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.

Toxigenic Endophyte-Infected Tall Fescue and Ergot Alkaloids.

"Fescue toxicosis" and reproductive ergotism present identical toxidromes in late-gestational mares and, likely, other equids. Both toxic syndromes are caused by ergopeptine alkaloids (EPAs) of fungal origin, and they are collectively referred to as equine ergopeptine alkaloid toxicosis (EEPAT). EPAs are produced by either a toxigenic endophyte (Epichloë coenophiala) in tall fescue and/or a nonendophytic fungus (Claviceps purpurea), infecting small grains and grasses. EEPAT can cause hypoprolactinemia-induced agalactia/dysgalactia, prolonged gestation, dystocia, and other reproductive abnormalities in mares, as well as failure of passive transfer in their frequently dysmature/overmature/postmature foals. Prevention relies on eliminating exposures and/or reversing hypoprolactinemia.

Ruminal ergovaline and volatile fatty acid dynamics: Association with poor performance and a key growth regulator in steers grazing toxic tall fescue.

Fescue toxicosis (FT) is produced by an ergot alkaloid (i.e., ergovaline [EV])-producing fungus residing in toxic fescue plants. Associations between EV, decreased weight gain and ruminal volatile fatty acids are unclear. Feces, rumen fluid, and blood were collected from 12 steers that grazed non-toxic (NT) or toxic (E +) fescue for 28 days. The E + group exhibited decreased propionate (P), increased acetate (A), and increased ruminal A:P ratio, with similar trends in feces. Plasma GASP-1 (G-Protein-Coupled-Receptor-Associated-Sorting-Protein), a myostatin inhibitor, decreased (day 14) only in E + steers. Ergovaline was present only in E + ruminal fluid and peaked on day 14. The lower ruminal propionate and higher A:P ratio might contribute to FT while reduced GASP-1 might be a new mechanism linked to E + -related weight gain reduction. Day 14 ergovaline zenith likely reflects ruminal adaptations favoring EV breakdown and its presence only in rumen points to local, rather than systemic effects.

Host Genetic Background Effect on Vertical Seed Transmission of Epichloë Endophyte Strains in Tall Fescue.

Tall fescue (Lolium arundinaceum (Schreb.) Darbysh.) is a cool-season perennial grass widely grown for forage and turf. Tall fescue lives in association with a fungal endophyte that helps the grass overcome abiotic and biotic stressors. The endophyte is asexual and transmits vertically from the tall fescue plant to the next generation through the seed. Producers of endophyte-infected tall fescue must have endophyte infection in at least 70% of their seed. Therefore, endophyte seed transmission is vital in breeding and seed production. Transfer of endophytes from their native host to different backgrounds of elite tall fescue cultivars can lead to a low seed transmission of the endophyte to the seed. This study screened 23 previously uncharacterized endophyte strains for transmissibility when artificially inoculated into continental and Mediterranean-type host tall fescue. We found no correlation between the rate of successful inoculation and the seed transmission rate of the endophyte in the new host. Nor did the seed transmission rate of the endophyte strains in their native host correlate with the seed transmission rate of the endophyte in the new host. Five strains exhibited seed transmission above 70% in both Mediterranean and Continental host backgrounds and will be characterized further for potential use in cultivar development.

Effect of arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) on microorganism of phenanthrene and pyrene contaminated soils.

A pot experiment was performed to investigate the effect of phytoremediation (CK, using tall fescue), fungi remediation (GV, using Glomus versiforme), bacterial remediation (PS, using Pseudomonas fluorescens Ps2-6), and microbial-phytoremediation (GVPS, using three species) on removing polycyclic aromatic hydrocarbons (PAHs) and the microbial diversity in soils. Inoculation with G. versiforme and P. fluorescens could increase the biomass of tall fescue and the accumulation of phenanthrene (PHE) and pyrene (PYR) in plants. Among them, the highest PHE and PYR removal efficiencies and highest biomass of tall fescue were observed in the GVPS treatment and the microbial diversity in contaminated soil was changed, the result revealed that Proteobacteria and Ascomycota were the dominant bacterial phylum and fungal phylum in all treatments, while more Proteobacteria were detected in GVPS treatment. At the genus level, the abundance of Sphingomonas (3.17%), Pseudomonas (2.05%), and Fusarium (8.65%) treated with GVPS increased compared with other treatments. These pieces of evidence contribute to a better understanding of the mechanisms involved in the combined microbial-phytoremediation strategies for PAHs-contaminated soils, especially the effects of microbial-phytoremediation on rhizosphere microbial diversity.

