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

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,

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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

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

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

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

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

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

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

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

LIST OF TABLES

microsporus VS-9 in 0.5% swollen chitin cultural medium 43

VS-9 crude chitinases 46

crude chitinase activity 47

R microsporus VS-9 49

and R microsporus VS-9 crude chitinases 52

aureoviride DY-59, and R microsporus VS-9 chitinases 55

culture filtrate of P variotii DG-3 after 12 days of growth in 0.5% swollen

P variotii DG-3 75

chitinase activity by N-terminal sequencing and matching with known

proteins in NCBI database 76

chitinases and commercial enzymes 82

shell and F solani cell walls 85

medium 102

antillanum B-3 107

chitinase 109

fractionated from DEAE-Sephadex chromatography 110

and reduced index after growth period 123

LIST OF FIGURES

plates isolated from different materials 23

fungi on chitin medium plates 23

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

fungi F solani with other chitinolytic fungi (A) T aureoviride DY-59,

(B) R microsporus VS-9, and P variotii DG-3 27

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

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

R microsporus VS-9 (○) These fungi were grown in 250 ml Erlenmeyer

flask containing CBM at 25oC, 150 rpm for 12 days 44

(●) and R microsporus VS-9 (○) crude chitinases 45

and R microsporus VS-9 crude chitinases, (A) standards of

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

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

DY-59 and R microsporus VS-9 chitinases The mixture of enzyme and

conidial suspension was incubated at 30oC for 20 hr (A) control, (B) DY-59 chitinase, and (C) VS-9 chitinase ICW, intact cell wall; DCW,

digested cell wall 51

rate, (A) T aureoviride DY-59 chitinase, (B) R microsporus VS-9

chitinase 52

900 µl of 1% hyphal biomass in sodium acetate buffer (pH 5) and 100 µl

VS-9 (○) 53

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 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

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

supernatant The protein was eluted stepwise with 20 mM Tris-HCl (pH

7.5) containing 0.0-0.5 M sodium chloride 70

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

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 (Amersham 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

swollen chitin was used as the substrate 73

of substrate (100µg ml-1) in 50 mM citrate buffer (pH 3) and 50 µl of enzymes were incubated at 37oC for 60 min The products (3 µl) were

separated by HPLC 78

the right) from chitin trimer (A, E), tetramer (B, F), pentamer (C, G), and

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

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

Brightener 28 81

(A), ungerminated conidia (UG) by Chi32; (B), short germinated conidia (SG) by Chi46; (C), normal germinated conidia (NG) in control (heated

enzyme) 83

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

(C) 86

heated enzyme at 100oC 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 37oC for 48 hours 87

Chi32 and Chi46 Reaction mixture consisting of 900 µl of 0.5 %

chitin (B) by Chi32 and Chi46 The chitin oligomers (2 µl) were separated by HPLC using NH2 P50 column 88

with L antilanum (L.an) and L fusisporium (L.fu) by software of website

http://bioinfo.genotoul.fr/multalin/- 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 other fungi from NCBI database by a rectangle tree-making software program (http://www.ncbi.nlm.nih.gov/BLAST/) 101

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

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 was eluted stepwise

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 Lane 1, molecular weight marker (Amersham Biosciences); lane 2, crude enzyme; lane 3, chitinase fractions from DEAE-Sephadex column, lane 4 activity staining of

chitinase fraction from DEAE-Sephadex column 104

chitinase 105

oligomer standard, (B) by L antillanum B-3 chitinase 106

hyphae bound to eggshells by SEM (A) and parasitized the second-stage

juvenile (B) 108

control egg, (B) an egg treated with B-3 purified chitinase Eggs were treated with the enzyme for 4 days and stained in lactoglycerol solution

as in Materials and Methods Scale bar: 18 µm 109

stage juvenile, (C) larva, (D) adult, and (E) root-knot symptom on cucumber 121

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

egg-parasitic fungi, more root galls were shown from root in 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

nematode-(Ne and A-1, Ne and B-3, Ne and A-1 plus B-3) 124

week-old cucumber in nematode-infected treatment 124

ABBREVIATIONS

SDS-PAGE: Sodium dodecyl sulfate polyacryl amine gel electrophoresis

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

F solani hyphae to produce chitin oligosaccharides, among which GlcNAc, (GlcNAc)2, and

