Catena 115 (2014) 96–103

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Catena
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Vegetation type affects soil enzyme activities and microbial functional

diversity following re-vegetation of a severely eroded red soil in
sub-tropical China
Rui Yin 1, Huan Deng ⁎,2, Hui-li Wang 3, Bin Zhang 4
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China

a r t i c l e i n f o a b s t r a c t

Article history: The objectives of this study were to evaluate the restorative effects of re-vegetation and vegetation type on soil
Received 17 March 2013 enzyme activities and microbial functional diversity of eroded red soil. Soil samples were collected by horizon
Received in revised form 1 November 2013 from eroded soils that had been restored for 18 years with Cinnamomum camphora, Pinus massoniana or Lespedeza
Accepted 27 November 2013
bicolor. Un-eroded soils planted with these vegetation types and an eroded bare soil served as references. Soil
microbial functional diversity was assessed by the community-level physiological profiles (CLPP) using BioLog
Keywords:
Soil restoration
Eco-plates. Re-vegetation improved soil enzyme activities and microbial functional diversity, compared with
Soil horizon the eroded bare soil. The soil restored with L. bicolor had the highest cellulase and β-glucosidase activities but
Microbial biomass the lowest urease activity. The soil restored with P. massoniana and C. camphora had the highest polyphenol
Physicochemical properties oxidase activity and microbial functional diversity, respectively. The microbial functional diversity and communi-
Community-level physiological profiles ty structure based on CLPP exhibited different patterns in O, A or B horizons. The varimax rotated component
matrix of CLPP further indicated that the polymers, phenolic compounds and carbohydrates largely affected the
microbial functional structure in O horizon, while amines, amino acids and carboxylic acids mainly affected the
microbial functional structure in B horizon. We suggest that the enzyme activities and microbial functional diver-
sity are determined by the quantity and quality of carbon inputs. Soil enzyme activities and microbial functional
diversity as well as soil microbial biomass and physicochemical properties in the restored eroded soil did not
completely recover to the levels of the un-eroded reference soil.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
Red soil, which is classified as Ultisols in the U.S. soil taxonomy sys-
tem, is a typical soil type in tropical and subtropical zones and occupies
45% of the world's land area (Shui et al., 2004). Red soil erosion is a
worldwide problem mainly due to its intrinsic nature, rainy climate and
hilly land shape (Carpenter et al., 2001; Okusami et al., 1997; Yassoglou
et al., 1997). In China, inappropriate soil management including intensive
agricultural practices and deforestation aggravated the red soil erosion.
This erosion area once reached 0.25 million km2, accounting for 22% of
the total red soil area. The eroded red soil is characterized by the loss of
O, A or even B horizons together with a reduction or depletion of soil
⁎ Corresponding author at: Institute of Urban Environment, Chinese Academy of
Sciences, Xiamen 361021, China. Tel.: +86 592 6190580; fax: +86 592 6190766.
E-mail address: hdeng@iue.ac.cn (H. Deng).
1
Current address: Nanjing Branch of the Chinese Academy of Sciences, Nanjing, China.
2
Current address: Institute of Urban Environment, Chinese Academy of Sciences,
Xiamen, China.
3
Current address: Guangxi Academy of Forestry, Nanning, China.
4
Current address: Chinese Academy of Agricultural Sciences, Beijing, China.
nutrients and microbial activity (Deng et al., 2010). Re-vegetation was
widely accepted as a useful way to dramatically increase soil fertilities
(Chen et al., 2002; Zhang and Xu, 2005) and reduce soil and water loss
of eroded red soil (Huang et al., 2010; Zheng et al., 2008). The remote
sensing data showed that the eroded area decreased from 0.25 million
km2 in 1986 to less than 0.2 million km2 in 2005 (Liang et al., 2010). In
addition, a small-scale field study showed that after a 20-year period of
re-vegetation the eroded area reduced while the soil microbial biomass
and soil organic carbon content dramatically recovered (Xu et al.,
2010). However, whether the restored eroded soil recovered to the
level of the un-eroded soil is seldom known.
A long-term study on the re-vegetation of eroded red soil has been
carried out since 1987 in the Ecological Experimental Station of the
Chinese Academy of Sciences. The native plants C. camphora, L. bicolor
and P. massoniana, grouped as evergreen and broad-leaf tree, leguminous
shrub and conifers, respectively, were adopted to restore the severely
eroded land, which was characterized by gullies with exposed parental
material. In addition, the experimental station preserved eroded bare
land and un-eroded land planted with native and secondary forests as
references. In this experimental station, a long term monitoring was

