Managing the Microbiome on the Surface of Tomato Fruit by Treatment of Tomato Plants with Non-Thermal Atmospheric-Pressure Plasma During Cultivation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors, you should address my comments highlighted across the text.

Comments for author File: Comments.pdf

Author Response

Response to the comments of Reviewer 1

Thank you so much for your great effort to review our manuscript and giving valuable suggestions and comments to improve it. I am sure that they have the point for improving the quality of the manuscript. Let us show you our response to each comment one by one.

 

Reviewer’s comments:

(1) L22: Please, use different words than those in the title.

Response to the comment: We replaced the keywords including the title to two keywords non-including the title: “long-term plasma-irradiation” and “ozone”.

 

(2) L74: “and Experimental Protocol”

Response to the comment: According to the reviewer's suggestion, we inserted "and Experimental Protocol" after a subheading "2.1. Plant Cultivation".

 

(3) L75: Research was carried out on tomato (Solanum lycopersicum L. cultivar Home-Momotaro) at ... in 20.. The experimental protocol was based on the comparison between ...

Response to the comment: According to the reviewer's suggestion, we rewrote the first paragraph of revised version.

 

(4) L157: The sub-section regarding the data statistical processing should be distincted from the previous sub-section.

Response to the comment: According to the reviewer's suggestion, we added the paragraph between the data statistical processing and the previous sub-section in revised version.

 

(5) L168 “by”

Response to the comment: According to the reviewer's suggestion, we rephrased “using” to “by”.

 

(6) L163 “by”

Response to the comment: According to the reviewer's suggestion, we rephrased “using” to “by”.

 

(7) Figure 1: I would recommend to perform a statistical processing to allow for a more reliable comparison between the experimental treatment, such as a two-way ANOVA with two experimental factors: irradiation treatment and treatment time (date).

Response to the comment: In each experiment, the average ± standard deviation (SD) of the values of 12 independent plasma-irradiated samples and 12 independent air-irradiated control samples was calculated. For statistical analysis, the comparison between two groups, plasma-irradiated sample and air-irradiated control sample, was subjected to Welch’s t-test, since a heteroscedasticity of two data variance was revealed by F-test.

 

(8) Figure 2: Please, read the previous comment.

Response to the comment: As well as Figure 1, the average ± standard deviation (SD) of the values of 12 independent plasma-irradiated samples and 12 independent air-irradiated control samples was calculated. For statistical analysis, the comparison between two groups, plasma-irradiated sample and air-irradiated control sample, was subjected to Welch’s t-test.

 

Reviewer 2 Report

Comments and Suggestions for Authors

This work evaluates the effects of non-thermal atmospheric-pressure plasma treatment on the growth of tomato plant as well as preservation of tomato fruits. The idea is interesting with some novelty. However, there are several points that need to be improved.

1.        As indicated in the manuscript, plasma treatment during cultivation could improve the preservation status of tomato fruits. However, only a picture showing the comparison of rot between the two treatments were presented. No data about the rot rate or rot index during preservation can be found. As is reported in literatures, plasma treatment could improve the preservation effect of fruits by regulating the respiration, softening process and other physiological activities except for the alteration of microbial community. In this sense, it may be better to determine some other quality and physiological indicators to interpret the improved preservation status by plasma treatment.

2.        Was there any effect on the chemical composition of the tomato fruits by plasma treatment?

3.        The intensities may affect the treatment of plasma. This work had not done the experiment with different intensities of plasma treatment. However, it can be included in the discussion.

Author Response

Response to the comments of Reviewer 2

Thank you so much for your great effort to review our manuscript and giving valuable suggestions and comments to improve it. I am sure that they have the point for improving the quality of the manuscript. Let us show you our response to each comment one by one.

 

(1) As indicated in the manuscript, plasma treatment during cultivation could improve the preservation status of tomato fruits. However, only a picture showing the comparison of rot between the two treatments were presented. No data about the rot rate or rot index during preservation can be found. As is reported in literatures, plasma treatment could improve the preservation effect of fruits by regulating the respiration, softening process and other physiological activities except for the alteration of microbial community. In this sense, it may be better to determine some other quality and physiological indicators to interpret the improved preservation status by plasma treatment.

Response to the comment: We would greatly appreciate your valuable comments. We completely agree with your suggestions.

