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Salt stress negatively affects plant growth, and the fungal endophyte Epichloëgansuensis increases the tolerance of its host grass species, Achnatherum inebrians, to abiotic stresses. In this work, we first evaluated the effects of E. gansuensis on glucose-6-phosphate dehydrogenase (G6PDH) and plasma membrane (PM) H + -ATPase activity of Achnatherum inebrians plants under varying NaCl concentrations. Our results showed that the presence of E. gansuensis increased G6PDH, PM H + -ATPase, superoxide dismutase and catalase activity to decrease O 2 •- , H 2 O 2 and Na + contents in A. inebrians under NaCl stress, resulting in enhanced salt tolerance. In addition, the PM NADPH oxidase activity and NADPH/NADP + ratios were all lower in A. inebrians with E. ganusensis plants than A. inebrians plants without this endophyte under NaCl stress. In conclusion, E. gansuensis has a positive role in improving host grass yield under NaCl stress by enhancing the activity of G6PDH and PM H + -ATPase to decrease ROS content. This provides a new way for the selection of stress-resistant and high-quality forage varieties by the use of systemic fungal endophytes.

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

The systemic fungal endophyte of the grass Achnatherum inebrians, Epichloë gansuensis, has important roles in enhancing resistance to biotic and abiotic stresses. In this work, we first evaluated the effects of E. gansuensis on nitrogen metabolism, nitrogen use efficiency, and stoichiometry of A. inebrians under varying nitrogen concentrations. The results demonstrated that E. gansuensis significantly improved the growth of A. inebrians under low nitrogen conditions. The fresh and dry weights, nitrogen reductase, nitrite reductase, and glutamine synthetase activity, NO 3 - , NH 4 + , N, and P content, and also the total N accumulation, N utilization efficiency, and N uptake efficiency were all higher in leaves of A. inebrians with E. ganusensis (E+) plants than A. inebrians plants without this endophyte (E-) under low nitrogen availability. In conclusion, E. gansuensis has positive effects on improving the growth of A. inebrians under low-nitrogen conditions by modulating the enzymes of nitrogen metabolism and enhancing nitrogen use efficiency.

Asexual cool-season grass endophytes of the genus Epichloë (Ascomycota: Clavicipitaceae) are strictly vertically disseminated. The hosts of these mutualistic fungi express no symptoms during the fungal lifecycle that takes place entirely within the plant, while their hosts receive beneficial outcomes. These fungi are distributed in two major locations within the mature seeds of their hosts; namely, within the embryo (including the scutellum, coleoptile, plumule, radicle, and coleorhiza tissues) and between the aleurone and pericarp layers, with the latter hyphae playing no role in transmission of the fungus to the next plant generation. Conflicting evidence remains in the literature on the timing of embryo colonization. In a detailed investigation, utilizing confocal microscopy to observe the distribution of Epichloë coenophiala strain AR601 in tall fescue (Lolium arundinaceum), we tracked endophyte hyphal colonization in the ovary (pre-fertilization) through to the fully mature seed stage. Confocal microscopy images revealed that at the early and mature developmental stages of the embryo sac, before host grass fertilization, there were large quantities of endophyte mycelium present, especially around the antipodal cells, indicating that this endophyte enters the embryo sac before the fertilization stage. After host fertilization, fungal hyphae could be seen in the true embryo and early nonstarchy endosperm. Understanding the mechanisms of transmission to the seed is important for commercial seed producers and end users.

Fungal endophytes belonging to the genus Epichloë form associations with temperate grasses belonging to the sub-family Poöideae that range from mutualistic through to pathogenic. We previously identified a novel endophyte gene (designated gigA for grass induced gene) that is one of the most abundantly expressed fungal transcripts in endophyte-infected grasses and which is distributed and highly expressed in a wide range of Epichloë grass associations. Molecular and biochemical analyses indicate that gigA encodes a small secreted protein containing an imperfect 27 amino acid repeat that includes a kexin protease cleavage site. Kexin processing of GigA liberates within the plant multiple related products, named here as epichloëcyclins, which we have demonstrated by MS/MS to be cyclic peptidic in nature. Gene deletion of gigA leads to the elimination of all epichloëcyclins with no conspicuous phenotypic impact on the host grass, suggesting a possible bioactive role. This is a further example of a ribosomal peptide synthetic (RiPS) pathway operating within the Ascomycetes, and is the first description of such a pathway from a mutualistic symbiotic fungus from this Phylum., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We have identified from the mutualistic grass endophyte Epichloë festucae a non-ribosomal peptide synthetase gene (sidN) encoding a siderophore synthetase. The enzymatic product of SidN is shown to be a novel extracellular siderophore designated as epichloënin A, related to ferrirubin from the ferrichrome family. Targeted gene disruption of sidN eliminated biosynthesis of epichloënin A in vitro and in planta. During iron-depleted axenic growth, ΔsidN mutants accumulated the pathway intermediate N(5)-trans-anhydromevalonyl-N(5)-hydroxyornithine (trans-AMHO), displayed sensitivity to oxidative stress and showed deficiencies in both polarized hyphal growth and sporulation. Infection of Lolium perenne (perennial ryegrass) with ΔsidN mutants resulted in perturbations of the endophyte-grass symbioses. Deviations from the characteristic tightly regulated synchronous growth of the fungus with its plant partner were observed and infected plants were stunted. Analysis of these plants by light and transmission electron microscopy revealed abnormalities in the distribution and localization of ΔsidN mutant hyphae as well as deformities in hyphal ultrastructure. We hypothesize that lack of epichloënin A alters iron homeostasis of the symbiotum, changing it from mutually beneficial to antagonistic. Iron itself or epichloënin A may serve as an important molecular/cellular signal for controlling fungal growth and hence the symbiotic interaction.

