Responses of fine roots and soil N availability to short-term nitrogen fertilization in a broad-leaved Korean pine mixed forest in northeastern China.

Abstract

Knowledge of the responses of soil nitrogen (N) availability, fine root mass, production and turnover rates to atmospheric N deposition is crucial for understanding fine root dynamics and functioning in forest ecosystems. Fine root biomass and necromass, production and turnover rates, and soil nitrate-N and ammonium-N in relation to N fertilization (50 kg N ha(-1) year(-1)) were investigated in a temperate forest over the growing season of 2010, using sequential soil cores and ingrowth cores methods. N fertilization increased soil nitrate-N by 16% (P<0.001) and ammonium-N by 6% (P<0.01) compared to control plots. Fine root biomass and necromass in 0-20 cm soil were 13% (4.61 vs. 5.23 Mg ha(-1), P<0.001) and 34% (1.39 vs. 1.86 Mg ha(-1), P<0.001) less in N fertilization plots than those in control plots. The fine root mass was significantly negatively correlated with soil N availability and nitrate-N contents, especially in 0-10 cm soil layer. Both fine root production and turnover rates increased with N fertilization, indicating a rapid underground carbon cycling in environment with high nitrogen levels. Although high N supply has been widely recognized to promote aboveground growth rates, the present study suggests that high levels of nitrogen supply may reduce the pool size of the underground carbon. Hence, we conclude that high levels of atmospheric N deposition will stimulate the belowground carbon cycling, leading to changes in the carbon balance between aboveground and underground storage. The implications of the present study suggest that carbon model and prediction need to take the effects of nitrogen deposition on underground system into account.

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Responses of Fine Roots and Soil N Availability to Short-
Term Nitrogen Fertilization in a Broad-Leaved Korean
Pine Mixed Forest in Northeastern China
Cunguo Wang1,2, Shijie Han1*, Yumei Zhou1, Caifeng Yan1,2, Xubing Cheng1,2, Xingbo Zheng1,2, Mai-He
Li1,3*
1State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China, 2Graduate University of Chinese Academy
of Sciences, Beijing, China, 3Swiss Federal Research Institute WSL, Zuercherstrasse, Birmensdorf, Switzerland
Abstract
Knowledge of the responses of soil nitrogen (N) availability, fine root mass, production and turnover rates to atmospheric N
deposition is crucial for understanding fine root dynamics and functioning in forest ecosystems. Fine root biomass and
necromass, production and turnover rates, and soil nitrate-N and ammonium-N in relation to N fertilization (50 kg N ha21
year21) were investigated in a temperate forest over the growing season of 2010, using sequential soil cores and ingrowth
cores methods. N fertilization increased soil nitrate-N by 16% (P,0.001) and ammonium-N by 6% (P,0.01) compared to
control plots. Fine root biomass and necromass in 0–20 cm soil were 13% (4.61 vs. 5.23 Mg ha21, P,0.001) and 34% (1.39
vs. 1.86 Mg ha21, P,0.001) less in N fertilization plots than those in control plots. The fine root mass was significantly
negatively correlated with soil N availability and nitrate-N contents, especially in 0–10 cm soil layer. Both fine root
production and turnover rates increased with N fertilization, indicating a rapid underground carbon cycling in environment
with high nitrogen levels. Although high N supply has been widely recognized to promote aboveground growth rates, the
present study suggests that high levels of nitrogen supply may reduce the pool size of the underground carbon. Hence, we
conclude that high levels of atmospheric N deposition will stimulate the belowground carbon cycling, leading to changes in
the carbon balance between aboveground and underground storage. The implications of the present study suggest that
carbon model and prediction need to take the effects of nitrogen deposition on underground system into account.
