Distribution of the fungal endophyte Neotyphodium lolii is not a major determinant of the distribution of fungal alkaloids in Lolium perenne plants

The relationships of the distributions of the insect and mammalian mycotoxins, lolitrem B and ergovaline, and the insect-feeding deterrent, peramine, with the distribution of fungal mycelium were investigated in three genotypes of perennial ryegrass (Lolium perenne L.) infected with the endophyte Neotyphodium lolii. In planta levels and distribution of the endophyte and of the three alkaloids were assessed in parallel, and different spatial or temporal concentration gradients were observed for each. Variation in the tissue distribution of the endophyte accounted only for 20%, 6%, and 31% of the variation in ergovaline, lolitrem B, and peramine, respectively. Alkaloid-endophyte ratios, determined in individual grass tissues, showed distinct in planta distribution patterns for each alkaloid and differed in magnitude among genotypes. The ergovaline-endophyte ratio was higher in the very basal plant tissues than in the apical tissues, while the lolitrem B and peramine ratios tended to be higher in apical tissues. The lolitrem B-endophyte ratio increased with leaf age, while no consistent temporal trends were detected for the other alkaloids. The results indicate that endophyte colonisation is a minor determinant of alkaloid levels, and that accumulation of the alkaloids relative to the endophyte mycelium is affected by plant genotype and tissue in a manner specific to each alkaloid. Possible factors in the regulation of alkaloid levels in the grass plant are discussed.     

Relationships between the genetic distance of Epichloë festucae isolates and the ergovaline and peramine contents of their Festuca rubra hosts

In semiarid grasslands of western Spain, plants of Festuca rubra are frequently infected by Epichloë festucae , an endophyte capable of producing the alkaloids ergovaline, toxic to mammals, and peramine, toxic to insects. The objectives of this paper were to estimate the variability in the content of ergovaline and peramine in plants of Festuca rubra from natural populations and to determine the relationship between the genetic distance among Epichloë strains and the alkaloid contents of plants infected by them. The results showed that a significant variation exits in the ergovaline and peramine contents of infected F. rubra plants, and that variation patterns are different for each alkaloid. Ergovaline content showed a significant variation between years. In contrast, most variation observed in the concentration of peramine occurred between populations. Mantel tests, constrained correspondence analysis (CCA) and generalised analysis of molecular variance (GAMOVA) all revealed a significant relationship between the genetic distances among 35 Epichloë isolates, and the differences in peramine content of their host plants. The relationship observed for ergovaline was more ambiguous. This indicates that plants infected by genetically close isolates of Epichloë festucae tend to be similar in terms of peramine content, while the ergovaline content of infected plants seems to be more dependent on external factors. The results of this study suggest that the selection of Epichloë festucae endophytes based on the alkaloid content of their host plants can be a successful strategy to select good or bad producers of peramine, but may not be a reliable method to detect fungal strains associated with particular ergovaline production profiles.     

Evaluation of endophyte toxin production and its interaction with transgenic perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) with altered expression of fructosyltransferases

Alkaloid concentration of perennial ryegrass herbage is affected by endophyte strain and host plant genotype. However, previous studies suggest that associations between host and endophyte also depends on environmental conditions, especially those affecting nutrient reserves and that water-soluble carbohydrate (WSC) concentration of perennial ryegrass plants may influence grass-endophyte associations. In this study a single transgenic event, with altered expression of fructosyltransferase genes to produce high WSC and biomass, has been crossed into a range of cultivar backgrounds with varying Epichloë endophyte strains. The effect of the association between the transgenic trait and alkaloid production was assessed and compared with transgene free control populations. In the vast-majority of comparisons there was no significant difference between alkaloid concentrations of transgenic and non-transgenic plants within the same cultivar and endophyte backgrounds. There was no significant difference between GOI+?(gene of interest positive) and GOI? (gene of interest negative) populations in Janthritrem response. Peramine concentration was not different between GOI+?and GOI? for 10 of the 12 endophytes-cultivar combinations. Cultivar Trojan infected with NEA6 and Alto with SE (standard endophyte) exhibited higher peramine and lolitrem B (only for Alto SE) concentration, in the control GOI? compared with GOI+. Similarly, cultivar Trojan infected with NEA6 and Alto with NEA3 presented higher ergovaline concentration in GOI?. Differences in alkaloid concentration may be attributable to an indirect effect in the modulation of fungal biomass. These results conclude that the presence of this transgenic insertion, does not alter the risk (toxicity) of the endophyte–grass associations. Endophyte–host interactions are complex and further research into associations with high WSC plant should be performed in a case by case basis.     

