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\begin{document}

\title{Effects of nitrogen and phosphorus addition on litter decomposition and
soil enzyme activities with Salix cupularis in an alpine desert
ecosystem}



\author[1]{Yu-Fu Hu}%
\author[1]{Gang Chen}%
\author[1]{Nairui Yang}%
\author[1]{Hongyu Qian}%
\author[1]{Wei Wang}%
\author[1]{Jian-kai Lu}%
\affil[1]{Sichuan Agricultural University - Chengdu Campus}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
Nitrogen (N) and phosphorus (P) are important factors controlling
biogeochemical cycling in terrestrial ecosystems and significantly
affect the decomposition process of litter. However, N and P addition
effects on litter decomposition, especially biological pathways in
alpine grassland in northwest Sichuan, remain unclear. Therefore, this
study explored the response mechanism of \emph{Salix cupularis} litter
decomposition and soil enzyme activity to different exogenous nitrogen
and phosphorus additions and the inner relationship, combined with field
in situ experiments and laboratory analysis methods. The results showed
that: (1) In general, N, P and NP treatments significantly promoted the
decomposition of litter, lignin and cellulose, and the addition of NP
had a stronger effect in the same concentration level. P addition could
alleviate the inhibitory effect of litter decomposition by high N
concentration. (2) Litter C, N and P basically showed a sustained
release pattern, and NP treatment had the strongest promoting effect.
(3) Each treatment significantly increased the activities of soil
invertase, cellulase, polyphenol oxidase, urease and phosphatase, and
the NP treatment had the best effect. The rate of litter decomposition
was significantly influenced by nutrient content as well as soil enzyme
activity, where cellulose content and invertase activity may be a key
factor controlling the rate of litter decomposition.%
\end{abstract}%



\sloppy


\textbf{Effects of nitrogen and phosphorus addition on litter
decomposition and soil enzyme activities with \emph{Salix cupularis} in
an alpine desert ecosystem}

Gang Chen \textsuperscript{1}, Nai-rui Yang \textsuperscript{1}, Yu-fu
Hu \textsuperscript{*}, Hong-yu Qian, Fan Yang, Wei Wang, Jian-kai Lu

College of Resources, Sichuan Agricultural University, Chengdu 611130,
China

* Corresponding author: Yu-fu Hu

E-mail address: huyufu@sicau.edu.cn (Y. Hu).

\textbf{Abstract:} Nitrogen (N) and phosphorus (P) are important factors
controlling biogeochemical cycling in terrestrial ecosystems and
significantly affect the decomposition process of litter. However, N and
P addition effects on litter decomposition, especially biological
pathways in alpine grassland in northwest Sichuan, remain unclear.
Therefore, this study explored the response mechanism of \emph{Salix
cupularis} litter decomposition and soil enzyme activity to different
exogenous nitrogen and phosphorus additions and the inner relationship,
combined with field in situ experiments and laboratory analysis methods.
The results showed that: (1) In general, N, P and NP treatments
significantly promoted the decomposition of litter, lignin and
cellulose, and the addition of NP had a stronger effect in the same
concentration level. P addition could alleviate the inhibitory effect of
litter decomposition by high N concentration. (2) Litter C, N and P
basically showed a sustained release pattern, and NP treatment had the
strongest promoting effect. (3) Each treatment significantly increased
the activities of soil invertase, cellulase, polyphenol oxidase, urease
and phosphatase, and the NP treatment had the best effect. The rate of
litter decomposition was significantly influenced by nutrient content as
well as soil enzyme activity, where cellulose content and invertase
activity may be a key factor controlling the rate of litter
decomposition.

\textbf{Keywords:} Salix cupularis; litter quality; decomposition rate;
nitrogen and phosphorus additions; soil enzyme activities; alpine desert
ecosystem

Funding

We are grateful to the anonymous reviewers for their critical review and
comments on previous drafts of this manuscript. This research was funded
by the National Natural Science Foundation of China (41771552).

