Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript investigates the modulation of host-dependent endophytes (HDEs) in grapevine callus under antibiotic treatments and explores associated physiological shifts. The integration of microbiome and plant physiological analyses is promising, but the manuscript requires major revisions before publication due to issues with methodological clarity, data interpretation, and figure quality.

The absence of Table 3, which describes antibiotic formulations, is a significant oversight. The authors should include detailed information on antibiotic concentrations, stock solution types, rationale for antibiotic selection, expected activity spectrum, and a clear description of the experimental design.

The timing of microbiome DNA extraction is not clearly specified, creating ambiguity regarding when DNA extraction occurred. Clarifying whether extraction took place at 45 days or at specific growth stages is crucial for interpreting the microbiome results accurately.

Several chemical formulas use normal-sized numbers instead of subscripts (e.g., H2O2, KH2PO4). These should be corrected (H₂O₂, KH₂PO₄). There are also spacing errors (e.g., lines 115, 137, and 274). A careful review for typographical consistency is recommended.

Acknowledging the large intra-treatment variability in alpha diversity values and the lack of significant differences among treatments in Tables 1–2 is essential. This variability may stem from long-term subculture effects, physiological heterogeneity, or technical variation and should be discussed.

Figures 1, 3, 4, and 7 lack clarity and resolution, hindering interpretation. Enhancing sharpness, labeling, and visual quality would greatly improve readability.

Line 209 should be corrected from “Bacteroidotateria” to Bacteroidota.

While the Results section is generally clear, some interpretations may need moderation. Distinguishing between statistically significant findings and descriptive trends would enhance accuracy.

The Discussion section provides valuable context and comparisons with previous studies but could benefit from moderating interpretations based on correlations or predictive analyses and acknowledging replicate variability to ensure alignment between conclusions and data.

ment variability in alpha diversity values and the lack of significant differences among treatments in Tables 1–2 is essential. This variability may stem from long-term subculture effects, physiological heterogeneity, or technical variation and should be discussed.

Figures 1, 3, 4, and 7 lack clarity and resolution, hindering interpretation. Enhancing sharpness, labeling, and visual quality would greatly improve readability.

Line 209 should be corrected from “Bacteroidotateria” to Bacteroidota.

While the Results section is generally clear, some interpretations may need moderation. Distinguishing between statistically significant findings and descriptive trends would enhance accuracy.

The Discussion section provides valuable context and comparisons with previous studies but could benefit from moderating interpretations based on correlations or predictive analyses and acknowledging replicate variability to ensure alignment between conclusions and data.

Author Response

Dear editor and reviewers,

The authors are greatly appreciated for those valuable comments, which were all helpful to improve our manuscript. The present version has been revised according to comments, and the point to point response to comments was attached as followed:

Response to Reviewer 1’ Comments

Comments 1: The manuscript investigates the modulation of host-dependent endophytes (HDEs) in grapevine callus under antibiotic treatments and explores associated physiological shifts. The integration of microbiome and plant physiological analyses is promising, but the manuscript requires major revisions before publication due to issues with methodological clarity, data interpretation, and figure quality.

Response 1: We are grateful for the reviewer’s encouraging remarks and constructive suggestions. We have thoroughly addressed each comment in the revised version of the manuscript. All changes have been highlighted for the reviewer’s convenience. Thank you for helping us improve the quality of this work.

Comments 2: The absence of Table 3, which describes antibiotic formulations, is a significant oversight. The authors should include detailed information on antibiotic concentrations, stock solution types, rationale for antibiotic selection, expected activity spectrum, and a clear description of the experimental design.

Response 2: We thank the reviewer for this important observation. As suggested, we have now included Table 3（Table 1 in revised manuscript）, which provides a detailed description of the antibiotic formulations used in this study. The new table includes the antibiotic names, concentrations, solvent types for stock solutions, the rationale for their selection, and their expected activity spectrum. We believe these additions significantly improve the clarity and reproducibility of our work.

Comments 3: The timing of microbiome DNA extraction is not clearly specified, creating ambiguity regarding when DNA extraction occurred. Clarifying whether extraction took place at 45 days or at specific growth stages is crucial for interpreting the microbiome results accurately.

Response 3: We thank the reviewer for highlighting this ambiguity. The timing has now been clearly specified in the revised manuscript. Specifically, we have added details stating that the callus samples for microbiome DNA extraction were collected and processed after 45 days of subculture on antibiotics-free medium,, upon reaching the biomass plateau.

