BGBiogeosciencesBGBiogeosciences1726-4189Copernicus PublicationsGöttingen, Germany10.5194/bg-15-4481-2018Understory vegetation plays the key role in sustaining soil microbial biomass and extracellular enzyme activitiesUnderstory vegetation plays the key role in sustaining soil
microbial biomassYangYangZhangXinyuzhangxy@igsnrr.ac.cnZhangChuangWangHuiminFuXiaoliChenFushengWanSongzeSunXiaominWenXuefaWangJifu13946004918@163.comCollege of Geographic Science, Harbin Normal University, Harbin, 150025, ChinaKey Laboratory of Ecosystem Network Observation and Modeling, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing, 100101, ChinaCollege of Resources and Environment, University of Chinese Academy of Sciences, Beijing, 100049, ChinaKey Laboratory of Agricultural Water Resources, Center for Agricultural Resources Research, Institute of Genetic and Developmental Biology, The Chinese Academy of Sciences, 286 Huaizhong Road, Shijiazhuang, 050021, Hebei, ChinaCollege of Forestry, Jiangxi Agricultural University, Nanchang, 330045, ChinaXinyu Zhang (zhangxy@igsnrr.ac.cn) and Jifu Wang (13946004918@163.com)24July201815144481449419December201716January201810June201810July2018This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/This article is available from https://bg.copernicus.org/articles/15/4481/2018/bg-15-4481-2018.htmlThe full text article is available as a PDF file from https://bg.copernicus.org/articles/15/4481/2018/bg-15-4481-2018.pdf
While we know that understory vegetation affects the soil
microbial biomass and extracellular enzyme activities in subtropical Chinese
fir (Cunninghamia lanceolata) forests, we are less certain about the
degree of its influence. We determined the degree to which the soil abiotic
and biotic properties, such as PLFAs and extracellular enzyme activities,
were controlled by understory vegetation. We established a paired treatment
in a subtropical Chinese fir plantation, which comprised one plot from which
the understory vegetation and litter were removed (None) and another from
which the litter was removed but the understory vegetation was left intact
(Understory). We evaluated how the understory vegetation influenced the soil
abiotic properties, the bacterial, fungal, and actinobacterial PLFAs, and the
activities of five hydrolases and two oxidative enzymes. The dissolved
organic carbon (DOC), particulate organic carbon, soil organic carbon,
ammonia nitrogen (NH4+–N), and total nitrogen contents and soil
moisture were 18 %, 25 %, 12 %, 34 %, 8 %, and 4 % lower in the
None treatments than in the Understory treatments, respectively (P<0.05).
Soil bacterial, fungal, and total PLFAs, and the potential activities of
β-1,4-glucosidase (βG), β-1,4-N-acetylglucosaminidase,
phenol oxidase, and peroxidase, were as much as 24 % lower in None
treatments than in Understory treatments (P<0.05). The specific activities
of C-acquiring enzymes were as much as 41 % higher (P<0.05), and the
ratio of C- to N-acquiring enzymes was also higher in the None treatments
than in the Understory treatments. This suggests that in the absence of
understory vegetation microbes invested more in C acquisition than N
acquisition because the carbon (C) inputs were less labile. The negative
relationship between DOC and AP shows that DOC is consumed when P-acquiring
enzymes are produced. The positive correlation between NH4+–N
and βG suggested the increased availability of N promoted the
decomposition of C. More extracellular enzymes that degrade soil organic
matter are produced when there is understory vegetation, which leads to
losses of soil C. On the other hand, the soil C sink is maintained by
increased inputs of C. We can therefore conclude that understory vegetation
contributes to C sequestration in Chinese fir forests and suggest that
understory should be maintained to sustain soil quality in subtropical
Chinese fir plantations.
Introduction
The interactions that occur between above-ground vegetation functional groups
and soil microbial communities are thought to be important drivers of
carbon (C) and nutrient cycling in terrestrial ecosystems (Murugan et al.,
2014). When understory vegetation is removed from forest ecosystems, soil
processes are influenced, such that above-ground plant diversity and biomass
decrease (Lamb et al., 2011; Fu et al., 2015) and the characteristics of the
below-ground rhizodeposits change (Li et al., 2013). The understory
vegetation absorbs water and nutrients from soil (Wang et al., 2014), and
also releases carbohydrates back to the soil as discarded root cap and border
cells, mucilage and exudates from roots (McNear Jr., 2013), and cellulose,
hemicelluloses, and lignin from leaf litter (Loeppmann et al., 2016a, b). The
net effect of understory vegetation on soil nutrients is determined by the
balance between its nutrient demand and capacity to release carbohydrates to
soil via the decomposition of understory-derived litter and rhizodeposits.
Soil extracellular enzymes produced by microorganisms or plant roots
catalyze the cycling of soil C, nitrogen (N), and phosphorus (P) (Burns et
al., 2013; Nannipieri et al., 2018). Because they respond rapidly to soil
environmental changes, soil enzyme activities are often used as indicators
of soil quality (Trasar-Cepeda et al., 2008; Burns et al., 2013). Individual
enzyme activities can reflect the substrate availability, the nutrient
requirements of microorganisms and plants, and the strategies used by
microbes and plants to maintain the nutrient balance when the soil
environment changes (Burns et al., 2013; Nannipieri et al., 2018). Because
it is difficult to know whether changes in the enzymatic activities reflect
changes in the soil microbial biomass or differences in the actual
activities (Trasar-Cepeda et al., 2008), we need to study the specific
enzyme activities, i.e., the activity normalized to the total PLFA contents
(Zhang et al., 2015, 2017). The enzyme ratio is used to
examine the relative allocation of energy versus nutrient acquisition, since
it intersects the metabolic theory of microbial ecology and the theory of
ecological stoichiometry (Stone et al., 2014; Loeppmann et al., 2016a; Xu et
al., 2017). By studying how the enzyme activities and ratios change when the
understory vegetation is removed, we hope to improve our understanding of
how the storage of C in soil is influenced by the understory vegetation, and
how microbial nutrient acquisition is affected by microbial biomass and soil nutrients.
