the role of fungal chitinases in biological control of plant root-knot nematode meloidogyne incognita on cucumber

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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




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 pr
oduced 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 fr
om 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.
Alignme
nt (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





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

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.





48


xi

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

48
Figure 3.7.
SDS-PAGE of the T. aureoviride DY-59 and R. microsporus VS-
9 crude
enzymes. Lane M1 and M2, standard protein marker, (A)
crude enzyme
of the DY-59 strain, (B) chitinase activity staining of the DY-59 strain
,
(C) crude enzyme of the VS-9 strain, (D) chitinase activity staining
of the
VS-9 strain





50
Figure 3.8.
Lysis of cell walls of F. solani macroconidia by the T. aureoviride DY-
59 and R. microsporus VS-9 chitinase
s. The mixture of enzyme and
conidial suspension was incubated at 30
o
C for 20 hr. (A) control,
(B)
DY-59 chitinase, and (C) VS-9 chitinase. ICW, intact cell wall; DCW,
digested cell wall

51
Figure 3.9.
Correlation between protein amount and F. solani
germination inhibition
rate, (A) T. aureoviride DY-59 chitinase, (B) R. microsporus VS-
9
chitinase


52
Figure 3.10.
Reducing sugar from F. solani
hyphal cell walls. A reaction mixture of
900 µl of 1% hyphal biomass in sodium acetate buffer (pH 5) and 100 µl
of crude enzyme from T. aureoviride DY-59 (●), and R. microsporus VS-
9 (○)




53
Figure 3.11.
Hydrolysis products from the cell walls of F. solani hyphae by DY-
59
and VS-9 crude chitinase. Reaction mixtu
re contained 1% hyphal
biomass in 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 (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
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 fungi by tree-
making program in NCBI



68
Figure 4.2.
DEAE-Sephadex chromatography of P. variotii DG-
3 culture
supernatant. The protein was eluted stepwise with 20 mM Tris-
HCl (pH
7.5) containing 0.0-0.5 M sodium chloride

70


xii

Figure 4.3.
Sephadex G-100 chromatography of F-1 chitinase fractions (F-1) and F-
3
chitinase fraction (F-3). The protein was eluted with in 20 mM Tris-
HCl
(pH 7.5)



71
Figure 4.4.
SDS-PAGE of Chi32 (A) and Chi46 (B). Proteins (40µl each)
were
loaded in each lane. The gels were stained with Coomassie brilliant blue
R-250 for 12% SDS-
PAGE and with Fluorescent Brightener 28 for
chitinase activity staining as in Materials and Methods
. In panel A: lane
1, molecular weight marker (Am
ersham Biosciences); lane 2, crude
enzyme; lane 3, flow-
through protein (no bound to DEAE column); lane

4, fractions F-1 from DEAE-Sephadex column; lane 5, purified
Chi32
chitinase from Sephadex G-100 column
; lane 6 to 8, activity staining of
crude enzyme, fraction F-1, and the
purified Chi32. In panel B: lane 1,
molecular weight marker; lane 2, fractions F-3 from DEAE-
Sephadex
column; lane 3, purified Chi46 chitinase from Sephadex G-100 column
;
and lane 4, activity staining of the purified Chi46












72
Figure 4.5.
Optimal temperature (A) and pH (B) of both Chi32 and Chi46. 1%
swollen chitin was used as the substrate


73

Figure 4.6.
Enzymatic hydrolysis prod
ucts of chitin oligomers by Chi32 (on the left)
and Chi46 (on the right). Standard chitin oligomers, [(GlcNAc)
n
n=1~6],
(A) hydrolysis products from trimer (GlcNAc)
3
, (B) tetramer (GlcNAc)
4
,
(C) pentamer (GlcNAc)
5
, and (D) hexamer (GlcNAc)
6
.

