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Doctor of Philosophy Dissertation The Role of Fungal Chitinases in Biological Control of Plant Root-Knot Nematode Meloidogyne incognita on Cucumber Department of Agricultural Chemistry, Graduate School Chonnam National University Nguyen Van Nam Directed by Professor Ro-Dong Park February, 2009 The Role of Fungal Chitinases in Biological Control of Plant Root-Knot Nematode Meloidogyne incognita on Cucumber Department of Agricultural Chemistry, Graduate School Chonnam National University NGUYEN VAN NAM This dissertation has been certified by the committee members February, 2009 i CONTENTS LIST OF TABLES viii LIST OF FIGURES x ABBREVIATIONS xvi ABSTRACT 1 CHAPTER I. GENERAL INTRODUCTION 1.1. General property and distribution of chitin 3 1.2. General characteristics of plant root-knot nematode 4 1.3. Biological control of plant parasitic nematode 5 1.4. Chitinases 7 1.4.1. Property and molecular structure of chitinases 7 1.4.2. Roles of chitinases in biological control of plant diseases 9 1.5. Study objectives 11 CHAPTER II. ISOLATION AND SCREENING OF ANTAGONISTIC CHITINOLYTIC FUNGI FROM SOIL ABSTRACT 12 2.1. INTRODUCTION 13 2.2. MATERIALS AND METHODS 16 ii 2.2.1. Sample site 16 2.2.2. Preparing of soil samples 16 2.2.3. Isolation, initial identification and maintenance of fungi 16 2.2.4. Composition of cultural medium 17 2.2.4.1. Swollen chitin mineral medium (SCM) 17 2.2.4.2. Peptone-rose bengal agar medium (PRBA) 17 2.2.5. Screening of chitinolytic fungi 18 2.2.6. Screening of M. incognita egg-parasitic fungi 18 2.2.7. Screening of mycoparasitic fungi against F. solani 18 2.2.8. Schale’s assay for determining of reducing sugar 19 2.2.9. Preparing of crab swollen chitin 19 2.2.10. Chitinase activity 20 2.2.11. Exochitinase assay 20 2.2.12. Characterizations of crude enzymes from chitinolytic fungi 20 2.3. RESULTS 21 2.3.1. Isolation of fungi and initial screening of chitinolytic fungi 21 2.3.2. Screening of M. incognita egg-parasitic fungi 25 2.3.3. Screening of antifungal fungi against F. solani 26 2.3.4. Characterization of crude chitinolytic enzymes from chitinase-producing fungi 28 2.4. DISCUSSION 30 CHAPTER III. THE CHARACTERISTICS AND ANTIFUNGAL ACTIVITY OF CHITINASES FROM Hypocrea aureoviride DY-59 AND Rhizopus microsporus VS-9 ON Fusarium solani iii ABSTRACT 32 3.1. INTRODUCTION 33 3.2. MATERIALS AND METHODS 34 3.2.1. DY-59 and VS-9 chitinolytic fungi and F. solani 34 3.2.2. Identification of chitinolytic fungal strains DY-59 and VS-9 34 3.2.3. Chemicals and preparation of enzyme substrates 35 3.2.4. Preparation of DY-59 and VS-9 crude chitinases 35 3.2.5. Enzyme assay 35 3.2.6. Substrate specificity and effect of cations on enzyme activity 35 3.2.7. Analysis of hydrolysis products by DY-59 and VS-9 chitinases 37 3.2.8. Electrophoresis and chitinase activity staining 37 3.2.9. Antifungal activity of DY-59 and VS-9 enzymes against F. solani 37 3.3 RESULTS 40 3.3.1. H. aureoviride DY-59 and R. microsporus VS-9 isolates and identification 40 3.3.2. Characteristics of DY-59 and VS-9 chitinases 43 3.3.3. Effects of DY-59 and VS-9 crude chitinases on F. solani in vitro 50 3.4. DISCUSSION 56 CHAPTER IV. PURIFICATION AND CHARACTERIZATION OF 32 kDa AND 46 kDa CHITINASES PRODUCED FROM FUNGUS Paecilomyces variotii DG-3 PARASITIZING ON Meloidogyne incognita EGGS ABSTRACT 60 iv 4.1. INTRODUCTION 61 4.2. MATERLALS AND METHODS 63 4.2.1. Fungal strain and maintenance 63 4.2.2. Preparation of crude chitinases and enzyme assay 63 4.2.3. Purification of chitinases from P. variotii DG-3 culture filtrate 63 4.2.4. Electrophoresis and activity staining of chitinases 64 4.2.5. Effect of temperature and pH on enzyme activity 64 4.2.6. Substrate specificity and effect of cations on enzyme activity 64 4.2.7. N-terminal amino-acid sequencing of chitinases and database searching 65 4.2.8. Enzymatic hydrolysis of chitooligosaccharides by Chi32 and Chi46 65 4.2.9. Effectiveness of Chi32 and Chi46 on M. incognita eggshells 66 4.2.10. Fluorescent observation of effected