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On-site decentralized urban stormwater management has gained significant momentum in urban planning. Recently, vegetated roofs have been recommended as a viable decentralized stormwater management system and nature-based solution to meet the challenge of urban floods. However, as another type of unconventional green infrastructure, vertical greenery systems (VGS), also known as vegetated facades, have received much less research attention. Even though some researchers suggest that stormwater management by VGS is comparable to that of vegetated roofs, empirical evidence to substantiate this claim is limited. In this study, we conducted rain simulations on newly-built vegetation containers with water storage compartments. These vegetation containers were designed to be incorporated into a VGS specifically for stormwater management. We tested variables that could influence water retention efficiency and evapotranspiration of the containers under field conditions, i.e., inoculation of plant growth-promoting microbes (PGPMs) (Rhizophagus irregularis and Bacillus amyloliquefaciens), different substrate types (sandy loam and reed-based substrate), simulated rain quantity, natural precipitation, substrate moisture, and air temperature. The inoculation of PGPMs significantly reduced runoff quantity from the vegetation containers. Meanwhile, the well-ventilated sandy-loam substrate significantly reduced the remaining water in the water storage compartments over 1-week periods between rain simulation events, achieving high water-use efficiency. The selected microbes were established successfully in the containers and promoted the growth of 2 out of 5 plant species. R. irregularis colonization responded to substrate type and host plant species, while B. amyloliquefaciens population density in the substrate did not respond to these factors. Environmental conditions, such as antecedent substrate moisture, air temperature, and natural precipitation also influenced the efficiency of stormwater retention and/or evapotranspiration. In conclusion, this study provides instructive and practical insights to reduce urban flood risk by using VGS.

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

Summary • Glutamine synthetase (GS) expression and activity is of central importance for cellular ammonium assimilation and recycling. Thus, a full characterization of this enzyme at the molecular level is of critical importance for a better understanding of nitrogen (N) assimilation in the mycorrhizal symbiosis. • Genomic and cDNA libraries of Suillus bovinus were constructed to isolate the GS gene, glnA, and corresponding cDNAs. The transcription initiation site was identified and transcription and enzyme activities were characterized in pure culture mycelium and mycorrhiza, and extramatrical mycelium samples harvested from Scots pine–Suillus bovinus microcosms grown on forest humus. • Pure culture mycelium, mycorrhiza and extramatrical mycelium all exhibited equivalent levels of GS transcription, translation and enzyme activities. However, levels of transcription and enzyme activity did not correlate as a large majority of detectable transcripts showed specific 5′-end truncation. • Our data suggest that GS is constitutively expressed and not directly affected by environmental conditions of the symbiotic N uptake. Any changes in the intracellular ammonium level are most likely handled by regulatory flexibility of GS at enzyme level.

Isozyme expression of nutrient-mobilizing and defence-related enzymes were examined in different functional components of intact Scots pine (Pinus sylvestris L.) ectomycorrhizal root systems developed in natural unsterilized forest humus. Scots pine seedlings colonized by Suillus bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch ex Fr.) strains were grown on humus in two-dimensional Perspex⌖ microcosms to allow development of complete ectomycorrhizal root systems. Soluble proteins from uncolonized short roots, whole mycorrhizal root tips or dissected mantle and core fractions, fungal strands and the outermost soil colonizing fine hyphae were subjected to native polyacrylamide gel electrophoresis and stained to detect esterase, acid phosphatase, peroxidase and different polyphenoloxidase isozyme activities. Tissue-specific esterase isozymes were detected in all components and most, including unique Hartig net isozymes, were of fungal origin. High fungal acid phosphatase activities were detected in mycorrhiza and fine hyphae of S. bovinus, supporting earlier findings of active phosphatase activity at the fungal interface in the Hartig net region and in the fine hyphal margins of extramatrical mycelium actively colonizing the humus. All compartments in the P. involutus mycorrhizosphere had weaker acid-phosphatase activities. Peroxidases formed a large part of the soluble-protein content in non-mycorrhizal short roots. The amount of peroxidases, on a tissue f. wt basis, was similar in mycorrhizal and non-mycorrhizal roots, but differential isozyme expression was detected, indicating a change in root peroxidase activities following mycorrhiza formation. The expression of DL-3,4-dihydroxyphenylalanine metabolizing polyphenol oxidase enzymes was reduced in mycorrhizas compared with non-mycorrhizal short roots. In the extramatrical mycelial components, fine hyphae expressed the highest polyphenoloxidase activity. P. involutus displayed some polyphenol-oxidizing isoenzymes not detected in S. bovinus systems. Laccase activity was not detected in the plant and fungal components examined. It is concluded that enzyme activities and isozyme expression are differentially regulated in the different functional components of Scots pine mycorrhizospheres.