Studies have shown that understory vegetation-induced changes in soil
properties are closely related to climate, soil type, plant species, and time
(Li et al., 2013; Nilsson and Wardle, 2005; Zhang et al., 2014). There is
however no consensus on how understory vegetation impacts the physical,
chemical, and biological properties of forest soils. For example, some
studies have reported decreases in the litter decomposition rate, soil
organic matter (SOM) content, and soil respiration rate (Wang et al., 2011,
2014; Liu et al., 2012), while others have reported little change in the soil
properties after understory vegetation was removed (Xiong et al., 2008; Zhao
et al., 2011). Wu et al. (2011) and Zhao et al. (2013) found that the fungal
biomass and the fungi to bacteria ratio decreased, but Murugan et al. (2014)
found that the bacterial and saprophytic fungal biomass increased, and
ectomycorrhizal fungi and arbuscular mycorrhizal fungi decreased after
understory vegetation was removed from eucalyptus plantations. In an alpine
shrubland, the soil arbuscular mycorrhizal fungal biomass decreased 5 months
after plant functional groups were removed, but this effect disappeared after
17 months (Urcelay et al., 2009). The effects of understory vegetation on
soil microbial biomass vary by ecosystem type. Huang et al. (2014) reported
that soil enzyme activities decreased in a subtropical alpine coniferous
forest, while Lin et al. (2012) found that they did not change in a
Pinus sylvestris var. mongolica plantation when understory
vegetation was removed. The current information about the responses of soil
enzyme activities to understory vegetation removal is therefore inconsistent.
Yu et al. (2014) reported that the average net ecosystem productivity of
Chinese subtropical forests (362±39 g C m-2 yr-1) was
approximately 82.6 % and 64.9 % higher than that of tropical and
temperate forests. To maintain soil fertility, it is important that C sinks
and tree growths are sustained in these forests. A valuable economic
resource, Chinese fir (Cunninghamia lanceolata) plantations are
widespread throughout southern China. They cover an area of
9.11×106 ha, and account for approximately 18 % of the total
plantation area in China (Huang et al., 2013). Understory vegetation and
litter are commonly removed from the forest floor in southern China and
elsewhere to facilitate seed germination; ensure survival of seedlings; avoid
the intense competition between understory vegetation and trees for water,
nutrients, and light; and for fuel for rural inhabitants (Xiong et al., 2008;
Wu et al., 2011; Liu et al., 2012).
Therefore, we established a long-term field experiment to assess how the
soil abiotic properties, PLFAs, and enzyme activities in a Chinese fir
plantation changed when the understory vegetation was removed. We
hypothesized that rhizodeposition, and therefore microbial biomass and
activity, would decrease when the understory vegetation was removed.
Material and methodsExperimental treatments
Our study site was in the Shixi forest plantation in Taihe County, Jiangxi
Province, China (115∘03′29.9′′ E, 26∘44′29.1′′ N). The area has a
subtropical monsoon climate with a mean annual temperature of
18.8 ∘C and a mean annual precipitation of 1340 mm. According to
the USDA-NRCS soil taxonomy (Soil Survey Staff, 1999), the soil
in this area is dominated by Udults, which forms from red sandstone and sandy
conglomerate and has moist and dry Munsell values of 7.5 YR 5/6 and
7.5 YR 6/6, respectively.
Paired-plot design treatments with understory vegetation and litter
removal (None), and understory vegetation intact and litter removal
(Understory). The same abbreviations are used below.
The study site is a second-generation Chinese fir plantation that was
planted in 1998. The average tree height and diameter at breast height
(measured at 1.3 m above ground level) were about 18 m and 17 cm,
respectively. The understory vegetation, including shrubs and herbs, is
dominated by Old World forked fern (Dicranopteris dichotoma Berth),
gambir (Uncaria), oriental blueberry
(Vaccinium bracteatum), nutgall tree (Rhus chinensis),
Chinese witch hazel (Loropetalum chinense), short shank robe oak
(Quercus glandulifera BI.), root of mayflower glorybower
(Clerodendron cyrtophyllum Turcz), and azalea (Rhododendron).
Three plots, measuring 30×30 m and separated by a buffer of a least
10 m to avoid any between-plot influence, were established in the plantation
in January 2013. One paired treatment with three replicates was established
within each of the three plots. Each plot was divided into four subplots
(15×15 m each) and contained two treatments, namely None, from which
both the understory vegetation and litter were removed, and Understory, from
which the litter was removed but the understory vegetation was left. The two
subplots in a plot with the same treatment were distributed across each plot
to avoid the effects of slope (Fig. 1) and their results were averaged. The
litter and understory were managed monthly. The amount of litter and
understory vegetation at the study site amounted to about 1020 and
6236 kg ha-1 yr-1, respectively, under natural conditions.
Soil sampling and analysis
Bulk soil samples were collected in the wet (April and November) and dry
(July) seasons in 2015. Five soil cores with an inner diameter of 5 cm were
collected randomly from between the surface and a depth of 10 cm from each
subplot and then mixed as one composite sample. All fresh soil samples were
sieved to 2 mm and stored at 4 ∘C until analysis.
Soil physical and chemical properties were determined as outlined by
Bao (2008). Soil temperature (ST) was determined at a depth of 10 cm with a
soil thermometer (TP101) during sampling. The soil moisture content (SMC) was
measured by drying aliquots of soil at 105 ∘C to constant weight.
Soil pH was measured at a soil-to-water ratio of 1:2.5 by a pH digital
meter. The contents of nitrate N (NO3-–N) and ammonia N
(NH4+–N) were measured with a continuous flow analyser (Bran
Luebbe, AA3) after extraction with a 2 M KCl solution (soil : solution
ratio of 1:10). Dissolved organic carbon (DOC) contents were measured with
a TOC analyser (Elementar, Liquid II) after
extraction with ultra-pure water (soil : solution ratio of 1:5) (Jones
and Willett, 2006). Particulate organic carbon (POC) was determined as
outlined by Garten et al. (1999). The contents of soil organic C (SOC) and
total nitrogen (TN) were measured with an elemental
analyser (Vario Max CN).
