EFFECT OF BIOLOGICAL CONTROL OF PHYTOPHTHORA

1 DISTRIBUTION, MOLECULAR IDENTIFICATION AND THE EFFECT OF BIOLOGICAL CONTROL OF PHYTOPHTHORA CINNAMOMI ON MACADAMIA IN THE LIMPOPO AND MPUMALANGA PROVINCES, SOUTH AFRICA By Nontokozo Sibahle Kunene Submitted in fulfillment of the requirements for the degree of 10 MASTER OF SCIENCE 11 12 In the Discipline of Plant Pathology 13 14 15 University of KwaZulu-Natal (UKZN), King Edward Avenue, Pietermaritzburg, Private Bag X01, Scottsville, 3209 16 17 And 18 19 Agricultural Research Council – Tropical and Subtropical Crops (ARC-TSC), Private 20 Bag X11208, Mbombela, Mpumalanga, 1200 21 Email: KuneneN@arc.agric.za 22 23 December 2017 DISSERTATION SUMMARY Macadamia is a nut-bearing tree belonging to the family Proteaceae Macadamia tetraphylla, and Macadamia integrifolia (also M ternifolia), and their hybrids are of importance in commercial cultivation Root rot and trunk canker are the most important diseases caused by P cinnamomi that could cause 60% yield losses and an estimated 10% of the annual gross value of macadamia The objective of this study was to determine the distribution, molecular identification and the effect of biological control of P cinnamomi in the main macadamia growing areas of South Africa The first part of this study assesses disease incidence and severity as well as the 10 distribution of P cinnamomi in the main macadamia growing areas of South Africa 11 Phytophthora cinnamomi was recovered from soil samples by baiting and from plant tissues 12 by plating on P cinnamomi selective medium Root rots and stem cankers were recorded in 13 52 % of the farms sampled No significant (P > 0.05) differences were observed in disease 14 incidence and severity between the sampled provinces The highest disease incidence 15 (64.2 %) was found in the Mpumalanga province Phytophthora cinnamomi had a wide 16 distribution in all the main macadamia growing areas 17 The second part of the study investigated DNA detection of P cinnamomi from soil 18 samples A nested PCR amplification protocol was optimised with both primary and nested 19 PCR specific for P cinnamomi detection The protocol improved both the specificity and 20 sensitivity of PCR amplification in comparison to the one-step PCR The application of 21 diagnostic nested PCR together with the DNA extracted using the baiting bioassay was 22 verified by comparison with DNA extracted using a kit The nested PCR using DNA 23 extracted by baiting was found to be more sensitive 24 The final part of the study examined two Trichoderma spp and eight unknown 25 Bacillus spp as potential biological control agents (BCAs) for management of P 26 cinnamomi The potential BCAs were evaluated for their in vitro growth inhibition of seven 27 P cinnamomi isolates All the isolates were sensitive to the ten potential bio-control agents 28 The Trichoderma spp and two best Bacillus spp (B 41b and NB 4) caused in vitro growth 29 inhibition of 22 – 90 % in the laboratory in vitro studies Depending on the mode of action, 30 these BCAs should be evaluated further for their potential use in the integrated 31 management of root rots and stem canker of macadamia i Declaration I declare that this thesis is my original work and has not been submitted for a degree at any other tertiary education institution To the best of my knowledge, this dissertation contains no material or work performed by others, published or unpublished without due reference 10 being made within the text 11 12 13 14 15 Signed………………………………… 16 Nontokozo Sibahle Kunene (Candidate) Date:…………………………… 17 18 Signed………………………………… 19 Dr AEC Jooste (Supervisor) Date:…………………………… 20 21 Signed………………………………… 22 Prof