Long-Term Nitrogen Addition Could Modify Degradation of Soil Organic Matter through Changes in Soil Enzymatic Activity in a Natural Secondary Forest
Round 1
Reviewer 1 Report
The article is is good, with data well described and visualization, with many interligated data of soil. The microbial anlysis is a importante intrument to study of soil suistanability, what justify the importance this work. The work was statistically planned, randomized and like seem right data analysis.
The introduction and results, however, need more substantiation by literature. Microorganisms need N to organic matter degradattion. In general, increasing N in the soil tends to increase microbial activity in the soil, especially in the case of organic N adittion.
Check in the cited studies and another studies if there is a restriction on microbial activity due to N addition for all types of N addition (includin organic and mineral) or in the most of case just by mineral form such as urea or another type.
Author Response
Dear Editor and Reviewers,
Thank you very much for giving us a chance to revise our manuscript (forests-2644459). We greatly appreciate the two reviewers for their valuable and constructive comments. We took these comments into full consideration when revising the manuscript. The reviewer’s comments are listed in black, and our responses are listed in blue. We hope our revision and responses can answer the reviewers’ questions and effectively raise the manuscript to the level of this journal. Thank you for all your help in processing and reviewing our manuscript. The point-by-point response to the reviewers’ comments is listed in the following.
Reviewer 1:
The article is is good, with data well described and visualization, with many interligated data of soil. The microbial anlysis is a importante intrument to study of soil suistanability, what justify the importance this work. The work was statistically planned, randomized and like seem right data analysis.
Response:
Thank you for your recognition of my work and agreement with the research direction.
The introduction and results, however, need more substantiation by literature. Microorganisms need N to organic matter degradattion. In general, increasing N in the soil tends to increase microbial activity in the soil, especially in the case of organic N adittion.
Check in the cited studies and another studies if there is a restriction on microbial activity due to N addition for all types of N addition (includin organic and mineral) or in the most of case just by mineral form such as urea or another type.
Response:
I completely agree with your viewpoint and have added relevant discussions in both the introduction and the discussion sections of the article.
“Furthermore, the form of N (e.g., NH4NO3 or urea) may also be a potentially important factor influencing the response of soil microorganisms to N addition. NH4NO3 and urea are among the most commonly used N fertilizers in N addition experiments. Yue et al. (2016) observed, in their experiments using urea as the N source, an increase in bacterial biomass upon urea addition. This effect could possibly be attributed to the augmented C input resulting from urea addition, which may introduce interference with the effects of N addition.”
Thank you again for your suggestions and comments.
Yours sincerely,
Qinggui Wang
Author Response File: 
Author Response.docx
Reviewer 2 Report
Dear editor and authors.
The authors discussed that N addition had a certain inhibitory effect on MBC, when in fact they mention that the effect is probably due to acidification from the N source used. This is what makes the most sense and not a deleterious effect due to the use of N, the authors need to improve the discussion a lot to make this clear.
I strongly suggest that authors create a correlation matrix to make the mechanisms clearer, especially the effect of pH.
For me, all the results found are related to pH, and the authors focus a lot on the harmful effect of N, when in fact it has to do directly with a property of the fertilizer used and not with N, as this is logical and supported in the literature, so the focus of the article should be changed, using the following theme Long-term nitrogen fertilizers addition with with acidifying capacity could modify degradation of soil organic matter through changes in soil enzymatic activity in a natural secondary forest. Because if the effect is really due to N, the authors need to carry out a new study controlling the pH and varying only the doses of N.
Therefore, I recommend that the article needs several changes to modify its focus, so that it can then be evaluated again. . Additionally, follow some comments below.
- Because the authors' hypothesis is that the application of N suppresses enzymes related to the C and N cycle, as this is not what is expected. The authors need to improve the introduction to create a basis for this hypothesis.
- Table with anova results is not necessary in the manuscript as presented, supplementary material must be made and included.
