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Peer-Review Record

Inhibitory Activity and Mechanism of Action with Thymol against the Blueberry Pathogenic Fungi Caused by Neopestalotiopsis clavispora

Horticulturae 2023, 9(9), 983; https://doi.org/10.3390/horticulturae9090983

by Shengjie Ye¹, Liang Shuai²

, Donglan Luo¹ and Liangjie Ba^1,*

Reviewer 1: Anonymous

Reviewer 2:

Hadeel M.M. Khalil Bagy

Reviewer 3: Anonymous

Horticulturae 2023, 9(9), 983; https://doi.org/10.3390/horticulturae9090983

Submission received: 20 July 2023 / Revised: 20 August 2023 / Accepted: 28 August 2023 / Published: 31 August 2023

(This article belongs to the Section Postharvest Biology, Quality, Safety, and Technology)

Round 1

Reviewer 1 Report (New Reviewer)

The paper takes into consideration the possible use of thymol against the fungus Neopestalotiopsis clavispora in blueberry trying also to underline the possible meachanism of action.

My main concern about this paper is the M&M section. Sometimes English is not used properly and, in my opinion, the methodologies are not described well and so result not clear to the reader.

I think that the authors have to describe briefly all the methodologies and do not have to simply cite the paper on which the methodology has been described previously. English has to be improved because in some cases is really unclear and, as a consequence, also the results section can not be revised in a proper way by the readers. I suggest the authors to rephrase the entire M&M section.

Some other minor remarks are as follows:

Line 15: in vitro has to be in italics

Line 29: I suggest “fungal activity” and not “fungus activity”

Line 50: I think you mean “antifungal” and not “antibacterial”.

M&M section: Did you consider replicates in the experiments? Please define this information.

Figure 1: graph C reports letters from the anova that result quite strange to me. For example, c and d letters belong to very close values. Are you sure about this statistical analysis?

Author Response

Dear Editor and Reviewers,

Thank you very much for your overall comprehensive and positive comments on this manuscript, and thank you for giving us the chance to revise. We have fully considered all the opinions and requirements in the letter, and have made detailed replies and modifications on an item-by-item basis.Notably, all my responses are marked in blue, the original contents are marked in green, and revised portions are marked in red. Please find my itemized responses below and my corrections in the “Manuscript File”.

Thanks again!

Responds to the Editor’s comments:

Comments:The paper takes into consideration the possible use of thymol against the fungus Neopestalotiopsis clavisporain blueberry trying also to underline the possible meachanism of action.

My main concern about this paper is the M&M section. Sometimes English is not used properly and, in my opinion, the methodologies are not described well and so result not clear to the reader.

Comments:Line 15: in vitro has to be in italics

Response:Thank you very much for your suggestion.Please find my itemized responses and my detailed modifications in the “Manuscript File”

Comments:Line 29: I suggest “fungal activity” and not “fungus activity”

Response:Thank you very much for your suggestion.Please find my itemized responses and my detailed modifications in the “Manuscript File”. Line 29: “ fungus activity”replaced by “fungal activity”.

Comments:Line 50: I think you mean “antifungal” and not “antibacterial”.

Response:Thank you very much for your suggestion.Please find my itemized responses and my detailed modifications in the “Manuscript File”. Line 29: “ antibacterial”replaced by “antifungal”.

Comments:Figure 1: graph C reports letters from the anova that result quite strange to me. For example, c and d letters belong to very close values. Are you sure about this statistical analysis?

Response: We are very grateful and agree with the important issues and suggestions you have pointed out. We have also changed figure 1-C, please check it in the newly submitted "manuscript file".The changed figure is shown below.

Author Response File: Author Response.docx

Reviewer 2 Report (Previous Reviewer 3)

I happy to revise this manuscript again in previously comments I have suggest some comments and I see authors nearly addressed all my comments but stile some comments need to improve before to accept in Horticulture

- in abstract ( you need to write some value of your results .)

- Keyword should be different than in title

- English language need to improve

- Most of figures need to improve even in most Fig did not mentioned what is (A, B,..) or what the meaning of bar or letter ………)

- Introduction need to modify and mentioned about the important of this disease in your region and some references is old you can update this references.

- Follow the instructions to authors on how they write the references.

- Make sure that all scientific names in the References list are italics.

- Please add the DOI for ALL the References.

- Maybe you can used some new references here

- https://doi.org/10.1007/s42161-021-00808-2

- https://doi.org/10.1016/j.biocontrol.2017.04.014

- https://doi.org/10.33687/phytopath.008.02.2967

Author Response

Dear Editor and Reviewers,

Thanks again!

Responds to the Editor’s comments:

Comments:in abstract ( you need to write some value of your results .)

Comments:Keyword should be different than in title

Response:Thank you very much for your suggestion.The specific modifications are in the reply below and the revised manuscript:

Keyword：“Blueberry; fungus activity;Neopestalotiopsis clavispora; Thymol;” with “Neopestalotiopsis clavispora; Thymol; Blueberry; Postharvest disease; Antifungal activity;”

Comments:English language need to improve

Response: Thank you for your advice, we have made meticulous modifications to this manuscript, therefore, the readers may understand our work more clearly. The corrected details are listed as hignlighted in the manuscript.

Comments:Most of figures need to improve even in most Fig did not mentioned what is (A, B,..) or what the meaning of bar or letter ………)

Response:Thank you very much for your suggestion.I have clearly explained the bar and letters under each picture. Please find my itemized responses and my detailed modifications in the “Manuscript File”

Comments:Introduction need to modify and mentioned about the important of this disease in your region and some references is old you can update this references.

