Abstract
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 (Cmic) and N (Nmic), 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, Cmic and Nmic 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 18:2ω6,9 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 Cmic and Nmic, 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.
1 Introduction
In field studies, birch species have often been found to raise soil pH, enhance the cycling of nutrients and stimulate microbial activities, whereas spruce species may do the opposite [1–4]. These effects are probably partly due to birch leaf litter containing more easily decomposable compounds than spruce needles [5,6]. However, the beneficial effect of birch may also arise from its root activities [7,8].
Roots of different tree species affect the surrounding soil profoundly. Usually, the additional C release by roots results in an increased microbial biomass and numbers in the rhizosphere [9], but plants can also compete with microbes for mineral nutrients, especially N [10–12]. Different microbial groups may be affected differently by roots: denitrification is often enhanced in the rhizospheres of annual plants, but nitrification may become less important [13,14]. The microbial community structure has also been shown to change in the rhizospheres of different tree species [15]. In a pot study where Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies (L.) Karst.) and silver birch (Betula pendula Roth) were grown from seeds for one growing season, the soil microbial biomass C and N and the C mineralization rate were increased by roots of pine and birch, but not by spruce roots [7]. The stimulatory effect of pine and birch seemed to be mostly due to their higher number of short roots and mycorrhizas compared to spruce and the higher amount of labile C which their roots release to soil, as suggested also by Bradley and Fyles [8] regarding paper birch (Betula papyrifera). Whether there are only these quantitative differences in roots and root exudates of pine, spruce and birch or whether there are also qualitative differences in their rhizodeposition is not known.
The aim of this study was to determine whether the microbial community structure and activity are different in the rhizospheres of pine, spruce and birch when the amount of roots does not influence the results. We also wanted to assess whether these possible effects vary in an organic and mineral soil.
2 Materials and methods
2.1 Experimental design
Seedlings of Scots pine, Norway spruce and silver birch were planted into pots of two soil types: an organic soil and a mineral soil. Pots without seedlings (unplanted) were also included. After one growing season, the following parameters were measured from the rhizospheres: the microbial biomass C and N (Cmic and Nmic), C mineralization, net ammonification, net nitrification, denitrification potential, denitrification enzyme activity, phospholipid fatty acid (PLFA) patterns, community level physiological profiles (CLPP), using Biolog plates, and plate counts of culturable microbes.
The soils were taken from a 60-years old Scots pine forest in southern Finland, representing a Vaccinium vitis-idaea type [16]. This field experiment has been described in detail by Priha and Smolander [4]. The soil type at the site was podsoil, the soil texture was sandy till and the humus type mor. Soil from the humus layer (organic soil) and separately from the 0–20-cm mineral soil below was collected from four areas of the plot. The soils were sieved (mesh size 6 mm for organic soil and 4 mm for mineral soil) and mixed thoroughly. The organic matter content of the organic soil was 64.8% of dry matter (d.m.) and the pH(CaCl2) 3.4 and those of the mineral soil 5.1% and 4.1, respectively.
Seedlings of southern Finnish provenance were obtained from the Suonenjoki Research Station of the Finnish Forest Research Institute. We aimed at having seedlings of approximately the same size, instead of the same age, and had 2-years old pine and spruce seedlings and 1-year old birch seedlings. The seedlings of pine, spruce and birch had, on average, a dry mass of 4.0, 3.2 and 3.6 g. The seedlings were growing in peat pots, but the roots were washed free of peat before planting. All of the seedlings were confirmed to be mycorrhizal by microscopic analysis of the roots [17].
The pot experiment was started at the beginning of June 1996. 100 ml of gravel, washed with water, was spread to the bottom of 1.5-l pots. Either 0.5 kg fresh weight (f.w.) of the organic soil or 1 kg f.w. of the mineral soil was put to the pots. The seedlings were planted and another 100 ml of washed gravel was added to the surface of the soil to prevent formation of moss and drying of the surface soil. Altogether, there were eight treatments: pine, spruce and birch growing in organic soil and in mineral soil and both soils without seedlings. There were 20 pots per treatment.
The temperature in the greenhouse was 22°C during the day (16 h) and 18°C during the night (8 h). Na-lamps (Elektro-valo) were used during the day when the light intensity decreased below 200 W m−2. Seedlings were watered to saturation approximately every other day and their positions were systematically changed every 2 weeks.
After 3 months, at the beginning of September 1996, five pots of each treatment were harvested for analyses. Before harvesting, the soils were watered into the same moisture content. In the laboratory, seedlings were removed from the pots and the roots were shaken to remove the adhering soil. The soil which remained on the roots was considered as rhizosphere soil and the rest of the soil was discarded. An attempt was made to leave a similar layer of soil on the roots of all tree species. This attempt was particularly successful in the organic soils, where the soil:root ratios, on a dry weight basis, were similar with different tree species. In the mineral soils, there were small differences in the soil:root ratios, which were highest with pine seedlings and lowest with birch seedlings. An additional three pots from each treatment were harvested for determining the PLFAs, CLPPs and plate counts and rhizosphere soil was separated as above. All of the analyses were done with fresh soil, kept at 4°C, 1–2 weeks from the harvest, except the PLFA analysis, for which soils were stored at −18°C.
2.2 Analyses of the seedlings
The shoots were dried at 40°C for 48 h and weighed. The needles/leaves were separated and ground and their total N concentration was measured with an automated CHN analyzer (Leco CHN-600). Roots were frozen in water and they were subsequently dried at 60°C for 48 h and weighed.
2.3 Chemical analyses of the soils
The d.m. content of the soil was determined by drying the samples at 105°C for 24 h. The soil organic matter content was measured as the loss on ignition from the dried samples at 550°C for 4 h. All soil results are shown per g organic matter. The water-holding capacity (WHC) was measured by soaking the soil samples in water for 2 h and then draining for 2 h. Soil pH was measured in soil:0.01 M CaCl2 suspensions (3:5 v/v). Total organic C and total N were measured from dried (40°C) samples using an automated CHN analyzer (Leco CHN-600).
2.4 Analyses of microbial biomass and activities of C and N cycles
The measurements of Cmic and Nmic with fumigation-extraction and substrate-induced respiration (SIR) methods have been described earlier [18]. These results are shown without the use of conversion factors. The rate of C mineralization was evaluated as CO2-C production at 14°C (basal respiration, [18]). The net ammonification and nitrification were measured by incubating the soil samples at 14°C for 40 days [13]. for all of these measurements, duplicate 1.5-g d.m. humus and 6-g d.m. mineral soil samples with the soil moisture content adjusted to 60% of the WHC were used.
The numbers of autotrophic nitrifiers (ammonium and nitrite oxidizers) were estimated by the most probable number (MPN) method, as described in [4]. The tubes were incubated in the dark at room temperature (22°C) for 14 weeks.
Denitrification activity was measured as N2O production, as described in [4]. Briefly, duplicate 1.5-g d.m. humus samples or 6-g d.m. mineral soil samples with the soil moisture content adjusted to 100% of the WHC were incubated at 14°C under 10 kPa partial pressure of acetylene. Results given are production rates of N2O-N between 1 and 2 days. Denitrification enzyme activity (DEA) aims at determining the activity of pre-existing denitrifying enzymes in soil, without allowing denitrifying organisms to proliferate [19]. DEA was measured with the same amounts of soil as above, but adding solutions of KNO3 and glucose and a N2 atmosphere [4]. The samples were incubated for approximately 5 h in the dark at 22°C and N2O was measured.
