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    The role of microorganisms in shaping the nectar chemistry of mānuka plants (Leptospermum scoparium) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Ecology at Massey University, Manawatū, New Zealand
    (Massey University, 2022) Bresciano Kauffman, Jorge Carlo
    Mānuka (Leptospermum scoparium) honey is one of the main export products from New Zealand. The distinctiveness of this honey comes from the floral nectar, which possesses dihydroxyacetone, a carbohydrate with UV protection capabilities, which converts to methylglyoxal, usually during the honey maturation process. Methylglyoxal gives mānuka honey its biological activity (antimicrobial and antioxidant properties) and is often used as a quality marker. While it is acknowledged that microorganisms play an important role in plant ecology, their abundance and impact on nectar chemistry remain understudied. Microorganisms can arrive in the nectar by pollinator activity or through the phloem and can change nectar chemical properties. This thesis aims to fill a gap in the knowledge of the microbial diversity of the nectar; the impact of pollinators in the microbial nectar community, and the effect of bacteria on dihydroxyacetone production and sugar content in mānuka nectar. To achieve this aim, a combination of metagenomics, bacterial cultivation, chemical analyses, and biological in vivo and in vitro assays was conducted. The results show that mānuka nectar bacterial communities were dominated by Pseudomonadales, followed by Rhizobiales, Sphingomonadales, Corynebacteriales, Baccillales, Enterobacteriales, Xanthomonadales, Clostridiales, and Lactobacillales, while nectar fungi communities were dominated by Microbotryomycetes, Dothideomycetes, Malasseziomycetes, Sordariomycetes, and Ustilaginomycetes. When comparing bagged (pollinator restricted) and unbagged (with pollinator access) flowers, a bioinformatic and statistical analysis showed the bacterial community did not differ in community composition. In contrast, the fungal community was affected by pollinator visitation, and by plant genotype. The inoculation of an important functional bacterial Pantoea agglomerans, that previously was identified as a biomarker for the bacterial community, determined by linear discriminant analysis and network analysis, showed a significant increase in DHA using natural nectar under the laboratory. However, P. agglomerans in vivo inoculation did not change the composition of the main nectar sugars, suggesting that bacterial inoculation can maximize important plant metabolites like DHA, with minor disruption to nectar sugar content. The cultivable bacterial community differed little from the main groups found through metabarcoding, meaning the stability or structure of a core bacterial community in the nectar can be maintained through cultivation. This thesis fills an important gap in the knowledge of microbial ecology of mānuka plants and provides insights into how to manipulate key bacteria to increase DHA nectar content.
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    The identification of genes involved in the degradation of polyphosphate in Chlamydomonas reinhardtii : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science at Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Oliveira da Rocha, Catarina Sofia
    Phosphate (P) is an essential nutrient which availability can limit the growth and survival of all organisms, including microalgae. Microalgae have been reported to absorb and accumulate P as polyphosphate (PolyP) intracellularly. This microalgal ability has interested engineers for years as it could prove valuable for P removal and recovery from wastewater. While a pathway for PolyP synthesis has been described in microalgae, little is known about the mechanisms involved during PolyP degradation in microalgae. In this study, a reproducible biochemical assay was designed to determine the kinetics of PolyP degradation in the model organism Chlamydomonas reinhardtii. For this purpose, C. reinhardtii wild-type 1690 was grown in minimal media low phosphorus (MM low-P) for 5 days prior to the addition of 10 mg L⁻¹ P. Biological triplicates were analysed, and we measured the changes in cellular P content and granular-PolyP. After the analysis of the bioassays, we extracted RNA from the treatment groups. Based on information reported in the literature regarding other organisms we selected vtc1, vtc4, ipy1, ipy3, ppa and nudix hydrolase as candidate genes encoding enzymes involved in PolyP degradation in C. reinhardtii. Quantitative PCR was used to measure the transcription of ipy1, ipy3, ppa and vtc1 genes and these were shown to be regulated after the addition of P, but we were unable to quantify vtc4 and nudix genes. To corroborate the transcriptomics data, we used insertional mutants knock down in IPY1, IPY3, PPA and Nudix hydrolase. The mutants were grown in ammonia acetate low phosphorus (low-P) media and we quantified the changes in cellular P content and granular-PolyP up to Day 13 (168-hours), after the addition of P. Based on our results, we selected Nudix hydrolase was the most likely candidate involved in the degradation of PolyP. For the first time, we took a step to better understand PolyP consumption in microalgae. This knowledge is critical for a better understanding of the function and regulation of PolyP in algae.
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    Environmental and genetic influences on growth, flowering, and nectar production in mānuka (Leptospermum scoparium J.R. Forst. & G. Forst.) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science, School of Agriculture and Environment, Massey University, Palmerston North, New Zealand
    (Massey University, 2019) Sheridan, Rachael Elizabeth
    Dihydroxyacetone (DHA) in floral nectar of the Mānuka shrub, Leptospermum scoparium (J.R. & G. Forst.) is a direct precursor to methylglyoxal (MGO) the bioactive compound of manuka honey. Accumulation of DHA in Mānuka nectars varies between trees, localities, and years. However, the reasons for this variability are largely unknown and its origins in nectar are unclear. Since high DHA to total sugar ratios (DHA/TSugar) in fresh honey result in high MGO in mature honey, it follows that nectars with high DHA/TSugar will produce high-value honey indicated by a high Unique Mānuka Factor (UMF®) attracting premium returns for the NZ honey industry and NZ economy. It is key to further optimise both nectar and DHA production by selecting for high producers. Selecting/developing varieties for maximum nectar potential (NP) needs an understanding of the relative influences of genotype (G), environment (E), and their interactions (GEI) on relevant trait expression. The responses of genetic clones from three high yielding Mānuka lines expressing varying levels of nectar DHA to temperature, light, and soil moisture were studied in controlled environments. The relative performances of the clones were evaluated and contributions of G, E, and GEI to aspects of their growth, flowering and nectar production quantified.--Shortened abstract