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.

Root-colonizing fungi can form mycorrhizal or endophytic associations with plant roots, the type of association depending on the host. We investigated the differences and similarities of the fungal communities of three boreal ericoid plants and one coniferous tree, and identified the community structure of fungi utilizing photosynthates from the plants studied. The fungal communities of roots and soils of Vaccinium myrtillus, Vaccinium vitis-idaea, Calluna vulgaris and Pinus sylvestris were studied in an 18-month-long experiment where the plants were grown individually in natural substrate. Photosynthates utilizing fungi were detected with DNA stable-isotope probing using 13 CO2 (13 C-DNA-SIP). The results indicated that the plants studied provide different ecological niches preferred by different fungal species. Those fungi which dominated the community in washed roots had also the highest 13 C-uptake. In addition, a common root endophyte without confirmed mycorrhizal status also obtained 13 C from all the plants, indicating close plant-association of this fungal species. We detect several fungal species inhabiting the roots of both ericoid mycorrhizal and ectomycorrhizal plants. Our results highlight that the ecological role of co-occurrence of fungi with different life styles (e.g. mycorrhizal or endophytic) in plant root systems should be further investigated.

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.

Decades of intensive discharge from industrial activities into coastal systems has resulted in the accumulation of a variety of persistent organic pollutants (POPs) in marine waters and sediments, having detrimental impacts on aquatic ecosystems and the resident biota. POPs are among the most hazardous chemicals originating from industrial activities due to their biotoxicity and resistance to environmental degradation. Bacterial communities are known to break down many of these aromatic compounds, and different members of naturally occurring bacterial consortia have been described to work in syntrophic association to thrive in heavily contaminated waters and sediments, making them potential candidates as bioindicators of environmental pollution. In this study environmental, sampling was combined with chemical analysis of pollutants and high-resolution sequencing of bacterial communities using Next Generation Sequencing molecular biology tools. The aim of the present study was to describe the bacterial communities from marine sediments containing high loads of POPs and to identify relevant members of the resident microbial communities that may act as bioindicators of contamination. Marine sediments were collected from a coastal bay area of the Baltic Sea historically influenced by intense industrial activity, including metal smelting, oil processing, and pulp and paper production. Different types of POPs were detected at high concentrations. Fiberbank sediments, resulting from historic paper industry activity, were found to harbour a clearly distinct bacterial community including a number of bacterial taxa capable of cellulolytic and dechlorination activities. Our findings indicate that specific members of the bacterial communities thrive under increasing levels of POPs in marine sediments, and that the abundances of certain taxa correlate with specific POPs (or groups), which could potentially be employed in monitoring, status assessment and environmental management purposes.

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.

In this study, the bacterial populations of roots and mycospheres of the boreal pine forest ericoid plants, heather (Calluna vulgaris), bilberry (Vaccinium myrtillus), and lingonberry (Vaccinium vitis-idaea), were studied by qPCR and next-generation sequencing (NGS). All bacterial communities of mycosphere soils differed from soils uncolonized by mycorrhizal mycelia. Colonization by mycorrhizal hyphae increased the total number of bacterial 16S ribosomal DNA (rDNA) gene copies in the humus but decreased the number of different bacterial operational taxonomic units (OTUs). Nevertheless, ericoid roots and mycospheres supported numerous OTUs not present in uncolonized humus. Bacterial communities in bilberry mycospheres were surprisingly similar to those in pine mycospheres but not to bacterial communities in heather and lingonberry mycospheres. In contrast, bacterial communities of ericoid roots were more similar to each other than to those of pine roots. In all sample types, the relative abundances of bacterial sequences belonging to Alphaproteobacteria and Acidobacteria were higher than the sequences belonging to other classes. Soil samples contained more Actinobacteria, Deltaproteobacteria, Opitutae, and Planctomycetia, whereas Armatimonadia, Betaproteobacteria, Gammaproteobacteria, and Sphingobacteriia were more common to roots. All mycosphere soils and roots harbored bacteria unique to that particular habitat. Our study suggests that the habitation by ericoid plants increases the overall bacterial diversity of boreal forest soils.

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.

