Ectomycorrhizal fungi increase the vitality of Norway spruce seedlings under the pressure of Heterobasidion root rot in vitro but may increase susceptibility to foliar necrotrophs.

We tested if root colonisation by ectomycorrhizal fungi (EMF) could alter the susceptibility of Norway spruce (Picea abies) seedlings to root rot infection or necrotic foliar pathogens. Firstly, spruce seedlings were inoculated by various EMF and challenged with Heterobasidion isolates in triaxenix tubes. The ascomycete EMF Meliniomyces bicolor, that had showed strong antagonistic properties towards root rot causing Heterobasidion in vitro, protected spruce seedlings effectively against root rot. Secondly, spruce seedlings, inoculated with M. bicolor or the forest humus, were subjected to necrotrophic foliar pathogens in conventional forest nursery conditions on peat substrates. Botrytis cinerea infection after winter was mild and the level of needle damage was independent of substrate and EMF colonisation. Needle damage severity caused by Gremminiella abietina was high in seedlings grown in substrates with high nutrient availability as well as in seedlings with well-established EMF communities. These results show that albeit M. bicolor is able to protect spruce seedlings against Heterobasidion root rot in axenic cultures it fails to induce systemic protection against foliar pathogens. We also point out that unsterile inoculum sources, such as the forest humus, should not be considered for use in greenhouse conditions as they might predispose seedlings to unintended needle damages.

Genetic host-tree effects on the ectomycorrhizal community and root characteristics of Norway spruce.

A greenhouse experiment was used to study the effects of host genotype on short root formation and ectomycorrhizal (ECM) fungal community structure in Norway spruce (Picea abies (L.) Karst.). Rooted cuttings representing 55 clones were inoculated with a mix of vegetative hyphae of five ECM fungal species (Laccaria sp., Amphinema byssoides, Piloderma sp., Cadophora finlandia, Paxillus involutus). After one growing season, the ECM fungal community structure was determined by amplifying the fungal internal transcribed spacer (ITS) of ribosomal DNA directly from ECM root tips. Restriction profiles of obtained amplicons were then compared to those of the inoculated strains. Spruce clones differed in their ECM fungal community composition; we found a statistically significant clone-specific effect on ECM fungal diversity and dominating fungal species. Nevertheless, the broad sense heritabilities of the levels of Laccaria sp., Piloderma sp. and A. byssoides colonisations as well as the ECM fungal community structure were low (H(2) = 0.04-0.11), owing to the high within-clone variation. As nitrogen concentration of needles correlated negatively with ECM fungal richness, our results imply that in the experimental conditions nutrient acquisition of young trees may benefit from colonisation with only one or two ECM fungal species. The heritability of short root density was moderate (H(2) = 0.41) and highest among all the measured shoot and root growth characteristics of Norway spruce cuttings. We suggest that the genetic component determining root growth and short root formation is significant for the performance of young trees in natural environments as these traits drive the formation of the below-ground symbiotic interactions.

Interactions between extraradical ectomycorrhizal mycelia, microbes associated with the mycelia and growth rate of Norway spruce (Picea abies) clones.

Despite their ecological relevance, field studies of the extraradical mycelia of ectomycorrhizal (ECM) fungi are rare. Here we examined in situ interactions between ECM mycelia and host vigour. Ectomycorrhizal mycelia were harvested with in-growth mesh bags buried under Norway spruce (Picea abies) clones planted in 1994 in a randomized block design. Mycelial biomass was determined and fungal species were identified by denaturing gradient gel electrophoresis (DGGE) and sequencing of the internal transcribed spacer 1 (ITS1) region. Microbial community structure in the mycelium was investigated by phospholipid fatty acid (PLFA) profiling. Compared to slow-growing spruce clones, fast-growing clones tended to support denser mycelia where the relative proportions of Atheliaceae fungi and PLFAs indicative of Gram-positive bacteria were higher. Ascomycetes and PLFAs representative of Gram-negative bacteria were more common with slow-growing clones. In general, the ECM mycelial community was similar to the ECM root-tip community. Growth rate of the hosts, the ECM mycelial community and the microbes associated with the mycelium were related, suggesting multitrophic interactions between trees, fungi and bacteria.

Ectomycorrhizal community structure varies among Norway spruce (Picea abies) clones.

In northern boreal forests, the diversity of ectomycorrhizal (ECM) species is much greater than that of their host trees. This field study investigated the role of individual trees in shaping the ECM community. We compared ECM communities of eight Norway spruce (Picea abies) clones planted in a clear-cut area in 1994 with a randomized block design. In 2003, the ECM fungi were identified from randomly sampled root tips using denaturing gradient gel electrophoresis (DGGE) and rDNA internal transcribed spacer (ITS) sequence similarity. ECM diversity varied among clone groups, showing twofold growth differences. Moreover, according to detrended correspondence analysis (DCA), ECM community structure varied not only among but also within slow-growing or fast-growing clones. Results suggest that ECM diversity and community structure are related to the growth rate or size of the host. A direct or indirect influence of host genotype was also observed, and we therefore suggest that individual trees are partly responsible for the high diversity and patchy distribution of ECM communities in boreal forests.

