Fusarium wilt had almost destroyed the banana export industry in Central America that was ...... Mannheim, Germany) and processed with Adobe Photoshop 7.0.
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
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
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 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.
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
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.
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: 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 MspI, RsaI, ScrFI, HindfI, and HaeIII.
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
