Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A PSYCHROMETRIC MODEL TO ASSESS THE BIOLOGICAL DECAY OF THE SARS-COV-2 VIRUS IN AEROSOLS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Sometimes the disease COVID-19 is used instead of the virus SARS-CoV-2, eg line 47 where the disease is compared with the influenza virus. The authors should check their manuscript and ensure that they use SARS-CoV-2 when referring to the virus, and COVID-19 when referring to the disease. Note, that it is the virus that transmits, not the disease.
Experimental design: no comment
Validity of the findings: No Comment
Additional comments: Line 42 – worth noting that Qi et al did not see similar relationships in other areas of mainland China
Sometimes the disease COVID-19 is used instead of the virus SARS-CoV-2, eg line 47 where the disease is compared with the influenza virus. The authors should check their manuscript and ensure that they use SARS-CoV-2 when referring to the virus, and COVID-19 when referring to the disease. Note, that it is the virus that transmits, not the disease.

Line 89, the authors might also reference the CDC in the US and PHE as two public bodies that have specifically recognised aerosol role in far field transmission of SARS-CoV-2, although to date, (with the exception of aerosol generating procedures) there is not an explicit recognition from WHO.
Line 90, consider referencing SAGE EMG paper too https://www.gov.uk/government/publications/emg-role-of-ventilation-in-controlling-sars-cov-2-transmission-30-september-2020
Line 157 – reference for the Breusch-Pagan test please
Line 241 – the authors should note that this linear relationship is shown for data within air temperature range of 10-40°C and VP of 0.2456 and 2.97 kPa. From the analysed data set we can not be confident that we can extrapolate this relationship outside these values. For example in Yang et al https://doi.org/10.1371/journal.pone.0046789 fig 1 the influenza viability is linear over c RH 50% to 100% but behaves differently at lower RH – obviously this needs translating into vapour pressure, but it is important to note that, in the absence of data, we don’t know if the relationship proposed by the authors remains for data outside the range of data used to generate the relationship.
On further reading I note that the authors do go some way to acknowledge this in lines 377 – 403, but an explicit note on the limits of this relationship would be useful earlier in the discussion around Line 241

Line 287 – a special character has not translated into the pdf correctly
Line 291 and 292 - a special character has not translated into the pdf correctly
Line 324 to 341, although the predicted  change in viability of the virus in different climatic conditions is interesting, the authors should note that most transmission is considered to occur indoors – nevertheless, for many indoor ventilation strategies the outside weather and vapour pressure conditions will affect the indoor conditions too. It would be helpful for the authors to note this.

A figure of chosen model showing predicted k changes with temp at fixed RH, and with RH at fixed temp would be a useful addition to this paper


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A PSYCHROMETRIC MODEL TO ASSESS THE BIOLOGICAL DECAY OF THE SARS-COV-2 VIRUS IN AEROSOLS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This reviewer found the paper interesting and enlightening, it was well written, with the English professional and simple enough to follow throughout.

The formatting of long reference lists within sentences made for difficult reading within some parts of the document. This I noted was not the case in the pre-print, which made use of numbered references. The following sentence is an example of difficult to read text due to multiple references interrupting the flow of the text. I am not entirely sure if this can be sorted by the authors or the journal?

“The reasons for the seasonal variations in the behaviour of COVID-19 are unclear, but may be related to variations in air temperature (Chin et al. 2020; Ma et al. 2020; Qi et al. 2020; Riddell et al. 2020; Wang et al. 2020), humidity (Ma et al. 2020; Qi et al. 2020; Wang et al. 2020) and ultraviolet (UV) radiation from sunlight (Merow & Urban 2020; Sfica et al. 2020; Tang et al. 2020; Whittemore 2020), as well as immunological (Moriyama et al. 2020; Whittemore 2020) and behavioural (Kanzawa et al. 2020) changes.”

However, the references provided were sufficient, of high quality, up to date and with enough context to enable to reader to review the subject in more detail, as required.

I personally found it a jump from the formula to Figures 1 and 2, and considered whether seeing a tables for these data might improve clarity, or see further explanation from the authors, for not including these data.

No further comments.
Experimental design: The paper has probably filled a knowledge gap and reviewed an area of aerosol science / microbiology, that has received perhaps just a cursory glance beforehand, certainly for COVID-19.

