A First Draft of the Core Fungal Microbiome of Schedonorus arundinaceus with and without Its Fungal Mutualist Epichloë coenophiala.

Tall fescue (Schedonorus arundinaceus) is a cool-season grass which is commonly infected with the fungal endophyte Epichloë coenophiala. Although the relationship between tall fescue and E. coenophiala is well-studied, less is known about its broader fungal communities. We used next-generation sequencing of the ITS2 region to describe the complete foliar fungal microbiomes in a set of field-grown tall fescue plants over two years, and whether these fungal communities were affected by the presence of Epichloë. We used the Georgia 5 cultivar of tall fescue, grown in the field for six years prior to sampling. Plants were either uninfected with E. coenophiala, or they were infected with one of two E. coenophiala strains: The common toxic strain or the AR542 strain (sold commerically as MaxQ). We observed 3487 amplicon sequence variants (ASVs) across all plants and identified 43 ASVs which may make up a potential core microbiome. Fungal communities did not differ strongly between Epichloë treatments, but did show a great deal of variation between the two years. Plant fitness also changed over time but was not influenced by E. coenophiala infection.

Climate sensitivity of the complex dynamics of the green spruce aphid-Spruce plantation interactions: Insight from a new mechanistic model.

Aphids can have a significant impact on the growth and commercial yield of spruce plantations. Here we develop a mechanistic deterministic mathematical model for the dynamics of the green spruce aphid (Elatobium abietum Walker) growing on Sitka spruce (Picea sitchensis (Bong.) Carr.). These grow in a northern British climate in managed plantations, with planting, thinning and a 60-year rotation. Aphid infestation rarely kills the tree but can reduce growth by up to 55%. We used the Edinburgh Forest Model (efm) to simulate spruce tree growth. The aphid sub-model is described in detail in an appendix. The only environmental variable which impacts immediately on aphid dynamics is air temperature which varies diurnally and seasonally. The efm variables that are directly significant for the aphid are leaf area and phloem nitrogen and carbon. Aphid population predictions include dying out, annual, biennual and other complex patterns, including chaos. Predicted impacts on plantation yield of managed forests can be large and variable, as has been observed; they are also much affected by temperature, CO2 concentration and other climate variables. However, in this system, increased CO2 concentration appears to ameliorate the severity of the effects of increasing temperatures coupled to worsening aphid infestations on plantation yield.

Effects of nutrient addition on endophyte-associated grass invasion in a long-term, old-field community experiment.

Strictly vertically transmitted (hereditary) Epichloë spp. fungal endophytes are symbionts with cool-season pooid host grasses. Such endophytes may increase host invasiveness in the non-native, introduced ranges. However, because costs and benefits for the host can vary with the growing conditions, the endophyte may become locally or temporally extinct when costs outweigh benefits. Our long-term field experiment involved the introduction of seven Schedonorus pratensis (meadow fescue) cultivars hosting Epichloë uncinata endophyte, which represent host-grass populations differing in genetic backgrounds and Epichloë infection frequencies, to an unmanaged old field. In the first 6 years, the host grasses persisted but did not become invasive in the plant community, regardless of their endophyte infection frequency. Subsequently, we hypothesized that increasing nutrient availability would decrease endophyte costs and thus increase the host's success and abundance. We fertilized half of the plots for four additional years and re-examined S. pratensis invasiveness. We predicted that increased nutrient availability would increase S. pratensis abundance and E. uncinata frequency and concentration, as well as decrease plant community diversity, relative to unfertilized plots. Fertilization increased endophyte concentrations in three low-endophyte host populations. However, E. uncinata did not enable S. pratensis populations to achieve high abundance or to reduce plant community diversity in the old field, with or without fertilization. Thus, nutrient availabililty and host invasiveness appear to be decoupled in this study system.

Comparison of Plant Metabolites in Root Exudates of Lolium perenne Infected with Different Strains of the Fungal Endophyte Epichloë festucae var. lolii.

