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The sclerotia of Pachyma hoelen are one of the traditional Chinese medicines and foods that are widely used in East Asian countries. The strains used for cultivation showed bad performance in recent years, and breeding of superior strains has become increasingly important for this fungus. Nevertheless, the mating system and life cycle of P. hoelen were still ambiguous. In this study, the methods for distinguishing between homokaryotic offspring with different mating types were established, as well as confirmation of strain hybridization based on allelic polymorphism at a locus of the rpb2 gene. The bipolar mating system was confirmed according to the mating results of homokaryotic SSIs. The fact that heterokaryotic parents produce homokaryotic meiospores proves that the life cycle is heterothallic. Combining scanning electron microscope observation and DAPI (4′,6-diamidino-2-phenylindole) fluorescent staining of hymenium and basidiospores in situ and ex situ, nuclear migration pattern from basidia to spores was revealed. The heterothallic life cycle was verified, revised, and supplemented step by step. This is the first report of systematic research on the mating system, life cycle, and outcrossing of homokaryotic offspring in P. hoelen. It will be helpful for the biological research, strain improvement, and development of the P. hoelen industry. [ABSTRACT FROM AUTHOR]

Cordyceps militaris (L.) Fr. Has long been recognized as a valuable functional food consumed in numerous countries. However, biosynthetic gene clusters of this species and safety regarding mycotoxin production remain largely unexplored. In this study, a ribosomally synthesized and post-translationally modified peptide (RiPP) cluster responsible for the production of cyclopeptide mycotoxins in Cordyceps was unveiled via genome mining. Ustiloxin B and a novel, predominant and Cordyceps specific ustiloxin I were confirmed by extraction and structural analysis. The difference between Ustiloxins I and B lied in the side chain at C19, where an additional methyl substituent in Ustiloxin I resulted in an alanine moiety substitution for glycine of Ustiloxin B. The simultaneous deletion of the two adjacent core genes, CmustYb and CmustYa , using a single guide RNA designed in the intergenic region, and subsequent in-situ complementation via AMA-mediated CRISPR/Cas9 system confirmed the RiPP cluster's responsibility for ustiloxin production. The cultivation of the edited strain yielded ustiloxin-free fruiting bodies without affecting agronomic characters. PCR and genome resequencing confirmed the absence of any off-target events or foreign sequence remnants. This study marks a significant advancement in utilizing CRISPR technology to control ustiloxins in food, underscoring its broader implications for food safety and quality improvement. [Display omitted] • Cordyceps militaris produce cyclopeptide mycotoxin, ustiloxins B and I. • Ustiloxin I was a new kind of ustiloxin, predominant and Cordyceps specific. • A RiPP was responsible for ustiloxin production confirmed by CRISPR/Cas9 editing. • Cultivation of the edited strain yielded ustiloxin-free fruiting bodies. • CRISPR-based technology is useful in improvement of food quality and safety. [ABSTRACT FROM AUTHOR]