Endophytes Alleviate Drought-Derived Oxidative Damage in Achnatherum inebrians Plants Through Increasing Antioxidants and Regulating Host Stress Responses.

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.

Microbial symbionts buffer hosts from the demographic costs of environmental stochasticity.

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.

Anti-herbivory defences delivered by Epichloë fungal endophytes: a quantitative review of alkaloid concentration variation among hosts and plant parts.

BACKGROUND AND AIMS: In the subfamily Poöideae (Poaceae), certain grass species possess anti-herbivore alkaloids synthesized by fungal endophytes that belong to the genus Epichloë (Clavicipitaceae). The protective role of these symbiotic endophytes can vary, depending on alkaloid concentrations within specific plant-endophyte associations and plant parts. METHODS: We conducted a literature review to identify articles containing alkaloid concentration data for various plant parts in six important pasture species, Lolium arundinaceum, Lolium perenne, Lolium pratense, Lolium multiflorum|Lolium rigidum and Festuca rubra, associated with their common endophytes. We considered the alkaloids lolines (1-aminopyrrolizidines), peramine (pyrrolopyrazines), ergovaline (ergot alkaloids) and lolitrem B (indole-diterpenes). While all these alkaloids have shown bioactivity against insect herbivores, ergovaline and lolitrem B are harmful for mammals. KEY RESULTS: Loline alkaloid levels were higher in the perennial grasses L. pratense and L. arundinaceum compared to the annual species L. multiflorum and L. rigidum, and higher in reproductive tissues than in vegetative structures. This is probably due to the greater biomass accumulation in perennial species that can result in higher endophyte mycelial biomass. Peramine concentrations were higher in L. perenne than in L. arundinaceum and not affected by plant part. This can be attributed to the high within-plant mobility of peramine. Ergovaline and lolitrem B, both hydrophobic compounds, were associated with plant parts where fungal mycelium is usually present, and their concentrations were higher in plant reproductive tissues. Only loline alkaloid data were sufficient for below-ground tissue analyses and concentrations were lower than in above-ground parts. CONCLUSIONS: Our study provides a comprehensive synthesis of fungal alkaloid variation across host grasses and plant parts, essential for understanding the endophyte-conferred defence extent. The patterns can be understood by considering endophyte growth within the plant and alkaloid mobility. Our study identifies research gaps, including the limited documentation of alkaloid presence in roots and the need to investigate the influence of different environmental conditions.

Inbred progeny of tall fescue infected with Epichloë fungal endophyte have a high defence compound content and no depression in seed set.

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.

Elucidating the Molecular Mechanisms by which Seed-Borne Endophytic Fungi, Epichloë gansuensis, Increases the Tolerance of Achnatherum inebrians to NaCl Stress.

Seed-borne endophyte Epichloë gansuensis enhance NaCl tolerance in Achnatherum inebrians and increase its biomass. However, the molecular mechanism by which E. gansuensis increases the tolerance of host grasses to NaCl stress is unclear. Hence, we firstly explored the full-length transcriptome information of A. inebrians by PacBio RS II. In this work, we obtained 738,588 full-length non-chimeric reads, 36,105 transcript sequences and 27,202 complete CDSs from A. inebrians. We identified 3558 transcription factors (TFs), 15,945 simple sequence repeats and 963 long non-coding RNAs of A. inebrians. The present results show that 2464 and 1817 genes were differentially expressed by E. gansuensis in the leaves of E+ and E- plants at 0 mM and 200 mM NaCl concentrations, respectively. In addition, NaCl stress significantly regulated 4919 DEGs and 502 DEGs in the leaves of E+ and E- plants, respectively. Transcripts associated with photosynthesis, plant hormone signal transduction, amino acids metabolism, flavonoid biosynthetic process and WRKY TFs were differentially expressed by E. gansuensis; importantly, E. gansuensis up-regulated biology processes (brassinosteroid biosynthesis, oxidation-reduction, cellular calcium ion homeostasis, carotene biosynthesis, positive regulation of proteasomal ubiquitin-dependent protein catabolism and proanthocyanidin biosynthesis) of host grass under NaCl stress, which indicated an increase in the ability of host grasses' adaptation to NaCl stress. In conclusion, our study demonstrates the molecular mechanism for E. gansuensis to increase the tolerance to salt stress in the host, which provides a theoretical basis for the molecular breed to create salt-tolerant forage with endophytes.

