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

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

A trade-off between growth and defence functions is commonly observed in plants. We propose that the association of plants with Epichloe fungal endophytes may eliminate this trade-off. This would be a consequence of the double role of these endophytes in host plants: the stimulation of plant growth hormones (e.g. gibberellins) and the fungal production of antiherbivore alkaloids. We put forward a model that integrates this dual effect of endophytes on plant growth and defence and test its predictions by means of meta-analysis of published literature. Our results support the notion that the enhanced plant resistance promoted by endophytes does not compromise plant growth. The limits and ecological benefits of this endophyte-mediated lack of plant growth-defence trade-off are discussed.

Plants secrete various defence-related proteins into the apoplast, including proteases. Papain-like cysteine proteases (PLCPs) are central components of the plant immune system. To overcome plant immunity and successfully colonize their hosts, several plant pathogens secrete effector proteins inhibiting plant PLCPs. We hypothesized that not only pathogens, but also mutualistic microorganisms interfere with PLCP-meditated plant defences to maintain endophytic colonization with their hosts. Epichloë festucae forms mutualistic associations with cool season grasses and produces a range of secondary metabolites that protect the host against herbivores. In this study, we performed a genome-wide identification of Lolium perenne PLCPs, analysed their evolutionary relationship, and classified them into nine PLCP subfamilies. Using activity-based protein profiling, we identified four active PLCPs in the apoplast of L. perenne leaves that are inhibited during endophyte interactions. We characterized the L. perenne cystatin LpCys1 for its inhibitory capacity against ryegrass PLCPs. LpCys1 abundance is not altered during the mutualistic interaction and it mainly inhibits LpCP2. However, since the activity of other L. perenne PLCPs is not sensitive to LpCys1, we propose that additional inhibitors, likely of fungal origin, are involved in the suppression of apoplastic PLCPs during E. festucae infection.

Epichloid fungal endophytes (Epichloe spp., Ascomycota: Clavicipitaceae) inhabit aerial tissues of several cool-season grasses, and enhance host growth and defence against herbivores. The presence of these symbionts can also affect interactions between the host and other non-epichloid plants. The role of an epichloid endophyte on interspecific competition has been tested using perennial grasses with contrasting results, but it has been scarcely tested using annual species in agroecosystems. We evaluated the impact of Epichloe-grass symbiosis on the competitive interaction between a non-host cereal crop (Triticum aestivum, wheat) and a host weed (Lolium multiflorum, ryegrass), growing in the presence of invertebrate herbivores (aphids) under no resource limitation. We conducted an outdoor mesocosm experiment with wheat plants growing in monoculture or in mixture with low or high proportions of ryegrass plants. Ryegrass plants presented either low (E-) or high (E+) incidence of Epichloe occultans (i.e. frequency of epichloid endophytic plants). We measured wheat vegetative and reproductive yield and its natural aphid infestation. Although epichloid endophyte incidence did not affect ryegrass biomass, wheat reproductive yield in mixtures (relative to wheat monocultures) was 45% higher when grown with E+ ryegrass plants than E- conspecific plants. Aphids preferred wheat plants grown with E- plants rather than wheat plants grown with E+ plants, but only in mixtures with high proportion of ryegrass. Our results demonstrate that epichloid endophyte incidence can decrease host competitive ability and confers associational protection to the non-endophytic neighbouring plants. Thus, ryegrass-endophyte symbiosis can increase crop yield by positive neighbourhood effects through different mechanisms probably related to the density of the weed. The benefits of this endosymbiont cannot be considered host-exclusive since they can be disseminated to non-endophytic plants. Furthermore, our results suggest that the epichloid endophyte incidence on annual weeds can contribute to agroecosystem sustainability by influencing pest management and increasing crop yield.

