Rhizoctonia -Scots pine interactions: detection, impact on seedling performance and host defence gene response

Rhizoctonia spp. are ubiquitous soil inhabiting fungi that enter into pathogenic or symbiotic associations with plants. In general Rhizoctonia spp. are regarded as plant pathogenic fungi and many cause root rot and other plant diseases which results in considerable economic losses both in agriculture and forestry. Many Rhizoctonia strains enter into symbiotic mycorrhizal associations with orchids and some hypovirulent strains are promising biocontrol candidates in preventing host plant infection by pathogenic Rhizoctonia strains. This work focuses on uni- and binucleate Rhizoctonia (respectively UNR and BNR) strains belonging to the teleomorphic genus Ceratobasidium, but multinucleate Rhizoctonia (MNR) belonging to teleomorphic genus Thanatephorus and ectomycorrhizal fungal species, such as Suillus bovinus, were also included in DNA probe development work. Strain specific probes were developed to target rDNA ITS (internal transcribed spacer) sequences (ITS1, 5.8S and ITS2) and applied in Southern dot blot and liquid hybridization assays. Liquid hybridization was more sensitive and the size of the hybridized PCR products could be detected simultaneously, but the advantage in Southern hybridization was that sample DNA could be used without additional PCR amplification. The impacts of four Finnish BNR Ceratorhiza sp. strains 251, 266, 268 and 269 were investigated on Scot pine (Pinus sylvestris) seedling growth, and the infection biology and infection levels were microscopically examined following tryphan blue staining of infected roots. All BNR strains enhanced early seedling growth and affected the root architecture, while the infection levels remained low. The fungal infection was restricted to the outer cortical regions of long roots and typical monilioid cells detected with strain 268. The interactions of pathogenic UNR Ceratobasidium bicorne strain 1983-111/1N, and endophytic BNR Ceratorhiza sp. strain 268 were studied in single or dual inoculated Scots pine roots. The fungal infection levels and host defence-gene activity of nine transcripts [phenylalanine ammonia lyase (pal1), silbene synthase (STS), chalcone synthase (CHS), short-root specific peroxidase (Psyp1), antimicrobial peptide gene (Sp-AMP), rapidly elicited defence-related gene (PsACRE), germin-like protein (PsGER1), CuZn- superoxide dismutase (SOD), and dehydrin-like protein (dhy-like)] were measured from differentially treated and un-treated control roots by quantitative real time PCR (qRT-PCR). The infection level of pathogenic UNR was restricted in BNR- pre-inoculated Scots pine roots, while UNR was more competitive in simultaneous dual infection. The STS transcript was highly up-regulated in all treated roots, while CHS, pal1, and Psyp1 transcripts were more moderately activated. No significant activity of Sp-AMP, PsACRE, PsGER1, SOD, or dhy-like transcripts were detected compared to control roots. The integrated experiments presented, provide tools to assist in the future detection of these fungi in the environment and to understand the host infection biology and defence, and relationships between these interacting fungi in roots and soils. This study further confirms the complexity of the Rhizoctonia group both phylogenetically and in their infection biology and plant host specificity. The knowledge obtained could be applied in integrated forestry nursery management programmes.

Communities of wood-inhabiting fungi : Ecological requirements and responses to forest management and fragmentation

Despite much research on forest biodiversity in Fennoscandia, the exact mechanisms of species declines in dead-wood dependent fungi are still poorly understood. In particular, there is only limited information on why certain fungal species have responded negatively to habitat loss and fragmentation, while others have not. Understanding the mechanisms behind species declines would be essential for the design and development of ecologically effective and scientifically informed conservation measures, and management practices that would promote biodiversity in production forests. In this thesis I study the ecology of polypores and their responses to forest management, with a particular focus on why some species have declined more than others. The data considered in the thesis comprise altogether 98,318 dead-wood objects, with 43,085 observations of 174 fungal species. Out of these, 1,964 observations represent 58 red-listed species. The data were collected from 496 sites, including woodland key habitats, clear-cuts with retention trees, mature managed forests, and natural or natural-like forests in southern Finland and Russian Karelia. I show that the most relevant way of measuring resource availability can differ to a great extent between species seemingly sharing the same resources. It is thus critical to measure the availability of resources in a way that takes into account the ecological requirements of the species. The results show that connectivity at the local, landscape and regional scales is important especially for the highly specialized species, many of which are also red-listed. Habitat loss and fragmentation affect not only species diversity but also the relative abundances of the species and, consequently, species interactions and fungal successional pathways. Changes in species distributions and abundances are likely to affect the food chains in which wood-inhabiting fungi are involved, and thus the functioning of the whole forest ecosystem. The findings of my thesis highlight the importance of protecting well-connected, large and high-quality forest areas to maintain forest biodiversity. Small habitat patches distributed across the landscape are likely to contribute only marginally to protection of red-listed species, especially if habitat quality is not substantially higher than in ordinary managed forest, as is the case with woodland key habitats. Key habitats might supplement the forest protection network if they were delineated larger and if harvesting of individual trees was prohibited in them. Taking the landscape perspective into account in the design and development of conservation measures is critical while striving to halt the decline of forest biodiversity in an ecologically effective manner.

