Field evaluation of the use of select entomopathogenic fungal isolates as microbial control agents of the soil-dwelling life stages of a key South African citrus pest, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) Submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY of RHODES UNIVERSITY by CANDICE ANNE COOMBES November 2015 Abstract The control of false codling moth (FCM), Thaumatotibia leucotreta (Meyrick, 1912) (Lepidoptera: Tortricidae), in citrus orchards is strongly reliant on the use of integrated pest management as key export markets impose stringent chemical restrictions on exported fruit and have a strict no entry policy towards this phytosanitary pest. Most current, registered control methods target the above-ground life stages of FCM, not the soil-dwelling life stages. As such, entomopathogenic fungi which are ubiquitous, percutaneously infective soil-borne microbes that have been used successfully as control agents worldwide, present ideal candidates as additional control agents. Following an initial identification of 62 fungal entomopathogens isolated from soil collected from citrus orchards in the Eastern Cape Province, South Africa, further laboratory research has highlighted three isolates as having the greatest control potential against FCM subterranean life stages: Metarhizium anisopliae G 11 3 L6 (Ma1), M. anisopliae FCM Ar 23 B3 (Ma2) and Beauveria bassiana G Ar 17 B3 (Bb1). These isolates are capable of causing above 80% laboratory-induced mycosis of FCM fifth instars. Whether this level of efficacy was obtainable under sub-optimal and fluctuating field conditions was unknown. Thus, this thesis aimed to address the following issues with regards to the three most laboratory-virulent fungal isolates: field efficacy, field persistence, optimal application rate, application timing, environmental dependency, compatibility with fungicides and the use of different wetting agents to promote field efficacy. Following fungal application to one hectare treatment blocks in the field, FCM infestation within fruit was reduced by 28.3% to 81.7%. Isolate Bb1 performed best under moderate to high soil moisture whilst Ma2 was more effective under low soil moisture conditions. All isolates, with the exception of Ma2 at one site, were recorded in the soil five months post-application. None of the wetting agents tested were found to be highly toxic to fungal germination and similar physical suspension characteristics were observed. Fungicide toxicity varied amongst isolates and test conditions. However, only Dithane (a.i. mancozeb) was considered incompatible with isolate Ma2. The implication of these results and the way forward is discussed. This study is the first report of the field efficacy of three laboratory-virulent fungal isolates applied to the soil of conventional citrus orchards against FCM soil-dwelling life stages. As such, it provides a foundation on which future research can build to ensure the development and commercialisation of a cost-effective and consistently reliable product. i Acknowledgements Thanks is extended to the following individuals and organisations for their assistance during the course of this study: Prof. Martin Hill and Dr Sean Moore, my two principal supervisors, for their continued assistance, valuable advice and motivation. Dr Jo Dames, my co-supervisor, for her inputs and willingness to help when necessary. Wayne Kirkman, Claire Love, Mellissa Peyper, Tara O’Neill, Sonnica Albertyn and especially Tamryn Marsberg for laboratory and field assistance. Thanks is also extended to fellow RU postgraduate students, interns and various individuals at CRI who assisted during field work. Citrus growers’ in whose orchards trials were conducted. Field trials were an integral part of this study and your willingness to allow us to conduct our research at your farms is greatly appreciated. Thank you also to those growers who supplied fungicides upon request. Funding for this work was provided by the South African Research Chairs Initiative of the Department of Science and Technology and the National Research Foundation of South Africa. Any opinion, finding, conclusion or recommendation expressed in this material is that of the authors and the NRF does not accept any liability in this regard. Citrus Research International for financial support Rhodes University for financial support ii Table of Contents Abstract…………………………………………………………………………..i Acknowledgements……………………………………………………………..ii Table of Contents………………………………………………………………iii List of Tables…………………………………………………………………...ix List of Figures………………………………………………………………….xi List of Abbreviations………………………………………………………...xvii CHAPTER 1 – GENERAL INTRODUCTION……………………………...1 1.1 SOUTH AFRICAN CITRUS……………………………………………….1 