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Abstract Background Lichens, traditionally considered as a simple partnership primarily between mycobiont and photobiont, are, in reality, complex holobionts comprised of a multitude of microorganisms. Lichen mycobiome represents fungal community residing within lichen thalli. While it is acknowledged that factors like the host lichen species and environmental conditions influence the structure of the lichen mycobiome, the existing research remains insufficient. To investigate which factor, host genus or location, has a greater impact on the lichen mycobiome, we conducted a comparative analysis of mycobiomes within Parmelia and Peltigera collected from both Turkey and South Korea, using high-throughput sequencing based on internal transcribed spacer region amplification. Results Overall, the lichen mycobiome was dominated by Capnodiales (Dothideomycetes), regardless of host or location. At the order level, the taxonomic composition was not significantly different according to lichen genus host or geographical distance. Hierarchical clustering of the top 100 abundant ASVs did not clearly indicate whether the lichen mycobiome was more influenced by host genus or location. Analyses of community similarity and partitioning variables revealed that the structure of the lichen mycobiome is more significantly influenced by location than by host genus. When analyzing the core mycobiome by host genus, the Peltigera mycobiome contained more ASV members than the Parmelia mycobiome. These two core mycobiomes also share common fungal strains, including basidiomycete yeast. Additionally, we used chi-squared tests to identify host genus-specialists and location-specialists. Conclusions By comparing lichen mycobiomes of the same genera across different countries, our study advances our comprehension of these microbial communities. Our study elucidates that, although host species play a contributory role, geographic distance exerts a more pronounced impact on the structure of lichen mycobiome. We have made foundational contributions to understanding the lichen mycobiome occupying ecologically crucial niches. We anticipate that broader global-scale investigations into the fungal community structures will provide more detailed insights into fungal residents within lichens.

A total of twenty four lichen species belonging to six families were collected from mountainous region of Nepal. The methanol extracts of each species were tested for antimicrobial and antioxidant activitiesin vitro. It was found that extracts of twenty one lichen species were active againstB. subtilis and seven species were active againstS. aureus. Similarly, in DPPH assay, three speciesPeltigera sp.,Cladonia sp., andCanoparmelia sp. showed comparable activity with commercial standard, BHA. In ABTS+ assay, extracts ofParmoterma sp.,Ramalina sp.,Peltigera sp. andCladonia sp. showed stronger activity than ascorbic acid. The observed data after comparison with previously published reports indicated that the high altitude lichens contain stronger antioxidant and antibacterial constituents. Similarly, the methanol extracts ofHeterodermia sp. andRamalina sp. showed comparable toxicity effect with commercial standard berberine chloride indicating a potent source of anticancer drugs.

In the original publication [...]

Abstract Background Lichens are symbiotic organisms composed of a fungus and a photosynthetic partner, which are key ecological bioindicators due to their sensitivity to environmental changes. The endolichenic fungi (ELF) living inside lichen thalli, are an important but understudied component of playing crucial ecological roles such as nutrient cycling and protection against environmental stressors. Therefore ELF community investigation is vital for fostering sustainable ecosystems and leveraging their ecological benefits. Deciphering the intricate relationships between ELF and their lichen hosts, alongside the influence of environmental factors on these communities, presents a significant challenge in pinpointing the underlying drivers of community structure and diversity. Results Our research demonstrated that locational factors were the main drivers of the ELF community structure, rather than host haplotype. Several climatic factors affected the diversity of the ELF community and contributed to the prevalence of different types of fungal residents within the ELF community. A decrease in isothermality was associated with a greater prevalence of pathotrophic and saprotrophic fungi within the ELF community, resulting in an overall increase in community diversity. By conducting a structural equation modeling analysis, we identified a robust link between climatic variables, fungal trophic mode abundance, and the species diversity of the ELF community. Conclusion This study’s discoveries emphasize the significance of examining climate-related factors when investigating ELF’s structure and function. The connection between fungi and climate is intricate and complex, and can be influenced by various other factors. Investigating the potential for ELF to adapt to changing climatic conditions, as well as the potential effects of changes in ELF communities on lichen function, would be valuable research areas. We anticipate that our research results will establish a basis for numerous future ELF research projects and have a significant impact on the field.

