Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer Report

Title: Effects of Different Sod-Seeding Patterns on Soil Properties, Nitrogen Cycle Genes, and N2O Mitigation in Peach Orchards

Recommendation: Accept after Minor Revisions

1. General Comments This manuscript presents a relevant and interesting study on the effects of sod-seeding patterns (intercropping) in peach orchards in the Beijing region. The topic is timely, addressing both soil health and greenhouse gas mitigation (N₂O) in intensive fruit production systems.

The experimental design, comparing clean tillage (CK) with three sod-seeding patterns (White clover, Ryegrass, and a mixture), is logical. The combination of analyzing soil physicochemical properties, N₂O fluxes, and microbial functional genes provides a comprehensive view of the mechanisms at play. The finding that the mixed sowing pattern (TPr) yields the best results for mitigation and soil quality is valuable for orchard management strategies.

However, the manuscript requires some clarifications regarding the methodology (specifically the genetic analysis) and some linguistic polishing before publication.

2. Specific Comments

- Abstract: The abstract is informative and covers the main findings well. However, please ensure that the specific reduction percentages for N₂O are consistent with the results section.

- Materials and Methods:

- Section 2.3.3: There is a slight confusion in the text regarding the genetic analysis. The Introduction mentions "qPCR and 16S rRNA sequencing," but Section 2.3.3 is titled "Soil metagenomic sequencing." Please clarify if you used qPCR for absolute quantification of specific genes (like nosZ, nirS) or if these abundances were derived solely from metagenomic data. If qPCR was used, the primer sequences and thermal cycling conditions should be briefly mentioned or cited.

- N₂O Sampling: Sampling was done "2 or 3 times every month." Did you perform additional sampling immediately after fertilization or heavy rainfall events? If not, please acknowledge in the Discussion that pulse emissions might have been missed, which is a common limitation in manual chamber methods.

- Results:

- Ensure that the statistical letters (a, b, c) on bar charts clearly match the description in the text.

-Discussion:

-The discussion on the mechanism of N2O reduction is good. The link between reduced ammonium pools and reduced nirS/nirK abundance is a strong argument.

-Section 4.5: The Implications section is well-written. I suggest adding a brief sentence on the practical cost-benefit for farmers. Is the TPr mixture significantly more expensive or labor-intensive to maintain than clean tillage?

3. Technical and Language Corrections

- English Language: The English is generally understandable, but there are several awkward phrasings and minor grammatical errors throughout the text. I recommend an English final proofread once again.

- Typos:

- There are hyphenation issues (likely due to formatting), e.g., "Ni-2 trogen," "de-90 composed." Please ensure the final clean version does not have these line-break artifacts.

- "htTPr" in the header seems to be a typo for "http".

Conclusion This is a solid study that contributes to sustainable orchard management. I recommend publication after the authors address the methodological clarification regarding the genetic analysis and polish the language.

 

Comments for author File: Comments.pdf

Author Response

Reviewer 1

Comments 1: - Abstract: The abstract is informative and covers the main findings well. However, please ensure that the specific reduction percentages for N₂O are consistent with the results section.

Response 1: Thanks for your comment. We have checked the data between abstract section (page 1 lines 29 to 30) and result section (page 7 lines 261 to 264), and ensured the reduction percentages of N₂O are consistent.

 

Comments 2: - Materials and Methods:- Section 2.3.3: There is a slight confusion in the text regarding the genetic analysis. The Introduction mentions "qPCR and 16S rRNA sequencing," but Section 2.3.3 is titled "Soil metagenomic sequencing." Please clarify if you used qPCR for absolute quantification of specific genes (like nosZ, nirS) or if these abundances were derived solely from metagenomic data. If qPCR was used, the primer sequences and thermal cycling conditions should be briefly mentioned or cited.