Archaea belong to the least well known major group of soil inhabiting microbes as the concept of the very existence of the archaea was introduced only in 1977 and the domain of Archaea established in 1990. The first reports of finding these organisms in soils were published even later. This paper will review the research carried out of the archaea in dry moderate soil environments. It will particularly consider the specific habitats where the archaea live in soils, as well as their associations with other organisms. There is thus far relatively little knowledge about the metabolism of the soil archaea, but the knowledge about their exact habitats and associations as well as their genetic potential point the way to discovering more about the different soil archaeal functions.

Over a 5 year period, we examined the influence of substrate and fertilization on nursery growth and outplanting performance of Norway spruce (Picea abies (L.) Karst.). We focused on the relative growth and development of roots and shoots and the colonization intensity and diversity of ectomycorrhizal (ECM) fungi. In the nursery, a conventional substrate (low-humified Sphagnum peat) supplemented with woody material (wood fibre and pine bark) and either mineral or organic fertilizers yielded shorter seedlings than those grown on the unmodified substrate. However, after outplanting, the growth rate of seedlings cultivated on modified substrates was higher than that of seedlings grown on the unmodified substrate. Seedlings cultivated in modified substrates had significantly higher root/shoot ratios and ECM diversity; the latter remained significant after ≥3 years of outplanting. Seedlings grown on a substrate containing 50% woody material and supplemented with organic fertilizer had the highest growth rate among all seedlings during the 3 year period of outplanting. Colonization intensity of ECM fungi was high in all seedlings except for those grown in heavily fertilized substrate. This study suggests that nursery techniques that produce seedlings with higher root/shoot ratios and ECM diversities could improve plantation success and growth rate for at least the first 3 years of outplanting.

Ectomycorrhizal fungi were characterised by restriction fragment length polymorphism (RFLP) and sequencing from Tilia cordata Mill root tips collected from forest, street and nursery habitats. Fungal colonisation intensity of roots was 80–90% in forest and street habitats and 60–70% at the nursery. On the whole, more Basidiomycota than Ascomycota were encountered in Tilia mycorrhizas, and soil horizons at depth of 5–10 cm contained more root tips and had higher diversity of mycorrhizal fungi than soils at depth of 20–25 cm. Healthy street trees and forest trees had the highest number of ectomycorrhizal morphotypes. Street and nursery trees did not have any common mycorrhizal fungal genera, whereas Boletus , Inocybe and Russula spp. were found from roots of both street and forest trees and Tuber spp. from roots of nursery and forest trees. Most of the genera found in this study do not have an earlier record of including Tilia root symbiotic species in Finland, although several but not all have been reported from other countries.

The effects of the transformation of a 4-coumarate:coenzyme A ligase (4CL) gene involved in lignin biosynthesis on the interactions between transgenic plants and soil microbiota were studied in silver birch (Betula pendula Roth). Although the genetic transformation did not lead to expected changes in wood chemistry, it had significant influence on root biomass and morphology. The transgenic lines had less root biomass and lower numbers of root tips than the wild-type birch. However, the transgenic lines formed normal ectomycorrhizas with Paxillus involutus (Batsch) Fr. and no differences between the transgenic lines and the wild-type birch were detected in the colonization by mycorrhizas in vitro. In a field trial, the decomposition of the transgenic and wild-type birch litter was equally rapid, and no marked differences were found between the transgenic lines and wild-type birch in total microbial biomass (SIR) or activity (basal respiration). The effects of the genetic transformation on the ergosterol content of the leaf litter were contradictory; the litter from one transgenic line had significantly higher ergosterol content and the litters from two other transgenic lines had significantly lower ergosterol content than the litter from wild-type birch. This study shows that the 4CL antisense transformation and the pleiotropic effects of the transformation on growth characteristics of birch did not disturb the formation of ectomycorrhizas with P. involutus and had no clear influence on the biodegradability of the leaf litter.

Mycorrhizal fungi contain chitin in their cell walls and may be influenced by transgenic chitinases. This study examined the ability of a transgenic tree, silver birch (Betula pendula Roth), constitutively expressing the sugar beet chitinase IV gene, to form ectomycorrhizae with Paxillus involutus (Batsch) Fr. Eight transgenic lines showing varying levels of sugar beet chitinase IV expression and the non-transgenic control plants were inoculated by P. involutus in vitro, and the morphology of the mycorrhizae, mycorrhization efficiency and shoot and root fresh weights were studied. All the transgenic birch lines were able to form normal ectomycorrhizae containing distinctive mantles and Hartig nets. The level of sugar beet chitinase IV expression had no significant influence on mycorrhizal colonization. The only significant differences between transgenic and control plants were detected in weight parameters. According to these results, the expression of sugar beet chitinase IV in silver birch is n...

