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Subject: search.filters.subject.Fungal diseases of plants

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    Identification and characterization of effector proteins from pine needle pathogens : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand
    (Massey University, 2022) Massoco Tarallo, Mariana
    Collectively, Dothistroma septosporum, Cyclaneusma minus and Phytophthora pluvialis cause serious foliar diseases on Pinus radiata in New Zealand and on many other pine species worldwide. Considering the ecological and economic importance of forest trees, understanding how these pathogens interact with their hosts on a molecular level is critical as it could lead to new and durable approaches to control the diseases they cause. Pathogens have the ability to deliver proteinaceous virulence factors, termed effectors, into the apoplast and cell cytoplasm of their host plants. Effectors typically promote host colonization through suppression of the plant immune system. However, in resistant host plants, one or more of these effectors can be recognized by corresponding immune receptors to activate the plant immune system. Often, one of the main outputs of this immune system is a localised cell death reaction, termed the hypersensitive response (HR), which renders the pathogen unable to cause disease (avirulent). The general goal of this thesis was to identify shared candidate effector (CE) proteins between the three foliar pine pathogens and to characterise their virulence (or avirulence) functions. This is important because disease resistance based on core effectors that are vital for a pathogen’s ability to cause disease is more likely to be durable. Using a combination of “omics” information and bioinformatic tools, two sets of orthologous CE proteins were identified between D. septosporum, C. minus and P. pluvialis, while several other sets were identified between the two fungal pathogens. Some of these CEs had the ability to trigger cell death responses in non-host Nicotiana plants, and some were shown to activate Nicotiana benthamiana genes involved in pathogen-associated molecular pattern-triggered immunity and HR. CEs were also screened in the host, Pn. radiata, using a method developed in this thesis, where it was determined that some of these CEs also trigger cell death. Two conserved cell death elicitor families, Ecp20 and Ecp32, were identified from D. septosporum and its close relative Fulvia fulvum, and the cell death triggered by some family members in N. benthamiana was shown to require membrane-localized receptor-like proteins. Tertiary structure predictions of CEs provided insights into the possible roles and host targets of these proteins during pine infection. Moreover, a shared β-trefoil fold was found between sequence-unrelated CE proteins from the three pine pathogens, along with evidence that they are also present in many other fungal species. A CRISPR/Cas9 gene editing methodology was applied to D. septosporum for the first time, which allowed for the functional characterization of three D. septosporum CE genes, two of which are also present in C. minus and P. pluvialis. Collectively, this thesis provides a significant advance in our understanding of pine-pathogen interactions at the molecular level and provides a blueprint for similar studies in other forest pathosystems.
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    'Whakapuputia mai o mānuka' : a case study on indigenous knowledge and mitigating the threat of myrtle rust (Austropuccinia psidii) : a research thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Horticultural Science, School of Agriculture and Environment, Massey University Te Kunenga ki Pūrehuroa, University of New Zealand, Palmerston North, New Zealand
    (Massey University, 2019) Tora, Mesulame J.
    This research centres on the recent myrtle rust (Austropuccinia psidii) incursion in New Zealand to review the literature on the disease specifically and to create a localised case study with Ngāi Tāneroa hapū of Ngāti Kahungunu ki Wairarapa. The case study focused on the importance of whakapapa, mātauranga Māori, tikanga Māori and the practices of kaitiaki to ethnobotany and the development of indigenous biosecurity measures (tools) to protect culturally important plant species within the Māori community The proverb stated in the title of this thesis whakapuputia mai o Mānuka, kia kore ai te whati – (cluster the branches of the Mānuka, so they will not break off) recognizes the status of plant knowledge in te Ao Māori. It provides a foundation of understanding how Māori can participate in resource management against biological threats, which are becoming increasingly common. The science around myrtle rust and the mitigation of any incursion threats is clearly aligned to western paradigms. The information presented in this thesis outlines an extensive understanding of the intricacies of the disease as understood by the science community. But this science alone has not been able to halt the spread or risk of myrtle rust into new geographical regions. Therefore, future management of the risk of myrtle rust incursions needs to look at alternative approaches for the development of suitable management tools. The holistic approach of traditional biodiversity management using mātauranga and tikanga Māori has much to offer to conservation of taonga resources, especially the mitigation of biological threats. The Māori worldview of the environment encompasses all elements beyond the physical attributes of an ecosystem that thrives through traditional kaitiaki inputs. The case study with Ngāi Tenaroa introduced several examples of how Māori can contribute to the mitigation of all threats on the ecosystem, not just fungal threats. Firstly, the role of whakapapa is explicit and cannot be ignored. This role consolidates the management tools across all generations at the very least. Secondly, the role of networks within Māori communities and inter-generational learning is also clear – and the risk that exists if this is lost is apparent. Lastly, examples of local knowledge such as the effect of hukahuka on plant health, companion trees and role of kaitiaki in decision-making have been identified and their importance conveyed from the hapū under study.