Plant-endophyte associations play important roles in grassland agricultural ecology. Many studies indicate that endophytic fungi can promote host growth and reduce the biotic and abiotic stresses of host plants. Epichloe endophytes of the family Clavicitaceae are fungal symbionts of Pooideae grasses. These endophytes usually do not express any obvious host symptoms. Many Epichloe endophytes can endow hosts with increased abiotic stress tolerance as a result of enhanced growth, tillering, reproduction and nutrient acquisition, particularly under conditions of drought, cold, salt and nutrient deficiencies and thus, have important roles in pastoral agricultural systems. Animal-safe grass-endophyte associations that confer bio-protective properties for increased pasture persistence and productivity have been developed and commercialized. Artificial inoculation with symbiotic Epichloe microbes is an important technique for the creation of novel germplasm. Selected Epichloe strains can add value to some grass-based forage systems by providing both biotic and abiotic stress resistance. In addition, they can improve and strengthen grass physiological functions and plant vigor. The Epichloe bromicola WBE1 endophyte of Hordeum brevisubulatum played an important role in maintaining the growth of host grass by promoting nutrient absorption and maintaining the ionic balance under salt stress. E. bromicola WBE1 can produce peramine, an alkaloid produced by Epichloe species, that protects host grasses from herbivorous insects. However, this strain was unable to synthesize the alkaloids that are toxic to livestock, such as ergine, ergonovine, ergovaline or lolitrem B. Barley ( Hordeum vulgare ) is one of the most important cereal crops. The importance of barley to human and animal nutrition, and indeed to the foundation and maintenance of human civilization, is well documented. Traditional barley breeding for improved environmental stress tolerance has resulted in good varieties. However, conventional breeding techniques neglect the microorganisms in plants. In this study, animal-safe endophytic E. bromicola WBE1 isolated from wild barley ( H. brevisubulatum ) was artificially inoculated into cultivated hulled barley ( H. vulgare cv. Yangsimai No. 1) and hull-less barley ( H. vulgare var. nudum cv. Chaiqing No. 1), creating novel barley germplasm. Plants that were inoculated with endophytes and those that were free of endophytes were evaluated for growth in the field. The plant height, tiller numbers, biomass and the grain weight per plant of endophyte-inoculated hulled barley plants (Yangsimai No. 1) were 5%, 20%, 46% and 22% higher than those of the control hulled barley plants, respectively, and they matured about 5 days earlier. The tiller numbers, biomass and grain weight per plant of endophyte-inoculated hull-less barley plants (Chaiqing No. 1) were 29%, 37% and 28% higher, respectively, than those of the control hull-less barley plants in the field. However, there was no significant difference between inoculated hull-less barley plants and control hull-less barley plants in the plant height and growth period. A novel germplasm of barley containing the endophyte was successfully created. E. bromicola improves plant characteristics and the growth of host. The results presented here provide evidence that the inoculation of barley with E. bromicola may be used to improve its germplasm. E. bromicola can be successfully inoculated into other phylogenetically close host species, and this technique has the potential to improve the growth of Hordeae cereal crops and serve as a broad application to produce agriculturally useful synthetic novel symbioses in germplasm.

The symbiotic relationship between Lolium perenne and Epichloe festucae var. lolii, a fungal endophyte that lives asymptomatically in its above-ground parts, benefits both the host and fungus. In an outdoor container experiment, the effects of three distinct endophyte haplotypes (AR1, AR37 and CT) on natural infestations of a root aphid, Aploneura lentisci and a mealybug Phenococcus graminicola were compared with an endophyte-free (Nil) control. Populations of these insects were suppressed in a genetically identical set of plants treated with insecticide. Over 18 months, foliar and root growth, tiller numbers and plant mortality were compared between the different endophyte treatments and between insecticide-treated and untreated plants. Hyphal densities were recorded once during the trial. Untreated plants infected with AR37 had the lowest populations of A. lentisci and P. graminicola, and the highest root and foliar growth compared with other treatments. Growth parameters of untreated plants infected with CT often exceeded that of AR1 and Nil due to low infestations of P. graminicola and lower populations of A. lentisci. Growth of Nil, AR1 and CT plants were higher in insecticide-treated than in corresponding untreated plants but was unaffected in AR37. Untreated Nil and AR1 plants had a lower survival than AR37 and CT plants. Cumulative root outgrowth for treated AR37 plants was significantly lower than for treated AR1 on three sampling occasions. These Epichloe haplotypes are primarily mutualists, although there are circumstances when they may be disadvantageous to their host. Opposing outcomes likely originate in the ecosystems in which associations evolved.

Fungal Epichloe endophytes form symbiotic associations with many temperate grasses, such as Lolium and Festuca, giving their host grasses an ecological advantage. The importance of specific Epichloe endophytes in providing varying levels of protection against invertebrate pests has been well documented. Similarly, but with fewer studies, the benefits of Epichloe to host grasses in drought events has been shown. Endophyte-infected grasses show an improved persistence against herbivore insect attack as well as resilience under drought. However, there are relatively few studies that investigate the interaction between drought and insect pressure, and yet it is these combined pressures that can prove detrimental for a ryegrass or fescue crop. This review examines the current state of knowledge on the effects of Epichloe on the interactions of insects and drought in temperate grasses.