Response:Thank you very much for your suggestion. We have added this part in the Introduction, which is as follows:In addition, Neopestalotiopsis clavispora is a pathogen that occurs in postharvest blueberries in the Majiang area of Guizhou Province. It can cause softening and decay of blueberries and cause economic losses. Please find my itemized responses and my detailed modifications in the “Manuscript File”

Comments:

Follow the instructions to authors on how they write the references.

Make sure that all scientific names in the References list are italics.

Please add the DOI for ALL the References.

Maybe you can used some new references here

https://doi.org/10.1007/s42161-021-00808-2

https://doi.org/10.1016/j.biocontrol.2017.04.014

https://doi.org/10.33687/phytopath.008.02.2967

Response:Thank you for your suggestion. We have written the references according to the author's instructions and have made sure that all scientific names in the list of references are in italics; We have updated the literature review with the latest research articles. Please see red fonts on Page 13. The added references are as follows:

Reference

[11]Bagy, H.M.M.K.; Abo-Elyousr, K.A.M. Antibacterial activity of some essential oils on bacterial spot disease of tomato plant caused by xanthomonas axonopodis pv. vesicatoria. Int. J. Phytopathology. 2019, 08(02), 53-61.

[16]Abdel-Rahim, I. R.; Abo-Elyousr, K.A.M. Using of endophytic Saccharomycopsis fibuligera and thyme oil for management of gray mold rot of guava fruits. Biol. Control. 2017, 110, 124-131.

[19]Sallam, N.M.A.; Ali, E.F.; Abo-Elyousr, K.A.M.; Bereika, M.F.F.; Seleim, M.A.A. Thyme oil treatment controls bacterial wilt disease symptoms by inducing antioxidant enzyme activity in solanum tuberosum. J. Plant Patho. 2021, 103(2), 563-572.

Reviewer 3 Report (Previous Reviewer 2)

The revised manuscript is improved and now it is suitable for publication. Authors need to check only a few issues:

Keywords: some words will start with small letters.

NEO (used as an abbreviation of Neopestalotiopsis clavispora): authors need to check it again. In my opinion, authors can use the full form or a short form of the scientific name N. clavispora

....... et al (in line 116, 124, 138, etc): authors need to check it again. It will be Wan et al. [22], Shao et al. [23], ......

Comments for author File: Comments.pdf

Author Response

Dear Editor and Reviewers,

Thanks again!

Responds to the Editor’s comments:

Comments:Keywords: some words will start with small letters.

Response:Thank you very much for your suggestion.Please find my itemized responses and my detailed modifications in the “Manuscript File”

Comments:NEO (used as an abbreviation of Neopestalotiopsis clavispora): authors need to check it again. In my opinion, authors can use the full form or a short form of the scientific name clavispora

Response:Thank you very much for your suggestion. I have modified the abbreviation of Neopestalotiopsis clavispora point by point.. Please find my itemized responses and my detailed modifications in the “Manuscript File”.“ NEO”replaced by “N. clavispora”.

Comments:....... et al (in line 116, 124, 138, etc): authors need to check it again. It will be Wan et al. [22], Shao et al. [23], ......

Response:Thank you very much for your suggestion.Please find my itemized responses and my detailed modifications in the “Manuscript File”

Author Response File: Author Response.docx

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The manuscript entitled “Inhibitory activity and mechanism of action with thymol against the blueberry pathogenic fungi caused by Neopestalotiopsis clavispora” (Horticulturae-2465790) brought results that need revision. However, the manuscript lacks some important details for the reader/reviewer to be fully understand. There are many confusing sentences throughout the manuscript, which are hard to understand. Please take into account that many sentences need to be rephrased, in my opinion. For these reasons, I strongly suggest to revise and improve this manuscript. I have listed some comments bellow:

Line 16: MIC” what is this

Abstract: abbreviations used in the abstract are not clear.

Introduction: There are two kinds of abbreviations used for species (ssp. And sp.), please keep uniformity in the text

The abbreviations used in the text may explain where firstly used.

Line 53: .It” please keep spacing

Line 64: The clear of the bacteriostatic activity and mechanism of action of natural fungicides 63 is essential for their application.” Please rephrase the sentence

Line 72: r NADPH/NADP+” abbreviations are not clear

Figure Line 198: please keep proper space between words inhibition rate c) Colony diameter

Conclusion: Taken together, thymol significantly inhibited both mycelial growth and spore” Please rephrase the sentence

References style used in the manuscript can be listed according to the journal style with consistency. Follow the journal style formatting for reference arrangements throughout all references. Please also correct the formatting style according to the author’s instructions of the journal. Please check for typos and missing italics, double spaces, abbreviations etc.

Minor editing of English language is required

Author Response

Please see the attachment.

Dear Editor and Reviewers,

Thanks again!

Responds to the Editor’s comments:

Comments:Line 16: MIC” what is this

Response: First, thank you very much for your overall approval and comprehensive comments on this manuscript. Based on the valuable suggestions and questions raised by the editors and reviewers, we have tried our best to improve and revise the shortcomings in the manuscript. The mass concentration of Neopestalotiopsis clavispora mycelial disks without mycelial growth in 48h was used as the minimum inhibitory concentration (MIC)." The mass concentration of Neopestalotiopsis clavispora mycelial disks without mycelial growth in 48 h was used as the minimum inhibitory concentration (MIC) " (Lines 114-116 Page 3) . Please find my itemized responses and my detailed modifications in the “Manuscript File”.

Comments: Abstract: abbreviations used in the abstract are not clear.

