Thymol Induces Cell Death of Fusarium oxysporum f. sp. niveum via Triggering Superoxide Radical Accumulation and Oxidative Injury In Vitro

Round 1

Reviewer 1 Report

The authors have investigated the effect of thymol over Fusarium oxysporum f. sp. niveum. Several analyses have been done in this respect, including inhibition of spore’s germination and development and different enzymes involved in the oxidative stress.

However, some controls are missing, such as comparison with a standard fungicide to verify if the thymol activity is similar or not!

There are several citations missing in the text (line 54, line 128). The manuscript should also be carefully checked for grammar and misspelling errors.

There should be space placed between the value (number) and symbol when describing temperatures (all over the text).

How do authors justify selecting photograph in Figure 3? Were all the hyphae looking damaged at 35 μg / mL thymol or it was picked just a random photograph with a damaged hypha?

Author Response

1. However, some controls are missing, such as comparison with a standard fungicide to verify if the thymol activity is similar or not!

Responses: Thanks for your suggestion. We’re sorry for the misunderstanding of using “fungicidal activity of thymol” in the manuscript. Actually, in this study, we detected the inhibitory effect of thymol against FON in vitro, without studying the real “fungicidal activity of thymol” that needs to be supported by greenhouse or field experiments. The effect of a standard fungicide is evaluated by field test, with many factors considered. So we have checked the whole manuscript and have rephased “fungicidal activity” or “antifungal activity” in order to focus on the inhibitory effect of thymol against FON in vitro and its mechanism.

2. There are several citations missing in the text (line 54, line 128). The manuscript should also be carefully checked for grammar and misspelling errors.

Responses: Thanks for your suggestion. We have checked and corrected the cited references and the write up throughout the manuscript.

3. There should be space placed between the value (number) and symbol when describing temperatures (all over the text).

Responses: Thanks for your suggestion. We have checked and supplied the missing spaces between number and symbol throughout the manuscript.

4. How do authors justify selecting photograph in Figure 3? Were all the hyphae looking damaged at 35 μg / mL thymol or it was picked just a random photograph with a damaged hypha?

Responses: Thanks for your suggestion. We have observed and checked the whole phenotype of mycelia under microscopy in order to confirm the changes of mycelia. For each treatment, all the hyphae had similar phenotypes. We have taken at least three microscopic photos for each treatment, which can be found as Figure S1 and S2 in supplementary material.

Reviewer 2 Report

the title should mentioned that this is ethanol extract 

Abstract should be have conclusion 

authors should mentioned why they selected these plants 

point 2.2 what you mean by Mycological materials 

is this isolates identified before 

how you prepare the concentration ?

table and figs need more details 

please see attach file 

 

 

 

Comments for author File: Comments.pdf

Author Response

1. the title should mentioned that this is ethanol extract.

Responses: Thanks for your comment. Thymol used in this study were not extracted from plants. They were purchased from Sigma-Aldrich at analytical grade (>98.5%). We have supplied these information in Materials and Methods in revised manuscript.

2. Abstract should be have conclusion

Responses: Thanks for your suggestion. We have revised the abstract to be have conclusion with some important data added.

3. authors should mentioned why they selected these plants 

Responses: Thanks for your comment. We didn’t use any plants in this study.

4. point 2.2 what you mean by Mycological materials

Responses: Thanks for your comment. We have provided the information about FON (race 2) used in this study. 

5. is this isolates identified before 

Responses: Thanks for your comment. The isolate of this FON was identified by Institute for Plant Protection in Jiangsu Academy of Agricultural Sciences. And we obtained this isolate from there. We have provided these information in revised manuscript.

6. how you prepare the concentration?

Responses: Thanks for your comment. Thymol was dissolved in ethanol to preparing a stocking solution at a very high concentration (10 mg/mL). Then it was diluted to different concentrations (10-50 µg/mL) with sterilized water or culturing medium. We have provided these information revised manuscript.

7. table and figs need more details

 Responses: Thanks for your comment. We have provided more details in figure legends in revised manuscript.

