Rhodiola rosea L. Essential Oil Reduces Postharvest Strawberry Decay by Disrupting Botrytis cinerea Cell Wall and Membrane Integrity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work presents novel results. From my point of view it can be published after some corrections (indicated in the attachment 

Comments for author File: Comments.pdf

Author Response

The findings of this work suggest REO exhibits antifungal activity against B. cinerea which reduces postharvest decay in strawberry. From my point of view this work presents novel results and can be published after some corrections.

Response: I appreciate your positive assessment of the novelty of our findings concerning the antifungal activity of REO against B. cinerea and its role in reducing postharvest decay in strawberries, as well as your support for its publication after revision. We have fully incorporated your suggestions and have carefully addressed all specific corrections in the revised manuscript to enhance the clarity and interpretation of the results. All modifications in the manuscript are marked in red font.

Comments 1: It is necessary to describe the origin of the fruit. Was it bought in the market or harvested? How long did it take between obtaining the fruit and the treatment application?

Response 1: Thank you for your valuable comment. The source of the experimental material and the timing of treatment are indeed critical to ensuring the consistency of the research results. We have added and refined the relevant information in the “Materials and Methods” section (Section 2.1) of the revised manuscript as follows:

1. The strawberries (Fragaria × ananassa cv. Akihime) used in this study were manually harvested in January 2025 from a local standardized strawberry plantation. At harvest, strawberries were strictly selected for uniform maturity (approximately 80% surface red coloration), size, and appearance, with no mechanical damage or visible disease.
2. The average daily temperature during the harvest period was 5-10℃, which was suitable for the short-term storage and transport of strawberries. Therefore, no active temperature control was used during transportation. The strawberries were transported to the laboratory within 1 hour after harvesting and were immediately sorted and prepared upon arrival. The essential oil preservation treatment was completed within 4 hours post-harvest to ensure consistent initial physiological status across all treatment groups and to minimize the influence of post-harvest physiological changes on the experimental outcomes.

These additions aim to enhance the transparency and reproducibility of the experimental methodology. We appreciate your careful review and constructive suggestions regarding this section.

Comments 2: Please check the citations indicated as Error! Reference source not found.

Response 2: Thank you for your comment. We have carefully revised the manuscript to ensure that all figure references in the Results section are correctly cited. References to “Error! Reference source not found” have been replaced with the appropriate figure labels.

Comments 3: Please indicate the name of the equipment used for the Analysis of Intracellular ROS accumulation.

Response 3: We thank the reviewer for raising this important point. In the revised manuscript, we have explicitly stated that intracellular ROS accumulation was analyzed using an inverted fluorescence microscope (TS100, Nikon, Shanghai, China). The text has been updated accordingly to ensure clarity regarding the instrumentation employed.

Comments 4: I suggest to use the term “fruit” as the plural of fruit, instead of “fruits”. Fruits, as plural, is used when it refers to different species.

Response 4: Thank you for your valuable suggestion. We have revised the manuscript according to your comment. In the revised version, we now consistently use “fruit” as the plural form of “fruit,” reserving “fruits” only for instances that specifically refer to different types or species of fruit. This adjustment has been applied throughout the entire text to ensure terminological accuracy and consistency.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Congratulations, the study is interesting and comprehensive, however, adjustments are needed in the document (see details in the attached document).

Comments for author File: Comments.pdf

Author Response

Congratulations, the study is interesting and comprehensive, however, adjustments are needed in the document.

Response: Thank you for your positive feedback and for acknowledging the interest and comprehensiveness of our study. We sincerely appreciate your encouraging comments. We have carefully addressed all the specific suggestions raised during the review and have made necessary revisions throughout the manuscript, particularly in improving the accuracy of the methodology and results description, to enhance its clarity and overall quality. All modifications in the manuscript are marked in red font.

Comments 1: L2 Rhodiola sp.???Italic (Rhodiola), L3 Italic, L14 fruits strawberries, L94 In vitro?, L99 days??, L120 rewrite this assay, L122 This? see line 120, L166 no italic (here in titles), L167, L180, L184 check, L406 standardize.

Response 1: Thank you for your helpful comment. We have revised them in the manuscript and all revisions in the paper are indicated in red font. We appreciate your attention to these details.