M. incognita eggs 66 4.2.11. Germination inhibition of F. solani conidia 66 4.2.12. Preparing of fungal mycelia and extraction of chitin and chitosan from F. solani cell walls 66 4.2.13. Infrared spectroscopy of fungal chitin 67 4.2.14. Hydrolysis of chitin from F. solani by Chi32 and Chi46 67 4.3. RESULTS 68 4.3.1. Identification of P. variotii DG-3 68 4.3.2. Preparing of crude chitinase from DG-3 isolate 69 4.3.3. Purification of chitinases from DG-3 70 4.3.4. Characterization of Chi32 and Chi46 73 4.3.5. N-Terminal amino acid sequencing of Chi32 and Chi46 75 4.3.6. Analysis of hydrolysis of chitooligosaccharides by Chi32 and Chi46 76 4.3.7. Parasitism of P. variotii on M. incognita eggs in vitro 80 v 4.3.8. Action of Chi32 and Chi46 on the structure of M. incognita eggshells 80 4.3.9. Inhibition of F. solani microconidial germination by Chi32 and Chi46 82 4.3.10. Extraction and determination of chitin and chitosan in F. solani cell walls 83 4.3.11. Lysis of cell walls and chitin from F. solani cell walls by Chi32 and Chi46 87 4.4. DISCUSSION 89 CHAPTER V. PARTIAL PURIFICATION AND CHARACTERIZATION OF CHITINASES FROM FUNGUS Lecanicillium antillanum B-3 PARASITISM TO ROOT-KNOT NEMATODE Meloidogyne incognita EGGS ABSTRACT 93 5.1. INTRODUCTION 94 5.2. MATERIALS AND METHODS 96 5.2.1. Screening and identification of B-3 chitinolytic-nematophagous isolate 96 5.2.2. Preparation of crude chitinases 96 5.2.3. Assay of enzyme activity and protein content 96 5.2.4. Partial purification of enzymes and characterization of B-3 chitinase 97 5.2.5. Extraction of M. incognita eggs 98 5.2.6. Assay of direct parasitism of fungus B-3 on nematode eggs 98 5.2.7. SEM observation for parasitism of fungi on M. incognita eggs 98 5.2.8. Effectiveness of crude and partially purified chitinase on nematode eggshells 99 5.2.9. Statistical analysis 99 5.3. RESULTS 100 vi 5.3.1. Fungal isolate and identification of B-3 fungus 100 5.3.2. Partial enzyme purification from B-3 culture filtrate 103 5.3.3. Characteristics of B-3 chitinase 105 5.3.4. Parasitism of B-3 isolate on M. incognita eggs in vitro 107 5.3.5. Effectiveness of enzymes on M. incognita eggs in vitro 108 5.4. DISCUSSION 111 CHAPTER VI. SUPPRESSION OF ROOT-KNOT NEMATODE Meloidogyne incognita ON CUCUMBER BY Lecanicillium psalliotae A-1 AND Lecanicillium antillanum B-3 CHITINOLYTIC FUNGI ABSTRACT 113 6.1. INTRODUCTION 114 6.2. MATERIALS AND METHODS 116 6.2.1. Extraction and identification of M. incognita from cucumber roots 116 6.2.2. Extraction of M. incognita eggs from cucumber roots 116 6.2.3. Maintenance of M. incognita in greenhouse 116 6.2.4. Isolation and identification of Fusarium solani from cucumber roots 117 6.2.5. Fungal isolates and preparation of inoculums of L. psalliotae A-1 and L. antillanum B-3 118 6.2.6. Pot preparation 118 6.2.7. Preparation of cucumber seedlings and plant growth condition 118 6.2.8. Soil amendment 118 6.2.9. Plant analysis 119 vii 6.2.10. Assessment of disease severity 119 6.2.11. Statistical analysis 119 6.3. RESULTS 110 6.3.1. Identification and determination of disease-causing factors 120 6.3.2. Analysis of growth parameters 122 6.3.3. Assessment of effects of fungi A-1 and B-3 against M. incognita on cucumber 124 6.4. DISCUSSION 127 CHAPTER VII. GENERAL DISCUSSION AND CONCLUSIONS 130 7.1. General discussion 130 7.2. General conclusions 133 CHAPTER VIII. REFERENCES 135 ABSTRACT IN KOREAN 147 ACKNOWLEDGEMENTS 149 PUBLICATIONS 151 BIOGRAPHICAL DATA 155 [...]