The expression patterns of the cytoskeletal proteins α-, β-, and γ-tubulin, actin, and myosin were investigated in young tomato roots and older roots at different intensities of mycorrhizal colonization. The relative level of cytoskeletal proteins was estimated by protein blotting and immunostaining. The contribution of plant α-, β-, and γ-tubulin to the total protein pool was higher in uncolonized 2-week-old roots than in 10-week-old roots, whereas the contribution of actin remained constant. The level of plant tubulin expression was clearly higher in mycorrhizal root systems than in uncolonized older root systems. These results indicate that tubulins are more involved in plant cell differentiation than actin. Myosin of approximately 230 kDa was expressed in the roots of 10-week-old wild-type tomato but not in young or mycorrhizal tomatoes. In contrast, a smaller ca. 170 kDa myosin was consistently present in all root samples. Indirect immunofluorescence microscopy showed that plant myosin was located particularly along the periarbuscular membranes surrounding the arbuscule branches. In uncolonized roots, myosin was associated mainly with membranes adjacent to plant cell walls. These data provide novel evidence that myosin expression and localization in root cells responds to mycorrhizal colonization.Key words: actin, myosin, arbuscular mycorrhizal fungi, protein expression, tubulin.

This study assessed the effect of mycorrhizal colonization by Glomus intraradices (Gi) and G. versiforme (Gv) on the bacterial community composition in the rhizosphere of canola, clover and two tomato genotypes (wild type (76R) and its mutant with reduced mycorrhizal colonization (rmc)). Additionally, the effect of light intensity on the rhizosphere bacterial community composition of the tomato genotypes was studied. The bacterial community composition was assessed by denaturing gradient gel electrophoresis (DGGE). In canola, which is considered to be a non-mycorrhizal species, inoculation with Gi increased the shoot dw compared to Gv and the non-mycorrhizal control plants and also induced changes in the bacterial community composition in the rhizosphere. These fungal effects were observed although less than 8% of the root length of canola was colonized. On the other hand, about 50% of the root length of clover was colonized and inoculation with Gv resulted in a higher shoot dw compared to Gi or the control plants but the rhizosphere bacterial community composition was not affected by inoculation. Plant growth, mycorrhizal colonization and bacterial community composition of the two tomato genotypes were affected by a complex interaction between tomato genotype, AM fungal species and light intensity. Low light intensity (photosynthetic photon flux 200–250 μmol m −2 s −1 ) increased the shoot–root ratio in both genotypes and reduced colonization in the wild type. The differences in bacterial community composition between the two genotypes were more pronounced at low than at high light intensity (550–650 μmol m −2 s −1 ).

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.

Fungi in the genus Tomentellopsis were subjected to molecular phylogenetic analyses in order to clarify species-level relationships and mycorrhiza-forming ability with coniferous and deciduous trees. Fungal nucleotide sequence data from the internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) region were obtained from fruitbodies, ectomycorrhiza and pure cultures. Maximum parsimony (MP), distance (neighbor joining, NJ) and maximum likelihood (ML) analyses of aligned ITS sequences highlighted three clades designated T. echinospora, T. submollis and T. bresadoliana. Sporocarp tissue and ectomycorrhizas, or isolated mycelia, previously described as Pinirhiza rosea, Piceirhiza rosea or Pink were clearly identified in a strongly supported T. submollis clade. Host-linked population variation in this clade was also noted that may reflect active speciation activity. A fungus isolated from Beige-type mycorrhizas formed on Scots pine seedlings appeared in the paraphyletic T. echinospora clade which included sequences showing greater divergence. The data provide a primary classification of Tomentellopsis species that is urgently needed in assessment of the importance of resupinate thelephoroid fungi in mycorrhizal communities associated with trees in coniferous and deciduous forest ecosystems.