However, there is no reality to combine a series of the data: the analysis of the respiration, softening process, and other physiological activities of tomato fruits, to the current manuscript, because the analysis of the relation between the respiration, softening process, and other physiological activities of tomato fruits during preservation and plasma treatment is a big experiment for us.

Indeed, we are now planning to conduct further detail analysis of multiple aspects of fruit preservation, including the respiration, softening process, and other physiological activities. Thus, it would be appreciated if you could allow us to address your requests in our next manuscript.

In current revised version, we would like to describe the importance of the subject in “Discussion section” of revised version.

 

(2) Was there any effect on the chemical composition of the tomato fruits by plasma treatment?

Response to the comment: It is included in our previous response because we are also planning metabolome analysis of tomato fruits harvested from long-term plasma-treated plants and air-treated control plants. Again, it would be appreciated if you could allow us to address the chemical composition of the tomato fruits by plasma treatment in our next manuscript.

 

(3) The intensities may affect the treatment of plasma. This work had not done the experiment with different intensities of plasma treatment. However, it can be included in the discussion.

Response to the comment: It is impossible to change the intensity of plasma of the PEGDS spray device in the manuscript. However, just in case, let me inform of you that the intensity of O₃ in plasma produced by the PEGDS spray device is nearly maximum. In our manuscript, the tomato plants were irradiated with plasma containing highest intensity of O₃ for long-term period.

Reviewer 3 Report

Comments and Suggestions for Authors

Abstract: It is recommended that after the introductory sentence of the subject, the following sentence should be the objective of the work. In reference to the material and methods, it is more interesting to address the design used rather than the variables evaluated.

It is recommended that the keywords be different from the words in the title.

Introduction: It is recommended that the introduction begins with a broader approach to the subject and becomes more specific until the last paragraph ends with the objective of the work in the last sentence.

Material and methods: It is recommended that the subtopics be better structured. The statistical analysis used should be presented in an isolated subtopic. It is recommended to describe the statistical design adopted. It is recommended to describe in more detail the statistical analysis performed.

Results: It is recommended to present the results obtained from the analyses performed. Phrases such as the first sentence written in the results section, for example, refer to material and methods. The differences obtained can be expressed as a % to make it more attractive to the reader and to draw attention to the difference obtained. When presenting an average value, it is recommended to present the standard error of the average in parentheses. The formatting of the figures could be improved, the numbers on the X axis are small, and the text identifying the X axis is in the reverse direction.

Discussion: it is recommended that the discussion be expanded and better structured with citations that help to explain the results obtained.

Appendix: some figures contained in this section would be interesting to present within the manuscript.

References: it is recommended to check whether all references are within the journal's standards and cited in the text.

Comments on the Quality of English Language

When restructuring the manuscript, it is recommended that the English be corrected by a fluent person with experience in scientific writing in the area of ​​study.

Author Response

Response to the comments of Reviewer 3

Thank you so much for your great effort to review our manuscript and giving valuable suggestions and comments to improve it. I am sure that they have the point for improving the quality of the manuscript. Let us show you our response to each comment one by one.

 

(1) Abstract: It is recommended that after the introductory sentence of the subject, the following sentence should be the objective of the work. In reference to the material and methods, it is more interesting to address the design used rather than the variables evaluated.

Response to the comment: We revised the abstract as bellow. It would be happy for us if it is suitable to the flow of the abstract which you could advice.

Abstract: Treatment of plants with non-thermal atmospheric-pressure plasma impacts several aspects of plant life. To investigate the effects of long-term plasma-irradiation on tomato cultivation, the plant growth and preservation status of harvested tomato fruits were compared between long-term plasma-irradiation and air-irradiation. The growth (plant height, numbers of leaves and fruit bunches, SPAD value, and plant dry weight) of plants, which were periodically irradiated with plasma from the three-leaf stage to the green enlarged-fruit stage, was same to air-irradiated control. However, the preservation status of tomato fruits harvested from plasma-irradiated plants was improved in comparison with the air-irradiated control. Analysis of the microbiome on the surface of the fruit indicated that long-term plasma-irradiation during cultivation promoted an increased bacterial diversity on the fruit surface. Thus, the effect of plasma irradiation on diversification of microbial population dynamics in tomato fruit may be associated with improved preservation status of harvested tomato fruits.

 

(2) It is recommended that the keywords be different from the words in the title.

Response to the comment: We replaced the keywords including the title to two keywords non-including the title: “long-term plasma-irradiation” and “ozone”.

