Review Reports - Phosphorus Input Threshold Drives the Synergistic Shift of Microbial Assembly and Phosphorus Speciation to Sustain Maize Productivity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Suggestions for the Authors
This is an important study that integrates a long-term field experiment with microbial ecology and data analysis. The work is relevant to the field, and its core—a 12-year phosphorus (P) gradient experiment—is the strength of the work. The manuscript has the potential to be an important contribution to Agronomy. The following suggestions aim to improve the manuscript so that this potential is achieved, focusing on strengthening the statistical analysis and balancing the interpretation of the results.

Introduction
The introduction presents the problem and hypotheses well, starting from the global "P dilemma" to the specific knowledge gap in purple soils. To improve the manuscript, I suggest two main improvements:
Expand the Literature Review: The current review is based on broad, globally scalable articles. I suggest incorporating more recent mechanistic studies on microbial P mobilization, particularly those from purple soils or similar subtropical systems. This will better contextualize your findings and reinforce the novelty claim.
Contextualizing the Threshold: It would be helpful to briefly discuss existing studies on the phosphorus (P) threshold in other subtropical regions. Comparing your findings with these studies will more clearly highlight the specific contribution of your work to purple soils.
Materials and Methods
The long-term experimental design is a differentiating part of your work, and the bioinformatic workflow is up-to-date. To improve clarity and rigor, consider the following:
Experimental Details: Could you clarify whether buffer strips or other methods were used to control edge effects in the plots? Also, since potassium (K) analysis was performed in the soil, a brief discussion of K dynamics would be important, as K can sometimes influence phosphorus responses in these soils.
Statistical Analysis: This is the area that requires the most attention.
ANOVA: I suggest replacing the LSD test with a more conservative post-hoc test, such as Tukey's HSD, to better control multiple comparisons. Although it may not be necessary to present all normality and homogeneity tests, a statement confirming that these assumptions have been met would reinforce confidence in the results, especially considering the minimum replication (n=3).
Structural Equation Modeling (SEM): The complexity of the SEM is ambitious for a sample size of n=15. As it stands, the model is likely overfitted. I suggest you reframe this as an exploratory path analysis, rather than a confirmatory SEM. You could also consider:
Simplifying the Model: Creating composite variables (e.g., a single "Labile P" variable from a PCA of its components) to reduce the number of paths to be estimated.
Avoiding the use of tools less sensitive to small sample sizes, such as TLI and SRMR. Another possibility is Bootstrapping: This technique would provide more realistic confidence intervals for the path coefficients, helping to distinguish stable relationships from potentially spurious relationships.
Network Analysis:
A simple correlation threshold (|ρ| > 0.7) can create non-true links. I would recommend using methods designed to infer direct interactions from compositional data, such as SPIEC-EASI (preferred) or SparCC. If it is essential to maintain Spearman's correlation, validate it with a null model approach to ensure that the connections are stronger than expected by chance.

Results
The figures and tables are clear and illuminating, especially the dose-response plots. Some points could improve clarity: Exercise caution in interpreting small numerical differences that may not be statistically significant (e.g., the small change in the bacterial Shannon index).

The yield plateau or grain yield is a key finding. It would be helpful to include confidence intervals around these values to clarify that they lie within the experimental margin of error (suggestion).

Discussion
The discussion integrates the complex datasets well. To make it even more compelling:

I suggest moderating the language around causality. For example, stating that Gemmatimonadetes "play essential functional roles" is too strong without direct evidence of functional gene expression. Framing this as a strong association or a hypothesis for future work would be more appropriate (suggestion).

The discussion of limitations should be expanded. Acknowledging the limitations of the small sample size and microbial sampling at a single point does not weaken the article; On the contrary, it demonstrates scientific transparency and honesty with the reader.
The discussion would greatly benefit from comparing its proposed threshold of 84 kg ha⁻¹ with those identified in other regions and from a brief mention of how alternative strategies, such as crop rotation, or the use of phosphate-solubilizing microbial inoculants, fit into this scenario.

