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Chapter 4 thesis

Fungi with dark-coloured mycelia - also known as black fungi - form a ubiquitous fraction of microbial communities on rock surfaces all over the world. These organisms show a high capacity to adapt to a wide range of ecological conditions, including those peculiar of extreme environments. Their high tolerance to multiple abiotic stresses, such as solar radiation and osmotic stress is assured by the production of melanin pigments and osmoprotective compounds. The cosmopolitan genera Lichenothelia and Saxomyces were taken as iconic representatives of polyextremotolerant rock-inhabiting black fungi. I investigated their phylogenetic relationships with an extended taxon sampling within the class Dothideomycetes, the most diverse and life-style rich fungal class in Ascomycota. The three loci phylogenetic inference I set up, considering both environmental samples and culture isolates, highlighted the paraphyly of the two genera. The integrative taxonomy approach based on morphological as well as phylogenetic evidences allowed the taxonomical revision of the genera and the description of three new taxa. Rock inhabiting fungi often share substrata with green algae and cyanobacteria and some of them are associated with lichen thalli. Lichenothelia is of a particular interest because it includes lichen parasites and species which are loosely associated with algae or which grow independently on rock. Given their life style plasticity, we chose it for an in vitro culture experiment, studying the development of three Lichenothelia species when co-cultured with two different subaerial algae isolated from lichens (i.e., lichen photobionts). The results showed that the presence of algae neither influence the growth rate of fungi nor the formation of any lichen-like structure. However, this standardized approach proved suitable for future investigations on fungal-algal interactions in other systems. Previous multi-locus phylogenies of Dothideomycetes have investigated evolutionary relationships at order and family level within the class but they often failed to resolve the early diverging nodes, which were generating inconsistent placements of some clades. Here, I applied a phylogenomic approach to resolve relationships in Dothideomycetes, adding the newly sequenced Lichenothelia and Saxomyces genomes, to a wide dataset comprised of 238 individuals. I explored the influence of tree inference methods, supermatrix vs. coalescent-based species tree, and the impact of varying amounts of genomic data. The phylogenomic reconstructions, based on up to three thousand genes, provide well-supported topologies for Dothideomycetes, recovering Lichenothelia and Saxomyces among the earliest diverging lineages in the class together with other rock inhabiting fungi and lichens and thus, suggesting the rock-inhabiting life style as ancestral in the class. Further studies will be necessary to shed light on the molecular bases of stress tolerance and latent capacity of establishing symbiosis of these fungi.

Lichen symbioses are complex, structured associations between fungi (mycobionts) and algae (photobionts). The extent to which these associations are more or less specific, is still largely unknown, and likely correlated with the dispersal mode of the partners. Variable patterns of mycobiont-photobiont associations have been detected in lichens and other algae-associated fungi. Here we present studies based on molecular and culture-dependent approaches of fungi and algae which form either well developed or loose associations. The lichen Tephromela atra is a geographically widespread species complex, which present a continuum of morphological diversity and different degrees of specificity towards Trebouxia photobionts. The fungus Schizoxylon albescens shows optional lichenization with algae of the genus Coccomyxa, occurring both as lichen and as saprobe when growing on different substrates. The species of Lichenothelia and Saxomyces, black, rock inhabiting fungi, form loose association with multiple algae. For these fungi we tested the capacity to form lichen-like relationships with algae using culture experiments. The experiments show various types of interactions with Trebouxia and Coccomyxa and shed new light on the life-style flexibility of fungi colonizing multiple ecological niches. This further suggests these fungi to be easily controlled study systems for the investigation of lichen symbiosis under lab conditions.

L‟analisi dei cambiamenti dell‟uso del suolo e la conseguente valutazione degli effetti che essi generano verso le diverse matrici (ambientale, paesaggistica, socia- le, di sicurezza e salute pubblica) assumono importanza sempre più riconosciuta nell‟ambito di una corretta pianificazione del territorio. All‟evidente urgenza di porre in atto misure di contrasto al consumo di suolo nei territori montani, si af- fianca, pertanto, la necessità di poter disporre di basi informative confrontabili che consentano il monitoraggio del fenomeno nello spazio e nel tempo. Con questo la- voro si esaminano i risultati desunti dall‟Inventario dell‟Uso delle Terre in Italia (IUTI), considerando anche potenzialità e limiti di utilizzo di questa base inventa- riale per analisi a scala regionale. Un particolare approfondimento riguarda i terri- tori montani della regione Veneto, per i quali si dispone di una base diacronica più estesa e di altre fonti di dati utilizzate per il confronto dei trend emersi.