Characterization of small GTPase Cdc42 from the ectomycorrhizal fungus Suillus bovinus and Agrobacterium tumefaciens-mediated transformation of fungi

Ectomycorrhizal formation between the host tree, Pinus sylvestris and fungal symbiont, Suillus bovinus was investigated at the molecular level by isolating genes regulating the organization of the actin cytoskeleton in the fungal partner S. bovinus. An Agrobacterium tumefaciens mediated transformation (ATMT) system was developed for the ectomycorrhizal fungi in order to assign specific functions to the cloned molecules. The developed ATMT system was also used to transform a plant pathogenic fungus, Helminthosporium turcicum, to hygromycin B resistance. Small GTPases Cdc42 and Rac1, the regulators of actin cytoskeleton in eukaryotes were isolated from S. bovinus. Sbcdc42 and Sbrac1, are both expressed in vegetative and in the symbiotic hyphae of S. bovinus . Using IIF microscopy, Cdc42 and actin were co-localized at the tips of vegetative hyphae and were visualized in association with the plasma membrane in swollen cells typical to the symbiotic hyphae. These results suggest that the small GTPases Cdc42 may play a significant role in the polarized growth of S. bovinus hyphae and regulate fungal morphogenesis during ectomycorrhiza formation through reorganization of the actin cytoskeleton. The functional equality of Cdc42 was tested in yeast complementation experiments using a Saccharomyces cerevisiae temperature sensitive mutant, cdc42-1ts. The genomic clone of CDC42 was isolated from S. bovinus genomic DNA via specific primers for Cdc42. The analogous S. cerevisiae cdc42 mutations, dominant active G12V and dominant negative D118A, were generated in the Sbcdc42 gene by in-vitro mutagenesis. The ectomycorrhizal fungi, S. bovinus, P. involutus and H. cylindroporum were transformed using ATMT and phleomycin as a selectable marker. PCR screeing suggested that the T-DNA was inserted in all the three fungal genomes but the fate of integration could not be proved by Southern blot analysis. An alternative Agrobacterium strain, AGL-1 and selection marker, hygromycin was used to transform our model fungus S. bovinus. PCR and Southern analysis suggested an improved efficiency of transformation. All the transformed fungal colonies selected for hygromycin gave positives in PCR and the Southerns showed multiple or single copy T-DNA integrations into the S. bovinus genome. Using the same Agrobacterium strain and the selectable marker, a maize pathogen, H. turcicum was also subjected to ATMT. The H. turcicum transformation data suggested the single copy T-DNA integrations into the genome of the screened transformants that further confirms wider applicability of the ATMT. The plasmids carrying the wild-type (pHGCDC42) and the mutated Sbcdc42 alleles (pHGGV; pHGDA) under Agaricus bisporus gpd promoter were constructed in an A. tumefaciens vector. ATMT was used to transform S. bovinus with the plasmids carrying the wild-type and mutated Sbcdc42 alleles. The isolation of Sbcdc42 and Sbrac1 genes and some other functionally related genes from ectomycorrhizal fungus, S. bovinus will form the basis of future work to resolve the signalling pathway leading to ectomycorrhizal symbiosis. The development of ATMT system will be a valuable tool in analysing the exact function of signalling pathway components in ectomycorrhizal symbiosis or in plant pathogenic interactions. The transformation frequency and broad applicability along with the simplicity of T-DNA integration make Agrobacterium a valuable, new and a powerfull tool for targeted and insertional mutagenesis in these plant associated fungi. The developed ATMT systems should therefore make it possible to generate large number of transformants with tagged genes which could then be screened for their specific roles in symbiosis and pathogenecity, respectively.

Traditional medicinal uses and biological activities of some plant extracts of African Combretum Loefl., Terminalia L. and Pteleopsis Engl. species

