Exposure to the Endophytic Fungi Regulates the Anthocyanin Profiles in the Post-Veraison Grape Berries of ‘Cabernet Sauvignon’
Round 1
Reviewer 1 Report
This work is suitable to publish in this journal. However, there are several points are needed to clarify and give more details. Additionally, some error of typing was found in several point. Please carefully check throughout the manuscript and correct them. I have made decision as major revision. The authors carefully read and all comments and questions are needed to response point by point. It can be accepted after revised MS is considered by editor. Finally, all my comments and questions are put on the MS in file attachment.
Comments for author File: 
Comments.pdf
Author Response
Dear Reviewer, the authors are greatly appreciated for those kindly raised valuable comments, which were all helpful to improve our manuscript. The present version has been revised accordingly, all comments and suggestions raised by each reviewer have been responded point to point.
Comments and Suggestions for Authors
This work is suitable to publish in this journal. However, there are several points are needed to clarify and give more details. Additionally, some error of typing was found in several point. Please carefully check throughout the manuscript and correct them. I have made decision as major revision. The authors carefully read and all comments and questions are needed to response point by point. It can be accepted after revised MS is considered by editor. Finally, all my comments and questions are put on the MS in file attachment.
Responses: Thank you for giving a chance to revise our manuscript, we have carefully learned all those kindly raised comments and suggestions and revised point by point accordingly.
Detailed comments
- Line 72: in vitro, italic?
Response: The point has mended in the text. Thank you!
- Line 78: What technique was used for isolation of fungal endophyte? There are any previous published paper from your own work, please give more detail and reference are needed to mention.
Response: Thanks for reminding. In the text, we have added the information and reference as follows: Twelve strains of fungal endophytes were used in this study, which isolated from grapevine leaves of ‘Rose Honey’ (RH) from local vineyards (Yunnan province, China) using a tissue patch method [15].
- Line 80: It would be describe for PCR condition and the universal primers sequence were used briefly.
Response: In the text, we have added the information as follows: The PCR mixture (50 μL) contained 1 μL ITS5 (10 μmol L–1), 1 μL ITS4 (10 μmol L–1), 5 μL 10×PCR buffer, 4 μL dNTPs, 1 μL DNA template, 0.3 μL Taq (5 U), and 37.7 μL ddH2O. The reaction conditions are as following: pre-denaturation at 94 °C for 4 min, with one cycle; denaturation at 94 °C for 1 min, renaturation at 54 °C for 1 min, and extension at 72 °C for 1 min, with 33 cycles; and extension at 72 °C for 3 min. PCR products were commercially sequenced (Shenggong, Shanghai) and the BLASTn search option of the NCBI database was used for species identification.
- Line 88：Table 1, How is accession no. of all fungal species? It should be mentioned.
Response: In the text, we have added the "GenBank Accession No." of the fungal endophytes in Table 1.
- Line 88：Table 1, All abbreviation (PAL, SPr, TF, TPh) are needed to mention in full name as the note under the table.
Response: In the text, we have added the full names of these mentioned abbreviations.
- Line109：Did you add tween-20 for preparation of fungal spore suspension?
Response: In our experiment, we did not add tween-20 for preparation of fungal spore suspension.
- Line115：PAL activity is needed to describe, how is it concern with anthocyanin or why this activity are needed to observe.
Response: In the text, we have added the relative information in the “Introduction” section.
- Line124：4 000, 4,000
Response: The point has mended in the text. Thank you!
- Line156,157, SPr, TF and TPh need to mention for full name
Response: Full name of these abbreviations have added in the text.
- Line188, put "104.07%, respectively."
Response: The point has mended accordingly. Thank you!
- Line208-211: letter size is differences.
Response: The point have revised.
- Line222: RH49, Is it RH48?
Response: We have checked the text, and it is RH49.
- Line225: RH44 to RH44,
Response: The point have revised. Thank you!