2.5 PLFA analysis
The phospholipid extraction and analysis of PLFAs was conducted as described by Frostegård et al. [20]. Briefly, 0.5-g f.w. organic soil and 2.5-g f.w. mineral soil samples were extracted with a chloroform:methanol:citrate buffer mixture (1:2:0.8) and the lipids were separated into neutral lipids, glycolipids and phospholipids in a silicic acid column. The phospholipids were subjected to mild alkaline methanolysis and the fatty acid methyl esters were separated by gas chromatography (Hewlett Packard 5890), equipped with a flame ionization detector and a HP-5 (phenylmethyl silicone) capillary column, 50 m in length, using He as a carrier gas. Peak areas were quantified by adding methyl nonadecanoate fatty acid (19:0) as an internal standard.
Fatty acids are designated in terms of the total number of carbon atoms:number of double bonds, followed by the position of the double bond from the methyl end of the molecule, indicated by ω and a number. The prefixes a, i and br indicate anteiso, iso and unknown branching, respectively. The prefix cy indicates a cyclopropane fatty acid and methyl branching (Me) is indicated as the position of the methyl group from the carboxyl end of the chain. The prefix C (C15:1) indicates that the PLFA has 15 carbon atoms and one double bond, but the arrangement of the carbon atoms (e.g. branching position) is not confirmed. The abbreviations t and c indicate trans and cis configuration of the double bonds.
The sum of PLFAs considered to be mainly of bacterial origin (i15:0, a15:0, 15:0, i16:0, 16:1ω9, 16:1ω7t, i17:0, a17:0, 17:0, cy17:0, 18:1ω7 and cy19:0) was chosen to represent the bacterial biomass (bacterial PLFAs) [21]. The quantity of 18:2ω6,9 was used as an indicator of the fungal biomass (fungal PLFA). The ratio fungal PLFA to bacterial PLFAs was also calculated.
2.6 CLPPs
CLPPs were conducted using Biolog plates, according to Campbell et al. [22]. Briefly, to extract the microbes, soil samples (10 g f.w.) were shaken in 100 ml 1/4 strength Ringers solution (Oxoid) for 10 min on an orbital shaker. A 10−4 dilution of each soil sample was centrifuged at 750×g for 10 min to remove soil and root particles which might introduce additional C into the wells. A 150-μl aliquot of the supernatant from the centrifuged samples was added to each well of a Biolog GN plate (Biolog, Hayward, CA, USA) and a MT plate with 30 additional carbon sources representing compounds reported in the literature to be plant root exudates [22]. Plates were incubated at 15°C and color development was measured as the absorbance at 590 nm (A590) using a microplate reader (Emax, Molecular Devices, Oxford, UK). The absorbance was measured at 0 h, then every 24 h for 5 days and then at 10 and 15 days.
We compared all 125 C sources with the 61 identified as being plant root exudates (30 from MT plate plus 31 from the GN plate, see [22]). When calculating the results, first, the 0-h reading was subtracted from each of the wells, as some compounds were initially colored. The blanks from both GN and MT plates were then subtracted and the average well color development (AWCD, [23]) was calculated for each time point. In order to eliminate variation in AWCD, which may arise from different cell densities in different samples, Garland [24] recommended comparison of samples of equivalent AWCD. We used on each sample the time point at which the AWCD was closest to the value of 0.75. This time point was either 10 or 15 days for all samples. The values from different time points were then all divided by their respective AWCDs.
2.7 Plate counts
The soil samples (10 g f.w.) were shaken in 100 ml 1/4 strength Ringers solution (Oxoid) for 10 min on an orbital shaker. The samples were then serially diluted to 10−7 in 1/4 strength Ringers solution and suspensions (0.1 ml) were spread, in duplicate, onto the following media: Tryptone Soy agar (1/10 Oxoid strength) plus cycloheximide (50 mg l−1) for enumeration of bacteria and actinomycetes, Pseudomonas Isolation agar (Oxoid) selective for populations of pseudomonads, Czapek Dox agar (Oxoid)+streptomycin sulfate (50 mg l−1)+tetracycline hydrochloride (50 mg l−1)+ampicillin (10 mg l−1) for enumeration of yeasts and fungi. The plates were incubated at 25°C and colonies were counted after 5–6 days and again after 13–14 days.
2.8 Statistical analyses
Means of the measured characteristics between treatments were compared with analysis of variance [25]. Organic soil and mineral soil were tested separately. Results were log- or log(x+1)-transformed when necessary for fulfilling the assumptions of variance analysis. Significant differences of the means were separated by Tukey's test (honestly significant difference) [25].
The mol percentages of PLFA values and the Biolog values divided by AWCD were subjected to principal component analysis (PCA) using a correlation matrix [26]. PCA was done separately for organic and mineral soil samples. The Systat 6.0.1 (SPSS, 1996) statistical software was used.
3 Results
3.1 Seedlings
Some characteristics of the seedlings at the time of harvest are shown in Table 1. The size of the shoots did not differ statistically significantly when growing in different soils, but seedlings of all tree species had more roots when growing in the mineral soil. The needles/leaves of all tree species had approximately two times higher N concentrations when growing in the organic soil as compared to mineral soil.
