Control of Neopestalotiopsis zimbabwana Using Origanum vulgare L. Essential Oil: Combined In Vitro, In Vivo and In Silico Approaches

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript horticulturae-3840721, titled “Control of Neopestalotiopsis zimbabwana Using Origanum vulgare L. Essential Oil: Combined in vitro, in vivo and in silico Approaches”, addresses an important paper investigating the effectiveness of an oregano essential oil (OEO)–based formulation against a plant disease in strawberries. However, in my opinion this paper must be revised in a major manner for reasons of forms and content.

Introduction

Could you briefly mention the recognized drawbacks of applying EOs in crops (e.g., volatility, photodegradation, phytotoxicity at elevated concentrations) in order to set the stage for why your in vivo formulation and efficacy data are especially significant.

Materials and Methods

You mention using the NIST library. Did you also use retention indices (e.g., from a standard alkane series) to confirm compound identities, or was identification based solely on mass spectral matching?

The in vivo formula consists of the hydrolate in the percentage of 67.7%. Please provide any characterization of HOV. Is there any water-soluble antimicrobial molecules from the process of distillation in it? How it is something other than a diluent has to be briefly explained.

Was there any phytotoxicity (foliar burn, chlorosis) noticed in the strawberry plants, particularly in the continuous weekly applications group for the formulation of OEO? It is an essential point for determining the practical application of the treatment.

Results

The table 2 is legible, but the compound numbers in the text (e.g., "the most abundant were.") are not quite the same as the # in the table 2. It's a minor point but verification for consistency would help smooth out flow. Also, compound #19 is "Unidentified"; could you give its tentative classification based on mass spectrum (e.g., "terpene hydrocarbon") or base peak?

Figure 3 and description are great. To make it even clearer, you may want to include a schematic or a panel in the figure that illustrates the treatment groups (1A, 1B, 2A, 2B, 3) in a visual manner.

Discussion

The paper must mention briefly the high in vitro MIC (40% v/v). Though the optimized formulation in vivo proved successful, what are the implications for cost-effectiveness, possible phytotoxicity, and commercial-scale field use? Is there a plan for synergizing OEO with other molecules to reduce the effective dosage?

While the in silico docking is a strength, the description could be moderated by recognition that this is a predictive model. Adding a sentence that this mechanism "needs additional biochemical verification (e.g., by means of enzyme inhibition assays)" would enhance the scientific rigor.

The extremely fast recrudescence of the disease upon termination of therapy is the main observation. You may wish to elaborate more on this aspect. Is this not evidence that OEO does not have any eradicant or systemic action but is solely protective? What are the consequences for its utilization in an integrated pest management (IPM) program (it has to be administered frequently and preventively)?

Conclusion

It is a very good conclusion. You can make it better by starting with a sentence looking into the future. E.g.: "Future research must be directed at formulation optimisation for maximum persistence and residual activity in the field, and at demonstrating the proposed mode of action by definitive biochemical tests."

Minor Revisions

• Abbreviations: "Anex" is provided but not defined in the text. It seems to be referring to the adjuvant/stabiliser (Tween-20?). It should be corrected for clarity.
• Overall: Perform a rigorous proofreading for subtle grammatical mistakes and flow (e.g., "The research investigates the assumption that." becomes "This study tested the hypothesis that.").

 

Author Response

Response to Reviewer 1 Comments

Introduction

Comment 1: Could you briefly mention the recognized drawbacks of applying EOs in crops (e.g., volatility, photodegradation, phytotoxicity at elevated concentrations) in order to set the stage for why your in vivo formulation and efficacy data are especially significant.

Response 1: Thank you very much for your valuable commentary. Following your suggestion, I revised the introduction to include both the advantages of low-dose essential-oil regimens and the drawbacks associated with higher concentrations or frequent applications. This addition contextualizes the importance of developing in vivo formulations that maximize efficacy while minimizing phytotoxicity L104-11.

Materials and Methods

Comment 2: You mention using the NIST library. Did you also use retention indices (e.g., from a standard alkane series) to confirm compound identities, or was identification based solely on mass spectral matching?]