Response:We are very grateful and agree with the important issues and suggestions you pointed out. After careful sorting and analysis, we have revised the content of the abstract. The specific modifications can be found in the following responses and revisions:

“Neopestalotiopsis clavispora caused rot is a significant blueberry postharvest disease that negatively impacts the fruit's commercial value. In this paper, we investigated the inhibitory activity and mechanism of action of thymol against pathogenic fungi of blueberry caused by Neopestalotiopsis clavispora. The results demonstrated that thymol administration could limit mycelial development; the inhibitory effect was positively connected with thymol mass concentrations, and the minimal inhibitory concentration was 100 mg/L. Further investigations revealed that MIC thymol treatment dramatically reduced the germination of pathogenic spores, and led to the increase of conductivity of outside the pathogen, the leakage of contents and the decrease of pH. Propidium iodide (PI) staining experiments demonstrated that MIC thymol caused damage to mycelial cell membranes. Additionally, MIC thymol treatment promoted mycelium MDA accumulation, inhibited SOD and CAT enzyme activities, decreased adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) content and energy charge levels, and the fluorescence intensity of mycelium caused by MIC thymol treatment were significantly increased by DCFH-DA assay. The results of this study indicate that thymol inhibits the growth of pathogenic bacteria by compromising the integrity of their cell membranes, promoting the accumulation of cellular ROS, and interfering with energy metabolism.”were replaced by “Neopestalotiopsis clavispora caused rot is a significant blueberry postharvest disease that negatively impacts the fruit's commercial value. In this paper, we investigated the inhibitory activity and mechanism of action of thymol against pathogenic fungi of blueberry caused by Neopestalotiopsis clavispora. The results demonstrated that thymol administration could limit mycelial development; the inhibitory effect was positively connected with thymol mass concentrations, and the minimal inhibitory concentration (MIC) was 100 mg/L. Further investigations revealed that MIC thymol treatment dramatically reduced the germination of pathogenic spores, and led to the increase of conductivity of outside the pathogen, the leakage of contents and the decrease of pH. Propidium iodide (PI) staining experiments demonstrated that MIC thymol caused damage to mycelial cell membranes. Additionally, MIC thymol treatment promoted mycelium malondialdehyde contents accumulation, inhibited superoxide dismutase (SOD) and catalase (CAT) enzyme activities, decreased adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) content and energy charge levels, and the fluorescence intensity of mycelium caused by MIC thymol treatment were significantly increased by 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) assay. The results of this study indicate that thymol inhibits the growth of pathogenic fungi by compromising the integrity of their cell membranes, promoting the accumulation of cellular reactive oxygen species (ROS), and interfering with energy metabolism.”Please find my itemized responses and my detailed modifications in the “Manuscript File”.

Comments: Introduction: There are two kinds of abbreviations used for species (ssp. And sp.), please keep uniformity in the text. The abbreviations used in the text may explain where firstly used.

Response:First of all, thank you very much for your overall approval and comprehensive evaluation of this manuscript. Based on the valuable suggestions and questions from the editors and reviewers, we have tried our best to improve and revise the deficiencies in the manuscript. We have revised the species-specific abbreviations for fungal diseases of blueberry in the newly submitted ‘manuscript file’. The details of the changes can be found in the following responses and revised manuscript:

“It is reported that there are many species of pathogenic fungi that can cause postharvest rot of blueberry fruits, mainly Penicillium sp., Botrytis cinerea, Colletotrichum sp., Alternaria sp. and Aspergillus sp.” replaced by “It is reported that there are many species of pathogenic fungi that can cause postharvest rot of blueberry fruits, mainly Penicillium spp., Botrytis cinerea, Colletotrichum spp., Alternaria spp. and Aspergillus spp.”Please find my itemized responses and my detailed modifications in the “Manuscript File”.

Comments: Line 53: .It” please keep spacing

Response:Thank you very much for your suggestion.Please find my itemized responses and my detailed modifications in the “Manuscript File”

Comments:Line 64: The clear of the bacteriostatic activity and mechanism of action of natural fungicides 63 is essential for their application.” Please rephrase the sentence

Comments:Line 72: “NADPH/NADP+” abbreviations are not clear

Response:Thank you very much for your suggestion.“ NADPH/NADP+”replaced by “Reduced nicotinamide adenine dinucleotide phosphate/Oxidized nicotinamide adenine dinucleotide phosphate(NADPH/NADP+)”. Please find my itemized responses and my detailed modifications in the “Manuscript File”.

Comments:Figure Line 198: please keep proper space between words inhibition rate c) Colony diameter

Response:Thank you very much for your suggestion.Please find my itemized responses and my detailed modifications in the “Manuscript File”

Comments:Conclusion: Taken together, thymol significantly inhibited both mycelial growth and spore” Please rephrase the sentence

Response:First, thank you very much for your overall approval and comprehensive comments on this manuscript. Based on the valuable suggestions and questions raised by the editors and reviewers, we have tried our best to improve and revise the shortcomings in the manuscript. “Taken together, thymol significantly inhibited both mycelial growth and spore germination of Neopestalotiopsis clavispora, with mycelial growth inhibition of only (88.925 ± 0.9)% when the MIC value was 100 mg/L.”replaced by“ In summary, thymol significantly inhibited Neopestalotiopsis clavispora mycelial growth and spore germination in vitro, with the rate of mycelial growth inhibition being only (88.925 ± 0.9)% at MIC of 100 mg/L.”Please find my itemized responses and my detailed modifications in the “Manuscript File”

Comments:References style used in the manuscript can be listed according to the journal style with consistency. Follow the journal style formatting for reference arrangements throughout all references. Please also correct the formatting style according to the author’s instructions of the journal. Please check for typos and missing italics, double spaces, abbreviations etc.

