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Forage plant breeding aims to deliver cultivars that increase on-farm productivity through improved seasonal and annual yield, persistence of yield in perennials, and nutritive and feeding value. Breeding is generally undertaken in highly controlled field trials with individual plants or rows. However, the correlation of trait values between spaced plant trials and sward plots of forages is often low and non-significant. Heritability of traits, a measure of likely gains that can be achieved from plant breeding, can be moderate to high for many traits. The upper level of genetic gains in forage yield measured in small plot trials ranges between 6% and 15% per decade depending on species, indicating significant and lasting improvements have been achieved. Results from some farmlet scale grazing trials measuring animal performance support this but some others, particularly with dairy cows, fail to show advantage to improved cultivars. Factors contributing to this include aspects of systems management, systems trial design, and/or breeding objectives and evaluation systems; occurrence of environmental stresses; insufficient statistical power; and scaling interactions that restrict or dilute trait expression. Several factors are identified that should be considered as evaluation systems evolve in response to the changing economic, climatic, and regulatory forces affecting grassland farming. [ABSTRACT FROM AUTHOR]

Exploitation of the symbiotic relationship between endophytic fungi and ryegrass is a crucial technique for reducing the incidence of insect pests. This is primarily due to the production of alkaloids, such as peramine, by the fungi. This alkaloid has been reported as both a deterrent and toxic to a variety of insects. However, insects have developed various strategies to counteract plant defenses. One of the most studied methods is their ability to sequester toxic compounds from plants. In this study, we examined the feeding preferences and adaptation to peramine in Chilesia rudis, a native Chilean larva. Using a no-choice assay, we assessed larval feeding preferences and mass gain on seven experimental lines and two commercial cultivars of endophyte-infected and non-infected ryegrass. Pupal development time and adult performance were evaluated post-assay. Additionally, we measured peramine content in larval carcasses, feces, and ryegrass leaves. Jumbo was the most preferred cultivar with 32 mm2 of leaf tissues consumed. The longest pupal development time was observed in L161 and ALTO AR1, both at 28 days. Wing length in adults was greatest in the Jumbo and L163 cultivars, measuring 1.25 cm and 1.32 cm, respectively. Peramine concentrations were detected in the bodies of C. rudis. In conclusion, this larva can adapt to endophyte-infected ryegrass and develop counter-adaptation mechanisms to mitigate the effects of peramine. [ABSTRACT FROM AUTHOR]

Plants employ various strategies to mitigate the impact of herbivory and one such strategy involves the associations with fungal endophytes. Some cool‐season forage grasses benefit from protection against herbivores by Epichloë fungal endophytes. However, since the maintenance of symbiosis relies on the plant, costs of hosting endophytes have been also observed. This becomes particularly crucial when forage varieties, inoculated with endophytes that are nontoxic to livestock but still protect host against insect herbivores, experience significant foliage loss (defoliation). We investigated the impact of a nontoxic Epichloë endophyte on the responses of tall fescue plants to defoliation across two morphotype varieties, Continental and Mediterranean, and different seasonal contexts. Under specific defoliation treatments, endophyte‐symbiotic plants showed reduced postdefoliation recovery, indicating the costs of hosting the endophyte for host plants. However, these conditions were not the most severe for plant growth. Regardless of the endophyte's presence, plants from Continental morphotype exhibited superior recovery than those from Mediterranean morphotype, an effect that was more pronounced in summer. Notably, the endophyte promoted plant growth after defoliation in winter for both morphotypes. Our study offers insights into mitigating endophyte‐associated costs on plant recovery postdefoliation by strategically managing cutting intensity and frequency, considering the seasonal variation. [ABSTRACT FROM AUTHOR]

Grasses hosting Epichloë endophytes are protected against herbivores due to the production of various fungal alkaloids. Previous research has found that high foliar phosphorus concentrations reduce the level of the alkaloid ergovaline, thereby reducing the endophyte‐mediated herbivore resistance. Yet, the impact of phosphorus on ergovaline biosynthesis versus its influence on endophyte growth and synthesis of other fungal alkaloids remains unresolved. Our objective was to elucidate these relationships. We grew endophyte‐symbiotic and non‐symbiotic Festuca arundinacea plants and fertilised them with different doses of phosphorus. Later, half of the plants from each treatment were challenged with larvae of the generalist chewing insect Spodoptera frugiperda. We assessed the relationships between foliar phosphorus levels, fungal mycelium and alkaloid concentrations, as well as their impacts on larvae performance, herbivore‐caused damage and plant biomass. Endophyte mycelial biomass in plant tissue was found to be independent of foliar phosphorus concentration. The alkaloids lolines and peramine showed a linear relationship with mycelial biomass but no correlation with foliar phosphorus. Surprisingly, high ergovaline concentrations were positively associated with an interaction between endophyte mycelial biomass and foliar phosphorus concentration. Although herbivory increased loline concentration, only high concentrations of ergovaline and peramine were related to reduced S. frugiperda larvae weight gain. However, endophyte presence did not reduce herbivory damage on plants. Contrary to expectation, we did not find a negative but a positive association between concentrations of foliar phosphorus and ergovaline alkaloid, through its interaction with endophyte mycelial biomass. Alternatively, our findings suggest that phosphorus plays a crucial role in modulating the Epichloë‐mediated defensive mutualism, primarily through its effects on ergovaline rather than on endophyte concentration or production of other alkaloids. Read the free Plain Language Summary for this article on the Journal blog. [ABSTRACT FROM AUTHOR]

Epichloë fungal endophytes hold promise in sustainable agriculture by fortifying cool-season grasses such as Elymus spp. against various stresses. Elymus spp. are widely distributed in Northwest China with a high incidence of endophyte infections. In this study, we identified 20 Epichloë endophytic fungal strains carried by five Elymus spp. from five areas of Northwest China and systematically characterized their morphology, molecular phylogeny, mating type, and alkaloid diversity for the first time. The morphological characterization underscores strain diversity, with variable colony textures and growth rates. A phylogenetic analysis confirms all strains are E. bromicola, emphasizing their taxonomic status. Alkaloid-encoding gene profiling delineates distinct alkaloid synthesis capabilities among the strains, which are crucial for host adaptability and resistance. A mating-type analysis reveals uniformity (mtAC) across the Epichloë strains, simplifying breeding strategies. Notably, the Epichloë strains exhibit diverse alkaloid synthesis gene profiles, impacting host interactions. This research emphasizes the ecological significance of Epichloë endophytes in Elymus spp. ecosystems, offering insights into their genetic diversity and potential applications in sustainable agriculture. [ABSTRACT FROM AUTHOR]

Abstract Background Perennial ryegrass (Lolium perenne) in New Zealand pastures is typically infected with the mutualist Epichloë fungal endophyte. This endophyte assists the plant in resisting biotic and abiotic stresses, but the standard strain of endophyte is toxic to livestock. Elite ryegrasses with selected endophytes have been developed to provide protective properties to the grass plant and lessen or eliminate the negative impacts on livestock. Methods Using immunology and molecular techniques, the presence of endophyte infection and endophyte strain in ryegrass tillers was determined for 24 dairy pastures sampled for up to 7 years in regions of the North and South Islands. Results In general, infection levels were high and showed small increases over time. Some pastures failed to reach 70% infection. The sown, selected endophytes were the dominant endophyte strains present and these were stable over time. Standard endophyte was the primary nonsown endophyte, and while generally low and so of little importance, it increased over time and for some pastures, this would have been detrimental to livestock. Pasture establishment technique influenced the level of contamination. Conclusions Results reinforce the importance of following best practice procedures in the seed industry and on‐farm. Researchers should monitor trials for contaminating nonsown standard endophyte.