Comments 2: L37: Could it be that things haven't changed after 5 years??If possible, please update the information/reference.

Response 2: Thank you for raising this important point regarding data timeliness. We have replaced the previously cited 2020 data with the latest strawberry production figures released by the Food and Agriculture Organization of the United Nations (2024) and updated the reference accordingly.

[1] Food and Agriculture Organization of the United Nations. Crops and Livestock Products: Strawberries. 2024. Available online: https://www.fao.org/faostat/zh/#data/QCL/visualize (accessed on 5 February 2026).

Comments 3: L84: isolation of Botitris cinerea fungus

Response 3: Thank you for your suggestion regarding the section heading. We have decided to retain the title “2.1. Materials” for the following reason: this subsection covers not only the origin of the fungal isolate but also describes the sources of the strawberry fruit and Rhodiola rosea L. essential oil used in the study. Using “Materials” as the heading allows for a comprehensive overview of all experimental materials included in this section. We appreciate your review and valuable comments.

Comments 4: L109: see line 97 correspond to some concentration diluted of the line 19 used?

Response 4: Thank you for your question regarding the concentration used. The 20 µL/L concentration for the in vivo fumigation does not correspond to a dilution from the in vitro assay, as the methods differ fundamentally (vapor-phase fumigation vs. liquid-phase agar dilution). This concentration was selected based on preliminary experiments. As shown in the Figure 1, REO vapor at different concentrations (10 µL/L, 20 µL/L, 40 µL/L) was tested on strawberries. Results indicated that the 20 µL/L concentration provided optimal control efficacy against gray mold without inducing phytotoxicity. We have clarified in the revised text that this is a headspace concentration for fumigation.

Figure 1. In vivo preliminary experiment. REO: Rhodiola rosea L. essential oil. The control group did not undergo essential oil vaporization. The REO-treated group underwent REO vaporization at headspace concentrations of 10, 20, and 40 µL/L, respectively. The area enclosed by the white circle in the image indicates where the essential oil caused phytotoxicity to the strawberries.

Comments 5: L112: Total N = 54 fruits? see line 106

Response 5: Thank you for raising this point for clarification. As suggested, we have revised the text to explicitly state the experimental design: Three independent biological replicates were employed in this experiment, with each replicate comprising 18 strawberries (54 strawberries in total). This information has been added to Section 2.3 in the revised manuscript.

Comments 6: L123: what period? (how many days)

Response 6: Thank you for your comment. We have clarified in the revised text (Section 2.4) that the natural storage experiment was conducted over a 3-day period.

Comments 7: L126: what treatments?? mentioned, please.

Response 7: Thank you for the comment. We have now specified the treatments in Section 2.4 as follows: a control group and an REO‑fumigated group. The control group did not undergo essential oil vaporization, while the REO‑fumigated group was vaporized with REO vapor at a headspace concentration of 20 μL/L.

Comments 8: L161: detail what values were exposed to respective statistic analisys, all results were by ANOVA?

Response 8: Thank you for your comment. We have revised Section 2.9 to detail the statistical approaches: For experiments involving three or more groups, one‑way analysis of variance (ANOVA) followed by Duncan’s multiple comparison test was applied to determine significant differences among treatments. For comparisons between two independent groups, Student’s t‑test for independent samples was used.

Comments 9: L172: references are commons in methodology, but this are results,

Response 9: Thank you for your comment. We have revised the manuscript accordingly by removing the citation from the Results section (3.1).

Comments 10: Table 1: the table should be digital here, not figure, print. build the table, please.

Response 10: Thank you for your comment. Table 1 has been revised as a properly formatted digital table and updated in the manuscript.

Comments 11: L188: in the respective text mentioned, please the figure (Figure 1A) or (Figure 1B); in the respective text mentioned, please the figure (Figure 2A) or (Figure 2B) or Figure 2C, this suggestion in all figures, and text please.

Response 11: Thank you for your comment, we have standardized all references to the subfigures of all Figures throughout the manuscript and figure captions to the consistent format "Figure A, Figure B, Figure C".

Comments 12: L191: what concentration??? write, please.

Response 12: Thank you for your question. The concentration of REO used in the in vivo fumigation experiment is 20 µL/L. We have indicated this in the figure.