Abstract Background Lichens represent not only the mutualism of fungal and photosynthetic partners but also are composed of microbial consortium harboring diverse fungi known as endolichenic fungi. While endolichenic fungi are known to exert a remarkable influence on lichen ecology through their crucial roles in nutrient cycling, bioprospecting and biodiversity, the enigmatic community structures of these fungal inhabitants remain shrouded in mystery, awaiting further exploration and discovery. To address knowledge gap, we conducted metabarcoding on two lichens using 18S gene amplification, Dirinara applanta and Parmotrema tinctorum, and compared their microbial communities to those found in the pine bark to which the lichens were attached. Our hypothesis was that the endolichenic communities would exhibit distinct diversity patterns, community structures, network structures, and specialist composition compared to the surrounding epiphytic community. Results Our investigation has shed light on the clear demarcation between the endolichenic and epiphytic fungal communities, as they exhibit markedly different characteristics that set them apart from each other. This research demonstrated that the endolichenic communities are less diverse as compared to the epiphytic communities. Through community similarity analysis, we observed that two endolichenic communities are more similar to each other in terms of community composition than with the adjacent epiphytic communities. Moreover, we unveiled a striking contrast in the network structures between the endolichenic and epiphytic communities, as the former displayed a more modular and less nested features that is evocative of a potent host-filtration mechanism. Conclusions Through our investigation, we have discovered that lichens harbor less intricate and interconnected fungal communities compared to the neighboring epiphytic environment. These observations provide valuable insights into the metagenomic architecture of lichens and offer a tantalizing glimpse into the unique mycobiome.

AbstractLichens play crucial roles in the ecosystems, contributing to soil formation and nutrient cycling, and being used in biomonitoring efforts to assess the sustainability of ecosystems including air quality. Previous studies on heavy metal accumulation in lichens have mostly relied on manipulated environments, such as transplanted lichens, leaving us with a dearth of research on how lichens physiologically respond to heavy metal exposure in their natural habitats. To fill this knowledge gap, we investigated lichens from two of South Korea’s geographically distant regions, Gangwon Province and Jeju Island, and examined whether difference in ambient heavy metal concentrations could be detected through physiological variables, including chlorophyll damage, lipid oxidation, and protein content. The physiological variables of lichens in response to heavy metals differed according to the collection area: Arsenic exerted a significant impact on chlorophyll degradation and protein content. The degree of fatty acid oxidation in lichens was associated with increased Cu concentrations. Our research highlights the value of lichens as a bioindicator, as we found that even small variations in ambient heavy metal concentrations can be detected in natural lichens. Furthermore, our study sheds light on which physiology variables that can be used as indicators of specific heavy metals, underscoring the potential of lichens for future ecology studies.

Abstract Endolichenic fungi are host organisms that live on lichens and produce a wide variety of secondary metabolites. Colorectal cancer stem cells are capable of self-renewal and differentiation into cancer cells, which makes cancers difficult to eradicate. New alternative therapeutics are needed to inhibit the growth of tumor stem cells. This study examined the ability of an extract of Jackrogersella sp. EL001672 (derived from the lichen Cetraria sp.) and the isolated compound 1′-O-methyl-averantin to inhibit development of cancer stemness. The endolichenic fungus Jackrogersella sp. EL001672 (KACC 83021BP), derived from Cetraria sp., was grown in culture medium. The culture broth was extracted with acetone to obtain a crude extract. Column chromatography and reverse-phase HPLC were used to isolate an active compound. The anticancer activity of the extract and the isolated compound was evaluated by qRT-PCR and western blotting, and in cell viability, spheroid formation, and reporter assays. The acetone extract of EL001672 did not affect cell viability. However, 1′-O-methyl-averantin showed cytotoxic effects against cancer cell lines at 50 μg/mL and 25 μg/mL. Both the crude extract and 1′-O-methyl-averantin suppressed spheroid formation in CRC cell lines, and downregulated expression of stemness markers ALDH1, CD44, CD133, Lgr-5, Msi-1, and EphB1. To further characterize the mechanism underlying anti-stemness activity, we examined sonic Hedgehog and Notch signaling. The results showed that the crude extract and the 1′-O-methyl-averantin inhibited Gli1, Gli2, SMO, Bmi-1, Notch-1, Hes-1, and the CSL complex. Consequently, an acetone extract and 1′-O-methyl-averantin isolated from EL001672 suppresses colorectal cancer stemness by regulating the sonic Hedgehog and Notch signaling pathways.

Lung cancer has the highest mortality rates worldwide. The disease is caused by environmental pollutants, smoking, and many other factors. Recent treatments include immunotherapeutics, which have shown some success; however, the search for new therapeutics is ongoing. Endolichenic fungi produce a whale of a lot of secondary metabolites, the therapeutic effects of which are being evaluated. Here, we used a crude extract and subfractions of the endolichenic fungus, Phoma sp. (EL006848), isolated from the Pseudevernia furfuracea. It was identified the fatty acid components, palmitic acid, stearic acid, and oleic acid, exist in subfractions E1 and E2. In addition, EL006848 and its fatty acids fractions suppressed benzo[a]pyrene (an AhR ligand)- induced expression of PD-L1 to inhibit the activity of multiple immune checkpoints. E2 subfraction, which had a higher fatty acid content than E1, downregulated expression of AhR/ARNT and several human transcription factors related to ESR1. Moreover, E2 showed a strong inhibitory effect on STAT3 expression and mild effect on NF-kB activity. These results suggest that fatty acids extracted from an endolichenic fungus can exert strong immunotherapeutic effects.

