Reproduction in selected New Zealand native ferns and their suitability for revegetation : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Plant Biology at Massey University, Palmerston North, New Zealand

The potential to use New Zealand native ferns for revegetation was assessed in laboratory , nursery and field experiments. Laboratory experiments indicated that the three native fern species. Blechnum novae-zelandiae. Cyathea medullaris and Dicksonia squarossa, had different maximum levels of spore germination. These differences also varied in response to seasonal changes in the environment. The effect of three soil conditioners on the germination of the same three species was minimal Gametophytes appeared to be tolerant of low levels of maceration, as they were able to continue to grow and develop normally. Additional laboratory experiments indicated that B. novae-zelandiae employs a mixed mating system, which utilizes an "antheridiogen" signal. The development of fern spores, laboratory propagated gametophytes and segmented rhizomes, was assessed in the nursery. Each experiment was applied with a hvdroseeding mix of paper fibre, tackifier. fertilizer and water Spore of B. novae-zelandiae. C. medullaris and D. squarossa failed to produce any long-lived gametophytes The survival of laboratory propagated gametophytes of B. novae-zelandiae. B. discolor and B. colensoi was low. However, a large proportion of surviving B. novae-zelandiae gametophytes produced sporophytes. B. novae-zelandiae rhizome segments produced healthy young ferns within 3 months of application. Field experiments were conducted on a sandstone/loess bank. 5 km east of Palmerston North. Aspects of the substrate were analysed including. pH. N, P and organic matter. The results indicated that the bank had a high soil pH, was deficient in several macronutrients and had no organic matter. Hydroseeding was applied using spore of the species B novae-zelandiae, C. medullaris and D. squarossa Hydroseeded spore failed to produce any visible gametophytes Rhizome experiments using B. novae-zelandiae and Microsorum pustulatum were also established. Low water availability resulted in poor rhizome establishment. The results suggest that there is great potential for utilizing native ferns in revegetation. Blechnum novae-zelandiae is the best species for revegetation in accordance to the results. Propagation via rhizome segmentation and gametophyte hydroseeding appear to be the most successful methods for establishing native ferns. This TIF project was carried out in conjunction with Rural Supply Technologies. Manaaki Whenua Landcare Research. Massey University and FoRST New Zealand

Intercomparison of Multiple UV-LIF Spectrometers using the Aerosol Challenge Simulator

Measurements of primary biological aerosol particles (PBAPs) have been conducted worldwide using ultraviolet light-induced fluorescence (UV-LIF) spectrometers. However, how these instruments detect and respond to known biological and non-biological particles, and how they compare, remains uncertain due to limited laboratory intercomparisons. Using the Defence Science and Technology Laboratory, Aerosol Challenge Simulator (ACS), controlled concentrations of biological and non-biological aerosol particles, singly or as mixtures, were produced for testing and intercomparison of multiple versions of the Wideband Integrated Bioaerosol Spectrometer (WIBS) and Multiparameter Bioaerosol Spectrometer (MBS). Although the results suggest some challenges in discriminating biological particle types across different versions of the same UV-LIF instrument, a difference in fluorescence intensity between the non-biological and biological samples could be identified for most instruments. While lower concentrations of fluorescent particles were detected by the MBS, the MBS demonstrates the potential to discriminate between pollen and other biological particles. This study presents the first published technical summary and use of the ACS for instrument intercomparisons. Within this work a clear overview of the data pre-processing is also presented, and documentation of instrument version/model numbers is suggested to assess potential instrument variations between different versions of the same instrument. Further laboratory studies sampling different particle types are suggested before use in quantifying impact on ambient classification.Peer reviewe

Contracting an element from a cocircuit

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Establishment of surface functionalization methods for spore-based biosensors and implementation into sensor technologies for aseptic food processing

Aseptic processing has become a popular technology to increase the shelf-life of packaged products and to provide non-contaminated goods to the consumers. In 2017, the global aseptic market was evaluated to be about 39.5 billion USD. Many liquid food products, like juice or milk, are delivered to customers every day by employing aseptic filling machines. They can operate around 12,000 ready-packaged products per hour (e.g., Pure-Pak® Aseptic Filling Line E-PS120A). However, they need to be routinely validated to guarantee contamination-free goods. The state-of-the-art methods to validate such machines are by means of microbiological analyses, where bacterial spores are used as test organisms because of their high resistance against several sterilants (e.g., gaseous hydrogen peroxide). The main disadvantage of the aforementioned tests is time: it takes at least 36-48 hours to get the results, i.e., the products cannot be delivered to customers without the validation certificate. Just in this example, in 36 hours, 432,000 products would be on hold for dispatchment; if more machines are evaluated, this number would linearly grow and at the end, the costs (only for waiting for the results) would be considerably high. For this reason, it is very valuable to develop new sensor technologies to overcome this issue. Therefore, the main focus of this thesis is on the further development of a spore-based biosensor; this sensor can determine the viability of spores after being sterilized with hydrogen peroxide. However, the immobilization strategy as well as its implementation on sensing elements and a more detailed investigation regarding its operating principle are missing. In this thesis, an immobilization strategy is developed to withstand harsh conditions (high temperatures, oxidizing environment) for spore-based biosensors applied in aseptic processing. A systematic investigation of the surface functionalization’s effect (e.g., hydroxylation) on sensors (e.g., electrolyte-insulator semiconductor (EIS) chips) is presented. Later on, organosilanes are analyzed for the immobilization of bacterial spores on different sensor surfaces. The electrical properties of the immobilization layer are studied as well as its resistance to a sterilization process with gaseous hydrogen peroxide. In addition, a sensor array consisting of a calorimetric gas sensor and a spore-based biosensor to measure hydrogen peroxide concentrations and the spores’ viability at the same time is proposed to evaluate the efficacy of sterilization processes

