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In a subarctic climate, the seasonal shifts in temperature, precipitation, and plant cover drive the temporal changes in the microbial communities in the topsoil, forcing soil microbes to adapt or decline. Many organisms, such as mound‐building ants, survive the cold winter owing to the favorable microclimate in their nest mounds. We have previously shown that the microbial communities in the nest of the ant Formica exsecta are significantly different from those in the surrounding bulk soil. In the current study, we identified taxa, which were consistently present in the nests over a study period of three years. Some taxa were also significantly enriched in the nest samples compared with spatially corresponding reference soils. We show that the bacterial communities in ant nests are temporally stable across years, whereas the fungal communities show greater variation. It seems that the activities of the ants contribute to unique biochemical processes in the secluded nest environment, and create opportunities for symbiotic interactions between the ants and the microbes. Over time, the microbial communities may come to diverge, due to drift and selection, especially given the long lifespan (up to 30 years) of the ant colonies., We have previously shown that the fungal and bacterial communities in nests of ants Formica exsecta are distinct from those in the surrounding soil. Now, the results from our three‐year study show that the nests also provide a temporally stable environment, where microbial taxa, less tolerant of climatic fluctuations, could survive through unfavorable seasons. The nest could thus act as a reservoir for such microbial inocula, promoting the divergence of the nest communities over time.

Arbuscular mycorrhizal fungi (AMF) enhance plant phosphorus uptake, increase soil water holding abilities, reduce soil erosion and can protect their hosts from soil-borne pathogens. Hence, AMF play an important part in improving sustainable agricultural practices, and information about the effects of different preceding crop species on the following crop's AMF well-being is crucial for designing crop rotations. We studied onion root and soil microbial diversity and onion root AMF colonization rates after being preceded by three AMF hosting and one non-hosting green manure crop species in a boreal climate organic field. One-season cultivation of different preceding green manure crops did not have a strong effect on AMF colonization or microbial diversity in onion roots nor in the surrounding soil. Onions had high AMF colonization and microbial diversity after all four preceding crops. The overall fungal and bacterial populations of the soil reacted more strongly to seasonal variations than preceding crops. The study suggests that one season is a too short time to influence the AMF community in boreal climate organic fields with conventional tillage. Thus, non-host preceding crops can also be used in rotations, especially together with AMF host crops.

Mycorrhizal fungi have a strong impact on soil biota. In this study, bacterial and archaeal populations in different parts of Suillus bovinus - Pinus sylvestris mycorrhizospheres in boreal forest were quantified and identified by DNA analysis. The numbers of bacterial and archaeal 16S rRNA gene copies were highest in uncolonized humus and lowest in fruiting bodies. The numbers of bacterial 16S rRNA gene copies varied from 1.3 × 107 to 3.1 × 109 copies g−1 fw and archaeal copies from 4.1 × 107 to 9.6 × 108 copies g−1 fw. The relatively high number of archaeal 16S rRNA gene copies was likely due to the cold and highly organic habitat. The presence of hyphae appeared to further promote archaeal numbers and the archaea:bacteria ratio was over one in samples containing only fungal material. Most detected archaea belonged to terrestrial Thaumarchaeota. Proteobacteria, Actinobacteria and Acidobacteria were predictably the dominating bacterial taxa in the samples with clear trend of Betaproteobacteria preferring the pine root habitats. acceptedVersion

In this study, the abundance and composition of prokaryotic communities associated with the inner tissue of fruiting bodies of Suillus bovinus, Boletus pinophilus, Cantharellus cibarius, Agaricus arvensis, Lycoperdon perlatum, and Piptoporus betulinus were analyzed using culture-independent methods. Our findings indicate that archaea and bacteria colonize the internal tissues of all investigated specimens and that archaea are prominent members of the prokaryotic community. The ratio of archaeal 16S rRNA gene copy numbers to those of bacteria was >1 in the fruiting bodies of four out of six fungal species included in the study. The largest proportion of archaeal 16S rRNA gene sequences belonged to thaumarchaeotal classes Terrestrial group, Miscellaneous Crenarchaeotic Group (MCG), and Thermoplasmata. Bacterial communities showed characteristic compositions in each fungal species. Bacterial classes Gammaproteobacteria, Actinobacteria, Bacilli, and Clostridia were prominent among communities in fruiting body tissues. Bacterial populations in each fungal species had different characteristics. The results of this study imply that fruiting body tissues are an important habitat for abundant and diverse populations of archaea and bacteria. acceptedVersion

