1. No category

Soil Biology & Biochemistry 71 (2014) 13e20
Contents lists available at ScienceDirect
Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
Response of organic carbon mineralization and microbial community
to leaf litter and nutrient additions in subtropical forest soils
Qingkui Wang a, b, *, Silong Wang a, b, Tongxin He a, c, Li Liu a, Jiabing Wu a
a
State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110164, PR China
Huitong Experimental Station of Forest Ecology, Chinese Academy of Sciences, Huitong 418307, PR China
c
University of Chinese Academy of Sciences, Beijing 100049, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2013
Received in revised form
31 December 2013
Accepted 7 January 2014
Available online 18 January 2014
Microorganisms are vital in soil organic carbon (SOC) mineralization. The deposition of atmospheric
nitrogen (N) and phosphorus (P), as well as leaf-litter addition, may affect SOC mineralization and microbial community structure by changing the availability of soil nutrients and carbon (C). In this study,
we added leaf-litters labeled by 13C (Pinus massoniana and Michelia macclurei) and nutrients (ammonium
chloride and monopotassium phosphate) alone and in combination to soils collected from a coniferous
forest in subtropical China. We aimed to investigate the effect of leaf-litter and nutrient addition on SOC
mineralization and soil microbial community. CO2 production was continuously measured during 120day laboratory incubation, and CO2 sources were partitioned using 13C isotopic techniques. The addition of P. massoniana and M. macclurei leaf-litters increased SOC mineralization by 7.4% and 22.4%,
respectively. N and P addition alone decreased soil respiration by 6.6% and 7.1%, respectively. Compared
with P addition, N addition exerted a higher inhibitory effect on SOC mineralization induced by leaf-litter
addition. Leaf-litter addition stimulated soil microbial activity and decreased the ratio of bacteria to fungi
as a result of greater promotion on fungal growth. Moreover, 16:0 and 18:1u9c phospholipid fatty acids
(PLFAs) had greater amount of 13C incorporation than other PLFAs, especially in nutrient-addition
treatments. These results suggested that increased C input through leaf litter can stimulate SOC
mineralization, whereas atmospheric N and P deposition can reduce this stimulatory effect and promote
soil C storage in subtropical forests. Our results also illustrated that the use of 13C-labeled leaf litter
coupled with 13C-PLFA profiling is a powerful tool for determining the microbial utilization of C.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Soil organic C mineralization
Litter addition
Nutrient availability
Nitrogen deposition
Subtropical forest
Priming effect
Phospholipid fatty acids
1. Introduction
Carbon (C) stored in soil comprises approximately threequarters of terrestrial C worldwide. This value is more than three
times the amount of C in the atmosphere (Schlesinger and
Andrews, 2000). However, the level of soil organic C (SOC) at a
particular time is controlled by the balance between C input from
litter and C output from SOC mineralization (Vesterdal et al., 2012).
SOC mineralization is affected by microbial activities, which were
controlled by the source of energy for microbes (Vanhala et al.,
2008). In forest ecosystems, in addition to roots, leaf litter represents a major source of SOC inputs. Thus, leaf litter may influence
SOC mineralization through the priming effect (Kuzyakov, 2010;
Zhang and Wang, 2012).
* Corresponding author. State Key Laboratory of Forest and Soil Ecology, Institute
of Applied Ecology, Chinese Academy of Sciences, Shenyang 110164, PR China.
Tel.: þ86 24 8397 0344; fax: þ86 24 8397 0300.
E-mail address: [email protected] (Q. Wang).
0038-0717/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.soilbio.2014.01.004
A positive priming effect has been defined as a short-term increase in the turnover of SOC induced by the addition of an external
organic substrate to the soil (Kuzyakov et al., 2000). The priming
effect of adding plant materials or easily decomposable substances
in order to simulate organic C input in natural ecosystems has been
extensively studied (Hamer and Marschner, 2005; Potthast et al.,
2010; Wang et al., 2013a). However, the directions of the priming
effect reported in different experiments are inconsistent, showing
positive priming (Fontaine et al., 2007; Zhang and Wang, 2012) and
negative or no priming effect (Hamer and Marschner, 2005;
Nottingham et al., 2009) induced by organic C addition. In forest
ecosystems, leaf litter serves as the main source of SOC and alters
the native SOC mineralization rate.
