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.

Two-speed genomes of Epichloe fungal pathogens show contrasting signatures of selection between species and across populations.

Antagonistic selection between pathogens and their hosts can drive rapid evolutionary change and leave distinct molecular footprints of past and ongoing selection in the genomes of the interacting species. Despite an increasing availability of tools able to identify signatures of selection, the genetic mechanisms underlying coevolutionary interactions and the specific genes involved are still poorly understood, especially in heterogeneous natural environments. We searched the genomes of two species of Epichloe plant pathogen for evidence of recent selection. The Epichloe genus includes highly host-specific species that can sterilize their grass hosts. We performed selection scans using genome-wide SNP data from seven natural populations of two co-occurring Epichloe sibling species specialized on different hosts. We found evidence of recent (and ongoing) selective sweeps across the genome in both species. However, selective sweeps were more abundant in the species with a larger effective population size. Sweep regions often overlapped with highly polymorphic AT-rich regions supporting the role of these genome compartments in adaptive evolution. Although most loci under selection were specific to individual populations, we could also identify several candidate genes targeted by selection in sweep regions shared among populations. The genes encoded small secreted proteins typical of fungal effectors and cell wall-degrading enzymes. By investigating the genomic signatures of selection across multiple populations and species, this study contributes to our understanding of complex adaptive processes in natural plant pathogen systems.

The growth promotion in endophyte symbiotic plants does not penalise the resistance to herbivores and bacterial microbiota.

A trade-off between growth and defence against biotic stresses is common in plants. Fungal endophytes of the genus Epichloë may relieve this trade-off in their host grasses since they can simultaneously induce plant growth and produce antiherbivore alkaloids that circumvent the need for host defence. The Epichloë ability to decouple the growth-defence trade-off was evaluated by subjecting ryegrass with and without Epichloë endophytes to an exogenous treatment with gibberellin (GA) followed by a challenge with Rhopalosiphum padi aphids. In agreement with the endophyte-mediated trade-off decoupling hypothesis, the GA-derived promotion of plant growth increased the susceptibility to aphids in endophyte-free plants but did not affect the insect resistance in endophyte-symbiotic plants. In line with the unaltered insect resistance, the GA treatment did not reduce the concentration of Epichloë-derived alkaloids. The Epichloë mycelial biomass was transiently increased by the GA treatment but at the expense of hyphal integrity. The response of the phyllosphere bacterial microbiota to both GA treatment and Epichloë was also evaluated. Only Epichloë, and not the GA treatment, altered the composition of the phyllosphere microbiota and the abundance of certain bacterial taxa. Our findings clearly demonstrate that Epichloë does indeed relieve the plant growth-defence trade-off.

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.

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.

Regulation of mineral elements in Hordeum brevisubulatum by Epichloë bromicola under Cd stress.

In this study, wild barley (Hordeum brevisubulatum) infected (E+) and uninfected (E-) by Epichloë bromicola were used for hydroponic experiments during the seedling stage. Various attributes, such as the effect of fungal endophyte on the growth and development of wild barley, the absorption of cadmium (Cd) and mineral elements (Ca, Mg, Fe, Mn, Cu, Zn), subcellular distribution, and chemical forms were investigated under CdCl2 stress. The results showed that the fungal endophy significantly reduced the Ca content and percentage of plant roots under Cd stress. The Fe and Mn content of roots, the mineral element content of soluble fractions, and the stems in the pectin acid or protein-chelated state increased significantly in response to fungal endophy. Epichloë endophyte helped Cd2+ to enter into plants; and reduced the positive correlation of Ca-Fe and Ca-Mn in roots. In addition, it also decreased the correlation of soluble components Cd-Cu, Cd-Ca, Cd-Mg in roots, and the negative correlation between pectin acid or protein-chelated Cd in stems and mineral elements, to increase the absorbance of host for mineral elements. In conclusion, fungal endophy regulated the concentration and distribution of mineral elements, while storing more Cd2+ to resist the damage caused by Cd stress. The study could provide a ground for revealing the Cd tolerance mechanism of endophytic fungal symbionts.

The present study is the first to study the effect of fungal endophy on essential mineral elements of plants under heavy metal stress, filling a gap in the existing research. The study could be helpful to reveal the mechanism of endophytic fungi to improve the host's tolerance to heavy metals and provide a foundation for the grass-endophyte symbionts to improve heavy metal-contaminated soils as ecological grasses.

Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density.

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

Genetic Covariation Between the Vertically Transmitted Endophyte Epichloë canadensis and Its Host Canada Wildrye.

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

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.