Conclusion: The conclusion is concise and provides a clear recommendation. We suggest moderating the certainty somewhat to reflect that the findings are from a single location. A brief mention of the need for future validation in different soil types or crop rotations would also be a good measure in closing the conclusion.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The English is generally understandable, and the manuscript can be followed without major difficulties. However, the text would benefit from careful linguistic revision.

Many sentences are too long, with multiple clauses and nested ideas. Dividing them into shorter sentences would greatly improve readability.

There are some grammatical problems (verb tense changes, subject-verb agreement) and poorly constructed sentences that could be corrected by a native English speaker or a professional editor.

Some obvious typographical and editing errors have already been pointed out in the appendix.

Abbreviations are occasionally used before being clearly defined in the text or figure captions; these have not been pointed out in the appendix.

I recommend a complete linguistic and stylistic revision, focusing on simplifying sentence structure, correcting typographical errors, and ensuring consistency in terminology and units.

Author Response

Response to Reviewer’s Comments

Manuscript number：agronomy-4008116

Title：Phosphorus input threshold drives the synergistic shift of microbial

assembly and phosphorus speciation to sustain maize productivity

Dear Editors and Reviewers：

We sincerely appreciate the opportunity to revise our manuscript entitled "Phosphorus input threshold drives the synergistic shift of microbial assembly and phosphorus speciation to sustain maize productivity" and are grateful for the insightful comments provided by the reviewers. These comments have been invaluable in refining and improving our paper, and they also hold significant guiding meaning for our research. In the following sections, we have provided detailed responses to each of the reviewers' comments. Additionally, we have conducted a comprehensive revision of the entire manuscript. In this response letter, the reviewers' comments are presented in italics, while our corresponding revisions and additions to the manuscript are highlighted in red text. We have made every effort to clearly document all modifications and hope that the revised manuscript meets the standards for publication.

Reviewer 1

Abstract

Introduction

The introduction presents the problem and hypotheses well, starting from the global "P dilemma" to the specific knowledge gap in purple soils. To improve the manuscript, I suggest two main improvements:

Comment 1: Expand the Literature Response eview: The current review is based on broad, globally scalable articles. I suggest incorporating more recent mechanistic studies on microbial P mobilization, particularly those from purple soils or similar subtropical systems. This will better contextualize your findings and reinforce the novelty claim.
Response 1: We sincerely thank the reviewer for this valuable suggestion. We fully agree that incorporating recent mechanistic studies on microbial P mobilization from specific systems like purple soils will significantly strengthen the background and novelty of our work.

Location: Line 47-50

Comment 2: Contextualizing the Threshold: It would be helpful to briefly discuss existing studies on the phosphorus (P) threshold in other subtropical regions. Comparing your findings with these studies will more clearly highlight the specific contribution of your work to purple soils.

Response 2: Following the reviewer's advice, we have now substantially revised the Introduction and Literature Review sections to include a more focused discussion on microbial mechanisms in subtropical systems, with particular emphasis on purple soils.

Location: Line 50-52

Comment 3: Here the authors present the hypotheses. It would be interesting to conclude by stating the objective of the work.

Response 3: We thank the reviewer for this valuable suggestion. As recommended, we have now added a clear statement of the research objectives immediately following the presentation of our hypotheses in the introduction section.

Location: Line 112-117

Materials and Methods

The long-term experimental design is a differentiating part of your work, and the bioinformatic workflow is up-to-date. To improve clarity and rigor, consider the following:

Comment 4: Experimental Details: Could you clarify whether buffer strips or other methods were used to control edge effects in the plots? Also, since potassium (K) analysis was performed in the soil, a brief discussion of K dynamics would be important, as K can sometimes influence phosphorus responses in these soils.
Response 4: We thank the reviewer for the valuable suggestions. We have revised the manuscript accordingly:The use of protective borders to control edge effects has been clarified in the Materials and Methods section.

