Impact of fertilisation on phenol content and growth rate of <Emphasis Type="Italic">Cladina stellaris</Emphasis>: a test of the carbon-nutrient balance hypothesis

Responses of concentrations of usnic (UA) and perlatolic (PA) acids and the relative growth rate (RGR) of a mat-forming lichen, Cladina stellaris, to enhanced N and P input were studied in a fertilisation experiment. It was predicted on the basis of carbon-nutrient balance (CNB) hypothesis that the concentrations of these phenolics would decline and the growth rate increase in response to increased nutrient uptake. The concentration of UA showed a convex response pattern to increased N input whereas the concentration of PA was non-responsive. An ecologically realistic, "moderate", N treatment clearly lowered the level of UA both with and without the P application. Applying P alone caused a significant increase in the level of UA. The RGR of C. stellaris did not respond to nutrient addition. The results indicate that even though the CNB hypothesis may be applicable in explaining concentrations of lichen secondary metabolites, it may be applied under a relatively narrow set of conditions. Especially inherited constraints in the growth of lichen fungi may seriously limit the responsiveness of lichens to short-time changes in the availability of resources. These limitations may also apply to other perennials adapted to nutrient-poor conditions.     

Lichen secondary metabolites affect growth of <Emphasis Type="Italic">Physcomitrella patens</Emphasis> by allelopathy

Lichen secondary metabolites can function as allelochemicals and affect the development and growth of neighboring bryophytes, fungi, vascular plants, microorganisms, and even other lichens. Lichen overgrowth on bryophytes is frequently observed in nature even though mosses grow faster than lichens, but there is still little information on the interactions between lichens and bryophytes.In the present study, we used extracts from six lichen thalli containing secondary metabolites like usnic acid, protocetraric acid, atranorin, lecanoric acid, nortistic acid, and thamnolic acid. To observe the influence of these metabolites on bryophytes, the moss Physcomitrella patens was cultivated for 5 weeks under laboratory conditions and treated with lichen extracts. Toxicity of natural mixtures of secondary metabolites was tested at three selected doses (0.001, 0.01, and 0.1 %). When the mixture contained substantial amounts of usnic acid, we observed growth inhibition of protonemata and reduced development of gametophores. Significant differences in cell lengths and widths were also noticed. Furthermore, usnic acid had a strong effect on cell division in protonemata suggesting a strong impact on the early stages of bryophyte development by allelochemicals contained in the lichen secondary metabolites.Biological activities of lichen secondary metabolites were confirmed in several studies such as antiviral, antibacterial, antitumor, antiherbivore, antioxidant, antipyretic, and analgetic action or photoprotection. This work aimed to expand the knowledge on allelopathic effects on bryophyte growth.     

Response of the lichen-eating moth <Emphasis Type="Italic">Cleorodes lichenaria</Emphasis> larvae to varying amounts of usnic acid in the lichens

Lichens are characterized by a great variety of secondary metabolites. The function of these substances remains partly unknown. In this study, we propose that some of these metabolites may expel insect herbivores. To test this hypothesis, we reared larvae of the lichenivorous moth Cleorodes lichenaria on three selected lichens, Cladonia arbuscula subsp. mitis, Usnea hirta, and Usnea dasypoga. In experimental setup, the secondary metabolite usnic acid was removed from the lichens with acetone prior to feeding, whereas a control was left untreated. On all three lichens, removal of usnic acid from the lichens using acetone significantly prolonged survival of larvae and increased their viability. Larvae reared on control lichens contained significantly more usnic acid than those reared on treated lichens, both in their biomass and their faeces. These results support the hypothesis that usnic acid serves as a repellent against insect feeding, besides its well established functions of UV protection and antimicrobial properties.     