8. please see attach file

 Responses: Thanks for your detailed comments. We have revised the whole manuscript based on the comments you marked in the manuscript, which includes abstract revision, errors in reference citation, missing citations, source of pathogen, source of thymol,  procedure of counting germinated conidia, some experimental procedures (fluorescent detection and measurement replicates), revisions of some results, and gramma mistakes.

Reviewer 3 Report

The manuscript "Thymol induces cell death of Fusarium oxysporum f. sp. niveum via triggering superoxide radical accumulation and oxidative injury" presents the activity of Thymol against Fusarium oxysporum f. sp. niveum. The research was well conducted and the results are interesting for an alternative control of this pathogenic fungus.

 

There are some points that need to be addressed by authors prior publication:

1. Abstract, Line 27. "Thymol induced the accumulation of superoxide radical...." - please clarify if this happened in mycelial cells.

2. Introduction, Line 44 - It is necessary to cite references for each Fusarium oxysporum subspecies. It is also necessary to put the names of formae speciales of F. oxysporum 

Line 54 - error in the reference, please correct.

Line 56-57 - Please put a reference in the sentence about the chemical fungicides.

Line 60 - Please clarify what organism have antagonistic action.

Materials and methods - Line 79 - Please specify the isolate of FON you have worked.

Line 84 - Please correct citation [15]

Line 86 - Please explain how Thymol was obtained.

Please specify the concentrations of thymol you performed the assays.

Line 128 - error in the reference, please correct.

Line 144 - What was the statistical software you performed the analysis?

Results - line 151 - Did you check if the linear regression was the best model to fit the data? Did you compare to logistic regression?

Line 213- 214 - Please rewrite: "CAT (catalase) activity showed similar changing pattern with that of SOD" I cannot understand.

Line 222 - please correct "memebrane"

Line 239 - It is necessary to apply a statistical test to compare the curves of conidial germination rate (figure 8B), e.g. Survival analysis.  It is also important to compare the treatments in the first time that the control reached 100%. This would be important to subside the discussion in the line 313.

 

It is necessary an english grammar and spell revision throughout the manuscript.

 

 

 

 

 

 

 

Author Response

1. Abstract, Line 27. "Thymol induced the accumulation of superoxide radical...." - please clarify if this happened in mycelial cells.

Responses: Thanks for your suggestion. Yes, this happened in mycelial cells. We have revised it.

2. Introduction, Line 44 - It is necessary to cite references for each Fusarium oxysporumsubspecies. It is also necessary to put the names of formae specialesof F. oxysporum

Responses: Thanks for your suggestion. We have provided the name of formae speciales of F. oxysporum for each crop with references.

3. Line 54 - error in the reference, please correct.

Responses: Thanks for your suggestion. We have corrected the reference here.

4. Line 56-57 - Please put a reference in the sentence about the chemical fungicides.

Responses: Thanks for your suggestion. We have provided a reference here.

5. Line 60 - Please clarify what organism have antagonistic action.

Responses: Thanks for your suggestion. We have listed these organisms and the references here.

 

6. Materials and methods - Line 79 - Please specify the isolate of FON you have worked.

Responses: Thanks for your suggestion. We have provided the information of FON (race 2) in revised manuscript.

7. Line 84 - Please correct citation [15]

Responses: Thanks for your suggestion. We have corrected the reference here.

8. Line 86 - Please explain how Thymol was obtained.

Responses: Thanks for your suggestion. Thymol was purchased from Sigma-Aldrich at analytical grade (>98.5%). We have supplied these information in Materials and Methods in revised manuscript.

9. Please specify the concentrations of thymol you performed the assays.

Responses: Thanks for your suggestion. We have provided the detailed information about thymol concentrations in Materials and Methods in revised manuscript.

10. Line 128 - error in the reference, please correct.

Responses: Thanks for your suggestion. We have corrected this citation.

11. Line 144 - What was the statistical software you performed the analysis?

Responses: Thanks for your suggestion. We used SPSS to perform the statistical analysis. We have provided this information in revised Materials and Methods.

12. Results - line 151 - Did you check if the linear regression was the best model to fit the data? Did you compare to logistic regression?