Location: Line 161-162

Comment 5: ANOVA: I suggest replacing the LSD test with a more conservative post-hoc test, such as Tukey's HSD, to better control multiple comparisons. Although it may not be necessary to present all normality and homogeneity tests, a statement confirming that these assumptions have been met would reinforce confidence in the results, especially considering the minimum replication (n=3).

Response 5: We thank the reviewer for the valuable suggestions. We have changed the LSD test to the Duncan test.

Location: Line 245-248

Comment 6: Structural Equation Modeling (SEM): The complexity of the SEM is ambitious for a sample size of n=15. As it stands, the model is likely overfitted. I suggest you reframe this as an exploratory path analysis, rather than a confirmatory SEM. You could also consider:

Simplifying the Model: Creating composite variables (e.g., a single "Labile P" variable from a PCA of its components) to reduce the number of paths to be estimated.

Avoiding the use of tools less sensitive to small sample sizes, such as TLI and SRMR. Another possibility is Bootstrapping: This technique would provide more realistic confidence intervals for the path coefficients, helping to distinguish stable relationships from potentially spurious relationships.

Response 6: We sincerely thank the reviewers for this important opinion on the sample size in the structural equation model. We have carefully considered the relevant recommendations and implemented key improvements : we redefined the analysis method as exploratory path analysis, rather than a confirmatory structural equation model. Although we have tried the proposed compound variable construction method, the current model structure best reflects the theoretical framework and biological processes studied. The re-sampling results show that the path coefficient is stable, which supports the robustness of the research conclusion under the experimental constraints.

Location: Line 262

Comment 7: Network Analysis:

A simple correlation threshold (|ρ| > 0.7) can create non-true links. I would recommend using methods designed to infer direct interactions from compositional data, such as SPIEC-EASI (preferred) or SparCC. If it is essential to maintain Spearman's correlation, validate it with a null model approach to ensure that the connections are stronger than expected by chance.

Response 7: We are sincerely grateful to the reviewer for raising this crucial point regarding the limitations of inferring microbial coexistence networks using simple correlation thresholds. We fully acknowledge that methods such as SPIEC-EASI or SparCC exhibit greater statistical robustness when handling compositional data, as they are specifically designed to infer direct interactions and minimise spurious correlations.

Within the specific context of this study, microbial network analysis serves as an exploratory tool to visualise the complexity of large-scale communities under varying phosphorus environments, rather than making robust causal inferences about specific microbial interactions. Whilst acknowledging that recommended methods are better suited to the latter, we contend that the conservative Spearman correlation threshold employed herein (|ρ| > 0.7), coupled with its widespread application and interpretability within the field, still provides an effective and reproducible preliminary overview for the present objectives.

Nevertheless, we place great value on the reviewer's valuable suggestions. We are confident these recommendations will significantly enhance the quality of future research and sincerely thank the reviewer for their insightful guidance on this important methodological issue.

Result

Comment 8: The figures and tables are clear and illuminating, especially the dose-response plots. Some points could improve clarity: Exercise caution in interpreting small numerical differences that may not be statistically significant (e.g., the small change in the bacterial Shannon index).

Response 8: We thank the reviewer for this reasonable opinion. We agree that the original expression may have over-interpreted the small numerical difference, and the sentence has been revised to weaken the conclusion.

Location: Line 341-344

Comment 9: The yield plateau or grain yield is a key finding. It would be helpful to include confidence intervals around these values to clarify that they lie within the experimental margin of error (suggestion).

Response 9: We thank the reviewers for their valuable suggestions. The confidence interval has been added to the yield data and clearly marked in the corresponding graph.

Location: Line 451

Discussion

The discussion integrates the complex datasets well. To make it even more compelling:

Comment 10: I suggest moderating the language around causality. For example, stating that Gemmatimonadetes "play essential functional roles" is too strong without direct evidence of functional gene expression. Framing this as a strong association or a hypothesis for future work would be more appropriate (suggestion).

