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.

Methane-oxidizing bacteria in a Finnish raised mire complex: effects of site fertility and drainage.

Methane-oxidizing bacteria (MOB) are the only biological sinks for methane (CH4). Drainage of peatlands is known to decrease overall CH4 emission, but the effect on MOB is unknown. The objective of this work was to characterize the MOB community and activity in two ecohydrologically different pristine peatland ecosystems, a fen and a bog, and their counterparts that were drained in 1961. Oligotrophic fens are groundwater-fed peatlands, but ombrotrophic bogs receive additional water and nutrients only from rainwater. The sites were sampled in August 2003 down to 10 cm below the water table (WT), and cores were divided into 10-cm subsamples. CH4 oxidation was measured by gas chromatography (GC) to characterize MOB activity. The MOB community structure was characterized by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) and sequencing methods using partial pmoA and mmoX genes. The highest CH4 oxidation rates were measured from the subsamples 20-30 and 30-40 cm above WT at the pristine oligotrophic fen (12.7 and 10.5 micromol CH4 dm-3 h-1, respectively), but the rates decreased to almost zero in the vicinity of WT. In the pristine ombrotrophic bog, the highest oxidation rate at 0-10 cm was lower than in the fen (8.10 micromol CH4 dm-3 h-1), but in contrast to the fen, oxidation rates of 4.5 micromol CH4 dm-3 h-1 were observed at WT and 10 cm below WT. Drainage reduced the CH4 oxidation rates to maximum values of 1.67 and 5.77 micromol CH4 dm-3 h-1 at 30-40 and 20-30 cm of the fen and bog site, respectively. From the total of 13 pmoA-derived DGGE bands found in the study, 11, 3, 6, and 2 were observed in the pristine fen and bog and their drained counterparts, respectively. According to the nonmetric multidimensional scaling of the DGGE banding pattern, the MOB community of the pristine fen differed from the other sites. The majority of partial pmoA sequences belonged to type I MOB, whereas the partial mmoX bands that were observed only in the bog sites formed a distinct group relating more to type II MOB. This study indicates that fen and bog ecosystems differ in MOB activity and community structure, and both these factors are affected by drainage.

Methanogen communities in a drained bog: effect of ash fertilization.

Forestry practises such has drainage have been shown to decrease emissions of the greenhouse gas methane (CH(4)) from peatlands. The aim of the study was to examine the methanogen populations in a drained bog in northern Finland, and to assess the possible effect of ash fertilization on potential methane production and methanogen communities. Peat samples were collected from control and ash fertilized (15,000 kg/ha) plots 5 years after ash application, and potential CH(4) production was measured. The methanogen community structure was studied by DNA isolation, PCR amplification of the methyl coenzyme-M reductase (mcr) gene, denaturing gradient gel electrophoresis (DGGE), and restriction fragment length polymorphism (RFLP) analysis. The drained peatland showed low potential methane production and methanogen diversity in both control and ash-fertilized plots. Samples from both upper and deeper layers of peat were dominated by three groups of sequences related to Rice cluster-I hydrogenotroph methanogens. Even though pH was marginally greater in the ash-treated site, the occurrence of those sequences was not affected by ash fertilization. Interestingly, a less common group of sequences, related to the Fen cluster, were found only in the fertilized plots. The study confirmed the depth related change of methanogen populations in peatland.

Pathways for methanogenesis and diversity of methanogenic archaea in three boreal peatland ecosystems.

The main objectives of this study were to uncover the pathways used for methanogenesis in three different boreal peatland ecosystems and to describe the methanogenic populations involved. The mesotrophic fen had the lowest proportion of CH4 produced from H2-CO2. The oligotrophic fen was the most hydrogenotrophic, followed by the ombrotrophic bog. Each site was characterized by a specific group of methanogenic sequences belonging to Methanosaeta spp. (mesotrophic fen), rice cluster-I (oligotrophic fen), and fen cluster (ombrotrophic bog).

Relationship between basal soil respiration rate, tree stand and soil characteristics in boreal forests.

