Plant Compartments Shape the Assembly and Network of Vallisneria natans-Associated Microorganisms
Round 1
Reviewer 1 Report
Review of Wan et al
The paper of Wan and collaborators deals with the very interesting subject of microorganisms associated to different plant compartments of the macrophyte Vallisneria natans. The authors provided a study which integrates different approaches. The manuscript is quite well written, even if it would be useful to be corrected because of some syntax errors. I fond some approaches like the co-occurrence nework analysis very instructive. However, I don’t understand points in the methodological procedure. In addition, some parts of the manuscript should be partially reorganized (some informations in the Results and Discussion part should be moved to the Introduction part). Therefore, I think that the paper needs subsequent revision before being suitable for publication.
First, in the Abstract part:
- - The authors should better formulate the objective of their study, in particular the importance of the use of Vallismeria natans as the model used here instead of the functional activities as well as the microbes closely related to wasterwater purification that are not so deeply discussed in the text relatively to the other parts (communities diversity, etc…). The main subjects of this paper, in this current form, are the diversity study and interaction networks and thus they should be more emphasized than the functional activities in the abstract.
- Replace by « … decreased from sediment to rhizosphere, rhizoplane, root endosphere until leaf endosphere. »
In the Introduction part:
- - Globally, I find the Introduction part interesting but not sufficiently documented and needs to be more focused on the objectives of the study.
- - Replace “Microorganisms capable of recognizing signal molecules and settling in specific…” by “Microorganisms able to recognize signal molecules and settle in specific…”
- - Could the authors develop more the “crucial role in improving water quality” in the Introduction please? If the authors want to develop and discuss this application in the Discussion part, it should be better mentioned in the Introduction.
- - I have the same remark concerning the “physiological requirements necessary to occupy these plant ecological niches”. I would need more details with references to better link the results findings concerning the bacterial functions in the Results and Discussion part.
- - The sentence about “Although the application value of V. natans in water environment…” is not correctly placed in the text. It doesn’t seem relevant here but maybe more accurate at the end of the Results and Discussion part or Conclusion.
- - Instead, the authors should better focus on the goal of the study (why it is important to focus on the diversity of the bacterial communities in the different parts of the plant) and more specifically explain the 3 points rapidly mentioned at the end on the Introduction part.
In the Material and Methods part, I have specific questions:
- - Please notice the names of genus and/or species in italic in the text.
- As it seems that the authors identify the “functional” part be an important finding of the study, the methodology part about the FAPROTAX database use should more deeply detailed (website, functioning of the packages, the different steps of the analysis…).
- Do the authors evaluated the option of performing a LEfSe analysis in complement of the others analyses they used to evaluate the bacteria strains that contribute the most to the distinction between the different samples analysed?
In the Results and Discussion part:
- - Please notice the names of genus and/or species in italic in the text.
- - Page 6: Could the authors detail more the “rigorous process” listed with reference #23?
- - Page 6: The description of the progression of the endophytic bacteria from the rhizosphere (sentences “Regarding the root-associated…” and “Before entering plants…”) should also be more suited with the reference #24 in the Introduction part. This would give more weight to the results.
- - Figure 1C seems no informative and could be removed.
- One sentence is exactly the same than in Introduction and should be modified “… possibly due to the physiological requirements necessary to occupy these plant niches…”
- Perhaps replace “endophytes” in the sentence “This is likely because endophytes…” by endophyte bacteria.
- Given that the authors work on submerged plants and that as they precise, water can contain “a large number of microorganisms that can enter the plant directly”, they should indicate the results concerning the diversity of bacteria found inside water samples if available.
- - Title of Figure 2: replace “Distribution proportion” by “Proportion”. The authors should add specific legend for the schemas represented above the histograms please.
- - Page 8: Replace “… in five samples had three groups.” By “… in five samples exhibited three groups”.
- - Page 8: Replace “In addition to sediment samples…” by “On the opposite,…”
- - Page 8: Replace “Specifically,…” by “More globally,…”
- - Legend of Figure 3: The authors should mention which kind of relative values are mentioned: abundance of bacterial genus? Enrichment in bacterial genus?
