title: Development of a Rhizosheath Selection Tool for screening perennial ryegrass (Lolium perenne L.) for reduced root competition against white clover (Trifolium repens L.) for soil phosphorus

authors: Yeates, Rebecca Maree

subjects: Agriculture

abstract: Perennial ryegrass (Lolium perenne L.) outcompetes white clover (Trifolium repens L.) for soil phosphorus [P], resulting in higher P fertiliser rates to overcome the competition. Perennial ryegrass has long, dense root hairs compared to white clover, which increase the root contact with soil (soil exploration) and root surface area for P uptake. This study was conducted to test whether the selection of perennial ryegrass with reduced root hair length and density would reduce root competition for P with no detrimental effect on the perennial ryegrass growth. 
A Rhizosheath Selection Tool [RST] was developed to select ryegrass populations with contrasting root hair length and density. The RST protocol used rhizosheath traits (the width and coverage of soil adhered to the extracted root system) along with the total seedling weight to select two distinct ryegrass populations. The low rhizosheath population had shorter, sparser root hairs on seminal roots and sparser root hairs on adventitious roots than the high rhizosheath population.
The two populations were then grown at five soil P levels in a glasshouse experiment and demonstrated that the rhizosheath trait selection had no detrimental effect on growth. There were no significant differences between the two population’s dry weight (g) and P concentration (mg P g-1 dry weight). However there was evidence to indicate that the high rhizosheath population was sometimes more efficient at P uptake with a larger total P content (mg P). 
The two perennial ryegrass populations were then grown with companion white clover in Olsen P 12 and 19 mg L⁻¹ soil with partitions separating the shoot systems to avoid shoot competition for light. The low rhizosheath population had reduced root competition with the companion white clover. In Olsen P 12 mg L⁻¹ soil, more P was partitioned to the white clover than the ryegrass, measured by total P content (mg P), and achieved a greater dry weight (g) than when grown with the HRS ryegrass. In Olsen P 19 mg L⁻¹ soil there was no rhizosheath selection effect on white clover growth, possibly because of the reduced need for soil exploration by roots under the higher soil P availability. 
The findings from this study confirmed that the RST for the selection of a perennial ryegrass population with a reduced capacity for root competition against white clover for soil P is effective. Furthermore, the RST selection had no detrimental effect to the ryegrass growth. This research highlights the importance of root traits in forage plant breeding, and the potential environmental and economic benefits a low rhizosheath perennial ryegrass could have in New Zealand’s pastoral farming system.

text: Development of a Rhizosheath Selection Tool for screening perennial ryegrass (Lolium perenne L.) for reduced root competition against white clover (Trifolium repens L.) for soil phosphorus Perennial ryegrass (Lolium perenne L.) outcompetes white clover (Trifolium repens L.) for soil phosphorus [P], resulting in higher P fertiliser rates to overcome the competition. Perennial ryegrass has long, dense root hairs compared to white clover, which increase the root contact with soil (soil exploration) and root surface area for P uptake. This study was conducted to test whether the selection of perennial ryegrass with reduced root hair length and density would reduce root competition for P with no detrimental effect on the perennial ryegrass growth. 
A Rhizosheath Selection Tool [RST] was developed to select ryegrass populations with contrasting root hair length and density. The RST protocol used rhizosheath traits (the width and coverage of soil adhered to the extracted root system) along with the total seedling weight to select two distinct ryegrass populations. The low rhizosheath population had shorter, sparser root hairs on seminal roots and sparser root hairs on adventitious roots than the high rhizosheath population.
The two populations were then grown at five soil P levels in a glasshouse experiment and demonstrated that the rhizosheath trait selection had no detrimental effect on growth. There were no significant differences between the two population’s dry weight (g) and P concentration (mg P g-1 dry weight). However there was evidence to indicate that the high rhizosheath population was sometimes more efficient at P uptake with a larger total P content (mg P). 
The two perennial ryegrass populations were then grown with companion white clover in Olsen P 12 and 19 mg L⁻¹ soil with partitions separating the shoot systems to avoid shoot competition for light. The low rhizosheath population had reduced root competition with the companion white clover. In Olsen P 12 mg L⁻¹ soil, more P was partitioned to the white clover than the ryegrass, measured by total P content (mg P), and achieved a greater dry weight (g) than when grown with the HRS ryegrass. In Olsen P 19 mg L⁻¹ soil there was no rhizosheath selection effect on white clover growth, possibly because of the reduced need for soil exploration by roots under the higher soil P availability. 
The findings from this study confirmed that the RST for the selection of a perennial ryegrass population with a reduced capacity for root competition against white clover for soil P is effective. Furthermore, the RST selection had no detrimental effect to the ryegrass growth. This research highlights the importance of root traits in forage plant breeding, and the potential environmental and economic benefits a low rhizosheath perennial ryegrass could have in New Zealand’s pastoral farming system.