0341-8162/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.catena.2013.11.015
 R. Yin et al. / Catena 115 (2014) 96–103
carried out in respect to soil fertility and soil loss after re-vegetation
(Zhang et al., 2004). Recently, microbial genetic diversity (Deng et al.,
2010) and soil functional resilience of carbon mineralization (Zhang
et al., 2010) was studied to evaluate restoration effect between different
vegetation types. However, these studies did not cover a series of bio-
chemical functionalities, which are important indicators of soil quality.
In this study, we investigated soil enzyme activities and microbial
functional diversity based on community-level physiological profiles
(CLPP). Soil enzyme activities and microbial functional diversity are fre-
quently used indicators to assess the effect of vegetative restoration on
soil quality since they mediate the processes of organic matter turnover
and are sensitive to soil management (Boyle et al., 2005; Carreira et al.,
2008; Giai and Boerner, 2007; Nannipieri et al., 2003). Principal compo-
nent analysis of CLPP reveals the shifts in microbial community and
such shifts can be associated with the utilization of specific carbon
sources (Wang et al., 2010). In this study, we hypothesized that vegeta-
tion type has a significant effect on soil enzyme activities and microbial
functional diversity since the quality and quantity of carbon input are
different between vegetation types. We aimed to answer two questions:
1) after 18 years of vegetative restoration, did the eroded soil recover to
the level of the un-eroded soil? 2) What are the characteristics of soil
enzyme activities and microbial functional diversity under different
vegetation types?
2. Material and methods
2.1. Sampling site
The sampling site was described in Deng et al (2010). Briefly, it was
located at the Red Soil Ecological Experiment Station in Jiangxi Province,
China (116°55′30″E, 28°15′20″N) with an area of approximately
113 ha. The climate is subtropical with an average annual precipitation
of 1785 mm. The average annual temperature is 17.8 °C. The soil tex-
ture in the site is loamy clay (Zhang et al., 2010). Prior to 1987, mainly
due to the excessive deforestation, some areas were left unplanted
and severely eroded while the other areas which were grown with
weeds dominated by Verbena officinalis and Imperata cylindrical, were
un-eroded. In 1987, P. massoniana, C. camphora and L. bicolor were
planted on both eroded and un-eroded land. Some eroded areas were
left un-planted.
2.2. Soil and foliage sampling
Sampling was carried out in December 2005. Soils and intact foliage
were collected from the same seven habitats as described in Deng et al
(2010). The seven habitats included three un-eroded habitats planted
with P. massoniana, L. bicolor or C. camphora, three eroded habitats re-
stored with the same three plants, and one un-planted eroded habitat.
Three similar stands with around 100 m apart from each other were
selected for each habitat and in each stand, soil and foliage were collect-
ed from three randomly selected plots each with an area of 50 cm ×
50 cm. Soil was collected from three horizons which were representa-
tives of the distribution of soil organic matter. The uppermost layer,
which was dark brown at a depth of 0–2 cm represented O horizon,
the sub-layer, which was dark red at 2–7 cm represented A horizon
and a lower layer, which was red at 7–13 cm represented B horizon.
Eroded bare soil which contained little organic matter and resembled
the B horizon, was collected at a depth of 0–13 cm. One core sample
was collected in each plot using cylindrical core (100 cm3 volume) at
horizons A and B for the measurement of bulk density. All soils were col-
lected after the above ground litters were removed. After sampling, soil