Endophyte isolate and host grass effects on Chaetocnema pulicaria (Coleoptera: Chrysomelidae) feeding

Endophytic fungi belonging to the genus Neotyphodium, confer resistance to infected host grasses against insect pests. The effect of host species, and endophtye species and strain, on feeding and survival of the corn flea beetle, Chaetocnema pulicaria Melsheimer (Coleoptera: Chrysomelidae) was investigated. The grass-endophyte associations included natural and artificially derived associations producing varying arrays of common endophyte-related alkaloids or alkaloid groups, peramine, lolitrem B, ergovaline, and the lolines. Preference and nonpreference tests showed that C. pulicaria feeding and survival were reduced by infection of tall fescue with the wild-type strain of N. coenophialum, the likely mechanism being antixenosis rather than antibiosis. In the preference tests, endophyte and host species effects were observed. Of the 10 different Neotyphodium strains tested in artificially derived tall fescue associations, eight strongly deterred feeding by C. pulicaria, whereas the remaining two strains had little or no effect on feeding. Infection of tall fescue with another fungal symbiont, p-endophyte, had no effect. Perennial ryegrass, Lolium perenne L., infected with six strains of endophyte, was moderately resistant to C. pulicaria compared with endophyte-free grass, but four additional strains were relatively inactive. Six Neotyphodium-meadow fescue, Festuca pratensis Huds., associations, including the wild-type N. uncinatum-meadow fescue combination, were resistant, whereas three associations were not effective. Loline alkaloids seemed to play a role in antixenosis to C. pulicaria. Effects not attributable to the lolines or any other of the alkaloids examined also were observed. This phenomenon also has been reported in tests with other insects, and indicates the presence of additional insect-active factors.     

Transformation of the ryegrass endophyte Neotyphodium lolii can alter its in planta mycelial morphology

The fungus Neotyphodium lolii grows in the intercellular spaces of perennial ryegrass as a mutualistic endosymbiont. One of the benefits it conveys to the plant is the production of alkaloids toxic to herbivores. We wanted to determine in planta expression patterns of the N. lolii 3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA reductase) gene, believed to be involved in the synthesis of two of these alkaloid toxins, lolitrem B and ergovaline. We transformed the N. lolii strain Lp19 with plasmids, in which DNA fragments upstream of the open reading frame of the N. lolii HMG CoA reductase gene controlled expression of the GUS (gusA; Escherichia coli β-glucuronidase) reporter gene. In exponentially growing cultures, the GUS gene was not expressed if the length of upstream sequence was less than 400 bp, and >1100 bp were required for maximum expression. When reintroduced into ryegrass plants, transformants often showed highly increased hyphal branching compared to the wild-type parent strain, although in culture their growth kinetics and morphology were indistinguishable from that of the wild-type. Deterioration of hyphae and the hypha–plant interface occurred and in one transformant reduced tillering (formation of new plants, referred to in agronomy as tillers) and death of infected plants. We found no evidence that these abnormalities were caused by interference of the construct with the function of the native gene, as judged by analysis of the site of integration of the promoter-GUS cassette, expression of the native gene and lolitrem B and ergovaline levels in infected plants. However, there was some correlation between GUS expression and the degree of hyphal branching, suggesting that high levels of β-glucuronidase may disturb the symbiotic interaction. Levels of another alkaloid, peramine, were also not significantly affected by transformation. In previous studies increased in planta branching of the endophyte has been shown to be associated with a severe reduction of alkaloid production. Our results show that a plant–endophyte association in which increased branching occurs is still able to produce alkaloids.     

