title: Exploring the mānuka phyllosphere microbiome

authors: Noble, Anya Sophia

subjects: manuka

abstract: The surface of plant leaves, termed phyllosphere, is a ubiquitous microbial habitat that harbours diverse communities of microorganisms. Although a growing body of experimental evidence demonstrates that these microorganisms can have an influential role in host physiology, the ecological processes that drive the assembly of natural phyllosphere communities remain poorly understood.

Leptospermum scoparium (mānuka) is an indigenous New Zealand tea tree widely known for the non-peroxide antibacterial properties of its honey. However, the host physiological traits associated with these properties exhibit variation that remain unexplained despite decades of research. Considering a preliminary study that identified spatially persistent host association in the mānuka phyllosphere microbiome with patterns congruent with those of a microbial community under strong host selection, the primary objective of this research was to generate a holistic understanding of the ecological processes underpinning community assembly in the natural mānuka phyllosphere.

Since the host specificity of the mānuka phyllosphere microbiome was unquantified, this PhD thesis research began with a multi-species, spatially hierarchical survey of a native forest to understand the relative influence of host species identity versus distance on the phyllosphere microbiome of mānuka and ecologically similar, adjacent native plant species. The results revealed that the relative influence of host species identity on the phyllosphere microbiome was quantitatively stronger in mānuka compared to other plant species, and mānuka species-specificity was not associated with leaf morphological traits.

Using a pair of morphologically indistinguishable and naturally co-occurring plant species (mānuka and Kunzea ericoides [kānuka]), I then explored the relative influence of host species identity and leaf morphology on inter-host dispersal. Specifically, I addressed a longstanding yet under-examined hypothesis that the relative strength of the phyllosphere microbiome as a source of dispersing microorganisms (i.e., source-strength) is contingent on leaf morphology. The results revealed considerable spatial heterogeneity among morphologically indistinguishable leaves at small spatial scales and suggested that mānuka may act as a stronger source of phyllosphere microorganisms than kānuka. These findings suggest that source-strength is determined by the quantitative difference in the relative strength of host selection among plant species.

To contextualise this apparent spatial stability and host species-specificity of the mānuka phyllosphere microbiome, temporal variation was investigated by replicate sampling in three different seasons. My results revealed that the relative influence of individual host trees was larger than the season, and specific phyllosphere taxa persisted across time. Additionally, my results revealed an increased core microbiome during summer flowering, suggesting an association between host selection strength and host phenology.

Lastly, I explored the relationship between the mānuka phyllosphere microbiome and mānuka honey quality. I sampled trees and honey from three adjacent mānuka populations known to exhibit visually discrete phenological traits (i.e., flowering time). My results revealed correlations between phyllosphere community composition and chemical properties of mānuka honey, including the primary constituent of mānuka’s non-peroxide antibacterial properties (i.e., methylglyoxal).

Through the incorporation of spatial and temporal sampling designs, as well as a multi-disciplinary case study, this thesis provides a holistic understanding of the relative influence of host selection (abiotic and biotic), dispersal (short- and long-distance), and climate, on the assembly of the mānuka phyllosphere microbiome. These results also provide new perspectives on prevailing controversies (e.g., host selection vs. dispersal), address unverified hypotheses (e.g., source-strength), and illustrate a path forward that will allow the emergence of a coherent and generalisable understanding of phyllosphere microbial ecology.

text: Exploring the mānuka phyllosphere microbiome The surface of plant leaves, termed phyllosphere, is a ubiquitous microbial habitat that harbours diverse communities of microorganisms. Although a growing body of experimental evidence demonstrates that these microorganisms can have an influential role in host physiology, the ecological processes that drive the assembly of natural phyllosphere communities remain poorly understood.

Leptospermum scoparium (mānuka) is an indigenous New Zealand tea tree widely known for the non-peroxide antibacterial properties of its honey. However, the host physiological traits associated with these properties exhibit variation that remain unexplained despite decades of research. Considering a preliminary study that identified spatially persistent host association in the mānuka phyllosphere microbiome with patterns congruent with those of a microbial community under strong host selection, the primary objective of this research was to generate a holistic understanding of the ecological processes underpinning community assembly in the natural mānuka phyllosphere.

