Shifts in the Soil Microbial Community and Enzyme Activity Under Picea crassifolia Plantations and Natural Forests

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study discussed the microbial influence and the enzymes produced to promote Picea crassifolia and natural forest plantations. If the corrections are effectively made, the study could contribute unique knowledge to the scientific community.

You could mention words not mentioned in the topic and arrange the Keywords alphabetically

Introduction

Line 69: Do other forest organisms and animals contribute to the environment's vulnerability?

I expected more discussion on this 

You could discuss how microbial diversity has improved afforestation and how their assistance has improved forest essential to humans

Line 88 - 90: Rephrase the sentence

Methodology

Line 96: What is asl? You could provide the coordinates of this region.

Line 100 - 102: Is there part of what you conducted, if so, this should appear in  the result section and not in the methodology

Line 115: How do you explain bulk soil?

Line 116: How many soil samples did you collect in all? Did you have control for this experiment?

Table 1: You could explain how you conducted this study in this section and further present and explain the table in the result section. Mixing the methods and results together makes the manuscript hard to understand

Line 130: Is this a ref?

Line 134: Ring method- Is this a type used to measure the surface tension of the liquid, if not, could you explain better?

Line 142: Is this the model of the Spectrophotometer, complete the model by adding the the model number and the country it was manufactured from

Line 143: What method did you adopt to isolate the microbes?

Line 149-15: Is this extracted directly from soil samples or from the isolated microbes

Line 159-161: This method is not well explained and do you have specific method you followed to get this done? ref

Line 172: You could remove this and add "and other analyses were conducted using R 4.4.1 software"

Result

Line 186: Remove (mean standard err) and insert in the table's footnote.

Table 2: all the letters should be in superscript

Line 189: Rephrase, you can start a sentence in this manner

Line 206 - 208: Remove here and place under the table as a footnote

Line 259: Why are some alphabets in uppercase while others are lower case? Put the alphabet in superscript

Line 260: What is RDA?

What is the difference between PCA and PCoA? How and when is it used to analyze data?

Line 263: You have lot of abbreviation which there meaning is not cleared. Could you provide the full meaning of these and other abbreviations

Comments for author File: Comments.pdf

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in the re-submitted files.

 

Comments 1: You could mention words not mentioned in the topic and arrange the Keywords alphabetically

Response 1: We have made the following revisions to the Keywords section:

• Deleted words that appear in the topic, specifically Picea crassifolia, plantation, natural forest, and microbial community, to avoid redundancy.
• Added new keywords relevant to the scope of this study, namely forest age, microbial abundance, and subalpine region, to better represent the content and focus of the research.
• Rearranged all keywords alphabetically for clarity and consistency.

These revisions can be found in the manuscript on Line 26-27.

 

Comments 2: Line 69: Do other forest organisms and animals contribute to the environment's vulnerability? I expected more discussion on this.

Response 2: We acknowledge the potential contribution of forest organisms and animals to environmental vulnerability. However, studies indicate that the vulnerability in Qinghai Province is primarily driven by geographical and climatic characteristics, making it sensitive to human activities. We have clarified this point and included a brief discussion of other contributing factors. Since “organisms” include “animals,” we did not single out animals separately to avoid misinterpretation, as they are not the focus of this study.

The updated text can be found on Line 78-80.

 

Comments 3: Line 83: You could discuss how microbial diversity has improved afforestation and how their assistance has improved forest essential to humans.

Response 3: We added the sentences to highlight how microbial diversity enhances soil fertility and nutrient cycling, supports tree establishment and growth, and improves tree resilience to environmental challenges, thereby contributing to the success of afforestation efforts.

The revised text can be found on Lines 56-60.

 

Comments 4: Line 88 - 90: Rephrase the sentence

Response 4: We have revised the sentence for clarity and precision to better convey the focus of the study.

The revised text can be found on Line 97-99.

 

Comments 5: Line 96: What is asl? You could provide the coordinates of this region.

Response 5: “asl” refers to Above Sea Level, which is a standard unit used to denote altitude or elevation in fields such as geography, geology, ecology, and atmospheric sciences. To address your suggestion, we have revised the sentence to replace “asl” with “altitude range” for clarity. Additionally, we have included the geographical coordinates of the study region as requested.