year: 2018

title: An evaluation of the NuroChek system for concussion assessment and management

authors: Treacy, Jennifer

abstract: Current methods of concussion assessment are subjective and vulnerable to error, and a missed concussion diagnosis could increase an athlete’s risk of further injury. An objective method of concussion assessment could provide valuable physiological data about injury severity and duration. The NuroChek system uses a flashing light stimulus to evoke electrophysiological activity, which is measured from occipital electrode sites and used to calculate a signal-to-noise ratio (SNR) and determine the magnitude of activity at the target location. Reductions in this activity are proposed to be associated with dysfunction or damage after concussion; in a previous study, the SNR was shown to decrease after concussion, and then return to baseline strength upon recovery.  

To examine the utility of the NuroChek system and SNR output in concussion assessment and management, 157 participants were assessed with the NuroChek system as rugby athletes (n = 121), combat athletes (n = 19), or non-athlete controls (n = 17). All athletes were assessed at multiple time points, while the non-athlete control group was only assessed at one time point. All participants completed two trials of the NuroChek headset and at least one cognitive measure.  

The first study (Chapter 4) examines the acute effects of concussion on the SNR, as well as any SNR changes after injury. Rugby athletes who sustained concussions (n = 21) were tested at multiple time points after their injuries with the NuroChek system and either the King-Devick (K-D) or the Sport Concussion Assessment Tool (SCAT-5). The post-injury and baseline SNR were compared to identify any changes within 3 days of concussion, as well as changes during the follow-up time points that might correspond with concussion recovery (up to 20 days post-injury and mid-season or end-of-season if available). No statistically significant changes were seen after concussion, W = 11.000, p = 0.612, d = -0.099, n = 8. When compared to the non-concussed male rugby athletes, the concussed athletes had significantly lower SNRs than the athletes who sustained regular repetitive impacts by the end-of-season, H(3) = 10.160, p = 0.017, d = 0.135, n = 76.  

The second study (Chapter 5) examines the different effects that concussion history, age, and sex have on the SNR and its trajectory over time in those experiencing repetitive impacts. While the SNR was not affected by concussion history or age, there was a main effect for sex: male athletes demonstrated significantly higher average SNRs than female athletes at all three time points. Additionally, the SNRs from female participants demonstrated a significantly greater proportion of a harmonic artefact (51.8%, compared to 15.1% of data from male participants) that decreased the quality of the female data. Sex and repetitive impacts were examined for any interaction, but the sample sizes were too small in some subgroups for statistical analysis. A main effect of repetitive impacts on the SNR was also seen in rugby and combat athletes.  This could indicate that repetitive impacts lead to higher SNR in athletes over time.  

Overall, the SNR in this study was not sensitive to changes after concussion or during recovery. No evidence was found to support NuroChek’s use in the assessment or management of concussion in rugby athletes. Additionally, the potential effect of repetitive impacts on the SNR confounds the relationship between the SNR and concussion. Future development of electrophysiological assessment methods for concussion should consider the effects of repetitive impacts and sex.

text: An evaluation of the NuroChek system for concussion assessment and management Current methods of concussion assessment are subjective and vulnerable to error, and a missed concussion diagnosis could increase an athlete’s risk of further injury. An objective method of concussion assessment could provide valuable physiological data about injury severity and duration. The NuroChek system uses a flashing light stimulus to evoke electrophysiological activity, which is measured from occipital electrode sites and used to calculate a signal-to-noise ratio (SNR) and determine the magnitude of activity at the target location. Reductions in this activity are proposed to be associated with dysfunction or damage after concussion; in a previous study, the SNR was shown to decrease after concussion, and then return to baseline strength upon recovery.  

