Meta-analysis of endophyte-infected tall fescue effects on cattle growth rates.

The objective of this study was to quantitatively summarize literature reporting endophyte-infected (Neotyphodium coenophialum) tall fescue (Festuca arundinacea) effects on cattle ADG. This meta-analysis evaluated endophyte infection level, climate, and forage yield using a literature dataset of 138 treatments from 20 articles. Three infection level measurements were tested: endophyte infection as a percentage of infected tillers (E%); ergovaline concentration in ppb ([E]); and total ergot alkaloid concentration ([TEA]). Three types of climate variables were used: base values (temperature, humidity, and relative humidity), climate indices (heat index and temperature-heat index [THI]), and novel climate variables accounting for duration of climate effects. Mixed effect models, weighted by 1/SEM, including a random effect of study were built for each factorial combination of measurement method and climate variable group. Because many studies were missing SEM, two datasets were used: one containing only data with SEM reported and one that also included missing-SEM data. For the complete-SEM dataset (CSD), models were weighted by 1/SEM. In the missing-SEM dataset (MSD) the mean reported 1/SEM was assigned as the weight for all missing SEM treatments. Although 18 initial models were created (2 × 3 × 3 factorial approach), the backward stepwise derivation resulted in models that included only endophyte infection level, suggesting a negative relationship between infection level and ADG. The CSD models predicted ADG to decrease 39 and 33 g/d with each increase of 100 ppb of [TEA] and [E], and by 39 g/d for each increase of 10% E%. In the MSD dataset, predicted ADG decreased by 39 and 33 g/d with each increase of 100 ppb of [TEA] and [E], and by 47 g/d for each increase of 10% E%. All relationships reported had P < 0.05. After visual inspection of the data, piecewise regression was used to identify an infection threshold (IT) of 60 ppb [E] and 11 E%, where the effect of infection level was constant on either side of the IT. The ADG was 40% and 49% greater for infection levels below the IT for [E] and E%, respectively. Across THI values in the analysis, ADG decreases ranged from 11.2% to 45.0% for cattle grazing endophyte-infected tall fescue compared to non-ergot alkaloid endophyte infected tall fescue. Pasture E%, [E], and [TEA] have a negative relationship with ADG in growing cattle, and increasing temperature decreases ADG when infection level is greater than the IT.

Animal physiology and genetic aspects of ryegrass staggers in grazing sheep.

Ryegrass staggers (RGS) is a metabolic disease of herbivores, caused by the ingestion of perennial ryegrass (Lolium perenne L.) containing a fungal endophyte (Neotyphodium lolii) which produces a tremorgenic toxin, lolitrem B. RGS has a major economic impact for agriculture in New Zealand as well as internationally. Management of RGS in grazing sheep can be problematic, and there is an incomplete knowledge of the interaction between the toxin and the grazing animal. This review is focused on recent advances in understanding the molecular physiology of RGS in the affected animal as well as the influence of animal genetics on the degree of susceptibility to RGS. Investigations to date suggest that the primary target for toxin is the large conductance, calcium-activated, potassium (BK) channel, resulting in disruption of neuromuscular junction signalling. Genetic investigation has established the existence of genes influencing resistance to RGS, however their identity has not been confirmed and their impact has not been established. Studies to date suggest that a multi-gene selection approach will be necessary in order to develop an effective selection tool for use in the agricultural industries.

Pathological changes seen in horses in New Zealand grazing Mediterranean tall fescue (Lolium arundinaceum) infected with selected endophytes (Epichloë coenophiala) causing equine fescue oedema.

