Effects of culture medium and temperature on fungal growth of Mycena and marasmioid isolates and in vitro symbiotic culture in mycoheterotrophic orchid, Gastrodia pubilabiata

Abstract

In vitro symbiotic culture of Gastrodia pubilabiata seeds were conducted with the fungal isolates from G. pubilabiata roots. We obtained five fungal isolates, which belonged to Mycena, Marasmiaceae and Omphalotaceae. Firstly, optimal temperature and culture medium for subculture of these fungal isolates were examined. All five isolates grew the fastest on malt extract agar medium. Mycelial growth rate was highest at 25 °C between 10 °C and 40 °C. Secondly, we evaluated suitable culture vessels and organic materials for symbiotic culture. Seeds germinated well in petri dishes with Quercus leaf disc on water agar medium, and the seed germination process was well observed without dense mycelium. The most developed seedlings were found in glass bottles filled with Japanese cedar leaves, but densely grew mycelium prevent accurate seedling counts. Leaves of Quercus, Japanese cedar and bamboo were used as organic materials for symbiotic culture. All three leaves induced seed germination with Mycena and Marasmiaceae fungi, but material types affected subsequent seedling growth. Our method will contribute to understanding the mycorrhizal association of Gastrodia species and also other mycoheterotrophic plants.

<i>In-vitro</i> symbiotic germination of seeds of five mycoheterotrophic <i>Gastrodia</i> orchids with <i>Mycena</i> and <i>Marasmiaceae</i> fungi

“…The plates were incubated at 25 °C under dark conditions for 3-7 d. Grown mycelium was subcultured on 2% malt extract agar medium (1.5% agar) at 25 °C. The latter culture condition previously yielded the best mycelial growth rates for fungi isolated from G. pubilabiata ( Shimazaki et al, 2021 ). The fungal isolates used in this study were deposited in the NITE Biological Resource Center ( Table 1 ; NBRC114082, NBRC114332, NBRC114083).…”

“…2. As the type of organic material affects seedling growth in G. pubilabiata ( Shimazaki et al, 2021 ), improvement of the substrate match could induce further growth of G. confusa cultured with Mycena sp. 2.…”

“…The fungal isolates were subcultured on a malt extract agar medium for 2-3 wk. Leaf discs were prepared following Park et al (2012) and Shimazaki et al (2021) . Dead leaves of Quercus glauca Thunb.…”

“…In-vitro symbiotic germination was achieved in G. nipponica ( Umata & Nishi, 2010 ) and G. pubilabiata ( Umata, Yamauchi, & Hashimoto, 2000 ) using unidentified fungal isolates from mycorrhizal roots and fruiting bodies. In our previous study, we successfully induced in-vitro symbiotic germination of G. pubilabiata with Mycena and marasmioid isolates ( Shimazaki, Higaki, Rammitsu, & Ogura-Tsujita, 2021 ). These results imply that in-vitro symbiotic culture systems occur in Gastrodia species other than G. elata .…”