Usnic acid

Since its first isolation in 1844, usnic acid [2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione] has become the most extensively studied lichen metabolite and one of the few that is commercially available. Usnic acid is uniquely found in lichens, and is especially abundant in genera such as Alectoria, Cladonia, Usnea, Lecanora, Ramalina and Evernia. Many lichens and extracts containing usnic acid have been utilized for medicinal, perfumery, cosmetic as well as ecological applications. Usnic acid as a pure substance has been formulated in creams, toothpaste, mouthwash, deodorants and sunscreen products, in some cases as an active principle, in others as a preservative. In addition to antimicrobial activity against human and plant pathogens, usnic acid has been shown to exhibit antiviral, antiprotozoal, antiproliferative, anti-inflammatory and analgesic activity. Ecological effects, such as antigrowth, antiherbivore and anti-insect properties, have also been demonstrated. A difference in biological activity has in some cases been observed between the two enantiomeric forms of usnic acid. Recently health food supplements containing usnic acid have been promoted for use in weight reduction, with little scientific support. The emphasis of the current review is on the chemistry and biological activity of usnic acid and its derivatives in addition to rational and ecologically acceptable methods for provision of this natural compound on a large scale.     

Influences of Nitrogen Sources on Usnic Acid Production in a Cultured Mycobiont of the Lichen Usnea hirta (L.) Wigg.

Effects of the nitrogen sources in the medium for the production of secondary metabolites in lichens were examined. The usnic acid production by a mycobiont of the lichen Usnea hirta was higher in the liquid medium containing ammonium and nitrate ions than in those containing amino acids.     

Influences of nitrogen sources on usnic acid production in a cultured Mycobiont of the lichen Usnea hirta (L.) Wigg

Effects of the nitrogen sources in the medium for the production of secondary metabolites in lichens were examined. The usnic acid production by a mycobiont of the lichen Usnea hirta was higher in the liquid medium containing ammonium and nitrate ions than in those containing amino acids.     

The antimicrobial activity of substances derived from the lichens Physcia aipolia, Umbilicaria polyphylla, Parmelia caperata and Hypogymnia physodes

In this study, in vitro antimicrobial activity of the physodic acid, usnic acid, atranorin and gyrophoric acid isolated from the lichens Hypogymnia physodes, Parmelia caperata, Physcia aipolia and Umbilicaria polyphylla, has been investigated. An antibiotic assessment was done against six bacteria (three Gram-positive and three Gram-negative) and eight fungi by determining the minimal inhibitory concentration (MIC) by the broth tube dilution method. The tested lichen substances inhibited growth of all the tested microorganisms. The bacteria showed a higher sensitivity against the tested fungi. The highest antimicrobial activity was found in the usnic acid of the Parmelia caperata lichen, where the lowest MIC was 0.0037 mg/ml against the Klebsiella pneumoniae (even lower than the one given by the streptomycin standard). The weakest antimicrobial activity was found in the physodic acid, which inhibited most of the microorganisms in the concentration of 1 mg/ml. Generally, all the components had relatively strong antimicrobial activity against the tested microorganisms, among which were human and animal pathogens. This could be of significance for their use for pharmaceutical purposes.     

Contrasting changes in palatability following senescence of the lichenized fungi Lobaria pulmonaria and L. scrobiculata

Epiphytic lichens can contribute significantly to ecosystem nutrient input because they efficiently accumulate atmospheric mineral nutrients and, in the case of cyanolichens, also fix nitrogen. The rate at which carbon and other nutrients gained by lichens enters the ecosystem is determined by lichen litter decomposability and by invertebrate consumption of lichen litter. In turn, these processes are driven by the secondary compounds present in senesced lichens. Therefore, we explored how lichen palatability and concentrations of secondary compounds change with tissue senescence for Lobaria pulmonaria, a green-algal lichen with cyanobacterial cephalodia, and Lobaria scrobiculata, a cyanobacterial lichen. During senescence both lichens lost 38–48 % of their stictic acid chemosyndrome, while m-scrobiculin and usnic acid in L. scrobiculata remained unchanged. Snails preferred senesced rather than fresh L. pulmonaria, while senesced L. scrobiculata were avoided. This provides evidence that species with labile secondary compounds will have higher turnover rates, through consumption and decomposition, than those producing more stable secondary compounds.     

