Symbiosis as a successful strategy in continental Antarctica: performance and protection of Trebouxia photosystem II in relation to lichen pigmentation

Lichens as symbiotic associations consisting of a fungus (the mycobiont) and a photosynthetic partner (the photobiont) dominate the terrestrial vegetation of continental Antarctica. The photobiont provides carbon nutrition for the fungus. Therefore, performance and protection of photosystem II is a key factor of lichen survival. Potentials and limitations of photobiont physiology require intense investigation to extend the knowledge on adaptation mechanisms in the lichen symbiosis and to clarify to which extent photobionts benefit from symbiosis. Isolated photobionts and entire lichen thalli have been examined. The contribution of the photobiont concerning adaptation mechanisms to the light regime and temperature conditions was examined by chlorophyll a fluorescence and pigment analysis focusing on the foliose lichen Umbilicaria decussata from North Victoria Land, continental Antarctica. No photoinhibition has been observed in the entire lichen thallus. In the isolated photobionts, photoinhibition was clearly temperature dependent. For the first time, melanin in U. decussata thalli has been proved. Though the isolated photobiont is capable of excess light protection, the results clearly show that photoprotection is significantly increased in the symbiotic state. The closely related photobiont of Pleopsidium chlorophanum, a lichen lacking melanin, showed a higher potential of carotenoid-based excess light tolerance. This fact discriminates the two photobionts of the same Trebouxia clade. Based on the results, it can be concluded that the successful adaptation of lichens to continental Antarctic conditions is in part based on the physiological potential of the photobionts. The findings provide information on the success of symbiotic life in extreme environments.     

Combined pre-seed treatment with microbial inoculants and Mo nanoparticles changes composition of root exudates and rhizosphere microbiome structure of chickpea (<Emphasis Type="Italic">Cicer arietinum L.</Emphasis>) plants

Lichenicolous (lichen-dwelling) fungi have been extensively researched taxonomically over many years, and phylogenetically in recent years, but the biology of the relationship between the invading fungus and the lichen host has received limited attention, as has the effects on the chemistry of the host, being difficult to examine in situ. Raman spectroscopy is an established method for the characterization of chemicals in situ, and this technique is applied to a lichenicolous fungus here for the first time. Xanthoriicola physciae occurs in the apothecia of Xanthoria parietina, producing conidia at the hymenium surface. Raman spectroscopy of apothecial sections revealed that parietin and carotenoids were destroyed in infected apothecia. Those compounds protect healthy tissues of the lichen from extreme insolation and their removal may contribute to the deterioration of the apothecia. Scytonemin was also detected, but was most probably derived from associated cyanobacteria. This work shows that Raman spectroscopy has potential for investigating changes in the chemistry of a lichen by an invading lichenicolous fungus.     

A study of the fungi occurring on 15th century frescoes in Florence,Italy

The role of fungi in the biodeterioration of frescoes has been investigated by means of transparent adhesive tape and by swabbing the surface of selected areas as well as by a study of the materials used in restoration. In the Ognissanti church at Florence a large fungal population was found on two 15th century frescoes, one by Botticelli of ‘St Augustine’ and the other by Ghirlandaio of ‘St Jerome’. Twenty-three species were identified, 15 on the Botticelli where the majority of the colony forming units (cfu) were Penicillium brevi-compactum Dierckx (61%) and P. purpurogenum Stoll (18%), and 13 on the Ghirlandaio where Aspergillus versicolor (Vuill.) Tiraboschi was the most common (74%), together with Cladosporium sphaerospermum Penz. (22%). The materials often used in restoration namely calcium caseinate, masonite and animal glue were tested and found to be suitable substrates for species of Aspergillus and Penicillium but not for Cladosporium cladosporioides and C. sphaerospermum.     

Testing Lobaria pulmonaria (L.) Hoffm. as an indicator of lichen conservation importance of Italian forests

In Italy, many lichen species rare at the national level share their ecological requirements with Lobaria pulmonaria. The aim of this work is to test this lichen as a potential indicator for assessing forest sites which are worthy of conservation for lichens in Italy. The hypothesis to be tested is that stands hosting large and viable populations of this lichen also host nationally rare and sensitive lichen species. The study was carried out in two large forested areas of Italy under different bioclimatic conditions and with different forest types: Cansiglio Forest, in NE Italy, and Cilento in SW Italy. Three types of stands characterized by different abundance levels of L. pulmonaria were identified in each site. Ten plots for each type of stand were surveyed in each site. Macrolichens were surveyed on a total of 180 trees. The abundance of L. pulmonaria proved to be a valuable indicator of forest stands hosting nationally rare lichen species and cyanolichens in different forest types and in different bioclimatic conditions. Significant differences in species richness, in the occurrence and abundance of nationally rare lichen species and cyanolichens, and in species composition were found in both sites among stands with different levels of L. pulmonaria. Results are expected to contribute to the development of more lichen-oriented criteria in nature protection policies.     

