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    Characterisation of the cell death-inducing activity of the conserved family of Ciborinia camelliae-like small secreted proteins (CCL-SSPs) of C. camelliae, Botrytis cinerea and Sclerotinia sclerotiorum : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology at Massey University, Manawatu, New Zealand
    (Massey University, 2019) McCarthy, Hannah
    Ciborinia camelliae, the causal agent of Camellia petal blight, is a necrotrophic fungus that sequesters nutrients from dead plant cells. Candidate effector proteins have been identified from the secretome as a highly conserved clade, termed C. camelliae-like small secreted proteins (CCL-SSPs). Notably, the CCL-SSPs are not unique to C. camelliae. Indeed, a single homolog of the CCL-SSP family has shown to be encoded by the genomes of the closely related necrotrophs, Botrytis cinerea and Sclerotinia sclerotiorum, (BcSSP and SsSSP, respectively). Previous work has identified the ability of BcSSP and SsSSP to induce cell death on Camellia ‘Nicky Crisp’ petals, whereas of the ten C. camellia CCL-SSPs (CcSSPs) tested, only one induced very weak cell death. The aim of this study was to determine what specific regions of the SsSSP protein confered cell death-inducing ability, and to further characterise the cell death-inducing capability of these CCL-SSPs. In this study it was shown, through generation of chimeric regionswapped proteins and infiltration into Camellia ‘Nicky Crisp’ petals and Nicotiana benthamiana leaves, that the region encoded by Exon 2 of SsSSP is essential for cell death-inducing activity. It was also discovered that BcSSP and SsSSP may induce cell death to different extents, as a significant different was shown in quantified cell death induced on Camellia ‘Nicky Crisp’ petals. It was also found that BcSSP can induce strong cell death on Arabidopsis thaliana leaves, while SsSSP does not. This research also investigated appropriate methods for characterising cell death of CCL-SSPs, and suggested addition of a C-terminus tag for future work. The results of this study have shed further light on the CCL-SSP family as candidate effector proteins and provided several avenues for future researchers to fully elucidate the function of CCL-SSPs and their role in virulence of these three necrotrophic fungi.
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    The epidemiology and pathology of Paranannizziopsis australasiensis in New Zealand reptiles : a thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science in Wildlife Health at Massey University, Palmerston North, New Zealand
    (Massey University, 2018) Webster, Rebecca Kirsty Elizabeth
    Paranannizziopsis australasiensis, has recently been diagnosed in tuatara at two captive facilities in New Zealand. This newly emerging fungal pathogen, is a member of the onygenalean fungal group formally known as Chrysosporium anamorph of Nannizziopsis vriesii (CANV). Fungi of this genera are thought to be obligate primary pathogens in reptiles, and closely related species such as Pphidiomyces ophiodiicola, and Nannizziopsis guarroi have caused significant morbidity and mortalities in captive and wild reptile populations. The detection of this disease raised concerns for wild and captive population health and resulted in a temporary cessation of tuatara breed and release programmes from affected facilities. Similar lesions have been reported in tuatara at multiple other captive facilities in New Zealand, but lack of veterinary assessment and, until recently, inadequate diagnostic capabilities has led to an inability to confirm the presence or absence of P. australasiensis in these populations. This research aimed to investigate the epidemiology of P. australasiensis in New Zealand wild and captive endemic reptiles. Skin samples were collected from nine captive, six wild and two ecosanctuary populations of tuatara across New Zealand. Skin samples from in contact geckos and skinks were opportunistically collected to determine the possible cross species infection of P. australasiensis. Samples were tested for presence of P. australasiensis by fungal culture followed by PCR, and by loop-mediated isothermal amplification (LAMP). Soil samples were collected from burrows, basking areas and captive enclosures and analysed by LAMP to determine the presence of P. australasiensis within the environment. Paranannizziopsis australasiensis was found to be wide spread in New Zealand captive and wild reptile populations. In populations where the pathogen was detected prevalence varied between 6.7% and 44.4% for tuatara, 3.8% and 40% for geckos and 6.7% and 66.7% for skinks. A low virulence of disease associated with infection was seen in tuatara across New Zealand, with many LAMP positive tuatara being asymptomatic. Increased severity of disease was seen in two captive tuatara, where other concurrent disease was present. One fatality was reported. In other reptile hosts, no disease was identified, and it is suspected these species act as reservoirs for the transmission of this organism to tuatara. Paranannizziopsis australasiensis was detected multiple times in soil samples and may survive as an environmental saprophyte. Paranannizziopsis australasiensis appears to have a close association with New Zealand reptiles. The prevalence, distribution and pathology of P. australasiensis observed in this study suggests that this organism is not a threat to tuatara or other endemic reptile populations in New Zealand. The findings of this study have enabled restrictions placed on tuatara translocations, based on P. australasiensis status, to be removed.