Inoculation with G. versiforme and P. fluorescens could increase PHE and PYR removal efficiencies, the biomass of tall fescue, microbial diversity, and the abundance of Proteobacteria, Sphingomonas, Pseudomonas, and Fusarium in (PHE + PYR)-contaminated soils.

Transcriptome analysis of Epichloë strains in tall fescue in response to drought stress.

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.

Differential gene expression in tall fescue tissues in response to water deficit.

Tall fescue (Festuca arundinacea Schreb.) is a popular pasture and turf grass particularly known for drought resistance, allowing for its persistence in locations that are unfavorable for other cool-season grasses. Also, its seed-borne fungal symbiont (endophyte) Epichloë coenophiala, which resides in the crown and pseudostem, can be a contributing factor in its drought tolerance. Because it contains the apical meristems, crown survival under drought stress is critical to plant survival as well as the endophyte. In this study, we subjected tall fescue plants with their endophyte to water-deficit stress or, as controls with normal watering, then compared plant transcriptome responses in four vegetative tissues: leaf blades, pseudostem, crown, and roots. A transcript was designated a differentially expressed gene (DEG) if it exhibited at least a twofold expression difference between stress and control samples with an adjusted p value of .001. Pathway analysis of the DEGs across all tissue types included photosynthesis, carbohydrate metabolism, phytohormone biosynthesis and signaling, cellular organization, and a transcriptional regulation. While no specific pathway was observed to be differentially expressed in the crown, genes encoding auxin response factors, nuclear pore anchors, structural maintenance of chromosomes, and class XI myosin proteins were more highly differentially expressed in crown than in the other vegetative tissues, suggesting that regulation in expression of these genes in the crown may aid in survival of the meristems in the crown.

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.

Antifeedant Effects and Repellent Activity of Loline Alkaloids from Endophyte-Infected Tall Fescue against Horn Flies, Haematobia irritans (Diptera: Muscidae).

Haematobia irritans is an obligate bloodsucking ectoparasite of cattle and is the global major pest of livestock production. Currently, H. irritans management is largely dependent upon broad-spectrum pesticides, which lately has led to the development of insecticide resistance. Thus, alternative control methods are necessary. Endophyte-infected grasses have been studied as an alternative due to their capability to biosynthesize alkaloids associated with anti-insect activities. Thus, the main aim of this study was to evaluate the antifeedant and repellent activity of lolines obtained from endophyte-infected tall fescue against H. irritans adults in laboratory conditions. The alkaloid extract (ALKE) was obtained by acid-base extraction. N-formyl loline (NFL) and N-acetyl loline (NAL) were isolated by preparative thin layer chromatography (pTLC) and column chromatography (CC), and the loline was prepared by acid hydrolysis of a NFL/NAL mixture. Loline identification was performed by gas chromatography coupled to mass spectrometry (GC/MS). Feeding behavior was evaluated by a non-choice test, and olfactory response was evaluated using a Y-tube olfactometer. Accordingly, all samples showed antifeedant activities. NFL was the most antifeedant compound at 0.5 µg/µL and 1.0 µg/µL, and it was statistically equal to NAL but different to loline; however, NAL was not statistically different to loline. NFL and NAL at 0.25 µg/µL were more active than loline. All samples except loline exhibited spatial repellency in the olfactometer. Thus, the little or non-adverse effects for cattle and beneficial activities of those lolines make them suitable candidates for horn fly management.

Rumen and Serum Metabolomes in Response to Endophyte-Infected Tall Fescue Seed and Isoflavone Supplementation in Beef Steers.