The inoculation of Epichloë endophytes into modern cereals, resulting in systemic infection, depends on the genetics of both the host and the endophyte strain deployed. Until very recently, the only modern cereal to have been infected with Epichloë, in which normal phenotype seed-transmitted associations were achieved, is rye (Secale cereale). Whilst minor in-roads have been achieved in infecting hexaploid wheat (Triticum aestivum), the phenotypes of these associations have all been extremely poor, including host death and stunting. To identify host genetic factors that may impact the compatibility of Epichloë infection in wheat, wheat–alien chromosome addition/substitution lines were inoculated with Epichloë, and the phenotypes of infected plants were assessed. Symbioses were identified whereby infected wheat plants were phenotypically like uninfected controls. These plants completed their full lifecycle, including the vertical transmission of Epichloë into the next generation of grain, and represent the first ever compatible wheat–Epichloë associations to be created. [ABSTRACT FROM AUTHOR]

Claviceptaceous endophytic fungi in the genus Epichloë mostly form a symbiotic relationship with cool-season grasses. Epichloë spp. are capable of producing bioactive alkaloids such as peramines, lolines, ergot alkaloids, and indole-diterpenes, which protect the host plant from herbivory by animals, insects, and nematodes. The host also benefits from enhanced tolerance to abiotic stresses, such as salt, drought, waterlogging, cold, heavy metals, and low nitrogen stress. The bioactive alkaloids produced can have both direct and indirect effects towards plant parasitic nematodes. Direct interaction with nematodes' motile stages can cause paralysis (nematostatic effect) or death (nematicidal effect). Indirectly, the metabolites may induce host immunity which inhibits feeding and subsequent nematode development. This review highlights the different mechanisms through which this interaction and the metabolites produced have been explored in the suppression of plant parasitic nematodes and also how the specific interactions between different grass genotypes and endophyte strains result in variable suppression of different nematode species. An understanding of the different grass–endophyte interactions and their successes and failures in suppressing various nematode species is essential to enable the proper selection of grass–endophyte combinations to identify the alkaloids produced, concentrations required, and determine which nematodes are sensitive to which specific alkaloids. [ABSTRACT FROM AUTHOR]

Background: Perennial ryegrass (Lolium perenne) in New Zealand pastures is typically infected with the mutualist Epichloë fungal endophyte. This endophyte assists the plant in resisting biotic and abiotic stresses, but the standard strain of endophyte is toxic to livestock. Elite ryegrasses with selected endophytes have been developed to provide protective properties to the grass plant and lessen or eliminate the negative impacts on livestock. Methods: Using immunology and molecular techniques, the presence of endophyte infection and endophyte strain in ryegrass tillers was determined for 24 dairy pastures sampled for up to 7 years in regions of the North and South Islands. Results: In general, infection levels were high and showed small increases over time. Some pastures failed to reach 70% infection. The sown, selected endophytes were the dominant endophyte strains present and these were stable over time. Standard endophyte was the primary nonsown endophyte, and while generally low and so of little importance, it increased over time and for some pastures, this would have been detrimental to livestock. Pasture establishment technique influenced the level of contamination. Conclusions: Results reinforce the importance of following best practice procedures in the seed industry and on‐farm. Researchers should monitor trials for contaminating nonsown standard endophyte. [ABSTRACT FROM AUTHOR]

Endophytes generally increase antioxidant contents of plants subjected to environmental stresses. However, the mechanisms by which endophytes alter the accumulation of antioxidants in plant tissues are not entirely clear. We hypothesized that, in stress situations, endophytes would simultaneously reduce oxidative damage and increase antioxidant contents of plants and that the accumulation of antioxidants would be a consequence of the endophyte ability to regulate the expression of plant antioxidant genes. We investigated the effects of the fungal endophyte Epichloë gansuensis (C.J. Li & Nan) on oxidative damage, antioxidant contents, and expression of representative genes associated with antioxidant pathways in Achnatherum inebrians (Hance) Keng plants subjected to low (15%) and high (60%) soil moisture conditions. Gene expression levels were measured using RNA-seq. As expected, the endophyte reduced the oxidative damage by 17.55% and increased the antioxidant contents by 53.14% (on average) in plants subjected to low soil moisture. In line with the accumulation of antioxidants in plant tissues, the endophyte increased the expression of most plant genes associated with the biosynthesis of antioxidants (e.g., MIOX, crtB, gpx) while it reduced the expression of plant genes related to the metabolization of antioxidants (e.g., GST, PRODH, ALDH). Our findings suggest that endophyte ability of increasing antioxidant contents in plants may reduce the oxidative damage caused by stresses and that the fungal regulation of plant antioxidants would partly explain the accumulation of these compounds in plant tissues. [ABSTRACT FROM AUTHOR]

Species' persistence in increasingly variable climates will depend on resilience against the fitness costs of environmental stochasticity. Most organisms host microbiota that shield against stressors. Here, we test the hypothesis that, by limiting exposure to temporally variable stressors, microbial symbionts reduce hosts' demographic variance. We parameterized stochastic population models using data from a 14‐year symbiont‐removal experiment including seven grass species that host Epichloë fungal endophytes. Results provide novel evidence that symbiotic benefits arise not only through improved mean fitness, but also through dampened inter‐annual variance. Hosts with "fast" life‐history traits benefited most from symbiont‐mediated demographic buffering. Under current climate conditions, contributions of demographic buffering were modest compared to benefits to mean fitness. However, simulations of increased stochasticity amplified benefits of demographic buffering and made it the more important pathway of host–symbiont mutualism. Microbial‐mediated variance buffering is likely an important, yet cryptic, mechanism of resilience in an increasingly variable world. [ABSTRACT FROM AUTHOR]

In nature, the symbiotic relationship between plants and microorganisms is crucial for ecosystem balance and plant growth. This study investigates the impact of Epichloë endophytic fungi, which are exclusively present aboveground, on the rhizosphere microbial functions of the host Melica transsilvanica. Using metagenomic methods, we analyzed the differences in microbial functional groups and functional genes in the rhizosphere soil between symbiotic (EI) and non-symbiotic (EF) plants. The results reveal that the presence of Epichloë altered the community structure of carbon and nitrogen cycling-related microbial populations in the host's rhizosphere, significantly increasing the abundance of the genes (porA, porG, IDH1) involved in the rTCA cycle of the carbon fixation pathway, as well as the abundance of nxrAB genes related to nitrification in the nitrogen-cycling pathway. Furthermore, the presence of Epichloë reduces the enrichment of virulence factors in the host rhizosphere microbiome, while significantly increasing the accumulation of resistance genes against heavy metals such as Zn, Sb, and Pb. This study provides new insights into the interactions among endophytic fungi, host plants, and rhizosphere microorganisms, and offers potential applications for utilizing endophytic fungi resources to improve plant growth and soil health. [ABSTRACT FROM AUTHOR]

Abstract Background Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. Results Expected relative increases in total fatty acids of 17–58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. Conclusions These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