Response 2: Compounds were identified solely by EI mass spectral matching against the NIST 2011 library (using match quality scores), For more clarity, we have included the NIST match scores in Table  L378-3771.

Comment 3: The in vivo formula consists of the hydrolate in the percentage of 67.7%. Please provide any characterization of HOV. Is there any water-soluble antimicrobial molecules from the process of distillation in it? How it is something other than a diluent has to be briefly explained.

Response 3: The HOV was not chemically characterized in our study. We now state in Materials and Methods that GC–MS analysis was performed exclusively on the oregano essential oil (OEO). We also clarify that HOV was obtained from the same distillation batch as OEO and describe its separation and storage (L189-19) In addition, we added one sentence in the Leaf phytotoxicity screen and formulations section to indicate why HOV is not a mere diluent and why its level was selected (L278-281) Finally, we recognize the absence of HOV compositional data as a limitation and state in the Conclusions (L520-525).

Comment 4: Was there any phytotoxicity (foliar burn, chlorosis) noticed in the strawberry plants, particularly in the continuous weekly applications group for the formulation of OEO? It is an essential point for determining the practical application of the treatment.

Response 4: Thank you for this critical observation regarding the potential phytotoxicity of OEO under continuous weekly applications. As noted in our response to Comment R1, effects such as foliar burns and leaf discoloration are recognized drawbacks when essential oils are applied at elevated concentrations. In our experiments, we did observe phytotoxicity when increasing the concentration during the justification of the 0.6% OEO used in our botanical formulation. Therefore, we have now integrated into the Materials and Methods section a detailed description of the phytotoxicity test, without explicitly stating the 0.6% OEO value in that section (L266-279) And present the result of the leaf phytotoxicity text (L 396-400)

Comment 5: Figure 3 and description are great. To make it even clearer, you may want to include a schematic or a panel in the figure that illustrates the treatment groups (1A, 1B, 2A, 2B, 3) in a visual manner.

Response 5: We sincerely thank the reviewer for this valuable suggestion. In response, we have included a new Figure 1 (L 299-300), which presents a schematic of the treatment allocation.

Results

Comment 6: The table 2 is legible, but the compound numbers in the text (e.g., "the most abundant were.") are not quite the same as the # in the table 2. It's a minor point but verification for consistency would help smooth out flow. Also, compound #19 is "Unidentified"; could you give its tentative classification based on mass spectrum (e.g., "terpene hydrocarbon") or base peak?

Response 6: We thank the reviewer for the careful reading. In the revised manuscript we have replaced the preliminary composition table with the final GC–MS table (Table 1, L376-377), which now reports 18 identified constituents in retention‑time order and includes library match scores. The earlier “Unidentified (#19)” entry was a mistake from an intermediate draft version and has been removed. We also corrected a typographical mislabeling: α-terpinolene is now correctly reported as α-terpineol (1.05%). Two minor percentage values were harmonized during this consolidation: hexadecanoic acid from 14.98% to 15.98%, and trans‑β‑ocimene from 0.47% to 0.40%; the total remains ~100% within rounding. We also corrected all internal references in the text from “Table 2” to “Table 1.” The Results paragraph was updated accordingly (L 369-375). In the Abstract now reflects hexadecanoic acid at 15.98% (L 21) to maintain full internal consistency. Finally, we standardized compound names (e.g., thymol methyl ether, (Z)-sabinene hydrate, p‑cymene) and retained the Rt‑ordered presentation, which is standard for GC–MS reporting (Table 1, L376-377),. These corrections do not affect the study’s conclusions.

Discussion

Comment 9.1: The paper must mention briefly the high in vitro MIC (40% v/v). Though the optimized formulation in vivo proved successful, what are the implications for cost-effectiveness, possible phytotoxicity, and commercial-scale field use?

Response 9.1: Thank you very much for your thoughtful and experienced perspective. In our assays, the in vitro MIC was relatively high (30% v/v, corrected an updated value, L23, 282), yet preventive in planta applications were effective at only 0.6% v/v (NOAEL). This gap underscores that practical performance depends on preventive exposure rather than matching the in vitro MIC, with important implications for cost-effectiveness, plant safety (L 387-400), but we recnigce at se didnen include an apotencial alpicacon . and potential commercial application.