A new species, Bacidia fuscopallida Lee & Heo and four new records, B. ekmaniana R. C. Harris, Ladd & Lendemer, B. friesiana (Hepp) Körb., B. heterochroa (Müll. Arg.) Zahlbr. and B. suffusa (Fr.) A. Schneid., are described from South Korea. Bacidia fuscopallida differs from B. diffracta S. Ekman, the most similar species, by warted but non-granular thallus, paler and smaller apothecia without pruina, proper exciple without crystals, over 11-septate ascospores and smaller pycnidia and pycnoconidia. Bacidia ekmaniana is recorded new to Asia, B. heterochroa is reported new to northeastern Asia and B. friesiana and B. suffusa are new to Korea. Molecular analyses employing internal transcribed spacer (ITS) sequences strongly support the classification of the five species of Bacidia. A surrogate key is provided to assist in the identification of all 19 taxa in Bacidia of Korea.

Abstract Lichen-forming fungi are mutualistic symbionts of green algae or cyanobacteria. We report the comparative analysis of six genomes of lichen-forming fungi in classes Eurotiomycetes and Lecanoromycetes to identify genomic information related to their symbiotic lifestyle. The lichen-forming fungi exhibited genome reduction via the loss of dispensable genes encoding plant-cell-wall-degrading enzymes, sugar transporters, and transcription factors. The loss of these genes reflects the symbiotic biology of lichens, such as the absence of pectin in the algal cell wall and obtaining specific sugars from photosynthetic partners. The lichens also gained many lineage- and species-specific genes, including those encoding small secreted proteins. These genes are primarily induced during the early stage of lichen symbiosis, indicating their significant roles in the establishment of lichen symbiosis.Our findings provide comprehensive genomic information for six lichen-forming fungi and novel insights into lichen biology and the evolution of symbiosis.

Plant pathogens pose major threats on agriculture and horticulture, causing significant economic loss worldwide. Due to the continuous and excessive use of synthetic pesticides, emergence of pesticide resistant pathogens has become more frequent. Thus, there is a growing needs for environmentally-friendly and selective antimicrobial agents with a novel mode of action, which may be used in combination with conventional pesticides to delay development of pesticide resistance. In this study, we evaluated the potentials of lichen substances as novel biopesticides against eight bacterial and twelve fungal plant pathogens that have historically caused significant phytopathological problems in South Korea. Eight lichen substances of diverse chemical origins were extracted from axenic culture or dried specimen, and further purified for comparative analysis of their antimicrobial properties. Usnic acid and vulpinic acid exhibited strong antibacterial activities against Clavibacter michiganensis subsp. michiganensis. In addition, usnic acid and vulpinic acid were highly effective in the growth inhibition of fungal pathogens, such as Diaporthe eres, D. actinidiae, and Sclerotinia sclerotiorum. Intriguingly, the growth of Rhizoctonia solani was specifically inhibited by lecanoric acid, indicating that lichen substances exhibit some degrees of selectivity to plant pathogens. These results suggested that lichen substance can be used as a selective biopesticide for controlling plant disease of agricultural and horticultural significance, minimizing possible emergence of pesticide resistant pathogens in fields.

Lichen is a symbiotic mutualism of mycobiont and photobiont that harbors diverse organisms including endolichenic fungi (ELF). Despite the taxonomic and ecological significance of ELF, no comparative investigation of an ELF community involving isolation of a pure culture and high-throughput sequencing has been conducted. Thus, we analyzed the ELF community in Parmotrema tinctorum by culture and metabarcoding. Alpha diversity of the ELF community was notably greater in metabarcoding than in culture-based analysis. Taxonomic proportions of the ELF community estimated by metabarcoding and by culture analyses showed remarkable differences: Sordariomycetes was the most dominant fungal class in culture-based analysis, while Dothideomycetes was the most abundant in metabarcoding analysis. Thirty-seven operational taxonomic units (OTUs) were commonly observed by culture- and metabarcoding-based analyses but relative abundances differed: most of common OTUs were underrepresented in metabarcoding. The ELF community differed in lichen segments and thalli in metabarcoding analysis. Dissimilarity of ELF community intra lichen thallus increased with thallus segment distance; inter-thallus ELF community dissimilarity was significantly greater than intra-thallus ELF community dissimilarity. Finally, we tested how many fungal sequence reads would be needed to ELF diversity with relationship assays between numbers of lichen segments and saturation patterns of OTU richness and sample coverage. At least 6000 sequence reads per lichen thallus were sufficient for prediction of overall ELF community diversity and 50,000 reads per thallus were enough to observe rare taxa of ELF.

