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Item Plasticity in reproduction and survival under dynamic socio-sexual environment : empirical evidence from Ephestia kuehniella Zeller (Lepidoptera: Pyralidae) : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Entomology at Massey University, Palmerston North, New Zealand(Massey University, 2017) Esfandi, KambizUsing an important pest of stored products, Ephestia kuehniella Zeller, I tested a number of theoretical predictions regarding strategies taken by males and females for resource allocations in response to dynamic socio-sexual environment. I demonstrate that males only respond to mean sperm competition levels and eupyrene sperm are produced both before and after emergence. Lifetime reproductive fitness in males depends on the number of copulations they can achieve, rather than the number of sperm ejaculated in each copulation. Regardless of whether males are exposed to rivals or not during their early adulthood, copulation duration and sperm allocation are not positively correlated, indicating that copulation duration cannot be used as a correct estimate of sperm allocation. Contrary to the previous prediction that males invest more in courting in the presence of rivals, my experiments demonstrate that males allocate more resource to courtship in the presence of additional females, which reduces their lifetime copulation frequency and fecundity. This finding offers a novel explanation for the success of mating disruption strategy using sex pheromones in pest management. Contradicting the previous prediction that females are more promiscuous under a female-biased condition and choosier in a male-biased sex ratio, my results show that perception of additional males makes females more receptive so that they mate more times and fertilise more eggs. Females call more when no additional mates or females are present than when either additional mates or females are present, suggesting that perception of no additional conspecifics by females may trigger them to allocate more energy for calling for further mating opportunities. Although virgin females lay similar numbers of eggs in all treatments, they start oviposition earlier and live shorter in the presence of conspecific males or females, supporting previous predictions that higher reproductive rate may accelerate senescence. Virgin females produce fewer eggs in male-biased than in female-biased sex ratio, suggesting that they reduce reproductive investment during their early life for mating opportunities under male-biased conditions. My studies provide insight into the plasticity in reproduction and survival under dynamic socio-sexual environment for animals with sexual reproduction in general and for this insect in particular.Item Molecular analysis of plant innate immunity triggered by secreted effectors from bacterial and fungal pathogens of apple : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Plant Science, Institute of Agriculture and Environment, Massey University, New Zealand(Massey University, 2017) Prokchorchik, MaximIn comparison to animals, plants do not have a dedicated immune system with mobile immune cells to protect themselves. Instead they rely on the innate immunity of each cell. Plant immunity branches into two classical layers: PTI (PAMP-triggered immunity) and ETI (Effector-triggered immunity). PTI detects the conserved molecular patterns (PAMPs) associated with pathogens and often can be overcome by pathogens translocating effector molecules into plant cells to inhibit the PTI. ETI, in turn, relies on intracellular receptors that can specifically recognize effectors or their activity and activate a rapid and robust response. The research presented in this thesis is focused on two pathogens of apple plants: the bacterial pathogen Erwinia amylovora (the causal agent of fire blight) and fungal pathogen Venturia inaequalis (the causal agent of apple scab disease). As both bacterial and fungal pathogens deliver effector molecules in order to promote their virulence, ETI engineering is a promising universal strategy to control these pathogens. In Chapter 3, the main aim was to elucidate the requirements and precise mechanism of how an important effector of E. amylovora, AvrRpt2, is recognized by the MR5 disease resistance (R) protein, derived from a hybrid apple Malus x robusta 5. I identified that a fragment of the guardee apple protein RIN4 was required and sufficient and required for MR5 activation. I further identified crucial amino acid residues responsible for this activation. Interestingly, cognate residues in RIN4 guardee homolog from Arabidopsis thaliana are responsible for suppression of the autoactivity of R protein RPS2. These findings led to the proposal of a novel hypothesis for evolutionary guardee adaption to the pool of R proteins present in plants. In Chapter 4, the main focus was to apply newly acquired whole-genome sequencing data of V. inaequalis for identifying the previously mapped AvrRvi8 effector, as well as several novel effectors predicted in silico. The sequences of these effectors were validated by amplification and resequencing of candidate genes from V. inaequalis cDNA. Further functional analysis of the selected gene candidates was performed. In addition, a library of constructs for generating V. inaequalis knock-out strains was prepared for future work. The findings from this thesis expected to be useful for breeders of apple to battle two economically important pathogens devastating the industry. Deployment of the MR5 system in apples should facilitate fire blight resistance in pipfruit and offers