In Africa various species of Combretum, Terminalia and Pteleopsis are used in traditional medicine. Despite of this, some species of these genera have still not been studied for their biological effects to validate their traditional uses. The aim of this work has been to document the ethnomedicinal uses of several species of Combretum and Terminalia in Mbeya region, south-western Tanzania, and to use this information for finding species with good antimicrobial and cytotoxic potential. During a five weeks expedition to Tanzania in spring 1999 sixteen different species of Combretum and Terminalia, as well as Pteleopsis myrtifolia were collected from various locations in the districts of Mbeya, Iringa and Dar-es-Salaam. Traditional healers in seven different villages in the Mbeya region were interviewed in Swahili and Nyakyusa on the medicinal uses of Combretum and Terminalia species shown to them. A questionnaire was used during the interviews. The results of the interviews correlated well between different villages, the same species being used in similar ways in different villages. Of the ten species shown to the healers six were frequently used for treatment of skin diseases, bacterial infections, diarrhea, oedema and wounds. The dried plants were most commonly prepared into hot water decoctions or mixed into maize porridge, Ugali. Infusions made from dried or fresh plant material were also common. Wounds and topical infections were treated with ointments made from the dried plant material mixed with sheep fat. Twenty-one extracts of six species of Combretum and four of Terminalia, collected from Tanzania, were screened for their antibacterial effects against two gram-negative and five gram-positive bacteria, as well as the yeast, Candida albicans, using an agar diffusion method. Most of the screened plants showed substantial antimicrobial activity. A methanolic root extract of T. sambesiaca showed the most potent antibacterial effects of all the plant species screened, and gave a MIC value of 0.9 mg/ml against Enterobacter aerogenes. Also root extracts of T. sericea and T. kaiserana gave excellent antimicrobial effects, and notably a hot water extract of T. sericea was as potent as extracts of this species made from EtOH and MeOH. Thus, the traditional way of preparing T. sericea into hot water decoctions seems to extract antimicrobial compounds. Thirty-five extracts of five species of Terminalia, ten of Combretum and Pteleopsis myrtifolia were screened for their antifungal effects against five species of yeast (Candida spp.) and Cryptococcus neoformans. The species differed from each other to their antifungal effects, some being very effective whereas others showed no antifungal effects. The most effective extracts showed antifungal effects comparable to the standard antibiotics itraconazol and amphotericin B. Species of Terminalia gave in general stronger antifungal effects than those of Combretum. The best effects were obtained with methanolic root extracts of T. sambesiaca, T. sericea and T. kaiserana, and this investigation indicates that decoctions of these species might be used for treatment of HIV-related fungal infections. Twenty-seven crude extracts of eight species of Combretum, five of Terminalia and Pteleopsis myrtifolia were evaluated for their cytotoxic effects against human cancer cell lines (HeLa, cervical carcinoma; MCF 7, breast carcinoma, T 24 bladder carcinoma) and one endothelial cell line (BBCE, bovine brain capillary endothelial cells). The most outstanding effects were obtained with a leaf extract of Combretum fragrans, which nearly totally inhibited the proliferation of T 24 and HeLa cells at a concentration of 25 ug/ml and inhibited 60 % of the growth of the HeLa cells at a concentration of 4.3 ug/ml. The species of Terminalia were less cytotoxically potent than the Combretum species, although T. sericea and T. sambesiaca gave good cytotoxic effects (< 30 % proliferation). In summary this study indicates that some of the species of Terminalia, Combretum and Pteleopsis, used in Tanzanian traditional medicine, are powerful inhibitors of both microbial and cancer cell growth. In depth studies would be needed to find the active compounds behind these biological activities.

Archaea in the Mycorrhizosphere of Boreal Forest Trees

Archaea were long thought to be a group of ancient bacteria, which mainly lived in extreme environments. Due to the development of DNA sequencing methods and molecular phylogenetic analyses, it was shown that the living organisms are in fact divided into three domains; the Archaea, Bacteria and the Eucarya. Since the beginning of the previous decade, it was shown that archaea generally inhabit moderate environments and that these non-extremophilic archaea are more ubiquitous than the extremophiles. Group 1 of non-extreme archaea affiliate with the phylum Crenarchaeota. The most commonly found soil archaea belong to the subgroup 1.1b. However, the Crenarchaeota found in the Fennoscandian boreal forest soil belong to the subgroup 1.1c. The organic top layer of the boreal forest soil, the humus, is dominated by ectomycorrhizal fungal hyphae. These colonise virtually all tree fine root tips in the humus layer and have been shown to harbour distinct bacterial populations different from those in the humus. The archaea have also been shown to colonise both boreal forest humus and the rhizospheres of plants. In this work, studies on the archaeal communities in the ectomycorrhizospheres of boreal forest trees were conducted in microcosms. Archaea belonging to the group 1.1c Crenarchaeota and Euryarchaeota of the genera Halobacterium and Methanolobus were detected. The archaea generally colonised fungal habitats, such as ectomycorrhizas and external mycelia, rather than the non-mycorrhizal fine roots of trees. The species of ectomycorrhizal fungus had a great impact on the archaeal community composition. A stable euryarchaeotal community was detected especially in the mycorrhizas, of most of the tested Scots pine colonising ectomycorrhizal fungi. The Crenarchaeota appeared more sporadically in these habitats, but had a greater diversity than the Euryarchaeota. P. involutus mycorrhizas had a higher diversity of 1.1c Crenarchaeota than the other ectomycorrhizal fungi. The detection level of archaea in the roots of boreal trees was generally low although archaea have been shown to associate with roots of different plants. However, alder showed a high diversity of 1.1c Crenarchaeota, exceeding that of any of the tested mycorrhizas. The archaeal 16S rRNA genes detected from the non-mycorrhizal roots were different from those of the P. involutus mycorrhizas. In the phylogenetic analyses, the archaeal 16S rRNA gene sequences obtained from non-mycorrhizal fine roots fell in a separate cluster within the group 1.1c Crenarchaeota than those from the mycorrhizas. When the roots of the differrent tree species were colonised by P. involutus, the diversity and frequency of the archaeal populations of the different tree species were more similar to each other. Both Cren- and Euryarchaeota were enriched in cultures to which C-1 substrates were added. The 1.1c Crenarchaeota grew anaerobically in mineral medium with CH4 and CO2 as the only available C sources, and in yeast extract media with CO2 and CH4 or H2. The crenarchaeotal diversity was higher in aerobic cultures on mineral medium with CH4 or CH3OH than in the anaerobic cultures. Ecological functions of the mycorrhizal 1.1c Crenarchaeota in both anaerobic and aerobic cycling of C-1 compounds were indicated. The phylogenetic analyses did not divide the detected Crenarchaeota into anaerobic and aerobic groups. This may suggest that the mycorrhizospheric crenarchaeotal communities consist of closely related groups of anaerobic and aerobic 1.1c Crenarchaeota, or the 1.1c Crenarchaeota may be facultatively anaerobic. Halobacteria were enriched in non-saline anaerobic yeast extract medium cultures in which CH4 was either added or produced, but were not detected in the aerobic cultures. They may potentially be involved in anaerobic CH4 cycling in ectomycorrhizas. The CH4 production of the mycorrhizal samples was over 10 times higher than for humus devoid of mycorrhizal hyphae, indicating a high CH4 production potential of the mycorrhizal metanogenic community. Autofluorescent methanogenic archaea were detected by microscopy and 16S rRNA gene sequences of the genus Methanolobus were obtained. The archaeal community depended on both tree species and the type of ectomycorrhizal fungus colonising the roots and the Cren- and Euryarchaeota may have different ecological functions in the different parts of the boreal forest tree rhizosphere and mycorrhizosphere. By employing the results of this study, it may be possible to isolate both 1.1c Crenarchaeota as well as non-halophilic halobacteria and aerotolerant methanogens from mycorrhizospheres. These archaea may be used as indicators for change in the boreal forest soil ecosystem due to different factors, such as exploitations of forests and the rise in global temperature. More information about the microbial populations with apparently low cell numbers but significant ecological impacts, such as the boreal forest soil methanogens, may be of crucial importance to counteract human impacts on such globally important ecosystems as the boreal forests.