\textbf{1.Introduction}

Litter plays an important role in maintains ecosystem productivity,
enhancing soil fertility, improving soil texture, and optimizing the
structure and metabolic characteristics of microbial communities in the
system (Canessa et al., 2021, Huangfu and Wei, 2018). Litter
decomposition is a crucial terrestrial ecosystem process which affects
the nutrients cycling. (Chapin et al., 2002; Handa et al., 2014; Liu et
al., 2016). Through material exchange and nutrient flow with soil,
litter decomposition connects aboveground plants and soil and offers an
essential way for plant-soil interaction (Chapin et al. 2002; Gregorich
et al., 2016; Xiao et al., 2014). The changes of litter quality,
including C, N, P concentration, cellulose and lignin can reflect the
decomposition rate of litter matter to some extent (Yang et al., 2017).
The decomposition rate is mainly controlled by climate, the plant
properties, soil environment and microorganism activities, and most of
these factors are affected by ecosystem nitrogen (N) deposition. (Van
Diepen et al., 2015; Zhou et al., 2017; Liu and Greaver 2010). In
addition, plant growth is usually limited by nitrogen or phosphorus, and
is often even limited by both phosphorus concentration and effectiveness
in the late stages of ecosystem regeneration, which will seriously
affect the decomposition process of litter. (Vitousek et al. 2010; Jiang
et al., 2016). Hence, understanding the effects of N and P input on
litter decomposition is imperative.

The process of decomposition of litter by extracellular enzymes produced
by soil microorganisms and plant roots is the key to promote
biogeochemical cycling in terrestrial ecosystems (Yue et al., 2020). By
changing soil physical and chemical properties, nitrogen and phosphorus
can affect the effective use of soil nutrient by extracellular enzymes
and regulate the nutrient uptake of plant, and ultimately affect the
results of litter decomposition and feedback process (Song et al., 2014;
Amend et al., 2015; Creamer et al., 2015; Waldrop et al. 2004). At the
same time, litter decomposition is a continuous biodegradation process,
the factors that dominate the mass loss of litter may change as the
decay time of litter progresses (Huo et al., 2019). The addition of
exogenous nutrients can usually accelerate the decomposition of litter
in the early stage by providing nutrients necessary for microbial
activities (Cuchietti et al., 2014), and slow the the decomposition of
litter in later stage by change the microbial community and reduce
enzyme activity (Cuchietti et al., 2014; Gill et al., 2021; Widdig et
al., 2020). Therefore, it is essential to investigate the dynamic
changes of litter decomposition and soil enzyme activity over time under
N and P addition.

As a high altitude and unique type of grassland ecosystem in the world,
alpine grasslands are extremely sensitive to global environmental
changes and play an important role in regulating the global ecological
environment (Chen et al., 2014). The alpine grassland of northwest
Sichuan is located in the alpine semi-humid region on the southeastern
edge of the Tibetan Plateau, with the world's largest plateau peat swamp
wetlands, and its regional ecological and environmental status is very
important (Hoover et al., 2016; Suseela et al., 2018). Over the years,
due to the intensity of human activities and overgrazing, the alpine
grasslands of northwest Sichuan have faced important ecological problems
such as the degradation of grassland functions and severe
desertification (Jiang et al., 2018). Grassland desertification control
has become an important task for regional ecological restoration.
Because of its cold tolerance and drought resistance, Salix cupularis
can prevent cold winds from fixing sand, improve the alpine ecological
environment, promote the growth of other herbaceous plants, and
gradually form a stable shrub ecosystem in the ecological restoration
process (Zhang et al., 2016; Yang et al., 2017). Therefore, we conducted
a N and P addition experiment in Salix cupularis ecosystem in alpine
sandy land in northwest Sichuan. We aim to reveal the response of Salix
cupularis litter decomposition and soil enzyme activities to different
exogenous nitrogen and phosphorus additions and their inner
relationship, and provide scientific basis for the formulation of
ecological restoration measures as well as natural resource management
and regional sustainable development in northwest Sichuan.

\textbf{2.Materials and methods}

2.1. Study site

The study area is in Hongyuan County, Aba Tibetan and Qiang Autonomous
Prefecture, northwest of Sichuan, China, on the southeastern edge of the
Tibetan Plateau, and is the largest plateau peat swamp wetland area in
the world, whose special ecological geographic location makes it an
important regulator of the global ecological environment. The study was
carried out in Wache Township, Hongyuan County (33\selectlanguage{ngerman}°10\selectlanguage{english}'N, 102deg37\selectlanguage{english}'E), a
typical demonstration area for sandy control in Sichuan Province. The
sample area is flat, with a relatively uniform topography and slope. The
topography slopes from southeast to northwest, and the landscape is
typical of the transition from mountain plain to hilly plateau, with an
altitude of 3600 m above sea level. The climate is continental plateau
cold temperate monsoon climate. The mean annual precipitation is 791.95
mm and temperature is 1.5\selectlanguage{english}. The plants are dominated by sea buckthorn,
Salix cupularis and medicinal plants, include Rhodiola rosea, Qianglong,
Songbei, Astragalus, Cordyceps, Kobresia setchwanensis, Leymus
secalinus, and Anaphalis flavescens. The area is fenced off from grazing
and the experiment was conducted in an ecological restoration area of
Salix cupularis.