except for the core samples was immediately sieved through a 2 mm
mesh. Soil aliquots were stored at 4 °C for a week before the measure-
ments of enzyme activities and microbial functional diversity; or air
dried for chemical analysis. The foliage was oven dried at 60 °C for
chemical analysis.
97
2.3. Soil and foliage properties
Soil pH was measured at 1:2.5 (soil/water); organic carbon content
and total nitrogen in foliage and soil was determined by wet oxidation
(K2CrO4) and Kjeldahl digestions, respectively (Lu, 2000). Microbial
biomass carbon (Cmic) was determined by CHCl3 fumigation–extraction
method as described by Vance et al (1987). Lignin content in foliage was
determined after hydrolysis with 72% H2SO4 (Newman et al., 1994).
Maximum water holding capacity (MWHC) was determined by the dif-
ference between dry and soaked soil weights (Walker and Austin,
2004). The bulk density was determined in situ by excavation method
(Black et al., 1965).
2.4. Soil enzyme activities
Soil enzyme activities were determined using spectrophotometry
(Alef and Nannipieri, 1995; Tabatabai, 1994). Acid phosphatase (EC
3.1.3.2) activity was determined by using disodium phenol phosphate
as a substrate. After incubation at 37 °C for 24 h, the amount of phenol
was measured at 510 nm. Urease (EC 3.5.1.5) activity was measured
using urea as a substrate. After incubation at 37 °C for 24 h, the amount
of NH+4 was measured at 578 nm. Invertase (EC 3.2.1.26) activity was
determined by using sucrose as a substrate. After incubation at 37 °C
for 24 h, the amount of glucose was measured at 550 nm. Cellulase
(EC 3.2.1.4) activity was measured using sodium carboxy methyl cellu-
lose as a substrate. After incubation at 37 °C for 72 h, the amount of glu-
cose was measured at 550 nm. Polyphenol oxidase (EC 1.10.3.1) activity
was determined by using pyrogallic acid as a substrate. After incubation
at 37 °C for 3 h, the amount of purpurogallin (PPG) was measured at
430 nm. β-Glucosidase (EC3.3.1.21) activity was measured using p-
nitrophenyl-β-D-glucopyranoside as a substrate. After incubation at
37 °C for 1 h, the amount of p-nitrophenol (PNP) was measured at
410 nm.
2.5. Community level physiological profile (CLPP)
CLPP of the soil microbial communities were analyzed by using
BioLog Eco-plates with 31 unique carbon substrates replicated three
times (Biolog Inc., Hayward, CA) (Garland and Mills, 1991). Briefly,
soil suspensions were prepared by suspending 10 g fresh soil in
100 mL sterile phosphate buffer solution (pH 7.0). After shaking, the
soil suspensions from horizon O were diluted in 1:500 while those
from horizons A and B in 1:100 so that the soil organic carbon content
from different horizons was adjusted to a similar level to reduce the ad-
ditional color development (Preston-Mafham et al., 2002). All diluted
suspensions were added to the BioLog Eco-plates using an 8-channel pi-
pette. The plates were incubated at 30 °C and the absorbance was read
at 590 nm using the MicroLog Rel 4.2 software (Biolog Inc., Hayward,
CA) every 12 h for one week.
2.6. Statistical analysis
The significance of factors of vegetation type, erosion and soil hori-
zon on soil enzyme activities and microbial functional diversity were
evaluated by three-way analysis of variance (ANOVA). Means within
each factor were compared using one-way ANOVA. Significance was
evaluated at P b 0.05 using Duncan's test. The BioLog data after 108 h
incubation with 0.6 average well color development (AWCD) were sub-
ject to principal component analysis (PCA) and Shannon diversity (H′)
analysis to show microbial functional structure and functional diversity,
respectively. H′ was calculated by using the formula (Zheng et al., 2005).
X
31
′
H ¼ − ðP i  ln P i Þ ð1Þ
i¼1
98 R. Yin et al. / Catena 115 (2014) 96–103