Fescue toxicosis impacts beef cattle production via reductions in weight gain and muscle development. Isoflavone supplementation has displayed potential for mitigating these effects. The objective of the current study was to evaluate isoflavone supplementation with fescue seed consumption on rumen and serum metabolomes. Angus steers (n = 36) were allocated randomly in a 2 × 2 factorial arrangement of treatments including endophyte-infected (E+) or endophyte-free (E-) tall fescue seed, with (P+) or without (P-) isoflavones. Steers were provided a basal diet with fescue seed for 21 days, while isoflavones were orally administered daily. Following the trial, blood and rumen fluid were collected for metabolite analysis. Metabolites were extracted and then analyzed by UPLC-MS. The MAVEN program was implemented to identify metabolites for MetaboAnalyst 4.0 and SAS 9.4 statistical analysis. Seven differentially abundant metabolites were identified in serum by isoflavone treatment, and eleven metabolites in the rumen due to seed type (p < 0.05). Pathways affected by treatments were related to amino acid and nucleic acid metabolism in both rumen fluid and serum (p < 0.05). Therefore, metabolism was altered by fescue seed in the rumen; however, isoflavones altered metabolism systemically to potentially mitigate detrimental effects of seed and improve animal performance.

Differentially expressed genes in cotyledon of ewes fed mycotoxins.

BACKGROUND: Ergot alkaloids (E+) are mycotoxins produced by the endophytic fungus, Epichloë coenophiala, in tall fescue that are associated with ergotism in animals. Exposure to ergot alkaloids during gestation reduces fetal weight and placental mass in sheep. These reductions are related to vasoconstrictive effects of ergot alkaloids and potential alterations in nutrient transport to the fetus. Cotyledon samples were obtained from eight ewes that were fed E+ (n = 4; E+/E+) or E- (endophyte-free without ergot alkaloids; n = 4; E-/E-) seed during both mid (d 35 to 85) and late (d 85-133) gestation to assess differentially expressed genes associated with ergot alkaloid induced reductions in placental mass and fetal weight, and discover potential adaptive mechanisms to alter nutrient supply to fetus. RESULTS: Ewes fed E+/E+ fescue seed during both mid and late gestation had 20% reduction in fetal body weight and 33% reduction in cotyledon mass compared to controls (E-/E-). Over 13,000 genes were identified with 110 upregulated and 33 downregulated. Four genes had a |log2FC| > 5 for ewes consuming E+/E+ treatment compared to controls: LECT2, SLC22A9, APOC3, and MBL2. REViGO revealed clusters of upregulated genes associated glucose, carbohydrates, lipid, protein, macromolecular and cellular metabolism, regulation of wound healing and response to starvation. For downregulated genes, no clusters were present, but all enriched GO terms were associated with anion and monocarboxylic acid transport. The complement and coagulation cascade and the peroxisome proliferator-activated receptor signaling pathway were found to be enriched for ewes consuming E+/E+ treatment. CONCLUSIONS: Consumption of ergot alkaloids during gestation altered the cotyledonary transcriptome specifically related to macronutrient metabolism, wound healing and starvation. These results show that ergot alkaloid exposure upregulates genes involved in nutrient metabolism to supply the fetus with additional substrates in attempts to rescue fetal growth.

Genome Resources for the Endophytic Fungus Paraphaeosphaeria sporulosa.

Paraphaeosphaeria genus includes plant pathogens or biocontrol agents as well as bioremediators and endophytic fungi. Paraphaeosphaeria sporulosa 10515 was isolated in 2013 as an endophyte of Festuca spp. collected on Mount Etna at 1,832 meters above sea level. Here, we present the first-draft whole-genome sequence of a P. sporulosa endophytic isolate. This data will be useful for future research on understanding the genetic bases of endophytism.

Mycelial biomass and concentration of loline alkaloids driven by complex population structure in Epichloë uncinata and meadow fescue (Schedonorus pratensis).

Many efforts have been made to select and isolate naturally occurring animal-friendly Epichloë strains for later reinfection into elite cultivars. Often this process involves large-scale screening of Epichloë-infected wild grass populations where strains are characterized and alkaloids measured. Here, we describe for the first time the use of genotyping-by-sequencing (GBS) on a collection of 217 Epichloë-infected grasses (7 S. arundinaceum, 4 L. perenne, and 206 S. pratensis). This genotyping strategy is cheaper than complete genome sequencing, is suitable for a large number of individuals, and, when applied to endophyte-infected grasses, conveniently genotypes both organisms. In total, 6273 single nucleotide polymorphisms (SNPs) in the endophyte data set and 38 323 SNPs in the host data set were obtained. Our findings reveal a composite structure with three distinct endophyte clusters unrelated to the three main S. pratensis gene pools that have most likely spread from different glacial refugia in Eurasia. All three gene pools can establish symbiosis with E. uncinata. A comparison of the endophyte clusters with microsatellite-based fingerprinting of the same samples allows a quick test to discriminate between these clusters using two simple sequence repeats (SSRs). Concentrations of loline alkaloids and mycelial biomass are correlated and differ significantly among the plant and endophyte subpopulations; one endophyte strain has higher levels of lolines than others, and one specific host genotype is particularly suitable to host E. uncinata. These findings pave the way for targeted artificial inoculations of specific host-endophyte combinations to boost loline production in the symbiota and for genome association studies with the aim of isolating genes involved in the compatibility between meadow fescue and E. uncinata.