The updated text can be found on Lines 106.

 

Comments 6: Line 100 - 102: Is there part of what you conducted, if so, this should appear in the result section and not in the methodology

Response 6: This section is intended to provide an overview of the study area, including the vegetation types of natural and plantation forests, to help readers understand the natural environment and baseline conditions of the research region. It does not present any research results but rather contextual information about the study site. We have revised the section title to “Site Description and Sampling” to clarify its purpose.

The updated text can be found on Lines 104.

 

Comments 7: Line 115: How do you explain bulk soil?

Response 7: We use “bulk soil” to describe undisturbed soil samples, as they provide comprehensive information about soil properties and processes in their natural state. To reduce ambiguity, we have revised the sentence to clarify the sampling method and avoid potential misinterpretation of “bulk soil”.

The updated text can be found on Lines 127.

 

Comments 8: Line 116: How many soil samples did you collect in all? Did you have control for this experiment?

Response 8: Thank you for your questions. To clarify, the sampling method description has been revised for better precision. A total of 36 soil analysis samples were collected by combining soil from three layers within each subplot into composite samples for each layer. Regarding the control, this study did not involve a traditional experimental control group, as the research focuses on natural forest and plantation under existing field conditions. We compare the difference between natural forests and plantations across different stand ages and soil layers. This approach is consistent with observational studies in forest soil science.

The updated text can be found on Lines 130.

 

Comments 9: Table 1: You could explain how you conducted this study in this section and further present and explain the table in the result section. Mixing the methods and results together makes the manuscript hard to understand.

Response 9: Thank you for pointing this out. We understand your concern regarding the mixing of methods and results. The table provides information about the study plots, and was intended to clarify the site background. To avoid confusion, we have removed the table from the main text and included it as supplementary material. This ensures that the methodology section focuses solely on describing how the study was conducted, while the supplementary material offers additional context about the study sites for interested readers.

 

Comments 10: Line 130: Is this a ref?

Response 10: The term “Kjeldahl” is not a reference but rather the name of the method used for determining total nitrogen concentrations. To avoid confusion, we have clarified this in the manuscript. The revised text now specifies the method without implying it as a citation.

The updated text can be found on Lines 138.

 

Comments 11: Line 134: Ring method- Is this a type used to measure the surface tension of the liquid, if not, could you explain better?

Response 11: We clarified that the “ring method” refers to the soil cutting ring method, commonly used to determine soil bulk density (SBD) and field water capacity (FWC). The revised text also cites the national standard of China (LY/T 1215–1999) for further reference.

The updated sentence can be found on Line 141-143.

 

Comments 12: Line 142: Is this the model of the Spectrophotometer, complete the model by adding the model number and the country it was manufactured from

Response 12: We have clarified the information and the updated sentence specifies that the spectrophotometer used was from Purkinje General, Beijing, China.

The updated sentence can be found on Line 151.

 

Comments 13: Line 143: What method did you adopt to isolate the microbes?

Response 13: The PLFA method does not involve directly isolating microbes but instead uses phospholipid fatty acid (PLFA) analysis to characterize the microbial community. PLFAs are extracted from microbial cell membranes and serve as biomarkers to indirectly represent the composition and dynamics of the microbial community. This approach is widely used in microbial ecology to study soil microbial communities without requiring direct isolation of individual microorganisms. We have added a statement in the methods section to explain this approach more clearly.

The updated sentence can be found on Line 153.

 

Comments 14: Line 149-150: Is this extracted directly from soil samples or from the isolated microbes

Response 14: Thank you for your question which gives me an opportunity to better explain the previous comment. The PLFAs were extracted directly from soil samples, not from isolated microbes. This method analyzes phospholipids in microbial cell membranes to indirectly characterize the soil microbial community without requiring microbial isolation. To better present the results, we have submitted a table categorizing the biomarkers of different types of microorganisms as supplementary material.

The updated information can be found in Table S2.