- - Page 9: Replace “similar to other submerged plants” by “in a similar way to other submerged plants”
- - Page 9: Replace “in the plant compartment except for sediment” by “in the plant compartment except in sediment”
- - The authors should modify this sentence “These bacteria are key to denitrification”. It is not well expressed.
- - Page 10: The authors should be more careful to not mix the different parts because sometimes the same conclusions appears in different parts like “These results indicated that the plant compartments have (instead of had) a significant effect on the distribution of bacterial OTUs.”
- - Same remark for the following part “The bacterial microbiomes of the different samples are (instead of were) largely composed of...”.
- - Perhaps be more explicit when the authors refer to the two emergent plants (reference #23) again.
- - Page 11: It is not clear for me from which part of the Table 2 are extracted the data about the percentages of the total degree of connection.
- - I don’t understand well the different parts of the Table 2: edge, modularity, etc… Perhaps these terms should be again explained in the legend of the Table please.
- - Correct “Desulfobacterot” by “Desulfobacterota”.
- - The co-occurrence network analysis is a very interesting work.
- - I have some difficulties with the last 3.5 part about the “Functional prediction of bacterial communities”. Although I think it is a very interesting component that could be add to the analysis, it is not as developed as the previous parts in its current form. As this part is relatively short, this leads to a disequilibrium (as well as in the abstract, one of my first remarks) between the different analyses. The authors should work to more deeply detail this part. It seems also that some results (Tables or Figures) are lacking although they are written in the text (like the lack of significant difference in relative abundance between the community types, p>0.05). Again, perhaps the discussion about the last paragraph should be reorganized to have more impact.
In the Conclusion part:
- - What the authors mean by “the mutualism between different part compartments of the plant”? Be careful that mutualism can have other meanings depending of the context…
- - The last sentence is too vague. Why the authors did not develop at any moment (Introduction or Results and Discussion parts) these ideas of technical applications about isolation of “efficient bacterial strains”? Perhaps this could be another idea the authors could use to validate the pertinence of their study but it is not sufficiently developed here.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer #1’s Comments
Review of Wan et al
The paper of Wan and collaborators deals with the very interesting subject of microorganisms associated to different plant compartments of the macrophyte Vallisneria natans. The authors provided a study which integrates different approaches. The manuscript is quite well written, even if it would be useful to be corrected because of some syntax errors. I fond some approaches like the co-occurrence nework analysis very instructive. However, I don’t understand points in the methodological procedure. In addition, some parts of the manuscript should be partially reorganized (some informations in the Results and Discussion part should be moved to the Introduction part). Therefore, I think that the paper needs subsequent revision before being suitable for publication.
We appreciate the reviewer’s constructive comments. This manuscript has been revised very carefully and discussion has been enriched.
First, in the Abstract part:
- The authors should better formulate the objective of their study, in particular the importance of the use of Vallisneria natans as the model used here instead of the functional activities as well as the microbes closely related to wasterwater purification that are not so deeply discussed in the text relatively to the other parts (communities diversity, etc…).
Accepting the reviewer's suggestion, “Macrophytes and … wastewater purification”has been revised as “The submerged plant …16S rRNA gene sequencing.”
- The main subjects of this paper, in this current form, are the diversity study and interaction networks and thus they should be more emphasized than the functional activities in the abstract.
Accepted reviewers' suggestions and added diversity study and co-occurrence network statements to the revision.
“The results showed that the diversity and network … distinct topological features.” has been supplemented.
- Replace by « … decreased from sediment to rhizosphere, rhizoplane, root endosphere until leaf endosphere. »
“The results showed … leaf endosphere” has been revised as “The results showed that the diversity and network complexity of bacterial community in the sed-iment was significantly higher than that in other plant compartments.”
In the Introduction part:
- Globally, I find the Introduction part is not sufficiently documented and needs to be more focused on the objectives of the study.
