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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. 

ABSTRACT Terpenes are among the oldest and largest class of plant-specialized bioproducts that are known to affect plant development, adaptation, and biological interactions. While their biosynthesis, evolution, and function in aboveground interactions with insects and individual microbial species are well studied, how different terpenes impact plant microbiomes belowground is much less understood. Here we designed an experiment to assess how belowground exogenous applications of monoterpenes (1,8-cineole and linalool) and a sesquiterpene (nerolidol) delivered through an artificial root system impacted its belowground bacterial and fungal microbiome. We found that the terpene applications had significant and variable impacts on bacterial and fungal communities, depending on terpene class and concentration; however, these impacts were localized to the artificial root system and the fungal rhizosphere. We complemented this experiment with pure culture bioassays on responsive bacteria and fungi isolated from the sorghum rhizobiome. Overall, higher concentrations (200 µM) of nerolidol were inhibitory toFerrovibriumand tested Firmicutes. While fungal isolates ofPenicilliumandPericoniawere also more inhibited by higher concentrations (200 µM) of nerolidol,Clonostachyswas enhanced at this higher level and together withHumicolawas inhibited by the lower concentration tested (100 µM). On the other hand, 1,8-cineole had an inhibitory effect onOrbiliaat both tested concentrations but had a promotive effect at 100 µM onPenicilliumandPericonia. Similarly, linalool at 100 µM had significant growth promotion inMortierella, but an inhibitory effect forOrbilia. Together, these results highlight the variable direct effects of terpenes on single microbial isolates and demonstrate the complexity of microbe-terpene interactions in the rhizobiome. ImportanceTerpenes represent one of the largest and oldest classes of plant-specialized metabolism, but their role in the belowground microbiome is poorly understood. Here, we used a “rhizobox” mesocosm experimental set-up to supply different concentrations and classes of terpenes into the soil compartment with growing sorghum for 1 month to assess how these terpenes affect sorghum bacterial and fungal rhizobiome communities. Changes in bacterial and fungal communities between treatments belowground were characterized, followed by bioassays screening on bacterial and fungal isolates from the sorghum rhizosphere against terpenes to validate direct microbial responses. We found that microbial growth stimulatory and inhibitory effects were localized, terpene specific, dose dependent, and transient in time. This work paves the way for engineering terpene metabolisms in plant microbiomes for improved sustainable agriculture and bioenergy crop production.