Rocks are among the oldest terrestrial niches hosting a multiplicity of life forms, of which diversity has been only partially uncovered. Endolithic metacommunities comprise all major groups of microorganisms, such as chemo-organotrophic, chemo-lithotrophic and phototrophic, represented by bacteria, microalgae and microfungi. Their diversity is often difficult to describe and may remain underestimated. Furthermore, knowledge about the diversity of microorganisms colonizing rocks in peculiar niches is even poorer due to the difficulty to retrieve environmental specimens. Here, we report the phylogenetic and phenotypic characterization of a few endolithic fungi and algae isolated from rock fragments collected at high elevation, i.e., on the top of two mountains over 6000 m altitude, Muztagh Ata (China) and Cerro Mercendario (Argentina). The identity of the strains was confirmed by sequencing the nuclear ITS and LSU, the plastidial rbcL loci and by morphological analysis. Three fungal strains belonging to the class Dothideomycetes and one algal strain belonging to the genus Trebouxia were isolated from Muztagh Ata, while six fungal strains belonging to the order Chaetothyriales and four algal strains belonging to the genus Myrmecia were isolated from Cerro Mercedario. The detected species diversity is discussed in an evolutionary and ecological context.

Kranner, Ilse/0000-0003-4959-9109; Holzinger, Andreas/0000-0002-7745-3978, PubMed: 33223597, Evidence is emerging that phytohormones represent key inter-kingdom signalling compounds supporting chemical communication between plants, fungi and bacteria. The roles of phytohormones for the lichen symbiosis are poorly understood, particularly in the process of lichenization, i.e. the key events which lead free-living microalgae and fungi to recognize each other, make physical contact and start developing a lichen thallus. Here, we studied cellular and extracellularly released phytohormones in three lichen mycobionts, Cladonia grayi, Xanthoria parietina and Tephromela atra, grown on solid medium, and the effects of indole-3-acetic acid (IAA) on their respective photobionts, Asterochloris glomerata, Trebouxia decolorans, Trebouxia sp. Using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) we found that mycobionts produced IAA, salicylic acid (SA) and jasmonic acid (JA). IAA represented the most abundant phytohormone produced and released by all mycobionts, whereas SA was released by X. parietina and T. atra, and JA was released by C. grayi only. With a half-life of 5.2 days, IAA degraded exponentially in solid BBM in dim light. When IAA was exogenously offered to the mycobionts' compatible photobionts at "physiological" concentrations (as released by their respective mycobionts and accumulated in the medium over seven days), the photobionts' water contents increased up to 4.4%. Treatment with IAA had no effects on the maximum quantum yield of photosystem II, dry mass, and the contents of photosynthetic pigments and alpha-tocopherol of the photobionts. The data presented may be useful for designing studies aimed at elucidating the roles of phytohormones in lichens., Austrian Science Fund (FWF)Austrian Science Fund (FWF); FWFAustrian Science Fund (FWF) [P32092-B32, I1951-B16], Open access funding provided by Austrian Science Fund (FWF).; We also thank the FWF for financial support (grants P32092-B32 to IK and I1951-B16 to AH).

Fungi represent relevant allergens and plant pathogens that can disperse on long ranges, potentially producing severe consequences on public health and agriculture. Up to 11% of the bioaerosol particles are fungal spores and mycelium fragments. Estimation of fungal species diversity in time and space is decisive but may be biased by abiotic conditions and sampling methods. Traditional morphological analyses of fungal spores have been widely applied in aerobiology in the past, while recently eDNA metabarcoding can complement these studies. Here, we used both morphological analysis (spore count and taxon identification) and high-throughput sequencing to disentangle spatio-temporal variation of fungi across Northern and Central Italy and to evaluate the detection efficiency of the two approaches. Our results showed that eDNA metabarcoding detects about three times more genera and has a higher detection efficiency than the morphological analyses. However, the efficiency is high in both spore count and eDNA metabarcoding methods when the most abundant or the rarest genera are considered but it can substantially vary between the two approaches when moderately abundant genera are analyzed. Furthermore, morphological spore determination resulted in higher variance explained by PERMANOVA analysis with respect to eDNA metabarcoding (26% and 13%, respectively), which leads to a better spatio-temporal characterization of the fungal genera. As both morphological analyses and eDNA metabarcoding methods capture significant interactions between seasons and sites, they could be preferably used as complementing approaches to reliably study airborne fungal diversity and variation.