Lolium perenne infected with the fungal endophyte Epichloë festucae var. lolii have specific, endophyte strain-dependent, chemical phenotypes in their above-ground tissues. Differences in these chemical phenotypes have been largely associated with classes of fungal-derived alkaloids which protect the plant against many insect pests. However, the use of new methodologies, such as various omic techniques, has demonstrated that many other chemical changes occur in both primary and secondary metabolites. Few studies have investigated changes in plant metabolites exiting the plant in the form of root exudates. As root exudates play an essential role in the acquisition of nutrients, microbial associations, and defense in the below-ground environment, it is of interest to understand how plant root exudate chemistry is influenced by the presence of strains of a fungal endophyte. In this study, we tested the influence of four strains of E. festucae var. lolii (E+ (also known as Lp19), AR1, AR37, NEA2), and uninfected controls (E-), on L. perenne growth and the composition of root exudate metabolites. Root exudates present in the hydroponic water were assessed by untargeted metabolomics using Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) liquid chromatography-mass spectrometry (LC-MS). The NEA2 endophyte strain resulted in the greatest plant biomass and the lowest endophyte concentration. We found 84 metabolites that were differentially expressed in at least one of the endophyte treatments compared to E- plants. Two compounds were strongly associated with one endophyte treatment, one in AR37 (m/z 135.0546 RT 1.17), and one in E+ (m/z 517.1987 RT 9.26). These results provide evidence for important changes in L. perenne physiology in the presence of different fungal endophyte strains. Further research should aim to connect changes in root exudate chemical composition with soil ecosystem processes.

Examining the Impacts of CO2 Concentration and Genetic Compatibility on Perennial Ryegrass-Epichloë festucae var lolii Interactions.

Perennial ryegrass (Lolium perenne) is the most cultivated cool-season grass worldwide with crucial roles in carbon fixation, turfgrass applications, and fodder for livestock. Lolium perenne forms a mutualism with the strictly vertically transmitted fungal endophyte, Epichloë festucae var lolii. The fungus produces alkaloids that protect the grass from herbivory, as well as conferring protection from drought and nutrient stress. The rising concentration of atmospheric CO2, a proximate cause of climatic change, is known to have many direct and indirect effects on plant growth. There is keen interest in how the nature of this plant-fungal interaction will change with climate change. Lolium perenne is an obligately outcrossing species, meaning that the genetic profile of the host is constantly being reshuffled. Meanwhile, the fungus is asexual implying both a relatively constant genetic profile and the potential for incompatible grass-fungus pairings. In this study, we used a single cultivar, "Alto", of L. perenne. Each plant was infected with one of four strains of the endophyte: AR1, AR37, NEA2, and Lp19 (the "common strain"). We outcrossed the Alto mothers with pollen from a number of individuals from different ryegrass cultivars to create more genetic diversity in the hosts. We collected seed such that we had replicate maternal half-sib families. Seed from each family was randomly allocated into the two levels of the CO2 treatment, 400 and 800 ppm. Elevated CO2 resulted in an c. 18% increase in plant biomass. AR37 produced higher fungal concentrations than other strains; NEA2 produced the lowest fungal concentrations. We did not find evidence of genetic incompatibility between the host plants and the fungal strains. We conducted untargeted metabolomics and quantitative proteomics to investigate the grass-fungus interactions between and within family and treatment groups. We identified a number of changes in both the proteome and metabalome. Taken together, our data set provides new understanding into the intricacy of the interaction between endophyte and host from multiple molecular levels and suggests opportunity to promote plant robustness and survivability in rising CO2 environmental conditions through application of bioprotective epichloid strains.

Effects of elevated CO2 on competition between native and invasive grasses.

Elevated atmospheric CO2 concentration increases the performance of invasive plants relative to natives when grown in monoculture, but it is unclear how that will affect the relative competitive abilities per se of invasive and native grasses grown together. We tested competitive outcomes for four native and four invasive perennial C3 and C4 grasses under ambient (390 ppm) and elevated (700 or 1000 ppm) CO2 concentrations in the greenhouse with non-limiting water and nutrients. We predicted that elevated CO2 would increase the competitive suppression of native grasses by invasive grasses. To test this, we determined the relative interaction intensity of biomass allocation for natives grown alone vs. those grown in native-invasive species pairs. We also measured photosynthetic traits that contribute to plant invasiveness and may be affected by elevated CO2 concentrations for species pairs in mixture to determine native-invasive relative performance. We found no effect of CO2 for the aboveground biomass and tiller production measures of interaction intensity or for relative performance for most of the measured photosynthetic traits. In competition, the invaders nearly always outperform natives in biomass and tiller production, regardless of CO2 level. The results suggest that increasing CO2 concentration alone has little effect on grass competitive outcomes under controlled conditions.