Accepting the reviewer's suggestion, we added some additional elements.
- Replace “Microorganisms capable of recognizing signal molecules and settling in specific…” by “Microorganisms able to recognize signal molecules and settle in specific…”
“Microorganisms capable of recognizing signal molecules and settling in specific…” has been revised as “Some microorganisms are capable of recognizing signal molecules and settling in specific compartmentalized niches,”
- Could the authors develop more the “crucial role in improving water quality” in the Introduction please? If the authors want to develop and discuss this application in the Discussion part, it should be better mentioned in the Introduction.
Two example “For example, Singh et al. used aquatic plants and root bacteria to remediate the chromium-containing wastewater, and after 72 hours, each pollution parameter was sig-nificantly reduced[10]. Also, macrophytes could affect sedimental biogeochemical cycling by increasing the activity of microbes, such as methanogens[11].” has been supplemented.
- I have the same remark concerning the “physiological requirements necessary to occupy these plant ecological niches”. I would need more details with references to better link the results findings concerning the bacterial functions in the Results and Discussion part.
“The movement of bacteria inside plants is supported by bacterial flagellum and plant transpiration stream. Migration along that intercellular space requires the secretion of cell wall degrading enzymes such as cellulases and pectinases” has been supplemented.
- The sentence about “Although the application value of V. natans in water environment…” is not correctly placed in the text. It doesn’t seem relevant here but maybe more accurate at the end of the Results and Discussion part or Conclusion.
The sentence about “Although the application value of V. natans in water environment…” has been inserted into the conclusion section.
- Instead, the authors should better focus on the goal of the study (why it is important to focus on the diversity of the bacterial communities in the different parts of the plant) and more specifically explain the 3 points rapidly mentioned at the end on the Introduction part.
“In recent years, important progress has been made in the research on the combined remediation technology of submerged plants and microorganisms. However, the application effect of functional bacteria is unstable, and further exploration of functional microbial germplasm resources is needed.” has been supplemented.
In the Material and Methods part, I have specific questions:
- Please notice the names of genus and/or species in italic in the text.
The names of genera and species have been carefully checked and corrected.
- As it seems that the authors identify the “functional” part be an important finding of the study, the methodology part about the FAPROTAX database use should more deeply detailed (website, functioning of the packages, the different steps of the analysis…).
The website and software script operation process used by the FAPROTAX database have been supplemented.
Do the authors evaluated the option of performing a LEfSe analysis in complement of the others analyses they used to evaluate the bacteria strains that contribute the most to the distinction between the different samples analysed?
Sorry, I didn't choose Lefse analysis to find the indicator species. We used veen diagram, phylogenetic tree and zi-pi model to find the dominant groups and key species in the related microorganisms of Vallisneria natans. Through these methods, we evaluate the bacteria strains that contribute the most to the distinction between the different samples analysed.
In the Results and Discussion part:
- Please notice the names of genus and/or species in italic in the text.
The names of genera and species have been carefully checked and corrected.
- Page 6: Could the authors detail more the “rigorous process” listed with reference #23?
This rigorous process is mainly caused by the plant immune defense mechanism, microorganisms must overcome it to survive and colonize in different plant compartments.
- Page 6: The description of the progression of the endophytic bacteria from the rhizosphere (sentences “Regarding the root-associated…” and “Before entering plants…”) should also be more suited with the reference #24 in the Introduction part. This would give more weight to the results.
In favor of the reviewer's opinion, this part has been deleted and the expression of relevant contents in the introduction part has been adjusted.
- Figure 1C seems no informative and could be removed.
Added content “Coverage index was greater than 0.99, indicating that the sequencing results covered almost all bacteria in the samples (Figure 1C).”
- One sentence is exactly the same than in Introduction and should be modified “… possibly due to the physiological requirements necessary to occupy these plant niches…”
“… possibly due to the physiological requirements necessary to occupy these plant niches…” has been revised as “Since most of the endophytic bacteria originated from the sediment and shared a certain similarity with the sediment microbial community, the diversity of root endophytic bacteria was higher than that of leaf tissue, which might be attributed to the fact that the root system was the main site for the interaction between plants and the sediment”
- Perhaps replace “endophytes” in the sentence “This is likely because endophytes…” by endophyte bacteria.