Background and aims: Low nitrogen negatively affects soil fertility and plant productivity. Glucose-6-phosphate dehydrogenase (G6PDH) and Epichloë gansuensis endophytes are two factors that are associated with tolerance of Achnatherum inebrians to abiotic stress. However, the possibility that E. gansuensis interacts with G6PDH in enhancing low nitrogen tolerance of host grasses has not been examined.Methods: A. inebrians plants with (E+) and without E. gansuensis (E−) were subjected to different nitrogen concentration treatments (0.1, 1, and 7.5 mM). After 90 days, physiological studies were carried out to investigate the participation of G6PDH in the adaption of host plants to low nitrogen availability.Results: Low nitrogen retarded the growth of A. inebrians. E+ plants had higher total dry weight, chlorophyll a and b contents, net photosynthesis rate, G6PDH activity, and GSH content, while having lower plasma membrane (PM) NADPH oxidase activity, NADPH/NADP+ ratios, and MDA and H2O2 than in E− A. inebrians plants under low nitrogen concentration.Conclusions: The presence of E. gansuensis played a key role in maintaining the growth of the A. inebrians plants under low nitrogen concentration by regulating G6PDH activity and the NADPH/NADP+ ratio and improving net photosynthesis rate. [ABSTRACT FROM AUTHOR]

The seed-transmitted fungal symbiont, Epichloë festucae, colonizes grasses by infecting host tissues as they form on the shoot apical meristem (SAM) of the seedling. How this fungus accommodates the complexities of plant development to successfully colonize the leaves and inflorescences is unclear. Since adenosine 3', 5'-cyclic monophosphate (cAMP)-dependent signaling is often essential for host colonization by fungal pathogens, we disrupted the cAMP cascade by insertional mutagenesis of the E. festucae adenylate cyclase gene (acyA). Consistent with deletions of this gene in other fungi, acyA mutants had a slow radial growth rate in culture, and hyphae were convoluted and hyper-branched suggesting that fungal apical dominance had been disrupted. Nitro blue tetrazolium (NBT) staining of hyphae showed that cAMP disruption mutants were impaired in their ability to synthesize superoxide, indicating that cAMP signaling regulates accumulation of reactive oxygen species (ROS). Despite significant defects in hyphal growth and ROS production, E. festucae ΔacyA mutants were infectious and capable of forming symbiotic associations with grasses. Plants infected with E. festucae ΔacyA were marginally less robust than the wild-type (WT), however hyphae were hyper-branched, and leaf tissues heavily colonized, indicating that the tight regulation of hyphal growth normally observed in maturing leaves requires functional cAMP signaling. [ABSTRACT FROM AUTHOR]

Background and aims: Salinization of soils causes severe problems to plant growth. With the important role of stoichiometry in many ecological processes, this study investigated the effect of an asexual Epichloë endophyte on the nutrient stoichiometry of wild barley ( Hordeum brevisubulatum) under salt stress. Methods: Plants with (E+) and without endophyte (E−) were subjected to different NaCl treatments (0, 150, 300, 450 and 600 mM). After 15 days, the dry weight as well as the carbon (C), nitrogen (N), phosphorus (P), sodium (Na) and potassium (K) contents in the plants were determined. Results: Salt stress significantly reduced the growth and nutrient absorption of wild barley. The biomass as well as the N, P and K contents were higher while the Na content was lower in E+ plants than in E− plants. However, there was no significant effect on C content between E+ and E− plants. The findings also showed that E+ plants had lower ratios of C:N, C:P, Na:K and a higher ratio of N:P than E− plants. Conclusions: The Epichloë endophyte played an important role in maintaining the growth of the host plants by promoting nutrient absorption and adjusting the ionic balance. The results have enhanced knowledge of the application of endophytes that will enable better crop production and ecological conservation. [ABSTRACT FROM AUTHOR]