Biotic and abiotic characteristics shape the microbial communities in the soil environment. Manipulation of soil, performed by ants when constructing their nests, radically changes the soil characteristics and creates a unique environment, which differs in its composition, frequency and abundance of microbial taxa, from those in the reference soils. We sampled nests of the mound-building ant Formica exsecta, and the surrounding reference soils over a three-month period, and generated NGS (Illumina MiSeq), and T-RFLP data of the bacterial and fungal communities. We used ordination techniques and network analysis to disclose the community structure, and we assessed the variation in diversity, evenness and enrichment of taxa between the two environments. We also used indicator analysis to identify the potential core microbiome of the nests. Our results show that the bacterial and fungal communities, in the rigorously curated nest environment, are significantly different from those in the reference soils, in terms of community structure and enrichment of characteristic indicator taxa. We demonstrate that the nests represent a niche, where microbial species can adapt and diverge from the communities in the surrounding soils. Our findings contribute to our understanding of the composition and function of microbiomes in fragmented habitats.

Microbes are ubiquitous and often occur in functionally and taxonomically complex communities. Unveiling these community dynamics is one of the main challenges of microbial research. Combining a robust, cost effective and widely used method such as Terminal Restriction Fragment Length Polymorphism (T-RFLP) with a Next Generation Sequencing (NGS) method (Illumina MiSeq), offers a solid alternative for comprehensive assessment of microbial communities. Here, these two methods were combined in a study of complex bacterial and fungal communities in the nest mounds of the antFormica exsecta, with the aim to assess the degree to which these methods can be used to complement each other. The results show that these methodologies capture similar spatiotemporal variations, as well as corresponding functional and taxonomical detail, of the microbial communities in a challenging medium consisting of soil, decomposing plant litter and an insect inhabitant. Both methods are suitable for the analysis of complex environmental microbial communities, but when combined, they complement each other well and can provide even more robust results. T-RFLP can be trusted to show similar general community patterns as Illumina MiSeq and remains a good option if resources for NGS methods are lacking.

BACKGROUND: Visualization of ericoid mycorrhizal colonization using traditional methods relies on either fresh or KOH stored samples. Increasing interest in studying ericoid mycorrhization has highlighted a need for methods which can be used for preserved samples and are simple to implement with commonly available equipment. OBJECTIVE: The aim of this study was to improve on traditional techniques for staining ericoid mycorrhizal fungi and microscopically visualizing ericoid mycorrhizal roots which have been preserved. METHODS: Ericoid mycorrhizal roots were placed in KOH or frozen at -20 degrees C for long-term storage. Traditional Trypan Blue staining methods were modified to reduce damage to fine mycorrhizal hyphae and cortical cells. A high light-intensity dark-field microscopy technique was applied to clearly visualize stained mycorrhizae. The novel application was compared to other commonly used practices. RESULTS: Trypan Blue staining without KOH storage or clearing allowed for successful staining of ericoid mycorrhizal roots stored at -20 degrees C. The application of high light-intensity dark-field microscopy provided high contrast visualization of mycorrhizal structures. CONCLUSIONS: The modified Trypan Blue staining method was effective on frozen root samples, with dark-field microscopy being particularly effective at visualizing dark colored roots. Advantages to this method are lowcost and relatively fast application time. Therefore, this method is a realistic option for large scale analyses with many samples which require long-term preservation.

Northern peatlands are often dominated by ericaceous shrub species which rely on ericoid mycorrhizal fungi (ERM) for access to organic sources of nutrients, such as nitrogen (N) and phosphorus (P), and host abundant dark septate endophytes (DSE). Relationships between hosts and fungal symbionts may change during deposition of anthropogenic N and P. We studied the long-term effects of N and P addition on two ericaceous shrubs, Calluna vulgaris and Erica tetralix, at Whim Bog, Scotland by analyzing fungal colonization of roots, enzymatic activity, and fungal species composition. Unexpectedly, the frequency of typical ERM intracellular colonization did not change while the occurrence of ERM hyphae tended to increase and DSE hyphae to decrease. Our findings indicate that altered nutrient limitations shift root associated fungal colonization patterns as well as affecting ericaceous root enzyme activity and thereby decomposition potential. Reduction of recalcitrant fungal biomass in melanized DSE may have implications for peatland C sequestration under nutrient addition.