Some characteristics of the seedlings and soils
| Soil | Tree species | Shoot, cm | Shoot, g d.m. | Roots, g d.m. | N in needles/leaves, mg g−1 d.m. | N in needles/leaves of the whole plant, mg | Soil pH (CaCl2) | Soil organic matter, % of d.m. | Total soil N, mg g−1 o.m. | |
| Organic soil | Pine | 34 (1)a | 6.0 (0.4)a | 1.6 (0.1)a | 20 (1)a | 61 (5)a | 3.3 (0.0)a | 69 (3)a | 1.9 (0.2)a | |
| Spruce | 34 (1)a | 4.1 (0.4)a | 2.1 (0.3)a | 23 (1)b | 35 (3)b | 3.4 (0.0)a | 68 (3)ab | 1.9 (0.1)a | ||
| Birch | 116 (6)b | 13.1 (0.8)b | 6.3 (0.5)b | 21 (1)ab | 52 (5)a | 3.2 (0.0)b | 59 (4)ab | 2.0 (0.1)a | ||
| No seedling | 3.6 (0.0)c | 55 (3)b | 1.9 (0.1)a | |||||||
| Mineral soil | Pine | 32 (1)a | 5.2 (0.8)a | 2.7 (0.5)a | 10 (0)a | 23 (4)a | 4.5 (0.0)a | 4.5 (0.0)a | 1.8 (0.1)a | |
| Spruce | 34 (1)a | 3.9 (0.5)a | 3.6 (0.6)a | 12 (1)a | 20 (1)a | 4.2 (0.0)b | 4.8 (0.1)bc | 1.9 (0.1)a | ||
| Birch | 90 (3)b | 10.5 (0.9)b | 10.0 (1.6)b | 11 (0)a | 20 (2)a | 4.4 (0.0)c | 5.1 (0.1)c | 1.9 (0.1)a | ||
| No seedling | 4.5 (0.0)a | 4.7 (0.1)ab | 1.8 (0.1)a |
| Soil | Tree species | Shoot, cm | Shoot, g d.m. | Roots, g d.m. | N in needles/leaves, mg g−1 d.m. | N in needles/leaves of the whole plant, mg | Soil pH (CaCl2) | Soil organic matter, % of d.m. | Total soil N, mg g−1 o.m. | |
| Organic soil | Pine | 34 (1)a | 6.0 (0.4)a | 1.6 (0.1)a | 20 (1)a | 61 (5)a | 3.3 (0.0)a | 69 (3)a | 1.9 (0.2)a | |
| Spruce | 34 (1)a | 4.1 (0.4)a | 2.1 (0.3)a | 23 (1)b | 35 (3)b | 3.4 (0.0)a | 68 (3)ab | 1.9 (0.1)a | ||
| Birch | 116 (6)b | 13.1 (0.8)b | 6.3 (0.5)b | 21 (1)ab | 52 (5)a | 3.2 (0.0)b | 59 (4)ab | 2.0 (0.1)a | ||
| No seedling | 3.6 (0.0)c | 55 (3)b | 1.9 (0.1)a | |||||||
| Mineral soil | Pine | 32 (1)a | 5.2 (0.8)a | 2.7 (0.5)a | 10 (0)a | 23 (4)a | 4.5 (0.0)a | 4.5 (0.0)a | 1.8 (0.1)a | |
| Spruce | 34 (1)a | 3.9 (0.5)a | 3.6 (0.6)a | 12 (1)a | 20 (1)a | 4.2 (0.0)b | 4.8 (0.1)bc | 1.9 (0.1)a | ||
| Birch | 90 (3)b | 10.5 (0.9)b | 10.0 (1.6)b | 11 (0)a | 20 (2)a | 4.4 (0.0)c | 5.1 (0.1)c | 1.9 (0.1)a | ||
| No seedling | 4.5 (0.0)a | 4.7 (0.1)ab | 1.8 (0.1)a |
Some characteristics of the seedlings and soils
| Soil | Tree species | Shoot, cm | Shoot, g d.m. | Roots, g d.m. | N in needles/leaves, mg g−1 d.m. | N in needles/leaves of the whole plant, mg | Soil pH (CaCl2) | Soil organic matter, % of d.m. | Total soil N, mg g−1 o.m. | |
| Organic soil | Pine | 34 (1)a | 6.0 (0.4)a | 1.6 (0.1)a | 20 (1)a | 61 (5)a | 3.3 (0.0)a | 69 (3)a | 1.9 (0.2)a | |
| Spruce | 34 (1)a | 4.1 (0.4)a | 2.1 (0.3)a | 23 (1)b | 35 (3)b | 3.4 (0.0)a | 68 (3)ab | 1.9 (0.1)a | ||
| Birch | 116 (6)b | 13.1 (0.8)b | 6.3 (0.5)b | 21 (1)ab | 52 (5)a | 3.2 (0.0)b | 59 (4)ab | 2.0 (0.1)a | ||
| No seedling | 3.6 (0.0)c | 55 (3)b | 1.9 (0.1)a | |||||||
| Mineral soil | Pine | 32 (1)a | 5.2 (0.8)a | 2.7 (0.5)a | 10 (0)a | 23 (4)a | 4.5 (0.0)a | 4.5 (0.0)a | 1.8 (0.1)a | |
| Spruce | 34 (1)a | 3.9 (0.5)a | 3.6 (0.6)a | 12 (1)a | 20 (1)a | 4.2 (0.0)b | 4.8 (0.1)bc | 1.9 (0.1)a | ||
| Birch | 90 (3)b | 10.5 (0.9)b | 10.0 (1.6)b | 11 (0)a | 20 (2)a | 4.4 (0.0)c | 5.1 (0.1)c | 1.9 (0.1)a | ||
| No seedling | 4.5 (0.0)a | 4.7 (0.1)ab | 1.8 (0.1)a |
| Soil | Tree species | Shoot, cm | Shoot, g d.m. | Roots, g d.m. | N in needles/leaves, mg g−1 d.m. | N in needles/leaves of the whole plant, mg | Soil pH (CaCl2) | Soil organic matter, % of d.m. | Total soil N, mg g−1 o.m. | |
| Organic soil | Pine | 34 (1)a | 6.0 (0.4)a | 1.6 (0.1)a | 20 (1)a | 61 (5)a | 3.3 (0.0)a | 69 (3)a | 1.9 (0.2)a | |
| Spruce | 34 (1)a | 4.1 (0.4)a | 2.1 (0.3)a | 23 (1)b | 35 (3)b | 3.4 (0.0)a | 68 (3)ab | 1.9 (0.1)a | ||
| Birch | 116 (6)b | 13.1 (0.8)b | 6.3 (0.5)b | 21 (1)ab | 52 (5)a | 3.2 (0.0)b | 59 (4)ab | 2.0 (0.1)a | ||
| No seedling | 3.6 (0.0)c | 55 (3)b | 1.9 (0.1)a | |||||||
| Mineral soil | Pine | 32 (1)a | 5.2 (0.8)a | 2.7 (0.5)a | 10 (0)a | 23 (4)a | 4.5 (0.0)a | 4.5 (0.0)a | 1.8 (0.1)a | |
| Spruce | 34 (1)a | 3.9 (0.5)a | 3.6 (0.6)a | 12 (1)a | 20 (1)a | 4.2 (0.0)b | 4.8 (0.1)bc | 1.9 (0.1)a | ||
| Birch | 90 (3)b | 10.5 (0.9)b | 10.0 (1.6)b | 11 (0)a | 20 (2)a | 4.4 (0.0)c | 5.1 (0.1)c | 1.9 (0.1)a | ||
| No seedling | 4.5 (0.0)a | 4.7 (0.1)ab | 1.8 (0.1)a |
Values are means of five pots, S.E.M.s in parentheses. Values with the same letter within one soil are not significantly (P≤0.05) different from each other.
d.m.=dry matter.
o.m.=organic matter.
Birch seedlings had a higher shoot and root biomass than pine and spruce in both soils. N concentrations in the needles/leaves did not differ much between different tree species within one soil, but on a basis of the whole seedling, pine and birch contained significantly more N than spruce when growing in the organic soil.
3.2 Physical and chemical characteristics of the soils
There were small, but significant, differences in the soil pH between tree species (Table 1). The soil organic matter content also varied between treatments, but the concentration of total soil N did not differ significantly between treatments.
3.3 Microbial biomass C and N, substrate-induced and basal respiration
In the organic soil, the flush of C from fumigation was higher in birch rhizosphere than in pine and spruce rhizosphere and unplanted soil did not differ statistically significantly from any of the soils (Fig. 1a). The flush of N from fumigation was also highest in birch rhizosphere (Fig. 1b). In the mineral soil, the flushes of C and N did not differ statistically significantly between treatments.