Mixture of 45
0 µl
of substrate (100µg ml
-1
) in 50 mM citrate buffer (pH 3) and 50 µl of
enzymes were incubated at 37
o
C for 60 min. The products (3 µl)
were
separated by HPLC

78
Figure 4.7.
Time course of product formation by Chi32 (on the left) and Chi46 (on
the right) from chitin trimer (A, E), tetramer (B, F), pentamer (C, G), and
hexamer (D, H). Mixture of 450 µl of substrate (500µg ml
-1
) in 50 mM
citrate buffer (pH 3) and 50 µl of enzymes were incubated at 37
o
C for 0,
30, 60, 90 and 120 min. The products (3 µl) were separated by HPLC. -
▲- (GlcNAc)
1
, -- (GlcNAc)
2
, -

- (GlcNAc)
3
, -- (GlcNAc)
4
, -
●
-
(GlcNAc)
5

, -
○
- (GlcNAc)
6








79
Figure 4.8.
The fungus P. variotii DG-3 invaded M. incognita eggs. T
he hyphae
bound to eggshell (arrow) by SEM (A) and by a microscope (B)


80
Figure 4.9.
M. incognita eggs damaged by Chi46. The intact egg (A)
untreated with



xiii

enzyme and the enzyme-treated egg (B) under light microscope. T
he

intact egg (C) untreated with enzyme and the enzyme-treated egg (D
)
under fluorescence microscope, after staining
with 0.01% Fluorescent
Brightener 28




81
Figure 4.10.
Inhibition of F. solani
microconidial germination by Chi32 and Chi46.
(A), ungerminated conidia (UG) by Chi32; (B), short germinated conidia
(SG) by Chi46; (C), n
ormal germinated conidia (NG) in control (heated
enzyme)




83
Figure 4.11.
Photograph of F. solani hyphae (x 400) (A) and hyphal chitin (B)

84
Figure 4.12.
FT-IR spectrum (KBr) Crab chitin (A), and F. solani chitin (B)

86

Figure 4.13.
FT-IR spectrum (KBr) F. solani cell walls (A), chitin (B),
and chitosan
(C)


86
Figure 4.14.
F. solani
cell walls digested by Chi46, intact cell walls in control (arrow),
heated enzyme at 100
o
C for 10 min as in control (A) and digested cell
walls by Chi46 (arrow), microconidia was
treated in Chi46 with 3.7 U
ml
-1
at 37
o
C for 48 hours




87
Figure 4.15.
Amount of reducing sugar released from F. solani
powder chitin by
Chi32 and Chi46. Reaction mixture con
sisting of 900 µl of 0.5 %

substrate and 100 µl of enzymes was incubated at 37
o
C for over 5 hours.



88
Figure 4.16.
Hydrolysis products from crab swollen chitin (A) and F. solani
powder
chitin (B) by Chi32 and Chi46. The chitin oligomers (2 µl) were
separated by HPLC using NH
2
P50 column



88
Figure 5.1.
Alignment of nucleotide sequence of 18S rRNA gene of B-3 isolate (B-
3)
with L. antilanum (L.an) and L. fusisporium
(L.fu) by software of website
multalin.html
, the similar nucleic
acid of B-
3 isolate and others are shown by red color letters and
phylogenetic tree of 18S rRNA gene of B-3 isolate and othe
r fungi from
NCBI database by a rectangle tree-

making software program
(







101
Figure 5.2.
Time course of chitinase activity and growth of L. antillanum B-
3 fungus
in 0.5% swollen chitin broth medium


102
Figure 5.3A.
DEAE-Sephadex column chromatography of L. antillanum B-3
culture
supernatant. Protease (Pro), glucanase 1 (Glu 1), glucanase 2 (Glu 2)
,
unidentified enzyme P4 and chitinase fractions were separated.
The
protein was eluted stepwise with 20 mM Tris-
HCl (pH 7.5) containing

xiv

0.0-0.5 M sodium chloride. Protein content (-○-
) and chitinase activity
(-●-)


103
Figure 5.3B.
DEAE-
Sephadex column chromatography of protein and protease from
culture supernatant L. antillanum B-3, the protein w
as eluted stepwise
with 20 mM Tris-HCl (pH 7.5) containing 0.0-0.1 M sodium chloride