Soil phospholipid fatty acids (PLFAs) were extracted following the procedure
outlined by Bossio and Scow (1998), and were determined with a gas
chromatograph (Agilent 6890N). Soil total PLFAs were represented by various
PLFA biomarkers; gram positive bacteria (G+) were represented by i14:0,
i15:0, a15:0, i16:0, i17:0, and a17:0, and gram negative bacteria (G-)
were represented by 16:1ω7c, cy17:0, 16:1ω9c, and cy19:0.
The total bacterial PLFAs were represented by biomarkers of G+ and
G-. The total fungi PLFAs were represented by arbuscular mycorrhizal
fungi (AMF) biomarkers 16:1ω5, as well as 18:1ω9c,
18:2ω6c, and 18:3ω6c, and the actinobacterial PLFAs were
represented by 10Me16:0, 10Me17:0, and 10Me18:0 (Bradley et al., 2007; Denef
et al., 2009).
Soil enzyme activities were measured following the methods of Saiya-Cork et
al. (2002). The specific substrates and functions of the enzymes assayed are
listed in Table A1. Five hydrolase activities, i.e., α-1,4-glucosidase (αG),
β-1,4-glucosidase (βG), β-1,4-N-acetylglucosaminidase (NAG),
β-1,4-xylosidase (βX) and acid phosphatase (AP)
were assayed using fluorogenically
labeled substrates. Briefly, a soil suspension was prepared by adding 1 g of
fresh soil to 125 mL of 50 mM acetate buffer. We added 200 µL of
the soil suspension and 50 µL of the substrate solution
(200 µM) to 96 microplates, making a total of eight analytical
replicates. Methylumbelliferone (MUB) was used to calibrate the hydrolase
activities. The microplates were incubated in the dark at 20 ∘C for
up to 4 h. After incubation, 10 µL of 1 M NaOH was added to each
well to terminate the enzymatic reaction. When the reactions had ended, the
fluorescence was measured using a microplate fluorometer (SynergyH4, BioTek)
with excitation and emission filters of 365 and 450 nm, respectively. We
calculated the specific enzyme activities by dividing the individual
potential hydrolase activities by the total PLFA contents (Zhang et al.,
2015, 2017). The total C-acquiring enzyme activity (Cenz) was
operationally defined as the sum of the αG, βG, and βX
activities (Stone et al., 2014) (Table A2).
The soil oxidase activities, i.e., polyphenol oxidase (PPO) and peroxidase (PER)
were assayed spectrophotometrically. We added 600 µL of the soil
suspension and 150 µL of the substrate solution to deep-well plates. We
also added 30 µL of 0.3 % H2O2 solution before determining
PER. After incubation in the dark at 20 ∘C for up to 5 h, the
deep-well plates were centrifuged for 3 min at 3000 r h-1. We then
transferred 250 µL of the supernatant to the microplates and measured
the absorbance at 450 nm with a microplate fluorometer (SynergyH4, BioTek) (DeForest, 2009).
Statistical analysis
Data are presented as the means ±standard errors. By applying the
one-sample Kolmogorov–Smirnov test within SPSS 17.0, we found that the data
satisfied the normal distribution criteria. We assessed the differences
between the soil abiotic properties, PLFA contents, and enzyme activities for
the understory treatments with a paired-sample t-test (SPSS 17.0). Where
two subplots within the same plot had the same treatment, we averaged the
data before analysis. We investigated the relationships between the soil
abiotic properties, PLFA contents, and enzyme activities for the two
treatments using redundancy analysis (RDA, CANOCO, version 4.5) and Pearson
correlation analysis (SPSS 17.0). We tested the significance of the variables
with the Monte Carlo permutation test before applying RDA. Figures were
generated using SigmaPlot (version 10.0). A significance level of P<0.05
was applied throughout.
ResultsSoil abiotic properties
The results suggest that the soil abiotic properties were influenced by the
understory vegetation management (Table 1). The contents of DOC, POC, SOC,
NH4+–N, and TN were 18 %, 25 %, 12 %, 34 %, and 8 %
lower in the None treatments than in the Understory treatments (P<0.05),
respectively. The SMC and POC/SOC were also 4 % and 15 %
lower in the None treatments than in the Understory treatments, respectively
(P<0.05). There were no significant differences between the contents of
NO3-–N, ST, pH, and the SOC/TN ratios in the
None and Understory treatments (P>0.05).
Soil PLFAs
The soil total PLFAs were 27 % lower in the None treatments than in the
Understory treatments (Fig. 2). Specifically, the bacterial and fungal PLFAs
were 26 % and 20 % lower (P<0.05) in the None treatments than in
the Understory treatments, respectively, but there were no significant
differences between the G+, G-, or actinobacterial PLFAs in the
two treatments (P>0.05). The fungi/bacteria ratios did not
change because the bacterial and fungal PLFAs were both lower in the None treatments.
Soil phospholipid fatty acids (PLFAs) in the different understory
vegetation treatments. Soil PLFA contents (a); ratio of PLFA
contents (b). G+/G-: ratio of gram positive
bacteria to gram negative bacteria; F/B: ratio of fungi to
bacteria. Different lowercases represent significant differences among the
None and Understory treatments (P<0.05). Data are the means
±standard errors. The same abbreviations apply to Fig. 4.
Soil enzyme activities
The soil enzyme activities varied as the understory vegetation management
varied. The potential activities of βG, NAG, PPO, and PER were
13 %, 24 %, 21 %, and 20 % lower in the None treatments than in the
Understory treatments (Fig. 3a and b) (P<0.05), respectively, but
the potential activities of acid phosphatases did not differ significantly
(P>0.05) between the two treatments. The ratio of
lnCenz/lnNAG was 6 % higher in the None treatments than in the
Understory treatments, but the ratios of lnCenz/lnAP were similar for
the different treatments. The trends were enzyme-specific when normalized by
the total PLFAs (Fig. 3d and e). The specific activities of the C-acquiring
enzymes, i.e., αGPLFAs, βGPLFAs and βXPLFAs,
were 40 %, 22 %, and 41 % higher, respectively, in the
None treatments than in the Understory treatments (P<0.05), but the
specific activities of N- (NAGPLFAs) and P-acquiring enzymes (APPLFAs)
were not significantly different between the two treatments (P>0.05).