- The quality of figure 1 needs to be improved, the color of the statistical test letters is bad to see.
- Insert statistical comparisons in the figures and enlarge the letters and numbers to improve data visualization.
- The discussion section should not be divided into topics, it should be done continuously.
Author Response
Dear Editor and Reviewers,
Thank you very much for giving us a chance to revise our manuscript (forests-2644459). We greatly appreciate the two reviewers for their valuable and constructive comments. We took these comments into full consideration when revising the manuscript. The reviewer’s comments are listed in black, and our responses are listed in blue. We hope our revision and responses can answer the reviewers’ questions and effectively raise the manuscript to the level of this journal. Thank you for all your help in processing and reviewing our manuscript. The point-by-point response to the reviewers’ comments is listed in the following.
Reviewer 2:
The authors discussed that N addition had a certain inhibitory effect on MBC, when in fact they mention that the effect is probably due to acidification from the N source used. This is what makes the most sense and not a deleterious effect due to the use of N, the authors need to improve the discussion a lot to make this clear.
Response:
I completely agree with your perspective. Soil acidification induced by nitrogen addition is indeed one of the factors contributing to the inhibition of microbial biomass carbon (MBC). However, the introduction of exogenous nitrogen may directly or indirectly affect the availability of carbon (C) and nitrogen (N) resources required for microbial growth. Additionally, inorganic nitrogen could potentially interact with soil organic matter to form recalcitrant compounds, all of which can exert inhibitory effects on MBC. I have provided a more detailed explanation of these aspects in the discussion section.
“In our study, we observed that N addition exhibited a certain inhibitory effect on MBC (Figure 1), consistent with findings in several previous studies [19-23]. The reasons behind the impact of N addition on MBC in field studies are multifaceted. This complexity arises because N addition can directly or indirectly affect the availability of C and N resources required for microbial growth. Additionally, N addition-induced soil acidification may also inhibit microbial growth (Chen et al., 2015). Furthermore, N addition leads to an increase in soil inorganic nitrogen content, which can react with soil organic matter, leading to the accumulation of recalcitrant compounds that may hinder microbial growth (Janssens et al., 2010; Treseder, 2008). Studies by Yue et al. (2016) using urea as an N source yielded results indicating increased bacterial biomass upon urea addition, possibly due to the enhanced C input resulting from urea addition, which may interfere with the effects of N addition. Tu et al. (2011) found in experiments conducted in artificial forest ecosystems that N addition could potentially lead to an increase in soil microbial biomass. However, research by McCrackin et al. in urban and desert ecosystems did not reveal any significant impact of nitrogen deposition on soil microbial biomass. These differing conclusions could be attributed to variations in the physicochemical properties of forest soils across different ecosystem types or variations in microbial requirements for nitrogen sources.”
I strongly suggest that authors create a correlation matrix to make the mechanisms clearer, especially the effect of pH.
For me, all the results found are related to pH, and the authors focus a lot on the harmful effect of N, when in fact it has to do directly with a property of the fertilizer used and not with N, as this is logical and supported in the literature, so the focus of the article should be changed, using the following theme Long-term nitrogen fertilizers addition with with acidifying capacity could modify degradation of soil organic matter through changes in soil enzymatic activity in a natural secondary forest. Because if the effect is really due to N, the authors need to carry out a new study controlling the pH and varying only the doses of N.
Response:
Following your suggestions, I have reorganized the discussion section. The discussion is structured based on the functional types of enzymes, and not all results are related to pH. In accordance with your advice, I conducted a correlation matrix analysis (Table S2). The analysis revealed that pH does not significantly impact the decomposition enzymes of lignin, whereas the influence of soil total nitrogen (TN) is more substantial. This result is consistent with the Redundancy Analysis (RDA) results presented in Figure 4. In the RDA plot, the angle between the environmental factor vector and enzyme activity vector represents the correlation, with 90 degrees indicating no correlation, close to 180 degrees indicating a negative correlation, and 0 degrees indicating a positive correlation.