Comments 4: Several chemical formulas use normal-sized numbers instead of subscripts (e.g., H2O2, KH2PO4). These should be corrected (H₂O₂, KH₂PO₄). There are also spacing errors (e.g., lines 115, 137, and 274). A careful review for typographical consistency is recommended.

Response 4: We are deeply grateful to the reviewer for their meticulous attention to detail in identifying the issues with chemical formulas and spacing. As suggested, we have revised all instances of chemical formulas to ensure proper subscript notation is used. Furthermore, we have corrected the spacing errors on the specified lines and have extended this careful review to the entire manuscript to guarantee overall typographical consistency.

Comments 5: Acknowledging the large intra-treatment variability in alpha diversity values and the lack of significant differences among treatments in Tables 1–2 is essential. This variability may stem from long-term subculture effects, physiological heterogeneity, or technical variation and should be discussed.

Response 5: In this version, we have briefly discussed the issue at the end of discussion section. Thank you!

Comments 6: Figures 1, 3, 4, and 7 lack clarity and resolution, hindering interpretation. Enhancing sharpness, labeling, and visual quality would greatly improve readability.

Response 6: We are grateful to the reviewer for pointing out the issues with the visual quality of the figures. In response, we have remade Figures 1, 3, 4, and 7 with a focus on increasing the resolution, sharpening the images, improving the clarity of all labels, and optimizing the overall layout. We believe these revisions have significantly improved their readability, and we thank the reviewer for the suggestion.

Comments 7: Line 209 should be corrected from “Bacteroidotateria” to Bacteroidota.

Response 7: We have corrected "Bacteroidotateria" to "Bacteroidota" on line 207 in the revised manuscript. Thank you!

Comments 8: While the Results section is generally clear, some interpretations may need moderation. Distinguishing between statistically significant findings and descriptive trends would enhance accuracy.

Response 8: In this revision, we have endeavoured to amend the descriptions of the results. Thank you!

Comments 9: The Discussion section provides valuable context and comparisons with previous studies but could benefit from moderating interpretations based on correlations or predictive analyses and acknowledging replicate variability to ensure alignment between conclusions and data.

Response 9: A brief discussion concerning the replicate variability have added in the discussion section, thank you!

Reviewer 2 Report

Comments and Suggestions for Authors

The characterization and manipulation of host-dependent endophytes (HDEs) in in vitro-cultured grapevine calli is a relevant topic that provides novel insights into an underexplored aspect of plant–microbiome interactions. The results, showing that antibiotic treatments can shape specific endophytic communities and affect callus physiological parameters, contribute valuable information to our understanding of the relationship between endophytic microenvironments and host metabolism.

I consider this manuscript to be solid and well-structured. The experimental design is good and appropriate to address the research questions. I would like to highlight the precision and rigor of the statistical analyses presented, supporting the conclusions.

For all these reasons, I recommend accept for publication in its current form since the manuscript meets the expected standards of scientific quality for publication.

Author Response

Response to Reviewer 2’ Comments

Comments 1: The characterization and manipulation of host-dependent endophytes (HDEs) in in vitro-cultured grapevine calli is a relevant topic that provides novel insights into an underexplored aspect of plant–microbiome interactions. The results, showing that antibiotic treatments can shape specific endophytic communities and affect callus physiological parameters, contribute valuable information to our understanding of the relationship between endophytic microenvironments and host metabolism.

I consider this manuscript to be solid and well-structured. The experimental design is good and appropriate to address the research questions. I would like to highlight the precision and rigor of the statistical analyses presented, supporting the conclusions.

For all these reasons, I recommend accept for publication in its current form since the manuscript meets the expected standards of scientific quality for publication.

Response 1: Thank you very much for your thorough review of our manuscript and for your highly positive and encouraging feedback. We are truly honored that you consider our work to be solid, well-structured, and relevant, and we sincerely appreciate your acknowledgment of the novelty and value of our findings. Your recognition of the experimental design and the precision of the statistical analyses is particularly encouraging.

We are delighted that you recommend acceptance of the manuscript in its current form, and we thank you once again for your time and supportive comments.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

 

In the manuscript titled “Identifying the physiological traits of host-dependent endophytes in grapevines using callus as the host material” you have performed a scientifically sound experiments and explored an important but relatively underexplored topic which is, the role of host-dependent endophytes in plant`s callus. In this case, in grapevine callus.

You described the antibiotic treatments designed to modulate endophytic communities and to check if there are correlations between these changes and physiological traits of plants. I find this approach innovative and relevant for plant microbiome research.

 

I have a few minor remarks:

 

Did you do the untreated callus baseline comparison for long-term culture effects? Could you describe it more clearly?

