Isolation and co-culturing of symbionts in the genus Usnea

Abstract

To understand the nature of a symbiotic relationship it is important to study the symbionts in different stages, from free-living to symbiosis. Lichens in the genus Usnea are mutualistic symbioses between Usnea spp., the mycobionts, and Trebouxia spp., the photobionts. In this study, in vitro experiments were conducted to isolate symbionts of several species of Usnea lichens and to produce axenic mycobiont and photobiont cultures. A co-culturing study was also carried out using the compatible, isolated symbionts to investigate the possibility of production of in vitro thalli. Usnea mycobionts were successfully isolated from different species, namely, U. articulata, U. ciliifera, U. inermis, U. pusilla, U. torulosa, and U. xanthopoga, and axenic cultures were prepared. Trebouxia photobionts were also isolated from the same species, except for U. articulata and U. xanthopoga. Although both dry and fresh lichen specimens were used in this study, the symbiont isolation was successful only from the fresh samples. The Usnea axenic cultures produced in this study were of two distinct morphological types. A phylogenetic analysis of fungal ITS data showed a clear relationship between the phylogenetic positions of cultured mycobionts and their morphological forms. In this study, the broth types of ‘Bold’s Basal’ and ‘Lilly and Barnett’ were the best growth media for the isolation of mycobionts and photobionts from Usnea thalli, respectively. These growth media were used in the co-culturing experiment and two in vitro thalli containing Usnea mycobionts and Trebouxia photobionts were produced. These isolation and co-culturing methods will be useful in future studies of the Usnea symbiosis.