Aspicilia humida Lee is described as a new lichen-forming fungus from a wetland forest, South Korea. The new species is distinguishable from Aspicilia aquatica (Fr.) Körb., the most similar species, by the absence of prothallus, black disk without green color in water, olive-brown epihymenium, shorter hymenium, hymenium I + yellowish blue-green, wider paraphysial tips without a vivid pigment, smaller asci, smaller ascospores, and the presence of stictic acid. Molecular analyses employing internal transcribed spacer (ITS) and mitochondrial small subunit (mtSSU) sequences strongly support A. humida as a distinct species in the A. cinerea group. A surrogate key is provided to assist in the identification of all 28 aspicilioid species of Korea.

Lichens are known to produce many novel bioactive metabolites. To date, approximately 1,000 secondary metabolites have been discovered, which are predominantly produced by the lichen mycobionts. However, despite the extensive studies on production of lichen secondary metabolites, little is known about the responsible biosynthetic gene clusters (BGCs). Here, we identified a putative BGC that is implicated in production of a red pigment, cristazarin (a naphthazarin derivative), in Cladonia metacorallifera. Previously, cristazarin was shown to be specifically induced in growth media containing fructose as a sole carbon source. Thus, we performed transcriptome analysis of C. metacorallifera growing on different carbon sources including fructose to identify the BGC for cristazarin. Among 39 polyketide synthase (PKS) genes found in the genome of C. metacorallifera, a non-reducing PKS (coined crz7) was highly expressed in growth media containing either fructose or glucose. The borders of a cristazarin gene cluster were delimited by co-expression patterns of neighboring genes of the crz7. BGCs highly conserved to the cristazarin BGC were also found in C. borealis and C. macilenta, indicating that these related species also have metabolic potentials to produce cristazarin. Phylogenetic analysis revealed that the Crz7 is sister to fungal PKSs that biosynthesize an acetylated tetrahydoxynaphthalene as a precursor of melanin pigment. Based on the phylogenetic placement of the Crz7 and putative functions of its neighboring genes, we proposed a plausible biosynthetic route for cristazarin. In this study, we identified a lichen-specific BGC that is likely involved in the biosynthesis of a naphthazarin derivative, cristazarin, and confirmed that transcriptome profiling under inducing and non-inducing conditions is an effective strategy for linking metabolites of interest to biosynthetic genes.

Background: Endolichenic fungi (ELF), which live the inside the lichen thallus, contain many secondary metabolites that show various biological activities. Recent studies show that lichen and ELF secondary metabolites have antioxidant, antibacterial, antifungal, cytotoxic, and anticancer activities.Purpose: Here, the effects of an ELF extract and its bioactive compounds were investigated on the H1975 cell line focusing on immune checkpoint marker inhibition.Methods: An ELF was isolated from the host lichen Bryoria fuscescens (Gyelnik) Brodo and D. Hawksw and identified the species as Nemania sp. EL006872. The fungus was cultured on agar medium and acetonic extracts were obtained. Secondary metabolites radianspenes C and D, and dahliane D, were isolated from the crude extract. The biological effects of both the crude extract and the isolated secondary metabolites were evaluated in cell viability, qRT-PCR assays, flow cytometry analysis and western blotting.Results: The cell viability assay revealed that extracts from Nemania sp. EL006872 and the isolated secondary compounds had low cytotoxicity. The crude extract, radianspenes C and D, and dahliane D, suppressed expression of mRNA encoding PD-L1 and aromatic hydrocarbon receptor (AhR), and surface expression of PD-L1 protein by cells exposed to benzo[a] pyrene. Radianspenes C and D, and dahliane D, reduced expression of AhR, PD-L1, ICOSL, and GITRL proteins by H1975 lung cancer cells, as well as exerting anti-proliferative effects.Conclusion: Radianspenes C and D, and dahliane D, bioactive compounds isolated from Nemania sp. EL006872 ELF, have the potential for use as immunotherapy and immunoncology treatments.

Lendemeriella luteoaurantia B.G. Lee is described as a new lichen species from South Korea. The new species is identified by smaller, yellow-orange apothecia, larger ascospores with wider septum width, and the absence of Cinereorufa-green pigment and teloschistin, different from the closest species, L. aureopruinosa I.V. Frolov, Vondrák, Arup, Konoreva, S. Chesnokov, Yakovczenko and Davydov in morphology and chemistry. Molecular phylogeny employing internal transcribed spacer (nuITS), nuclear large subunit ribosomal RNA (nuLSU), and mitochondrial small subunit (mtSSU) sequences strongly supports the new species as nonidentical in the genus Lendemeriella. A preliminary key is provided to assist in the identification of all 10 species of Lendemeriella.