Soil respiration is considered to represent the overall microbial activity reflecting mineralisation of organic matter in soil. It is the most commonly used biological variable in soil studies. In long-term monitoring of forested areas, there is a need for reference values for soil microbiological variables in different forest ecosystems. In this study we describe the relationship between soil respiration rate, tree stand and humus chemical characteristics of boreal coniferous forests stands. Soil respiration rate was higher in pine dominated than in spruce dominated study sites when the result was calculated on dry matter bases. However, when calculated on area bases, the result was opposite and no difference was found when the soil respiration rate was calculated on organic carbon bases. Irrespective of the main tree species, the soil respiration rate was equal in different development classes but not equal in soil fertility classes, i.e. within forest site types based on differences in ground vegetation. Respiration rates were clearly higher in mesic sites when calculated on dry matter, C(org) or area bases. However, soil respiration rate did not correlate with soil chemical variables indicating site fertility. Soil respiration rate on dry matter basis was at a lower level in the south and on more fertile sites, and on the other hand at a higher level in older stands and on sites with a thicker organic layer.

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.

Effect of Cd-containing wood ash on the microflora of coniferous forest humus.

The use of wood ash in forestry has been questioned because the cadmium (Cd) concentration of ash, which varies between 1 and 20 mg kg(-1) ash, exceeds the level allowed for fertilizers (3 mg kg(-1)) used in agriculture. To investigate the combined and separated effects of Cd and ash on the forest humus microflora, pumice or wood ash, spiked with a water-soluble (CdCl(2)) or -insoluble (CdO) form of Cd at three levels (0, 400 and 1000 mg kg(-1)), were applied at a fertilization level of 5000 kg ha(-1) in a laboratory microcosm study. The trial consisted of 60 microcosms (five replications per treatment), which were incubated in darkness at +20 degrees C and a constant relative air humidity of 60%. After two months the humus in the microcosms was sampled. Analyses of CO(2) evolution to measure the overall microbial activity and of phospholipid fatty acid (PLFA) pattern to measure microbial community structure were performed. The substrate-use patterns of Biolog EcoPlates were analyzed as a measure of bacterial functionality. Finally the bacterial (3)H-thymidine incorporation in the presence of different concentrations of Cd and the number of colony forming units (cfu) of bacteria on nutrient agar in the presence of 0, 5 and 20 mg Cd l(-1) agar were applied to measure Cd tolerance. The use of pumice (pH of humus under the pumice 4.0) did not induce any changes in the above variables compared to two untreated microcosms (humus pH 3.9). Pumice was therefore used to distribute the Cd evenly over the humus surface in order to estimate the possible effect of Cd without ash (pH of humus under the ash 7.0). The application of ash increased the microbial activity, changed the PLFA and substrate-use patterns and increased cfu compared to the humus under pumice. The form and level of Cd in the ash had no further effect on this result. In the humus under pumice the level, but not the form of Cd decreased the microbial activity and changed the PLFA pattern compared to the unspiked pumice. None of the treatments induced bacterial tolerance to Cd. Ash thus protected the humus microflora from the harmful effects of Cd.

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.

Vitality fertilization of Scots pine stands growing along a gradient of heavy metal pollution: short-term effects on microbial biomass and respiration rate of the humus layer.

In 1992 forest vitality fertilization experiments were established on a heavy metal deposition gradient with four treatments in three replications at distances of 0.5, 4 and 8 km from a Cu-Ni smelter in order to estimate their impact on the disturbed forest ecosystem. The increase in Cu concentration in the humus (F/H) layer of the Calluna site type Scots pine (Pinus sylvestris) stands from ca. 300 to 8000 mg kg(-1) d.m. (dry matter) along the 8 km long transect towards the smelter resulted in declining soil microbial biomass and soil respiration activity. Three independent measurements of microbial biomass: C(mic)-FE (fumigation extraction), C(mic)-SIR (substrate induced respiration), and ATP have been used together with an indicator of fungal biomass (ergosterol) and microbial activity (soil respiration). Within this Cu pollution range, all the measured microbial biomass levels declined to 10%-28% of the control plot values and activity assessed by respiration was lowered to 16%. Liming has increased the C(mic)-SIR and respiration rate. Treatments with test fertilizer, made from grounded apatite, did not result in different microbial biomass and respiration rate values compared to the respective controls along the whole gradient. Nitrogen + lime treatments resulted in similar changes to lime alone. No changes, as compared to the respective control, could be detected with nitrogen fertilization at the less polluted end of the gradient.

