Gregory Bonito

Pezizales are a diverse and economically important order of fungi. They are common in the environment, having epigeous form, such as morels and hypogeous, forms called truffles. The mature ascospores of most epigeous Pezizales are forcibly discharged through an opening at the ascus apex created with the lifting of the operculum, a lid-like structure specific to Pezizales. The axenic cultures of Pezizales fungi isolated from single ascospores are important for understanding the life cycle, development, ecology, and evolution of these fungi. However, obtaining single-spore isolates can be challenging, particularly for collections obtained in locations where sterile work environments are not available. In this paper, we introduce an accessible method for harvesting ascospores from fresh ascomata in the field and laboratory for obtaining single-spore isolates. Ascospores are harvested on the inside cover of Petri plate lids in the field, air dried, and stored. At a later date, single-spore isolates are axenically cultured through serial dilution and plating on antibiotic media. With this approach, we were able to harvest ascospores and obtain single-spore isolates from 12 saprotrophic and 2 ectomycorrhizal species belonging to six Pezizales families: Discinaceae, Morchellaceae, Pezizaceae, Pyronemataceae, Sarcosomataceae, and Sarcoscyphaceae. This method worked well for saprotrophic taxa (12 out of 19 species, 63%) and was even effective for a few ectomycorrhizal taxa (2 out of 13 species, 15%). This process was used to study the initial stages of spore germination and colony development in species across several Pezizales families. We found germination often commenced with the swelling of the spore, followed by the emergence of 1–8 germ tubes. This method is sufficiently straightforward that, provided with sterile Petri dishes, citizen scientists from distant locations could use this approach to capture spores and subsequently mail them with voucher specimens to a research laboratory for further study. The generated single-spore Pezizales isolates obtained through this method were used to generate high-quality genomic data. Isolates generated in this fashion can be used in manipulative experiments to better understand the biology, evolution, and ecogenomics of Pezizales.