Comments 13: L218: what concentration here??? in each figure added the concentration used, please; L242 REO (what concentration)?

Response 13: Thank you for your question. The concentration of REO used in the in vivo fumigation experiment is 20 µL/L. We have indicated this in the figure.

Comments 14: L321: see observations above about write of results citing the figures, please.

Response 14: Thank you for your comment. We have carefully revised the manuscript to ensure that all figure references in the Results section are correctly cited. In Section 3.8, references to “Error! Reference source not found” have been replaced with the appropriate figure labels.

Comments 15: L387: see style of journal, please.

Response 15: Thank you for your comment. We have carefully reviewed and revised the reference list to align with the journal’s required style.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Rhodiola essential oil reduces postharvest strawberry decay by disrupting Botrytis cinerea cell wall and membrane integrity” addresses an important and timely topic in postharvest pathology, namely the search for natural, environmentally friendly alternatives to synthetic fungicides for the control of gray mold in strawberries. The overall experimental framework, combining in vitro antifungal assays, in vivo fruit trials, and mechanistic investigations at the cellular level, is logically structured and generally appropriate for the scope of a horticultural science journal. However, while the study presents coherent results, several conceptual, methodological, and interpretative shortcomings need to revised and substantial revisions are required to strengthen the manuscript.

 

From the perspective of novelty, the research question is moderately original. Essential oils have been extensively studied as antifungal agents against Botrytis cinerea, and the proposed mode of action - disruption of cell wall and membrane integrity - is a well-established paradigm in essential oil research. Although the use of Rhodiola rosea essential oil (REO) is relatively less explored compared with more common oils (e.g., thyme, oregano, clove), the manuscript does not sufficiently articulate how REO differs mechanistically or functionally from previously studied essential oils. To improve the novelty and scientific positioning of the study, the authors should explicitly define what new conceptual or mechanistic insight is provided beyond confirming a broadly known antifungal mechanism. A clearer hypothesis-driven framework in the Introduction, rather than a largely descriptive rationale, would significantly enhance the originality of the work.

 

Regarding scope, the study fits well within the aims of journals focusing on postharvest biology, horticultural quality preservation, and natural plant protection strategies. The integration of disease control efficacy with fruit quality parameters is appropriate and relevant.

 

In terms of significance and interpretation, the results demonstrate that REO can reduce gray mold severity and delay quality deterioration in strawberries, and the conclusions are generally consistent with the presented data. Nevertheless, the biological and practical significance of the observed effects appears somewhat overstated. Even after REO treatment, disease severity and incidence remain relatively high, and the manuscript does not provide a benchmark comparison with either conventional fungicides or other essential oils. The authors are therefore encouraged to temper claims related to “effective suppression” and “marketability preservation,” or alternatively to include comparative controls that would allow a more robust evaluation of REO performance.

 

The overall quality of presentation is acceptable. The manuscript is generally readable, and the structure is clear; however, numerous formatting and referencing errors (e.g., repeated “Error! Reference source not found”). These errors needs to be corrected. Furthermore, the manuscript should clearly distinguish between biological and technical replicates, as this is currently ambiguous.

 

Finally, the English language is generally understandable but requires editing. Some sentences are awkwardly phrased, and occasional grammatical inaccuracies reduce clarity.

 

Specific Suggestions and Comments

L3: Botrytis cinerea – should be in italic

L34-82: Introduction

The introduction provides a comprehensive background on strawberry postharvest losses, the economic importance of strawberries, and the potential of essential oils as natural antifungal agents. The novelty and hypothesis-driven aspect should be more emphasized and provide clear research gaps.

 

L165: Results and Discussion section should be divided. Discussion is superficial and a more deep discussion should be provided with comparison with relevant literatures. Also provide the future aspects and limitation of the study.

 

L193: Figures 1-8. Give in full REO

L360: Figure 8: The title is too short.

 

L361-367: The Conclusion should be rewritten giving more details and also provide bullet points

 

Formatting of References should be done.

