 1998;101-127.
69.Lorito M. Chitinolytic enzymes and their genes. In: Harman GE, Kubicek CP, eds. Trichoderma and Gliocladium. Vol. 2. London: Taylor & Francis, 1998;73-99.
70.de la Cruz J, Hidalgo-Gallego A, Lora JM, Benitez T, Pintor-Toro JA, Llobell A. Isolation and characterization of three chitinases from Trichoderma harzianum. Eur J Biochem 1992;206(3):859-67.
71.Carsolio C, Gutierrez A, Jimenez B, Montagu MV, Herrera-Estrella A. Characterization of ech42, a Trichoderma harzianum endochitinase gene expressed during mycoparasitism. Microbiology 1994;91:10903-10907.
72.Lima LH, Ulhoa CJ, Fernandes AP, Felix CR. Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rolfsii and Rhizoctonia solani cell walls. J Gen Appl Microbiol 1997;43(1):31-37.
73.Woo SL, Donzelli B, Scala F, Mach RL, Harman GE, Kubicek CP, Del Sorbo G, Lorito M. Disruption of ech42 (endochitinase-encoding) gene affects biocontrol activity in Trichoderma harzianum strain P1. Mol Plant-Microbe Interact. 1999;12:419-429.
74.Baek JM, Howell CR, Kenerley CM. The role of an extracellular chitinase from Trichoderma virens Gv29-8 in the biocontrol of Rhizoctonia solani. Curr Genet 1999;35(1):41-50.
75.Mach RL, Peterbauer CK, Payer K, Jaksits S, Woo SL, Zeilinger S, Kullnig CM, Lorito M, Kubicek CP. Expression of two major chitinase genes of Trichoderma atroviride (T. harzianum P1) is triggered by different regulatory signals. Appl Environ Microbiol 1999;65(5):1858-63.
76.de las Mercedes Dana M, Limon MC, Mejias R, Mach RL, Benitez T, Pintor-Toro JA, Kubicek CP. Regulation of chitinase 33 (chit33) gene expression in Trichoderma harzianum. Curr Genet 2001;38(6):335-42.
77.El-Katatny MH, Gudelj M, Robra KH, Elnaghy MA, Gubitz GM. Characterization of a chitinase and an endo-beta-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii. Appl Microbiol Biotechnol 2001;56(1-2):137-43.
78.Omumasaba CA, Yoshida N, Ogawa K. Purification and characterization of a chitinase from Trichoderma viride. J Gen Appl Microbiol 2001;47(2):53-61.
79.Brunner K, Peterbauer CK, Mach RL, Lorito M, Zeilinger S, Kubicek CP. The Nag1 N-acetylglucosaminidase of Trichoderma atroviride is essential for chitinase induction by chitin and of major relevance to biocontrol. Curr Genet 2003;43(4):289-95.
80.Hoell IA, Klemsdal SS, Vaaje-Kolstad G, Horn SJ, Eijsink VG. Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1. Biochim Biophys Acta 2005;1748(2):180-90.
81.Jinzhu S, Qian Y, Beidong L, Dianfu C. Expression of the chitinase gene from Trichoderma aureoviride in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 2005;69(1):39-43.
82.Ike M, Nagamatsu K, Shioya A, Nogawa M, Ogasawara W, Okada H, Morikawa Y. Purification, characterization, and gene cloning of 46 kDa chitinase (Chi46) from Trichoderma reesei PC-3-7 and its expression in Escherichia coli. Appl Microbiol Biotechnol 2005:1-10.
83.Klemsdal SS, Clarke JL, Hoell IA, Eijsink VG, Brurberg MB. Molecular cloning, characterization, and expression studies of a novel chitinase gene (ech30) from the mycoparasite Trichoderma atroviride strain P1. FEMS Microbiol Lett 2006;256(2):282-9.
84.Bamforth CW. The adaptability, purification and properties of exo-beta 1,3-glucanase from the fungus Trichoderma reesei. Biochem J 1980;191(3):863-6.