Nutrient availability may be important in explaining the tremendous differences in the extent of the priming effect observed in
literature (Kuzyakov, 2010). Numerous studies have assessed
the influence of nitrogen (N) availability by N addition on C mineralization. However, no general conclusion has been drawn yet, and
increases (Cleveland and Townsend, 2006; Tu et al., 2013), decreases
14
Q. Wang et al. / Soil Biology & Biochemistry 71 (2014) 13e20
(Craine et al., 2007; Bradford et al., 2008; Mo et al., 2008), and no
change (Thirukkumaran and Parkinson, 2000) in soil respiration after
N addition have been observed. In China, the mean annual N deposition increased from 13.2 kg N ha1 in the 1980s to 21.1 kg N ha1 in
the 2000s (Liu et al., 2013). This may increase soil N availability and
consequently influence SOC mineralization and priming effect. N
deposition can also aggravate phosphorus (P) limitation in plant and
microbial processes, particularly in acidic soil (Vitousek et al., 2010),
but this limitation may be weakened by P addition to soils. However,
the results of studies through laboratory incubation on the effects of P
addition on microbial activities remain controversial, showing stimulatory (Allen and Schlesinger, 2004; Bradford et al., 2008), inhibitory
(Thirukkumaran and Parkinson, 2000), or no (Groffman and Fisk,
2011) effects on soil respiration. Moreover, the previous studies
were mainly conducted in boreal and temperate forests, resulting in
little information on response of SOC mineralization to combining
addition of leaf litter and nutrients in subtropical forests.
The important role of biotic factors (e.g., microbial community
structure and activity) in SOC mineralization is now being recognized
(Strickland et al., 2009; Garcia-Pausas and Paterson, 2011; Tavi et al.,
2013). Several experimental studies have demonstrated that external
substrate addition can alter soil microbial community structure (de
Vries et al., 2006; Moore-Kucera and Dick, 2008; Denef et al., 2009;
Dungait et al., 2011), which may consequently affect the magnitude
and direction of SOC mineralization and change C flow within the soil
microbial community (Williams et al., 2006; Garcia-Pausas and
Paterson, 2011; Yao et al., 2012). Recently, some studies have used 13C
stable isotopic technology to successfully trace C flow from 13Clabeled substrates into soil microbial community in agricultural and
grassland soils (Dungait et al., 2011; Yao et al., 2012; Zhang et al.,
2013). These studies have provided important information on
groups of microbes utilizing a given substrate through GC-C-IRMS
analyses of individual phospholipid fatty acids (PLFAs). However, information on this issue is limited in forest ecosystems, particularly in
subtropics, although some studies have been conducted in temperate
forests (Moore-Kucera and Dick, 2008; Rubino et al., 2010).
In the present study, we used the 13C-labeled Pinus massoniana
(coniferous tree species) and Michelia macclurei (broadleaved tree
species) leaf litter to investigate the response of native SOC mineralization and soil microbial community to the addition of leaf-litter,
N, and P, alone and in combination, in a subtropical forest soil. We
hypothesized that (1) an increase in native SOC mineralization occurs after leaf-litter addition, and this increase is greater in soils with
leaf-litter addition at a high C:P ratio (M. macclurei); (2) N and P
addition decreases the priming effect induced by leaf-litter addition; and (3) N and P addition changes the impact of leaf litter supply
on the microbial community structure and the 13C incorporation
into different groups of microorganisms. This study aimed to
investigate the effects of leaf-litter and nutrient addition on the
mineralization of native SOC and how soil microbial community
composition and 13C flow within soil community respond to litter
and nutrient addition. To the best of our knowledge, this study is the
first quantitative research on the effects of N and P addition on the
priming effect and 13C flow with soil microbial community in subtropical forests, which favors to better understand effect of N and P
deposition on the C cycle in forest ecosystems.
to 110 080 E). The soil samples were taken to the laboratory, passed
through a 2 mm sieve and root and other residues in soil samples
were removed by hand. The total C and N concentrations in the soil
samples were 17.5 g kg1 and 1.45 g kg1, respectively. The soil
1
mineral N (NHþ
4 eN and NO3 eN) concentration was 11.2 mg$kg .
In the experiment, the used soil had low available P with
1.04 mg kg1, limiting the growth of plants and microbes. Soil pH
was 4.35. The sand, silt, and clay contents in the soil were 11.2%,
46.1%, and 42.7%. The soil bulk density was 1.26 g cm3. A pulsechase technique was used to label P. massoniana and M. macclurei
seedlings with 13CO2 gas with an abundance of 99.9% in a growth
chamber. The seedlings were labeled with 99.9 atom % 13CO2 at
every 7 days. At the end of the three-month labeling period, the
seedlings were harvested, rinsed with deionized water, dried, and
separated into leaves, stems, and roots. These components had
different d13C. In this experiment, leaves are used and their
chemical properties are shown in Table 1.