Response 2: Thank you for pointing this out. We agree with this comment. We used metagenomic data for absolute quantification of specific genes (like nosZ, nirS). Therefore, we have changed “identify microbial drivers (via qPCR and 16S rRNA sequencing) of N₂O production pathways (nitrification, denitrification) and their interaction with soil physicochemistry” to “to investigate the microbial drivers of N₂O production pathways (nitrification, denitrification) and their interaction with soil physicochemistry using metagenomic sequencing” in introduction section (page 2 lines 80 to 82) and provided more information concerning metagenomic sequencing in section 2.3.3 (pages 5 lines 167 to 189).

 

Comments 3: - Materials and Methods: - N2O Sampling: Sampling was done "2 or 3 times every month." Did you perform additional sampling immediately after fertilization or heavy rainfall events? If not, please acknowledge in the Discussion that pulse emissions might have been missed, which is a common limitation in manual chamber methods.

Response 3: Agree. We have, accordingly, revised as “Measurement of [N₂O] was conducted 2 to 3 times monthly, with supplementary sampling performed promptly following fertilization and intense rainfall events.” (page 4 lines 158 to 159) to emphasize this point.

 

Comments 4: - Results:- Ensure that the statistical letters (a, b, c) on bar charts clearly match the description in the text.

Response 4: Agree. We have checked and revised 3.1 section(pages 5 to 7 lines 198 to 237) and ensured that the statistical letters (a, b, c) on bar charts clearly match the description in the text.

 

Comments 5: -Discussion:

-The discussion on the mechanism of N2O reduction is good. The link between reduced ammonium pools and reduced nirS/nirK abundance is a strong argument.

-Section 4.5: The Implications section is well-written. I suggest adding a brief sentence on the practical cost-benefit for farmers. Is the TPr mixture significantly more expensive or labor-intensive to maintain than clean tillage?

Response 5: Thank you for pointing this out. We agree with this comment. Therefore, we have added a brief sentence on the labor-intensive maintenance of TPr mixture (page 13 lines 475 to 476).

 

Comments 6: Technical and Language Corrections

- English Language: The English is generally understandable, but there are several awkward phrasings and minor grammatical errors throughout the text. I recommend an English final proofread once again.

Response 6: Thank you for pointing this out. We have proofread and revised the phrases and grammatical errors.

 

Comments 7: - Typos:

- There are hyphenation issues (likely due to formatting), e.g., "Ni-2 trogen," "de-90 composed." Please ensure the final clean version does not have these line-break artifacts.

- "htTPr" in the header seems to be a typo for "http".

Response 7:  Thank you for pointing this out. We have revised these typos.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript Number: agronomy- 3978337

Title: Effects of Different Sod-Seeding Patterns on Soil Properties, Nitrogen Cycle Genes, and N₂O Mitigation in Peach Orchards

 

Comment Summary:

            This manuscript presents valuable work on how different sod-seeding patterns (Tr, Pr, TPr) influence soil physicochemical properties, N-cycle genes, and N₂O emissions in peach orchards. The study is original, relevant to sustainable orchard management, and supported by substantial field and metagenomic data. However, several areas need clearer presentation, better methodological transparency, stronger statistical interpretation, and careful refinement of the narrative to avoid over-interpretation.  However, some important information for result and discussion are missing as detailed below.

 

1. Manuscript may be further improved by considering to include and discuss more on the following issues;
   • Introduction should be conducted to highlight the research gaps and the novel in your research. Concepts are repeated (e.g., need to study sod-seeding effects in Asian orchards). In addition, the objectives of the study are not clearly distinguished from background.

1. Results and Discussion: Please explain the sequencing procedures in accordance with standard boilerplate descriptions. In particular, clarify which thresholds, databases, and functional gene annotation pipelines were used to identify nitrogen cycling genes. In addition, some nutrient-related results require clearer justification. For example:

• Why does the Pr treatment substantially increase NH₄⁺–N?
• Why does the Tr treatment lead to a decrease in SON?