Epichloë Endophyte Infection Changes the Root Endosphere Microbial Community Composition of Leymus Chinensis Under Both Potted and Field Growth Conditions.

Epichloë endophytes can not only affect the growth and resistance of the host plant but also change the biotic and abiotic properties of the soil where the host is situated. Here, we used endophyte-infected (EI) and endophyte-free (EF) Leymus chinensis as plant materials, to study the microbial diversity and composition in the host root endosphere and rhizosphere soil under both pot and field conditions. The results showed that endophyte infection did not affect the diversity of either bacteria or fungi in the root zone. There were significant differences in both bacterial and fungal communities between the root endosphere and the rhizosphere, and between the field and the pot, while endophytes only affected root endosphere microbial communities. The bacterial families affected by endophyte infection changed from 29.07% under field conditions to 40% under pot conditions. In contrast, the fungal families affected by endophyte infection were maintained at nearly 50% under both field and pot conditions. That is to say, bacterial communities in the root endosphere were more strongly affected by environmental conditions, and in comparison, the fungal communities were more strongly affected by species specificity. Endophytes significantly affected the fungal community composition of the host root endosphere in both potted and field plants, only the effect was more obvious in potted plants. Endophyte infection increased the abundance of three fungal families (Thelebolaceae, Herpotrichiellaceae and Trimorphomycetaceae) under both field and potted conditions. In potted plants, endophytes also altered the dominant fungi from pathogenic Pleosporales to saprophytic Chaetomiaceae. Endophyte infection increased the relative abundance of arbuscular mycorrhizal fungi and saprophytic fungi, especially under potted conditions.Overall, endophytes significantly affected the fungal community composition of the host root endosphere in both potted and field plants. Endophytes had a greater impact on root endosphere microorganisms than the rhizosphere, a greater impact on fungal communities than bacteria, and a greater impact on root endosphere microorganisms under potted conditions than at field sites.

Mechanisms of Epichloë bromicola to Promote Plant Growth and Its Potential Application for Coix lacryma-jobi L. Cultivation.

Endophytic fungi play important roles in regulating plant growth and development and usually used as a promising strategy to enhance the biosynthesis of host valuable secondary metabolite, but the underlying growth-promoting mechanisms are only partly understood. In this study, the wild-type Arabidopsis thaliana seedlings co-cultured with fungal endophyte Epichloë bromicola showed auxin (IAA)-stimulated phenotypes, and the growth-promoting effects caused by E. bromicola were further verified by the experiments of spatially separated co-culture and fungal extract treatment. IAA was detected and identified in the extract of E. bromicola culture by LC-HRMS/MS, whereas 2,3-butanediol was confirmed to be the predominant volatile active compound in the diethyl ether and ethyl acetate extracts by GC-MS. Further study observed that IAA-related genes including synthesis key enzyme genes (CYP79B2, CYP79B3, NIT1, TAA1 and YUCCA1) and controlling polar transport genes (AUX1, BIG, EIR1, AXR3 and ARF1), were highly expressed at different periods after E. bromicola inoculation. More importantly, the introduction of fungal endophyte E. bromicola could effectively promote the growth and accumulation of coixol in Coix under soil conditions. Our study showed that endophytic fungus E. bromicola might be considered as a potential inoculant for improving medicinal plant growth.

Crude Extracts and Secondary Metabolites of Epichloë bromicola against Phytophthora infestans.

Potato late blight caused by Phytophthora infestans is still one of the main factors limiting potato production. Epichloë spp. can provide host plants with various resistances, which makes them show great potential in the biological control of diseases. In this study, we explored the potential biological activity of crude extracts of 20 strains of Epichloë bromicola to control P. infestans. The crude extracts of strains 1 and 8 showed significant antifungal activity with an inhibition rate of 88 % and 81 %, respectively, and showed different effects on the mycelium morphology of P. infestans observed by scanning electron microscopy. Moreover, the two crude extracts demonstrated an interesting therapeutic and protective effect on potato late blight, and none of the extracts had an adverse effect against zebrafish embryos. A total of 13 metabolites were isolated from the crude extract of strain 8, and these tested compounds showed a weak antifungal effect and the inhibition rate was less than 80 %. These findings suggested that strains 1 and 8 have potential for biocontrol of late potato blight.

Localisation of phosphoinositides in the grass endophyte Epichloë festucae and genetic and functional analysis of key components of their biosynthetic pathway in E. festucae symbiosis and Fusarium oxysporum pathogenesis.

Phosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signaling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localize the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P2, PI[4]P and PI[4,5]P2 in hyphae of the endophyte Epichloë festucae in axenic culture and during interaction with its grass host Lolium perenne. We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homolog of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did enable the detection of PI[3,4,5]P3 in septa and mycelium of E. festucae and showed that TepA is required for chemotropism in F. oxysporum. The identification of PI[3,4,5]P3 in ΔtepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P3 and that fungal PTEN homologs are functional lipid phosphatases. The F. oxysporum chemotropism defect suggests a conserved role of PTEN homologs in chemotaxis across protists, fungi and mammals.

Can Aphid Herbivory Induce Intergenerational Effects of Endophyte-conferred Resistance in Grasses?

Plants have evolved mechanisms to survive herbivory. One such mechanism is the induction of defences upon attack that can operate intergenerationally. Cool-season grasses (sub-family Pooideae) obtain defences via symbiosis with vertically transmitted fungal endophytes (genus Epichloë) and can also show inducible responses. However, it is unknown whether these herbivore-induced responses can have intergenerational effects. We hypothesized that herbivory by aphids on maternal plants induces the intergenerational accumulation of endophyte-derived defensive alkaloids and resistance intensification in the progeny. We subjected mother plants symbiotic or not with Epichloë occultans, a species known for its production of anti-insect alkaloids known as lolines, to the aphid Rhopalosiphum padi. Then, we evaluated the progeny of these plants in terms of loline alkaloid concentration, resistance level (through herbivore performance), and shoot biomass. Herbivory on mother plants did not increase the concentration of lolines in seeds but it tended to affect loline concentration in progeny plants. There was an overall herbivore-induced intergenerational effect increasing the endophyte-conferred defence and resistance. Symbiotic plants were more resistant to aphids and had higher shoot biomass than their non-symbiotic counterparts. Since maternal herbivory did not affect the loline concentrations in seeds, the greater resistance of the progeny could have resulted from an inherited mechanism of epigenetic regulation. It would be interesting to elucidate the origin of this regulation since it could come from the host or the fungal symbiont. Thus, endophyte-driven differential fitness between symbiotic and non-symbiotic plants might be higher as generations pass on in presence of herbivores.

Infection by Endophytic Epichloë sibirica Was Associated with Activation of Defense Hormone Signal Transduction Pathways and Enhanced Pathogen Resistance in the Grass Achnatherum sibiricum.

Epichloë endophytes can improve the resistance of host grasses to pathogenic fungi, but the underlying mechanisms remain largely unknown. Here, we used phytohormone quantifications, gene expression analysis, and pathogenicity experiments to investigate the effect of Epichloë sibirica on the resistance of Achnatherum sibiricum to Curvularia lunata pathogens. Comparison of gene expression patterns between endophyte-infected and endophyte-free leaves revealed that endophyte infection was associated with significant induction of 1,758 and 765 differentially expressed genes in the host before and after pathogen inoculation, respectively. Functional analysis of the differentially expressed genes suggested that endophyte infection could activate the constitutive resistance of the host by increasing photosynthesis, enhancing the ability to scavenge reactive oxygen species, and actively regulating the expression of genes with function related to disease resistance. We found that endophyte infection was associated with induction of the expression of genes involved in the biosynthesis pathways of jasmonic acid, ethylene, and pipecolic acid and amplified the defense response of the jasmonic acid/ethylene co-regulated EIN/ERF1 transduction pathway and Pip-mediated TGA transduction pathway. Phytohormone quantifications showed that endophyte infection was associated with significant accumulation of jasmonic acid, ethylene, and pipecolic acid after pathogen inoculation. Exogenous phytohormone treatments confirmed that the disease index of plants was negatively related to both jasmonic acid and ethylene concentrations. Our results demonstrate that endophyte infection can not only improve the constitutive resistance of the host to phytopathogens before pathogen inoculation but also be associated with enhanced systemic resistance of the host to necrotrophs after C. lunata inoculation.

Efficient nonenzymatic cyclization and domain shuffling drive pyrrolopyrazine diversity from truncated variants of a fungal NRPS.