Phylogenetic studies of cyanobacterial lichens

Phylogenetic studies of cyanobacterial lichens Lichens are symbiotic assemblages between fungi (mycobiont) and green algae (phycobiont) or/and cyanobacteria (cyanobiont). Fossil records show that lichen-like symbioses occurred already 600 million years ago. Lichen symbiosis has since then become an important life strategy for the Fungi, particularly for species in the phylum Ascomycota as approximately 98% of the lichenized fungal species are ascomycetes. The taxonomy of lichen associations is based on the mycobiont. We reconstructed, using DNA sequence data, hypotheses of phylogenetic relationships of lichen-forming fungi that include species associated with cyanobacteria. These hypotheses of phylogeny should form the basis for the taxonomy. They also allowed studies of the origin and the evolution of specific symbioses. Genetic diversity and phylogenetic relationships of symbiotic cyanobionts were also studied in order to examine selectivity of cyanobionts and mycobionts as well as possible co-evolution between partners involved in lichen associations. The suggested circumscription of the family Stereocaulaceae to include Stereocaulon and Lepraria is supported. The recently described crustose Stereocaulon species seem to be correctly placed in the genus, although Stereocaulon traditionally included only fruticose species. The monospecific crustose genus Muhria is also shown to be best placed in Stereocaulon. Family Lobariaceae as currently delimited is monophyletic. Within Lobariaceae genus Sticta including Dendriscocaulon dendroides form a monophyletic group while the genera Lobaria and Pseudocyphellaria are non-monophyletic. A new classification of Lobariaceae is obviously needed. Further studies are however required before a final proposal for a new classification can be made. Our results show that the cyanobacterial symbiotic state has been gained repeatedly in the Ascomycota while losses of symbiotic cyanobacteria appear to be rare. The symbiosis with green algae is confirmed to have been gained repeatedly in Ascomycota but also repeatedly lost. Cyanobacterial symbioses therefore seem to be more stable than green algal associations. Cyanobacteria are perhaps more beneficial for the lichen fungi and therefore maintained. The results indicate a dynamic association of the lichen symbiosis. This evolutionary instability will perhaps be important for the lichen fungi as the utilization of options will perhaps enable lichens to colonize new substrates and survive environmental changes. Some cyanobacterial lichen genera seem to be highly selective towards the cyanobiont while others form symbioses with a broad spectrum of cyanobacteria. No evidence of co-evolution between fungi and cyanobacteria in cyanolichens could be demonstrated.