Response:Thank you very much for your suggestion. I have revised my references one by one. Please find my itemized responses and my detailed modifications in the “Manuscript File”

Author Response File: Author Response.docx

Reviewer 2 Report

Minor revision is needed to make it suitable for acceptance. My specific comments are given below (also see the attached PDF file):

Line 23: DCFH-DA--- use full form

Line 25: ROS---- use full form

line 27: Keywords: arrange alphabetically

line 38-39: mention specific epithet. If numerous species are under a genus, then use 'spp.' instead of 'sp.'

line 51-52: italicize the genus of scientific names.

line 71: ROS----- full form

line 73: NEO---- full form

line 155: SOD, CAT--- full form

Line 180-183: statistical analysis: if the p-value is significant and more than two groups are there, then use a post hoc test.

Figure 2C: Dry weight at 20 mg/L & 80 mg/L of thymol denoted by the same letter 'b'. I think there is a significant difference between the dry weights in the two concentrations. Recheck it. Which test was performed by the authors? Mention in the Figure ligand.

References: use abbreviations of the journal names (line 438, 445, 449, etc.).

Comments for author File: Comments.pdf

Author Response

Please see the attachment

Dear Editor and Reviewers,

Thanks again!

Responds to the Editor’s comments:

1.Comments:Minor revision is needed to make it suitable for acceptance. My specific comments are given below (also see the attached PDF file):

Line23:“DCFH-DA” replaced by “2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA)”
line 25:“ROS” replaced by “reactive oxygen species (ROS)”
line 27:Keywords for specific changes, see the following replies and the revised manuscript: “Thymol; Neopestalotiopsis clavispora; Blueberry; Fungi activity;” replaced by “Blueberry; Fungi activity;Neopestalotiopsis clavispora; Thymol; ”
line 38-39:Based on your comments, the specific modifications are in the reply below and the revised manuscript: “It is reported that there are many species of pathogenic fungi that can cause postharvest rot of blueberry fruits, mainly Penicillium , Botrytis cinerea, Colletotrichum sp., Alternaria sp. and Aspergillus sp.” replaced by “It is reported that there are many species of pathogenic fungi that can cause postharvest rot of blueberry fruits, mainly Penicillium spp., Botrytis cinerea, Colletotrichum spp., Alternaria spp. and Aspergillus spp. ”
line 51-52:We are very grateful and agree with the important issues and suggestions you have pointed out. The specific modifications are in the reply below and the revised manuscript: “Thymol (2-isopropyl-5-methylphenol) is the main monoterpene phenol occurring in essential oils isolated. from plants belonging to the Lamiaceae family (Thymus, Ocimum, Origanum, and Monarda genera),” replaced by “ Thymol (2-isopropyl-5-methylphenol) is the main monoterpene phenol occurring in essential oils isolated. from plants belonging to the Lamiaceae family (Thymus, Ocimum, Origanum, and Monarda genera), ”
line 71:“ROS”replaced by “reactive oxygen species (ROS)”
line 73:“NEO” replaced by “Neopestalotiopsis clavispora (NEO)”
line 155: “SOD, CAT ” replaced by“ superoxide dismutase (SOD) and catalase (CAT)”

2.Comments:Line 180-183: statistical analysis: if the p-value is significant and more than two groups are there, then use a post hoc test.

Lines 190-194: statistical analysis: :“ The experimental data were analyzed by analysis 0f variance (ANOVA) using Excel 2016 and SPSS Statistics 25 statistical software and expressed as mean±standard deviation (n≥3) with at least three replications. p<0.05 indicated a significant difference; plotting was performed using Origin 2021 (OriginLab Ltd., Northampton, MA, USA)software.” were replaced by “All treatments involved in the study were repeated 3 times, and the differential significance analysis was conducted using Duncan's multiple range test or one way analysis of variance (ANOVA) with SPSS software (IBM SPSS Statistics 25.0). Data were expressed as mean ± standard deviation (SD). plotting was performed with Origin 2021 (OriginLab Ltd., Northampton, MA, USA) software.”

Comments:Figure 2C: Dry weight at 20 mg/L & 80 mg/L of thymol denoted by the same letter 'b'. I think there is a significant difference between the dry weights in the two concentrations. Recheck it. Which test was performed by the authors? Mention in the Figure ligand.

Response: Thank you for your reminder. According to your suggestion, Figure 2C has been modified, and the specific modification is shown in the following reply and the revised original manuscript:

4.Comments:References: use abbreviations of the journal names (line 438, 445, 449, etc.).

Response: Thank you for your reminder. The abstract has been revised one by one according to your suggestions. The specific modifications are in the revised manuscript

Author Response File: Author Response.docx

Reviewer 3 Report

I have some points that need to be addressed as follows.

- Authors in many places in the article wrote bacterial pathogen, antibacterial if authors did not know what the different between fungi and bacteria I cannot recommended to publish this article

e.g in abstract ( The results of this study indicate that thymol inhibits the growth of pathogenic bacteria by…..)

in material “The pathogenic bacteria were stored on potato…. “

English language need to improve

I did not see that authors repeated his experiment or how many replicate they used

Most of figures need to improve even in most Fig did not mentioned what is (A, B,..) or what the meaning of bar or letter ………)

Figure 4. Effect of thymol treatment on mycelium cell membrane integrity analyzed by PI staining what is the different between 4 fig

Figure 5. Effect of thymol on MDA (A), CAT (B), and SOD (C) activity of Neopestalotiopsis clav-298 ispora; what is the letter and bar …….