Epichlo? festucae and related mutualistic symbionts of grasses.

Epichlo? and Neotyphodium species (Ascomycota) are mutualistic symbionts (endophytes) of temperate grasses, to which they impart numerous and profound fitness benefits. Epichlo? festucae, a common symbiont of Festuca, Lolium,and Koeleria spp., is a model for endophyte research that is amenable to Mendelian and molecular genetic analysis. Characteristics of E. festucae include: (i) production of the anti-insect alkaloids peramine and lolines, (ii) production of the anti-vertebrate alkaloids lolitrem B and ergovaline, (iii) efficient vertical transmission via host seeds, (iv) a mildly pathogenic state associated with the E. festucae sexual cycle, and (v) a clear role in enhancing survival of host plants. Genetic analysis of alkaloid production has recently begun. Also, physiological and ultrastructural studies suggest that signals communicated between E. festucae and host plants ensure an exquisitely balanced interaction to the mutual benefit of both partners. Several mutualistic Neotyphodium species are hybrids between E. festucae and other endophyte species.     

Contribution of ergot alkaloids to suppression of a grass-feeding caterpillar assessed with gene knockout endophytes in perennial ryegrass

Neotyphodium and Epichloë species (Ascomycota: Clavicipitaceae) are fungal symbionts (endophytes) of grasses. Many of these endophytes produce alkaloids that enhance their hosts’ resistance to insects or are toxic to grazing mammals. The goals of eliminating from forage grasses factors such as ergot alkaloids that are responsible for livestock disorders, while retaining pasture sustainability, and of developing resistant turf grasses, require better understanding of how particular alkaloids affect insect herbivores. We used perennial ryegrass Lolium perenne L. (Poaceae) symbiotic with Neotyphodium lolii × Epichloë typhina isolate Lp1 (a natural interspecific hybrid), as well as with genetically modified strains of Lp1 with altered ergot alkaloid profiles, to test effects of ergot alkaloids on feeding, growth, and survival of the black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), a generalist grass‐feeding caterpillar. Neonates or late instars were provided clippings from glasshouse‐grown plants in choice and rearing trials. Wild‐type endophytic grass showed strong antixenosis and antibiosis, especially to neonates. Plant‐endophyte symbiota from which complex ergot alkaloids (ergovaline and lysergic acid amides such as ergine) or all ergot alkaloids were eliminated by endophyte gene knockout retained significant resistance against neonates. However, this activity was reduced compared to that of wild‐type Lp1, providing the first direct genetic evidence that ergot alkaloids contribute to insect resistance of endophytic grasses. Similarity of larval response to the two mutants suggested that ergovaline and/or ergine account for the somewhat greater potency of wild‐type Lp1 compared to the knockouts, whereas simpler ergot alkaloids contribute little to that added resistance. All of the endophyte strains also produced peramine, which was probably their primary resistance component. This study suggests that ergot alkaloids can be eliminated from an endophyte of perennial ryegrass while retaining significant insect resistance.     

Peramine and other fungal alkaloids are exuded in the guttation fluid of endophyte-infected grasses

