Mycobiota Associated with Symptomatic and Asymptomatic Fraxinus excelsior in Post-Dieback Forest Stands

Round 1

Reviewer 1 Report

 

Review report

The aim of the study presented in the article "Mycobiota Associated with Symptomatic and Asymptomatic Fraxinus excelsior in Post-Dieback Forest Stands" by R. Bakys et al. was to determine the composition and structure of the fungal community of ash trees in stands established either from natural regeneration or artificially as progeny trials. The results showed that the fungal communities associated with F. excelsior in young stands established on sites previously affected by the disease were complex and consisted of 1487 fungal taxa. The authors found that Hymenoscyphus fraxineus, the fungus that causes ash dieback, was present in all stands but was not common. It was also highlighted that Cladosporium sp. could be the limiting factor for the infection rate or spread of H. fraxineus. All this determines the importance and relevance of the topic discussed.

A few comments below point to deficiencies in the manuscript to which I would like to draw the authors' attention and which I believe should be corrected.

1.      Introduction.

- The chapter contains only one paragraph dealing with the main theme of the work (lines 73-89). Moreover, the literature cited in this paragraph is only mentioned but not discussed. In my opinion, information on mycobiota diversity of healthy and H. fraxineus-infested ash stands is very important and should be discussed in more detail, citing the main findings, i.e. traits associated with promoting or limiting disease development and spread.

- No research hypotheses were formulated and no clear objectives of the study were stated.

2.      Materials and Methods

Just two brief comments on this chapter, which is generally well organised and described.

- I noticed a contradiction between the age of the trees mentioned in the text (20-30 years, line 105) and the information in Table 1 (7-25 years). Please correct this where necessary.

- I also recommend changing the order and descriptors of the sites studied to make a clear distinction between self-regenerated stands and progeny trials.

3.      Discussion.

The chapter contains parts that do not relate directly to the results obtained and should, in my opinion, rather be placed in other chapters (Introduction, Material and Methods) or simply removed from the manuscript. The examples are the lines: 306-313; 314-319; 323-325; 326-331; 356-361; 362-371; 391-402; 422-425.  

4.      Conclusions.

This chapter is missing from the manuscript. This seems to be a consequence of the lack of a research hypothesis and clearly stated aims of the study.

The last part of the chapter Discussion (from line 425) cannot serve as a conclusion, as its form is still more like a discussion.

5.      References and other errors

Line 327 there is “crows” instead of crowns

Reference no. 5, 9 – provide full reference to cited articles;

Reference no. 40 – remove repetition;

Reference no. 54 – correct author’s name

Author Response

Reviewer #1

The aim of the study presented in the article "Mycobiota Associated with Symptomatic and Asymptomatic Fraxinus excelsior in Post-Dieback Forest Stands" by R. Bakys et al. was to determine the composition and structure of the fungal community of ash trees in stands established either from natural regeneration or artificially as progeny trials. The results showed that the fungal communities associated with F. excelsior in young stands established on sites previously affected by the disease were complex and consisted of 1487 fungal taxa. The authors found that Hymenoscyphus fraxineus, the fungus that causes ash dieback, was present in all stands but was not common. It was also highlighted that Cladosporium sp. could be the limiting factor for the infection rate or spread of H. fraxineus. All this determines the importance and relevance of the topic discussed.

Response: we acknowledge these observations.

A few comments below point to deficiencies in the manuscript to which I would like to draw the authors' attention and which I believe should be corrected.

 

1. Introduction.

- The chapter contains only one paragraph dealing with the main theme of the work (lines 73-89). Moreover, the literature cited in this paragraph is only mentioned but not discussed. In my opinion, information on mycobiota diversity of healthy and H. fraxineus-infested ash stands is very important and should be discussed in more detail, citing the main findings, i.e. traits associated with promoting or limiting disease development and spread.

 

Response: we agree that information on mycobiota diversity associated with asymptomatic and symptomatic ash tissues is important. However, to our opinion it is already discussed in Discussion section.

 

- No research hypotheses were formulated and no clear objectives of the study were stated.

 

Response: aims and the hypothesis were included at the end of Introduction.

 

2. Materials and Methods

Just two brief comments on this chapter, which is generally well organised and described.