Response 10: We thank the reviewer for this precise and constructive suggestion. We agree that our initial language overstepped in implying direct causality from compositional data. We have now moderated the language throughout the relevant section as suggested, framing the relationship as a strong association and presenting the functional role of specific taxa like Gemmatimonadetes as a compelling hypothesis for future work, rather than a confirmed conclusion. We believe this revision significantly improves the accuracy and appropriateness of our interpretation.

Location: Line 594-597

Comment 11: The discussion of limitations should be expanded. Acknowledging the limitations of the small sample size and microbial sampling at a single point does not weaken the article; On the contrary, it demonstrates scientific transparency and honesty with the reader.

Response 11: We thank the reviewer for prompting a more comprehensive discussion of the limitations. In response, we have added a concise statement to the “Limitations” section that explicitly acknowledges the constraints related to sample size and single time-point microbial sampling, as suggested.

Location: Line 640-643

Comment 12: The discussion would greatly benefit from comparing its proposed threshold of 84 kg ha⁻¹ with those identified in other regions and from a brief mention of how alternative strategies, such as crop rotation, or the use of phosphate-solubilizing microbial inoculants, fit into this scenario.

Response 12: We thank the reviewers for their valuable suggestions. Based on your comments, we have now revised the discussion section to directly compare the phosphate fertilizer thresholds we have determined with those reported in other regions, with special reference to other relevant issues. In addition, we more clearly described the role of legume-maize rotation and phosphate-solubilizing microbial inoculants in optimizing phosphate fertilizer application around the proposed threshold.

Location: Line 628-631

Conclusion:

Comment 13: The conclusion is concise and provides a clear recommendation. We suggest moderating the certainty somewhat to reflect that the findings are from a single location. A brief mention of the need for future validation in different soil types or crop rotations would also be a good measure in closing the conclusion.

Response 13: We sincerely thank the reviewer for this constructive suggestion. We have now revised the conclusion accordingly. Specifically, we have toned down the language by using more tentative terms (e.g., "tentatively," "appears to," "approximately") to reflect the site-specific nature of our study. Furthermore, we have added a final sentence to explicitly state the need for future validation across different soil types, climates, and cropping systems. We believe these changes have improved the precision and balance of our conclusion.

Location: Line 647-665

Comment 14: The English is generally understandable, and the manuscript can be followed without major difficulties. However, the text would benefit from careful linguistic revision. Many sentences are too long, with multiple clauses and nested ideas. Dividing them into shorter sentences would greatly improve readability. There are some grammatical problems (verb tense changes, subject-verb agreement) and poorly constructed sentences that could be corrected by a native English speaker or a professional editor.

Response 14: We thank the reviewers for their positive evaluation of the readability of the manuscript and constructive suggestions for English expression. We fully agree that improving readability is crucial. In response to this opinion, we have commissioned English-speaking professional experts to edit the full text, focusing on correcting long sentence structure, grammatical errors, tense consistency and sentence construction.

Comment 15: Some obvious typographical and editing errors have already been pointed out in the appendix.

Response 15: We thank the reviewers for their comments and have amended the appendix as requested.

Comment 16: Abbreviations are occasionally used before being clearly defined in the text or figure captions; these have not been pointed out in the appendix.

Response 16: We thank the reviewers for their comments and have included definitions of abbreviations in the appendix.

Location: Line 693-695

Comment 17: I recommend a complete linguistic and stylistic revision, focusing on simplifying sentence structure, correcting typographical errors, and ensuring consistency in terminology and units.

Response 17: We thank the reviewer for this critical assessment. In direct response to this comment, the entire manuscript has undergone a comprehensive linguistic and stylistic revision by a professional editing service.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Review
Phosphorus input threshold drives the synergistic shift of mi-crobial assembly and phosphorus speciation to sustain maize productivity

The introduction clearly presents the role of phosphorus in agriculture, but it would benefit from a more coherent structure. The main idea, identifying an optimal threshold for phosphorus application appears only toward the end and should be introduced earlier.
There are repetitions regarding phosphorus solubility and mobility, which could be condensed into one concise sentence. The transition to the applied case of “purple soil” is abrupt and lacks a logical connection. A clearer transition and an explicit formulation of the scientific problem are needed: what is not yet known and what the study aims to clarify.