Correlations between soil enzyme activities, soil PLFAs, and soil abiotic properties
The first (RD1) and second (RD2) ordination axes explained 62.0 % and
15.5 % of the total variability in the different PLFAs, respectively. Soil
temperature, SMC, NO3-–N, NH4+–N, DOC, SOC, and
SOC/TN were mainly correlated with RD1 (Fig. 4a). Ammonia
nitrogen and DOC were positively correlated with bacterial, actinobacterial,
and total PLFAs, and SOC was positively correlated with G-, bacterial,
fungal, and total PLFAs (P<0.05) (Table A3).
Soil enzyme activities in the different understory vegetation
treatments. Soil potential hydrolase activities (a), soil potential
oxidase activities (b), enzyme activity ratios (c), and
soil hydrolase activities normalized by total PLFAs (d).
αG: α-1,4-glucosidase, βG: β-1,4-glucosidase,
NAG: β-1,4-N-acetylglucosaminidase, βX: β-1,4-xylosidase,
AP: acid phosphatase, PPO: phenol oxidase, and PER: peroxidase. The same
abbreviations apply to Fig. 4.
Redundancy analysis of all soil abiotic properties and PLFA
contents (a), and potential enzyme activities (b).
SMC: soil moisture content, pH: soil pH, NO3-–N: soil nitrate
nitrogen, NH4+–N: soil ammonia nitrogen, TN: soil total
nitrogen, DOC: soil dissolved organic carbon, POC: soil particulate organic
carbon, SOC: soil organic carbon, POC/SOC: ratio of POC to
SOC, and SOC/TN: ratio of SOC to TN.
The first (RD1) and second (RD2) ordination axes explained 50.1 % and
19.9 % of the total variability in the potential enzyme activities,
respectively. The contents of DOC, NO3-–N, and
NH4+–N were mainly related to RD2 (Fig. 4b). Dissolved organic
carbon was positively correlated with αG and negatively correlated
with βX and AP, and NH4+–N was positively correlated with
αG and βG (P<0.05; Table A3). Bacterial and total PLFAs were
positively correlated with αG, βG, NAG, PPO, and PER, and fungal
PLFAs were positively correlated with αG, βG, and NAG (P<0.05;
Table A4).
Discussion
Consistent with our hypothesis, the contents of organic C (including DOC,
POC, and SOC) and N (including NH4+–N and TN) were lower in the
plots from which the understory vegetation was removed than in those with
intact understory vegetation (Table 1), which suggests that understory
vegetation promotes C and N storage in soil. Other researchers reported
minimal changes in the soil physical and chemical properties when the
understory vegetation was removed (Xiong et al., 2008; Zhao et al., 2011),
and the different results may reflect the variable composition of the
understory vegetation (Nilsson and Wardle, 2005). In our study, we removed
the litter from all treatments. The roots of the Chinese fir trees may take
over the space previously occupied by the understory vegetation and may
increase their exudation to compensate for the reduced C inputs (Li et al.,
2016), and the residues from the roots of understory vegetation may also
decompose in the soil (Li et al., 2013). However, the increased quantities of
labile C from Chinese fir roots and understory vegetation root residues may
not fully compensate for the C loss when the understory vegetation is
removed. Additionally, soil C tends to be higher when the plant functional
diversity is high (Zhou et al., 2016). When the understory vegetation is
removed, the plant diversity decreases, and the soil C content also
decreases. Previously, researchers found that soil N contents increased when
the amount of N taken up by plants decreased during tree girdling experiments
(Kaiser et al., 2010) and in soils without live roots (Loeppmann et al.,
2016a). However, we found that the soil N increased when the understory
vegetation remained intact, which suggests that the amount of available N
released from plant roots and SOM degradation exceeded the amount taken up by
plants. The POC/SOC ratios were lower for the understory
vegetation removal plots than for the plots with intact understory vegetation
(Table 1), which suggests that POC declined more strongly than SOC when the
understory vegetation was removed. Since POC is related to aggregate
stability, the soil in Chinese fir plantations will be more productive when
the understory vegetation remains intact (Bouajila and Gallali, 2010). As
also reported by Wang et al. (2014), the SMC decreased when the understory
vegetation was removed (Table 1), which shows that the understory vegetation
has benefits for soil moisture.
Soil abiotic properties in the different understory vegetation treatments.
Values in the table are the means ±standard error.
ST: soil temperature, SMC: soil moisture, pH: soil pH,
NO3-–N: soil nitrate nitrogen, NH4+–N: soil
ammonia nitrogen, TN: soil total nitrogen, DOC: soil dissolved organic
carbon, POC: soil particulate organic carbon, SOC: soil organic carbon,
POC/SOC: ratio of POC to SOC, and
SOC/TN: ratio of SOC to TN. Different lowercase letters
represent significant differences between None and Understory treatments
(P<0.05). Data were means ±standard errors. The same
abbreviations are used below.
Consistent with our hypothesis, total PLFAs declined when the understory
vegetation was removed (Fig. 2). It is known that fungal biomass decreases
when understory vegetation was removed (Wu et al., 2011; Liu et al., 2012;
Zhao et al., 2013). The PLFAs in AMF were lower in the plots with no
understory vegetation (Fig. A1), which reflects the reduced plant diversity.
Since certain AMF may only grow when specific plants are present, changes in
the plant communities over time will result in changes in their mycorrhizal
partners (Hart et al., 2001). Other studies have suggested that decreases in
the fungal PLFAs were mainly related to a reduction in mycorrhizal fungi, as
mycorrhizal fungi are more dependent on below-ground C allocation by plants
than other fungi (Kaiser et al., 2010). Mycorrhizal species in the
understory vegetation included Dicranopteris dichotoma,
Vaccinium bracteatum, Loropetalum chinense, and Rhododendron. Chinese fir (arbuscular mycorrhizal
plant) monocultures may support fewer fungi biomass than other plantations
where the understory vegetation is left intact. The bacterial biomass also
decreased after the understory vegetation was removed, mainly because of the
decreases in the soil C and N (Table A3) and plant diversity (Lamb et al.,
2011). Actinobacteria promote the decomposition of recalcitrant C compounds
and, while Brant et al. (2006) considered that they might increase when the
available nutrient contents were low, we did not observe such a tendency
(Fig. 2), perhaps because of the high variability in the actinobacterial
PLFAs in the soils.