2.2. Experimental design

In August 2018, an area of Salix cupularis with basically the same
growth condition was selected to delineate a test sample site for
exogenous N and P addition. The soil basic physicochemical properties,
site conditions and initial values of the chemical composition of litter
in the test plots are shown in Table 1. The topography and climate types
in western China are complex, and the atmospheric N deposition is lower
compared with other regions, with an annual N deposition of 3.62
g*m\textsuperscript{-2}*year\textsuperscript{-1} in western Sichuan
(Yang et al., 2018). A According to the N deposition and N, P ecological
stoichiometric ratio, control group without fertilization, three
fertilization types (N, P, and NP) and three fertilization gradients
were set up in this experiment for a total of 10 treatments: low N (N1,
3 g*m\textsuperscript{-2}*year\textsuperscript{-1}), medium N (N2, 6
g*m\textsuperscript{-2}*year\textsuperscript{-1}) and high N (N3, 9
g*m\textsuperscript{-2}*year\textsuperscript{-1}); low P (P1, 1
g*m\textsuperscript{-2}*year\textsuperscript{-1}), medium P (P2, 3
g*m\textsuperscript{-2}*year\textsuperscript{-1}), and high P (P3, 6
g*m\textsuperscript{-2}*year\textsuperscript{-1}); low NP (N1+P1),
medium NP (NP2, N2+P2) and high NP (NP3, N3+P3). Urea
(CO(NH\textsubscript{2})2, N: 46.67\%) and sodium dihydrogen phosphate
(NaH\textsubscript{2}PO\textsubscript{4}, P: 44.65\%) were used as
fertilizers. A completely randomized design was used to randomly select
three Salix cupularis for each treatment in test plot, for a total of 30
Salix cupularis. A 1mx1m sample square was randomly laid within the
vertical projection of the canopy of the thickets, and the litter was
collected, removed from the thickets, and brought back to the laboratory
for drying and shredding. A portion of the shredded litter was used for
initial nutrient concentration analysis, and three samples (50g) of each
treatment were accurately weighed, mixed with each addition treatment,
and packed into 10cmx15cm nylon mesh decomposition bags. The nylon mesh
bags were randomly placed in the 1mx1m sample square of the thickets,
sealed with sealing clips to ensure that soil animals could not enter,
and the four corners of each litter bag were fixed with stainless steel
wire ties.

2.3. Soil sampling and laboratory analyses

The experiment was conducted on August 12, 2018 for in situ simulation
of the litter decomposition of exogenous N and P addition, and the nylon
net bags were retrieved on the 90th, 270th and 360th days of
decomposition, respectively. After retrieval, the surface of the net
bags was gently brushed, and plant fine roots and gravel were picked out
from the bags, followed by washing the soil in the bags with ultrapure
water. Each treatment (3 bags) was mixed well and dried in an oven at 60
\selectlanguage{english} for 72 h to a constant weight, then weighed and recorded after passing
through a 1 mm steel sieve, and crushed through a 0.149 mm steel sieve
for determination of total carbon, total nitrogen, total phosphorus,
lignin and cellulose concentration of litter. At the same time, soil was
collected from the 0-10 cm layer under the nylon net bags. Three bags of
soil samples from each sample were mixed into one bag, totaling 30 bags.

Plant total carbon was determined by concentrated sulfuric
acid-potassium dichromate oxidation method, total nitrogen by
concentrated sulfuric acid-hydrogen peroxide-boiling-Kaeschner method,
and total phosphorus by molybdenum antimony anti-colorimetric method
(Lu, 1999); lignin and cellulose were determined by acid detergent fiber
method (ADF method); soil organic carbon (SOC) was determined using
potassium dichromate external heating method, total nitrogen (TN) by
Kaeschner method, total phosphorus (TP) by molybdenum antimony
anti-colorimetric method. 3,5-dinitrosalicylic acid colorimetric method
for the determination of soil invertase (INV) and cellulase (CBH),
sodium phenol-sodium hypochlorite colorimetric method for the
determination of urease (UG), sodium hydrogen phosphate colorimetric
method for the determination of alkaline phosphatase (AP), and
o-phenylene triol colorimetric method for the determination of
polyphenol oxidase (POX) (Blair et al., 1994).