Comments 9.2: Is there a plan for synergizing OEO with other molecules to reduce the effective dosage?

Response 9.2: Thank you for this constructive suggestion. Yes, our next phase explicitly includes testing synergistic combinations of oregano essential oil (OEO) with other essential oils , to lower the minimal effective dose while maintaining (or improving) efficacy. We have already stated in the Conclusion section (L 507-535)

Comments 10: While the in silico docking is a strength, the description could be moderated by recognition that this is a predictive model. Adding a sentence that this mechanism "needs additional biochemical verification (e.g., by means of enzyme inhibition assays)" would enhance the scientific rigor.

Response 10: We appreciate this helpful suggestion and agree that the mechanistic interpretation drawn from docking should be explicitly framed as predictive and contingent on experimental validation. To strengthen this point, we added a final sentence in the docking results section that refers to how such verification could be carried out experimentally (L511-515).

Comments 11: The extremely fast recrudescence of the disease upon termination of therapy is the main observation. You may wish to elaborate more on this aspect. Is this not evidence that OEO does not have any eradicant or systemic action but is solely protective? What are the consequences for its utilization in an integrated pest management (IPM) program (it has to be administered frequently and preventively)?

Response 11. Belo the MFC the OE nless the minimum fungicidal concentration (MFC) is reached, OEO acts in a fungistatic manner: it suppresses growth only under continuous exposure, but does not eradicate the pathogen line 470. Relatedo to IPM programs, We emphasized this point in the the dicus seccion as the ulitizaicon (469-471), but we need to introduce the ocmbept ien te lines 27-28, 63-69, an using

Comment 12: It is a very good conclusion. You can make it better by starting with a sentence looking into the future. E.g.: "Future research must be directed at formulation optimisation for maximum persistence and residual activity in the field, and at demonstrating the proposed mode of action by definitive biochemical tests."

Response 12: Thank you for the helpful suggestion. We have revised and improved the Conclusions to begin with a forward‑looking paragraph that outlines our planned work: on (486-493).

Minor Revisions

Comment 1.1. Abbreviations: "Anex" is provided but not defined in the text. It seems to be referring to the adjuvant/stabiliser (Tween-20?). It should be corrected for clarity.

Response m.1.1. Thank you for catching this oversight, and we apologize for the confusion. Anex was a residual term from an earlier draft and is not used in the present study. The only adjuvant/stabilizer employed here is Tween 20 at 0.6% v/v in the fixed vehicle (Section 2.2). We have removed “Anex” from the Abbreviations list.

Comment m.1.2 Overall: Perform a rigorous proofreading for subtle grammatical mistakes and flow (e.g., "The research investigates the assumption that." becomes "This study tested the hypothesis that.").

Response m.1.2. We have carefully revised the section to adopt a more hypothesis-driven language (L130–134) and explicitly included a statement on future research directions (L486–493).

5. Additional clarifications

[Here, mention any other clarifications you would like to provide to the journal editor/reviewer.]

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript, entitled: "Control of Neopestalotiopsis zimbabwana Using Origanum vulgare L. Essential Oil: Combined in vitro, in vivo and in silico Approaches", presents the results of a study investigating alternative control options against an emerging, polyphagous pathogenic fungus. In addition to in vitro and in vivo efficacy testing, it also attempts to examine the effect of the essential oil inhibitor on the level of fungal mitochondrial proteins. The synthetic fungicidal agent captan is used as a positive control.

The introductory part is sufficiently detailed. It presents the occurrence of Neopestalotiopsis zimbabwana and its known host plants.
The material and methods chapter is also detailed, but the clarity is greatly impaired by the lack of subchapters. I suggest that the material and methods chapter be adapted to the structure of the results chapter.

I also recommend italicizing scientific names, e.g. in the case of Origanum vulgare (e.g. in line 177 of the manuscript). I also recommend giving the year of publication in the materials and methods section for the virtual grid method adapted from Feng et al. (line 273 of the manuscript).

The results section is sufficiently detailed and well illustrated. In the case of Figure 2, I suggest that the definitions of the abbreviations be published in the figure legend. I also suggest italicizing the term in planta (e.g., in line 440 of the manuscript). 