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    A study of the alternaria leafspot complex on potatoes and tomatoes in the Manawatu : this thesis is presented as partial fulfillment of the requirements for the M. Agr. Sc. degree of Massey University College of the Manawatu.
    (Massey University, 1963) Hawthorne, Brian Tredinnick
    The fungus Alternaria (Macrosporium) solani is associated with a foliage disease of potatoes and tamatoes throughout the world. Although there are often several phases of attack on these hosts by the fungus e.g. tuber damage in the potato and seedling loss with tomatoes, the disease has been named on the basis of the foliage symptoms which are characteristic. Two names are commonly used (l) Target Spot (2) Early Blight, and of the two, 'Target sport' is the more descriptive since foliage lesions are circular to irregular dark brown areas with a very characteristic zonation effect due to a series of more or less concentric rings within the lesion. [From Introduction]
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    The development and significance of fungi in poultry rearing sheds : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University
    (Massey University, 1984) Mylvaganam, Indira A
    A study was conducted at the Poultry Research Centre, Massey University, to determine the numbers, types and patterns of development of fungi that occur in poultry rearing sheds. Two sheds, a broiler shed and a layer shed were analysed for the fungal population by weekly sampling of litter, feed and air. Particular emphasis was placed on the presence of Penicillium and Aspergillus as these genera contain species of potential significance to flock health as infectious or toxigenic agents. Use of the dilution plating method with modified Potato Dextrose Agar and Aspergillus Differential Medium for culture resulted in successful isolation of these genera. Further selectivity was attained with the use of a modification of the strip-bait method of Luttrell (1967}. Air sampling was by the exposed agar plate method. The poultry houses were found to contain high levels of viable fungal propagules. Of the total fungi, Penicillium, Aspergillus and Scopulariopsis were the major genera. Other genera included Rhizopus, Mucor, Cladosporium and Geotrichum. Penicillium was found throughout the trials. Scopulariopsis was present at low levels in fresh litter and feed but counts increased greatly with time. Counts of species of Aspergillus, (A. flavus and A. fumigatus) also showed increases mainly towards the end of the trials. Other genera (Geotrichum, Cladosporium etc.) were found on certain days only. As litter aged, its pH and moisture content increased and these increases were correlated to the increase in total fungal numbers during the period of housing. Patterns of fungal succession in the litter were similar to those in air. For some species the increases in numbers in litter were preceded by a similar peak in the air (eg. A. flavus) whereas for others the peak in litter was earlier than the peak in air (S. brevicaulis). The correlation between patterns in air and those in feed and litter was more obvious in the layer shed, where a more active flock created greater air movement than in the broiler shed where the birds were docile and caused very little circulation of air. A. flavus isolates were screened for the production of aflatoxin on coconut agar. Approximately 30% of all A. flavus isolates tested were toxigenic. Also, it was possible to extract the toxin after growth in semisynthetic liquid medium from isolates which had been strongly fluorescent on coconut agar, but not those which had been weakly fluorescent. Feed and litter samples tested for the presence of aflatoxin and other mycotoxins (T-2, ochratoxin and zearalenone) were negative for these mycotoxins. The fungal population of poultry houses has been shown to include species which may be of economic importance. The large amounts of A. flavus (potentially toxigenic) and A. fumigatus (infectious), in particular, should not be ignored as they may well affect poultry health and productivity.