2.4. Statistical analyses

The mass remaining of litter (\emph{M\textsubscript{r}} ) (\%) during
the N and P addition test was calculated using the formula:

\emph{M\textsubscript{r}} =\emph{M\textsubscript{t}}
/\emph{M\textsubscript{0}} \selectlanguage{english}?100\% (1)

Where \emph{M\textsubscript{t}} and \emph{M\textsubscript{0}} refer to
the mass (g) of litter at time \emph{t} of decomposition and the initial
mass (g) of litter, respectively.

The value of the rate constant \emph{k} for decomposition of litter
matter was calculated by (Olson, 1963):

\emph{M\textsubscript{t}} /\emph{M\textsubscript{0}} =\emph{e}
\textsuperscript{--\emph{kt}} (2)

Where \emph{k} refers to the annual decomposition rate constant of
litter.

The time to decompose 50\% (\emph{T\textsubscript{0.5}} ) and 95\%
(\emph{T\textsubscript{0.95}} ) of the litter was calculated (Olson,
1963) as:

\emph{T\textsubscript{0.5}} = --ln (1 -- 0.5)/\emph{k} (3)

\emph{T\textsubscript{0.95}} = --ln (1 -- 0.95)/\emph{k} (4)

The lignin, cellulose, total carbon, total nitrogen and total phosphorus
remaining rates (\emph{M\textsubscript{cr}} ) (\%) during decomposition
of litter were calculated as:

\emph{M\textsubscript{cr}} =(\emph{C\textsubscript{t}}
\selectlanguage{english}?\emph{M\textsubscript{t}} )/(\emph{C\textsubscript{0}}
\selectlanguage{english}?\emph{M\textsubscript{0}} )\selectlanguage{english}?100\% (5)

Where \emph{C\textsubscript{t}} and \emph{C\textsubscript{0}} refer to
the litter nutrient concentration (g*kg\textsuperscript{-1}) at
time\emph{t} and the initial litter nutrient concentration
(g*kg\textsuperscript{-1}), respectively.

The results of the study were processed and analyzed using SPSS 22.0 and
R 3.6.3. One-way ANOVA was used to analyze the differences in the
decomposition constants (k), the mass remaining of litter, lignin,
cellulose, total carbon, total nitrogen and total phosphorus remaining
rate, and soil enzyme activities under different treatments at the same
decomposition time, and three-way repeated measures ANOVA (RM-ANOVA) was
used to investigate the reciprocal effects of N and P addition for each
index and their synergistic effects with decomposition time. Pearson
correlation analysis and linear regression analysis were preformed to
evaluate the correlation between the decomposition rate of litter,
litter endogenous nutrient concentration and soil enzyme activity. To
compare the relative importance of litter endogenous nutrient
concentration and soil enzyme activity on K-value, the relative
influence analysis was calculated using the ''gbm'' package.The results
were plotted by Origin 2022 and R 3.6.3.

\textbf{3.Results}

3.1. Litter mass remaining and decomposition rate constant

The results showed that N, P and NP additions significantly contributed
to the decomposition of litter matter (Fig.1). At 360 days, the mass
remaining of litter was 53.62\%, 62.36\% and 65.16\% in N1, N2 and N3
treatment; 55.56\%, 53.02\% and 51.35\% in P1, P2 and P3 treatment;
51.05\%, 47.72\% and 49.23\% in NP1, NP2 and NP3 treatment,
respectively. One-way ANOVA showed that compared to CK, there were
significant differences in mass remaining of litter in different
concentrations of N, P and NP treatments, respectively (P \textless{}
0.001). Under N addition, N1, N2 and N3 treatments significantly reduced
mass remaining in the early stages of decomposition, and only N1
treatment had a significant effect in the middle and late stages of
decomposition; under P addition, P1, P2 and P3 significantly reduced
mass remaining in each decomposition period; under NP addition, NP1, NP2
and NP3 treatments significantly reduced mass remaining in the middle
and late stages of decomposition, and no significant difference was
found between NP2 and NP3 treatments. The RM-ANOVA showed that the
interaction of N and P additions and their synergistic effect with
decomposition time had a significant effect on the mass remaining of
litter (P \textless{} 0.001) (Table S1).