Prokaryotes colonize decaying wood and contribute to the degradation process, but the dynamics of prokaryotic communities during wood decay is still poorly understood. We studied the abundance and community composition of Bacteria and Archaea inhabiting naturally decaying Picea abies logs and tested the hypothesis that the variations in archaeal and bacterial abundances and community composition are coupled with environmental parameters related to the decay process. The data set comprises >500 logs at different decay stages from five geographical locations in south and central Finland. The results show that Bacteria and Archaea are an integral and dynamic component of decaying wood biota. The abundances of bacterial and archaeal 16S rRNA genes increase as wood decay progresses. Changes in bacterial community composition are clearly linked to the loss of density of wood, while specific fungal-bacterial interactions may also affect the distribution of bacterial taxa in decaying wood. Thaumarchaeota were prominent members of the archaeal populations colonizing decaying wood, providing further evidence of the versatility and cosmopolitan nature of this phylum in the environment. The composition and dynamics of the prokaryotic community suggest that they are an active component of biota that are involved in processing substrates in decaying wood material.

Catabolism of amino acids is a central process in cellular nitrogen turnover, but only a few of the mechanisms involved have been described from basidiomycete fungi. This study identified one such mechanism, the l-amino acid oxidase (Lao1) enzyme of Hebeloma cylindrosporum, by 2D gel separation and MS. We determined genomic DNA sequences of lao1 and part of its upstream gene, a putative pyruvate decarboxylase (pdc2), and cloned the cDNA of lao1. The two genes were also identified and annotated from the genome of Laccaria bicolor. The lao1 and pdc2 gene structures were conserved between the two fungi. The intergenic region of L. bicolor possessed putative duplications not detected in H. cylindrosporum. Lao1 sequences possessed dinucleotide-binding motifs typical for flavoproteins. Lao1 was less than 23 % identical to Lao sequences described previously. Recombinant Lao1 of H. cylindrosporum was expressed in Escherichia coli, purified and refolded with SDS to gain catalytic activity. The enzyme possessed broad substrate specificity: 37 l-amino acids or derivatives served as effective substrates. The highest activities were recorded with l-glutamate, but positively charged and aromatic amino acids were also accepted. Michaelis constants for six amino acids varied from 0.5 to 6.7 mM. We have thus characterized a novel type of Lao-enzyme and its gene from the basidiomycete fungus H. cylindrosporum.

The distribution of heterotrophic flagellates, naked amoebae, testate amoebae and ciliates was investigated in habitats created by Scots pine-Paxillus involutus and -Suillus bovinus ectomycorrhizospheres. The protozoa living on plant and fungal surfaces preferred the non-mycorrhizal pine roots over mycorrhizal roots or external mycelium. The testate amoebae were more abundant on external mycelium than on mycorrhizae regardless of the mycorrhizal fungal species. Numbers of protozoa were higher in the different habitats provided by S. bovinus mycorrhizospheres when compared with P. involutus mycorrhizospheres. Interestingly, the quality of the bacterial flora as food for the protozoa was affected by the mycorrhizal fungi even in the soils adjacent to non-mycorrhizal root tips of pine. These results demonstrate that mycorrhizal fungi create habitats differently suitable for protozoa living in boreal forest soil.

Archaea colonising defined compartments of Scots pine Suillus bovinus or Paxillus involutus mycorrhizospheres developed in forest humus-containing microcosms were investigated by nested polymerase chain reaction (PCR), cloning, restriction fragment length polymorphism (RFLP) and sequencing. Archaea representing six RFLP groups were detected in the system. Sequence analysis of clones representing the different RFLP types confirmed the presence of novel Finnish forest soil Crenarchaeota. Archaeal sequences were identified from mycorrhizas of both P. involutus and S. bovinus, at the margins of the external mycelium and in uncolonised humus but not from non-mycorrhizal short roots. Fungal and compartment-specific crenarchaeal occupation of mycorrhizospheres is discussed in relation to bacterial community distribution in similar systems.