the opportunity for further engineering of MR5 to detect other pathogens. Furthermore, the effector library developed for V. inaequalis offers a novel tool for studying both virulence and avirulence mechanisms present in the applescab pathosystem. It is envisaged that further effector research will elucidate authentic targets critical for resistance development in apple.Item Investigating the evolutionary changes in Crabtree-negative yeasts during a long-term evolution experiment : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Albany, New Zealand(Massey University, 2017) Morley, AnnabelThe Crabtree effect is a metabolic strategy that allows yeast to ferment in the presence of oxygen. This is of interest as not all yeasts display this strategy, and nearly 100 years after its discovery it is still unclear what the overall benefit is. Two key theories attempt to explain the emergence of this phenomenon, the make-accumulate-consume theory and the rate/yield trade-off theory. The aim of this thesis was to investigate whether a trade-off between rate and yield develops in Crabtree-negative yeasts over the course of 1500 generations in a high sugar environment. Chapter Two demonstrates that growth rate is more likely to increase than decrease while growth yield is more likely to decrease than increase in the isolate-derived populations of yeast. We find that species that started out relatively fast, changed little while the slower species had more significant gains in growth rate. With growth yield, the species with initially high yield lost more significantly than the already low yield species. This could suggest there is an overall optimum growth rate and growth yield, that the species are evolving towards. In Chapter Three, ethanol production was measured using colorimetric tests and no change was observed to support the development of the Crabtree effect in these populations after 1500 generations. In Chapter Four growth yield was investigated using flow cytometry and it was found that several yeast populations both increased in cell size and decreased in growth yield. This is an interesting observation that has been observed in several previous experimental evolution experiments. In Chapter Five, as cell size is often associated with ploidy changes, DNA content was measured using DAPI and SYTOX DNA stains, detected by flow cytometry. This did not provide any statistically significant conclusions but highlighted the importance of employing further techniques to analyse the DNA content of these populations. This thesis has illustrated the importance of studying the competitive behaviours of microorganisms in isolation, where selfish traits appear to thrive.Item Epigenetic regulation of Epichloë festucae secondary metabolite biosynthesis and symbiotic interaction with Lolium perenne : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Palmerston North, New Zealand(Massey University, 2017) Lukito, YonathanHistone methylation is one of several epigenetic layers for transcriptional regulation. Most studies on the importance of this histone modification in regulating fungal secondary metabolite gene expression and pathogenicity have focussed on the role of histone methyltransferases, while few studies have focussed on the role of histone demethylases that catalyse the reversal of the modification. Epichloë festucae (Ascomycota) is an endophyte that forms a mutualistic interaction with perennial ryegrass. The fungus contributes to the symbiosis by the production of several classes of secondary metabolites, these have anti-insect and/or anti-mammalian activity. The EAS and LTM clusters in E. festucae are located subtelomerically and contain the biosynthetic genes for two of these important metabolites which are only synthesised in planta. Thus, in the host plant these genes are highly expressed, but they are tightly silenced in culture conditions. Previous study has shown that histone H3K9 and H3K27 methylation and their corresponding histone methyltransferases are important for this process. In this study, the role of histone lysine demethylases (KDMs) in regulating these genes and the symbiotic interaction is described. Eight candidate histone demethylases (Jmj1-Jmj8) were identified in E. festucae, among these proteins are homologues of mammalian KDM4, KDM5, KDM8, JMDJ7, and N. crassa Dmm-1. The genes for the proteins were overexpressed in E. festucae and histone methylation levels were determined in the strains. Overexpression of the genes was not observed to cause any change to the culture and symbiotic phenotypes of the fungus. Western blot analysis subsequently identified one of the proteins, KdmB, as the histone H3K4me3 demethylase. Further analysis by ChIP- and RT-qPCR showed that demethylation of H3K4me3 by KdmB at the eas/ltm genes is crucial for the activation of these genes in planta. The full expression of several other telomeric genes was similarly found to require kdmB. On the other hand, the COMPASS H3K4 methyltransferase complex subunit CclA that is required for H3K4 trimethylation in E. festucae represses the eas/ltm genes in culture conditions by maintaining H3K4me3 levels at the loci. Thus, these findings suggest a repressive role for H3K4me3 at these Subtelomeric secondary metabolite loci and are consistent with the role of H3K4me3 in yeast telomeric silencing. Disruption of kdmB did not affect the symbiotic interaction of E. festucae with the host grass but severely reduced the levels of lolitrem B, an animal neurotoxin. At the same time, the levels of ergovaline, another animal toxin, and peramine, an insect feeding deterrent, were not