Responses: Thanks for your suggestion. Yes, we have compared these two methods. The linear regression is better.  The EC₅₀ (21 µg/mL) of thymol was calculated with linear regression based on colony diameter on plate.  Thymol at this concentration also result in the decrease in mycelial fresh weight by 50% at liquid medium (Figure 2). This confirms that EC₅₀ derived from linear regression is appropriate in this study.

13. Line 213- 214 - Please rewrite: "CAT (catalase) activity showed similar changing pattern with that of SOD" I cannot understand.

Responses: Thanks for your suggestion. We have rephrased this sentence in revised manuscript.

14. Line 222 - please correct "memebrane"

Responses: Thanks for your suggestion. We have made correction here in revised manuscript.

15. Line 239 - It is necessary to apply a statistical test to compare the curves of conidial germination rate (figure 8B), e.g. Survival analysis.  It is also important to compare the treatments in the first time that the control reached 100%. This would be important to subside the discussion in the line 313.

Responses: Thanks for your suggestion. We have provided the statistical analysis in revised Figure 8B.

It is necessary an english grammar and spell revision throughout the manuscript.

Responses: Thanks for your suggestion. We have checked and have revised the English writing throughout the manuscript, especially avoiding using passive voice.

Reviewer 4 Report

This is an in vitro study carried out conscientiously on the different effects that Thymol essential oil has on the mycelium of FOX niveum.

The work seems to be done with a single isolate (this is not clear in material and methods), and there are several references that do not appear in the document ([Error! Reference source 54 not found.]

But my biggest criticism is regarding the reality of cultivation and the need for this study:

The authors do not seem to know the reality of this pathogen in the field, the introduction is very deficient and speaks of generalities about Fusarium, it does not make a true review of the state of the art of the special way that affects watermelons. For example, he does not talk about physiological races (there are four races of this fungus (Fon race 0, 1, 2 and 3). Nor does he talk about the commercial varieties of watermelons having different degrees of resistance to these races.

But above all, this disease is treated in the field in the vast majority of producing regions through the use of grafting plants. This management is enough to stop the disease and the use of phytosanitary products against FOX niveum is minimal.

The work presented is made up of in vitro analysis, they do not present any in vivo test with plants or in the field or greenhouse. The biology of the pathogen means that once the fungus has entered the watermelon plant, it develops in the vascular system, where thymol treatments would never act on the pathogen. That is why it has been used in postharvest against Aspergillus but not against vascular diseases and therefore I do not believe that this paper deserves to be published in a journal with the impact of Agronomy without real verification that the results can be effective in controlling the pathology on field.

Author Response

This is an in vitro study carried out conscientiously on the different effects that Thymol essential oil has on the mycelium of FOX niveum.

The work seems to be done with a single isolate (this is not clear in material and methods), and there are several references that do not appear in the document ([Error! Reference source 54 not found.]

But my biggest criticism is regarding the reality of cultivation and the need for this study:

The authors do not seem to know the reality of this pathogen in the field, the introduction is very deficient and speaks of generalities about Fusarium, it does not make a true review of the state of the art of the special way that affects watermelons. For example, he does not talk about physiological races (there are four races of this fungus (Fon race 0, 1, 2 and 3). Nor does he talk about the commercial varieties of watermelons having different degrees of resistance to these races.

But above all, this disease is treated in the field in the vast majority of producing regions through the use of grafting plants. This management is enough to stop the disease and the use of phytosanitary products against FOX niveum is minimal.

The work presented is made up of in vitro analysis, they do not present any in vivo test with plants or in the field or greenhouse. The biology of the pathogen means that once the fungus has entered the watermelon plant, it develops in the vascular system, where thymol treatments would never act on the pathogen. That is why it has been used in postharvest against Aspergillus but not against vascular diseases and therefore I do not believe that this paper deserves to be published in a journal with the impact of Agronomy without real verification that the results can be effective in controlling the pathology on field.

Responses: Thanks for your comments.

First, we have provided the information of FON used in this study, which is FON race 2.

Second, some errors of reference citation have been corrected in revised manuscript.

