New Forests (2017) 48:699–717 DOI 10.1007/s11056-017-9593-5

Weed control increases nitrogen retranslocation and growth of white spruce seedlings on a reclaimed oil sands soil Prem Pokharel1 Scott X. Chang1

•

Woo-Jung Choi2 • Ghulam M. Jamro1

•

Received: 21 November 2016 / Accepted: 17 June 2017 / Published online: 19 June 2017 Ó Springer Science+Business Media B.V. 2017

Abstract Early establishment of seedlings in reclaimed oil sand areas is often limited by low nutrient and water availability due to factors such as strong understory vegetation competition. Management practices such as nursery fertilization and field weed control could help early establishment of planted seedlings and reclamation success. We investigated the effect of nursery nutrient loading and field weed control on the growth, nitrogen (N) retranslocation within seedling components, and plant N uptake from the soil for white spruce (Picea glauca [Moench] Voss) seedlings planted on a highly competitive reclaimed oil sands site for two years. Exponential fertilization during nursery production increased the root biomass but not the nutrient reserve in the seedling. In the field experiment, on average across the treatments, 78 and 49% of the total N demand of new tissue growth in the first and second year were met by N retranslocation, respectively. Though exponential fertilization did not affect N retranslocation, it increased the percent height and root collar diameter growth. Weed control increased not only the growth of seedlings by increasing soil N availability, but also N retranslocation within the seedlings in the second year after outplanting. We conclude that vegetation management by weed control is feasible in

Electronic supplementary material The online version of this article (doi:10.1007/s11056-017-9593-5) contains supplementary material, which is available to authorized users. & Scott X. Chang [email protected] Prem Pokharel [email protected] Woo-Jung Choi [email protected] Ghulam M. Jamro [email protected] 1

Department of Renewable Resources, University of Alberta, 442 Earth Sciences Building, Edmonton T6G 2E3, Canada

2

Department of Rural & Biosystems Engineering, Chonnam National University, Gwangju 500-757, Republic of Korea

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improving the early growth of white spruce seedlings planted on reclaimed soils and facilitate tree establishment in the oil sands region. Optimization of the nursery exponential N fertilization regime for white spruce may further help with early revegetation of reclaimed oil sands sites. Keywords Picea glauca  Revegetation  competition  Nutrient loading

15

N feeding  Foliar d13C  Understory

Introduction Reclaimed oil sands areas in northern Alberta, Canada are often revegetated with white spruce (Picea glauca [Moench] Voss), one of the most commonly distributed species in the mixedwood boreal region (Fung and Macyk 2000). However, survival and growth of nursery grown coniferous seedlings are often affected by outplanting stress (Rietveld 1989; Grossnickle 2000) which has been noted as a major factor resulting in the failure of early establishment of the seedlings (Close et al. 2005b). Understory competition for resources such as nutrients and water can add outplanting stress and thus further limit the early growth of seedlings (Matsushima and Chang 2006; Sloan and Jacobs 2013). In this context, building up a nutrient reserve in seedlings during nursery production (Timmer and Munson 1991) and understory vegetation control (Rietveld 1989; Matsushima and Chang 2007) in the field can reduce the stresses and thus help seedling establishment after outplanting. The rationale of nutrient loading in nursery seedling production is to store nutrients in the seedling that can consequently be used during early growth through retranslocation before the root system of the seedling is established (Timmer 1997). For this reason, the internal remobilization of nutrients is an important pathway through which seedlings are provided with nutrients for initial growth and survival when seedlings grow slowly and thus have limited root systems (van den Driessche 1985; Burdett 1990). The importance of internal nutrient cycling in supplying nutrients for new tissues of coniferous trees has been extensively reported (Millard and Grelet 2010; Villar-Salvador et al. 2015). Exponential fertilization is a method for loading nutrients into seedlings, designed to supply nutrients to seedlings at an exponential rate in excess of the demand for the current growth during nursery production (Timmer and Aidelbaum 1996) Therefore, exponential fertilization can induce luxury uptake of nutrients, resulting in an increased nutrient reserve in seedlings (Timmer and Aidelbaum 1996; Salifu and Timmer 2003). One of the advantages of nursery nutrient loading over field fertilization is that it prevents inadvertent supply of nutrients to understory vegetation that competes with the seedlings (Chang et al. 1996). Nursery nutrient loading has been used in a number of coniferous (Imo and Timmer 2001; Jonsdottir et al. 2013) and broadleaf species (Close et al. 2005a; Birge et al. 2006) in forest restoration. Schott et al. (2016) demonstrated the advantages of nutrient loading over field fertilization and vegetation management to increase the growth and survival of trembling aspen (Populus tremuloides Michx.) seedlings after outplanting on boreal reclamation sites. For white spruce, however, studies are limited to nursery production of seedlings (Hu 2012), seedlings growth experiments in the greenhouse (Hu et al. 2015) and growth experiments in abandoned farm fields (McAlister and Timmer 1998), and thus no relevant study is available for planting on reclaimed oil sands sites which often have harsh growth conditions. Although disturbed oil sands sites