X
31
P i ¼ ðC i −RÞ= ðC i −RÞ
i¼1
where Ci is the color production within each well, R is the absorbance
value of the plate's control well. Data analysis was performed using
SPSS 14.0 software (LEAD Technologies Inc., Chicago, IL). The varimax
rotated component matrix was applied to determine the carbon sources
with high factor loading (N 0.6) on principle components (Kline, 1994).
3. Result
3.1. Soil and foliage properties
Vegetative restoration significantly increased (P b 0.05) soil organic
material content (SOC), total nitrogen (TN), maximum water holding
capacity (MWHC) and microbial biomass carbon (Cmic) while it de-
creased soil pH and bulk density (BD), compared with the eroded bare
soil. However, SOC, TN and Cmic in restored eroded soil were generally
lower than those in un-eroded soil (Table 1). SOC and TN were
the highest in L. bicolor habitat. In O horizon soil pH was higher in
C. camphora habitat than in P. massoniana and L. bicolor habitats. Soil
pH decreased from O to B horizon in C. camphora habitat while it in-
creased from O to B horizon in P. massoniana and L. bicolor habitats.
The C/N ratios of foliage of P. massoniana, C. camphora and L. bicolor
were 40:1, 30:1 and 27:1, respectively, and the lignin contents were
33%, 24% and 20%, respectively.
3.2. Soil enzyme activities
Soil enzyme activities varied among vegetation types and between
un-eroded and restored eroded soils, and decreased from O to B horizons
(Fig. 1). The enzyme activities in eroded bare soil was un-detectable
except for acid phosphatase activity (0.16 μmol phenol g−1 h−1),
which was significantly lower (P b 0.05) than that of vegetated soil
in B horizon. The activity of polyphenol oxidase was the highest in
P. massoniana habitat. In L. bicolor habitat, the activities of cellulase and
β-glucosidase were the highest while urease activity was the lowest.
The three-way ANOVA showed that the factors of horizon, vegetation
type and erosion (un-eroded or restored) were significant (P b 0.05)
for the enzyme activities except that erosion was not significant for β-
glucosidase activity (Table 2). It also showed significant interactions
between vegetation type and horizon, horizon and erosion, and between
ð2Þ
vegetation type and erosion (Table 2). The correlation analysis (Table 3)
showed that the enzyme activities were positively correlated (P b 0.05)
with SOC, TN and Cmic.
3.3. Microbial functional diversity
Shannon index (H′) of the vegetated soil was significantly higher
than H′ of eroded bare soil except for L. bicolor and P. massoniana habi-
tats in O horizon (Fig. 2). In O horizon, H′ of C. camphora habitat was sig-
nificantly higher (P b 0.05) than that of L. bicolor and P. massoniana
habitats and H′ of un-eroded C. camphora habitat was significantly
higher than eroded C. camphora habitat. However, in A and B horizons,
there was no significant difference between vegetation types. The
three-way ANOVA demonstrated that factors of horizon, vegetation
type and erosion and interactions between any two of the three factors
were significant for H′. The correlation analysis (Table 3) showed that H′
was positively correlated with soil pH but negatively correlated (P b
0.05) with SOC, TN and Cmic.
3.4. Soil microbial functional structure
Principle component analysis illustrated the microbial functional
structure based on their carbon substrate utilization patterns (Fig. 3).
In O horizon, the L. bicolor habitat was separated from the C. camphora
habitat. In O and A horizons, there was no distinct separation between
un-eroded and restored soils except that eroded P. massoniana habitat
separated from the un-eroded habitat along PC1. In B horizon, the
eroded P. massoniana habitat and the eroded C. camphora habitat sepa-
rated from the un-eroded habitats along PC1 and PC2, respectively. The
eroded P. massoniana habitat, eroded L. bicolor habitat and eroded C.
camphora habitat were separated from each other along PC1. Eroded
bare soil was separated from planted soil in B horizon.
The rotated component matrix (Table 4) showed a trend that the
number of carbon sources with high loading (N0.6) for PC1 and PC2
belonging to amines, amino acids and carboxylic acids in B horizon
was higher than in O horizon, while the number of high-loading carbon
sources belonging to phenolic compounds, carbohydrates and polymers
in O horizon was higher than in B horizon.