 

(3) Introduction: It is recommended that the introduction begins with a broader approach to the subject and becomes more specific until the last paragraph ends with the objective of the work in the last sentence.

Response to the comment: Thank you for your valuable suggestion. We have reconfirmed “Introduction section”. We are sorry but we are wondering that the flow seems to be fine, because it begins with a broader approach to the subject and becomes more specific until the last paragraph ends.

 

(4) Material and methods: It is recommended that the subtopics be better structured. The statistical analysis used should be presented in an isolated subtopic. It is recommended to describe the statistical design adopted. It is recommended to describe in more detail the statistical analysis performed.

Response to the comment: Thank you for your valuable suggestion. Reviewer #1 could recommend same point.

In the sub-section: “Analysis of Plant Growth”, we added the paragraph and described the statistical analysis: the comparison between two groups, plasma-irradiated sample and air-irradiated control sample, was subjected to Welch’s t-test, since a heteroscedasticity of two data variance was revealed by F-test.

Regarding the analysis of the microbial community on the surface of tomato fruits, we added the paragraph between the data statistical processing and the previous sub-section in revised version.

 

(5) Results: It is recommended to present the results obtained from the analyses performed. Phrases such as the first sentence written in the results section, for example, refer to material and methods. The differences obtained can be expressed as a % to make it more attractive to the reader and to draw attention to the difference obtained. When presenting an average value, it is recommended to present the standard error of the average in parentheses. The formatting of the figures could be improved, the numbers on the X axis are small, and the text identifying the X axis is in the reverse direction.

Response to the comment: Thank you for your valuable suggestions.

(a) Phrases such as the first sentence written in the results section, for example, refer to material and methods.

→ We deleted redundant sentences which were referred to “Material and methods section”.

(b) The differences obtained can be expressed as a % to make it more attractive to the reader and to draw attention to the difference obtained.

→ We are sorry, but we could not find the difference which may be appropriate to be expressed as a “percentage” in the “Result section” of the manuscript.

(c) When presenting an average value, it is recommended to present the standard error of the average in parentheses.

→ We are sorry, but we could not find presenting an average value in the text of the “Result section” of the manuscript.

(d) The formatting of the figures could be improved, the numbers on the X axis are small, and the text identifying the X axis is in the reverse direction.

→ Figure 6 only has the number on the X axis. So, we revised the size of the numbers are bigger. Regarding “the text identifying the X axis is in the reverse direction”, we are wondering if it is not X axis but Y axis. We understand as it is and revised the direction of the text of Y axis.

 

(6) Discussion: it is recommended that the discussion be expanded and better structured with citations that help to explain the results obtained.

Response to the comment: Thank you for your valuable suggestions, and I am so sorry to submit incomplete manuscript. When I look close at it, I realized that the discission section was not well organized at all and should be restructured and improved.

We revised it with additional citations as possible.

 

(7) Appendix: some figures contained in this section would be interesting to present within the manuscript.

Response to the comment: Thank you for your valuable suggestions.

We have reconfirmed “Appendix A and “Appendix B”. The photograph of the facility, timetable of the experimental and schematic of plasma device should be included in “Appendix A”.

In “Appendix B”, Figure A5, Table A2 and Figure A6 are the data to evaluate the potential of plasma device, but not the data resulting any new finding. Thus, Figure A5, Table A2 and Figure A6 should be presented in “Appendix B”.

Figure A7, A8, A9 and A10 are the data of second and third experiments out of triplicated experiments. Since first set of data has been presented within the manuscript, the data of second and third experiments should be included in “Appendix B”, because the results are same among three experiments.

Finally, main purpose of microbiome analysis is to understand alpha- and beta-diversity of the population structure of microbiome at the surface of tomato fruits, but not focus on special bacteria. So, Figure A11 and A12 seem to be appropriate to present in “Appendix B”.   

 

(8) References: it is recommended to check whether all references are within the journal's standards and cited in the text.

Response to the comment: Thank you for your valuable suggestions.

We have reconfirmed all references are appropriate to be cited in the text.

 

(9) When restructuring the manuscript, it is recommended that the English be corrected by a fluent person with experience in scientific writing in the area of study.

Response to the comment: I am sorry my poor English. Indeed, the manuscript has been reviewed for English. However, I have check it again.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors, the manuscript can be accepted in present form for publication in Horticulturae, in my opinion.

