219 L. J. MORSE, S. H. FAETH and T.A. DAY Arizona State University, School of Life Sciences, P.O. Box 874501, Tempe, AZ 85287-4501 lauracucci@gmail.com Abstract We examined the effect of endophyte infection, endophyte and host plant maternal genotype on traditional growth parameters. We also measured leaf water potential, leaf rolling, and stomatal density to provide explanations for differences in biomass production and relative growth rates. Our general findings show that Neotyphodium infection, Neotyphodium haplotype and its interaction with host maternal genotype influence Arizona fescue growth, and biomass production. Endophyte haplotype and its interaction with host maternal genotype is the most critical and consistent factor in influencing growth and physiological outcomes. Endophyte-host interactions are likely to be enormously complex because of the genetic and environmental variation that exists in natural populations. The outcome of these interactions in natural grass-endophyte systems is exceedingly difficult to predict based simply on the presence or absence of the endophyte. Keywords: growth rate, haplotype, leaf rolling, plant biomass, water potential, stomatal density Introduction Increasing evidence suggests that the outcome of the interaction between Neotyphodium endophyte and environmental factors depends on the genotype of the host grass and the haplotype of the endophyte (systemic endophytes are haploid) (Saikkonen et al. 2004; Müller & Kraus 2005). This dependency is even evident in agronomic grasses, where genetic variation of both host and endophyte are limited due to bottleneck effects and selective breeding. Far less is known about the influence of Neotyphodium infections on wild populations of native grasses than on agronomic grasses (e.g. Faeth 2002). Festuca arizonica Vasey (Arizona fescue) is a native perennial bunchgrass, that is widespread in semi-arid ponderosa pine (Pinus ponderosa)/grassland communities above 2000 m elevation in the southwest USA (Kearney & Peebles 1960). Neotyphodium infection frequencies in wild populations of Arizona fescue are usually high. Surveys show the frequency of infected plants in Arizona fescue populations range from 50-100% (Schulthess & Faeth 1998; Saikkonen et al. 1999). Despite high frequencies, however, the infection by asexual Neotyphodium does not appear to benefit the host, as predicted for Neotyphodium symbionts of tall fescue and ryegrass (Wilkinson & Schardl 1997). Genetic variation in Neotyphodium endophytes also potentially influences interaction outcomes. Different Neotyphodium strains that have been intentionally manipulated or transferred to agronomic tall fescue may alter growth and physiological properties of the host (e.g. Assuero et al. 2000, 2002). To our knowledge, there have been no tests of how endophyte haplotype in conjunction with host genotype and environmental factors, alters host growth and biomass production, gas exchange, and water relations in native grasses infected with Neotyphodium. We assessed the relative performance in terms of growth and biomass production of uninfected (E-) and Neotyphodium infected (E+) plants with two endophyte haplotypes within four maternal genotypes of Arizona fescue. We measured traditional growth analysis parameters, including relative growth rates, above- and below-ground biomass and below-ground:above-ground biomass ratios. We also measured water potential, leaf rolling, and stomatal density to provide explanations for differences in biomass production and relative growth rates. Our purpose here is to incorporate not just infection status but also variation in endophyte haplotypes within hosts and the environment to determine how these factors interact to influence host growth. Methods The host plant – Festuca arizonica Festuca arizonica Vasey (Arizona fescue) is a native perennial bunchgrass that is widespread in semi-arid ponderosa pine (Pinus ponderosa)/grassland communities above 2000 m elevation in the southwest USA (Kearney & Peebles 1960)). Neotyphodium infection frequencies in wild populations of Arizona fescue are usually high. The endophyte – Neotyphodium Arizona fescue harbours at least three distinct forms of Neotyphodium, each likely a unique species (Sullivan & Faeth 2004). Two of the four maternal plants use in our experiments (MD 1 and MD 49) harboured one non-hybrid endophyte haplotype (termed H1) and the other two maternal plants (MD 44 and MD 46) harboured another non-hybrid endophyte haplotype (termed H2). Haplotypes of the experimental plants were confirmed by microsatellite DNA analyses of multiple loci (Sullivan & Faeth 2004). Seed sources To test the effect of infection, host maternal genotype and endophyte haplotype and environmental factors on host growth and physiological parameters, we used Neotyphodium infected (E+) and uninfected (E-) Arizona fescue seeds from four naturally-infected maternal plants (MD 1, MD 44, MD 46, and MD 49) from the same population at Merritt Draw. Merritt Draw is a drainage meadow on the Mogollon Rim (elevation 2500 m) in Arizona. Maternal plants were randomly selected in 1997 from a pool of about 50 infected plants in the population. To remove the endophyte, maternal plants were split into ramets and half were treated hydroponically with low levels of the fungicide propiconazole while the other half were treated the same except without fungicide (Faeth & Sullivan 2003). All ramets were planted individually into 16 oz cups with native soil (Brolliar stony clay loam) and continually split and re- potted as they grew for about 1 year to provide cloned replicates after hydroponic treatment. The clones were then transplanted to a plot at the Arboretum in Flagstaff in 1998. After growing for 3 years in the field, E+ and E- (endophyte experimentally removed) seeds from each of the four maternal plant genotypes used in this experiment were collected from the clones. Seeds were collected from E- and E+ plants in September 2001, cold-treated for 30 days at 5°C and then stored at room temperature.