I also suggest expanding the discussion and conclusion chapters by analyzing the results of other related studies studying the phytopathological efficacy of essential oils.

After implementing the modifications and additions suggested above, I recommend publishing the manuscripts in the form of a scientific article.

Author Response

Comments 1: The material and methods chapter is also detailed, but the clarity is greatly impaired by the lack of subchapters. I suggest that the material and methods chapter be adapted to the structure of the results chapter.

Response 1: Thank you for your suggestion. We have now sectioned the text accordingly, as follows:. “2.1 Morphological and molecular identification (L132); 2.2 Plant material & hydrodistillation: OEO/HOV co collection, separation (L169); 2.3 GC–MS analysis of OEO (L184); 2.4 In vitro assay, MIC and MFC definitions (L198); 2.5 Leaf phytotoxicity screen and formulations (L143); 2.6 In vivo assays in strawberry plants (growth chamber): treatments, inoculation and disease assessment (L266); 2.7 In silico docking against cytochrome b (PDB: 5TL8) (L311). “

This restructuring directly addresses your request for parallelism with Results and clarifies methods chapter at first sight.

Comments 2: I also recommend italicizing scientific names, e.g. in the case of Origanum vulgare (e.g. in line 177 of the manuscript). I also recommend giving the year of publication in the materials and methods section for the virtual grid method adapted from Feng et al. (line 273 of the manuscript).

Response 2: Thank you for pointing this out. We have standardized the in-text citation format to "Author et al. [numeral]"; thus, “Bhattarai et al. [46]” This now appears correctly (L302). Additionally, we have ensured that scientific names, such as Origanum vulgare (L180), are consistently italicized throughout the manuscript. The previous mention of “Feng” was incorrect and has been replaced (L460).

Comments 3: The results section is sufficiently detailed and well-illustrated. In the case of Figure 2, I suggest that the definitions of the abbreviations be published in the figure legend. I also suggest italicizing the term in planta (L440 of the manuscript).

Response 3: We appreciate this clarity‑oriented recommendation. We revised the Figure 1 (in the actualized manuscript is now the Figure 1, corresponding to the in vitro assay, the legend to define all abbreviations actually used, and it was corrected (L 366-371).

We applied the same principle to the following figure other legends, trying so each one is self‑contained and unambiguous (L393).

“Finally, we attended to your suggestion to italicizing “in planta” terms (L 460).

Comments 4: I also suggest expanding the discussion and conclusion chapters by analyzing the results of other related studies studying the phytopathological efficacy of essential oils.

Response 4: Thank you for this suggestion. Following your recommendation, we expanded the Discussion section to include direct comparisons with previous studies that evaluated the antifungal activity of essential oils—particularly those targeting Neopestalotiopsis and related phytopathogenic fungi. Each paragraph in the Discussion now integrates references to published literature, emphasizing similarities or contrasts between our findings and those of other researchers [L421-471].

 

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The paper by Gómez-Yáñez et al. tests the efectivity of Origanum vulgare essential oil against the fungal pathogen Neopestalotiopsis zimbabwana, comparing it to commercial synthetic fungicide Captan. The fungal isolate used for testing is natural isolate collected on symptomatic Watsonia borbonica in Mexico. The efficacy of essential oil was tested in vitro on PDA plates and in vivo on strawberry plants. Furthermore, the composition of essential oil was determined by GC-MS, and the efficacy of binding of its active compounds to cytochrome b was assessed in silico. All in all, this research provides valuable information and builds on the existing knowledge on effectiveness of natural essential oils, showing their equivalence with synthetic fungicides.

Unfortunately, the paper has some major issues that need to be addressed to become of publication quality:

1. My biggest concern is Discussion that is simply too weak. The researchers did not comment or discuss the relevance of their results in practise and in the context of existing literature. The discussion section cites only five literature references, which is insufficient for a comprehensive literature review. For instance, their valuable result presented in Fig 3 – that botanical pesticide is of equal effectiveness as synthetic fungicide – needs to be highlighted and discussed how it is beneficial, especially considering that they concluded that constant application of either treatments is needed for effective plant protection. This is just one example but all other results should be discussed like that and put in practical and literature context; Discuss your MIC and MFC in comparison with literature, if not on the same fungus than on a related species, etc.
2. There are some inconsistencies in Results section, regarding the interpretation of data obtained in Figure 2. Since 30% OEO has only “d” it is statistically different than H2O, EtOH, Tw20, Captan and DCM. Also, the result that biopesticide had statistically stronger effect than Captan in vitro, but they were the same in vivo (even though the same concentration was used, if I understood correctly) needs to be discussed.