The litter decomposition rate constant (\emph{K} ) increased
significantly with increasing P addition concentration with the highest
value in the P3 treatment (0.67 year\textsuperscript{-1}), and showed a
trend of increasing and then decreasing with increasing N and NP
addition concentrations with the highest values in the N1 (0.62
year\textsuperscript{-1}) and NP2 (0.73 year\textsuperscript{-1})
treatments, respectively (Table 2). In the CK treatment, the time
required for 50\% (T\textsubscript{0.5}) and 95\%
(T\textsubscript{0.95}) of litter decomposition was 1.67 and 7.21 years,
respectively. Under N addition, T\textsubscript{0.5} and
T\textsubscript{0.95} were 1.11 and 4.81 years for the N1 treatment,
1.47 and 6.34 years for the N2 treatment, and 0.62 and 6.99 years for
the N3 treatment, respectively. Under P addition,
T\textsubscript{0.5}and T\textsubscript{0.95} were 1.18 and 5.10 years
for the P1 treatment, 1.09 and 4.72 years for the P2 treatment, and 4.49
and 1.04 years for the P3 treatment, respectively. Under NP addition,
T\textsubscript{0.5}and T\textsubscript{0.95} were 1.03 and 4.46 years
for NP1 treatment, 0.94 and 4.05 years for NP2 treatment, and 0.98 and
4.12 years for NP3 treatment, respectively. The RM-ANOVA showed that the
interaction of N and P additions and their synergistic effect with
decomposition time had a significant effect on the decomposition rate
constant (P \textless{} 0.001) (Table S1).

3.2. Litter lignin, cellulose and nutrient remaining rate

Overall, the lignin remaining rate showed a trend of decreasing and then
increasing under N, P and NP addition, and the cellulose remaining rate
continued to decrease with increasing decomposition time (Fig.2). Low
concentrations of N, P and NP addition could promote the decomposition
of lignin and cellulose, while high concentration treatment inhibited
this effect, and lignin was accumulated under N3 treatment (105.61\%).
The one-way ANOVA showed that compared to CK, the lignin and cellulose
remaining rates of N, P and NP treatments were significantly different
(P \textless{} 0.001). After 360 days of decomposition, the lignin
remaining rate was the lowest in N2 (68.46\%), P1 (60.73\%) and NP1
(57.84\%) treatments, respectively, and the cellulose remaining rate in
N1 (27.65\%), P2 (29.72\%) and NP1 (25.06\%) treatments, respectively.
It was noteworthy that the lignin remaining rate was significantly
higher in both N3 and NP3 treatments than CK during litter decomposition
(P \textless{} 0.05). The RM-ANOVA showed that the interaction of N and
P addition and its synergistic effect with decomposition time had a
significant effect on both lignin and cellulose remaining rates (P
\textless{} 0.001) (Table S2).

The results showed that N, P and NP additions could significantly
promote the release of litter carbon (C) compared to CK treatment
(Fig.3). During the litter decomposition, the litter C remaining rate
was not significantly different among different N addition
concentrations, and the lowest in N3 treatment (38.30\%) in late stage;
in the late stage, the P3 (24.69\%) and P2 (24.35\%) treatments were
significantly lower than P1 (26.63\%); in the early and middle stages,
NP3 treatment was significantly lower than other treatments, and in the
late stage the NP2 (14.92\%) treatment was significantly lower than
other treatments. Compared with CK, N addition significantly inhibited
the release of litter nitrogen (N) during the decomposition process, and
the effect was strengthened with increasing N concentration and the
highest N remaining rate (70.91\%) was in N3 treatment; P addition
significantly reduced the N remaining rate and the lowest value
(25.06\%) was in P3 treatment; NP2a and NP3 treatments significantly
increased the N remaining rate in the early and middle stages, and NP1
was significantly lower than CK treatment with the lowest value of
34.29\% in late stage. Compared with CK treatment, N addition
significantly inhibited the release of litter phosphorus (P) in the
early and late stages of decomposition, and the lowest P remaining rate
(35.94\%) was in N2 treatment; P3 addition significantly increased the
litter P remaining rate in early and late decomposition, while P1
treatment significantly reduced the P remaining rate with the lowest
value of 54.67\% in the middle and late stages ; NP1 treatment
significantly reduced the P remaining rate in the middle and late stages
with the lowest value of 29.31\%. The RM-ANOVA showed that the
interaction of N and P addition and its synergistic effect with
decomposition time had a significant effect on the litter C, N and P
remaining rates (P \textless{} 0.001) (Table S2).