To examine the utility of the NuroChek system and SNR output in concussion assessment and management, 157 participants were assessed with the NuroChek system as rugby athletes (n = 121), combat athletes (n = 19), or non-athlete controls (n = 17). All athletes were assessed at multiple time points, while the non-athlete control group was only assessed at one time point. All participants completed two trials of the NuroChek headset and at least one cognitive measure.  

The first study (Chapter 4) examines the acute effects of concussion on the SNR, as well as any SNR changes after injury. Rugby athletes who sustained concussions (n = 21) were tested at multiple time points after their injuries with the NuroChek system and either the King-Devick (K-D) or the Sport Concussion Assessment Tool (SCAT-5). The post-injury and baseline SNR were compared to identify any changes within 3 days of concussion, as well as changes during the follow-up time points that might correspond with concussion recovery (up to 20 days post-injury and mid-season or end-of-season if available). No statistically significant changes were seen after concussion, W = 11.000, p = 0.612, d = -0.099, n = 8. When compared to the non-concussed male rugby athletes, the concussed athletes had significantly lower SNRs than the athletes who sustained regular repetitive impacts by the end-of-season, H(3) = 10.160, p = 0.017, d = 0.135, n = 76.  

The second study (Chapter 5) examines the different effects that concussion history, age, and sex have on the SNR and its trajectory over time in those experiencing repetitive impacts. While the SNR was not affected by concussion history or age, there was a main effect for sex: male athletes demonstrated significantly higher average SNRs than female athletes at all three time points. Additionally, the SNRs from female participants demonstrated a significantly greater proportion of a harmonic artefact (51.8%, compared to 15.1% of data from male participants) that decreased the quality of the female data. Sex and repetitive impacts were examined for any interaction, but the sample sizes were too small in some subgroups for statistical analysis. A main effect of repetitive impacts on the SNR was also seen in rugby and combat athletes.  This could indicate that repetitive impacts lead to higher SNR in athletes over time.  

Overall, the SNR in this study was not sensitive to changes after concussion or during recovery. No evidence was found to support NuroChek’s use in the assessment or management of concussion in rugby athletes. Additionally, the potential effect of repetitive impacts on the SNR confounds the relationship between the SNR and concussion. Future development of electrophysiological assessment methods for concussion should consider the effects of repetitive impacts and sex.

year: 2024

title: Studies on the health and bacterial symbionts of  toheroa (Paphies ventricosa)