Aerobic carbon-cycle related microbial communities in boreal peatlands: responses to water-level drawdown

Boreal peatlands represent a considerable portion of the global carbon (C) pool. Water-level drawdown (WLD) causes peatland drying and induces a vegetation change, which affects the decomposition of soil organic matter and the release of greenhouse gases (CO2 and CH4). The objective of this thesis was to study the microbial communities related to the C cycle and their response to WLD in two boreal peatlands. Both sampling depth and site type had a strong impact on all microbial communities. In general, bacteria dominated the deeper layers of the nutrient-rich fen and the wettest surfaces of the nutrient-poor bog sites, whereas fungi seemed more abundant in the drier surfaces of the bog. WLD clearly affected the microbial communities but the effect was dependent on site type. The fungal and methane-oxidizing bacteria (MOB) community composition changed at all sites but the actinobacterial community response was apparent only in the fen after WLD. Microbial communities became more similar among sites after long-term WLD. Litter quality had a large impact on community composition, whereas the effects of site type and WLD were relatively minor. The decomposition rate of fresh organic matter was influenced slightly by actinobacteria, but not at all by fungi. Field respiration measurements in the northern fen indicated that WLD accelerates the decomposition of soil organic matter. In addition, a correlation between activity and certain fungal sequences indicated that community composition affects the decomposition of older organic matter in deeper peat layers. WLD had a negative impact on CH4 oxidation, especially in the oligotrophic fen. Fungal sequences were matched to taxa capable of utilizing a broad range of substrates. Most of the actinobacterial sequences could not be matched to characterized taxa in reference databases. This thesis represents the first investigation of microbial communities and their response to WLD among a variety of boreal peatland habitats. The results indicate that microbial community responses to WLD are complex but dependent on peatland type, litter quality, depth, and variable among microbes.

Mycorrhizal fungi and nitrogen dynamics in drained peatland

Fungi have a fundamental role in carbon and nutrient transformations in the acids soils of boreal regions, such as peatlands, where high amounts of carbon (C) and nutrients are stored in peat, the pH is relatively low and the nutrient uptake of trees is highly dependent on mycorrhizae. In this thesis, the aim was to examine nitrogen (N) transformations and the availability of dissolved N compounds in forestry-drained peatlands, to compare the fungal community biomass and structure at various peat N levels, to investigate the growth of ectomycorrhizal fungi with variable P and K availability and to assess how the ectomycorrhizal fungi (ECM) affect N transformations. Both field and laboratory experiments were carried out. The peat N concentration did not affect the soil fungal community structure within a site. Phosphorus (P) and potassium (K) deficiency of the trees as well as the degree of decomposition and dissolved organic nitrogen (DON) concentration of the peat were shown to affect the fungal community structure and biomass of ECMs, highlighting the complexity of the below ground system on drained peatlands. The biomass of extrametrical mycorrhizal mycelia (EMM) was enhanced by P and/or K deficiency of the trees, and ECM biomass in the roots was increased by P deficiency. Thus, PK deficiency in drained peatlands may increase the allocation of C by the tree to ECMs. It was also observed that fungi can alter N mineralization processes in the rhizosphere but variously depending on fungal species and fertility level of peat. Gross N mineralization did not vary but the net N mineralization rate significantly increased along the N gradient in both field and laboratory experiments. Gross N immobilization also significantly increased when the peat N concentration increased. Nitrification was hardly detectable in either field or laboratory experiments. During the growing season, dissolved inorganic N (DIN) fluctuated much more than the relatively stable DON. Special methodological challenges associated with sampling and analysis in microbial studies on peatlands are discussed.

