Crossing the species barrier: investigating vertical transmission of a fungal endophyte from tall fescue within a novel ryegrass association

Many Poaceae grasses form a symbiosis with Epichloë fungal endophytes. Initially a serious problem in agriculture due to their production of alkaloids toxic to livestock, endophyte strains were identified that were less toxic to livestock whilst also possessing advantageous traits, including insect deterrent alkaloids. Selection and transfer of these endophytes into elite grass cultivars has resulted in pastures with improved persistence with no, or reduced, livestock toxicosis. These novel associations are now successfully marketed in New Zealand (NZ), Australia, USA and South America. Epichloë species exhibit strong host specificity and the asexual form is exclusively vertically transmitted through maternal lines via the seed embryo. In New Zealand, the dominant commercially available agricultural associations are between perennial ryegrass and E. festucae var. lolii strains that rely on the production of peramine, epoxy-janthitrems and ergovaline for their insect deterrent properties. However, AgResearch Ltd. and PGG Wrightson Seeds Ltd. have developed a novel association between a tall-fescue-derived endophyte (designated strain AR501) and a perennial ryegrass host. This brings a new set of secondary metabolites to the New Zealand agriculture scenario based on perennial ryegrass pastures, namely the loline alkaloids. An association formed between this fungus and perennial ryegrass would provide the grass sward excellent protection against ASW and grass grub, without creating any animal health problems. However, this process is far from perfect because of endophyte transmission failures. Possible reason for these failures include endophyte and host genotype incompatibility, environmental factors and crop management regimes. This study was an investigation of two of the factors that may affect the vertical transmission of AR501 and the maintenance of endophyte viability in stored seeds. A controlled environment was used to determine the influence of temperature on endophyte transmission frequency, on fungal biomass and on concentrations of loline alkaloids, in tall fescue and perennial ryegrass. Seedlings of both associations were arranged in a controlled environment at four temperature regimes ranging from 6-25°C. After three and six weeks, endophyte transmission frequencies and endophyte biomass were quantified. After three weeks plants from both associations were transferred from a 12/6°C to a 25/16°C environment and their endophyte biomass re-calculated. For perennial ryegrass, the endophyte transmission frequency was significantly higher in plants kept at a cold (day/night 12/6°C) temperature regime compared to the warm (day/night 25/16°C) regime. This was not observed in tall fescue. The endophyte biomass concentrations of both associations increased when after three weeks plants were transferred from the cold to warm temperature regime. To better understand how the endophyte and its host behaved under different temperature regimes, the cardinal temperature for AR501 and the host was determined. The cardinal temperatures for AR501 were higher in perennial ryegrass (Tb of -1.82°C, Topt of 25.26°C and Tm of 54.94°C) than in tall fescue (Tb of -0.04°C, Topt of 15.90°C and Tm of 29.04°C). Cardinal temperatures were also higher for perennial ryegrass than for tall fescue. However the induction of thermodormancy in tall fescue seeds interfered with the calculation of cardinal temperatures for the endophyte and tall fescue host, and it is probable that the optimal temperature for AR501 in the tall fescue host may be higher than determined in this study. Storage factors such as temperature, seed moisture content and length of storage can affect the viability of both seed and endophyte. Seeds of both associations were stored for 12 months under four storage temperature regimes (5°C, 10°C, 20°C and 30°C) and at two seed moisture contents (10% and 14%) to investigate whether temperature and/or seed moisture content would affect endophyte viability in stored seeds. In both associations, endophyte in seeds stored at 30°C and 14% SMC had died after 12 months of storage, but in seeds stored in moisture proof packaging at lower temperature (5 or 10°C), endophyte viability was maintained throughout the 12 months. In addition, the thermal time model was used to predict time for AR501 viability in the stored seeds. The prediction was that AR501 viability would remain above 70% for 13 months longer in tall fescue seeds than in perennial ryegrass seeds if they were stored at low temperature (≤10°C) and at 10% SMC. The effect of seed crop management factors, such as nitrogen (N) fertiliser, fungicide and plant growth regulator application on AR501 infection frequency in perennial ryegrass and Italian ryegrass was also investigated. None of the three seed production management factors affected the transmission of AR501 from plant to seed in perennial ryegrass, but did so in Italian ryegrass. This requires further investigation