Consistent with our hypothesis and the results of Huang et al. (2014), we
found that the potential extracellular enzyme activities were lower when
there was no understory vegetation (Fig. 3). However, Lin et al. (2012) did
not observe any changes in soil enzyme activities when understory vegetation
was removed. The soil rhizosphere is a hotspot of microbial activities
(Kuzyakov and Blagodatskaya, 2015). Decreases in the quantity and diversity
of root exudates in the understory vegetation, and changes in the soil
abiotic and biotic properties may cause direct and indirect changes in soil
enzyme activities (Liu et al., 2012; Huang et al., 2014). The potential C
hydrolase activity was higher when the understory remained intact, indicating
the high soil microbial demand for C. The specific C hydrolase activities
normalized by PLFAs were lower when the understory vegetation remained intact
than when it was removed, which may reflect opportunistic microorganisms
(microorganisms that use enzyme products rather than produce enzymes) that
emerged in response to an increase in the labile C input (Allison, 2005), and
a subsequent decline in the ability of microorganisms to produce C-acquiring
enzymes. The ratio of C- to N-acquiring enzymes increased when the understory
vegetation was removed, perhaps because the microbes produced enzymes that
acquired C rather than N when the labile C inputs were lower. There are
various explanations for the changes observed in the potential enzyme
activities, as follows: (1) mycorrhizal fungi vanish when understory
vegetation is removed (Fekete et al., 2011), which means there are fewer
microorganisms to produce enzymes, so the total amount of enzymes decreases.
(2) When the understory vegetation remains intact, root exudates are
continuously released to soil, but when the understory vegetation is removed,
below-ground root residues are the main source of C for the understory
vegetation. Thus, the inputs of C with different chemical compositions may
have influenced the enzyme activities (Li et al., 2013).
The activities of αG and βG were positively correlated with the
contents of the soil inorganic N fractions (Table A3), which suggests that
the decomposition of C decreased because of the reduced availability of N
when the understory vegetation was removed. The size of the soil C pool
reflects the balance between the inputs and outputs of C (De Deyn et al.,
2008). When understory vegetation is removed, both the soil C inputs,
including root exudates, fine root turnover (Liu et al., 2012), and SOM
decomposition rate (Wu et al., 2011; Liu et al., 2012; Zhao et al., 2013),
and soil C outputs, such as soil respiration (Wang et al., 2013), decrease.
The lower SOC contents in the plots from which the understory vegetation was
removed therefore indicate that the removal of understory vegetation had more
effect on the outputs of soil C than on the inputs. Polyphenols are mainly
decomposed by PPO, so the decrease in PPO activity may result in an increase
in the content of polyphenols that have toxic effects on soil microbes and
inhibit hydrolase activities (Sinsabaugh, 2010). Oxidative enzymes are
responsible for the degradation of poor-quality, chemically complex
compounds, such as lignin, aromatic compounds, and phenolic compounds
(Sinsabaugh, 2010). Therefore, the lower activities of PPO and PER observed
after the understory vegetation was removed may result in an increase in the
content of refractory compounds in SOM.
Phosphorus is generally the most limiting element in the highly weathered red
soils in southern China. Soil P is generally present in an organic form or is
immobilized when the contents of Al and Fe are high (Margalef et al., 2017).
Of all the enzymes we assayed, the activity of AP was the highest (Fig. 3),
which may reflect the fact that P was the limiting nutrient in the red soils.
Soil microorganisms may produce more phosphatase to mineralize organic P to
meet their demand for P (Allison and Vitousek, 2005). Loeppmann et
al. (2016a, b) reported that N-degrading enzymes in the rhizosphere of
maize-planted soil increased when the available N decreased because of plant
N uptake, which suggests that N demand in the rhizosphere might be regulated
by a similar mechanism in the cultivated field; we however did not find any
evidence of such a control in our study. The soil nutrient availability
affects rhizosphere priming (Dijkstra et al., 2013). The higher potential NAG
activity and higher contents of NH4+–N in the treatments with
the intact understory vegetation suggest that the energy-rich C compounds
released through the roots promoted the production of N-acquiring enzymes
that released available N from SOM. The low potential activity of NAG in the
treatments from which the understory vegetation was removed was related to
the reduction in the fungal biomass, and reflects the fact that chitin, a
major structural component of fungal cell walls (Loeppmann et al., 2016b),
can be degraded by NAG (Mganga et al., 2015). We did not observe any change
in the AP activities when the understory vegetation was removed. Because
Chinese firs coexist with fungi and form mycorrhizal associates (Li et al.,
2011), and mycorrhizal fungi produce soil acid phosphatase (Rosling et al.,
2016), these enzymes were most likely produced by Chinese firs. The negative
relationships between the potential activity of AP and DOC suggest that DOC
was the substrate for microbes, and that large amounts of DOC were consumed
when producing P-acquiring enzymes.
Conclusions
Our results demonstrate that understory vegetation plays an important role in
enhancing the soil C and N contents and the soil potential activities of C-
and N-hydrolase and oxidase, but does not influence the P-hydrolase activity.
The ratio of C- to N-acquiring enzymes increased after the understory
vegetation was removed, which implies that, under lower inputs of labile C,
microbes invest more in C-acquiring enzymes than N-acquiring enzymes. The
positive relationship between the activities of C-degrading enzymes and the
soil inorganic N contents suggests that C decomposition was inhibited by the
lower available N contents after understory vegetation was removed. It could
be expected that less N taken up by plants may increase soil N content after
understory vegetation was removed; however, soil N inputs decrease with
reduced understory vegetation root material inputs, which leads to inorganic
N decreases over time. The potential activity of AP was negatively correlated
with the content of DOC, which indicates that large amounts of DOC, an energy
source, were consumed when producing P-acquiring enzymes. Therefore,
understory vegetation can contribute to C sequestration by enhancing C inputs
to soil, even though C may be lost from soil with understory vegetation
through the degradation of SOM by enzymes. We suggest that, as part of
routine forestry management, understory vegetation should not be removed from
subtropical Chinese fir plantations.
Requests for data or other materials should be directed to
Xinyu Zhang (zhangxy@igsnrr.ac.cn).
