Enhanced culturing techniques for the mycobiont isolated from the lichen Xanthoria parietina

Abstract

Lichens and their isolated symbionts are potentially valuable resources for biotechnological approaches. Especially mycobiont cultures that produce secondary lichen products are receiving increasing attention, but lichen mycobionts are notoriously slow-growing organisms. Sufficient biomass production often represents a limiting factor for scientific and biotechnological investigations, requiring improvement of existing culturing techniques as well as methods for non-invasive assessment of growth. Here, the effects of pH and the supplement of growth media with either D-glucose or three different sugar alcohols that commonly occur in lichens, D-arabitol, D-mannitol and ribitol, on the growth of the axenically cultured mycobiont isolated from the lichen Xanthoria parietina were tested. Either D-glucose or different sugar alcohols were offered to the fungus at different concentrations, and cumulative growth and growth rates were assessed using two-dimensional image analysis over a period of 8 weeks. The mycobiont grew at a pH range from 4.0 to 7.0, whereas no growth was observed at higher pH values. Varying the carbon source in Lilly-Barnett medium (LBM) by replacing 1% D-glucose used in the originally described LBM by either 1%, 2% or 3% of D-mannitol, or 3% of D-glucose increased fungal biomass production by up to 26%, with an exponential growth phase between 2 and 6 weeks after inoculation. In summary, we present protocols for enhanced culture conditions and non-invasive assessment of growth of axenically cultured lichen mycobionts using image analysis, which may be useful for scientific and biotechnological approaches requiring cultured lichen mycobionts.

The Temporal Variation of Secondary Metabolites in the Mycobiont Culture and Thallus of Parmelina carporrhizans and Parmelina quercina Analyzed using High-Performance Liquid Chromatography

“…Furthermore, the study of the changes in secondary metabolites over time enriches the catalogue of substances produced by mycobionts. Our study also shows that it is possible to maintain cultures for long periods of time (up to 550 days), and current research is solving problems such as pH variation over time in cultures by improving growth [43].…”

“…However, there are some limitations of mycobiont cultures, including the inverse relationships between mycobiont growth and the production of secondary metabolites [4]. This can be resolved, at least in some species, by modulating culture conditions, i.e., changing the carbon source from glucose to sugar alcohols that commonly occur in lichens, such as ribitol, arabitol, and mannitol [43]. Another problem is the loss of the viability of cultures over time; somewhere between 100 and 300 days, the mycobiont culture stops the culture's growth [4,44,45].…”

How to build a lichen: from metabolite release to symbiotic interplay

“…For example, ribitol stimulated growth of the Ramalina farinacea and Ramalina fastigiata mycobionts (Wang et al, 2009), and mannitol and glucose that of the Xanthoria parietina mycobiont, although in the latter growth rates decreased with increasing ribitol ), the phytohormones IAA, salicylic acid (SA), JA, and ET, lectins with arginase (ARG) activity, and the phenolic polyketides usnic acid (UA), vulpinic acid (VA), and various depsides (DS), and depsidones (DSD). See text and Tables 1 and 2 (Pichler et al, 2021). Thus, the preference for specific sugars and sugar alcohols as carbon sources may depend on mycobiont species, and sugars and sugar alcohols can affect mycobiont growth in a concentration-dependent manner (Fig.…”

“…Moreover, exogenous treatment with sugars and sugar alcohols affects the growth of cultured lichen mycobionts. For example, ribitol stimulated growth of the Ramalina farinacea and Ramalina fastigiata mycobionts (Wang et al ., 2009 ), and mannitol and glucose that of the Xanthoria parietina mycobiont, although in the latter growth rates decreased with increasing ribitol concentrations (Pichler et al ., 2021 ). Thus, the preference for specific sugars and sugar alcohols as carbon sources may depend on mycobiont species, and sugars and sugar alcohols can affect mycobiont growth in a concentration‐dependent manner (Fig.…”

Lichen cell factories: methods for the isolation of photobiont and mycobiont partners for defined pure and co-cultivation

“…After obtaining axenic (i.e. pure) cultures of the lichen partners, isolates are normally cultivated on various solid and soft agar media with different nutrients, C- and N-source concentrations [ 10 , 15 , 16 , 26 ]. Lichen symbionts grow much faster as cell aggregates in lab conditions than in nature, e.g.…”