Many grasses live in association with asymptomatic fungi (Neotyphodium spp. endophytes), which grow in the intercellular spaces of the grass. These endophytes produce a range of alkaloids that protect the grass against grazing by mammals and insects. One of these alkaloids is an unusual pyrrolopyrazine, peramine. Peramine appears to be continuously produced by the endophyte, but does not progressively accumulate. No mechanism for the removal of peramine by its further metabolism or any other process has been reported. Our aim was to detect peramine or peramine metabolites in plant fluids to determine if peramine is mobilized, metabolized or excreted by the plant. We also wanted to determine if other fungal metabolites are mobilized by the plant, as has been proposed for the loline alkaloids.We developed a highly sensitive method for the analysis of peramine, using a linear ion trap mass spectrometer. We studied the fragmentation pathway of peramine using ESI MSⁿ and ESI FTICRMS. Based on these results we developed a single reaction monitoring method using the fragmentation of the guanidinium moiety. Cut leaf fluid and guttation fluid of different grass endophyte associations (Lolium perenne with Neotyphodium lolii, Festuca arundinacea with Neotyphodium coenophialum, and Elymus sp. with Epichloë sp.) were analysed. Peramine was detected in the cut leaf fluid of all grass-endophyte associations, but not in the guttation fluid of all associations. In some associations we also detected lolines and ergot peptide alkaloids. This is the first report showing the mobilization of fungal alkaloids into plant fluids by the host plant in grass-endophyte associations.     

The determinant step in ergot alkaloid biosynthesis by an endophyte of perennial ryegrass

Many cool-season grasses harbor fungal endophytes in the genus Neotyphodium, which enhance host fitness, but some also produce metabolites--such as ergovaline--believed to cause livestock toxicoses. In Claviceps species the first step in ergot alkaloid biosynthesis is thought to be dimethylallyltryptophan (DMAT) synthase, encoded by dmaW, previously cloned from Claviceps fusiformis. Here we report the cloning and characterization of dmaW from Neotyphodium sp. isolate Lp1, an endophyte of perennial ryegrass (Lolium perenne). The gene was then disrupted, and the mutant failed to produce any detectable ergovaline or simpler ergot and clavine alkaloids. The disruption was complemented with the C. fusiformis gene, which restored ergovaline production. Thus, the biosynthetic role of DMAT synthase was confirmed, and a mutant was generated for future studies of the ecological and agricultural importance of ergot alkaloids in endophytes of grasses.     

Near-term impacts of elevated CO2, nitrogen and fungal endophyte-infection on Lolium perenne L. growth, chemical composition and alkaloid production

Carbon dioxide has been rapidly accumulating in the atmosphere and is expected to continue to do so. This accumulation is presumed to have important direct effects on plant growth. The interacting affects of a small increase in CO₂ concentration (466 p.p.m., approximately 30% increase from current ambient conditions), nitrogen fertilization and fungal endophyte (Neotyphodium lolii) infection on the growth and chemical composition of perennial ryegrass (Lolium perenne) were investigated. It was found that dry mass production was approximately 50% greater under elevated CO₂ than under ambient CO₂, but only in conditions of high soil N. High molecular weight carbohydrates and total carbohydrates (LMW + HMW CHO) depended on an interaction between CO₂ and endophyte infection. Infected plants contained significantly more carbohydrate than endophyte-free plants, and the difference was greatest in ambient CO₂ conditions. Protein concentrations were also influenced by the interaction between CO₂ and endophyte-infection. Endophyte-free plants had 40% lower concentrations of soluble protein under elevated CO₂ than under ambient CO₂, but this CO₂ effect on soluble protein was largely absent in endophyte-infected plants. CO₂, endophyte-infection and nitrogen interacted to influence the total chlorophyll concentration of the grass such that chlorophyll concentration was always lower in elevated CO₂ but this decline was much greater in endophyte-free plants, particularly in conditions of high soil N. In the endophyte-infected plants, the concentrations of the pyrrolopyrazine alkaloid peramine depended on the interaction between CO₂ and N fertilization such that peramine concentrations declined with increasing N at ambient CO₂ but remained roughly constant across N levels at elevated CO₂. A similar pattern was seen for the ergot alkaloid ergovaline. The biochemical responses of perennial ryegrass to elevated CO₂ are clearly modified by the presence of endophytic fungi.     