2.2. Experimental design and soil incubation
The experiment was set up to have nine treatments with three
replicates. The details are provided in Table 2. In this experiment,
P. massoniana and M. macclurei leaf litters had similar C concentration. P. massoniana litter had higher P concentration and lower
C:P ratio. We expected that the difference in chemical quality of the
two litters would result in different responses of SOC mineralization and soil microbial community.
For incubation, 240 g of soil (dry weight) for each replicate of each
treatment was placed in a 500 mL Mason jar. The 13C-labeled leaflitter, ammonium chloride solution, and potassium dihydrogen
phosphate solution were then added to the soil according to the
experimental design. The grounded leaf-litter was evenly incorporated with the soil to make a homogeneous mixture with the soil.
Finally, the water content of the soil in each treatment was adjusted
to 60% of water holding capacity by adding deionized water. A glass
vial containing 20 mL of 0.1 M NaOH solution was placed in each
Mason jar to trap evolved CO2 from the soil, and the Mason jars were
then sealed. All the Mason jars with soil were incubated in the dark
for 120 d at 28 C. Three additional Mason jars with a beaker containing 20 mL of 0.1 M NaOH were sealed. These jars served as controls to account for the CO2 trapped from the air. New beakers
containing the 20 Ml NaOH solution were added on each collection
date. The collected NaOH solution in each beaker was immediately
transferred to sample flask and sealed with lid. Beakers with NaOH
solution trapped CO2 were collected at 1, 3, 6, 12, 23, 41, 62, 83, 102,
and 120 d after incubation. To determine the d13C of released CO2,
10 mL of NaOH solution was collected from the glass vial containing
20 mL of NaOH solution on each collection date. The remaining 10 mL
of NaOH solution was used to determine the amount of released CO2.
The released CO2 was measured using alkali-trapping techniques.
2.3. Soil chemical analysis
The concentrations of C and N in the soil and leaf litter samples
were determined using an element analyzer. To measure the P
concentration, 0.2 g of litter samples were digested in 10 mL of
triacid mixture (nitric, perchloric, and sulfuric acids; 5:1:1), and
2. Materials and methods
2.1. Soil and
13
C-labeled leaf litter
The soil used in this experiment was collected at a layer of
0 cme10 cm from a coniferous forest located at the Huitong National Research Station of Forest Ecosystem in Huitong county,
Hunan province (latitude 26 400 to 27 90 N and longitude 109 260
Table 1
Chemical properties of the labeled Pinus massoniana (PM) and Michelia macclurei
(MM) leaf-litter used in the experiment.
C (g kg1)
PM
MM
477.1
476.7
N
(g kg1)
P
(g kg1)
C/N
19.2
22.2
1.51
0.70
24.9
21.5
C/P
316
681
Ca
(g kg1)
Mg
(g kg1)
d13C
1.61
4.0
2.64
3.37
1318
2107
(&)
Q. Wang et al. / Soil Biology & Biochemistry 71 (2014) 13e20
Table 2
Total amount of Pinus massoniana (PM) and Michelia macclurei (MM) leaf-litter, N,
and P added to the soil samples from 0 cm to 10 cm depth increment in the nine
substrate-addition treatments.
Soil only
þN
þP
þPM
þPM þ N
þPM þ P
þMM
þMM þ N
þMM þ P
Leaf litter (g C kg1
dry soil)
N (mg kg1
dry soil)
P (mg kg1
dry soil)
0
0
0
1.43
1.43
1.43
1.43
1.43
1.43
0
100
0
0
100
0
0
100
0
0
0
50
0
0
50
0
0
50
then cooled. The P concentration was determined colorimetrically
in the digested samples using the ammonium molybdate stannus
chloride method (Olsen and Sommers, 1982). The evolved CO2 from
the soil was measured by titration with 0.05 M HCl. The evolved
CO2 from the soil samples was calculated from the difference in
value of the evolved CO2 in the Mason jars with and without soil.
The d13C of CO2 in the NaOH solution was measured using a stable
isotope-ratio mass spectrometer.
2.4. Partitioning CO2 sources and quantifying the priming effect
To calculate the amount of CO2eC derived from leaf-litter and
soil under incubation, the following equations were used:
CL ¼ Ct ðdt dS Þ=ðdL dS Þ
(1)
CS ¼ Ct ðdL dt Þ=ðdL dS Þ
(2)
In the Equations (1) and (2), Ct (Ct ¼ CL þ CS) is the total amount
of CO2eC during the considered time interval and dt is the corresponding isotopic composition. CL is the amount of C derived from
the added leaf-litter and dL is its isotopic composition in the leaflitter. CS is the amount of C derived from SOC and dS is its isotopic
composition in SOC.