Collema Weber ex F.H. Wigg is scarcely scattered throughout South Korea. However, a detailed taxonomic or revisionary study has not been carried out for almost two decades. The present study was based on specimens deposited in the lichen herbarium at Korean Lichen Research Institute, which were identified using recent literature. In this revisionary study, a total of sixteen species of Collema, including five new records, C. furfuraceum var. luzonense, C. pulchellum var. subnigrescens, C. rugosum, C. subconveniens, C. tenax, and one variety C. leptaleum var. leptaleum were documented. Further, detailed descriptions of each species and their morphological, anatomical and chemical characteristics and a key to all known Collema species of South Korea were reported.

Two new species, Thelopsis ullungdoensis and Phylloblastia gyeongsangbukensis are described from Ullung-Do (Island), South Korea. The closest relatives from Europe and Korea are epiphytic Thelopsis flaveola which differs by their immersed or semi-immersed yellow ascomata, ascospores without halo and their habitat of smooth bark (mainly Fagus) in humid and cold climates. Thelopsis gangwondoensis differs by its bigger semi-immersed ascomata (600–700 µm in diam.), oblong halonate ascospores (8–12 × 6–8 µm) and its habitat of smooth bark of deciduous trees. P. gyeongsangbukensis differs from its relatives within the genus in having 5-septate ascospores (22–26 × 6–8 µm), semi-immersed, subglobose ascomata with a flattened top (250–400 µm) and a thin, matt, uneven gray-brown to gray-green continuous thallus without isidia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Atraric acid (AA) is a phenolic compound isolated from Stereocaulon japonicum that has demonstrated anti-androgen properties and was used to design an alternative formulation for the treatment of alopecia. This new topical formulation was designed using a solvent mixture system composed of ethanol as a volatile vehicle, oleic acid as a permeation enhancer, and water for skin hydration. The ideal topical AA formulation (AA–TF#15) exhibited an 8.77-fold higher human skin flux and a 570% increase in dermal drug deposition, compared to 1% (w/w) AA in ethanol. In addition, compared to other formulations, AA–TF#15 (1% [w/w] AA) activated keratinocytes and human dermal papilla cell proliferation at a concentration of 50 µM AA, which is equivalent to 50 µM minoxidil. Moreover, AA–TF#15 treatment produced a significant increase in hair regrowth by 58.0% and 41.9% compared to the 1% (w/w) minoxidil and oral finasteride (1 mg/kg)-treated mice. In addition, AA–TF#15 showed a higher expression level of aldehyde dehydrogenase 1, β-catenin, cyclin D1, and pyruvate kinase M2 proteins in the skin of AA–TF#15-treated mice compared to that of those treated with minoxidil and oral finasteride. These findings suggest AA–TF#15 is an effective formulation for the treatment of scalp androgenic alopecia.

Colorectal cancer (CRC) is the third most deadly type of cancer in the world and continuous investigations are required to discover novel therapeutics for CRC. Induction of apoptosis is one of the promising strategies to inhibit cancers. Here, we have identified a novel compound, Libertellenone T (B), isolated from crude extracts of the endolichenic fungus from Pseudoplectania sp. (EL000327) and investigated the mechanism of action. CRC cells treated by B were subjected to apoptosis detection assays, immunofluorescence imaging, and molecular analyses such as immunoblotting and QRT-PCR. Our findings revealed that B induced CRC cell death via multiple mechanisms including G2/M phase arrest caused by microtubule stabilization and caspase-dependent apoptosis. Further studies revealed that B induced the generation of reactive oxygen species (ROS) attributed to activating the JNK signaling pathway by which apoptosis and autophagy was induced in Caco2 cells. Moreover, B exhibited good synergistic effects when combined with the well-known anticancer drug, 5-FU, and another cytotoxic novel compound D, which was isolated from the same crude extract of EL000327. Overall, Libertellenone T induces G2/M phase arrest, apoptosis, and autophagy via activating the ROS/JNK pathway in CRC. Thus, B may be a potential anticancer therapeutic against CRC that is suitable for clinical applications.

Lichens play significant roles in ecosystem function and comprise about 20% of all known fungi. Polyketide-derived natural products accumulate in the cortical and medullary layers of lichen thalli, some of which play key roles in protection from biotic and abiotic stresses (e.g., herbivore attacks and UV irradiation).

Psoroglaena humidosilvae Lee is described as a new lichen species from a wetland forest in South Korea. The new species is distinct from P. stigonemoides (Orange) Henssen by little projections locally present on the thallus; smaller, paler, and globose perithecia; smaller asci; and smaller ascospores generally 3-septate. Molecular analyses employing internal transcribed spacer (ITS), mitochondrial small subunit (mtSSU), and nuclear large subunit ribosomal RNA (LSU) sequences strongly support P. humidosilvae as a nonidentical species in the genus Psoroglaena. A surrogate key is provided to assist in the identification of all 22 species of Psoroglaena.