- I noticed a contradiction between the age of the trees mentioned in the text (20-30 years, line 105) and the information in Table 1 (7-25 years). Please correct this where necessary.

 

Response: the indicated text on indicated line was removed.

 

- I also recommend changing the order and descriptors of the sites studied to make a clear distinction between self-regenerated stands and progeny trials.

 

Response: the order of site descriptors was changed as suggested.

 

3. Discussion.

The chapter contains parts that do not relate directly to the results obtained and should, in my opinion, rather be placed in other chapters (Introduction, Material and Methods) or simply removed from the manuscript. The examples are the lines: 306-313; 314-319; 323-325; 326-331; 356-361; 362-371; 391-402; 422-425.

 

Response: we consider that these parts provide support to our study and place it in a broader context, and therefore, we would like to keep this information intact.

 

4. Conclusions.

This chapter is missing from the manuscript. This seems to be a consequence of the lack of a research hypothesis and clearly stated aims of the study.

The last part of the chapter Discussion (from line 425) cannot serve as a conclusion, as its form is still more like a discussion.

 

Response: conclusions were included at the end of the manuscript.

 

5. References and other errors

Line 327 there is “crows” instead of crowns

Reference no. 5, 9 – provide full reference to cited articles;

Reference no. 40 – remove repetition;

Reference no. 54 – correct author’s name

 

Response: changed as indicated.

Author Response File: Author Response.pdf

Reviewer 2 Report

The paper is very well written and structured and brings new and interesting information on Fraxinus mycobiota.

The introduction is comprehensive and gives an appropriate review of previous research. Authors should only add a sentence or two on aims of this particular research. Which questions were raised and what were the expected answers, that is, what did this research aim to accomplish?

Materials and methods are very well written as well, only few details should be added (please see the comments in the PDF document).

Results are clear and backed-up with diagrams and detailed tables. I suggest adding a short paragraph on differences in mycobiota between natural stands and progeny trials.

Since the discussion is quite long and brings a lot of different data, I strongly suggest adding a Conclusion paragraph to highlight the main findings of this extensive and interesting research.

 

Comments for author File: Comments.pdf

Author Response

Reviewer #2

The paper is very well written and structured and brings new and interesting information on Fraxinus mycobiota.

Response: we are grateful for these observations.

The introduction is comprehensive and gives an appropriate review of previous research. Authors should only add a sentence or two on aims of this particular research. Which questions were raised and what were the expected answers, that is, what did this research aim to accomplish?

Response: aims and the hypothesis were added at the end of Introduction.

Materials and methods are very well written as well, only few details should be added (please see the comments in the PDF document).

Response: suggestions in the PDF were followed and changes were made accordingly. Regarding the observation that “The diagram for roots is missing :)”, only one root per tree was sampled as it is not possible to separate between asymptomatic and symptomatic root samples, and therefore, the diagram is not included.

Results are clear and backed-up with diagrams and detailed tables. I suggest adding a short paragraph on differences in mycobiota between natural stands and progeny trials.

Response: results on natural stands and progeny trials are discussed in lines 454-479.

Since the discussion is quite long and brings a lot of different data, I strongly suggest adding a Conclusion paragraph to highlight the main findings of this extensive and interesting research.

Response: the conclusion section was added to the manuscript.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