The source of CO2 production from SOC was fitted to a singleexponential model using the following equation:
Ct ¼ C0 1 ekt
(3)
15
performed as described by Wang et al. (2013b). Briefly, 5 g of freezedried soil was extracted for 2 h with chloroform:methanol: phosphate buffer (1:2:0.8), and the phospholipids were separated from
other lipids on a silicic acid column. The resultant fatty acid methyl
esters were separated, quantified, and identified on an Agilent 6890
gas chromatograph equipped with a flame ionization detector and
an Ultra-2 column. The fatty acid methyl ester and BAME controls
were used to identify the peaks. Methyl nonadecanoate was used as
the internal standard for quantifying the PLFAs.
For the analyses of microbial group abundance and distribution
of litter-derived C within microbial groups, the following PLFA
designations were used: Gram-positive bacteria i15:0, a15:0, i16:0,
i17:0, and a17:0; Gram-negative bacteria 17:0cy, 16:1u7c 16:1u9c,
and 19:0cy; fungi 18:1u9c, 18:1u9t, and 18:2u9,12c; actinomycetes
10Me16:0, 10Me17:0 and 10Me18:0 (Moore-Kucera and Dick,
2008).
The d13C values of individual PLFA were determined using
isotope ratio mass spectrometry, as described by Williams et al.
(2006). The proportion of litter-derived labeled C in each PLFA
was determined using a mass balance approach (Rubino et al.,
2010; Yao et al., 2012).
Pi ¼
d13 Ct d13 Cc
.
d13 Cl d13 Cc
(5)
where d13Ct is the d13C enrichments (&) of individual PLFA in the
soils with leaf-litter at the end of incubation; d13Cc is the d13C enrichments (&) of individual PLFA in the control soils; and d13Cl is
the d13C of the labeled leaf litters (&). The total labeled leaf-litterderived C in each PLFA was calculated by multiplying each Pi by the
individual PLFA abundances. Moreover, the specific microbial
respiration (SMR) was expressed as 13CeCO2 mineralized/total 13C
in PLFA.
2.6. Statistical analysis
All statistical analyses were performed using SPSS version 17.0
for Windows. A one-way analysis of variance (ANOVA), followed by
Tukey HSD test, was used to analyze the effects of the added leaf
litter and inorganic N and P on the CO2 production of total derivedlitter and eSOC, priming effect, soil microbial community, SMR and
percentage distribution of litter-derived 13C among PLFAs. The
significant difference was considered at P < 0.05.
where Ct is the cumulative C mineralized (mg C kg1) at time t, C0 is
the potentially mineralizable C (mg C kg1), and k is the first-order
rate constant (d1).
In the incubation experiment, the priming effect induced by the
added leaf-litter was calculated by comparing the amount of CO2 in
the leaf-litter-containing soil samples with the amount of CO2 in
the soil samples without leaf-litter addition (Hamer and Marschner,
2005). The priming effect (PE) during the 120 d incubation period
was calculated using the following equation:
PE ¼ 100 ðCO2 Ctreatment CO2 Ccontrol Þ=CO2 Ccontrol
(4)
where Ctreatment is the accumulated amount of CO2 derived from
SOC in the treatments with leaf-litter addition and Ccontrol is the
amount of CO2 derived from SOC without leaf-litter addition.
2.5. PLFA analysis
At the end of incubation, part of the soil was collected and
immediately freeze-dried. Lipid extraction and PLFA analyses were
Fig. 1. Effects of N, P and P. massoniana (PM) and M. macclurei (MM) leaf-litter addition
on the cumulative amount of CO2eC derived from native SOC. The vertical bars are
standard errors (n ¼ 3).
16
Q. Wang et al. / Soil Biology & Biochemistry 71 (2014) 13e20
Table 3
Effects of leaf litter and nutrient addition on soil potentially mineralizable C and the
rate constant estimated using first-order kinetics from C mineralization data obtained from 120-d incubation.
Soil only
þN
þP
þPM
þPM þ N
þPM þ P
þMM
þMM þ N
þMM þ P
Potentially mineralizable
C (mg kg1)
Rate constant (d1)
166.6b
154.0a
154.5a
182.3c
151.7a
169.9b
200.6d
173.4b
180.9c
0.038b
0.044c
0.043c
0.027a
0.035b
0.036b
0.038b
0.057d
0.047c
Different letters following the data in the same column denote significance.