Response of sheep rumen fermentation and microbial communities to feed infected with the endophyte Epichloë gansuensis as evaluated with rumen-simulating technology.

Achnatherum inebrians, a perennial grass, is widely distributed in China. When infected by the endophyte Epichloë gansuensis, A. inebrians produces an abundance of alkaloids that enhance plant survival but are toxic to animals. Here we used in vitro fermentation to study the impact of endophyte- infected A. inebrians (E+) addition on rumen fermentation characteristics and on microbial community and diversity as assessed with amplicon sequencing technology. We examined E+ addition at five levels, E0, E25, E50, E75, and E100, corresponding to 0%, 25%, 50%, 75%, and 100% of the fermentation substrate, respectively. Both the fermentation characteristics and rumen microbial community structure differed significantly among treatments. E100 resulted in the highest values for pH, the Shannon index, Kiritimatiellaeota, and Lentisphaerae levels relative to the other treatments. In contrast, E25 was associated with higher levels of ammonia nitrogen, total volatile fatty acid, propionate, butyrate, isobutyrate, valerate, of the phyla Bacteroidetes and Firmicutes, and of the genus Prevotella_1, Succiniclasticum, Family_XIII_AD3011_group, Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG-001, and Pyramidobacter as compared with other treatments. E50 resulted in the greatest values for the abundance-based coverage estimator (ACE) and the Chao1 index as compared with other treatments. E0 resulted in the greatest values for digestibility of dry matter, gas production, acetate, and Ruminobacter as compared with other treatments. This approach avoided animal toxicity experiments and confirmed that rumen fermentation characteristics and rumen microbiota were affected by E+ toxin. Therefore, E25 showed higher abundance in Prevotella_1, Prevotellaceae_ UCG-001, and Lachnospiraceae_XPB1014_group that implied they should play significant roles in E+ alkaloids degradation. And then, we can infer that rumen microorganisms should function as an antidote with respect to this poisoning reaction at moderate dietary percentages of E+.

Getting ready for the ozone battle: Vertically transmitted fungal endophytes have transgenerational positive effects in plants.

Ground-level ozone is a global air pollutant with high toxicity and represents a threat to plants and microorganisms. Although beneficial microorganisms can improve host performance, their role in connecting environmentally induced maternal plant phenotypes to progeny (transgenerational effects [TGE]) is unknown. We evaluated fungal endophyte-mediated consequences of maternal plant exposure to ozone on performance of the progeny under contrasting scenarios of the same factor (high and low) at two stages: seedling and young plant. With no variation in biomass, maternal ozone-induced oxidative damage in the progeny that was lower in endophyte-symbiotic plants. This correlated with an endophyte-mediated higher concentration of proline, a defence compound associated with stress control. Interestingly, ozone-induced TGE was not associated with reductions in plant survival. On the contrary, there was an overall positive effect on seedling survival in the presence of endophytes. The positive effect of maternal ozone increasing young plant survival was irrespective of symbiosis and only expressed under high ozone condition. Our study shows that hereditary microorganisms can modulate the capacity of plants to transgenerationally adjust progeny phenotype to atmospheric change.

Ontogenetic and trans-generational dynamics of a vertically transmitted fungal symbiont in an annual host plant in ozone-polluted settings.

Tropospheric ozone is an abiotic stress of increasing importance in the context of global climate change. This greenhouse gas is a potent phytotoxic molecule with demonstrated negative effects on crop yield and natural ecosystems. Recently, oxidative stress has been proposed as a mechanism that could regulate the interaction between cool-season grasses and Epichloë endophytes. We hypothesized that exposure of Lolium multiflorum plants, hosting endophytes to an ozone-polluted environment at different ontogenetic phases, would impact the trans-generational dynamics of the vertically transmitted fungal symbiont. Here, we found that the ozone-induced stress on the mother plants did not affect the endophyte vertical transmission but it impaired the persistence of the fungus in the seed exposed to artificial ageing. Endophyte longevity in seed was reduced by exposure of the mother plant to ozone. Although ozone exposure did not influence either the endophyte mycelial concentration or their compound defences (loline alkaloids), a positive correlation was observed between host fitness and the concentration of endophyte-derived defence compounds. This suggests that fungal defences in grass seeds were not all produced in situ but remobilized from the vegetative tissues. Our study reveals ozone trans-generational effects on the persistence of a beneficial symbiont in a host grass.

