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Characterization of Trichoderma Species Isolated in Ecuador and Their Potential As a Biocontrol Agent Against Phytopathogenic Fungi from Ecuador and Japan

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Characterization of Trichoderma Species Isolated in Ecuador and Their Potential As a Biocontrol Agent Against Phytopathogenic Fungi from Ecuador and Japan Characterization of Trichoderma Species Isolated in Ecuador and Their Potential as a Biocontrol Agent Against Phytopathogenic Fungi from Ecuador and Japan （エクアドルにおいて分離された Trichoderma 属菌の同定・機能解析と エクアドルおよび日本産植物病原菌に対する生物防除剤としての可能性） Galarza Romero Luis Lenin 2015 CONTENST CONTENTS i LIST OF TABLES v LIST OF FIGURES vi Chapter 1 General Introduction 1 1.1 Trichoderma morphology 1 1.2 Identification of Trichoderma species 2 1.3 Ecology 3 1.4 Trichoderma species as biocontrol agent 5 1.5 Mechanism of biocontrol of Trichoderma species 6 1.6 Lytic enzymes 9 1.7 Genes involved in the mycoparasitism 10 1.8 Goals of this study 14 Chapter 2 Identification of Trichoderma strains to species level 16 2.1 Introduction 16 2.2 Materials and Method 19 2.2.1. Isolation and identification of Trichoderma species 19 2.2.2. Pathogens 22 2.2.3. DNA sequencing and phylogenetic analysis of Trichoderma species 24 2.2.4. In vitro mycoparasitism assay 25 i 2.3 Result 27 2.3.1 Molecular identification of Trichoderma species 27 2.3.2 Phylogenetic analysis of Trichoderma species 34 2.3.3 Growth inhibition 40 2.3.4 Mycoparasitism 44 2.4 Discussion 49 Chapter 3 Microscopy interaction of Trichoderma harzianum T36 55 using Ds-red and green fluorescent protein reporter systems 3.1 Introduction 55 3.2 Materials and Methods 57 3.2.1. Fungal samples 57 3.2.2. Plasmid and Fungal protoplast preparation and transformation 57 3.2.3. In vitro mycoparasitism interactions assay 58 3.3 Result 59 3.3.1. Ds-red and GFP expression and stability in transformants strains 59 3.3.2. Morphology of T. harzianum T36 (ThDsred) and F. oxysporum f. sp. cubense Fo-01 (FocGFP) 61 3.3.3. Interactions between T. harzianum T36 (ThDsred) and F. 63 oxysporum f. sp. cubense Fo-01 (FocGFP) 70 3.3 Discussion ii Chapter 4 Involvement of ThSNF1 in development and virulence of a biocontrol 73 agent Trichoderma harzianum 4.1 Introduction 73 4.2 Materials and Method 75 4.2.1 Fungal strains and culture conditions 75 4.2.2 Isolation and gene targeting of ThSNF1 75 4.2.3 Gene expression analysis 77 4.2.4 Morphology and colony growth 77 4.2.5 In vitro mycoparasitism assay 78 4.3 Result 80 4.3.1. Cloning and targeted disruption of ThSNF1 in T. harzianum 80 4.3.2. Phenotypic characterization of the ThSNF1-targeted strain 84 4.3.3. The expression of the genes encoding wall-degrading enzymes in 87 the ThSNF1-targeted strain 4.3.4. Mycoparasitism ability of the ThSNF1-targeted strain 88 4.4 Discussion 91 Chapter 5 Compressive Discussion 95 5.1 Identification of Trichoderma isolates 95 5.2 Trichoderma genus as biocontrol agent 97 5.3 Genes involved in the mycoparasitism 99 iii ACKNOWLEDGMENTS 102 REFERENCE 103 APPENDIX 127 SUMMARY 137 和文摘要 141 145 List of Publications iv LIST OF TABLES Table 2.1. Morphological classification of Ecuadorian Isolated 21 Table 2.2 List of pathogenic fungi used in this study 23 Table 2.3. Molecular classification of the Ecuadorian isolated. 28 Table 2.4. Inhibitory effects of Trichoderma sp. against pathogenic fungi 42 Table 4.1. Primers used in this study 79 Table S1. List of media and buffer 127 Table S2. Primers used in this study 130 Table S3. Mix PCR using in this study 131 132 Table S4. PCR conditions using in this study Table S5. Ecuadorian Trichoderma isolates, morphological and molecular information 133 Table S6. Inhibition activity of Trichoderma strains (+) indicted more that 70% of 134 inhibition (-) indicated less than 70% of inhibition. v LIST OF FIGURES Fig. 2.1. Ecuador sampling map for Trichoderma isolates (a-coastal region, b- 20 highland region) Fig. 2.2. Colonies of pathogenic fungi on PDA, (Foc) Fusarium oxysporum f. sp. 23 cubense (Fo-01), (Mf) Mycosphaerella fijiensis (Ec-01), (Mr) Moniliophthora roreri (Cp-01), (Mp) M. perniciosa (MrEO-1), (Fol) F. oxysporum f. sp. lycopersici (Chz1-A), (Aa) Alternaria alternata tomato pathotype (As-27), (Rn) Rosellinia necatrix (ES-0601). Fig. 2.3. Graphic illustration of antagonistic test, pathogen in contrast with 26 Trichoderma strains (R1) and growth of the pathogen in control dishes (R2). Based in the formula PIRGP = (R1 – R2)/R1 x 100. Fig 2.4. T. harzianum strains isolated in different region of Ecuador. T1, T3 and 30 T36 Coast Region and T15, T19 and T20 Highland Region. 