85.de la Cruz J, Pintor-Toro JA, Benitez T, Llobell A, Romero LC. A novel endo-beta-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum. J Bacteriol 1995;177(23):6937-45.
86.Noronha EF, Ulhoa CJ. Characterization of a 29-kDa beta-1,3-glucanase from Trichoderma harzianum. FEMS Microbiol Lett 2000;183(1):119-23.
87.Kulminskaya AA, Thomsen KK, Shabalin KA, Sidorenko IA, Eneyskaya EV, Savel'ev AN, Neustroev KN. Isolation, enzymatic properties, and mode of action of an exo-1,3-beta-glucanase from Trichoderma viride. Eur J Biochem 2001;268(23):6123-31.
88.Bara MT, Lima AL, Ulhoa CJ. Purification and characterization of an exo-beta-1,3-glucanase produced by Trichoderma asperellum. FEMS Microbiol Lett 2003;219(1):81-5.
89.Monteiro VN, Ulhoa CJ. Biochemical characterization of a beta-1,3-glucanase from Trichoderma koningii induced by cell wall of Rhizoctonia solani. Curr Microbiol 2006;52(2):92-6.
90.de la Cruz J, Pintor-Toro JA, Benitez T, Llobell A. Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism. J Bacteriol 1995;177(7):1864-71.
91.de la Cruz J, Llobell A. Purification and properties of a basic endo-beta-1,6-glucanase (BGN16.1) from the antagonistic fungus Trichoderma harzianum. Eur J Biochem 1999;265(1):145-51.
92.Montero M, Sanz L, Rey M, Monte E, Llobell A. BGN16.3, a novel acidic beta-1,6-glucanase from mycoparasitic fungus Trichoderma harzianum CECT 2413. Febs J 2005;272(13):3441-8.
93.Geremia RA, Goldman GH, Jacobs D, Ardiles W, Vila SB, Van Montagu M, Herrera-Estrella A. Molecular characterization of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum. Mol Microbiol 1993;8(3):603-13.
94.Dunaevsky YE, Gruban TN, Beliakova GA, Belozersky MA. Enzymes secreted by filamentous fungi: regulation of secretion and purification of an extracellular protease of Trichoderma harzianum. Biochemistry (Mosc) 2000;65(6):723-7.
95.Uchikoba T, Mase T, Arima K, Yonezawa H, Kaneda M. Isolation and characterization of a trypsin-like protease from Trichoderma viride. Biol Chem 2001;382(10):1509-13.
96.De Marco JL, Felix CR. Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches' broom disease. BMC Biochem 2002;3:3.
97.Delgado-Jarana J, Rincon AM, Benitez T. Aspartyl protease from Trichoderma harzianum CECT 2413: cloning and characterization. Microbiology 2002;148(Pt 5):1305-15.
98.Pozo MJ, Baek JM, Garcia JM, Kenerley CM. Functional analysis of tvsp1, a serine protease-encoding gene in the biocontrol agent Trichoderma virens. Fungal Genet Biol 2004;41(3):336-48.
99.Suarez B, Rey M, Castillo P, Monte E, Llobell A. Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity. Appl Microbiol Biotechnol 2004;65(1):46-55.
100.Suarez MB, Sanz L, Chamorro MI, Rey M, Gonzalez FJ, Llobell A, Monte E. Proteomic analysis of secreted proteins from Trichoderma harzianum. Identification of a fungal cell wall-induced aspartic protease. Fungal Genet Biol 2005;42(11):924-34.
101.Howell CR. Mechanisms employed by Trichoderma species in the biological control of plant diseases: The history and evolution of current concepts. Plant Disease 2003;87(1):4-10.
102.Kapteyn JC, Montijn RC, Vink E, delaCruz J, Llobell A, Douwes JE, Shimoi H, Lipke PN, Klis FM. Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer. Glycobiology 1996;6(3):337-345.
103.Lorito M, Woo SL, Donzelli B, Scala F. Synergistic, antifungal interactions of chitinolytic enzymes from fungi, bacteria and plants. In: Muzzarelli RAA, ed. Chitin Enzymology II. Italy: Atec, Grottammare (AP), 1996;157-164.