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are reclaimed by bringing in off-site soil materials such as peat mineral soil mix or forest floor mineral soil mix as sources of organic matter and nutrients (Macdonald et al. 2015), the growth of seedlings can still be limited by low nutrient availability (Sloan and Jacobs 2013; Sloan et al. 2016), particularly nitrogen (N) for white spruce growth (Duan and  Chang 2015). Specifically, available N (NHþ 4 N and NO3 N) in reclaimed soils is lower than that in adjacent natural forest soils (Jung and Chang 2012; Jamro et al. 2014), implying that seedling growth in reclaimed soils might benefit from improved N nutrition by nutrient loading. However, studies on the effect of N loading on white spruce growth on reclaimed soils are lacking. Control of understory vegetation, which either colonizes the site after disturbance or is grown purposefully in sloping areas for soil erosion control, can further help seedling growth by alleviating competition for resources such as nutrients, water and light (Brand 1991; Munson et al. 1993; Matsushima and Chang 2006). However, experimental data on the effect of understory vegetation control on the growth of planted white spruce seedlings on reclaimed oil sands soils are still lacking. Therefore, studying the effect of two common silvicultural treatments (nursery nutrient loading and understory vegetation control) on seedling growth in reclaimed oil sands areas will provide insights into the implementation of the treatments at a larger scale. In the present study, we investigated the growth and N uptake responses of white spruce seedlings to nursery nutrient loading and understory vegetation control over two growing seasons after outplanting. We hypothesized that (1) exponential fertilization would increase seedling growth after outplanting through internal remobilization of N stored in the reserve and (2) understory vegetation control would also improve seedling growth by increasing soil water availability and N uptake from the soil. As the mechanisms (i.e., CO2 supply through stomata and CO2 consumption by carboxylation) underlying the influences of water and N availability on seedling growth can be deciphered by the carbon isotope ratio (13C/12C, expressed as d13C) (Livingston et al. 1999; Choi et al. 2005), the foliar d13C of seedlings was also determined.

Materials and methods Nursery production of seedlings Containerized white spruce seedlings were produced in styroblocks (615A) with 45 cavities per block, 336 mL volume per cavity (Beaver Plastics Ltd., Acheson, AB, Canada) filled with a mixture (pH 5.5) of unfertilized peat moss and perlite (9:1, v:v) in a greenhouse at the Smoky Lake Forestry Nursery (54°050 N, 112°140 W) in Alberta, following the conventional and exponential fertilization procedures described in Hu et al. (2015). The larger size of container (similar to the size used by Sloan et al. 2016) than what would normally be used for white spruce seedling production was chosen in this experiment to help seedlings better acclimatize after outplanting in oil sands restoration. A commercial water-soluble fertilizer [Plant-Prod with 20:20:20 N (a combination of 5.9% nitrate, 3.9% ammonium and 10.2% urea N):P2O5:K2O plus micronutrients, Plant Products Co. Ltd., Brampton, ON, Canada] was applied for 22 weeks at a total rate of 300 mg N seedling-1 for conventional fertilization, the rate used by Smoky Lake Forestry Nursery. For a modified exponential fertilization regime, 450 mg N seedling-1 was used following the results of Hu (2012) who tested 300, 450, 750, 900 and 1050 mg N seedling-1 in a

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modified exponential model and recommended 450 mg N seedling-1 as the optimum rate for exponential fertilization without causing any toxicity. The high rate of fertilizer used in this experiment was partly due to the larger size of containers used for seedling production. For conventional fertilization, an equal amount of fertilizer was applied each week, and for the modified exponential fertilization regime, the weekly fertilization rate decreased from 24 mg in the first week to 0.77 mg in the ninth week and then increased to 101.09 mg per seedling in the twenty-second week following the fertilization model in Birge et al. (2006). To label the seedlings with 15N that would allow the quantification of N transfer from old tissues to newly grown tissues via remobilization of N, the seedlings were fed with 15Nurea (60.0 15N atom%) in week 16, with the amount of 15N-urea added replacing 20% of the fertilizer (14.4 mg seedling-1 in conventional and 16.4 mg seedling-1 in exponential fertilization). During the 22 weeks, the seedlings were grown at 65–85% humidity and 18–30 °C temperature under a 20 h daily photoperiod using sodium vapor lamps at a light intensity 250 lmol m-2s-1. Seedlings were hardened for two weeks from week 22 in the greenhouse without fertilizer application by lowering the photoperiod to 8 h before the seedlings were harvested (Blgras and D’aoust 1992). The harvested seedlings were then kept in cold storage at -2 °C until they were planted to the field.