3. Results
3.1. CO2 derived from native SOC and leaf litter
Without leaf-litter addition, the addition of N or P to the soils
decreased the CO2 production from native SOC mineralization by an
average of 6.6% and 7.1%, respectively, compared with the soils
without any addition (Fig. 1). Nutrient addition also decreased the
pool size of mineralizable C, but increased the rate constant
(Table 3). When leaf litter was added individually, CO2 production
from native SOC was increased by 7.4% and 22.4%, respectively, for
P. massoniana and M. macclurei leaf-litter. The potentially mineralizable C pool size increased with leaf-litter addition (Table 3),
whereas the rate constant decreased with P. massoniana leaf-litter
addition.
When leaf-litter and N were added together, the CO2 derived
from native SOC in the þPM þ N treatment was significantly lower
than that in the soils only treatment, whereas the CO2 derived from
native SOC in the þMM þ N treatment was higher than that in the
soils only treatment. When leaf-litter and P were added together,
the CO2 derived from native SOC in the þPM þ P and þMM þ P
treatments was increased. The addition of N and P decreased the
priming effect induced by leaf-litter addition (Fig. 2). After N
addition, the increase in the native SOC mineralization induced by
P. massoniana and M. macclurei leaf-litter addition decreased to
8.6% and 8.2%, respectively. Moreover, P addition caused the increase in the native SOC mineralization induced by P. massoniana
and M. macclurei leaf-litter to decrease to 2.6% and 13.2%,
respectively.
When only leaf-litter was added, the amount of CO2 derived
from P. massoniana leaf-litter was greater than that from
M. macclurei (Fig. 3). The addition of N and P did not affect the CO2
production from P. massoniana leaf-litter, but decreased that from
M. macclurei leaf-litter. Considering the total litter decomposition
during the 120-d incubation period, approximately 14.1% of the
added 13C-labeled P. massoniana leaf-litter was decomposed, and
not affected by N and P addition (Fig. 4). For the M. macclurei leaflitter, approximately 11.5% was decomposed, which was significantly reduced to 9.3% by N addition, but not affected by P addition.
Moreover, the addition of N and P did not affect the SMR in the
P. massoniana leaf-litter treatment, but decreased the SMR in the
M. macclurei leaf-litter treatment with 29.7% and 26.3%,
respectively.
3.2. Soil microbial community and
13
Fig. 2. Effects of N, P, P. massoniana (PM) and M. macclurei (MM) leaf-litter addition on
the priming effect on native SOC mineralization after 120-d incubation. The vertical
bars are standard errors (n ¼ 3). The asterisks denote the significant effects of N and P
addition on leaf litter decomposition.
biomass determined by PLFA (Table 4). The addition of M. macclurei
leaf litter combined with nutrients also significantly increased the
fungal biomass. However, leaf-litter or nutrient addition did not
affect bacterial, Gram-positive bacterial, and actinomycetous
biomass. The ratio of bacteria to fungi was significantly decreased
by leaf-litter and nutrient addition, but the ratio of Gram-positive to
Gram-negative bacteria was decreased by leaf-litter addition alone.
In addition, nutrient addition decreased the effect of P. massoniana
leaf-litter addition on the ratio of Gram-positive to Gram-negative
bacteria.
Most labeled leaf-litter-derived C was incorporated into 16:0
and 18:1u9c, followed by i15:0 and 19:0cy (Fig. 5). When leaf litter
alone was added to the soils, the incorporation of litter-derived C
into 19:0cy was greater in the P. massoniana addition treatment,
whereas the incorporation into i15:0 was greater in the
M. macclurei addition treatment. The N addition decreased the
incorporation of P. massoniana litter-derived C into 19:0cy and
18:1u9t and the incorporation of M. macclurei litter-derived C into
i15:0, a15:0, 16:1u9c, and 18:1u9t, but increased the incorporation
of 13C derived from both leaf litters into 15:0, 17:0, 18:2u9, 12c, and
10Me17:0. The P addition decreased the incorporation of
C distribution in the PLFAs
The addition of P. massoniana leaf-litter with or without P
significantly increased the soil total microbial biomass and fungal
Fig. 3. Effects of N, P and P. massoniana (PM) and M. macclurei (MM) leaf-litter addition
on the cumulative amount of CO2eC derived from leaf litters. The vertical bars are
standard errors (n ¼ 3).
Q. Wang et al. / Soil Biology & Biochemistry 71 (2014) 13e20
17
Fig. 4. Effects of N and P addition on the decomposition of the added 13C-labeled P. massoniana (PM) and M. macclurei (MM) leaf-litter and the specific microbial respiration (SMR)
after 120-d incubation. The vertical bars are standard errors (n ¼ 3). The asterisks denote the significant effects of N and P addition on leaf litter decomposition.