104
Figure 5.4.
SDS-PAGE of chitinase fraction from B-3 culture filtrate. Proteins (40µl
each)
were loaded in each lane. The gels were stained with Coomassie
brilliant blue R-250 for 12% SDS-
PAGE and with Fluorescent
Brightener 28 for chitinase activity staining. L
ane 1, molecular weight
marker (Amersham Biosciences); lane 2, crude enzyme; lane 3, chitinase
fractions from DEAE-Sephadex column, lane 4 activity stai

ning of
chitinase fraction from DEAE-Sephadex column







104
Figure 5.5.
The temperature (A) and pH (B) profiles of the L. antillanum B-
3
chitinase

105
Figure 5.6.
Hydrolysis products from swollen chitin by chitinases, (A) chitin
oligomer standard, (B) by L. antillanum B-3 chitinase


106
Figure 5.7.
The fungus-invaded M. incognita eggs and juvenile by fungus B-3
. The
hyphae bound to eggshells by SEM (A) and parasitized the second-
stage
juvenile (B)

108
Figure 5.8.
The effect of the L. antillanum B-3 chitinase on M. incognita eggs.
(A)
control egg, (B) an egg treated with B-3 purified chitina
se. Eggs were
treated with the enzyme for 4 days and stained in lactoglycerol solution
as in Materials and Methods. Scale bar: 18 µm




109
Figure 6.1.
Cucumber root-knot causing factor, (A) M. incognita egg, (B)
second
stage juvenile, (C) larva, (D) adult, and (E) root-
knot symptom on
cucumber



121
Figure 6.2.
Shoot and root growth of 5 week-old cucumber treated with
M. incognita
and egg-parasitic fungi A-1 and B-
3. Cucumber shoot and root growth
were different in case of each treatment, in Ne and B-

3 fungus and
control (sterilized soil) treatment, shoot and root growth is higher than
those in other treatments





122
Figure 6.3.
Root galls on 3 week-old cucumber treated with nematode and egg-
parasitic fungi, more root galls were shown from root in nematode-
infected treatment (Ne), no
root gall was shown from root in control
(Control) and fewer galls were shown from root in A-1 and B-
3 treatment






xv

(Ne and A-1, Ne and B-3, Ne and A-1 plus B-3)

124
Figure 6.4.
The root galls and M. incognita eg
g masses were shown on root of 5

week-old cucumber in nematode-infected treatment


124
Figure 6.5.
Number of cucumber root-galls per plant in case of deferent treatments

125

xvi

ABBREVIATIONS

AIM: Alkaline insoluble material
BSA: Bovine serum albumin
CBM: Chitin broth medium
CT: Chitin
CTS: Chitosan
DA: Degree of acetylation
DD: Degree of deacetylation
DEAE: Diethylaminoethyl (OCH
2
CH
2
NH(CH
2
CH
3
)
2
)
DNS: Dinitrosalysilic acid
FT-IR: Fourier transform infrared spectroscopy
GlcNAc: N-acetyl-D-glucosamine
(GlcNAc)

2
:

Chitobiose, N,N’-di-N-acetylchitobiose;
(GlcNAc)
3
:

Chitotriose, N,N‘,N’’-tri-N-acetylchitotriose;
(GlcNAc)
4
:

Chitotetraose, N,N’,N’’,N’’’-tetra-N-acetylchitotetraose;
(GlcNAc)
5
:

Chitopentaose, N,N’,N’’,N’’’,N’’’’-penta-N-acetylchitopentaose;
(GlcNAc)
6
:

Chitohexaose, N,N’,N’’,N’’’,N’’’’,N’’’’’-hexa-N-
acetylchitohexaose;
HPLC: High performance of liquid chromatography
J2: Second-stage juvenile
PNP: ρ-nitrophenol.
ρNP-GlcNAc: ρ-nitrophenyl-N-acetyl-D-glucosaminine
PCR: Polymerase chain reaction

PDA: Potato dextrose agar
PDB: Potato dextrose broth
SA: Sodium acetate


xvii

SDS-PAGE: Sodium dodecyl sulfate polyacryl amine gel electrophoresis
SCM: Swollen chitin mineral medium
SEM: Scanning electronic microscope
TLC: Thin layer chromatography
TCA: Trichloro acetic acid
U ml
-1
: Unit/ ml

- 1 -

THE ROLE OF FUNGAL CHITINASES IN BIOLOGICAL CONTROL
OF PLANT ROOT-KNOT NEMATODE Meloidogyne incognita
ON CUCUMBER

NGUYEN VAN NAM

Department of Agricultural Chemistry
Graduate School, Chonnam National University,
Gwangju, 500-757, Korea
(Directed by Professor Ro-Dong Park)

ABSTRACT

Chitin layer, a major composition of fungal cell walls of Fusarium solani and eggshells
of Meloidogyne incognita that are disease-causing factors of root galls of cucumber, is a
target for fungal chitinases considered to be involved in the parasitism process. This study
was focused on an important function and elucidation of fungal chitinases in parasitism
process. Among fungal chitinases, chitinases produced from Hypocrea aureoviride DY-59
and Rhizopus microsporus VS-9 were used for antifungal activity against F. solani.