It is an area of I have briefly reviewed and now upon reading this paper I realise I fundamentally misunderstood some of the effects of energy, humidity and temperature on (enveloped) viruses, as did those perhaps writing the articles I read.

As the authors themselves say, it requires someone now to do some research to fill experimental gaps. However, I believe the methodology and investigation has been thorough and seems to fit the datasets available. Brief working through the data suggest a user could reproduce or further develop the models, k and input temperatures & humidity data and come up with half-life survival times for another data-set.

No further comments.
Validity of the findings: Overall, the paper is well rounded and does indeed indicate the SARS-CoV-2 virus is similar to influenza A, another enveloped RNA virus, with seasonal behaviour, and is influenced by variations in air temperature and humidity.

The authors give explanation to the viral characteristics and behaviour that would be effected by lower temperatures and humidity, with an elongated and well referenced discussion.

I was pleased the authors clearly explained the limitation of the work and would hope to see them or others take up the mantle with future work, some thoughts on this include:

- Extending the work beyond October for the Milan, Paris & London.
- Extending the work beyond the above cities.
- Perhaps the authors could consider typical half-lives of the virus within typical indoor scenarios (a typical office, home, etc.), if this is plausible.

No further comments.
Additional comments: Insightful and interesting paper, although I would be keen to see the paper published sooner rather than later, the extensions I have discussed would enhance it still further. Perhaps these extensions could be published as a follow up piece of work, rather than delay the publication of this paper.

No further comments.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A PSYCHROMETRIC MODEL TO ASSESS THE BIOLOGICAL DECAY OF THE SARS-COV-2 VIRUS IN AEROSOLS
Review round: 1
Reviewer: 3

Output:
Basic reporting: Well written paper, generally easy to follow and relevant information provided. However, I suggest that more information would be useful on the experiments upon which the statistical analysis is based. What were the experimental setups, typically? How similar are these experiments to each other? Do they all follow the same/similar procedure? If not are there differences that may affect the decay constant? Maybe the size range of aerosol droplets generated, for example?

While I am able to understand the meaning of the different statistical analyses used in the paper, I can’t say that I’m that familiar with many of these methods, e.g. Breusch-Pagan test. I’m sure readers who are familiar with statistical methods will be familiar with these, however for those who aren’t, like myself, suitable references would be useful when discussing such methods.
Experimental design: The experimental design seems reasonable to me. However my knowledge of statistics is not particularly in depth so I can’t say I’m well placed to critique some of the statistical methods used.
Validity of the findings: The main finding of the paper is that there seems to be a definite, predictable relationship between the psychometric data considered (mainly temperature and humidity) and the value of the decay constant. The authors provide a convincing case regarding this result.
The authors then attempt to apply the derived relationship to historical meteorological data, providing estimates of the virus survival time outdoors. However, airborne transmission is likely to occur mostly indoors. Could the authors comment on the expected correlation between outdoor conditions and indoor conditions, and subsequently the decay constant? For example, indoor temperatures in winter will be significantly higher than outdoors. AH is likely to be more closely correlated between indoors and outdoors. Given the strong correlation between k and AH, I would expect the trend in fig 4 to look similar for indoor conditions? However the half-life periods may be considerably shorter in winter when considering the indoor environment? Is there a simple, indicative way that indoor conditions could be modelled for different seasons?

Given that airborne transmission outdoors is likely to be very low for all seasons, how relevant is the outdoor half life of the virus? Which is what the model is used to predict in this case. With this in mind, I do question the meaning of the comparison between case rates and predicted outdoor decay constant. The authors do acknowledge the limitations of this comparison and some of the other parameters which are likely to be significant such as seasonal variations in sunlight and indoor occupancy.  They also state, referring to the decay constant, “it is important to note that we cannot say to what extent this contributes to the overall transmission of COVID-19”. In which case, why try to link case rates and the decay constant? The comparison is further complicated by the gradual increase in testing over the period of the pandemic.
Additional comments: The paper shows a clear link between the psychometric parameters and the decay constant of the virus, which is an important contribution to the literature. It also provides a very interesting and insightful discussion of the possible mechanisms by which these parameters affect the viability of the virus. The authors use the derived model to predict the decay constant using meteorological data since the start of the pandemic. This provides an interesting illustration of the degree with which the decay constant can vary under different conditions, with an order of magnitude difference between seasons. A comparison is made between the number of cases reported in the UK, France and Italy and the predicted decay constants, roughly showing an inverse relation. This comparison is at this point tenuous due to many other factors which are yet to be understood. However the authors do acknowledge this. Overall I think this is a good paper, however is there a more meaningful analysis to be made to illustrate the significance of the derived model?