... analyzed by HPLC (A) chitinoligomer standard (1~6), (B) hydrolysis products from the DY-59 chitinases (hyphae + DY-59 enzyme), and (C) hydrolysis products from VS-9 chitinases (hyphae + VS-9 enzyme) Figure 4.1 54 Morphology of colony and conidiophores of P variotii DG-3 on PDA medium on seventh day following culture (A), (B) and a phylogenic tree of 18S rRNA gene (C) of P variotii DG-3 and other... cellulose and is now regarded as a renewable resource The polymer is hydrolyzed by chitinase to oligomers, mainly dimers, which are subsequently degraded to N-acetyl-glucosamine by β-Nacetylglucosaminidase In addition, the chitin polymer is deacetylated by deacetylase to chitosan, which is hydrolyzed by chitosanase to chitooligomers Chitinoligosaccharides and chitosanoligosaccharides are regarded as biologically... hyphal biomass in sodium acetate buffer (pH 5) and 100 µl of crude enzyme from T aureoviride DY-59 (●), and R microsporus VS9 (○) Figure 3.11 53 Hydrolysis products from the cell walls of F solani hyphae by DY-59 and VS-9 crude chitinase Reaction mixture contained 1% hyphal biomass in 50 mM sodium acetate buffer (pH 5) and crude enzyme (ratio 2:1, v/v) Hydrolysis products were analyzed... endochitinase of 46 kDa (Chi46) were purified from Paecilomyces variotii DG-3 by DEAE Sephadex A-50 and Sephadex G-100 chromatography The N-terminal amino acid sequences of Chi32 and Chi46 enzymes were determined to be DPYQTNVVYTGQDFVSPDLF and DAXXYRSVAYFVNWA, respectively The structural degradation of M incognita eggshells was shown by light and fluorescent microscopes Both chitinases showed also the inhibition... Germany) using n-propanol/water/ NH4OH (70:30:1, by vol.) as developing solvent x 48 Figure 3.6 Enzymatic hydrolysis products from crab swollen chitin by T aureoviride DY-59 and R microsporus VS-9 chitinases (A) chitin oligomer standard, (B) hydrolysis product by T aureoviride DY-59 chitinase, and (C) hydrolysis products by R microsporus VS-9 chitinases Figure 3.7 48 SDS-PAGE of the T aureoviride DY-59... 25oC, 150 rpm for 12 days Figure 3.4 Optimal pH (A) and optimal temperature (B) of T aureoviride DY-59 (●) and R microsporus VS-9 (○) crude chitinases Figure 3.5 44 45 TLC of hydrolysis products of swollen chitin by T aureoviride DY-59 and R microsporus VS-9 crude chitinases, (A) standards of (NAcGlc)n1~6, (B) hydrolysis products by DY-59 and VS-9 chitinases The analysis was done on silica gel... chitinase activity ranged from 0.0 to 11.8 U ml-1 Of chitinolytic fungi screened, six fungal isolates, DY-2, DY-16 DY-19, A-1, B-3, and DG-3, showed the parasitism on Meloidogyne incognita eggs and five fungal isolates DY-16, DY-19, DY-59, VS-9, and DG-3, showed the competitive interaction with Fusarium solani on PDA medium The functional fungi were selected for further study on chitinase production, parasitism... eggs parasitized by chitinolytic fungi, the hyphae invaded inside the eggs: DY-2 (A), DY-16 (B), DY-19 (C), A-1 (D) , B-3 (E) and DG-3 (F) Figure 2.4 25 Agar plate assay for competitive interaction between phytopathogenic fungi F solani with other chitinolytic fungi (A) T aureoviride DY-59, (B) R microsporus VS-9, and P variotii DG-3 Figure 3.1 27 Alignment (above) and phylogenetic tree... similarity between the two families of proteins suggest that they have different folds The two families of chitinases are called family 18 and family 19 in a general classification of glycosyl hydrolases (Dahiya et al 2006, Patil et al 2000) These chitinases can be classified as endochitinases and exochitinases, and exochitinases can be subdivided to chitobiosidase and N-acetyl-β -D- glucosaminidase Endochitinases... chitinases exclusively belong to glycoside hydrolase (GH) family 18 according to the CAZy classification Family 18 enzymes are widely found in fungi, bacteria, animals, viruses and plants and are traditionally subdivided into classes III and V (Nakamura et al 2007) Recently, the project applicant analyzed the chitinase-encoding genes in H jecorina (T reesei) at the genomic level Based on this analysis a novel . 50 mM sodium acetate buffer (pH 5) and crude enzyme (ratio 2:1, v/v). Hydrolysis products were analyzed by HPLC. (A) chitinoligomer standard (1~6), (B) hydrolysis products from the DY- 59 chitinases. chitinases (hyphae + DY-59 enzyme), and (C) hydrolysis products from VS-9 chitinases (hyphae + VS-9 enzyme) 54 Figure 4.1. Morphology of colony and conidiophores of P. variotii DG-3 . 2.3. The fungus-invaded M. incognita eggs parasitized by chitinolytic fungi, the hyphae invaded inside the eggs: DY-2 (A), DY-16 (B), DY- 19 (C), A-1 (D) , B-3 (E) and DG-3 (F) 25 Figure