AIM: To investigate whether Mediterranean tall fescue (Lolium arundinaceum (Schreb.) Darbysh. (syn Festuca arundinacea)) infected with selected fungal endophytes (Epichloë coenophiala (formerly Neotyphodium coenophialum)) caused equine fescue oedema when grown in New Zealand, and to examine the pathological changes associated with this intoxication. METHODS: Horses were grazed on Mediterranean tall fescue that was infected with the endophytes AR542 (n=2), or AR584 (n=3), or Mediterranean tall fescue that was endophyte-free (n=2). Blood samples were taken up to 7 days after the start of feeding to detect changes in concentrations of total protein in serum and packed cell volume. Any horse showing clinical evidence of disease was subject to euthanasia and necropsy. RESULTS: Within 6 days, both horses grazing fescue infected with AR542 became depressed and lethargic. One horse grazing fescue infected with endophyte AR584 became depressed within a 5-day feeding period while another horse in this group died shortly after being removed from the AR584 pasture. The third horse in this group did not develop clinical signs within the 5-day feeding period. However, haemoconcentration and hypoproteinaemia was detected in all horses grazing Mediterranean tall fescue that was infected by AR542 or AR584 endophyte. No abnormalities were observed in horses grazing fescue that was endophyte-free. Necropsy examination was performed on two horses grazing fescue infected with AR542 and one horse grazing fescue infected with AR584. All three horses had marked oedema of the gastrointestinal tract. Histologically, the oedema was accompanied by large numbers of eosinophils, but no necrosis. CONCLUSIONS: Horses grazing Mediterranean tall fescue that was infected by AR542 or AR584 developed hypoproteinaemia and haemoconcentration, most likely due to leakage of plasma proteins into the gastrointestinal tract. This suggests that these selected endophytes produce a compound that is toxic to horses, although the toxic principle is currently unknown. CLINICAL RELEVANCE: Mediterranean tall fescue that is infected by AR542 or AR584 should not be fed to horses in New Zealand. This intoxication should be considered in horses in which a rapid onset of hypoproteinaemia and haemoconcentration is detected. This intoxication should also be considered if marked gastrointestinal oedema is observed.

Aboveground Epichloë coenophiala-Grass Associations Do Not Affect Belowground Fungal Symbionts or Associated Plant, Soil Parameters.

Cool season grasses host multiple fungal symbionts, such as aboveground Epichloë endophytes and belowground arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSEs). Asexual Epichloë endophytes can influence root colonization by AMF, but the type of interaction-whether antagonistic or beneficial-varies. In Schedonorus arundinaceus (tall fescue), Epichloë coenophiala can negatively affect AMF, which may impact soil properties and ecosystem function. Within field plots of S. arundinaceus that were either E. coenophiala-free (E-), infected with the common, mammal-toxic E. coenophiala strain (CTE+), or infected with one of two novel, non-toxic strains (AR542 NTE+ and AR584 NTE+), we hypothesized that (1) CTE+ would decrease AMF and DSE colonization rates and reduce soil extraradical AMF hyphae compared to E- or NTE+, and (2) this would lead to E- and NTE+ plots having greater water stable soil aggregates and C than CTE+. E. coenophiala presence and strain did not significantly alter AMF or DSE colonization, nor did it affect extraradical AMF hypha length, soil aggregates, or aggregate-associated C and N. Soil extraradical AMF hypha length negatively correlated with root AMF colonization. Our results contrast with previous demonstrations that E. coenophiala symbiosis inhibits belowground AMF communities. In our mesic, relatively nutrient-rich grassland, E. coenophiala symbiosis did not antagonize belowground symbionts, regardless of strain. Manipulating E. coenophiala strains within S. arundinaceus may not significantly alter AMF communities and nutrient cycling, yet we must further explore these relationships under different soils and environmental conditions given that symbiont interactions can be important in determining ecosystem response to global change.

Effects of Tall Fescue and Its Fungal Endophyte on the Development and Survival of Tawny-Edged Skippers (Lepidoptera: Hesperiidae).

Invasive, exotic grasses are increasing in tallgrass prairie and their dominance may be contributing to the decline of grassland butterflies through alterations in forage quality. Tall fescue (Schedonorus arundinaceus (Schreb.) Dumort.), an exotic grass covering millions of acres in the United States, can host a fungal endophyte, Epichloë coenophiala (Morgan-Jones & Gams). Alkaloids produced by the endophyte are known to be toxic to some foliar-feeding pest insects. Endophyte-infected tall fescue is commonly planted in hayfields, pastures, lawns, and is invading natural areas, but effects of the endophyte on nonpest insects such as butterflies are relatively unknown. Our objective was to investigate the role that tall fescue and its endophyte might play in the decline of grass skippers (Hesperiidae). We examined growth and survival parameters of tawny-edged skippers (Polites themistocles (Latreille)) that were reared on endophyte-infected tall fescue (E+), endophyte-free tall fescue (E-), and Kentucky bluegrass (KBG). KBG was included as a comparison because it is a cool season grass known to be palatable to P. themistocles larvae. Interestingly, results showed that the endophyte did not affect growth and survival of larvae compared to uninfected tall fescue, even though significant amounts of loline alkaloids (average 740 ppm) were measured in endophyte-infected plant material. Larvae feeding on KBG grew faster with greater survival rates than larvae on both tall fescue treatments. These results confirm that tall fescue invasion and dominance may be deteriorating the quality of grassland habitats for native pollinators; however, this effect does not appear to be linked to endophyte infection.

