Fungi Detected in the Previous Year’s Leaf Petioles of Fraxinus excelsior and Their Antagonistic Potential against Hymenoscyphus fraxineus

Round 1

Reviewer 1 Report

Research article revised, is a study with actual topic bringing interesting results generated with extensive material, pertaining ash dieback - the very economically important disease in Europe. 

Literary introduction is well written, contains essential data on the issue, I found it exhaustive. The aims are clearly described.

The methodology is appropriate for the analysis, individual methodological approaches are described in details.

Table 1- Italicised latin names of trees: Alnus glutinosa, ...

Chapter 2.4, lines 197-200: PCR conditions – 1. denaturation step was 98C? usually 95C. 2. Annealing temperature ranged from 52-64C, how it was calculated for different fungal species

Results section is extensive, long and therefore not easy to follow in same parts. In some cases, general data are included and presented, that in my opinion are not relevant for this study, I suggest to critically review and delete these parts.

For example, in subchapter 3.2 parts started in line 407 about type of fruiting bodies, or explain why these parts are essential for this study. Or lines 421-423 are not strictly results, belong to the Discussion.

Discussion is divided in two subchapters, first – fungal communities, second competition between H. fraxineus and saprotrophs, what entered an idea to rephrase the aims or at least to consider to reformulate similarly in two main aims: 1. Fungal species diversity..... (merge (i) and (iii).

The data are appropriately analysed and interpreted, but in summarising the results in subchapter 4.2. I miss a clear and unambiguous new outcome from obtained experiment.

Supplementary material, tables, figures:

Too many tables, figures, and supplementary material, try to reduce them.

Tables S2 and S3 should be merged in one table, you can include one more column and mark there, if the species or taxonomic rank was detected in situ or isolated, or both

Then if you compare data in Table S3 and Fig 1, their content is almost the same, presented in other way, I suggest to combine it in one form of presentation.

Or table 2 contains similar data as Table S2, please try present the same data once, chose the most suitable form which is more relevant for analysis and outputs presentation.

References

It will be right and useful if the authors could allow to reviewers to have look in their accepted paper to check the originality of two manuscripts that have very similar names:

Bilański, P.; Kowalski, T. Fungal endophytes in Fraxinus excelsior petioles and their in vitro antagonistic potential against the ash dieback pathogen Hymenoscyphus fraxineus. Microbiol. Res. 2021

Author Response

We would like to thank for the time and effort given to review our manuscript and for comprehensive comments and suggestions. We believe that the manuscript has been significantly improved, thanks to Your valuable contribution. We express our gratitude to both Reviewers in the manuscript in Acknowledgements.

We took into account Your many suggestions and answer all your questions.

Question 1: Table 1- Italicised latin names of trees: Alnus glutinosa, ...

Answer 1: We corrected the style of Latin names of trees to italics.

Question 2: Chapter 2.4, lines 197-200: PCR conditions – 1. denaturation step was 98C? usually 95C. 2. Annealing temperature ranged from 52-64C, how it was calculated for different fungal species

Answer 2: Ad 1 PCR was performed according to the manufacturer's protocol https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0012395_PhusionGreen_HighFidelity_DNAPolymerase_UG.pdf

Ad 2 For optimal results, the Tm calculator was used as recommended by the manufacturer

https://www.thermofisher.com/pl/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html#instruction

Question 3: Results section is extensive, long and therefore not easy to follow in same parts. In some cases, general data are included and presented, that in my opinion are not relevant for this study, I suggest to critically review and delete these parts.

For example, in subchapter 3.2 parts started in line 407 about type of fruiting bodies, or explain why these parts are essential for this study. Or lines 421-423 are not strictly results, belong to the Discussion.

Answer 3:

We deleted the text in lines 310-313

We deleted the text in lines 387-391 

We deleted the text in lines 408- 413

We deleted the text in lines 418-419

We deleted the text in line 438

We deleted the text in lines 496-498

We deleted the text in lines 500-502

We would like to leave the text in lines 422-424  because we have a figure attached to this text and this is the only place in Results with information about this process 

Question 4: Discussion is divided in two subchapters, first – fungal communities, second competition between H. fraxineus and saprotrophs, what entered an idea to rephrase the aims or at least to consider to reformulate similarly in two main aims: 1. Fungal species diversity..... (merge (i) and (iii).