The rest of fig like this

Discussion part need to improve here some new references maybe you can used for improve your discussion. If found useful, cite these recent references

Junior O.J.C.,Youssef K., Koyama R., Ahmed S., Dominguez A.R.,Mühlbeier D.T., Roberto S.R. 2019. Control of Gray Mold on Clamshell-Packaged ‘Benitaka’ Table Grapes Using Sulphur Dioxide Pads and Perforated Liners. Pathogens, 8, 271.https://doi.org/10.3390/pathogens8040271

Hadeel M. M. Khalil Bagy, Badawy F. M. Ibtesam, Eman A. A. Abou-Zaid, Badawy M. Sabah & Sallam, Nashwa, M. A (2021) Control of green mold disease using chitosan and its effect on orange properties during cold storage, Archives of Phytopathology and Plant Protection, 54 (11-12): 570-585 DOI: 10.1080/03235408.2020.1847568

English very difficult to understand/incomprehensible

Author Response

Please see the attachment.

Dear Editor and Reviewers,

Thanks again!

Responds to the Editor’s comments:

Comments:Authors in many places in the article wrote bacterial pathogen, antibacterial if authors did not know what the different between fungi and bacteria I cannot recommended to publish this article

Comments:English language need to improve

Response:Thank you for your advice, we have made meticulous modifications to this manuscript, therefore, the readers may understand our work more clearly. The corrected details are listed as hignlighted in the manuscript.

Comments:I did not see that authors repeated his experiment or how many replicate they used

Lines 190-194: statistical analysis: :“ The experimental data were analyzed by analysis 0f variance (ANOVA) using Excel 2016 and SPSS Statistics 25 statistical software and expressed as mean±standard deviation (n≥3) with at least three replications. p<0.05 indicated a significant difference; plotting was performed using Origin 2021 (OriginLab Ltd., Northampton, MA, USA)software.” were replaced by “All treatments involved in the study were repeated 3 times, and the differential significance analysis was conducted using t-test or one way analysis of variance (ANOVA) with SPSS software (IBM SPSS Statistics 25.0). Data were expressed as mean ± standard deviation (SD). plotting was performed with Origin 2021 (OriginLab Ltd., Northampton, MA, USA) software.”

Comments:Most of figures need to improve even in most Fig did not mentioned what is (A, B,..) or what the meaning of bar or letter ………)

Response: Thank you very much for your overall approval and comprehensive comments on this manuscript. We have made modifications to the inappropriate figures, and the revised sentences will be highlighted in red in the submitted 'manuscript file'. The specific modifications are as follows:

Lines 211-214, : This sentence provides a comprehensive explanation, replacing “Inhibitory effect of thymol on NEO A : in vitro growth of NEO under the effect of different concentrations of thymol; B: inhibition rate of thymol on NEO(*P<0.05, ***P<0.001 ) ; C: colony diameter of NEO. Lowercase letters indicate significant differences, P<0.05;” with “Inhibitory effect of thymol on Neopestalotiopsis clavispora (NEO). (A) in vitro growth of NEO under the effect of different concentrations of thymol; (B) inhibition rate of thymol on NEO(*P < 0.05, ***P < 0.001 ) ; (C) colony diameter of NEO. Lowercase letters indicate significant differences, P < 0.05;”

Lines 225-229, : This sentence provides a comprehensive explanation, replacing “Effect of 0 and 100 mg/L thymol treatments on spore germination at different times; B: inhibition of spore germination for treatments 0, 4, 8 and 12 h; C: change in mycelial dry weight for different concentrations of thymol treatments.”with “Effect of 0 and 100 mg/L thymol treatments on spore germination at different times; B: inhibition of spore germination for treatments 0, 4, 8 and 12 h; C: change in mycelial dry weight for different concentrations of thymol treatments. Error bars represent the standard errors of the means. Different letters above bars indicate significant differences in the mean for that sampling period (P < 0.05).”

Lines 262-266, : This sentence provides a comprehensive explanation, replacing “Extracellular conductivity (A), extracellular pH (B), intracellular nucleic acid release (C), extracellular soluble protein (D), and reducing sugars (E) of NEO under the effect of different concentrations of thymol (P<0.05);”with “A: Extracellular conductivity (A), extracellular pH (B), intracellular nucleic acid release (C), extracellular soluble protein (D), and reducing sugars (E) of NEO under the effect of different concentrations of thymol. Values are expressed as mean ± Standard deviation , error bars represent standard deviations of the means, and different letters at any given time indicate significance of difference according to Duncan’s multiple range test (P < 0.05). ”

Lines 289-291 :“ Effect of thymol treatment on mycelium cell membrane integrity analyzed by PI staining”with “Fluorescence microscopy of Neopestalotiopsis clavispora after treatment with thymol. Effect of thymol treatment on mycelium cell membrane integrity analyzed by PI staining.(Bright: Bright field; PI: propidium iodide) ”

Lines 320-323 :“ Effect of thymol on MDA (A), CAT (B), and SOD (C) activity of Neopestalotiopsis clavispora;”with “Effect of thymol on malondialdehyde (MDA) (A), catalase (CAT) (B), and superoxide dismutase (SOD) (C) activity of Neopestalotiopsis clavispora; Error bars represent the standard errors of the means. Different letters above bars indicate significant differences in the mean for that sampling period (P < 0.05). ”

Lines 330-333 :“ROS levels as expressed by DCFH-DA staining”with “Fluorescence microscopy images showing accumulation of reactive oxygen species in Neopestalotiopsis clavispora . green fluorescence indicates presence of ROS , Red arrows in Fig. 6 point to the clearly visible septae separating constituent cells within the filaments(Bright: Bright field; DCF: 2,7-Dichlorodi-hydrofluorescein diacetate).”