[Phylogeny of Neotyphodium endophyte from western Chinese Elymus species based on act sequences].

OBJECTIVE: We analyzed phylogenetic relationships between asexual endophytes isolated from western Chinese Elymus species. METHODS: Fifteen act sequences were cloned from asexual endophytes of the western Chinese Elymus species. The phylogenetic tree and network topology structure were constructed using act sequences obtained and Genbank. RESULTS: The act sequences from asexual endophyte were single-copy gene. The western Chinese asexual endophye (Neotyphodium spp.) has a different origination from the North American sexual endophyte ( Epichloë elymi) from Elymus species, but there is a closer relationship between Neotyphodium spp. and Neotyphodium chisosum from North American Achnatherum eminens. Further, endophyte gene-flow exists between sexual Neotyphodium chisosum from North American Achnatherum eminens ( Hap 8) and asexual Neotyphodium sp. from western Chinese Elymus species (Hap 3).

Is the vertical transmission of Neotyphodium lolii in perennial ryegrass the only possible way to the spread of endophytes?

Endophytic fungi live their whole life within host tissues usually without any visible symptoms. Their vertical transmission (seed-plant-seed) has been well described and documented. However, horizontal transmission (plant-plant) needs more clarification. The aim of this study was to assess the extent to which endophytes move vertically in ecotypes of perennial ryegrass and whether there is evidence for the horizontal transmission of endophytes. Ecotypes from grasslands in Poland were collected in the form of living plants and used for vertical transmission analysis. Plants, the seed collected from these plants and plants grown from this collected seed were tested for endophytic infection. Provided that all produced seeds were viable and able to germinate and produce seedlings, the vertical transmission of Neotyphodium endophytes in perennial ryegrass ecotypes was nearly complete. For the horizontal transmission experiment, endophyte-hosting plants (E+) and endophyte-free plants (E-) of four cultivars were planted in the field in close proximity on small plots that were frequently mown. These studies revealed that after 7 months of growth next to E+ plants, the characteristic Neotyphodium spp. mycelia were found in E- plants, which was especially true for plants growing in close proximity to the infected plants. The occurrence of horizontal transmission of endophytes has not been previously demonstrated.

Aboveground endophyte affects root volatile emission and host plant selection of a belowground insect.

Plants emit specific blends of volatile organic compounds (VOCs) that serve as multitrophic, multifunctional signals. Fungi colonizing aboveground (AG) or belowground (BG) plant structures can modify VOC patterns, thereby altering the information content for AG insects. Whether AG microbes affect the emission of root volatiles and thus influence soil insect behaviour is unknown. The endophytic fungus Neotyphodium uncinatum colonizes the aerial parts of the grass hybrid Festuca pratensis × Lolium perenne and is responsible for the presence of insect-toxic loline alkaloids in shoots and roots. We investigated whether endophyte symbiosis had an effect on the volatile emission of grass roots and if the root herbivore Costelytra zealandica was able to recognize endophyte-infected plants by olfaction. In BG olfactometer assays, larvae of C. zealandica were more strongly attracted to roots of uninfected than endophyte-harbouring grasses. Combined gas chromatography-mass spectrometry and proton transfer reaction-mass spectrometry revealed that endophyte-infected roots emitted less VOCs and more CO2. Our results demonstrate that symbiotic fungi in plants may influence soil insect distribution by changing their behaviour towards root volatiles. The well-known defensive mutualism between grasses and Neotyphodium endophytes could thus go beyond bioactive alkaloids and also confer protection by being chemically less apparent for soil herbivores.

Neotyphodium Endophyte Changes Phytoextraction of Zinc in Festuca arundinacea and Lolium perenne.