The flush of (a) extractable C and (b) extractable N from fumigation of the soils, (c) substrate-induced respiration and (d) basal respiration of the soils. Values are means of five pots, bars show S.E.M. Values with the same letter within one soil are not significantly (P≤0.05) different from each other.
The flush of (a) extractable C and (b) extractable N from fumigation of the soils, (c) substrate-induced respiration and (d) basal respiration of the soils. Values are means of five pots, bars show S.E.M. Values with the same letter within one soil are not significantly (P≤0.05) different from each other.
In the organic soil, SIR did not differ statistically significantly between different treatments, but in the mineral soil, SIR was lowest in unplanted soil, although the difference was statistically significant only for spruce (Fig. 1c). In the organic soil, the basal respiration was not significantly affected by the tree species, but was higher in unplanted soil than in pine rhizosphere (Fig. 1d). In the mineral soil, the basal respiration was lowest in unplanted soil and did not differ between rhizospheres of different tree species.
3.4 Ammonification, nitrification and numbers of nitrifiers
In the organic soil, the concentration of mineral N and both ammonium and nitrate separately were the highest in unplanted soil and lowest in birch rhizosphere (Table 2). Also in the mineral soil, the concentrations were highest in pots without seedlings. All soils contained negligible or small amounts of nitrate. In the organic soil, the net formation of mineral N did not differ between treatments, but in the mineral soil, it was significantly greater in the spruce rhizosphere. A considerable net nitrification occurred only in the unplanted mineral soil.
Initial concentrations of mineral N, net formation of mineral N in an aerobic incubation and numbers of ammonium and nitrite oxidizers determined by the MPN method
| Soil | Tree species | Initial, μg g−1 o.m. | Net formation, μg g−1 o.m. 40 days−1 | MPN, g−1 o.m. | |||||
| NH4+-N | (NO2−+NO3−)-N | (NH4++NO2−+NO3−)-N | NH4+-N | (NO2−+NO3−)-N | (NH4++NO2−+NO3−)-N | NH4+-oxidizers | NO2−-oxidizers | ||
| Organic soil | Pine | 301 (31)a | 1 (1)a | 302 (31)a | 238 (31)a | 0 (0)a | 238 (31)a | <10a | 7.0×102 (2.2×102)ac |
| Spruce | 508 (45)b | 4 (1)b | 512 (45)b | 235 (26)a | −1 (0)a | 234 (27)a | <10a | 1.8×103 (2.0×102)b | |
| Birch | 76 (23)c | 0 (0)a | 76 (23)c | 235 (16)a | 0 (0)a | 235 (16)a | <10a | 2.4×102 (1.2×102)c | |
| No seedling | 834 (19)d | 10 (2)c | 844 (20)d | 193 (8)a | −1 (1)a | 192 (9)a | <10a | 1.4×103 (3.0×102)ab | |
| Mineral soil | Pine | 64 (19)a | 1 (1)a | 64 (18)a | −5 (18)a | 0 (1)a | −4 (18)a | 520 (460)ab | 3.2×104 (1.2×104)a |
| Spruce | 55 (9)a | 1 (0)a | 55 (8)a | 129 (18)b | 2 (1)a | 131 (18)b | 130 (70)a | 1.3×104 (3.7×103)a | |
| Birch | 58 (17)a | 0 (0)a | 59 (17)a | 11 (11)a | 0 (1)a | 11 (11)a | 140 (60)ab | 3.3×104 (1.0×104)a | |
| No seedling | 237 (50)b | 29 (4)b | 266 (50)b | −143 (12)c | 168 (29)b | 26 (21)a | 2910 (780)b | 2.5×105 (9.1×104)b | |
| Values are means of five pots, S.E.M.s in parentheses. Values with the same letter within one soil are not significantly (P≤0.05) different from each other. | |||||||||
| o.m.=organic matter. | |||||||||
| Soil | Tree species | Initial, μg g−1 o.m. | Net formation, μg g−1 o.m. 40 days−1 | MPN, g−1 o.m. | |||||
| NH4+-N | (NO2−+NO3−)-N | (NH4++NO2−+NO3−)-N | NH4+-N | (NO2−+NO3−)-N | (NH4++NO2−+NO3−)-N | NH4+-oxidizers | NO2−-oxidizers | ||
| Organic soil | Pine | 301 (31)a | 1 (1)a | 302 (31)a | 238 (31)a | 0 (0)a | 238 (31)a | <10a | 7.0×102 (2.2×102)ac |
| Spruce | 508 (45)b | 4 (1)b | 512 (45)b | 235 (26)a | −1 (0)a | 234 (27)a | <10a | 1.8×103 (2.0×102)b | |
| Birch | 76 (23)c | 0 (0)a | 76 (23)c | 235 (16)a | 0 (0)a | 235 (16)a | <10a | 2.4×102 (1.2×102)c | |
| No seedling | 834 (19)d | 10 (2)c | 844 (20)d | 193 (8)a | −1 (1)a | 192 (9)a | <10a | 1.4×103 (3.0×102)ab | |
| Mineral soil | Pine | 64 (19)a | 1 (1)a | 64 (18)a | −5 (18)a | 0 (1)a | −4 (18)a | 520 (460)ab | 3.2×104 (1.2×104)a |
| Spruce | 55 (9)a | 1 (0)a | 55 (8)a | 129 (18)b | 2 (1)a | 131 (18)b | 130 (70)a | 1.3×104 (3.7×103)a | |
| Birch | 58 (17)a | 0 (0)a | 59 (17)a | 11 (11)a | 0 (1)a | 11 (11)a | 140 (60)ab | 3.3×104 (1.0×104)a | |
| No seedling | 237 (50)b | 29 (4)b | 266 (50)b | −143 (12)c | 168 (29)b | 26 (21)a | 2910 (780)b | 2.5×105 (9.1×104)b | |
| Values are means of five pots, S.E.M.s in parentheses. Values with the same letter within one soil are not significantly (P≤0.05) different from each other. | |||||||||
| o.m.=organic matter. | |||||||||
Initial concentrations of mineral N, net formation of mineral N in an aerobic incubation and numbers of ammonium and nitrite oxidizers determined by the MPN method
| Soil | Tree species | Initial, μg g−1 o.m. | Net formation, μg g−1 o.m. 40 days−1 | MPN, g−1 o.m. | |||||
| NH4+-N | (NO2−+NO3−)-N | (NH4++NO2−+NO3−)-N | NH4+-N | (NO2−+NO3−)-N | (NH4++NO2−+NO3−)-N | NH4+-oxidizers | NO2−-oxidizers | ||