Eq 8. 0.6931 comes from ln(2). It would clarify the meaning of this equation to write ln(2).

L145 What was the alternative to artificial saliva? It would be useful to comment on the significance of the solution used.

L191. Why control for artificial saliva if no statistical difference was seen between experiments which did and didn’t use it?
L280. should be lower case p?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A HEAT-SHOCK 20 PROTEIN ISOLATED FROM WATERMELON (CLHSP22.8) NEGATIVELY REGULATES THE RESPONSE OF ARABIDOPSIS TO SALT STRESS VIA MULTIPLE SIGNALING PATHWAYS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Aims and objectives are clearly defined. Clear outline of research. References are relevant and properly cited.
Experimental design: Clear description of methods. Sound experimental design.
Validity of the findings: The findings reported here have a potency to open new research avenues in the field.
Additional comments: The manuscript submitted by He et al., presents a study focusing on gene expression regulation of HSP20 – ClHSP22.8 from watermelon. The authors show that the expression of this gene is repressed under stress conditions like exogenous abscisic acid (ABA) and salt stress and it acts as a negative regulator of plant stress response.
Although the overall data are interesting, there are a few limitations that need to be addressed. Here are my comments that might help authors to improve the manuscript quality.

1) Clear and convincing data for the role of ClHSP22.8 in ‘stress induced repression/activation’ of target genes are missing. Figure 5 and lines 206-224 describe the effects on the levels of ABA- and stress-related genes under salt treatment upon overexpression of ClHSP22.8. These claims need to be confirmed by normalizing the effects seen in WT cells. All the changes in the levels of the various genes tested, are observed in both WT and OE22.8 cell lines. In order to confirm the reduction/elevation is more pronounced in the OE22.8 lines, authors should normalize the changes observed in WT cells and then check if OE22.8 lines show statistically significant difference in regulation.
2) Figure 5: Did authors employ the rest of the two OE22.8 lines? Are these results reproducible?
3) Lines 179:182: Authors describe that there are two ABA responsiveness elements (ABREs) and one anaerobic induction element (ARE) from the ClHSP22.8 promoter (Figure 3C). The biochemical validation of the significance of these elements is necessary.
4) Did authors employ any negative control or empty vector in the experiments described in Figure 2B?
5) Figure 4: Growth of the transgenic and WT plants under ’mock’ treatment is an important control and needs to be shown like in Figures 4A and 4B.
6) Authors need to comment on the result where there is around 4-5 fold induction in the levels of ClHSP22.8 expression after 12 hours of NaCl treatment.
7) Figure 4B: Authors claim to observe a reduction in root growth in WT plants by 29.61% and 8.87% by ABA and salt treatment respectively. Are these reduction values statistically  significant?
8) Figure S2: Is this image acquired from the same gel? The lane 1 and the other lanes seem to be spliced together. Also, what was the loading and/or normalization control used here?
9) Figure 4A: The data seem to be normalized to OE22.8-1 expression, is that correct? In my opinion, they should be normalized to WT levels.
10) Figure 4B: The data for OE22.8-3 are missing.
11) Discussion includes many details regarding the implications of the findings and speculates possible mechanisms. Can authors include a schematic model?
12) Figures 3A, 3B: The Y axis label is missing. Also, an asterisk (*) representing statistical significance is missing.
13) The legends for supplementary figures are missing.
14) The font size of the data labels in many figures is too small.
15) The quality of some the figures is poor; so high resolution images are needed.
16) A grammar check would be helpful.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A HEAT-SHOCK 20 PROTEIN ISOLATED FROM WATERMELON (CLHSP22.8) NEGATIVELY REGULATES THE RESPONSE OF ARABIDOPSIS TO SALT STRESS VIA MULTIPLE SIGNALING PATHWAYS
Review round: 1
Reviewer: 2