authors: Bennion, Matthew Joseph

subjects: Shellfish health

abstract: Disease outbreaks in marine species can have devastating consequences for host populations, host-associated species assemblages, ecosystem functioning, and for human users of these marine resources. At present, the risk factors associated with disease outbreaks, such as warming and pollution, are increasing. Given the increasing risk of disease outbreaks and the role that parasites play in marine ecosystems, there is a vital need to gain knowledge of marine diseases for species conservation, and the monitoring, management, and forecasting of disease. The field of marine disease ecology is growing, but many facets of our understanding remain in their infancy. Faced with a lack of baseline health data, efforts to combat disease outbreak in the ocean are impeded, even though the future of food security hinges on an ability to source food from the ocean. With a shift towards aquaculture, in particular bivalve aquaculture, knowledge of ambient health of wild and farmed species will dictate both yield, and our ability to protect wild stocks. 
Disease outbreak poses added risk to species that are already in peril. The toheroa (Paphies ventricosa) is a threatened surf clam endemic to Aotearoa (New Zealand). Once bountiful, overharvesting and population collapse put an end to the large-scale fisheries of the early and mid-1900s. The protections enacted on toheroa populations should have ensured their recovery, but recovery has not occurred. Disease has been suggested as one possible explanation. However, without knowledge of disease dynamics in toheroa populations, restorative efforts risk failure, or worse, inflicting further damage. Intracellular microcolonies of bacteria (IMCs) have been linked to numerous shellfish mass mortality events (MMEs) in Aotearoa in recent years, and were first detected in toheroa in 2017. This discovery raised questions regarding the role IMCs might be playing in preventing the recovery of toheroa populations. 
To address this uncertainty, this thesis set out to investigate what pathogens and parasites are present in toheroa, and determine what effect they might be having on toheroa health. A histopathology survey was conducted to gather fundamental baseline health information on toheroa populations across their entire distribution. IMCs were found to be the only potential pathogens of note. Histology data coupled with Bayesian models indicated that the probability of higher IMC intensity was associated with decreased toheroa condition. Using PCR, DNA sequencing, and in situ hybridisation, IMCs were characterised as bacteria in the genus Endozoicomonas. In many marine organisms, Endozoicomonas spp. are cosmopolitan members of associated-bacterial communities, often fulfilling biogeochemical cycling functions in host tissues. In toheroa populations, Endozoicomonas spp. abundance was found to be seasonal, but site specificity indicated habitat characteristics (specifically, the presence of freshwater outflows) might be a significant driver of their abundance. The bacterial community of toheroa was subsequently investigated using 16S rRNA gene sequencing, and candidates common to other coastal bivalve molluscs were found to be dominant, including Spirochaetaceae, Mycoplasmataceae, and Endozoicomonadaceae (in order of relative dominance). Freshwater outflows appeared to be such a significant driver of microbiome taxonomic dissimilarity, that specimens collected from sites 1240 km apart hosted more similar bacterial communities than specimens from sites with and without streams on the same beach and within the same region. The key indicator taxa driving microbiome dissimilarity were found to be Endozoicomonas spp. and sulfate-reducing bacteria in the phylum Desulfobacterota. 
Gas bubbles were also observed on the shells of toheroa at the same time that IMCs were first detected. At that stage, temperature and total dissolved gaseous pressure was thought to contribute to their manifestation. To gain a better understanding of this phenomenon, a spatio-temporal survey was conducted which revealed an association of high gas bubble intensity with the austral-autumn/winter and freshwater outflows. A new hypothesis is therefore presented that links gas bubbles to sulfate-reducing bacteria and hydrogen sulfide (H₂S), a by-product of anaerobic digestion. Given the apparent influence of freshwater on toheroa intrinsic health, the capacity for streams to deliver pollutants to toheroa beds was assessed. Results gained from inductively coupled plasma-mass spectrometry (ICP-MS) suggest that despite the modified nature of adjacent land, streams do not deliver significant loads of bioavailable trace metals to toheroa beds in the intertidal zone. Instead, nutrition in the form of large phytoplankton blooms in autumn/winter is thought to be the primary source of bioavailable trace metals for toheroa.
No observations of significant pathogenesis, associated with Endozoicomonas species, were made throughout this research. Furthermore, during the timeframe of this research (apart from intermittent mortality events reported), there is no evidence of an ongoing disease epidemic in toheroa populations. While not providing evidence for the factor preventing the recovery of toheroa, this research does have implications for the ongoing conservation and management of toheroa. It seems reasonable to assume that infectious disease is not a major factor contributing to the limited recovery of toheroa throughout Aotearoa. Instead, associations made between freshwater outflows, nutrition, and detritus indicate that habitat requirements provided by freshwater streams are having significant effects on toheroa health, survival, and homeostasis. Furthermore, evidence presented suggests that the Endozoicomonas spp. present in toheroa and other Aotearoa shellfish are likely to be an important endosymbiont. In the future, studies on remaining toheroa populations should prioritise nutritional health. Namely, what role symbionts play in toheroa nutrition, and how niche requirements shape symbiont community composition and function in toheroa tissues. Answers to these questions could profoundly increase the success of toheroa restoration and conservation efforts.

