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Identification of Blueberry Leaf Rust Pathogen and Quantification of Disease Infection Levels in A 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. Identification of Blueberry Leaf Rust Pathogen and Quantification of Disease Infection Levels in a Blueberry Plantation in Hastings, NZ A thesis presented in partial fulfilment of the requirements for the degree of Master of AgriScience in Horticulture At Massey University, Turitea Campus, Palmerston North, New Zealand Xiaoying Chen 2019 Abstract Blueberries (Vaccinium spp.) are a favourite fruit and they are produced worldwide. In New Zealand, blueberries are the main export berry fruit and contribute greatly to export income. More than 2,800 tonnes of blueberries were produced in the 2017/2018 harvesting season and earned $34.8 million export income in 2018. Currently, 740 ha of the blueberry plantations can be found in both the South Island and North Island. Otautau is the main growing region in the South Island while Waihopo, the Waikato regions of Ngatea and Ohaupo, the Bay of Plenty and Hastings are the regions in the North Island, producing most of the fresh blueberries in New Zealand. However, blueberry leaf rust has become a prevalent disease in blueberry production and a concerning issue for blueberry growers. In Hastings production sites, serious infections have been found in recent years. Although fungicides were applied to control blueberry leaf rust, this form of control is incomplete and unsustainable for blueberry production. The deployment of varieties that are naturally resistant would be a better option for managing blueberry leaf rust disease. Currently, few cultivars are available for this purpose, but breeding for rust resistance can address this demand. The main issues preventing the production of resistant varieties are insufficient knowledge about this rust pathogen in New Zealand, and the lack of resistant germplasm sources and efficient resistance screening procedures. In this study, using the morphological characteristics and genome sequencing results based on the Internal Transcribed Spacer (ITS) regions, Thekospora minima was identified as the causal organism of blueberry leaf rust disease in Hastings, Hawke’s Bay, New Zealand. Additionally, a field assessment was used for understanding the blueberry rust disease resistance levels in current blueberry cultivars. The disease incidence and disease severity of 23 blueberry cultivars, including five rabbiteye, three northern highbush and fifteen southern highbush, were assessed using Fiji software during the 2019 harvesting season. Based on a Tukey Honest Significant Differences i (TukeyHSD) analysis, these observed blueberry cultivars were divided into four infection levels of blueberry leaf rust using the percentage of the infected area on the leaf (PIAL). ‘Scintilla’ was highly susceptible to blueberry leaf rust disease, while ‘Blue Moon’ and ‘Southern Splendour’ were moderately susceptible. Nineteen blueberry cultivars, made up of ‘Rahi’, ‘Centra Blue’, ‘Centurion’, ‘Titan’, ‘Sky Blue’, ‘Nui’, ‘Duke’, ‘Camellia’, ‘Misty’, ‘Springhigh’, ‘Snowchaser’, ‘Miss Jackie’, ‘Miss Lily’, ‘Georgia Dawn’, ‘Suziblue’, ‘Kestrel’, ‘Flicker’, ‘Sweetcrisp’ and ‘Palmetto’, showed susceptibility to this rust disease, and ‘‘O’ Neal’ was the one that showed partial resistance to the blueberry rust infection. Furthermore, using 1.5×104 concentration inoculum, an inoculation test was completed in a temperature-controlled room at the Plant Growth Unit of Massey University. The inoculum was sprayed on the healthy leaves from detached branches of the ‘Sky Blue’ blueberry cultivar and they were grown in reverse osmosis water for a 35-day observation on rust symptom development. Fiji software was applied in the assessment of disease severity in this inoculation test. A strong correlation (>0.99) was found between the increase in lesion area (ILA) from the inoculation test and the PIAL from field assessment. A preliminary prediction equation was established by a simple linear regression model. This equation can be used to predict the blueberry leaf rust level on different blueberry cultivars and breeding materials under field conditions by using the results from an inoculation test. This model would be an efficient approach for assisting the screening on blueberry leaf rust of blueberries. ii Acknowledgements I wish to sincerely acknowledge several groups of people and individuals who have contributed with their help, guidance, support and patience to the completion of this thesis. First and foremost, I would like to express profound appreciation to my supervisors, Svetla Sofkova-Bobcheva, Huub Kerckhoffs and Carl Mesarich, without your enthusiastic recommendation, consistent encouragement, help, and patient guidance, it would be much harder for me to accomplish this thesis. I am really appreciated Svetla for enlightening my thoughts and guiding me through this difficult journey. I also extend my appreciation to Huub for his enthusiastic recommendations have assisted me from the start to the final submission. I would also like to thank Carl for his dedication and time on guiding my laboratory experiments and evaluating my thesis. I sincerely appreciate Gourmet Blueberries. Ltd supported me blueberry cultivars and gave me much useful information about blueberry production issues, especially grateful to Daniel Hutchins for his great suggestions and attention to my research. I am very grateful to Lesley Taylor and Kay Sinclair for the arrangement of laboratory facilities throughout my research. Thanks to Plant Growth Unit at Massey University for the use of the greenhouse and the temperature-controlled room to conduct my research. Lastly, I am very grateful to my parent and friends for all their support expressed in numerous ways. They always encouraged me when I was upset and believed in me all the time even when I didn’t believe in myself. iii Table of Contents Abstract ........................................................................................................................... i Acknowledgements ...................................................................................................... iii Table of Contents .......................................................................................................... iv List of Figures .............................................................................................................. vii List of Tables ................................................................................................................. ix Abbreviations ................................................................................................................ xi Chapter 1 Introduction ................................................................................................... 1 1.1 Introduction ....................................................................................................... 1 1.2 Literature review ............................................................................................... 3 1.2.1 Taxonomy of blueberry ........................................................................... 3 1.2.2 Botanical characteristics of blueberries .................................................. 4 1.2.3 Phenological stages ................................................................................. 7 1.2.4 Blueberry breeding history ..................................................................... 9 1.2.5 Current breeding progress ..................................................................... 11 1.2.6 Cultivation requirements for various blueberry types .......................... 16 1.2.7 Global blueberry production ................................................................. 23 1.2.8 Challenges of New Zealand blueberry industry.................................... 27 1.2.9 Solutions for blueberry rust control ...................................................... 28 1.2.10 The lack of blueberry rust-resistant cultivars and the issues impede on the breeding of rust-resistance varieties ......................................................... 29 1.2.11 Blueberry rust disease ......................................................................... 31 1.2.12 Blueberry rust species in New Zealand .............................................. 40 iv 1.2.13 Identification of blueberry rust species in Hastings ............................ 41 1.2.14 Issues from current screening procedures on blueberry rust disease .. 45 1.2.15 Development of efficient resistance screening procedures for blueberry rust disease ..................................................................................................... 47 1.3 The aim of this study and research objectives ................................................ 51 Chapter 2 Identification of rust species from Hastings blueberry production regions 53 2.1 Introduction and aims ..................................................................................... 53 2.2 Morphological identification .......................................................................... 54 2.2.1 Materials ............................................................................................... 54 2.2.2 Methods................................................................................................. 56 2.2.3 Results ..................................................................................................
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