3.3. soil enzyme activity

The results showed that N, P and NP additions significantly increased
the activities of INV, CBH, POX, UG and AP compared to CK treatment
(P\textless{}0.05) (Fig.4). During the litter decomposition, the
activities of INV, UG and AP decreased in the middle stage and increased
in the late stage. The CBH activity was highest in 90 days of
decomposition and then continued to decline, and the POX activity showed
an increasing trend. In the late stage, with increasing N addition, both
CBH and AP activities increased significantly, INV and POX activities
decreased significantly, while UG showed a trend of first increase and
then decrease. With the increase of P addition, the activities of INV,
CBH and AP decreased significantly, and the activities of POX showed a
trend of first increase and then decrease, while P addition had no
significant effect on soil UG activity. With increasing NP addition, AP
activity decreased significantly, INV and CBH activities showed a trend
of first increase and then decrease, while NP addition had no
significant effect on polyphenol oxidase and urease. The RM-ANVON showed
that the interaction of N and P addition and its synergistic effect with
decomposition time had significant effects on INV, CBH, POX, UG, and AP
(P \textless{} 0.001) (Table S3).

3.4. Influencing factors in the decomposition of litter

The Pearson's correlation analyses and linear regression analysis showed
that the litter \emph{K} -value was positively correlated with cellulose
content (R\textsuperscript{2} = 0.217, \emph{P} \textless{} 0.001), INV
(R\textsuperscript{2} = 0.242, \emph{P} \textless{} 0.001), and CBH
(R\textsuperscript{2} = 0.1, \emph{P} \textless{} 0.01) activity, and
negatively correlated with N/P (R\textsuperscript{2} = 0.069,
\emph{P}\textless{} 0.05) (Fig.5 and 7). Moreover, the contributions of
litter cellulose content (28.53\%) and soil INV activity (26.06\%) to K
were significantly higher than other indicators (Fig.6).

\textbf{4.Discussion}

4.1. Effects of nitrogen and phosphorus addition on litter decomposition
rate

The input of N into the ecosystem changes processes such as microbial
community structure and extracellular enzyme activity, which can affect
the decomposition of litter matter (Hernandez et al., 2019; Wu et al.,
2013). The mass remaining of litter in the early stage of decomposition
was significantly lower than CK at low N concentration (Fig.1),
indicating that the moderate N addition in the early stage was
beneficial to litter decomposition, while too much was inhibitory, which
is consistent with previous reports (Chen et al., 2013; Song et al
2015). Similarly, the low concentration N treatment had higher
\emph{k}values and was able to accelerate the decomposition of litter
(Table 2). This is because moderate N input reduces the litter C/N ratio
and adjusts the suitable conditions for litter decomposition, while high
N input significantly reduces the soil pH, which is not conducive to
microbial decomposition activities and tends to break the N/P balance
and enhance ecosystem phosphorus limitation (Chen et al., 2013; Song et
al 2015; Heuck et al., 2018). During the decomposition process, the high
P concentration treatment had a larger \emph{k} value and lower mass
remaining of litter, indicating that P addition significantly stimulated
litter decomposition, and the higher the concentration the stronger the
stimulating effect. This could be explained by the fact that, on the one
hand, P addition relieved the P nutrient limitation of ecosystem in the
early stage of litter decomposition, and on the other hand, it increased
the soil nutrient effectiveness, thus improving microbial activity as
well as phosphorus use efficiency (Lin et al., 2019). The mass remaining
rate of litter in all concentrations of NP treatment was lower than N
and P treatment during the litter decomposition, indicating that NP
addition had the strongest effect on litter decomposition. The results
also indicated that N and P addition had an interactive effect on litter
decomposition, and P addition may alleviate the inhibitory effect of
high N on litter decomposition (Hao et al., 2013). In the study,
increasing NP concentration slowed down the decomposition rate of
litter, which may be related to the chemical regulation of phosphorus
content in litter (Zheng et al., 2017). Overall, the decomposition rate
of litter was higher in the early stage than in the middle and late
stages, which could be because the study area is in highland alpine
region, and rapid reproduction and growth of soil microorganisms would
consume a large amount of nutrients and accelerate the litter
decomposition in the early stage (July-October).