Appendix information
Contents of arbuscular mycorrhizal fungi in the different understory
vegetation treatments.
Soil enzymes and their corresponding substrates and functions.
EnzymeE. CAbbreviationSubstrateFunctionPeroxidase1.11.1.7PERL-DOPAOxidizes lignin and aromatic compounds using H2O2or secondary oxidants as an electron acceptor (Sinsabaugh, 2010).Phenol oxidase1.10.3.2PPOL-DOPAOxidizes phenolic compounds using oxygen as anelectron acceptor (Sinsabaugh, 2010).α-1,4-glucosidase3.2.1.20αG4-MUB-α-D-glucosideReleases glucose from starch (Stone et al., 2014).β-1,4-glucosidase3.2.1.21βG4-MUB-β-D-glucosideReleases glucose from cellulose (Stone et al., 2014).β-1,4-xylosidase3.2.1.37βX4-MUB-β-D-xylosideReleases xylose from hemicellulose (Stone et al., 2014).β-1,4-N-3.2.1.14NAG4-MUB-N-acetyl-β-D-Releases N-acetyl glucosamine from oligosaccharidesacetylglucosaminidaseglucosaminide(Stone et al., 2014).Acid phosphatase3.1.3.1AP4-MUB-phosphateReleases phosphate groups (Stone et al., 2014).
Enzyme indexes: the potential enzyme
activity and the total PLFA contents were used to calculate different enzyme
indexes. α-1,4-glucosidase (αG), β-1,4-glucosidase
(βG), and β-1,4-xylosidase (βX) represented C-acquiring
enzymes, whereas β-1,4-N-acetylglucosaminidase(NAG) represented
N-cycling enzymes. Acid phosphatase (AP) represented P-acquiring enzymes.
Enzyme indexesDescriptionReferenceαGPLFAs; βGPLFAs; βXPLFAsSpecific enzyme activity of C-acquiring enzymesZhang et al. (2015, 2017)(enzyme activities to total PLFAs)NAGPLFAsSpecific enzyme activity of N-acquiring enzymesZhang et al. (2015, 2017)(enzyme activities to total PLFAs)APPLFAsSpecific enzyme activity of P-acquiring enzymesZhang et al. (2015, 2017)(enzyme activities to total PLFAs)lnCenz/lnNAGRatio of C- to N-acquiring enzymesStone et al. (2014), Loeppmann et al.(2016a), Xu et al. (2017)lnCenz/lnAPRatio of C- to P-acquiring enzymesStone et al. (2014), Loeppmann et al.(2016a), Xu et al. (2017)
Cenz: the total C-acquiring enzyme activity (the
potential activities of αG+βG+βX).
Pearson correlation coefficients between soil abiotic properties, PLFA
contents, and potential enzyme activities.
Values are Pearson
r value. * indicates a significant difference at P<0.05;
** indicates a significant difference at P<0.01. G+: gram
positive bacteria, G-: gram negative bacteria, PLFAs: total PLFAs,
G+/G-: ratio of G+ and G-,
F/B: ratio of fungi to bacteria.
αG: α-1,4-glucosidase, βG: β-1,4-glucosidase,
NGA: β-1,4-N-acetylglucosaminidase, βX: β-1,4-xylosidase,
AP: acid phosphatase, PPO: phenol oxidase,
PER: peroxidase. These abbreviations apply to Tables A4–A7.
Pearson correlation coefficients between PLFA contents and
potential enzyme activities.
Different lowercase letters represent significant differences between
different treatments, and different uppercase letters represent significant
differences among different months in the same treatment (P<0.05). The same
abbreviations apply to Tables A6 and A7.
XYZ, HMW, XLF, FSC, XMS, XFW designed the study; YY, CZ, and
SZW performed the study and analyzed data; YY, XYZ, and JFW wrote the paper.
The authors declare that they have no conflict of interest.
Acknowledgements
This work was jointly financed by the National Natural Science Foundation of
China (nos. 41571251 and 41571130043).
Edited by: Anja Rammig
Reviewed by: three anonymous referees
References
Allison, S. D.: Cheaters, diffusion and nutrients constrain decomposition by
microbial enzymes in spatially structured environments, Ecol. Lett., 8, 626–635, 2005.
Allison, S. D. and Vitousek, P. M.: Responses of extracellular enzymes to simple
and complex nutrient inputs, Soil Biol. Biochem., 37, 937–944, 2005.
Bao, S. D.: Soil and Agricultural Chemistry Analysis, 3rd Edn., Agricultrue
Press, Beijing, 2008.
Bossio, D. A. and Scow, K. M.: Impacts of carbon and flooding on soil microbial
communities: Phospholipid fatty acid profiles and substrate utilization patterns,
Microb. Ecol., 35, 265–278, 1998.
Bouajila, A. and Gallali, T.: Land use effect on soil and particulate organic
carbon, and aggregate stability in some soils in Tunisia, Afr. J. Agric. Res.,
5, 764–774, 2010.Bradley, K., Hancock, J. E., Giardina, C. P., and Pregitzer, K. S.: Soil microbial
community responses to altered lignin biosynthesis in Populus tremuloides
vary among three distinct soils, Plant Soil, 294, 185–201, 2007.
Brant, J. B., Myrold, D. D., and Sulzman, E. W.: Root controls on soil microbial
community structure in forest soils, Oecologia, 148, 650–659, 2006.
Burns, R. G., DeForest, J. L., Marxsen, J., Sinsabaugh, R. L., Stromberger,
M. E., Wallenstein, M. D., Weintraub, M. N., and Zoppini, A.: Soil enzymes in
a changing environment: current knowledge and future directions, Soil Biol.
Biochem., 58, 216–234, 2013.
De Deyn, G. B., Cornelissen, J. H. C., and Bardgett, R. D.: Plant functional
traits and soil carbon sequestration in contrasting biomes, Ecol. Lett.,
11, 516–531, 2008.
DeForest, J. L.: The influence of time, storage temperature, and substrate age
on potential soil enzyme activity in acidic forest soils using MUB-linked
substrates and L-DOPA, Soil Biol. Biochem., 41, 1180–1186, 2009.
