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Epichloë is a genus of filamentous fungal endophytes that have co-evolved with cool-season grasses with which they form long-term, symbiotic associations. In natural ecosystems Epichloë have implications for species diversity, food web structures and fundamental ecological processes. Epichloë produce a range of secondary metabolites that can have negative effects on herbivores, be they invertebrates or vertebrates. In many managed pastoral systems, selected asexual Epichloë strains are purposefully associated with grass cultivars (termed novel endophyte-grass associations) as they confer beneficial traits that protect the host from abiotic stresses (e.g. drought) and biotic stresses (e.g. herbivory from invertebrates) while having minimal impact on the health and welfare of ruminant livestock. As well as conferring beneficial traits, agriculturally selected strains of Epichloë can also increase plant biomass and seed yield. Epichloë endophyte-technology has and continues to be a scientific, agricultural, and marketing success with selected asexual strains of Epichloë now essential components of many pasture ecosystems, within New Zealand and abroad. An estimated 90% of proprietary ryegrass now sold in New Zealand contains a selected endophyte strain while the economic impact in New Zealand of AR37 alone has been estimated at NZ$3.6 billion over 20 years. With a changing climate, agriculture will continue to experience increasing temperatures, elevated atmospheric CO2 levels and changing precipitation patterns that will likely increase the geographic range and overwintering survival rates of pests. Furthermore, plant disease outbreaks are expected to intensify due to increases in the severity of existing phytopathogens and/or invasions by new phytopathogens. Functional symbioses, such as those between temperate grasses and Epichloë, are among the most successful mechanisms by which plants can improve their ability to tolerate stress. Future research is likely to utilise targeted genetic manipulation techniques to broaden the biocontrol ability of Epichloë strains, particularly to mitigate climate related stresses. [ABSTRACT FROM AUTHOR]

In identifying endophytes for use in pastures in New Zealand there have been two strategies used, either exclusion of ergopeptine and lolitrem alkaloids or, retaining some ergopeptine expression to enhance the stability of natural defences against invertebrate pests. Both have their strengths and weaknesses. It is a matter of balancing these to ensure the end-user has access to a product that satisfies their needs – a persistent pasture with low to nil animal toxicity in terms of animal production and welfare. The range and intensity of ergot alkaloids in grazed pasture on both pasture and ruminants is reviewed, with emphasis on New Zealand. Ergot alkaloids associated with pasture are produced by associations between certain Epichloë endophyte strains and temperate grasses. Ergot alkaloids have been shown to improve persistence of pasture through providing resistance/deterrence to insect pests as well as deterring grazing animals. However, ergovaline is toxic to grazing animals. Some commercially available ryegrass-endophyte associations can produce ergovaline concentrations close to those found in associations between ryegrass and the standard endophyte. It is feasible to eliminate ergot alkaloids from pasture grasses in New Zealand as endophyte strains are commercially available that do not express ergovaline and yet still provide excellent pest resistance. [ABSTRACT FROM AUTHOR]

Definition: 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. [ABSTRACT FROM AUTHOR]

Asexual cool-season grass endophytes of the genusEpichloë(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 ofEpichloë coenophialastrain 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. [ABSTRACT FROM PUBLISHER]

Brassica species display enormous diversity and subsequently provide the widest assortment of products used by man from a single plant genus. Many species are important for agriculture, horticulture, in bioremediation, as medicines, soil conditioners, composting crops, and in the production of edible and industrial oils such as liquid fuels and lubricants. Many wild Brassica relatives possess a number of useful agronomic traits, including beneficial microbial endophytes that could be incorporated into breeding programs. Endophytes of Brassica , and/or their metabolites, have been demonstrated to improve and promote plant growth; increase yield; reduce disease symptoms caused by plant pathogens; reduce herbivory from insect pests; remove contaminants from soil; improve plant performance under extreme conditions of temperature and water availability; solubilise phosphate and contribute assimilable nitrogen to their hosts. Brassica napus (oilseed rape) and Brassica oleracea var. botrytis (broccoli and cauliflower) are the most economically important species of Brassica worldwide. These commercial crops are attacked by a wide range of pathogens and insect pests that are responsible for millions of dollars in lost revenue, with current control options offering little mitigation. No alternative control products are available for the Brassica industry, although it has been well documented in the literature that the use of endophytic microorganisms can offer beneficial traits to their host plants, including pest and disease resistance. The aim of this review is to describe the literature concerning beneficial microbial endophytes and their prospects to enhance or provide additional traits to their Brassica host species. [ABSTRACT FROM AUTHOR]