An endophytic fungus, Phoma sp. NG-25, produces a set of structurally related polyketides including cercosporamide, phomodione, and usnic acid, among which, cercosporamide has been reported to have strong antifungal and anticancer activities. In this study, Phoma sp. NG-25 was grown in seven growth media to determine the optimal culture condition conducive for cercosporamide production. Cercosporamide production peaked on the eighteenth day of incubation in beef peptone dextrose (BPD) broth media. The cercosporamide titer reached to an average of 77.5 µg/mL in BPD. Paper disk diffusion assay revealed that culture filtrate containing cercosporamide as a major constituent inhibited the growth of taxonomically diverse plant pathogens, including ascomycetous, basidiomycetous, and oomycete fungi. Cercosporamide exhibited strong antifungal activities against two pepper anthracnose pathogens, Colletotrichum gloeosporioides and C. scovillei with EC50 values of 3.8 and 7.0 µg/mL, respectively. This study suggests the potential application of cercosporamide as an effective antifungal agent in controlling anthracnose in pepper. [ABSTRACT FROM AUTHOR]

Lichens are a natural source of bioactive compounds. Cladonia metacorallifera var. reagens KoLRI002260 is a rare lichen known to produce phenolic compounds, such as rhodocladonic, thamnolic, and didymic acids. However, these metabolites have not been detected in isolated mycobionts. We investigated the effects of six carbon sources on metabolite biosynthesis in the C. metacorallifera mycobiont. Red pigments appeared only in Lilly and Barnett’s media with fructose at 15 °C after 3 weeks of culture and decreased after 6 weeks. We purified these red pigments using preparative-scale high performance liquid chromatography and analyzed them via nuclear magnetic resonance. Results indicated that 1% fructose-induced cristazarin and 6-methylcristazarin production under light conditions. In total, 27 out of 30 putative polyketide synthase genes were differentially expressed after 3 weeks of culture, implying that these genes may be required for cristazarin production in C. metacorallifera. Moreover, the white collar genes Cmwc-1 and Cmwc-2 were highly upregulated at all times under light conditions, indicating a possible correlation between cristazarin production and gene expression. The cancer cell lines AGS, CT26, and B16F1 were sensitive to cristazarin, with IC50 values of 18.2, 26.1, and 30.9 μg/mL, respectively, which highlights the value of cristazarin. Overall, our results suggest that 1% fructose under light conditions is required for cristazarin production by C. metacorallifera mycobionts, and cristazarin could be a good bioactive compound.

Lichens are prolific producers of natural products of polyketide origin. We previously described a culture of lichen-forming fungus (LFF) Cladonia macilenta that produces biruloquinone, a purple pigment that is a phenanthraquinone rarely found in nature. However, there was no genetic information on the biosynthesis of biruloquinone. To identify a biosynthetic gene cluster for biruloquinone, we mined polyketide synthase (PKS) genes from the genome sequence of a LFF isolated from thalli of C. macilenta. The 38 PKS in C. macilenta are highly diverse, many of which form phylogenetic clades with PKS previously characterized in non-lichenized fungi. We compared transcriptional profiles of the 38 PKS genes in two chemotypic variants, one producing biruloquinone and the other producing no appreciable metabolite in vitro. We identified a PKS gene (hereafter PKS21) that was highly upregulated in the LFF that produces biruloquinone. The boundaries of a putative biruloquinone gene cluster were demarcated by co-expression patterns of six clustered genes, including the PKS21. Biruloquinone gene clusters exhibited a high degree of synteny between related species. In this study we identified a novel PKS family responsible for the biosynthesis of biruloquinone through whole-transcriptome analysis.

Abstract Lichens are symbiotic organisms that produce various secondary metabolites. Here, different lichen extracts were examined to identify secondary metabolites with anti-migratory activity against human lung cancer cells. Everniastrum vexans had the most potent inhibitory activity, and atranorin was identified as an active subcomponent of this extract. Atranorin suppressed β-catenin-mediated TOPFLASH activity by inhibiting the nuclear import of β-catenin and downregulating β-catenin/LEF and c-jun/AP-1 downstream target genes such as CD44, cyclin-D1 and c-myc. Atranorin decreased KAI1 C-terminal interacting tetraspanin (KITENIN)-mediated AP-1 activity and the activity of the KITENIN 3′-untranslated region. The nuclear distribution of the AP-1 transcriptional factor, including c-jun and c-fos, was suppressed in atranorin-treated cells, and atranorin inhibited the activity of Rho GTPases including Rac1, Cdc42, and RhoA, whereas it had no effect on epithelial-mesenchymal transition markers. STAT-luciferase activity and nuclear STAT levels were decreased, whereas total STAT levels were moderately reduced. The human cell motility and lung cancer RT² Profiler PCR Arrays identified additional atranorin target genes. Atranorin significantly inhibited tumorigenesis in vitro and in vivo. Taken together, our results indicated that E. vexans and its subcomponent atranorin may inhibit lung cancer cell motility and tumorigenesis by affecting AP-1, Wnt, and STAT signaling and suppressing RhoGTPase activity.