30 Fig 2.5. T. asperellum strains from different region of Ecuador. T2, T4, T9 and T10 32 Coast Region. T5, T13 and T18 Highland Region. 32 Fig 2.6. T. reesei (T29) and T. virens (T43) isolated from different region of Ecuador 33 vi Fig. 2.7. Phylogenetic relations of Trichoderma taxa based on neighbor-joining 35 analysis of ITS sequence data. The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 0.11417193 is shown. The percentage of replicate trees in which the associated taxa are clustered together in the bootstrap test (2000 replicates) are shown next to the branches [2]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method [3] and are in the units of the number of base substitutions per site. The analysis involved 25 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 500 positions in the final dataset. Evolutionary analyses were conducted in MEGA 5.1 [4]. Fig. 2.8. Phylogenetic relations of Trichoderma taxa based on neighbor-joining 37 analysis of EF-1α sequence data. The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 1.18257330 is shown. The percentage of replicate trees in which the associated taxa are clustered together in the bootstrap test (2000 replicates) are shown next to the branches [2]. The evolutionary distances were computed using the Kimura 2- parameter method [3] and are in the units of the number of base substitutions per site. The analysis involved 25 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 60 positions in the final dataset. Evolutionary analyses were conducted in MEGA 5.1 [4]. Fig. 2.9. Phylogenetic relations of Trichoderma taxa based on neighbor-joining 39 analysis of RPB2 sequence data. The evolutionary history was inferred using the Neighbor-Joining method [1]. The optimal tree with the sum of branch length = 0.39840624 is shown. The percentage of replicate trees in which the associated taxa are clustered together in the bootstrap test (2000 replicates) are shown next to the branches [2]. The evolutionary distances were computed using the Kimura 2- parameter method [3] and are in the units of the number of base substitutions per site. The analysis involved 23 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 317 positions in the final dataset. Evolutionary analyses were conducted in MEGA 5.1 [4]. vii Fig. 2.10. Percentage of inhibition of radial growth of pathogens, Trichoderma 43 strains against several pathogen fungi (a) T. harzianum strains, (b) T. asperellum strains (c) T. reesei and T. virens. Fig. 2.11. Mycoparasitism index of Trichoderma strains against several pathogens 45 fungi (Foc) Fusarium oxysporum f. sp. cubense (Fo-01), (Mf) Mycosphaerella fijiensis (Ec-01), (Mr) Moniliophthora roreri (Cp-01), (Mp) Mo. perniciosa (MrEO-1), (Fol) F. oxysporum f. sp. lycopersici (Chz1-A), (Aa) Alternaria alternata tomato pathotype (As-27), (Rn) Rosellinia necatrix (ES-0601). Fig. 2.12. Antagonism test of T. harzianum strains (T1, T3, T15, T19, T20, T36). 46 Photo taken after ten days of incubation. (Foc) F. oxysporum f. sp. cubense Fo-01, (Mf) M. fijiensis Ec-01, (Mr) M. roreri CP-01, (Mp) M. perniciosa MrEO-1, (Fol) F. oxysporum f. sp. lycopersici Chz1-A, (Aa) A. alternate As-27, (Rn) R. necatrix ES-0601. Fig. 2.13. Antagonism test of T. asperellum strains (T2, T4, T5, T9, T10, T13, 47 T18). Photo taken after ten days of incubation. (Foc) F. oxysporum f. sp. cubense Fo-01, (Mf) M. fijiensis Ec-01, (Mr) M. roreri CP-01, (Mp) M. perniciosa MrEO- 1, (Fol) F. oxysporum f. sp. lycopersici Chz1-A, (Aa) A. alternate As-27, (Rn) R. necatrix ES-0601. Fig 2.14. Antagonism test of T. reesei strain (T29) and T. virens strain (T43). Photo 48 taken after ten days of incubation. (Foc) F. oxysporum f. sp. cubense Fo-01, (Mf) M. fijiensis Ec-01, (Mr) M. roreri CP-01, (Mp) M. perniciosa MrEO-1, (Fol) F. oxysporum f. sp. lycopersici Chz1-A, (Aa) A. alternate As-27, (Rn) R. necatrix ES-0601. Fig. 3.1. Graphic representation of fungi interaction on petri dish. 58 viii Fig. 3.2. Hyphae from (a) T. harzianum T36 (ThDsred) and (b) F. oxysporum f. sp. 60 cubense Fo-01 (FocGFP) using fluorescent microscopy 40x. Fig. 3.3. T. harzianum T36 (ThDsred) morphology, (a) conidiophores, (b) 62 phyliades and (c) conidia. Fig. 3.4. F. oxysporum f. sp. cubense Fo-01 (FocGFP) morphology, (a) hypha, (b) 62 conidia. Fig. 3.5. T. harzianum T36 (ThDsred) (a) mycelia growth alongside F. oxysporum 64 f. sp. cubense Fo-01 (FocGFP) after 24h of co-cultivation. (b) Arrows indicate T. harzianum T36 (ThDsred) make damage to F.
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