“endophytes” has been revised as “endophyte bacteria”.
- Given that the authors work on submerged plants and that as they precise, water can contain “a large number of microorganisms that can enter the plant directly”, they should indicate the results concerning the diversity of bacteria found inside water samples if available.
Unfortunately, we can't provide relevant information for reviewers. Because no water samples were collected in this study, it is impossible to analyze the relationship between microorganisms and V.natans. We will carefully consider your valuable suggestions in future research.
- Title of Figure 2: replace “Distribution proportion” by “Proportion”. The authors should add specific legend for the schemas represented above the histograms please.
Figure 2 has been revised. I have added specific legend for the schemas represented above the histograms please.
- Page 8: Replace “… in five samples had three groups.” By “… in five samples exhibited three groups”.
“… in five samples had three groups.” has been revised as “… in five samples exhibited three groups”.
- Page 8: Replace “In addition to sediment samples…” by “On the opposite,…”
“In addition to sediment samples, other samples pos-sessed more species belonging to Rhizobium, Flavobacterium, Pseudomonas, Massilia, and Polaromonas.” has been revised as “Species belonging to Rhizobium, Flavobacterium, Pseudomonas, Massilia, and Polaromonas were particularly en-riched in the rhizosphere, rhizoplane as well as in root and leaf endosphere, when compared to sediment samples.”
- Page 8: Replace “Specifically,…” by “More globally,…”
“Specifically,…” has been revised as “More globally,…”
- Legend of Figure 3: The authors should mention which kind of relative values are mentioned: abundance of bacterial genus? Enrichment in bacterial genus?
“The relative values…” has been revised as “The relative abundance values…”
- Page 9: Replace “similar to other submerged plants” by “in a similar way to other submerged plants”
“similar to other submerged plants” has been revised as “in a similar way to other submerged plants”
- Page 9: Replace “in the plant compartment except for sediment” by “in the plant compartment except in sediment”
“in the plant compartment except for sediment” has been revised as “in the plant compartment except in sediment”
- The authors should modify this sentence “These bacteria are key to denitrification”. It is not well expressed.
“These bacteria are key to denitrification in wastewater management” has been revised as “These bacteria are key to nitrogen removal in the management of wastewater”
- Page 10: The authors should be more careful to not mix the different parts because sometimes the same conclusions appears in different parts like “These results indicated that the plant compartments have (instead of had) a significant effect on the distribution of bacterial OTUs.”
Thank you for reminding me. I have carefully checked these omissions and corrected them.
“plant compartments had a significant effect” has been revised as “plant compartments have a significant effect”
- Same remark for the following part “The bacterial microbiomes of the different samples are (instead of were) largely composed of...”.
Modified
- Perhaps be more explicit when the authors refer to the two emergent plants (reference #23) again.
“Phragmites australis and Triarrhena lutarioriparia” has been supplemented.
- Page 11: It is not clear for me from which part of the Table 2 are extracted the data about the percentages of the total degree of connection.
Table 3 of the percentage of total connectivity has been supplemented.
- I don’t understand well the different parts of the Table 2: edge, modularity, etc… Perhaps these terms should be again explained in the legend of the Table please.
“a: Degree of nodes tending to differentiate into different network modules. b: Degree of nodes tending to cluster together. c: Network path distance is the length of the shortest path between two nodes within the network.” had been explained in the legend of the Table please.
- Correct “Desulfobacterot” by “Desulfobacterota”.
Modified
- The co-occurrence network analysis is a very interesting work.
Thank you for agreeing with our research methods.