Furthermore, I do not think that Figure 1 is relevant and necessary. The phylogenetic tree with only one isolate, reference and outgroup is redundant, since it all can be summed in one sentence.

On the other hand, maybe a picture of inhibitory effect on PDA plates and damage on tested plants (comparison between treatments in some extent) would be informative, and could be added to the paper.

3. There are some big errors in literature cited – the numbers do not correspond to the authors, and some authors names written in the text are not in the list of references. I tried to mark all the errors I could find, but the citations must be thoroughly checked and corrected.
4. The introduction is disproportionally long in relation to the rest of the paper and could benefit from some shortening. In particular, the paragraphs concerning the negative effects of synthetic fungicides (rows 57-80), even though very important, could be more concise.
5. I do not feel that this paper needs separate list of abbreviations. The majority of the abbreviations in your list are the really common things that do not need special explanation (e.g. in vivo, spp. measurement units…). The rest of them you should define in full wording on first mention, put the abbreviation in parentheses and use the abbrev. further on in text.

 

All other smaller issues are marked and commented in attached pdf document.

Comments for author File: Comments.pdf

Author Response

Comment 1: My biggest concern is Discussion that is simply too weak. The researchers did not comment or discuss the relevance of their results in practise and in the context of existing literature. The discussion section cites only five literature references, which is insufficient for a comprehensive literature review. For instance, their valuable result presented in Fig 3 – that botanical pesticide is of equal effectiveness as synthetic fungicide – needs to be highlighted and discussed how it is beneficial, especially considering that they concluded that constant application of either treatments is needed for effective plant protection. This is just one example but all other results should be discussed like that and put in practical and literature context; Discuss your MIC and MFC in comparison with literature, if not on the same fungus than on a related species, etc.

Response 1 We appreciate the reviewer’s suggestion. In the revised version, we expanded the comparison by presenting the results in terms of MIC and MFC and by contrasting them with recent studies, in the lines L434-497

Comment 2: Furthermore, I do not think that Figure 1 is relevant and necessary. The phylogenetic tree with only one isolate, reference and outgroup is redundant, since it all can be summed in one sentence.

Response 2: We thank the reviewer for the suggestion. Following this advice, we removed Figure 1 and summarized the identification in a single paragraph L357--364.

Comment 2.1: The in vivo formula consists of the hydrolate in the percentage of 67.7%. Please provide any characterization of HOV. Is there any water-soluble antimicrobial molecules from the process of distillation in it? How it is something other than a diluent has to be briefly explained.

Response 2.2: We are truly grateful for the reviewer’s insightful suggestion. While the manuscript focuses on quantitative data, we could provide representative images as Supplementary.

Comment 3: There are some big errors in literature cited – the numbers do not correspond to the authors, and some authors names written in the text are not in the list of references. I tried to mark all the errors I could find, but the citations must be thoroughly checked and corrected.

Response 3: We apologize for the oversight. The revised manuscript has been carefully reviewed to ensure that all citations are correctly included and properly formatted throughout the entire text

 

Comment 4: The introduction is disproportionally long in relation to the rest of the paper and could benefit from some shortening. In particular, the paragraphs concerning the negative effects of synthetic fungicides (rows 57-80), even though very important, could be more concise.

Response 4: Thank you for the suggestion. We have condensed the section on synthetic fungicide drawbacks has been reformulated only in four lines (L 61-65).

Comment 5: I do not feel that this paper needs separate list of abbreviations. The majority of the abbreviations in your list are the really common things that do not need special explanation (e.g. in vivo, spp. measurement units…). The rest of them you should define in full wording on first mention, put the abbreviation in parentheses and use the abbrev. further on in text.