(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

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

CHAPTER I

GENERAL INTRODUCTION

1.1 General property and distribution of chitin

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,

-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)

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)

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,

(iii) growth at suitable pH and temperature ranges, (iv) growth on artificial media, (v) identifiable and with mode of action known, (vi) formulation in usable form, competition with other soil microorganisms Many soil-borne fungi have been demonstrated to be antagonists of nematodes These include nematode-trapping or predacious fungi, endoparasitic fungi, parasites of nematode eggs and cysts, and fungi that produce metabolites toxic to nematodes The endoparasitic fungi are often obligate parasites and have a limited saprophytic phase They produce almost no mycelium in soil and complete their life cycle within the body of their hosts The endoparasites with encysting zoospores belong to the

Chytridiomycetes and Oomycetes and their infective propagule is a flagellated zoospore

Zoosporic endoparasitic fungi depend on free water for their activity, their limited growth in culture Moreover, they are poor competitive saprophytic ability (Siddiqui and Mahmood 1996)

More than 50 species of predacious fungi, which capture and kill nematodes in soil is

found in several genera of Hyphomycetes and some species in Zoopagales With age, these

fungi exhibit reduction in nematode-trapping efficiency For effective control, it is necessary that their limited period of activity coincides with the period of nematode invasion of crop roots Peak fungal activity is reached only 12-15 days after introduction It is believed that the activity of these organisms might be stimulated by the addition of organic matter to the soil Predacious fungi form different types of traps, such as adhesive branches, adhesive-network traps, adhesive knobs, non-constricting rings and constricting rings, to capture nematodes Predacious fungi have been shown to be poor competitive saprophytes and are susceptible to antagonism from other soil fungi Opportunistic fungi can colonize nematode reproductive structures and have the ability to deleteriously affect them Nematodes belonging to the heterogenic group and at the sedentary stages of their life-cycle are vulnerable to be attacked by these fungi either within the host roots or when exposed on the root surface or within the soil Fungal growth of these fungi is known to be enhanced in the rhizosphere Once in contact with cysts or egg masses, these fungi grow rapidly and

eventually parasitize all eggs that are in early embryonic developmental stages Apparently, when juveniles are formed the parasitic activities of these fungi are generally reduced (Casas-Flores and Herrera-Estrella 2007, Dong and Zhang 2006)

1.4 Chitinases

1.4.1 Property and molecular structure of chitinases

Chitinases (EC 3.2.1.14) are glycosyl hydrolases that catalyze the degradation of chitin,

a component chemical in nematode eggshells and fungal cell walls Chitinolytic enzymes are found in a variety of organisms, not only fungi and insects, but also bacteria and higher plants Recently chitinases have received renewed attention since they play a role in plant defense against chitin-containing pathogens The catalytic domains of chitinases can be grouped in two families based on amino acid sequence similarities and the lack of 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

Endochitinases randomly cleave chitin polymers, producing chitinoligomers; chitobiosidases

terminal nonreducing ends, thus producing GlcNAc (Duo-Chuan 2006, Gooday 1994, Mavromatis et al 2003) Most of fungal chitinases are multi-domain structures with the molecular masses range widely from 27 to 190 kDa (Dahiya et al 2006, Fukamino 2000).The optimal pH ranges from between 4.0 and 7.0, and optimal temperature for most fungal