Grasses of the tribe Triticeae were screened to determine the presence of mutualistic epichloae fungal endophytes. Over 1500 accessions, from more than 250 species, encompassing 22 genera within the Triticeae were screened using immunodetection and direct staining/microscopy techniques. Only two genera, Elymus and Hordeum, were identified as harbouring epichloae endophytes with accessions native to a range of countries including Canada, China, Iran, Kazakhstan, Kyrgyzstan, Mongolia, Russia and the USA. Genetic analysis based on simple sequence repeat data revealed that the majority of endophytes cluster according to geographical regions rather than to host species; many strains isolated from Hordeum grouped with those derived from Elymus, and amongst the Elymus-derived strains, there was no clear correspondence between clustering topology and host species. This is the first detailed survey demonstrating the genetic diversity of epichloae endophytes within the Triticeae and highlights the importance of germplasm centres for not only preserving the genetic diversity of plant species but also the beneficial microorganisms they may contain. [ABSTRACT FROM AUTHOR]

Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium lolii and N. coenophialum, symbiotic endophytes of the Pooideae grasses Lolium perenne and Festuca arundinacea respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxyfluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor white M2R, were assessed for staining of endophyte hyphae in vitro from axenic fungal cultures and in planta, including epidermal leaf sheath peels, nodes, ovaries, embryos and meristems. CMFDA produced the greatest intensity of staining of fnngal hyphae and gave excellent contrast in planta compared to the plant cells. Compared to the other dyes, CMFDA was also the least affected by photo bleaching and continued to fuoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed. [ABSTRACT FROM AUTHOR]

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. Summary statement: A debate has been raised if fungal endophytes can relieve the plant growth‐defence trade‐off. Epichloë endophytes decoupled the trade‐off: the growth‐mediated penalisation of plant defences was observed in E− but not in E+ plants. The Epichloë production of alkaloids was likely the decoupling mechanism. [ABSTRACT FROM AUTHOR]

Many bacteria form symbiotic associations with plant-associated fungi. The effects of these symbionts on host fitness usually depend on symbiont or host genotypes and environmental conditions. However, bacterial endosymbionts, that is those living within fungal cells, may positively regulate host performance as their survival is often heavily dependent on host fitness. Contrary to this, bacteria that establish ectosymbiotic associations with fungi, that is those located on the hyphal surface or in close vicinity to fungal mycelia, may not have an apparent net effect on fungal performance due to the low level of fitness dependency on their host. Our analysis supports the hypothesis that endosymbiotic bacteria of fungi are beneficial symbionts, and that effects of ectosymbiotic bacteria on fungal performance depends on the bacterial type involved in the interaction (e.g. helper versus pathogen of fungi). Ecological scenarios, where the presence of beneficial bacterial endosymbionts of fungi could be compromised, are also discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plants interacting with mutualistic fungi (MF) or antagonistic fungi (AF) can form associations with bacteria. We assessed whether the performance gain conferred by mutualistic bacteria to fungal‐associated plants is affected by the interaction between symbiont traits, type of bacterial‐protective traits against AF and abiotic/biotic stresses. Results showed that (A) performance gain conferred by bacteria to MF‐associated plants was greater when symbionts promoted distinct rather than similar plant functions, (B) bacterial‐based alleviation of the AF's negative effect on plants was independent of the type of protective trait, (C) bacteria promoted a greater performance of symbiotic plants in presence of biotic, but not abiotic, stress compared to stress‐free situations. The plant performance gain was not affected by any fungal‐bacterial trait combination but optimised when bacteria conferred resistance traits in biotic stress situations. The effects of bacteria on fungal‐associated plants were controlled by the interaction between the symbionts' functional traits and the relationship between bacterial traits and abiotic/biotic stresses. [ABSTRACT FROM AUTHOR]

