Optimizing Dothistroma Septosporum Infection of Pinus Radiata and the Development of Red-Band Disease
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Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Optimizing Dothistroma septosporum Infection Of Pinus radiata And The Development Of Red-Band Disease A thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand. Naydene Barron 2006 i Abstract The filamentous fungus Dothistroma septosporum infects pine species throughout the world causing red-band disease, one of the most serious diseases of conifer species. In NZ, a clonally derived asexual strain of D. septosporum was identified in 1964, and has spread throughout the country. There are conflicting accounts on the environmental conditions required for infection, which has lead to difficulties in optimizing a laboratory-based system for infection. The pathogen is spread naturally through rain-splashed inoculum of conidiospores from mature stromata that have erupted through the pine needle tissue. Diseased needles become necrotic, often with a red band due to the mycotoxin dothistromin produced by the hyphae. Dothistromin has the chemical structure of a difuranoanthraquinone and shows similarity to the aflatoxin precursor, versicolorin B produced by Aspergillus parasiticus. The role of dothistromin in pathogenicity has not yet been determined, although experiments have shown injecting toxin into pine needles results in the characteristic red band lesion. In this study it was found that fluctuating temperature (16°C/24°C), a 12 h diurnal cycle (white and ultraviolet light), high relative humidity and continuous moisture are conditions conducive to development of red-band disease on inoculated pine trees in an artificial environment. A higher rate of infection was obtained using pine seedlings as opposed to pine cuttings, and using a spore suspension containing a yeast extract. A dothistromin minus mutant was able to infect pine needles, indicating that dothistromin is not a pathogenicity factor, though it may be a virulence factor. The use of GFP-expressing isolates allowed the initial infection process to be monitored with both wild type and mutant isolates. Additionally, a PCR-based diagnostic procedure to confirm infection was developed. The production of aflatoxin by Aspergillus species is regulated by nutritional parameters and extracellular pH, which affect both growth and aflatoxin gene expression. D. septosporum similarly has enhanced growth at acidic pH, but it does not appear that pH has a strong influence on physiological processes as toxin biosynthesis and gene expression do not appear to be pH regulated. Different carbon and nitrogen sources also affect the morphology of D. septosporum. ii Acknowledgements I would like to thank both my supervisors Dr Rosie Bradshaw and Dr Peter Long for their dedication to this research project. Rosie you have been both supportive and understanding during the course of my Masters. You have been an outstanding supervisor who believed in my ability to complete this research, in addition to being approachable and appreciative, and with your dedication and guidance you made the completion of this task so much easier. Peter, thank you for your time and ideas and especially for the humor and making sure my pronunciation of fungal species in addition to data analysis was up to scratch! You have both been inspirational and a pleasure to work with. I have gained so much knowledge from both of you in the last two and half years and enjoyed working on this research project. To my lab colleagues, both past and present, it has been great to be associated with such a dedicated group of people who helped make this journey possible. Arne, thank you for your technical assistance, sharing of your knowledge and ideas and having a great sense of humor. Shugang thank you for sharing your knowledge and your willingness to listen and provide new information. I wish everyone in the lab the best of luck in completion of their studies and success for the future. To my partner, thank you for being so supportive, encouraging and understanding over the duration of my studies, in addition to helping me with revision before exams, and making sure I got it right. To my daughter, you have been patient over the last few years, but understand the importance of studying. I hope you have learnt that even though you can’t see the forest for the trees, with a lot of hard work and dedication there is always light at the end of the tunnel. To both of you, we have missed