Metabolic profiles of Lolium perenne are differentially affected by nitrogen supply, carbohydrate content, and fungal endophyte infection

Lolium perenne cultivars differing in their capacity to accumulate water soluble carbohydrates (WSCs) were infected with three strains of fungal Neotyphodium lolii endophytes or left uninfected. The endophyte strains differed in their alkaloid profiles. Plants were grown at two different levels of nitrogen (N) supply in a controlled environment. Metabolic profiles of blades were analyzed using a variety of analytical methods. A total of 66 response variables were subjected to a principle components analysis and factor rotation. The first three rotated factors (46% of the total variance) were subsequently analyzed by analysis of variance. At high N supply nitrogenous compounds, organic acids and lipids were increased; WSCs, chlorogenic acid (CGA), and fibers were decreased. The high-sugar cultivar 'AberDove' had reduced levels of nitrate, most minor amino acids, sulfur, and fibers compared to the control cultivar 'Fennema', whereas WSCs, CGA, and methionine were increased. In plants infected with endophytes, nitrate, several amino acids, and, magnesium were decreased; WSCs, lipids, some organic acids, and CGA were increased. Regrowth of blades was stimulated at high N, and there was a significant endophyte x cultivar interaction on regrowth. Mannitol, a fungal specific sugar alcohol, was significantly correlated with fungal biomass. Our findings suggest that effects of endophytes on metabolic profiles of L. perenne can be considerable, depending on host plant characteristics and nutrient supply, and we propose that a shift in carbon/N ratios and in secondary metabolite production as seen in our study is likely to have impacts on herbivore responses.     

High nitrogen supply and carbohydrate content reduce fungal endophyte and alkaloid concentration in Lolium perenne

The relationship between cool-season grasses and fungal endophytes is widely regarded as mutualistic, but there is growing uncertainty about whether changes in resource supply and environment benefit both organisms to a similar extent. Here, we infected two perennial ryegrass (Lolium perenne) cultivars (AberDove, Fennema) that differ in carbohydrate content with three strains of Neotyphodium lolii (AR1, AR37, common strain) that differ intrinsically in alkaloid profile. We grew endophyte-free and infected plants under high and low nitrogen (N) supply and used quantitative PCR (qPCR) to estimate endophyte concentrations in harvested leaf tissues. Endophyte concentration was reduced by 40% under high N supply, and by 50% in the higher sugar cultivar. These two effects were additive (together resulting in 75% reduction). Alkaloid production was also reduced under both increased N supply and high sugar cultivar, and for three of the four alkaloids quantified, concentrations were linearly related to endophyte concentration. The results stress the need for wider quantification of fungal endophytes in the grassland-foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes or increasing N inputs affect the role of endophytes in grassland ecosystems.     

Isolate-dependent impacts of fungal endophytes in a multitrophic interaction

Neotyphodium lolii , an endophytic fungus of perennial ryegrass, deters Argentine stem weevil, Listronotus bonariensis , an important insect pest of pastures in New Zealand. Deterrence is apparently due to several alkaloids the fungus produces. We asked if the fungus also affects the third trophic level. Specifically, we tested if several different isolates of the fungus altered the growth and survival of the parasitoid, Microctonus hyperodae . Adult weevils were collected from paddocks near Lincoln, New Zealand and maintained in an environmental chamber. Weevils were assorted into treatment groups and fed perennial ryegrass (cv. Nui) lacking endophyte infection (nil) or containing one of the following endophyte strains: ARW, AR1, AR6, AR37. All endophyte strains differed from one another with respect to the profile of alkaloids they produce. Following two weeks of acclimation, weevils were placed with M. hyperodae to allow for parasitization. Weevils were then placed into Petri dishes (10 per dish) and fed grass clippings from the appropriate treatment group. We collected weevils as they died and dissected them to assess the development of parasitoids. Emergence of prepupae from hosts and survival to adulthood were also recorded. Fungal isolates did not differ in their influence on weevil feeding or survival. In contrast, the effect of the endophyte on the parasitoid varied among isolates of the fungus. Isolates ARW and AR6 reduced parasitoid adult emergence relative to nil endophyte. In contrast, AR37 had no negative effect on survival of the parasitoid. Furthermore, an index of developmental rate showed that parasitoids developed more slowly when reared from hosts fed grass containing any of the strains of N. lolii , except AR37, compared to endophyte-free grass. Negative effects of the endophyte on parasitoid survival were associated with the presence of ergovaline while effects on parasitoid development rate were associated with the presence of any alkaloid.     