Site description and soil properties The study was conducted at a reclaimed site (56°580 N and 111°190 W) in the Athabasca Oil Sands region in northern Alberta where land disturbed for oil sands extraction has been reclaimed after open-pit mining activities. The study area belongs to a continental boreal climate with long cold winters and short warm summers; the mean annual temperature in the last 30 years was 1 °C and the mean annual precipitation was 418.6 mm (316.3 mm as rainfall and 102.3 mm as snowfall) (Environment Canada 2015). The mean temperature and total precipitation in the growing season (May–September) were 13.8 °C and 297.7 mm, respectively, in 2014 and were 13.8 °C and 194.9 mm, respectively, in 2015 (Environment Canada 2016). The vegetation of the forests in the study sites were composed of coniferous trees such as black spruce (Picea mariana [Mill.] BSP), white spruce, jack pine (Pinus banksiana Lamb.) and tamarack (Larix lariciana [Du Roi] K. Koch) and deciduous species including trembling aspen, white birch (Betula papyrifera Marshall), and balsam poplar (Populus balsamifera L.) in pure as well as in mixed-wood stands (Fung and Macyk 2000). During open pit mining, the forest was clear-cut, and surface soil, overburden and oil sands were removed from the area. After oil sands extraction, overburden substrates were brought back to the area as part of the reclamation process, and peat mineral soil mix was then placed on these substrates to complete land reclamation in 2008. The site was seeded with barley (Hordeum vulgare L.) in 2009 to control erosion and to stabilize the reclaimed soil. The percent coverage by understory vegetation (excluding moss and lichen) was assessed using two 50 9 50 cm sized quadrat per plot and was 63.7 ± 9.3% (mean ± SE) and 68.2 ± 11.4% in August of the first (2014) and the second (2015) growing seasons, respectively. The dominant understory species were Calamogrostis canadensis [Michx.] Beauv., Agropyron trachycaulum [Link] Malte, Hordeum vulgare L., Carex spp., Epilobium angustifolium L., Melilotus alba Desr, Taraxacum officinale Weber, Lathyrus venosus Muhl., Sonchus arvensis L., and Erigeron canadensis L. The soils of natural ecosystems adjacent to the study site are xeric to sub-hydric Brunisols and Luvisols in uplands and Gleysols and Organic soils in lowlands (Oil Sands Vegetation Reclamation Committee 1998). The soil of our study site was further

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6.71 (0.06)

6.85 (0.07)

0–10

10–30

1.17 (0.14)

1.22 (0.15)

Electrical conductivity (ds m-1)

0.69 (0.15)

0.59 (0.12)

Bulk density (g cm-3)

Values are means with standard errors in the parentheses (n = 16)

pH

Depth (cm)

Table 1 Selected properties of the soil in the study site

88.70 (28.65)

93.12 (19.22)

Total C (g kg-1)

3.45 (1.35)

3.71 (0.9)

Total N (g kg-1)

29.45 (2.72)

26.77 (2.22)

C:N

5.30 (1.16)

7.20 (0.9)

NHþ 4 N (mg kg-1)

3.62 (1.24)

6.05 (1.38)

NO 3 N (mg kg-1)

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characterized by analyzing physical and chemical properties after collecting soil samples from plots (see below for the establishment of the plots) (Table 1). Soil samples were collected in June 2014 (about midseason) using an auger at two depths (0–10 and 10– 30 cm) from five randomly selected points in each plot and composited for each depth. The samples were analyzed for total carbon (TC) and total N (TN) by the combustion method using an automated elemental analyzer (NA-1500 series, Carlo Erba, Milan, Italy),  NHþ 4 N with the indophenol blue method (Keeney and Nelson 1982), NO3 N with the vanadium oxidation method (Miranda et al. 2001), pH at 1:2 (w/v) of soil:CaCl2 solution (0.01 mol L-1) using a pH meter (Orion, Thermo Fisher Scientific Inc., Beverly, MA, USA), and electrical conductivity at 1:1 (w/v) of soil:water using an AP75 portable waterproof conductivity/TDS meter (Thermo Fisher Scientific Inc., Waltham, MA, USA). The bulk density of the soils was measured using a steel corer (98.1 cm3). The soil moisture content in the upper 10 cm soil was measured at three points in each plot once every month by TDR 300 soil moisture meter (Spectrum Technologies, INC., IL, USA) in the active growing season (mid May to mid September) of 2014 and 2015. The average volumetric water content of the soil for the entire active growing season was 36.73 ± 9.6% (v/v) and 30.43 ± 7.2% (v/v) in the first and second growing season, respectively.

