Biological control of Fusarium oxysporum f.sp ...

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Biological control of Fusarium oxysporum f.sp. cubense using non-pathogenic F. oxysporum endophytes by

Aneen Belgrove

Submitted in partial fulfilment of the requirements for the degree of Magister Scientiae In the Faculty of Natural and Agricultural Science University of Pretoria Pretoria

Date October 2007

PROMOTOR:

Prof. A. Viljoen

CO-PROMOTOR:

Dr. C. Steinberg

I © University of Pretoria

Declaration

I, the undersigned, declare that the work contained in this thesis is my own and original work and that it has not previously in its entirety or part submitted for a degree to any other university.

_________________________________

II

TABLE OF CONTENTS Acknowledgements

XII

Preface

XIII

Chapter 1: Biological control of Fusarium wilt diseases ABSTRACT

2

INTRODUCTION

3

THE FUSARIUM WILT PATHOGEN

4

THE DISEASE

6

CONTROL OF FUSARIUM WILT

7

Chemical control

7

Cultural control

9

Disease resistance

10

Biological control

12

BIOLOGICAL CONTROL OF FUSARIUM WILT

12

Suppressive soils

12

Mechanisms of biological control

13

Antibiosis

13

Competition

14

Induced resistance

15

Biological control agents

16

Pseudomonas and Bacillus spp.

16

Non-pathogenic Fusarium oxysporum

17

Other microorganisms reducing Fusarium wilt

19

Mycorrhizae

20

Endophytic biological control organisms

21

Combining different biological control agents

22

Combining biological control agents with other control strategies 23 Factors affecting biological control

23

CONCLUSION

25

REFERENCES

26 III

Chapter 2: Evaluation of non-pathogenic Fusarium oxysporum endophytes from banana for biological control of Fusarium oxysporum f.sp. cubense Abstract

51

Introduction

52

Materials and Methods

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Results

60

Discussion

62

References

65

Chapter 3: Phenolic acid production in Cavendish banana roots following colonization by non-pathogenic Fusarium oxysporum and Pseudomonas fluorescens Abstract

82

Introduction

83

Materials and Methods

85

Results

90

Discussion

93

References

98

Chapter 4: Transformation of a non-pathogenic Fusarium oxysporum endophyte with the green (GFP) and red (DsRed-Express) fluorescent protein genes Abstract

114

Introduction

115

Materials and Methods

116

Results

122

Discussion

123

References

125

Chapter 5: Histological investigation of the interaction between pathogenic and non-pathogenic isolates of Fusarium oxysporum, and banana IV

Abstract

133

Introduction

134

Materials and Methods

136

Results

139

Discussion

140

References

143

Summary

154

V

LIST OF TABLES:

Chapter 2: Table 1. Intergenic spacer region (IGS) genotype groups obtained with restriction fragment length polymorphism analysis of Fusarium oxysporum isolates collected from healthy banana roots in Fusarium wilt suppressive soils, and their pathogenicity status.

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Table 2. The number of Fusarium oxysporum isolates obtained from banana roots planted in Fusarium wilt suppressive soils in Kiepersol, South Africa. The isolates were grouped according to their PCR-restriction fragment length polymorphisms of the intergenic spacer region.

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VI

LIST OF FIGURES:

Chapter 2: Figure 1. Morphological characteristics of Fusarium oxysporum: A) Microconidia borne in false head, and B) A single chlamydospore produced apically on a fungal hyphae.

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Figure 2. Identification of Fusarium oxysporum isolates by using PCR primers FOF1 and FOR1. Lanes 1: 100bp DNA marker; 2: Water used as negative control; 3: Fusarium solani; 4: Fusarium oxysporum f.sp. cubense; 5-8: Endophytic F. oxysporum isolates CAV 552, 553, 557 and 563. The sizes of the molecular weight marker and the size of the band are indicated to the left of the figure.

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Figure 3. PCR amplification products of the intergenic spacer region of the ribosomal DNA of Fusarium oxysporum isolates. PCR products were visualized on a 0.8% agarose gel stained with ethidium bromide. Lanes 1:  molecular weight marker; 2-10: Fusarium oxysporum isolates; and 11: water control.

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Figure 4. Restriction fragment length polymorphism (RFLP) patterns obtained for Fusarium oxysporum isolates from healthy banana roots. Each of the illustrations represent the RFLP pattern produced when the intergenic spacer region of the ribosomal DNA was digested with the restriction enzymes MspI, RsaI, ScrFI, HindfI, and HaeIII.

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Figure 5: The mean incidence of Fusarium wilt of banana caused by the pathogen Fusarium oxysporum f. sp. cubense in the greenhouse, as affected by treatments with various isolates of non-pathogenic F. oxysporum. The control treatment is CAV 092 and received water only. Bars presented with the same letter are not significantly different at P