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    Genetic studies of Phytophthora on Theobroma cacao from East New Britain and Bougainville (Papua New Guinea) : a thesis presented in partial fulfilment of the requirements for the degree of Master of AgriScience (Horticulture) at Massey University, Palmerston North, New Zealand
    (Massey University, 2016) Butubu, James
    DNA was extracted from 14 Phytophthora isolates from Theobroma cacao plants (SG2 hybrids, hybrid derived clones and Trinitario varieties) collected from New Britain and Bougainville in Papua New Guinea (PNG). A fragment of the mitochondrial genome cytochrome b (cytb) region was amplified from these DNAs using the polymerase chain reaction (PCR) and compared to cytochrome b sequences from Phytophthora palmivora and other Phytophthora (two isolates previously isolated from cocoa lesions and two P. palmivora isolates obtained from culture collection-Australia). All isolates were identical in their cytb gene sequence and similar to P. palmivora. Additionally, we sequenced the mitochondrial genomes of four isolates from PNG. The syntenic arrangement of genes in one complete assembly was compared with other published mitochondrial genomes. The sequences of four mitochondrial genes (COII, nad2, rps10 and SecY) from the four PNG isolates were aligned with orthologues from accessions of P. palmivora and other Phytophthora species available in the NCBI Genbank reference database. A concatenated data matrix was produced with 2,295 homologous sequence positions. 34 accessions of Phytophthora (including 14 P. palmivora) were used to construct a maximum likelihood tree of phylogenetic relationships. This reconstruction recovered all 10 major clades of Phytophthora previously reported. In this phylogenetic reconstruction, the four PNG isolates were clearly identifiable as P. palmivora and these were closely related to the Clade 4 Phytophthora species P. megakarya and P. quercetora. Of the genes analysed, COII showed greatest variability, resolving P. palmivora into three sub groups. COII was sequenced in all P. palmivora isolates from PNG and used to reconstruct an ML tree. The phylogenetic analyses suggested a potential origin for the PNG strain of P. palmivora in Samoa. Syntenic comparisons of P. palmivora and other clade 4 species identified a potential target for developing a Loop Mediated Amplification (LAMP) assay for P. palmivora near the atpH gene region. DNA amplification primers were designed for this region using PrimerExplorer, V4, Eiken Chemical CO.Ltd) and validated against available DNAs for Clade 4 and other Phytophthora species.
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    Studies on black scurf of potato caused by Rhizoctonia solani Khun : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science at Massey University
    (Massey University, 1973) Mackintosh, Brian Leslie
    The potato, Solanum tuberosum L. is second only to wheat in importance as a food crop in New Zealand. During the last five years the total area of potatoes grown has fluctuated between 8,000 and 10,000 hectares, and the yield has increased steadily to over 25 tonnes per hectare (Table I). Table I Potato production in New Zealand. Area and production for several recent seasons*. Season Area (ha) Total Yield (tonnes) Yield (tonnes/ha) 1966-67 8,020 187,267 23.35 1967-68 9,517 235,831 24.78 1968-69 10,132 256,263 25.32 1969-70 9,928 253,263 25.51** 1970-71 7,689 (est.) * New Zealand Official Year Book, 1972. (Yield figures converted to metric equivalents). **= 10.15 tons/acre Although potatoes are grown in all parts of New Zealand, the bulk of the market is supplied from three areas. The Pukekohe district supplies the early markets from September to December and also substantial quantities of mid-season and main-crop potatoes. The February to July market is supplied principally from the Manawatu-Rangitikei district, and winter supplies (May to November) are drawn mainly from Canterbury, Otago and Southland (Claridge, 1972; Baxter, 1972). [FROM PREFACE]
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    The development of diagnostic tools for the grapevine pathogen Eutypa lata : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University
    (Massey University, 2001) Jones, Paula Elizabeth
    Eutypa lata is the causal agent of Eutypa dieback on grapevines. The fungus invades the vine and grows there unnoticed, possibly for several years, causing discolouration and deformation of the vine shoots and leaves. Most berries fail to establish on these shoots and the fungus eventually kills the vine. The damaging effects of this fungus have had a notable financial impact on the grape and wine industry world wide and E. lata is at present the primary constraint on vineyard longevity in many places including California and Australia. Little is known about the occurrence and distribution of Eutypa dieback within New Zealand. This is due mainly to difficulties associated with identification of the disease in grapevines. To develop a molecular probe for the identification of E. lata from grapevine wood the Polymerase Chain Reaction (PCR) amplified the Internal Transcribed Spacers (ITS1 and ITS2) and the intervening 5.8S gene of ribosomal DNA (rDNA) from representative isolates. The sequences of the E. lata ITS regions were used to design two pairs of primers, each of which was subsequently shown to be specific for the amplification of predicted-size