The stromata of Epichloe fungi are structures covering part of the stem of grasses. Under the fungal layer, still green tissues of the plant survive, although the development of the new leaves is inhibited. Stromata are the places where conidia and ascospores develop. Also, here Botanophila flies dine on mycelium, lay the eggs, defecate, and the larvae develop. The interaction of the three symbionts was analyzed concerning the organisms' adaptation to understand the differences in physiology and ecology of this microenvironment that support stable symbiosis spreading presently in Europe since the beginning of the XXI century. For analysis of the infrared radiation emitted by stromata, a high-resolution infrared camera FLIR E50 was used. The visualization of stromata temperature profiles was shown in the form of pseudo-colored (false) infrared images. The 13C discrimination was used to characterize photosynthesis of the plant tissue enclosed within the stromata. The stromata had a substantially lower temperature than the green plant tissues. The difference reached ~5.6°C during midday hours, whereas it was smaller in the evening, reaching only ~3.6°C. The mycelium of Epichloe cultivated on agar showed about 2°C lower temperature in comparison to the surrounding. The plant tissues enclosed within the stroma were photosynthetically active, although this activity was of phosphoenolpyruvate carboxylase (PEPC) type and less involved in heat dissipation during the day. The stromata, built by fungal hyphae, on which fungal reproductive structures develop, form a cool shelter. This shelter provides a place for the larvae of Botanophila flies.

26 páginas, 4 figuras y 1 tabla. -- The final version is available at http://www.elsevier.com, Plants harbor a wide diversity of microorganisms in their tissues. Some of them have a longco-evolutionary history with their hosts, likely playing a pivotal role in regulating the plantinteraction with other microbes such as pathogens. Some cool-season grasses are symbi-otic withEpichlo€efungal endophytes that grow symptomless and systemically in above-ground tissues. Among the many benefits that have been ascribed to endophytes, theirrole in mediating plant interactions with pathogens has been scarcely developed. Here,we explored the effects ofEpichlo€efungal endophytes on the interaction of host grasseswith fungal pathogens. We made a meta-analysis that covered a total of 18 host grass spe-cies, 11 fungal endophyte species, and 22 fungal pathogen species. We observedendophyte-mediated negative effects on pathogensin vitroandin planta. Endophyte nega-tive effects on pathogens were apparent not only in laboratory but also in greenhouse andfield experiments.Epichlo€efungal endophytes had negative effects on pathogen growth andspores’ germination. On living plants, endophytes reduced both severity and incidence ofthe disease as well as colonization and subsequent infection of seeds. Symbiosis with en-dophytes showed an inhibitory effect on debilitator and killer pathogens, but not on castra-tors, and this effect did not differ among biotrophic or necrotrophic lifestyles. We foundthat this protection can bedirectthrough the production of fungistatic compounds, thecompetition for a common resource, or the induction of plant defenses, andindirectasso-ciated with endophyte-generated changes in the abiotic or the biotic environment. Severalmechanisms operate simultaneously and contribute differentially to the reduction of dis-ease within grass populations., This work was funded by the University of Buenos Aires (UBA), National Scientific and Technical Research Council (CONICET), and the National Agency for Scientific and Technological Promotion (ANPCyT - PICT 2633-2016)

The endophytic fungus Epichloë festucae systemically colonizes the intercellular spaces of cool-season grasses to establish a mutualistic symbiosis. Hyphal growth of the endophyte within the host plant is tightly regulated and synchronized with the growth of the host plant. A genetic screen to identify symbiotic genes identified mutant FR405 that had an antagonistic interaction with the host plant. Perennial ryegrass infected with the FR405 mutant were stunted and underwent premature senescence and death. The disrupted gene in FR405 encodes a nuclear-localized protein, designated as NsiA for nuclear protein for symbiotic infection. Like previously isolated symbiotic mutants the nsiA mutant is defective in hyphal cell fusion. NsiA interacts with Ste12, a C2H2 zinc-finger transcription factor, and a MAP kinase MpkB. Both are known as essential components for cell fusion in other fungal species. In E. festucae, MpkB, but not Ste12, is essential for cell fusion. Expression of several genes required for cell fusion and symbiosis, including proA/adv-1, pro41/ham-6, ham7, ham8, and ham9 were downregulated in the nsiA mutant. However, the NsiA ortholog in Neurospora crassa was not essential for hyphal cell fusion. These results demonstrate that the roles of NsiA and Ste12 orthologs in hyphal cell fusion are distinctive between fungal species.

Systemic fungal endophytes may increase the adaptive potential of their hosts to different environmental conditions. However, the mechanisms involved in the adjustment are still an area of active inquiry. Considering that anatomical features in plants are the result of adaptation to different environments, this aspect of plants response to the symbionts deserves further research. In this work we investigated whether an asexual Epichloe endophyte modifies the foliar anatomy of symbiotic plants (E+) of its host grass with respect to endophyte-free plants (E-), using the Epichloe occultans–Lolium multiflorum system. The experiment was carried out in a greenhouse and plants were grown under benign, non-stressful (controlled) conditions. Symbiotic plants showed thinner leaf blades and lower mesophyll thickness. Likewise, the endophyte is associated with a greater number of vascular bundles. The stomatal density on the lower epidermis was higher in symbiotic plants than non-symbiotic plants. Our study reveals that Epichloe occultans modifies the foliar anatomy of Lolium multiflorum. Considering that leaf anatomy exhibits characters that were selected as a result of evolution in relation to specific environmental conditions, these results are promising since they can offer information about the mechanisms involved in the adaptive adjustment of symbiotic plants.