Cellular interactions and catabolic activities of mycorrhizal root associated non-sporulating bacteria were investigated in a simplified phytoremediation simulation involving a woody plant species. Mycorrhizal Scots pine ( Pinus sylvestris ) seedlings pre-colonised by Suillus bovinus or Paxillus involutus were grown in forest humus containing microcosms amended with petroleum hydrocarbon (PHC) contaminated soil. Fungal hyphae of both species, emanating from mycorrhizal roots, colonised the PHC contaminated soil over a 16-week period and dense long-lived patches of S. bovinus hyphae formed on the PHC contaminated soil. Transmission electron microscopy revealed a microbial biofilm at the PHC soil-fungal interface composed of differentiated pseudoparenchymous patch hyphae supporting a morphologically diverse bacterial population. Certain non-sporulating bacterial isolates closely associated with the S. bovinus patch hyphae or P. involutus `web' hyphae from the PHC soil harboured similar sized mega-plasmids (approx. 150 kb). Isolates of Pseudomonas fluorescens from the `patch' mycorrhizospheres represented different biovars, displayed similar REP-PCR genomic fingerprints, grew on e.g. m -toluate and m -xylene as sole carbon sources, cleaved catechol, and harboured plasmid-borne catabolic marker genes, xylE and xylMA , involved in degradation of mono-aromatics. The plasmids were transmissible in vitro, and Pseudomonas putida transconjugants retained a similar catabolic profile. The identification of microbial biofilms containing catabolic bacteria in the external mycorrhizosphere is discussed in relation to both phytoremediation mechanisms and normal efficient nutrient mobilisation from highly lignin-rich forest soils.

Bacteria were isolated and characterized from uncolonized soil, nonmycorrhizal and mycorrhizal short roots, and soil-colonizing external mycelium from intact Pinus sylvestris - Suillus bovinus and Pinus sylvestris - Paxillus involutus mycorrhizospheres developed in microcosms containing dry pine forest humus or nursery peat. Total numbers of colony-forming units (CFU/mg dry weight) in the different locations from all ectomycorrhizospheres indicated an overall bacterial-enrichment gradient towards the roots, whereas sporeformers were more evenly distributed. Fluorescent pseudomonads were commonly isolated from all mycorrhizosphere locations in nursery peat, but they were nearly absent from the forest humus community. In contrast, sporeformers were more abundant at all locations in the latter growth substrate. The bacterial species composition of forest and nursery mycorrhizospheres was clearly divergent when characterized according to their carbon source utilization patterns in Biolog®GN or GP microplates. Factorial-designed ANOVA of a principal component analysis of the carbon source utilization data showed significant differences between isolates from the two soil types and, to a lesser extent, between S. bovinus and Paxillus involutus mycorrhizospheres. Bacterial communities from mycorrhizospheres and uncolonized soil were distinguished by their preferential utilization of carbohydrates and organic and amino acids, respectively. Suillus bovinus associated bacteria appeared to favour mannitol and Paxillus involutus associated bacteria appeared to favour fructose as carbon sources. This study demonstrates the combined effect of soil type, fungal symbiont, and precise location on bacterial communities associated with Pinus sylvestris ectomycorrhizospheres.Key words: Biolog, carbon source utilization, ectomycorrhiza, Scots pine, soil bacteria.

The bacterial populations associated with different plant and fungal habitats of intact Pinus sylvestris – Suillus bovinus or Pinus sylvestris – Paxillus involutus ectomycorrhizospheres grown in natural forest soil were examined by scanning and transmission electron microscopy. Surfaces of nonmycorrhizal Pinus sylvestris roots hosted large numbers of morphologically distinct bacteria. Bacteria were detected on the mantle surfaces and at inter- and intra-cellular locations in the mantle and Hartig net of Suillus bovinus mycorrhizas. The fungal strands were colonized by only a few bacteria unlike the outermost external fine hyphae on which extensive monolayers of bacteria were attached. The mycorrhizas of Paxillus involutus were mostly devoid of bacteria, but the intact external mycelium supported both bacterial colonies and solitary bacteria. Intracellular bacteria were not present in Paxillus involutus hyphae. In both mycorrhizal systems, bacterial aggregation and attachment to hyphae were mediated with electron-dense or -translucent material. Our study shows that the Pinus sylvestris mycorrhizospheres formed by two different ectomycorrhizal fungi are clearly dissimilar habitats for mycorrhizosphere-associated bacteria. Additionally, the spatially and physiologically defined mycorrhizosphere habitats were shown to host distinct populations of bacteria.Key words: ectomycorrhiza, intracellular bacteria, Paxillus involutus, soil bacteria, Suillus bovinus.