To further establish that the changes in enzyme activity are indeed due to nitrogen addition, we performed a partial correlation analysis to reevaluate the relationship between soil TN and enzyme activity, while controlling for the effects of pH changes. The results indicate that even after accounting for pH variations, TN remains significantly correlated with enzyme activity (Table S3).
Both tables have been added to Appendix A.
Table S2: Correlation aalysis of evironmental fctors and sil ezyme ativity
|
　 |
POD |
ACP |
SC |
UE |
PPO |
LAP |
NAG |
|
TN |
-0.44 *** |
0.42 ** |
-0.47 *** |
-0.56 *** |
0.09 |
0.42 ** |
-0.80 *** |
|
TP |
-0.17 |
0.46 *** |
-0.47 *** |
-0.16 |
0.02 |
0.39 ** |
-0.27 |
|
T5cm |
-0.62 *** |
-0.14 |
-0.29 * |
-0.01 |
-0.16 |
0.42 ** |
-0.20 |
|
W5cm |
-0.21 |
0.14 |
0.09 |
0.16 |
0.09 |
0.32 * |
0.07 |
|
pH |
0.02 |
-0.53 *** |
0.40 ** |
0.52 *** |
-0.07 |
-0.20 |
0.27 * |
Table S3: Partial crrelation aalysis of ttal ntrogen on soil enzyme activity controlling for pH
|
　 |
r |
p |
statistic |
n |
Method |
|
POD |
-0.46 |
0.0005 |
-3.73 |
54 |
pearson |
|
ACP |
0.28 |
0.0404 |
2.10 |
54 |
pearson |
|
SC |
-0.38 |
0.0049 |
-2.94 |
54 |
pearson |
|
UE |
-0.46 |
0.0005 |
-3.73 |
54 |
pearson |
|
PPO |
0.06 |
0.6552 |
0.45 |
54 |
pearson |
|
LAP |
0.38 |
0.0048 |
2.95 |
54 |
pearson |
|
NAG |
-0.78 |
0.0000 |
-8.83 |
54 |
pearson |
Because the authors' hypothesis is that the application of N suppresses enzymes related to the C and N cycle, as this is not what is expected. The authors need to improve the introduction to create a basis for this hypothesis.
Response:
Fully accepted and already modified.
The introduction has been revised to establish the foundation for the hypothesis that 'the application of N suppresses enzymes related to the C and N cycles.
“Furthermore, according to the economic theory of microbial metabolism (resource allo-cation theory, Allison et al., 2011), enzyme production increases when nutrients are limited (Allison and Vitousek, 2005; Koch, 1985). Conversely, when nutrients are no longer limiting, enzyme production decreases (Chróst, 1991; Pelletier and Sygusch, 1990; Sinsabaugh and Moorhead, 1994). Therefore, the activity of soil enzymes related to the C and N cycles typically reflects microbial demand for energy and nutrients (Turner and Wright, 2014). After N addition, soil nutrient availability may shift towards P limitation, potentially leading to the inhibition of soil enzymes associated with C and N cycles and an increase in soil enzymes related to P cycling.”
- Table with anova results is not necessary in the manuscript as presented, supplementary material must be made and included.
Response:
Fully accepted and already modified.
The table with the analysis of variance results from the manuscript has been relocated to Appendix A (Table S1).
