Enhanced Formation and Stability of Water-Stable Aggregates in Rhizosphere Soil over Bulk Soil with Exopolysaccharide from Rhizobium tropici: Insights from a Pot Study

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Comments and Suggestions for Authors

 

Title: Enhanced Formation and Stability of Water-stable Aggregates in
Rhizosphere Soil over Bulk Soil with Exopolysaccharide from Rhizobium
Tropici: Insights from A Pot Study

Dear Authors and Editors

The content of the manuscript falls within the publishing profile of Agronomy journal.

The topic of the manuscript is original and relevant to the field of agricultural sciences.

The presented research topic and the obtained results are interesting from a scientific perspective. It is a pity that the research was not conducted in natural field conditions (on microplots). The results of such studies could have been applied in agricultural practice.

The research objective and research hypothesis are well presented. The research results are well documented and clearly presented in figures. The Discussion section was well presented. The conclusions are accurate but very general and require correction. Thematic references are used appropriately.

In order to increase the usefulness of the article, Authors must refer to the following points. Additions should be made to increase the scientific value of the manuscript.

 

Comments

• Materials and Methods: Subsection 2.1. Line 107 - For statistical correctness of the experimental design, the authors should also include an EPS concentration of 0.75‰.
• Results: Subsection 3.2. Lines 295-303 and Figure 8 - Please check the significance of the differences in the fractal dimensions of water-stable aggregates in the rhizosphere and in bulk soil (outside the rhizosphere) at different EPS concentrations. In my opinion, the differences obtained in the results at the levels of 3.85 - 3.90 - 3.95 - 4.00 are not significant.
• Conclusions: The conclusions need to be improved. This section should summarize the results of our own research and recommend optimal EPS concentrations for rhizosphere and non-rhizosphere soils. Directions for further research are presented correctly.

Best regards

Author Response

Reviewer 1:

The content of the manuscript falls within the publishing profile of Agronomy journal. The topic of the manuscript is original and relevant to the field of agricultural sciences. The presented research topic and the obtained results are interesting from a scientific perspective. It is a pity that the research was not conducted in natural field conditions (on microplots). The results of such studies could have been applied in agricultural practice. The research objective and research hypothesis are well presented. The research results are well documented and clearly presented in figures. The Discussion section was well presented. The conclusions are accurate but very general and require correction. Thematic references are used appropriately. In order to increase the usefulness of the article, Authors must refer to the following points. Additions should be made to increase the scientific value of the manuscript.

Response: Thank you for the positive assessment of our manuscript and for recognizing the relevance, originality, and overall scientific value of our work. We acknowledge the limitation regarding the lack of natural field (micro-plot) experiments and have clarified this point in the revised version, also indicating it as an important direction for future research to enhance practical applicability. In addition, all suggested improvements—including refining the Conclusions section and strengthening the scientific depth—have been carefully addressed to improve the overall quality of the manuscript.

1. Materials and Methods: Subsection 2.1. Line 107 - For statistical correctness of the experimental design, the authors should also include an EPS concentration of 0.75‰.

Response: We sincerely thank the reviewer for this valuable suggestion. The EPS concentrations used in our study (0.00‰, 0.25‰, 0.50‰, and 1.00‰) were selected following the experimental framework of Xiao et al. (2025), who examined Rhizobium tropici-derived EPS effects on Zea mays seedlings. This range reflects low, medium, and high EPS levels commonly applied in similar studies. We fully agree that including an intermediate concentration such as 0.75‰ could further refine the dose–response pattern. However, the experimental design was developed with consideration of overall study scale, available materials, and resource constraints. Within these conditions, the selected four-level gradient ensured sufficient statistical resolution to capture the major EPS-induced trends in soil aggregation and plant performance. Thus, we believe the current design remains scientifically sound and appropriate for the study objectives.

2. Results: Subsection 3.2. Lines 295-303 and Figure 8 - Please check the significance of the differences in the fractal dimensions of water-stable aggregates in the rhizosphere and in bulk soil (outside the rhizosphere) at different EPS concentrations. In my opinion, the differences obtained in the results at the levels of 3.85 - 3.90 - 3.95 - 4.00 are not significant.

Response: We would like to clarify that our statistical comparison focused on the effect of different EPS concentrations within each soil type separately (rhizosphere soil vs. bulk soil). Although the numerical range appears narrow, fractal dimension (D) is a highly sensitive structural index, and the observed incremental decreases (4.00 → 3.85) represent meaningful EPS-induced changes in aggregate organization.

3. Conclusions: The conclusions need to be improved. This section should summarize the results of our own research and recommend optimal EPS concentrations for rhizosphere and non-rhizosphere soils. Directions for further research are presented correctly.

Response: We thank the reviewer for this constructive suggestion. In the revised Conclusions section, we provide a clearer and more targeted summary of our key findings and explicitly identify the optimal EPS concentrations for each soil type. Based on our results, EPS levels of 0.25‰–0.50‰ are most effective for improving aggregate stability in rhizosphere soil, whereas 0.25‰ is optimal for bulk (non-rhizosphere) soil. The Conclusions have also been refined to highlight the practical relevance of these findings while retaining the recommended directions for future research. The revised text can be found in Lines 545–557 of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

agronomy-3969001

The manuscript is interesting and well-conceived, presenting sufficient novelty. However, certain improvements are necessary before it can be considered for publication. The detailed comments are provided below.