Citation: Wang C, Han S, Zhou Y, Yan C, Cheng X, et al. (2012) Responses of Fine Roots and Soil N Availability to Short-Term Nitrogen Fertilization in a Broad-
Leaved Korean Pine Mixed Forest in Northeastern China. PLoS ONE 7(3): e31042. doi:10.1371/journal.pone.0031042
Editor: Ben Bond-Lamberty, DOE Pacific Northwest National Laboratory, United States of America
Received September 12, 2011; Accepted December 30, 2011; Published March 6, 2012
Copyright: ? 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Financial support was obtained from National Basic Research Program of China (NO. 2011CB403200), National Natural Science Foundation of China
(NO. 40930107) and Knowledge Innovation Program of Chinese Academy of Sciences (NO. KSCX-YW-Z-1022). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: hansj@iae.ac.cn (SH); maihe.li@wsl.ch (MHL)
Introduction
Fine roots (,2 mm in diameter) are important nutrient sources
and sinks, and play important roles in water and nutrient uptake in
terrestrial ecosystems [1,2]. Fine root biomass represents less than
2% of total ecosystem biomass, whereas fine root production may
account for up to 30–75% of total net primary production [1,3,4].
In addition, fine root turnover can serve as a modulator of soil C
and N cycling and can contribute equivalent or greater C and N
than aboveground litterfall to soil organic matter pools [3,5].
Anthropogenic N deposition significantly increases biologically
available N in ecosystems [6,7], and soil N availability is one of the
crucial factors affecting fine root dynamics [4,8,9]. During the past
decades, the effects of N availability on fine root production and
turnover have been studied extensively. However, knowledge of
potential effects of N availability on fine root dynamics is still
insufficienttodevelopaconclusiveexplanationoftheNeffectsonfine
rootdynamics[8,10].Previousstudiesfoundthattherewasanegative
relationship between fine root (including mycorrhizal) biomass and
soil N availability [8,11,12]. Four possible relationships among fine
root production, turnover rates and increased soil N availability have
been reported [8,13]: (1) fine root production and turnover rates
increased; (2) fine root production and turnover rates decreased; (3)
fine root production increased, while fine root turnover rates
decreased; and (4) fine root production decreased, while fine root
turnoverratesincreasedwithincreaseinsoilNavailability.Aberetal.
[14] found that the form of N may be more important to fine root
mass than the total amount of N, and they [14] reported that the
seasonal dynamics of fine root biomass in stands with ammonium-N
as the dominant N form significantly differed from those in stands
withnitrate-NasthedominantNform.However,onlyafewattempts
have been made to confirm those relationships between different N
forms and fine root mass in different forest ecosystems with different
soil properties, tree species, and ecosystem productivity.
Current techniques do not allow us to directly measure the fine
root production and turnover rates in forest ecosystems, but a
combination of sequential soil cores and ingrowth cores may
credibly quantify fine root dynamics [15,16,]. The sequential soil
cores approach has been commonly used to estimate fine root
production and turnover rates [17], and the ingrowth cores
method is recognized to be suitable for comparing root growth
among treatments, sites or species, because the growth of fine roots
into ingrowth cores represents the ‘‘current growth potential’’
rather than the absolute fine root production [17,18,19].
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The broad-leaved Korean pine (Pinus koraiensis Siebold & Zucc)
mixed forest is the dominant vegetation type in northeastern
China. Wet and dry N deposition in that area has reached
23 kg N ha21year21exceeding the critical load in the broad-
leaved Korean pine mixed forest [20,21]. We used sequential soil
cores combined with ingrowth cores method to investigate fine
roots in relation to soil N availability after N fertilization in the
broad-leaved Korean pine mixed forest. Soil nitrate-N and
ammonium-N contents, fine root biomass, necromass, production,
and turnover rates were determined. Our primary objective was to
understand effects of different forms of N on fine root dynamics.
We hypothesized that: (1) N fertilization decreases fine root
biomass and necromass; (2) fine root production and turnover
rates increase with N fertilization, and (3) fine root mass is
correlated with soil nitrate-N and ammonium-N contents.
Materials and Methods
No specific permits were required for the described field studies.
The study was carried out within the research forest of Changbai
Forest Ecosystem Research Station (CBFERS) established in 1979.
Research activities within that research forest do not need any
specific permissions from any government levels, but need to
inform Professor Han SJ (Director CBFERS, co-author of the
present paper).