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.

Microfungal species composition and fungal biomass in a coniferous forest soil polluted by alkaline deposition.

Isolations of soil microfungi from the humus (F/H-layer) of a coniferous forest soil which was either unpolluted (pH 4.1) or polluted (pH 6.6) for 25 years by deposition of alkaline dust, were made by soil washing and spore plating. Both techniques revealed similar changes in species composition. Alkaline dust exposure caused a reduction in overall species numbers, but led to higher relative isolation frequencies of Mortierella alpina, Oidiodendron tenuissimum, Penicillium montanese, Sagenomella verticillata, and Trichosporiella sporotrichioides. The incidence of M. isabellina, O. cf. clamydosporium, P. spinulosum, Penicillium sp. 1, P. sclerotiorum, Trichoderma viride, and Verticillium bulbillosum was reduced on polluted sites. The amount of the mainly fungal-derived phospholipid fatty acid 18 : 2ω6 decreased by 23%, while the amount of ergosterol increased by 9% in the polluted soil.

Soil Bacterial Biomass, Activity, Phospholipid Fatty Acid Pattern, and pH Tolerance in an Area Polluted with Alkaline Dust Deposition.

Soil bacterial biomass, phospholipid fatty acid pattern, pH tolerance, and growth rate were studied in a forest area in Finland that is polluted with alkaline dust from an iron and steel works. The pollution raised the pH of the humus layer from 4.1 to 6.6. Total bacterial numbers and the total amounts of bacterial phospholipid fatty acids in the humus layer did not differ between the unpolluted control sites and the polluted ones. The number of CFU increased by a factor of 6.4 in the polluted sites compared with the controls, while the bacterial growth rate, measured by the thymidine incorporation technique, increased about 1.8-fold in the polluted sites. A shift in the pattern of phospholipid fatty acids indicated a shift in the bacterial species composition. The largest proportional increase was found for the fatty acid 10Me18:0, which indicated an increase in the number of actinomycetes in the polluted sites. The levels of the fatty acids i14:0, 16:1omega5, cy17:0, 18:1omega7, and 19:1 also increased in the polluted sites while those of fatty acids 15:0, i15:0, 10Me16:0, 16:1omega7t, 18:1omega9, and cy19:0 decreased compared with the unpolluted sites. An altered pH tolerance of the bacterial assemblage was detected either as a decrease in acid-tolerant CFU in the polluted sites or as altered bacterial growth rates at different pHs. The latter was estimated by measuring the thymidine incorporation rate of bacteria extracted from soil by homogenization-centrifugation at different pHs.

Complement activation by cold agglutinins.

Purified monoclonal human IgM cold agglutinins (CA) of different specificities (anti-I, anti-i, anti-Pr) were investigated for their complement-activating capacity in a homologous system. Incubation of human RBC with excess of IgM CA in the cold, and subsequently with human serum at 37 degrees C, resulted in striking differences in hemolysis. Hemolysis did not correlate to the amount of antibodies bound to RBC at 4 or 20 degrees C. Despite the hemolytic inefficiency of anti-i and anti-Pr CA tested, C1 fixation and subsequent activation of the classical pathway of complement could be assessed in all cases. Absolute numbers of C3 molecules bound to RBC, exceeding the critical level to initiate the terminal sequence of the complement cascade, could not fully explain the differences in the hemolytic activity of the CA. Since C8 binding protein (C8bp) carries I determinants it is hypothesized that anti-I-induced complement-mediated hemolysis might also be favored by the binding of the autoantibody to and probably steric hindrance of this major regulatory protein of the terminal complement sequence. The prominent role of homologous restriction of complement-mediated lysis as a protective mechanism can also be deduced from the fact that rabbit as well as rat serum as a source of heterologous complement lysed cold agglutinin-sensitized red blood cells more efficiently than human serum.