Since the host specificity of the mānuka phyllosphere microbiome was unquantified, this PhD thesis research began with a multi-species, spatially hierarchical survey of a native forest to understand the relative influence of host species identity versus distance on the phyllosphere microbiome of mānuka and ecologically similar, adjacent native plant species. The results revealed that the relative influence of host species identity on the phyllosphere microbiome was quantitatively stronger in mānuka compared to other plant species, and mānuka species-specificity was not associated with leaf morphological traits.

Using a pair of morphologically indistinguishable and naturally co-occurring plant species (mānuka and Kunzea ericoides [kānuka]), I then explored the relative influence of host species identity and leaf morphology on inter-host dispersal. Specifically, I addressed a longstanding yet under-examined hypothesis that the relative strength of the phyllosphere microbiome as a source of dispersing microorganisms (i.e., source-strength) is contingent on leaf morphology. The results revealed considerable spatial heterogeneity among morphologically indistinguishable leaves at small spatial scales and suggested that mānuka may act as a stronger source of phyllosphere microorganisms than kānuka. These findings suggest that source-strength is determined by the quantitative difference in the relative strength of host selection among plant species.

To contextualise this apparent spatial stability and host species-specificity of the mānuka phyllosphere microbiome, temporal variation was investigated by replicate sampling in three different seasons. My results revealed that the relative influence of individual host trees was larger than the season, and specific phyllosphere taxa persisted across time. Additionally, my results revealed an increased core microbiome during summer flowering, suggesting an association between host selection strength and host phenology.

Lastly, I explored the relationship between the mānuka phyllosphere microbiome and mānuka honey quality. I sampled trees and honey from three adjacent mānuka populations known to exhibit visually discrete phenological traits (i.e., flowering time). My results revealed correlations between phyllosphere community composition and chemical properties of mānuka honey, including the primary constituent of mānuka’s non-peroxide antibacterial properties (i.e., methylglyoxal).

Through the incorporation of spatial and temporal sampling designs, as well as a multi-disciplinary case study, this thesis provides a holistic understanding of the relative influence of host selection (abiotic and biotic), dispersal (short- and long-distance), and climate, on the assembly of the mānuka phyllosphere microbiome. These results also provide new perspectives on prevailing controversies (e.g., host selection vs. dispersal), address unverified hypotheses (e.g., source-strength), and illustrate a path forward that will allow the emergence of a coherent and generalisable understanding of phyllosphere microbial ecology.

year: 2024

title: Constructing a focused taxonomy from a document collection

authors: Medelyan, Olena

subjects: computer science

abstract: We describe a new method for constructing custom taxonomies from document collections. It involves identifying relevant concepts and entities in text; linking them to knowledge sources like Wikipedia, DBpedia, Freebase, and any supplied taxonomies from related domains; disambiguating conflicting concept mappings; and selecting semantic relations that best group them hierarchically. An RDF model supports interoperability of these steps, and also provides a flexible way of including existing NLP tools and further knowledge sources. From 2000 news articles we construct a custom taxonomy with 10,000 concepts and 12,700 relations, similar in structure to manually created counterparts. Evaluation by 15 human judges shows the precision to be 89% and 90% for concepts and relations respectively; recall was 75% with respect to a manually generated taxonomy for the same domain.

text: Constructing a focused taxonomy from a document collection We describe a new method for constructing custom taxonomies from document collections. It involves identifying relevant concepts and entities in text; linking them to knowledge sources like Wikipedia, DBpedia, Freebase, and any supplied taxonomies from related domains; disambiguating conflicting concept mappings; and selecting semantic relations that best group them hierarchically. An RDF model supports interoperability of these steps, and also provides a flexible way of including existing NLP tools and further knowledge sources. From 2000 news articles we construct a custom taxonomy with 10,000 concepts and 12,700 relations, similar in structure to manually created counterparts. Evaluation by 15 human judges shows the precision to be 89% and 90% for concepts and relations respectively; recall was 75% with respect to a manually generated taxonomy for the same domain.