| Organic soil | Pine | 301 (31)a | 1 (1)a | 302 (31)a | 238 (31)a | 0 (0)a | 238 (31)a | <10a | 7.0×102 (2.2×102)ac |
| Spruce | 508 (45)b | 4 (1)b | 512 (45)b | 235 (26)a | −1 (0)a | 234 (27)a | <10a | 1.8×103 (2.0×102)b | |
| Birch | 76 (23)c | 0 (0)a | 76 (23)c | 235 (16)a | 0 (0)a | 235 (16)a | <10a | 2.4×102 (1.2×102)c | |
| No seedling | 834 (19)d | 10 (2)c | 844 (20)d | 193 (8)a | −1 (1)a | 192 (9)a | <10a | 1.4×103 (3.0×102)ab | |
| Mineral soil | Pine | 64 (19)a | 1 (1)a | 64 (18)a | −5 (18)a | 0 (1)a | −4 (18)a | 520 (460)ab | 3.2×104 (1.2×104)a |
| Spruce | 55 (9)a | 1 (0)a | 55 (8)a | 129 (18)b | 2 (1)a | 131 (18)b | 130 (70)a | 1.3×104 (3.7×103)a | |
| Birch | 58 (17)a | 0 (0)a | 59 (17)a | 11 (11)a | 0 (1)a | 11 (11)a | 140 (60)ab | 3.3×104 (1.0×104)a | |
| No seedling | 237 (50)b | 29 (4)b | 266 (50)b | −143 (12)c | 168 (29)b | 26 (21)a | 2910 (780)b | 2.5×105 (9.1×104)b | |
| Values are means of five pots, S.E.M.s in parentheses. Values with the same letter within one soil are not significantly (P≤0.05) different from each other. | |||||||||
| o.m.=organic matter. | |||||||||
| Soil | Tree species | Initial, μg g−1 o.m. | Net formation, μg g−1 o.m. 40 days−1 | MPN, g−1 o.m. | |||||
| NH4+-N | (NO2−+NO3−)-N | (NH4++NO2−+NO3−)-N | NH4+-N | (NO2−+NO3−)-N | (NH4++NO2−+NO3−)-N | NH4+-oxidizers | NO2−-oxidizers | ||
| Organic soil | Pine | 301 (31)a | 1 (1)a | 302 (31)a | 238 (31)a | 0 (0)a | 238 (31)a | <10a | 7.0×102 (2.2×102)ac |
| Spruce | 508 (45)b | 4 (1)b | 512 (45)b | 235 (26)a | −1 (0)a | 234 (27)a | <10a | 1.8×103 (2.0×102)b | |
| Birch | 76 (23)c | 0 (0)a | 76 (23)c | 235 (16)a | 0 (0)a | 235 (16)a | <10a | 2.4×102 (1.2×102)c | |
| No seedling | 834 (19)d | 10 (2)c | 844 (20)d | 193 (8)a | −1 (1)a | 192 (9)a | <10a | 1.4×103 (3.0×102)ab | |
| Mineral soil | Pine | 64 (19)a | 1 (1)a | 64 (18)a | −5 (18)a | 0 (1)a | −4 (18)a | 520 (460)ab | 3.2×104 (1.2×104)a |
| Spruce | 55 (9)a | 1 (0)a | 55 (8)a | 129 (18)b | 2 (1)a | 131 (18)b | 130 (70)a | 1.3×104 (3.7×103)a | |
| Birch | 58 (17)a | 0 (0)a | 59 (17)a | 11 (11)a | 0 (1)a | 11 (11)a | 140 (60)ab | 3.3×104 (1.0×104)a | |
| No seedling | 237 (50)b | 29 (4)b | 266 (50)b | −143 (12)c | 168 (29)b | 26 (21)a | 2910 (780)b | 2.5×105 (9.1×104)b | |
| Values are means of five pots, S.E.M.s in parentheses. Values with the same letter within one soil are not significantly (P≤0.05) different from each other. | |||||||||
| o.m.=organic matter. | |||||||||
In the organic soil, there were negligible numbers of ammonium oxidizers in all treatments (Table 2). Numbers of nitrite oxidizers tended to be the lowest in birch rhizosphere. In the mineral soil, numbers of both ammonium and nitrite oxidizers were significantly higher in unplanted soil, but did not differ in the rhizospheres of different tree species.
3.5 Denitrification and denitrification enzyme activity
Denitrification activity was low in all of the soils. In the organic soil, the denitrification rate was lowest and DEA highest in birch rhizosphere (Fig. 2). In the mineral soil, denitrification activity was significantly higher in unplanted soil than in the rhizosphere soils, but DEA did not differ significantly between different treatments. DEA correlated positively (r=0.79, P<0.00) with the plant dry weight in the organic soils, but not in the mineral soils (r=0.24, P=0.39).
(a) Denitrification potential and (b) denitrification enzyme activity of the soils. Values are means of five pots, bars show S.E.M. Values with the same letter within one soil are not significantly (P≤0.05) different from each other.
(a) Denitrification potential and (b) denitrification enzyme activity of the soils. Values are means of five pots, bars show S.E.M. Values with the same letter within one soil are not significantly (P≤0.05) different from each other.
3.6 PLFA profiles
In the organic soil, the total amount of microbial lipids did not differ significantly between treatments, but the ratio of fungal to bacterial PLFAs was significantly higher in the birch rhizosphere than in the other soils (Table 3). PLFA profiles from the rhizospheres of all three tree species and the unplanted soil were distinct (Fig. 3a). Birch rhizosphere was most clearly separated from the unplanted soil along PC axis 1, which explained 42% of the variation. There were more of the fungal specific fatty acid 18:2ω6,9 and more branched fatty acids i16:1, i16:0, 10Me16:0, br17:0, br18:0 and 10Me17:0 in the birch rhizosphere (Fig. 4). PLFAs i17:0, 20:4 and C17:0 were relatively most abundant in the pine rhizosphere compared to the other samples. The amounts of 16:1ω7c and 18:1ω7 were also high in the pine rhizosphere compared to birch. The PLFAs common in the spruce rhizosphere, a15:0, 16:1ω5, 16:1ω7c and 18:1ω7, were similar to those abundant in the unplanted soil. The ratio of trans-unsaturated 16:1ω7 to cis-unsaturated 16:1ω7 was significantly higher in the birch rhizosphere (Table 3).