Chitinase produced from Lecanicillium antillanum B-3 was used for degradation of M.
incognita eggshells. Chitinases produced from Paecilomyces variotii DG-3 were elucidated
their role in antifungal activity against F. solani and degradation of M. incognita eggshells.
Chitinase of 51.9 kDa from H. aureoviride DY-59 and chitinases of 64.1 kDa and 59.0 kDa
from R. microsporus VS-9 were detected on SDS-PAGE gel. Chitinases of DY-59 and VS-9
inhibited more than 60% for F. solani microconidial germination in 4.9 µg and 27.0 µg
protein ml
-1
at 20 hrs after treatment, respectively. The chitinases degraded the cell walls of
F. solani hyphae to produce chitin oligosaccharides, among which GlcNAc, (GlcNAc)
2
, and


- 2 -

(GlcNAc)
5
were determined by HPLC. Exochitinase of 32 kDa (Chi32) and 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 of F. solani conidial germination. Chitin dimer is a major final product for fungal
chitin extracted from cell walls of F. solani mycelia by both enzymes Chi32 and Chi46.
Chitinase of 37 kDa was purified from Lecanicillium antillanum B-3 by DEAE-Sephadex
chromatography. The enzyme was responsible for degradation of M. incognita eggshell
structures with damaging ratio of 30% after 5 days of incubation. Two egg-parasitic fungi
Lecanicillium psalliotae A-1 and Lecanicillium antillanum B-3 were used for suppression of

M. incognita on cucumber. The result showed that the fungal treatments reduced root galls
by 71.8%, 60.3%, and 47.4% in B-3, A-1 plus B-3, and A-1 treated cucumber roots,
respectively. In addition, nematode population of cucumber root was reduced by 79.8%,
62.0%, and 37.9% in B-3, A-1 plus B-3, and A-1 treatments after 7 weeks of growth period.
The results demonstrate that the fungus B-3 showed effectiveness of biological control of M.
incognita on cucumber in pot experiment. In conclusion, chitinase of 51.9 kDa produced
from H. aureoviride DY-59, and chitinases of 64.1 and 59.0 kDa produced from R.
microsporus VS-9 were responsible enzymes for degradation of fungal cell walls of F. solani.
Chitinases of 32 kDa and 46 kDa purified from P. variotii DG-3 and chitinase of 37 kDa
purified from L. antilanum B-3 were considered to be a key role in parasitism process of
nematophagous fungi against M. incognita eggs.






- 3 -

CHAPTER I

GENERAL INTRODUCTION


1.1. General property and distribution of chitin
Chitin, a linear polysaccharide of β-(1,4)-linked N-acetylglucosamine (GlcNAc)
residues, is the second most abundant biopolymer in nature next to 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-N-

acetylglucosaminidase. 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 protective agent against plant
pathogenic fungi (Park et al. 2002, Yamaoka et al. 1999).
Resources of chitin for industrial processing are crustacean shells. Traditionally, chitin
is isolated from crustacean shells by demineralization with diluted acid and deproteinization
in a hot base solution. Furthermore, chitin is converted to chitosan by deacetylation in
concentrated NaOH solution. The chemical process of chitin extraction causes changes in
molecular weight, degree of deacetylation of the product, degradation of nutritionally
valuable proteins and environmental pollution. Thus, enzymatic procedures for all steps in
processing crude material have been investigated (Jung et al. 2006, Kuk et al. 2005).
Recent investigations confirm the suitability of chitin and its derivatives in chemistry,
biotechnology, medicine, veterinary, dentistry, agriculture, food processing, environmental
protection, and textile production. The development of technologies based on the utilization
of chitin derivatives is caused by their polyelectrolite properties, the presence of reactive
functional groups, gel-forming ability, high adsorption capacity, biodegradability and
bacteriostatic, fungistatic and antitumour influence (Synowiecki and Al-Khateeb 2003).