The bacterial aetiology of rosy discoloration of ancient wall paintings

The inventory of microorganisms responsible for biological deterioration of ancient paintings has become an integral part of restoration activities. Here, the microbial agent of rosy discoloration on medieval frescoes in the Crypt of the Original Sin (Matera, Italy) was investigated by a combination of microscopic, molecular and spectroscopic approaches. The bacterial community from three rosy-discoloured painting sites was characterized by 16S rRNA gene-based techniques. The eubacterial population was prevalently composed of Actinobacteria, among which Rubrobacter radiotolerans-related bacteria accounted for 63-87% of the 16S rRNA gene pool per sampled site. Archaea, with prevalence of Haloarchaea-related species, were detected in one of the three sites where they accounted for < 0.1% of the total 16S rRNA gene pool. Raman spectroscopy confirmed the identity between R. radiotolerans carotenoids (bacterioruberins) and pigments responsible for colour alteration of frescoes. This investigation provides the first evidence of a causal relationship between heavy contamination by Rubrobacter-related bacterioruberin-producing bacteria and rosy discoloration of ancient wall paintings.     

Influence of different lights of mixed cultures of microalgae from ancient frescoes

Deterioration of frescoes inside a hypogean room of the Domus aurea in Rome was mainly caused by the growth of an algal association which persisted on the frescoes even in darkness. Following the proposal to utilize a new illumination system, the present research was undertaken to ascertain the effect of an incandescent lamp on the whole association in culture. For comparison a fluorescent lamp was also employed, and two low light intensities were tested. Observations on the development and morphology of the various algal species and analysis of the photosynthetic pigment content indicate that the Lyngbya spp. dominant in the association appeared to be highly adaptable to both lamps and intensities. Carefully limited use of the new lamp was advisable.     

New siphulin derivatives from the lichen Siphula ceratites

From the lichen, Siphula ceratites, two new metabolities, protosiphulin and oxysiphulin, were isolated besides the known compound, siphulin. The structures of the two new chromenones were proved by ¹H NMR and ¹³C NMR spectral analysis in comparison with those of siphulin.     

Secondary chemistry of cultured mycobionts: formation of a complete chemosyndrome by the lichen fungus of Lobaria spathulata

Abstract:The study aimed to optimize culture conditions and nutrient requirements for the production of secondary metabolites by the cultured mycobiont Lobaria spathulata. This species proved to be an excellent model system for such studies, as the complete chemosyndrome found in the natural lichen thallus was repeatedly formed in the cultured mycobiont with differentiated, aerial mycelia. Nutrient media containing the disaccharide, sucrose, were found to favour both rapid growth and the production of typical lichen substances. Higher proportions of the secondary compounds were detected in the developing mycobiont than in mature lichen thalli.     

Sequence Variation of the tRNALeu Intron as a Marker for Genetic Diversity and Specificity of Symbiotic Cyanobacteria in Some Lichens

We examined the genetic diversity of Nostoc symbionts in some lichens by using the tRNA^Leu (UAA) intron as a genetic marker. The nucleotide sequence was analyzed in the context of the secondary structure of the transcribed intron. Cyanobacterial tRNA^Leu (UAA) introns were specifically amplified from freshly collected lichen samples without previous DNA extraction. The lichen species used in the present study were Nephroma arcticum, Peltigera aphthosa, P. membranacea, and P. canina. Introns with different sizes around 300 bp were consistently obtained. Multiple clones from single PCRs were screened by using their single-stranded conformational polymorphism pattern, and the nucleotide sequence was determined. No evidence for sample heterogenity was found. This implies that the symbiont in situ is not a diverse community of cyanobionts but, rather, one Nostoc strain. Furthermore, each lichen thallus contained only one intron type, indicating that each thallus is colonized only once or that there is a high degree of specificity. The same cyanobacterial intron sequence was also found in samples of one lichen species from different localities. In a phylogenetic analysis, the cyanobacterial lichen sequences grouped together with the sequences from two free-living Nostoc strains. The size differences in the intron were due to insertions and deletions in highly variable regions. The sequence data were used in discussions concerning specificity and biology of the lichen symbiosis. It is concluded that the tRNA^Leu (UAA) intron can be of great value when examining cyanobacterial diversity.     

Evaluation of Antimicrobial Properties of Lichen Substances against Plant Pathogens

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.     