Phosphorus uptake and translocation through intact mycelial systems of Paxillus involutus and Suillus variegatus infecting Pinus contorta seedlings was monitored non-destructively using a beta-scanner. Mycorrhizal plants were grown in flat perspex chambers (20 x 6 cm(2)) and root growth was restricted to the upper portion of each chamber enabling mycelial translocation to be studied over distances of up to 15 cm. P-32 was supplied, either directly to distal parts of the extending mycelium, or to single, cut mycelial strands in feeding dishes. Two-dimensional patterns of activity were accumulated as scans with a lateral resolution of 5 mm and a longitudinal resolution of 3-4 mm. No distinct translocation front could be detected but patterns of accumulation of label in the mycorrhizal roots were not consistent with movement by simple diffusion. Activity in translocating hyphae became visible only after the activity in mycorrhizal root lips had been visible for a few days. In all cases there was a lag period of 20-50 hours before P-32 could be detected in mycorrhizal root tips. Pre-feeding with unlabelled phosphate had no effect on this lag period. This implies continuous translocation of phosphate at low concentrations and a lag period due to the time needed for detectable levels of phosphate to accumulate in mycorrhizal roots. Thus the minimum velocity of phosphate movement in the hyphae would be 7.5 mm/h, if the first molecules of P-32 arriving at the roots could be detected and the transport distance is 15 cm. Accumulation of phosphate to the roots was fairly constant, but not linear. The phosphorus uptake rate by intact mycelial margins was nearly four orders of magnitude higher than the uptake rate of cut mycorrhizal strands. The results indicate that the fine, foraging hyphae are better suited for nutrient uptake than mycelial strands and that phosphorus translocation in the hyphae occurs by active translocation of small amounts rather than by mass flow. (Less)

Bacterial isolations were carried out on Pinus sylvestris – Suillus bovinus mycorrhizospheres obtained directly from boreal pine forest. When samples were taken during dry weather, the numbers of bacterial colony-forming units were significantly higher in uncolonized short roots and external mycelia than in mycorrhizal roots and soil outside the mycorrhizosphere. In contrast, the colony-forming unit counts were similar in all hypogeous samples after rainy weather. Culturable bacteria were absent from most Suillus bovinus sporocarps. The bacteria isolated from all types of mycorr hizo sphere samples, i.e. short roots, mycorrhizal roots, and external mycelia, consisted primarily of Burkholderia spp., whereas most isolates from soil outside the mycorrhizosphere were identified as Paenibacillus spp. This study shows that mycorrhizal external mycelia can expand the habitat favourable for common rhizosphere bacteria into the soil far from the immediate rhizosphere. Some of these bacteria may help the trees with nitrogen acquisition, since potentially diazotrophic bacteria harbouring nitrogenase reductase (nifH) genes were isolated from mycorrhizal root tips.Key words: boreal forest soil, Burkholderia, ectomycorrhiza, nitrogen fixation, Paenibacillus.

In the sedimental organic matter of eutrophic continental seas, such as the largest dead zone in the world, the Baltic Sea, bacteria may directly participate in nutrient release by mineralizing organic matter or indirectly by altering the sediment's ability to retain nutrients. Here, we present a case study of a hypoxic sea, which receives riverine nutrient loading and in which microbe-mediated vicious cycles of nutrients prevail. We showed that bacterial communities changed along the horizontal loading and vertical mineralization gradients in the Gulf of Finland of the Baltic Sea, using multivariate statistics of terminal restriction fragments and sediment chemical, spatial and other properties of the sampling sites. The change was mainly explained by concentrations of organic carbon, nitrogen and phosphorus, which showed strong positive correlation with Flavobacteria, Sphingobacteria, Alphaproteobacteria and Gammaproteobacteria. These bacteria predominated in the most organic-rich coastal surface sediments overlain by oxic bottom water, whereas sulphate-reducing bacteria, particularly the genus Desulfobacula, prevailed in the reduced organic-rich surface sediments in the open sea. They correlated positively with organic nitrogen and phosphorus, as well as manganese oxides. These relationships suggest that the bacterial groups participated in the aerobic and anaerobic degradation of organic matter and contributed to nutrient cycling. The high abundance of sulphate reducers in the surficial sediment layers reflects the persistence of eutrophication-induced hypoxia causing ecosystem-level changes in the Baltic Sea. The sulphate reducers began to decrease below depths of 20 cm, where members of the family Anaerolineaceae (phylum Chloroflexi) increased, possibly taking part in terminal mineralization processes. Our study provides valuable information on how organic loading affects sediment bacterial community compositions, which consequently may maintain active nutrient recycling. This information is needed to improve our understanding on nutrient cycling in shallow seas where the dead zones are continuously spreading worldwide.