Rapid advances in the genome sequencing programmers over last five years provided first insights into the chitinolytic potential of microorganisms (http://genome.jgi-

psf.org/mic_home.html) Information about their hydrolytic enzymes is compiled in the Carbohydrate Active Enzymes database (CAZy, http://www.cazy.org) Filamentous fungi have developed more complex machinery of chitinolytic enzymes than bacteria and fungal genomes contain in general between 10 and 25 different chitinases The reasons why fungi have so many chitinases are not well understood Analysis of up to 25 fungal genomes so far has shown that fungal 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 subgroup of chitinases was

detected, which had not been previously described in filamentous fungi This subgroup is phylogenetically different from the class III and class V chitinases and consequently GH 18 chitinases were (re)classified into subgroup A (class V), subgroup B (class III) and subgroup

C (i.e the novel subgroup of fungal chitinases) (Figure 1.1) Up to now only chitinases belonging to subgroups A and B had been characterized This novel subgroup of chitinases displays several interesting properties which clearly distinguish them from other fungal chitinases Subgroup A and B chitinases comprise proteins of 30-50 kDa whereas about a molecular mass of 140-170 kDa is typical for subgroup C These chitinases contain N-terminal signal peptides, which suggest that they are targeted to the secretive pathway They contain a chitin-binding domain (CBM 18 according to the CAZy classification) and tandem lysin motifs (LysMs), which are short peptide domains implicated in binding of peptidoglycan and structurally related molecules in bacterial and eukaryotic proteins, respectively (Seidl et al 2005) Most chitinases characterized from filamentous fungi have

no CBMs Only in a few cases a CBM, more precisely a cellulose-binding domain (CellBD)

- CBM1 in the CAZy classification - was found (Choquer et al 2007, Takaya et al 1998)

Figure 1.1 Domain organization of fungal chitinases, BD, binding domain

(Seidl et al 2005)

1.4.2 Roles of chitinases in biological control of plant diseases

Among the chitinase-producing organisms, fungi are believed to produce various isoforms of chitinases with different biophysical functions In fungi, chitinases play an important roles in biological and physiological such as lysis of the cell walls (separation of cells after division, hyphal autolysis), nutritional requirements, morphogenetic formation (sporulation, spore germination, hyphal growth) and antagonistic actions against other microorganisms (Sahai and Manocha 1993)

Several extracellular enzymes from nematophagous fungi, including protease (especially the subtilisin family of serine proteases), chitinase and collagenase involved in the infection of nematodes, have been identified, cloned, and homologously or heterologously expressed, respectively The molecular mechanisms of the sequences are not well explained, but based on research on entomopathogens, whose infectious mode was believed to be somewhat similar to that of nematophagous fungi, it is likely that hydrolytic enzymes participate in several steps of host infection Moreover, ultrastructural and histochemical studies have suggested that penetration of the nematode cuticle involves the

activity of hydrolytic enzymes In the last decade, extracellular enzymes as virulence factors

in the infection process have been intensively studied, and the identification of numerous enzymes has confirmed their involvement in the molecular mechanism of infection (Huang

et al 2004)

Described by Huang et al (2004) chitinases, which have been assumed to be required for hyphal growth and participate in infection of mycoparasitic, entomopathogenic or nematopathogenic fungi are inducible enzymes catalyzing chitin, which is one of the important components of invertebrate cuticles Fungal chitinases and its potential role in infecting nematode eggs were first put forward (Godoy et al 1982, 1983, Miller and Sands

1975, Stirling and Mankau 1979) Then, chitinase activity was detected by enzymatic assay

in Verticillium spp isolated from infected nematode eggs It was also revealed that the

proportion of infected nematode eggs increased concurrently with enhancement of chitinase activity (Khan et al 2003, Khan et al 2004, Nguyen et al 2007, Tikhonov et al 2002) Much evidence has been shown that fungal chitinases can degrade fungal cell walls and

inhibit fungal growth in vitro (Duo-Chuan 2006) The antagonistic Trichoderma induces the

production of extracellular hydrolytic enzymes, which are responsible for the direct attack

β-D-glucosaminidase, which is essential for triggering chitinase gene expression (Brunner et al 2003) When produced at a low level, this enzyme diffuses into the soil and catalyses the release of cell-wall-oligomers from the target fungi (Harman et al 2004, Limon et al 2004)

