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    THẠC SĨ The role of fungal chitinases in biological control of plant root-knot nemato

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  6. The role of fungal chitinases in biological control of plant root-knot nemato

    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


    viii
    LIST OF TABLES

    List Title Page
    Table 2.1. Composition of basal mineral chitin medium 17
    Table 2.2. Pepton-rose bengal agar medium . 17
    Table 2.3. Number of fungal isolates producing clear zone on 0.5% swollen chitin
    medium plates

    22
    Table 2.4. Reducing sugar, chitinase and exochitinase produced by fungal isolates in
    0.5% CMB .

    24
    Table 2.5. Percentage of M. incognita egg parasitized by chitinolytic fungi over 3
    days

    26
    Table 2.6. Competitive interaction between chitinolytic fungi and F. solani on PDA
    plates

    27
    Table 2.7. Reducing sugar from F. solani hyphal cell walls digested by fungal
    chitinases

    28
    Table 2.8. Characteristics of crude chitinases from chitinolytic fungi . 29
    Table 3.1. Characteristics of chitinolytic fungi T. aureoviride DY-59 and R.
    microsporus VS-9 in 0.5% swollen chitin cultural medium

    43
    Table 3.2. Substrate specificity of the T. aureoviride DY-59 and R. microsporus VS-
    9 crude chitinases .

    46
    Table 3.3. Effect of cation ions on T. aureoviride DY-59 and R. microsporus VS-9
    crude chitinase activity .

    47
    Table 3.4. K m and V max of chitinase from chitinolytic fungi T. aureoviride DY-59 and
    R. microsporus VS-9

    49
    Table 3.5. Inhibition of F. solani microconidial germination by T. aureoviride DY-59
    and R. microsporus VS-9 crude chitinases

    52
    Table 3.6. Production of chitin monomer and dimer from hyphae of F. solani by T.
    aureoviride DY-59, and R. microsporus VS-9 chitinases

    55
    Table 4.1 Fungal growth, pH, reducing sugar, protein and chitinase activity from
    culture filtrate of P. variotii DG-3 after 12 days of growth in 0.5% swollen
    chitin medium


    69
    Table 4.2. Substrate specificity of the purified Chi32 and Chi46 from P. variotii
    DG-3 .

    74
    Table 4.3. Effect of metal ions on the activity of the purified Chi32 and Chi46 from
    ix
    P. variotii DG-3 . 75
    Table 4.4. Identification of the proteins F-1 and F-3 from P. variotii expressing
    chitinase activity by N-terminal sequencing and matching with known
    proteins in NCBI database .


    76
    Table 4.5. Effected rate (%) of M. incognita eggs treated with P. variotii DG-3
    chitinases and commercial enzymes

    82
    Table 4.6. Yield and composition of material obtained during extraction of chitinous
    material from F. solani cultured in one litter of YPG medium

    84
    Table 4.7. Assignment of the relevant bands of FT-IR spectra of chitin from crab
    shell and F. solani cell walls

    85
    Table 5.1 Characteristics of chitinolytic fungus L. antillanum B-3 in culture broth
    medium .

    102
    Table 5.2. Substrate specificity of L. antillanum B-3 chitinase 106
    Table 5.3. Effect of metal ions (10 mM) on L. antillanum B-3 chitinase . 106
    Table 5.4. Rate (%) of parasitized M. incognita eggs by chitinolytic fungus L.
    antillanum B-3 .

    107
    Table 5.5. Rate (%) of damaged M. incognita eggs by L. antillanum B-3 crude
    chitinase .

    109
    Table 5.6. Percent damage of M. incognita eggs by L. antillanum B-3 enzymes
    fractionated from DEAE-Sephadex chromatography

    110
    Table 6.1. Root-knot causing agents isolated and extracted from cucumber roots . 121
    Table 6.2. Change in cucumber growth parameters in nematode-infected and
    nematode-fungus A-1 and B-3 treatment for growth period

    123
    Table 6.3. Egg number and total number of M. incognita per gram of cucumber root
    and reduced index after growth period .

    123








    x
    LIST OF FIGURES

    List Title Page
    Figure 1.1. Domain organization of fungal chitinase . 9
    Figure 2.1. Number of clear-zone-producing isolates on swollen chitin medium
    plates isolated from different materials .

    23
    Figure 2.2. Surrounding colony (A) and inside colony (B) clear zone produced by
    fungi on chitin medium plates .

    23
    Figure 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 2.4. 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


    27
    Figure 3.1. Alignment (above) and phylogenetic tree (bottom) of 18S rRNA from
    DY-59 fungal strains and other fungal strains from NCBI database, the
    similar nucleic acid of 18S rRNA gene from DY-59 and others are shown
    as the same red color letters, phylogenetic tree was made by a rectangle
    tree-making software program http://www.ncbi.nlm.nih.gov/BLAST .




    41
    Figure 3.2. Alignment (above) and phylogenetic tree (bottom) of 18S rRNA from
    VS-9 fungal strains and other fungal strains from NCBI database, the
    similar nucleic acid of 18S rRNA gene from VS-9 and others are shown
    as the same red color letters, phylogenetic tree was made by a rectangle
    tree-making software program http://www.ncbi.nlm.nih.gov/BLAST




    42
    Figure 3.3. Time courses of chitinase production from T. aureoviride DY-59 ( ● ) and
    R. microsporus VS-9 ( ○ ). These fungi were grown in 250 ml Erlenmeyer
    flask containing CBM at 25
    o
    C, 150 rpm for 12 days


    44
    Figure 3.4. Optimal pH (A) and optimal temperature (B) of T. aureoviride DY-59
    ( ● ) and R. microsporus VS-9 ( ○ ) crude chitinases

    45
    Figure 3.5. TLC of hydrolysis products of swollen chitin by T. aureoviride DY-59
    and R. microsporus VS-9 crude chitinases, (A) standards of
    (NAcGlc)n 1~6 , (B) hydrolysis products by DY-59 and VS-9 chitinases.
    The analysis was done on silica gel 60F 254 plates (Merck, Germany)
    using n-propanol/water/ NH 4 OH (70:30:1, by vol.) as developing solvent.

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