Study area and experimental design
The study was conducted in an old growth forest (,200 years
old) of broad-leaved Korean pine mixed forest in Changbai
Mountain (42u249N, 128u59E), northeastern China. Mean annual
temperature is 3.5uC, with the highest monthly mean temperature
of 20.5uC occurring in August and the lowest monthly mean
temperature of 216.5uC occurring in January. The mean annual
precipitation is 700 mm, 70–80% of which falls during the
growing season from May to October. The soil, developed from
volcanic ash, is classified as Eutric cambisol (FAO classification)
with high organic matter content in surface layer. The soil is sandy
loam in 3–8 cm and gravelly sand in 8–24 cm depth. Soil bulk
density is 0.35 g cm3in 0–10 cm, 0.68 g cm3in 10–20 cm layer.
The other soil characteristics in the experimental site are shown in
Table 1. The dominant tree species are P. koraienssis, Fraxinus
mandschurica, Acer mono, and Tilia amurensis. The mean canopy
height and diameter at breast height were 15 m and 34.2 cm,
respectively. The tree density was 560 trees ha21. The above-
ground tree litterfall is 4.03 Mg ha21, and aboveground produc-
tivity is 10.02 Mg ha21. The main shrub species are Philadelphus
schrenkii, Euonymus alatus, Lonicera japonica, Corylus mandshurica, Deutzia
scabra, and the main herbaceous species are Anemone raddeana,
Anemone cathayensis, Cyperus microiria, Funaria officinalis, Adonis vernalis,
Brachybotrys paridiformis, and Filipendula palmate.
Six 50650 m plots (3 N-addition plots and 3 control plots) with
a buffer zone of .20 m between any two plots were randomly
established in September 2009. NH4NO3was diluted in 40 L of
deionized water. The additions were done monthly (six times from
May to October 2010) with a sprayer. The application rates of
50 kg N ha21year21were about double the annual total N
deposition (23 kg N ha21year21) in this area [21]. Three control
plots were simultaneously supplied with the same amount of
deionized water.
Soil collection and separation of roots
Two weeks after each N fertilization date, twenty soil cores were
collected randomly at each plot from May to October (May 20,
June 20, July 25, August 25, September 20, October 18), 2010.
Soil cores with 5 cm internal diameter were taken to a soil depth of
20 cm and separated into 0–10 cm and 10–20 cm soil samples.
According to Yang and Li [23], a sampling depth of 20 cm can
cover 80% of the total fine roots in this forest. The distance
between any two sampling locations was not less than 5 m. Each
sampled location was marked with a wooden stake to avoid
repeated sampling at the same or nearby locations. Soil cores were
placed immediately into plastic bags and kept at 24uC until later
processing (within one week). In the laboratory, ten out of the
twenty samples were washed free of soil by deionized water (1–
2uC) through a 0.5 mm mesh sieve. Roots ,2 mm in diameter
were separated into live and dead roots according to visible
morphological features (colour, luster, elasticity, degree of
cohesion of cortex, periderm and stele) through microscopic
inspection [9,24,25]. Roots were dried at 65uC to a constant mass
and weighed. Ash content was determined for a composite of fine
root samples for each plot and each sampling date.
The other ten soil cores were used to measure nitrate-N and
ammonium-N content. About 20 g of fresh soils were extracted
with 100 mL of 2.0 mol L21KCl solution and shaken for one
hour. Extracts were settled for approximately 20 minutes and
filtered. Ammonium-N content was determined using the
indophenol blue colorimetric method [26], and nitrate-N was
determined using the cadmium reduction method [27].
Ingrowth cores
Sixty polyethylene (2 mm mesh) bags (20 cm in length65 cm in
diameter) served as root ingrowth cores. Root-free soil was
collected outside the plots but within the same forest, sieved
through a 2 mm mesh and air-dried. Each bag filled with root-free
soil was put into a hole of 20 cm depth and 5 cm diameter in both
N fertilization and control plots in September 2009, and the
distance between any two ingrowth cores was not less than 5 m.