                        Providing mechanistic explanations or supporting references for these patterns will improve the clarity and scientific rigor of the Results and Discussion section.

1. The quality of some Figures is not similarly scale.

• Figure 2: combines too many variables; separate panels or grouping by categories would improve readability.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

Comments 1:Manuscript may be further improved by considering to include and discuss more on the following issues;

Introduction should be conducted to highlight the research gaps and the novel in your research. Concepts are repeated (e.g., need to study sod-seeding effects in Asian orchards). In addition, the objectives of the study are not clearly distinguished from background.

Response 1: Thanks for your helpful suggestion. We have modified the introduction section by highlighting the research gaps and the novel in this research (page 2 lines 56 to 62 and lines.82 to 85), by removing the repeated concepts (page 2 line 56), and by giving the objectives of the study clearly from back ground (page 2 lines 79 to 82).

 

Comments 2:Results and Discussion: Please explain the sequencing procedures in accordance with standard boilerplate descriptions. In particular, clarify which thresholds, databases, and functional gene annotation pipelines were used to identify nitrogen cycling genes.

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have provided detailed information about sequencing procedures in page 5 lines 167 to 189.

 

Comments 3:In addition, some nutrient-related results require clearer justification. For example:

Why does the Pr treatment substantially increase NH₄⁺–N?

Why does the Tr treatment lead to a decrease in SON?

                        Providing mechanistic explanations or supporting references for these patterns will improve the clarity and scientific rigor of the Results and Discussion section.

Response 3: Thanks for your suggestion. We have provided explanation and supporting reference for increase of NH₄⁺-N in Pr treatment in page 11 lines 348 to 351. Decrease in SON occurred in Pr treatment but not in Tr treatment (page 6 figure 2h and page 6 lines 227 to 228).

 

Comments 4:The quality of some Figures is not similarly scale.

Figure 2: combines too many variables; separate panels or grouping by categories would improve readability.

Response 4: Agree. We have, accordingly, separated Figure 2 into 3 groups, Panel A soil physicochemical indices, Panel B soil nutrient indices and Panel C soil enzymatic activities (page 6 lines 202 to 210) to improve readability.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Overall rating

The paper has significant publication potential and provides valuable findings, but requires technical and stylistic refinement, as well as focusing on key scientific messages. The topic is very current and scientifically significant — N₂O emissions and sustainable management of orchards. The experimental design is straightforward, with multiple treatments and replicates. Combining physico-chemical analyses, enzyme activities, metagenomics and structural modeling is a great methodological plus. The integration of sod-seeding systems with N₂O mitigation is relevant and relatively poorly investigated for Asian orchards, which gives this study its importance. Metagenomics + SEM add special value. A strong argument is given about N₂O emissions and problems in Chinese orchards and a good reference base is given. The results are largely consistent and clearly demonstrate the superiority of TPr treatment.

Disadvantages:

The hypothesis could have been more clearly indicated. The wording proposal can be as follows: "We hypothesized that the leguminous-grass mixture (TPr) will most improve soil properties and reduce N₂O emissions due to complementary nitrogen nutrition and balanced microbial activity."

The results are clear, but special attention should be paid to the discussion, which is too long and repeats the results, rather than analyzing the mechanisms deeply enough.

The SEM model is not sufficiently explained or visually optimally interpreted.

The link between functional genes and specific microbiological processes could be explained more precisely.

Treatments should be tabulated (TPr, Tr, Pr, CK—with seed amounts).

There is a lack of information on the number of samples per treatment for metagenomics and the soil sampling season.

The methodology for N₂O measurement is missing: soil temperature, WFPS assessment, standardization of the time of day when the measurements were made.

Sentences like "In summary, sod-seeding patterns comprehensively improved soil quality", as well as explanations of mechanisms, should be transferred to the discussion.

