The Seagrass Holobiont: What We Know and What We Still Need to Disclose for Its Possible Use as an Ecological Indicator
, Alice Rotini 3, Loredana Manfra 3,4, Marco Maria D’Andrea 2, Gidon Winters 5,6 and Luciana Migliore 2,*
Round 1
Reviewer 1 Report
This article is a comprehensive and systematic review of past studies on bacterial microflora associated with seagrasses (marine submerged plants). Although there are two preceding reviews on the same topic (seagrass holobiont) published recently (Ugarelli et al. 2017; Tarquino et al. 2019), the present review still have significant originality in focusing the usefulness of seagrass microbiome as an indicator of environmental stresses (e.g., high temperatures, low light, low pH) and ecophysiological responses of seagrasses to them. The authors also critically reviewed available methods for microbiome analysis, proposed recommendable protocols, and suggested most important fields for future studies, which adds further merits to this article. I will recommend it to my colleagues once it is published.
I would like to suggest only some minor revision as follows:
- L.217 (p.5): marked –> labeled
- L.319 (p.7): What do you mean by "other types of nitrogen" if they are not dissolved nitrogen? Please suggest a few examples.
- L.330 (p.7): What do you mean specifically by "anthropogenic pressure"?
- p.16: Table 1 is divided inappropriately.
- L.818 (p.20): Conclusions should be Section 5.
- Information of Refs. #25 and #79 seems incomplete.
- Please double-check thoroughly the correspondence between citations in the main text and literature listed in References. There are a few reference numbers in the main text that seemed wrong to me.
Author Response
General comments
This article is a comprehensive and systematic review of past studies on bacterial microflora associated with seagrasses (marine submerged plants). Although there are two preceding reviews on the same topic (seagrass holobiont) published recently (Ugarelli et al. 2017; Tarquino et al. 2019), the present review still have significant originality in focusing the usefulness of seagrass microbiome as an indicator of environmental stresses (e.g., high temperatures, low light, low pH) and ecophysiological responses of seagrasses to them. The authors also critically reviewed available methods for microbiome analysis, proposed recommendable protocols, and suggested most important fields for future studies, which adds further merits to this article. I will recommend it to my colleagues once it is published.
Reply to general comments: First of all, we would like to thank very much Referee #1 for the positive evaluation of our manuscript. We also would like to thank her/him for the useful suggestions regarding both the English/text editing and for pointing out some conceptual issues. All of them have been accepted, and the manuscript has been modified as requested. Please, find below a complete point by point rebuttal to the observations.
Point 1: marked –> labeled 

Response 1: Modified as suggested by the Reviewer
Point 2: What do you mean by "other types of nitrogen" if they are not dissolved nitrogen? Please suggest a few examples.
Response 2: Sorry, the concept was poorly explained: we meant that different microbes that can provide nitrogen to the plant, in different conditions or plants parts. Thank you very much for this comment. The section has been rephrased and examples added
Point 3: What do you mean specifically by "anthropogenic pressure"?

Response 3: As "anthropogenic pressure", we meant the anthropic activities along the coast that can impact the environment, like fish farming activities, commercial maritime facilities, etc. This explanation has been introduced in the text.
Point 4: Table 1 is divided inappropriately
Response 4: The Table spread across pages has been reorganized
Point 5: marked –> labeled 