Exploitation of the symbiotic relationship between endophytic fungi and ryegrass is a crucial technique for reducing the incidence of insect pests. This is primarily due to the production of alkaloids, such as peramine, by the fungi. This alkaloid has been reported as both a deterrent and toxic to a variety of insects. However, insects have developed various strategies to counteract plant defenses. One of the most studied methods is their ability to sequester toxic compounds from plants. In this study, we examined the feeding preferences and adaptation to peramine in Chilesia rudis, a native Chilean larva. Using a no-choice assay, we assessed larval feeding preferences and mass gain on seven experimental lines and two commercial cultivars of endophyte-infected and non-infected ryegrass. Pupal development time and adult performance were evaluated post-assay. Additionally, we measured peramine content in larval carcasses, feces, and ryegrass leaves. Jumbo was the most preferred cultivar with 32 mm2 of leaf tissues consumed. The longest pupal development time was observed in L161 and ALTO AR1, both at 28 days. Wing length in adults was greatest in the Jumbo and L163 cultivars, measuring 1.25 cm and 1.32 cm, respectively. Peramine concentrations were detected in the bodies of C. rudis. In conclusion, this larva can adapt to endophyte-infected ryegrass and develop counter-adaptation mechanisms to mitigate the effects of peramine.

Epichloë fungal endophytes hold promise in sustainable agriculture by fortifying cool-season grasses such as Elymus spp. against various stresses. Elymus spp. are widely distributed in Northwest China with a high incidence of endophyte infections. In this study, we identified 20 Epichloë endophytic fungal strains carried by five Elymus spp. from five areas of Northwest China and systematically characterized their morphology, molecular phylogeny, mating type, and alkaloid diversity for the first time. The morphological characterization underscores strain diversity, with variable colony textures and growth rates. A phylogenetic analysis confirms all strains are E. bromicola, emphasizing their taxonomic status. Alkaloid-encoding gene profiling delineates distinct alkaloid synthesis capabilities among the strains, which are crucial for host adaptability and resistance. A mating-type analysis reveals uniformity (mtAC) across the Epichloë strains, simplifying breeding strategies. Notably, the Epichloë strains exhibit diverse alkaloid synthesis gene profiles, impacting host interactions. This research emphasizes the ecological significance of Epichloë endophytes in Elymus spp. ecosystems, offering insights into their genetic diversity and potential applications in sustainable agriculture.

Background: Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. Results: Expected relative increases in total fatty acids of 17–58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. Conclusions: These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species. [ABSTRACT FROM AUTHOR]

The interaction between Botanophila flies and Epichloë fungi is one of the few interactions in which grass-associated fungi are 'pollinated' by flies. We analysed the gains and losses for both interaction partners and the time factors determining cost dynamics. A hypothesis regarding the relationship between changes in the severity of grass infection with Epichloë fungus, size of fungal sexual structures (stromata), and fly presence (expressed as egg and larvae numbers) was tested. During a 3-year study conducted on permanent field plots, fungal infection of wild grass tussocks of Holcus lanatus and fly visits to fungal stromata were monitored. An increase in the number of shoots with stromata and a reduction in stromata size were observed. From one year to another, flies deposited fewer eggs on stromata, and larval body mass decreased. We explain these results by the deterioration of grass condition due to increased Epichloë infection, which resulted in higher maintenance costs for the fungal parasite. [ABSTRACT FROM AUTHOR]

Symbiotic mutualisms are thought to be stabilized by correlations between the interacting genotypes which may be strengthened via vertical transmission and/or reduced genetic variability within each species. Vertical transmission, however, may weaken interactions over time as the endosymbionts would acquire mutations that could not be purged. Additionally, temporal variation in a conditional mutualism could create genetic variation and increased variation in the interaction outcome. In this study, we assessed genetic variation in both members of a symbiosis, the endosymbiotic fungal endophyte Epichloë canadensis and its grass host Canada wildrye (Elymus canadensis). Both species exhibited comparable levels of diversity, mostly within populations rather than between. There were significant differences between populations, although not in the same pattern for the two species, and the differences were not correlated with geographic distance for either species. Interindividual genetic distance matrices for the two species were significantly correlated, although all combinations of discriminant analysis of principle components (DAPC) defined multilocus genotype groups were found suggesting that strict genotype matching is not necessary. Variation in interaction outcome is common in grass/endophyte interactions, and our results suggest that the accumulation of mutations overtime combined with temporal variation in selection pressures increasing genetic variation in the symbiosis may be the cause. [ABSTRACT FROM AUTHOR]

Claviceptaceous endophytic fungi in the genus Epichloë mostly form a symbiotic relationship with cool-season grasses. Epichloë spp. are capable of producing bioactive alkaloids such as peramines, lolines, ergot alkaloids, and indole-diterpenes, which protect the host plant from herbivory by animals, insects, and nematodes. The host also benefits from enhanced tolerance to abiotic stresses, such as salt, drought, waterlogging, cold, heavy metals, and low nitrogen stress. The bioactive alkaloids produced can have both direct and indirect effects towards plant parasitic nematodes. Direct interaction with nematodes’ motile stages can cause paralysis (nematostatic effect) or death (nematicidal effect). Indirectly, the metabolites may induce host immunity which inhibits feeding and subsequent nematode development. This review highlights the different mechanisms through which this interaction and the metabolites produced have been explored in the suppression of plant parasitic nematodes and also how the specific interactions between different grass genotypes and endophyte strains result in variable suppression of different nematode species. An understanding of the different grass–endophyte interactions and their successes and failures in suppressing various nematode species is essential to enable the proper selection of grass–endophyte combinations to identify the alkaloids produced, concentrations required, and determine which nematodes are sensitive to which specific alkaloids.

The inoculation of Epichloë endophytes into modern cereals, resulting in systemic infection, depends on the genetics of both the host and the endophyte strain deployed. Until very recently, the only modern cereal to have been infected with Epichloë, in which normal phenotype seed-transmitted associations were achieved, is rye (Secale cereale). Whilst minor in-roads have been achieved in infecting hexaploid wheat (Triticum aestivum), the phenotypes of these associations have all been extremely poor, including host death and stunting. To identify host genetic factors that may impact the compatibility of Epichloë infection in wheat, wheat–alien chromosome addition/substitution lines were inoculated with Epichloë, and the phenotypes of infected plants were assessed. Symbioses were identified whereby infected wheat plants were phenotypically like uninfected controls. These plants completed their full lifecycle, including the vertical transmission of Epichloë into the next generation of grain, and represent the first ever compatible wheat–Epichloë associations to be created.

In nature, the symbiotic relationship between plants and microorganisms is crucial for ecosystem balance and plant growth. This study investigates the impact of Epichloë endophytic fungi, which are exclusively present aboveground, on the rhizosphere microbial functions of the host Melica transsilvanica. Using metagenomic methods, we analyzed the differences in microbial functional groups and functional genes in the rhizosphere soil between symbiotic (EI) and non-symbiotic (EF) plants. The results reveal that the presence of Epichloë altered the community structure of carbon and nitrogen cycling-related microbial populations in the host’s rhizosphere, significantly increasing the abundance of the genes (porA, porG, IDH1) involved in the rTCA cycle of the carbon fixation pathway, as well as the abundance of nxrAB genes related to nitrification in the nitrogen-cycling pathway. Furthermore, the presence of Epichloë reduces the enrichment of virulence factors in the host rhizosphere microbiome, while significantly increasing the accumulation of resistance genes against heavy metals such as Zn, Sb, and Pb. This study provides new insights into the interactions among endophytic fungi, host plants, and rhizosphere microorganisms, and offers potential applications for utilizing endophytic fungi resources to improve plant growth and soil health.