A specific citation error appears in the Introduction (‘Error! Reference source not found.’). This indicates a missing or broken reference and should be corrected before publication to ensure manuscript consistency and technical accuracy.

Materials and Methods
2.1. Experimental Site Description and Design
The location is clearly described, with coordinates, soil type and climatic context. The information is useful, but it would be appropriate to include a sentence explaining the rationale for choosing this area its direct link with the scientific problem addressed in the study.
2.2. Soil Sampling and Analysis
The randomized design is correctly presented, with replications and fertilization levels.
However, the section is quite dense. A schematic diagram, table, or visual summary of the treatments and applied doses would improve clarity.
2.3. Plant Sample Collection and Determination
The methods for soil analysis are standard but overly detailed and fragmented. A clearer grouping into physical–chemical analyses, biological analyses and phosphorus analyses would make the section easier to follow.
2.5. DNA Extraction, Illumina NovaSeq Sequencing, and Data Processing
The protocol is complete and well structured. It is recommended to justify the choice of the genetic markers (16S and ITS) and explain why genomic analysis is relevant to establishing the phosphorus threshold.
2.6. Co-occurrence Network Analysis of Bacteria and Fungi
An introductory sentence would be helpful to connect the network analysis to the study’s main objective. Network indicators (degree, centrality, etc.) should also be briefly defined for readers less familiar with these concepts.
Results
3.2. Response of phosphorus application to soil phosphorus classification
The term “transformation patterns and ecological risks” is used, but the ecological risks are not sufficiently explained. It would be useful to specify whether there is a risk of leaching, fixation or nutrient imbalance.
3.3. Alpha/beta-diversity and community structure of bacterial and fungal communities
The higher sensitivity of fungi is mentioned, but an ecological explanation is missing. What mechanism could explain this more pronounced response?
3.4. Effects of Phosphorus Application on Soil Microbial Network Complexity
It is stated that “network robustness” is highest at P75, but the criteria defining robustness for agricultural soils are not clearly explained.

Author Response

Response to Reviewer’s Comments

Manuscript number：agronomy-4008116

Title：Phosphorus input threshold drives the synergistic shift of microbial

assembly and phosphorus speciation to sustain maize productivity

Dear Editors and Reviewers：

Review 2

Comment 1: The introduction clearly presents the role of phosphorus in agriculture, but it would benefit from a more coherent structure. The main idea, identifying an optimal threshold for phosphorus application appears only toward the end and should be introduced earlier.

Response 1: We thank the reviewers for their constructive comments on the structure of the introduction. We agree that the core goal of determining the optimal threshold for phosphate fertilizer application is elaborated in advance, which can enhance the coherence of the narrative. We have modified the introduction.

Location: line 52-54

Comment 2: There are repetitions regarding phosphorus solubility and mobility, which could be condensed into one concise sentence. The transition to the applied case of “purple soil” is abrupt and lacks a logical connection. A clearer transition and an explicit formulation of the scientific problem are needed: what is not yet known and what the study aims to clarify.

Response 2: We thank the reviewer for these constructive suggestions. We have revised the introduction to condense repetitive information on P availability, improve the logical transition to the "purple soil" context, and more explicitly state the specific scientific gaps and research aims.

Location: line 58-70

Comment 3: A specific citation error appears in the Introduction (‘Error! Reference source not found.’). This indicates a missing or broken reference and should be corrected before publication to ensure manuscript consistency and technical accuracy.

Response 3: We are most grateful for the reviewer's suggestions and have now revised the literature citations in the introduction accordingly.

Location: line 36-37

Materials and Methods
Comment 4: 2.1. Experimental Site Description and Design
The location is clearly described, with coordinates, soil type and climatic context. The information is useful, but it would be appropriate to include a sentence explaining the rationale for choosing this area its direct link with the scientific problem addressed in the study.