Response 5: Modified as suggested by the Reviewer
Point 6: Conclusions should be Section 5.
Response 6: Modified as suggested by the Reviewer
Point 7: Information of Refs. #25 and #79 seems incomplete
Response 7: These references have been integrated with the lacking parts
Point 8: Please double-check thoroughly the correspondence between citations in the main text and literature listed in References. There are a few reference numbers in the main text that seemed wrong to me.
Response 8: The references have been double-checked thoroughly, so as the correspondence between citations in the main text and the list in References.
As last points:
New references have been included: Khon et al., 2020 with few lines dedicated to this paper (lines 265-270), Garcia-Bonet et al., 2020 (line 407), Schloss et al., 2009 (line 683), Bolyen et al., 2019 (line 684).
The English language has been revised thorough the entire ms, and all the changes are traced and readable in the attached revised version.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The seagrass holobiont as an ecological indicator in a changing 2
environment: what we know and what we still need to disclose 3 Chiara Conte 1,2, Alice Rotini 3, Loredana Manfra 3,4, Marco Maria D’Andrea 2, Gidon Winters 5,6 and Luciana 4
Migliore 2,*
The review deals with an interesting topic and seems well documented in all the aspects of the seagrass holobiont. The main part of the review concerns the knowledge of the microbiome of seagrass, the way to study them and the parameters that have been demonstrated to affect them. The ecological indicator part is minor, and the tittle should be improved to better fit to the content of the review.
I would suggest to improve also the structure of the article mainly the first part to avoid repetition (see below).
I have only minor comments.
Line 248-251 : « This colonization process may follow different pathways: microbial communities associated with leaves generally mirror the microbes present in the surrounding seawater column, while microbial communities associated with the rhizome/root usually strongly differs from the sediment microbial community [25,39,103,120]. : When read the quoted paper, it appeared that only ref 103 is focused on that assumption and the comparison of xater, leaves, roots and sediments . For the other references, not all the compartments are studied. Pleases removed the other references
The reference 25 is incomplete, Line 899 : reference “ 25 Martin, B.C.; Sanchez Alcaron, M.; Gleeson, D.; Middleton, J.A. Root microbiomes as indicators of seagrass health. 2020. ,”
Improve the structure of the paper to avoid repetitions:
The same idea is indicated several time:, indicated 3 times: microbial communities associated with leaves mirror the microbes present in the surrounding seawater (line 250, line 314, line 399, )
Other idea indicated 2 times: Line 250: “while microbial communities associated with the rhizome/root usually strongly differs from the 251 sediment microbial community [25,39,103,120]. “
And
Line 414 “The belowground associated microbial communities were found to be less variable than those associated with the aboveground compartment [22-24,26,91] and usually differ strongly from the surrounding sediment microbes “
Line 495: typping error (lower script “vent” in composition due to the proximity of the vents,
Author Response
General comments:
The review deals with an interesting topic and seems well documented in all the aspects of the seagrass holobiont. The main part of the review concerns the knowledge of the microbiome of seagrass, the way to study them and the parameters that have been demonstrated to affect them.
The ecological indicator part is minor, and the title should be improved to better fit to the content of the review.
I would suggest to improve also the structure of the article mainly the first part to avoid repetition (see below).
I have only minor comments.
Reply to general comments: We warmly thank Referee #2 for the positive evaluation of our manuscript and for the consideration given to our work. We would like to thank her/him for the useful comments. All of them have been accepted, and the manuscript has been modified as requested, starting from the title change. Please, find below a complete point by point rebuttal to the observations.
Point 1: The ecological indicator part is minor, and the title should be improved to better fit the content of the review.
Response 1: The title has been modified
Point 2: I would suggest improving the structure of the article mainly the first part to avoid repetition (see below).
Response 2: Thank you for the comment, the repetition was avoided. All the requested changes have been made, as you can see in the next points
Point 3: «This colonization […] sediment microbial community [25,39,103,120]» When reading the quoted paper, it appeared that only ref 103 is focused on that assumption and the comparison of water, leaves, roots, and sediments. For the other references, not all the compartments are studied. Pleases removed the other references.
Response 3: Modified as suggested by the Referee
Point 4: Reference 25 is incomplete, Line 899: reference “ 25 Martin, B.C.; Sanchez Alcaron, M.; Gleeson, D.; Middleton, J.A. Root microbiomes as indicators of seagrass health. 2020.
Response 4: The reference has been completed and checked thoroughly
Point 5: The same idea is indicated several time: indicated 3 times: microbial communities associated with leaves mirror the microbes present in the surrounding seawater (line 250, line 314, line 399).
Response 5: The concept has been maintained at line 262
Point 6: Other idea indicated 2 times: Line 250: “while microbial communities associated with the rhizome/root usually strongly differs from the sediment microbial community [25,39,103,120]. “ And Line 418 “The belowground associated microbial communities were found to be less variable than those associated with the aboveground compartment [22-24,26,91] and usually differ strongly from the surrounding sediment microbes “.
Response 6: The concept has been maintained at line 263
Point 7: Typing error (lower script “vent” in composition due to the proximity of the vents.
Response 7: The typing error has been corrected
As last points:
New references have been included: Khon et al., 2020 with few lines dedicated to this paper (lines 265-270), Garcia-Bonet et al., 2020 (line 407), Schloss et al., 2009 (line 683), Bolyen et al., 2019 (line 684).
The English language has been revised thorough the entire ms, and all the changes are traced and readable in the attached revised version.
Author Response File: Author Response.pdf