A complex ergovaline gene cluster in epichloe endophytes of grasses

Clavicipitaceous fungal endophytes of the genera Epichlo? and Neotyphodium form symbioses with grasses of the subfamily Pooideae, in which they can synthesize an array of bioprotective alkaloids. Some strains produce the ergopeptine alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyte-infected grasses. Cloning and analysis of a nonribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii revealed a putative gene cluster for ergovaline biosynthesis containing a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Despite conservation of gene sequence, gene order is substantially different between the N. lolii, C. purpurea, and A. fumigatus ergot alkaloid gene clusters. Southern analysis indicated that the N. lolii cluster was linked with previously identified ergovaline biosynthetic genes dmaW and lpsA. The ergovaline genes are closely associated with transposon relics, including retrotransposons and autonomous and nonautonomous DNA transposons. All genes in the cluster were highly expressed in planta, but expression was very low or undetectable in mycelia from axenic culture. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.     

Importance of host plant species, Neotyphodium endophyte isolate, and alkaloids on feeding by Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae

Three grass host species--tall fescue, Festuca arundinacea Schreber; meadow fescue, Festuca pratensis Hudson; and perennial ryegrass, Lolium perenne L.--each infected with a number of different Neotyphodium endophyte isolates, were investigated for their effects on fall armyworm, Spodoptera frugiperda (J.E. Smith). Alkaloid profiles varied among associations. Choice and no-choice tests comparing feeding and early development of S. frugiperda larvae on endophyte-infected and endophyte-free leaf blade material were performed. Endophyte-mediated resistance to S. frugiperda was greatest in meadow fescue and weakest in tall fescue. Some endophyte isolates, particularly in perennial ryegrass and meadow fescue, had a major effect on feeding and development of S. frugiperda, whereas others had no effect or were only weakly efficacious. In tall fescue, some associations deterred S. frugiperda from feeding in choice tests but had no effect on development, whereas larvae reared on other associations weighed significantly more than control larvae fed endophyte-free grass. It was concluded that the deleterious consequences of endophyte infection were easily masked by other factors in tall fescue. Relative leaf age had no effect on feeding preferences in the three host species. Chemical analysis of herbage from the plants used, and results from a no-choice study using spiked artificial diets, failed to individually implicate any of the major known alkaloids (peramine, lolitrem B, ergovaline, and lolines) in the observed effects on S. frugiperda. Hypotheses explaining these observations, and their impact on creating desirable grass-endophyte associations for use in pastures, are discussed.     

Fungal endosymbionts affect aphid population size by reduction of adult life span and fecundity

The symbiosis between grasses and endophytic fungi is a common phenomenon and can affect herbivore performance through acquired, chemical plant defence by fungal alkaloids. In laboratory experiments, two species of common grass aphids, Rhopalosiphum padi and Metopolophium dirhodum were tested, in a population experiment (on four plant cultivars) and individually (on one plant cultivar) for the effects of the endophyte, Neotyphodium lolii, that forms symbiotic associations with perennial ryegrass Lolium perenne. In the population experiment that lasted for four aphid generations both aphid species showed decreased population sizes when feeding on each of the four endophyte-infected cultivars. Individuals of R. padi tested individually showed reduced adult life span and fecundity when feeding on infected plants. Individuals of M. dirhodum showed no response in any of the traits measured. This suggests that R. padi individuals are more sensitive to endophyte infection than M. dirhodum individuals. However, all infected grass cultivars reduced population sizes of both aphid species over four generations. Therefore, fungal endophytes can reduce populations of aphid herbivores independent of plant cultivars.     

Can Epichloë endophytes enhance direct and indirect plant defence?