Table S1. Effects of nitrogen addition on abiotic soil variables in different seasons (mean ± SD)
|
Month |
Treatment |
TN (g kg-1) |
TP (g kg-1) |
T5cm (℃) |
W5cm (m3/m3) |
pH |
|
May |
CK |
21.46±0.06aA |
0.71±0.04aA |
7.66±0.20aA |
0.41±0.06aAB |
5.55±0.01cB |
|
LN |
28.42±1.07bA |
1.39±0.03bC |
7.90±0.34aA |
0.44±0.03aB |
5.44±0.02bA |
|
|
HN |
31.71±1.75bAB |
1.42±0.07bB |
7.64±0.37aA |
0.50±0.02aD |
5.35±0.01aAB |
|
|
June |
CK |
26.71±0.75aB |
0.88±0.02aB |
11.32±0.19aB |
0.49±0.02aB |
5.78±0.02bC |
|
LN |
30.56±0.37bAB |
1.37±0.03bC |
11.86±0.91aB |
0.41±0.06aB |
5.46±0.01aB |
|
|
HN |
29.56±0.77bA |
1.38±0.02bB |
10.65±0.58aB |
0.42±0.03aBCD |
5.41±0.02aAB |
|
|
July |
CK |
26.56±1.67aB |
0.90±0.02aB |
17.03±0.28aD |
0.34±0.05aA |
5.94±0.01cD |
|
LN |
33.74±1.49bBC |
0.94±0.04aA |
17.61±0.16aD |
0.38±0.02aAB |
5.50±0.03bA |
|
|
HN |
35.18±1.18bBC |
1.09±0.13aA |
16.17±0.69aD |
0.39±0.02aABC |
5.35±0.01aB |
|
|
August |
CK |
29.55±0.73aBC |
0.92±0.03aB |
16.70±0.20aD |
0.50±0.00aB |
5.89±0.03cCD |
|
LN |
33.71±2.16aBC |
1.26±0.08bBC |
16.59±0.30aD |
0.47±0.02aB |
5.62±0.02bC |
|
|
HN |
39.99±1.36bD |
1.00±0.06aA |
16.57±0.11aD |
0.45±0.05aCD |
5.53±0.03aB |
|
|
September |
CK |
31.570.91aCD |
0.98±0.06aB |
13.32±0.39aC |
0.32±0.02aA |
5.63±0.02bB |
|
LN |
35.09±0.44bC |
1.19±0.01bB |
13.68±0.07aC |
0.30±0.01aA |
5.47±0.02bAB |
|
|
HN |
39.81±1.01cD |
1.18±0.09abAB |
13.64±0.08aC |
0.32±0.00aA |
5.42±0.02aAB |
|
|
October |
CK |
32.75±0.86aD |
0.98±0.06aB |
7.38±0.15aA |
0.30±0.03aA |
5.58±0.01cA |
|
LN |
34.86±1.11aC |
0.95±0.06aA |
7.91±0.52aA |
0.29±0.01aA |
5.47±0.02bC |
|
|
HN |
36.31±1.54aCD |
1.01±0.08aA |
7.70±0.39aA |
0.34±0.02aAB |
5.38±0.02aA |
Notes: CK, do not add N; HN, high level of N (5.0 g N·m–2·yr–1); LN, low level of N (2.5 g N·m–2·yr–1); TN represents soil total nitrogen; TP represents soil total phosphorus; T5cm represents soil temperature at 5cm soil depth; W5cm represents soil volume water content at 5cm soil depth. Lowercase letters for a given variable indicate significant difference (P < 0.05) among different N addition treatments in same season based，and capital letter for a given variable indicate significant difference (P < 0.05) among different season in same N addition treatments based on one-way ANOVA (N=3), followed by Tukey’s HSD test.
- The quality of figure 1 needs to be improved, the color of the statistical test letters is bad to see.
Response:
Fully accepted and already modified.
In Figure 1, the white the statistical test letters have been replaced with clearer black ones.
- Insert statistical comparisons in the figures and enlarge the letters and numbers to improve data visualization.
Response:
Fully accepted and already modified.
The text in the figures has been enlarged for better visibility.
- The discussion section should not be divided into topics, it should be done continuously.
Response:
Fully accepted and already modified.
The discussion section has been revised to be continuous and no longer segmented into topics.
Thank you again for your suggestions and comments.
