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

by

Aneen Belgrove 动感之星

Submitted in partial fulfilment of the requirements for the degree of Magister Scientiae In the Facul

ty 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 [pic]

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.

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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

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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 54

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

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Abstract 133

Introduction

134

Materials and Methods 136 Results 139 Discussion 140 References 143

Summary

154

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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. 72 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.

74

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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. 75 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.

76 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: λ molecular weight marker; 2-10: Fusarium oxysporum isolates; and 11: water control. 77 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 Msp I, Rsa I, ScrF I, Hindf I, and Hae III. 78 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