Insight into bacterial and archaeal community structure of Suaeda altissima and Suaeda dendroides rhizosphere in response to different salinity level

ABSTRACT Suaeda as a halophyte with wide adaptability of salinization level plays an important role in saline soil improvement and management. To some extent, the strong salt tolerance of Suaeda is influenced by rhizobacteria. However, the effect of different Suaeda species and salinization level on the microbial community diversity still unknown. In our study, high-throughput sequencing technology was used to explore the difference in the bacterial and archaeal community diversity of Suaeda altissima and Suaeda dendroides rhizosphere under high (EC, 8–16 dS/m) and severe (EC > 16 dS/m) saline soil. The result showed that complex soil environment and Suaeda species co-shaped bacterial and archaeal community structure, and pH was one of the most important driving factors. In addition, the increase of soil salinity significantly decreased the complexity of bacterial and archaeal co-occurrence network. We explored Halomonas and Candidatus Nitrocosmicus as core microorganisms in the Suaeda rhizosphere, which may play a key ecological role in improving salt tolerance and promoting growth of Suaeda. Our study improves the macroscopic understanding of the interrelationships between soil environments-microbes-plants. IMPORTANCE Suaeda play an important ecological role in reclamation and improvement of agricultural saline soil due to strong salt tolerance. At present, research on Suaeda salt tolerance mainly focuses on the physiological and molecular regulation. However, the important role played by microbial communities in the high-salinity tolerance of Suaeda is poorly studied. Our findings have important implications for understanding the distribution patterns and the driving mechanisms of different Suaeda species and soil salinity levels. In addition, we explored the key microorganisms that played an important ecological role in Suaeda rhizosphere. We provide a basis for biological improvement and ecological restoration of salinity-affected areas.

parameters? 9. Line 400.Cyanobacteria et.al.? Is it correct?10.Line 414.Where is the Table 5B??? Discussion: Discussion section is too lengthy and need to be largely revised.The authors should delete many descriptive expressions.Most importantly, authors need to strengthen the logicality of the discussion.Additionally, the discussion section does not require so many sub-titles, short and concise titles should be summarized in this section.1. Line 427.microbes 2. Line428.various species?There are only two different types here.3. Line 448.Reword this sentence.4. Lines 449-452.Why did the authors get this conclusion?Add some related results and references to enhance your statements. 5. Lines 452-454.The authors need to elucidate why and how the previous studies' findings consistent with your results.6. Line 465.What is the purpose of introducing "core microbiome" here?Dose it relevant to your results? 7. Line 474.Why did the authors discuss the pathogen suppressive of Actinobacteria?I did not see any results about the pathogens in your results.Thus, I suggest to carefully reconsider the logicality of these sentences.8. Line 477-478.While Actinobacteria and Proteobacteria are well-known dominant bacterial taxa in soils and are known to contribute to plant growth and health, this study focuses on specific bacterial groups identified as keystone taxa in saline soils.These keystone taxa have the potential to play critical functional roles, and we are particularly interested in exploring their potential functions in promoting plant growth and health in these challenging environments.9. Line 501-502.should include the relevant results and discuss more about this taxa to explain the importance.10.Line 508."Marmoricola" should be italic.11.Line 519.They are often found in... 12. Line 522-526.Reword this sentence.13.Line 529-530.Cited the relevant reference(s).14.Line 552-554.It's hard to understand the purpose of this paragraph and its connection with above.15.Line 557-568.Soil pH is an important factor that driving soil microbial community assembly.However, in this study, authors should focus on the soil salinization.16.Line 574.What is "t microbial network"?17.Line 579-580.What is the relationship between theses physical constraints and microbial networks?These sentences are vague and need to be reword.18. Lines 584-590.The authors' hypotheses require further elaboration and deeper discussion.It is unclear how they arrived at these hypotheses, and more statements and evidence are needed to support their claims.19.Line 595.soil's network is vague.

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The study from Wang et al. investigated the bacterial and archaeal community composition and diversity in the two types of Suaeda rhizosphere along different salinity gradient.The authors focused on how soil properties regulated the bacterial and archaeal communities diversity and networks, which is interesting.However, there are a number of issues that the authors need to address and revise carefully, as well as more in-deep discussion needs to be added for a better elucidation of the results.Moreover, the results and discussion section were not well organized, and there are many writing errors in the manuscript and all citation formats are incorrect.I provide the authors with specific and textual comments below, I hope they find them useful in the further improvement of their manuscript.