Output:
Basic reporting: This manuscript is well-prepared and nicely presented with good scientific quality. However, below I have highlighted some issues, which need to be fixed before further consideration. Also, see the attached file for some minor corrections.
Experimental design: See the below comments.
Validity of the findings: See the below comments.
Additional comments: 	Correct the spelling of multiple in the title.
	The word Arabidopsis should always be in italic in the title and main text body. Please check the entire text and fix it.
	Add a space before the citation. There are many places where spaces need to be inserted. Please carefully check the entire text for this error and fix it.
	Line 56, 57, define the ABA on the first appearance.
	Line 60-65, we need strong references for these statements. Please check and add a suitable reference.
	Line 121, 136, use the standard style for units, i.e., 100 mg L-1. Check the entire text/figures and modify the unit.
	Line 153-160, please rearrange the text and cite the figure 1 accordingly. Currently, Fig 1B is not cited in the text; probably authors forget to write 1B.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A HEAT-SHOCK 20 PROTEIN ISOLATED FROM WATERMELON (CLHSP22.8) NEGATIVELY REGULATES THE RESPONSE OF ARABIDOPSIS TO SALT STRESS VIA MULTIPLE SIGNALING PATHWAYS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The article is interesting. It analyzed the expression patterns and roles of ClHSP22.8 from watermelon in response to salt stress. The article found that ClHSP22.8 is a negative regulator in plant response to salt stress via ABA-dependent and -independent, and other stress responsive signaling pathways. But it still needs some editing.
Experimental design: Generally I think the experimental design is proper.
Validity of the findings: no comment
Additional comments: The manuscript (MS) by He et al. titled " A heat-shock 20 protein isolated from watermelon (ClHSP22.8) negatively regulates the response of Arabidopsis to salt stress via multiple signaling pathways " characterized an HSP20 gene (ClHSP22.8) from watermelon. Quantitative real-time PCR (qRT-PCR) and promoter::β-glucuronidase (GUS) assays showed that the expression of ClHSP22.8 was repressed by exogenous ABA and salt treatment. Meanwhile, Arabidopsis lines that over-expressed ClHSP22.8 were successfully constructed and used to investigate the sensitivity to ABA and salt tolerance. The expression profiles of genes related to ABA and the stress response of plants that over-expressed ClHSP22.8 under salt treatment were analyzed. The MS is well written, which not only provide a better understanding of the specific roles of HSP20s in watermelon adaption to salt stress, but may also provide insight into the potential signaling processes in response to stressful conditions. There are several minor issues that should be corrected:
1. In the title ” A heat-shock 20 protein isolated from watermelon (ClHSP22.8) negatively regulates the response of Arabidopsis to salt stress via mutiple signaling pathways ” ‘mutiple’ should be ‘multiple’.
2. In the Figure 3A, the legend of At, Os… was absent.
3. In the Figure 4, the name of the over-expressed ClHSP22.8 lines (OE22.8-1, OE22.8-2, and OE22.8-4) should be uniform.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A HEAT-SHOCK 20 PROTEIN ISOLATED FROM WATERMELON (CLHSP22.8) NEGATIVELY REGULATES THE RESPONSE OF ARABIDOPSIS TO SALT STRESS VIA MULTIPLE SIGNALING PATHWAYS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The manuscript meets basic criteria of reporting.
Experimental design: Sound experimental design has been employed. I do not have any additional comments on the revised version of the manuscript.
Validity of the findings: No additional comments on the revised manuscript.
Additional comments: The revised version of the manuscript submitted by He, Yao et al., addressed the reviewers' concerns raised in the earlier version of the manuscript. I recommend this article for publication in PeerJ.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: A HEAT-SHOCK 20 PROTEIN ISOLATED FROM WATERMELON (CLHSP22.8) NEGATIVELY REGULATES THE RESPONSE OF ARABIDOPSIS TO SALT STRESS VIA MULTIPLE SIGNALING PATHWAYS
Review round: 2
Reviewer: 2

Output:
Basic reporting: Dear Authors,
Thank you for revising the MS according to the proposed comments and suggestions. Notably, the MS has been improved; thus, I am endorsing the submission for publication in PeerJ. Congratulations!
Experimental design: no comment
Validity of the findings: no comment
Additional comments: no comment


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HOMEOSTATIC RESPONSES AND GROWTH OF LEYMUS CHINENSIS UNDER INCREMENTALLY INCREASING SALINE-ALKALI STRESS
Review round: 1
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1.-it was unclear to me in your methods how exactly you harvest. Did you clip to some residual height, or did you clip to ground level? Since residual height related to L. chinensis regrowth.