fragments from genomic DNA of E. lata. The primer pairs were further tested using template DNA extracted from healthy grapevines and from other fungi commonly isolated from dieback diseased grapevines but no PCR amplification was observed. Simple DNA extraction protocols, leading to the rapid release of DNA, were tested to enable identification of E. lata from pure culture and grapevine wood; however, a suitable DNA extraction method from these materials was not found. Currently the only known source of inoculum is ascospores, which are released from perithecia during and immediately after rainfall. However, few perithecia have been found in New Zealand vineyards. This has prompted the study of the mating habits of E. lata. As the sexual stage of E. lata cannot be obtained in culture at present, the analysis of its mating system must be performed in natural populations. Molecular characterisation of the mating type at the outset of a mating project allows significant savings in time and effort as it drastically reduces the number of crosses that must be set up. So far, cloning of mating type (MAT) genes from fungi has been hampered by low conservation among them. Most ascomycete fungi have one mating type gene with two alternative forms or idiomorphs (MAT1-1 and MAT1-2). One of the pair of MAT genes. MAT1-2, encodes a protein with a conserved DNA binding motif called the high mobility group (HMG) box. There is sufficient sequence conservation at the borders of the HMG box to allow PCR amplification. New Zealand isolates of E. lata, including sixteen single ascospore isolates from one perithecium, were tested for the presence of a MAT1-2 idiomorph using this PCR based approach. Five different sets of primers were used which were designed to anneal at different target sites with different specificities. PCR products of the expected size were obtained and sequenced, but despite exhaustive attempts to optimise PCR specificity, none of these had convincing homology to fungal mating type genes. Progress on the basic aspects of the genetics of E. lata will continue to be hampered until the organism is induced to complete its life cycle in culture. Molecular studies into the mating type genes which regulate sexual compatibility and sexual reproduction in the fungus should lead to a deeper understanding of the life-cycle of E. lata and the critical influence of sex on population genetics. In addition, it will provide a scientific basis for a management program urgently needed to minimise the impact of this disease.
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    Development of a pathogenicity testing system for Dothistroma pini infection of Pinus radiata : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 2004) West, Phillip John
    Dothistroma pini is a fungal pathogen of pine species around the world and can be found in most parts of New Zealand. Infection by D. pini causes a disease commonly known as Dothistroma needle blight. Dothistroma needle blight has a significant financial impact on New Zealand's forestry industry. Although control of infection by D. pini is currently very successful there is a possibility that a new strain introduced from another country could be a lot more damaging and overcome current control measures. In recent years both the incidence and severity of the disease have increased in the northern hemisphere and other parts of the world. A distinctive characteristic of Dothistroma needle blight is the production in the infected needle of a toxic red pigment called dothistromin. Dothistromin is produced as a secondary metabolite by D. pini and has known phytotoxic properties as well as clastogenic and mutagenic properties towards human cells. Purified dothistromin toxin injected into pine needles has been shown to reproduce symptoms similar to those observed during D. pini infection. Because of this production, dothistromin is thought to play an important role in the infection process. Mutants of D. pini that are deficient in dothistromin production have been made recently that will allow this role to be investigated. The aim of this study was to develop a pathogenicity testing system under PC2 containment (required for dothistromin deficient mutant) and to develop microscopy methods required to monitor both epiphytic and endophytic growth of the fungus on the needle D. pini requires high light intensity, continuous leaf moisture and a specific temperature range in order to infect pine needles. Progress was made towards developing a robust pathogenicity testing system. This study has also developed several microscopy techniques for the visualisation of epiphytic growth including a fluorescent microscopy technique. Other bright field and fluorescent staining techniques were investigated with some success. Staining techniques were not successful for the visualisation of endophytic D. pini growth but a green fluorescent protein (sgfp) reporter construct was obtained and two gfp plasmid contracts were developed for the transformation of D. pini for use as biomarkers. Successful introduction of the gfp constructs into D. pini will allow in situ visualisation of endophytic and epiphytic D. pini growth. The work done in this study will be useful for the further investigation into the role of dothistromin toxin, which may lead to new or more efficient methods of controlling D. pini as well as possibly providing information about other polyketide molecules of economic or medical significance.