Lines 348-352 :“ Effect of thymol on intracellular ATP (A), AMP (B), ADP (C) contents and EC (D) of Neopestalotiopsis clavispora”with “Effect of thymol on intracellular adenosine triphosphate (ATP) (A), adenosine diphosphate (ADP) (B), adenosine monophosphate (AMP) (C) contents and energy charge (EC) levels (D) of Neopestalotiopsis clavispora. Values are expressed as mean ± Standard deviation , error bars represent standard deviations of the means, and different letters at any given time indicate significance of difference according to Duncan’s multiple range test (P < 0.05). ”

Comments:Discussion part need to improve here some new references maybe you can used for improve your discussion. If found useful, cite these recent references

Response:Thank you for your suggestion. We have updated the literature review with the latest research articles. Please see red fonts on Page 13. The added references are as follows:

Reference

Junior, O.J.C. ; Youssef, K.; Koyama, R.; Ahmed, S.;Dominguez, A.R.; Mühlbeier,T.; Roberto S.R. Control of gray mold on clamshell-packaged 'benitaka' table grapes using sulphur dioxide pads and perforated liners. Pathogens. 2019, 8(4), 217.

[15] Khalil Bagy Hadeel, M.M.; Ibtesam Badawy, F.M.; Abou Zaid Eman, A.A.; Sabah Badawy, M.; Nashwa Sallam, M.A. Control of green mold disease using chitosan and its effect on orange properties during cold storage. Arch. Phytopathol. Plant Protect. 2020, 54(11-12), 570-585.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

I see authors covered all my comments

Moderate editing of English language required

Author Response

Dear Editor and Reviewers,

非常感谢您对这份稿件的全面和积极的评论，并感谢您给我们修改的机会。我们充分考虑了函中的所有意见和要求，并逐项进行了详细的答复和修改。值得注意的是，我所有的回答都用蓝色标记，原始内容用绿色标记，修改后的部分用红色标记。请在下面找到我的逐项回复，并在“手稿文件”中找到我的更正。

再次感谢！

回应编辑的评论：

评论：在我看来，所呈现的结果存在一个主要的方法学错误：不存在阴性对照（即真菌的生长而不添加任何乙醇或百里酚的酒精溶液）。在第 192-192 行中，您声明乙醇对菌丝体生长没有影响，但与正常生长相比，您没有显示这一点，仅显示乙醇处理的培养物。您如何确定乙醇（作用于微生物的正常消毒剂）没有效果？我不是在争论百里酚的作用，但乙醇的存在也可能产生一些影响。应用数据证明缺乏影响。

响应： 首先，非常感谢您对本文稿件的全面认可和全面评论。根据编辑和审稿人提出的宝贵建议和问题，我们尽力改进和修改稿件中的不足。我很抱歉这个问题。该方法中没有解释清楚。在10mg/ml母夜，乙醇含量为0.05%。在先前的实验中，0.05%乙醇对菌丝体没有抑制作用，本文未对此进行说明。

评论：此外，手稿还需要修改英文风格和语法。请请资深同事或专业读者提供帮助。

响应：谢谢你的建议。关于整篇文章的语法和表达方式，我请我的母语为英语的朋友帮助我对修订版进行改进。

注释：第11行：“造成腐烂”应写在句子中的其他位置或不同位置

响应：谢谢你的建议。我们修改了新提交的“手稿文件”中的第 11 行。具体修改见以下回复及修改稿件：

第11行：“锁骨孢子虫引起的腐烂是一种严重的蓝莓采后疾病，会对水果的商业价值产生负面影响。取而代之的是“锁骨新孢子虫引起的腐烂是蓝莓重要的采后病害，严重影响蓝莓果实的商业价值。

注释：第13行：真菌（单数）不是真菌（复数）（这个错误在全文中重复了好几次）

响应：感谢您的建议，我们根据您的建议进行了许多修改，如手稿中突出显示的那样。具体修改见以下回复和修改稿件。

注释：第14行：指定分析是在体外还是体内进行

响应：谢谢你的建议。我们修改了新提交的“手稿文件”中的第 14 行。具体修改见以下回复及修改稿件：

第11行：“结果表明，麝香草酚给药可以限制菌丝体生长。取而代之的是“结果表明，麝香酚给药可以限制体外菌丝体生长。

注释：第 25 行：指定真菌的名称，因为您的写作方式似乎对蓝莓的任何致病真菌起作用

响应：谢谢你的建议。我们修改了新提交的“手稿文件”中的第 25 行。具体修改见以下回复及修改稿件：

第11行：“结果表明，麝香草酚给药可以限制菌丝体生长。这项研究的结果表明，麝香草酚通过损害病原真菌细胞膜的完整性，促进细胞活性氧（ROS）的积累，干扰能量代谢来抑制病原真菌的生长。取而代之的是“这项研究的结果表明，百里酚通过损害其细胞膜的完整性、促进细胞活性氧（ROS）的积累和干扰能量代谢来抑制锁骨新孢子虫的生长。

注释：第 34-35 行：句子的主语不是您所指的“哪个”。请更正

响应：谢谢你的建议。我们修改了新提交的“手稿文件”中的第 25 行。具体修改见以下回复及修改稿件：

第34-35行：“然而，蓝莓果实在收获后容易受到机械损伤34，因为它们的皮薄，肉多汁，质地柔软，可以被病原微生物感染。取而代之的是“然而，蓝莓果实由于皮薄、果肉多汁、质地柔软，被病原微生物感染，在采后容易发生机械损伤。

Comments:lines 66-71: I suggest to add at least a refernce to support the statement

Response:Thank you for your suggestion. Regarding this issue, we have specifically used an example to support this view in line 71-76, so I am sorry that I don't think it is necessary to add a literature to support the statement.

Comments:Section 2.1: no need to have a specific section for these chemicals. Add the relevant info when you mention them in the methodological section/s

Response:Thank you for your suggestion.I have deleted section 2.1 and added relevant information to the methodological section referring to the corresponding chemicals.The specific modifications are in the reply below and the revised manuscript.