- Line228: Figure 2, Could not found "a" and "b" in the figure. please clarify this
Response: Thank you for reminding. In the text, we have mended the Figure 2 in the present version.
Line245: put the comma "15.53 mg/100g, "
Response: The point have revised. Thank you!
- Line358: The conclusion should be showed the significant point of this finding in particular novel result of anthocyanin found after co-culture with fungal endophyte. Which isolate should be the best candidate for future application.
Response: Thanks so much for the kind suggestion, and the section of "Conclusions" has been rewritten to more emphasize our novel result of the research, as well as those potential candidate isolates in future utilizations.
Reviewer 2 Report
General comments The manuscript entitled “Exposures to the Endophytic Fungi Regulate the Anthocyanin Profiles in Post-veraison Grape Berries of ‘Cabernet Sauvignon’ “contains interesting findings of great scientific importance. There are no significant critical comments regarding the research methodology, the quality of chemical analyzes and the method of interpreting the results. Comments concern the stylistic and editorial side of the manuscript. The weakest part of the manuscript requiring major revisions is the 'Conclusions'.
Detailed comments
l. 15-17 and l. 70-72. “For validating the effects, our present work further assessed the impacts of the same fungal endophytes on grape berries of another worldwide planting grapevine cultivar ‘Cabernet Sauvignon’”. This sentence suggests that the local variety 'Rose Honey' is widely cultivated around the world. Therefore, it is advisable to correct the text, e.g. “For validating the effects, our present work further assessed the impacts of the same fungal endophytes on berries of another grapevine cultivar ‘Cabernet Sauvignon’, which is widely cultivated around the world”. A similar change of meaning can be achieved by using a comma: “For validating the effects, our present work further assessed the impacts of the same fungal endophytes on berries of another, worldwide planting grapevine cultivar ‘Cabernet Sauvignon’”. l. 19. Before using the abbreviation “PAL” for the first time in an abstract, its full meaning should be given. ‘Conclusions’ is a section of the manuscript that should be rewritten. It's meant to be a standalone summary. It should not include addresses to literature (references) or tables available in other sections of the text. Such addresses are appropriately placed in the Introduction, Discussion or Results sections. Conclusions should not contain controversial content, but findings resulting from the present research. l. 371. “quality of certain or different grape varieties”, should be replaced with another term, e.g. “quality of chosen (selected) grape varieties”.
Date of manuscript received: 13 January 2023
Date of this review: 17 January 2023
Comments for author File: Comments.pdf
Author Response
Dear Reviewer, the authors are greatly appreciated for those kindly raised valuable comments, which were all helpful to improve our manuscript. The present version has been revised accordingly, all comments and suggestions raised by each reviewer have been responded point to point.
Comments and Suggestions for Authors
General comments
The manuscript entitled “Exposures to the Endophytic Fungi Regulate the Anthocyanin Profiles in Post-veraison Grape Berries of ‘Cabernet Sauvignon’ “contains interesting findings of great scientific importance. There are no significant critical comments regarding the research methodology, the quality of chemical analyzes and the method of interpreting the results. Comments concern the stylistic and editorial side of the manuscript. The weakest part of the manuscript requiring major revisions is the 'Conclusions'.
Response: Thanks so much for the kind suggestion. We have revised the "Conclusions'' section accordingly.
Detailed comments
- 15-17 and l. 70-72. “For validating the effects, our present work further assessed the impacts of the same fungal endophytes on grape berries of another worldwide planting grapevine cultivar ‘Cabernet Sauvignon’”. This sentence suggests that the local variety 'Rose Honey' is widely cultivated around the world. Therefore, it is advisable to correct the text, e.g. “for validating the effects, our present work further assessed the impacts of the same fungal endophytes on berries of another grapevine cultivar ‘Cabernet Sauvignon’, which is widely cultivated around the world”. A similar change of meaning can be achieved by using a comma: “For validating the effects, our present work further assessed the impacts of the same fungal endophytes on berries of another, worldwide planting grapevine cultivar ‘Cabernet Sauvignon’”.