Response 4: We thank the reviewer for this suggestion. We have now added a sentence to explicitly state the rationale for selecting the Ya'an Experimental Station, highlighting that its representative purple soil and farming system make it an ideal model for studying the core scientific questions of this research.

Location: line 123-127

Comment 5: 2.2. Soil Sampling and Analysis
The randomized design is correctly presented, with replications and fertilization levels. However, the section is quite dense. A schematic diagram, table, or visual summary of the treatments and applied doses would improve clarity.

Response 5: We are most grateful for the reviewer's comments. We have now visualised the experimental design in tabular form and included it in the supplementary materials.

Comment 6:2.3. Plant Sample Collection and Determination
The methods for soil analysis are standard but overly detailed and fragmented. A clearer grouping into physical–chemical analyses, biological analyses and phosphorus analyses would make the section easier to follow.

Response 6:We would like to express our gratitude to the reviewers for their valuable suggestions and have made the necessary revisions in accordance with their requirements.

Location: line 153-176

Comment 7:2.5. DNA Extraction, Illumina NovaSeq Sequencing, and Data Processing
The protocol is complete and well structured. It is recommended to justify the choice of the genetic markers (16S and ITS) and explain why genomic analysis is relevant to establishing the phosphorus threshold.

Response 7: We thank the reviewer for the suggestion. We have now briefly clarified the rationale for selecting 16S/ITS markers and the relevance of genomic analysis to P threshold determination in the revised 'Materials and Methods' section.

Location: line 213-215 and line 222-224

Comment 8: 2.6. Co-occurrence Network Analysis of Bacteria and Fungi
An introductory sentence would be helpful to connect the network analysis to the study’s main objective. Network indicators (degree, centrality, etc.) should also be briefly defined for readers less familiar with these concepts.

Response 8: We thank the reviewer for this constructive suggestion. We have now added an introductory sentence at the beginning of the network analysis section to clearly state its purpose in relation to our research objectives. Furthermore, we have included concise explanations for the key global and node-level network metrics to improve accessibility for a broader readership.

Location: line 242-247

Results
Comment 9: 3.2. Response of phosphorus application to soil phosphorus classification
The term “transformation patterns and ecological risks” is used, but the ecological risks are not sufficiently explained. It would be useful to specify whether there is a risk of leaching, fixation or nutrient imbalance.

Response 9: We thank the reviewer for this valuable suggestion. We have now revised the results section to explicitly articulate the specific ecological risks implied by the data.

Location: line 311-318

Comment 10: 3.3. Alpha/beta-diversity and community structure of bacterial and fungal communities
The higher sensitivity of fungi is mentioned, but an ecological explanation is missing. What mechanism could explain this more pronounced response?

Response 10: We thank the reviewer for raising this important point. We have now revised the results section to include a potential ecological mechanism explaining the observed greater sensitivity of fungal communities. Specifically, we suggest that their complex hyphal morphology and narrower niche breadth may make them more vulnerable to disruption from P-induced changes in the soil environment (e.g., pH shifts) compared to bacteria.

Location: line 350-353

Comment 11:3.4. Effects of Phosphorus Application on Soil Microbial Network Complexity
It is stated that “network robustness” is highest at P75, but the criteria defining robustness for agricultural soils are not clearly explained.

Response 11: We thank the reviewer for this insightful comment. We have now clarified the definition of "network robustness" in the agricultural context within the revised manuscript. Specifically, we have elaborated that the topology observed at P75—featuring streamlined connections coupled with high modularity and path length—is theorized to enhance the microbial system's ability to resist environmental disturbances and maintain functional stability, thereby contributing to sustained soil health.

Location: line 401-404

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors,

Please make the corrections to the formatting of the numbers in the brackets.

Comments on the Quality of English Language

Dear authors,

Please check if any corrections are needed in the English.