4.2. The dynamics of litter nutrient concentration during litter
decomposition

Lignin, a recalcitrant component of litter, is difficult to be
decomposed by various types of hydrolytic enzymes in soil due to its
complex aromatic structure, and its degradation is an important process
in the decomposition of plant litter. As a whole, in each treatment the
lignin remaining rate was decreased faster in the early and middle
stages than in late stage, which indicated that both N and P could
significantly affect lignin degradation in the early and middle litter
decomposition process (Fig.2), which was consistent with previous
reports (Song et al., 2017; He et al., 2019). This may be because the
main influencing factor of early lignin degradation is the initial
nutrient content of litter, and the input of N and P increased the
integrated level of initial effective nutrients and reduced the critical
point of decomposition of lignin. (Song et al., 2017; He et al., 2019).
The study showed that in the late stage P addition could accelerate the
lignin decomposition, while the high concentration of N and NP addition
would accumulated the lignin, which indicated that high concentration of
N input inhibits lignin degradation and the alleviating effect of P
input on the limitation of energy supply for soil microbial activity
diminishes with increasing concentration of N input, and the high
concentration of NP treatment reduces the synergistic effect of N and P
for litter decomposition (Jiang et al., 2019). High concentrations of N
input altered microbial habitat and community composition, inhibited
lignin-related decomposition enzyme activities, and reduced microbial
decomposition capacity (Rinkes et al., 2016; Gao et al., 2017). Plant
cellulose is an important carbon source for soil microbial activity and
significantly influenced by soil enzyme activity as a relatively
abundant biopolymer in the ecosystem (Thomson et al., 2013). During the
decomposition process, the cellulose remaining rate of litter showed a
decreasing trend and the decrease of cellulose under NP treatment was
higher than N and P treatment alone. This was due to the fact that N and
P inputs reduced C/N and C/P ratios, increased microbial activity and
cellulase activity, resulting in a decrease of cellulose content (Jiang
et al., 2014; Talbot et al., 2012). The correlation and relative
influence analysis showed that the cellulose content had a significant
positive correlation with the litter decomposition rate, and its
contribution to litter decomposition was the largest (Fig.5-7). This may
be because cellulose is the most abundant polysaccharide compound in
litter, and the exogenous nutrients addition change the community
structure of decomposers, enabling them to decompose more cellulose to
obtain energy materials (\selectlanguage{polish}Š\selectlanguage{english}tursov\selectlanguage{ngerman}á et al., 2012; Talbot and Treseder,
2012). At the same time, cellulose mainly require decomposition by
cellulase and invertase (Xiao et al., 2018), thus cellulase and
invertase activities were also significantly positively correlated with
litter decomposition rate.

Litter nutrients are key factors affecting the composition of bacterial
communities during decomposition, and C, N, and P stoichiometry
significantly affect the structure and function of microorganisms, thus
altering the fixation and mineralization processes of litter N and P
(Barantal et al., 2014; Xu et al., 2020). The results showed that N, P
and NP addition significantly promoted the degradation of carbon (C) in
litter (Fig.3), because the nutrient input compensated for the
environmental nutrient deficiency of microbial growth in the study area
and accelerate the consuming of carbon sources by microorganisms (Ren et
al., 2018). In the late stage of decomposition, the NP treatment had a
significantly lower C remaining rate than the N and P additions alone,
and had the highest contribution to the litter C release, which
indicated that N and P additions had interactive effects on litter C
release (Wang et al., 2017).However, the synergistic effect of high N
and P addition might lead to microbial carbon utilization above the
critical level, resulting in an increase of litter C remaining rate in
the late stage of decomposition. In the study, N and P addition were
able to accelerate the litter P and N release , while causing the
accumulation of N and P, respectively, and the changes were aggravated
with the increase of concentration. This is because, on the one hand, N
and P addition reduced litter C/N and C/P, increased N and P hydrolase
activity, which accelerated the decomposition of litter N and P (Jian et
al., 2016); on the other hand, long-term N and P addition alone
increased nutrient limitation in the ecosystem and produced nutrient
enrichment with the increase of N and P input (Jie et al., 2014; Chen et
al., 2012), while NP treatment could alleviate this nutrient limitation
to some extent. During the decomposition process, the gradual decrease
of litter N/P reflected that the study area was mainly N-limited and the
litter N release rate of \emph{Salix cupularis} after removing N, P
limitation was higher than P.