Seeds are involved in the transmission of microorganisms from one plant generation to the next, acting as initial inoculum for the plant microbiome, therefore provide a key source of variability in plants. This study aimed to characterize the seed bacteria and fungi communities in Elymus nutans, a dominant perennial grass growing in the Qinghai Tibet Plateau (QTP) and explore the effects of plant growth location on the seed microbiome. Seeds were collected from plants growing in four locations in the QTP. The seed microbial community was examined by Illumina MiSeq sequencing of DNA extracted from the surface sterilized seeds. The seed bacterial community was dominated by the bacteria phylum Proteobacteria (98%) and fungal phyla Ascomycota (83%) and Basidiomycota (15%). At the lower taxonomic level, the bacterial genus Pseudomonas dominated in all four locations with an average relative abundance of 83% whereas the fungal genera that dominated the seed microbiome was diverse, the most prominent being Epichloe, Pyrenophora, Mycosphaerella and Bullera. Ecologically important bacterial family Nitrosomonadaceae (nitrifiers) and fungal phylum Glomeromycota (arbuscular mycorrhizal fungi) were detected in this study for the first time as seed endophytes. The Elymus nutans seed bacterial community was not impacted by the plant growth location, in contrast, the seed fungal community varied significantly in four locations. The seeds of Elymus nutans host diverse endophytic bacteria and fungi. Unlike the bacteria, the host plant selection of seed fungal endophytes was observed to have been affected by plant growth location. Positive and negative associations in the Elymus nutans seed microbiome were observed.

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

Plants are expected to face novel challenges as consequence of human-driven global change. Outbreaks of pests and higher incidence of contaminants are increasing. Plants can improve tolerance to stress factors through associations with symbiotic microorganisms. Certain grasses establish persistent and asymptomatic symbioses with Epichloe fungal endophytes, which are known to confer protection against Herbivores and improve plant tolerance to abiotic stress factors. Nonetheless, accumulating evidence suggests the symbiosis outcome is context-dependent. We evaluated the capacity of the endophyte fungus E. occultans in protecting the annual grass Lolium multiflorum against a spontaneous larva attack of the generalist herbivore Agrotis ipsilon under episodic exposure of plants to ozone. Symbiotic and non-symbiotic plants were individually grown outdoors and exposed to ozone at different stages resulting in four treatments: control (plant never exposed to ozone), plant exposed to ozone at the vegetative stage, plant exposed to ozone at reproductive stage, and plant exposed to ozone at both stages. After the last exposure, there was an outbreak of A. ipsilon larvae. We evaluated herbivore damage, seed production per plant, and endophyte transmission to the seeds. Frequency of attacked plants was irrespective of both the endophyte and ozone exposure. However, the damage level per plant was only reduced by the endophyte. Seed production was slightly lower in endophyte-symbiotic plants and not affected by ozone. Interestingly, herbivore damaged and undamaged endophyte-symbiotic plants contributed equally to seed production. However, in plants exposed to ozone once at the vegetative or reproductive stage, endophyte-free undamaged plants had higher seed production than endophyte-free damaged plants. Ozone treatments did not affect the transmission efficiency of endophytes to the seeds. Mean endophyte transmission efficiency per plant was 95%. Medium doses of ozone seem to have undetectable effects on grass-endophyte symbiosis, not affecting the defensive mutualism nor the persistence of the symbiont across generations.

Summary Epichloë festucae is an endophytic fungus that forms a symbiotic association with Lolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae.A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from uninfected and infected L. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem MS was used to identify a novel product of a secondary metabolite gene cluster.Metabolites likely to be present in the apoplast were identified using marvis in combination with the BioCyc and KEGG databases, and an in‐house Epichloë metabolite database. We were able to identify the known endophyte‐specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers.To determine whether these methods can be applied to the identification of novel Epichloë‐derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative MS analysis of apoplastic wash fluid from wild‐type‐ vs mutant‐infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

Lolium perenne (perennial ryegrass) is a common temperate pasture grass species and is favored by dairy farmers. Commercially, the known association with the mutualistic endophytic fungus Epichloë festucae subsp. lolii is used to enhance insect resistance of host plants. Knowledge of other members of the microbiome and their functions are limited. Sequencing of the V4 region of the 16S ribosomal RNA gene was used to examine the bacterial microbiome of perennial ryegrass (Alto) seed (generation 1 [G1]), subsequent mature plants grown in soil and sand, and seed (generation 2 [G2]) from crosses of these mature plants. The G1 microbiome was dominated by the class Gammaproteobacteria. The mature plant microbiomes were far more diverse, comprising up to 37 classes inclusive of Gammaproteobacteria. Different growth media yielded different microbiome profiles in mature plants. The G2 microbiome, similar to the G1 microbiome, was dominated by Gammaproteobacteria as the primary constituent, with additional supplementation from class Bacilli. This suggests the continuation of a core microbiome which persists from seed through plant maturation to seed. This study sheds new light on the hereditability of perennial ryegrass bacterial microbiomes and has identified some operational taxonomic units of potential commercial significance due to their seed transmissibility, and their roles are currently being explored.