Response: Thanks so much for giving the good suggestion to better improve our manuscript. We have revised the sentences accordingly in the text.
- Before using the abbreviation “PAL” for the first time in an abstract, its full meaning should be given. ‘Conclusions’ is a section of the manuscript that should be rewritten. It's meant to be a standalone summary. It should not include addresses to literature (references) or tables available in other sections of the text. Such addresses are appropriately placed in the Introduction, Discussion or Results sections. Conclusions should not contain controversial content, but findings resulting from the present research.
Response: The full name of "PAL" in the abstract has been added. And the section of ‘Conclusions’ has been rewritten. Thank you!
- 371. “quality of certain or different grape varieties”, should be replaced with another term, e.g. “quality of chosen (selected) grape varieties”.
Response: This sentence has been rewritten accordingly. Thank you!
Reviewer 3 Report
Manuscript ID: horticulturae-2135554
Manuscript title: Exposures to the Endophytic Fungi Regulate the Anthocyanin Profiles in Post-veraison Grape Berries of ‘Cabernet Sauvignon’
Authors: Xiaoxia Pan, Chunxiao Chen, Yutao Wang, Youyong Zhu and Mingzhi Yang
The present work deals with the effects of the infection brought about by endophytic fungi on the biosynthesis of phenylpropanoid molecules in grape berries
The novelty and originality appear to be adequate. The topic is also adequate and of interest, for the Journal readership.
There are however a number of relevant points I would ask the Authors to take into account, before their manuscript can be further considered for publication. In the following, I will first concentrate on the major points, and then I will move to the minor ones.
Major points:
1) The rationale behind the experiment: in order to increase the attractiveness of their study, the Authors should make clear its practical implications, in agronomic/horticultural terms: do they believe it would be possible to carry out artificial infection of a vineyard with endophytic fungi, in order to influence the anthocyanin profile of a given grape cultivar? Or instead the present one is a purely theoretical study, illustrating the potential biological effects of endophytic fungi?
2) Materials and methods: a) please specify how was obtained the “berry powder”, alluded to at line 122, from which all the anthocyanin metabolites were extracted; b) please specify which extraction method was adopted for measuring enzymatic activities (PAL); c) please clarify why one discovers only in the Conclusions (line 322) that anthocyanin were measured also in leaves, and not only in fruits: supplementary material (Table S3, in the present case) cannot be intended to introduce additional experimental details and results, not previously cited and described in the main text.
3) Statistics: please state clearly how many biological replicates have been analyzed for each experimental variant, and how many technical replicates for each biological replicate
4) Results: in the present work, results concerning anthocyanin are presented in quantitative terms (e.g. mg per 100 g of sample matrix). In this respect, and to the best on my knowledge, the only manner for correctly estimating quantities, in a chromatographic separation, is the use of standards, either internal or external. Did the Authors use anthocyanin standards, in their chromatographic separations? If yes, which ones? If not, then the Authors should only compare the relative abundance of anthocyanin molecules among each other, on the basis of their mass spectra, in a semi-quantitative manner.
5) Results presentation: Figure 1 is not clear at all. And in general, many figures and tables here lack appropriate legends, allowing the reader to smoothly understand what is being presented without recurring to the text. As the rule of thumb, each table and figure should be self-consistent, i.e. clearly understandable without any reference to the text.
6) Language: albeit with much sympathy from my side for the Authors, being myself a non-native speaker, the English used in the present manuscript is rather below an acceptable standard, especially in terms of syntax (but also grammar, at times). So that, I strongly recommend the Authors to have their whole manuscript inspected and corrected by a competent writer in English, either a colleague of theirs, or, even better a professional editing service.