Table 1
Mean and standard error (in parentheses) of soil pH, soil organic carbon (SOC), total nitrogen (N), bulk density (BD), maximum water holding capacity (MWHC) and microbial biomass
carbon (Cmic) in the three horizons and between un-eroded (U) and restored eroded (E) soil. Bold numbers in the B horizon are significantly different from eroded bare soil (P b 0.05).

Horizon Vegetation pH SOC TN BD MWHC Cmic

(g kg−1) (g kg−1) (g cm−3) (g g−1) (g kg−1)

U E U E U E U E U E U E

O P. massoniana 4.05 4.10 34.12 27.20 1.88 1.69 – – 0.54 0.52 0.49 0.38
(0.06) (0.03) (2.05) (1.56) (0.16) (0.09) (0.04) (0.03) (0.06) (0.04)
C. camphora 4.55 4.57 32.25 30.41 1.64 1.74 – – 0.55 0.52 0.42 0.42
(0.03) (0.02) (1.80) (1.02) (0.08) (0.05) (0.03) (0.01) (0.04) (0.02)
L. bicolor 3.86 3.95 41.68 36.93 2.71 2.39 – – 0.63 0.59 0.44 0.43
(0.03) (0.05) (1.52) (0.70) (0.15) (0.11) (0.02) (0.01) (0.05) (0.03)
A P. massoniana 4.38 4.28 13.05 6.72 0.89 0.56 1.23 1.30 0.46 0.55 0.20 0.10
(0.09) (0.04) (0.65) (0.43) (0.10) (0.05) (0.04) (0.05) (0.03) (0.02) (0.02) (0.01))
C. camphora 4.38 4.50 12.77 7.05 0.90 0.76 1.27 1.14 0.51 0.56 0.20 0.13
(0.07) (0.06) (0.50) (0.35) (0.08) (0.06) (0.04) (0.06) (0.04) (0.01) (0.02) (0.02)
L. bicolor 4.17 4.24 17.79 9.42 1.09 0.91 1.12 1.18 0.56 0.51 0.19 0.14
(0.06) (0.05) (0.26) (0.18) (0.05) (0.02) (0.07) (0.03) (0.04) (0.04) (0.02) (0.01)
B P. massoniana 4.34 4.49 6.93 2.76 0.41 0.38 1.19 1.33 0.59 0.55 0.10 0.05
(0.02) (0.14) (0.25) (0.27) (0.04) (0.03) (0.05) (0.03) (0.02) (0.03) (0.02) (0.00)
C. camphora 4.29 4.29 8.06 3.58 0.66 0.46 1.38 1.36 0.47 0.57 0.10 0.05
(0.04) (0.04) (0.19) (0.10) (0.03) (0.07) (0.05) (0.09) (0.02) (0.01) (0.02) (0.01)
L. bicolor 4.25 4.34 10.09 3.87 0.74 0.69 1.20 1.32 0.57 0.56 0.10 0.09
(0.04) (0.06) (0.30) (0.07) (0.05) (0.02) (0.04) (0.01) (0.01) (0.04) (0.01) (0.01)
Eroded bare soil 4.77 1.94 0.37 1.43 0.34 0.02
(0.03) (0.01) (0.01) (0.01) (0.00) (0.01)
 R. Yin et al. / Catena 115 (2014) 96–103 99

P. massoniana C. camphora L. bicolor

4 8

µmol p-nitrophenol g-1 h-1

µmol purpurogallin g-1 h-1

β-glucosidase Polyphenol Oxidase
3 6

2 4

1 2

0 0
4 0.3
Invertase Cellulase
µmol glucose g-1 h-1

µmol glucose g-1 h-1

Enzyme activities

3
0.2
2

0.1
1

0 0.0
10 4
Acid phosphatase Urease
µmol phenol g-1 h-1

8
µmol NH3 g-1 h-1 3
6
2
4
1
2

0 0
U E U E U E U E U E U E
O horizon A horizon B horizon O horizon A horizon B horizon

Fig. 1. Soil enzyme activities of restored eroded (E) and un-eroded (U) soils with different vegetation types and in different horizons. Bars represent standard error of means value of
enzyme activities (n = 3).

4. Discussion example, there was no significant difference between the restored soil
and the un-eroded soil for invertase and acid phosphatase activities in
This study clearly showed that after 18 years of restoration, enzyme O horizon of P. massoniana habitat. Cole and Spildie (2007) estimated
activities and microbial functional diversity, as well as Cmic and soil time needed to recover degraded soil under a series of measures includ-
physiochemical properties greatly improved except re-vegetation acid- ing transplanting (19 years), seeding (31 years) and applying compost
ified soil, compared with eroded bare soil. The significant effect of the (56 years). Our results suggest that a longer period than 18 years is
factor of erosion indicates that the enzyme activities and microbial func- needed for complete recovery.
tional diversity in the restored soil did not recover to the level of those in In this study, SOC, TN, Cmic and enzyme activities decreased from O
the un-eroded soil. Nevertheless, the strong interactions between hori- horizon to B horizon. It is likely because soil carbon substrates and nitro-
zon and erosion and between vegetation type and erosion for enzyme gen were mostly derived from foliage rather than root deposits. The de-
activities and microbial functional diversity indicate that the degree of crease of enzyme activities was possibly due to the decreased substrates
restoration varied with horizons and with vegetation types. The com- and microbial biomass (Ralte et al., 2005). The correlation analysis
plete restoration appeared in some horizons and vegetation types, for showed a positive relationship between microbial functional diversity

Table 2
Three-way ANOVA of enzyme activities and Shannon index (H′) for soil microbial functional diversity.