My assessment would be accept with minor modification with the following remarks: The manuscript 1928364 submitted to Forests describes the fungal communities associated with shoots, leaves and roots of ash affected by H. fraxineus using NGS (PacBio), with a main emphasis on comparing symptomatic leaves / shoots with asymptomatic leaves / shoots from the same trees. In Lithuanian forests affected by ash dieback since at least 25 years, some young ash stands remain relatively healthy. Investigating the fungal communities in those stands and how they are affected by ash dieback symptoms is thus of high interest. Moreover the work accumulated a nice dataset, although some methodological precision should be added (see latter). The sampling is very large, especially for leaves / shoot (720 samples from 12*10 trees in 10 stands) which makes the work very robust. I however felt that the very large dataset gathered would have benefited to be more thoroughly analysed (see further comment). Further comment: - If I well understood, the leave/shoot/root samples were analysed individually, not pooled by symptom status per tree or per stand. So the 720 leaf samples with each an individual extraction and NGS analysis? I was however not sur about it and I feel it is important to make that completely clear (L141-151). - Shannon index are given in Table2 which would suggest that this diversity index is higher in asymptomatic versus symptomatic tissues. But, no statistical test is given although it would greatly strengthen the result. Again, I was not sure at which level the data are available (original sampled leaf/shoot, tree or plot). But, anyhow, even if samples are aggregated at the plot level, enough repeat are available for formal statistical comparison. L303-304: give the correlation and associated pvalue between H. fraxineus read and Shannon index in symptomatic leaves and shoots, even if not significant. - Table 4-7 are quite difficult to read. I would suggest replacing them by table with data aggregating the data at the symptomatic / asymptomatic level. While having the plot variability is nice, this could be done by putting the data as supplemental data. - The NMDS results for shoots / leaves is very nice. It would be greatly strenghten by a statistical analysis. I would suggest to perform permutational multivariate variance analysis. This is straightforward with the adonis function of R Vagan package (can take into account the plot / tree dependence), although I am sur some SAS function can do the work. - As indicated, I feel a more thorough analysis would be nice. In particular to assess which taxa may be different between symptomatic / asymptomatic tissues owing the extensive and well designed sampling. I know in R some package are very well adapted to analyze this type of sampling (DESeq2) although originally design to look differential gene expression between 2 different treatments.

 

Author Response

Reviewer #3

 

My assessment would be accept with minor modification with the following remarks: The manuscript 1928364 submitted to Forests describes the fungal communities associated with shoots, leaves and roots of ash affected by H. fraxineus using NGS (PacBio), with a main emphasis on comparing symptomatic leaves / shoots with asymptomatic leaves / shoots from the same trees. In Lithuanian forests affected by ash dieback since at least 25 years, some young ash stands remain relatively healthy. Investigating the fungal communities in those stands and how they are affected by ash dieback symptoms is thus of high interest.

 

Response: we acknowledge these observations.

 

Moreover the work accumulated a nice dataset, although some methodological precision should be added (see latter). The sampling is very large, especially for leaves / shoot (720 samples from 12*10 trees in 10 stands) which makes the work very robust. I however felt that the very large dataset gathered would have benefited to be more thoroughly analysed (see further comment). Further comment: - If I well understood, the leave/shoot/root samples were analysed individually, not pooled by symptom status per tree or per stand. So the 720 leaf samples with each an individual extraction and NGS analysis? I was however not sur about it and I feel it is important to make that completely clear (L141-151).

 

Response: within the same site, samples of the same type (e.g. symptomatic leaves) were pooled together, which is now clarified in Materials and Methods.

 

 - Shannon index are given in Table2 which would suggest that this diversity index is higher in asymptomatic versus symptomatic tissues. But, no statistical test is given although it would greatly strengthen the result. Again, I was not sure at which level the data are available (original sampled leaf/shoot, tree or plot). But, anyhow, even if samples are aggregated at the plot level, enough repeat are available for formal statistical comparison.

 

Response: the suggested statistical analyses were added to the manuscript.

 

 L303-304: give the correlation and associated pvalue between H. fraxineus read and Shannon index in symptomatic leaves and shoots, even if not significant.

 

Response: p-values were included in the manuscript.

 

 - Table 4-7 are quite difficult to read. I would suggest replacing them by table with data aggregating the data at the symptomatic / asymptomatic level. While having the plot variability is nice, this could be done by putting the data as supplemental data.

 

Response: we also consider that the plot variability data is useful and would like to keep the tables intact.

 

 - The NMDS results for shoots / leaves is very nice. It would be greatly strenghten by a statistical analysis. I would suggest to perform permutational multivariate variance analysis. This is straightforward with the adonis function of R Vagan package (can take into account the plot / tree dependence), although I am sur some SAS function can do the work.

 

Response: the analysis was performed, and p-values were added to the manuscript. The description of statistical method used was added to the Materials and Methods section.

 

- As indicated, I feel a more thorough analysis would be nice. In particular to assess which taxa may be different between symptomatic / asymptomatic tissues owing the extensive and well designed sampling. I know in R some package are very well adapted to analyze this type of sampling (DESeq2) although originally design to look differential gene expression between 2 different treatments.

 

Response: to highlight the differences in relative abundance of fungal taxa, we have applied the conditional colour formatting of in the Supplementary Tables 1-4.

 

Author Response File: Author Response.pdf