Perennial ryegrass (Lolium perenne L.) remains the predominant grazed grass in New Zealand’s pastoral agriculture. Brought in from Europe in the early nineteenth century, locally adapted populations or ecotypes developed in a number of regions. Government plant breeders, employed to provide elite cultivars for farmer use as a public good, exploited an ecotype from the Hawke’s Bay region to produce ‘Grasslands Ruanui’. In the1950s Trevor Ellett, a dairy farmer in Mangere, Auckland noted that Grasslands Ruanui did not persist as well as ryegrass sown with seed harvested from 40 to 60 year old paddocks on his own farm. This ecotype, referred to as Mangere ryegrass, has since become the basis of many ryegrass cultivars bred by both government and commercial plant breeders over the past 50 years. This review documents how Mangere ryegrass became an important part of ryegrass cultivar development, the part it played in commercial plant breeders becoming involved in cultivar development, and coincidently in the discovery that the Epichloë fungal endophyte is crucial for perennial ryegrass persistence under drought, heat and insect pressure. [ABSTRACT FROM AUTHOR]

Anthropogenic nitrogen (N) enrichment can have complex effects on plant communities. In low-nutrient, primary successional systems such as sand dunes, N enrichment may alter the trajectory of plant community assembly or the dominance of foundational, ecosystem-engineering plants. Predicting the consequences of N enrichment may be complicated by plant interactions with microbial symbionts because increases in a limiting resource, such as N, could alter the costs and benefits of symbiosis. To evaluate the direct and interactive effects of microbial symbiosis and N addition on plant succession, we established a long-term field experiment in Michigan, USA, manipulating the presence of the symbiotic fungal endophyte Epichloë amarillans in Ammophila breviligulata, a dominant ecosystem-engineering dune grass species. From 2016 to 2020, we implemented N fertilization treatments (control, low, high) in a subset of the long-term experiment. N addition suppressed the accumulation of plant diversity over time mainly by reducing species richness of colonizing plants. However, this suppression occurred only when the endophyte was present in Ammophila. Although Epichloë enhanced Ammophila tiller density over time, N addition did not strongly interact with Epichloë symbiosis to influence vegetative growth of Ammophila. Instead, N addition directly altered plant community composition by increasing the abundance of efficient colonizers, especially C4 grasses. In conclusion, hidden microbial symbionts can alter the consequences of N enrichment on plant primary succession. [ABSTRACT FROM AUTHOR]

Vertically transmitted endophytic fungi can mitigate the negative effects of salinity encountered by their host grass and alter the competitive interactions between plant individuals. To experimentally study the interactive effects of the fungal endophyte Epichloë coenophiala on salt tolerance and intraspecific competition of its host plant, tall fescue Festuca arundinacea, we subjected 15 maternal lines of each Epichloë associated (E+) and Epichloë free (E−) tall fescue to salt treatment and competition in the greenhouse and common garden. Then, to explore variation in endophyte incidence in natural populations of tall fescue, we surveyed 23 natural populations occurring on or near the Baltic Sea coast in Aland islands in southwestern Finland for endophyte incidence, distance to shore, and competitive environment. Under salinity in the greenhouse, E− plants grew larger than E+ plants, but there was no size difference in the control treatment. E− plants grew taller and were more likely to flower than E+ plants when grown in benign conditions in the common garden but not with salinity or competition. The frequency of Epichloë incidence was high (90%) in natural populations, and it decreased towards the shore and risk of salt exposure. These results demonstrate a negative effect of Epichloë endophyte on the salt tolerance of its host. The high incidence of Epichloë in natural populations of tall fescue in the northern part of the species distribution range is likely due to factors other than salinity. [ABSTRACT FROM AUTHOR]

Plants harbor a variety of fungal symbionts both above- and belowground, yet little is known about how these fungi interact within hosts, especially in a world where resource availability is changing due to human activities. Systemic vertically transmitted endophytes such as Epichloë spp. may have particularly strong effects on the diversity and composition of later-colonizing symbionts such as root fungal endophytes, especially in primary successional systems. We made use of a long-term field experiment in Great Lakes sand dunes to test whether Epichloë colonization of the dune-building grass, Ammophila breviligulata, could alter fungal root endophyte species richness or community composition in host plants. We also tested whether nitrogen addition intensified the effects of Epichlöe on the root endophyte community. We found that Epichloë increased richness of root endophytes in Ammophila by 17% overall, but only shifted community composition of root endophytes under nitrogen-enriched conditions. These results indicate that Epichlöe acts as a key species within Ammophila, changing richness and composition of the root mycobiome and integrating above- and belowground mycobiome interactions. Further, effects of Epichloë on root endophyte communities were enhanced by N addition, indicating that this fungal species may become even more important in future environments. [ABSTRACT FROM AUTHOR]

Asexual Epichloë are endophytic fungi that form mutualistic symbioses with cool-season grasses, conferring to their hosts protection against biotic and abiotic stresses. Symbioses are maintained between grass generations as hyphae are vertically transmitted from parent to progeny plants through seed. However, endophyte transmission to the seed is an imperfect process where not all seeds become infected. The mechanisms underpinning the varying efficiencies of seed transmission are poorly understood. Host gene expression in response to Epichloë sp. LpTG-3 strain AR37 was examined within inflorescence primordia and ovaries of high and low endophyte transmission genotypes within a single population of perennial ryegrass. A genome-wide association study was conducted to identify population-level single nucleotide polymorphisms (SNPs) and associated genes correlated with vertical transmission efficiency. For low transmitters of AR37, upregulation of perennial ryegrass receptor-like kinases and resistance genes, typically associated with phytopathogen detection, comprised the largest group of differentially expressed genes (DEGs) in both inflorescence primordia and ovaries. DEGs involved in signaling and plant defense responses, such as cell wall modification, secondary metabolism, and reactive oxygen activities were also abundant. Transmission-associated SNPs were associated with genes for which gene ontology analysis identified "response to fungus" as the most significantly enriched term. Moreover, endophyte biomass as measured by quantitative PCR of Epichloë non-ribosomal peptide synthetase genes, was significantly lower in reproductive tissues of low-transmission hosts compared to high-transmission hosts. Endophyte seed-transmission efficiency appears to be influenced primarily by plant defense responses which reduce endophyte colonization of host reproductive tissues. [ABSTRACT FROM AUTHOR]

Abstract Here we describe a new, haploid and stroma forming species within the genus Epichloë, as Epichloë scottii sp. nov. The fungus was isolated from Melica uniflora growing in Bad Harzburg, Germany. Phylogenetic reconstruction using a combined dataset of the tubB and tefA genes strongly support that E. scottii is a distinct species and the so far unknown ancestor species of the hybrid E. disjuncta. A distribution analysis showed a high infection rate in close vicinity of the initial sampling site and only two more spots with low infection rates. Genetic variations in key genes required for alkaloid production suggested that E. scottii sp. nov. might not be capable of producing any of the major alkaloids including ergot alkaloid, loline, indole-diterpene and peramine. All isolates and individuals found in the distribution analysis were identified as mating-type B explaining the lack of mature stromata during this study. We further release a telomere-to-telomere de novo assembly of all seven chromosomes and the mitogenome of E. scottii sp. nov.