4.3. The dynamics of soil enzyme activities during litter decomposition

Invertase and cellulase are produced by microorganism and play important
roles in the decomposition of litter, and their activities are limited
by the number and structural function of the microbial community, the
chemical nature of the litter substrate, and the elemental stoichiometry
and nutrient effectiveness (Wang et al., 2010).The results showed that
in each nutrient addition invertase and cellulase activities were
significantly increased with significant effect in the early stage of
decomposition, which corresponded to the rapid degradation of
polysaccharide compounds(e.g. cellulose) in the early stage (Fig.4).
Cellulose is an easily decomposable substrate at the initial stage and N
and P addition effectively stimulated the increase in carbon hydrolytic
enzyme activities and the exudation of substrates for enzymes (Wang et
al., 2010; Song et al., 2018). The decrease of INV activity with the
increase of N addition concentration might be related to the inhibition
of litter decomposition and reduction of the amount of substrate for INV
action under high N concentration treatment (Ge 2017). In the late stage
of decomposition, the CBH activity of NP treatment showed a decreasing
trend. This may be due to the fact that in the early stage, the
cellulose-like polysaccharide content of litter increased with the input
of NP and the limitation of litter N and P was removed, while litter
carbon gradually became the main influencing factor in the later stage,
and thus cellulase activity was inhibited (Chen et al., 2014). POX
activity is an important indicator of lignin degradation. In the study,
POX activity decreased with the increase of N addition, which
corresponded to the change of lignin. N input promoted the
polymerization of lignin-like aromatic compounds with phenols into
refractory substances and limited the activity of POX (Wang et al.,
2019; Song et al., 2018). Meanwhile, POX activity was significantly
higher in middle concentrations of P and NP addition than in low and
high concentrations. This suggests that the moderate amount P input
might disrupt this bio polymerization and alleviate P limitation (Kunito
et al., 2009; Jiang et al., 2014). This was also verified by the results
of the correlation analysis in this study, in which the rate of litter
decomposition was negatively correlated with lignin/P and positively
correlated with litter P content.

Soil UG and AP are important drivers of the carbon, nitrogen and
phosphorus cycle in the ecosystem, and significantly affected by changes
of litter decomposition conditions such as exogenous N, P addition
(Zhang et al., 2017). Compared to CK, UG activity showed a significant
increase in all treatments, while the effect was inhibited in high N
addition (Fig.4), which was inconsistent with the previous study (Guan
et al., 2019). This is because the substrate exudation rate was
decreased by the slowing down of the degradation process of litter under
high N treatment, resulting in lower UG activity (Zeng et al., 2019).
However, there was no significant difference between different
concentrations of P, NP additions, which might be due to the fact that
the UG on topsoil was more sensitive to N addition than P addition
(Jiang et al., 2014). In the study, AP activity was increased with
increasing of N and P concentrations, which is similar to the previous
study (Calvo-Fernández et al., 2018), because reduction of litter C/N
and C/P ratio promoted the production of microbial extracellular
phosphatases (Chen et al., 2018). However, AP activity decreased with
increasing NP concentration, which may be closely related to changes in
microbial habitat conditions caused by high concentrations of N and P
additions in the short term (Jiang et al., 2018). With the extension of
decomposition time, the soil enzyme activities of each treatment showed
a trend of decreasing and then increasing, which was probably related to
the change of climatic conditions in the study area. After the growing
season, the rapidly decreases temperature and water content in the
alpine region reduced the microbial activity, resulting in the decrease
of the type and activity of litter decomposition enzymes.

5. Conclusion

Our result showed that N, P and NP addition could significantly affect
the decomposition of litter matter, and P addition could alleviate the
inhibitory effect of litter decomposition by high N concentration. N and
P had an interactive effect on the decomposition of litter matter. The
lignin remaining rate of litter was slightly enriched in the late stage
of decomposition, which was most obvious in high N treatment, while the
cellulose remaining rate decreased continuously with decomposition time.
Litter C, N and P basically showed a continuous release pattern, with
the strongest promoting effect in NP treatment. All additive treatments
significantly increased soil invertase, cellulase, polyphenol oxidase,
urease, and phosphatase activities, which were generally higher in the
NP treatment than the other treatments. The rate of litter decomposition
was significantly influenced by nutrient content as well as soil enzyme
activity, where cellulose content and invertase activity may be a key
factor controlling the rate of litter decomposition.

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