Dissociation and metal-binding characteristics of yellow lichen substances suggest a relationship with site preferences of lichens

Background and Aims: Many species of lichen-forming fungi contain yellow or orangeextracellular pigments belonging to the dibenzofurans (usnicacid), anthraquinones (e.g. parietin) or pulvinic acid group.These pigments are all equally efficient light screens, leadingus to question the potential ecological and evolutionary significanceof diversity in yellow and orange lichen substances. Here thehypothesis is tested that the different pigments differ in metal-bindingcharacteristics, which suggest that they may contribute to adaptationto sites differing in pH and metal availability. Methods: UV spectroscopy was used to study the dissociation and the pHdependence of the metal-binding behaviour of seven isolatedlichen substances in methanol. Metals applied were selectedmacro- and micro-nutrients (Cu²⁺, Fe²⁺, Fe³⁺, Mg²⁺, Mn²⁺ andZn²⁺). Key Results: All the pigments studied are strong to moderate acids with pK_a1values between 2·8 and 4·5. Metal complexationis common in the lichen substances studied. Complexation takesplace under acidic conditions with usnic acid, but under alkalineconditions with parietin and most compounds of the pulvinicacid group. The pulvinic acid derivative rhizocarpic acid formsmetal complexes both in the acidic and the alkaline range. Conclusions: Metal complexation by lichen substances could be a prerequisitefor lichen substance-mediated control of metal uptake. Assumingsuch an effect at pH values where the affinity of the metalfor the lichen substance is intermediate would explain the strongpreference of lichens with usnic or rhizocarpic acids to acidicsubstrata. Moreover, it would explain the preference of lichenswith parietin and some lichens with compounds of the pulvinicacid group either for nutrient-rich substrata at low pH or forcalcareous substrata.     

Neuroprotective activity and cytotoxic potential of two Parmeliaceae lichens: Identification of active compounds

BackgroundLichens are symbiotic organisms capable of producing unique secondary metabolites, whose pharmacological activities are attracting much interest.PurposeThe present study aimed to investigate the in vitro neuroprotective effects and anticancer potential of methanol extracts of two Parmeliaceae lichens: Cetraria islandica and Vulpicida canadensis. The chemical composition of the two lichens was also determined.MethodsNeuroprotective activity was studied with respect to the antioxidant properties of the extracts; radical scavenging tests (ORAC and DPPH assays) were performed and oxidative stress markers (intracellular ROS production, caspase-3 activity, MDA and glutathione levels) were assessed in a hydrogen peroxide-induced oxidative stress model in astrocytes. Cytotoxic activity was tested against human HepG2 (hepatocellular carcinoma) and MCF-7 (breast adenocarcinoma) cell lines.ResultsCell viability studies identified a single concentration for each extract that was subsequently used to measure oxidative stress markers. Lichen extracts were able to reverse the oxidative damage caused by hydrogen peroxide, thus promoting astrocyte survival. Both lichen extracts also had anticancer activity in the cell lines, with IC₅₀ values of 19.51–181.05 µg/ml. The extracts had a high total phenolic content, and the main constituents identified by HPLC were fumarprotocetraric acid in Cetraria islandica, and usnic, pinastric and vulpinic acids in Vulpicida canadensis. The biological activities of the lichen extracts can be attributed to these secondary metabolites.ConclusionThe lichen species studied are promising sources of natural compounds with neuroprotective activity and cytotoxic potential, and warrant further research.     