The effect of Neotyphodium endophytes on growth parameters and zinc (Zn) tolerance and uptake was studied in two grass species of Festuca arundinacea and Lolium perenne. Plants were grown under different Zn concentrations (control, 200, 400, 800, and 1800 mg kg(-1)) in potted soil for 5 months. The results showed that the number of plant tillers was 85 and 51% greater in endophyte infected Festuca (FaEI) and Lolium (LpEI), respectively, compared to their endophyte free (EF) plants. Roots and shoots dry weights in infected Festuca were 87 and 9% greater than non-infected counterparts but in opposite, EF Lolium had 47 and 8% greater root and shoot dry weights than LpEI. Endophyte infected Festuca and Lolium improved chlorophyll fluorescence as Fv/Fm at high concentrations of Zn, showing their better chlorophyll functions and significant reduction of Zn stress in endophyte infected plants. Shoots of endophyte infectedFestuca had 82% greater concentration of Zn than EF Festuca when grown in soil containing 1800 mg kg(-1) Zn. Festuca and Lolium may tolerate high Zn concentration in soil without reduction in shoot and root growth. Endophyte infection in Festuca may help the grass accumulate and transport more Zn in aboveground parts under Zn-stress, thereby aiding phytoremediation of contaminated soils.

Competitive outcomes depend on host genotype, but not clavicipitaceous fungal endophytes, in Lolium perenne (Poaceae).

UNLABELLED: • PREMISE OF THE STUDY: Cool-season grasses infected by endophytic fungal symbionts sometimes show improved tolerance of herbivory and abiotic stresses, but the potential effects of endophytes on host competitive ability are unclear. This study examined intraspecific competition among variable genotypes of Lolium perenne with and without the endophyte Epichloë festucae var. lolii (Clavicipitaceae, Ascomycota) to assess the effect of infection on competitive outcomes.• METHODS: Eleven host genotypes distributed among infected plants of four cultivars of L. perenne were used for two pairwise competition experiments. In the greenhouse, an infected or uninfected ramet of each genotype was grown alone and paired in competition with a ramet of an uninfected genotype of a native accession of L. perenne. Tiller numbers and root and shoot dry mass were determined for targets and competitor plants after 15 wk.• KEY RESULTS: Competition and host genotype had highly significant effects on all measured variables. However, endophyte-mediated effects on hosts were minor, and those that occurred depended on genotype. In addition, endophytic status of target plants did not influence tiller number or dry mass in 10 of 11 competitor genotypes, although these native genotypes did significantly vary in their competitive performance.• CONCLUSIONS: Genotypic variation among cultivars and accessions from native populations of L. perenne determines the effect of both intraspecific competition and infection by a fungal endophyte. However, in the absence of herbivory, asexual clavicipitaceous endophytes may not be as important to host success in competitive environments as previously thought in this globally important grass species.

The bull sperm microRNAome and the effect of fescue toxicosis on sperm microRNA expression.

Tall fescue [Schedonorus phoenix (Scop.) Holub] accounts for nearly 16 million hectares of pasture in the Southeastern and Mid-Atlantic U.S. due to its heat, drought, and pest resistance, conferred to the plant by its symbiotic relationship with the endophyte Neotyphodium coenophialum. The endophyte produces ergot alkaloids that have negative effects on the growth and reproduction of animals, resulting in the syndrome known as fescue toxicosis. The objectives of our study were to identify microRNA (miRNA) present in bovine sperm and to evaluate the effects of fescue toxicosis on sperm miRNA expression. Angus bulls were assigned to treatments of either toxic or non-toxic fescue seed diets. Semen was collected and subjected to RNA isolation. Three samples from each treatment group were chosen and pooled for deep sequencing. To compare miRNA expression between treatment groups, a microarray was designed and conducted. For each of the top ten expressed miRNA, target prediction analysis was conducted using TargetScan. Gene ontology enrichment was assessed using the Database for Annotation, Visualization and Integrated Discovery. Sequencing results elucidated the presence of 1,582 unique small RNA present in sperm. Of those sequences, 382 were known Bos taurus miRNA, 22 were known but novel to Bos taurus, and 816 were predicted candidate miRNA that did not map to any currently reported miRNA. Of the sequences chosen for microarray, twenty-two showed significant differential expression between treatment groups. Gene pathways of interest included: regulation of transcription, embryonic development (including blastocyst formation), Wnt and Hedgehog signaling, oocyte meiosis, and kinase and phosphatase activity. MicroRNA present in mature sperm appears to not only be left over from spermatogenic processes, but may actually serve important regulatory roles in fertilization and early developmental processes. Further, our results indicate the possibility that environmental changes may impact the expression of specific miRNA.