The total amount of microbial PLFAs, the ratio of fungal to bacterial PLFAs and ratio of trans-unsaturated 16:1ω7 to cis-unsaturated 16:1ω7
| Soil | Tree species | Microbial PLFAs, μmol g−1 o.m. | 18:2ω6,9:bacterial PLFAs | 16:1ω7t:16:1ω7c | |||||
| Organic soil | Pine | 1.4 (0.2)a | 0.15 (0.01)a | 0.29 (0.02)a | |||||
| Spruce | 1.4 (0.1)a | 0.14 (0.02)a | 0.23 (0.01)ac | ||||||
| Birch | 1.7 (1.1a | 0.29 (0.01)b | 0.43 (0.03)b | ||||||
| No seedling | 1.8 (0.1)a | 0.12 (0.01)a | 0.20 (0.01)c | ||||||
| Mineral soil | Pine | 1.7 (0.2)a | 0.11 (0.01)ab | 0.17 (0.01)a | |||||
| Spruce | 1.8 (0.2)a | 0.17 (0.02)a | 0.17 (0.02)a | ||||||
| Birch | 1.9 (0.2)a | 0.18 (0.03)a | 0.20 (0.02)a | ||||||
| No seedling | 1.7 (0.2)a | 0.07 (0.02)b | 0.15 (0.00)a | ||||||
| Values are means of three pots, S.E.M.s in parentheses. Values with the same letter within one soil are not significantly (P≤0.05) different from each other. | o.m.=organic matter. | ||||||||
| Soil | Tree species | Microbial PLFAs, μmol g−1 o.m. | 18:2ω6,9:bacterial PLFAs | 16:1ω7t:16:1ω7c | |||||
| Organic soil | Pine | 1.4 (0.2)a | 0.15 (0.01)a | 0.29 (0.02)a | |||||
| Spruce | 1.4 (0.1)a | 0.14 (0.02)a | 0.23 (0.01)ac | ||||||
| Birch | 1.7 (1.1a | 0.29 (0.01)b | 0.43 (0.03)b | ||||||
| No seedling | 1.8 (0.1)a | 0.12 (0.01)a | 0.20 (0.01)c | ||||||
| Mineral soil | Pine | 1.7 (0.2)a | 0.11 (0.01)ab | 0.17 (0.01)a | |||||
| Spruce | 1.8 (0.2)a | 0.17 (0.02)a | 0.17 (0.02)a | ||||||
| Birch | 1.9 (0.2)a | 0.18 (0.03)a | 0.20 (0.02)a | ||||||
| No seedling | 1.7 (0.2)a | 0.07 (0.02)b | 0.15 (0.00)a | ||||||
| Values are means of three pots, S.E.M.s in parentheses. Values with the same letter within one soil are not significantly (P≤0.05) different from each other. | o.m.=organic matter. | ||||||||
The total amount of microbial PLFAs, the ratio of fungal to bacterial PLFAs and ratio of trans-unsaturated 16:1ω7 to cis-unsaturated 16:1ω7
| Soil | Tree species | Microbial PLFAs, μmol g−1 o.m. | 18:2ω6,9:bacterial PLFAs | 16:1ω7t:16:1ω7c | |||||
| Organic soil | Pine | 1.4 (0.2)a | 0.15 (0.01)a | 0.29 (0.02)a | |||||
| Spruce | 1.4 (0.1)a | 0.14 (0.02)a | 0.23 (0.01)ac | ||||||
| Birch | 1.7 (1.1a | 0.29 (0.01)b | 0.43 (0.03)b | ||||||
| No seedling | 1.8 (0.1)a | 0.12 (0.01)a | 0.20 (0.01)c | ||||||
| Mineral soil | Pine | 1.7 (0.2)a | 0.11 (0.01)ab | 0.17 (0.01)a | |||||
| Spruce | 1.8 (0.2)a | 0.17 (0.02)a | 0.17 (0.02)a | ||||||
| Birch | 1.9 (0.2)a | 0.18 (0.03)a | 0.20 (0.02)a | ||||||
| No seedling | 1.7 (0.2)a | 0.07 (0.02)b | 0.15 (0.00)a | ||||||
| Values are means of three pots, S.E.M.s in parentheses. Values with the same letter within one soil are not significantly (P≤0.05) different from each other. | o.m.=organic matter. | ||||||||
| Soil | Tree species | Microbial PLFAs, μmol g−1 o.m. | 18:2ω6,9:bacterial PLFAs | 16:1ω7t:16:1ω7c | |||||
| Organic soil | Pine | 1.4 (0.2)a | 0.15 (0.01)a | 0.29 (0.02)a | |||||
| Spruce | 1.4 (0.1)a | 0.14 (0.02)a | 0.23 (0.01)ac | ||||||
| Birch | 1.7 (1.1a | 0.29 (0.01)b | 0.43 (0.03)b | ||||||
| No seedling | 1.8 (0.1)a | 0.12 (0.01)a | 0.20 (0.01)c | ||||||
| Mineral soil | Pine | 1.7 (0.2)a | 0.11 (0.01)ab | 0.17 (0.01)a | |||||
| Spruce | 1.8 (0.2)a | 0.17 (0.02)a | 0.17 (0.02)a | ||||||
| Birch | 1.9 (0.2)a | 0.18 (0.03)a | 0.20 (0.02)a | ||||||
| No seedling | 1.7 (0.2)a | 0.07 (0.02)b | 0.15 (0.00)a | ||||||
| Values are means of three pots, S.E.M.s in parentheses. Values with the same letter within one soil are not significantly (P≤0.05) different from each other. | o.m.=organic matter. | ||||||||
Principal component scores for PLFA data in (a) organic soil and (b) mineral soil.
Principal component scores for PLFA data in (a) organic soil and (b) mineral soil.
Loading values for the individual PLFAs from the principal component analysis of the organic soils.
Loading values for the individual PLFAs from the principal component analysis of the organic soils.
In the mineral soil, the total amount of microbial lipids did not differ between different treatments, but the ratio of fungal:bacterial PLFAs tended to be lowest in the unplanted soil (Table 3). Tree species did not clearly discriminate from each other in the PCA (Fig. 3b).
3.7 CLPPs
In the organic soil, the CLPPs separated birch rhizosphere from the other soils (Fig. 5). All 125 C sources from GN and MT plates and 61 root exudate C sources did not differ in their separation power. The AWCD in the birch rhizosphere was considerably lower than in the other soils (results not shown). The use of many phenolic acids was characteristic to birch rhizosphere. When using the C sources only from the MT plate, microbial communities from pine and spruce rhizosphere also had a tendency of separating from the unplanted soil (Fig. 5c). The substrates on MT plates had overall lower utilization rates than the ones on GN plates. In the mineral soil, CLPPs from different tree species did not separate from each other in PCA with any substrate combination (Fig. 6).
Principal component scores for CLPP profiles in organic soils done with (a) all 125 C sources, (b) 61 root exudate C sources and (c) 31 C sources from MT plates.
Principal component scores for CLPP profiles in organic soils done with (a) all 125 C sources, (b) 61 root exudate C sources and (c) 31 C sources from MT plates.
Principal component scores for CLPP profiles in mineral soils done with (a) all 125 C sources and (b) 61 root exudate C sources.
Principal component scores for CLPP profiles in mineral soils done with (a) all 125 C sources and (b) 61 root exudate C sources.
In some samples, there were significant, but not consistent, differences in the blank control wells and glucose containing wells in the GN and MT plates (results not shown).
3.8 Plate counts
In the organic soil, numbers of colony-forming bacteria were lower in birch rhizosphere than in spruce rhizosphere (Table 4). There were negligible numbers of colony-forming pseudomonads in birch rhizosphere. Numbers of colony-forming fungi and yeasts did not differ between treatments.