 

Comments 15: Line 159-161: This method is not well explained and do you have specific method you followed to get this done? Ref

Response 15: The enzyme activity measurements were performed following the method described in reference [49], which provides detailed information on the buffer composition, substrate names, and incubation times. We followed this protocol closely and included the relevant reference for readers seeking additional details.

The updated sentence can be found on Line 171.

 

Comments 16: Line 162: complete the model description

Response 16: Thank you for pointing out. We have revised the text to include the complete model description

The updated sentence can be found on Line 172.

 

Comments 17: Line 172: You could remove this and add "and other analyses were conducted using R 4.4.1 software"

Response 17: We have revised the text accordingly to streamline the description.

The updated sentence can be found on Line 182.

 

Comments 18: Line 186: Remove (mean standard err) and insert in the table's footnote.

Response 18: We have revised the table caption by removing (means ± standard error) and included this information in the table footnote for clarity and consistency.

The updated sentence can be found on Line 197-198.

 

Comments 19: Table 2: all the letters should be in superscript

Response 19: We have revised the table to ensure that all letters are presented in superscript format. The updated table can be found in the revised manuscript.

 

Comments 20: Line 189: Rephrase, you can start a sentence in this manner

Response 20: Thank you for your suggestion. We have rephrased the sentence to improve readability.

The updated sentence can be found on Line 201.

 

Comments 21: Line 206 - 208: Remove here and place under the table as a footnote

Response 21: We have removed the text from above the table and included it as a footnote to ensure proper placement and clarity.

The updated sentence can be found on Line 223.

 

Comments 22: Line 259: Why are some alphabets in uppercase while others are lower case? Put the alphabet in superscript

Response 22: Thank you for pointing this out. We have clarified the distinction between uppercase and lowercase letters in the table notes and ensured that all letters are presented in superscript format as requested.

The updated sentence can be found on Line 286-288.

 

Comments 23: Line 260: What is RDA? What is the difference between PCA and PCoA? How and when is it used to analyze data?

Response 23: Thank you for your question.

RDA Explanation:

• Redundancy Analysis (RDA) is a constrained ordination method that evaluates the relationships between response variables such as microbial community composition and explanatory variables such as abiotic factors. It can quantify the extent to which environmental factors explain variations in microbial community composition.

Difference Between PCA and PCoA:

• Principal Component Analysis (PCA): PCA is an unconstrained ordination method that reveals the variation patterns within microbial communities but cannot directly assess whether these variations are related to soil environmental factors. However, PCA cannot explain the relationships between microbial community data and environmental factors.
• Principal Coordinate Analysis (PCoA): The goal of PCoA is to analyze sample similarities or differences using a distance matrix (e.g., Bray-Curtis). PCoA is suitable for examining similarities or differences between samples. However, it also cannot directly quantify the influence of environmental factors on microbial communities.

How and When to Use RDA:

• RDA is used when the goal is to directly assess correlations between explanatory and response variables. It introduces environmental factors into the analysis to quantitatively reveal their effects on microbial communities, making it a more suitable method for exploring the relationships between soil environmental factors and microbial communities.
• In this study, RDA was applied to analyze the correlations between abiotic factors and microbial community composition as well as enzyme activities, enabling us to identify the drivers influencing these parameters in forest soils.

 

Comments 24: Line 263: You have lot of abbreviation which there meaning is not cleared. Could you provide the full meaning of these and other abbreviations

Response 24: We appreciate your feedback. The full names for all abbreviations are provided in the Methods section of the manuscript. Additionally, we added the whole name of the abbreviations in the notes of the tables, which helps maintain clarity and conciseness in the Results section. For consistency, abbreviations are also used in figures and tables to ensure concise representation.

Reviewer 2 Report

Comments and Suggestions for Authors

I have reviewed the manuscript titled
"Shifts in the Soil Microbial Community and Enzyme Activity under Picea crassifolia Plantations and Natural Forests," which examines the impact of forest type on soil microbial communities and enzyme activity. The topic is relevant and addresses a notable gap in understanding the ecological differences between natural forests and plantations. However, several concerns warrant attention.