Minor points:
7) Acronyms: please be sure that every single uncommon acronym is spelled out in full, upon its first mention (see for example “ITS DNA” on line 80). Pease avoid using acronyms in figures and tables without having them defined in figure legends and table headings.
8) Please use the exponential notation, for numerical values, and not the fractional one: e.g mg g-1, and not mg/g.
9) Please check the layout of tables, because most of the times the columns headings are truncated
All the above considering, I recommend major revision of the present manuscript accurately addressing all the points raised above.
Author Response
Dear Reviewer, the authors are greatly appreciated for those kindly raised valuable comments, which were all helpful to improve our manuscript. The present version has been revised accordingly, all comments and suggestions raised by each reviewer have been responded point to point.
The present work deals with the effects of the infection brought about by endophytic fungi on the biosynthesis of phenylpropanoid molecules in grape berries
The novelty and originality appear to be adequate. The topic is also adequate and of interest, for the Journal readership.
There are however a number of relevant points I would ask the Authors to take into account, before their manuscript can be further considered for publication. In the following, I will first concentrate on the major points, and then I will move to the minor ones.
Major points:
- The rationale behind the experiment: in order to increase the attractiveness of their study, the Authors should make clear its practical implications, in agronomic/horticultural terms: do they believe it would be possible to carry out artificial infection of a vineyard with endophytic fungi, in order to influence the anthocyanin profile of a given grape cultivar? Or instead the present one is a purely theoretical study, illustrating the potential biological effects of endophytic fungi?
Response: Thank you for raising the kind consideration to our study. Presently, our study is to theoretically validate the potential application of endophytic fungi in regulating the anthocyanin of grape berries. In addition, the significance of the study is to provide method for screening candidate endophytic fungal isolates which could be utilized the practices. Our following researches will consider how to practically apply these candidate fungal isolates in agronomic/horticultural systems. In present version, we have added these information in the sections "Introduction" and "Conclusions'' accordingly, which we hope the revision have made your kind consideration more clearly. Thank you again!
2) Materials and methods: a) please specify how was obtained the “berry powder”, alluded to at line 122, from which all the anthocyanin metabolites were extracted; b) please specify which extraction method was adopted for measuring enzymatic activities (PAL); c) please clarify why one discovers only in the Conclusions (line 322) that anthocyanin were measured also in leaves, and not only in fruits: supplementary material (Table S3, in the present case) cannot be intended to introduce additional experimental details and results, not previously cited and described in the main text.
Response: Thanks so much for the kind reminding. a) and b): we have added accordingly the information in the sections "Materials and methods". c): The section of ‘Conclusions’ has been rewritten, and the text of ‘Comparative analysis of total contents of detected metabolites in cells, leaves and fruits of ‘Cabernet Sauvignon’ after co-cultured with different endophytic fungal strains’, and supplementary Table S3 has been deleted in this present version, due to the unnecessary.
3) Statistics: please state clearly how many biological replicates have been analyzed for each experimental variant, and how many technical replicates for each biological replicate
Response: Thanks so much for reminding. In the text, we have added the information accordingly.
4) Results: in the present work, results concerning anthocyanin are presented in quantitative terms (e.g. mg per 100 g of sample matrix). In this respect, and to the best on my knowledge, the only manner for correctly estimating quantities, in a chromatographic separation, is the use of standards, either internal or external. Did the Authors use anthocyanin standards, in their chromatographic separations? If yes, which ones? If not, then the Authors should only compare the relative abundance of anthocyanin molecules among each other, on the basis of their mass spectra, in a semi-quantitative manner.
Response: Our study used malvidin-3-O-glucoside chloride (Sigma-Aldrich, USA) as an external standard, and we have added in the present version.
- Results presentation: Figure 1 is not clear at all. And in general, many figures and tables here lack appropriate legends, allowing the reader to smoothly understand what is being presented without recurring to the text. As the rule of thumb, each table and figure should be self-consistent, i.e. clearly understandable without any reference to the text.