Limon et al (2004) reported that endochitinases Chit 42 and Chit 33 from T harzianum have

an essential role in the antagonistic activity to numerous fungal phytopathogens although this activity involved several mechanisms Using green fluorescent protein, direct reporter of

chitinase gene expression during mycoparasitic interaction between Trichoderma harzianum and Rhizoctonia solani, Zeilinger et al (1999) implied that 42 kDa endochitinase gene expression was induced by soluble production of enzymatic digestion of R solani cell walls

1.5 Study objectives

The potential roles of fungal chitinases in exogenous chitin degradation, in defense and attacking mechanisms against other fungi, nematode, and arthropods raise a number of questions whether more logical background information is not known For that purpose, the objectives of our study are about: (1) isolation of chitinolytic fungi having antifungal activity

and nematode-egg parasitism, (2) characterization of H aureoviride DY-59 and R

microsporus VS-9 chitinases and elucidating the role of DY-59 and VS-9 chitinases on the

suppression of a F solani fungus (3) purification and characterization of two chitinases (Chi32 and Chi46) from M incognita egg-parasitic fungus P variotii DG-3, (4) analysis of the mode of action of these chitinases on chitooligomers, (5) extraction of chitin from F

solani cell walls and analysis of hydrolysis products from fungal chitin by Chi32 and Chi46,

(6) initial elucidation of the role of the 37kDa chitinases from L antillanum B-3 in the infection process to M incognita eggs, (7) suppression of root-knot nematode M incognita

on cucumber by chitinolytic fungi L psalliotae A-1 and L antillanum B-3 in pot experiment

CHAPTER II

ISOLATION AND SCREENING OF

ANTAGONISTIC-CHITINOLYTIC FUNGI FROM SOIL

ABSTRACT

Chitinases play an important role in parasitism of fungi into some plant pathogens containing chitin in cell walls such as fungi, nematodes, or insects This chapter describes procedure of screening of chitinase-producing fungi, and then these chitinolytic fungi were sequentially screened for antifungal activity and nematode egg parasitism One hundred forty-one of fungal isolates were isolated from soil samples in Korea, Vietnam, and Thailand Twenty-five percentage of fungi isolated produced the clear zone on swollen chitin medium plates When the fungi were cultured in chitin medium broth, the 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 on M incognita eggs and antifungal activity against F

solani.

2.1 INTRODUCTION

Soil fungal communities play biodiversity and have a critical role in biogeochemistry

cycles as well as ecosystem functioning They play a central role in decomposing organic

matter, in determining the release of mineral nutrients, and in nutrient cycling, affect soil nutrient contents, chemical-physical properties, and consequently primary productivity

because they must rely on dead organic matter as their source of carbon and energy (Lucas et

application as biological control agents such as genus: Trichoderma, Verticillium,

Paecilomyces and more Genus Trichoderma is opportunistic, avirulent plant symbiont, and

function as parasites and antagonists of many phytopathogenic fungi, thus protecting plants

from diseases So far, Trichoderma spp are among the most studied fungal biological

control agents and commercially marketed as biopesticides, biofertilizers and soil

fungi of root knot and cyst nematode (Sharon et al 2001, Tikhonov et al 2002)

The complex process of mycoparasitism consists of several events, including recognition of the host, attack and subsequent penetration and killing of hosts During this

process Trichoderma secretes cell wall degrading enzymes that hydrolyze the cell walls of

the host fungus, subsequently released oligomers from the pathogen cell walls triggering fungi to produce antibiotics and lysis enzymes to kill hosts (Howell 2003) It is believed that

Trichoderma secretes hydrolytic enzymes at a constitutive level and detects the presence of

another fungus by sensing the molecules released from the host by enzymatic degradation (Harman et al 2004)

In nature, fungi continuously suppress virtually nematodes all soils because of their constant association with nematodes in the rhizosphere A large number of fungi are known

to trap, prey or parasite on nematodes but the most important genera include Paecilomyces,