The study investigates differences in microbial communities and enzymatic activities between Picea crassifolia plantations and natural forests, emphasizing the effects of plantation age and soil layers.
The study is relevant for understanding forest management impacts in Qinghai Province. While it highlights microbial and enzymatic trends, its originality is limited due to similar existing studies. The focus on taxon-specific microbial dynamics under a single tree species is valuable but could benefit from broader taxonomic and functional comparisons.

Key Findings:

- Natural forests exhibit superior microbial abundance and enzymatic activity compared to plantations.
- Microbial communities in plantations improve with age.
- These findings support known ecological patterns but offer limited novel insights.

Limitations and Concerns:

Methodology:
- Relies on PLFA analysis, which lacks the resolution of DNA-based techniques.
- Limited temporal scope and insufficient age replication reduce robustness.

Interpretation:
Contradictory results, such as anomalously high fungal-to-bacterial (FUN/BAC) ratios in plantations, lack mechanistic explanations.
Limited analysis of nitrogen-cycling microbes and functional enzymes.

Visualization:
Figures lack error bars and statistical annotations; tables could better integrate cross-references to figures.

Recommendations for Improvement:
Advanced Techniques: Incorporate metagenomics or qPCR to analyze functional genes like nifH, amoA, and nirK, focusing on nitrogen-cycling microbes.
Expanded Scope: Study younger and older stands, seasonal dynamics, and additional regions for generalizability.
Ecosystem Functions: Evaluate nitrogen-cycling enzymes (e.g., urease, nitrate reductase) and microbial contributions to ecosystem services.

The study provides insights into microbial dynamics in Picea crassifolia forests but requires methodological enhancements, a broader scope, and deeper mechanistic analysis to justify publication. Strengthening controls, integrating nitrogen-related microbial analyses, and improving visualization will enhance its scientific impact.

Questions:
1.How do nitrogen-cycling microbes vary between plantations and natural forests?
2.Can metagenomics validate trends in microbial composition and FUN/BAC ratios?
3. What effects does plantation age have on ecosystem services like the cycling of nitrogen and carbon?
4. What seasonal microbial shifts occur in these forest types?
5. How can these findings inform sustainable afforestation practices?

Considering the above-mentioned points will improve the quality of the manuscript and warrant its publication.

Author Response

Thank you for your thorough evaluation and constructive suggestions. We appreciate your feedback, which has helped us improve the clarity and scope of our study. Below, we address your comments:

 

Comment 1:

Methodology:

• Relies on PLFA analysis, which lacks the resolution of DNA-based techniques.
• Limited temporal scope and insufficient age replication reduce robustness.

Response 1:

• While we acknowledge the limitations of PLFA analysis compared to DNA-based techniques, it remains a sensitive and effective method for distinguishing microbial groups, aligning well with the objectives of our study. To address the limitations, we have included future research directions in the manuscript, proposing the use of advanced techniques such as metagenomics or qPCR.

The updated sentence can be found on Line 436-438.

• While this research provides valuable insights into microbial dynamics across different stand ages, we have included suggestions in the Conclusion section for future studies to expand the temporal scope and include seasonal dynamics to enhance the robustness and generalizability of the findings

The updated sentence can be found on Line 449-450.

 

Comment 2:

Interpretation:

• Contradictory results, such as anomalously high fungal-to-bacterial (FUN/BAC) ratios in plantations, lack mechanistic explanations.
• Limited analysis of nitrogen-cycling microbes and functional enzymes.

Response 2:

• We have revised the discussion to better address the anomalously high fungal-to-bacterial (FUN/BAC) ratios observed in plantations. Based on our findings and relevant literature, we propose that these differences are primarily driven by variations in soil EC and C and N content, as supported by our correlation analysis. These points have been clarified in the discussion section.

The updated sentence can be found on Line 434-436.

• This study employed PLFA analysis, which does not directly address nitrogen-cycling microbes or related enzyme activities. However, we recognize the importance of these aspects and have included them as future research directions in the manuscript.

The updated sentence can be found on Line 445-449.

 

Comment 3:

Visualization:

Figures lack error bars and statistical annotations; tables could better integrate cross-references to figures.