- I have some difficulties with the last 3.5 part about the “Functional prediction of bacterial communities”. Although I think it is a very interesting component that could be add to the analysis, it is not as developed as the previous parts in its current form. As this part is relatively short, this leads to a disequilibrium (as well as in the abstract, one of my first remarks) between the different analyses. The authors should work to more deeply detail this part. It seems also that some results (Tables or Figures) are lacking although they are written in the text (like the lack of significant difference in relative abundance between the community types, p>0.05). Again, perhaps the discussion about the last paragraph should be reorganized to have more impact.
We appreciate the reviewer’s constructive comments. We have supplemented Figure 7 to illustrate the significant differences of the top ten functional groups in relative abundance. At the same time, the discussion part is also strengthened to explain the role of bacterial communities related to V.natans.
In the Conclusion part:
- What the authors mean by “the mutualism between different part compartments of the plant”? Be careful that mutualism can have other meanings depending of the context…
“This effect extends to all aspects of the microbiota, including the α diversity index and OTU distribution of the microbial community, the interactions between microorganisms, the potential function of the microbiota, and the mutualism between different compartments of the plant.” has been revised as “These effects extend to all aspects of the bacterial community, including the OTU distribution, diversity, community composition, co-occurrence networks, and the mutualism between the potential function of the microbiome and different plant compart-ments of the V. natans.”
- The last sentence is too vague. Why the authors did not develop at any moment (Introduction or Results and Discussion parts) these ideas of technical applications about isolation of “efficient bacterial strains”? Perhaps this could be another idea the authors could use to validate the pertinence of their study but it is not sufficiently developed here.
“This study provides theoretical and technical support for the further isolation and acquisition of efficient bacterial strains.” has been revised as “This study provides theoretical and technical data for the future work of isolating beneficial bacteria from plants.”
Author Response File: 
Author Response.docx
Reviewer 2 Report
The manuscript by Wan et al. investigates the diversity of bacteria on different compartments of the aquatic plant Vallisneria natans. As stated by the authors, V. natans is common in freshwater ecosystems and may play a relevant role in phytoremediation. The experimental setup allowed the comparison of the bacterial communities along several compartments, from the outermost sediment to the innermost root and leaf endospheres.
General comments
-The absence of line numbers makes it much more complicated to point out the comments/suggestions.
-The work mentioned in reference 22 is a recent study performed on the same plant species, so I would expect a more detailed comparison of the findings.
Minor comments
PAGE 1
Title – “compartment niches” does not add information, I would suggest “Plant compartments shape”
Abstract
- “This study provides theoretical and technical support for the further isolation and acquisition of efficient bacterial strains.” – efficient in what? I would suggest simplifying to “This study provides theoretical and technical data that is useful for the further isolation of plant beneficial bacteria.” (this sentence should be also altered on the conclusions section)
Introduction
-“colonize different plant compartments such as the rhizosphere, rhizoplane, root endophytes, and leaf endophytes” – should be “root endosphere, and leaf endosphere”
- “Microorganisms capable of recognizing signal molecules and settling in specific compartmentalized niches are preferentially enriched, while other microorganisms are filtered out [6]. This enrichment process is…”- could be altered to “Some microorganisms are capable of recognizing signal molecules and settling in specific compartmentalized niches, while other microorganisms are filtered out [6]. This compartment enrichment in particular species is”
-“Studies have demonstrated” – should be “Previous studies have demonstrated”
PAGE 2
Introduction
-“hierarchical filtration mechanism” - could be altered to “hierarchical selection mechanism”
- “endophytic bacteria spread” - should be “endophytic bacteria may spread”
-“It is often” - could be altered to “This species is often”
Materials and Methods
-“Chloroplast excluding primers” (no comma)
-Please clarify which reverse primer was used
PAGE 3
Materials and Methods
-the information about circos plot, heatmap and Venn diagrams could be removed (these are common displays of the data on this type of studies)
-in general, references should be provided for the specific software/database used.
PAGE 4
In order to state that “The α-diversity indexes of the bacterial community significantly differed among plant compartments” (this sentence is repeated on this page), a statistical analysis should be performed. Figures 1D,1E and table 1 seem to indicate (at least) no differences between rhizosphere and rhizoplane.