Cool season grasses often form reciprocal symbiotic relationships with endophytic fungal species in genus Epichloë. In this study, we characterized three fungal endophytes isolated from the grass Hordeum bogdanii native to northwest China. Based on morphological characteristics and phylogenetic analyses of tefA, tubB, and actG sequences, we identified them as Epichloë sp. HboTG-2 (H. bogdanii Taxonomic Group 2: E. bromicola × E. typhina). Alkaloid synthesis related genes analysis showed that Epichloë sp. HboTG-2 may have the ability only to produce peramine which is toxic to insects but not to animals. In the process of this study, we did not observe sexual structures or epiphyllous growth on leaves of infected plants. [ABSTRACT FROM AUTHOR]

Context. Tall fescue (Schedonorus arundinaceus) is one of the most important cultivated forage grasses in temperate regions but its association with some Epichloë fungal endophytes usually makes it unsuitable for livestock feeding due to toxic alkaloids. However, re-inoculation of plants with non-toxic endophytes can result in positive effects for livestock feeding. Aims. Assess the effects of the non-toxic AR584 endophyte on seed viability and germination in two tall fescue cultivars. Methods. The effects of AR584 endophyte (presence/absence) and tall fescue cultivar (INIA Aurora/Taita) were evaluated on seed viability and germination responses across a range of temperature and water availability regimes. Response to constant temperature and water availability was characterised by the thermal-time and hydro-time models, respectively. Key results. Under accelerated aging conditions, endophyte presence reduced seed viability in cultivar Taita. For both cultivars, endophyte-infected seeds showed a lower germination rate than endophyte-free seeds, except for INIA Aurora under low incubation temperatures. Also, endophyte-infected seeds in both cultivars displayed lower germination rates and final germination as the water potential was lowered. Conclusions. A strong cultivar-by-endophyte interaction for seed germination indicates the need to understand how specific cultivarendophyte combinations best fit a given environmental condition, as defined by temperature and water availability. Implications. The information we present can be valuable not only for farmers who have to decide what to cultivate but also for seed companies that have to preserve seed quality. [ABSTRACT FROM AUTHOR]

Substantial evidence is available on the capacity of particular strains of Epichloë fungal endophyte to remove the barriers of self‐pollination in host grasses. We hypothesized that this might open up new methods to obtain inbred lines for specific turf hybrids and genetic studies.In the present study, we evaluated the first generation of self‐pollination derived plants of endophyte infected clones (EIS1) from putative genotypes 75B+ and 75C+ and those from the first generation of self‐pollination in six commercial tall fescue clones plus their hybrids with 75B− and 75C− for growth, seed yield and polyphenolic content as an index for biosynthesis of defence compounds under field conditions.The results showed that EIS1 had high hyphal density within leaf sheaths and higher growth and seed‐related traits in at least one genotype. There were higher amounts of flavonoid and phenolic compounds (up to twofold) in both genotypes than in their hybrid counterparts and endophyte‐free progeny. Selected genotypes within EIS1 contained significantly more chlorogenic acid, p‐coumaric acid and rutin than the best non‐infected genotypes.We conclude that phenotypic selection of individuals from the S1 population is feasible for improving fitness and stress resistance in novel inbred lines of tall fescue for development of new turf cultivars with the desired ecophysiological traits. [ABSTRACT FROM AUTHOR]

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Epichloë endophytes in grass associations express a myriad of secondary metabolites which can affect the health of grazing animals and reduce the impact of insect pests on pasture. The ideal endophyte-grass association must have a favourable chemical profile such that the impact on animal health is minimised while the beneficial, deterrent effect on insect pests is maximised. A number of endophyte-perennial ryegrass associations have been successfully commercialised but research is on-going to further improve production in farming systems. Secondary metabolites expressed by endophyte-infected tall fescue include lolines, an animal-safe class of compound which imparts a potent effect on insects. Since endophyte-infected perennial ryegrass does not express lolines, a tall fescue endophyte, AR501, was inoculated into perennial ryegrass in an attempt to improve the insect resistance of this pasture type. In addition to animal safety, it is imperative that consideration is given to the safety of humans consuming animal products derived from livestock grazing the novel pasture. Although pure loline alkaloids have previously been tested on mice it is essential that the entire AR501 endophyte-infected perennial ryegrass matrix is tested since this will result in the exposure of both known and unknown secondary metabolites to mice. Three treatment groups each containing 6 male and 6 female mice were fed diets containing AR501 endophyte-infected perennial ryegrass seed (30%), perennial ryegrass seed containing no endophyte (30%) or a diet without seed (control) for 3 weeks. Mice fed control diet ate more than either of the treatment groups fed a diet containing seed. Male mice fed diet containing Nil endophyte seed ate more than those eating AR501 endophyte-infected perennial ryegrass seed although there was no difference observed in the food intake of female mice. While a few statistically significant differences were observed in the haematology and serum biochemical data, in every instance the difference was restricted to only one gender so is considered unlikely to be of toxicological significance. Mice fed AR501 endophyte-infected perennial ryegrass seed remained healthy throughout the experimental period despite consuming 62,000 mg/kg lolines and 4600 mg/kg peramine per day as well as the wide array of other unknown secondary metabolites expressed by this endophyte. Although animal products may contain additional metabolites as a result of animal metabolism, this experiment raises no food safety concerns for AR501 endophyte-infected perennial ryegrass. [Display omitted] • Endophytes are used to confer beneficial insect pest protection to their host plants. • Endophytes produce a myriad of known and unknown secondary metabolites. • Secondary metabolites consumed by animals could go up the food chain to be consumed by humans. • AR501 endophyte-infected ryegrass seed was fed to mice for 21 days. • The lack of toxicity observed raises no food safety concerns for these compounds. [ABSTRACT FROM AUTHOR]

Epichloë coenophiala, a systemic fungal symbiont (endophyte) of tall fescue (Lolium arundinaceum), has been documented to confer to this grass better persistence than plants lacking the endophyte, especially under stress conditions such as drought. The response, if any, of the endophyte to imposition of stress on the host plant has not been characterized previously. Therefore, we investigated effects on gene expression by E. coenophiala and a related endophyte when plant-endophyte symbiota were subjected to acute water-deficit stress. Plants harboring different endophyte strains were grown in sand in the greenhouse, then half were deprived of water for 48 h and the other half were watered controls. RNA was isolated from different plant tissues, and mRNA sequencing (RNA-seq) was conducted to identify genes that were differentially expressed comparing stress treatment with control. We compared two different plants harboring the common toxic E. coenophiala strain (CTE) and two non–ergot-alkaloid-producing Epichloë strains in tall fescue pseudostems, and in a second experiment we compared responses of E. coenophiala CTE in plant pseudostem and crown tissues. The endophytes responded to the stress with increased expression of genes involved in oxidative stress response, oxygen radical detoxification, C-compound carbohydrate metabolism, heat shock, and cellular transport pathways. The magnitude of fungal gene responses during stress varied among plant-endophyte symbiota. Responses in pseudostems and crowns involved some common pathways as well as some tissue-specific pathways. The fungal response to water-deficit stress involved gene expression changes in similar pathways that have been documented for plant stress responses, indicating that Epichloë spp. and their host plants either coordinate stress responses or separately activate similar stress response mechanisms that work together for mutual protection. [ABSTRACT FROM AUTHOR]