Response 3:

We would like to clarify that our figures already include error bars and statistical annotations to present the data accurately. If there are specific concerns about certain figures, we would be happy to address them. Regarding the suggestion that “tables could better integrate cross-references to figures,” while the exact concern is unclear, we have added explanatory notes under both tables and figures to enhance clarity and improve the connection between them. We hope these changes make the data presentation more comprehensive and easier to interpret.

The updated sentence can be found on Line 257-268.

 

Comment 4:

Recommendations for Improvement:

• Advanced Techniques: Incorporate metagenomics or qPCR to analyze functional genes like nifH, amoA, and nirK, focusing on nitrogen-cycling microbes.
• Expanded Scope: Study younger and older stands, seasonal dynamics, and additional regions for generalizability.
• Ecosystem Functions: Evaluate nitrogen-cycling enzymes (e.g., urease, nitrate reductase) and microbial contributions to ecosystem services.
  Response 4:
• We have included future research directions in the manuscript, proposing the use of advanced techniques such as metagenomics or qPCR.

The updated sentence can be found on Line 436-438.

• While we acknowledge that studying younger and older stands, seasonal dynamics, and additional regions would improve the generalizability of our findings, we have incorporated this recommendation into the discussion as a suggestion for future research.

The updated sentence can be found on Line 449-450.

• Furthermore, the evaluation of nitrogen-cycling enzymes and their contributions to ecosystem services has also been included in the future research outlook to address this important aspect.

The updated sentence can be found on Line 445-449.

 

Once again, we thank you for your insightful comments, which have strengthened the scientific rigor and clarity of our manuscript. Please find the detailed answers of your questions below and the corresponding revisions in the re-submitted files.

 

Question 1: How do nitrogen-cycling microbes vary between plantations and natural forests?

Response 1: The PLFA analysis used in this study cannot directly identify nitrogen-cycling microbes. However, our findings suggest that microbial communities play a role in nitrogen cycling. Specifically, as stated in line 394, the increase in carbon and nitrogen content enhances microbial PLFA abundance, particularly by promoting microbial growth. This indicates a potential linkage between microbial community composition and nitrogen transformation processes.

 

Question 2: Can metagenomics validate trends in microbial composition and FUN/BAC ratios?

Response 2: While our study employed PLFA analysis to investigate microbial composition and FUN/BAC ratios, metagenomics could indeed provide more detailed insights by directly identifying microbial taxa and functional genes. Future studies integrating metagenomics could validate and expand on our findings, offering a deeper understanding of the functional roles and ecological significance of microbial communities in plantations and natural forests.

 

Question 3: What effects does plantation age have on ecosystem services like the cycling of nitrogen and carbon?

Response 3: Our findings indicate that plantation age significantly influences microbial composition and enzyme activities, which are closely tied to the cycling of nitrogen and carbon. We have revised the discussion to include an explanation of how plantation age affects carbon and nitrogen cycling. The revised text highlights the role of increased litter input and microbial activity in promoting robust nutrient cycling as plantation age advances.

 

Question 4: What seasonal microbial shifts occur in these forest types?

Response 4: Thank you for your insightful question. This study does not involve a seasonal comparison but focuses on the microbial community and activity differences between plantations and natural forests of Picea crassifolia across different stand ages. Seasonal variations in microbial dynamics are indeed an important aspect for understanding forest ecosystem processes. Future research could explore how microbial communities and their functions respond to seasonal changes. In response, we have added a statement to highlight the importance of future research in this area. Specifically, we emphasize the necessity of seasonal sampling to capture microbial dynamics and their contributions to ecosystem functions.

 

Question 5: How can these findings inform sustainable afforestation practices?

Response 5: Our findings demonstrate the importance of conserving natural forests for maintaining soil microbial diversity and functionality, while also highlighting the potential for enhancing microbial health in plantations through age management and improved soil conditions. To address this, we have added a statement at the end of the conclusion to emphasize how these results can guide sustainable afforestation practices.

These revisions can be found in the manuscript on Line 464-469.

Reviewer 3 Report

Comments and Suggestions for Authors

1.      First of all, in the abstract, the background is not adequate enough to lead to the objective statement. Please change it to a more persuasive background statement with good novelty and necessity.

2.      “focused” in line 9.

3.      “in its plantations and natural forests” This is too confusing. Please rewrite.