text: Studies on the health and bacterial symbionts of  toheroa (Paphies ventricosa) Disease outbreaks in marine species can have devastating consequences for host populations, host-associated species assemblages, ecosystem functioning, and for human users of these marine resources. At present, the risk factors associated with disease outbreaks, such as warming and pollution, are increasing. Given the increasing risk of disease outbreaks and the role that parasites play in marine ecosystems, there is a vital need to gain knowledge of marine diseases for species conservation, and the monitoring, management, and forecasting of disease. The field of marine disease ecology is growing, but many facets of our understanding remain in their infancy. Faced with a lack of baseline health data, efforts to combat disease outbreak in the ocean are impeded, even though the future of food security hinges on an ability to source food from the ocean. With a shift towards aquaculture, in particular bivalve aquaculture, knowledge of ambient health of wild and farmed species will dictate both yield, and our ability to protect wild stocks. 
Disease outbreak poses added risk to species that are already in peril. The toheroa (Paphies ventricosa) is a threatened surf clam endemic to Aotearoa (New Zealand). Once bountiful, overharvesting and population collapse put an end to the large-scale fisheries of the early and mid-1900s. The protections enacted on toheroa populations should have ensured their recovery, but recovery has not occurred. Disease has been suggested as one possible explanation. However, without knowledge of disease dynamics in toheroa populations, restorative efforts risk failure, or worse, inflicting further damage. Intracellular microcolonies of bacteria (IMCs) have been linked to numerous shellfish mass mortality events (MMEs) in Aotearoa in recent years, and were first detected in toheroa in 2017. This discovery raised questions regarding the role IMCs might be playing in preventing the recovery of toheroa populations. 
To address this uncertainty, this thesis set out to investigate what pathogens and parasites are present in toheroa, and determine what effect they might be having on toheroa health. A histopathology survey was conducted to gather fundamental baseline health information on toheroa populations across their entire distribution. IMCs were found to be the only potential pathogens of note. Histology data coupled with Bayesian models indicated that the probability of higher IMC intensity was associated with decreased toheroa condition. Using PCR, DNA sequencing, and in situ hybridisation, IMCs were characterised as bacteria in the genus Endozoicomonas. In many marine organisms, Endozoicomonas spp. are cosmopolitan members of associated-bacterial communities, often fulfilling biogeochemical cycling functions in host tissues. In toheroa populations, Endozoicomonas spp. abundance was found to be seasonal, but site specificity indicated habitat characteristics (specifically, the presence of freshwater outflows) might be a significant driver of their abundance. The bacterial community of toheroa was subsequently investigated using 16S rRNA gene sequencing, and candidates common to other coastal bivalve molluscs were found to be dominant, including Spirochaetaceae, Mycoplasmataceae, and Endozoicomonadaceae (in order of relative dominance). Freshwater outflows appeared to be such a significant driver of microbiome taxonomic dissimilarity, that specimens collected from sites 1240 km apart hosted more similar bacterial communities than specimens from sites with and without streams on the same beach and within the same region. The key indicator taxa driving microbiome dissimilarity were found to be Endozoicomonas spp. and sulfate-reducing bacteria in the phylum Desulfobacterota. 
Gas bubbles were also observed on the shells of toheroa at the same time that IMCs were first detected. At that stage, temperature and total dissolved gaseous pressure was thought to contribute to their manifestation. To gain a better understanding of this phenomenon, a spatio-temporal survey was conducted which revealed an association of high gas bubble intensity with the austral-autumn/winter and freshwater outflows. A new hypothesis is therefore presented that links gas bubbles to sulfate-reducing bacteria and hydrogen sulfide (H₂S), a by-product of anaerobic digestion. Given the apparent influence of freshwater on toheroa intrinsic health, the capacity for streams to deliver pollutants to toheroa beds was assessed. Results gained from inductively coupled plasma-mass spectrometry (ICP-MS) suggest that despite the modified nature of adjacent land, streams do not deliver significant loads of bioavailable trace metals to toheroa beds in the intertidal zone. Instead, nutrition in the form of large phytoplankton blooms in autumn/winter is thought to be the primary source of bioavailable trace metals for toheroa.
No observations of significant pathogenesis, associated with Endozoicomonas species, were made throughout this research. Furthermore, during the timeframe of this research (apart from intermittent mortality events reported), there is no evidence of an ongoing disease epidemic in toheroa populations. While not providing evidence for the factor preventing the recovery of toheroa, this research does have implications for the ongoing conservation and management of toheroa. It seems reasonable to assume that infectious disease is not a major factor contributing to the limited recovery of toheroa throughout Aotearoa. Instead, associations made between freshwater outflows, nutrition, and detritus indicate that habitat requirements provided by freshwater streams are having significant effects on toheroa health, survival, and homeostasis. Furthermore, evidence presented suggests that the Endozoicomonas spp. present in toheroa and other Aotearoa shellfish are likely to be an important endosymbiont. In the future, studies on remaining toheroa populations should prioritise nutritional health. Namely, what role symbionts play in toheroa nutrition, and how niche requirements shape symbiont community composition and function in toheroa tissues. Answers to these questions could profoundly increase the success of toheroa restoration and conservation efforts.

year: 2021