A stronger focus is needed on two key relationships:

NH₄⁺ → N₂O

nirK/nirS → N₂O

The conclusion clearly summarizes the results and confirms the importance of TPr treatment, but lacks recommendations for practical production (eg optimal seed quantities or expected benefits per hectare).

Comments for author File: Comments.pdf

Author Response

Reviewer 3

Comments 1:The hypothesis could have been more clearly indicated. The wording proposal can be as follows: "We hypothesized that the leguminous-grass mixture (TPr) will most improve soil properties and reduce N₂O emissions due to complementary nitrogen nutrition and balanced microbial activity."

Response 1: Agree. We have, accordingly, added “Our hypothesis is that the leguminous-grass mixture (TPr) will enhance soil properties and decrease N₂O emissions by providing complementary nitrogen nutrition and promoting balanced microbial activity.” in page 2 lines 82 to 85.

 

Comments 2:The results are clear, but special attention should be paid to the discussion, which is too long and repeats the results, rather than analyzing the mechanisms deeply enough.

Response 2: Thank you for pointing this out. We agree with this comment. Therefore, we have analyzed the mechnisms more deeply (page 11 lines 348 to 351 and page 12 lines 391 to 421).

 

Comments 3:The SEM model is not sufficiently explained or visually optimally interpreted.

Response 3: Thanks for your suggestion. We have explained the SEM model more sufficiently in page 10 lines 325 to 327.

 

Comments 4:The link between functional genes and specific microbiological processes could be explained more precisely.

Response 4: Agree. We have, accordingly, explained more precisely between functional genes and specific microbiological processes in page 12 lines 391 to 421.

 

Comments 5:Treatments should be tabulated (TPr, Tr, Pr, CK—with seed amounts).

Response 5: Agree. We have, accordingly, added a table (page 3 line 114 Table 1).

 

Comments 6:There is a lack of information on the number of samples per treatment for metagenomics and the soil sampling season.

Response 6: Thanks for your suggestion. We have provided information on the number of samples per treatment for metagenomics and the soil sampling season. “Surface soil samples were collected from experimental plots at a depth of 0–10 cm, with 3 replicates per treatment. The samples were obtained using a sterilized auger, homogenized, and then stored at -80°C until DNA extraction in August.” (page 4 lines 163 to 166)

 

Comments 7:The methodology for N₂O measurement is missing: soil temperature, WFPS assessment, standardization of the time of day when the measurements were made.

Response 7: Thanks for your suggestion. We have provided the information of soil temperature, WFPS assessment, standardization of the time of day when the measurements were made (page 4 lines 147 to 150).

 

Comments 8:Sentences like "In summary, sod-seeding patterns comprehensively improved soil quality", as well as explanations of mechanisms, should be transferred to the discussion.

Response 8: Agree. We have, accordingly, transferred sentences “In summary, sod-seeding patterns comprehensively improved soil quality through optimizing physicochemical properties, nutrient supply and enzyme activity. The soil quality improvement underscored the dominance of TPr in enzyme kinetics and nutrient mobilization and the ascendancy of Pr in in organic matter retention and pH moderation.” to the discussion section (page 11 lines 369 to 373).

 

Comments 9:A stronger focus is needed on two key relationships:

 

NH₄⁺ → N₂O

 

nirK/nirS → N₂O

Response 9: Agree. We have, accordingly, emphasized key relationships from NH₄⁺ to N₂O and from nirK/nirS to N₂O in section 4.3 (page 12 lines 389 to 429).

 

Comments 10:The conclusion clearly summarizes the results and confirms the importance of TPr treatment, but lacks recommendations for practical production (eg optimal seed quantities or expected benefits per hectare).

Response 10: Agree. We have, accordingly, added the recommendation about seed quantities for practical production in page 14 lines 496 to 497. “The recommended seed amounts for Trifolium repens and Lolium perenne in TPr pattern are 0.5 and 1 kg·667 m^-2, respectively”

Author Response File: Author Response.pdf