General comments:
1. Title: The current version is too normal and is not attractive.Please revise it appropriately.
2. Since this study aims to investigate the variations in the rhizosphere microbial community of two types of Suaeda plants under different levels of soil salinity, it is important for the authors to provide a clear introduction to the differences in how these plants respond to soil salinization.How these two Suaeda types response to salt stress?Why they need to investigate the rhizosphere microbial communities?Were there any important research gaps/hypotheses that need to be elucidated so that make the mechanisms behind more clear?It may be difficult to understand the observed differences in microbial communities between the two plant types and the purpose of this study if lack these information.Therefore, the authors should provide a thorough explanation of these two plant responses to soil salinity to support their findings.
3. The archaeal community is extracted from 16S amplicon sequencing data using bacteria-specific primers, thus there are very few archaea detected using this method.This is important and need to be discussed carefully.Moreover, I did not find any results related to functional prediction in the study, although the authors mentioned it in the M&M section.Therefore, it is unclear whether functional prediction was actually performed and, if so, what the results were.The authors should provide more clarity on this matter and clearly state whether functional prediction was performed and include the results in their manuscript.
4. As mentioned in results, soil pH and salinity level are all key factor influencing microbial community assembly, however, why the authors declare that "soil salinity and Suaeda species co-shaped bacterial and archaeal community structure"?There is no bulk soil samples here.More direct proof should be provided.
5. The discussion and conclusion sections of the manuscript are somewhat vague and lack clear conclusions, especially in the discussion section, the authors display many descriptive expressions and repeat their results, which does not provide a satisfactory analysis of their findings.Therefore, it is recommended that the discussion and conclusion be rewritten with a more critical and concise method that focuses on interpreting the results in light of the research questions and objectives.This will help to provide a clearer and more convincing conclusion and make the manuscript higher quality.

Grammar and writing:
The writing of the text needs to be carefully checked.So many incorrect wordings/phrases, font format, punctuation mark, and citation format.Please find more specific comments below.

General comments:
I believe the main issue with this part is the language accuracy and conciseness.The authors used many short sentences that lack strong connections between each other, and the Abstract is not concise.To improve the readability and logicality of the text, the authors should consider adding conjunctions and reducing descriptive statements.
Although I have pointed out some places, there are still many areas that need to be revised.Additionally, it would be beneficial to add some clear conclusions at the end of the Abstract.

Specific comments:
7. Line 19.Which kind of agricultural soil?It is better to emphasize "the improvement of saline soil".rhizosphere microbiomes?Were there any important research gaps/hypotheses that need to be elucidated so that make the microbial mechanisms behind more clear?Specific comments: 1. Line 55. Whose functions?
5. Line 97.Providing some references to support your statements.12. Line 122.… strong and very strongly salinity levels?Please change the expression.

Materials and methods:
1. Line 141. S. altissima was found in B and D according to Table S2?
2. If possible, I suggest using figures to clearly express your field experiment and sampling strategy.