Verticillium, Hirsutella, Nematophthora, Arthrobotrys, Drechmeria, Fusarium and Monacrosporium Application of some of these fungi has given very interesting results

(Siddiqui and Mahmood 1999) Chitinases are produced by some mycoparasitic, nematophagous or entomopathogenic fungi (Suresh and Chandrasekaran 1999), and some chitinase-producing fungi are capable of parasitizing phytopathogenic fungi, plant-parasitic

nematodes and insects Trichoderma harzianum has been reported as a biocontrol agent for

phytopathogenic fungi and plant-parasitic nematodes (Limon et al 1999, Sharon et al 2001)

Verticillium lecanii is the famous entomopathogenic fungus and is reported as agent to

parasitize plant-parasitic nematodes (Sugimoto et al 2003) Chitinases are considered as playing a key role in the parasitic mechanism of fungi into host cells containing chitin (e.g cell walls of fungi, eggshells of nematode eggs, exoskeletons of insects)

The protection of plant roots against infection by any microbial agents and plant nematodes is mainly dependent upon the introduction of hazardous pesticides into environment (El-Fiky et al 2003) Applications of fungicides and nematicide fumigants can have drastic effects on the environment and consumer and they are often applied in lager quantities Chemical methods are not economical in the long run because they pollute the atmosphere, damage the environment, leave harmful residues, and can lead to the development of resistant strains among the target organisms with repeated use

Using chitinolytic fungi as nematophagous and mycoparasitic agents have capability to

control plant pathogens, like Verticillium spp Trichoderma spp provide an alternative

means of reducing the incidence of plant diseases without negative aspects of pesticide application Interaction between biocontrol agents and plant pathogens have been studied extensively and application of bicontrol agents to protect some commercially important crops is promising (El-Fiky et al 2003)

One of the ways by which biocontrol agents can supper the plant pathogen is production

of hydrolytic enzymes Degradation of cell walls of some pathogenic organisms, especially fungi, via hydrolytic enzymes is an important mechanism in biological control Chitinases

are a one of hydrolytic enzymes produced by fungi and the role of chitinases in biocontrol of some pathogenic fungi has received increased attention for its effect on fungal pathogens and

it is encouraging to use chitinase-producing organisms as biological control agents (Dahiya

et al 2006)

For that purpose, the objective of this study was to isolate fungi having chitinolytic activity and having antifungal activity and plant nematode parasitism Enzyme characterizations of chitinases from selected fungi were determined

2.2 MATERIALS AND METHODS

2.2.1 Sample site

Field works were conducted in the agricultural fields of Korea, Vietnam, and Thailand Soil samples were collected and kept in plastic bags and transferred to the biochemistry

2.2.2 Preparing of soil samples

Soil samples were dried at room temperature for few days, grounded to powder by mortar and pestle, and then soil samples were separated and collected through 150 µm sieves One gram of prepared soil was diluted in 50 ml of 0.9% NaCl solution, shaken at 150 rpm,

dilution plate technique One ml of soil solution was transferred to PRBA (Burgess et al 1994)

2.2.3 Isolation, initial identification and maintenance of fungi

The fungi growing on PRBA medium were isolated to pure isolates The single fungal colony was isolated and transferred to new potato dextrose agar (PDA) plates The isolates were subcultured until to become a pure fungus on PDA medium

For initial identification of fungi, the morphological characteristics of fungi grown on PDA medium were observed Colony morphology, spore, and conidiophore shape, culture medium color and so on were observed and these characteristics were compared with identification keys (Barnett 1962, Burgess et al 1994, Samuels 2004, Watanable 2002) The

time maintenance

2.2.4 Composition of cultural medium for normal use

2.2.4.1 Swollen chitin mineral medium (SCM)

Table 2.1 Composition of basal mineral chitin medium

2.2.4.2 Peptone-rose bengal agar medium (PRBA)

Table 2.2 Pepton-rose bengal agar medium

(Onkar and James 1985)