PAGE 5
The compartments should be mentioned in the general sentence of the Figure 1 legend (and not in the (B) specific information.
PAGE 6
-Vallisneria natans needs to be formatted to italic font
-“Regarding the root-associated bacterial community, the α-diversity gradually decreased from the rhizosphere to the rhizoplane and then to the root endosphere.” This is true to the Chao1 index only; it is the opposite for the Shannon index. Please revise the discussion in order to reflect the results of the analyses performed for alpha-diversity.
PAGE 7
-In Figure 2, the * for the statistical analysis should be located on the column that is significantly different from the remaining (no need to indicate the statistical value)
PAGE 8
- “In addition to sediment samples, other samples possessed more species belonging to Rhizobium, Flavobacterium, Pseudomonas, Massilia, and Polaromonas.” Instead of this sentence, I would suggest “Species belonging to Rhizobium, Flavobacterium, Pseudomonas were particularly enriched in the rhizosphere, rhizoplane as well as in root and leaf endosphere, when compared to sediment samples.”
-please revise the sentence “The relative abundance of unclassified_f__Oxalobacteraceae was higher in root endosphere samples than in the other samples.” Did the authors meant “unclassified_f__Comamonadaceae”?
PAGE 9
-Please remove “can survive in nutrient-rich and sunny environments” or provide a reference for this information.
PAGE 10
-the bar plot of figure 4A repeats the data already shown on Table 1 (please remove this duplication)
-The following sentences are repeated; please remove “This study shows that the plant compartment determines the differential distribution of the subsets of common bacterial communities. The bacterial microbiomes of the different samples were largely composed of Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Actinobacteria, accounting for 66.44% of the relative abundance.” The remaining sentences of this paragraph may need to be relocated.
-It is interesting that the vast majority of the non-sediment OTUs present in other compartment are found in rhizoplane. Maybe the authors would like to explore this finding. (do these correspond to any of the connectors found on this compartment?)
PAGE 11
-the second mention to Table 2 in the text seems inappropriate (the percentages for each taxa are data not shown)
- “We discovered that the network complexity of different V. natans samples changed consistently with the changing trend of alpha diversity, gradually decreasing from sediment to leaf endosphere.” As mentioned previous, the trend in alpha diversity does not differentiate rhizosphere from rhizoplane; figure 5A seems to point in the same direction. However, figure 5B indicates that the structure of the bacterial population is quite different between the two compartments (and this is worth discussing). Does this indicate a higher redundancy (and higher resilience) on the rhizoplane community?
PAGE 14
-Table 2 repeats information already displayed in Figure 5; please avoid these repetitions.
PAGE 15
Conclusions could mentioned the which plant compartments were the most similar and the most divergent.
Author Response
Response to Reviewer #2’s Comments
The manuscript by Wan et al. investigates the diversity of bacteria on different compartments of the aquatic plant Vallisneria natans. As stated by the authors, V. natans is common in freshwater ecosystems and may play a relevant role in phytoremediation. The experimental setup allowed the comparison of the bacterial communities along several compartments, from the outermost sediment to the innermost root and leaf endospheres.
General comments
-The absence of line numbers makes it much more complicated to point out the comments/suggestions.
I apologize for the inconvenience caused to your manuscript review due to my negligence.
-The work mentioned in reference 22 is a recent study performed on the same plant species, so I would expect a more detailed comparison of the findings.
This part has been supplemented “Yan et al. used V.natans for rhizodegradation to repair PAH contaminated sediments, which showed that the shannon indices gradually decreased from sediment bacterial community to endosphere until rhizoplane[27].”
Minor comments
PAGE 1
Title – “compartment niches” does not add information, I would suggest “Plant compartments shape”
“compartment niches” has been revised as “Plant compartments shape”
Abstract
- “This study provides theoretical and technical support for the further isolation and acquisition of efficient bacterial strains.” – efficient in what? I would suggest simplifying to “This study provides theoretical and technical data that is useful for the further isolation of plant beneficial bacteria.” (this sentence should be also altered on the conclusions section)
“This study provides theoretical and technical support for the further isolation and acquisition of efficient bacterial strains.” has been revised as “These results strongly suggested how the microbial community adapted to different plant compartments and provided theoretical and technical data for isolating beneficial bacteria from mac-rophytes in the future.”