Mycorrhizal and non-mycorrhizal Scots pine (Pinus sylvestris L.) seedling combinations were grown in two-dimensional Perspex® microcosms containing forest humus or nursery Sphagnum peat, without additional fertilization. Spatial and temporal patterns of mycorrhizal fungal colonization of roots were assessed over a 3-month period, through visual morphotyping and polymerase chain reaction assisted rDNA fingerprinting of developed mycorrhizas. Six distinct morphotypes of mycorrhiza developed on non-mycorrhizal seedlings grown in forest humus. Three of the morphotypes (white1-, black-, and brown-type) were, respectively, associated with the fungi Suillus bovinus (L. ex Fr.) O. Kuntze, Cenococcum geophilum Fr., and Thelephora terrestris Ehrh.:Fr. (= Tomentella radiosa (P. Karst.) Rick). A fourth pink-type morphotype displayed features indicating root colonization by Tomentellopsis submollis (Svrcek) Hjortstam, but this could not be confirmed because of a lack of source restriction fragment length polymorphism (RFLP) data. Brown-type mycorrhiza were the first to appear after seedling transplantation and black-type mycorrhizas showed local and dispersed root colonization dynamics. Mycorrhiza development in the unfertilized nursery peat substrate was restricted to a single unidentifiable brown-type morphotype, which appeared after 44 days. Rapid colonization of adjacent non-mycorrhizal seedlings by Tomentellopsis submollis, but not S. bovinus, was detected following the introduction of preinoculated seedlings into the humus or peat microcosms. The biomass of seedlings grown in nursery peat, regardless of mycorrhizal status, was significantly lower than that of mycorrhizal seedlings grown in humus. These findings support and extend previous bait seedling studies, as they provide a primary in situ characterization of distinct root colonization strategies of mycorrhizal species in forest humus and nursery peat.

In this study the presence and orientation of fungal microtubules were recorded in arbuscular mycorrhizal symbiosis for the first time. Visualization of the fungal microtubules was achieved by using a protocol specifically labelling only fungal tubulins. Microtubules of external mycelium, intraradical hyphae, arbuscules, and vesicles of the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith were examined when in symbiosis with tomato (Lycopersicon esculentum Mill.). Microtubules were organized as bundles in both external and intraradical hyphae. The bundles of microtubules extended directly from intraradical hyphae into the arbuscules, where the microtubules remained as bundles in the larger hyphae. In the fine fungal branches of the arbuscules, microtubules were seen as thinner filaments. Fungal microtubules were seen to connect the intraradical hyphae and arbuscules. In addition, microtubules of adjacent arbuscules could continue directly from one arbuscule to another. Microtubules reached to the basal cone of each vesicle, but the live vesicles, containing many nuclei, seemed devoid of any microtubular labelling.Key words: cytoskeleton, endomycorrhiza, filamentous fungi, tomato, tubulin, Zygomycota.

Heterogeneous ethylene polymerization catalysts were prepared by supporting tetrakis(dimethylamino)titanium, Ti[N(CH 3 ) 2 ] 4 , on chemically modified silica surface. The modification of silica was made using a silane coupling agent, Cp(CH 2 ) 3 Si(OCH 2 CH 3 ) 3 and atomic layer chemical vapor deposition (ALCVD). Titanium amide was also immobilized straight on the silica and on a methyllithium-treated modified silica. The aim of methyllithium treatment was to alkylate the unreacted ethoxy groups of the silane coupling agent on the silica. Methyllithium methylates, besides ethoxy groups, also silica surface and surface cyclopentadienyl groups. A model compound for these heterogeneous catalysts was prepared using a reaction of Ti[N(CH 3 ) 2 ] 4 with trimethylsilylcyclopentadienyl yielding a monocyclopentadienyl titanium compound, Me 3 SiCpTi[N(CH 3 ) 2 ] 3 . The preliminary results of ethylene polymerization using the prepared heterogeneous and homogeneous titanocene amide complexes and the effect of amount of MAO are presented.

Heterogeneous ethylene polymerization catalysts were prepared by supporting tetrakis(dimethylamino)zirconium on chemically modified silica surface. The silica was modified with a silane coupling agent, Cp(CH 2 ) 3 Si(OEt) 3 , using ALCVD-technique. On the resulting cyclopentadienyl surface zirconium amide, Zr(NMe 2 ) 4 , was immobilized with and without using methyllithium. Depending on the use of methyllithium the immobilization resulted in either mono- or bis-cyclopentadienyl zirconium complexes, which were characterized using chemical analysis, FT-IR, 13 C- and 29 Si-NMR results. Moreover, a homogeneous model compound, (Me 3 SiCp) 2 Zr(NMe 2 ) 2 , was synthesized with a reaction between Zr(NMe 2 ) 4 and Me 3 SiCp. The preliminary results of ethylene polymerization with these supported and unsupported organometallic complexes are presented.