2.- Did harvest frequency disturb the effect of pH on L. chinensis growth? If yes, then it seems like you can't make any conclusions about the effect of the saline-alkali stress, because when the pH reached to 9.3 in 14-day salinity stress intervals, L. chinensis had been harvested 4 times (Table 1).

3.- Line 99: what did you mean adulthood? If L. chinensis had grown to adulthood on 7 August, did the leaf wilting when you harvested it on 28 August in 23-day salinity stress intervals, then how did you handle them?


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HOMEOSTATIC RESPONSES AND GROWTH OF LEYMUS CHINENSIS UNDER INCREMENTALLY INCREASING SALINE-ALKALI STRESS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The study provides information pertaining to homeostatic responses and growth of Leymus chinensis under incrementally increasing saline-alkali stress. In abstract a meaningful conclusion may be added to make it more informative but brief. Literature cited is sufficient; however, more literature may be added to strengthen the relationship of saline-alkali with nutrients (NP) on the basis of ecological stoichiometric and growth model in introduction. The results are relevant and prove the hypothesis.
Experimental design: The experimental design has not been made clear and needs more details. However, statistical data analysis is well.
Validity of the findings: The findings are novel and add to existing knowledge.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HOMEOSTATIC RESPONSES AND GROWTH OF LEYMUS CHINENSIS UNDER INCREMENTALLY INCREASING SALINE-ALKALI STRESS
Review round: 1
Reviewer: 3

Output:
Basic reporting: The research analyzed the homeostatic responses and growth to incrementally increasing saline-alkali stress in Leymus chinensis. It is providing some new supports for current theoretical research. However, the paper exists a lot of flaws, thus need to done a major revision.

Abstract: the present is not good, need to deep revise it. Detail comments are seen in attached pdf file.

Introduction: introduction needs more detail. I suggest that you add the description at lines 59- 61 to provide more justification to expand upon the knowledge gap being filled.
In addition, the research no clear hypothesis. Need to add it.
Experimental design: Methods described with un-sufficient detail and information to replicate. Detail as follows: 1. Where the plants were grow during experiment? Growing house? Need details; 2. Line 78-79. Why you sealed bottom hole? It is will lead the soil very salty; 3. Most importantly, line 88-89, Hoagland’s nutrient solution contains KNO3, NH4H2PO4, and the N concentration is about 4 times than field condition. It is will decrease your N treatment effect in experiment. Need to explain the relationship between of them; Same question for P.
Validity of the findings: Data analysis: 1. which model you used in analysis? Need details; 2. Add error bars in all figures.

Results: only presented N, P, N:P, why no any information about biomass or growth rate et al?

Discussion: Should revise it based on revised results.

Conclusion: not good. Need to re-write it.

Refs: too much. 30 enough.
Additional comments: The research analyzed the homeostatic responses and growth to incrementally increasing saline-alkali stress in Leymus chinensis. It is providing some new supports for current theoretical research. However, the paper exists a lot of flaws, thus need to done a major revision.

Abstract: the present is not good, need to deep revise it. Detail comments are seen in attached pdf file.

Introduction: introduction needs more detail. I suggest that you add the description at lines 59- 61 to provide more justification to expand upon the knowledge gap being filled.
In addition, the research no clear hypothesis. Need to add it.

Methods: 1. Where the plants were grow during experiment? Growing house? Need details; 2. Line 78-79. Why you sealed bottom hole? It is will lead the soil very salty; 3. Most importantly, line 88-89, Hoagland’s nutrient solution contains KNO3, NH4H2PO4, and the N concentration is about 4 times than field condition. It is will decrease your N treatment effect in experiment. Need to explain the relationship between of them; Same question for P.

Data analysis: 1. which model you used in analysis? Need details; 2. Add error bars in all figures.

Results: only presented N, P, N:P, why no any information about biomass or growth rate et al?

Discussion: Should revise it based on revised results.

Conclusion: not good. Need to re-write it.