out on a lot of time together, but that sacrifice has enabled me to meet my goal, and complete this part of my life, thank you. To mum and dad, thank you for believing in me, your support, financial assistance and helping with Chantel. Lastly, I would like to thank Rosie for sourcing financial assistance, the CoRE, Bio- protection Centre, NZ Plant Protection Society, C. Alma Baker Trust and Massey University for providing financial assistance for myself and this project. iii Table of Contents Abstract i Acknowledgements ii Table of Contents iii List of Figures vii List of Tables ix Abbreviations x Chapter One – Introduction 1 1.1 Dothistroma Needle Blight 1 1.1.1 Environmental conditions conductive to infection 1 1.1.2 Mode of infection 3 1.1.3 Lifecycle in the forest environment 4 1.1.4 Dothistroma needle blight control 5 1.1.5 Dothistroma mycotoxin 6 1.1.6 Fungal gene clusters 8 1.2 Pathogenicity Assay 11 1.3 Green Fluorescence Protein 13 1.3.1 The use of GFP to follow infection 15 1.4 Ambient pH Regulates Physiological Processes 16 1.5 Summary of Current Research 18 1.5.1 Hypothesis 1 18 1.5.2 Aim 1 18 1.5.3 Objectives 18 1.5.4 Hypothesis 2 18 1.5.5 Aim 2 19 1.5.6 Objectives 19 Chapter Two – Materials and Methods 20 2.1 Biological Strains 20 2.1.1 Fungal isolates 20 2.1.2 Plant species 20 2.1.3 Isolation of fresh wild-type D. septosporum 20 2.2 Growth of Cultures and Culturing Techniques 21 2.2.1 Fungal cultures 21 iv 2.2.2 Maintenance of D. septosporum stocks 21 2.2.3 Obtaining D. septosporum conidia and quantification 22 2.2.4 Germination assay 22 2.2.5 McIlvaine buffered solid media 23 2.2.6 McIlvaine buffered liquid media 23 2.2.7 Harvesting mycelium from liquid media 24 2.3 DNA Extraction, Quantification and Analysis 24 2.3.1 DNA extraction: CTAB method 24 2.3.2 Fluorometric quantification 25 2.3.3 Nano-drop quantification 25 2.3.4 Agarose gel electrophoresis 25 2.4 RNA Isolation, Analysis and cDNA Synthesis 26 2.4.1 RNA extraction 26 2.4.2 cDNA synthesis 26 2.5 Polymerase Chain Reaction (PCR) 26 2.5.1 rDNA primers 27 2.5.2 Purification of DNA from PCR products 27 2.5.3 Real-time PCR 28 2.6 Automated Sequencing 28 2.7 Quantification of Dothistromin 29 2.7.1 Preparation of DOTH-MSA conjugate ELISA plates 29 2.7.2 Preparation of samples for ELISA 29 2.7.3 DOTH-MSA plates 30 2.8 Pathogen Assay 30 2.8.1 Assay chamber 30 2.8.2 Ultraviolet light (UV) 31 2.8.3 GMO suite 31 2.8.4 Mycelia as plant inoculum 31 2.8.5 Pine seedling and cutting inoculation 32 2.8.6 Diseased pine needle analysis 32 2.8.7 Diagnostic procedure confirming disease 32 2.9 Statistical Analysis 33 Chapter Three – Red-band Disease Pathogen Assay 34 3.1 Introduction 34 3.2 Results 38 3.3 Environmental Variables 38 3.3.1 Ultraviolet light 38 3.3.2 Temperature and humidity 38 3.3.3 Water source 39 3.3.4 Scoring infection 40 v 3.3.5 Natural inoculum validates developed pathogen assay system 42 3.4 Host Factors 43 3.4.1 Use of detached plant material in pathogen assay 43 3.4.2 Seedlings versus cuttings 44 3.5 Pathogen Factors 45 3.5.1 Type of inoculum 45 3.5.2 The effect of conidia suspension on infection 46 3.5.3 The effect of external nutrients on infection 47 3.5.4 The effect of conidia growth media on disease incidence 49 3.5.5 Double inoculation with two D. septosporum isolates 50 3.5.6 Does infection occur in the absence of DOTH? 50 3.6 Diagnostic Procedure to Confirm Red-band Disease 51 3.6.1 Use of universal primers to confirm infection 51 3.6.2 Specific mating type primers confirm presence of D. septosporum 53 3.7 Discussion 55 3.7.1 Environmental conditions 55 3.7.2 P. radiata and D. septosporum factors 56 3.7.3 Developed diagnostic procedure 59 Chapter Four – Does pH Affect D. septosporum Physiological Processes? 60 4.1 Introduction 60 4.2 Results 64 4.2.1 Growth on different media 64 4.2.2 Growth and sporulation on pH adjusted media 66 4.2.3 Liquid media inoculated with D. septosporum conidia 68 4.2.4 Liquid media inoculated with D. septosporum mycelium 73 4.3 Discussion 79 4.3.1 Colony growth and morphology on different media 79 4.3.2 How does the pH of solid media effect growth and sporulation? 80 4.3.3 Inoculation of spores into liquid medium at adjusted pH 81 4.3.4 Analysis of mycelium as inoculum in pH adjusted liquid medium 83 Chapter Five – Green Fluorescent Proteint as a Marker to Monitor Infection 86 5.1 Introduction 86 5.2 Results 89 5.2.1 Conidia germination 89 5.2.2 Effect of pH on conidia germination 90 5.2.3 Survival and distribution of GFP conidia on pine needles 91 vi 5.2.4 Germination of D.