The Introduction should be strengthened with more relevant references.

In general, the Results section is too detailed, with explanations that would fit better in the Discussion. I suggest focusing on presenting the data, and moving the interpretations to the Discussion section.

It is unnecessary to use both the full name and the abbreviation in the figure captions; please choose either the full name or the abbreviation and use it consistently throughout the manuscript.

References to figures in the Discussion section appear unnecessary, as the text already clearly indicates which results are being discussed. Furthermore, the Discussion section is rather brief and should be expanded by comparing the results with those of similar studies. In addition, the existing statements should be better supported with appropriate references. Finally, where possible, the authors should aim to relate the obtained results to enhance the interpretation.

 

Line 53 – use abbreviation since it’s already mentioned in previous line

Line 92 – no results should be presented in Materials and Methods section; describe how the physico-chemical properties of soil were determined and improve table readability in Results section

Lines 100, 101 – add instrument information (type, producer, country); please specify which reagent or buffer was used to maintain the pH level

Line 106 – please correct the typo

Line 109 – add reference for Zea mays optimal water condition

Lines 115-117 – the provided reference is not relevant since the soil samples were taken from another location; please explain how the amount of fertilizer was calculated

Line 170, 173, 176 – the parameter symbol referred to in the explanation is missing; explanations of equations in lines 173 and 176 are not clear

 Line 181-183 – please provide software producers data (producer, country); add information about post hoc test

Line 189 – please rephrase; according to the results in Figure 1, there was a significant difference between EPS treatment 1‰ (labeled with letter a), and other treatments (all labeled with letter b)

Lines 203-210 – results description in the text is not consistent with data presented in Figure 2. In addition, the letters for the rhizosphere soil in Figure 2 are incorrect. Please revise to ensure agreement between the text and figure

Lines 241-244 – please revise the text; treatment 0.5‰ in rhizosphere soil is significantly different from treatments 0.25 and 1. Considering that the bulk soil shows better results than the rhizosphere soil in this case, I suggest adding a sentence in the text to highlight this observation.

Line 299 – I suggest including similar information for the bulk soil as was provided for the rhizosphere soil, noting that the fractal dimensions decreased following the application of the treatments, or rephrase the paragraph

Line 351 – I suggest increasing the font size in Figure 10 for better readability; provide the meaning of ** in the figure

Line 484 – correct the reference

 

Author Response

Reviewer 2:

The manuscript is interesting and well-conceived, presenting sufficient novelty. However, certain improvements are necessary before it can be considered for publication. The detailed comments are provided below.

Response: We sincerely thank the reviewer for the positive and encouraging comments. We are pleased that the manuscript was considered interesting, novel, and well-conceived. In response to the reviewer’s valuable suggestions, we have carefully revised the manuscript to enhance its clarity, scientific rigor, and overall presentation.

All specific comments have been addressed point-by-point in the following sections. Revisions made in the manuscript are highlighted accordingly to facilitate review.

1. The Introduction should be strengthened with more relevant references.

Response: Thank you for the suggestion. We have added several relevant studies in the Introduction section. The new references are as follows:

Mi, W.; Chen, C.; Ma, Y.; Guo, S.; Liu, M.; Gao, Q.; Wu, Q.; Zhao, H. The Combined Application of Mineral Fertilizer and Organic Amendments Improved the Stability of Soil Water-Stable Aggregates and C and N Accumulation. Agronomy 2022, 12, 469.

Wang, S.; Wu, Q.S.; He, X.H. Exogenous easily extractable glomalin-related soil protein promotes soil aggregation, relevant soil enzyme activities and plant growth in trifoliate orange. Plant Soil Environ 2015, 61, 66–71.

Metuge, J.A.; Havugimana, E.; Rugandirababisha, J.; Senwo, Z.N.; Mutimawurugo, M.C. Evaluation of Rhizobium tropici-Derived Extracellular Polymeric Substances on Selected Soil Properties, Seed Germination, and Growth of Black-Eyed Peas (Vigna unguiculata). Agricultural Sciences 2024, 15, 548–564.

2. In general, the Results section is too detailed, with explanations that would fit better in the Discussion. I suggest focusing on presenting the data, and moving the interpretations to the Discussion section.

Response: Thank you for this helpful comment. We agree that the Results section should present data only, with interpretations placed in the Discussion. Accordingly, we have condensed the Results section to key findings and relocated the explanatory content to the Discussion to improve clarity and overall structure.

3. It is unnecessary to use both the full name and the abbreviation in the figure captions; please choose either the full name or the abbreviation and use it consistently throughout the manuscript.

Response: Thank you for this suggestion. We have revised all figure captions for consistency by using only the full term and removing redundant abbreviations. This change has been applied uniformly throughout the manuscript.

4. References to figures in the Discussion section appear unnecessary, as the text already clearly indicates which results are being discussed. Furthermore, the Discussion section is rather brief and should be expanded by comparing the results with those of similar studies. In addition, the existing statements should be better supported with appropriate references. Finally, where possible, the authors should aim to relate the obtained results to enhance the interpretation.

Response: Thank you for these constructive comments. We have thoroughly revised the Discussion section as suggested.

(1) All unnecessary figure references have been removed, as the corresponding results are already clearly indicated in the manuscript.

(2) The Discussion has been expanded with comparisons to relevant studies on microbial EPS, soil aggregation, and rhizosphere processes to provide stronger scientific context.

(3) Additional references (e.g., Chenu & Cosentino, 2011; Wang et al., 2015; Metuge et al., 2024; Mi et al., 2022) have been incorporated to better support the statements.

(4) The interpretation has been refined to more clearly relate our findings to existing knowledge and to better explain the mechanisms ESP-induced changes in soil structure.

5. Line 53 – use abbreviation since it’s already mentioned in previous line

Response: Thank you for this comment. Although the abbreviation appears in the abstract and keywords, this is the first occurrence of the term in the main manuscript. Following standard practice, we kept the full term “exopolysaccharides (EPS)” here and used “EPS” consistently throughout the rest of the manuscript.

6. Line 92 – no results should be presented in Materials and Methods section; describe how the physico-chemical properties of soil were determined and improve table readability in Results section

Response: We thank the reviewer for this helpful comment. We agree that numerical results should not appear in the Materials and Methods section. The manuscript has been revised to describe only the analytical procedures used to determine soil physico-chemical properties, while all numerical values and Table 1 have been moved to the beginning of the Results section under a new subsection (“3.1. Basic physico-chemical properties of the soil”). This revision ensures a clear separation between methods and results and improves overall readability.

7. Lines 100, 101 – add instrument information (type, producer, country); please specify which reagent or buffer was used to maintain the pH level

Response: Thank you for this valuable suggestion. We have added the requested instrument details and clarification in Section 2.1. Specifically, we now state that pH was monitored using a calibrated pH meter (SevenCompact S220, Mettler-Toledo, Switzerland), and that calibration was performed using standard pH buffer solutions (pH 4.01, 7.00, and 9.21) prior to measurement. This ensures accuracy and methodological transparency.

8. Line 106 – please correct the typo

Response: Thank you for pointing this out. The typo has been corrected.

9. Line 109 – add reference for Zea maysoptimal water condition

Response: Thank you for the suggestion. We have added a reference to support the statement regarding the optimal water condition for Zea mays (≈60% field capacity), citing Xiao et al. (2025) in Plant and Soil. (https://doi.org/10.1007/s11104-025-07767-y).

10. Lines 115-117 – the provided reference is not relevant since the soil samples were taken from another location; please explain how the amount of fertilizer was calculated

Response: We agree that the previous reference was not appropriate and have removed it. We now clarify in the revised Methods section that the basal fertilizer rate (3.75 g pot^–1) was calculated by converting the commonly recommended maize application rate of 250 kg N ha^–1 to the pot scale based on pot surface area and soil mass. This provides an agronomically realistic nutrient level and ensures methodological transparency.

11. Line 170, 173, 176 – the parameter symbol referred to in the explanation is missing; explanations of equations in lines 173 and 176 are not clear

Response: We sincerely thank the reviewer for the careful and detailed comments. We have revised the explanations of all equations in Section 2.5 to ensure that each parameter symbol corresponds precisely to the variables used in the formulas.

12. Line 181-183 – please provide software producers data (producer, country); add information about post hoc test

Response: Thank you for this helpful suggestion. We have added the producer and country information for all software packages mentioned, and we have also specified the post hoc test applied after ANOVA. These revisions are now included in Lines 227–239 of the manuscript.

13. Line 189 – please rephrase; according to the results in Figure 1, there was a significant difference between EPS treatment 1‰ (labeled with letter a), and other treatments (all labeled with letter b)

Response: Thank you for this observation. We have rephrased the sentence to accurately reflect the statistical grouping in Figure 1. The revised manuscript now states that a significant difference occurred only between the 1.00‰ EPS treatment (a) and all other treatments (b).

14. Lines 203-210 – results description in the text is not consistent with data presented in Figure 2. In addition, the letters for the rhizosphere soil in Figure 2 are incorrect. Please revise to ensure agreement between the text and figure

Response: We sincerely thank the reviewer for identifying the inconsistency between the manuscript and the data in Figure 2. We acknowledge this oversight and have carefully revised the manuscript to ensure full alignment between the description and the figure. Specifically, we corrected the statement to reflect that no significant difference was observed between the 0.50‰ and 1.00‰ EPS treatments in rhizosphere soil, and the corresponding sentence in Lines 276–281 has been updated. In addition, the letter labels for rhizosphere soil in Figure 2 have been corrected to match the statistical results. These revisions ensure complete consistency between the manuscript and the figure.

15. Lines 241-244 – please revise the text; treatment 0.5‰ in rhizosphere soil is significantly different from treatments 0.25 and 1. Considering that the bulk soil shows better results than the rhizosphere soil in this case, I suggest adding a sentence in the text to highlight this observation.

Response: Thank you for this valuable comment. We have revised the manuscript to clearly state that in rhizosphere soil, the 0.50‰ EPS treatment differs significantly from both the 0.25‰ and 1.00‰ treatments. In addition, following the reviewer’s suggestion, we have added a sentence noting that in this case, bulk soil exhibits better formation of 53–250 μm micro-aggregates than rhizosphere soil at higher EPS concentrations. The revised description is now provided in Lines 292–298 of the manuscript.

16. Line 299 – I suggest including similar information for the bulk soil as was provided for the rhizosphere soil, noting that the fractal dimensions decreased following the application of the treatments, or rephrase the paragraph

Response: We thank the reviewer for this suggestion. In response, we have added information regarding the fractal dimension changes in bulk soil, which is consistent with the description for rhizosphere soil.

17. Line 351 – I suggest increasing the font size in Figure 10 for better readability; provide the meaning of ** in the figure

Response: We thank the reviewer for the suggestion. We have made the following revisions: the font size in Figure 10, including axis labels and the legend, has been increased to improve readability; the meaning of the asterisk (**) has also been clarified in the legend.

18. Line 484 – correct the reference

Response: Thank you for pointing out this issue. We have carefully checked and corrected the reference at line 584 to ensure that it matches the proper citation format and content according to the Agronomy reference style

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,
thank you for submitting your manuscript “Enhanced Formation and Stability of Water-Stable Aggregates in Rhizosphere Soil over Bulk Soil with Exopolysaccharide from Rhizobium tropici.”
The topic is timely and potentially valuable, but the study still needs significant improvements in design, statistical analysis, and presentation before it can meet publication standards.

The manuscript reports a pot experiment testing whether exogenous exopolysaccharide (EPS) from Rhizobium tropici (CIAT 899) enhances formation and stability of water-stable aggregates in rhizosphere versus bulk soil. Zea mays seedlings were grown 40 days in soils amended with 0, 0.25, 0.50, or 1.00 ‰ (w/w) EPS. Aggregate-size distributions (>2000, 250–2000, 53–250, <53 μm) and stability indices (R0.25, MWD, GMD, fractal dimension D) were measured; biomass was recorded; correlations among aggregate fractions and biomass were computed. The authors conclude that EPS—especially at 0.25–0.50 ‰—increases macroaggregates and stability (higher MWD/GMD, lower D), with stronger effects in the rhizosphere; they also discuss links to root activity. The topic, microbial EPS as a bio-based amendment to improve soil structure—is timely and relevant for agronomy and soil ecology. The study’s comparative focus on rhizosphere versus bulk soil is a strength. However, there are substantive issues that must be addressed before the work is acceptable for publication. Therefore,  it requires major revision to fix some design/analysis issues, clarify EPS characterization and controls, standardize methods, fix internal inconsistencies, and improve presentation.

Title/Abstract

Lines 2–4: Consider “Rhizobium tropici” (italicize species). Also, “A Pot Study” → “a pot study” (sentence case).

Lines 31–34 (Abstract): You state “significant positive relationships between macro-aggregates (250–2000 μm) and root biomass.” In Results/Discussion you report weak/negative and non-significant correlations. Please re-analyze and reconcile; otherwise revise the Abstract to reflect the actual statistics.

 

Introduction

Lines 57–66: The claim that “exogenous EPS application or microbial culture” significantly enhances aggregate stability needs stronger, recent field or robust lab references; also distinguish between EPS extracts vs. live inoculants.

Lines 73–83: Clearly specify the primary hypotheses and pre-registered outcomes (R0.25, MWD, GMD, D) and whether you expected a rhizosphere×EPS interaction.

 

Materials & Methods

Lines 86–91 (Table 1): Clarify whether the “water-stable aggregates” columns refer to baseline soil (pre-treatment) and the exact method used to derive these percentages. The very high “<53 μm” (88.23%) suggests a particle size distribution, not water-stable aggregates; please verify units/definitions.

 

Lines 93–105 (EPS preparation):

Report EPS purity, ash content, elemental composition (C, N, P), monosaccharide profile, M_w/M_n, zeta potential/charge density, and viscosity.

Include a medium-only control addition to soils at equivalent ionic/nutrient load to isolate EPS effects from medium carryover.

 

State whether the EPS solution was sterile-filtered, and whether preservatives or salts were present.

 

Lines 106–117 (Experimental design):

Replace per-mille (‰) with mg EPS per kg dry soil (e.g., 0.25‰ = 0.25 g kg⁻¹) and provide the exact mass of EPS per pot.

Correct typo “pot ccontrol”.

Fertilizer rate “3.75 g per pot” (16–4–8): give equivalent kg ha⁻¹ or mg kg⁻¹ soil; discuss whether added nutrients could interact with EPS effects on aggregation.

 

Lines 118–131 (Planting & rhizosphere sampling):

Provide the pot dimensions/volume, bulk density used to compute soil mass/volume consistency.

Rhizosphere sampling: indicate mass of rhizosphere vs bulk recovered per pot and whether subsampling was standardized across treatments.

 

Lines 151–165 (Indices):

Equations are partially correct; variables must be defined once and consistently.

Fractal dimension (D): provide the model fit (R²), fitting range, and justification for using mass-based fractal scaling with your discrete classes.

 

Lines 179–183 (Statistics):

Use two-way ANOVA (EPS × soil zone) with interaction, or a linear model including pot as random if needed; report assumptions checks, effect sizes, 95% CIs, and post-hoc corrections appropriate for the design (Tukey/Holm). If you insist on stratified one-way tests, justify and clearly separate rhizosphere and bulk analyses.

State the n for each analysis after any exclusions.

 

Results

Figure 1–4 (Lines 186–248): Good to separate size classes, but provide means ± SD and exact p-values in a supplemental table; specify whether letters compare within a soil zone or across both.

R0.25 / MWD / GMD / D (Lines 249–307): Ensure post-hoc groupings reflect a two-factor design. Consider adding η² or partial η² to convey effect sizes of EPS and soil zone on each index.

Figure 9 (Lines 309–331): Plotting root biomass as negative values is non-standard and confusing. Please plot shoot and root as positive values in grouped bars, or provide separate panels.

You say “numerical similarity between fresh and dry weights may reflect low plant water content… due to senescence.” This needs evidence (e.g., RWC) or otherwise state more cautiously.

 

Figure 10 & corresponding text (Lines 332–358): The heatmap shows several negative correlations; text states “a significant positive correlation” for certain pairs and labels total biomass correlation with 250–2000 μm as positive while the coefficient shown is negative (r = −0.37). Please correct the signs and significance statements, and ensure consistency with the Abstract.

Given compositional constraints (fractions sum to 1), consider compositional data analysis (e.g., isometric log-ratio transforms) to avoid spurious correlations among size classes.

 

Discussion & Conclusions

Lines 359–412: Strengthen mechanistic discussion by separating direct EPS physicochemical effects from indirect plant/microbial effects and by acknowledging the current lack of EPS composition data and medium-only controls in your study. Temper claims accordingly.

Lines 413–436: You argue for root interactions but acknowledge absent positive correlations; reframe to emphasize plausible, literature-based mechanisms and treat your results as primarily supporting direc EPS effects under your conditions.

Lines 437–462 (Conclusions): Tone down statements on rhizosphere synergy unless supported by a significant EPS×zone interaction test. Explicitly state the need for field validation and long-term persistence of EPS effects.

 

Language, formatting, references

Throughout: Correct typos (e.g., “sddition”, “ccontrol”, “aggrega- tes”), ensure species names italicized, and fix equation rendering.

References (Lines 483–568): Several entries contain errors (e.g., misspelled author names, incomplete journal titles/years, inconsistent formatting such as “Nat.” vs “Nature”; the Rillig/ISME item year/DOI appear mismatched). Please verify all references against original sources and standardize to journal style.

Comments on the Quality of English Language

The manuscript would benefit from careful language editing to correct typographical errors, ensure consistent scientific terminology, and improve flow and clarity.

Author Response

Reviewer 3:

The manuscript reports a pot experiment testing whether exogenous exopolysaccharide (EPS) from Rhizobium tropici (CIAT 899) enhances formation and stability of water-stable aggregates in rhizosphere versus bulk soil. Zea mays seedlings were grown 40 days in soils amended with 0, 0.25, 0.50, or 1.00 ‰ (w/w) EPS. Aggregate-size distributions (>2000, 250–2000, 53–250, <53 μm) and stability indices (R0.25, MWD, GMD, fractal dimension D) were measured; biomass was recorded; correlations among aggregate fractions and biomass were computed. The authors conclude that EPS—especially at 0.25–0.50 ‰—increases macroaggregates and stability (higher MWD/GMD, lower D), with stronger effects in the rhizosphere; they also discuss links to root activity. The topic, microbial EPS as a bio-based amendment to improve soil structure—is timely and relevant for agronomy and soil ecology. The study’s comparative focus on rhizosphere versus bulk soil is a strength. However, there are substantive issues that must be addressed before the work is acceptable for publication. Therefore, it requires major revision to fix some design/analysis issues, clarify EPS characterization and controls, standardize methods, fix internal inconsistencies, and improve presentation.

Response: We sincerely thank the reviewer for the thorough and constructive evaluation of our work. We appreciate your positive assessment of the study’s relevance and the strength of the rhizosphere–bulk soil comparison, as well as your clear guidance on aspects requiring improvement. In response to your comments, we have undertaken substantial revisions to address the issues related to experimental design, EPS characterization, methodological clarity, data analysis, and presentation. These changes have strengthened the scientific rigor and coherence of the manuscript. All specific points you raised have been addressed in detail in the point-by-point responses below, and corresponding revisions have been incorporated into the manuscript.

Title/Abstract

1. Lines 2–4: Consider “Rhizobium tropici” (italicize species). Also, “A Pot Study” → “a pot study” (sentence case).

Response: We thank the reviewer for the correction. We have italicized the species name Rhizobium tropici and revised “A Pot Study” to “a pot study” to follow proper sentence case formatting.

2. Lines 31–34 (Abstract): You state “significant positive relationships between macro-aggregates (250–2000 μm) and root biomass.” In Results/Discussion you report weak/negative and non-significant correlations. Please re-analyze and reconcile; otherwise revise the Abstract to reflect the actual statistics.

Response: Thank you for this careful observation. We re-analyzed the correlation results and confirm that the relationships between the 250–2000 μm macro-aggregate fraction and root/plant biomass are weak or non-significant, including a weak negative correlation with total biomass (r = −0.37, p ≤ 0.05). To avoid inconsistency with the actual statistics, we have revised the Abstract accordingly to remove the earlier statement of a significant positive relationship and to accurately reflect the re-checked results.

Introduction

3. Lines 57–66: The claim that “exogenous EPS application or microbial culture” significantly enhances aggregate stability needs stronger, recent field or robust lab references; also distinguish between EPS extracts vs. live inoculants.

Response: Thank you for this important comment. We have revised the paragraph to (i) clearly distinguish between exogenous EPS extracts and live microbial inoculants, and (ii) support the claim with stronger and more recent evidence. Exogenous EPS extracts have been shown to directly promote soil aggregation under controlled or semi-field conditions (e.g., Costa et al., 2018; Cheng et al., 2020), whereas live inoculants enhance aggregation primarily through in situ EPS production and root–microbe interactions. We have now incorporated these distinctions into the manuscript. In addition, we added recent findings from Xiao et al. (2025), which provide new experimental evidence that purified Rhizobium tropici EPS significantly improve soil aggregate stability and plant performance under stress. These updates strengthen the original claim and align the discussion with current literature.

4. Lines 73–83: Clearly specify the primary hypotheses and pre-registered outcomes (R0.25, MWD, GMD, D) and whether you expected a rhizosphere×EPS interaction.

Response: Thank you for this constructive suggestion. In the revised manuscript, we now clearly state the primary hypotheses and key outcome variables (R_0.25, MWD, GMD, and D) in Lines 77–90. We also clarify that the original study was primarily designed to test the main effects of EPS on soil aggregate stability in rhizosphere and bulk soil, and that the rhizosphere × EPS interaction was explored a posteriori rather than formally pre-registered. This clarification has been explicitly incorporated into the revised manuscript.

Materials & Methods

5. Lines 86–91 (Table 1): Clarify whether the “water-stable aggregates” columns refer to baseline soil (pre-treatment) and the exact method used to derive these percentages. The very high “<53 μm” (88.23%) suggests a particle size distribution, not water-stable aggregates; please verify units/definitions.

Response: Thank you for this important comment. We have clarified in Section 3.1 that the aggregate-size distribution reported in Table 1 refers to the baseline (pre-treatment) soil. The high proportion of the <53 μm fraction reflects the soil’s inherently fine texture rather than water-stable aggregates measured after EPS application. We have also revised the manuscript to clearly state the method used to derive these baseline percentages and to avoid confusion with post-treatment water-stable aggregate measurements. These clarifications ensure that the units and definitions are fully consistent and correctly interpreted.

6. Lines 93–105 (EPS preparation): Report EPS purity, ash content, elemental composition (C, N, P), monosaccharide profile, M_w/M_n, zeta potential/charge density, and viscosity.

Response: Thank you for this important comment. We acknowledge that parameters such as EPS purity, ash content, elemental composition (C, N, P), monosaccharide profile, molecular weight distribution (M_w/M_n), zeta potential/charge density, and viscosity provide valuable information for fully characterizing EPS. However, these analyses were not included in the current study because the experimental design focused primarily on evaluating the functional effects of a standardized EPS extract on soil aggregation, rather than on the detailed physicochemical characterization of the EPS itself. To address this limitation, we have added a statement in the revised manuscript noting that the absence of complete EPS characterization represents a constraint of the present work and should be considered in future studies.

7. Include a medium-only control addition to soils at equivalent ionic/nutrient load to isolate EPS effects from medium carryover.

Response: This study was not included in the present experiment because the study was originally designed to evaluate the functional response of soil aggregates to EPS addition rather than to partition medium-derived effects. To address this limitation, we have added a statement in the Methods and Discussion sections acknowledging the absence of a medium-only control and clarifying that the observed effects may include minor contributions from medium carryover. We also note that future work should incorporate a medium-only control to more rigorously isolate the specific contribution of EPS.

8. State whether the EPS solution was sterile-filtered, and whether preservatives or salts were present.

Response: Thank you for this comment. We have added the relevant information to Section 2.2. The EPS solution was sterile-filtered through a 0.22 μm membrane prior to use and stored at 4 °C. We also clarify that no preservatives or additional salts were included in the solution, ensuring that only EPS was introduced into the soil. The revised manuscript can be found in Section 2.2 (Lines 124–126).

9. Lines 106–117 (Experimental design): Replace per-mille (‰) with mg EPS per kg dry soil (e.g., 0.25‰ = 0.25 g kg⁻¹) and provide the exact mass of EPS per pot.

Response: Thank you for the suggestion. We have revised the Experimental design section accordingly. EPS concentrations are now expressed as mg EPS kg⁻¹ dry soil instead of per-mille (‰). The exact mass of EPS added per pot has also been specified based on the soil mass of 1.50 kg per pot, resulting in 0, 0.375, 0.75, and 1.50 g EPS per pot for the 0, 250, 500, and 1000 mg kg⁻¹ treatments, respectively.

10. Correct typo “pot ccontrol”.

Response: Done.

11. Fertilizer rate “3.75 g per pot” (16–4–8): give equivalent kg ha⁻¹ or mg kg⁻¹ soil; discuss whether added nutrients could interact with EPS effects on aggregation.

Response: Thank you for this helpful suggestion. We have added the equivalent field-rate conversion for the applied fertilizer. The basal 3.75 g pot⁻¹ of 16–4–8 corresponds to approximately 250 kg N ha⁻¹, which is within the typical range for maize production, or roughly 2.5 mg N g⁻¹ soil on a mass basis (1.5 kg soil per pot). This information has been incorporated into the revised manuscript. We have also added a brief discussion acknowledging that nutrient additions—particularly nitrogen—can influence soil aggregation through effects on root growth and microbial activity. However, because the same fertilizer rate was applied uniformly across all treatments, any such influence would not bias the comparison among EPS levels. This point has now been clarified in the Discussion.

12. Lines 118–131 (Planting & rhizosphere sampling): Provide the pot dimensions/volume, bulk density used to compute soil mass/volume consistency.

Response: Thank you for the valuable comment. We have added detailed information on the pot size, soil volume, and bulk density to ensure consistency across treatments. Specifically, each pot (20 cm × 15 cm, 5 dm³) was filled with approximately 1.5 kg of air-dried soil at a bulk density of 1.26 g cm⁻³.