Differential effects of lichens,mosses and grasses on respiration and nitrogen mineralization in soils of the New Jersey Pinelands

In the New Jersey Pinelands, severely disturbed areas often do not undergo a rapid succession to forest; rather, a patchy cover of lichens, mosses and grasses persists for decades. We hypothesized that these plant covers affect soil microbial processes in different ways, and that these effects may alter the successional dynamics of the patches. We predicted that the moss and grass covers stimulate soil microbial activity, whereas lichens inhibit it, which may in turn inhibit succession. We collected soil cores from beneath each type of cover plus bare soil within two types of highly disturbed areas—sites subjected to hot wildfires, and areas mined for sand. Organic matter (OM) content, soil respiration and potential N mineralization were measured in the cores. Soils under mosses were similar to those under grasses; they accumulated more OM and produced more mineral N, predominantly in the form of ammonium, than either the bare soils or the soils beneath lichens. Mineralization under lichens, like that of the bare soils but unlike the soils beneath mosses or grasses, was dominated by net nitrification. These patterns were reproduced in experimentally transplanted moss and lichen mats. Mosses appear to create high-nutrient microsites via high rates of OM accumulation and production of ammonium, whereas lichens maintain low-nutrient patches similar to bare soil via low OM accumulation rates and production of mineral N predominantly in the mobile nitrate form. These differences in soil properties may explain the lack of vascular plant invasion in lichen mats, in contrast to the moss-dominated areas.     

Lichen Secondary Metabolites in Flavocetraria cucullata Exhibit Anti-Cancer Effects on Human Cancer Cells through the Induction of Apoptosis and Suppression of Tumorigenic Potentials

Lichens are symbiotic organisms which produce distinct secondary metabolic products. In the present study, we tested the cytotoxic activity of 17 lichen species against several human cancer cells and further investigated the molecular mechanisms underlying their anti-cancer activity. We found that among 17 lichens species, F. cucullata exhibited the most potent cytotoxicity in several human cancer cells. High performance liquid chromatography analysis revealed that the acetone extract of F. cucullata contains usnic acid, salazinic acid, Squamatic acid, Baeomycesic acid, d-protolichesterinic acid, and lichesterinic acid as subcomponents. MTT assay showed that cancer cell lines were more vulnerable to the cytotoxic effects of the extract than non-cancer cell lines. Furthermore, among the identified subcomponents, usnic acid treatment had a similar cytotoxic effect on cancer cell lines but with lower potency than the extract. At a lethal dose, treatment with the extract or with usnic acid greatly increased the apoptotic cell population and specifically activated the apoptotic signaling pathway; however, using sub-lethal doses, extract and usnic acid treatment decreased cancer cell motility and inhibited in vitro and in vivo tumorigenic potentials. In these cells, we observed significantly reduced levels of epithelial-mesenchymal transition (EMT) markers and phosphor-Akt, while phosphor-c-Jun and phosphor-ERK1/2 levels were only marginally affected. Overall, the anti-cancer activity of the extract is more potent than that of usnic acid alone. Taken together, F. cucullata and its subcomponent, usnic acid together with additional component, exert anti-cancer effects on human cancer cells through the induction of apoptosis and the inhibition of EMT.     

Spatial trends in usnic acid concentrations of the lichen<Emphasis Type="Italic"> Flavocetraria nivalis</Emphasis> along local climatic gradients in the Arctic (Kongsfjorden,Svalbard)

The dibenzofuran usnic acid is an important secondary lichen metabolite, having many postulated biological roles, but evidence from field surveys is scanty. Trends in usnic acid concentrations and lobe width in the arctic lichen Flavocetraria nivalis were analysed along local longitudinal and altitudinal gradients in the Kongsfjorden area, northwestern Spitsbergen. Temperature was measured along the same gradients during 1 year. Other environmental variables were also estimated. The model that best explains the variability in usnic-acid levels includes the parameters effective temperature sum, frost sum and temperature range. Temperature range indicates a relationship between high usnic acid levels and humidity, whereas the two first parameters indicate a relationship with low temperatures, which could be direct or indirect. Much of the variability in usnic acid levels and lobe width could not be explained by the selected models. Thus, the secondary metabolism and lobe growth in this lichen is a complex matter, involving numerous environmental and possibly also intrinsic factors.     