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    Transcriptional regulation during appressorium formation and function in Glomerella cingulata : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology at Massey University, Palmerston North, New Zealand
    (Massey University, 2006) Tong, XingZhang
    Glomerella cingulata, anamorph Colletotrichum gloeosporioides, causes bitter rot in apples and fruit rot in other subtropical fruits. In response to environmental cues such as contact with the host, Glomerella cingulata forms a special structure called an appressorium, which accumulates glycerol and thereby generates a sufficiently high turgor pressure to push an infection peg into the host tissue. It is known that the cAMP and MAPK signalling transduction pathways control appressorium formation and function in Colletotrichum species and other appressorium-forming fungi. This process is accompanied by a global change in gene expression. Little is known of transcriptional regulation during this process. The aim of this project was to study the transcriptional regulation of appressorium formation and function in G. cingulata. The G cingulata SAP gene had previously been shown to be expressed as a longer transcript during the early stage of appressorium differentiation. It was considered possible that the transcription factors that regulated expression of the longer transcript may be also involved in the regulation of appressorium differentiation. Identification of the transcription factor involved may help to understand the mechanisms that regulate appressorium differentiation. The plan was to use the yeast one-hybrid system to isolate the transcription factor. This required identification of the promoter regions responsible for expression of the longer SAP transcript. Therefore, the G. cingulata SAP promoter was characterized by mapping the transcription start point. Three transcription start points were determined by RLM-RACE. To further characterise the promoters, SAP-GFP reporter plasmids were constructed and transformed into G. cingulata. Even though a reasonable level of GFP expression was observed in RT-PCR experiments, however, no differences in fluorescence intensity were seen between the wild type and GFP reporter transformants. Therefore, no further attempts to study the sap promoter were made. The candidate gene approach was chosen as an alternative way to study the transcriptional regulation of appressorium formation and function in G. cingulata. The G. cingulata StuA gene was cloned using degenerate PCR, single specific primer PCR, subgenomic library screening and plasmid rescue from a disruption mutant. Targeted gene deletion of the G. cingulata StuA gene was successful. Deletion mutants display many phenotypic changes. Complementation mutants were constructed to confirm the function of this gene. A full length copy of this gene together with a second selection marker was reintroduced into the deletion mutant and the wild type phenotype was restored. Deletion mutants form appressoria at the normal rate and with unaltered morphology. In comparison with the wild type, these appressoria did not generate high turgor pressure as shown by a cytorrhysis assay. This resulted in a defect in appressorium penetration of onion epidermal cells. Nor were these mutants able to invade unwounded apples. Therefore, the G. cingulata StuA gene is required for appressorium function. In addition, deletion mutants displayed stunted aerial hyphae, "wettable" mycelium, reduced conidia production, and a defect in conidiophore and perithecium formation. These results suggested that the G. cingulata StuA gene has multiple roles in fungal development.
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    Sex has no detectable net benefits for Candida albicans : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Microbiology at Massey University, Palmerston North, New Zealand
    (Massey University, 2012) Zhang, Ningxin
    Like many other important opportunistic human fungal pathogens, for more than a century Candida albicans was thought to be strictly asexual until a parasexual cycle was recently discovered in the laboratory. It is uncertain, however, whether sex is still a viable reproductive strategy for C. albicans. In this study I tested whether or not mating enhanced survival of parental genes in this yeast, by mating 10 clinical isolates and testing recombinants’ fitness. Clinical isolates of C.albicans usually are diploid, carrying both the MTLa and MTLα mating type alleles, each on a different copy of chromosome 5. These strains are apparently incapable of meiosis and cannot mate with each other, because the a1-α2 heterodimer suppresses mating. Through selection on sorbose-containing agar I induced loss of MTL-heterozygosity and generated 5 MTLa and 5 MTLα derivatives of clinical isolates. Existing mating techniques involve the use of auxotrophic markers, requiring time-consuming sequential disruption of two copies of biosynthetic genes if wild-type clinical isolates are to be mated. Furthermore, auxotrophy affects the virulence of a strain, and this can potentially interfere with comparing the fitness of recombinants with that of their parents. I therefore developed a method for mating clinical isolates marked with two drug resistance markers, the mycophenolic acid (MPA) resistance-conferring allele of IMH3 and the nourseothricin (NAT) resistance gene CaNAT1, allowing selection of recombinants on the basis of resistance to both agents. I marked all MTLa strains with the MPA resistance gene and all MTLα strains with the NAT resistance gene. This allowed 25 combinations for mating. Recombinants were obtained from 15 combinations of 9 strains. It was found that not all C. albicans clinical isolates could mate. Using growth rate as the criterion, I tested the fitness of clinical isolates, MTLhomozygous derivatives with and without resistance markers and recombinants during adaptation to a novel environment (YPD medium), maximizing the potential benefits of sex. After computationally correcting for the impact of experimental manipulations, I calculated the net benefit of sex as the difference in the number of offspring from two cells that become mating competent and engage in sex compared to the offspring they could have produced by continued clonal reproduction. My results indicated that, as a rule, engaging in sex reduces the chances of survival of C. albicans’ genes, in part because MTL homozygosis significantly reduced growth rates. Through fitness increase after recombination, sex may eventually confer a net benefit for some strain combinations in the laboratory, but this probably occurs too late to prevent elimination of recombinants by competition and genetic drift in nature. Sex in C. albicans therefore diminished parents’ chances to pass on their genes to future generations. These findings have a significant impact on the assessment of the role of sex in C. albicans and other “asexual” human fungal pathogens. Recent loss of the function of sex and incomplete decay of the sex machinery are the most likely explanation of C. albicans’s residual ability to mate, and one that also needs to be considered in other fungal pathogens.