Response 5 Thank you for your thoughtful comment. In accordance with your suggestion, we have removed the list of abbreviations. Common terms such as in vivo, spp., and measurement units are now left without special emphasis, while the remaining abbreviations are introduced in full upon their first mention in the text and used consistently thereafter.

Additional note: All other smaller issues are marked and commented in attached pdf document.

Additional note: All other minor issues are marked and commented in the attached PDF document.

We addressed each of them point by point and provided the corresponding answers that follows.

In the revised version, we corrected the values of the percentage compounds (L22), specified the MFC (L24), replaced “fungi” with “pathogenic fungi” (L41), and removed the incorrect comma (L82). We also ensured that all references were properly linked, resolving bibliography numbering issues and correcting the citation to Al-Rashed (L90). The first mention of Neopestalotiopsis zimbabwana in the main body was expanded (L44), and the wording was improved to “phytopathogenicity fungi” in accordance with the prior suggestion (L60). We reintroduced and defined MIC and MFC in the main text (L98), corrected a citation by adding the missing et al. (L103), reformulated the last paragraph of the Introduction to present the objectives clearly (L137–139), changed the wording to “laboratory” (L127), and summarized the paragraph at L132–149 to avoid unnecessary detail. We completed the phrase at L170–171 with the appropriate citation; expanded L176–178 to include methodological details—explicitly stating the commercial extraction kit, tissue mass, and other relevant information; and added citation details to the sentence “The plant was harvested in the early morning” (L183–188). The extraction section was reformulated to avoid excessive information and improve concision (L189–194). We unified the missing reference at the end of the mass-analysis paragraph (L206); corrected the use of the °C symbol (L201); and corrected the expression to “glass rod with rounded ends” (L2014). We improved the wording at L211 (“…OEO was dissolved in …”), omitted the sentence at L212, resolved the bibliography at L222, moved the information at L224–225 to the new L55, and added the supporting bibliography at L226 (now located at L235). We removed the line at L245; at L251 we added the location for the preparation of Steiner’s solution and the corresponding reference. The sentence on foliar inoculation of N. zimbabwana originally at L269 was relocated and clarified at L283 (“The inoculum was prepared…”). We clarified the details and quantity of inoculum (1 mL) at L285–288. The first paragraph originally at L298 was integrated into the new Introduction paragraph (now L326), and that line was correctly integrated at L345; the paragraph now reads more fluidly (L341–350).

We removed the phylogenetic figure (former Figure 1) and described it instead in the new Section 3.1 (L365–368). We corrected the table numbering issue around L370-375 and presented the table appropriately at L376, now including retention time and ordering compounds by retention time. The prior paragraph was adjusted so that compound formulas, retention times, and percentages align with the new ordering, while the text continues to highlight major compounds and provides list numbers to guide the reader. The information at L349–352 is shown only in the description of Figure 2; the paragraph at (old versión L340–343) was deleted; and at L378–4382 we rewrote the paragraph to describe the results textually, following the reviewer’s suggestion. As pointed out by the reviewer, the figure concerning N. zimbabwana required a complete presentation; this has now been corrected and expanded at L380–385. In response to the reviewer’s specific question about fungistatic versus fungicidal effects, we clarified our reasoning and rewrote the relevant paragraph (now L384–389) for greater concision. For, we revised the figure description to avoid asserting an ANOVA, retained the use of HSD (to indicate comparisons across two time points), and clarified the figure nomenclature. We removed the paragraph at old versión L389–395 and placed the essential information in the paragraph preceding Figure 2; the discussion now properly begins at L487–489. We also ordered Table 2 in descending order (L427) and, finally, removed the separate list of abbreviations, expanding each abbreviation appropriately throughout the document (L482).

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I am writing to follow up on my review of manuscript horticulturae-3840721, entitled : Control of Neopestalotiopsis zimbabwana Using Origanum vulgare L. Essential Oil: Combined in vitro, in vivo and in silico Approaches. I'm pleased to inform you that the authors have addressed all my comments thoughtfully and respectfully.

Their revisions demonstrate a clear understanding of the points I raised, and the changes they have made significantly improve the quality of the manuscript. I believe the paper is now much stronger and ready for further consideration.