Characterization of deletions at the retinoblastoma locus in patients with bilateral retinoblastoma.

DNA samples from 92 unrelated patients with bilateral retinoblastoma were analyzed by Southern blot hybridization with cDNA and genomic clones of the retinoblastoma (RB-1) gene. Qualitative and quantitative evaluation of the Southern blot patterns showed a deletion of all or part of the RB-1 gene in 15 patients. Deletion hot spots were not detected. The study shows that 16% of germ cell mutations are detectable by Southern blot hybridization, but that densitometric analysis is required in most cases.

Long-term treatment effects in patients with bilateral retinoblastoma: ocular and mid-facial findings.

A total of 99 patients with bilateral retinoblastoma who had been treated between 1965 and 1982 were reexamined in April 1988 to study the late effects of treatment. Their median age at the follow-up visit in 1988 was 16 years (range, 6-27 years), and the median follow-up was 15 years (range, 6-26 years). All patients underwent a full eye examination, morphometric measurements of the mid-face and genetic counselling. Each eye or orbit and the corresponding side of the patient's mid-face were evaluated separately, resulting in 198 data sets from 99 individuals. Subjects were divided into four treatment groups according to whether photo- and cryo-coagulation, enucleation, radiation therapy or various combinations thereof were used. In all, 81 eyes had a visual acuity of greater than 0.4 (in 23 of these, however, only with low-vision aids). Within a dose range of 36-51 Gy, the location of the tumor (36%) or cataract (15%) were the main factors responsible for poor visual acuity, whereas radiation retinopathy and/or optic neuropathy occurred in only three cases. Cataracts were more frequently observed following orthovoltage as compared with megavoltage therapy (P = 0.012). A total of 72 eyes had been enucleated and had not received radiation therapy at any time. Cosmetic results (as measured by several parameters) in these cases were significantly better then those in 28 subjects who underwent combined radiation therapy and enucleation. As defined by various subjective as well as objective findings, mid-facial hypoplasia occurred significantly more often following orthovoltage as compared with megavoltage therapy.

[The question of midface growth inhibition following retinoblastoma treatment in early childhood]. / Zur Frage der Wachstumshemmung im Mittelgesicht nach frühkindlicher Retinoblastomtherapie.

99 patients treated for bilateral retinoblastomas in the University Hospital of Essen between 1965 and 1983 were followed up for midface growth inhibition for an average period of 15 1/2 years. Depending on the type of treatment employed the patients were divided into four groups: Local tumor treatment using cryotherapy or laser techniques, ophthalmectomy alone, radiotherapy alone, and combined ophthalmectomy and radiotherapy. Statistically significant differences between the treatment groups were found regarding such symptoms as hypoplasia of the zygomatic bone, hypoplasia of the transversal orbit, hypoplasia of the temporal fossa, hypoplasia of the root of the nose as well as hypotelorism and enophthalmos. Combined surgery and radiotherapy caused the most severe growth disorders, followed by radiotherapy alone and ophthalmectomy alone. With local treatment detectable growth disorders were observed, if at all, only as a result of contralateral radiation scatter. Orthovoltage radiotherapy was found to produce growth disorders of significantly higher severity than megavoltage radiotherapy.

The effects of acidic irrigation on soil microorganisms at Kevo, Northern Finland.

Effects of simulated acid rain (a mixture of H(2)SO(4) and HNO(3); pH(3)) on soil microbiology were studied in a field experiment in northern Finland. Irrigated control plots received the same amount of spring water (pH 6) as the acid treated plots. Fungal lengths and total bacterial numbers were studied after the treatments had continued for three growing seasons. The numbers of bacteria in five physiological groups (those utilizing starch, protein, pectin, xylan, or cellulose) were measured by MPN (most probable number) techniques. The lengths of total and FDA active fungal hyphae were not significantly different between the acid treated and the control plots. The counts of total bacteria were not significantly different between treatments, but the MPNs of all five physiological groups of bacteria were approximately 60% lower in the acid treated plots than in the controls.