Variables DF F-value

H′ β-Glucosidase Polyphenol oxidase Invertase Cellulase Acid phosphatase Urease

Horizon (H) 2 30.3⁎⁎⁎ 385⁎⁎⁎ 623⁎⁎⁎ 294⁎⁎⁎ 186⁎⁎⁎ 105⁎⁎⁎ 262⁎⁎⁎

Erosion (R) 1 8.08⁎⁎ 2.88 56.2⁎⁎⁎ 41.4⁎⁎⁎ 21.2⁎⁎⁎ 43.3⁎⁎⁎ 56.8⁎⁎⁎
Vegetation types (V) 2 3.99⁎ 48.2⁎⁎⁎ 121⁎⁎⁎ 12.4⁎⁎⁎ 183⁎⁎⁎ 21.9⁎⁎⁎ 43.4⁎⁎⁎
H∗R 2 4.91⁎ 0.38 29.7⁎⁎⁎ 7.56⁎⁎ 12.0⁎⁎⁎ 2.36 3.04
H∗V 4 7.56⁎⁎ 30.2⁎⁎⁎ 45.5⁎⁎⁎ 5.75⁎⁎ 77.0⁎⁎⁎ 5.73⁎⁎ 14.5⁎⁎⁎
R∗V 2 3.42⁎ 0.04 7.80⁎⁎ 1.52 14.9⁎⁎⁎ 0.61 5.29⁎⁎
H∗R∗V 4 0.33 0.10 5.75⁎⁎ 2.77⁎ 7.56⁎⁎⁎ 2.28 3.91⁎⁎
⁎ P b 0.05.
⁎⁎ P b 0.01.
⁎⁎⁎ P b 0.001.
100 R. Yin et al. / Catena 115 (2014) 96–103

Table 3
Correlation coefficient matrix of soil physical–chemical properties, microbial biomass carbon (Cmic), enzyme activities and Shannon index (H′) for soil microbial functional diversity.

pH SOC TN MWHC Cmic H′ β-Glucosidase Polyphenol oxidase Invertase Cellulase Acid phosphatase

SOC −0.50⁎
TN −0.59⁎ 0.98⁎⁎⁎
MWHC −0.49⁎ 0.25 0.30
Cmic −0.40 0.98⁎⁎⁎ 0.94⁎⁎⁎ 0.12
H′ 0.61⁎⁎ −0.57⁎ −0.61⁎⁎ −0.18 −0.55⁎
β-Glucosidase −0.49⁎ 0.94⁎⁎⁎ 0.96⁎⁎⁎ 0.40 0.87⁎⁎⁎ −0.54⁎
Polyphenol oxidase −0.39 0.77⁎⁎⁎ 0.70⁎⁎ 0.04 0.85⁎⁎⁎ −0.55⁎ 0.55⁎
Invertase −0.47⁎ 0.90⁎⁎⁎ 0.86⁎⁎⁎ 0.15 0.93⁎⁎⁎ −0.61⁎⁎ 0.75⁎⁎⁎ 0.93⁎⁎⁎
Cellulase −0.71⁎⁎ 0.72⁎⁎ 0.81⁎⁎⁎ 0.59⁎ 0.58⁎ −0.43 0.83⁎⁎⁎ 0.29 0.55⁎
Acid phosphatase −0.47⁎ 0.78⁎⁎⁎ 0.71⁎⁎ 0.08 0.82⁎⁎⁎ −0.49⁎ 0.57⁎ 0.91⁎⁎⁎ 0.94⁎⁎⁎ 0.41
Urease −0.10 0.76⁎⁎⁎ 0.65⁎⁎ 0.15 0.85⁎⁎⁎ −0.36 0.55⁎ 0.89⁎⁎⁎ 0.87⁎⁎⁎ 0.16 0.87⁎⁎⁎

SOC: soil organic carbon; TN: total nitrogen; MWHC: maximum water holding capacity.
⁎ P b 0.05.
⁎⁎ P b 0.01.
⁎⁎⁎ P b 0.001.

and soil pH. A similar result was obtained in a previous study in which red soil, the mixed plantation of L. bicolor with C. camphora may im-
the diversity of bacterial 16S rRNA gene positively correlated with the prove high fertility and microbial functional diversity but avoid soil
soil pH (Deng et al., 2010). The possible reason was discussed, briefly, acidification.
that more acidic than neutral condition tends to exert more stress on
the microbial community and leave only the most resistant species. 2 O horizon
Both studies confirmed the consistency of functional diversity and
genetic diversity in their responses to soil pH variation. The possible rea- 1
sons for the lower soil pH in the vegetated soil than in the eroded bare

PC2 (15%)
soil include organic acids released by plant roots (Giddens et al., 1997) 0
and soil microorganisms (Wei et al., 2006) and loss of exchangeable
cations in soil due to plant uptake (Alfredsson et al., 1998). -1
The factor of vegetation type influences soil properties and enzyme
activities and microbial communities mainly through the amount and -2
chemical composition of foliage. We previously showed that litter accu-
mulation was the highest in L. bicolor habitat (Deng et al., 2010). The -3
present study further showed that the content of lignin, which is hard -2 -1 0 1 2
to decompose (Herman et al., 2008), was the lowest in the foliage of PC1 (25%)
L. bicolor. In addition, L. bicolor is a nitrogen fixing plant. As a result,
1.0
the soil organic carbon and nitrogen content was higher in L. bicolor A horizon
habitat than other habitats. Since the nitrogen fixing species could dra-
matically improve soil fertility and microbial activity, they were widely
0.5
PC2 (17%)

used in the restoration of degraded ecosystem (Sheoran et al., 2010).

