The study was conducted on a 4-yr-old clearcut plot (1 ha) on Mount Mara, located near the town of Sicamous (51° N, 119° W) in the southern interior of British Columbia (BC). The site is located within the Wet Cold Engelmann Spruce – Subalpine Fir (ESSFwc) bio-geo-climatic zone (Meidinger & Pojar, 1991). Mean annual temperature is 1.2°C, and mean annual precipitation is 930 mm, of which about two-thirds falls as snow between late October and late May The surrounding forest is dominated by a 300-yr-old stand of Engelmann spruce and subalpine fir (Abies lasiocarpa (Hook.) Nutt.). Undisturbed soils are Humo-Ferric Podzols overlain by a 4 cm deep forest floor. The understory shrub and herb community includes white-flowered rhododendron (Rhododendron albiflorum Hook.), black huckleberry (Vaccinium mebranaceum Dougl. ex. Torr.), oval-leaved blueberry (V. ovalifolium Sm.), Sitka valerian (Valeriana sitchensis Bong.), oak fern (Gymnocarpium dryopteris (L.) Newman), foamflower (Tiarella unifoliata Hook.) and mountain arnica (Arnica latifolia Bong.).
Seedlings used in the present study were prepared as part of a larger investigation on the effects of ECM diversity on the nutrition of conifer seedlings after disturbance. The strategy involved planting nonmycorrhizal Engelman spruce seedlings in a clearcut, and allowing naturally occurring ECM fungi to colonize the roots. Briefly, seeds of spruce were surface sterilized and planted in a sterilized peat and vermiculite mixture (1 : 1 v/v). Seedlings were grown with photoperiod extension to 18 h in a glasshouse for c. 6 wk, and then transferred to the study site in early July 1999. The 800 nonmycorrhizal seedlings were planted in rows at regular 0.5 m spacing, at least 10 m from the edge of the clearcut. Seedlings were carefully removed from the potting mix and planted in a small furrow using a hand spade. The depth of the forest floor in which each seedling was planted varied between 0 and 2 cm, because of soil disturbance that had occurred during harvest operations. At time of planting, the shoots of seedlings (1–2 cm high) were covered by 10 cm high cages made of 5 mm wire mesh to protect them from herbivores. Seedlings were left to grow for 1 yr.
In situ15N labelling
In mid-June 2000, immediately following snowmelt, a plastic sleeve (10 cm dia. × 15 cm high) was inserted into the soil around 150 randomly chosen seedlings using a Gidding corer (Giddings Machine Co., Ft. Collins, CO, USA). Each soil core, complete with seedling and sleeve, was lifted out of the ground, the bottom covered with a plastic cap, and the capped core was replaced into the hole with soil packed around it to eliminate any air space. The purpose of the sleeve and cap was to contain the 15N-labelled solution that would be subsequently applied, within a defined soil volume. At the time of coring, the shoots of plants growing within a 15 cm radius of each seedling were excised to eliminate any shading. The coring process caused little disturbance to the soil around the seedlings because the soil was soft and moist. Seedlings were left to grow for another 4 wk before labelling, to allow roots and hyphae that may have been damaged during the coring process to re-establish within the core. By mid-July, when isotope solutions were applied, all seedlings had broken bud.
Fifty cores were injected with 20 ml of aqueous 1 mm (15NH4)2SO4 solution (139.6 mg l−1 at 99 atom %15N), another 50 cores with 20 ml of aqueous 2 mm K15NO3 solution (216.6 mg l−1 at 99 atom %15N), and another 50 cores with 20 ml distilled water. Solutions in each core were injected in five 4-ml aliquots at points located equidistant between the stem and the edge of the core. In order to distribute the solutions evenly throughout the cores, we used a syringe equipped with a 15-cm needle having four openings pointing outward. The needle was inserted 10 cm deep and the plunger was depressed gently as the needle was slowly pulled back up through the soil.
Fifteen minutes following injection (t = 0), half the cores allocated to each solution were destructively sampled. The cores were removed from the ground, the soil was extruded onto clean plastic sheeting using disposable rubber gloves, seedlings were carefully extracted from the soil, the shoots were excised from the roots, and both were rinsed with 0.05 m CaCl2 and placed in separate plastic bags on ice. The remaining soil was sieved through a 5.6-mm mesh and placed in a plastic bag on ice. All instruments were rinsed with 0.05 m CaCl2 and deionized water between the sampling of each core. The procedure was repeated for the remaining cores at 26 h after injection (t = 26). Soil and tissue samples were returned to the laboratory and stored at 4°C overnight.
Gross mineral N production rates
Gross production rates of NH4+ and NO3− were estimated following the principles of isotope dilution (Kirkham & Bartholomew, 1954). Approximately 30 g of fresh soil from each core was weighed in an aluminum dish, dried at 105°C for 48 h, and reweighed to determine moisture content. A second 30-g subsample from each core was weighed in a 500-ml Mason jar, 100 ml of 1 m KCl solution was added to the jar, the jar was sealed and the mixture placed on a reciprocal shaker (130 cycles min−1) for 60 min. The supernatant was filtered through a Whatman 42 cellulose filter paper than had been preleached with 1 m KCl. Mineral N concentration in each soil extract was measured colorimetrically using a Technicon II AutoAnalyser (Pulse Instrumentation Ltd, Saskatoon, Canada), with nitroprusside-salicylate reagent for determining NH4 ± N, and sulfanilamide color reagent with a Cu-coated Cd reduction column for determining NO3–N. Extracts were prepared for mass spectrometry by diffusion on acid traps according to Brooks et al. (1989), with the amount of N diffused adjusted to 300 µg-N with expected enrichment of c. 1% N, as suggested by Bradley & Fyles (1996). Devarda's alloy was added to extracts from soil samples injected with 15NO3− before diffusion to convert NO3− to NH4+. Samples were encapsulated in Sn capsules and sent to the Stable Isotope Laboratory, University of California at Davis, for isotopic analysis.