Análisis ecológico de las comunidades liquénicas de la Catedral de León (España)

Ecological analysis of Leon Cathedral lichen communities (Spain).A study of the lichen communities present on Leon Cathedral showed there to be 39 species present, mainly crustose forms of the Teloschistaceae and Lecanoraceae. Two lichen communities were found on calcareous stonework and on highly nitrified habitats. Species distribution depends mainly on nitrophytism, hygrophytism, photophytism and pH indexes, and to a lesser extent on angle of inclination, exposure and substrate type.     

Biological colonization patterns on the ruins of Angkor temples (Cambodia) in the biodeterioration vs bioprotection debate

The biological colonization present on the temples of the UNESCO World Heritage site of Angkor is wide and relevant, but a debate on its biodeteriorative and bioprotective effects is now developing. We investigated the biological patterns observed on two temples (Ta Nei and Ta Keo) exposed to different microclimatic conditions, in order to assess the damage caused by the communities present on the stone. We analyzed the penetration (depth and spread) into the stone, and the degree of decohesion of seven communities (green algae, cyanobacteria, lichens and mosses). The microscopic analyses highlighted a clear interaction between organism and stone, displaying a trend of increasing harmfulness from the community of the green algae (Trentepohlia) up to the moss communities. All the lichen communities show biodeterioration abilities: the Pyxine community seems more aggressive than the Lepraria and Cryptothecia communities, and more also than the cyanobacterial communities. The positive effects of the lichen cover in reducing dangerous evaporation processes cannot outweigh the negative effects of their hyphal penetration. Light forest cover seems beneficial for the conservation of the Angkor monuments since it reduces evaporation processes, but further studies should be carried out so as to find an optimal balance between contrasting factors.     

The occurrence of biomineralization products in four lichen species growing on sandstone in western Norway

High performance thin-layer chromatography/thin-layer chromatography, X-ray diffraction, and scanning electron microscopy analysis of thallus and lichen-rock interface samples, were undertaken to characterize biomineralization products in Fuscidea cyathoides, Ochrolechia tartarea, Ophioparma ventosa, and Pertusaria corallina, growing on sandstone in western Norway. Whewellite (monohydrate form of Ca oxalate) was found in the thallus of all species, but not in any of the weathering rinds beneath the species. A significantly higher amount of whewellite was detected in the thalli of F. cyathoides and O. ventosa than in the other two species. There were only a few differences in whewellite occurrence between the thallus edge and centre samples in the four species. HPTLC/TLC and SEM analysis indicate that lichen compounds occur within the rock beneath some of the lichen specimens. Only divaricatic acid was observed within the weathering rind beneath O. ventosa. No lichen substances were found in the weathering rind beneath F. cyathoides and P. corallina, whereas gyrophoric and lecanoric acids were found in the weathering rind beneath O. tartarea.     

POST-FIRE COLONIZATION OF A MEDITERRANEAN FOREST STAND BY EPIPHYTIC LICHENS

The post-fire colonization of a Quercus ilex forest by epiphytic lichens has been studied in Catalonia (NE Spain), eleven years after a fire. Specific richness and lichen biomass have been studied separately on Quercus ilex , Erica arborea , Rosmarinus officinalis . and Cistus albidus , to reveal possible differences among phorophytes in facilitating lichen establishment. Shrubs play an important role in colonization by common species whereas the stools of Quercus ilex offer a suitable substratum for rarer species. Cistus albidus , with the highest lichen diversity and 98·2% of the total biomass of macrolichens, is the most suitable phorophyte for lichen establishment.     

Lichen substance concentrations in the lichen Hypogymnia physodes are correlated with heavy metal concentrations in the substratum

Lichen substances (i.e. lichen-specific carbon-based secondary compounds) are known to be involved in the uptake and immobilization of metal ions, though the biochemical mechanisms of this interaction are largely unexplained. Previous research on potential effects of lichen substances on heavy metal uptake and tolerance mostly focused on lichens in heavily polluted areas with exceptionally high metal concentrations. In the present study, we aimed at gathering information as to whether lichen substances might be involved in the fine-tuning of metal uptake even at not or low-polluted sites. Therefore, we studied lichen substance concentrations in the epiphytic lichen Hypogymnia physodes and metal concentrations in its substratum in a montane spruce forest of Germany. H. physodes produces two depsides and five depsidones, which had been shown to be involved in metal homeostasis, namely in Cu and Mn uptake, in previous laboratory experiments. The amount of lichen substances increased with increasing heavy metal concentration in the substratum, though the latter varied only in the range of a few μmol g⁻¹ between the sample trees. Variability of lichen substance concentrations in H. physodes within the individual trees was low. Among the different lichen substances of H. physodes, the amount of the depsidone physodalic acid relative to the total of lichen substances was most closely correlated to the concentrations of Cu and Mn in the substratum, whereas the amount of the depsidone 3-hydroxyphysodic acid decreased both with increasing concentrations of these two metals and physodalic acid. Thus, our data suggest that lichen substances contribute to metal homeostasis not only in heavy metal-rich habitats, but also at not or low-polluted sites where the lichen substances apparently help to maintain constant intracellular metal concentrations despite of spatially varying availabilities of metal ions.     