- 4 -

Chitin is also constructed as major layer in cell walls of fungi and plant-parasitic
nematode eggshells therefore chitin layer is a target of chitinases for biological control of
plant parasitic nematode and phytopathogenic fungi (Bird and Mcclure 1975, Nguyen et al.
2007, 2008a).

1.2. General characteristics of plant root-knot nematode
Plant-parasitic nematodes, the important agricultural pests, have been reported to cause
damage amounting to more than 100 billion US dollars per year throughout the world,

placing them behind fungi but ahead of bacteria and viruses in terms of damage (Sharon et al.
2001). The root-knot nematodes (Meloidogyne spp.) are sedentary endoparasites and are
among the most damaging agricultural pests, attacking a wide range of crops. The infection
starts with root penetration of second-stage juveniles (J2), hatched in soil from eggs stored in
egg masses that have been laid by the females on the infected roots (Huang et al. 2004).
Root-knot nematodes have a life cycle consisting of five developmental stages. The first
and second juvenile stages (J1 and J2) occur in the egg. Motile J2s hatch from eggs in the
soil and locate host plants by following gradients of chemical cues. Following invasion of
the host plant roots, a permanent feeding site is established within the root and the
nematodes feed, grow and mould three more times to the adult stages. Adult males emerge
from the root while the females remain in the roots and lay eggs at various stages of
development into a gelatinous matrix, which extrudes from the root. The eggs are surrounded
by the eggshells whose strength are provided by a chitinous layer (Fanelli et al. 2005).
Among nematode developing stages, second-stage juveniles and eggs are more
susceptible to fungi. The shell of nematode egg belonging to the order Tylenchida consists
three layers; vitelline, chitin and lipit. Vitelline layer is about 10 - 40 nm. Chitin layer, 50-
400 nm, is the most obvious as it is visible under the light microscope, which is composed of
protein matrix (50–60% of the composition) embedding chitin microfibrils. This chitinous
layer is the thickest and probably the major barrier to infection (Bird and Mcclure 1975).


- 5 -

1.3. Biological control of plant parasitic nematode
It has been accepted for decades that effective control of plant-parasitic nematodes is
dependent on chemical nematicides. Nematicides, though efficient and working quickly, are
now being reappraised with respect to the environmental hazards that they pose, their high
costs and limited availability in many developing countries. De-registration of some of the
more hazardous nematicides has emphasized the need for new methods to control nematodes
(Fanelli et al. 2005). Control of plant-parasitic nematodes can be by improvements of soil

structure and fertility, alteration of the level of plant-resistance, release of nematode-toxic
compounds, parasites (fungi and bacteria) and other nematode antagonists (biological control
agents) (Akhtar and Malik 2000).
Today, numerous microorganisms are recognized as antagonists of plant-parasitic
nematodes. Biological control agents that act beneficially against nematodes are widespread
in cultivated soils worldwide and can help to control nematodes. These organisms have been
found on a variety of nematode hosts and in many different climates and environmental
conditions (Siddiqui and Mahmood 1996). However, nematicidal efficacy of naturally
occurring antagonists is likely to be affected by environmental conditions. Interactions
between plant-parasitic nematodes and fungal, bacterial and invertebrate antagonists are
influenced differently by several biotic and abiotic factors (Siddiqui and Mahmood 1996).
Biocontrol agents of nematodes are unlikely to be as fast-acting as nematicides, and to obtain
lasting reduction in nematode numbers it is likely that they will have to be integrated with
other methods (Kerry 1980)
.
Of the organisms, fungi, bacteria, viruses, predators, nematodes, insects, mites and
some vertebrates, those parasitize or prey on nematodes or reduce nematode population by
their antagonistic behavior, fungi hold important position and some of them have shown
great potential as biological control agents (Siddiqui and Mahmood 1996). According to Kim
et al. (1998) the characteristics of good biological control agents are: (i) high parasitism to
nematodes, (ii) parasitism of nematode but not pathogenic to crop plants or higher animals,