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.

Physconia hokkaidensis methanol extract (PHE) was studied to identify anticancer effects and reveal its mechanism of action by an analysis of cytotoxicity, cell cycles, and apoptosis biomarkers. PHE showed strong cytotoxicity in various cancer cells, including HL-60, HeLa, A549, Hep G2, AGS, MDA-MB-231, and MCF-7. Of these cell lines, the growth of MDA-MB-231 was concentration-dependently suppressed by PHE, but MCF-7 was not affected. MDA-MB-231 cells, triple-negative breast cancer (TNBC) cells, do not express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2), whereas MCF-7 cells are ER-positive, PR-positive, and HER-2-negative breast cancer cells. The number of cells in sub-G1 phase was increased after 24 h of treatment, and annexin V/PI staining showed that the population size of apoptotic cells was increased by prolonged exposure to PHE. Moreover, PHE treatment downregulated the transcriptional levels of Bcl-2, AMPK, and p-Akt, whereas it significantly upregulated the levels of cleaved caspase-3, cleaved caspase-9, and cleaved-PARP. In conclusion, it was confirmed that the PHE exhibited selective cytotoxicity toward MDA-MB-231, not toward MCF-7, and its cytotoxic activity is based on induction of apoptosis.

Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from Cladonia macilenta. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with Agrobacterium cells, and incubation temperature, were tested to identify an optimized ATMT condition for the C. macilenta mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in C. macilenta, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis.

Oxidative stress, mitochondrial dysfunction, and neuroinflammation are strongly associated with the pathogenesis of Parkinson’s disease (PD), which suggests that anti-oxidative and anti-inflammatory compounds might provide an alternative treatment for PD. Here, we evaluated the neuroprotective effects of evernic aid (EA), which was screened from a lichen library provided by the Korean Lichen Research Institute at Sunchon National University. EA is a secondary metabolite generated by lichens, including Ramalina, Evernia, and Hypogymnia, and several studies have described its anticancer, antifungal, and antimicrobial effects. However, the neuroprotective effects of EA have not been studied. We found that EA protected primary cultured neurons against 1-methyl-4-phenylpyridium (MPP+)-induced cell death, mitochondrial dysfunction, and oxidative stress, and effectively reduced MPP+-induced astroglial activation by inhibiting the NF-κB pathway. In vivo, EA ameliorated MPTP-induced motor dysfunction, dopaminergic neuronal loss, and neuroinflammation in the nigrostriatal pathway in C57BL/6 mice. Taken together, our findings demonstrate that EA has neuroprotective and anti-inflammatory effects in PD models and suggest that EA is a potential therapeutic candidate for PD.

Lichen-derived extracts are a potential source for skin health products due to their antioxidant activity that can prevent damage caused by ultraviolet (UV) rays. The slow natural growth and limited reproduction of lichens limit their use. However, endolichenic fungi (ELF) from lichen thalli can serve as a more efficient, rapid, and standardized biological resource for the cosmetic industry. We aimed to investigate the UV protection of Lobaria pulmonaria, Bryoria capillaris, and Usnea sp., and isolated ELF extracts. Total antioxidant, phenolic- flavonoid content, sun protection factor (SPF), and tyrosinase inhibitory activities of lichen and ELF extracts were also investigated. As a result, ELF extracts were found to have antioxidant content close to, and in some cases, much higher than lichen species. While SPF values of lichen species varied between 31.45-31.80, results close to lichens were found in the range of 6.54-32.01 values of ELF extracts. Interestingly, lichen extracts did not have tyrosinase inhibitory activity, while some ELF extracts had tyrosinase inhibitory activity in the range of 2.49-38.44. The results of this study suggest that ELF extracts may have the potential to be used as innovative UV protectants in the cosmetic industry. [ABSTRACT FROM AUTHOR]

