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Abstract BackgroundTruffles are subterranean fungal fruiting bodies that are highly prized for their culinary value. Cultivation of truffles was pioneered in Europe and has been successfully adapted in temperate regions throughout the globe. Truffle orchards have been established in North America since the 1980s, and while some are productive, there are still many challenges that must be overcome to develop a viable North American truffle industry. These challenges include extended delays between establishment and production, comparatively low yields, high spatial heterogeneity in yield distribution, and orchard contamination with lower-value truffle fungi. AimHere we review known requirements for truffle production including necessary environmental conditions, reproductive biology, and effective agronomic practices. ContentWe consider the potential limitations of importing exotic host-fungal associations into North America where there is already a rich community of competing ectomycorrhizal fungi, host pests and pathogens. We also describe the status of the North American truffle industry with respect to market potential, including production costs, pricing, and biological and socioeconomic risk factors. A critical aspect of modern trufficulture involves monitoring with genetic tools that supply information on identity, abundance and distribution of fungal symbionts, abundance of competitive and contaminating fungi, and insight into the interactions between fungal mating types that are fundamental to the formation of truffle primordia. ImplicationsCultivation of the ectomycorrhizal truffle symbiosis requires application of pragmatic agronomic practices, adopting rigorous quality control standards, and an understanding of fungal biology, microbiology, and molecular biology. Consequently, significant interdisciplinary collaboration is crucial to further develop the North American truffle industry. 

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. 

The genus Rugosporella is proposed to accommodate Peziza atrovinosa in the modern classification scheme of the Pezizaceae. We used DNA sequences of ITS, LSU and RPB2 to resolve the phylogenetic placement of this taxon. The species occupies a distinct position within a large and diverse clade of hypogeous and epigeous taxa, all of which are either known to be or presumed to be ectomycorrhizal. Although no DNA sequences of this species have been identified from ectomycorrhizal roots, we provide isotopic data supporting its ectomycorrhizal lifestyle. Peziza atrovinosa is distinctive in its moderately large vinaceous brown apothecia with thick flesh and the relatively small ascospores that are ornamented with a high reticulum and that become yellow-brown at maturity. This species is found across eastern North America.