P. massoniana and M. macclurei litter-derived C into 19:0cy and
18:1u9t, but increased the incorporation into 18:2u9, 12c, and
18:1u9c. Moreover, P addition also increased the incorporation of
P. massoniana litter-derived C into i16:0, 15:0, and 16:0.
4. Discussions
Leaf-litter addition to soils increased the native SOC mineralization, but the chemical quality of the substrate added to soils
affected its magnitude (Blagodatskaya and Kuzyakov, 2008;
Potthast et al., 2010; Wang et al., 2013a). As our hypothesis,
M. macclurei leaf-litter with higher C:P ratio can cause greater
mineralization of native SOC compared with P. massoniana, suggesting that lower-quality litters can induce higher positive priming effect. This result is in agreement with the findings of other
researchers (Nottingham et al., 2009; Wang et al., 2013a; Zhang and
Wang, 2012). One possible mechanism is that the available fresh
leaf litters acted as an energy source for the production of extracellular enzymes by microbes with the subsequent increase in the
mineralization of soil organic matter (SOM), leading to priming
effect (Schimel and Weintraub, 2003). During the decomposition of
M. macclurei leaf litter with rich C and poor P, soil microbes would
have to mine for P from more SOM, being supported by the
reduction in priming effect after P addition. Soil microbial community structure has an important role in mineralizing SOM
(Strickland et al., 2009; Garcia-Pausas and Paterson, 2011). Our
results that leaf-litter addition led to a shift in soil microbial community structure measured by PLFA analysis also supported the
theory (Table 4). However, the different responses of soil microbial
community to the addition of M. macclurei and P. massoniana litters
maybe explained by the differences in the magnitude of priming
effect.
Similar to some previous studies (Thirukkumaran and Parkinson,
2000; Craine et al., 2007; Bradford et al., 2008), decreases in SOC
mineralization following the addition of N or P alone suggest that
available N and P is deficit in the subtropical soils and SOC mineralization was suppressed when nutrient availability is high, which
was supported by the low amount of available N and P. This is also
confirmed by other studies in subtropical or tropical regions
(Cleveland and Townsend, 2006; Mo et al., 2008; Ouyang et al.,
2008). There have been numerous studies which, like ours, monitored the effect of nutrient additions on soil respiration in the laboratory or field. In the literature, N or P addition have been shown to
have inhibitory (Thirukkumaran and Parkinson, 2000; Bradford
et al., 2008; Ouyang et al., 2008; Mo et al., 2008), stimulatory
(Fierer et al., 2003; Allen and Schlesinger, 2004; Tu et al., 2013), and
no (Yoshitake et al., 2007; Groffman and Fisk, 2011) effects. In
summary, our results and those of other studies imply that N or P
additions do not consistently accelerate SOC mineralization, even in
ecosystems with low soil N or P availability.
As our expectation, N and P addition also decreased the positive
priming effect, indicating that priming effect is also dependent on
the amount of available nutrients in soils (Cheng, 2009). Our findings were in agreement with some previous results (Hartley et al.,
2010; Zhang and Wang, 2012), supporting the observation of
Fontaine et al. (2011) who noted that priming effect is low when
nutrient availability is high. These results suggest that an increase
in N and P availability is helpful in diminishing SOC mineralization
induced by leaf litter input, and may likely increase soil C deposit to
a certain extent. Our result confirms a preferential substrate utilization of soil microorganisms if the primary nutrients (e.g. N and P)
are present, resulting in lower SOM mineralization. Meanwhile,
similar to some previous studies (Conde et al., 2005; Hamer and
Marschner, 2005), N or P addition with M. macclurei leaf-litter
produced a positive priming effect. This result is probably due to
the high C:N or C:P ratio despite N or P addition. Another important
finding was that N addition altered the direction of the priming
effect from positive to negative, indicating that atmospheric N
deposition in subtropical P. massoniana forests may increase the C
storage in soil by decreasing SOC mineralization.
Generally, leaf-litter addition affects soil microbial community,
consistent with our hypothesis and some previous findings
(Waldrop and Firestone, 2004; Moore-Kucera and Dick, 2008;
Nottingham et al., 2009; Wang et al., 2013b). The decline in the ratio
of bacteria to fungi indicates that leaf-litter addition has greater
promotion on the growth of fungi than bacteria. This result is in
agreement with the finding of Rousk and Bååth (2007), wherein N
addition stimulated fungal activity. This result also supports the
fact that saprophytic fungi have a predominant role in litter
decomposition (Meidute et al., 2008). Meanwhile, de Vries et al.