Yours sincerely,
Qinggui Wang
Dear Editor and Reviewers,
Thank you very much for giving us a chance to revise our manuscript (forests-2644459). We greatly appreciate the two reviewers for their valuable and constructive comments. We took these comments into full consideration when revising the manuscript. The reviewer’s comments are listed in black, and our responses are listed in blue. We hope our revision and responses can answer the reviewers’ questions and effectively raise the manuscript to the level of this journal. Thank you for all your help in processing and reviewing our manuscript. The point-by-point response to the reviewers’ comments is listed in the following.
Reviewer 2:
The authors discussed that N addition had a certain inhibitory effect on MBC, when in fact they mention that the effect is probably due to acidification from the N source used. This is what makes the most sense and not a deleterious effect due to the use of N, the authors need to improve the discussion a lot to make this clear.
Response:
I completely agree with your perspective. Soil acidification induced by nitrogen addition is indeed one of the factors contributing to the inhibition of microbial biomass carbon (MBC). However, the introduction of exogenous nitrogen may directly or indirectly affect the availability of carbon (C) and nitrogen (N) resources required for microbial growth. Additionally, inorganic nitrogen could potentially interact with soil organic matter to form recalcitrant compounds, all of which can exert inhibitory effects on MBC. I have provided a more detailed explanation of these aspects in the discussion section.
“In our study, we observed that N addition exhibited a certain inhibitory effect on MBC (Figure 1), consistent with findings in several previous studies [19-23]. The reasons behind the impact of N addition on MBC in field studies are multifaceted. This complexity arises because N addition can directly or indirectly affect the availability of C and N resources required for microbial growth. Additionally, N addition-induced soil acidification may also inhibit microbial growth (Chen et al., 2015). Furthermore, N addition leads to an increase in soil inorganic nitrogen content, which can react with soil organic matter, leading to the accumulation of recalcitrant compounds that may hinder microbial growth (Janssens et al., 2010; Treseder, 2008). Studies by Yue et al. (2016) using urea as an N source yielded results indicating increased bacterial biomass upon urea addition, possibly due to the enhanced C input resulting from urea addition, which may interfere with the effects of N addition. Tu et al. (2011) found in experiments conducted in artificial forest ecosystems that N addition could potentially lead to an increase in soil microbial biomass. However, research by McCrackin et al. in urban and desert ecosystems did not reveal any significant impact of nitrogen deposition on soil microbial biomass. These differing conclusions could be attributed to variations in the physicochemical properties of forest soils across different ecosystem types or variations in microbial requirements for nitrogen sources.”
I strongly suggest that authors create a correlation matrix to make the mechanisms clearer, especially the effect of pH.
For me, all the results found are related to pH, and the authors focus a lot on the harmful effect of N, when in fact it has to do directly with a property of the fertilizer used and not with N, as this is logical and supported in the literature, so the focus of the article should be changed, using the following theme Long-term nitrogen fertilizers addition with with acidifying capacity could modify degradation of soil organic matter through changes in soil enzymatic activity in a natural secondary forest. Because if the effect is really due to N, the authors need to carry out a new study controlling the pH and varying only the doses of N.
Response:
Following your suggestions, I have reorganized the discussion section. The discussion is structured based on the functional types of enzymes, and not all results are related to pH. In accordance with your advice, I conducted a correlation matrix analysis (Table S2). The analysis revealed that pH does not significantly impact the decomposition enzymes of lignin, whereas the influence of soil total nitrogen (TN) is more substantial. This result is consistent with the Redundancy Analysis (RDA) results presented in Figure 4. In the RDA plot, the angle between the environmental factor vector and enzyme activity vector represents the correlation, with 90 degrees indicating no correlation, close to 180 degrees indicating a negative correlation, and 0 degrees indicating a positive correlation.