Fungal symbionts, present in above and in belowground tissues, such as that of Epichloe endophytes and arbuscular mycorrhizal (AM) fungi, respectively, can modify the responses of host plants to environmental changes. Individual grass plants of the subfamily Pooideae can be host to both a foliar Epichloe endophytic fungus and root-associated AM fungi. Understanding the multiple interactions among above- and belowground symbionts and their host is an important step in understanding terrestrial ecosystems. A field experiment was conducted to study the effects of E. gansusensis endophyte and soil moisture on the belowground AM fungal biodiversity associated with Achnatherum inebrians, through amplicon sequencing technology. Soil properties were compared among stands using standard techniques. Our results show that E. gansusensis increased root-associated AM fungal diversity under drought conditions, while decreasing diversity under the water addition treatment. Water addition and water stress treatments decreased the diversity and richness of the AM fungal community in rhizosphere soil compared to the normal treatment. The E. gansusensis altered the composition of the root-associated AM fungal community. Aboveground biomass was closely positively related to the abundance of Funneliformis in the root and the diversity of the rhizosphere soil AM fungal community was positively related to the soil total nitrogen and phosphorus. This study suggested that soil moisture regimes shifted the effects of E. gansuensis on the diversity of the root-associated AM fungal community from positive to negative; moreover, soil moisture and foliar E. gansusensis altered soil properties, thereby affecting belowground AM fungi.

In ascomycetes and basidiomycetes, iron-responsive GATA-type transcriptional repressors are involved in regulating iron homeostasis, notably to prevent iron toxicity through control of iron uptake. To date, it has been unknown whether this iron regulator contributes toward mutualistic endosymbiosis of microbes with plants, a system where the endophyte must function within the constraints of an in-host existence, including a dependency on the host for nutrient acquisition. Functional characterization of one such protein, SreA from Epichloë festucae, a fungal endosymbiont of cool-season grasses, indicates that regulation of iron homeostasis processes is important for symbiotic maintenance. The deletion of the sreA gene (ΔsreA) led to iron-dependent aberrant hyphal growth and the gradual loss of endophyte hyphae from perennial ryegrass. SreA negatively regulates the siderophore biosynthesis and high-affinity iron uptake systems of E. festucae, similar to other fungi, resulting in iron accumulation in mutants. Our evidence suggests that SreA is involved in the processes that moderate Epichloë iron acquisition from the plant apoplast, because overharvesting of iron in ΔsreA mutants was detected as premature chlorosis of the host using a hydroponic plant growth assay. E. festucae appears to have a tightly regulated iron management system, involving SreA that balances endophyte growth with its survival and prevents overcompetition with the host for iron in the intercellular niche, thus promoting mutualistic associations. Mutations that interfere with Epichloë iron management negatively affect iron-dependent fungal growth and destabilize mutualistic Epichloë –ryegrass associations.

Cool-season grasses are the most common forage types in livestock operations and amenities. Several of the cool-season grasses establish mutualistic associations with an endophytic fungus of the Epichloe genus. The grasses and endophytic fungi have evolved over a long period of time to form host-fungus specific relationships that confer protection for the grass against various stressors in exchange for housing and nutrients to the fungus. This review provides an overview of the mechanisms by which Epichloe endophytes and grasses interact, including molecular pathways for secondary metabolite production. It also outlines specific mechanisms by which the endophyte helps protect the plant from various abiotic and biotic stressors. Finally, the review provides information on how Epichloe infection of grass and stress affect the rhizosphere environment of the plant.

Recent studies have revealed presence of fungus-originated genes in genomes of cool-season grasses, suggesting occurrence of multiple ancestral gene transfer events between the two distant lineages. The current article describes identification of glucanase-like and monooxygenase-like genes from creeping bent grass, as lateral gene transfer candidates. An in silico analysis suggested presence of the glucanase-like gene in Agrostis, Deyeuxia, and Polypogon genera, but not in other species belonging to the clade 1 of the Poeae tribe. Similarly, the monooxygenase-like gene was confined to Agrostis and Deyeuxia genera. A consistent result was obtained from PCR-based screening. The glucanase-like gene was revealed to be ubiquitously expressed in young seedlings of creeping bent grass. Although expression of the monooxygenase-like gene was suggested in plant tissues, the levels were considerably lower than those of the glucanase-like gene. A phylogenetic analysis revealed close relationships of the two genes between the corresponding genes in fungal endophyte species of the Epichloë genus, suggesting that the genes originated from the Epichloë lineage.