Microbial communities in Pb contaminated boreal forest soil

Lead contamination in the environment is of particular concern, as it is a known toxin. Until recently, however, much less attention has been given to the local contamination caused by activities at shooting ranges compared to large-scale industrial contamination. In Finland, more than 500 tons of Pb is produced each year for shotgun ammunition. The contaminant threatens various organisms, ground water and the health of human populations. However, the forest at shooting ranges usually shows no visible sign of stress compared to nearby clean environments. The aboveground biota normally reflects the belowground ecosystem. Thus, the soil microbial communities appear to bear strong resistance to contamination, despite the influence of lead. The studies forming this thesis investigated a shooting range site at Hälvälä in Southern Finland, which is heavily contaminated by lead pellets. Previously it was experimentally shown that the growth of grasses and degradation of litter are retarded. Measurements of acute toxicity of the contaminated soil or soil extracts gave conflicting results, as enchytraeid worms used as toxicity reporters were strongly affected, while reporter bacteria showed no or very minor decreases in viability. Measurements using sensitive inducible luminescent reporter bacteria suggested that the bioavailability of lead in the soil is indeed low, and this notion was supported by the very low water extractability of the lead. Nevertheless, the frequency of lead-resistant cultivable bacteria was elevated based on the isolation of cultivable strains. The bacterial and fungal diversity in heavily lead contaminated shooting sectors were compared with those of pristine sections of the shooting range area. The bacterial 16S rRNA gene and fungal ITS rRNA gene were amplified, cloned and sequenced using total DNA extracted from the soil humus layer as the template. Altogether, 917 sequenced bacterial clones and 649 sequenced fungal clones revealed a high soil microbial diversity. No effect of lead contamination was found on bacterial richness or diversity, while fungal richness and diversity significantly differed between lead contaminated and clean control areas. However, even in the case of fungi, genera that were deemed sensitive were not totally absent from the contaminated area: only their relative frequency was significantly reduced. Some operational taxonomic units (OTUs) assigned to Basidiomycota were clearly affected, and were much rarer in the lead contaminated areas. The studies of this thesis surveyed EcM sporocarps, analyzed morphotyped EcM root tips by direct sequencing, and 454-pyrosequenced fungal communities in in-growth bags. A total of 32 EcM fungi that formed conspicuous sporocarps, 27 EcM fungal OTUs from 294 root tips, and 116 EcM fungal OTUs from a total of 8 194 ITS2 454 sequences were recorded. The ordination analyses by non-parametric multidimensional scaling (NMS) indicated that Pb enrichment induced a shift in the EcM community composition. This was visible as indicative trends in the sporocarp and root tip datasets, but explicitly clear in the communities observed in the in-growth bags. The compositional shift in the EcM community was mainly attributable to an increase in the frequencies of OTUs assigned to the genus Thelephora, and to a decrease in the OTUs assigned to Pseudotomentella, Suillus and Tylospora in Pb-contaminated areas when compared to the control. The enrichment of Thelephora in contaminated areas was also observed when examining the total fungal communities in soil using DNA cloning and sequencing technology. While the compositional shifts are clear, their functional consequences for the dominant trees or soil ecosystem remain undetermined. The results indicate that at the Hälvälä shooting range, lead influences the fungal communities but not the bacterial communities. The forest ecosystem shows apparent functional redundancy, since no significant effects were seen on forest trees. Recently, by means of 454 pyrosequencing , the amount of sequences in a single analysis run can be up to one million. It has been applied in microbial ecology studies to characterize microbial communities. The handling of sequence data with traditional programs is becoming difficult and exceedingly time consuming, and novel tools are needed to handle the vast amounts of data being generated. The field of microbial ecology has recently benefited from the availability of a number of tools for describing and comparing microbial communities using robust statistical methods. However, although these programs provide methods for rapid calculation, it has become necessary to make them more amenable to larger datasets and numbers of samples from pyrosequencing. As part of this thesis, a new program was developed, MuSSA (Multi-Sample Sequence Analyser), to handle sequence data from novel high-throughput sequencing approaches in microbial community analyses. The greatest advantage of the program is that large volumes of sequence data can be manipulated, and general OTU series with a frequency value can be calculated among a large number of samples.

Self-assembly of hydrophobin proteins from the fungus Trichoderma reesei

Hydrophobins are small surface active proteins that are produced by filamentous fungi. The surface activity of hydrophobin proteins leads to the formation of a film at the air-water interface and adsorption to surfaces. The formation of these hydrophobin films and coatings is important in many stages of fungal development. Furthermore, these properties make hydrophobins interesting for potential use in technical applications. The surfactant-like properties of hydrophobins from Trichoderma reesei were studied at the air-water interface, at solid surfaces, and in solution. The hydrophobin HFBI was observed to spontaneously form a cohesive film on a water drop. The film was imaged using atomic force microscopy from both sides, revealing a monomolecular film with a defined molecular structure. The use of hydrophobins as surface immobilization carriers for enzymes was studied using fusion proteins of HFBI or HFBII and an enzyme. Furthermore, sitespecifically modified variants of HFBI were shown to retain their ability to selfassemble at interfaces and to be able to bind a second layer of proteins by biomolecular recognition. In order to understand the function of hydrophobins at interfaces, an understanding of their overall behavior and self-assembly is needed. HFBI and HFBII were shown to associate in solution into dimers and tetramers in a concentration-dependent manner. The association dynamics and protein-protein interactions of HFBI and HFBII were studied using Förster resonance energy transfer and size exclusion chromatography. It was shown that the surface activity of HFBI is not directly dependent on the formation of multimers in solution.

The Emerging Phenotype: Ecological Genetics of Color, Form and Sex in the Common Frog

One of the main aims of evolutionary biology is to explain why organisms vary phenotypically as they do. Proximately, this variation arises from genetic differences and from environmental influences, the latter of which is referred to as phenotypic plasticity. Phenotypic plasticity is thus a central concept in evolutionary biology, and understanding its relative importance in causing the phenotypic variation and differentiation is important, for instance in anticipating the consequences of human induced environmental changes. The aim of this thesis was to study geographic variation and local adaptation, as well as sex ratios and environmental sex reversal, in the common frog (Rana temporaria). These themes cover three different aspects of phenotypic plasticity, which emerges as the central concept for the thesis. The first two chapters address geographic variation and local adaptation in two potentially thermally adaptive traits, namely the degree of melanism and the relative leg length. The results show that although there is an increasing latitudinal trend in the degree of melanism in wild populations across Scandinavian Peninsula, this cline has no direct genetic basis and is thus environmentally induced. The second chapter demonstrates that although there is no linear, latitudinally ordered phenotypic trend in relative leg length that would be expected under Allen s rule an ecogeographical rule linking extremity length to climatic conditions there seems to be such a trend at the genetic level, hidden under environmental effects. The first two chapters thus view phenotypic plasticity through its ecological role and evolution, and demonstrate that it can both give rise to phenotypic variation and hide evolutionary patterns in studies that focus solely on phenotypes. The last three chapters relate to phenotypic plasticity through its ecological and evolutionary role in sex determination, and consequent effects on population sex ratio, genetic recombination and the evolution of sex chromosomes. The results show that while sex ratios are strongly female biased and there is evidence of environmental sex reversals, these reversals are unlikely to have caused the sex ratio skew, at least directly. The results demonstrate that environmental sex reversal can have an effect on the evolution of sex chromosomes, as the recombination patterns between them seem to be controlled by phenotypic, rather than genetic, sex. This potentially allows Y chromosomes to recombine, lending support for the recent hypothesis suggesting that sex-reversal may play an important role on the rejuvenation of Y chromosomes.