(2006) found that N fertilizer addition decreased the ratio of
18:2u6,9 to bacterial PLFA, whereas Denef et al. (2009) found no
effect of N addition on the relative abundance of saprotrophic
fungal PLFAs. Meidute et al. (2008) noted that the addition of
glucose mainly favored bacterial growth, and fungi were more
favored by the addition of cellulose, indicating that the response of
bacteria and fungi to an external C source was substratedependent. This result may partly explain that the addition of
P. massoniana leaf-litter has greater influence on soil microbial
community structure than the addition of M. macclurei leaf-litter.
18
Q. Wang et al. / Soil Biology & Biochemistry 71 (2014) 13e20
Table 4
Changes in the concentrations (nmol g1 soil) of PLFAs and two PLFA ratios in soils with P. massoniana (PM), M. macclurei (MM), N and P additions at the end of 120 d of
incubation.
Microbial biomass
Soil only
þN
þP
þPM
þPM þ N
þPM þ P
þMM
þMM þ N
þMM þ P
25.2
26.7
32.2
37.3
33.5
37.4
29.6
32.8
37.6
1.4a
6.9 ab
3.1 ab
4.4b
1.8 ab
3.4b
6.6 ab
1.3 ab
2.1b
Bacteria
18.0
18.8
22.7
25.3
22.7
25.3
20.4
22.1
25.5
1.0a
4.7a
2.1a
3.0a
1.3a
2.3a
4.5a
0.8a
1.4a
Fungi
4.4
5.1
6.3
8.5
7.4
8.7
6.4
7.3
8.6
Actinomycete
0.2a
1.5 ab
0.7abc
0.9c
0.5bc
0.8c
1.5abc
0.3bc
0.5c
2.82
2.81
3.24
3.54
3.48
3.49
2.77
3.31
3.46
0.22a
0.68a
0.31a
0.50a
0.15a
0.34a
0.59a
0.16a
0.22a
Bacteria:Fungi
4.08
3.75
3.63
2.98
3.08
2.92
3.18
3.03
2.97
0.06c
0.21b
0.16b
0.06a
0.16a
0.02a
0.05a
0.04a
0.08a
Gþ
6.89
7.44
8.37
8.56
8.44
8.72
7.56
7.48
9.27
G
0.48a
1.62a
0.80a
0.78a
0.35a
0.79a
1.58a
0.29a
0.37a
3.02
3.13
4.03
5.39
3.79
4.31
3.78
3.47
4.30
Gþ:G
0.07a
0.90a
0.46 ab
0.79c
0.27a
0.39abc
0.84 ab
0.15a
0.20bc
2.28
2.41
2.08
1.60
2.23
2.02
2.00
2.16
2.15
0.11bcd
0.21d
0.04bc
0.14a
0.08bcd
0.01bc
0.04b
0.09bcd
0.07bcd
Data expressed as mean SD (n ¼ 3) are reported for different taxa and two PLFA ratios (fungi/bacteria and Gramepositive/Gramenegative bacteria) under different
treatments at the end of 120-d incubation. Gþ and G- indicate the Gramenegative and Gramepositive bacteria, respectively. Different letters following the data in the same
column denote significance.
Fig. 5. Percentage distribution of 13C among the PLFAs (A: Gram-negative bacteria, B: Gram-positive bacteria, C: other bacteria except A and B, D: fungi, E: actinomycetes) at the end
of 120-d incubation. Bars represent the standard deviation of the mean (n ¼ 3). Different letters on the bars denote the significance.
Q. Wang et al. / Soil Biology & Biochemistry 71 (2014) 13e20
Differences in the substrate chemical quality and the relative
abundance of bacteria and fungi in soils can also explain their responses to the addition of different leaf litters in soil (Meidute et al.,
2008; Zhang et al., 2013).
Leaf-litter addition also affects bacterial community composition. The lower ratio of Gram-positive to Gram-negative bacteria in
soils with 13C-labeled leaf-litter addition alone suggests that leaflitter addition stimulated the growth of Gram-negative bacteria
more effectively than Gram-positive bacteria. This result is probably due to the fact that the leaf litter C added to soils was utilized
to a greater extent by the Gram-negative community with faster
growth rates (Kuzyakov et al., 2000; Garcia-Pausas and Paterson,
2011) because Gram-negative bacteria have a preferential utilization of plant biomass and have important roles in processing
compounds derived from plant residues (Kramer and Gleixner,
2008; Tavi et al., 2013). Significant increase in the ratio of Grampositive to Gram-negative bacteria due to P. massoniana leaf-litter
addition disappeared when nutrients were added. This observation indicates a tight coupling of soil bacterial community to
nutrient availability, which was consistent with findings by Denef
et al. (2009), where N fertilization modified the bacterial community in a grassland soil.