Physiological response, function of sweat glands, and hair follicle cycling in cattle in response to fescue toxicosis and hair genotype.

Fescue toxicosis is a syndrome that results when cattle consume toxic endophyte-infected tall fescue. The objective of this study was to compare the response in physiological variables, sweat gland function, hair follicle cycling, and gene expression to feeding a total mixed ration that included tall fescue haylage and tall fescue seed containing a toxic endophyte (EI) or tall fescue haylage containing a nontoxic novel endophyte (EN) in beef heifers (Angus × Senepol heifers, n = 31) with 2 different hair genotypes. Numbers in each subgroup were as follows: novel endophyte, heterozygous slick (EN-S; n = 8), novel endophyte, homozygous hairy (wild type, EN-W; n = 7), endophyte-infected, heterozygous slick (EI-S; n = 10), and endophyte-infected, homozygous hairy (wild type, EI-W; n = 6). Physiological measurements were taken weekly for 7 wk. Data were analyzed using the MIXED procedure of SAS including dietary fescue treatment (EN vs. EI) and hair genotype (S vs. W) as main effects, day as a repeated measure, and temperature-humidity index (THI) as a covariate. Skin biopsies were taken before treatment initiation and on day 37 of treatment. Average surface temperature (ST) increased as the THI increased (P < 0.0001). Average ST was greater (P < 0.01) for animals fed EI than for animals fed the EN fescue diet, and greater (P < 0.01) for animals with the W genotype compared with animals with the S genotype. The difference between heifers with the S and W genotype was greater at greater THI (genotype × day interaction, P < 0.01). Transepidermal water loss (TEWL) was greater (P < 0.05) for animals with the S genotype compared with the W genotype and greater (P < 0.05) for heifers with the S genotype than for heifers with the W genotype when fed EI (36.7, 38.5, 30.0, and 38.7 g/m2 per hour for EN-W, EN-S, EI-W, and EI-S, respectively). The fraction of follicles in telogen in plucked hair samples for heifers fed EI was greater for animals with the S genotype than the W genotype (fraction in telogen: 0.456, 0.565, 0.297, 0.702 for EN-W, EN-S, EI-W, and EI-S, respectively; diet × genotype interaction, P < 0.05). Fraction of follicles in anagen was the opposite. EI fescue resulted in increased ST, changes in hair follicle cycling that support greater hair growth, and decreased TEWL for heifers with the W genotype compared with S genotype, suggesting greater heat stress in response to EI.

Soil organic matter prevails over heavy metal pollution and vegetation as a factor shaping soil microbial communities at historical Zn-Pb mining sites.

This study examined the effects of soil heavy metals, macronutrients, texture and pH as well as plant species richness and composition on soil respiration, enzymatic activity, microbial biomass, metabolic quotient (qCO2) and arbuscular mycorrhizal fungi (AMF) at sites of historical Zn-Pb mining. The study was conducted both on a large scale (65 heaps scattered over the area of 750 km2) and on a small scale (25 plots along two 48 m transects extending from heaps to adjacent fallow fields). Total concentrations of metals exceeded 400 (Cd), 20,000 (Pb) and 80,000 (Zn) mg kg-1 at the most polluted sites. Although they decreased along the heap-fallow direction, they still remained above environmental standards in fallow soils. In contrast, some soluble metal forms increased with the increasing distance from heaps. Soil organic matter had the strongest positive effect on most microbial parameters. Total and/or available heavy metals exhibited significant negative effects on microbial biomass, enzymatic activity and AMF, and a positive effect on qCO2. Organic matter alleviated negative effects of heavy metals on microorganisms; they were not observed where the increase in the contamination was accompanied by the increase in organic matter content. Plant species richness affected positively enzymatic activity and mycorrhization level. Plant species composition possibly contributed to the formation of soil microbial communities, but its effect was entangled in that of heavy metals as plant communities changed along pollution gradients (from metal-tolerant grasslands dominated by Festuca ovina to calcareous grasslands and ruderal communities at less polluted sites).

