Ascosporic Fertilization of Epichloë typhina in Dactylis glomerata Seed Production Fields in Oregon and Implications for Choke Management

Epichloe typhina, causal agent of choke, is a heterothallic, endophytic fungus that systemically infects the crown and foliage of Dactylis glomerata (orchardgrass). The sexual stage, which develops on reproductive tillers as 4 to 16 cm long, whitish, felt-like, cylindrical stromata, restricts inflorescence development, resulting in seed yield loss. Conidia produced on stromata function as spermatia. Fertilization is facilitated by flies (1), which transfer conidia while feeding, defacating, and ovipositing on the stromata. Conidia are not known to be wind disseminated (1). Following fertilization, stromata thicken and turn orange as perithecia develop and mature. Ascospores are forcibly discharged and are disseminated by wind. Although the process of infection of orchardgrass by E. typhina has not been fully established, ascospores are believed to be responsible for new plant infections. About 90% of US orchardgrass seed production occurs in the Willamette Valley of Oregon. Choke, first observed in orchardgrass in the Willamette Valley in the mid-1990s, spread rapidly among orchardgrass fields. Up to 30% of plants within a field can be infected by the third or fourth year of production (2). Although fertilization of stromata by flies is a well established fact (1), recent exclusion experiments suggest that a mechanism other than insects may be responsible for fertilization in E. typhina (4). The objective of this study was to determine if ascospores could serve as spermatia and fertilize stromata of E. typhina. Source material included 50 naturally E. typhina-infected orchardgrass plants from commercial fields. Plants were maintained via clonal propagation of tillers and vernalized in a growth chamber to initiate stromata. At tiller elongation and just prior to stromatal emergence, plants were transferred to the laboratory and each stroma was enclosed in a plastic tube (2.5 cm diameter × 30 cm long). Aquarium pumps, 0.45-μm filters, and flasks with deionized water were used to supply each tube with clean, humidified air. For ascosporic inoculum, a fertile stroma was excised and placed on a wire mesh screen over a microscope slides in a covered petri dish containing moist tissue. After 20 to 40 min, ascospores ejected onto the glass slide were suspended in drops of DI water + surfactant (2 drops of Tween 20 per 100 ml). The drops were examined at 200× to confirm presence of ascospores and that no conidia were present. A 5-μl drop of ascospore suspension was placed at the top, base, and center of each of five replicate stromata. Five-μl drops of conidia (1 × 102 conida/ml) or water were similarly transferred to stromata (five replicate stromata per treatment) as control inoculations. Conidia were collected from unfertilized stromata among a separate set of plants with an artist paint brush and suspended in 10-ml DI water + surfactant. There were 15 plants in the experiment, and the experiment was repeated twice.