In situ bioremediation through mulching of soil polluted by a copper-nickel smelter.

Bioremediation of a heavy metal-polluted soil was investigated in a 3-yr field experiment by adding mulch to a polluted forest floor. The mulch consisted of a mixture of compost and woodchips. The remediation treatment decreased the toxicity of the soil solution to bacteria as determined by the [3H]-thymidine incorporation technique, that is, by measuring the growth rate of soil bacteria extracted from unpolluted humus after exposing them to soil solution containing heavy metals from the experimental plots. Canonical correlation analysis was performed in order to identify the chemical and microbiological changes in the soil. The pH of the mulched organic layer increased by one unit. The concentration of complexed Cu increased and that of free Cu2+ decreased in the soil solution from the mulch treatment. According to basal respiration and litter decomposition, microbial activity increased during the 3 yr following the remediation treatment. The [3H]-thymidine incorporation technique was also used to study the growth rate and tolerance of bacteria to Cu. The bacterial growth rate increased and the Cu tolerance decreased on the treated plots. The structure of the microbial community, as determined by phospholipid fatty acid (PLFA) analysis, remained unchanged. The results indicate that remediation of the polluted soil had occurred, and that adding a mulch to the forest floor is a suitable method for remediating heavy metal-polluted soil.

Microbial activities related to C and N cycling and microbial community structure in the rhizospheres of Pinus sylvestris, Picea abies and Betula pendula seedlings in an organic and mineral soil.

The aim of this study was to determine whether Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies (L.) Karst.) and silver birch (Betula pendula Roth) seedlings have a selective influence on the soil microbial community structure and activity and whether this varies in different soils. Seedlings of pine, spruce and birch were planted into pots of two soil types: an organic soil and a mineral soil. Pots without seedlings were also included. After one growing season, microbial biomass C (C(mic)) and N (N(mic)), C mineralization, net ammonification, net nitrification, denitrification potential, phospholipid fatty acid (PLFA) patterns and community level physiological profiles (CLPPs) were measured in the rhizosphere soil of the seedlings. In the organic soil, C(mic) and N(mic) were higher in the birch rhizosphere than in pine and spruce rhizosphere. The C mineralization rate was not affected by tree species. Unplanted soil contained the highest amount of mineral N and birch rhizosphere the lowest, but rates of net N mineralization and net nitrification did not differ between treatments. The microbial community structure, measured by PLFAs, had changed in the rhizospheres of all tree species compared to the unplanted soil. Birch rhizosphere was most clearly separated from the others. There was more of the fungal specific fatty acid 18:2omega6,9 and more branched fatty acids, common in Gram-positive bacteria, in this soil. CLPPs, done with Biolog GN plates and 30 additional substrates, separated only birch rhizosphere from the others. In the mineral soil, roots of all tree species stimulated C mineralization in soil and prevented nitrification, but did not affect C(mic) and N(mic), PLFA patterns or CLPPs. The effects of different tree species did not vary in the mineral soil. Thus, in the mineral soil, the strongest effect on soil microbes was the presence of a plant, regardless of the tree species, but in the organic soil, different tree species varied in their influence on soil microbes.

Structure of the Microbial Communities in Coniferous Forest Soils in Relation to Site Fertility and Stand Development Stage.

> Abstract The structure, biomass, and activity of the microbial community in the humus layer of boreal coniferous forest stands of different fertility were studied. The Scots pine dominated CT (Calluna vulgaris type) represented the lowest fertility, while VT (Vaccinium vitis-idaéa type), MT (Vaccinium myrtillus type), and OMT (Oxalis acetocella-Vaccinium myrtillus type) following this order, were more fertile types. The microbial community was studied more closely by sampling a succession gradient (from a treeless area to a 180-years-old Norway spruce stand) at the MT type site. The phospholipid fatty acid (PLFA) analysis revealed a gradual shift in the structure of the microbial community along the fertility gradient even though the total microbial biomass and respiration rate remained unchanged. The relative abundance of fungi decreased and that of bacteria increased with increasing fertility. The structure of the bacterial community also changed along the fertility gradient. Irrespective of a decrease in fungal biomass and change in bacterial community structure after clear-cutting, the PLFA analysis did not show strong differences in the microbial communities in the stands of different age growing on the MT type site. The spatial variation in the structure of the microbial community was studied at a MT type site. Semivariograms indicated that the bacterial biomass, the ratio between the fungal and bacterial biomasses, and the relative amount of PLFA 16:1omega5 were spatially autocorrelated within distances around 3 to 4 m. The total microbial and fungal biomasses were autocorrelated only up to 1 m. The spatial distribution of the humus microbial community was correlated mainly with the location of the trees, and consequently, with the forest floor vegetation.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p168.html