1.1.1 The taxonomy of citrus……………………………………………..1 1.1.2 Citrus production and export……………………………………….2 1.1.3 Citrus pests and their control……………………………………….3 1.2 TARGET PEST: THAUMATOTIBIA LEUCOTRETA……………………...5 1.2.1 Classification……………………………………………………….5 1.2.2 Distribution and host range………………………………………...5 1.2.3 Morphology of life stages…………………………………………..7 1.2.3.1 The egg……………………………………………………7 1.2.3.2 The larva…………………………………………………..7 1.2.3.3 The pupa…………………………………………………...8 1.2.3.4 The adult…………………………………………………...9 1.2.4 Life cycle………………………………………………………….10 1.2.5 Economic importance……………………………………………..11 1.2.6 Control options……………………………………………………12 iii 1.2.6.1 Monitoring………………………………………………..12 1.2.6.2 Cultural control…………………………………………..13 1.2.6.3 Chemical control…………………………………………14 1.2.6.4 Behavioural control………………………………………15 1.2.6.5 Sterile insect technique…………………………………...15 1.2.6.6 Biological control………………………………………...16 1.2.6.7 Post-harvest treatment…………………………………...18 1.3 ENTOMOPATHOGENIC FUNGI (EPF).………………………………...18 1.3.1 General biology………………………………………………………18 1.3.2 EPF as control agents……………………………………………..…..20 1.3.3 Efficacy affecting factors…………………………......………………22 1.3.3.1 Soil temperature………………………………………….22 1.3.3.2 Soil moisture……………………………………………...23 1.3.3.3 Soil amendments………………………………………….23 1.3.3.4 Soil structure and texture………………………………...25 1.3.3.5 Soil biota…………………………………………………25 1.3.4 Genera Beauveria and Metarhizium………………………………26 1.3.5 EPF against FCM in South African citrus……………..………….28 1.4 RESEARCH AIMS………………………………………………………...31 CHAPTER 2 – THE EFFECT OF EPF ON FCM FIFTH INSTAR MORTALITY DURING FIELD-CAGE TRIALS…………………………33 2.1 INTRODUCTION…………………………………………………………33 iv 2.2 MATERIALS AND METHODS…………………………………………..36 2.2.1 Site details………………………………………………………...36 2.2.2 Fungal isolates…………………………………………………….36 2.2.3 Insect culture………………………………………………………37 2.2.4 Treatments………………………………...………………………37 2.2.5 Experimental procedure…………………………………………...37 2.2.6 Fungal concentration in cages…………………………………….38 2.3 RESULTS………………………………………………………………….41 2.3.1 Fungal efficacy……………………………………………………41 2.3.2 Fungal concentration in cages…………………………………….43 2.4 DISCUSSION……………………………………………………………...44 CHAPTER 3 – SOIL APPLICATION OF EPF IN CONVENTIONAL CITRUS ORCHARDS: EFFICACY AGAINST FCM…………………….48 3.1 INTRODUCTION…………………………………………………………48 3.2 MATERIALS AND METHODS…………………………………………..49 3.2.1 Fungal isolates…………………………………….………………49 3.2.2 Field sites………………………………………………………….50 3.2.3 Fungal application………………………………………………...51 3.2.4 Treatments………………………………………………………...54 3.2.5 Monitoring fungal efficacy………………………………………..56 3.3 RESULTS………………………………………………………………….58 3.3.1 Early application trials (Atmar 1, Atmar 2 and Oranjelus)……….58 3.3.2 Late application trials (Marwell and Stenhope)…………………..63 v 3.4 DISCUSSION……………………………………………………………...66 CHAPTER 4 – FIELD PERSISTENCE OF SELECT EPF IN CONVENTIONAL CITRUS ORCHARD SOIL…………………………...73 4.1 INTRODUCTION…………………………………………………………73 4.2 MATERIALS AND METHODS…………………………………….…….74 4.2.1 Fungal isolates…………………………………………………….74 4.2.2 Monitoring fungal persistence…………………………………….75 4.2.2.1 Soil sampling……………………………………………75 4.2.2.2 CFU analysis……………………………………………..76 4.3 RESULTS………………………………………………………………….77 4.4 DISCUSSION……………………………………………………………...80 CHAPTER 5 – PHYSICAL SUSPENSION CHARACTERISTICS AND TOXICITY OF FIVE ADJUVANTS ON THE SPORES OF FUNGAL ISOLATES Bb1 AND Ma2…………………………………………………..87 5.1 INTRODUCTION........................................................................................87 5.2 MATERIALS AND METHODS…………………………………………..88 5.2.1 Fungal isolates…………………………………………………….88 5.2.2 Adjuvants………………………………………………………….89 5.2.3 Physical suspension characteristics of each suspension concentrate …………………………………………………………………………..89 5.2.4 Toxicity of adjuvants to Ma2 and Bb1 after 24 h exposure………91 vi 5.3 RESULTS………………………………………………………………….91 5.3.1 Physical suspension characteristics of each suspension concentrate …………………………………………………………………………..91 5.3.2 Toxicity of adjuvants to Ma2 and Bb1 after 24 h exposure……..102 5.4 DISCUSSION…………………………………………………………….104 CHAPTER 6 – IN VITRO IMPACT OF FUNGICIDES ON THE GROWTH, VIABILITY AND SPORULATION OF TWO FUNGAL ISOLATES…………………………………………………………………..108 6.1 INTRODUCTION………………………………………………………..108 6.2 MATERIALS AND METHODS…………………………………………110 6.2.1 Fungicides………………………………………………………..110 6.2.2 Fungal suspensions………………………………………………110 6.2.3 Agar medium…………………………………………………….111 6.2.4 Experimental treatments…………………………………………111 6.2.4.1 Vegetative growth……………………………………….112 6.2.4.2 Viability…………………………………………………112 6.2.4.3 Sporulation……………………………………………...113 6.2.5 Compatibility of Ma2 and Bb1 with tested fungicides: Alves’s model…………………………………………………………………..113 6.3 RESULTS………………………………………………………………...113