 

Author Response

The manuscript entitled “Rhodiola essential oil reduces postharvest strawberry decay by disrupting Botrytis cinerea cell wall and membrane integrity” addresses an important and timely topic in postharvest pathology, namely the search for natural, environmentally friendly alternatives to synthetic fungicides for the control of gray mold in strawberries. The overall experimental framework, combining in vitro antifungal assays, in vivo fruit trials, and mechanistic investigations at the cellular level, is logically structured and generally appropriate for the scope of a horticultural science journal. However, while the study presents coherent results, several conceptual, methodological, and interpretative shortcomings need to revised and substantial revisions are required to strengthen the manuscript.

Response: Thank you for your positive feedback on our research. Based on your comments, we have carefully revised the issues in the manuscript, with particular attention to improving the accuracy of the results description and enhancing the detail in the conclusions. All modifications are marked in red font.

Comments 1: From the perspective of novelty, the research question is moderately original. Essential oils have been extensively studied as antifungal agents against Botrytis cinerea, and the proposed mode of action - disruption of cell wall and membrane integrity - is a well-established paradigm in essential oil research. Although the use of Rhodiola rosea essential oil (REO) is relatively less explored compared with more common oils (e.g., thyme, oregano, clove), the manuscript does not sufficiently articulate how REO differs mechanistically or functionally from previously studied essential oils. To improve the novelty and scientific positioning of the study, the authors should explicitly define what new conceptual or mechanistic insight is provided beyond confirming a broadly known antifungal mechanism. A clearer hypothesis-driven framework in the Introduction, rather than a largely descriptive rationale, would significantly enhance the originality of the work.

Response 1: Thank you for your valuable suggestions regarding enhancing the originality and hypothesis-driven nature of this study. In response to your comments, we have revised the manuscript as follows:

In the Introduction, we have restructured the research background to establish a clear research hypothesis as the driving force. Building on the documented bioactivity of specific components (geraniol and linalool) in Rhodiola rosea L. essential oil reported in the literature, we explicitly propose the scientific hypothesis that it may exhibit antifungal effects against Botrytis cinerea and involve related mechanisms, thereby strengthening the logical foundation and exploratory direction of the study. In the Discussion sections, beyond validating the classic mechanism of disrupting cell membrane/wall integrity, we further integrate relevant literature to explore additional potential antifungal pathways and unique modes of action associated with this essential oil. This provides a more comprehensive conceptual perspective for understanding its antimicrobial activity. The specific modifications have been highlighted in red font in the manuscript for your convenience. Thank you once again for your guidance.

Comments 2: In terms of significance and interpretation, the results demonstrate that REO can reduce gray mold severity and delay quality deterioration in strawberries, and the conclusions are generally consistent with the presented data. Nevertheless, the biological and practical significance of the observed effects appears somewhat overstated. Even after REO treatment, disease severity and incidence remain relatively high, and the manuscript does not provide a benchmark comparison with either conventional fungicides or other essential oils. The authors are therefore encouraged to temper claims related to “effective suppression” and “marketability preservation,” or alternatively to include comparative controls that would allow a more robust evaluation of REO performance.

Response 2: Thank you for your rigorous and meticulous suggestions on the manuscript. We have revised the wording in the relevant results sections to ensure greater objectivity and precision. Regarding the issue that strawberries treated with REO still showed severe disease on day 3 (as shown in Figure 1A), this may primarily be attributed to the use of mycelial disc inoculation in our experiment, which resulted in a very rapid disease progression and partially masked the treatment effect of REO. In the experiment, we attempted to inoculate using B. cinerea spore suspensions; however, we were unable to obtain an ideal quantity of spores and thus ultimately opted for mycelial disc inoculation. Nevertheless, the efficacy of REO in preserving strawberries was quite evident in controlling disease under natural storage conditions (as shown in Figure 2A). To maintain precise expression, we have adjusted the wording to more appropriate terms such as "effective suppression" and "marketability preservation" throughout the text, to better align with the experimental outcomes. To better contextualize the efficacy of REO, we have added a comparative discussion in the revised manuscript (lines 273-276), comparing its antifungal performance with that of other well-studied essential oils reported in the literature. This provides a clearer framework for assessing REO's potential and limitations. All modifications have been marked in red text in the manuscript. We appreciate your constructive feedback, which has strengthened the clarity and rigor of this study.