The ingrowth cores were re-sampled in September 2010, and
separated into 0–10 cm and 10–20 cm soil layer.
Calculation of fine root production and turnover rates
The fine root production was calculated using (1) sequential soil
cores in combination with the maximum-minimum method
(SC-MM) [17,18,24], and (2) ingrowth cores method (IC)
[10,18], respectively. In the SC-MM method, the production
was calculated from the differences between maximum and
minimum of the fine root biomass from May to October [28].
Table 1. Characteristics of the 0–20 cm soil in the studied forest sites.*
Total C g?kg21
Total N g?kg21
Total K g?kg21
Total P g?kg21
Soil pH (H2O) Organic matter g?kg21
C/N
Mean 156.60 7.1712.20 0.97 5.85270.0021.84
SE9.90 0.650.380.080.0617.10 0.96
*adapted from Zhang et al. [22]. Note g kg21=g per kg soil.
doi:10.1371/journal.pone.0031042.t001
Responses of Fine Roots to N Fertilization
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Mean values of fine root biomass per plot were calculated for each
sampling date. For the IC method, fine root biomass in the cores
refers directly to the fine root production [17]. Fine root turnover
rates were estimated as the ratio of the total fine root produced in
the growing season to the fine root biomass [14,29].
Statistical analysis
Homogeneity of variances was tested by Levene’s test. Fine root
biomass and necromass were Square-Root-transformed, and
contents of soil nitrate-N and ammonium-N were log-transformed.
Effects of N treatment, sampling dates, soil layers, and their
interactions on fine roots, soil nitrate-N and ammonium-N were
analyzed using three-way ANOVAs. Differences in production
and turnover rates of fine roots between N fertilization and control
plots, and between 0–10 cm and 10–20 cm soil layers were
analyzed by independent samples t-test. Pearson correlation was
used to detect the relationships among fine root biomass,
necromass, soil nitrate-N and ammonium-N content. All statistical
analyses were conducted with SPSS 17.0 (SPSS Inc., USA).
Results
Soil nitrate-N and ammonium-N
Soil nitrate-N and ammonium-N were affected significantly by
N fertilization (P,0.01), soil layers (P,0.001), and sampling dates
(P,0.001) (Table 2). N fertilization significantly increased soil
nitrate-N by 16% (9.33 vs. 10.82 mg kg21) and ammonium-N by
6% (8.58 vs. 9.09 mg kg21) in 0–20 cm soil (Fig. 1a, Table 2).
Nitrate-N and ammonium-N in 0–10 cm soil were significantly
higher than those in 10–20 cm soil for both N fertilization and
control plots (Fig. 1a, Table 2). Soil nitrate-N contents with the
highest level in the early growing season (May) decreased from
May to October (Fig. 2a). Compared to nitrate-N, high
ammonium-N contents in 0–10 cm soil were observed during
the late growing season (August to October), whereas ammonium-
N contents in 10–20 cm soil did not exhibit clear seasonal
fluctuation (Fig. 2b).
Fine root biomass and necromass
Both fine root biomass and necromass were influenced
significantly by N fertilization, sampling dates, and soil layers,
respectively (Table 2). Fine root biomass in 0–10 cm soil layer in N
fertilization plots was 19% less than that in control plots (3.35 vs.
3.98 Mg ha21) across the sampling dates (Fig. 1b). The fine root
biomass and necromass in both N fertilization and control plots
showed significant seasonal fluctuation, with two peaks in July and
September in N fertilization plots and one peak in August in
control plots (Fig. 2c). Fine root biomass in 10–20 cm soil
exhibited the same seasonal pattern in plots for both treatments,
peaking in July (Fig. 2c).
Mean fine root necromass in 0–10 cm soil in N fertilization
plots was 31% less than that in control plots (1.05 vs.
1.38 Mg ha21) (Fig. 1b). Fine root necromass in both N
fertilization and control plots showed significant seasonal fluctu-
ation, with the maximum values occurring in September in control
plots but in August in N fertilization plots. The fine root necromass
in N treated and control plots was lowest in May (Fig. 2d). Fine
root necromass in 10–20 cm soil exhibited similar seasonal pattern
to that in 0–10 cm soil, and the fine root necromass in control
plots was 41% greater than that in N fertilization plots (0.48 vs.