Sipha maydis sensitivity to defences of Lolium multiflorum and its endophytic fungus Epichloë occultans.

BACKGROUND: Plants possess a sophisticated immune system to defend from herbivores. These defence responses are regulated by plant hormones including salicylic acid (SA) and jasmonic acid (JA). Sometimes, plant defences can be complemented by the presence of symbiotic microorganisms. A remarkable example of this are grasses establishing symbiotic associations with Epichloë fungal endophytes. We studied the level of resistance provided by the grass' defence hormones, and that provided by Epichloë fungal endophytes, against an introduced herbivore aphid. These fungi protect their hosts against herbivores by producing bioactive alkaloids. We hypothesized that either the presence of fungal endophytes or the induction of the plant salicylic acid (SA) defence pathway would enhance the level of resistance of the grass to the aphid. METHODS: Lolium multiflorum plants, with and without the fungal endophyte Epichloë occultans, were subjected to an exogenous application of SA followed by a challenge with the aphid, Sipha maydis. RESULTS: Our results indicate that neither the presence of E. occultans nor the induction of the plant's SA pathway regulate S. maydis populations. However, endophyte-symbiotic plants may have been more tolerant to the aphid feeding because these plants produced more aboveground biomass. We suggest that this insect insensitivity could be explained by a combination between the ineffectiveness of the specific alkaloids produced by E. occultans in controlling S. maydis aphids and the capacity of this herbivore to deal with hormone-dependent defences of L. multiflorum.

Current and potential future distribution of the American dog tick (Dermacentor variabilis, Say) in North America.

The American dog tick (Dermacentor variabilis) is medically and economically important in North America. This species is found across central and eastern North America from the Gulf Coast of Mexico through southern Canada. In parts of this region, D. variabilis is a vector for pathogens that cause diseases in humans and animals. Our aim was to determine whether climate change would affect the distribution of the climatically suitable area for D. variabilis in North America, to aid monitoring for potential future spread of tick-borne pathogens. We developed a species distribution model for D. variabilis to project where climate will likely be suitable for the tick in North America using a maximum entropy method, occurrence records from museum and laboratory archives, and 10 environmental variables relevant to climate requirements for the tick. We used four emissions scenarios from the Intergovernmental Panel on Climate Change's Fifth Assessment Report and 10 climate models from the Coupled Model Intercomparison Project (phase 5) to estimate potential future climate suitability and determine how the tick's distribution could change. Our consensus model projected that the area of suitable climate in North America could increase from present by approximately 50% by 2070. In areas beyond the current northern limit of D. variabilis, climate could become more suitable for the tick than at present, possibly resulting in a northward expansion in Canada, but the potential suitability of the southern range of D. variabilis could decrease, depending on the region and climate model. Due to the ability of D. variabilis to harbor and transmit pathogens, a change in the distribution of this species could also affect the risk of human and animal diseases throughout North America, particularly in the northern range of the tick.

The plant hormone salicylic acid interacts with the mechanism of anti-herbivory conferred by fungal endophytes in grasses.

The plant hormone salicylic acid (SA) is recognized as an effective defence against biotrophic pathogens, but its role as regulator of beneficial plant symbionts has received little attention. We studied the relationship between the SA hormone and leaf fungal endophytes on herbivore defences in symbiotic grasses. We hypothesize that the SA exposure suppresses the endophyte reducing the fungal-produced alkaloids. Because of the role that alkaloids play in anti-herbivore defences, any reduction in their production should make host plants more susceptible to herbivores. Lolium multiflorum plants symbiotic and nonsymbiotic with the endophyte Epichloë occultans were exposed to SA followed by a challenge with the aphid Rhopalosiphum padi. We measured the level of plant resistance to aphids, and the defences conferred by endophytes and host plants. Symbiotic plants had lower concentrations of SA than did the nonsymbiotic counterparts. Consistent with our prediction, the hormonal treatment reduced the concentration of loline alkaloids (i.e., N-formyllolines and N-acetylnorlolines) and consequently decreased the endophyte-conferred resistance against aphids. Our study highlights the importance of the interaction between the plant immune system and endophytes for the stability of the defensive mutualism. Our results indicate that the SA plays a critical role in regulating the endophyte-conferred resistance against herbivores.

