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    Identification and functional characterisation of glycoside hydrolases from the kauri dieback pathogen, Phytophthora agathidicida : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science at Massey University, Manawatū, New Zealand
    (Massey University, 2022) Bradley, Ellie Lynn
    The survival of kauri, an ancient conifer species endemic to New Zealand, is currently threatened by kauri dieback disease, caused by the oomycete plant pathogen Phytophthora agathidicida. As P. agathidicida continues to spread throughout kauri forests in the northern North Island of New Zealand, encouraging research has indicated there may be natural tolerance to the disease within the kauri population. This resistance is likely governed, in part, by the plant immune system, which is activated upon recognition of pathogen invasion patterns such as microbe-associated molecular patterns (MAMPs), damage-associated molecular patterns (DAMPs), and effectors (virulence factors required for host colonisation), which are recognised at the plant cell surface by plant immune receptors. To better understand how P. agathidicida interacts with its plant host on a molecular level, pathogenproduced proteinaceous invasion patterns need to be identified and characterized to aid in the identification of cognate immune receptors in the plant host, which may be involved in activation of the plant immune system. As the role of glycoside hydrolase (GH) proteins in virulence and pathogenicity of fungal and oomycete plant pathogens is well established (Chapter two), an effectoromics approach was used to identify six P. agathidicida GH12 proteins that appear to act as MAMPs in both Nicotiana benthamiana and Nicotiana tabacum (Chapter three). Furthermore, nuclear magnetic resonance was used to identify considerable changes in kauri leaf apoplastic wash fluid of approximately 17 metabolites, including sucrose and glucose, in response to P. agathidicida inoculation (Chapter four), thus suggesting a role for GH proteins in the hydrolysis of some of these metabolites. Finally, proteomic analysis of P. agathidicida culture filtrates via liquid chromatography-mass spectrometry (LC–MS) was used to validate the expression of predicted P. agathidicida proteins and to investigate the capacity of this method to identify candidate invasion patterns for future analysis. Chapter five established that LC–MS analysis of Phytophthora culture filtrate was an effective method for the identification of putative apoplastic invasion pattern candidates and confirmed the production of all six P. agathidicida GH12 cell death elicitors in culture. Collectively, this thesis has advanced our understanding of the molecular mechanisms underpinning the interaction of P. agathidicida with its host and has contributed to the identification of candidate apoplastic effectors.
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    Development of an in vitro assay to screen Agathis australis (kauri) for resistance to Phytophthora agathidicida : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Massey University, Manawatū, New Zealand
    (Massey University, 2017) Herewini, Echo
    The iconic Agathis australis (kauri) of New Zealand, is under serious threat from kauri dieback disease caused by the soil-borne pathogen Phytophthora agathidicida. Infected kauri express symptoms of root and collar rot, bleeding resins at the base of the trunk, yellowing of foliage, canopy thinning, and tree mortality. Phytophthora agathidicida was first associated with kauri decline in 1972, where it was initially identified as P. heveae however, there was some uncertainty about its significance and taxonomy. The pathogen was officially identified as a new organism in 2008 and was called Phytophthora taxon Agathis until its formal description as Phytophthora agathidicida in 2015. This pathogen is easily vectored through root to root contact and mobile zoospores. Management and research has focused on mapping pathogen distribution, reducing spread, improving detection, ex situ conservation and clonal production using tissue culture techniques. In order to gain better understanding of the disease epidemiology and to develop better breeding programmes, a reliable in vitro resistance screening assay is required. This research focused on the development of a screening assay using detached leaves from tissue culture material as a means of accelerating screening assays compared to the more labour-intensive root inoculation assays. Foliar inoculations and assessment techniques were initially optimised on kauri leaves from tissue culture lines. The most successful inoculation method involved placing P. agathidicida-colonised agar plugs on wounded detached leaves. The assay was further tested on 2 year old kauri seedlings. Variation in susceptibility across kauri genotypes and leaf age, and variation in virulence among P. agathidicida isolates was observed. To further investigate the impact of leaf age on lesion extension, an assay was conducted on detached leaves from six rooted kauri saplings over 5 years of age, across three leaf age groups with P. agathidicida, P. multivora, and P. cinnamomi. Variation in virulence among these Phytophthora species was observed. Leaf necrosis was most severe with young tissue and susceptibility tended to decrease with increasing leaf age. Preliminary studies with 50 kauri clones identified different levels of susceptibility and tolerance across the different genotypes to P. agathidicida. The methods developed within this study have increased our understanding of the overall response of kauri to P. agathidicida foliar inoculations. This study demonstrated variation in the susceptability of kauri foliage to Phytophthora inoculation, although no complete resistance was observed. Further work is required to determine if there is a relationship between root and leaf responses which will help establish if in vitro genotypic variation can accurately predict natural genotypic variation seen within kauri forests.
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    Discontinuous distributions of iconic New Zealand plant taxa and their implications for southern hemisphere biogeography : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2007) Knapp, Michael
    New Zealand has long been regarded as a key to understanding discontinuous distributions in the Southern Hemisphere. The archipelago is a fragment of the ancient super continent Gondwana. It has been isolated for 80 million years, has an excellent fossil record, and some of its most ancient biota such as the Southern Beeches (Nothofagus) and the Araucariaceae show disjunct distribution patterns with relatives on other fragments of Gondwana. Some of the most controversial problems of Southern Hemisphere biogeography with wide ranging implications involve New Zealand taxa. Three of them have been addressed in this thesis. The transoceanic relationships of the genus Nothofagus have long been regarded as an iconic example of a distribution pattern resulting from the break up of Gondwana. Phylogenetic analyses presented here show that, though most of the extant distribution of the genus is indeed shaped by tectonic events, Southern Beeches have crossed the Tasman Sea between Australia and New Zealand at least twice during the Tertiary period These results, together with findings of studies on other plant and animal taxa, emphasise the importance of dispersal but at the same time raise the question of whether any New Zealand taxa can be considered Gondwanan relicts. There is no geological evidence for the continuous existence of land throughout the Tertiary in the New Zealand area. However, molecular clock analyses presented in this thesis indicate that Agathis australis (New Zealand Kauri) diverged from its closest Australian relative prior to the Oligocene, or period of greatest submergence during the Tertiary. Thus these findings reject the hypothesis of the complete drowning of the New Zealand landmass during the Tertiary. They cannot reject the hypothesis of Stöckler et al. (2002) that the New Zealand Kauri lineage has persisted on the archipelago since its separation from Gondwana. Explanations for forest distribution patterns within the New Zealand islands themselves are diverse. New Zealand Nothofagus species show distribution gaps that are not explained by recent environmental factors alone. Early Miocene tectonic events and alternatively Pleistocene climates have been proposed as causes for this disjunct distribution pattern. Phylogeographic analyses reported in this thesis suggest that severe Pliocene and Pleistocene climates as well as Pliocene and Pleistocene tectonic events have shaped present day distribution and diversification of Nothofagus species in New Zealand.