Comments 3: The overall quality of presentation is acceptable. The manuscript is generally readable, and the structure is clear; however, numerous formatting and referencing errors (e.g., repeated “Error! Reference source not found”). These errors needs to be corrected. Furthermore, the manuscript should clearly distinguish between biological and technical replicates, as this is currently ambiguous.

Response 3: Thank you for your positive feedback on the readability, structure, and overall quality of our manuscript. We have carefully addressed the issues you raised and made the following revisions accordingly:

1. All citation errors (including instances of “Error! Reference source not found”) have been corrected. The reference list has been thoroughly reviewed to ensure consistent formatting and accurate information.
2. We have revised the sections on “biological replicates” and “technical replicates” in the Methods chapter to eliminate potential ambiguities in the original text.

All modifications have been marked in red text in the manuscript. Thank you again for your thorough and professional review.

Comments 4: Finally, the English language is generally understandable but requires editing. Some sentences are awkwardly phrased, and occasional grammatical inaccuracies reduce clarity.

Response 4: Thank you for your suggestion regarding the need for language improvement. In response, we have carefully revised the entire manuscript to correct grammatical inaccuracies and rephrase awkward sentences, thereby enhancing the overall clarity and readability. On this basis, we have commissioned Elsevier Author Services to professionally revise the language issues present in the manuscript, and we hope that the quality after revision will meet your requirements. All changes have been highlighted in red font in the document for your convenience during review.

Comments 5: L3: Botrytis cinerea – should be in italic.

Response 5: Thank you for your careful review. We apologize for our oversight. We have now consistently displayed Botrytis cinerea in italics.

Comments 6: L34-82: Introduction The introduction provides a comprehensive background on strawberry postharvest losses, the economic importance of strawberries, and the potential of essential oils as natural antifungal agents. The novelty and hypothesis-driven aspect should be more emphasized and provide clear research gaps.

Response 6: Thank you for your valuable feedback. We have revised the Introduction section to better highlight the hypothesis-driven nature and novelty of our study (lines 60-78). In the revised version, we first note that systematic research on Rhodiola rosea L. essential oil in the field of postharvest preservation is rarely reported. It then emphasizes that its main components (such as geraniol and linalool) have been proven to effectively extend the shelf life of fruits and vegetables after harvest. Based on this, we clearly state the hypothesis of this study: as a composite system containing known active ingredients, Rhodiola rosea L. essential oil may inhibit postharvest diseases and maintain quality in strawberries through synergistic effects. The specific modifications have been highlighted in red font in the manuscript for your convenience. Thank you once again for your guidance.

Comments 7: L165: Results and Discussion section should be divided. Discussion is superficial and a more deep discussion should be provided with comparison with relevant literatures. Also provide the future aspects and limitation of the study.

Response 7: Thank you for your suggestion. We have restructured the manuscript by separating the original combined "Results and Discussion" section into two distinct sections. In the Discussion section, we have systematically referenced and compared our findings with relevant prior research to better contextualize the outcomes. The Conclusion section now concisely summarizes the key findings of the study and further outlines potential directions for future research as well as acknowledged limitations. The specific modifications have been highlighted in red font in the manuscript for your convenience.

Comments 8: L193: Figures 1-8. Give in full REO

Response 8: Thank you for your suggestion. We have indicated the full term of REO in the figure caption.

Comments 9: L360: Figure 8: The title is too short.

Response 9: Thank you for your suggestion. We have amended the figure caption for Figure 8 to: The effect of different concentrations of REO treatment for 120 min on reactive oxygen species (ROS) accumulation in B. cinerea. REO: Rhodiola rosea L. essential oil. Green fluorescence indicates ROS levels, with enhanced intensity reflecting elevated accumulation.

Comments 10: L361-367: The Conclusion should be rewritten giving more details and also provide bullet points.

Response 10: Thank you for your suggestion. We have rewritten the conclusion section and supplemented it with the key findings derived from this research. We have also added a brief outlook on potential future research directions to enhance the completeness and forward-looking perspective of this section. All revisions in the paper are indicated in red font.

Comments 11: Formatting of References should be done.

Response 11: Thank you for pointing this out, we have reviewed and revised all references to ensure compliance with the journal’s formatting requirements.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The study is improved. It can be accepted.