The impacts of climate change on the abundance and distribution of the Spotted Wing Drosophila (Drosophila suzukii) in the United States and Canada.

D. suzukii is a relatively recent and destructive pest species to the North American soft-skinned fruit industry. Understanding this species' potential to shift in abundance and range due to changing climate is an important part of an effective mitigation and management strategy. We parameterized a temperature-driven D. suzukii population dynamics model using temperature data derived from several Global Circulation Models (CMIP5) with a range of relative concentration pathway (RCP) predictions. Mean consensus between the models suggest that without adaptation to both higher prolonged temperatures and higher short-term temperature events D. suzukii population levels are likely to drop in currently higher-risk regions. The potential drop in population is evident both as time progresses and as the severity of the RCP scenario increases. Some regions, particularly in northern latitudes, may experience increased populations due to milder winter and more developmentally-ideal summer conditions, but many of these regions are not currently known for soft-skinned fruit production and so the effects of this population increase may not have a significant impact.

Spatial Competition: Roughening of an Experimental Interface.

Limited dispersal distance generates spatial aggregation. Intraspecific interactions are then concentrated within clusters, and between-species interactions occur near cluster boundaries. Spread of a locally dispersing invader can become motion of an interface between the invading and resident species, and spatial competition will produce variation in the extent of invasive advance along the interface. Kinetic roughening theory offers a framework for quantifying the development of these fluctuations, which may structure the interface as a self-affine fractal, and so induce a series of temporal and spatial scaling relationships. For most clonal plants, advance should become spatially correlated along the interface, and width of the interface (where invader and resident compete directly) should increase as a power function of time. Once roughening equilibrates, interface width and the relative location of the most advanced invader should each scale with interface length. We tested these predictions by letting white clover (Trifolium repens) invade ryegrass (Lolium perenne). The spatial correlation of clover growth developed as anticipated by kinetic roughening theory, and both interface width and the most advanced invader's lead scaled with front length. However, the scaling exponents differed from those predicted by recent simulation studies, likely due to clover's growth morphology.

Effects of elevated CO2 on photosynthetic traits of native and invasive C3 and C4 grasses.

BACKGROUND: Rising CO2 is expected to result in changes in plant traits that will increase plant productivity for some functional groups. Differential plant responses to elevated CO2 are likely to drive changes in competitive outcomes, with consequences for community structure and plant diversity. Many of the traits that are enhanced under elevated CO2 also confer competitive success to invasive species, and it is widely believed that invasive species will be more successful in high CO2. However, this is likely to depend on plant functional group, and evidence suggests that C3 plants tend to respond more strongly to CO2. RESULTS: We tested the hypothesis that invasive species would be more productive than noninvasive species under elevated CO2 and that stronger responses would be seen in C3 than C4 plants. We examined responses of 15 grass species (eight C3, seven C4), classified as noninvasive or invasive, to three levels of CO2 (390, 700 and 1000 ppm) in a closed chamber experiment. Elevated CO2 decreased conductance and %N and increased shoot biomass and C/N ratio across all species. Differences between invasive and noninvasive species depended on photosynthetic mechanism, with more differences for traits of C3 than C4 plants. Differences in trait means between invasive and noninvasive species tended to be similar across CO2 levels for many of the measured responses. However, noninvasive C3 grasses were more responsive than invasive C3 grasses in increasing tiller number and root biomass with elevated CO2, whereas noninvasive C4 grasses were more responsive than invasive C4 grasses in increasing shoot and root biomass with elevated CO2. For C3 grasses, these differences could be disadvantageous for noninvasive species under light competition, whereas for C4 grasses, noninvasive species may become better competitors with invasive species under increasing CO2. CONCLUSIONS: The ecophysiological mechanisms underlying invasion success of C3 and C4 grasses may differ. However, given that the direction of trait differences between invasive and noninvasive grasses remained consistent under ambient and elevated CO2, our results provide evidence that increases in CO2 are unlikely to change dramatically the competitive hierarchy of grasses in these functional groups.

Thermal Tolerances of the Spotted-Wing Drosophila Drosophila suzukii (Diptera: Drosophilidae).

