Thạc Sĩ 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
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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
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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
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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
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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
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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
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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


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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








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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.