Comments:line 93-94: add reference about the identification of the species (if available), otherwise you need to specify the method you used for this and present the results of these analyses.

Comments:Section 2.2: there are several typos to be corrected

Response:Thank you for your advice, we have made many modifications based on your proposal as highlighted in the manuscript. The specific modifications are in the reply below and the revised manuscript:

Sectoin 2.2: “The Neopestalotiopsis clavispora strain was isolated from post-harvest naturally decayed blueberries in Majiang, Guizhou province,China. which had been morphologically and molecularly identified, and was obtained from the Biology and Medicine (Guizhou, China) Laboratory of Guiyang College. The pathogenic fungi were stored on potato dextrose agar (PDA) at 25 ± 2 ℃. The total spore suspension concentration was adjusted to 1× 10⁶CUF/mL by hematocrit, inoculated in potato dextrose broth(PDB) per 20 mL, and incubated for 48 hours. The mycelium was then rinsed three times with phosphate buffer sodium (PBS, 0.1 mol/L, pH=7.0) before being utilized for further research.” replaced by“The strain of Neopestalotiopsis clavispora was isolated from post-harvest naturally decayed blueberries in Majiang, Guizhou province, China. It has been isolated and preserved by the Laboratory of Biology and Medicine (Guizhou, China) of Guiyang University through morphological and molecular identification. The pathogen was preserved on potato dextrose agar ( PDA, Shanghai Bo Microbiology Technology Co., Ltd.) at 25±2℃. The total spore suspension concentration was adjusted to 1×10⁶ CUF/mL by blood cell plate counting method, and inoculated into 20mL potato dextrose broth(PDB) and cultred for 48 hours. Then the mycelium was washed three times with phosphate buffer sodium (PBS, 0.1 mol/L, pH = 7.0) before being utilized for further research.”

Comments:line 109: I believe it is "after 48 hours", not "in"

Response:Thank you for your suggestion.We have amended line 109 in our newly submitted "Manuscript File". The specific modifications are in the reply below and the revised manuscript:

line 109: “The mass concentration of Neopestalotiopsis clavispora (NEO) mycelial disks without mycelial growth in 48 h was used as the minimum inhibitory concentration (MIC)”replaced by “The mass concentration of Neopestalotiopsis clavispora (NEO) mycelial disks without mycelial growth after 48 h was used as the minimum inhibitory concentration (MIC) ”

Comments:Sectoin 2.4: not clear the title, not clear if you used petri or slides, not clear what you mean with "coated", and how you proceeded. Please rewrite

Response:Thank you very much for your overall approval and comprehensive comments on this manuscript. We described the methods of spore germination in section 2.4, but these descriptions may not be clear enough, so we have revised them. Specific modifications are as follows:

Sectoin 2.4: “Referring to the method of Run [21] to determine the spore germination of NEO, 50μL(1 × 10⁶CUF/mL) of NEO spore suspension was taken and evenly coated with a final mass concentration of 0 (containing the same volume of ethanol solution), 100 mg/L of thymol on the surface of PDA medium. spores were cultured in the dark at 25 ℃ for 0, 4, 8 and 12 h, and the 4 corners and central areas of the medium were selected for sectioning (8mm × 8mm). All spores on the sections were counted under the light microscope (BX53, OLYMPUS), and the spore germination rate was calculated by dividing the germinated spores by all spores.” replaced by“Referring to the method of Run [21] to determine the spore germination of NEO, 50μL(1×10⁶CUF/mL) of NEO spore suspension was prepared and coated on the surface of PDA medium supplemented with thymol and ethanol. spores were cultured in the dark at 25 ℃ for 0, 4, 8 and 12 h, and the 4 corners and central areas of the medium were selected for sectioning (8mm × 8mm). All spores on the sections were counted under the light microscope (BX53, OLYMPUS), and the spore germination rate was calculated by dividing the germinated spores by all spores.”

Comments:Section 2.5: Brielfy describe the method you refer to. Not clear what you determined: maybe the effect of thymol on mycelial growth?

Response:Thank you very much for your overall approval and comprehensive comments on this manuscript.We described thymol's approach to NEO mycelial weight in section 2.5, but these descriptions may not be clear enough, so we have revised them.Specific modifications are as follows:

Sectoin 2.5: “ Referring to the method reported by Wan et al [22] to determine the mycelial dry weight, mycelium was treated with thymol at final mass concentrations of 0, 20, 40, 60, 80, and 100 mg/L, and incubated at 25 ℃ for 24 h. Then the mycelium was washed three times in sodium phosphate buffer, and the results were expressed as g/100mLPDB.” replaced by “Referring to the method reported by Wan et al [22] to determine the mycelial dry weight, mycelium was treated with thymol at final mass concentrations of 0, 20, 40, 60, 80, and 100 mg/L, and incubated at 25 ℃ for 24 h. Then the mycelium was washed three times in sodium phosphate buffer, the mycelia were dried at 70℃ for 12 h, and the dry weights were then measured using an analytical balance (Ms105, Mettler Toledo, Greifensee, Switzerland). the results were expressed as g/100mLPDB.”