Role of Epichloë Endophytes in Improving Host Grass Resistance Ability and Soil Properties.

The past decade has witnessed significant advances in understanding the interaction between grasses and systemic fungal endophytes of the genus Epichloë, with evidence that plants have evolved multiple strategies to cope with abiotic stresses by reprogramming physiological responses. Soil nutrients directly affect plant growth, while soil microbes are also closely connected to plant growth and health. Epichloë endophytes could affect soil fertility by modifying soil nutrient contents and soil microbial diversity. Therefore, we analyze recent advances in our understanding of the role of Epichloë endophytes under the various abiotic stresses and the role of grass-Epichloë symbiosis on soil fertility. Various cool-season grasses are infected by Epichloë species, which contribute to health, growth, persistence, and seed survival of host grasses by regulating key systems, including photosynthesis, osmotic regulation, and antioxidants and activity of key enzymes of host physiology processes under abiotic stresses. The Epichloë endophyte offers significant prospects to magnify the crop yield, plant resistance, and food safety in ecological systems by modulating soil physiochemical properties and soil microbes. The enhancing resistance of host grasses to abiotic stresses by an Epichloë endophyte is a complex manifestation of different physiological and biochemical events through regulating soil properties and soil microbes by the fungal endophyte. The Epichloë-mediated mechanisms underlying regulation of abiotic stress responses are involved in osmotic adjustment, antioxidant machinery, photosynthetic system, and activity of key enzymes critical in developing plant adaptation strategies to abiotic stress. Therefore, the Epichloë endophytes are an attractive choice in increasing resistance of plants to abiotic stresses and are also a good candidate for improving soil fertility and regulating microbial diversity to improve plant growth.

Effects of the Clonostachys epichloë fungal hyperparasite on the symbiotic interaction between Botanophila flies and Epichloë fungus.

Botanophila flies are associated with Epichloë fungi in a symbiotic relationship in which the flies benefit from stromata as a food source for both adults and larvae, and the fungus benefits from the transfer of conidia by the flies, resulting in fertilization. Derogations from this pattern indicate that the Epichloë-Botanophila interaction cannot be clearly defined. The situation may be complicated by reports of new elements of the interaction, e.g., Wolbachia bacteria present in Botanophila larvae. The present study investigates the impact of Clonostachys epichloë (Speg.) Schroers, the fungal hyperparasite of Epichloë stromata, on the Botanophila-Epichloë interaction. The interaction between C. epichloë and Botanophila flies associated with Epichloë typhina subsp. clarkii (J.F. White) Leuchtm. & Schardl stromata was studied in the Holcus lanatus L. grass population. C. epichloë was present on 76.5% of stromata, covering on average 44.8 ± 32.1% of its surface and influencing the final perithecial coverage to the same extent as larval feeding. C. epichloë began to appear on stromata much later than the fly eggs and did not affect the preference for Botanophila egg laying. On the other hand, C. epichloë reduced larval hatching success and increased the mortality of the larvae. Clonostachys was responsible for 76.0% of all deaths, overgrowing brood chambers, and its mycelium was present both on and within larvae in all cases. Overall, as a result of the presence of C. epichloë, the number of Botanophila fly offspring decreased by 52.7%. Of the 26 surviving larvae, 10 (38.5%) were affected by C. epichloë, and their weight was significantly lower than that of unaffected larvae. Results show that C. epichloë, a new element of the interaction between E. typhina fungus and Botanophila flies, negatively affects both fungal reproduction and the offspring success of flies. This is the first report on the entomopathogenic activity of C. epichloë against Epichloë-associated Botanophila flies.

Phosphatidic acid produced by phospholipase D is required for hyphal cell-cell fusion and fungal-plant symbiosis.

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyze the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloë festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne. Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analyzed by light-, confocal- and electron microscopy. Changes in PA distribution were analyzed in E. festucae using a PA biosensor and the impact of these changes on the endocytic recycling and superoxide production investigated. We found that E. festucae PldB, and the N. crassa ortholog, PLA-7, are required for polarized growth and cell fusion and contribute to ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network for hyphal morphogenesis and growth of filamentous fungi.

Novel bioassay to assess antibiotic effects of fungal endophytes on aphids.