Secondary metabolites in Cladina stellaris in relation to reindeer grazing and thallus nutrient content

We analysed concentrations of phenols and key macro-nutrients in a dominant mat-forming lichen, Cladina stellaris, in dry heath forests subjected to heavy reindeer grazing in Finnish Lapland. Lichen samples were collected in the beginning of two growth seasons from six to eight sites with old reindeer exclosures that served as control plots within sites. The concentration of perlatolic acid in lichen apices (top 10 mm) was higher in grazed plots than in control ones, whereas usnic acid did not seem to respond unequivocally to grazing. Moreover, there was a strong negative relationship between nitrogen and phenolic content of Cladina stellaris in intact plots but this relationship was absent in grazed ones. Changes in nitrogen and phosphorus contents caused by reindeer grazing were not correlated with changes in the level of phenolics in lichen thalli. The present result demonstrated that the carbon-nutrient balance (CNB) hypothesis may well explain some of the variation in lichen phenolic content but is not sufficient for explaining changes caused by reindeer grazing. We hypothesized that physical changes in the lichen microenvironment induced by reindeer have more profound impact on lichen phenolic content than alterations in thallus nutrient content.     

Removal of secondary compounds increases invertebrate abundance in lichens

We investigated how lichen carbon-based secondary compounds (CBSCs) affect abundance of invertebrates in five lichen species growing on the forest floor (Cladonia rangiferina, Cladonia stellaris) or on tree trunks (Evernia prunastri, Hypogymnia physodes, Pseudevernia furfuracea). To do this, CBSCs were removed by rinsing lichen thalli in acetone (which has no adverse effects on the lichens) and the lichens were re-transplanted in their natural habitat. After 4 months there was higher abundance of mites, springtails and spiders in the three epiphytic lichens that had their CBSC concentrations reduced. The increase in predatory spiders following CBSC reduction suggests that the compounds have multitrophic consequences. The acetone treatment reduced the number of nematodes in four of the lichen species. Given that lichens serve as important habitats for a diverse range of invertebrates, increased knowledge of how lichen CBSCs may regulate their abundance helps us to better understand the role that lichens and their defence compounds play in structuring forest food webs.     

Antioxidant and antimicrobial activity of some lichen species

The aim of the research is to explore the overall in vitro antioxidant activity, total phenol content, reduction power and antimicrobial activity of the methanol extracts of the lichens Cetraria pinastri, Cladonia digitata, Cladonia fimbriata, Fulgensia fulgens, Ochrolechia parella and Parmelia crinita. The methanol extract of the Cetraria pinastri showed a strong antioxidant activity, whereas the extracts of the species Fulgenesi fulgens, Cladonia fimbriata and Parmelia crinita showed the moderate one and the extract of the species Ochrolechia parella and Cladonia digitata the weak one. The methanol extract of the lichen Cetraria pinastri had the biggest total phenol content (32.9 mg/g of the dry extract). A certain correlation was established between the antioxidant activity and the total phenol content for the researched lichen extracts. The work also explores the antimicrobial activity of the methanol extracts of the mentioned species of lichens against six bacterial and eleven fungi species by the disc-diffusion method and by establishing the minimum inhibitory concentration (MIC). The methanol extracts of the lichens Cetraria pinastri and Parmelia crinita showed the strongest both antibacterial and antifungal activity against most of the tested microorganisms. These researches suggest that the lichens Cetraria prunastri can be used as new sources of the natural antioxidants and the substances with antimicrobial features.     