Desertification is currently a major concern, and vast regions have already been devastated in the arid zones of many countries. Combined application of cyanobacteria with soil fixing chemicals is a novel method of restoring desertified areas. Three cyanobacteria, Nostoc sp. Vaucher ex Bornet & Flahault, Phormidium sp. Kützing ex Gomont and Scytonema arcangeli Bornet ex Flahault were isolated and tested in this study. Tacki-SprayTM (TKS7), which consists of bio-polysaccharides and tackifiers, was used as a soil fixing agent. In addition, superabsorbent polymer (SAP) was applied to the soil as a water-holding material and nutrient supplement. Application of cyanobacteria with superabsorbent polymer and TKS7 (CST) remarkably improved macro-aggregate stability against water and erodibility against wind after 12 months of inoculation when compared to the control soil. The mean weight diameter and threshold friction velocity of the CST treated soil were found to be 75% and 88% of those of the approximately 20-year-old natural cyanobacterial crust (N-BSC), respectively, while these values were 68% and 73% of those of the N-BSC soil after a single treatment of cyanobacteria alone (CY). Interestingly, biological activities of CST were similar to those of CY. Total carbohydrate contents, cyanobacterial biomass, microbial biomass, soil respiration, carbon fixation and effective quantum yield of CST treated soil were enhanced by 50-100% of the N-BSC, while those of control soil were negligible. Our results suggest that combined application of cyanobacteria with soil fixing chemicals can rapidly develop cyanobacterial crust formation in the field within 12 months. The physical properties and biological activities of the inoculated cyanobacterial crust were stable during the study period. The novel method presented herein serves as another approach for combating desertification in arid regions.

The genus Phaeophyscia Moberg, which belongs to the family Physciaceae, includes about 50 species, with 17 species reported in South Korea. This genus is characterized by a foliose thallus, Physcia/Pachysporaria-type ascospores, a paraplectenchymatous-type lower cortex, and lacking atranorin. In this study, about 650 specimens of Phaeophyscia aligned with the atranorin-absent groups collected from South Korea were re-examined. The taxonomy of these groups in South Korea requires revision based on the analyses of the morphology, chemistry, and molecular phylogeny. We infer that (1) each genus of the main foliose groups of Physciaceae forms a monophyletic clade, which also supports the separation of Phaeophyscia species with a prosoplectenchymatous lower cortex into the genus Physciella; (2) three atranorin-lacking genera were confirmed in South Korea: Hyperphyscia, Phaeophyscia, and Physciella, including a new combination named Physciella poeltii (Frey) D. Liu and J.S. Hur, and three new records from South Korea of Phaeophyscia hunana, P. leana, and P. sonorae; and (3) four species should be excluded from the lichen flora of South Korea: Hyperphyscia adglutinata, Phaeophyscia endococcina, Phaeophyscia erythrocardia, and Phaeophyscia imbricata.

Balloon flower (Platycodon grandiflorum) is a kind of mountain herbs whose roots have restorative properties and the cultivating acreage of balloon flower has been steadily increasing in Korea. More frequent rain and high amount of rainfalls as a result of climate changes predisposed balloon flower to the outbreaks of root rot at high-density cultivation area in recent years. Root crowns were usually discolored into brown to blackish brown at first and the infected plants showed slight wilting symptom at early infection stage. Severely infected roots were entirely rotted and whole plants eventually died at late infection stage. The overall disease severities of root rot of balloon flower were quite variable according to the surveyed fields in Jeonnam, Gyeongnam and Jeju Provinces, which ranged from 0.1% to 40%. The root rot occurred more severely at the paddy or clay soils than the sandy soils and their severities were much higher at lowland than upland in the same localty. The disease increased with aging of the balloon flower. The causal fungi were identified as Fusarium solani and F. oxysporum on the basis of their mycological characteristics. The optimum temperature ranges of their mycelial growths was found to be 24°C. The pathogenic characters of F. solani and F. oxysporum treated by artificial wounding inoculation on healthy roots of balloon flower revealed that F. solani was more virulent than F. oxysporum. This study identified the causal agents of root rot of balloon flower as Fusarium solani and F. oxysporum, probably for the first time.

The aim of this study was to contribute to the knowledge of the richness and distribution of the lichens in the Bío-Bío Region. A total of 120 samples were collected in the surroundings of Termas de Chillán and Las Trancas, Bio-Bio region. The sampling was realized at random considering the high diversity of available substrates. Taxonomical identification was performed on the basis of the analysis of morphological, reproductive and chromatographic (TLC) characters. A total of 41 species were identified, which include 18 families and 31 genera. Out of the total of identified species, 49 % are corticolous, 34 % saxicolous and 17 % terricolous. Of the total of species found, four are new records for Chile: Menegazzia confusa P. James, M. pertransita (Stirt.) R. Sant., Rinodina sophodes (Ach.) A. Massal. and Usnea sinensis Motyka. For each species, information on lichen name, family, substratum, altitude, global distribution, and sampling site is provided. Also, keys to identify the genera found in the study area are included. Pictures for the new records of lichens for the country are also presented. The work contributes to extend the knowledge on the taxa’s distribution in the region. The results suggest that the lichenological expeditions should be continued in unexplored localities of Chile in order to increase the knowledge of the richness, ecology and distribution of lichen biodiversity.