Denef, K., Roobroeck, D., Wadu, M. C. W. M., Lootens, P., and Boeckx, P.:
Microbial community composition and rhizodeposit-carbon assimilation in
differently managed temperate grassland soils, Soil Biol. Biochem., 44, 144–153, 2009.
Dijkstra, F. A., Carrillo, Y., Pendall, E., and Morgan, J. A.: Rhizosphere
priming: a nutrient, Front. Microbiol., 4, 1–8, 2013.
Fekete, I., Varga, C., Kotroczó, Z., Tóth, J. A., and Várbíró,
G.: The relation between various detritus inputs and soil enzyme activities in
a Central European deciduous forest, Geoderma, 167, 15–21, 2011.
Fu, X. L., Yang, F. T., Wang, J. L., Di, Y. B., Dai, X. Q., Zhang, X. Y., and
Wang, H. M.: Understory vegetation leads to changes in soil acidity and in
microbial communities 27 years after reforestation, Sci. Total Environ.,
502, 280–286, 2015.
Garten, C. T., Post, W. M., Hanson, P. J., and Cooper, L. W.: Forest soil carbon
inventories and dynamics along an elevation gradient in the southern Appalachian
Mountains, Biogeochemistry, 42, 115–145, 1999.
Hart, M. M., Reader, R. J., and Klironomos, J. N.: Life-history strategies of
arbuscular mycorrhizal fungi in relation to their successional dynamics,
Mycologia, 93, 1186–1194, 2001.
Huang, Y. M., Yang, W. Q., Zhang, J., Lu, C. T., Liu, X., Wang, W., Guo, W.,
and Zhang, D. J.: Response of soil microorganism and soil enzyme activity to
understory plant removal in the subalpine coniferous plantation of western
Sichuan, Acta Ecol. Sin., 34, 4183–4192, 2014.
Huang, Z. Q., He, Z. M., Wan, X. H., Hu, Z. H., Fan, S. H., and Yang, Y. S.:
Harvest residue management effects on tree growth and ecosystem carbon in a
Chinese fir plantation in subtropical China, Plant Soil, 364, 303–314, 2013.
Jones, D. L. and Willett, V. B.: Experimental evaluation of methods to quantify
dissolved organic nitrogen (DON) and dissolved organic carbon (DOC) in soil,
Soil Biol. Biochem., 38, 991–999, 2006.
Kaiser, C., Koranda, M., Kitzler, B., Fuchslueger, L., Schnecker, J., Schweiger,
P., Rasche, F., Zechmeister-Boltenstern, S., Sessitsch, A., and Richter, A.:
Belowground carbon allocation by trees drives seasonal patterns of extracellular
enzyme activities by altering microbial community composition in a beech forest
soil, New Phytol., 187, 843–858, 2010.
Kuzyakov, Y. and Blagodatskaya, E.: Microbial hotspots and hot moments in soil:
Concept & review, Soil Biol. Biochem., 83, 184–199, 2015.
Lamb, E. G., Kennedy, N., and Siciliano, S. D.: Effects of plant species richness
and evenness on soil microbial community diversity and function, Plant Soil,
338, 483–495, 2011.
Li, L., Zhou, G. Y., Liu, J. A., and Li, H.: The resource investigation and
community structure characteristics of mycorrhizal fungi associated with
Chinese fir, Afr. J. Biotechnol., 10, 5719–5724, 2011.
Li, M. H., Du, Z., Pan, H. L., Yan, C. F., Xiao, W. F., and Lei, J. P.: Effects
of neighboring woody plants on target trees with emphasis on effects of
understory shrubs on overstory physiology in forest communities: a mini-review,
Commun. Ecol., 13, 117–128, 2016.
Li, Y. F., Zhang, J. J., Chang, S. X., Jiang, P. K., Zhou, G. M., Fu, S. L.,
Yan, E. R., Wu, J. S., and Lin, L.: Long-term intensive management effects on
soil organic carbon pools and chemical composition in Moso bamboo (Phyllostachys
pubescens) forests in subtropical China, Forest Ecol. Manage., 303, 121–130, 2013.Lin, G., Zhao, Q., Zhao, L., Li, H. C., and Zeng, D. H.: Effects of understory
removal and nitrogen addition on the soil chemical and biological properties of
Pinus sylvestris var. mongolica plantation in Keerqin Sandy
Land, Chin. J. Appl. Ecol., 23, 1188–1194, 2012.
Liu, Z. F., Wu, J. P., Zhou, L. X., Lin, Y. B., and Fu, S. L.: Effect of
understory fern (Dicranopteris dichotoma) removal on substrate utilization
patterns of culturable soil bacterial communities in subtropical Eucalyptus
plantations, Pedobiologia, 55, 7–13, 2012.
Loeppmann, S., Biagodatskaya, E., Pausch, J., and Kuzyakov, Y.: Enzyme properties
down the soil profile – A matter of substrate quality in rhizosphere and
detritusphere, Soil Biol. Biochem., 103, 274–283, 2016a.
Loeppmann, S., Semenov, M., Blagodatskaya, E., and Kuzyakov, Y.: Substrate
quality affects microbial- and enzyme activities in rooted soil, J. Plant Nutr.
Soil Sci., 179, 39–47, 2016b.Margalef, O., Sardans, J., Fernandez-Martinez, M., Molowny-Horas, R., Janssens,
I. A., Ciais, P., Goll, D., Richter, A., Obersteiner, M., Asensio, D., and
Penuelas, J.: Global patterns of phosphatase activity in natural soils, Sci.
Rep., 7, 1337, 10.1038/s41598-017-01418-8, 2017.
McNear Jr., D. H.: The rhizosphere roots soil and everything in between, Nat.
Educ. Know., 4, 1, 2013.
Mganga, K. Z., Razavi, B. S., and Kuzyakov, Y.: Microbial and enzymes response
to nutrient additions in soils of Mt. Kilimanjaro region depending on land use,
Eur. J. Soil Biol., 69, 33–40, 2015.
Murugan, R., Beggi, F., and Kumar, S.: Belowground carbon allocation by trees,
understory vegetation and soil type alter microbial community composition and
nutrient cycling in tropical Eucalyptus plantation, Soil Biol. Biochem.,
76, 257–267, 2014.