Experimental design and plot establishment The experiment used a completely randomized block design with two treatments: nursery fertilization regime (conventional vs. exponential fertilization) and understory vegetation (i.e., weed) control (weed-intact vs. weed-removed) in four replicated blocks to block out possible environmental gradients such as slope and weed density. In June 2014 (six years after reclamation), 16 plots (each plot size was 6 9 6 m) were established with a 2 m buffer zone between adjacent plots. In each plot, 36 seedlings were transplanted with 1 m spacing on June 12, three days after being taken out of cold storage. In total, 576 seedlings (2 fertilization regimes 9 2 weed control treatments 9 36 seedlings per plot 9 4 replications) were planted in the plots. Measurements of growth and N uptake were conducted with the 25 seedlings located at the center of each plot to avoid the edge effect. In the weed removal treatment, weeds were controlled manually by cutting the aboveground vegetation every three weeks during the active growing season from May to September. Understory vegetation was cut short around the seedlings in the weed-intact plots to just below the seedlings to eliminate potential light limitation for seedling growth. Seedlings were grown until the end of the active growing season of 2015 under natural conditions except for the weed control.

Measurement of seedling growth and N uptake Seedling survival rate was measured at the end of the growing season in 2014 and 2015. Seedling height and root collar diameter (RCD) at ground level were measured at the time of outplanting in June and at the end of the active growing season in September 2014 and 2015. Shoot height was measured with a meter stick (1 mm accuracy) and RCD was measured with a Vernier caliper (0.05 mm accuracy) in two directions perpendicular to each other and the average value was used to represent the RCD of seedlings. Absolute growth was calculated as the difference between final and initial size, and percentage growth increment was calculated as the change in size at the end of the first and second growing seasons relative to the initial size.

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Biomass, total N and 15N in seedling components were also determined at the time of outplanting to the field and at the end of the first and second growing seasons. At the time of outplanting, five seedlings of each nursery fertilization regime were randomly selected from each of three storage boxes representing different replications in the cold storage. Seedlings were composited per replication separately for the conventional and exponential fertilization treatments and were used for measurement of biomass and total N (concentration and 15N abundance). At the end of the growing season, two (the first growing season) or four (the second growing season) seedlings were randomly selected and destructively harvested by cutting the seedling at ground level and roots were excavated intact with a shovel. A smaller number of seedlings were harvested at the first sampling to minimize site disturbance and to leave seedlings for the second harvest. The roots were washed onto a sieve (0.5 mm mesh) to remove soil and peat materials. The shoots and roots were then rinsed three times with deionized water. For seedlings sampled from the plots, shoots were separated into new (current year) and old (previous year) shoots. The shoots were further separated into stems and needles. Seedling component samples were composited per plot and dried at 70 °C for 72 h to determine biomass. The plant samples were mixed thoroughly and a portion of the samples were ground to fine powder with a Mixer Mill MM 200 ball mill (Thomas Scientific, Swedesboro, NJ, USA) for N analysis. The N concentration, 15N atom %, and 13C atom % (d13C) were analyzed using a stable isotope ratio mass spectrometer (Thermo Delta Plus XP IRMS, Waltham, MA, USA) linked to a CN analyzer (Costech 4010, Valencia, CA, USA) at the Stable Isotope Facility of the University of Wyoming, Laramie, WY, USA. The N in the newly formed seedling components (stem and needle) was separated into N derived from old plant tissue (NDFPold) and that from soil (NDFS). The NDFPold (mg N) was calculated using the following equation (Hauck and Bremner 1976; Dele´ens et al. 1994): NDFPold ¼ TNnew  ðAnew =Aold Þ

ð1Þ

where TNnew is the total N (mg) content in the new tissue calculated as N concentration (mg g-1) 9 dry weight of biomass (g), Anew is 15N atom % excess of N (15N atom % - 0.3663) in the new tissue, and Aold is 15N atom % excess of N in the old tissue. The Aold was calculated as the biomass-weighted mean 15N atom % of needles, stems and roots of seedlings at the time of outplanting to the field for the first growing season, and as that of current and 1-year-old needle and stem and root of the seedlings measured at the end of the first growing season for the second growing season. The NDFS (mg N) was calculated as, NDFS ¼ TNnew  NDFPold

ð2Þ

The amount of N remobilized from seedling components (needles, stems and roots) in the first growing season was calculated by mass balance using 15N abundance, N concentration and biomass of old tissues at nursery production and at the end of first growing season.