Microbial diversity in the municipal composting process and development of detection methods

Composting refers to aerobic degradation of organic material and is one of the main waste treatment methods used in Finland for treating separated organic waste. The composting process allows converting organic waste to a humus-like end product which can be used to increase the organic matter in agricultural soils, in gardening, or in landscaping. Microbes play a key role as degraders during the composting-process, and the microbiology of composting has been studied for decades, but there are still open questions regarding the microbiota in industrial composting processes. It is known that with the traditional, culturing-based methods only a small fraction, below 1%, of the species in a sample is normally detected. In recent years an immense diversity of bacteria, fungi and archaea has been found to occupy many different environments. Therefore the methods of characterising microbes constantly need to be developed further. In this thesis the presence of fungi and bacteria in full-scale and pilot-scale composting processes was characterised with cloning and sequencing. Several clone libraries were constructed and altogether nearly 6000 clones were sequenced. The microbial communities detected in this study were found to differ from the compost microbes observed in previous research with cultivation based methods or with molecular methods from processes of smaller scale, although there were similarities as well. The bacterial diversity was high. Based on the non-parametric coverage estimations, the number of bacterial operational taxonomic units (OTU) in certain stages of composting was over 500. Sequences similar to Lactobacillus and Acetobacteria were frequently detected in the early stages of drum composting. In tunnel stages of composting the bacterial community comprised of Bacillus, Thermoactinomyces, Actinobacteria and Lactobacillus. The fungal diversity was found to be high and phylotypes similar to yeasts were abundantly found in the full-scale drum and tunnel processes. In addition to phylotypes similar to Candida, Pichia and Geotrichum moulds from genus Thermomyces and Penicillium were observed in tunnel stages of composting. Zygomycetes were detected in the pilot-scale composting processes and in the compost piles. In some of the samples there were a few abundant phylotypes present in the clone libraries that masked the rare ones. The rare phylotypes were of interest and a method for collecting them from clone libraries for sequencing was developed. With negative selection of the abundant phylotyps the rare ones were picked from the clone libraries. Thus 41% of the clones in the studied clone libraries were sequenced. Since microbes play a central role in composting and in many other biotechnological processes, rapid methods for characterization of microbial diversity would be of value, both scientifically and commercially. Current methods, however, lack sensitivity and specificity and are therefore under development. Microarrays have been used in microbial ecology for a decade to study the presence or absence of certain microbes of interest in a multiplex manner. The sequence database collected in this thesis was used as basis for probe design and microarray development. The enzyme assisted detection method, ligation-detection-reaction (LDR) based microarray, was adapted for species-level detection of microbes characteristic of each stage of the composting process. With the use of a specially designed control probe it was established that a species specific probe can detect target DNA representing as little as 0.04% of total DNA in a sample. The developed microarray can be used to monitor composting processes or the hygienisation of the compost end product. A large compost microbe sequence dataset was collected and analysed in this thesis. The results provide valuable information on microbial community composition during industrial scale composting processes. The microarray method was developed based on the sequence database collected in this study. The method can be utilised in following the fate of interesting microbes during composting process in an extremely sensitive and specific manner. The platform for the microarray is universal and the method can easily be adapted for studying microbes from environments other than compost.

Multifunctional RNA silencing pathway polymerase QDE-1 of Neurospora crassa

Double-stranded RNA and associated proteins are known to regulate the gene expression of most eukaryotic organisms. These regulation pathways have different components, outcomes and distinct nomenclature depending on the model system, and often they are referred to collectively as RNA silencing. In many cases, RNA-dependent RNA polymerases (RdRPs) are found to be involved in the RNA silencing, but their targets, activities, interaction partners and reaction products remain enigmatic. In the filamentous fungus Neurospora crassa, the RdRP QDE-1 is critical for silencing of transgenes a phenomenon known as quelling. In this thesis the structure, biochemical activities and biological functions of QDE-1 were extensively studied. This dimeric RdRP was shown to possess five distinct catalytic in vitro activities that could be dissected by mutagenesis and by altering reaction conditions. The biochemical characterization implied that QDE-1 is actually an active DNA-dependent RNA polymerase that has additional RdRP activity. It also provided a structural explanation for the dimerization and suggested a biological framework for the functions of QDE-1 in vivo. (I) QDE-1 was also studied in a broader context along with the other components of the quelling pathway. It was shown that DNA damage in Neurospora causes a dramatic increase in the expression level of the Argonaute protein QDE-2 as well as the synthesis of a novel class of small RNAs known as qiRNAs. The accumulation of qiRNAs was shown to be dependent on several quelling components, and particularly to be derived from an aberrant ssRNA (aRNA) molecule that is synthesized by QDE-1 in the nucleus. The genomic distribution of qiRNA targets was analyzed and the possible biological significance of qiRNAs was studied. Importantly, qiRNAs are the first class of small RNAs that are induced by DNA damage. (II) After establishing that QDE-1 is a multifunctional RNA polymerase with several activities, template specificities and subcellular locations, the focus was turned onto its interaction partners. It had been previously known that QDE-1 associates with Replication Protein A (RPA), but the RecQ helicase QDE-3 was now shown to regulate this interaction. RPA was also observed to promote QDE-1 dependent dsRNA synthesis in vitro. By characterizing the interplay between QDE-1, QDE-3 and RPA, a working model of quelling and qiRNA pathways in Neurospora was presented. (III) This work sheds light on the complexity of the various RNA silencing pathways of a fungal model system. It shows how an RdRP can regulate gene expression on many levels, and suggests novel lines of research in other eukaryotic organisms.