This study explored the effects of the Epichloë&nbsp, sinensis endophyte on growth, photosynthesis, ionic content (K+ and Ca2+), phytohormones (abscisic acid—ABA, cytokinin—CTK, indolE−3-acetic acid—IAA, and gibberellin—GA), and elements—C, N, P (in the shoot and root) in two ecotypes of Festuca sinensis (ecotypes 111 and 141) under different soil water conditions (35% and 65% relative saturation moisture content (RSMC)). The results showed that 35% RSMC inhibited the plants’ growth, and compared with 65% RSMC, there was a significant (p &lt, 0.05) decrease in the growth and photosynthesis indices, the contents of CTK and GA, Ca2+ concentration, and the contents of C, N, and P (in both the aboveground and underground parts) under 35% RSMC. E.&nbsp, sinensis had beneficial effects on host growth and stress tolerance. Under both 35% and 65% RSMC, the presence of E.&nbsp, sinensis significantly (p &lt, 0.05) increased host plant height, tiller number, root length, root volume, shoot dry weight, chlorophyll content, and the rate of photosynthesis of both ecotypes. Furthermore, the shoot C, N, and P contents in plants infected with E.&nbsp, sinensis (E+) from the two ecotypes, under both conditions of RSMC, were significantly (p &lt, 0.05) higher than those in corresponding plants that were not infected with E.&nbsp, sinensis (E−). Under 35% RSMC, the contents of ABA, K+, Ca2+, and root P contents in E+ plants were significantly (p &lt, 0.05) higher than those in corresponding E− plants in both ecotypes. However, under 65% RSMC, root C, N, and P contents in E+ plants of ecotype 111 and 141 were significantly (p &lt, 0.05) higher than those in corresponding E− plants. In addition, the host ecotype also had effects on host growth and stress tolerance, the growth and photosynthetic indices of ecotype 141 were significantly (p &lt, 0.05) higher than those of ecotype 111 under 35% RSMC, which suggested that ecotype 141 is more competitive than ecotype 111 under water deficiency conditions. These findings suggest that the endophyte improved the host plant resistance to water deficiency by maintaining the growth of the plant, improving photosynthesis, accumulating K+ and Ca2+, promoting nutrient absorption, and adjusting the metabolism of plant hormones.

The study of the contribution of the plant defence hormones, salicylic acid (SA) and jasmonic acid (JA), in the resistance against pathogens of plants associated with Epichloë fungal endophytes has been scanty. We hypothesised that Epichloë spp., capable of inducing host plant SA-dependent defences, would increase the levels of plant resistance against biotrophic pathogens. Plants of Achnatherum inebrians, with and without the fungal endophyte Epichloë gansuensis, were inoculated with the biotrophic fungal pathogen Blumeria graminis. We measured the status of plant defences (associated with SA and JA signalling pathways) and the levels of resistance to the pathogen. Plants associated with the endophyte showed less disease symptoms caused by the biotrophic pathogen than plants without the endophyte. In agreement with our hypothesis, the Epichloë endophyte increased the plant production of SA and enhanced the expression levels of plant genes of synthesis and response to the SA hormone. The elevated expression of SA-related genes coding for putative plant enzymes with anti-fungal activities promoted by the endophyte may explain the enhanced resistance to the pathogen. The present study highlights that interaction between the plant immune system and Epichloë fungal endophytes can contribute significantly to the resistance of endophyte-symbiotic plants against pathogens.

The phenomenon of plant mutualistic symbiosis with microbes may have a positive effect on the improvement of plant tolerance to environmental stresses as well as on the ability of plants to accumulate heavy metal (HM) ions from soil. The influence of Epichloë fungal endophyte (Ascomycota, Clavicipitaceae) on perennial ryegrass (Lolium perenne L.) plants grown in the presence of elevated concentrations of HM ions (Cd2+, Pb2+, and Cu2+) in soil was studied. The presence of Epichloë in the host grass tissues resulted in different accumulation of HM ions in the aboveground parts of the plants. In some cases, endophyte infection positively affected ryegrass ability to accumulate HM ions from soil. In plants with (E +) and without (E −) endophytes, the hormesis effect was induced by the elevated concentration of Cu2+ ions, resulting in better growth and photosynthesis, as examined by measurements of Chl a fluorescence. The obtained results indicate that based on the laboratory evaluation of the efficiency of HM accumulation, we were able to choose the best associations of perennial ryegrass with endophytes for HM phytoremediation.