Introduction
-“colonize different plant compartments such as the rhizosphere, rhizoplane, root endophytes, and leaf endophytes” – should be “root endosphere, and leaf endosphere”
“root endophytes, and leaf endophytes” has been revised as “root endosphere, and leaf endosphere”
- “Microorganisms capable of recognizing signal molecules and settling in specific compartmentalized niches are preferentially enriched, while other microorganisms are filtered out [6]. This enrichment process is…”- could be altered to “Some microorganisms are capable of recognizing signal molecules and settling in specific compartmentalized niches, while other microorganisms are filtered out [6]. This compartment enrichment in particular species is”
“Microorganisms capable of recognizing signal molecules and settling in specific compartmentalized niches are preferentially enriched, while other microorganisms are filtered out [6]. This enrichment process is…” has been revised as “Some microorganisms are capable of recognizing signal molecules and settling in specific compartmentalized niches, while other microorganisms are filtered out [6]. This compartment enrichment in particular species is…”
-“Studies have demonstrated” – should be “Previous studies have demonstrated”
“Studies have demonstrated” has been revised as “Previous studies have demonstrated”
PAGE 2
Introduction
-“hierarchical filtration mechanism” - could be altered to “hierarchical selection mechanism”
“hierarchical filtration mechanism” has been revised as “hierarchical selection mechanism”
- “endophytic bacteria spread” - should be “endophytic bacteria may spread”
“endophytic bacteria spread” has been revised as “endophytic bacteria may spread”
-“It is often” - could be altered to “This species is often”
“It is often…” has been revised as “This species is often…”
Materials and Methods
-“Chloroplast excluding primers” (no comma)
Modified
-Please clarify which reverse primer was used
The chimeric sequences such as host mitochondria and chloroplasts were removed, and the V5–V7 hypervariable regions of the 16S rDNA of the microorganism were am-plified by using nested primers 799F (5'-AACMGGATTAGATACCCKG-3') and 1392R (5'-ACGGGCGGTGTGTRC-3'), 799F (5'-AACMGGATTAGATACCCKG-3') and 1193R (5'-ACGTCATCCCCACCT-TCC-3')
PAGE 3
Materials and Methods
-the information about circos plot, heatmap and Venn diagrams could be removed (these are common displays of the data on this type of studies)
Agree with the reviewer's opinion, this part has been deleted.
-in general, references should be provided for the specific software/database used.
The relevant software or website used has been supplemented.
PAGE 4
In order to state that “The α-diversity indexes of the bacterial community significantly differed among plant compartments” (this sentence is repeated on this page), a statistical analysis should be performed. Figures 1D,1E and table 1 seem to indicate (at least) no differences between rhizosphere and rhizoplane.
“The α-diversity indexes of the bacterial community significantly differed among plant compartments” has been revised as “To investigate the effect of plant compartments on bacterial communities, α-diversity indices of bacterial communities in different samples were calculated”
Statistical analysis is supplemented “In figure 1D, bacterial richness (Chao 1 index) was significantly greater in sediment (755.950 ± 7.417) than other plant compartments (rhizosphere, 722.446 ± 28.159; rhizoplane, 709.794 ± 54.305; root endosphere, 515.799 ± 44.392; leaf endosphere, 311.312 ± 51.223). A similar result was found in bacterial diversity (Shannon index, figure 1E)”.
PAGE 5
The compartments should be mentioned in the general sentence of the Figure 1 legend (and not in the (B) specific information.
The concrete has been modified to the general sentence pattern of the legend.
PAGE 6
-Vallisneria natans needs to be formatted to italic font
Thank you for pointing out that it has been revised.