Nannipieri, P., Trasar-Cepeda, C., and Dick, R. P.: Soil enzyme activity: a
brief history and biochemistry as a basis for appropriate interpretations and
meta-analysis, Biol. Fertil. Soils, 54, 11–19, 2018.
Nilsson, M. C. and Wardle, D. A.: Understory vegetation as a forest ecosystem
driver: evidence from the northern Swedish boreal forest, Front. Ecol. Environ.,
3, 421–428, 2005.
Rosling, A., Midgley, M. G., Cheeke, T., Urbina, H., Fransson, P., and Phillips,
R. P.: Phosphorus cycling in deciduous forest soil differs between stands
dominated by ecto- and arbuscular mycorrhizal trees, New Phytol., 209, 1184–1195, 2016.
Saiya-Cork, K. R., Sinsabaugh, R. L., and Zak, D. R.: The effects of long term
nitrogen deposition on extracellular enzyme activity in an Acer saccharum forest
soil, Soil Biol. Biochem., 34, 1309–1315, 2002.
Sinsabaugh, R. L.: Phenol oxidase, peroxidase and organic matter dynamics of
soil, Soil Biol. Biochem., 24, 391–401, 2010.
Soil Survey Staff: Soil taxonomy: a basic system of soil classification for
making and interpreting soil surveys, 2nd Edn., Government Printing Office,
Washington, D.C., 1999.
Stone, M. M., DeForest, J. L., and Plante, A. F.: Changes in extracellular
enzyme activity and microbial community structure with soil depth at the
Luquillo Critical Zone Observatory, Soil Biol. Biochem., 75, 240–241, 2014.
Trasar-Cepeda, C., Leiros, M. C., and Gil-Sotres, F.: Hydrolytic enzyme
activities in agricultural and forest soils. Some implications for their use as
indicators of soil quality, Soil Biol. Biochem., 40, 2146–2155, 2008.
Urcelay, C., Diaz, S., Gurvich, D. E., Chapin, F. S., Cuevas, E., and Dominguez,
L. S.: Mycorrhizal community resilience in response to experimental plant
functional type removals in a woody ecosystem, J. Ecol., 97, 1291–1301, 2009.
Wang, F. M., Zou, B., Li, H. F., and Li, Z. A.: The effect of understory removal
on microclimate and soil properties in two subtropical lumber plantations, J.
Forest Res., 19, 238–243, 2014.
Wang, Q. K., He, T. X., Wang, S. L., and Liu, L.: Carbon input manipulation
affects soil respiration and microbial community composition in a subtropical
coniferous forest, Agr. Forest. Meteorol., 178, 152–160, 2013.
Wang, X. L., Zhao, J., Wu, J. P., Chen, H., Lin, Y. B., Zhou, L. X., and Fu, S.
L.: Impacts of understory species removal and/or addition on soil respiration
in a mixed forest plantation with native species in southern China, Forest Ecol.
Manage., 261, 1053–1060, 2011.
Wu, J. P., Liu, Z. F., Wang, X. L., Sun, Y. X., Zhou, L. X., Lin, Y. B., and Fu,
S. L.: Effects of understory removal and tree girdling on soil microbial community
composition and litter decomposition in two Eucalyptus plantations in South
China, Funct. Ecol., 25, 921–931, 2011.Xiong, Y. M., Xia, H. P., Li, Z. A., Cai, X. A., and Fu, S. L.: Impacts of
litter and understory removal on soil properties in a subtropical Acaciamangium plantation in China, Plant Soil, 304, 179–188, 2008.
Xu, Z. W., Yu, G. R., Zhang, X. Y., He, N. P., Wang, Q. F., Wang, S. Z., Wang,
R. L., Zhao, N., Jia, Y. L., and Wang, C. Y.: Soil enzyme activity and
stoichiometry in forest ecosystems along the North-South Transect in eastern
China (NSTEC), Soil Biol. Biochem., 104, 152–163, 2017.
Yu, G. R., Chen, Z., Piao, S. L., Peng, C. H., Ciais, P., Wang, Q. F., Li, X. R.,
and Zhu, X. J.: High carbon dioxide uptake by subtropical forest ecosystems in
the East Asian monsoon region, P. Natl. Acad. Sci. USA, 111, 4910–4915, 2014.Zhang, C., Zhang, X.-Y., Zou, H.-T., Kou, L., Yang, Y., Wen, X.-F., Li, S.-G.,
Wang, H.-M., and Sun, X.-M.: Contrasting effects of ammonium and nitrate
additions on the biomass of soil microbial communities and enzyme activities
in subtropical China, Biogeosciences, 14, 4815–4827, 10.5194/bg-14-4815-2017, 2017.
Zhang, J. J., Li, Y. F., Chang, S. X., Jiang, P. K., Zhou, G. M., Liu, J., Wu,
J. S., and Shen, Z. M.: Understory vegetation management affected greenhouse
gas emissions and labile organic carbon pools in an intensively managed Chinese
chestnut plantation, Plant Soil, 376, 363–375, 2014.
Zhang, X. Y., Dong, W. Y., Dai, X. Q., Schaeffer, S., Yang, F. T., Radosevich,
M., Xu, L. L., and Sun, X. M.: Responses of absolute and specific soil enzyme
activities to long term additions of organic and mineral fertilizer, Sci. Total
Environ., 536, 59–67, 2015.
Zhao, J., Wang, X. L., Shao, Y. H., Xu, G. L., and Fu, S. L.: Effects of
vegetation removal on soil properties and decomposer organisms, Soil Biol.
Biochem., 45, 954–960, 2011.
Zhao, J., Wan, S. Z., Fu, S. L., Wang, X. L., Wang, M., Liang, C. F., Chen, Y.
Q., and Zhu, X. L.: Effects of understory removal and nitrogen fertilization
on soil microbial communities in Eucalyptus plantations, Forest Ecol. Manage.,
310, 80–86, 2013.
Zhou, L. L., Cai, L. P., He, Z. M., Wang, R. W., Wu, P. F., and Ma, X. Q.:
Thinning increases understory diversity and biomass, and improves soil properties
without decreasing growth of Chinese fir in southern China, Environ. Sci. Pollut.
Res., 23, 24135–24150, 2016.