The higher distribution of 13C in the bacterial PLFAs than fungal
PLFAs indicates that leaf litter-derived C was more efficiently
incorporated into bacteria than into fungi, which is in agreement
with some previous findings, wherein the amounts of labeled C in
bacterial PLFAs was greater than that in fungi PLFAs (Yao et al.,
2012; Zhang et al., 2013). Lower incorporation of leaf litterderived C into fungi than bacteria maybe provides indirect evidence that fungi were important in metabolizing soil C and the
major organisms involved in priming (Fontaine et al., 2011). Cellulose without any nutrients added by Fontaine et al. (2011) primarily promoted fungal growth, but in our experiment we added
leaf litters with some nutrients. This was likely to explain why
Fontaine et al. (2011) found that fungi are main actors of the
priming effect. The highest distribution of leaf litter-derived 13C in
the 16:0 and 18:1u9c fatty acids suggest that the two groups of
microorganisms preferentially utilize the fresh C. The highest 13C
distribution is simply due to their higher abundance in soils (data
not shown) and preferential utilization of the leaf litter-derived C,
which was confirmed by the higher d13C-PLFA. The 13C incorporated
into Gram-positive bacteria was twice as much as that into Gramnegative bacteria, resulting to higher concentrations of Grampositive bacteria than Gram-negative bacteria. This result supports the fact that 13C derived from exudates and glucose was
primarily incorporated into Gram-positive bacteria rather than
Gram-negative bacteria (Rubino et al., 2010; Dungait et al., 2011).
The above results suggest that Gram-positive bacteria have primary
role in C cycles. Meanwhile, Rubino et al. (2010) found a similar d13C
in Gram-positive and Gram-negative bacteria, and fungal PLFA,
suggesting that all microorganisms were incorporating similar
component of litter C at the same rate. With regard to conflicting
findings, how soil bacterial community responds to fresh C supply
in forest soils still needs further study.
Some studies have demonstrated that N addition reduced 13C
incorporated into the amount of fungal PLFAs (Crossman et al.,
2006; Denef et al., 2009). As our third hypothesis, in our study, N
and P addition also decreased the incorporation of labeled 13C into
the 18:1u9t PLFA as an indicator of fungi. The decrease in the
incorporation of 13C into Gram-negative bacterial PLFAs due to N
and P addition suggests that nutrient addition reduced the Gramnegative bacterial activity. The N addition increased the distribution of 13C-derived from P. massoniana leaf-litter in Gram-positive
bacteria, but decreased the distribution of 13C-derived from
M. macclurei leaf-litter in Gram-positive bacteria, suggesting that
19
the effect of N availability on the Gram-positive bacteria is
substrate-dependent. Meanwhile, no increase in 13C enrichment in
gram-positive bacterial PLFAs was observed after N fertilization
(Denef et al., 2009). Moreover, different response of the SMF to
nutrient addition between P. massoniana and M. macclurei leaflitters maybe was due to difference in litter quality. M. macclurei
leaf-litter had higher quality and its labile C was lost quickly than
P. massoniana leaf-litter. The lower SMF detected in the M. macclurei
leaf-litter treatment after N and P addition was due to the decrease
in the labile C pool, suggesting that shifts occurred in the C utilization efficiency of the soil microbial community (Thirukkumaran
and Parkinson, 2000).
In conclusion, as our hypothesis, the addition of M. macclurei
leaf-litter with relatively higher C:P ratio produced higher priming
effect than P. massoniana leaf-litter addition, suggesting that P
availability is vital to priming effect in acid soils from subtropical
forests. The N and P addition decreased the CO2 production derived
from native SOC and priming effect induced by leaf-litter addition,
suggesting that atmospheric N and P deposition may increase the C
deposit in soil by suppressing SOC mineralization in subtropical
forests. Leaf-litter addition and nutrients to soils had greater promotion on fungal growth, resulting in the reduction of bacteria:fungi ratio. Higher amount of fresh litter C incorporated into the
16:0 and 18:1u9c PLFAs suggests that these two microorganisms
have greater role in fresh C cycling.
Acknowledgments
This work was conducted at Huitong National Research Station
of Forest Ecosystem, and financially supported by the National Basic
Research Program of China (973 Program, Grant no.
2012CB416905), and the National Natural Science Foundation of
China (Grant nos. 41030533, 31070436 and 41201254). We are
tremendously grateful to Micai Zhong and Xiaojun Yu for their
assistance in collecting and analyzing the samples. We thank the
anonymous reviewers for helpful comments on revision of this
manuscript.
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