Statistical analyses Data were tested for normality of distribution and homogeneity of variance with Shapiro– Wilk and Levene’s tests, respectively. A log-transformation (base 10) was applied for NDFS of new needle and new stem of the first growing season data and NDFPold of new needle and NDFS of new needle and stem of the second growing season data to meet the

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assumption of normality, and back-transformed data are reported. The effect of fertilization regime during nursery production on seedling size, biomass, total N and 15N was analyzed by a one-way analysis of variance (ANOVA) using the Proc MIXED model (SAS 9.2, SAS Institute, Cary, NC, USA). For the field experiment, three-way ANOVA was conducted to examine the effect of fertilization regime and weed removal over two seasons. Since there was no significant interaction between time and other factors, we used two-way ANOVA results to examine the fixed effects of fertilization regime and weed removal and the random effect of block on dependent variables such as seedling growth, N retranslocation and N uptake from the soil. Regression analyses were conducted to investigate the relationship between variables such as foliar d13C and NDFPold versus growth parameters and N concentration of seedlings. The means were separated by Tukey’s test. The level of significance for all statistical tests was set at a = 0.05.

Results Seedling growth and N uptake in the nursery stage Total seedling biomass did not differ between conventional and exponential fertilization treatments; but exponential fertilization produced greater (P = 0.045) root biomass (Table 2). Height and RCD were greater (P \ 0.001 for both) in conventionally than in exponentially fertilized seedlings. Exponential fertilization increased (P = 0.003) N concentration of stem by 36% as compared to conventional fertilization but did not affect those of needle and root (Table 2). The N content in the seedling did not differ between fertilization regimes regardless of the seedling component (Table 2); and thus exponential fertilization in the nursery was not successful in increasing nutrient content in the seedlings produced.

Seedling growth and N status after outplanting Survival rates of planted seedlings were 92 and 89% in the first and second growing seasons, respectively, not being different between fertilization and weed removal treatments (data not shown). The effects of the treatments on seedling biomass were not significant at the end of the first growing season (data not shown). At the end of the second growing season, however, the total and each component biomass of the seedlings were affected by either nursery fertilization, field weed control or both, without interaction between the treatments (Table 3). Seedlings nursed with exponential fertilization had lower old needle and root biomass than those nursed with conventional fertilization. Weed control increased biomass of new needles by 54%, new stems by 97%, roots by 46%, and total biomass by 29% over the weed-intact treatment. The height and percent height increment were affected by nursery fertilization in both growing seasons while weed control had no effect on height growth (Fig. 1). The height was greater in conventional fertilization than in exponential fertilization in both growing seasons (Fig. 1a). The percent height increment of seedlings during the two growing seasons was greater (P = 0.001 in the first and P \ 0.001 in the second growing seasons) for exponentially (87 and 135% in the first and second seasons, respectively) than for conventionally fertilized seedlings (57 and 89%, respectively).

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1.36 b (0.15)

5.38 (0.68)

Root

Whole plant

2.09 (0.08)

5.24 (0.27)

1.79 a (0.10)

1.35 (0.12)

30.15 (0.91)

24.59 (0.78)

22.25 (1.10)

16.61 b (0.38)

34.07 (3.55)

27.43 (1.98)

23.02 (0.96)

22.53 a (0.86) 133.51 (11.77)

30.65 (2.82)

23.54 (2.50)

79.32 (7.16)

CF

143.07 (4.43)

41.31 (1.56)

30.45 (1.88)

71.30 (4.48)

EF

N content (mg N seedling-1)

0.98 b (0.01)

0.95 b (0.02)

0.96 (0.01)

0.91 b (0.03)

1.14 a (0.04)

1.02 (0.07)

1.15 a (0.02)

1.19 a (0.04)

EF

N abundance (%) CF

15

Values are means with standard errors in the parentheses (n = 3). Different letters indicate significant differences in the values between fertilization regimes

2.61 (0.35)

1.41 (0.24)

Stem

EF

CF

CF

EF

N concentration (mg N g-1)

Dry mass (g seedling-1)

Needle

Seedling component

Table 2 Biomass and N concentration, N content and 15N abundance (%) in different organs/components of seedlings with different fertilization regimes (conventional, CF; exponential, EF) before transplanting at the end of the nursery phase

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123

Exponential

0.754

F9W

6.96 (0.64)

0.813

0.011

0.520

7.78 a (0.62)

5.83 b (0.24)

6.55 (0.52)

0.295

0.003

0.808

7.06 a (0.55)

4.58 b (0.17)

5.74 (0.64)

0.791

0.010

0.865

2.49 a (0.32)

1.26 b (0.06)

1.91 (0.32)

1.84 (0.33)