Toxicity of soil labile aluminum fractions and aluminum species in soil water extracts on the rhizosphere bacterial community of tall fescue.

Different forms of aluminum (Al) in soil can be toxic to plants and the bacterial community. In our previous study, the distribution and toxicity to plants of soil Al species and soil labile Al fractions were examined. However, the toxicity of different forms of Al on the bacterial community has not been completely studied. In this study, five soil samples (pH: 4.92, 6.17, 6.62, 6.70, 8.51) were collected from Lichuan, China. Tall fescue was planted in rhizosphere boxes with those soils for 120 days. The toxicity of soil Al species and soil labile Al fractions on the bacterial community of near-rhizosphere (NR) soils and far-rhizosphere (FR) soils were analyzed. The effect of different forms of Al on bacterial community between NR and FR soils was small, but the difference was obvious according to the different spatial distribution of samples. An individual bacterial community has eosinophilia, and most bacterial communities are tolerant of heavy metals (e.g., Cu, Zn, Cd). The toxicity of exchangeable Al has a strong effect on the bacterial community. Meanwhile, the toxicity of Al3+ to the bacterial community is strong. In this study, the key finding was that the toxicity of the Al-F- complex toward the bacterial community and plants was different. AlF2+, AlF2+, AlF3, and AlF4- are toxic for the bacterial community, and the correlation decreases with the addition of F-. This finding is of considerable significance to the treatment of acid-contaminated soil and the study of the tolerance mechanism of plants toward Al.

Comparative physiological and metabolomic analyses reveal mechanisms of Aspergillus aculeatus-mediated abiotic stress tolerance in tall fescue.

Aspergillus aculeatus has been shown to stimulate plant growth, but its role in plants abiotic stress tolerance and the underlying mechanisms are not fully documented. In this study, we investigated the mechanisms of A.aculeatus-mediated drought, heat and salt stress tolerance in tall fescue. The results showed that A.aculeatus inoculation improved drought and heat stress tolerance in tall fescue as observed from its effect on turf quality (TQ) and leaf relative water content (LWC). In the same stress conditions, A.aculeatus alleviated reactive oxygen species (ROS) induced burst and cell damage, as indicated by lower H2O2, electrolyte leakage (EL) and malondialdehyde (MDA) levels. Additionally, the A.aculeatus inoculated plants exhibited higher photosynthetic efficiency than uninoculated plants under drought, heat and salt stress conditions. The fungus reduced the uptake of Na+, and inoculated plants showed lower Na+/K+, Na+/Ca2+and Na+/Mg2+ ratios compared to uninoculated ones under salt stress. Furthermore, comparative metabolomic analysis showed that A.aculeatus exclusively increased amino acid (such as proline and glycine) and sugar (such as glucose, fructose, sorbose, and talose) accumulation under drought and heat stress. However, there were no differences between inoculated and uninoculated plants except for changes in H2O2 level, Na+ in the root and photosynthetic efficiency under salt stress. Taken together, this study provides the first evidence of the protective roles of A.aculeatus in the tall fescue response to abiotic stresses, partially via protection of photosynthesis and modulation of metabolic homeostasis.

Physiological consequences of gamma ray irradiation in tall fescue with elimination potential of Epichloë fungal endophyte.