Perennial ryegrass is an important feed base for the dairy and livestock industries around the world. It is often infected with mutualistic fungal endophytes that confer protection to the plant against biotic and abiotic stresses. Bioassays that test their antibiotic effect on invertebrates are varied and range from excised leaves to whole plants. The aim of this study was to design and validate a "high-throughput" in-planta bioassay using 7-day-old seedlings confined in small cups, allowing for rapid assessments of aphid life history to be made while maintaining high replication and treatment numbers. Antibiosis was evaluated on the foliar and the root aphid species; Diuraphis noxia (Mordvilko) and Aploneura lentisci (Passerini) feeding on a range of perennial ryegrass-Epichloë festucae var. Lolii endophyte symbiota. As expected, both D. noxia and A. lentisci reared on endophyte-infected plants showed negatively affected life history traits by comparison to non-infected plants. Both species exhibited the highest mortality at the nymphal stage with an average total mortality across all endophyte treatments of 91% and 89% for D. noxia and A. lentisci respectively. Fecundity decreased significantly on all endophyte treatments with an average total reduction of 18% and 16% for D. noxia and A. lentisci respectively by comparison to non-infected plants. Overall, the bioassay proved to be a rapid method of evaluating the insecticidal activity of perennial ryegrass-endophyte symbiota on aphids (nymph mortality could be assessed in as little as 24 and 48 hours for D. noxia and A. lentisci respectively). This rapid and simple approach can be used to benchmark novel grass-endophyte symbiota on a range of aphid species that feed on leaves of plants, however we would caution that it may not be suitable for the assessment of root-feeding aphids, as this species exhibited relatively high mortality on the control as well.

Assortative mating in sympatric ascomycete fungi revealed by experimental fertilizations.

Mate recognition mechanisms resulting in assortative mating constitute an effective reproductive barrier that may promote sexual isolation and speciation. While such mechanisms are widely documented for animals and plants, they remain poorly studied in fungi. We used two interfertile species of Epichloë (Clavicipitaceae, Ascomycota), E. typhina and E. clarkii, which are host-specific endophytes of two sympatrically occurring grasses. The life cycle of these obligatory outcrossing fungi entails dispersal of gametes by a fly vector among external fungal structures (stromata). To test for assortative mating, we mimicked the natural fertilization process by applying mixtures of spermatia from both species and examined their reproductive success. Our trials revealed that fertilization is non-random and preferentially takes place between conspecific mating partners, which is indicative of assortative mating. Additionally, the viability of hybrid and non-hybrid ascospore offspring was assessed. Germination rates were lower in E. clarkii than in E. typhina and were reduced in ascospore progeny from treatments with high proportions of heterospecific spermatia. The preferential mating between conspecific genotypes and reduced hybrid viability represent important reproductive barriers that have not been documented before in Epichloë. Insights from fungal systems will deepen our understanding of the evolutionary mechanisms leading to reproductive isolation and speciation.

Infection Rates and Alkaloid Patterns of Different Grass Species with Systemic Epichloë Endophytes.

Symbiotic Epichloë species are fungal endophytes of cool-season grasses that can produce alkaloids with toxicity to vertebrates and/or invertebrates. Monitoring infections and presence of alkaloids in grasses infected with Epichloë species can provide an estimate of possible intoxication risks for livestock. We sampled 3,046 individuals of 13 different grass species in three regions on 150 study sites in Germany. We determined infection rates and used PCR to identify Epichloë species diversity based on the presence of different alkaloid biosynthesis genes, then confirmed the possible chemotypes with high-performance liquid chromatography (HPLC)/ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) measurements. Infections of Epichloë spp. were found in Festuca pratensis Huds. (81%), Festuca ovina L. aggregate (agg.) (73%), Lolium perenne L. (15%), Festuca rubra L. (15%) and Dactylis glomerata L. (8%). The other eight grass species did not appear to be infected. For the majority of Epichloë-infected L. perenne samples (98%), the alkaloids lolitrem B and peramine were present, but ergovaline was not detected, which was consistent with the genetic evaluation, as dmaW, the gene encoding the first step of the ergot alkaloid biosynthesis pathway, was absent. Epichloë uncinata in F. pratensis produced anti-insect loline compounds. The Epichloë spp. observed in the F. ovina agg. samples showed the greatest level of diversity, and different intermediates of the indole-diterpene pathway could be detected. Epichloë infection rates alone are insufficient to estimate intoxication risks for livestock, as other factors, like the ability of the endophyte to produce the alkaloids, also need to be assessed.IMPORTANCE Severe problems of livestock intoxication from Epichloë-infected forage grasses have been reported from New Zealand, Australia, and the United States, but much less frequently from Europe, and particularly not from Germany. Nevertheless, it is important to monitor infection rates and alkaloids of grasses with Epichloë fungi to estimate possible intoxication risks. Most studies focus on agricultural grass species like Lolium perenne and Festuca arundinacea, but other cool-season grass species can also be infected. We show that in Germany, infection rates and alkaloids differ between grass species and that some of the alkaloids can be toxic to livestock. Changes in grassland management due to changing climate, especially with a shift toward grasslands dominated with Epichloë-infected species such as Lolium perenne, may result in greater numbers of intoxicated livestock in the near future. We therefore suggest regular monitoring of grass species for infections and alkaloids and call for maintaining heterogenous grasslands for livestock.