However, our study showed that the soil pH and microbial functional di-
versity were lower in L. bicolor habitat than in C. camphora habitat. The
mixed plantation of nitrogen fixing shrub with trees was favorable for 0.0
tree growing and reducing soil erosion (Nichols et al., 2001). For eroded

P. massoniana C. camphora
-0.5
3.5 L. bicolor eroded bare soil -1 0 1 2 3
PC1 (26%)
a
2
a B horizon
Shannon index

3.0 b b
1
PC2 (15%)

b
c 0
2.5

-1

2.0 -2
U E U E U E -3 -2 -1 0 1 2
O horizon A horizon B horizon PC1 (21%)

Fig. 2. Shannon index of the microbial functional diversity in restored eroded soil (E) and
un-eroded (U) soil with different vegetation types and in different horizons. Bars repre-
sent standard error. In O horizon, same letters above bars are not significantly different
(P b 0.05, Duncan's test, n = 3) between vegetation types from un-eroded soil (U) or re-
stored eroded soil (E).
Fig. 3. Principal component analysis of the BioLog data after 108 h incubation from O, A
and B horizons of un-eroded (filled) and eroded (open) soils planted with: P. massoniana
(triangles); C. camphora (circles); and L. bicolor (squares). The eroded bare soil (cross) was
dotted with planted soil of B horizon. Bars represent standard error of mean values
(n = 3) along PC1 and PC2.
Table 4
Varimax rotated component matrix of loadings of carbon sources.
O horizon A horizon B horizon
Carbon sources Sort PC1 PC2 PC1 PC2 PC1 PC2
phenyl ethylamine AN 0.55 0.07 0.65 –0.24 –0.27 0.70
putrescine AN 0.15 –0.02 –0.12 –0.55 –0.04 0.23
L–arginine AA 0.31 –0.10 0.15 –0.66 0.21 –0.48
L–asparagine AA 0.34 0.63 –0.93 0.21 –0.15 –0.22
L–phenylalanine AA 0.79 –0.02 0.24 –0.83 –0.68 0.24
L–serine AA –0.10 0.64 0.31 –0.11 0.80 –0.24
L–threonine AA 0.53 0.29 0.62 –0.02 –0.25 0.05
glycyl–L–glutamic acid AA –0.01 0.31 0.44 0.22 0.73 0.05
beta–methyl–D–glucoside CH –0.61 –0.37 0.06 0.58 0.36 0.31
D–galactonic acid r–lactone CH 0.47 0.00 –0.58 0.55 0.02 –0.72

R. Yin et al. / Catena 115 (2014) 96–103

D–xylose CH –0.80 0.24 –0.73 0.35 –0.04 0.30
L–erythritol CH –0.67 0.33 0.32 0.11 –0.18 0.04
D–mannitol CH 0.06 –0.90 –0.61 –0.43 –0.25 –0.49
N–acetyl–D–glucosamine CH 0.52 0.21 –0.02 –0.66 –0.51 –0.12
D–cellobiose CH –0.66 0.27 –0.40 0.57 0.28 0.61
glucose–1–phosphate CH 0.17 0.40 –0.15 0.61 0.35 0.36
alfa–D–lactose CH 0.11 –0.64 –0.31 0.74 0.03 0.57
D,L–alfa–glycerolphosphate CH 0.08 –0.85 0.41 –0.14 0.54 0.48
pyruvatic acid methyl ester CA –0.61 0.12 –0.52 –0.57 –0.35 –0.74
D–galacturonic acid CA 0.53 –0.28 –0.37 0.57 –0.82 –0.10
r–hydroxybutyric acid CA 0.33 –0.01 0.21 –0.07 0.00 0.36
D–glucosaminic acid CA 0.05 –0.15 –0.90 0.08 –0.85 –0.02
itaconic acid CA 0.29 0.10 0.41 0.05 0.36 0.43
alfa–ketobutyric acid CA 0.63 0.03 0.42 –0.29 –0.50 –0.41
D–malic acid CA –0.12 0.55 0.62 0.19 0.15 0.47
2–hydroxybenzoic acid PC 0.50 0.16 0.61 –0.31 0.56 0.32
4–hydroxybenzoic acid PC 0.85 –0.01 0.14 0.41 –0.34 0.44
tween 40 PM 0.02 –0.75 0.61 0.14 0.36 –0.42
tween 80 PM –0.63 0.27 –0.06 –0.33 0.38 –0.48
alfa–cyclodextrin PM 0.78 –0.09 0.04 0.89 0.21 0.73
glycogen PM –0.63 0.35 –0.26 0.60 0 .43 0.15

AN: amines; AA: amino acids; CH: carbohydrates; CA: carboxylic acids; PC: phenolic compounds; PM: polymers. Factor loadings above 0.6 are highlighted with gray background.