Nonribosomal peptide synthetases (NRPSs) generate the core peptide scaffolds of many natural products. These include small cyclic dipeptides such as the insect feeding deterrent peramine, which is a pyrrolopyrazine (PPZ) produced by grass-endophytic Epichloë fungi. Biosynthesis of peramine is catalyzed by the 2-module NRPS, PpzA-1, which has a C-terminal reductase (R) domain that is required for reductive release and cyclization of the NRPS-tethered dipeptidyl-thioester intermediate. However, some PpzA variants lack this R domain due to insertion of a transposable element into the 3' end of ppzA We demonstrate here that these truncated PpzA variants utilize nonenzymatic cyclization of the dipeptidyl thioester to a 2,5-diketopiperazine (DKP) to synthesize a range of novel PPZ products. Truncation of the R domain is sufficient to subfunctionalize PpzA-1 into a dedicated DKP synthetase, exemplified by the truncated variant, PpzA-2, which has also evolved altered substrate specificity and reduced N-methyltransferase activity relative to PpzA-1. Further allelic diversity has been generated by recombination-mediated domain shuffling between ppzA-1 and ppzA-2, resulting in the ppzA-3 and ppzA-4 alleles, each of which encodes synthesis of a unique PPZ metabolite. This research establishes that efficient NRPS-catalyzed DKP biosynthesis can occur in vivo through nonenzymatic dipeptidyl cyclization and presents a remarkably clean example of NRPS evolution through recombinant exchange of functionally divergent domains. This work highlights that allelic variants of a single NRPS can result in a surprising level of secondary metabolite diversity comparable to that observed for some gene clusters.

Chromosome-level genomes provide insights into genome evolution, organization and size in Epichloe fungi.

Epichloe fungi are endophytes of cool season grasses, both wild species and commercial cultivars, where they may exhibit mutualistic or pathogenic lifestyles. The Epichloe-grass symbiosis is of great interest to agricultural research for the fungal bioprotective properties conferred to host grasses but also serves as an ideal system to study the evolution of fungal plant-pathogens in natural environments. Here, we assembled and annotated gapless chromosome-level genomes of two pathogenic Epichloe sibling species. Both genomes have a bipartite genome organization, with blocks of highly syntenic gene-rich regions separated by blocks of AT-rich DNA. The AT-rich regions show an extensive signature of RIP (repeat-induced point mutation) and the expansion of this compartment accounts for the large difference in genome size between the two species. This study reveals how the rapid evolution of repeat structure can drive divergence between closely related taxa and highlights the evolutionary role of dynamic compartments in fungal genomes.

Increase in Cd Tolerance through Seed-Borne Endophytic Fungus Epichloë gansuensis Affected Root Exudates and Rhizosphere Bacterial Community of Achnatherum inebrians.

Soil cadmium (Cd) pollution is a serious environmental problem imperiling food safety and human health. The endophyte Epichloë gansuensis can improve the tolerance of Achnatherum inebrians to Cd stress. However, it is still unknown whether and how the endophyte helps host plants build up a specific bacterial community when challenged by CdCl2. In this study, the responses of the structure and function of bacterial community and root exudates of E+ (E. gansuensis infected) and E- (E. gansuensis uninfected) plants to Cd stress were investigated. Analysis of bacterial community structure indicated that the rhizosphere bacterial community predominated over the root endosphere bacterial community in enhancing the resistance of CdCl2 in a host mediated by E. gansuensis. E+ plant strengthened the interspecific cooperation of rhizosphere bacterial species. Moreover, the analysis of root exudates demonstrated E. gansuensis and increased the contents of organic acids and amino acids under Cd stress, and most root exudates were significantly correlated with rhizosphere bacteria. These results suggested that E. gansuensis employed a specific strategy to recruit distinct rhizosphere bacterial species and relevant functions by affecting root exudates to improve the tolerance of the host to Cd stress. This study provides a firm foundation for the potential application of symbionts in improving phytostabilization efficiency.

Animal safety of a tall fescue endophyte (Epichloë sp.) in a perennial ryegrass (Lolium perenne) host.

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

Chitin Deacetylases Are Required for Epichloë festucae Endophytic Cell Wall Remodeling During Establishment of a Mutualistic Symbiotic Interaction with Lolium perenne.

Epichloë festucae forms a mutualistic symbiotic association with Lolium perenne. This biotrophic fungus systemically colonizes the intercellular spaces of aerial tissues to form an endophytic hyphal network and also grows as an epiphyte. However, little is known about the cell wall-remodeling mechanisms required to avoid host defense and maintain intercalary growth within the host. Here, we use a suite of molecular probes to show that the E. festucae cell wall is remodeled by conversion of chitin to chitosan during infection of L. perenne seedlings, as the hyphae switch from free-living to endophytic growth. When hyphae transition from endophytic to epiphytic growth, the cell wall is remodeled from predominantly chitosan to chitin. This conversion from chitin to chitosan is catalyzed by chitin deacetylase. The genome of E. festucae encodes three putative chitin deacetylases, two of which (cdaA and cdaB) are expressed in planta. Deletion of either of these genes results in disruption of fungal intercalary growth in the intercellular spaces of plants infected with these mutants. These results establish that these two genes are required for maintenance of the mutualistic symbiotic interaction between E. festucae and L. perenne.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.