affected. Therefore, disruption or inhibition of KdmB may also serve as a promising approach for future endophyte improvement programmes. The E. festucae homologue of KDM8 (an H3K36me2 demethylase), Jmj4, was further investigated in this study but no H3K6 demethylase activity was found for the protein. Both disruption and overexpression of the gene encoding Jmj4 similarly had no effect on the culture and symbiotic phenotypes of E. festucae. However, deletion of setB, encoding the homologue of yeast Set2 (H3K36 methyltransferase) specifically reduced histone H3K36me3 levels in E. festucae. This contrasts with deletion of Set2 in other fungi which affected H3K36 mono-, di- and trimethylation. The ΔsetB mutant was severely impeded in development, and was unable to establish infection of the host plant. Introduction of the wild-type setB gene reversed these phenotypes. This study shows that H3K4 trimethylation controlled by CclA and KdmB is an important regulator of subtelomeric secondary metabolite genes in E. festucae but not for the symbiotic interaction of the fungus with perennial ryegrass. On the other hand, the histone H3K36 methyltransferase SetB specifically controls H3K36 trimethylation in E. festucae and is required for normal vegetative growth and ability of the fungus to infect the host plant.Item Ecological factors affecting the establishment of the biological control agent Gargaphia decoris Drake (Hemiptera: Tingidae) : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand(Massey University, 2017) Falla, Cecilia MaríaThe Brazilian lace bug (Gargaphia decoris Drake (Hemiptera:Tingidae)) was released in New Zealand in 2010 for the biological control of the invasive weed woolly nightshade (Solanum mauritianum Scopoli (Solanaceae)). Currently there is scarce information about the potential effect of ecological factors on the establishment of this biological control agent. This study investigated: 1) the effect of maternal care and aggregation on nymphal survival and development; 2) the effect of temperature, photoperiod and humidity on G. decoris performance; and 3) the effect of light intensity on S. mauritianum and G. decoris performance. Maternal care and aggregation are characteristic behaviours of G. decoris. These behaviours have an adaptive significance for the offspring and are key determinants for the survival of the species under natural conditions. Maternal care is reported to increase the survival and development of offspring under field conditions, and higher aggregations to increase the survival of the offspring. However, in this study, maternal care negatively affected the survival and development of the offspring, and higher aggregations had no significant impact on offspring survival. The availability of host plants under laboratory conditions may have influenced the expression of these behaviours. Climate is a factor that constrains insect development and therefore establishment. In this study, temperature affected the survival, nymphal development, life cycle, adult longevity, female reproductive success (i.e. total number of eggs, number of eggs laid per female, number of egg batches, number of eggs per batch, pre-oviposition period, percent females that oviposited successfully, number of eggs in the first batch and percentage of eggs that hatched from the first batch) and population growth parameters (i.e. life table). Temperatures between 20 – 25 °C were the optimal temperatures for G. decoris establishment. Photoperiod affected the mean percentage of egg hatch (i.e. emergence of nymphs in egg batch collected from colony) and total nymphal survival (i.e. egg to adult emergence), adult longevity and population growth parameters. The photoperiod 16L:8D was the optimal photoperiod for insect establishment. Humidity affected the mean percentage of egg hatch, adult longevity and population growth parameters. G. decoris population growth was highest at 70 ± 10% RH but the population growth was faster at 50 ± 10%. The CLIMEX model predicted that G. decoris could occupy broader regions not only on its native range (i.e. Brazil and Argentina) but also other regions where S. mauritianum is considered invasive (i.e. New Zealand and South Africa). G. decoris is predicted to be able to establish optimally in most of New Zealand North Island, except in regions with altitudes higher than 1300 meters above sea level. Most of the South Island is considered unsuitable for G. decoris establishment, except parts of the West Coast, Nelson and the Tasman region, which are predicted to be moderately to marginally suitable. Light intensity and plant age (i.e. day of harvest) affected host plant quality and had an indirect impact on insect establishment. Light intensity and plant age affected key physiological, morphological and defensive traits of S. mauritianum. Three compounds appeared to be involved, and were positively identified as glycoalkaloids: α-solamargine/β-solamarine, solauricine/solasonine, and unknown-954. The reproductive performance of G. decoris was affected because females avoided ovipositing on unshaded plants. The presence of trichomes and an increase in concentration of glycoalkaloids in the second harvest affected the nymphal performance and was reflected in adults, which had smaller bodies and wings. The results of my study have implications for using the Brazilian lace bug G. decoris in biological control programmes. The ecological factors included in this study work synergistically rather than independently and are important to consider when deciding the best locations in which the insect could be liberated.Item Physiology of rumen bacteria