Answer 4:

Following your suggestion, we have now formulated two goals; we combined goal (i) and (iii)

Besides, we renamed subchapter 4.1, now it is 4.1 Fungal Diversity

Question 5: The data are appropriately analysed and interpreted, but in summarising the results in subchapter 4.2. I miss a clear and unambiguous new outcome from obtained experiment.

Answer 5:

In our opinion, in subchapter 4.2, the most important achievements are clearly listed:

line 732  … „The results show that some of the saprotrophs found can be considered as very effective antagonists of H. fraxineus”.

line 744  …“The dual-culture assay is expected to demonstrate the different strategies used by various groups of to gain occupation.”

line 747 first group: …. “One of these groups includes the fast growing fungi…, which strongly inhibited growth of H. fraxineus in vitro”.

line 759  second group: …. “The most potentially valuable saprotrophs were those that inhibited growth of H. fraxineus from the greatest distance and produced wide inhibition zones.”

line 797  third group: … “Only a few fungi overgrew and covered the H. fraxineus colony in dual cultures, an effect that may indicate their potential for antagonism in nature.”

For each group, we list the saprotrophs which, according to the research results, play the greatest antagonistic role.

Supplementary material, tables, figures:

Question 6: Too many tables, figures, and supplementary material, try to reduce them.

Tables S2 and S3 should be merged in one table, you can include one more column and mark there, if the species or taxonomic rank was detected in situ or isolated, or both

Answer 6: According your suggestion, we merged tables S2 and S3.

Question 7: Then if you compare data in Table S3 and Fig 1, their content is almost the same, presented in other way, I suggest to combine it in one form of presentation. Or table 2 contains similar data as Table S2, please try present the same data once, chose the most suitable form which is more relevant for analysis and outputs presentation.

Answer 7: As mentioned above,  we reduced tables S2 and S3 to one common table

References

Question 8: It will be right and useful if the authors could allow to reviewers to have look in their accepted paper to check the originality of two manuscripts that have very similar names:

Bilański, P.; Kowalski, T. Fungal endophytes in Fraxinus excelsior petioles and their in vitro antagonistic potential against the ash dieback pathogen Hymenoscyphus fraxineus. Microbiol. Res. 2021

Answer 8: We enclose the manuscript published in Microbiological Research for Reviewers (author-coverletter-14399057.v1.docx). As the titles of both works show, there is a fundamental difference. The present work is devoted to fungi in dead previous years petioles in the litter. The paper in Microbiological Research is devoted to fungal endophytes, that is, in living symptomless petioles in leaves in tree crown.

Author Response File: Author Response.docx

Reviewer 2 Report

I enjoyed reading the manuscript. It is well-written and has a logical flow.

The results are presented in a very good way, in addition to scientific soundness of results and proper analysis approaches, the use of different types of diagrams such as PCA and network analysis adds to the value of the study. 

Discussion part properly refer to similar studies and the findings are critically analysed.

It would have been great to shorten the manuscript and move some parts to the supplementary materials.

Author Response

We would like to thank for the time and effort given to review our manuscript and for comprehensive comments and suggestions. We believe that thanks to Your valuable contribution the manuscript has been significantly improved. We express our gratitude  to both Reviewers in the manuscript in Acknowledgements.

We took into account Your suggestions.

Question 1: It would have been great to shorten the manuscript and move some parts to the supplementary materials.

Answer 1: We reduced the number of Figures in the main text and moved two of them to Supplementary materials. In addition, we combined two tables (S2 and S3) in Supplementary, and now created one table from them. The text on the research objectives was shortened, we removed a small fragment from the chapter 'Results'. In general, it is difficult to significantly shorten the text because we received a lot of results, and we did not want to present them in two separate papers.