A Gubba (Co-supervisor) Date:…………………………… 23 ii Acknowledgements Abba Baba, Mvelinqangi… Ngiphakamisa udumo Kuwe I wish to express my appreciation to my Supervisor, Dr Elize Jooste, for her valuable supervision I know I frustrated you a lot but thank you for pushing and not giving up on me Thank you for your guidance, assistance and always making time to help I am extremely thankful to Associate Professor, Prof G, and my supervisor, who has been my inspiration and has pushed me to work hard even when I felt the heat was a bit too much Thank you for believing in me from the very moment I entered that Plant Pathology 10 305 lecture You had my back, been a friend and a dad when I needed you 11 A special thank you to my Angels, my Grandmother, and Mother You have been my 12 unwavering support system from the word go Your prayers have carried me through, and 13 just know that this is only the beginning, the best is yet to come I will continue making you 14 proud Thanks for raising a Queen 15 Thanks are also due to my colleagues at the ARC, Department of Plant Pathology: you 16 made life a whole lot easier Zama, thank you for being a sister and a friend, the love you 17 have shown me is astounding Thabs, thanks for being a friend and a confidant, and always 18 making sure that I am okay A special shout-out to “Bra Tom,” who helped me a great deal 19 Thank you for sharing your vast knowledge and willingness to help at any given time 20 I am grateful to Subtrop as well as the macadamia growers and farmers who allowed me to 21 collect samples from their orchards 22 I thank and appreciate everyone who contributed towards this study Those who helped me 23 during sample collection to data analyses Everyone who supported me throughout this 24 project, you are highly appreciated 25 I wish to express my highest appreciation to NRF and the ARC-PDP program for the 26 financial assistance throughout the study I would not have been here had it not been for the 27 funding from these two companies iii Dedication 10 11 {To iNdlovukazi, Jobe kaMatshane, Mthembu weGubazi… Gogo, this one is for you} 12 13 14 15 16 17 18 19 20 21 iv Table of Contents DISSERTATION SUMMARY i Declaration ii Acknowledgements iii Dedication iv INTRODUCTION TO DISSERTATION CHAPTER LITERATURE REVIEW 1.1 Macadamia 10 1.1.1 History and Distribution 11 1.1.2 Botany 12 1.1.3 Economic Importance 13 1.2 The Macadamia Industry 10 14 1.2.1 Global Macadamia Industry 10 15 1.2.2 The South African Macadamia Industry 12 16 1.3 Insect Pests and Diseases of Macadamia 14 17 1.3.1 The Macadamia Stinkbug (Nezara viridula) 15 18 1.3.2 The Nut Borer 15 19 1.3.3 Raceme Blight 15 20 1.3.4 Macadamia husk spot 16 21 1.3.5 Phytophthora Blight 16 22 1.4 The Genus Phytophthora 16 23 1.5 Phytophthora cinnamomi 18 24 1.5.1 Epidemiology and Aetiology 20 25 1.5.2 Mechanism of Infection 20 26 1.5.3 Disease Development on Macadamia 22 27 1.5.4 Control 24 28 1.5.4.1 Cultural 25 29 1.5.4.2 Chemical 25 30 1.5.4.3 Resistance 26 31 1.5.4.4 Biological Control 27 32 1.6 Detection techniques for Phytophthora cinnamomi 28 33 1.6.1 Isolation 28 v 1.6.2 Baiting 29 1.6.3 Immunological Detection 30 1.7 DNA Detection Methods 31 1.7.1 PCR Detection of P cinnamomi 32 1.8 References 34 CHAPTER 55 OPTIMISATION OF A NESTED PCR PROTOCOL FOR THE DETECTION OF PHYTOPHTHORA CINNAMOMI ON MACADAMIA IN THE LIMPOPO AND MPUMALANGA PROVINCES, SOUTH AFRICA 55 10 Abstract 55 11 2.1 Introduction 56 12 2.2 Materials and Methods 57 13 2.2.1.1 P cinnamomi isolates 57 14 2.2.1.2 P cinnamomi isolates from soil samples 57 15 2.2.2 Maintenance of P cinnamomi isolates 57 16 2.2.3 DNA extraction from mycelial cultures 58 17 2.2.4 DNA extraction from soil