associated with low methane emitting sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Palmerston North, New Zealand(2017, 2017) Kumar, SandeepThe fermentation of feed and formation of methane (CH4) by ruminant animals occur in the rumen, and both are microbial processes. There is a natural variation in CH4 emissions among sheep, and this variation is heritable. Therefore, breeding for sheep that naturally produce less CH4 is a viable strategy to reduce anthropogenic greenhouse gas emissions. Rumen bacteria play a major role in feed fermentation and in the formation of hydrogen (H2) or formate, which are converted to CH4 by other rumen microbes called methanogens. It has been shown that rumen bacterial community compositions in low CH4 emitting sheep differ to those in high CH4 emitting sheep. This led to the hypothesis that the metabolism of dominant rumen bacteria associated with low CH4 emitting sheep should explain the lower CH4 yield, for example by producing less H2 or formate than bacteria associated with high CH4 emitting sheep. In this project, the diversity and physiology of members of the bacterial genera Quinella, Sharpea and Kandleria, which are major bacterial groups associated with low-CH4 emitting sheep, were investigated. It appeared that the genus Quinella is more diverse than previously suspected, and might contain at least eight potential species, although to date none have been maintained in laboratory culture. Sharpea and Kandleria contain two and one species respectively. Experiments with Sharpea and Kandleria showed that these behave like classical lactic acid bacteria that produce lactate as their major end product and did not change their fermentation pattern to produce more H2 or formate when grown in the presence of methanogens. This strengthens a previous hypothesis that sought to explain low CH4 emissions from sheep with Sharpea and Kandleria in their rumens, in which this invariant production of lactate was a key assumption. Quinella is another bacterium found in larger numbers in the rumen of some low CH4 sheep. Virtually nothing is known about its metabolism. FISH probes and cell concentration methods were developed which helped in its identification and resulted in construction of four genome bins of Quinella that were more than 90% complete with as little as 0.20% contaminated. Bioinformatic analyses of the proteins encoded by these genomes showed that Quinella has the enzymes for lactate formation and for the randomising pathway of propionate formation. This indicated that lactate and propionate might be major fermentation end products of Quinella. Additionally, the presence of an uptake hydrogenase in the Quinella genomes opens up the new possibility that Quinella might even use free H2 in the rumen. In all these possible pathways, little or no H2 would be produced, explaining why an increased abundance of Quinella in the rumen would lead to lower CH4 emissions from those sheep with high abundances of this bacterium.Item Using marine ecoengineering to mitigate biodiversity loss on modified structures in the Waitematā Harbour : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Conservation Biology at Massey University, Albany, New Zealand(Massey University, 2017) McKenzie, Connor JamesThe construction of infrastructure on the foreshore is an unavoidable consequence of an ever-expanding human population. Traditionally, this infrastructure has replaced softsubstrates with hard substrates. Furthermore, even for native biota which occupy hard substrates, the flat, featureless construction of most marine infrastructure provides little habitat heterogeneity and results in depauperate communities with little biotic resistance against non-indigenous species. Marine ecoengineering provides a possible solution to this global phenomenon by using intelligent construction techniques that promote the accumulation of native biodiversity. Here, I used eco-engineered settlement plates to examine the effect of habitat complexity on the biodiversity of communities inhabiting existing. Additionally, we examined the effects of climate change driven increases in rainfall on the performance of ecoengineered substrates in the mid-intertidal zone. Last, we reviewed and synthesised the available literature on the species present in The Waitematā Harbour and, to the best of my knowledge, provide the most complete species lists to date. In chapter two, we transplanted eco-engineered settlement plates seeded with local bivalve, Perna canaliculus, onto an existing seawall and monitored the accumulation of biodiversity. Overall, we show that both structural and biological habitat heterogeneity enhanced the biodiversity of the seawall community. Additionally, we found that the cemented pavement of volcanic rock that constituted the existing seawall, accumulated biodiversity faster than flat concrete settlement plates, supporting the use of this type of seawall construction over flat concrete seawalls. However, benefits to biodiversity could be further enhanced by explicitly adopting ecoengineering designs that provide crevices for intertidal organisms. In chapter three, we examined the performance of ecoengineered substrates under the prediction that climate change will enhance rainfall by 20% in the Auckland region. While no effect of increased rainfall was observed for the mobile invertebrate community or the flat plates, increased rainfall did influence the biodiversity of the fouling community on the ridged plates, likely as a consequence of reduced desiccation stress. Although this was only a short-term experiment we predict that given time to