 

Please include a statistical significance for observed differences (e.g., alpha diversity, physiological traits)

 

A functional predictions: BugBase and FUNGuild are presented, but validation of functional roles (e.g., metabolite assays, gene expression) is missing. If I am right please state clearly tat those are only predictions.

 

I would recommend to avoid too speculative sentences like, e.g. “risks to host health” based on predicted pathogenicity.

 

Please verify if figures concerning e.g. correlation networks, need more clear legends and quantitative interpretation.

 

You should summarize briefly the supplementary tables in the text. You have referenced it, but I lack mentioning it in the text.

 

In the abstract, and in the manuscript in general, please highlight or emphasize more, the key findings and their significance, e. g. that:

Antibiotic treatments reshape host-dependent bacterial communities.

That those changes correlate with physiological traits of grapevine callus, and that that the system provides a model for studying host dependent bacteria and their functional roles.

 

Kind regards

 

Author Response

Dear editor and reviewers,

The authors are greatly appreciated for those valuable comments, which were all helpful to improve our manuscript. The present version has been revised according to comments, and the point to point response to comments was attached as followed:

Response to Reviewer 3’ Comments

Comments 1: In the manuscript titled “Identifying the physiological traits of host-dependent endophytes in grapevines using callus as the host material” you have performed a scientifically sound experiments and explored an important but relatively underexplored topic which is, the role of host-dependent endophytes in plant`s callus. In this case, in grapevine callus.

Response 1: Thank you very much for your thoughtful review and for your generous positive feedback on our manuscript. We are truly encouraged by your assessment that our work is "scientifically sound" and explores an "important but relatively underexplored topic." Your recognition of the significance of studying host-dependent endophytes in the grapevine callus system is deeply appreciated and serves as a great motivation for our continued research in this area. We are delighted that you found value in our approach and findings.

Comments 2: You described the antibiotic treatments designed to modulate endophytic communities and to check if there are correlations between these changes and physiological traits of plants. I find this approach innovative and relevant for plant microbiome research.

I have a few minor remarks:

Did you do the untreated callus baseline comparison for long-term culture effects? Could you describe it more clearly?

Please include a statistical significance for observed differences (e.g., alpha diversity, physiological traits)

Response 2: In this experiment, we compared differently treated callus over the same period of subculture without preparing primary callus that had not undergone subculture. Comparing the HDE dynamics in callus before and after long-term subculture presents a fascinating research direction, which we may consider incorporating into future experimental designs. Thank you very much!

Comments 3: A functional predictions: BugBase and FUNGuild are presented, but validation of functional roles (e.g., metabolite assays, gene expression) is missing. If I am right please state clearly tat those are only predictions.

I would recommend to avoid too speculative sentences like, e.g. “risks to host health” based on predicted pathogenicity.

Response 3: We sincerely thank the reviewer for raising this important point. We fully agree that the functional insights should be clearly presented as computational predictions. In response, we have thoroughly revised Section 3.2 to explicitly state that the findings from BugBase and FUNGuild are strictly in silico predictions, not validated by functional assays. The language has been carefully modified throughout to be more cautious and speculative, removing overly strong conclusions to ensure the narrative accurately reflects the predictive nature of these analyses.

Comments 4: Please verify if figures concerning e.g. correlation networks, need more clear legends and quantitative interpretation.

Response 4: We extend our sincere thanks to the reviewer for this valuable comment. In direct response to it, we have replaced the original figure with a new, clearer version (Figure 7) that significantly enhances readability. Furthermore, we have thoroughly revised the accompanying legend to provide a more detailed and quantitative interpretation of the data, as suggested. We believe these revisions have substantially improved the figure, and we welcome the reviewer's further assessment.

Comments 5: You should summarize briefly the supplementary tables in the text. You have referenced it, but I lack mentioning it in the text.

Response 5: We thank the reviewer for pointing out the need for better presentation of the supplementary tables and figures. In response, we have now consolidated all supplementary tables and figures into a dedicated Appendix section at the end of the manuscript. We hope that having the tables readily available in the appendix facilitates a smoother review process. Thank you for your attention to this matter.

Comments 6: In the abstract, and in the manuscript in general, please highlight or emphasize more, the key findings and their significance, e. g. that:

Antibiotic treatments reshape host-dependent bacterial communities.

That those changes correlate with physiological traits of grapevine callus, and that that the system provides a model for studying host dependent bacteria and their functional roles.

Response 6: Thank you for these good suggestions. The relevant sections have been revised to the best of our ability to highlight the key findings and conclusions of the research.