Characterisation of dairy strains of Geobacillus stearothermophilus and a genomics insight into its growth and survival during dairy manufacture : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

The thermophilic bacilli, such as G. stearothermophilus, are an important group of contaminants in the dairy industry. Although these bacilli are generally not pathogenic, their presence in dairy products is an indicator of poor hygiene and high numbers are unacceptable to customers. In addition, their growth may result in milk product defects caused by the production of acids or enzymes, potentially leding to off-flavours. These bacteria are able to grow in sections of dairy manufacturing plants where temperatures reach 40 – 65 °C. Furthermore, because they are spore formers, they are difficult to eliminate. In addition, they exhibit a fast growth rate and tend to readily form biofilms. Many strategies have been tested to prevent the formation of thermophilic bacilli biofilms in dairy manufacture, but with limited success. This is, in part, because little is known about the diversity of strains found in dairy manufacture, the structure of thermophilic bacilli biofilms and how these bacteria have adapted to grow in a dairy environment. In Chapters 2 and 3, phenotypic approaches were taken to understand the diversity of strains within a manufacturing plant. Specifically in Chapter 2, strains of the most dominant thermphilic bacilli, G. stearothermophilus, were isolated from the surface of various locations within the evaporator section and ten strains were evaluated for different phenotypic characteristics. Biochemical profiling, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fatty profiling demonstrated that the population was diverse. In Chapter 3, it was shown that the same ten strains varied in their ability to form biofilms and produce spores. Three strains of G. stearothermophilus, A1, P3 and D1, were selected for further analysis. SEM demonstrated that there were differences in biofilm morphologies between the three strains, particularly D1 versus the other two strains, A1 and P3. In Chapters 4, 5 and 6 a comparative genomics approach was taken to determine how these bacteria are able to grow and survive within a dairy manufacturing environment, as well as how they differ from other strains of Geobacillus. In Chapter 4 draft genome sequences were generated for three strains of G.stearothermophilus. Identification of a putative lactose operon in the three dairy strains provided evidence of dairy adaptation. In Chapter 5 a phylogenomics approach was taken to resolve relationships within the Geobacillus genus and to identify differences within the G. stearothermophilus group itself. Finally in Chapter 6 comparison with the model organism B. subtilis, gave a genomics insight into the potential mechanisms of sporulation for Geobacillus spp

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

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

Burmese amber fossils bridge the gap in the Cretaceous record of polypod ferns

publisher: Elsevier articletitle: Burmese amber fossils bridge the gap in the Cretaceous record of polypod ferns journaltitle: Perspectives in Plant Ecology, Evolution and Systematics articlelink: http://dx.doi.org/10.1016/j.ppees.2016.01.003 content_type: article copyright: Copyright © 2016 Elsevier GmbH. All rights reserved.Copyright © 2016 Elsevier GmbH. All rights reserved. This document is the authors' final accepted version of the journal article. You are advised to consult the publisher's version if you wish to cite from it

Methods of inoculating cypress with Seridium species to screen for resistance and pathogen variability : a thesis presented in partial fulfilment of the requirements for the degree of Master of Applied Science in Plant Health at Massey University, Palmerston North, New Zealand

The cypress species are grown for their timber value, ornamental beauty and shelter. Their existence is threatened by the presence of cypress canker disease caused by fungal pathogens of the genus Seiridium. The long term solution for controlling this disease is to breed for cypress clones that are resistant to cypress canker. Screening for resistance is conducted by artificially inoculating cypress plants with the pathogen's inoculum. This study aimed at developing reliable methods of artificial inoculation that are suitable for New Zealand's climatic conditions. Infection of cypress plants in nature is caused by conidia but mycelial inocula are more commonly used in artificial inoculation. Several methods of inducing sporulation of Seiridium species were investigated Addition of plant substrates was shown to increase sporulation of cultures of Seiridium isolates. Studies comparing the two types of inocula (mycelial plugs and conidial suspensions) showed that mycelium inocula caused a higher percentage of canker lesions than spore inocula. Conidial inocula offer a more consistent pathogenicity. Experiments to determine the effective spore load revealed that the percentage of canker increased with the increase of inoculum load. Pathogenicity varied between species and individual colonies of Seiridium isolates. Infection of cypress in nature is thought to occur through wounds and in this work, wounding was required for infection under both glasshouse and outdoor conditions. Inoculation of the main stem and side branches showed disease symptoms develop more rapidly on side branches than on the main stem. Investigations on in vitro inoculation of tissue cultured plants and excised side shoots showed the possibility of screening cypress ramets under different environmental conditions. Temperature and percentage relative humidity were found to influence the percentage of successful inoculations on cypress plants

Human Microsporidial Infections

Microsporidia are eukaryotic, spore forming obligate intracellular parasites, first recognised over 100 years ago. Microsporidia are becoming increasingly recognised as infectious pathogens causing intestinal and extra-intestinal diseases in both immuno-competent and immuno-suppressed patients. They are characterised by the production of resistant spores that vary in size depending on the species; and poses a unique organelle, the polar tubule (polar filament), which is coiled inside the spore as demonstrated by its ultra structure. Other unusual characteristics are the lack of mitochondria and the prokaryotic-like ribosomes, which indicate the primitive nature of the group. Presently there are seven genera, Enterocytozoon, Encephalitozoon, Nosema, Pleistophora, Trachi pleistophora, Brachiola, vittaforma species which have been reported from human hosts as agents of systemic, ocular, intestinal and muscular infections, are described and the diagnosis, treatment, and source of infections discussed