develop, a distinct fouling community could influence the diversity mobile invertebrate community, shifting the whole community vertically up the seawall. The review of the Waitematā taxonomy presented in chapter four, provides a reference for future studies of the biodiversity of the Waitematā harbour as well as identifying several gaps in our understanding, a cause for concern. Specifically, we show that non-indigenous species make up a considerable proportion of the fouling species listed for the Harbour and suggest that some of this could have been avoided by the adoption of ecoengineering techniques. Overall, this thesis recognises that habitat heterogeneity, be it natural or man-made, is a vital driver of biodiversity. Each chapter provides additional insight, supporting the benefits of marine ecoengineering. These positive results within the Waitematā Harbour show potential for larger scale experimental trials and for the broader application of these techniques in other locations. By implementing intelligent design and eco-friendly materials in marine infrastructure, we can reduce the impact on local intertidal communities and indirectly reduce the spread of non-indigenous species.Item Neighbours at war : aggressive behaviour and spatial responsiveness in the anemone, Actinia tenebrosa : this thesis is completed in part of a Masters of Conservation Biology degree(Massey University, 2017) Balfour, GeorgiaHabitable space is precious and animals have developed a wide variety of mechanisms to acquire and defend favourable space. Aggression is considered any animal behaviour that involves actual or potential harm to another animal of either the same or different species. Agonistic behaviours must also be considered as it is any social behaviour related to fighting. Both aggressive and agnostic behaviours are observed in many animal species as resources including light exposure, nutrients and mates are often limited. Although agonistic behaviour varies among species, agonistic interactions can be partitioned into three specific types of behaviours: threat, aggression, and submission or avoidance. While any one of these behaviours can be observed in isolation, in an interaction between two animals, there is normally a sequence of behaviours which can culminate in combat. Anemones have unique adaptions such as clubs, fighting tentacles, bundles of stinging cells, sweeper tentacles and acrorhagi that allow them to defend themselves from competitors. Previous research also suggests that anemone populations are a collection of clusters of genetically similar which assemble via limited dispersal and locomotion. I chapter two I examined the effect of aggression on individuals at varying distances and predicted that those anemones that are initially located in closest proximity (<1 centimetre) in the field will be less aggressive towards each other than those anemones found further way from each other. Overall, my results suggest that Actinia tenebrosa have an obvious sequence of aggressive behaviours, and that indeed, aggressive behaviours were less common and less severe between nearest neighbours than among individuals sampled at greater distances. My results also show that aggressive behaviours are typically only expressed when individuals are within close proximity of each other <10cm. This behaviour is important to understand as it aids in fully understanding how aggressive behaviours determine dominance hierarchies and the spatial arrangement in A. tenebrosa. I chapter three, I investigated whether there was evidence for an ideal spatial arrangement of individuals in the field by testing whether individuals return to a similar spatial arrangement if randomised. The results from this chapter suggest that there is no single ideal spatial arrangement of individuals but rather individuals will find a spatial arrangement that is stable. I also observed that there appears to be an acclimation between individuals that resulted in a favourable position within the cluster. Lastly, I observed that instead of trying to return to a specific aggregation, individuals acclimate each other and move relative to those individuals surrounding them, much like stars in the sky. The results from this study would suggest that spatial structure of individuals in the field is dependent on intraspecies interactions and the recognition of individuals.Item The time course of corticosterone responses in kororā (little penguin, Eudyptula minor) : a thesis presented in partial fulfilment of the requirements of the degree of Master of Science in Zoology at Massey University(Massey University, 2017) Long, Kar HuiWhen birds and other vertebrates perceive a situation to be threatening the hypothalamopituitary-adrenal (HPA) axis is activated and glucocorticoid hormones are secreted from the adrenal gland. Activation of the HPA axis in response to a stimulus perceived to be threatening is called a stress response. The main glucocorticoid hormone in birds is corticosterone. Corticosterone responses of birds are typically measured by the collection of an initial blood sample when a bird is captured or picked up, then the collection of further blood samples until 30 to 60 minutes has elapsed, at which time the bird is released. Whilst this standard sampling protocol provides information on the size of the corticosterone response, it does not provide any indication of how long it takes for corticosterone concentrations to return to initial values. The main objective of this thesis was to characterise the total duration of the corticosterone response of free-living kororā (little penguins, Eudyptula minor). Little