The Impact of Endophyte Infection, Seed Aging, and Imbibition on Selected Sugar Metabolite Concentrations in Seed.

This study investigated effects of seed aging and imbibition on sugar metabolite concentrations in Epichloë endophyte-infected and endophyte-free seed of tall fescue ( Festuca arundinacea Schreb.). Two treatments, namely, accelerated aging and imbibition, were applied to the seeds, with embryo and endosperm tissues analyzed separately. Gas chromatography with flame ionization detection was employed for analysis of sugar metabolites within the seed tissues. Mannitol, ribitol, and trehalose were more abundant in embryo than endosperm tissues and were identified at consistently higher concentrations within endophyte-infected compared to endophyte-free seeds. The ratio of raffinose to sucrose significantly increased with seed aging in both endophyte-free and endophyte-infected embryo tissues, while significantly lower concentrations of trehalose were detected in tissues dissected from aged-seed regardless of endophyte status. This research provides fundamental insight into the metabolic details of endophyte survival in seed and provides a first evaluation of key carbohydrates present in the fungal-plant symbiosis.

The LaeA orthologue in Epichloë festucae is required for symbiotic interaction with Lolium perenne.

LaeA is a conserved global regulator of secondary metabolism and development in fungi. It is often required for successful pathogenic interactions. In this study, the laeA homologue in the fungal grass endophyte E. festucae was deleted and functionally characterised in vitro and its role in the mutualistic E. festucae interaction with Lolium perenne (perennial ryegrass) was determined. We showed that laeA in E. festucae is required for normal hyphal morphology, resistance to oxidative stress, and conidiation under nutrient-limited in vitro conditions. In planta studies revealed that laeA is expressed in a tissue-specific manner and is required to form a compatible plant interaction, with the majority of seedlings inoculated with a laeA deletion mutant either dying or being uninfected. In mature infected plants no difference was observed in the number or morphology of endophytic hyphae. However, the number of epiphyllous hyphae were greatly increased. Comparative transcriptomics analyses suggested roles for plant cell wall degradation, fungal cell wall composition, secondary metabolism and small-secreted proteins in Epichloë foliar symbiosis.

Local adaptation in natural European host grass populations with asymmetric symbiosis.

Recent work on microbiomes is revealing the wealth and importance of plant-microbe interactions. Microbial symbionts are proposed to have profound effects on fitness of their host plants and vice versa, especially when their fitness is tightly linked. Here we studied local adaptation of host plants and possible fitness contribution of such symbiosis in the context of abiotic environmental factors. We conducted a four-way multi-year reciprocal transplant experiment with natural populations of the perennial grass Festuca rubra s.l. from northern and southern Finland, Faroe Islands and Spain. We included F. rubra with and without transmitted symbiotic fungus Epichloë that is vertically transmitted via host seed. We found local adaptation across the European range, as evidenced by higher host fitness of the local geographic origin compared with nonlocals at three of the four studied sites, suggesting that selection pressures are driving evolution in different directions. Abiotic factors did not result in strong fitness effects related to Epichloë symbiosis, indicating that other factors such as herbivory are more likely to contribute to fitness differences between plants naturally occurring with or without Epichloë. Nevertheless, in the case of asymmetric symbiosis that is obligatory for the symbiont, abiotic conditions that affect performance of the host, may also cause selective pressure for the symbiont.

Heritable Epichloë symbiosis shapes fungal but not bacterial communities of plant leaves.

Keystone microbial species have driven eco-evolutionary processes since the origin of life. However, due to our inability to detect the majority of microbiota, members of diverse microbial communities of fungi, bacteria and viruses have largely been ignored as keystone species in past literature. Here we tested whether heritable Epichloë species of pooidae grasses modulate microbiota of their shared host plant.