Response: The legends of Figure 1 have revised accordingly in the text. Thank you!
6) Language: albeit with much sympathy from my side for the Authors, being myself a non-native speaker, the English used in the present manuscript is rather below an acceptable standard, especially in terms of syntax (but also grammar, at times). So that, I strongly recommend the Authors to have their whole manuscript inspected and corrected by a competent writer in English, either a colleague of theirs, or, even better a professional editing service.
Response: The present version has been fixed the the syntax and grammatical errors by a professional editing service, American Journal Experts (AJE).
Minor points:
- Acronyms: please be sure that every single uncommon acronym is spelled out in full, upon its first mention (see for example “ITS DNA” on line 80). Please avoid using acronyms in figures and tables without having them defined in figure legends and table headings.
Response: Thanks so much for reminding. We have carefully checked our manuscript thoroughly and added the information.
- Please use the exponential notation, for numerical values, and not the fractional one: e.g mg g-1, and not mg/g.
Response: We have checked and revised the points carefully in the present version.
9) Please check the layout of tables, because most of the times the columns headings are truncated
Response: The concerned points have checked and mended in the text. Thank you!
All the above considering, I recommend major revision of the present manuscript accurately addressing all the points raised above.
Response: Thanks so much for raising all these valuable comments and suggestions. We have checked the all those concerns and revised accordingly.
Round 2
Reviewer 3 Report
Manuscript ID: horticulturae-2135554_v2
Manuscript title: Exposures to the Endophytic Fungi Regulate the Anthocyanin Profiles in Post-veraison Grape Berries of ‘Cabernet Sauvignon’
Authors: Xiaoxia Pan, Chunxiao Chen, Yutao Wang, Youyong Zhu and Mingzhi Yang
In the present revised version, several of the comments raised from my side on the original submission have been met in a satisfactory manner.
There are, however, still some points which deserve adequate consideration on the part of the Authors:
1) Materials and methods: in order to be a bit more credible, the sentence concerning the use of the anthocyanin (external) standard should be not associated to the tissues extraction procedure (unless it was instead an internal standard), as in the present revised version, but rather it should be associated to the UPLC/MS analysis
2) Results: the way in which the RI index was calculated is obscure, and needs adequate clarification
3) Results presentation: Figure 1 continues to be not clear at all, at least to me. Please explain (in the text or in the figure legend) or change the type of presentation for the results shown in Figure 1.
All the above considering, I recommend major revision of the present manuscript accurately addressing all the points raised above.
Author Response
Dear Reviewer, thank you so much for pointing out all these inadequacies that remaining in our manuscript. We have learned the considerations carefully and revised them point by point as best as we can. All concerned points have also responded accordingly. Thank you and many wishes!
1) Materials and methods: in order to be a bit more credible, the sentence concerning the use of the anthocyanin (external) standard should be not associated to the tissues extraction procedure (unless it was instead an internal standard), as in the present revised version, but rather it should be associated to the UPLC/MS analysis
Responses: Thanks so much for pointing out the improper description in M & M section, concerning the quantitation of anthocyanin by using external standard. The sentence have rewritten and moved to the end of the subsection as a separate paragraph.
2) Results: the way in which the RI index was calculated is obscure, and needs adequate clarification
Response: The detailed information to describe the response index (RI) have added in M & M section of the manuscript, including the calculation method and the significance of using the index. In results section, sentences related to RI have rewrote. We hope the revisions could have well clarified the index of response index (RI) in the manuscript.
3) Results presentation: Figure 1 continues to be not clear at all, at least to me. Please explain (in the text or in the figure legend) or change the type of presentation for the results shown in Figure 1.
Responses: We are so sorry to present a difficult-understand figure, owing to the imperfect plots, and the unclear figure tittle and and legends as well. In this version, Figure 1 has revised completely, by adding the axis tittles, rewriting the figure tittle and the corresponding legends. We hope the revision have made the figure more understandable. Thank you!