0.34 Mg ha21).
Fine root mass in relation to soil nitrate-N and
ammonium-N
Both fine root biomass and necromass in 0–10 cm soil were
significantly negatively correlated with soil nitrate-N, and total N
availability (nitrate-N+ammonium-N), but not with soil ammoni-
um-N contents (Table 3). No statistically significant correlations
between fine root mass and soil nitrate-N or ammonium-N in 10–
20 cm soil layer were found. There was significantly positive
relationship between fine root biomass and necromass, and
negative relationship between soil nitrate-N and ammonium-N
in both 0–10 and 10–20 cm soil layer, respectively (Table 3).
Production and turnover rates of fine roots
Production and turnover rates of fine roots estimated by
sequential soil cores method differed from that estimated by
ingrowth cores method (Table 4). Compared to control treatment,
N fertilization did not affect production and turnover rates of fine
roots estimated by sequential soil cores (Table 4). However,
production and turnover rates of fine roots were found to
significantly increased by N fertilization using ingrowth cores
method (Table 4). Using ingrowth cores method, fine root
production in 0–10 and 10–20 cm soil in N fertilization plots
was 42% and 55% greater than those in control plots (0.64 vs.
0.45 Mg ha21and 0.17 vs. 0.11 Mg ha21, respectively. Table 4),
and turnover rates of fine roots in 0–10 and 10–20 cm soil in N
fertilization plots was 73% and 44% greater than those in control
Table 2. Effects of N treatment, sampling dates, soil layers, and their interactions on fine root biomass, necromass, soil nitrate-N
and ammonium-N (Amm-N), analyzed using 3-way ANOVAs.
Factorsd. f. BiomassNecromass Nitrate-N N Amm-N
Treatment113.766*** 34.990***274.713***8.672**
Sampling dates522.152*** 12.494***3863.843***139.503***
Soil layers1 1268.616***482.044*** 3907.011***1381.976***
Treatment6Sampling dates1 4.705**2.597**68.435***48.029***
Treatment6Soil layers112.608** 0.929NS
65.901***8.604**
Sampling dates6Soil layers5 19.557***20.682*** 318.376***74.999***
Treatment6Sampling dates6Soil layers57.787***3.372* 158.253*** 7.320***
Error5656 114 114
d.f.: degree of freedom. Significance level: NS: not significant P.0.05; *P,0.05; **P,0.01; ***P,0.001. The biomass and necromass are gained from sequential soil cores.
F-values are given.
doi:10.1371/journal.pone.0031042.t002
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plots (0.19 vs. 0.11 year21and 0.13 vs. 0.09 year21, respectively,
Table 4).
Discussion
Seasonal dynamics of fine root biomass and necromass
A substantial seasonal variation of fine root biomass and
necromass was observed in each of the two soil layers (i.e. 0–10 cm
and 10–20 cm) during the growing season (Fig. 2c, d). Vogt et al.
[30] found modal or bimodal peaks of fine root biomass followed
by periods of high necromass in forest ecosystems. However, the
largest fine root biomass in an Asian white birch forest was found
in August, while the largest necromass occurred in June [29]. In
the present study, the largest fine root biomass and necromass in
0–10 cm soil were found in August and September, respectively,
which is consistent with the seasonal pattern that the peaks of fine
root biomass is followed by a period of high necromass [30,31,32].
The average fine root biomass (0–20 cm soil) in control plots
was 5.23 Mg ha21, within the range of 2.59 to 8.28 Mg?ha21
reported for the broad-leaved Korean pine mixed forest in
Changbai Mountain [23,33,34]. The fine root necromass of
1.86 Mg ha21was lower than the results (1.88–3.42 Mg ha21)
reported by Shan et al. [33] and Yang and Li [23] for the same
forest type. Fine root mass displayed a large variability between
years investigated, indicating a highly temporal heterogeneity
associated with inter-annual environmental variations in the
broad-leaved Korean pine mixed forest [34].