New stem

0.748

0.362

0.007

5.40 (0.42)

5.84 (0.51)

4.84 b (0.26)

6.41 a (0.46)

Old needle

0.995

0.043

0.072

9.36 a (0.59)

7.76 b (0.40)

7.87 (0.66)

9.25 (0.39)

Old stem

0.514

0.005

0.034

6.36 a (0.52)

4.35 b (0.42)

4.65 b (0.65)

6.06 a (0.42)

Root

0.914

0.010

0.074

30.67 a (2.15)

23.79 b (1.09)

25.01 (2.32)

29.46 (1.55)

Total

a

Values in bold indicate significance at P \ 0.05

Values are means with standard errors in the parentheses (n = 4). Different letters indicate significant differences in the values within fertilization regime and weed control treatments

0.609

0.007

F

W

ANOVA (P [ F)a

12.21 a (1.27)

Weed-removed

Effects

7.85 b (0.52)

Weed-intact

Weed control (W)

10.32 (1.30)

9.64 (1.21)

Conventional

5.89 (0.61)

New needle

New needle

New stem

Biomass (g)

N concentration (mg N g-1)

Fertilization regime (F)

Treatment

Table 3 Effects of fertilization regime (conventional and exponential in nursery production) and weed control (weed-intact and weed-removed in the field) on nitrogen concentration and biomass of seedling components in the second growing season after outplanting

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Fig. 1 Seedling height and RCD (root collar diameter) at the end of nursery production and over two growing seasons after outplanting in the field. Different letters represent significant differences between the treatments at P \ 0.05. Vertical bars are standard errors of means (n = 4)

Weed removal significantly affected absolute as well as percent RCD increment in the second but not in the first growing season (Fig. 1b), but nursery fertilization did not affect RCD in both growing seasons. Weed removal resulted in 25% greater (P \ 0.001) absolute RCD growth compared to that of the weed-intact factor in the second growing season. The percent RCD increment in weed removed plots (152%) was significantly greater (P \ 0.001) than that in the weed-intact plots (104%) in the second growing season. The N concentration in seedling components decreased in the second growing season regardless of the nursery fertilization and field weed control treatments (Table 3). In the first growing season, N concentration in seedling components was not affected by the nursery fertilization and weed control. In the second growing season, however, weed removal increased N concentration by 58% in new needles and by 34% in new stems (Table 3). The pattern of N content followed that of N concentration (Fig. 2). The nursery fertilization treatment did not affect N content in both growing seasons. With marginal significance (P \ 0.100), weed removal increased N content of new needles by 26% and the total N in the seedling by 25% in the first growing season. In the second growing season, the increment was 152% (P = 0.005) for new needles and 175% (P = 0.010) for new stems.

Foliar d13C, NDFPold and NDFS Nursery fertilization significantly affected foliar d13C in new needles in the first growing season (P = 0.048) but not in the second growing season (P = 0.794); meanwhile, weed

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Fig. 2 Effects of weed removal treatments on total nitrogen (N) in seedling components over two growing seasons: a the first growing season and b the second growing season. Different letters represent significant differences between the treatments at P \ 0.05. Vertical bars are standard errors of means (n = 4)

control affected foliar d13C in new needles in both growing seasons and in old needles in the first growing season (Fig. 3). Specifically, weed removal decreased d13C in new needles in the first (P \ 0.001) and second growing seasons (P = 0.014) and old needle in the first growing season (P = 0.039) as compared to the weed intact treatment. Regression analyses of the relationship between foliar d13C and seedling growth parameters in the second growing season showed that d13C was positively correlated with seedling biomass and foliar N concentration (Fig. 4). The amount of NDFPold and thus the internal remobilization of N were not affected by the nursery fertilization treatment in both growing seasons but were increased by weed removal in the second growing season regardless of the nursery fertilization regime used to produce the seedlings (Fig. 5a, b). In the second growing season, weed removal increased NDFPold by 40% (P = 0.040) in needles and by 50% (P = 0.005) in the stem. On the other hand, NDFS was affected by fertilization in the first growing season and by weed removal treatment in both growing seasons (Fig. 5c, d). Exponential fertilization increased NDFS in needles by 134% (P = 0.042) and weed removal increased NDFS in needles in both the first (P = 0.043) and second (P = 0.006) growing seasons, and in stems only in the first growing season (P = 0.008) as compared to weed-intact plots. The contributions of NDFPold and NDFS to total N in new tissues changed over the growing seasons (Fig. 5; Table S1). The contribution of NDFPold in new needles was much greater (71% of total N demand) in the first than in the second growing season (46%) in the weed-removed plots. However, in the weed-intact plots, the NDFPold was greater than that of NDFS in both

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711

Fig. 3 Foliar d13C of seedlings in the first and second growing seasons. Different letters represent significant differences between the treatments at P \ 0.05. Vertical bars are standard errors of means (n = 4)

growing seasons. The percent contribution of NDFPold to total N in new tissues was decreased (P = 0.005) only by field weed control but not affected by nursery fertilization (Table S1). The NDFPold in the second growing season was positively correlated with biomass and height growth (Fig. S1).