Comparative and functional genome analysis of fungi for development of the protein production host Trichoderma reesei

Filamentous fungi of the subphylum Pezizomycotina are well known as protein and secondary metabolite producers. Various industries take advantage of these capabilities. However, the molecular biology of yeasts, i.e. Saccharomycotina and especially that of Saccharomyces cerevisiae, the baker's yeast, is much better known. In an effort to explain fungal phenotypes through their genotypes we have compared protein coding gene contents of Pezizomycotina and Saccharomycotina. Only biomass degradation and secondary metabolism related protein families seem to have expanded recently in Pezizomycotina. Of the protein families clearly diverged between Pezizomycotina and Saccharomycotina, those related to mitochondrial functions emerge as the most prominent. However, the primary metabolism as described in S. cerevisiae is largely conserved in all fungi. Apart from the known secondary metabolism, Pezizomycotina have pathways that could link secondary metabolism to primary metabolism and a wealth of undescribed enzymes. Previous studies of individual Pezizomycotina genomes have shown that regardless of the difference in production efficiency and diversity of secreted proteins, the content of the known secretion machinery genes in Pezizomycotina and Saccharomycotina appears very similar. Genome wide analysis of gene products is therefore needed to better understand the efficient secretion of Pezizomycotina. We have developed methods applicable to transcriptome analysis of non-sequenced organisms. TRAC (Transcriptional profiling with the aid of affinity capture) has been previously developed at VTT for fast, focused transcription analysis. We introduce a version of TRAC that allows more powerful signal amplification and multiplexing. We also present computational optimisations of transcriptome analysis of non-sequenced organism and TRAC analysis in general. Trichoderma reesei is one of the most commonly used Pezizomycotina in the protein production industry. In order to understand its secretion system better and find clues for improvement of its industrial performance, we have analysed its transcriptomic response to protein secretion stress conditions. In comparison to S. cerevisiae, the response of T. reesei appears different, but still impacts on the same cellular functions. We also discovered in T. reesei interesting similarities to mammalian protein secretion stress response. Together these findings highlight targets for more detailed studies.

The effect of lignin content and lignin modification on Norway spruce wood properties and decay resistance

Three different Norway spruce cutting clones growing in three environments with different soil and climatic conditions were studied. The purpose was to follow variation in the radial growth rate, wood properties and lignin content and to modify wood lignin with a natural monolignol, coniferyl alcohol, by making use of inherent wood peroxidases. In addition, the incorporation of chlorinated anilines into lignin was studied with synthetic model compounds and synthetic lignin preparations to show whether unnatural compounds originating from pesticides could be bound in the lignin polymer. The lignin content of heartwood, sapwood and earlywood was determined by applying Fourier transform infrared (FTIR) spectroscopy and a principal component regression (PCR) technique. Wood blocks were treated with coniferyl alcohol by using a vacuum impregnation method. The effect of impregnation was assessed by FTIR and by a fungal decay test. Trees from a fertile site showed the highest growth rate and sapwood lignin content and the lowest latewood proportion, weight density and modulus of rupture (MOR). Trees from a medium fertile site had the lowest growth rate and the highest latewood proportion, weight density, modulus of elasticity (MOE) and MOR. The most rapidly growing clone showed the lowest latewood proportion, weight density, MOE and MOR. The slowest growing clone had the lowest sapwood lignin content and the highest latewood proportion, weight density, MOE and MOR. Differences between the sites and clones were small, while fairly large variation was found between the individual trees and growing seasons. The cutting clones maintained clone-dependent wood properties in the different growing sites although variation between trees was high and climatic factors affected growth. The coniferyl alcohol impregnation increased the content of different lignin-type phenolic compounds in the wood as well as wood decay resistance against a white-rot fungus, Coriolus versicolor. During the synthetic lignin preparation 3,4-dichloroaniline became bound by a benzylamine bond to β-O-4 structures in the polymer and it could not be released by mild acid hydrolysis. The natural monolignol, coniferyl alcohol, and chlorinated anilines could be incorporated into the lignin polymer in vivo and in vitro, respectively.