Effect of Isolation Conditions on Diversity of Endolichenic Fungal Communities from a Foliose Lichen, Parmotrema tinctorum

Abstract

Endolichenic fungi (ELF) are emerging novel bioresources because their diverse secondary metabolites have a wide range of biological activities. Metagenomic analysis of lichen thalli demonstrated that the conventional isolation method of ELF covers a very limited range of ELF, and the development of an advanced isolation method is needed. The influence of four variables were investigated in this study to determine the suitable conditions for the isolation of more diverse ELF from a radially growing foliose lichen, Parmotrema tinctorum. Four variables were tested: age of the thallus, severity of surface-sterilization of the thallus, size of a thallus fragment for the inoculation, and nutrient requirement. In total, 104 species (1885 strains) of ELF were isolated from the five individual thalli of P. tinctorum collected at five different places. Most of the ELF isolates belong to Sordariomycetes. Because each part of lichen thallus (of different age) has unique ELF species, the whole thallus of the foliose lichen is needed to isolate diverse ELF. Moderate sterilization is appropriate for the isolation of diverse ELF. Inoculation of small fragment (1 mm2) of lichen thallus resulted in the isolation of highest diversity of ELF species compared to larger fragments (100 and 25 mm2). Moreover, ELF species isolated from the small thallus fragments covered all ELF taxa detected from the medium and the large fragments in this study. The use of two media—Bold’s basal medium (nutrient poor) and potato dextrose agar (nutrient rich)—supported the isolation of diverse ELF. Among the tested variables, size of thallus fragment more significantly influenced the isolation of diverse ELF than other three factors. Species composition and richness of ELF communities from different lichen thalli differed from each other in this study.

Endolichenic Fungal Community Analysis by Pure Culture Isolation and Metabarcoding: A Case Study ofParmotrema tinctorum

“…Several efforts have been made to isolate diverse ELF. We previously reported the use of small lichen segments increased the richness of ELF isolates because it prevented fast-growing fungal hyphae covering slow-growing fungal colonies [ 40 ]. Moreover, Muggia et al.…”

“…Because if ELF community is greatly affected by vertical transmission, ELF community dissimilarities would be constant regardless of distances between lichen segments. Furthermore, considerable numbers of ELF have been reported to be endophytes [ 9 , 12 , 13 , 34 , 40 ]. Hence, horizontal transmission would be reasonable that several endophytes invaded lichen thalli after epiphytic lichen had attached to tree bark.…”

“…Surface organic residues of thalli were removed using a syringe tip and running tap water, and thalli surfaces were sterilized with 70% ethanol followed by 0.4% sodium hypochlorite (both for 90 sec) as described by Yang et al. [ 40 ]. After rinsing with sterilized distilled water (SDW), lichen thalli were divided into 10 equally sized segments (1 cm 2 ), which were subjected to culture isolation and metabarcoding of their ELF ( Figure 1 ).…”

“…In recent years, studies on ELF diversity have been conducted mainly by culture-based analysis [ 14 , 34–36 ] and metabarcoding [ 33 , 37 , 38 ], but the structures of ELF communities determined by these two approaches were different from each other. Xylariaceous fungi (Sordariomycetes), which might play the role of an endophyte or late decomposer [ 39 ], were reported repetitively that they possessed a large amount of ELF as determined by culture-based studies [ 14 , 34 , 40–42 ]. However, ELF belonging to Sordariomycetes were not frequently detected in metabarcoding-based studies [ 33 , 38 ].…”

Transcriptional Divergence Underpinning Sexual Development in the Fungal Class Sordariomycetes

“…For more than 70 years, N. crassa has been a model system for studies in genetics and biochemistry, serving as the study organism for Beadle and Tatum’s “one gene, one enzyme” hypothesis ( 39 ). Chaetomium globosum is a cosmopolitan fungus that is a common contaminant on plant debris and often associated with terrestrial plants, marine algae, and lichens as endophytes ( 40 – 42 ). These five species all develop perithecia on carrot agar medium, a “common garden” environment that was established to create uniform growth conditions across species for comparative fungal transcriptome analysis ( 10 ).…”