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Studies on Guava Wilt Disease STUDIES ON GUAVA WILT DISEASE Nigel Mark Grech Submitted in partial fulfillment of the academic requirements for the degree of Doctor of Philosophy in Plant Pathology Discipline of Plant Pathology School of Agricultural, Earth and Environmental Sciences College of Agriculture, Engineering and Science University of KwaZulu-Natal Pietermaritzburg South Africa November, 2015 I THESIS SUMMARY i ACKNOWLEDGEMENTS I would like to thank the following: Professor Frits Rijkenberg for getting me started on this undertaking; Professor Mark Laing, my major advisor, for his encouragement, sound advice, editing skills and friendship in getting me through to the end; The College of Agriculture, Engineering and Science for their long patience whilst I balanced work and graduate studies; Mr Ron Frean (deceased) who assisted me considerably in the field; The Institute for Tropical and Sub Tropical Crops, ARC, for allowing me the opportunity to work on this exciting pathogen and truly fascinating crop; and special thanks to Dr John Moll, who is now sadly deceased but is fondly remembered, for his enthusiasm, support, awful fishing and excellent sense of humor. ii DECLARATION I, Nigel Mark Grech, hereby declare that: 1. This document is a token of my own work and has not previously, in its entirety or in part, been submitted to any other institution for any academic award. 2. All sources of information used in this literature review have been duly acknowledged. 3. This thesis does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other scientists. Where other written sources have been quoted, then: i) Their words have been rewritten but the general information attributed to them has been referenced. ii) Where their exact words have been used, then their writing has been placed in italics and inside quotation marks, and referenced. 4. This thesis does not contain text, graphics or tables copied and pasted from the internet unless specifically acknowledged, and the source being detailed in the thesis and in the references sections. Nigel Mark Grech (Candidate) Signature:………………………………………………………………………………………… As the candidate’s supervisor, I agree to the submission of this thesis: Supervisor: Prof Mark Laing Signature: Date:………………………………….. iii TABLE OF CONTENTS THESIS SUMMARY ................................................................................................................................... I ACKNOWLEDGEMENTS ....................................................................................................................... II DECLARATION ....................................................................................................................................... III CHAPTER 1: INTRODUCTION, LITERATURE REVIEW AND GLOBAL DISTRIBUTION OF GUAVA WILT DISEASE .......................................................................................................................... 1 1.1 Guava .............................................................................................................................................................. 1 1.2 Guava Production ........................................................................................................................................... 4 1.4 The Identity of the Pathogen Causing Guava Wilting Disease ...................................................................... 13 1.5 Thesis Research ............................................................................................................................................. 16 REFERENCES ......................................................................................................................................................... 17 CHAPTER 2: ISOLATION OF THE CAUSAL ORGANISM OF GUAVA WILT DISEASE, AND FULFILLMENT OF KOCH’S POSTULATES ..................................................................................... 25 ABSTRACT ............................................................................................................................................................ 25 2.1 INTRODUCTION........................................................................................................................................ 25 2.2 MATERIALS AND METHODS ..................................................................................................................... 26 2.2.1 Collection procedures .............................................................................................................................. 26 2.2.2 Isolation procedures ................................................................................................................................ 26 2.2.3 Fulfilment of Koch’s Postulates ................................................................................................................ 28 2.2.4 Preliminary pathogenicity tests of different conidial types ...................................................................... 30 2.3 DATA ANALYSES ....................................................................................................................................... 33 2.4 RESULTS ................................................................................................................................................... 33 2.4.1 Isolation of the causal organism .............................................................................................................. 33 2.4.2 Pathogenicity tests and fulfillment of Koch’s Postulate ........................................................................... 35 2.4.3 Preliminary pathogenicity tests of different conidial types ...................................................................... 38 2.5 DISCUSSION ............................................................................................................................................. 39 REFERENCES ......................................................................................................................................................... 40 iv CHAPTER 3: IDENTIFICATION, TYPE CULTURE COMPARISONS, MICROSCOPY AND GENOMIC ANALYSIS OF THE PATHOGEN, NALANTHAMALA PSIDII .................................. 42 ABSTRACT ............................................................................................................................................................ 42 3.1 INTRODUCTION........................................................................................................................................ 42 3.2 MATERIALS AND METHODS ..................................................................................................................... 46 3.2.1 Fungal type cultures ................................................................................................................................. 46 3.2.2 Microscopy .............................................................................................................................................. 49 3.2.3 Maintenance of isolates and type cultures, morphological characterization of the causal organism of GWD 51 3.2.4 Genomic analysis of the South African type N. psidii isolate (CSF 221) ..................................................... 52 3.2.5 Type Culture Pathogenicity Tests ............................................................................................................. 53 3.3 DATA ANALYSES ....................................................................................................................................... 53 3.4 RESULTS ................................................................................................................................................... 54 3.4.1 Taxonomic characteristics of the causal fungi of GWD and TGWD, N. psidii ............................................. 54 3.4.3 Pathogenicity tests .................................................................................................................................. 76 3.5 DISCUSSION ............................................................................................................................................. 78 3.5.1 Morphological Taxonomic Studies .............................................................................................................. 78 3.5.2 Genomic Taxonomic Studies ....................................................................................................................... 85 3.5.3 Pathogenicity Studies .................................................................................................................................. 86 3.5.4 Genus Level Taxonomy ............................................................................................................................... 87 3.5.5 Species Level Taxonomy .......................................................................................................................... 88 REFERENCES ......................................................................................................................................................... 89 CHAPTER 4: PHYSIOLOGICAL COMPARISON BETWEEN SOUTH AFRICAN AND TAIWANESE ISOLATES OF NALANTHAMALA PSIDII AND SPECIFIC COMPARISONS WITH GLIOCLADIUM VERMOESENII ............................................................................................... 95 ABSTRACT ...........................................................................................................................................................
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