Phytohormones are pivotal signaling compounds in higher plants, in which they exert their roles intracellularly, but are also released for cell‐to‐cell communication. In unicellular organisms, extracellularly released phytohormones can be involved in chemical crosstalk with other organisms. However, compared to higher plants, hardly any knowledge is available on the roles of phytohormones in green algae. Here, we studied phytohormone composition and extracellular release in aero‐terrestrial Trebouxiophyceae. We investigated (a) which phytohormones are produced and if they are released extracellularly, and if extracellular phytohormone levels are (b) affected by environmental stimuli, and (c) differ between lichen‐forming and non‐lichen‐forming species. Three free‐living microalgae (Apatococcus lobatus, Chloroidium ellipsoideum, and Myrmecia bisecta) and three lichen‐forming microalgae (Asterochloris glomerata, Trebouxia decolorans, and Trebouxia sp.) were studied. Algae were grown on solid media and the following cellular phytohormones were identified by ultra‐high‐performance liquid chromatography coupled with tandem mass spectrometry (UHPLC‐MS/MS): indole‐3‐acetic acid (IAA), indole‐3‐butyric acid (IBA), abscisic acid (ABA), gibberellin A4 (GA4), and zeatin (ZT). Furthermore, IAA, IBA, ABA, jasmonic acid (JA), gibberellin A3 (GA3), and GA4 were found to be released extracellularly. IAA and ABA were released by all six species, and IAA was the most concentrated. Phytohormone release was affected by light and water availability, especially IAA in A. glomerata, Trebouxia sp., and C. ellipsoideum. No clear patterns were observed between lichen‐forming and non‐lichen‐forming species. The results are envisaged to contribute valuable baseline information for further studies into the roles of phytohormones in microalgae.

Fungal spores and mycelium fragments are particles which become and remain airborne and have been subjects of aerobiological studies. The presence and the abundance of taxa in aerobiological samples can be very variable and impaired by changeable climatic conditions. Because many fungi produce mycotoxins and both their mycelium fragments and spores are potential allergens, monitoring the presence of these taxa is of key importance. So far data on exposure and sensitization to fungal allergens are mainly based on the assessment of few, easily identifiable taxa and focused only on certain environments. The microscopic method used to analyze aerobiological samples and the inconspicuous fungal characters do not allow a in depth taxonomical identification. Here, we present a first assessment of fungal diversity from airborne samples using a DNA metabarcoding analysis. The nuclear ITS2 region was selected as barcode to catch fungal diversity in mixed airborne samples gathered during two weeks in four sites of North-Eastern and Central Italy. We assessed the taxonomic composition and diversity within and among the sampled sites and compared the molecular data with those obtained by traditional microscopy. The molecular analyses provide a tenfold more comprehensive determination of the taxa than the traditional morphological inspections. Our results prove that the metabarcoding analysis is a promising approach to increases quality and sensitivity of the aerobiological monitoring. The laboratory and bioinformatic workflow implemented here is now suitable for routine, high-throughput, regional analyses of airborne fungi.

Black, rock inhabiting fungi (RIF) are polyextremotolerant, oligotrophic organisms which colonize bare rocks and are specialized to grow in niches precluded to other microorganisms in the harshest environments. In many cases RIF share this environment with green algae and cyanobacteria forming subaerial biofilms; some of them have also been found to be associated with lichen thalli. The RIF genus Lichenothelia is of particular interest because it includes lichen parasites and species which are loosely associated with algae or grow independently on rocks. Here, in vitro culture experiments studied the development of three Lichenothelia species when co-cultured with two different lichen photobionts on growth media differing in nutrient content. The growth rates of these fungi were statistically evaluated and the structure of the mixed cultures was analyzed by light and scanning electron microscopy. The results show that the presence of algae neither influence the growth rate of fungi nor the formation of any lichen-like structure; tight contacts between the hyphae and the algal cells were also not detected. Since multiple trials were carried out on well characterized species of microcolonial fungi and the methodological procedures were established, standardized and coupled with morphological analyses, this approach proves suitable for future investigations on fungal-algal interactions in other systems.