penguins at Oamaru were picked up from their nestboxes and initial blood samples collected. Birds were handled and then restrained by being placed in a box. Further blood samples were collected 15, 30 and 60 min after the birds were first picked up. Birds were then returned to their nest boxes and an additional blood sample collected 15, 30, 60, 120, 240, or 360 min later. Mean corticosterone concentrations declined to initial values two hours after birds were returned to nest boxes. The rates at which corticosterone concentrations increased when a stressor was present and then decreased when the stressor was no longer present were positively correlated. Seasonal changes in corticosterone responses in little penguins were also investigated in this study. Mean corticosterone responses were similar in winter and in the pre-laying period, whereas mean responses were lower in birds during early chick rearing. Corticosterone responses during the pre-laying period were greater in male than female little penguins. The current study is the first to document the complete corticosterone responses of free-living penguins and provides information about changes of corticosterone concentrations after a stressor is removed from the free-living individuals. It is also the first to reveal that free-living penguins with relatively high corticosterone responses to a stressor had relatively high rates of corticosterone decline.Item Metabarcoding of the rhizosphere microbiome of perennial ryegrass in response to Epichloë festucae var. lolii infection : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand(Massey University, 2017) Mahoney-Kurpe, SamEpichloë endophytes inhabit the intercellular spaces of cool-season pasture grasses, and can confer upon their hosts agriculturally desirable benefits such as heightened resistance to biotic and abiotic stresses. The mechanisms underlying many of these benefits are not well understood. Previously observed Epichloë-associated impacts towards the rhizosphere microbiome of their hosts could be a contributing factor, however the overall extent to which specific taxa in the rhizosphere microbiome of perennial ryegrass are affected by Epichloë festucae var. lolii infection remains to be elucidated. To assess this, two independent experiments were carried out in which clonal perennial ryegrass (NuiD) plants inoculated or uninoculated with E. festucae var. lolii (Lp19) originating from sterile tissue culture were grown in soil collected from a natural ryegrass pasture. After approximately two months of growth under controlled conditions in a growth cabinet, their prokaryotic and fungal rhizosphere microbiomes were compared using high-throughput metabarcoding. For prokaryotes, endophyte infection had no significant impact on species richness or evenness of the rhizosphere microbiome of their hosts in either experiment. A very minor but significant shift in overall community composition was shown in the first experiment but not the second. At the level of phyla, aside from a minor 1.1% increase in the relative abundances of Bacteroidetes in the rhizosphere of infected compared with uninfected plants in the first experiment but not the second, there were no other significantly differentially abundant prokaryotic phyla due to endophyte infection. At the genus level rhizospheres of infected and uninfected plants showed a high degree of similarity in both experiments, with little variability between replicates within treatments. At the level of operational taxonomic units (OTUs), in the first experiment there was only one significantly differentially abundant OTU in the rhizosphere depending on endophyte infection, and nine in the second. However, all of which had relatively low abundances (<0.3%), and none were consistently significantly differentially abundant in both experiments. For fungi, there were no significant impacts of endophyte infection on species richness or evenness of the rhizosphere in either experiment, nor were there any significant endophyte-associated shifts detected in overall rhizosphere community composition. Taxonomic analyses found that in both experiments endophyte infected plants had decreased abundances of a single abundant OTU compared with uninfected plants, which was found to be significant across both experiments (P= 0.026). The OTU sequence mapped with moderate (76-90%) homology to a number of reference sequences assigned as belonging to the class Sordariomycetes. Given previously observed endophyte-associated effects on arbuscular mycorrhizal (AM) fungi, reads assigned as belonging to AM were filtered and analysed separately. This showed that there were no significant effects of endophyte infection towards AM diversity nor overall community composition in both experiments, although there was an endophyte-associated increase in the abundance of the AM family Acaulosporaceae in the first experiment but not the second. Thus, aside from an endophyte-associated antagonism towards an abundant OTU in the rhizosphere likely of the class Sordariomycetes, E. festucae var. lolii had an otherwise minor impact on the prokaryotic and fungal rhizosphere microbiome of their perennial ryegrass hosts. The minor magnitude of endophyte-associated effects was further emphasized by analyses consistently showing that both prokaryotic and fungal rhizosphere community composition differed to a greater extent between plants of each experiment irrespective of endophyte infection than between plants of differing endophyte status within each experiment- at least in this cultivar-endophyte strain interaction under the conditions of this study.