The role of the Oregon State University Endophyte Service Laboratory in diagnosing clinical cases of endophyte toxicoses.

The Oregon State University Colleges of Veterinary Medicine and Agricultural Sciences instituted the Endophyte Service Laboratory to aid in diagnosing toxicity problems associated with cool-season grasses in livestock. The endophyte (Neotyphodium coenophalum) present in tall fescue (Festuca arundinacea) produces ergopeptine alkaloids, of which ergovaline is the molecule used to determine exposure and toxicity thresholds for the vasoconstrictive conditions "fescue foot" and "summer slump". Another vasoconstrictive syndrome, "ergotism," is caused by a parasitic fungus, Claviceps purpurea, and its primary toxin, ergotamine. "Ryegrass staggers" is a neurological condition that affects livestock consuming endophyte (Neotyphodium lolii)-infected perennial ryegrass (Lolium perenne) with high levels of lolitrem B. HPLC-fluorescent analytical methods for these mycotoxins are described and were used to determine threshold levels of toxicity for ergovaline and lolitrem B in cattle, sheep, horses, and camels. In addition, six clinical cases in cattle are presented to illustrate diagnosis of these three diseases.

Neotyphodium coenophialum-infected tall fescue and its potential application in the phytoremediation of saline soils.

The growth response of endophyte-infected (EI) and endophyte-free (EF) tall fescue to salt stress was investigated under two growing systems (hydroponic and soil in pots). The hydroponic experiment showed that endophyte infection significantly increased tiller and leaf number, which led to an increase in the total biomass of the host grass. Endophyte infection enhanced Na accumulation in the host grass and improved Na transport from the roots to the shoots. With a 15 g l(-1) NaCl treatment, the phytoextraction efficiency of EI tall fescue was 2.34-fold higher than EF plants. When the plants were grown in saline soils, endophyte infection also significantly increased tiller number, shoot height and the total biomass of the host grass. Although EI tall fescue cannot accumulate Na to a level high enough for it to be termed a halophyte, the increased biomass production and stress tolerance suggested that endophyte/plant associations had the potential to be a model for endophyte-assisted phytoextraction in saline soils.

Species diversity of Epichloë symbiotic with two grasses from southern Argentinean Patagonia.

In this work we performed morphological and molecular phylogenetic analyses (based on sequences of calmodulin M [calM], translation-elongation factor 1-α [tefA] and ß-tubulin [tubB] genes) to characterize the diversity of Epichloë endophytes in Bromus setifolius and Phleum alpinum. The phylogenies obtained from the three genes were congruent and allowed differentiation of three lineages of endophytes that also presented morphological differences. One lineage corresponds to the previously described species Epichloë tembladerae, which is present in a wide range of native grasses from Argentina including B. setifolius and P. alpinum. Another genotype isolated only from B. setifolius is a non-hybrid endophyte, a rare condition for the South American Epichloë endophytes. Isolates of this genotype, described herein as a new variety, Epichloë typhina var. aonikenkana, presented waxy colonies at maturity and a low production of conidia. The third lineage, exclusively found in isolates from P. alpinum, is a hybrid between E. typhina and a common ancestor of E. amarillans and E. baconii. Isolates of this lineage produce abundant conidia that are variable in shape and size. Based on its unique phylogenetic position and morphology, we propose the new species, Epichloë cabralii for this lineage. The new combinations Epichloë tembladerae and E. pampeana also are proposed for the previously described Neotyphodium tembladerae and Neotyphodium pampeanum species.

Cytotoxic effect of ergot alkaloids in Achnatherum inebrians infected by the Neotyphodium gansuense endophyte.

Ergonovine or ergonovinine was isolated from the aerial parts of endophyte (Neotyphodium gansuense) infected (E+) drunken horse grass (Achnatherum inebrians), neither of which existed in endophyte-free (E-) plants. Both of these ergot alkaloids had a cytotoxic effect on animal smooth muscle cells and increased cell growth inhibition with greater concentrations, in a significantly (P < 0.05) positive correlation. The median inhibitory concentrations (IC50) for ergonovine and ergonovinine were 71.95 and 72.75 µg/mL, respectively. These results indicate that endophytic ergot alkaloids may be the cause of drunken horse grass poisoning.

