Rhizopogon roseolus, an ectomycorrhizal mushroom, has a symbiotic relationship with the Japanese black pine tree (Pinus thunbergii). Several studies on bacterial endosymbionts in the ectomycorrhizal fungal mycelium have been reported in Amanita, Laccaria, and Lyophyllum. However, reports on bacterial endosymbionts in R. roseolus mycelium are not available. This study aimed to isolate and identify the bacteria from R. roseolus. Bacterial isolation was conducted using a Luria-Bertani medium. Three bacterial strains were successfully isolated from R. roseolus TUFC 10010 and H9 mycelia as strains MBRr6a, MBRr6b, and MBRrH9. Only R. roseolusstrain H1 did not harbor any bacteria. The fungal mycelia were treated using the LIVE/DEAD BacLight bacterial viability kit to detect bacteria. Hyphal bacteria were confirmed by SYTO staining, with a green spot indicating a live bacterium in or on the mycelium. Molecular identification was conducted for the 16S rRNA region. The phylogenetic tree was reconstructed using MEGA X software. Bacterial strain MBRr6a was identified as Bacillus tequilensis, while MBRr6b and MBRrH9 were identified as B. subtilis. This study is the first to report R. roseolus harboring two Bacillus species, B. tequilensis and B. subtilis, in its mycelium.

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A new variety of mushroom was produced by cross-breeding Pleurotus eryngii and Pleurotus nebrodensis. When single basidiospore isolation was performed, 40 strains of monokaryon were obtained from each of the parental dikaryotic strains. The monokaryon was crossed using 1600 combinations. Nine dikaryotic strains were selected based on mycelial characteristics on plate medium and cultivation tests on cedar sawdust medium. There was no correlation between the mycelial growth rate on plate medium and the fruiting yields on cedar sawdust medium. Cultivation tests were performed multiple times on two selected strains, ON26 and ON27, and ON26 was confirmed to have superior stability. The average yield of ON26 was 155-160 g and the growth period was 13 days. ON26 did not require low-temperature incubation after completion of the mycelial running, which is usually necessary in P. nebrodensis cultivation, and the cultivation period of ON26 was almost identical to that of P. eryngii, while retaining the pale color of the pilei surface. The hybrid strain ON26 of P. eryngii and P. nebrodensis was selected as a new type of mushroom.

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