Author Response

Dear Reviewer 1

I are so happy to know that you could agree with the revised manuscript can be accepted for publication in Horticulturae.

Thank you very much for your great effort and valuable comments to improve our manuscript.

With my best regards,

Hideki Takahashi

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made some revisons according to the comments, and the questions were answered.

Author Response

Dear Reviewer 2

Thank you very much for your great effort and valuable comments to improve our manuscript.

Actually, we are now conducting the analysis of the relation between the respiration, softening process, and other physiological activities of tomato fruits during preservation and plasma treatment, and fruit metabolome analysis. When a series of results and conclusion was obtained, we would like to submit our second manuscript to "Horticulturae", again. In that case, we would appreciate it if you could peer review it.

Many thanks,

Hideki Takahashi

 

Reviewer 3 Report

Comments and Suggestions for Authors

It is recommended that the manuscript be adapted to the template format.

Abstract: It is recommended that after the introductory sentence of the subject, the following sentence be the objective of the work. Addressing the statistical design used in the material and methods is more interesting.

Introduction: It is recommended that the introduction begin with a broader approach to the subject and become more specific until the last paragraph ends with the objective of the work in the previous sentence.

Material and methods: The subtopics should be better structured, the statistical analysis should be presented in an isolated subtopic, and the statistical design adopted should be described.

Results: It is recommended that the results obtained from the analyses performed be presented. The differences obtained can be expressed in % to make it more attractive to the reader and draw attention to the difference obtained. When presenting an average value, it is recommended to show the standard error of the mean in parentheses.

Appendix: Some figures in this section would be interesting to include in the manuscript. For example, figures A2 and A3 help draw the reader's attention. Figure A11 needs to have its font size increased as it is unreadable.

Author Response

Response to the comments of Reviewer #3

Thank you so much for your great effort to review our manuscript and giving valuable suggestions and comments to improve it. I am sure that they have the point for further improving the quality of the manuscript. Let us show you our response to each comment one by one.

 

Reviewer’s comments:

(1) Abstract: It is recommended that after the introductory sentence of the subject, the following sentence be the objective of the work. Addressing the statistical design used in the material and methods is more interesting.

Response to the comment: According to the reviewer #3's suggestion, we further improved.

 

(2) Introduction: It is recommended that the introduction begin with a broader approach to the subject and become more specific until the last paragraph ends with the objective of the work in the previous sentence.

Response to the comment: Thank you for your valuable suggestion. We have further revised “Introduction section” and further improved the flow of the introduction from a broader approach to the subject and becomes more specific until the last paragraph ends.

 

(3) Material and methods: The subtopics should be better structured, the statistical analysis should be presented in an isolated subtopic, and the statistical design adopted should be described.

Response to the comment: According to the reviewer #3's suggestion, we inserted "2.4 Statistical analysis" as a subheading in "2. Materials and Methods” section.

 

(4) Results: It is recommended that the results obtained from the analyses performed be presented. The differences obtained can be expressed in % to make it more attractive to the reader and draw attention to the difference obtained. When presenting an average value, it is recommended to show the standard error of the mean in parentheses.

Response to the comment: First, I would like to apologize if I made a mistake or misunderstand your comment regarding the expression of data. Regarding growth parameters: plant height, numbers of leaves and fruit bunches, SPAD value, and plant dry weight, there were not significant difference between long-term periodical plasma treatment and control air-treatment. Thus, I am wondering if the expression in % does not make sense, because there was not difference in each parameter between two treatments. In case of microbiome analysis, the obtained results in figures 7 and 8 of second revised version can not be expressed in %. It would be appreciated if you could allow me to resubmit this revised version.

 

(5) Appendix: Some figures in this section would be interesting to include in the manuscript. For example, figures A2 and A3 help draw the reader's attention. Figure A11 needs to have its font size increased as it is unreadable.

Response to the comment: According to the reviewer's suggestion, we remove figures A2 and A3 of previous manuscript to new figures 1 and 2 of second revised version, and I have renumbered the other figures accordingly. In addition, the font sizes in figures A10 and A11 of previous manuscript were improved to become readable and renumbered new figures A9 and A10 in second revised version. Since still there is a limitation of the size in each name of “phylum” in new figures A9 and A10, it seems to be difficult to further improved due to technical reason. It would be appreciated if you could accept this revised version.