year: 2013

title: Elucidating the origin and transmission of tetrodotoxin in New Zealand bivalves

authors: Biessy, Laura

subjects: Tetrodotoxin

abstract: Tetrodotoxin (TTX) is a potent neurotoxin that acts by selectively targeting sodium channels, blocking propagation of action potentials and causing paralysis. It has been responsible for countless human intoxications and deaths around the world, particularly in Japan, from consumption of pufferfish. The distribution of TTX and its analogues is remarkably diverse, and the toxin has been detected in organisms from marine, freshwater and terrestrial environments. Increasing detections of TTX in aquaculture species not typically associated with TTX, such as edible bivalves and gastropods, has drawn considerable attention to the toxin, reinvigorating scientific interest and regulatory concerns. There have been reports of TTX in 18 species of edible shellfish species from ten countries since the 1980’s, with some reports above the safe threshold established by the European Food Safety Authority (EFSA) of 44 μg of TTX per kg of shellfish.
Despite decades of research, the exact origin of TTX remains a mystery. Current literature supports three hypotheses: endogenous production, symbiotic bacteria, or direct bioaccumulation through a dietary source. In 2009, the sea slug Pleurobranchaea maculata was found to contain high concentrations of TTX in New Zealand and an extensive research programme to explore the origin of TTX in P. maculata in New Zealand was launched. During this research, Paphies australis, an endemic clam, was found to accumulate high concentrations of TTX (800 μg kg⁻¹). The aim of this thesis was to elucidate the source of TTX in these clams that are a common food source and are culturally important in New Zealand. Paphies australis are largely sessile and found in subtidal habitats making them a highly amedable organisms to investigate the source of TTX.
In this thesis, I used a multiple line of evidence approach to investigate potential TTX producers. This included histological and analytical techniques to explore the micro-distribution of TTX within the organs of P. australis, aquaria studies to investigate the depuration and uptake of TTX, field studies to explore the variations in TTX concentrations from different P. australis populations and other bivalve species in New Zealand, and molecular analyses. Immunohistological analysis of
P. australis tissues employing a TTX monoclonal antibody demonstrated that TTX was present in the outer cells of the siphons, but also in their digestive system, foot and gill tissues. These results were also supported by chemical analysis using liquid chromatography with tandem mass spectrometry. Observing TTX in organs involved in feeding provided initial evidence to support the hypotheses of an exogenous source in P. australis and presence in siphon tips supports the hypothesis that TTX is used as a chemical defence against predators.
The TTX depuration rate in P. australis was then assessed by maintaining TTX-bearing individuals in captivity and feeding them a non-toxic diet for 150 days. The bivalves significantly depurated the toxin (0.4% of total toxin content per day) and there was a rapid decline of the toxin in their digestive glands with only traces amounts remaining after 21 days. This result provides evidence to support the hypothesis that P. australis do not endogenously produce TTX.
During field surveys, P. australis from 10 different sites around New Zealand were harvested and tested for TTX. There were significant differences in TTX concentrations from the different sites. All P. australis contained TTX but bivalves from the warmer North Island sites contained significantly higher concentrations than those from the South Island. These results provided further evidence to support the proposition that the source of TTX in P. australis is likely exogenous, varying in abundance by location. I hypothesised that the source is a warm-water-adapted TTX producer that is mostly present or more prevalent in warmer climates, or that TTX production is triggered by warmer temperatures.
To explore this possibility further, I used metabarcoding to investigate the bacteria present in the digestive glands and siphons of P. australis from the 10 sites with different TTX concentrations and collected monthly over a year from one site with high TTX concentrations. Marine cyanobacteria, in particular picocyanobacteria (e.g., Cyanobium, Synechococcus, Pleurocapsa, and Prochlorococcus), were found in all samples collected from sites containing the highest amount of TTX and were present in the core microbiome of TTX-bearing P. australis. Cyanobacteria are well known for producing a wide range of marine and freshwater toxins and this finding warrants further investigation.
Lastly, I assessed if wild P. australis could sequester and accumulate TTX from a dietary source. I developed a novel method to artificially feed a controlled amount of TTX to bivalves. The micro-encapsulation method incorporated humic acid so that the water-soluble TTX bound to a solid substance. The main finding from this study was that P. australis can rapidly accumulate of TTX from an external dietary source and concentrations were much higher (> 100 μg kg⁻¹) than the safe threshold in bivalves established by the EFSA (44 μg kg⁻¹) in under two weeks. This further strengthens the hypothesis that bivalves most likely accumulate TTX from their diet.
This thesis has addressed several critical knowledge gaps in our understanding of the source of TTX in bivalves, particularly in the clam P. australis. A detailed account of each study is presented in this thesis and future research to elucidate the origin of TTX in bivalves are suggested.