Tropospheric ozone is among the global change factors that pose a threat to plants and microorganisms. Symbiotic microorganisms can assist plants to cope with stress, but their role in the tolerance of plants to ozone is poorly understood. Here, we subjected endophyte-symbiotic and non-symbiotic plants of Lolium multiflorum, an annual species widely distributed in temperate grasslands, to high and low (i.e., charcoal-filtered air) ozone levels at vegetative and reproductive phases. Exposure to high ozone reduced leaf photochemical efficiency and greenness in both symbiotic and non-symbiotic plants. However, ozone-induced oxidative damage at biochemical level (i.e., lipid peroxidation) was mostly detected in symbiotic plants. Ozone exposure at the vegetative phase did not affect the reproductive investment in seeds, indicating full recovery from stress. Ozone exposure at the reproductive phase reduced biomass and seed production only in symbiotic plants indicating a symbiont-associated cost. At low ozone, endophyte-symbiotic plants showed a steeper slope in the relationship between seed number and seed weight (i.e., a number-weight trade-off) compared to non-symbiotic plants. However, when plants were treated at the reproductive phase, ozone increased the imbalance between seed number and seed weight in both endophyte-symbiotic and non-symbiotic plants. Plants with endophytes at the reproductive stage produced fewer seeds, which were not compensated by increased seed weight. Thus, fungal mycelium growing within ovaries or ozone-induced antioxidant systems may result in costs that finally depress the fitness of plants. Despite ozone pollution could destabilize plant-endophyte mutualisms and render them dysfunctional, other endophyte-mediated benefits (e.g., resistance to herbivory, tolerance to drought) could over-compensate these losses and explain the high incidence of the symbiosis in nature.

2018-2019 UNCG University Libraries Open Access Publishing Fund Grant Winner. Aim: The endophyte Epichloe alsodes, with known insecticidal properties, is found in a majority of Poa alsodes populations across a latitudinal gradient from North Carolina to New York. A second endophyte, E. schardlii var. pennsylvanica, with known insect-deterring effects, is limited to a few populations in Pennsylvania. We explored whether such disparate differences in distributions could be explained by selection from biotic and abiotic environmental factors. Location: Along the Appalachian Mountains from North Carolina to New York, USA. Taxon: Fungi. Methods: Studied correlations of infection frequencies with abiotic and biotic environmental factors. Checked endophyte vertical transmission rates and effects on overwintering survival. With artificial inoculations for two host populations with two isolates per endophyte species, tested endophyte–host compatibility. Studied effects of isolates on host performances in greenhouse experiment with four water-nutrients treatments. Results: Correlation analysis revealed positive associations of E. alsodes frequency with July Max temperatures, July precipitation, and soil nitrogen and phosphorous and negative associations with insect damage and soil magnesium and potassium. Plants infected with E. alsodes had increased overwintering survival compared to plants infected with E. schardlii or uninfected (E-) plants. Artificial inoculations indicated that E. alsodes had better compatibility with a variety of host genotypes than did E. schardlii. The experiment with reciprocally inoculated plants grown under different treatments revealed a complexity of interactions among hosts, endophyte species, isolate within species, host plant origin, and environmental factors. Neither of the endophyte species increased plant biomass, but some of the isolates within each species had other effects on plant growth such as increased root:shoot ratio, number of tillers, and changes in plant height that might affect host fitness. Main conclusion: In the absence of clear and consistent effects of the endophytes on host growth, the differences in endophyte-mediated protection against herbivores may be the key factor determining distribution differences of the two endophyte species.

Summary Epichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with the grass host Lolium perenne. Endophytic hyphae exit the host by an appressorium‐like structure known as an expressorium. In plant‐pathogenic fungi, the tetraspanin Pls1 and the NADPH oxidase component Nox2 are required for appressorium development. Previously we showed that the homologue of Nox2, NoxB, is required for E. festucae expressorium development and establishment of a mutualistic symbiotic interaction with the grass host. Here we used a reverse genetics approach to functionally characterize the role of the E. festucae homologue of Pls1, PlsA. The morphology and growth of ΔplsA in axenic culture was comparable to wild‐type. The tiller length of plants infected with ΔplsA was significantly reduced. Hyphae of ΔplsA had a proliferative pattern of growth within the leaves of L. perenne with increased colonization of the intercellular spaces and the vascular bundles. The ΔplsA mutant was also defective in expressorium development although the phenotype was not as severe as for ΔnoxB, highlighting potentially distinct roles for PlsA and NoxB in signalling through the NoxB complex. Hyphae of ΔplsA proliferate below the cuticle surface but still occasionally form an expressorium‐like structure that enables the mutant hyphae to exit the leaf to grow on the surface. These expressoria still form a septin ring‐like structure at the point of cuticle exit as found in the wild‐type strain. These results establish that E. festucae PlsA has an important, but distinct, role to NoxB in expressorium development and plant symbiosis.