Genome rearrangements in filamentous fungi are prevalent but little is known about the modalities of their evolution, in part because few complete genomes are available within a single genus. To address this, we have generated and compared 15 complete telomere-to-telomere genomes across the phylogeny of a single genus of filamentous fungi, Epichloë. We find that the striking distinction between gene-rich and repeat-rich regions previously reported for isolated species is ubiquitous across the Epichloë genus. We built a species phylogeny from single-copy gene orthologs to provide a comparative framing to study chromosome composition and structural change through evolutionary time. All Epichloë genomes have exactly seven nuclear chromosomes, but despite this conserved ploidy, analyses reveal low synteny and substantial rearrangement of gene content across the genus. These rearrangements are highly lineage-dependent, with most occurring over short evolutionary distances, with long periods of structural stasis. Quantification of chromosomal rearrangements shows they are uncorrelated with numbers of substitutions and evolutionary distances, suggesting that different modes of evolution are acting to create nucleotide and chromosome-scale changes. [ABSTRACT FROM AUTHOR]

To assess animal health and production in sheep grazing perennial ryegrass (Lolium perenne) infected with a tall fescue endophyte (Epichloë sp.). Three replicates of pure perennial ryegrass pastures infected with AR501 tall fescue endophyte (AR501 HE), AR1, AR37 or standard endophyte (STD) (all >85% infection) and a low endophyte control (AR501 LE) were grazed by 10 lambs for 7 weeks during late summer/early autumn, 2017. AR501 HE and AR501 LE were in the same tetraploid cultivar and the remaining treatments were in the same diploid cultivar. Lambs were weighed on Days 0, 19, 37 and 48 and assessed for ryegrass staggers on a 0–4 tremor scale nine times from Day 13. On Days 14 and 20, rectal temperatures and respiration rates were measured in lambs grazing AR501 HE, AR501 LE and STD under heat load. Pasture samples from each treatment were taken regularly (8 times) for analysis of known endophyte secondary metabolites. Peak mean ryegrass staggers scores for lambs grazing STD and AR37 treatments were 3.9 and 2.7, respectively, indicating environmental conditions were conducive to toxicity. Lambs grazing AR501 HE showed no ryegrass staggers at any date. The liveweight of STD lambs was up to 6.6 kg less than some or all other treatments on Days 19, 34 and 48, but there was no evidence of a difference between the liveweight of AR501 HE lambs and AR501 LE, AR37 or AR1 lambs on any date. Rectal temperatures and respiration rates of STD lambs were higher than AR501 HE lambs by 0.2°C and 26 breaths/minute, respectively. Apart from lower rectal temperature of AR501 HE lambs on Day 14, there was no evidence of differences between the AR501 HE and AR501 LE lambs. N-Formylloline was the only loline present in AR501 HE herbage (350–860 mg/kg). Peramine in AR501 HE herbage (42–77 mg/kg) was greater than that in STD and AR1 herbage on five and seven of the eight sampling dates, respectively. Lambs grazing AR501 HE showed no ryegrass staggers, exacerbated heat stress or suppressed liveweight gain compared with lambs grazing AR501 LE in a season when endophyte-induced toxicity was evident in STD and AR37 pastures. This suggests AR501 tall fescue endophyte does not produce toxic concentrations of secondary metabolites. This study suggests that there are no secondary metabolites produced by the tall fescue AR501 endophyte-perennial ryegrass association which affect animal health or production. [ABSTRACT FROM AUTHOR]

Fungi from the genus Epichloë form systemic endobiotic infections of cool season grasses, producing a range of host-protective natural products in return for access to nutrients. These infections are asymptomatic during vegetative host growth, with associations between asexual Epichloe? spp. and their hosts considered mutualistic. However, the sexual cycle of Epichloe? spp. involves virulent growth, characterized by the envelopment and sterilization of a developing host inflorescence by a dense sheath of mycelia known as a stroma. Microscopic analysis of stromata revealed a dramatic increase in hyphal propagation and host degradation compared with asymptomatic tissues. RNAseq was used to identify differentially expressed genes in asymptomatic vs stromatized tissues from 3 diverse Epichloë -host associations. Comparative analysis identified a core set of 135 differentially expressed genes that exhibited conserved transcriptional changes across all 3 associations. The core differentially expressed genes more strongly expressed during virulent growth encode proteins associated with host suppression, digestion, adaptation to the external environment, a biosynthetic gene cluster, and 5 transcription factors that may regulate Epichloë stroma formation. An additional 5 transcription factor encoding differentially expressed genes were suppressed during virulent growth, suggesting they regulate mutualistic processes. Expression of biosynthetic gene clusters for natural products that suppress herbivory was universally suppressed during virulent growth, and additional biosynthetic gene clusters that may encode production of novel host-protective natural products were identified. A comparative analysis of 26 Epichloë genomes found a general decrease in core differentially expressed gene conservation among asexual species, and a specific decrease in conservation for the biosynthetic gene cluster expressed during virulent growth and an unusual uncharacterized gene. [ABSTRACT FROM AUTHOR]

Global warming is expected to increase drought severity in diverse environments, impacting plant performance. Plants acclimate to drought by mechanisms like stomatal closure and osmotic adjustment. Maternally inherited symbiotic microorganisms with capacity to regulate these mechanisms have the potential to influence intergenerational plant drought responses. We studied how a seedborne endophyte affects maternal drought effects on seed germination water requirements and specialized metabolites. Isotopic analysis of seed cellulose indicated that drought led to improved water use efficiency (WUE; higher δ13C) in endophyte-free mother plants, seemingly without affecting stomatal conductance (non-significant δ18O change). Alternatively, endophyte-symbiotic plants did not exhibit a change in WUE but apparently an increase in stomatal conductance (significant δ18O decrease). Regardless of the symbiosis, drought reduced seed production but not seed size. Endophyte symbiosis improved the seed concentration of mannitol and sorbitol, but this increment was higher under drought. Maternal plant responses to drought did not increase seed germination under reduced water potential but induced dormancy in seeds from endophyte-free but not endophyte-symbiotic plants. Our findings suggest that although differential accumulation of metabolites in seeds results from how endophyte-symbiotic plants perceive and respond to drought, this response may not form part of a mechanism that would enhance seed performance of progeny under drought. • Drought events will increase under global warming, impacting plant performance. • Plant responses to drought involve mechanisms like stomatal closure and osmotic adjustment. • Maternally inherited fungal endophytes influence intergenerational plant drought responses. • Endophyte-symbiotic plants show different water use efficiency and stomatal conductance under drought. • Seedborne endophytes modulate maternal drought effects on seed germination and metabolites. [ABSTRACT FROM AUTHOR]