Perennial plants and their associated microorganisms grow in the areas that may be contaminated with long-lived gamma-emitting radionuclides. This will induce gamma stress response in plants and their accompanying microorganisms. The present work investigated the growth and physiological responses of Epichloe endophyte infected tall fescue to gamma radiation, as well as whether the endophyte could persist and infect the host plant once exposed to gamma radiation. Seeds of Iranian native genotype of 75B+ of tall fescue were exposed to different doses, including 5.0, 10.0, 15.0, 20.0, 30.0 and 40.0 krad of gamma ray from a 60Co source. Irradiated and unirradiated seeds were sown in pots and grown under controlled conditions in the greenhouse. The growth and physiological parameters associated with plant tolerance to oxidative stress of host plants, as well as endophytic infection frequency (% of plants infected) and intensity (mean number of endophytic hyphae per the field of view), were examined in 3 months-old seedlings. The results indicated that all gamma radiation doses (except 5.0 kr) significantly reduced the height and survival percentage of the host plant. Days to the emergence of seedling increased gradually as gamma doses rose. A dose-rate dependent induction was seen for photosynthetic pigments and proline content. Malondialdehyde (MDA) content grew with elevation of irradiation doses. Depending on the dose and time, the activities of antioxidant enzymes in the host plant responded differently to gamma radiation. Gamma radiation altered the enzyme activities with sever decline in SOD and CAT activities. However, it had barely any effect on in APX and POD activities. The results also revealed that the persistence and intensity of endophyte were affected after gamma-ray irradiation. The initial percentage of tall fescue seeds infected with the endophyte was 91% in un-irradiated seeds. Presence of the viable endophyte started to decline significantly (23%) at 5.0 kr of gamma radiation. A dramatic reduction in the presence and intensity of endophyte occurred at 10.0 to 40.0 kr intensities. Gamma radiation × trait (GT)-biplot analysis indicated positive correlations between the endophyte symbiosis and antioxidant enzyme activities. Also, negative correlations were observed between the endophyte and MDA content in the host plant. Our results suggest that radiation stress (doses over 5.0 kr) caused reduction in the growth and antioxidant enzyme activities of the host plant that accompanied by a dramatic reduction in the persistence and intensity of endophyte fungi. Our findings have provided the basic information for future studies on the effect of gamma irradiation on the interaction between endophytic fungi and its host plant.

Transcriptome Analysis and Differential Expression in Tall Fescue Harboring Different Endophyte Strains in Response to Water Deficit.

Two tall fescue [Lolium arundinaceum (Schreb.) Darbysh. = Schedonorus arundinaceus (Schreb.) Dumort. = Festuca arundinacea var. arundinacea Schreb.] plant genotypes with an Epichloë coenophiala (Morgan-Jones & W. Gams) C.W. Bacon & Schardl common toxic endophyte (CTE), one with a nontoxic strain (NTE19) and one with another Epichloë species (FaTG-4) were evaluated and compared with their respective endophyte-free clones for responses to water-deficit stress in the greenhouse. One of the plant genotypes (P27) showed a positive effect of its CTE strain on tiller production after stress and resumed watering. In transcriptome analysis of the pseudostems (leaf sheath whorls), differentially expressed genes (DEGs) were defined as having at least twofold expression difference and false discovery rate (FDR) < 0.05 in comparisons of water treatment (stressed or watered), endophyte presence or absence, or both. Stress affected 38% of the plant transcripts including those for the expected stress-response pathways. The DEGs affected by endophyte in stressed plants were unique to individual plant genotypes. In unstressed plants, endophyte presence tended to reduce expression of genes putatively for defense against fungi, but in unstressed P27 endophyte presence there was enhanced expression of dehydrin and heat shock protein genes. Our results indicated subtle and variable effects of endophytes on tall fescue gene expression; where the endophyte confers protection, its effects on plant gene expression may help prime the plant for stress resistance.

Arbuscular mycorrhizal fungal community composition determines the competitive response of two grassland forbs.

We performed a greenhouse experiment to assess how differences in AM fungal community composition affect competitive response of grassland plant species. We used a full factorial design to determine how inoculation with natural AM fungal communities from different habitats in Western Estonia affects the growth response of two grassland forbs (Leontodon hispidus L., Plantago lanceolata L.) to competition with a dominant grass (Festuca rubra L.). We used AM fungal inocula that were known to differ in AM fungal diversity and composition: more diverse AM fungal communities from open grasslands and less diverse AM fungal communities from former grassland densely overgrown by pines (young pine forest). The presence of AM fungi balanced competition between forb and grass species, by enhancing competitive response of the forbs. The magnitude of this effect was dependent on forb species identity and on the origin of the AM fungal inoculum in the soil. The grassland inoculum enhanced the competitive response of the forb species more effectively than the forest inoculum, but inoculum-specific competitive responses varied according to the habitat preference of the forb species. Our findings provide evidence that composition and diversity of natural AM fungal communities, as well as co-adaptation of plant hosts and AM-fungal communities to local habitat conditions, can determine plant-plant interactions and thus ultimately influence plant community structure in nature.