Induced or constitutive production of secondary metabolites is a successful plant defence strategy against herbivores which can be mediated by plant associated micro-organisms. Several grass species can be associated with an endophytic fungus of the genus Epichloe which produces herbivore toxic or deterring alkaloids. Besides these direct defences, herbivorous insects are controlled via indirect plant defence mechanisms by attracting predators. Recent studies indicate that Epichloe endophytes can improve the grass emitted volatile organic compounds towards herbivore deterrence. Due to their defensive mutualistic function, we hypothesize that Epichloe altered plant volatiles can attract aphid predators and contribute to an increased indirect plant defence. With a common garden study, we show that hoverfly (Syrphidae) larvae and pupae were more abundant on endophyte-infected plants compared to uninfected plants. Our results indicate that the Epichloe endophyte provides, besides direct defence (alkaloid), indirect plant defence by improving the plant odor attracting more olfactory foraging aphid predators. Future research is needed in order to understand: (I) whether endophyte-mediated changes in plant volatiles are induced herbivore specific, (II) whether there is a trade-off between endophyte-mediated direct and indirect plant defence, (III) whether the endophyte produces volatiles or induces a change in plant-derived volatiles, (IV) the role of plant signals in endophyte-mediated plant defence.

Both arbuscular mycorrhizal fungi (AMF) Claroideoglomus etunicatum and grass endophytes (Epichloe) could improve disease tolerance. Research is limited on the dual infection by AMF and Epichloe on plant diseases. Bipolaris sorokiniana is a fungal species that causes leaf spot in cereals, and also in perennial ryegrass (Lolium perenne), impacting its growth and production. This experiment tested the dual infection of AMF and Epichloe on perennial ryegrass growth and the physiological and biochemical indexes under limited soil conditions occurs in nature ecosystem. The results showed that infection with B. sorokiniana significantly decreased plant growth and shoot N concentration (P

Systemic Epichloe endophytes are common fungal symbionts of many cool-season grasses. They are known for their capability of increasing host plant tolerance against biotic and abiotic stressors, in...

Symbiotic fungi commonly increase plant acquisition of soil nutrients. Because prior work has focused on root fungi, we examined how leaf endophytes (Epichloe) influenced plant responses to fertilization and altered plant traits that may cascade to food webs and ecosystem processes. We manipulated endophyte presence/absence in two populations of Trisetum spicatum, a wild relative of oat, under a 2 × 2 addition of soil nitrogen (N) and phosphorus (P) in the greenhouse. Endophyte symbiosis altered how plant biomass responded to soil N and how plant traits responded to soil P. Endophytes boosted the biomass gains from N-fertilization in one population. Plants from a second population had weak benefits of symbiosis, but the endophyte altered plant traits, by increasing specific leaf area under P-fertilization, root diameter under low P, and concentration of the fungal alkaloid AcAP under N fertilization. Endophyte presence suppressed the typically observed increase in root hair density in response to soil P limitation. Under low P, symbiotic plants from both populations had improved forage quality relative to symbiont-free plants, although N-fertilization had a larger effect size on forage quality than did symbiosis. Finally, the two populations differed in production of fungal alkaloids, which generally increased in response to fertilization. Predicting how microbial symbionts mediate plant acquisition of nutrients requires understanding how much their effects vary among plant and endophyte genotypes. Here, the magnitude and direction of leaf symbionts’ effects on plant yield and traits varied between populations and with soil nutrient availability.

Fungi of the genus Epichloë (Clavicipitaceae, Ascomycota) cause choke disease in many grass species. The disease manifests itself as fungal stromata that form around developing inflorescences, thereby suppressing their maturation. Economic losses in agricultural production due to choke have long been known in the U.S.A. and France, but attempts to control choke disease have not been successful. The interaction between Epichloë typhina (Pers.) Tul. & C. Tul. and its naturally occurring fungal hyperparasite, Clonostachys epichloë (Speg.) Schroers (sexual morph Bionectria epichloë) was investigated in populations of the grass Puccinellia distans (L.) Parl. Fungal hyperparasites occur widely in nature, and many are successfully used commercially as biological control agents against plant pathogenic fungi. Microscopy of Epichloë stromata infected with C. epichloë revealed a lack of asci with ascospores in perithecia and damage to mycelia at sites colonized by C. epichloë. The ability of C. epichloë to colonize E. typhina was confirmed via two in vitro experiments. The percent inhibition of growth of E. typhina strains by C. epichloë varied from 18.40 to 46.50%, and the mycoparasite colonized up to 100% of Epichloë mycelia in a precolonization experiment. We discuss the possibility of using C. epichloë to control choke disease caused by E. typhina in grass populations.

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.