text: Elucidating the origin and transmission of tetrodotoxin in New Zealand bivalves Tetrodotoxin (TTX) is a potent neurotoxin that acts by selectively targeting sodium channels, blocking propagation of action potentials and causing paralysis. It has been responsible for countless human intoxications and deaths around the world, particularly in Japan, from consumption of pufferfish. The distribution of TTX and its analogues is remarkably diverse, and the toxin has been detected in organisms from marine, freshwater and terrestrial environments. Increasing detections of TTX in aquaculture species not typically associated with TTX, such as edible bivalves and gastropods, has drawn considerable attention to the toxin, reinvigorating scientific interest and regulatory concerns. There have been reports of TTX in 18 species of edible shellfish species from ten countries since the 1980’s, with some reports above the safe threshold established by the European Food Safety Authority (EFSA) of 44 μg of TTX per kg of shellfish.
Despite decades of research, the exact origin of TTX remains a mystery. Current literature supports three hypotheses: endogenous production, symbiotic bacteria, or direct bioaccumulation through a dietary source. In 2009, the sea slug Pleurobranchaea maculata was found to contain high concentrations of TTX in New Zealand and an extensive research programme to explore the origin of TTX in P. maculata in New Zealand was launched. During this research, Paphies australis, an endemic clam, was found to accumulate high concentrations of TTX (800 μg kg⁻¹). The aim of this thesis was to elucidate the source of TTX in these clams that are a common food source and are culturally important in New Zealand. Paphies australis are largely sessile and found in subtidal habitats making them a highly amedable organisms to investigate the source of TTX.
In this thesis, I used a multiple line of evidence approach to investigate potential TTX producers. This included histological and analytical techniques to explore the micro-distribution of TTX within the organs of P. australis, aquaria studies to investigate the depuration and uptake of TTX, field studies to explore the variations in TTX concentrations from different P. australis populations and other bivalve species in New Zealand, and molecular analyses. Immunohistological analysis of
P. australis tissues employing a TTX monoclonal antibody demonstrated that TTX was present in the outer cells of the siphons, but also in their digestive system, foot and gill tissues. These results were also supported by chemical analysis using liquid chromatography with tandem mass spectrometry. Observing TTX in organs involved in feeding provided initial evidence to support the hypotheses of an exogenous source in P. australis and presence in siphon tips supports the hypothesis that TTX is used as a chemical defence against predators.
The TTX depuration rate in P. australis was then assessed by maintaining TTX-bearing individuals in captivity and feeding them a non-toxic diet for 150 days. The bivalves significantly depurated the toxin (0.4% of total toxin content per day) and there was a rapid decline of the toxin in their digestive glands with only traces amounts remaining after 21 days. This result provides evidence to support the hypothesis that P. australis do not endogenously produce TTX.
During field surveys, P. australis from 10 different sites around New Zealand were harvested and tested for TTX. There were significant differences in TTX concentrations from the different sites. All P. australis contained TTX but bivalves from the warmer North Island sites contained significantly higher concentrations than those from the South Island. These results provided further evidence to support the proposition that the source of TTX in P. australis is likely exogenous, varying in abundance by location. I hypothesised that the source is a warm-water-adapted TTX producer that is mostly present or more prevalent in warmer climates, or that TTX production is triggered by warmer temperatures.
To explore this possibility further, I used metabarcoding to investigate the bacteria present in the digestive glands and siphons of P. australis from the 10 sites with different TTX concentrations and collected monthly over a year from one site with high TTX concentrations. Marine cyanobacteria, in particular picocyanobacteria (e.g., Cyanobium, Synechococcus, Pleurocapsa, and Prochlorococcus), were found in all samples collected from sites containing the highest amount of TTX and were present in the core microbiome of TTX-bearing P. australis. Cyanobacteria are well known for producing a wide range of marine and freshwater toxins and this finding warrants further investigation.
Lastly, I assessed if wild P. australis could sequester and accumulate TTX from a dietary source. I developed a novel method to artificially feed a controlled amount of TTX to bivalves. The micro-encapsulation method incorporated humic acid so that the water-soluble TTX bound to a solid substance. The main finding from this study was that P. australis can rapidly accumulate of TTX from an external dietary source and concentrations were much higher (> 100 μg kg⁻¹) than the safe threshold in bivalves established by the EFSA (44 μg kg⁻¹) in under two weeks. This further strengthens the hypothesis that bivalves most likely accumulate TTX from their diet.
This thesis has addressed several critical knowledge gaps in our understanding of the source of TTX in bivalves, particularly in the clam P. australis. A detailed account of each study is presented in this thesis and future research to elucidate the origin of TTX in bivalves are suggested.

year: 2021