Results:
1.All the figures are fuzzy, the font size is too small.The authors should revise to make the figures clearer.
3. Line 251.It is better to change "the soil of GIP-3 has…" as "soil samples from GIP-3 have…".Discussion section is too lengthy and need to be largely revised.The authors should delete many descriptive expressions.Most importantly, authors need to strengthen the logicality of the discussion.Additionally, the discussion section does not require so many sub-titles, short and concise titles should be summarized in this section.Dear reviewers 1: Thank you for giving us the opportunity to submit a revised draft of the manuscript "Analysis of bacterial and archaeal community structure in Suaeda altissima and Suaeda dendroides rhizosphere under different salinity level" (Spectrum01649-23R1) for publication in Microbiology Spectrum.We appreciate the time and effort that you dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper.We have incorporated the suggestions made by the reviewers.Please see below for a point-by-point response to the reviewers' comments and concerns.
General comments: 1. Title: The current version is too normal and is not attractive.Please revise it appropriately.
Reply：Thanks for your suggestions, we have changed the title from "Analysis of bacterial and archaeal community structure in Suaeda altissima and Suaeda dendroides rhizosphere under different salinity level" to "Insight into bacterial and archaeal community structure of Suaeda altissima and Suaeda dendroides rhizosphere in response to different salinity level".
2. Since this study aims to investigate the variations in the rhizosphere microbial community of two types of Suaeda plants under different levels of soil salinity, it is important for the authors to provide a clear introduction to the differences in how these plants respond to soil salinization.How these two Suaeda types response to salt stress?Why they need to investigate the rhizosphere microbial communities?Were there any important research gaps/hypotheses that need to be elucidated so that make the mechanisms behind more clear?It may be difficult to understand the observed differences in microbial communities between the two plant types and the purpose of this study if lack these informations.Therefore, the authors should provide a thorough explanation of these two plant responses to soil salinity to support their findings.Reply: Thanks for your suggestions.We have added the Suaeda plants respond to soil salinization and the importance and reasons of rhizosphere microorganisms for plant salt tolerance and growth in introduction section.
3. The archaeal community is extracted from 16S amplicon sequencing data using bacteria-specific primers, thus there are very few archaea detected using this method.This is important and need to be discussed carefully.Moreover, I did not find any results related to functional prediction in the study, although the authors mentioned it in the M&M section.Therefore, it is unclear whether functional prediction was actually performed and, if so, what the results were.The authors should provide more clarity on this matter and clearly state whether functional prediction was performed and include the results in their manuscript.
Reply: Thanks for your suggestion.We referred to common primers for bacteria and archaea involved in some studies.We accept your feedback and will use archaea specific primers for more accurate research in future studies.Thank you very much.For the functional prediction in the result, we did not conduct functional prediction analysis, but due to my negligence added this method, we have removed its description.
4. As mentioned in results, soil pH and salinity level are all key factor influencing microbial community assembly, however, why the authors declare that "soil salinity and Suaeda species co-shaped bacterial and archaeal community structure"?There is no bulk soil samples here.More direct proof should be provided.
Reply: Thanks for your suggestions.We have modified this section.
5. The discussion and conclusion sections of the manuscript are somewhat vague and lack clear conclusions, especially in the discussion section, the authors display many descriptive expressions and repeat their results, which does not provide a satisfactory analysis of their findings.Therefore, it is recommended that the discussion and conclusion be rewritten with a more critical and concise method that focuses on interpreting the results in light of the research questions and objectives.This will help to provide a clearer and more convincing conclusion and make the manuscript higher quality.
Reply: Thanks for your suggestions.We've rewritten the discussion and conclusion sections.
6. Grammar and writing: The writing of the text needs to be carefully checked.So many incorrect wordings/phrases, font format, punctuation mark, and citation format.Please find more specific comments below.
Reply: Thanks for your suggestions.We have corrected the text for incorrect grammar and citation formatting according to specific comments below.We have tried our best to polish the language in the revised manuscript.
Abstract: General comments: I believe the main issue with this part is the language accuracy and conciseness.The authors used many short sentences that lack strong connections between each other, and the Abstract is not concise.To improve the readability and logicality of the text, the authors should consider adding conjunctions and reducing descriptive statements.Although I have pointed out some places, there are still many areas that need to be revised.Additionally, it would be beneficial to add some clear conclusions at the end of the Abstract.Specific comments: Reply: Thanks for your suggestion.We have checked the language accuracy and conciseness.We have adding conjunctions and reducing descriptive statements.We made a big modify to the Abstract.7. Line 19.Which kind of agricultural soil?It is better to emphasize "the improvement of saline soil".
Reply: Thanks for your suggestion.We have emphasized "saline soil" in Abstract section.
Reply: Thanks for your suggestion.We have reworded the abstract.9. Line 20.However, … Reply: Thanks for your suggestion.We have added the "However".
10. Line 24. "strongly and very strongly…" Please reword this sentence.If possible, the authors can add the specific level of salinity in two soil types to provide a more detailed understanding of the impact of salinity on the soil microbial community.