地衣型真菌次级代谢产物抗菌活性初步研究

地衣是一种传统的民族药物,能产生多种具有活性的物质。该研究对地衣型真菌(Xanthoria elegans,Myelochroa indica,Ramalina peruviana,Cladonia macilenta,Nephromopsis pallescens,Cladonia coccifera)进行液体培养,2个月后,培养液用乙酸乙酯萃取后获得初提物。该研究采用抑菌圈法评价地衣型真菌初提物对7种致病细菌(Bacillus subtilis,Bacillus cereus,Vibrio parahaemolyticus,Straphylococcus haemolyticus,Pseudomonas aeruginosa,Staphylococcus aureus,Micrococcus luteus)的抗菌活性,并测定最低抑菌浓度(MIC)。结果表明:6种地衣型真菌的初提物均具有一定的抗菌活性,且不同培养基对地衣型真菌产生抗菌物质有显著影响。其中,R.peruviana在MY液体培养基中所产生的次级代谢产物对金黄色葡萄球菌、藤黄微球菌、溶血性葡萄球菌、铜尿假单胞菌具有抑制效果,但在YMG培养基中所得初提物对供试7种致病细菌不具有抑菌效果。X.elegans在YMG培养基中所得初提物对枯草芽孢杆菌具有明显抗菌活性,其抑菌圈直径可达17.77 mm。该研究证实不同地衣型真菌液体培养初提物具有抗菌活性,不同的培养基也直接影响地衣型真菌抗菌效果。该研究结果为地衣型真菌的进一步研究及民族药的开发利用奠定了基础。     

Dietary Supplementation of Usnic Acid,an Antimicrobial Compound in Lichens,Does Not Affect Rumen Bacterial Diversity or Density in Reindeer

Reindeer (Rangifer tarandus tarandus) may include large proportions of lichens in their winter diet. These dietary lichens are rich in phenolic secondary compounds, the most well-known being the antimicrobial usnic acid. Previous studies have shown that reindeer host rumen bacteria resistant to usnic acid and that usnic acid is quickly detoxified in their rumen. In the present study, reindeer (n = 3) were sampled before, during, and after usnic acid supplementation to determine the effect on their rumen microbial ecology. Ad libitum intake of usnic acid averaged up to 278 mg/kg body mass. Population densities of rumen bacteria and methanogenic archaea determined by real-time PCR, ranged from 1.36 × 10⁹ to 11.8 × 10⁹ and 9.0 × 10⁵ to 1.35 × 10⁸ cells/g wet weight, respectively, and the two populations did not change significantly during usnic acid supplementation (repeated measures ANOVA) or vary significantly between the rumen liquid and particle fraction (paired t test). Rumen bacterial community structure determined by denaturing gradient gel electrophoresis did not change in response to intake of usnic acid. Firmicutes (38.7 %) and Bacteriodetes (27.4 %) were prevalent among the 16S rRNA gene sequences (n = 62) from the DGGE gels, but representatives of the phyla Verrucomicrobia (14.5 %) and Proteobacteria (1.6 %) were also detected. Rapid detoxification of the usnic acid or resistance to usnic acid may explain why the diversity of the dominant bacterial populations and the bacterial density in the reindeer rumen does not change during usnic acid supplementation.     

Determinants of Lichen Diversity in a Rain Forest Understory

Change in lichen diversity is often used as a bioindicator to estimate effects of atmospheric pollution, but natural variation in lichen cover and species richness can be very high. We examined the top-down effects of spore-consuming ants and the bottom-up effects of nutrient and light availability on lichen diversity associated with the leaf surface of the rain forest understory plant, Piper cenocladum. Plots containing P. cenocladum were randomly assigned to treatments in factorial experiments that included high and low light levels, nutrient enrichment, and presence and absence of the ant mutualist, Pheidole bicornis . At the conclusion of the experiments, plants were harvested and size of leaves, secondary metabolite content (amides), epiphyll cover, and the species richness of the lichens (which comprised 85% of the epiphyll community) were quantified. Epiphyll cover (mosses, liverworts, and lichens) was greater on plants that had ant-mutualists and balanced resources. Lichen species richness was greater for plants with balanced resources, particularly for those with high light availability. Relationships between toxins and lichen cover and richness were weak and unclear. In this system, natural sources of variation were reliable determinants of lichen diversity and both biotic and abiotic influences were important.