The spotted-wing drosophila (Drosophila suzukii Matsumura) is an invasive species of Asian origin that is now widely distributed in North America and Europe. Because of the female's serrated ovipositor, eggs are laid in preharvest fruit, causing large economic losses in cultivated berries and stone fruit. Modeling D. suzukii population dynamics and potential distribution will require information on its thermal tolerance. Large summer populations have been found in regions with severe winter conditions, though little is known about responses to prolonged low-temperature exposure. We used controlled chambers to examine D. suzukii fecundity, development rate, and mortality across a range of temperatures encompassing the upper and lower thresholds (5­35°C). Optimal temperatures (Topt) were found to be 28.2°C for the development of the egg-to-adult stage, and 22.9°C for reproductive output. No adult eclosion occurred below 8.1°C (Tlower) or above 30.9°C (Tupper). We also investigated survival outcomes following prolonged (42-d) low-temperature exposure to a simulated cold winter (−5, −3, −1, 1, 3, and 5°C). Adult survival was dependent on temperature, with a mean LT50 of 4.9°C. There were no effects of sex, mating status, geographic strain, and photoperiod preexposure on overwintering survival. Thirty-eight percent of females that were mated prior, but not after, prolonged low-temperature exposure produced viable offspring, suggesting that this species may undergo sperm storage. This study provides data on the thermal tolerances of D. suzukii, which can be used for models of D. suzukii population dynamics, degree-day, and distribution models.

Mixed-strain housing for female C57BL/6, DBA/2, and BALB/c mice: validating a split-plot design that promotes refinement and reduction.

BACKGROUND: Inefficient experimental designs are common in animal-based biomedical research, wasting resources and potentially leading to unreplicable results. Here we illustrate the intrinsic statistical power of split-plot designs, wherein three or more sub-units (e.g. individual subjects) differing in a variable of interest (e.g. genotype) share an experimental unit (e.g. a cage or litter) to which a treatment is applied (e.g. a drug, diet, or cage manipulation). We also empirically validate one example of such a design, mixing different mouse strains -- C57BL/6, DBA/2, and BALB/c -- within cages varying in degree of enrichment. As well as boosting statistical power, no other manipulations are needed for individual identification if co-housed strains are differentially pigmented, so also sparing mice from stressful marking procedures. METHODS: The validation involved housing 240 females from weaning to 5 months of age in single- or mixed- strain trios, in cages allocated to enriched or standard treatments. Mice were screened for a range of 26 commonly-measured behavioural, physiological and haematological variables. RESULTS: Living in mixed-strain trios did not compromise mouse welfare (assessed via corticosterone metabolite output, stereotypic behaviour, signs of aggression, and other variables). It also did not alter the direction or magnitude of any strain- or enrichment-typical difference across the 26 measured variables, or increase variance in the data: indeed variance was significantly decreased by mixed- strain housing. Furthermore, using Monte Carlo simulations to quantify the statistical power benefits of this approach over a conventional design demonstrated that for our effect sizes, the split- plot design would require significantly fewer mice (under half in most cases) to achieve a power of 80%. CONCLUSIONS: Mixed-strain housing allows several strains to be tested at once, and potentially refines traditional marking practices for research mice. Furthermore, it dramatically illustrates the enhanced statistical power of split-plot designs, allowing many fewer animals to be used. More powerful designs can also increase the chances of replicable findings, and increase the ability of small-scale studies to yield significant results. Using mixed-strain housing for female C57BL/6, DBA/2 and BALB/c mice is therefore an effective, efficient way to promote both refinement and the reduction of animal-use in research.

Effects of the Epichloë fungal endophyte symbiosis with Schedonorus pratensis on host grass invasiveness.