104.Burges HD. Formulation of mirobial pesticides. Dordrecht, The Netherlands: Kluwer Academic Publishers, 1998.
105.Butt TM, Copping L. Fungal biological control agents. Pesticide Outlook 2000;11:186-191.
106.Jarvis WR. Botryotinia and Botrytis species: taxonomy, physiology, and pathogenicity. Monogr Res Branch Can Dept Agric 1977;15.
107.De Curtis F, Torriani S, Rossi E, De Cicco V. Selection and use of Metchnikowia pulcherrima as a biological control agent for postharvest rots of peaches and table grapes. Ann. Microbiol. Enzimol. 1996;4:45-55.
108.Janisiewicz WJ. Enhancement of Biocontrol of Blue Mold with the Nutrient Analog 2-Deoxy-d-Glucose on Apples and Pears. Appl Environ Microbiol 1994;60(8):2671-2676.
109.Jarvis WR. Managing diseases in greenhouse crops. Plant Dis. 1989;73:190-194.
110.Yunis H, Elad Y, Mahrer Y. The effect of air temperature, relative humidity and canopy wetness on gray mold of cucumbers in unheated greenhouses. Phytoparasitica 1990;18:203-215.
111.Verhoeff K. Spotting of tomato fruits caused by Botrytis cinerea. Neth. J. Plant Pathol. 1970;76:219-226.
112.Elad Y. Latent infection of Botrytis cinerea in rose flowers and combined chemical and physiological control of the disease. Crop Protect. 1988;7:361-366.
113.Hammer PE, Yang SF, Reid MS, Marois J. Postharvest control of Botrytis cinerea infections on cut roses using fungistatic storage atmospheres. J. Americ. Soc. Horticult. Sci. 1990;115:102 107.
114.Honda Y, Mizumura Y. Light and temperature dependent conidium and sclerotium formation in Botrytis spp. Bulletin Faculty Agricult. Shimane Univ. 1991;25:27-35.
115.Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680-5.
116.Neuhoff V, Arold N, Taube D, Ehrhardt W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988;9(6):255-62.
117.Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 1992;203(1):173-9.
118.Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 1996;68(5):850-8.
119.Ye XY, Ng TB, Cheng KJ. Purification and characterization of a cellulase from the ruminal fungus Orpinomyces joyonii cloned in Escherichia coli. Int J Biochem Cell Biol 2001;33(1):87-94.
120.Wood PJ. The interaction of direct dyes with water soluble substituted celluloses and cereal β-glucans. Ind. Eng. Chem. Prod. Res. Dev. 1980;19:19-23.
121.Vinale F, Marra R, Scala F, Ghisalberti EL, Lorito M, Sivasithamparam K. Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens. Letters in Applied Microbiology 2006;43:143-148.
122.Lukasheva EV, Berezov TT. L-Lysine alpha-oxidase: physicochemical and biological properties. Biochemistry (Mosc) 2002;67(10):1152-8.
123.Donzelli BG, Harman GE. Interaction of ammonium, glucose, and chitin regulates the expression of cell wall-degrading enzymes in Trichoderma atroviride strain P1. Appl Environ Microbiol 2001;67(12):5643-7.
124.Lora JM, De la Cruz J, Llobell A, Benitez T, Pintor-Toro JA. Molecular characterization and heterologous expression of an endo-beta-1,6-glucanase gene from the mycoparasitic fungus Trichoderma harzianum. Mol Gen Genet 1995;247(5):639-45.
125.Peberdy JF. Protein secretion in filamentous fungi--trying to understand a highly productive black box. Trends Biotechnol 1994;12(2):50-7.
126.Fryksdale BG, Jedrzejewski PT, Wong DL, Gaertner AL, Miller BS. Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis. Electrophoresis 2002;23(14):2184-93.
127.Packer NH, Keatinge L. Glycobiology and proteomics. In: Pennington SR, Dunn MJ, eds. Proteomics from protein sequence to function. Oxford: BIOS Scientific Publishers, 2001.