Detection and quantification of three distinct Neotyphodium lolii endophytes in Lolium perenne by real time PCR of secondary metabolite genes.

Perennial ryegrass (Lolium perenne) is a widely used pasture grass, which is frequently infected by Neotyphodium lolii endophytes that enhance grass performance but can produce alkaloids inducing toxicosis in livestock. Several selected endophyte strains with reduced livestock toxicity, but that confer insect resistance, are now in common use. Little is known regarding the survival and persistence of these endophytes when in competition with common toxic endophytes. This is mainly because there are currently no assays available to easily and reliably quantify different endophytes in pastures or in batches of seeds infected with multiple strains. We developed real time PCR assays, based on secondary metabolite genes known to differ between N. lolii endophyte strains, to quantify two selected endophytes, AR1 and AR37, and a common toxic ecotype used in New Zealand. A duplex PCR allowed assessment of endophyte:grass DNA ratios with high sensitivity, specificity and precision. Endophyte specific primers/probes could detect contamination of AR37 seeds with other endophytes down to a level of 3-25%. We demonstrated that it is possible to quantify different endophyte strains simultaneously using multiplex PCR. This method has potential applications in management of endophytes in pastures and in fundamental research into this important plant-microbe symbiosis.

Evidence of dehydration and electrolyte disturbances in cases of perennial ryegrass toxicosis in Australian sheep.

CASE REPORT: Perennial ryegrass toxicosis (PRGT) is a common disease entity in Australia, presenting as an association of clinical signs including alterations in normal behavioural, ataxia ('staggers'), ill thrift and gastrointestinal dysfunction ('scours'). Clinical signs can range in severity from mild (gait abnormalities and failure to thrive) to severe (seizures, lateral recumbency and death). Presentation across the flock is usually highly variable. PRGT is caused by toxins produced by the endophytic fungus Neotyphodium lolii, a symbiont of perennial ryegrass that is present in pastures across the temperate regions of Australia and Tasmania. A particular feature of PRGT in Australia is the occasional occurrence of large-scale sheep losses, suggesting other factors are influencing mortality rates compared with other PRGT risk zones such as North America and New Zealand. During 2011, producers in the state of Victoria experienced a mild outbreak of PRGT that affected large numbers of animals but with limited mortalities. Clinical samples taken from affected sheep showed a high incidence of dehydration and electrolyte abnormalities. CONCLUSION: We speculate that changes in hydration status may be a contributory aetiological factor in those years in which high numbers of deaths are associated with PRGT outbreaks in Australia.

Does fungal endophyte infection improve tall fescue's growth response to fire and water limitation?

Invasive species may owe some of their success in competing and co-existing with native species to microbial symbioses they are capable of forming. Tall fescue is a cool-season, non-native, invasive grass capable of co-existing with native warm-season grasses in North American grasslands that frequently experience fire, drought, and cold winters, conditions to which the native species should be better-adapted than tall fescue. We hypothesized that tall fescue's ability to form a symbiosis with Neotyphodium coenophialum, an aboveground fungal endophyte, may enhance its environmental stress tolerance and persistence in these environments. We used a greenhouse experiment to examine the effects of endophyte infection (E+ vs. E-), prescribed fire (1 burn vs. 2 burn vs. unburned control), and watering regime (dry vs. wet) on tall fescue growth. We assessed treatment effects for growth rates and the following response variables: total tiller length, number of tillers recruited during the experiment, number of reproductive tillers, tiller biomass, root biomass, and total biomass. Water regime significantly affected all response variables, with less growth and lower growth rates observed under the dry water regime compared to the wet. The burn treatments significantly affected total tiller length, number of reproductive tillers, total tiller biomass, and total biomass, but treatment differences were not consistent across parameters. Overall, fire seemed to enhance growth. Endophyte status significantly affected total tiller length and tiller biomass, but the effect was opposite what we predicted (E->E+). The results from our experiment indicated that tall fescue was relatively tolerant of fire, even when combined with dry conditions, and that the fungal endophyte symbiosis was not important in governing this ecological ability. The persistence of tall fescue in native grassland ecosystems may be linked to other endophyte-conferred abilities not measured here (e.g., herbivory release) or may not be related to this plant-microbial symbiosis.