Discussion Effects of exponential fertilization on nutrient reserve and growth of seedling Exponential fertilization of seedlings during nursery production aims to increase the nutrient reserve in seedling components to take advantage of luxury uptake of nutrients (Timmer 1997; Salifu and Timmer 2003). Many studies reported an increase in the nutrient reserve in exponentially fertilized seedlings of coniferous species such as black spruce (Malik and Timmer 1996; Salifu and Timmer 2003) and Chinese-fir (Cunninghamia lanceolata [Lamb] Hook) (Xu and Timmer 1999). In our previous studies, we also found that the nutrient reserve of the seedling was significantly increased for trembling aspen

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Fig. 4 Relationships between foliar d13C with growth parameters and foliar N concentration of seedlings in the second growing season (n = 16)

Fig. 5 Effects of nursery fertilization and weed removal treatments on nitrogen (N) derived from older components of the plant (NDFPold) and N derived from the soil (NDFS) by new tissues in the first and second growing seasons: a, b NDFPold, and c, d NDFS. Different letters represent significant differences between the treatments at P \ 0.05. Vertical bars are standard errors of means (n = 4)

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(Pokharel and Chang 2016) and jack pine (Pokharel et al. 2017) by using a similar exponential fertilization used in the present study. However, in this experiment, the modified exponential fertilization technique did not increase the N reserve in white spruce seedlings (Table 2). This is probably due to the very low efficiency of a few last applications of fertilizer because of high fertilizer rate used in the experiment. Despite exponential fertilization in the nursery did not increase N reserve and total biomass of the seedling, a greater relative height growth was observed in the exponentially fertilized seedlings with smaller initial height compared to the conventionally fertilized ones. The greater relative height growth for seedlings with a smaller initial height is a wellknown allometric principle that works under many circumstances for coniferous seedlings (van den Driessche and van den Driessche 1991). This is because the ratio of non-assimilatory versus assimilatory biomass increases with seedling size (Brand et al. 1987) and larger seedlings may experience planting check especially on harsh sites (South and Zwolinski 1997). The NDFPold was not affected by nursery fertilization (Fig. 5a) and this is inconsistent with our finding with a different tree species, where N retranslocation in exponentially fertilized jack pine seedlings was 2.5-fold greater than that in the conventionally fertilized seedlings on the same reclaimed soil (Pokharel et al. 2017). The lack of exponential fertilization effect on N retranslocation in this study was due to the similar nutrient reserves in the needles of seedlings at the end of nursery production regardless of fertilizer treatment (Table 2). The higher N concentration in the stem of exponentially fertilized seedlings did not affect NDFPold, indicating that the N stored in the stem was not retranslocated to newly growing tissues of white spruce seedlings (Grossnickle 2000). Therefore, exponential fertilization did not increase N reserve at the nursery and thus NDFPold was not changed after field planting; however, the increased root biomass led to greater height growth which may be beneficial in terms of competition for the light resource (Claveau et al. 2002).

Effects of weed control on seedling growth, N retranslocation and soil N uptake Weed competition has been widely reported to affect the growth of seedlings in various environments such as in the clear-cut area (Munson et al. 1995) and on reclaimed oil sands sites (Pokharel and Chang 2016). This is because removing competitive weeds can increase the availability of resources such as water, nutrients and light to benefit the growth of crop plants (Davis et al. 1999; Franklin et al. 2012). In our study, the greater biomass (Table 3) and RCD growth (Fig. 1) of seedlings in weed-removed plots supports the beneficial effects of weed control on seedling growth (Man et al. 2008). Sloan et al. (2016) suggested that resources other than N could limit plant growth in reclaimed soils; the resource limitation varies with site quality, time since reclamation and fertilization history of the reclamation site. In the study site, however, soil available N (particularly NO 3 N) was low (Table 1) and thus N was likely to be the growth limiting factor. In a study for identifying growth limiting factors of white spruce on reclaimed sites near our study site with similar reclamation practices and soil characteristics, Duan and Chang (2015) also found that soil N availability was the dominant growth limiting factor. Therefore, weed control might eliminate the competition to seedlings for soil N as evidenced by the higher foliar N concentration in weed-removed plots (Table 3). Though weed removal may also