101
102 R. Yin et al. / Catena 115 (2014) 96–103
Soil enzyme activities were positively correlated with SOC content.
The input of organic carbons (OC) improves soil enzyme activities in
ways that 1) OC act as substrates and stimulate enzyme releasing;
2) OC increase microbial biomass and activity; and 3) OC bind with
enzymes to form humus–protein complex which protects enzymes
(Allisona and Jastrow, 2006). Soil enzyme activities were also affected
by the quality of carbon input. The most active polyphenol oxidase in
P. massoniana habitat could be largely due to the relatively high lignin
content in foliage since polyphenol oxidase catalyzes the oxidation of
phenolic compounds including lignin and its derivatives (Shukla and
Varma, 2011). Cellulase and β-glucosidase carry out the hydrolytic con-
version of cellulose. The two enzymes were the most active in L. bicolor
habitats. Exocellulase is a class of enzymes including cellobiohydrolase,
endoglucanase and β-glucosidase that catalyzes cellulolysis. β-glucosi-
dase catalyzes the conversion of cellubiose to glucose, which is the last
step of cellulose hydrolysis and relieves the inhibition of cellubiose to
the other two enzymes (Chauve et al., 2010). Therefore, cellulase activ-
ity was closely related with β-glucosidase activity. The urease activity in
L. bicolor habitat was significantly lower than that in P. massoniana and
C. camphora habitats. This is probably because the high N availability in
L. bicolor habitat inhibited the synthesis of N-degrading enzyme
through a negative feedback mechanism (Allison et al., 2006).
The microbial functional structure differed between vegetation
types, especially in O horizon, indicating that the quality of carbon
input was a major factor affecting microbial composition. It can be con-
firmed by rotated component matrix, which clearly showed that the uti-
lization of polymers, phenolic compounds and carbohydrates largely
affected the microbial functional structure in O horizon. The polymers
like cellulose and lignin were major components of foliage while carbo-
hydrates and phenolic compounds were likely the decomposition prod-
ucts of cellulose and lignin, respectively (Lorenz et al., 2004). The
utilization of amines, amino acids and carboxylic acids mainly affected
microbial community in B horizon. It indicates that the microbial func-
tional structure was largely affected by the ability of micro-organisms
to degrade root exudates, which are composed of small organic mole-
cules including carbonic acids, amino acids and sugars (Uren, 2007).
In the three horizons, the microbial functional structure of eroded
P. massoniana habitat exhibited distinct separation from that of the
un-eroded habitat. The possible reason could be that the un-eroded
C. camphora and L. bicolor habitats were previously dominated by
V. officinalis and I. cylindrical while the un-eroded P. massoniana habitat
by P. massoniana itself. Therefore, after the plantation of C. camphora
and L. bicolor in 1987, soil microbial community developed simulta-
neously between the eroded and un-eroded habitats while the soil
microbial community in eroded P. massoniana habitat developed
decades later than that in the un-eroded P. massoniana habitat.
5. Conclusions
This study clearly shows that re-vegetation is an effective way to
restore eroded red soil, though maybe more than 18 years is needed
for complete restoration. Soil enzyme activities and microbial functional
diversity varied between vegetation types and such variation is closely
related to the quality of carbon sources that input to the soil through
foliage. We suggest that L. bicolor is the most suitable for restoration be-
cause it performed best in the accumulation of soil organic carbon and
nitrogen, which can further support other plant species. C. camphora
can increase soil pH and microbial functional diversity and a mixture
of L. bicolor and C. camphora may restore eroded red soil more effec-
tively than single vegetation type. We therefore suggest the studying
of restoration effect of a mixture of plant species.
Acknowledgments
This programme is supported by the Asia-Link Programme Asia-
Link-001(81468) and by the Natural Science Foundation of China
(NSFC) (Grant No. 405220130223). We thank Valerie Gibson from the
Queen's University Belfast for her kind help to improve the English
writing.
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