PHYSIOLOGICAL EFFECT OF LICHEN SECONDARY METABOLITES ON THE LICHEN PARASITE MARCHANDIOMYCES CORALLINUS

It has been suggested that the host specificity exhibited by some lichenicolous fungi depends on their ability to tolerate the secondary chemistry of potential host lichens. For example, the lichen parasite Marchandiomyces corallinus is able to degrade the tissues of the lichen Flavoparmelia baltimorensis irrespective of the presence or absence of endogenous phenolic compounds. In contrast, the degradation of tissues from the lichen Lasallia papulosa is suppressed when endogenous phenolics are not removed. We have investigated the physiological basis of this inhibition in order to understand more about how lichen chemistry influences host preference in lichenicolous fungi. Results showed that the secondary compounds from L. papulosa inhibit the overall growth of M. corallinus , but not the catalytic activity of its tissue-degrading polysaccharidases. This effect is different from that shown by another lichen parasite, Nectria parmeliae , where lichen compounds specifically inhibited polysaccharidase activity. Compared with the compounds of L. papulosa , the endogenous phenolics of F. baltimorensis inhibited the growth of M. corallinus substantially less and exhibited little or no inhibition of polysaccharidases. For M. corallinus , host preference appears to be associated with physiological adaptation to the chemistry of F. baltimorensis .     

Effects of forest continuity and tree age on epiphytic lichen biota in coniferous forests in Estonia

Epiphytic lichen biota on Picea abies and Pinus sylvestris in Estonia was studied. Twenty-one spruce and 21 pine sample plots were located in old forests with long forest continuity, and 12 spruce and 12 pine sample plots in young first-generation forests (<100 years). Altogether 103 lichen species were recorded on the 330 sampled trees. Lichen species richness per plot was significantly higher in old forests in case of both tree species; 31 lichen species, including red-listed and protected species, were found only in old forests. Tree age had a positive effect on lichen species richness on tree stem in old and young spruce forests and in young pine forests. Tree age also had an effect on the presence of several species. Both tree age and forest continuity affected lichen species composition. Arthonia leucopellaea, Chrysothrix spp. and Lecanactis abietina were found in at least every third old spruce or pine forest and in no young forests, and can be regarded as good indicators of old coniferous forests with long continuity in Estonia.     

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.     

Isolation and characterization of β-d-glucan,heteropolysaccharide, and trehalose components of the basidiomycetous lichen Cora pavonia

Cora pavonia, a lichen having a basidiomycetous mycobiont, is rich in protein (36%), of which 10% is tyrosine. α,α-Trehalose is present and was isolated in 4.4% yield. The lichen was found to contain polysaccharides typical of basidiomycetes and different from those of ascomycetes and ascomycetous lichens. Isolated and characterized were a β-d-glucan and a heteropolysaccharide containing l-rhamnose, l-fucose, d-xylose, d-mannose, d-glucose, and d-galactose. The β-d-glucan was highly branched with 21% of nonreducing end-groups, contained 3-O-, 6-O-, and 3,6-di-O-substituted β-d-glucopyranosyl units, and had a main chain consisting of (1→3)- and (1→6)-links. The heteropolysaccharide component contained mainly mannose and xylose, having a mannose-containing nucleus and a main chain with preponderant (1→3)-linked α-d-mannopyranosyl residues. These were unsubstituted (10%), and 4-O- (10%) and 2,4-di-O-substituted (10%) with residues of β-d-xylopyranose. On methylation analysis of the heteropolysaccharide, a capillary column of DB-210 proved to be particularly useful for gas-liquid chromatographic resolution of partially O-methylated alditol acetates.     

Lichen chemistry and selective grazing by the coleopteran Lasioderma serricorne

Part of the Lichenological Herbarium of the University of Trieste (TSB) was infested by Lasioderma serricorne, a polyphagus coleopteran. 1440 samples of 50 lichen species with different chemistry (Parmelia s.lat., Physcia s.lat.) were analyzed in order to assess whether the lichen diet of the insect was species- and especially lichen-chemistry-selective. Multivariate analysis indicates a significant negative correlation between the presence of some lichen substances and grazing phenomena. Atranorin, calcium oxalate, fumarprotocetraric acid, gyrophoric acid, lecanoric acid, skyrin, usnic acid and zeorin play an antiherbivorous role.