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change soil moisture conditions, there was no difference in soil moisture content between weed-intact and weed-removed plots (data not shown). The less negative foliar d13C in weed-removed than in weed-intact treatments (Fig. 3) indicates that weed control affected stomatal conductance or carboxylation rate due to changed water or N availability (Choi et al. 2005). According to the 13C discrimination model (Farquhar et al. 1989), foliar d13C becomes less negative by either decreased stomatal conductance due to water limitation or increased carboxylation under high N availability. In our study, however, the lack of differences in soil moisture content between weed control treatments suggests that N availability rather than water availability had affected the foliar d13C (Livingston et al. 1999; Choi et al. 2005); this is further supported by the positive correlation between foliar d13C and N concentration (Fig. 4c). In our study, water availability was less likely to be a limiting factor on sites reclaimed with overburden substrate covered with peat mineral soil mix because of the high water holding capacity of the peat mineral soil mix. In addition, the positive correlation between foliar d13C and seedling growth (Fig. 4a, b) further confirms that foliar d13C increased due to increased carboxylation since if water stress had limited seedling growth there should be a negative correlation between foliar d13C and tree growth (Choi et al. 2005). Based on the mass balance calculation of 15N in seedling components, N remobilization from needles was five times greater than that from the root in the first growing season (data not shown) while N remobilization from the stem was negative, indicating that nutrient stored and remobilized from needles was more important than that from root and stem in white spruce, similar to findings in other evergreen species (Millard and Grelet 2010; Uscola et al. 2015). On average of all treatments, N retranslocation provided about 78% of the total N demand of new needles in the first year growth, indicating that the new growth was sustained mainly by internal N remobilization. But in the second growing season, the contribution of NDFPold to growth demand of new needles dropped to below 50% in the weed-removed plots. This indicates that N retranslocation in white spruce seedling was very important to sustain new growth immediately after outplanting (Villar-Salvador et al. 2015); however, its contribution decreased sharply in subsequent years. Coniferous seedlings are known to have slow initial root development, and thus the interaction between roots and rhizosphere soils are not likely to develop enough to exploit soil N in the first year after transplanting (Grossnickle 2000). In a white spruce stand established with bare-root seedlings, McAlister and Timmer (1998) also observed very small nutrient uptake from the soil by the plants in the first growing season after outplanting and concluded that nutrient accumulation in new growth was met mostly by internal rather than external nutrient sources. When the N demand of new tissue growth cannot be met by N uptake from the soil, plants require increasing N remobilization to meet the demand. Our result suggests that the N retranslocation in white spruce was related to the growth rate of new tissue (Villar-Salvador et al. 2015). In a pot study of N retranslocation in Sitka spruce [Picea sitchensis (Bong.) Carr.] Millard and Proe (1993) also showed that N retranslocation was controlled by the sink strength of current-year growth. However, positive relationships between NDFPold and growth parameters such as biomass and RCD growth in our study (Fig. S1) are opposite to the results of Munson et al. (1995), in which N retranslocation was controlled by the short-term imbalance in supply versus the demand for nutrients rather than nutrient demand by the current year growth.

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Conclusions Exponential fertilization in the nursery increased the percent height growth but not the component biomass and N retranslocation in white spruce seedlings after outplanting. Weed removal increased soil N availability to and N uptake by, N retranslocation within and growth of the outplanted seedlings. The N requirement for new tissue growth after outplanting was mainly sustained by NDFPold. We conclude that internal nutrient cycling in white spruce seedlings planted on reclaimed oil sands soils was important during early establishment (i.e., the first year) but soil nutrient availability played a more significant role in subsequent years. Vegetation management in the form of weed control was effective in increasing soil N availability and improving white spruce seedling growth. Further research on the optimization of exponential fertilization to build up the nutrient reserve in white spruce seedlings during nursery production is needed and expanding the research to reclaimed sites with different cover soils or different reconstruction techniques will provide data on the applicability of the nutrient and vegetation management techniques in oil sands reclamation. Acknowledgements We would like to thank the Land Reclamation International Graduate School (LRIGS), which was funded through a CREATE (Collaborative Research and Training Experience) grant from the National Science and Engineering Research Council of Canada (NSERC), for financial support in the form of a fellowship to the senior author and the Environmental Reclamation Research Group (ERRG) of the Canadian Oil Sands Network for Research and Development (CONRAD) for funding this research. We would like to acknowledge Suncor Energy Inc. for logistic support. We would also like to thank Dr. Jin Hyeob Kwak, Ms. Kangyi Lou and Ms. Stephanie Ibsen for their great help in field and laboratory work and Drs. Francis Salifu, Phil Comeau, Douglass Jacobs, Xiao Tan and Carmela Arevalo for discussions and guidance.

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