Line 27 & 29, Its all S. altissima?
Reply: Thanks for your reminder.We have rewritten the Abstract section.
Reply: Thanks for your suggestion.We have reworded this sentence.
13. Line 35-36.Why soil salinity influence rhizosphere microbial community?Based on the previous results, soil pH was the main factor.Please explain this.
Reply: In our study, soil types were categorized as heavily saline and very heavily saline based on soil electrical conductivity.Soil salinity level is judged by the magnitude of soil EC, which includes all the ions and electrolytes in the soil.Under natural conditions, changes in soil salinity levels are accompanied by changes in soil pH, and these changes in pH are partly due to the presence of Na 2 CO 3 and NaHCO 3 , which complement each other.We therefore wanted to investigate the main factors that alter the composition and structure of plant rhizosphere microbial communities.Therefore, we measured some physicochemical parameters related to soil salinity, including soil pH.By Monte Carlo analysis and RDA analysis, we identified the most relevant factors on community composition, among which pH also was significantly correlated with bacterial archaeal community structure.In broad terms, the large differences of microbial community structure were caused by different salinity levels.In detail, it was due to changes in some ion contents in the soil and changes in pH that drove changes in plant inter-root microbial community structure.
Reply: Thanks for your suggestion.We have changed "important implication" to "important ecological roles".
Reply: We have reworded this sentence.Our study has important ecological roles for fully understanding soil salinization and halophyte species on the spatial distribution and ecological diversity of bacteria and archaea, and we provide a basis for biological improvement and ecological restoration of salinity-affected areas.

Introduction:
General comments: Since this study introduce the importance of bacterial and archaeal community of two Suaeda types under different salt stress and aimed to analyze the variations in community diversity and composition, the authors should clearly introduce the effects of soil salinity on rhizosphere microbiomes.How the rhizosphere bacterial and archaeal community influence the plant fitness to soil salinization?Why they need to be investigated?What's the difference between halophytes and glycophytes rhizosphere microbiomes?Were there any important research gaps/hypotheses that need to be elucidated so that make the microbial mechanisms behind more clear?Reply: Thanks for your suggestion.We have rewritten the introduction section, added the interaction between the rhizosphere bacterial and archaeal community and the plant fitness to soil salinization, the reasons of them need to be investigated.We added the research hypotheses in the last graph of instruction section.
Specific comments: 1. Line 55. Whose functions?Reply: we added "soil productivity" in this sentence.
Reply: Thanks for your suggestion.We have rewritten this paragraph.

8 .
Line 19-20.Reword the sentence.9. Line 20.However, … 10.Line 24. "strongly and very strongly…" Please reword this sentence.If possible, the authors can add the specific level of salinity in two soil types to provide a more detailed understanding of the impact of salinity on the soil microbial community.11.Line 27 & 29, Its all S. altissima?12. Line 31."showed declined".Reword this sentence, it's too chinglish.13.Line 35-36.Why soil salinity influence rhizosphere microbial community?Based on the previous results, soil pH was the main factor.Please explain this.14.Line 46. … important ecological roles.15.Line 45-46.Reword the sentence.Introduction: General comments: Since this study introduce the importance of bacterial and archaeal community of two Suaeda types under different salt stress and aimed to analyze the variations in community diversity and composition, the authors should clearly introduce the effects of soil salinity on rhizosphere microbiomes.How the rhizosphere bacterial and archaeal community influence the plant fitness to soil salinization?Why they need to be investigated?What's the difference between halophytes and glycophytes

3 . 4 . 5 .
Line 148-150.Suggest rewording this sentence.Line 202.The authors predict the functions of bacterial community using Tax4Fun.However, I did not see any relevant results about microbial functions in the manuscript.It is inappropriate and more direct analyses should be provided.Line 217-219.Delete this sentence.6.Line 212 and 225, vegan package needs to be cited.7. Line 228-230.How you build the networks?What package you used?How the networks visualized?Please add the detailed expression.
• Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred 4. Line 270.Please add the cited Figure or Table to support your statement.5. Line 346-348.Add the related Figures or Tables.
7. Line 393.The network edges is degree?8. Line 396.How you define the complexity of microbial networks?The number of edges and nodes?Or some other parameters?9. Line 400.Cyanobacteria et.al.? Is it correct?10.Line 414.Where is the Table 5B??? Discussion: