Rhizobial Inoculation Improves Soil Properties and Microbial Network Stability to Support Medicago sativa L. Production in Cold Arid Regions

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents useful field-based data on rhizobial inoculation effects on Medicago sativa under cold arid conditions. The integration of soil properties, microbial community analysis, ecological networks, and SEM modeling is a strength of the study. The work is relevant for sustainable forage production and microbial-assisted agriculture in plateau ecosystems. However, several interpretations should be made more cautiously and some methodological details require clarification before publication.

Major Comments

1. As the yield gain was very small (only 2.4–3.2%), it should be justified that how this small gain is significant in todays time.  
2. The manuscript repeatedly states that inoculation improved microbial stability and resilience. However, α-diversity, β-diversity, RAVD, cohesion, and composite stability indices were mostly non-significant. The conclusions should better match the actual analysis.
3. The strong reduction in NH₄⁺–N and NO₃⁻–N is interesting, but the explanation is too speculative. Since nodulation, nitrogen fixation activity, or plant N accumulation were not quantified, the claim that biological nitrogen fixation caused these reductions should be discussed more carefully.
4. The inoculant used was not only rhizobia; it also contained phosphate-solubilizing bacteria and Bacillus subtilis. Therefore, the observed effects cannot be attributed solely to rhizobia. Therefore, the use of inoculant should be preferred over any specific group.
5. The microbial network analysis is interesting, but important methodological details are missing:
• filtering criteria for ASVs,
• number of ASVs used for network construction,
• and whether networks were built from pooled or individual samples.

These details are important for reproducibility.

1. Positive and negative correlations in co-occurrence networks should not be directly interpreted as cooperation or competition. Correlation does not necessarily indicate ecological interaction. Please modify the discussion accordingly considering the limitation of obtained data.
2. The SEM results are interesting because the inoculant showed a weak negative direct effect on yield but positive indirect effects through soil factors. This point deserves deeper discussion rather than only emphasizing positive effects.
3. The discussion sometimes overinterprets the findings. For example, claims related to “ecosystem stability,” “resilience,” and “sustainable productivity” appear stronger than supported by the data. The authors are advised to make the statements based on real data obtained.

Methodology Comments

1. Please clarify whether this was a single-season experiment and provide baseline soil properties before treatment application.
2. Information on sequencing depth, rarefaction, and coverage statistics should be added.
3. The statement “field as a random effect” is confusing because the study appears to have been conducted at a single experimental site. Please clarify the mixed-model structure.
4. The reason for using Bonferroni correction instead of a less conservative approach (e.g., FDR) should be explained.

Minor Comments

1. Several typographical and formatting issues are present throughout the manuscript (e.g., “maĴer”, “aĴributable”, double punctuation). Careful English editing is needed.
2. Some figure legends are too long and repetitive and can be simplified.

 

Comments on the Quality of English Language

Can be improved further with the aim to have better clarity of presentation.

Author Response

Responses to comments from reviewers

(Microorganisms-4316392)

Dear Prof.

Thank you very much for handling our manuscript “Rhizobial Inoculation Improves Soil Properties and Microbial Network Stability to Support Medicago sativa L Production in Cold Arid Regions”. We highly appreciate the insightful comments from the anonymous reviewers. After fully considering these comments, we made thorough revisions to our manuscript.

Below, each reviewer comment appears in bold, followed by our detailed response in italics. All changes made in the revised manuscript are highlighted in red for easy reference. Should any additional modifications be required, please let us know—we will be pleased to address them promptly.

Once again, thank you for your time and effort.

Qianqian Zhao

The manuscript presents useful field-based data on rhizobial inoculation effects on Medicago sativa under cold arid conditions. The integration of soil properties, microbial community analysis, ecological networks, and SEM modeling is a strength of the study. The work is relevant for sustainable forage production and microbial-assisted agriculture in plateau ecosystems. However, several interpretations should be made more cautiously and some methodological details require clarification before publication..

Comment 1. As the yield gain was very small (only 2.4–3.2%), it should be justified that how this small gain is significant in todays time.

Response: We thank the reviewer for this important comment. We agree that the observed yield increase was modest and should not be overstated as a strong yield-promoting effect. In the revised manuscript, we have clarified that the yield response was small and statistically non-significant, and we now interpret it more cautiously.

Our emphasis has therefore been revised from “yield improvement” to “maintenance of yield with a modest increase under cold and arid field conditions”. We also clarified that the relevance of this response lies not only in the small biomass gain itself, but also in its concurrence with significant increases in soil organic matter and marked changes in mineral nitrogen pools. In nutrient-poor, high-elevation dryland forage systems, even small yield gains may be agronomically relevant when accompanied by improved soil functioning and reduced mineral nitrogen availability. However, we fully agree that the practical and economic significance of such a modest yield response requires validation through multi-year field trials and cost-benefit assessment.

The sentence now reads（Lines 29-32）: The composite microbial inoculant maintained Medicago sativa L. yield, with only modest and non-significant increases in some treatments. In contrast, soil organic matter increased significantly with application rate (p < 0.001), suggesting a stronger short-term effect on soil properties than on yield.

The sentence now reads（Lines 238-241）: The composite microbial inoculant did not significantly increase Medicago sativa L. yield under the present short-term field conditions (Fig. 2a). Yield was generally main-tained across treatments, with only modest increases observed under E1 and E4, corre-sponding to gains of 2.4% and 3.2%, respectively.

The sentence now reads（Lines 377-380）: In the present short-term field experiment, the composite microbial inoculant did not significantly increase yield, although modest increases of 2.4% and 3.2% were observed under E1 and E4, respectively. These responses were accompanied by significant increases in soil organic matter and changes in mineral nitrogen pools.

Comment 2. The manuscript repeatedly states that inoculation improved microbial stability and resilience. However, α-diversity, β-diversity, RAVD, cohesion, and composite stability indices were mostly non-significant. The conclusions should better match the actual analysis.

Response: We appreciate this helpful comment. We agree that the previous wording overstated the microbial stability results. In the revised manuscript, we have substantially tempered these statements. We now distinguish between two levels of response: first, the overall bacterial community structure and diversity, which were largely unchanged; and second, the co-occurrence network topology, which was reconfigured by inoculation.

Specifically, α-diversity, β-diversity, RAVD, cohesion and the composite network stability index did not differ significantly among treatments. Therefore, we no longer claim that inoculation broadly enhanced microbial stability or resilience. Instead, we state that inoculation reshaped network architecture and partially affected network robustness, while the overall community-level stability remained largely unchanged.

The sentence now reads（Lines 33-34）: Although network vulnerability was lowest in E4, the differences among treatments were not statistically significant.

The sentence now reads（Lines 325-326）: Overall, inoculation increased network dissimilarity and altered network topological properties, indicating a restructuring of rhizosphere bacterial network architecture.

Comment 3. The strong reduction in NH₄⁺–N and NO₃⁻–N is interesting, but the explanation is too speculative. Since nodulation, nitrogen fixation activity, or plant N accumulation were not quantified, the claim that biological nitrogen fixation caused these reductions should be discussed more carefully.

Response: We agree with the reviewer. The previous explanation was too causal given the measurements available in this study. Because nodule number, nitrogenase activity and plant nitrogen accumulation were not quantified, we cannot directly attribute the decreases in NH₄⁺–N and NO₃⁻–N to enhanced biological nitrogen fixation.

We have revised the Discussion to present biological nitrogen fixation only as a possible mechanism, rather than as a demonstrated cause. We now interpret the reductions in mineral nitrogen more conservatively as the possible result of changes in plant uptake, microbial immobilization, nitrogen transformation processes and inoculant-mediated rhizosphere interactions. We also added this limitation and proposed future measurements, including nodulation traits, nitrogenase activity, plant nitrogen accumulation and functional genes involved in nitrogen cycling.

The sentence now reads（Lines 398-404）: The significant reductions in NH₄⁺–N and NO₃⁻–N indicate substantial changes in the rhizosphere mineral nitrogen pool. The rhizobial inoculant may have altered the balance among plant nitrogen uptake, microbial immobilization, and nitrogen transformation processes, while biological nitrogen fixation may represent only one of several potential contributing pathways. Future studies should incorporate assessments of nodulation traits, nitrogenase activity, plant nitrogen accumulation, and functional genes involved in nitrification and mineralization to further elucidate the underlying mechanisms.

Comment 4. The inoculant used was not only rhizobia; it also contained phosphate-solubilizing bacteria and Bacillus subtilis. Therefore, the observed effects cannot be attributed solely to rhizobia. Therefore, the use of inoculant should be preferred over any specific group.

Response: We thank the reviewer for this valuable comment. We agree that the commercial seed-coating product used in this study was a composite microbial inoculant containing rhizobia, phosphate-solubilizing bacteria, and Bacillus subtilis. Therefore, the observed responses cannot be exclusively attributed to rhizobia alone.

However, rhizobia constituted the principal functional component of the inoculant and were specifically included to promote symbiotic nitrogen fixation in Medicago sativa L. Consequently, we retained the term “rhizobial inoculant” in parts of the manuscript where the focus is on the dominant functional component of the product. At the same time, we have revised the manuscript to clearly describe the inoculant composition and to avoid attributing the observed effects solely to rhizobia. Relevant sections of the manuscript have been modified accordingly.

The sentence now reads（Lines 115-118）: The seed-coating product used in this study was a rhizobial-based composite microbial inoculant, primarily composed of rhizobia and supplemented with phosphate-solubilizing bacteria and Bacillus subtilis. The inoculant contained ≥ 5 × 10⁸ CFU·mL⁻¹ viable cells and ≥ 6% total nutrients (N + P₂O₅ + K₂O).

Comment 5. The microbial network analysis is interesting, but important methodological details are missing:

filtering criteria for ASVs,

number of ASVs used for network construction,

and whether networks were built from pooled or individual samples.

Response: （Lines 134-135）：We thank the reviewer for highlighting this important methodological issue. We have revised the Methods section to provide additional details on ASV processing and network construction. Specifically, we clarified the sequence quality-control procedures, ASV generation workflow, the number of ASVs used for network analysis, and the network construction strategy. We also specified that networks were constructed separately for each treatment using eight independent biological replicates and that samples were not physically pooled.

The sentence now reads（Lines 155-162）: Sequences shorter than 50 bp were removed. Paired-end reads were merged only when the overlap length was ≥10 bp and the maximum mismatch rate was ≤0.2, followed by chimera removal. High-quality sequences were denoised using UNOISE and resolved into amplicon sequence variants (ASVs). The resulting ASV abundance table was used for subsequent analyses of community structure, diversity, and network construction.

The sentence now reads（Lines 190-195）: Molecular ecological networks were constructed under the Random Matrix Theory (RMT) framework using treatment-specific ASV abundance matrices. Networks for CK, E1, E2, E3, and E4 were built separately from eight independent biological replicates per treatment, without physical pooling of samples. Associations with |r| > 0.8 and FDR-adjusted p < 0.05 were retained for network construction, and the resulting networks were used for subsequent topological analyses.

Comment 6. Positive and negative correlations in co-occurrence networks should not be directly interpreted as cooperation or competition. Correlation does not necessarily indicate ecological interaction. Please modify the discussion accordingly considering the limitation of obtained data.

Response: We sincerely thank the reviewer for this important and constructive comment. We fully agree that positive and negative correlations in microbial co-occurrence networks should not be directly interpreted as ecological cooperation or competition. Such associations may arise from shared environmental preferences, common responses to unmeasured soil variables, indirect relationships, or the inherent limitations of compositional data, rather than from direct biological interactions. Accordingly, we have revised the relevant statements in the Methods, Results, Discussion, and figure legends to avoid overinterpreting correlation-based network patterns. Terms such as “cooperative linkages” and “competitive linkages” have been replaced with “positive associations” and “negative associations”. We have also added a limitation statement in the Discussion to clarify that the network analysis reflects community association patterns rather than direct evidence of ecological interactions.

The sentence now reads（Lines 202-206）: Cohesion metrics were partitioned into positive cohesion and absolute negative cohesion, which quantify the relative strength of positive and negative co-occurrence associations, respectively. Because these associations are correlation-based, they should not be interpreted as direct evidence of cooperation or competition among taxa.

The sentence now reads（Lines 310-314）: As the inoculant application rate increased, the proportion of negative associations decreased, whereas the proportion of positive associations increased. These changes indicate that the correlation structure of the rhizosphere bacterial network was altered, but they do not necessarily reflect direct ecological interactions among taxa

The sentence now reads（Lines 467-469）: Overall, network dissimilarity increased, whereas cohesion metrics remained largely stable. These results indicate that rhizobial inoculant treatment altered the network structure of the rhizosphere bacterial community.

Comment 7. The SEM results are interesting because the inoculant showed a weak negative direct effect on yield but positive indirect effects through soil factors. This point deserves deeper discussion rather than only emphasizing positive effects.

Response: We appreciate this insightful suggestion. We agree that the weak negative direct path from inoculant dose to yield is an important result and should not be overlooked. In the revised Discussion, we now explicitly discuss this finding. After accounting for soil and microbial mediators, the inoculant dose itself showed a weak negative direct effect on yield, suggesting that inoculation did not directly translate into immediate yield improvement within a single growing season. Instead, its contribution to yield was mainly mediated through changes in soil properties. We have therefore revised the discussion to present a more balanced interpretation, emphasizing both the possible short-term trade-offs and the positive soil-mediated pathway.

The sentence now reads（Lines 482-491）: The SEM provided a more nuanced interpretation of the inoculant effect. After soil and microbial variables were included in the model, the direct pathway from inoculant application rate to yield was weakly negative (-0.17), whereas the indirect pathway mediated by soil properties was positive. This result indicates that, after accounting for changes in soil properties, the inoculant did not directly increase yield within a single growing season. One possible explanation is that rhizobial inoculation initially altered nutrient allocation, rhizosphere resource competition, or microbial community adjustment, thereby weakening its immediate direct contribution to aboveground biomass. Meanwhile, the positive soil-mediated pathway suggests that the main benefit of inoculation was expressed more through changes in soil nutrient status than through a direct stimulation of yield.

Comment 8. The discussion sometimes overinterprets the findings. For example, claims related to “ecosystem stability,” “resilience,” and “sustainable productivity” appear stronger than supported by the data. The authors are advised to make the statements based on real data obtained.

Response: We thank the reviewer for pointing out this issue. We agree that some statements in the original Discussion and Conclusions were stronger than what could be supported by the data obtained in this study. This study was conducted during a single growing season and focused mainly on yield, soil physicochemical properties, bacterial community composition, and network metrics. Therefore, we have toned down broad conclusions related to ecosystem stability, resilience, and sustainable productivity. In the revised manuscript, we now use more cautious and data-supported wording, such as “network robustness”, “short-term soil responses”, “rhizosphere bacterial association patterns”, and “potential implications for forage management”. We have also added a limitation statement, noting that long-term and multi-site experiments are needed before drawing conclusions about ecosystem resilience or sustainable productivity.

The sentence now reads（Lines 516-520）: Overall, this study provides field-based evidence that rhizobial seed coating can alter soil nutrient status and rhizosphere bacterial co-occurrence patterns in Medicago sativa L. production systems. However, longer-term and multi-site studies are required to determine whether these short-term responses translate into stable yield benefits or broader ecosystem-level effects on the Qinghai-Tibet Plateau.

Comment 9. Please clarify whether this was a single-season experiment and provide baseline soil properties before treatment application.

Response: We thank the reviewer for this helpful comment. We have clarified in the Materials and Methods section that this study was conducted as a single-season field experiment. In addition, the relevant data have been included in a new supplementary table. This addition helps define the temporal scope of the study and provides a clearer background for interpreting the treatment effects.

The sentence now reads（Lines 105-108）: This experiment was conducted in 2023 at the National Forage Variety Regional Testing Station in Gahai Town, Delingha City, Qinghai Province, as a single-season field experiment. Before treatment application, composite topsoil samples were collected from the experimental field to characterize the baseline soil conditions(Table S1).

Comment 10. Information on sequencing depth, rarefaction, and coverage statistics should be added.

Response: We thank the reviewer for this helpful suggestion. We would like to clarify the sequencing depth, rarefaction, and coverage statistics used in this study. According to the sequencing report, the final ASV table comprised 7,147 ASVs from 40 rhizosphere soil samples. After quality filtering and rarefaction, all samples were normalized to 27,795 sequences per sample, resulting in a total of 1,111,800 sequences used for downstream analyses. The rarefaction curves approached saturation, and Good’s coverage values ranged from 0.9991 to 0.9999, indicating that the sequencing depth was sufficient to capture the dominant bacterial diversity in the rhizosphere soil samples. These results support the reliability of the subsequent microbial community diversity and network analyses.

Comment 11. The statement “field as a random effect” is confusing because the study appears to have been conducted at a single experimental site. Please clarify the mixed-model structure.

Response: We thank the reviewer for pointing out this unclear statement. This study was conducted at a single experimental site; therefore, the phrase “field as a random effect” was inaccurate and has now been revised. In the revised manuscript, we clarify that treatment was considered a fixed effect. Where mixed-effects models were applicable, the block or replicate structure was included as a random intercept to account for the randomized complete block design. The Statistical Analysis section has been revised accordingly.

The sentence now reads（Lines 233-239）: Because the experiment was conducted at a single field site, field was not included as a random effect. Treatment was considered a fixed effect. Where mixed-effects models were used, the experimental block or replicate structure was included as a random intercept to account for the randomized complete block design. All-subset model selection was performed using the MuMIn package, and the relative contributions of selected predictors to explained yield variation were further partitioned using the glmm.hp package.

Comment 12. The reason for using Bonferroni correction instead of a less conservative approach (e.g., FDR) should be explained.

Response: We thank the reviewer for this valuable comment. We would like to clarify that the Bonferroni correction was used only for planned pairwise comparisons among a limited number of treatment groups. Because our study included five predefined treatments, the number of treatment-level comparisons was relatively small. In this context, Bonferroni correction was chosen as a conservative approach to control the family-wise error rate and reduce the risk of Type I error.

By contrast, FDR correction was applied in the construction of microbial co-occurrence networks, where a large number of pairwise ASV correlations were tested simultaneously. In such high-dimensional correlation analyses, FDR is more appropriate because it controls the expected proportion of false discoveries while retaining reasonable statistical power.

Therefore, the two correction methods were used for different analytical purposes: Bonferroni correction was applied to limited planned treatment comparisons, whereas FDR correction was used for high-dimensional microbial network inference. We appreciate the reviewer’s suggestion, which allowed us to clarify the rationale behind our statistical choices.

Comment 13. Several typographical and formatting issues are present throughout the manuscript (e.g., “maĴer”, “aĴributable”, double punctuation). Careful English editing is needed.

Response: We thank the reviewer for noting these issues. We have carefully checked the manuscript and corrected typographical, formatting, and punctuation errors throughout the text. Examples include correcting abnormal character encoding, removing repeated punctuation, standardizing spaces around units and mathematical symbols, and improving sentence flow.

The sentence now reads（Lines 263-265）: Collectively, rhizobial inoculation may have enhanced biological nitrogen fixation and nutrient uptake, thereby increasing nitrogen assimilation by plants and associated mi-crobes while promoting soil organic matter accumulation.

The sentence now reads（Lines 531）: Table S2. Rhizosphere soil bacterial network topology parameters.

The sentence now reads（Lines 243-245）: Data visualization was performed using the ggplot2 package. The selected variables were then incorporated into a piecewise structural equation model (SEM) using the piecewiseSEM package.

Comment 14. Some figure legends are too long and repetitive and can be simplified.

Response: We thank the reviewer for this useful suggestion. We have shortened and simplified the figure legends to reduce repetition while retaining the essential information needed to interpret each figure. Definitions of abbreviations and treatment codes are now provided consistently and concisely.

The sentence now reads（Lines 381-385）: Figure 8. Direct and indirect pathways linking rhizobial inoculation, soil properties, microbial variables, and Medicago sativa L. yield. (a) Piecewise structural equation model. Values beside arrows are standardized path coefficients; solid and dashed arrows indicate significant and non-significant paths, respectively. (b) Standardized direct, indirect, and total effects on yield. TP, total phosphorus; TK, total potassium; NO₃⁻–N, nitrate nitrogen. *p < 0.05, **p < 0.01, ***p < 0.001.

All changes made in the revised manuscript are highlighted in red for easy reference.

We sincerely thank the editors and reviewers for their valuable comments and dedicated efforts in evaluating our work.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

This study explored the dose-dependent effect of a rhizobial inoculant on increasing the yield of the forage plant Medicago sativa L., as well as on agrochemical and microbiological parameters of the soil. Although the results were not as remarkable as may be expected, the authors were able to identify interesting consequences of using different inoculant doses. Specifically, they demonstrated an increase in soil organic matter content, as well as an increase in the complexity and stability of rhizosphere bacterial networks, depending on the inoculant dose used. A significant influence of soil and microbial factors on alfalfa yield was substantiated. It was shown that the inoculant dose influenced yield primarily indirectly, through changes in soil parameters.

The organization of the article is satisfactory and well structured, and the length of the manuscript is appropriate to the content. The title and the abstract adequately convey the content of the manuscript. The methods used by authors are appropriate and accurately described. I would like to emphasize the competent application of mathematical statistics and modeling methods, which ensured a reliable interpretation of the results obtained.

While reading the manuscript, I would like to clarify some points given below:

Materials and Methods

Paragraph 2.1, page 3, lines 116-127. This text does not correspond to the paragraph title.

lines 116-118. Was a liquid microbial preparation used to treat the seeds? Please, clarify.

Paragraph 2.2, page 4, lines135. Authors provide the date of soil sampling, but there was no date of experiment starting.

lines 145. Please clarify the sonication parameters. This is important. Please, provide some information about duration of experiment.

Results

Figure 4. Caption. “…Different lowercase letters indicate significant differences among treatments (p < 0.05).” It seems that in relation to this figure it would be more correct to indicate that “The same letters indicate the absence of significant differences between the treatments”

In my opinion, this manuscript may be published after minor revision. 

Author Response

Responses to comments from reviewers

(Microorganisms-4316392)

Dear Prof.

Thank you very much for handling our manuscript “Rhizobial Inoculation Improves Soil Properties and Microbial Network Stability to Support Medicago sativa L Production in Cold Arid Regions”. We highly appreciate the insightful comments from the anonymous reviewers. After fully considering these comments, we made thorough revisions to our manuscript.

Below, each reviewer comment appears in bold, followed by our detailed response in italics. All changes made in the revised manuscript are highlighted in red for easy reference. Should any additional modifications be required, please let us know—we will be pleased to address them promptly.

Once again, thank you for your time and effort.

Qianqian Zhao

This study explored the dose-dependent effect of a rhizobial inoculant on increasing the yield of the forage plant Medicago sativa L., as well as on agrochemical and microbiological parameters of the soil. Although the results were not as remarkable as may be expected, the authors were able to identify interesting consequences of using different inoculant doses. Specifically, they demonstrated an increase in soil organic matter content, as well as an increase in the complexity and stability of rhizosphere bacterial networks, depending on the inoculant dose used. A significant influence of soil and microbial factors on alfalfa yield was substantiated. It was shown that the inoculant dose influenced yield primarily indirectly, through changes in soil parameters.

In my opinion, this manuscript may be published after minor revision.

Comment 1. Paragraph 2.1, page 3, lines 116-127. This text does not correspond to the paragraph title.

Response: We thank the reviewer for this helpful comment. We agree that the original title of Section 2.1, “Study area and site description”, did not fully cover the experimental design, inoculant information, seed coating procedure, fertilization, and field management described in this section. To improve structural clarity, we have revised the subsection title to more accurately reflect its content. We have made the requested revisions and indicated them in red text （Lines 104）.

Comment 2. lines 116-118. Was a liquid microbial preparation used to treat the seeds? Please, clarify.

Response: We thank the reviewer for raising this point. The microbial preparation used in this study was a liquid rhizobial biostimulant, with a viable cell count of ≥5 × 10⁸ CFU mL⁻¹. It was applied as a seed-coating inoculant rather than as a soil drench. For each treatment, the inoculant was thoroughly mixed with the seeds at the assigned application rate to ensure uniform adhesion. The coated seeds were then air-dried in the shade and sown within 12 h. We have clarified this information in the revised manuscript.

The sentence now reads（Lines 117-121）: The seed-coating product used in this study was a rhizobial-based composite mi-crobial inoculant, primarily composed of rhizobia and supplemented with phos-phate-solubilizing bacteria and Bacillus subtilis. The inoculant contained ≥ 5 × 10⁸ CFU·mL⁻¹ viable cells and ≥ 6% total nutrients (N + P₂O₅ + K₂O). The seed coating content and ratio were strictly adhered to according to industry standards (NY/T 798-2006) and previous optimization studies

Comment 3. Paragraph 2.2, page 4, lines135. Authors provide the date of soil sampling, but there was no date of experiment starting.

Response: We thank the reviewer for this helpful suggestion. We agree that the experimental duration should be clearly stated. In the revised manuscript, we have added the sowing date and clarified that the experiment was conducted during a single growing season in 2023, from sowing to physiological maturity. This addition defines the temporal scope of the study and helps readers interpret the short-term treatment effects.

The sentence now reads（Lines 105-107）: The field experiment was initiated on May 1, 2023, and continued for one growing season until Medicago sativa L. reached physiological maturity in October 2023. At physiological maturity, five sampling points were randomly selected along the diagonal of each plot.

Comment 4. lines 145. Please clarify the sonication parameters. This is important. Please, provide some information about duration of experiment.

Response:We thank the reviewer for this important comment. In the original manuscript, we only stated that the samples were sonicated for 10 min, without providing sufficient methodological clarification. The rhizosphere soil detachment procedure followed an established internal protocol developed by our research group. As some detailed optimization parameters of this protocol are part of an unpublished methodological procedure, we are not able to disclose all technical details at this stage.

Nevertheless, to improve the transparency and reproducibility of the manuscript, we have clarified the essential information in the revised Methods section. Briefly, fresh roots with attached rhizosphere soil were suspended in sterile PBS buffer containing 0.1% Tween-80, vigorously shaken, and then sonicated for 10 min under low-temperature conditions to facilitate rhizosphere soil detachment while minimizing DNA degradation. The same sonication procedure was applied consistently to all samples. We have also added the duration of the field experiment, which lasted from May 1, 2023, to physiological maturity in October 2023.

The sentence now reads（Lines 144-147）: Fresh roots with attached rhizosphere soil were suspended in sterile PBS buffer containing 0.1% Tween-80, vigorously shaken, and then sonicated for 10 min under low-temperature conditions to facilitate rhizosphere soil detachment while minimizing DNA degradation. The same procedure was applied consistently to all samples.

Comment 5. Figure 4. Caption. “…Different lowercase letters indicate significant differences among treatments (p < 0.05).” It seems that in relation to this figure it would be more correct to indicate that “The same letters indicate the absence of significant differences between the treatments”

Response: We thank the reviewer for this careful observation. We agree that, because the panels in Figure 4 show the same lowercase letter among treatments, the caption should emphasize the absence of significant differences rather than significant differences. We have revised the figure caption accordingly to avoid ambiguity. We have made the requested revisions and indicated them in red text （Lines 314）.

All changes made in the revised manuscript are highlighted in red for easy reference.

We sincerely thank the editors and reviewers for their valuable comments and dedicated efforts in evaluating our work.

 

Author Response File: Author Response.pdf