-“Regarding the root-associated bacterial community, the α-diversity gradually decreased from the rhizosphere to the rhizoplane and then to the root endosphere.” This is true to the Chao1 index only; it is the opposite for the Shannon index. Please revise the discussion in order to reflect the results of the analyses performed for alpha-diversity.
This part has been revised and discussed again.
PAGE 7
-In Figure 2, the * for the statistical analysis should be located on the column that is significantly different from the remaining (no need to indicate the statistical value)
Figure 2 has been revised.
PAGE 8
- “In addition to sediment samples, other samples possessed more species belonging to Rhizobium, Flavobacterium, Pseudomonas, Massilia, and Polaromonas.” Instead of this sentence, I would suggest “Species belonging to Rhizobium, Flavobacterium, Pseudomonas were particularly enriched in the rhizosphere, rhizoplane as well as in root and leaf endosphere, when compared to sediment samples.”
“In addition to sediment samples, other samples possessed more species belonging to Rhizobium, Flavobacterium, Pseudomonas, Massilia, and Polaromonas.” has been revised as “Species belonging to Rhizobium, Flavobacterium, Pseudomonas were particularly enriched in the rhizosphere, rhizoplane as well as in root and leaf endosphere, when compared to sediment samples.”
-please revise the sentence “The relative abundance of unclassified_f__Oxalobacteraceae was higher in root endosphere samples than in the other samples.” Did the authors meant “unclassified_f__Comamonadaceae”?
I' m sorry, this is my negligence. It's really the "unclassified_f__Comamonadaceae " you say, not the "unclassified_f__Oxalobacteraceae ".
PAGE 9
-Please remove “can survive in nutrient-rich and sunny environments” or provide a reference for this information.
“can survive in nutrient-rich and sunny environments” has been deleted.
PAGE 10
-the bar plot of figure 4A repeats the data already shown on Table 1 (please remove this duplication)
The bar plot of figure 4A has been deleted.
-The following sentences are repeated; please remove “This study shows that the plant compartment determines the differential distribution of the subsets of common bacterial communities. The bacterial microbiomes of the different samples were largely composed of Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes, and Actinobacteria, accounting for 66.44% of the relative abundance.” The remaining sentences of this paragraph may need to be relocated.
“This study shows … relative abundance.” has been revised as “By defining shared OTUs from different plant compartments in overlapping areas of Venn diagrams, we found that the dominant phyla/class were mainly Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, and Actinobacteria.”
“These common groups are dominant microorganisms in V. natans.” has been revised as “These common groups were defined as the key groups shared among the microbial communities in V.natans.”
-It is interesting that the vast majority of the non-sediment OTUs present in other compartment are found in rhizoplane. Maybe the authors would like to explore this finding. (do these correspond to any of the connectors found on this compartment?)
Agree with the reviewer's opinion, this section has been modified.
PAGE 11
-the second mention to Table 2 in the text seems inappropriate (the percentages for each taxa are data not shown)
Table 3 has been revised as a new supplement here.
- “We discovered that the network complexity of different V. natans samples changed consistently with the changing trend of alpha diversity, gradually decreasing from sediment to leaf endosphere.” As mentioned previous, the trend in alpha diversity does not differentiate rhizosphere from rhizoplane; figure 5A seems to point in the same direction. However, figure 5B indicates that the structure of the bacterial population is quite different between the two compartments (and this is worth discussing). Does this indicate a higher redundancy (and higher resilience) on the rhizoplane community?
Sorry, due to negligence, the idea of “We discovered that the network complexity of different V. natans samples changed consistently with the changing trend of alpha diversity, gradually decreasing from sediment to leaf endosphere.” is wrong and has been revised.
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-Table 2 repeats information already displayed in Figure 5; please avoid these repetitions.
Table 2 and Figure 5 have been modified.
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Conclusions could be mentioned as to which plant compartments were the most similar and the most divergent.
“There were significant differences between sediment and other plant compartments, although two endosphere samples (root and leaf endosphere) had some similarities. It was proven that different plant compartments of V. natans greatly influenced its microbial community in sediment.” has been supplemented.
Author Response File: Author Response.docx