To further establish that the changes in enzyme activity are indeed due to nitrogen addition, we performed a partial correlation analysis to reevaluate the relationship between soil TN and enzyme activity, while controlling for the effects of pH changes. The results indicate that even after accounting for pH variations, TN remains significantly correlated with enzyme activity (Table S3).
Both tables have been added to Appendix A.
Table S2: Correlation aalysis of evironmental fctors and sil ezyme ativity
|
　 |
POD |
ACP |
SC |
UE |
PPO |
LAP |
NAG |
|
TN |
-0.44 *** |
0.42 ** |
-0.47 *** |
-0.56 *** |
0.09 |
0.42 ** |
-0.80 *** |
|
TP |
-0.17 |
0.46 *** |
-0.47 *** |
-0.16 |
0.02 |
0.39 ** |
-0.27 |
|
T5cm |
-0.62 *** |
-0.14 |
-0.29 * |
-0.01 |
-0.16 |
0.42 ** |
-0.20 |
|
W5cm |
-0.21 |
0.14 |
0.09 |
0.16 |
0.09 |
0.32 * |
0.07 |
|
pH |
0.02 |
-0.53 *** |
0.40 ** |
0.52 *** |
-0.07 |
-0.20 |
0.27 * |
Table S3: Partial crrelation aalysis of ttal ntrogen on soil enzyme activity controlling for pH
|
　 |
r |
p |
statistic |
n |
Method |
|
POD |
-0.46 |
0.0005 |
-3.73 |
54 |
pearson |
|
ACP |
0.28 |
0.0404 |
2.10 |
54 |
pearson |
|
SC |
-0.38 |
0.0049 |
-2.94 |
54 |
pearson |
|
UE |
-0.46 |
0.0005 |
-3.73 |
54 |
pearson |
|
PPO |
0.06 |
0.6552 |
0.45 |
54 |
pearson |
|
LAP |
0.38 |
0.0048 |
2.95 |
54 |
pearson |
|
NAG |
-0.78 |
0.0000 |
-8.83 |
54 |
pearson |
Because the authors' hypothesis is that the application of N suppresses enzymes related to the C and N cycle, as this is not what is expected. The authors need to improve the introduction to create a basis for this hypothesis.
Response:
Fully accepted and already modified.
The introduction has been revised to establish the foundation for the hypothesis that 'the application of N suppresses enzymes related to the C and N cycles.
“Furthermore, according to the economic theory of microbial metabolism (resource allo-cation theory, Allison et al., 2011), enzyme production increases when nutrients are limited (Allison and Vitousek, 2005; Koch, 1985). Conversely, when nutrients are no longer limiting, enzyme production decreases (Chróst, 1991; Pelletier and Sygusch, 1990; Sinsabaugh and Moorhead, 1994). Therefore, the activity of soil enzymes related to the C and N cycles typically reflects microbial demand for energy and nutrients (Turner and Wright, 2014). After N addition, soil nutrient availability may shift towards P limitation, potentially leading to the inhibition of soil enzymes associated with C and N cycles and an increase in soil enzymes related to P cycling.”
- Table with anova results is not necessary in the manuscript as presented, supplementary material must be made and included.
Response:
Fully accepted and already modified.
The table with the analysis of variance results from the manuscript has been relocated to Appendix A (Table S1).