Initial studies of grass-endophyte mutualisms using Schedonorus arundinaceus cultivar Kentucky-31 infected with the vertically transmitted endophyte Epichloë coenophiala found strong, positive endophyte effects on host-grass invasion success. However, more recent work using different cultivars of S. arundinaceus has cast doubt on the ubiquity of this effect, at least as it pertains to S. arundinaceus-E. coenophiala. We investigated the generality of previous work on vertically transmitted Epichloë-associated grass invasiveness by studying a pair of very closely related species: S. pratensis and E. uncinata. Seven cultivars of S. pratensis and two cultivars of S. arundinaceus that were developed with high- or low-endophyte infection rate were broadcast seeded into 2 × 2-m plots in a tilled, old-field grassland community in a completely randomized block design. Schedonorus abundance, endophyte infection rate, and co-occurring vegetation were sampled 3, 4, 5, and 6 years after establishment, and the aboveground invertebrate community was sampled in S. pratensis plots 3 and 4 years after establishment. Endophyte infection did not enable the host grass to achieve high abundance in the plant community. Contrary to expectations, high-endophyte S. pratensis increased plant richness relative to low-endophyte cultivars. However, as expected, high-endophyte S. pratensis marginally decreased invertebrate taxon richness. Endophyte effects on vegetation and invertebrate community composition were inconsistent among cultivars and were weaker than temporal effects. The effect of the grass-Epichloë symbiosis on diversity is not generalizable, but rather specific to species, cultivar, infection, and potentially site. Examining grass-endophyte systems using multiple cultivars and species replicated among sites will be important to determine the range of conditions in which endophyte associations benefit host grass performance and have subsequent effects on co-occurring biotic communities.

Predicting potential global distributions of two Miscanthus grasses: implications for horticulture, biofuel production, and biological invasions.

In many regions, large proportions of the naturalized and invasive non-native floras were originally introduced deliberately by humans. Pest risk assessments are now used in many jurisdictions to regulate the importation of species and usually include an estimation of the potential distribution in the import area. Two species of Asian grass (Miscanthus sacchariflorus and M. sinensis) that were originally introduced to North America as ornamental plants have since escaped cultivation. These species and their hybrid offspring are now receiving attention for large-scale production as biofuel crops in North America and elsewhere. We evaluated their potential global climate suitability for cultivation and potential invasion using the niche model CLIMEX and evaluated the models' sensitivity to the parameter values. We then compared the sensitivity of projections of future climatically suitable area under two climate models and two emissions scenarios. The models indicate that the species have been introduced to most of the potential global climatically suitable areas in the northern but not the southern hemisphere. The more narrowly distributed species (M. sacchariflorus) is more sensitive to changes in model parameters, which could have implications for modelling species of conservation concern. Climate projections indicate likely contractions in potential range in the south, but expansions in the north, particularly in introduced areas where biomass production trials are under way. Climate sensitivity analysis shows that projections differ more between the selected climate change models than between the selected emissions scenarios. Local-scale assessments are required to overlay suitable habitat with climate projections to estimate areas of cultivation potential and invasion risk.

Climate and host plant availability impact the future distribution of the bean leaf beetle (Cerotoma trifurcata).

The bean leaf beetle, Cerotoma trifurcata, has become a major pest of soybean throughout its North American range. With a changing climate, there is the potential for this pest to further expand its distribution and become an increasingly severe pest in certain regions. To examine this possibility, we developed bioclimatic envelope models for both the bean leaf beetle, and its most important agronomic host plant, soybean (Glycine max). These two models were combined to examine the potential future pest status of the beetle using climate change projections from multiple general circulation models (GCMs) and climate change scenarios. Despite the broad tolerances of soybean, incorporation of host plant availability substantially decreased the suitable and favourable areas for the bean leaf beetle as compared to an evaluation based solely on the climate envelope of the beetle, demonstrating the importance of incorporating biotic interactions in these predictions. The use of multiple GCM-scenario combinations also revealed differences in predictions depending on the choice of GCM, with scenario choice having less of an impact. While the Norwegian model predicted little northward expansion of the beetle from its current northern range limit of southern Ontario and overall decreases in suitable and favourable areas over time, the Canadian and Russian models predict that much of Ontario and Quebec will become suitable for the beetle in the future, as well as Manitoba under the Russian model. The Russian model also predicts expansion of the suitable and favourable areas for the beetle over time. Two predictions that do not depend on our choice of GCM include a decrease in suitability of the Mississippi Delta region and continued favourability of the southeastern United States.

Transcript profiling of fructan biosynthetic pathway genes reveals association of a specific fructosyltransferase isoform with the high sugar trait in Lolium perenne.