4.      Please statement the importance of PLFA.

5.      Please define “NF” in line 18, natural forests. Moreover, the methodology should be improved by presenting treatments along with their abbreviations. This could help the results readable.

6.      Significant numeric data should be added to the results in the abstract.

7.      The conclusion is not well proposed. The conclusion suggests the effects of the two factors on the soil microbes, but how about the differences between the two factors mentioned in the results of the abstract? Moreover, what further applications or research can be benefited from the study?

8.      The keywords should be arranged alphabetically.

9.      The importance of the soil microbes is the key reason for the study. Therefore, it should be presented more clearly with more efforts in the introduction.

10.  Based on the last paragraph of the introduction, I would suggest another title for the study which is “Investigation of the Soil Microbial Community and Enzyme Activity in Picea crassifolia Plantations and Natural Forests by phospholipid fatty acid analysis”.

11.  Please match the objective statements in the abstract and the introduction.

12.  There should be sources for the information in lines 95–104.

13.  PF in line 109 should be plantations forests

14.  “Total concentrations of carbon (LOC), nitrogen (LTN), and phosphorus (LTP) concentrations” remove one “concentrations” in lines 128–129.

15.  The sampling methods for the soil and liter samples should be better presented. Perhaps, an image should be added to the supplementary data.

16.  Tables 3–6 and Figures 3 and 4 should have a note defining the components within them.

17.  The significant contribution and the applicability of the study should be presented at the end of the conclusion based on its significant results.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions in the re-submitted files.

 

Comments 1: First of all, in the abstract, the background is not adequate enough to lead to the objective statement. Please change it to a more persuasive background statement with good novelty and necessity.

Response 1: Thank you for your valuable suggestion. We have revised the abstract’s background to emphasize the importance of soil microbes in regulating biogeochemical cycles and maintaining forest ecosystem sustainability. Additionally, we have highlighted the limited understanding of microbial communities and enzyme activity in natural and plantation forests within Plateau regions, underscoring the novelty and necessity of our study.

These revisions can be found in the manuscript on Line 11-13.

 

Comments 2: “focused” in line 9.

Response 2: Thank you for your observation. We have revised the sentences and deleted this word.

 

Comments 3: “in its plantations and natural forests” This is too confusing. Please rewrite.

Response 3: Thank you for your comment. We agree that the original sentence could lead to confusion. To better express the focus of the study, we have removed this sentence. Instead, we added some words to emphasis the role of microbes in natural and plantation forests at the beginning of Abstract.

These revisions can be found in the manuscript on Line 14.

 

Comments 4: Please statement the importance of PLFA.

Response 4: We have added a statement to highlight the importance of the PLFA method, emphasizing its sensitivity to structural changes in microbial communities.

The revised text can be found on Line 16-17.

 

Comments 5: Please define “NF” in line 18, natural forests. Moreover, the methodology should be improved by presenting treatments along with their abbreviations. This could help the results readable.

Response 5: We have defined “NF” as natural forests when it first appears in the Abstract (line 12). Additionally, we have improved the presentation of abbreviations by referencing similar published studies and revising the methodology section to clearly present treatments alongside their abbreviations, enhancing the readability of the results

The updated text can be found on Lines 14.

 

 

Comments 6: Significant numeric data should be added to the results in the abstract.

Response 6: Thank you for your comment. We have added a statement to the abstract that highlights the relative proportions of microbial groups identified in the study. This provides foundational data for understanding the composition and ecological functions of microbial communities and serves as a baseline for evaluating their responses to environmental changes.

The updated text can be found on Lines 17-18.

 

Comments 7: The conclusion is not well proposed. The conclusion suggests the effects of the two factors on the soil microbes, but how about the differences between the two factors mentioned in the results of the abstract? Moreover, what further applications or research can be benefited from the study?

Response 7: We have revised the conclusion in the abstract to clearly present the differences between natural forests and plantations in terms of microbial PLFA and enzyme activities. Additionally, we have emphasized the implications of these findings for forest management practices and soil microbial conservation.

The updated text can be found on Lines 23-25.

 

Comments 8: The keywords should be arranged alphabetically.