Seasonal variations in fine root biomass were correlated with
seasonal variations in soil N availability [4,35,36]. In a Larix gmelini
plantation in northeastern China, 58–73% seasonal variation in
fine root biomass is explained by changes in soil N availability
[37]. But the amount of live and dead fine roots depends on a
variety of abiotic and biotic factors [25,29]. A meta analysis
showed that the fine root biomass in the boreal forests were
significantly correlated with soil nutrient availability, temperature,
and moisture [38]. Moreover, stand age was also found to have
marked effects on fine root biomass [25,31]. Yang et al. [31]
reported diverse seasonal dynamics of fine root biomass and
necromass along a successional chronosequences. However, Fine ´r
et al. [2] analyzed a database of fine root biomass of 512 forest
stands from the literature, and found that environmental factors
(latitude, mean annual precipitation, elevation, temperature) could
not explain a significant amount of the variation in fine root
biomass, whereas the mean basal area of the forest stand could
explain 49% of the fine root biomass variation at stand level.
Responses of fine root mass, production and turnover
rates to N fertilization
The present study indicated that N fertilization led to a
significant decrease in fine root biomass and necromass in top soil
layers, which supported our hypothesis 1 that N fertilization
decreases fine root biomass and necromass.. Cost-benefit analysis
indicated that more resources are devoted to aboveground when
soil resource availability was high [39,40]. The relatively less C
allocated to roots led to a decline in fine roots with increasing soil
N availability [41]. However, in a Fraxinus mandshurica plantation in
northeastern China and a Pinus resinosa forest in Wisconsin, USA,
N fertilization reduced fine root biomass, but dramatically
increased the fine root necromass [28,42]. Pregitzer et al. [43]
also found that fine root biomass of Populus tremuloides significantly
Figure 1. The averaged soil nitrate-N and ammonium-N (amm-N) (a), and fine root biomass and necromass (b) in 0–10 and 10–
20 cm soil in N fertilization and control plots across the study period from May to October, 2010 (Mean ± SE, n=3). The biomass and
necromass are gained from sequential soil cores.
doi:10.1371/journal.pone.0031042.g001
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increased in high N soil compared to low N soil. The variations in
fine root biomass and necromass in response to N fertilization
were highly tree species-specific [10]. Furthermore, the response of
fine roots to N fertilization may also depend on soil type, forest age
and productivity, land use history, etc.
The present study found that, compared to control plots, N
fertilization did not alter fine root production and turnover rates in
0–10 and 10–20 cm soil estimated by sequential soil cores
approach (Table 4). However, fine root production and turnover
rates estimated by ingrowth cores approach were found to be
Figure 2. Seasonal changes in soil nitrate-N (a), ammonium-N (Amm-N) (b), fine root biomass (c), and necromass (d), in 0–10 and
10–20 cm soil in N fertilization and control plots (Means ± SE, n=3). The biomass and necromass are from sequential soil cores.
doi:10.1371/journal.pone.0031042.g002
Table 3. Pearson correlations among fine root biomass, necromass, soil nitrate-N and ammonium-N (Amm-N) in 0–10 cm and 10–
20 cm soil layer during the sampling period, 2010.
Necromass
Biomass and
necromass Nitrate-N Amm-NN availability (nitrate-N+ +amm-N)
0–10 cm soil layer
Biomass0.379* 0.913**
20.629**0.203
20.574**
Necromass 0.722**
20.553*0.229
20.471**
Biomass and necromass
20.714** 0.253
20.637**
Nitrate -N
20.477**0.811**
Amm-N0.127
10–20 cm soil layer
Biomass0.416** 0.959** 0.022 0.2860.112
Necromass0.654** 0.118
20.100 0.093
Biomass and necromass 0.0460.2090.114
Nitrate -N
20.335* 0.955**
Amm-N
20.042
The biomass and necromass are gained from sequential soil cores.
doi:10.1371/journal.pone.0031042.t003
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