Induced or constitutive production of secondary metabolites is a successful plant defence strategy against herbivores which can be mediated by plant associated micro-organisms. Several grass species can be associated with an endophytic fungus of the genus Epichloë which produces herbivore toxic or deterring alkaloids. Besides these direct defences, herbivorous insects are controlled via indirect plant defence mechanisms by attracting predators. Recent studies indicate that Epichloë endophytes can improve the grass emitted volatile organic compounds towards herbivore deterrence. Due to their defensive mutualistic function, we hypothesize that Epichloë altered plant volatiles can attract aphid predators and contribute to an increased indirect plant defence. With a common garden study, we show that hoverfly (Syrphidae) larvae and pupae were more abundant on endophyte-infected plants compared to uninfected plants. Our results indicate that the Epichloë endophyte provides, besides direct defence (alkaloid), indirect plant defence by improving the plant odor attracting more olfactory foraging aphid predators. Future research is needed in order to understand: (I) whether endophyte-mediated changes in plant volatiles are induced herbivore specific, (II) whether there is a trade-off between endophyte-mediated direct and indirect plant defence, (III) whether the endophyte produces volatiles or induces a change in plant-derived volatiles, (IV) the role of plant signals in endophyte-mediated plant defence.     

Novel perennial ryegrass-Neotyphodium endophyte associations: relationships between seed weight,seedling vigour and endophyte presence

Two forage cultivars of perennial ryegrass (Lolium perenne) colonised by the mutualistic fungal endophyte Neotyphodium lolii, strain AR37, were used to investigate relationships between seed weight, seed vigour and endophyte presence. Seed was separated into six fractions according to weight, with each fraction divided into two further groups with the first being subject to accelerated ageing. Seed germination rates and proportions of viable and total endophyte frequencies were assessed for each fraction. Heavier fractions of seed produced a higher number of endophyte infected seedlings sooner than the lighter fractions for both cultivars. The highest proportion of viable endophyte was found in the fastest germinating perennial ryegrass seed for all weight fractions, from both cultivars, indicating a strong relationship between endophyte presence and seed germination rate. For one of the cultivars, after accelerated ageing, as seed weight increased the proportion of viable endophyte increased and the discrepancy between the proportion of endophyte in fresh and accelerated aged seed was reduced. This implies that for this cultivar heavier ryegrass seed provides a more favourable habitat to the dormant endophyte than lighter seed during storage and/or allowed for a greater biomass of endophyte hyphae to proliferate in the seed tissues prior to seed dormancy, thus allowing the fungus to develop more propagules and greater nutritional reserves.     

Endophytes inconsistently affect plant communities across Schedonorus arundinaceus hosts

Fungal endophytes in cool-season grasses may affect communities at multiple trophic levels. However, it is unclear whether community-scale endophyte effects arise due to the endophyte itself or as a result of unique, endophyte–host interactions. We used a long-term field experiment to test whether common-toxic (CT) and non-ergot alkaloid-producing (novel) endophytes in Schedonorus arundinaceus (tall fescue) forage cultivars consistently affect communities across tall fescue hosts. Tilled plots (2 × 2 m; Guelph, ON) were seeded with Georgia 5 and Jesup cultivars containing either the CT or AR542 (novel) endophyte and allowed to be re-colonized by plant species from the local propagule pool. Non-seeded control plots were included to assess effects of seeding the non-native grass. We assessed plant, invertebrate, soil moisture, and soil nutrient responses to the endophyte–cultivar treatments after four growing seasons. Seeding tall fescue affected plant species abundances, but not richness, and did not consistently alter soil moisture and nutrient pools. Endophyte identity in the tall fescue cultivars affected the communities, but effects were not consistent between cultivars. Within Georgia 5, the AR542 endophyte reduced tall fescue abundance and altered the invertebrate community relative to CT plots. Within Jesup, the AR542 endophyte reduced species evenness and decreased soil moisture during dry periods relative to CT plots. Endophyte effects were not consistent between cultivars, and it is probable that the community-scale effects of endophyte infection in tall fescue cultivars arise due to unique interactions between cultivar and endophyte.