Table S1. Effects of nitrogen addition on abiotic soil variables in different seasons (mean ± SD)
|
Month |
Treatment |
TN (g kg-1) |
TP (g kg-1) |
T5cm (℃) |
W5cm (m3/m3) |
pH |
|
May |
CK |
21.46±0.06aA |
0.71±0.04aA |
7.66±0.20aA |
0.41±0.06aAB |
5.55±0.01cB |
|
LN |
28.42±1.07bA |
1.39±0.03bC |
7.90±0.34aA |
0.44±0.03aB |
5.44±0.02bA |
|
|
HN |
31.71±1.75bAB |
1.42±0.07bB |
7.64±0.37aA |
0.50±0.02aD |
5.35±0.01aAB |
|
|
June |
CK |
26.71±0.75aB |
0.88±0.02aB |
11.32±0.19aB |
0.49±0.02aB |
5.78±0.02bC |
|
LN |
30.56±0.37bAB |
1.37±0.03bC |
11.86±0.91aB |
0.41±0.06aB |
5.46±0.01aB |
|
|
HN |
29.56±0.77bA |
1.38±0.02bB |
10.65±0.58aB |
0.42±0.03aBCD |
5.41±0.02aAB |
|
|
July |
CK |
26.56±1.67aB |
0.90±0.02aB |
17.03±0.28aD |
0.34±0.05aA |
5.94±0.01cD |
|
LN |
33.74±1.49bBC |
0.94±0.04aA |
17.61±0.16aD |
0.38±0.02aAB |
5.50±0.03bA |
|
|
HN |
35.18±1.18bBC |
1.09±0.13aA |
16.17±0.69aD |
0.39±0.02aABC |
5.35±0.01aB |
|
|
August |
CK |
29.55±0.73aBC |
0.92±0.03aB |
16.70±0.20aD |
0.50±0.00aB |
5.89±0.03cCD |
|
LN |
33.71±2.16aBC |
1.26±0.08bBC |
16.59±0.30aD |
0.47±0.02aB |
5.62±0.02bC |
|
|
HN |
39.99±1.36bD |
1.00±0.06aA |
16.57±0.11aD |
0.45±0.05aCD |
5.53±0.03aB |
|
|
September |
CK |
31.570.91aCD |
0.98±0.06aB |
13.32±0.39aC |
0.32±0.02aA |
5.63±0.02bB |
|
LN |
35.09±0.44bC |
1.19±0.01bB |
13.68±0.07aC |
0.30±0.01aA |
5.47±0.02bAB |
|
|
HN |
39.81±1.01cD |
1.18±0.09abAB |
13.64±0.08aC |
0.32±0.00aA |
5.42±0.02aAB |
|
|
October |
CK |
32.75±0.86aD |
0.98±0.06aB |
7.38±0.15aA |
0.30±0.03aA |
5.58±0.01cA |
|
LN |
34.86±1.11aC |
0.95±0.06aA |
7.91±0.52aA |
0.29±0.01aA |
5.47±0.02bC |
|
|
HN |
36.31±1.54aCD |
1.01±0.08aA |
7.70±0.39aA |
0.34±0.02aAB |
5.38±0.02aA |
Notes: CK, do not add N; HN, high level of N (5.0 g N·m–2·yr–1); LN, low level of N (2.5 g N·m–2·yr–1); TN represents soil total nitrogen; TP represents soil total phosphorus; T5cm represents soil temperature at 5cm soil depth; W5cm represents soil volume water content at 5cm soil depth. Lowercase letters for a given variable indicate significant difference (P < 0.05) among different N addition treatments in same season based，and capital letter for a given variable indicate significant difference (P < 0.05) among different season in same N addition treatments based on one-way ANOVA (N=3), followed by Tukey’s HSD test.
- The quality of figure 1 needs to be improved, the color of the statistical test letters is bad to see.
Response:
Fully accepted and already modified.
In Figure 1, the white the statistical test letters have been replaced with clearer black ones.
- Insert statistical comparisons in the figures and enlarge the letters and numbers to improve data visualization.
Response:
Fully accepted and already modified.
The text in the figures has been enlarged for better visibility.
- The discussion section should not be divided into topics, it should be done continuously.
Response:
Fully accepted and already modified.
The discussion section has been revised to be continuous and no longer segmented into topics.
Thank you again for your suggestions and comments.
Yours sincerely,
Qinggui Wang
Author Response File: Author Response.docx
Round 2
Reviewer 2 Report
Dear Editor and authors, the manuscript was improved and can be accepted in the current version.
