The pink color of Pleurotus salmoneostramineus is due to a complex of pink color protein (PsPCP) and 2-dehydro-3-deoxylaetiporic acid A, which is a polyene pigment with the yellow color of Laetiporus sulphureus. In this study, we used the Cas9 ribonucleoprotein as a plasmid-free genome-editing tool to perform gene destruction in P. salmoneostramineus. Gene disruption via split-marker recombination by targeting the PsPCP gene using a pre-assembled Cas9/single-guide RNA system was introduced into the protoplasts of the wild-type monokaryotic P. salmoneostramineus strain N13 (A1B1) or N83 (A2B2). Disruptions at the target site were identified using genomic PCR, followed by sequencing. Strains N13 ∆PsPCP and N83 ∆PsPCP were matured on PDA agar plates and then cultivated on sawdust media to form the fruit body of the dikaryotic mutant N13 ∆PsPCP × N83 ∆PsPCP. In addition, compatible mutants of N13 ∆PsPCP × N83 and N13 × N83 ∆PsPCP were cultivated, and the pink coloring of these fruiting bodies was compared. N13 ∆PsPCP × N83 ∆PsPCP showed completely white fruiting bodies. In vitro color-developing reaction using 2-dehydro-3-deoxylaetiporic acid A extracted from the mutant with recombinant PsPCP, which showed no color, suggested that the amount of the compound was trace or degraded during fruit body formation.

A macroconidial synanamorph, namely a phragmosporous phialidic morph of Kendrickiella phycomyces (Pezizomycotina incertae sedis), was newly observed in authentic cultures isolated from the Kitora and Takamatsuzuka Tumuli in Japan. The synanamorph was sparsely produced throughout of the colonies on PDA after a few days at room temperature in cultures revived from lyophiles or in streak cultures, both derived from germinated Kendrickiella-type aseptate microconidia. After 1-2 wk, the microconidial synanamorph (the Kendrickiella-type synanamorph) and creeping hyphae completely overgrew the macroconidial morph produced on the colony surface. This sparsely produced morph appears to be a common cultural character in K. phycomyces with pleomorphic conidiation. The morphology of the macroconidial morph is described and the taxonomy of K. phycomyces is briefly reviewed and discussed.

The genus Vezdaea consists of lichenized or alga-associated fungi and is characterized by small apothecia without exciple. Unidentified Vezdaea was collected in Japan and morphological observation was performed. The collected samples differed from any known species of the genus Vezdaea in the combination of net-like morphology of paraphyses and the size of the asci and ascospores. Fungal isolates were established from ascospores and goniocysts, and the phylogenetic analysis was performed. The isolates from ascospores and adjacent goniocysts were shown to be conspecific and grouped within the genus Vezdaea. Vezdaea minima is established to accommodate the collected fungus. Species of Vezdaea with and without net-like paraphyses were shown to be not monophyletic respectively. An algal isolate was obtained from the goniocyst, which generally contains a single algal cell, and was nested within Coccomyxa viridis clade as the result of the phylogenetic analysis. Our result indicates that the photobiont of Vezdaea needs further investigation since it has been considered as Leptosira based on a single observation.

Two novel yeast species belonging to the genus Metschnikowia were isolated from flowers and young fruits in Okinawa, Japan. Five strains (JCM 35539, JCM 36740^T, JCM 36743, JCM 36744, and JCM 36747) and four strains (JCM 35542, JCM 35544^T, JCM 36745, and JCM 38279) were repeatedly isolated from samples collected over 2-3 y. Because BLAST analysis of D1/D2 large subunit ribosomal DNA sequences did not clearly resolve their phylogenetic placement and intragenomic polymorphisms were detected in the internal transcribed spacer region, their relationships were further assessed by whole-genome phylogenetic analysis using available genome data from Metschnikowia and related taxa. This analysis showed that the two novel species formed a distinct clade with Candida danieliae, the closest among described species in the dataset. Analysis of MAT-locus gene collinearity revealed that each novel species possessed strains of opposite mating types, and mating assays between compatible strains produced elongated asci containing needle-shaped ascospores, which is a characteristic feature of the genus Metschnikowia. By integrating phylogenomic, mating, morphological, and physiological evidence, we conclude that these strains represent two novel yeast species, for which the names Metschnikowia okinawaensis sp. nov. and Metschnikowia ryukyuensis sp. nov. are proposed.