2.2.5 Screening of chitinolytic fungi

Chitinolytic fungi were screened by clear zone exhibited on swollen chitin mineral plates (SCM), by amount of reducing sugar in broth medium and by the chitinase activity A pure fungus was grown on SCM plate to produce clear zone during 5-10 days Clear-zone-producing fungi were selected and sequentially cultured in 250 ml Erlenmeyer flasks containing 100 ml swollen chitin broth medium (CBM) to assay chitinase activity and amount of reducing sugar The clear-zone fungi producing chitinase with high enzyme activity were selected as chitinolytic fungi for sequential screening of antifungal activity

against Fusarium solani and nematode-egg parasitism on Meloidogyne incognita (Nguyen et

al 2007, 2008a)

2.2.6 Screening of Meloidogyne incognita egg-parasitic fungi

The M incognita (female, male and juvenile) and eggs were extracted and identified

following Materials and Methods described in Chapter VI To screen of egg-parasitic fungi, chitinolytic fungi selected were used to assay of direct parasitism of fungi on nematode eggs Fungal isolates were cultured on PDA medium for a time belonged mycelial growth (normally from 3 to 9 days) Five-mm-diameter fungal blocks were excised and transferred up-side-down to 8.7 x 15 cm Petri plates containing 1.5% water agar medium (WA) Four fungal blocks were placed in a group as four replications for observation per day The preparations, three fungal-block groups as a multiple pieces on a Petri dish, were incubated

approximately 1500 eggs ml-1 were directly placed on the top of each fungal piece and

(Olympus SZ 4045 Japan), and microscope (Motic BA, Japan) to count the number of fungus-invaded eggs at 1, 2, 3, 4, and 5 days after treatment (Nguyen et al 2007)

2.2.7 Screening of mycoparasitic fungi against Fusarium solani

F solani was isolated and identified as described in Materials and Methods of Chapter

VI Inhibition of mycelial growth, hyphal extension-inhibition, and conidial inhibition assay were carried out to select chitinase-producing fungi as mycoparasitic fungi

germination-F solani isolated from cucumber root was cultured on PDA medium at 28oC for 9 days Five

mm fungal piece from 9-days-old cultures was placed at center of 9 cm PDA medium disks

treatment Then three of 5 mm fungal pieces of chitinolytic fungi were put as a rectangular

form surrounding F solani colony Growth inhibition was observed and recorded for colony

growth inhibition and competition The second criterion for select antifungal activity is conidial germination inhibition and lysis fungal cell walls (Chapter III)

2.2.8 Schale’s assay for determining of reducing sugar

The assay was carried out as described by (Imoto and Yamashita 1971) Briefly, 1 ml of

a 0.5 M sodium carbonate solution containing 0.5 g potassium ferricyanide was mixed with

density was determined at 420 nm Amount of reducing sugar was determined basing on

standard curve of N-acetyl glucosamine, N-glucosamine or glucose

2.2.9 Preparing of crab swollen chitin

Swollen chitin was prepared from chitin (Shin-yong Chitosan Ltd Korea) by method previously described (Monreal and Reese 1969) Twenty grams of chitin power was slowly added into 200 ml of phosphoric acid, stirred several hours at room temperature, and kept this preparation at 4oC for 24 h The mixture was added to 5 liters of distilled water with rapid stirring and kept overnight at 4oC The precipitate was collected by centrifugation at 6,000g for 30 min at 4oC The precipitate was washed with distilled water until the pH of swollen chitin solution reach to neutral The suspension was blended to small pieces and

prepared as 2% swollen chitin suspension in desired pH buffer (pH 4, or 5) and stored at 4oC until use

2005)

2.2.11 Exochitinase assay

min (Kuk et al 2005)

2.2.12 Characterizations of crude enzymes from chitinolytic fungi

The basic characteristics of chitinases such as optimal temperature, pH, substrate specificity, hydrolysis products and so on were analyzed following methods described in Materials and Methods of Chapter III, IV, and V