Response 8: Thank you for your suggestion. We have revised the keywords to ensure they are arranged alphabetically in the manuscript.

The updated text can be found on Lines 26-27.

 

Comments 9: The importance of the soil microbes is the key reason for the study. Therefore, it should be presented more clearly with more efforts in the introduction.

Response 9: Thank you for your valuable suggestion. We have revised the introduction to provide a more comprehensive explanation of the importance of soil microbes, emphasizing their roles in maintaining soil quality and forest ecosystem sustainability. The revised text also highlights the significance of understanding soil microbial dynamics for sustainable forest management.

The updated text can be found on Lines 31-34.

 

Comments 10: Based on the last paragraph of the introduction, I would suggest another title for the study which is “Investigation of the Soil Microbial Community and Enzyme Activity in Picea crassifolia Plantations and Natural Forests by phospholipid fatty acid analysis”

Response 10: Thank you for your thoughtful suggestion. While phospholipid fatty acid (PLFA) analysis is indeed a key method in our study, we believe including it in the title may create confusion due to “enzyme activity”. As PLFA analysis is not the primary contribution of this study, we have decided to keep the current title. However, to highlight the importance of this method, we have added “phospholipid fatty acid analysis” to the keywords section.

The updated text can be found on Lines 26.

 

Comments 11: Please match the objective statements in the abstract and the introduction.

Response 11: We have clarified the research objectives and ensured consistency between the abstract and the introduction. The revised text expresses the study’s goals as clearly and coherently as possible.

The updated sentence can be found on Line 100-102.

 

Comments 12: There should be sources for the information in lines 95–104.

Response 12: Thank you for your comment. We have added the Huzhu Tu Autonomous County Yearbook as the source for the information presented in lines 104-114. This yearbook provides detailed records of the natural characteristics of the study area. The reference has been included in the revised manuscript.

The updated sentence can be found on Line 109.

 

Comments 13: PF in line 109 should be plantations forests

Response 13: We have revised the expression of abbreviations to ensure clarity and improved readability.

The updated sentence can be found on Line 121.

 

Comments 14: “Total concentrations of carbon (LOC), nitrogen (LTN), and phosphorus (LTP) concentrations” remove one “concentrations” in lines 128–129.

Response 14: Thank you for your observation. We have removed the redundant “concentrations” to improve clarity.

The updated sentence can be found on Line 138.

 

Comments 15: The sampling methods for the soil and liter samples should be better presented. Perhaps, an image should be added to the supplementary data.

Response 15: We didn’t take clear images of the sampling process to include as supplementary data. However, we have revised the description of the sampling methods to make them more explicit and precise for better understanding.

The updated sentence can be found on Line 127-130.

 

Comments 16: Tables 3–6 and Figures 3 and 4 should have a note defining the components within them.

Response 16: Based on feedback from other reviewers, we moved Table 1 to the supplementary materials for clarity. Notes were added under the original Tables 3 and 4 to define their components. As the elements in original Tables 5 and 6 overlap with content already explained in previous tables, no additional notes were added to avoid redundancy. We have also included supplementary explanations under Figures 3 and 4 for improved clarity.

The updated sentence can be found on Line 195-198, 219-225, 257-268, 285-291, 311-315，319, 323 and 331-333.

 

Comments 17: The significant contribution and the applicability of the study should be presented at the end of the conclusion based on its significant results

Response 17: We have revised the conclusion to clearly highlight the study’s significant contributions and practical applicability. The updated conclusion emphasizes the importance of conserving natural forests and managing plantations to enhance soil microbial health and ecosystem sustainability.

The updated sentence can be found on Line 464-469.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript discussed the rhizosphere microbiomes of Picea crassifolia and natural forest shift and their enzymatic activity in a particular plateaux location. The study aimed to investigate differences in soil microbial communities and enzyme activity across three soil layers between natural forests and identify major driving factors for the differences in soil microbial characteristics. The comments have been addressed 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have effectively addressed all concerns raised during the review process. As a result, the manuscript has been significantly improved and is now suitable for acceptance in its current form.

Reviewer 3 Report

Comments and Suggestions for Authors

All comments have been addressed. I suggested that the manuscript should be submitted