The tolerance to, and degradation of m-toluate by Scots pine (Pinus sylvestris), a symbiotic mycorrhizal fungus (Suillus bovinus) and Pseudomonas fluorescens strains, with or without m-toluate-degrading capacity, was determined individually and in all symbiotic/associative plant-microbe combinations. Fungal survival on medium with m-toluate was increased in co-culture with the degradative bacterial strains on agar plates (up to 0.02%, w/v). When fungi were grown in mycorrhizal association with Scots pine seedlings in test-tube microcosms containing expanded clay pellets and growth media, the fungus was able to withstand m-toluate concentrations up to 2.0%, w/v in all treatments. The seedling tolerance remained unaltered regardless of the presence or absence of mycorrhizal fungi or biodegradative bacteria. Reduction in m-toluate levels was only detected in treatments inoculated with bacterial strains harbouring TOL catabolic plasmids. The plant and fungus, alone or in mycorrhizal symbiosis, were unable to cleave m-toluate. The presence of easily available plant-derived carbon sources did not impede m-toluate degradation by the bacteria in the mycorrhizosphere.

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.

A novel heterogeneous α-olefin polymerization catalyst is prepared by the immobilization of CpZrCl 3 on a chemically modified silica support. The surface modification of silica was carried out by utilizing the saturated gas-solid reactions of a silane coupling agent with a hydrocarbon spacer, Cp(CH 2 ) 3 Si(OCH 2 CH 3 ) 3, and a partially dehydroxylated silica. In gas-solid reactions all reactive surface hydroxyl groups were utilized in bonding the silane coupling agent on silica. The immobilization of CpZrCl 3 on the cyclopentadienyl surface formed on the silica was done using liquid-phase reactions. The prepared materials were characterized by FT-IR spectroscopy. The supported catalyst system has a high activity in ethylene polymerization and can be used to produce polyethylene with a narrow molecular weight distribution in the presence of methylaluminoxane. The catalytic activity was doubled when CpZrCl 3 was immobilized on the cyclopentadienyl surface of SiO 2 compared with the activity of CpZrCl 3 in homogeneous catalysis. However, the direct heterogenization of CpZrCl 3 onto unmodified silica produced a catalyst with a very low activity. The effect of surface modification reactions and the surface structure of a support on the catalyst performance is presented and discussed.

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.

cf thèse de doctorat (PhD); Arbuscular mycorrhizal (AM) symbioses may alter the competitive abilities of plant species and facilitate positive interactions such as nutrient transfer between plants. They are therefore particularly interesting components in agroforestry systems. We studied spatial variation of AM colonisation on a cut-and-carry fodder production site (agroforestry plot) of the legume tree [i]Gliricidia sepium[/i] and the fodder grass [i]Dichanthium aristatum[/i]. Roots of the two plant species were sampled under the tree canopies and on the adjacent grass plot at 1 and 3.5 m from the first tree row where [i]G. sepium[/i] roots also occur. Roots of [i]D. aristatum[/i] were also sampled on a nearby grass monocrop. Colonisation of arbuscules, vesicles and hyphae in root samples was visually determined, and AM fungal species were identified by DNA sequencing. Colonisation and frequency of types of AM formations varied statistically significantly between the species and sampling points. Arbuscular colonisation in [i]G. sepium[/i] roots was higher under the tree canopies than on the adjacent grass plot. Soil nutrient content, particularly P and N, and interspecies competition are the most probable explanations for the observed variation in AM colonisation. Both arbuscular colonisation and arbuscule: vesicle ratio in [i]D. aristatum[/i] roots was lower on the [i]D. aristatum[/i] monocrop than on the agroforestry plot under or near the tree canopies. Intercropping could stimulate AM symbiosis in [i]D. aristatum[/i]. Both plant species formed symbiosis with [i]Rhizophagus intraradices[/i], indicating potential for interplant N transfer via common mycelial networks of AM-forming fungi.

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)

Sections of Pinus sylvestris-Suillus bovinus ectomycorrhiza were used to test the applicability of time-resolved fluorescence microscopy for studying plant and fungal material. The autofluorescence problems encountered with standard indirect immunofluorescence microscopy when studying specialized plant material were eliminated by the technique. The europium-labeled structures of both plant and fungal cells were the only visible areas in the sections. However, fine details were lost due to the coarse pixel size of the digital camera used to transfer the image on screen. Even with current resolution, time-resolved imaging can be an efficient method for detection of targets in both plant and fungal tissues with autofluorescence problems.