Comments:Section 2.6: not clear if you treated the mycelium (why you use the plural?) on petri or liquid cultures

Response:Thank you very much for your overall approval and comprehensive comments on this manuscript.We described the effects of thymol on the relative conductivity, nucleic acid release, and pH of NEO thymol in section 2.6, but these descriptions may not be clear enough to explain that they were performed under liquid medium, so we have revised them. Specific modifications are as follows:

Sectoin 2.6: “Relative conductivity and pH were determined with reference to the method of Shao et al [23] . Mycelia were treated with a final mass concentration of 0 (control) and 100 mg/L thymol, and pH and extracellular conductivity were determined at 0, 2, 4, 6 and 8h of incubation in a portable pH meter/potentiometer (PHBJ-260, 0.01 pH resolution, Shanghai Precision Scientific Instruments, Shanghai, China) and conductivity meter (DDS-11A, Shanghai Yidian Scientific Instruments Co, Shanghai, China)in a shaker, and relative conductivity was calculated. Mycelial nucleic acid leakage was determined using an ultraviolet-visible spectrophotometer (60UV-Vis, Agilent) as described by Shi et al [24] , and the results were expressed as absorbance values at 260 nm.” replaced by “Relative conductivity and pH were determined with reference to the method of Shao et al [23] . NEO mycelia from 20mL PDB culture were gained through centrifugating for 20 min at 4000×g, rinsed three times, and resuspendedin 20mL of pH 7.0 phosphate buffered saline. Mycelia were treated with a final mass concentration of 0 (control) and 100 mg/L thymol, and pH and extracellular conductivity were determined at 0, 2, 4, 6 and 8h of incubation in a portable pH meter/potentiometer (PHBJ-260, 0.01 pH resolution, Shanghai Precision Scientific Instruments, Shanghai, China) and conductivity meter (DDS-11A, Shanghai Yidian Scientific Instruments Co, Shanghai, China)in a shaker, and relative conductivity was calculated. Mycelial nucleic acid leakage was determined using an ultraviolet-visible spectrophotometer (60UV-Vis, Agilent) as described by Shi et al [24] , and the results were expressed as absorbance values at 260 nm.”

Comments:line 136: what is it "thymol control"? Please correct (also line 141 and following sections)

Response:Thank you for your suggestion.We have amended line 136 and line 141 in our newly submitted "Manuscript File". The specific modifications are in the reply below and the revised manuscript:

line 109: “Mycelia were treated with thymol control, 100mg/L”replaced by “The suspensions were treated with thymol at various concentrations (0, MIC) ”

Comments:line 182: you repeated the treatment 3 times or you performed three independent experiments?Section 2.13: The description of the statistical analysis is completely wrong. Please correct as it should be.

Response:Thank you for your suggestion.We have amended section 2.13 in our newly submitted "Manuscript File". The specific modifications are in the reply below and the revised manuscript:

section 2.13: “All treatments involved in the study were repeated 3 times, and the differential significance analysis was conducted using Duncan's multiple range test or one way analysis of variance (ANOVA) with SPSS software (IBM SPSS Statistics 25.0). Data were expressed as mean± standard deviation (SD). plotting was performed with Origin 2021 (OriginLab Ltd., Northampton, MA, USA) software.” replaced by “Statistical analysis of experimental data was conducted using SPSS 25.0 software. Analysis of variance was applied followed by Duncan’s multiple range tests to separate means. A value of p < 0.05 was considered significant.”

Comments:line 190-192: fig 1 does not provide data that support the statement about mass-concentration dependent effect

Response: Thank you for your suggestion. The colony diameter of mycelium after 2d of treatment was 23.62, 19.47, 6.267, 3.9, 1.11, and 0 mm for the control and 20, 40, 60, 80, and 100 mg/L treatments;The colony diameter of mycelium after 7d of treatment was 80, 67.87,50.64, 48.39, 17.78, and 8.86 mm for the control and 20, 40, 60, 80, and 100 mg/L treatments; Since the results of Section 3.1 mainly describe several groups with large differences, not all data are listed, and relevant data can be added to the appendix.

Comments:section 3.3: no need to repeat all the data you present in figure. The first sentence is not understandable. Last sentence not needed, it is a conclusion.

Response:Thank you for your suggestion.We have amended section 3.3 in our newly submitted "Manuscript File". The specific modifications are in the reply below and the revised manuscript:

section 3.3: “Different mass concentrations of thymol treatment had varying degrees of influence on the dry weight of mycelium as compared to the control group, and the concentration of thymol displayed an opposing trend shift with the dry weight of mycelium (Fig. 2-C). The dry weight of mycelium after 24 hours of treatment was 0.1191, 0.0564, 0.0517, 0.0316, 0.0221, and 0.0211 g/100mLPDB for the control and 20, 40, 60, 80, and 100 mg/L treatments, respectively, and the dry weight of mycelium was considerably lower than the control (P < 0.05). Thymol had a considerable influence on NEO mycelial development, according to the findings.” replaced by “The mycelial weights of NEO in thymol treatment and control groups are shown in Fig. 2-C. The data showed that mycelial growth biomass w as strongly inhibited with increasing the thymol concentration. Initially, the dry weights was 0.1191 g/100mL at the no thymol concentration. At higher thymol concentrations (20, 40, 60 and 80 mg/mL), the effect on mycelial weights was recorded at a significant level (p < 0.05) in comparison with the control group.”

Comments:figure 3: I do not see SD bars

Response: We are very grateful and agree with the important issues and suggestions you have pointed out. Figure 3 Each graph has an SD bar, which is not noticeable in the graph because the error is relatively small.

Comments:lines239-240,lines259-261,line273-274,line 283-285,line 292-295,line 325-327: no references have to be included in the results. The sentence must be moved to discussion.

Response: We are very grateful and agree with the important issues and suggestions you have pointed out. We have made large-scale modifications in the manuscript file . Please check in the newly submitted manuscript file.

Author Response File: Author Response.docx

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Inhibitory Activity and Mechanism of Action with Thymol against the Blueberry Pathogenic Fungi Caused by Neopestalotiopsis clavispora

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