Ergot alkaloid exposure during gestation alters. I. Maternal characteristics and placental development of pregnant ewes1.

Tall fescue [Lolium arundinaceum (Scheyreb.) Darbysh] is the primary cool season forage grass in the Southeastern United States. Most tall fescue contains an endophytic fungus (Epichloë coenophiala) that produces ergot alkaloids and upon ingestion induces fescue toxicosis. The objective of this study was to assess how exposure to endophyte-infected (E+; 1.77 mg hd-1 d-1 ergovaline and ergovalinine) or endophyte-free (E-; 0 mg hd-1 d-1 ergovaline and ergovalinine) tall fescue seed fed during 2 stages of gestation (MID, days 35-85/LATE, days 86-133) alters placental development. Thirty-six, fescue naïve Suffolk ewes were randomly assigned to 1 of 4 fescue treatments: E-/E-, E-/E+, E+/E-, or E+/E+. Ewes were individually fed the same amount of E+ or E- seed mixed into total mixed ration during MID and LATE gestation. Terminal surgeries were conducted on day 133 of gestation. Ewes fed E+ fescue seed had elevated (P < 0.001) ergot alkaloid excretion and reduced (P < 0.001) prolactin levels during the periods when fed E+ seed. Ewes switched on day 86 from E- to E+ seed had a 4% reduction (P = 0.005) in DMI during LATE gestation, which translated to a 2% reduction (P = 0.07) in DMI overall. Average daily gain was also reduced (P = 0.049) by 64% for E-/E+ ewes during LATE gestation and tended to be reduced (P = 0.06) by 33% overall. Ewes fed E+ seed during LATE gestation exhibited a 14% and 23% reduction in uterine (P = 0.03) and placentome (P = 0.004) weights, respectively. Caruncle weights were also reduced by 28% (P = 0.003) for E-/E+ ewes compared with E-/E- and E+/E-. Ewes fed E+ seed during both MID and LATE gestation exhibited a 32% reduction in cotyledon (P = 0.01) weights, whereas ewes fed E+ seed only during MID gestation (E+/E-) had improved (P = 0.01) cotyledon weights. The percentage of type A placentomes tended to be greater (P = 0.08) for E+/E+ ewes compared with other treatments. Other placentome types (B, C, or D) did not differ (P > 0.05). Total fetal weight per ewe was reduced (P = 0.01) for ewes fed E+ seed during LATE gestation compared with E-; however, feeding E+ seed during MID gestation did not alter (P = 0.70) total fetal weight per ewe. These results suggest that exposure to ergot alkaloids during LATE (days 86-133) gestation has the greatest impact on placental development by reducing uterine and placentome weights. This, in turn, reduced total fetal weight per ewe by 15% in ewes fed E+ seed during LATE gestation (E-/E+ and E+/E+).

Transcriptomics of Epichloë-Grass Symbioses in Host Vegetative and Reproductive Stages.

Epichloë species are fungal symbionts (endophytes) of cool-season grasses that transmit vertically via inflorescence primordia (IP), ovaries (OV), and ultimately, embryos. Epichloë coenophiala, an endophyte of tall fescue (Schedonorus arundinaceus), provides multiple protective benefits to the grass. We conducted transcriptome analysis of the tall fescue-E. coenophiala symbiosis, comparing IP, OV, vegetative pseudostems (PS), and the lemma and palea (LP) (bracts) of the young floret. Transcriptomes of host OV and PS exhibited almost no significant differences attributable to endophyte presence or absence. Comparison of endophyte gene expression in different plant parts revealed numerous differentially expressed genes (DEGs). The 150 endophyte DEGs significantly higher in PS over OV included genes for alkaloid biosynthesis and sugar or amino acid transport. The 277 endophyte DEGs significantly higher in OV over PS included genes for protein chaperones (including most heat-shock proteins), trehalose synthesis complex, a bax inhibitor-1 protein homolog, the CLC chloride ion channel, catalase, and superoxide dismutase. Similar trends were apparent in the Brachypodium sylvaticum-Epichloë sylvatica symbiosis. Gene expression profiles in tall fescue IP and LP indicated that the endophyte transcriptome shift began early in host floral development. We discuss possible roles of the endophyte DEGs in colonization of reproductive grass tissues.