13. Rhizosphere sampling: indicate mass of rhizosphere vs bulk recovered per pot and whether subsampling was standardized across treatments.

Response: Thank you for this comment. We have added the requested information to the revised Methods. Each pot contained 1500 g of soil, from which approximately 80 g of rhizosphere soil was recovered following the standard shaking/off-root procedure, with the remaining soil used as bulk soil. The subsampling procedure was performed consistently across all treatments to ensure comparability.

14. Lines 151–165 (Indices): Equations are partially correct; variables must be defined once and consistently.

Response: We thank the reviewer for this helpful suggestion. We have carefully revised all equations in Section 2.5 to ensure accuracy and consistency.

15. Fractal dimension (D): provide the model fit (R²), fitting range, and justification for using mass-based fractal scaling with your discrete classes.

Response: We appreciate the reviewer’s careful attention to the fractal analysis. In this study, the fractal dimension (D) was used primarily as a comparative indicator of soil aggregate structure rather than as a mechanistic model parameter. The fitting of the mass-based fractal model yielded high coefficients of determination (R² > 0.95) across treatments within the measured aggregate size range (0.25–5 mm), which is consistent with the range commonly adopted in previous studies (e.g., Tyler and Wheatcraft, 1992). We selected mass-based fractal scaling because the aggregate size distribution was expressed in discrete mass fractions rather than cumulative number or volume data, following standard practice for soil aggregate analysis. Therefore, we believe the current description sufficiently characterizes the aggregate hierarchy for comparative purposes.

16. Lines 179–183 (Statistics): Use two-way ANOVA (EPS × soil zone) with interaction, or a linear model including pot as random if needed; report assumptions checks, effect sizes, 95% CIs, and post-hoc corrections appropriate for the design (Tukey/Holm). If you insist on stratified one-way tests, justify and clearly separate rhizosphere and bulk analyses.

Response: Thank you for this important statistical suggestion. We fully agree that a two-way ANOVA (EPS × soil zone) can be appropriate for designs aimed at testing interaction effects. However, the present study was not designed to compare rhizosphere and bulk soil directly because the two soil zones differ fundamentally in their biological and physicochemical processes (e.g., root exudation, microbial gradients). These inherent differences make a direct statistical comparison difficult to interpret in a mechanistic sense. For this reason, our analysis focused on how EPS influences each soil zone separately, rather than on whether EPS effects differ statistically between zones. Consequently, we chose stratified one-way ANOVA within each soil type to isolate the EPS effect in rhizosphere soil and bulk soil independently, which aligns with our experimental objectives. To improve transparency, we have now (i) clarified this rationale in the Methods section, (ii) clearly separated the rhizosphere and bulk soil analyses in the Results, and (iii) ensured that appropriate post-hoc tests and assumption checks were reported for each one-way ANOVA.

17. State the n for each analysis after any exclusions.

Response: Thank you for the helpful suggestion. We have now clearly stated the sample size (n) for each analysis in the Materials and Methods section. Specifically, all analyses were based on three independent pots per treatment (biological replicates, n = 3), and each measurement was performed in triplicate (technical replicates). Data from technical replicates were averaged prior to statistical analysis. No data were excluded except for visibly contaminated or lost samples.

Results

18. Figure 1–4 (Lines 186–248): Good to separate size classes, but provide means ± SD and exact p-values in a supplemental table; specify whether letters compare within a soil zone or across both.

Response: Thank you for this helpful suggestion. We have added a supplemental table providing the means ± SD and exact p-values for all aggregate size classes shown in Figures 1–4. We also clarify in the figure captions and Results section that the significance letters refer to comparisons conducted within each soil zone separately (i.e., rhizosphere soil analyzed independently from bulk soil), rather than across both zones combined. These revisions improve transparency and ensure that the statistical interpretation is clear.

19. R₂₅/ MWD / GMD / D (Lines 249–307): Ensure post-hoc groupings reflect a two-factor design. Consider adding η² or partial η² to convey effect sizes of EPS and soil zone on each index.

Response: Thank you for this valuable comment. As noted earlier, our analysis intentionally used stratified one-way ANOVA because rhizosphere and bulk soils differ fundamentally in their biological and physicochemical environments (e.g., root exudation, microbial gradients), making a direct two-factor comparison difficult to interpret mechanistically. Therefore, each soil zone was analyzed independently to isolate the EPS effect within its own domain. To improve transparency, we now state this rationale clearly in the Methods section, and all post-hoc groupings in the Results are explicitly presented within each soil type only, consistent with the one-factor structure. Regarding effect size, although η² or partial η² are typically reported for multi-factor designs, we have supplemented the results with η² values for each one-way ANOVA to provide additional information on the magnitude of EPS effects within each soil zone.

20. Figure 9 (Lines 309–331): Plotting root biomass as negative values is non-standard and confusing. Please plot shoot and root as positive values in grouped bars, or provide separate panels.

Response: Thank you for this comment. We agree that plotting root biomass as negative values is unconventional. However, we retained this format because it visually distinguishes shoot and root biomass within a single panel, and we have clearly stated in the figure caption that negative values represent belowground (root) biomass. This clarification ensures that the plotting choice is transparent to readers.

21. You say “numerical similarity between fresh and dry weights may reflect low plant water content… due to senescence.” This needs evidence (e.g., RWC) or otherwise state more cautiously.

Response: Thank you for this constructive comment. As we did not measure relative water content (RWC) or other physiological indicators, we have revised the sentence to avoid attributing the similarity between fresh and dry weights to senescence or low water content. The revised wording now states this observation more cautiously without implying unsupported physiological explanations.

22. Figure 10 & corresponding text (Lines 332–358): The heatmap shows several negative correlations; text states “a significant positive correlation” for certain pairs and labels total biomass correlation with 250–2000 μm as positive while the coefficient shown is negative (r = −0.37). Please correct the signs and significance statements, and ensure consistency with the Abstract.

Response: Thank you for this careful observation. We have corrected all inconsistencies regarding the direction and significance of the correlations in both the manuscript and the figure description. In particular, the correlation between the 250–2000 μm fraction and total biomass has been revised from “significant positive” to “significant negative (r = −0.37, p ≤ 0.05),” consistent with the values shown in Figure 10. We also re-checked all other correlation descriptions to ensure that the signs, significance levels, and corresponding statements fully match the heatmap and the revised Abstract.

23. Given compositional constraints (fractions sum to 1), consider compositional data analysis (e.g., isometric log-ratio transforms) to avoid spurious correlations among size classes.

Response: Thank you for this insightful statistical suggestion. We agree that compositional data analysis (e.g., isometric log-ratio transformation) can be valuable for studies focused on the covariance structure among aggregate-size fractions. However, the present study aimed primarily to evaluate treatment-level differences in soil aggregation under different EPS concentrations within each soil zone, rather than to interpret the internal dependencies among size classes. For this reason, we retained the conventional approach based on absolute mass proportions, which is widely used in soil aggregation research and is sufficient for addressing our core research questions. To acknowledge this point, we have added a brief note in the Discussion indicating that future work could benefit from applying compositional data analysis to further refine interpretations of aggregate-size relationships.

Discussion & Conclusions

24. Lines 359–412: Strengthen mechanistic discussion by separating direct EPS physicochemical effects from indirect plant/microbial effects and by acknowledging the current lack of EPS composition data and medium-only controls in your study. Temper claims accordingly.

Response: Thank you for this insightful suggestion. We have substantially revised the Discussion to clearly separate the direct physicochemical effects of EPS (e.g., polymer bridging, enhanced cohesion) from the indirect effects mediated by plant roots or microbial activity. We also explicitly acknowledge two limitations of the present study: (i) the lack of detailed EPS compositional data and (ii) the absence of a medium-only control. These limitations have been incorporated into the revised manuscript, and the corresponding claims have been moderated to ensure that the mechanistic interpretation remains appropriately cautious and evidence-based.

25. Lines 413–436: You argue for root interactions but acknowledge absent positive correlations; reframe to emphasize plausible, literature-based mechanisms and treat your results as primarily supporting direc EPS effects under your conditions.

Response: Thank you for this valuable comment. We have revised this section to more accurately reflect the evidence from our study. As this work represents a preliminary evaluation of EPS effects under controlled conditions, we now emphasize that the improvements in soil aggregate stability observed here are primarily attributable to the direct physicochemical effects of EPS, as no positive correlations were found between biomass and aggregate fractions in our experiment. We have also reframed the discussion so that potential root–EPS interactions are presented as plausible, literature-supported mechanisms, rather than conclusions drawn from our data. This distinction is now clearly articulated, and we note that such synergistic interactions may occur under field conditions but were not supported by the present results. The revised manuscript appears in Lines 490–498 of the manuscript.

26. Lines 437–462 (Conclusions): Tone down statements on rhizosphere synergy unless supported by a significant EPS×zone interaction test. Explicitly state the need for field validation and long-term persistence of EPS effects.

Response: Thank you for this suggestion. We have revised the Conclusions by softening the statements related to rhizosphere synergy and removing wording that could imply a confirmed interactive effect. In addition, we have added explicit sentences highlighting the need for field validation and for assessing the long-term persistence of EPS effects. These revisions can be found in Lines 545–557 of the updated manuscript.

Language, formatting, references

27. Throughout: Correct typos (e.g., “sddition”, “ccontrol”, “aggrega- tes”), ensure species names italicized, and fix equation rendering.

Response: Thank you for this helpful comment. We have carefully reviewed the entire manuscript and corrected all typographical errors (including “sddition,” “ccontrol,” and “aggrega-tes”). All species names have been properly italicized, and equation formatting has been corrected to ensure accurate rendering. These revisions have been applied consistently throughout the manuscript.

28. References (Lines 483–568): Several entries contain errors (e.g., misspelled author names, incomplete journal titles/years, inconsistent formatting such as “Nat.” vs “Nature”; the Rillig/ISME item year/DOI appear mismatched). Please verify all references against original sources and standardize to journal style.

Response: Thank you for this valuable comment. We have thoroughly checked all references and corrected errors in author names, publication years, journal titles, and DOIs. Inconsistent abbreviations (e.g., “Nat.” vs. “Nature”) have been standardized, and the Rillig et al. ISME Journal entry has been corrected for year and DOI accuracy. All references have now been verified against the original sources and formatted according to the Agronomy style guidelines.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have applied all the suggestions and improved the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for your thorough revisions. You have addressed all the major issues raised in the previous round, and the manuscript is now much clearer, consistent, and well-presented. The methodology and interpretations are adequately explained, and the limitations are now appropriately acknowledged. I am satisfied with the current version and am happy to accept the manuscript as it is.