Third, we have revised the “Introduction” in order to provide more information about FON, such as formae speciales, physiological races, watermelon resistance, controlling management, etc.

Fourth, in this study, we focus on the inhibitory effect of thymol against FON in vitro, without studying the real “fungicidal activity of thymol” that needs to be supported by greenhouse or field experiments. So we’re sorry for the misunderstanding of using “fungicidal activity of thymol” in the manuscript. We have checked the whole manuscript and have rephased “fungicidal activity” or “antifungal activity” in order to focus on the inhibitory effect of thymol against FON in vitro and its mechanism. Using fungicide to treat the root of watermelon seedlings before transplanting is also effective to control Fusarium wilt. In the future, we aim to develop appropriate formulation thymol that can be used to test the real fungicidal activity of thymol against FON.

Round 2

Reviewer 1 Report

The manuscript is now adequate for publication. Thank you for your corrections.

Author Response

 Thanks for your advice and review.

Reviewer 3 Report

The authors improved the manuscript after the suggestions of the reviewers. There are some minor points to authors correct, presented below:

 

Line 64- error in reference

Line 65-70 - Please improve this part. Please show how the fungus could overcome this management practices and would cause the disease, point out a connection to the subject of the research.

Line 103 - please correct [30]

Line 128 - Please put a period after "...Japan)"

Line 131 - Please correct fluorescent

Line 296 - The presentation of the graph (B) with letters to demonstrate statistical difference is confusing. I suggest to put an adittional table to show these data.

 

Author Response

1. Line 64- error in reference

Response: Thanks for your comment. We have corrected the citation here.

2. Line 65-70 - Please improve this part. Please show how the fungus could overcome this management practices and would cause the disease, point out a connection to the subject of the research.

Response: Thanks for your suggestion. We have revised this part by adding the possible limitations of listed approaches in order to connect the subject of current research.

3. Line 103 - please correct [30]

Response: Thanks for your suggestion. We have corrected the citation here.

4. Line 128 - Please put a period after "...Japan)"

Response: Thanks for your suggestion. A period has been added here.

5. Line 131 - Please correct fluorescent

Response: Thanks for your suggestion. We have corrected this word here.

6. Line 296 - The presentation of the graph (B) with letters to demonstrate statistical difference is confusing. I suggest to put an adittional table to show these data.

Response: Thanks for your suggestion. We have revised the caption of Figure 8 in order to make it understandable for the letters for statistical analysis. In (B), the different lowercase letters indicated the significant difference among different treatment at each time point. And the colors of letters correspond to the colors of legends indicating different treatment.

Reviewer 4 Report

Although the work is correct from a technical point of view, it is irrelevant from the point of view of the actual application of Thymol in the field. As I have indicated, it is a pathogen that would never be directly affected by the substance analyzed because the pathogen is in the soil or in the vascular system of the plant) and whose control in the field has already been resolved without the use of chemical products.

It is not an article to be published in Agronomy, although it is a special issue on Phytoalexins, Resistance Inducers and Sustainable Control Measures in Crop Protection Strategies, it lacks a true practical application, authors are recommended to send it to another less applied journal.

Author Response

Although the work is correct from a technical point of view, it is irrelevant from the point of view of the actual application of Thymol in the field. As I have indicated, it is a pathogen that would never be directly affected by the substance analyzed because the pathogen is in the soil or in the vascular system of the plant) and whose control in the field has already been resolved without the use of chemical products.

It is not an article to be published in Agronomy, although it is a special issue on Phytoalexins, Resistance Inducers and Sustainable Control Measures in Crop Protection Strategies, it lacks a true practical application, authors are recommended to send it to another less applied journal.

Response: Thanks for your comment. We agree with you that the disease can be effectively controlled by grafting or intercropping, but the use of fungicides is also applicable (we have cited some new references in the part of Introduction). In some areas where grafting and intercropping are not applicable, people are still trying to use fungicides to control the disease. Identifying new chemicals still has some kind of significance. And we are trying our best to develop the formulation of thymol in order to test its effect in the field, which may be a long way to go. But we are trying.