Structure of a microbial community in soil after prolonged addition of low levels of simulated acid rain

Humus samples were collected 12 growing seasons after the start of a simulated acid rain experiment situated in the subarctic environment. The acid rain was simulated with H2SO4, a combination of H2SO4 and HNO3, and HNO3 at two levels of moderate acidic loads close to the natural anthropogenic pollution levels of southern Scandinavia. The higher levels of acid applications resulted in acidification, as defined by humus chemistry. The concentrations of base cations decreased, while the concentrations of exchangeable H+, Al, and Fe increased. Humus pH decreased from 3.83 to 3.65. Basal respiration decreased with decreasing humus pH, and total microbial biomass, measured by substrate-induced respiration and total amount of phospholipid fatty acids (PLFA), decreased slightly. An altered PLFA pattern indicated a change in the microbial community structure at the higher levels of acid applications. In general, branched fatty acids, typical of gram-positive bacteria, increased in the acid plots. PLFA analysis performed on the bacterial community growing on agar plates also showed that the relative amount of PLFA specific for gram-positive bacteria increased due to the acidification. The changed bacterial community was adapted to the more acidic environment in the acid-treated plots, even though bacterial growth rates, estimated by thymidine and leucine incorporation, decreased with pH. Fungal activity (measured as acetate incorporation into ergosterol) was not affected. This result indicates that bacteria were more affected than fungi by the acidification. The capacity of the bacterial community to utilize 95 different carbon sources was variable and only showed weak correlations to pH. Differences in the toxicities of H2SO4 and HNO3 for the microbial community were not found.

Phospholipid Fatty Acid Composition and Heavy Metal Tolerance of Soil Microbial Communities along Two Heavy Metal-Polluted Gradients in Coniferous Forests.

The effects of long-term heavy metal deposition on microbial community structure and the level of bacterial community tolerance were studied along two different gradients in Scandinavian coniferous forest soils. One was near the Harjavalta smelter in Finland, and one was at Ronnskar in Sweden. Phospholipid fatty acid (PLFA) analysis revealed a gradual change in soil microbial communities along both pollution gradients, and most of the individual PLFAs changed similarly to metal pollution at both sites. The relative quantities of the PLFAs br18:0, br17:0, i16:0, and i16:1 increased with increasing heavy metal concentration, while those of 20:4 and 18:2(omega)6, which is a predominant PLFA in many fungi, decreased. The fungal part of the microbial biomass was found to be more sensitive to heavy metals. This resulted in a decreased fungal/bacterial biomass ratio along the pollution gradient towards the smelters. The thymidine incorporation technique was used to study the heavy metal tolerance of the bacteria. The bacterial community at the Harjavalta smelter, exposed mainly to Cu deposition, exhibited an increased tolerance to Cu but not to Cd, Ni, and Zn. At the Ronnskar smelter the deposition consisting of a mixture of metals increased the bacterial community tolerance to all tested metals. Both the PLFA pattern and the bacterial community tolerance were affected at lower soil metal concentrations than were bacterial counts and bacterial activities. At Harjavalta the increased Cu tolerance of the bacteria and the change in the PLFA pattern of the microbial community were found at the same soil Cu concentrations. This indicated that the altered PLFA pattern was at least partly due to an altered, more metal-tolerant bacterial community. At Ronnskar, where the PLFA data varied more, a correlation between bacterial community tolerance and an altered PLFA pattern was found up to 10 to 15 km from the smelter. Farther away changes in the PLFA pattern could not be explained by an increased community tolerance to metals.

Quantification of frankia strains and other root-associated bacteria in pure cultures and in the rhizosphere of axenic seedlings by high-performance liquid chromatography-based muramic Acid assay.

Application of a high-performance liquid chromatography-based muramic acid assay with precolumn fluorescence derivatization to quantification of root-associated bacteria was studied both in pure cultures and in the rhizosphere of axenic Festuca rubra seedlings. Quantities of muramic acid from acid-hydrolyzed cells of Frankia strains, Streptomyces griseoviridis, Enterobacter agglomerans, Klebsiella pneumoniae, Pseudomonas sp., and Bacillus polymyxa were mostly proportional to the respective cell protein and carbon quantities, but in some strains, culture age and particularly sporulation affected these ratios considerably. The muramic acid/cell protein ratio was generally 2 to 4 times higher in strains of the two actinomycete genera, Frankia and Streptomyces, than in the rest of the strains. Quantification of Frankia strains, S. griseoviridis, E. agglomerans, and Pseudomonas sp. was also attempted from the rhizosphere of F. rubra seedlings which had been inoculated with pure cultured bacteria and incubated briefly. It was possible to quantify Frankia cells by use of the muramic acid assay from both the root and the growth medium, whereas cells of the rest of the bacterial genera could only be detected in the medium. The detection limit for muramic acid was about 10 ng/ml hydrolysis volume, and from the Festuca rhizosphere, 28 to 63% of the muramic acid in the Frankia inoculum was recovered.