Lolium perenne cultivars with elevated levels of fructans in leaf blades (high sugar-content grasses) have been developed to improve animal nutrition and reduce adverse environmental impacts of pastoral agricultural systems. Expression of the high sugar trait can vary substantially depending on genotype×environment (G×E) interactions. We grew three potential high sugar-content and a control cultivar in three temperature regimes and quantified water soluble carbohydrates (WSCs) and the expression of all functionally characterised L. perenne fructan pathway genes in leaf tissues. We also analysed the distribution, expression and sequence variation of two specific isoforms of Lp6G-FFT (fructan: fructan 6G-fructosyltransferase). Our study confirmed a significant G×E interaction affecting the accumulation of fructans in the high sugar-content cultivar AberDart, which accumulated higher levels of high DP (degree of polymerisation) fructans in blades compared to the control cultivar only when grown at 20°C (day)/10°C (night) temperatures. The cultivar Expo on the other hand accumulated significantly higher levels of high DP fructans in blades independent of temperature. Fructan levels in pseudostems were higher than in blades, and they increased markedly with decreasing temperature, but there was no consistent effect of cultivar in this tissue. The expression of the high sugar trait was generally positively correlated with transcript levels of fructosyltransferases. Presence and expression of only one of the two known 6G-FFT isoforms was positively correlated with high fructan biosynthesis, while the second isoform was associated with low fructan concentrations and positively correlated with fructan exohydrolase gene expression. The presence of distinct 6G-FFT sequence variants appears to be associated with the capacity of high sugar-content grasses to accumulate higher fructan levels particularly at warmer temperatures. These findings might be exploited for the selection and breeding of 'warm-effective' high sugar-content grasses to overcome some of the limitations of current high sugar-content ryegrass cultivars.

The effect of endophyte presence on Schedonorus arundinaceus (tall fescue) establishment varies with grassland community structure.

The endophyte Neotyphodium coenophialum in Schedonorus arundinaceus (tall fescue) may alter host interactions with specific resident species or shift the host's niche. These effects can be quantified by assessing tall fescue responses to, and effects on, the variation among resident species (selection) and resident species interactions (complementarity). To determine how N. coenophialum affects tall fescue, grassland microcosms containing 16 transplants of two, four, or eight resident species were seeded with endophyte-infected (E+) or endophyte-free (E-) Kentucky-31 (KY-31) tall fescue. All resident species were also grown in monoculture. Aboveground biomass was harvested 9 weeks after tall fescue was added (18 weeks' total growth). At harvest, more E+ than E- individuals were present and they represented a larger portion of the aboveground biomass across richness treatments, despite similar germination in concurrent trials. Tall fescue individuals were larger in microcosms dominated by more productive resident species (greater selection). In contrast, fewer E-, but not E+, individuals were present in microcosms with more facilitative interactions among the resident species (greater complementarity). E- and E+ tall fescue also affected resident species differently. High-richness E+ microcosms were more diverse and less dominated by productive species (lower selection) than E- microcosms. Thus, E+ KY-31 may more readily establish in, and affect, species-rich, functionally diverse communities as a result of niche shifts during establishment and negative effects on specific resident species. Although results need to be further tested under field conditions, it appears that endophyte presence may only facilitate KY-31 invasion into a limited suite of community types.

Metabolite analysis of the effects of elevated CO2 and nitrogen fertilization on the association between tall fescue (Schedonorus arundinaceus) and its fungal symbiont Neotyphodium coenophialum.

Atmospheric CO2 is expected to increase to between 550 ppm and 1000 ppm in the next century. CO2-induced changes in plant physiology can have ecosystem-wide implications and may alter plant-plant, plant-herbivore and plant-symbiont interactions. We examined the effects of three concentrations of CO2 (390, 800 and 1000 ppm) and two concentrations of nitrogen fertilizer (0.004 g N/week versus 0.2 g N/week) on the physiological response of Neotyphodium fungal endophyte-infected and uninfected tall fescue plants. We used quantitative PCR to estimate the concentration of endophyte under altered CO2 and N conditions. We found that elevated CO2 increased the concentration of water-soluble carbohydrates and decreased the concentration of plant total amino acids in plants. Fungal-derived alkaloids decreased in response to elevated CO2 and increased in response to nitrogen fertilization. Endophyte concentration (expressed as the number of copies of an endophyte-specific gene per total genomic DNA) increased under elevated CO2 and nitrogen fertilization. The correlation between endophyte concentration and alkaloid production observed at ambient conditions was not observed under elevated CO2. These results suggest that nutrient exchange dynamics important for maintaining the symbiotic relationship between fungal endophytes and their grass hosts may be altered by changes in environmental variables such as CO2 and nitrogen fertilization.