Author Response

Response 1:
We sincerely thank Reviewer 1 for this constructive assessment. We appreciate the reviewer’s recommendation to proceed with the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors,

this version of the manuscript has been significantly improved since the first version. The authors have addressed all my bigger and smaller issues from the last review. In the current version there are only several minor issues to address, including still some mismatches of the literature cited. All the corrections needed are marked in the attached pdf document.

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 3

We sincerely appreciate your careful and constructive review, which provided valuable suggestions to improve our manuscript. Accordingly, we have made the following changes.

Comment 1: (old version L46–47) Watsonia borbonica L. written outside the parentheses.
Response: Corrected; now appears inside the parentheses (L46–47).

Comment 2: (L58–59) Citation number needs adjustment.
Response: Now reads “…phytopathogenic fungi [1, 11–13]” (L58–59).

Comment 3: (L83) Expand OEO.
Response: Now appears “In particular, Origanum vulgare essential oil (OEO) stands …” (L83).

Comment 4: (old version L90–91) Relocation of “better idea in the Ov L103–107.”
Response: Moved and rewritten at L102; citations carefully reviewed.

Comment 5: (old version L105) Remove period.
Response: Corrected at L100.

Comment 6: (old version L139) Delete “controlled conditions.”
Response: Removed; no longer appears (L134).

Comment 7: (old version L150) Correct style of “spp.”
Response: Adjusted to normal (non-italic) style (L144).

Comment 8: (old version L160) “omit the cite 33.”
Response: Citation removed (L154).

Comment 9: (old version L166) Begin new sentence after citation.
Response: Period added and sentence split (“Chroma…”) (L160).

Comment 10: (old version L183) Unclosed parenthesis.
Response: Deleted; punctuation corrected (L177).

Comment 11: (old version L188) Remove “Shi et al.”
Response: Now uses numeric citation only (L182).

Comment 12: (old version L193) Remove “Walasek et al.”
Response: Corrected (L187).

Comment 13: (old version L194–195) Redundant idea.
Response: Deleted.

Comment 14: (old version L197) Remove manual reference “46.”
Response: Deleted (L191).

Comment 15: (old version L221) Missing period.
Response: Added (L213).

Comment 16: (old version L277–279) Redundant lines.
Response: Deleted.

Comment 17: (old version L294) Abbreviation “CRD.”
Response: Standardized to “CRD.”

Comment 18: (old version L358) Expand abbreviation “OEO.”
Response: Expanded (L348).

Comment 19: (old version L362) “botanical formulation” → “botanical pesticide.”
Response: Corrected (L352).

Comment 20: (old version L363) Delete “response.”
Response: Omitted (L353).

Comment 21: (old version L364) Remove “3 μL OEO” and fix punctuation.
Response: Corrected (L353–355).

Comment 22: (old version L408) Wrong period.
Response: Changed to comma (L407).

Comment 23: (old version L421) Parenthesis and period.
Response: Deleted (L412).

Comment 24: (old version L438) Wording improvement.
Response: Revised to “In the case of HOV” (L427).

Comment 25: (old version L439) Add Darapanit et al. (2021) [60].
Response: Added to reference list (L661–663).

Comment 26: (old version L445) Change to “slightly below 20%.”
Response: Corrected (L435).

Comment 27: (old version L447) Add Cid-Pérez et al. (2019).
Response: Included in reference list (L659–661).

Comment 28: (old version L451) MIC correction and comparison check.
Response: Corrected to 30% (L445).

Comment 29: (old version L463) Omission “(1-A y 2-B,)”.
Response: Corrected to “…(2-A and 2-B)” (L455).

Comment 30: (old version L470) Add “which.”
Response: Now reads “which agrees” (L462).

Comment 31: (old version L480–481) Paragraph unclear.
Response: Rewritten for clarity (L470–473).

Comment 32: (old version L485) Italicize Saccharomyces cerevisiae.
Response: Corrected (L478).

Comment 33: (old version L487) Remove “Haque et al.”
Response: Deleted (L479).

Comment 34: (old version L534–537) Bibliography update.
Response: Changed to suitable sources (L216, L630–632, L640–642).

Author Response File: Author Response.pdf