Results from plate counts of the soils
| Soil | Tree species | Bacteria, cfu g−1 o.m. soil | Pseudomonads, cfu g−1 o.m. soil | Fungi, cfu g−1 o.m. soil | Yeasts, cfu g−1 o.m. soil |
| Organic soil | Pine | 1.3×108 (1.1×107)ab | 9.9×105 (4.6×105)a | 1.5×106 (2.4×104)a | <103a |
| Spruce | 2.3×108 (7.3×107)a | 9.1×105 (2.7×105)a | 1.5×106 (1.3×105)a | 2.9×105 (2.7×105)a | |
| Birch | 4.2×107 (3.4×107)b | <103c | 8.9×105 (2.3×105)a | 1.9×105 (1.1×105)a | |
| No seedling | 6.7×107 (1.5×107)ab | 2.0×105 (4.3×104)b | 9.3×105 (2.6×105)a | 7.5×104 (2.0×104)a | |
| Mineral soil | Pine | 7.9×107 (1.1×107)a | 3.3×106 (7.8×105)a | 2.5×106 (4.3×105)a | 1.1×106 (4.1×105)a |
| Spruce | 5.8×107 (1.5×107)a | 1.4×106 (6.8×105)a | 1.9×106 (3.1×104)a | 8.5×105 (2.4×105)a | |
| Birch | 2.1×107 (6.4×106)b | 3.4×106 (7.0×105)a | 2.2×106 (3.0×105)a | 2.6×105 (1.9×105)a | |
| No seedling | 6.1×106 (8.5×105)c | 1.9×106 (3.6×105)a | 2.7×106 (2.4×105)a | 1.2×105 (5.8×104)a |
| Soil | Tree species | Bacteria, cfu g−1 o.m. soil | Pseudomonads, cfu g−1 o.m. soil | Fungi, cfu g−1 o.m. soil | Yeasts, cfu g−1 o.m. soil |
| Organic soil | Pine | 1.3×108 (1.1×107)ab | 9.9×105 (4.6×105)a | 1.5×106 (2.4×104)a | <103a |
| Spruce | 2.3×108 (7.3×107)a | 9.1×105 (2.7×105)a | 1.5×106 (1.3×105)a | 2.9×105 (2.7×105)a | |
| Birch | 4.2×107 (3.4×107)b | <103c | 8.9×105 (2.3×105)a | 1.9×105 (1.1×105)a | |
| No seedling | 6.7×107 (1.5×107)ab | 2.0×105 (4.3×104)b | 9.3×105 (2.6×105)a | 7.5×104 (2.0×104)a | |
| Mineral soil | Pine | 7.9×107 (1.1×107)a | 3.3×106 (7.8×105)a | 2.5×106 (4.3×105)a | 1.1×106 (4.1×105)a |
| Spruce | 5.8×107 (1.5×107)a | 1.4×106 (6.8×105)a | 1.9×106 (3.1×104)a | 8.5×105 (2.4×105)a | |
| Birch | 2.1×107 (6.4×106)b | 3.4×106 (7.0×105)a | 2.2×106 (3.0×105)a | 2.6×105 (1.9×105)a | |
| No seedling | 6.1×106 (8.5×105)c | 1.9×106 (3.6×105)a | 2.7×106 (2.4×105)a | 1.2×105 (5.8×104)a |
Results from plate counts of the soils
| Soil | Tree species | Bacteria, cfu g−1 o.m. soil | Pseudomonads, cfu g−1 o.m. soil | Fungi, cfu g−1 o.m. soil | Yeasts, cfu g−1 o.m. soil |
| Organic soil | Pine | 1.3×108 (1.1×107)ab | 9.9×105 (4.6×105)a | 1.5×106 (2.4×104)a | <103a |
| Spruce | 2.3×108 (7.3×107)a | 9.1×105 (2.7×105)a | 1.5×106 (1.3×105)a | 2.9×105 (2.7×105)a | |
| Birch | 4.2×107 (3.4×107)b | <103c | 8.9×105 (2.3×105)a | 1.9×105 (1.1×105)a | |
| No seedling | 6.7×107 (1.5×107)ab | 2.0×105 (4.3×104)b | 9.3×105 (2.6×105)a | 7.5×104 (2.0×104)a | |
| Mineral soil | Pine | 7.9×107 (1.1×107)a | 3.3×106 (7.8×105)a | 2.5×106 (4.3×105)a | 1.1×106 (4.1×105)a |
| Spruce | 5.8×107 (1.5×107)a | 1.4×106 (6.8×105)a | 1.9×106 (3.1×104)a | 8.5×105 (2.4×105)a | |
| Birch | 2.1×107 (6.4×106)b | 3.4×106 (7.0×105)a | 2.2×106 (3.0×105)a | 2.6×105 (1.9×105)a | |
| No seedling | 6.1×106 (8.5×105)c | 1.9×106 (3.6×105)a | 2.7×106 (2.4×105)a | 1.2×105 (5.8×104)a |
| Soil | Tree species | Bacteria, cfu g−1 o.m. soil | Pseudomonads, cfu g−1 o.m. soil | Fungi, cfu g−1 o.m. soil | Yeasts, cfu g−1 o.m. soil |
| Organic soil | Pine | 1.3×108 (1.1×107)ab | 9.9×105 (4.6×105)a | 1.5×106 (2.4×104)a | <103a |
| Spruce | 2.3×108 (7.3×107)a | 9.1×105 (2.7×105)a | 1.5×106 (1.3×105)a | 2.9×105 (2.7×105)a | |
| Birch | 4.2×107 (3.4×107)b | <103c | 8.9×105 (2.3×105)a | 1.9×105 (1.1×105)a | |
| No seedling | 6.7×107 (1.5×107)ab | 2.0×105 (4.3×104)b | 9.3×105 (2.6×105)a | 7.5×104 (2.0×104)a | |
| Mineral soil | Pine | 7.9×107 (1.1×107)a | 3.3×106 (7.8×105)a | 2.5×106 (4.3×105)a | 1.1×106 (4.1×105)a |
| Spruce | 5.8×107 (1.5×107)a | 1.4×106 (6.8×105)a | 1.9×106 (3.1×104)a | 8.5×105 (2.4×105)a | |
| Birch | 2.1×107 (6.4×106)b | 3.4×106 (7.0×105)a | 2.2×106 (3.0×105)a | 2.6×105 (1.9×105)a | |
| No seedling | 6.1×106 (8.5×105)c | 1.9×106 (3.6×105)a | 2.7×106 (2.4×105)a | 1.2×105 (5.8×104)a |
Values are means of three pots, S.E.M.s in parentheses. Values with the same letter within one soil are not significantly (P≤0.05) different from each other.
cfu=colony-forming units.
o.m.=organic matter.
In the mineral soil, numbers of colony-forming bacteria were significantly lower in unplanted soil compared to all rhizosphere soils and significantly lower in birch rhizosphere than in pine and spruce rhizosphere (Table 4). Numbers of colony-forming pseudomonads, fungi and yeasts did not significantly differ between tree species.
4 Discussion
Microbial biomass C and N and the C mineralization rate most often did not differ between different tree species (Fig. 1). An exception was birch rhizosphere in the organic soil, where Cmic and Nmic were significantly higher than in the rhizospheres of pine and spruce. At the time of harvest, birch seedlings were clearly larger than those of pine and spruce. However, because approximately similar layers of soil on the roots were studied and the soil:root ratios were similar, the size differences between the seedlings should not have influenced the results. This indicates that there may be some qualitative differences in the rhizodeposition of birch versus conifers.
SIR and basal respiration were significantly lower in unplanted mineral soil than in the rhizospheres of all tree species (Fig. 1). This was probably because seedlings had provided soil microbes with an extra input of substrate, which had stimulated their growth, as was also seen by the significantly higher numbers of culturable bacteria in the rhizosphere soils than in the unplanted soil (Table 4). This is in agreement with the results of Parmelee et al. [11], who found that in the organic soil, pine roots and microbes competed with each other for moisture and N, but in nutrient-poor mineral soil, roots provided the main input of substrate, which was more significant than the adverse effect of roots.