We have identified from the mutualistic grass endophyte Epichloë festucae a non-ribosomal peptide synthetase gene (sidN) encoding a siderophore synthetase. The enzymatic product of SidN is shown to be a novel extracellular siderophore designated as epichloënin A, related to ferrirubin from the ferrichrome family. Targeted gene disruption of sidN eliminated biosynthesis of epichloënin A in vitro and in planta. During iron-depleted axenic growth, ΔsidN mutants accumulated the pathway intermediate N5-trans-anhydromevalonyl-N5-hydroxyornithine (trans-AMHO), displayed sensitivity to oxidative stress and showed deficiencies in both polarized hyphal growth and sporulation. Infection of Lolium perenne (perennial ryegrass) with ΔsidN mutants resulted in perturbations of the endophyte-grass symbioses. Deviations from the characteristic tightly regulated synchronous growth of the fungus with its plant partner were observed and infected plants were stunted. Analysis of these plants by light and transmission electron microscopy revealed abnormalities in the distribution and localization of ΔsidN mutant hyphae as well as deformities in hyphal ultrastructure. We hypothesize that lack of epichloënin A alters iron homeostasis of the symbiotum, changing it from mutually beneficial to antagonistic. Iron itself or epichloënin A may serve as an important molecular/cellular signal for controlling fungal growth and hence the symbiotic interaction. [ABSTRACT FROM AUTHOR]

Abstract: Selected Neotyphodium sp. endophytes are now commonly used to enhance pasture persistence and livestock productivity, with seed of perennial ryegrass and tall fescue cultivars with these selected endophytes being commercially available. In a large population of perennial ryegrass plants infected with a Neotyphodium sp. endophyte that was being grown for seed production a small percentage of inflorescences were distorted and covered with a conspicuous white mycelial growth. Within individual plants only a small number of inflorescences were affected and the amount of distortion differed between affected inflorescences. This Neotyphodium sp. is an interspecific hybrid of Epichloë typhina and Neotyphodium. lolii and like nearly all other Neotyphodium spp is symptomless in host grasses. The fungus isolated from distorted inflorescences had colonies that were identical to those isolated from symptomless inflorescences and these were characteristic of this Neotyphodium sp. This is the first report of distorted inflorescences covered with epiphytic hyphal growth on host grasses infected with an interspecific hybrid Neotyphodium sp. [Copyright &y& Elsevier]

Abstract: A fundamental hallmark of fungal growth is that vegetative hyphae grow exclusively by extension at the hyphal tip. However, this model of apical growth is incompatible with endophyte colonization of grasses by the symbiotic Neotyphodium and Epichloë species. These fungi are transmitted through host seed, and colonize aerial tissues that develop from infected shoot apical meristems of the seedling and tillers. We present evidence that vegetative hyphae of Neotyphodium and Epichloë species infect grass leaves via a novel mechanism of growth, intercalary division and extension. Hyphae are attached to enlarging host cells, and cumulative growth along the length of the filament enables the fungus to extend at the same rate as the host. This is the first evidence of intercalary growth in fungi and directly challenges the centuries-old model that fungi grow exclusively at hyphal tips. A new model describing the colonization of grasses by clavicipitaceous endophytes is described. [Copyright &y& Elsevier]

Although much is known about the signals and mechanisms that lead to pathogenic interactions between plants and fungi, comparatively little is known about fungus-plant mutualistic symbioses. We describe a novel role for reactive oxygen species (ROS) in regulating the mutualistic interaction between a clavicipitaceous fungal endophyte, Epichloë festucae, and its grass host, Lolium perenne. In wild-type associations, E. festucae grows systemically in intercellular spaces of leaves as infrequently branched hyphae parallel to the leaf axis. A screen to identify symbiotic genes isolated a fungal mutant that altered the interaction from mutualistic to antagonistic. This mutant has a single-copy plasmid insertion in the coding region of a NADPH oxidase gene, noxA. Plants infected with the noxA mutant lose apical dominance, become severely stunted, show precocious senescence, and eventually die. The fungal biomass in these associations is increased dramatically, with hyphae showing increased vacuolation. Deletion of a second NADPH oxidase gene, noxB, had no effect on the E. festucae-perennial ryegrass symbiosis. ROS accumulation was detected cytochemically in the endophyte extracellular matrix and at the interface between the extracellular matrix and host cell walls of meristematic tissue in wild-type but not in noxA mutant associations. These results demonstrate that fungal ROS production is critical in maintaining a mutualistic fungus-plant interaction. [ABSTRACT FROM AUTHOR]