Bromus auleticus is a winter wild species with promissory characteristics for domestication as forage that inhabits temperate‐warm grasslands from a wide region from South America. Populations from regions with different environmental conditions are considered as ecotypes and are associated with different seedborne Epichloë fungal endophytes. Association with Epichloë provides B. auleticus with increased growth capacity and resistance against pathogens. The seeds mature at the end of spring and germinate in autumn, remaining dormant during wet and warm summer. As the persistence of Epichloë endophytes in the seed is highly sensitive to temperature and humidity, we studied how the endophytes modulate seed germination in response to these factors in two B. auleticus ecotypes named La Pampa (LP) and El Palmar (EP). We also studied how environmental factors during germination affect endophyte survival. In the LP ecotype, adapted to coldest temperatures in autumn, Epichloë improved final germination under alternating temperatures, but had no effects on germination inhibition imposed by low water availability. In the EP ecotype, adapted to warm humid summer and autumn, the endophyte promoted germination at constant 25°C and alternating 15/25°C temperatures, and prevented germination at low water potentials. At 30°C, seed germination was inhibited and whereas seeds remained viable, endophyte survival was significantly lowered. The effect of Epichloë on B. auleticus seed germination was ecotype‐specific: promoting germination at alternating temperatures in La Pampa ecotype or at high temperatures in El Palmar ecotype, while inhibiting it at low water availabilities, could be an endophyte‐mediated ecotypic strategy minimising endophyte mortality and increasing seedling establishment. [ABSTRACT FROM AUTHOR]

Here we describe a new, haploid and stroma forming species within the genus Epichloë, as Epichloë scottii sp. nov. The fungus was isolated from Melica uniflora growing in Bad Harzburg, Germany. Phylogenetic reconstruction using a combined dataset of the tubB and tefA genes strongly support that E. scottii is a distinct species and the so far unknown ancestor species of the hybrid E. disjuncta. A distribution analysis showed a high infection rate in close vicinity of the initial sampling site and only two more spots with low infection rates. Genetic variations in key genes required for alkaloid production suggested that E. scottii sp. nov. might not be capable of producing any of the major alkaloids including ergot alkaloid, loline, indole-diterpene and peramine. All isolates and individuals found in the distribution analysis were identified as mating-type B explaining the lack of mature stromata during this study. We further release a telomere-to-telomere de novo assembly of all seven chromosomes and the mitogenome of E. scottii sp. nov. [ABSTRACT FROM AUTHOR]

In identifying endophytes for use in pastures in New Zealand there have been two strategies used, either exclusion of ergopeptine and lolitrem alkaloids or, retaining some ergopeptine expression to enhance the stability of natural defences against invertebrate pests. Both have their strengths and weaknesses. It is a matter of balancing these to ensure the end-user has access to a product that satisfies their needs – a persistent pasture with low to nil animal toxicity in terms of animal production and welfare. The range and intensity of ergot alkaloids in grazed pasture on both pasture and ruminants is reviewed, with emphasis on New Zealand. Ergot alkaloids associated with pasture are produced by associations between certain Epichloë endophyte strains and temperate grasses. Ergot alkaloids have been shown to improve persistence of pasture through providing resistance/deterrence to insect pests as well as deterring grazing animals. However, ergovaline is toxic to grazing animals. Some commercially available ryegrass-endophyte associations can produce ergovaline concentrations close to those found in associations between ryegrass and the standard endophyte. It is feasible to eliminate ergot alkaloids from pasture grasses in New Zealand as endophyte strains are commercially available that do not express ergovaline and yet still provide excellent pest resistance. [ABSTRACT FROM AUTHOR]

Argentine stem weevil adults (ASW, Listronotus bonariensis) feed on the leaves of agricultural grasses and their larvae mine the pseudostem, causing extensive damage that can result in plant death. Plants emit volatiles that serve as signals to host-searching insects and these odours can be altered by both herbivory and fungal endophyte-infection. This study investigated whether ASW adults utilise olfaction to identify their host plants, perennial ryegrass (Lolium perenne), and if conspecific herbivory or the presence of Epichloë festucae var. lolii fungal endophytes (strain wild-type or AR1) influenced such responses. Results from olfactometer bioassays established that ASW adults were able to utilise their olfactory response to orient towards volatiles released by perennial ryegrass and further, the weevils displayed a preference for plants previously damaged by conspecific weevils. However, there was no evidence that weevils had the ability to distinguish between endophyte-infected and endophyte-free plants using olfaction alone. Using a push–pull extraction technique, thirteen volatile compounds were identified in the blend released by perennial ryegrass. Endophyte and herbivory were found to alter these volatile compounds and quantities emitted by this forage grass. This study suggests that despite observing differences in the plant volatile blend, ASW do not perceive endophyte (wild-type and AR1) using olfaction alone and must rely on other cues, e.g. contact chemoreception or post-ingestional malaise, to detect the presence of a bioactive endophyte in an otherwise acceptable host plant. [ABSTRACT FROM AUTHOR]

Definition: Asexual Epichloë are obligate fungal mutualists that form symbiosis with many temperate grass species, providing several advantages to the host. These advantages include protection against vertebrate and invertebrate herbivores (i.e., grazing livestock and invertebrate pests, respectively), improved resistance to phytopathogens, increased adaptation to drought stress, nutrient deficiency, and heavy metal-containing soils. Selected Epichloë strains are utilised in agriculture mainly for their pest resistance traits, which are moderated via the production of Epichloë-derived secondary metabolites. For pastoral agriculture, the use of these endophyte infected grasses requires the balancing of protection against insect pests with reduced impacts on animal health and welfare. [ABSTRACT FROM AUTHOR]

Abstract Variation in plant communities is likely to modulate the feeding and oviposition behavior of herbivorous insects, and plant‐associated microbes are largely ignored in this context. Here, we take into account that insects feeding on grasses commonly encounter systemic and vertically transmitted (via seeds) fungal Epichloë endophytes, which are regarded as defensive grass mutualists. Defensive mutualism is primarily attributable to alkaloids of fungal origin. To study the effects of Epichloë on insect behavior and performance, we selected wild tall fescue (Festuca arundinacea) and red fescue (Festuca rubra) as grass–endophyte models. The plants used either harbored the systemic endophyte (E+) or were endophyte‐free (E−). As a model herbivore, we selected the Coenonympha hero butterfly feeding on grasses as larvae. We examined both oviposition and feeding preferences of the herbivore as well as larval performance in relation to the presence of Epichloë endophytes in the plants. Our findings did not clearly support the female's oviposition preference to reflect the performance of her offspring. First, the preference responses depended greatly on the grass–endophyte symbiotum. In F. arundinacea, C. hero females preferred E+ individuals in oviposition‐choice tests, whereas in F. rubra, the endophytes may decrease exploitation, as both C. hero adults and larvae preferred E− grasses. Second, the endophytes had no effect on larval performance. Overall, F. arundinacea was an inferior host for C. hero larvae. However, the attraction of C. hero females to E+ may not be maladaptive if these plants constitute a favorable oviposition substrate for reasons other than the plants' nutritional quality. For example, rougher surface of E+ plant may physically facilitate the attachment of eggs, or the plants offer greater protection from natural enemies. Our results highlight the importance of considering the preference of herbivorous insects in studies involving the endophyte‐symbiotic grasses as host plants.

Vertically transmitted endophytic fungi can mitigate the negative effects of salinity encountered by their host grass and alter the competitive interactions between plant individuals. To experimentally study the interactive effects of the fungal endophyte Epichloë coenophiala on salt tolerance and intraspecific competition of its host plant, tall fescue Festuca arundinacea, we subjected 15 maternal lines of each Epichloë associated (E+) and Epichloë free (E−) tall fescue to salt treatment and competition in the greenhouse and common garden. Then, to explore variation in endophyte incidence in natural populations of tall fescue, we surveyed 23 natural populations occurring on or near the Baltic Sea coast in Aland islands in southwestern Finland for endophyte incidence, distance to shore, and competitive environment. Under salinity in the greenhouse, E− plants grew larger than E+ plants, but there was no size difference in the control treatment. E− plants grew taller and were more likely to flower than E+ plants when grown in benign conditions in the common garden but not with salinity or competition. The frequency of Epichloë incidence was high (90%) in natural populations, and it decreased towards the shore and risk of salt exposure. These results demonstrate a negative effect of Epichloë endophyte on the salt tolerance of its host. The high incidence of Epichloë in natural populations of tall fescue in the northern part of the species distribution range is likely due to factors other than salinity.