samples 59 18 2.2.5 Polymerase chain reaction (PCR) amplification 59 19 2.2.6 Gel electrophoresis 61 20 2.3 Results 61 21 22 2.3.1 Optimized PCR amplification with nested primers CIN3A/CINITS4 and CIN3B/CIN2R 61 23 2.3.2 PCR amplification with LPV3 primers 61 24 25 2.3.3 Comparison between DNA isolated with the soil kit and DNA isolated by the baiting bioassay using nested PCR amplification 62 26 2.4 Discussion 64 27 2.5 References 65 28 CHAPTER 68 29 30 THE INCIDENCE AND DISTRIBUTION OF PHYTOPHTHORA CINNAMOMI IN THE MAIN MACADAMIA GROWING AREAS OF SOUTH AFRICA 68 31 Abstract 68 32 3.1 Introduction 69 33 3.2 Materials and Methods 70 34 3.2.1 Disease surveys 70 35 3.2.2 Recovery of P cinnamomi 70 vi 3.2.3 Detection of P cinnamomi 71 3.2.4 Data analysis 71 3.3 Results 72 3.3.1 Disease surveys 72 3.3.2 P cinnamomi isolates recovered 72 3.3.3 Detection of P cinnamomi 73 3.4 Discussion 74 3.5 References 76 CHAPTER 80 10 11 IN VITRO EVALUATION OF BIOLOGICAL CONTROL AGENTS AGAINST PHYTOPHTHORA CINNAMOMI ON MACADAMIA IN LIMPOPO AND MPUMALANGA, SOUTH AFRICA 80 12 Abstract 80 13 4.1 Introduction 81 14 4.2 Materials and Methods 82 15 4.2.1 Phytophthora cinnamomi isolates 82 16 4.2.2 Screening of P cinnamomi isolates for pathogenicity and virulence on green apples 83 17 4.2.3 Selected potential biological control agents 83 18 4.2.4 In vitro antagonistic bioassays 84 19 4.2.5 Data Analysis 85 20 4.3 Results 85 21 4.3.1 Pathogenicity and virulence tests on green apples 85 22 4.3.2 In vitro antagonistic bioassays of Trichoderma against P cinnamomi isolates 86 23 4.3.3 In vitro antagonistic bioassays of Bacillus against P cinnamomi isolates 87 24 4.3 Discussion 91 25 4.5 References 93 26 CHAPTER 98 27 GENERAL DISCUSSION 98 28 5.1 Overview of major outcomes 98 29 5.2 Implications of outcomes 98 30 5.3 Recommendations 99 31 5.4 References 100 32 PROJECT OUTCOMES 101 33 APPENDICES 102 34 vii INTRODUCTION TO DISSERTATION Macadamia is a nut-bearing tree that belongs to the family Proteaceae that consists of evergreen woody plants (Augstburger et al., 2002) The aboriginal tribes used the nuts as a staple food and as a base for medicines and cosmetics in Australia, where the nut originated from (de Villiers, 2003) Two different species of macadamia, namely Macadamia integrifolia (Maiden and Betche) (also M ternifolia), and Macadamia tetraphylla (L.A.S Johnson), together with their hybrids, are of importance in commercial cultivation (Augstburger et al., 2002) Macadamia production is limited to the tropical and subtropical 10 regions of the world, but the nut is exported worldwide In South Africa, macadamia 11 production is mainly confined to the Limpopo, Mpumalanga, and KwaZulu-Natal provinces; 12 and to a lesser extent to the Eastern and Western Cape provinces (DAFF, 2015) 13 Plant pathogens (fungi, stramenopiles, bacteria, viruses, nematodes) are a major 14 threat to plant production since they result in quality and quantity reduction of commercial 15 crops worldwide (Bailey, 2010) There are major losses in agriculturally essential crops due 16 to these pathogens, and they, therefore, remain important constraints in agricultural 17 production (Bailey, 2010) A number of factors affect macadamia production, but those of 18 great economic importance are the disease-causing and quality-reducing pathogens The 19 main and most severe diseases in macadamia are caused by Phytophthora spp These are 20 capable of reducing vigour, production, and may cause complete trunk death (Akinsanmi 21 and Drenth, 2010) Phytophthora cinnamomi (Rands) has been the chief limiting cause to 22 successful macadamia production in countries such as Hawaii (Ko, 2009), Australia 23 (Rosengarten, 2004), California (Zentmyer, 1980), Kenya (Sikinyi, 1993) and South Africa 24 (Manicom, 2003) It causes stem canker, root rot and quick decline in macadamia 25 worldwide (Serfontein et al., 2007) Root rot and trunk canker are major diseases that could 26 cause 60% yield losses and an estimated 10% of the annual gross value of macadamia 27 (Muthoka et al., 2005) 28 P cinnamomi is the most important and destructive oomycete of not only 29 macadamia worldwide, but over 1000 plant types (Zentmyer, 1980), including avocados, 30 eucalyptus, kiwi fruit, chestnut, peach, pineapple, pear, and many native Australian and 1 South Africa plants (Pegg et al., 2002) The pathogen is of specific importance to the avocado (Persea americana Mill.) and macadamia industries because of its potential to destroy avocado and macadamia orchards in a short time frame It infects and kills trees of all ages, from nursery trees to large fruit-bearing trees through the destruction of feeder roots (Bekker, 2007) P cinnamomi is responsible for the widespread damage of macadamia trees worldwide It infects the feeder roots Infection occurring through the fine feeder roots results in root rots whereas infection occuring through wounded trunks of mature trees results in the development of trunk cankers (Mbaka, 2011) Infected trees die in three to five years; this, however, depends on the management of the orchard 10 11 Significance of Research 12 More than 95 % of produced macadamia nuts are exported internationally to Europe, Japan 13 and the United States of America (DAFF, 2015) The industry has the capability to enrich 14 rural livelihoods of macadamia growers However, root rot and stem cankers caused by P 15 cinnamomi are a notable macadamia production constraint in South Africa To develop 16 effective management strategies for root rot and trunk cankers, the distribution of P 17 cinnamomi in macadamia growing areas of the country needs to be established 18 Early and reliable detection is fundamental to developing appropriate control 19 strategies for plant diseases and limit their further spread Routine methods that are 20 currently used for the detection and identification of P cinnamomi entail isolating the 21 pathogen directly from soil samples onto antibiotic media (Anderson, 2006) The potential 22 for improved control of this pathogen requires development of molecular detection 23 techniques to confirm morphological identification of P cinnamomi 24 Chemical control is the most effective control measure for P cinnamomi, and to this 25 end, phosphate-based fungicides play the prime role (Bekker, 2007) Pathogens, however, 26 have the potential to overcome chemicals by developing resistance Biological control 27 agents (BCAs) such as Trichoderma and Bacillus spp have been reported to control 28 several soil-borne diseases Their effectiveness in control of P cinnamomi induced root 29 rots, and trunk cankers of macadamia need to be established This will structure a 30 foundation for their addition in the integrated management of the two diseases ... value of macadamia The objective of this study was to determine the distribution, molecular identification and the effect of biological control of P cinnamomi in the main macadamia growing areas of. .. Efficacy of Water Soluble Silicon for Control of Phytophthora cinnamomi Root Rot of Avocado MSc thesis Department of Applied Sciences, University of Pretoria, South Africa, pp 1-9 Department of Agriculture,... developing resistance Biological control 27 agents (BCAs) such as Trichoderma and Bacillus spp have been reported to control 28 several soil-borne diseases Their effectiveness in control of P cinnamomi