- (Eqn 1)
where GPR = gross production rate
APE0 = atom percentage excess of the labelled pool at t = 0
APEt = atom percentage excess of the labelled pool at t = 26
[N]0 = N concentration of the labelled pool at t = 0
[N]t = N concentration of the labelled pool at t = 26.
However, for this equation to be valid, each t = 0 sample must be paired to one t = 26 sample, and both must be subsamples of the same experimental unit. In our study, given that t = 0 and t = 26 samples were from different cores, we estimated APE0 and [N]0 for each sample extracted at t = 26 as follows. First, we calculated the fraction of 15N recovered () in all t = 0 cores according to the following equation (Hart et al., 1994):
- (Eqn 2)
- ( Eqn 3)
Similarly, we used [N]t as an estimate of [N]0 for each core extracted at t = 26, given that prior soil tests had shown very little net mineralization over the assay period.
- (Eqn 4)
where M0 = Mass of tracer plus nontracer of the labelled pool
H0 = Mass of tracer of the labelled pool at t = 0.
H = Mass of tracer of the labelled pool at t = 26.
The concentration of microbial N was measured by the chloroform fumigation – direct extraction method (Brookes et al., 1985). For chloroform fumigation, 50 ml of glass-distilled CHCl3 (prefiltered through 2.5 g AlO3) was placed with boiling chips in a glass dish on the bottom of a pressure cooker. A 30 g (f. wt) subsample of soil from each core was placed in a Mason jar, the jars were placed inside the pressure cooker on shelves lined with moist paper towel and fumigated under vacuum (21.3 kPa) for 24 h. After removal from the pressure cooker, fumigated as well as nonfumigated soils from each core were extracted in 1 m KCl solution, as described above, and the extracts were immediately frozen at −20°C until analyzed. Upon thawing, 5-ml aliquots of fumigated and nonfumigated soil extracts were syringe filtered through a 0.45-µm membrane filter, oxidized with 10 ml of alkaline K2S2O8, and autoclaved at 121°C for 45 min converting all N present in a sample to NO3− (D’Elia et al., 1977). Total NO3− N was measured colorimetrically, as described above, and microbial N in each core was calculated as the difference between fumigated and nonfumigated subsamples divided by the total soil dry mass in the core.
Mineral N uptake rates
Seedling roots and shoots, including ECM fungal sheaths, were oven dried at 60°C for 72 h, ground in a Wiley Mill and passed through a 800 µm sieve. Subsamples were packaged in Sn capsules and sent to the Stable Isotope Laboratory (UC at Davis) for 15N analysis by continuous flow mass spectrometry. Assuming that isotope discrimination by the roots would be minor relative to the enrichment caused by labelling of the soil, plant uptake (PU) of each mineral N form was calculated as:
- (Eqn 5)
where SNP = soil N pool = (soil dry mass) × [N]t
EIP = excess isotope in the plant = (plant dry mass) × (atom percentage 15N excess in the plant).
Each PU value of each mineral N form was then converted to plant uptake rate (PUR) by dividing by time (i.e. 26 h = 1.083 d). Given that PUR may be biased by the amount of roots, we generated a new variable, which we called the relative plant uptake rate (RPUR), by dividing PUR by the dry mass of roots in each core.
Given the one-time sampling opportunity that was implicit to this novel experimental protocol, 18 potential data points were lost as a result of low [N]t-values. Four additional data points were lost due to a manipulation error during microbial-N determinations, eight data points were deleted from percentage ECM counts because seedlings had less than one fully colonized root tip, and three data points representing either PUR or RPUR were deleted because of failed analyses at the isotope laboratory. The remaining 264 data points were used to determine the dependence of PUR and RPUR on the four independent variables. Simple linear regressions between each pair of variables were estimated independently for NH4+ and NO3− by the least squares difference method using SPSS 11.0 software (SPSS Inc, Chicago, IL, USA). Regression parameters were considered different from zero when P < 0.05. Before analysis, all measured variables were log transformed to meet assumptions of normality and homoscedasticity, except for % ECM root tips, which was arcsin–square root transformed.
When two or more variables related significantly to plant N uptake, exploratory path analysis was used to investigate possible causal structures between the variables, using the SGS algorithm (Spirtes et al., 1993; Shipley, 2000a) as implemented in the EPA2 program (Shipley, 1997). A detailed explanation of the algorithm is given by Shipley (2000b) and only an intuitive description is given here. The first part of the algorithm constructs an undirected dependency graph, which links two variables if, and only if, they are dependent when all possible subsets of other variables in the model are mathematically fixed. The second part of the algorithm attempts to orient the undirected dependency graph based on ‘unshielded colliders’ in the graph. This is described in detail in Shipley (2000a) and the mathematical proof is given in Spirtes et al. (1993).