Plants harbor a variety of fungal symbionts both above- and belowground, yet little is known about how these fungi interact within hosts, especially in a world where resource availability is changing due to human activities. Systemic vertically transmitted endophytes such as Epichloë spp. may have particularly strong effects on the diversity and composition of later-colonizing symbionts such as root fungal endophytes, especially in primary successional systems. We made use of a long-term field experiment in Great Lakes sand dunes to test whether Epichloë colonization of the dune-building grass, Ammophila breviligulata, could alter fungal root endophyte species richness or community composition in host plants. We also tested whether nitrogen addition intensified the effects of Epichlöe on the root endophyte community. We found that Epichloë increased richness of root endophytes in Ammophila by 17% overall, but only shifted community composition of root endophytes under nitrogen-enriched conditions. These results indicate that Epichlöe acts as a key species within Ammophila, changing richness and composition of the root mycobiome and integrating above- and belowground mycobiome interactions. Further, effects of Epichloë on root endophyte communities were enhanced by N addition, indicating that this fungal species may become even more important in future environments.

Epichloë endophytes are biotrophic fungi that establish mutualistic symbiotic relationship with grasses and affect performance of the host under different environments. Wild barley (Hordeum brevisubulatum) is an important forage grass and often infected by Epichloë bromicola, thus showing tolerances to stresses. Since the plant growth correlates with both microbial infection and nutrient stoichiometry, this study was performed to investigate whether the function of Epichloë bromicola endophyte to improve host growth depend upon the nitrogen (N), phosphorus (P) fertilization. Epichloë-infected (E+) and Epichloë-free (E-) wild barley plants were subjected to nine types of mixed N (0.2 mM, 3 mM, 15 mM) and P (0.01 mM, 0.1 mM, 1.5 mM) levels treatments for 90 d to collect plant samples and determine multiple related indexes. We found that E. bromicola and N, P additions positively affected seed germination. Further, E. bromicola significantly enhanced chlorophyll content and root metabolic activity under N-deficiency, and meanwhile, might alter allocation of photosynthate under different conditions. The contents of N, P and stoichiometry of C:N:P of E+ plants were significantly higher than that of E- under nutrient deficiency, but contrary results were observed under adequate nutrients. Therefore, we propose that the growth-promoting ability of E. bromicola is closely correlated with N and P additional levels. Under low N, P additions, positive roles of endophyte are significant as opposed to negative roles under high N, P additions. [ABSTRACT FROM AUTHOR]

Festuca rubra subsp. pruinosa is a perennial grass that inhabits sea cliffs, a habitat where salinity and low nutrient availability occur. These plants have a rich fungal microbiome, and particularly common are their associations with Epichloë festucae in aboveground tissues and with Fusarium oxysporum and Periconia macrospinosa in roots. In this study, we hypothesized that these fungi could affect the performance of F. rubra plants under salinity, being important complements for plant habitat adaptation. Two lines of F. rubra , each one consisting of Epichloë -infected and Epichloë -free clones, were inoculated with the root endophytes (F. oxysporum and P. macrospinosa) and subjected to a salinity treatment. Under salinity, plants symbiotic with Epichloë had lower Na+ content than non-symbiotic plants, but this effect was not translated into plant growth. P. macrospinosa promoted leaf and root growth in the presence and absence of salinity, and F. oxysporum promoted leaf and root growth in the presence and absence of salinity, plus a decrease in leaf Na+ content under salinity. The growth responses could be due to functions related to improved nutrient acquisition, while the reduction of Na+ content might be associated with salinity tolerance and plant survival in the long term. Each of these three components of the F. rubra core mycobiome contributed with different functions, which are beneficial and complementary for plant adaptation to its habitat in sea cliffs. Although our results do not support an obvious role of Epichloë itself in FRP salt tolerance, there is evidence that Epichloë can interact with root endophytes, affecting host plant performance. [ABSTRACT FROM AUTHOR]

Epichloë endophytes have been shown to be mutualistic symbionts of cool-season grasses under most environmental conditions. Although pairwise interactions between hemiparasites and their hosts are heavily affected by host-associated symbiotic microorganisms, little attention has been paid to the effects of microbe–plant interactions, particularly endophytic symbiosis, in studies examining the effects of parasitic plants on host performance. In this study, we performed a greenhouse experiment to examine the effects of hereditary Epichloë endophyte symbiosis on the growth of two host grasses (Stipa purpurea and Elymus tangutorum) in the presence or absence of a facultative root hemiparasite (Pedicularis kansuensis Maxim). We observed parasitism of both hosts by P. kansuensis: when grown with a host plant, the hemiparasite decreased the performance of the host while improving its own biomass and survival rate of the hemiparasite. Parasitized endophyte-infected S. purpurea plants had higher biomass, tillers, root:shoot ratio, and photosynthetic parameters and a lower number of functional haustoria than the endophyte-free S. purpurea conspecifics. By contrast, parasitized endophyte-infected E. tangutorum had a lower biomass, root:shoot ratio, and photosynthetic parameters and a higher number of haustoria and functional haustoria than their endophyte-free counterparts. Our results reveal that the interactions between the endophytes and the host grasses are context dependent and that plant–plant interactions can strongly affect their mutualistic interactions. Endophytes originating from S. purpurea alleviate the host biomass reduction by P. kansuensis and growth depression in the hemiparasite. These findings shed new light on using grass–endophyte symbionts as biocontrol methods for the effective and sustainable management of this weedy hemiparasite. [ABSTRACT FROM AUTHOR]

Festuca rubra subsp. pruinosa is a perennial grass that inhabits sea cliffs, a habitat where salinity and low nutrient availability occur. These plants have a rich fungal microbiome, and particularly common are their associations with Epichloë festucae in aboveground tissues and with Fusarium oxysporum and Periconia macrospinosa in roots. In this study, we hypothesized that these fungi could affect the performance of F. rubra plants under salinity, being important complements for plant habitat adaptation. Two lines of F. rubra, each one consisting of Epichloë-infected and Epichloë-free clones, were inoculated with the root endophytes (F. oxysporum and P. macrospinosa) and subjected to a salinity treatment. Under salinity, plants symbiotic with Epichloë had lower Na+ content than non-symbiotic plants, but this effect was not translated into plant growth. P. macrospinosa promoted leaf and root growth in the presence and absence of salinity, and F. oxysporum promoted leaf and root growth in the presence and absence of salinity, plus a decrease in leaf Na+ content under salinity. The growth responses could be due to functions related to improved nutrient acquisition, while the reduction of Na+ content might be associated with salinity tolerance and plant survival in the long term. Each of these three components of the F. rubra core mycobiome contributed with different functions, which are beneficial and complementary for plant adaptation to its habitat in sea cliffs. Although our results do not support an obvious role of Epichloë itself in FRP salt tolerance, there is evidence that Epichloë can interact with root endophytes, affecting host plant performance.