In a field study during two growing seasons in northern Sweden, we assessed meadow fescue ( Festuca pratensis Huds.) with and without Neotyphodium uncinatum to determine whether the endophyte affects plant performance regarding tiller and biomass production. In addition, we measured loline alkaloids produced in infected grasses. Approximately 1000 plants grew at two different levels of nitrogen fertilization: the normal amount applied in this region and one-half that amount. Increasing the amount of nitrogen increased the total number of tillers on the plants. Endophyte-infected plants produced more tillers per plant than plants without the endophyte; there was, however, no significant difference in proportion of reproductive tillers, winter survival, or total biomass production between endophyte-infected and noninfected plants. Infected plants produced loline alkaloids in different quantities without indication of being influenced by different nitrogen fertilization levels. [ABSTRACT FROM AUTHOR]

Culture‐independent survey techniques are fundamental tools when assessing plant microbiomes. These methods rely on DNA that is carefully preserved after collecting samples to achieve meaningful results. Immediately freezing samples to −80°C after collection is considered one of the most robust methods for preserving samples before DNA extraction but is often impractical. Preservation solutions can solve this problem, but commercially available products are expensive, and there is limited data comparing their efficacy with other preservation methods. In this study, we compared the impact of three methods of sample preservation on plant microbiome surveys: (1) RNAlater, a proprietary preservative, (2) a home‐made salt‐saturated dimethyl sulphoxide preservation solution (DESS), and (3) freezing at −80°C. DESS‐preserved samples, stored at room temperature for up to four weeks, did not show any significant differences to samples frozen at −80°C, while RNAlater inflated bacterial alpha diversity. Preservation treatments did not distinctively influence fungal alpha diversity. Our results demonstrate that DESS is a versatile and inexpensive preservative of DNA in plant material for diversity analyses of fungi and bacteria. [ABSTRACT FROM AUTHOR]

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

Few studies have evaluated the effects of bacteria that form associations with fungi on plant-microbial symbioses. We investigated the effects of a novel symbiotic bacterium (E226) of Epichloë , a well-researched mutualistic fungal endophyte of grasses, on perennial ryegrass (Lolium perenne) also associated with Epichloë. We hypothesised that E226 would promote plant growth but would not interfere with the in-planta growth of Epichloë and its production of antiherbivore alkaloids. E226, identified as Micrococcus luteus, formed an ectosymbiotic relationship with Epichloë when cultured on artificial media (i.e., bacterial cells were located on the hyphal surface) and possessed plant-growth promoting traits identified through whole genome analysis. Seeds of L. perenne , already colonised by an Epichloë strain, were inoculated with E226. The bacterium established within the seedlings, and the abundance of M. luteus populations initially increased with plant age. In agreement with our hypotheses, plant inoculation with E226 enhanced the production of leaves and did not affect the growth of Epichloë or the concentration of Epichloë -derived alkaloids. The putative abilities of E226 to solubilise phosphate and produce vitamins and metabolic cofactors may explain the observed plant growth promotion. • Micrococcus luteus strain E226 was an ectosymbiont of Epichloë endophytes. • E226 promoted the growth of plants. • E226 did not compromise the Epichloë growth and alkaloid production. • E226 promoted the plant growth likely via enhancing the host nutrition. [ABSTRACT FROM AUTHOR]

