Pre-empting Maize Lethal Necrosis Disease in South Africa: potyviruses of maize (Zea mays) by Susanna Elizabeth (Azille) Schulze Submitted in partial fulfilment of the requirements for the degree Magister Scientiae in the Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria August 2018 Supervisor: Professor Gerhard Pietersen Declaration I, Susanna Elizabeth Schulze declare that the dissertation, which I hereby submit for the degree Magister Scientiae at the University of Pretoria, is my own work and has not previously been submitted by me for a degree at this or any other tertiary institution. ______SE Schulze______ _____6 August 2018______ Susanna Elizabeth Schulze Date i Pre-empting Maize Lethal Necrosis Disease in South Africa: potyviruses of maize (Zea mays) By Susanna Elizabeth Schulze Supervisor: Professor Gerhard Pietersen Department: Microbiology Degree: MSc (Microbiology) Summary The devastating Maize Lethal Necrosis Disease (MLND) hampers the production of maize, especially in developing countries. It causes mottling, necrosis and chlorosis of host plants, which results in severely reduced growth and yield and in extreme cases plant death in Zea mays (maize). Maize is regarded as one of three main staple food crops worldwide and in sub-Saharan Africa and South America it provides nutrition for over 1.2 billion people. Therefore, this disease is a severe food security threat and needs to be managed where it is present or pre-empted where it is predicted to spread to. MLND is caused by the co-infection of Maize chlorotic mottle virus (MCMV) and a cereal-infecting member of the Potyviridae family such as Sugarcane mosaic virus (SCMV), Johnsongrass mosaic virus (JGMV) or Maize dwarf mosaic virus (MDMV). MCMV is not present in South Africa, but was recently introduced into East Africa. The disease is predicted to disperse to neighbouring countries including South Africa (SA) in the near future. A number of potyviruses of maize have, however, been reported from SA, although the status, distribution and their diversity of these viruses has not been studied and a molecular tool to simultaneously detect these viruses has not yet been developed. ii In this study the status of potyviruses on maize in SA was determined, the diversity of the major maize potyviruses including those from SA and Tanzania was investigated, and an assay to simultaneously detect four maize-infecting viruses that are likely to be involved in MLND was developed. The results from this study allow us to pre-empt the introduction of MLND into SA by enabling us to 1) gain insight into which potyviruses are most likely to be present in potential MLND infections in SA and predict which areas are likely to be most affected, 2) better understand the evolution and diversity of the most common potyvirus on South African maize, SCMV isolates, in Africa and produce evidence that the MLN viral complex (or epidemiology thereof) will be more similar in SA to that seen in Asian countries rather than that seen in other African countries, and 3) verify that rapid, accurate and cost effective disease diagnosis can be made using the tool developed in this study. The conclusions of this study will contribute to future research regarding the viral components and epidemiology of MLND, especially in Africa. It also highlights some factors to consider in the development of genetically modified virus-resistant maize towards a durable control strategy to curb the impact of this disease. The maize industry, seed companies, quarantine services and research facilities working on MLN will benefit from the knowledge generated as well as the assay developed here. The study will also be useful in the subsequent selection of future research directions. iii Acknowledgements Sincere gratitude on my behalf is gladly expressed to the following people and organisations for their integral role in the completion of this study: To the ARC-PPRI, Grain SA and University of Pretoria as well as the National Research Foundation Scarce-skills program for either research-related or personal financial assistance. To my supervisor, Professor Gerhard Pietersen, thank you for the ample opportunities that I was provided with to gain first-hand plant virology experience on many memorable field trips, for ingraining in me enthusiasm and passion for research, for instilling in me a desire to produce thorough work, for your guidance in both practical and written aspects of my dissertation as well as for making the privilege possible of having experienced laboratory research abroad. To my lifelong support-network, Heinrich, Beana and Geor Schulze – I appreciate you for your abundant love, patience, infallible guidance and for encouraging me to stay hopeful and faithful in trying circumstances. Thank you for being my greatest supporters and for being proud of my accomplishments, as though I achieved them all on my own. I am deeply thankful to you for inspiring me to have pride in my work, for nurturing curiosity as well as analytical thinking (as a scientist requires), and for the financial support throughout my academic career. To another irreplaceable member of my support-network, Fuzzy Schulze, thank you for your unconditional companionship and comic relief that made long hours of work throughout my academic career both endurable and memorable. To Isebrand Naudé, thank you for your positivity which has kept me zealous in my endeavours, urging me not to underestimate myself as well as for inspiring me to both live with vision and to believe unceasingly. I am appreciative to you for always speaking kind words, lending a listening ear and helping me to keep perspective – and for never getting tired of doing that. To my laboratory colleagues: Elrea Strydom and Shaina Facey, thank you for being generous with your time to assist on my field trips and for your invaluable camaraderie. Elrea Strydom, I am especially thankful to you for your guidance in the lab, for your friendship, for all the shared experiences that have allowed us to grow iv as people, for imparting your wisdom in all facets of life and for being a sister in faith and fellowship. Friends in the department of Microbiology: Kinosha Moodley and Nadine Laubser, thank you for inspiring me with your diligence and for your valuable friendship. To Khumbuzile Bophela, thank you for teaching me to have faith when it comes to lab work, to work conscientiously when it comes to writing and for mentoring me long after your official duties were fulfilled. To Professor Bradley Flett, Professor Dave Berger, Moses Ramusi and Wilmarie Kriel for collecting and sending samples that meaningfully contributed to this study. To Jasper Reese and Ronel Roberts for receiving, extracting and sequencing samples from Tanzania under permit. To others who have aided this study by providing accommodation on the various field trips or other forms of assistance or information: Hotze and Rita Riekert, Stompie du Plessis, Michelle Pietersen, Jean du Preez, The South African Weather Service, Chris Mulders and Kobus Beer, I am exceptionally grateful for your contributions. v Table of contents Declaration ................................................................................................................. i Summary .................................................................................................................... ii Acknowledgements .................................................................................................. iv Table of contents...................................................................................................... vi List of acronyms....................................................................................................... xi List of tables ........................................................................................................... xiv List of figures .......................................................................................................... xv Preface ...................................................................................................................... 1 Chapter 1: Relevant literature ................................................................................. 2 1.1 Introduction ....................................................................................................... 3 1.2 Pathogens present in the Maize Lethal Necrosis Disease-complex .................. 8 1.2.1 The potyviruses .......................................................................................... 8 1.2.1.1 Potyvirus species already present in South Africa and potentially present on maize ............................................................................................ 11 1.2.1.1.1 Sugarcane mosaic virus ............................................................... 11 1.2.1.1.2 Maize dwarf mosaic virus ............................................................. 12 1.2.1.2 Other Poaceae-infecting potyviruses potentially present in South Africa on maize ......................................................................................................... 13 1.2.1.2.1 Johnsongrass mosaic virus .......................................................... 13 1.2.1.2.2 Pennisetum mosaic virus ............................................................. 14 1.2.1.2.3 Sorghum mosaic virus .................................................................. 14 1.2.1.2.4 Zea mosaic virus .........................................................................