The concentration of mineral N in soil was dependent on the microbial and plant uptake of N, concluded from both the Nmic values and concentrations of N in the needles/leaves of the seedlings (Tables 1 and 2Fig. 1). The net formation of mineral N did not differ between different tree species in the organic soil, but in the mineral soil, it was highest in spruce rhizosphere (Table 2). In other studies, roots of trees have been shown both to increase and decrease N mineralization in soil [8,11]. Nevertheless, the higher net rate of mineral N formation in spruce rhizosphere could be due to a higher microbial N uptake during the incubation in the other soils. The net nitrification was only evident in unplanted mineral soil and the numbers of ammonium and nitrite oxidizers were highest in that soil (Table 2). This may be due to the fact that autotrophic nitrifiers are poor competitors with heterotrophic microbes. Therefore, in the organic soil and in the rhizosphere soils, they have been out-competed, but in the plantless mineral soil, where there are less heterotrophic microbes, they are active.
Denitrification, as a process, is dependent on available C and a low partial pressure of O2, which is why denitrification is often enhanced in the rhizosphere [13,14]. However, in our study, denitrification was only stimulated in spruce rhizosphere in the organic soil. Availability of electron acceptor seemed to be the main factor controlling denitrification in these soils, because patterns of denitrification activity followed the concentration of nitrate especially in the organic soils (Fig. 2a, Table 2). The DEA in soil did not differ substantially between different tree species, except that it was higher in birch rhizosphere than in those of pine and spruce in the organic soil (Fig. 2b). DEA measurements aim at determining the activity of pre-existing denitrifying enzymes in soil, but it may also be that the way it was measured here, it really more described the potential for denitrification. Denitrifying activity in the rhizosphere has been found to correlate with photosynthetic activity [27] and plant dry weight [28]. In our study, there was a positive correlation between DEA and plant dry weight in the organic soils, but not in the mineral soils.
There were shifts in the soil microbial community structure in response to different tree species in the organic soil, but not in the mineral soil (Figs. 35 and 6). One reason for this could be that in the organic soil, there was more diversity to start with, which makes it possible that different groups are enriched in different conditions, whereas the original microbial community in the mineral soil probably was less diverse. Not much is known about the microbial communities in the rhizospheres of trees [29]. It has to be borne in mind that it are not only the roots of the trees that affect the microbial communities in soil, but also different mycorrhizal species and their exudates have been found to change the soil bacterial communities [30,31]. We did not determine how many and what kind of mycorrhizal infections the seedlings had in this study, but it may have been that seedlings were more mycorrhizal in the organic soil, as it is known that the highest concentration of mycorrhizal propagules is in the humus (organic) layer of soils [32]. If this was true, then, the influence of mycorrhizas on bacterial communities would also have been stronger in the organic soil.
In the organic soil, the fatty acids more common in birch rhizosphere than in those of pine and spruce or in unplanted soil were the fungal specific 18:2ω6,9 (Fig. 4) and branched fatty acids, which have commonly been found in Gram-positive bacteria [33]. The increasing amount of 18:2ω6,9 and the increased ratio of fungal to bacterial PLFAs from unplanted soil to birch rhizosphere was possibly due to higher populations of mycorrhizal fungi rather than saprophytes.
The PLFA pattern of the pine rhizosphere in the organic soil separated slightly from the PLFA patterns of spruce and unplanted soil, but the changes in the individual PLFAs could not be clearly associated to certain groups of bacteria (Fig. 4). The PLFA patterns of the spruce rhizosphere and the unplanted soil were relatively similar. The PLFAs more common in those samples were mostly mono-unsaturated, typical to Gram-negative bacteria [34], even though the abundance of branched a15:0, common in Gram-positive bacteria, was also high. The relative amount of bacterial PLFA 16:1ω5 [35] was highest in the unplanted organic soil and also higher in the spruce rhizosphere than under the other tree species. In the study of Frostegård et al. [36], 16:1ω5 decreased during incubation and they suggested that this PLFA may reflect the dynamics of organisms that are responding to changes in the C status of soil. It could be that pine and birch were taking up more nutrients from the soil than the spruce seedlings, which had caused more competition between microbes and the plants and also a less favorable C status of the soil towards the end of the growing season.
The CLPPs clearly differentiated only birch rhizosphere from the other soils (Figs. 5 and 6). There were negligible amounts of colony-forming pseudomonads in the birch rhizosphere in the organic soil (Table 4) and the AWCD in Biolog plates from organic birch rhizospheres was very low, in spite of the bacterial inoculum densities being approximately the same as with other soils. This could influence the strong discrimination of birch by CLPPs, as the numbers of pseudomonads have been found to be directly correlated with color development in Biolog wells [37]. The low number of Pseudomonas species in birch rhizosphere was surprising, given that this species is particularly stimulated in the rhizosphere (reviewed by Bolton et al. [38]). Nevertheless, fluorescent pseudomonads were commonly isolated from Scots pine mycorrhizospheres in nursery peat, but they were almost absent from outer mycorrhizospheres in pine forest humus, where Bacillus species were more important [30]. Because birch roots filled the pots almost totally, birch rhizosphere samples probably contained also soil around external hyphae of mycorrhizas, whereas pine and spruce rhizosphere samples included only soil around the roots. Thus, there might have been a higher dominance of Bacillus species in birch rhizosphere samples compared to those of pine and spruce, as indicated also by the PLFA profiles, which showed a high amount of Gram-positive bacteria in birch rhizosphere.
It is plausible to think that using ecologically relevant C sources, such that can be found from soils, would give the best separation with CLPPs. Campbell et al. [22] found the separation of microbial communities to be more distinct using the 61 exudate C sources than the 125 C sources in GN and MT plates together. In this study, however, this was not the case (Fig. 5). The C sources in the MT plate alone, however, had a tendency of separating also pine and spruce rhizosphere from the unplanted soil. This might indicate that the substrates in MT plates were more ecologically relevant for these samples than the ones in GN plates.
In conclusion, in the organic soil, Cmic and Nmic were higher in birch rhizosphere than in pine and spruce rhizosphere. Also the microbial community structure in the rhizospheres of pine, spruce and birch and in the unplanted soil were distinct. In spite of this, rates of C and N mineralization were not different between treatments. In the mineral soil, all tree roots stimulated C mineralization in soil and prevented nitrification, compared to unplanted soil. In addition, the size of the microbial biomass and microbial community structure did not differ in the rhizospheres of different tree species. Thus, in the mineral soil, the strongest effect on soil microbes was the presence of a plant, but in the organic soil, different tree species had a significant influence on soil microbes.
Acknowledgments
We thank the staff at the Ruotsinkylä field station for taking care of the plants. Anneli Rautiainen, Terhi Hallantie, Eileen J. Reid and Ruth MacDougall are thanked for help in the laboratory. We are grateful for the Academy of Finland and the foundations Metsämiesten säätiö and Emil Aaltosen säätiö for supporting this work financially.