The most studied mechanism of protection against herbivores in grasses associated with Epichloë fungal endophytes has been the fungal production of alkaloids. However, the contribution of the plant immune response on the level of resistance to herbivores in symbiotic grasses has been poorly explored. We studied the relationship between the plant hormone, jasmonic acid (JA) and Epichloë fungal endophytes on herbivore defences in symbiotic grasses. We hypothesized that an exogenous application of methyl jasmonate (MeJA), an activator of the plant JA defence response, would increase the level of resistance of endophyte‐symbiotic and non‐symbiotic plants to a chewing insect. As Epichloë endophytes produce alkaloids, an enhancement of the JA defence would complement the resistance given by these alkaloids.Lolium multiflorum plants symbiotic and non‐symbiotic with the endophyte Epichloë occultans were subjected to an exogenous application of MeJA followed by a challenge with the generalist chewing insect Spodoptera frugiperda. We measured the level of plant resistance to chewing insects, and the defences conferred by host plants and fungal endophytes.Symbiotic plants were more resistant to S. frugiperda than their non‐symbiotic counterparts. However, despite the fact that the concentration of JA significantly increased in all plants exposure to MeJA, neither endophyte‐symbiotic nor non‐symbiotic plants showed an enhanced resistance to insects. Unexpectedly, the exposure of endophyte‐symbiotic plants to MeJA led to a reduction in the concentration of loline alkaloids (i.e. N‐formyllolines and N‐acetylnorlolines), consequently decreasing the level of plant resistance to the herbivore.Synthesis. Our results suggest that, rather than complementing the alkaloid‐based defence, the jasmonic acid hormone weakens the anti‐herbivore mechanism conferred by Epichloë endophytes. The present study highlights that the interaction between the jasmonic acid hormone and the presence of leaf fungal endophytes can be of importance for the effectiveness of the anti‐herbivore defences of symbiotic plants. Our results suggest that, rather than complementing the alkaloid‐based defence, the jasmonic acid hormone weakens the anti‐herbivore mechanism conferred by Epichloë endophytes. The present study highlights that the interaction between the jasmonic acid hormone and the presence of leaf fungal endophytes can be of importance for the effectiveness of the anti‐herbivore defences of symbiotic plants. [ABSTRACT FROM AUTHOR]

Perennial ryegrass is an important pasture, forage and turf plant, and in New Zealand, it is commonly and intentionally inoculated with selected beneficial endophyte strains. These endophytes provide enhanced traits to their host grasses with AR37, a market leading strain, conferring a broad spectrum of insect deterrence coupled with excellent animal performance. The effect of different nitrogen forms, with and without potassium, on a range of seed quality parameters, and presence of AR37, was determined. TSW and seed germination did not respond to nitrogen application. However, a combination of nitrogen and potassium reduced TSW and seed germination and increased empty seed percentage. There was no influence of spikelet position on any of the seed quality parameters measured. However, within a spikelet, there was an effect of floret position on TSW, empty seed and endophyte presence. The effect varied depending on whether nitrogen was applied as nitrate or ammonium. [ABSTRACT FROM AUTHOR]

Abstract: The plant hormone salicylic acid (SA) is recognized as an effective defence against biotrophic pathogens, but its role as regulator of beneficial plant symbionts has received little attention. We studied the relationship between the SA hormone and leaf fungal endophytes on herbivore defences in symbiotic grasses. We hypothesize that the SA exposure suppresses the endophyte reducing the fungal‐produced alkaloids. Because of the role that alkaloids play in anti‐herbivore defences, any reduction in their production should make host plants more susceptible to herbivores. Lolium multiflorum plants symbiotic and nonsymbiotic with the endophyte Epichloë occultans were exposed to SA followed by a challenge with the aphid Rhopalosiphum padi. We measured the level of plant resistance to aphids, and the defences conferred by endophytes and host plants. Symbiotic plants had lower concentrations of SA than did the nonsymbiotic counterparts. Consistent with our prediction, the hormonal treatment reduced the concentration of loline alkaloids (i.e., N‐formyllolines and N‐acetylnorlolines) and consequently decreased the endophyte‐conferred resistance against aphids. Our study highlights the importance of the interaction between the plant immune system and endophytes for the stability of the defensive mutualism. Our results indicate that the SA plays a critical role in regulating the endophyte‐conferred resistance against herbivores. [ABSTRACT FROM AUTHOR]