Background External nutrient discharges have caused eutrophication in many estuaries and coastal seas such as the Baltic Sea. The sedimented nutrients can affect bacterial communities which, in turn, are widely believed to contribute to release of nutrients such as phosphorus from the sediment. Methods We investigated relationships between bacterial communities and chemical forms of phosphorus as well as elements involved in its cycling in brackish sediments using up-to-date multivariate statistical methods. Bacterial community composition was determined by terminal restriction fragment length polymorphism and cloning of the 16S rRNA gene. Results and Conclusions The bacterial community composition differed along gradients of nutrients, especially of different phosphorus forms, from the estuary receiving agricultural phosphorus loading to the open sea. This suggests that the chemical composition of sediment phosphorus, which has been affected by riverine phosphorus loading, influenced on bacterial communities. Chemical and spatial parameters explained 25% and 11% of the variation in bacterial communities. Deltaproteobacteria, presumptively sulphate and sulphur/iron reducing, were strongly associated to chemical parameters, also when spatial autocorrelation was taken into account. Sulphate reducers correlated positively with labile organic phosphorus and total nitrogen in the open sea sediments. Sulphur/iron reducers and sulphate reducers linked to iron reduction correlated positively with aluminium- and iron-bound phosphorus, and total iron in the estuary. The sulphate and sulphur/iron reducing bacteria can thus have an important role both in the mineralization and mobilization of nutrients from sediment. Significance Novelty in our study is that relationships between bacterial community composition and different phosphorus forms, instead of total phosphorus, were investigated. Total phosphorus does not necessarily bring out interactions between bacteria and phosphorus chemistry since proportions of easily usable mobile (reactive) phosphorus and immobile phosphorus forms in different sediments can vary. Our study suggested possible feedbacks between different forms of phosphorus and bacterial community composition.

How entire microbial communities are structured across stratified sediments from the historical standpoint is unknown. The Baltic Sea is an ideal research object for historical reconstruction, since it has experienced many fresh- and brackish water periods and is depleted of dissolved oxygen, which increases the sediment's preservation potential. We investigated the bacterial communities, chemical elements (e.g. Cr, Pb Na, P, Sr and U) and sediment composition in a stratified sediment core dated by radiocarbon and spanning 8000 years of Baltic Sea history, using up-to-date multivariate statistics. The communities were analysed by 16S rRNA gene terminal restriction fragment length polymorphism. The communities of the deep Early Litorina and surface Late Litorina Sea laminae were separated from the communities of the middle Litorina Sea laminae, which were associated with elevated concentrations of U and Sr trace elements, palaeo-oxygen and palaeosalinity proxies. Thus, the Litorina Sea laminae were characterized by past oxygen deficiency and salinity increase. The communities of the laminae, bioturbated and homogeneous sediments were differentiated, based on the same historical sea phases, with correct classifications of 90%. Palaeosalinity was one of the major parameters that separated the bacterial communities of the stratified sediments. A discontinuous spatial structure with a surprising increase in community heterogeneity was detected in Litorina Sea sediments from 388 to 422 cm deep, which suggests that a salinity maximum occurred in the central Gulf of Finland app. 6200–6600 years ago. The community heterogeneity decreased from the surface down to 306 cm, which reflected downcore mineralization. The plateau of the decrease was in the app. 2000-year-old sediment layers. Bacterial community data may be used as an additional tool in ocean-drilling projects, in which it is important to detect mineralization plateaus both to determine historically comparable portions of sediment samples and historical events, such as sea-level rise culminations.