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ABSTRACTEpichloëis 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 Epichloëhave implications for species diversity, food web structures and fundamental ecological processes. Epichloëproduce a range of secondary metabolites that can have negative effects on herbivores, be they invertebrates or vertebrates. In many managed pastoral systems, selected asexual Epichloëstrains 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 Epichloëcan also increase plant biomass and seed yield. Epichloëendophyte-technology has and continues to be a scientific, agricultural, and marketing success with selected asexual strains of Epichloënow 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 CO2levels 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 Epichloëstrains, particularly to mitigate climate related stresses.

This study investigated effects of seed aging and imbibition on sugar metabolite concentrations in Epichloëendophyte-infected and endophyte-free seed of tall fescue (Festuca arundinaceaSchreb.). Two treatments, namely, accelerated aging and imbibition, were applied to the seeds, with embryo and endosperm tissues analyzed separately. Gas chromatography with flame ionization detection was employed for analysis of sugar metabolites within the seed tissues. Mannitol, ribitol, and trehalose were more abundant in embryo than endosperm tissues and were identified at consistently higher concentrations within endophyte-infected compared to endophyte-free seeds. The ratio of raffinose to sucrose significantly increased with seed aging in both endophyte-free and endophyte-infected embryo tissues, while significantly lower concentrations of trehalose were detected in tissues dissected from aged-seed regardless of endophyte status. This research provides fundamental insight into the metabolic details of endophyte survival in seed and provides a first evaluation of key carbohydrates present in the fungal-plant symbiosis.

ABSTRACTAsexual 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 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.

Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium loliiand N. coenophialum,symbiotic endophytes of the Pooideae grasses Lolium perenneand Festuca arundinacea, respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxy-fluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor while 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 fungal 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 fluoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed.

Birds and other forms of wildlife are a major issue for airport authorities worldwide, as they can create hazards to operating aircraft. Wildlife “strikes,” the majority caused by birds, can cause damage to operating aircraft and in severe cases lead to a loss of human life. Many airfields contain large areas of ground cover herbage alongside their runways that consist of mixtures of grasses, legumes, and weeds that can harbor many invertebrates. Many airfields use insecticides to control insect populations; however, mounting pressure from regional councils and water boards aim to reduce this practice due to ground water runoff and contamination concerns. Avanex Unique Endophyte Technology, a product specifically developed to reduce the attractiveness of airports and surrounding areas to birds, is based on a novel association between a selected strain of Epichloe&x0308; endophyte and a turf-type tall fescue cultivar. This grass–endophyte association acts through a direct mechanism whereby a negative response in birds is created through taste aversion and postingestion feedback as well as an indirect mechanism by deterring many invertebrates, a food source of many bird species.