Refs: too much. 30 enough.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: HOMEOSTATIC RESPONSES AND GROWTH OF LEYMUS CHINENSIS UNDER INCREMENTALLY INCREASING SALINE-ALKALI STRESS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Most of comments have been addressed in the revision. Language also fine. However, next few place need to revised. 1. Abstract: the first sentence not good. Leymus has grown salinty and alkalinity environment thousands and thousands years, that is, it has well adopted sal-alk environment. Thus, we cannot concluded that Leymus threatened by increasing.... 2. No any hypotheses were found in introduction. I strong suggest authors add it in revision.
Experimental design: 1. Experimental site description is useless in the study. The author should give some details information where the plant was placed. If placed in greenhouse, what about temperature, moisture, and PAR?
2. Lack of experimental design method like RCBD and so on. Need to add it in next revision.
Validity of the findings: no comment
Additional comments: Most of comments have been addressed in the revision. Language also fine. However, next few place need to revised. 1. Abstract: the first sentence not good. Leymus has grown salinty and alkalinity environment thousands and thousands years, that is, it has well adopted sal-alk environment. Thus, we cannot concluded that Leymus threatened by increasing.... 2. No any hypotheses were found in introduction. I strong suggest authors add it in revision. 3. Experimental site description is useless in the study. The author should give some details information where the plant was placed. If placed in greenhouse, what about temperature, moisture, and PAR? 4. Lack of experimental design method like RCBD and so on. Need to add it in next revision.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSIS DURING SEEDS DEVELOPMENT BETWEEN TWO SOYBEAN CULTIVARS
Review round: 1
Reviewer: 1

Output:
Basic reporting: In this paper, Peng et al. reported a differential gene expression analysis between low and high oil soybean cultivars at three time points during seed development. The manuscript is well structured, the documentation of methods is sufficient. The figures and tables are in well shape and readable.
Experimental design: However, my main concern is the lack of replicates for each time points, which makes the differential gene expression analysis less reliable. The whole analysis is very descriptive and failed to identify novel genes/pathways involved in the oil and protein content differences between these two soybean cultivars.

1.	What is the phylogenic relationship between these two soybean cultivars? How different are they genetically?
2.	Do the author think other than gene expression differences, genetic differences between these two soybean cultivars could contribute to the oil and protein content differences?
3.	The authors should try to call SNPs/Indels using RNA-Seq data. It could be a useful resource and may gain new insight in into the phenotypic differences.
Validity of the findings: 1.	Figure 1b, what does the percentage of Y axis mean in the bar plot? In the main text, the authors state “The total content of proteins and oil was measured in mature seeds”. Is this seed stage included in one of the three timepoints surveyed by RNA-Seq?
2.	Based on the method described in the manuscript, it is unclear that whether the determination of oil content was normalized based on seed weight.
3.	Figure 2, how do these differentially expressed genes identified overlap with different comparison groups? To be more biological reverent, it is better to show whether time point or sample specific differentially expressed genes can be detected. But duo to the lack of replicates at each time point, the time point comparison results would be less reliable.
4.	Figure 3, it is unclear whether all DEGs are used or only time point specific highly expressed genes are used for the GO enrichment analysis.
5.	Figure 4, comparing the numbers of DEGs identified could be misleading. Genes up-regulated in one comparison could be down-regulated in another comparison. Just showing the numbers is not sufficient, or biological meaningful. It is better to use volcano plots to show the degrees of differences of expression and highlight known genes involved in the oil content pathways.
6.	Figure 5, without replicates at each time point, these comparisons have no statistical power.
7.	Figure 7, it seems only DEGs are shown. It should include negative controls. For example, some housekeeping genes.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSIS DURING SEEDS DEVELOPMENT BETWEEN TWO SOYBEAN CULTIVARS
Review round: 1
Reviewer: 2

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: 1. The DEGs of the synthesis and elongation of fatty acids, FAD2, KCS and LOX, have been revealed might contribute to the oil content difference in A7 and A35, while SSPs might be due to the transcription regulators such as ABI3 and LEC2.
The authors should provide evidence for molecular detection of the expression levels of these genes in A7 and A35 seeds at different stages of development.
2, Line 103: beans should be soybean.
3, Line 109: cConstruction should be Construction.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSIS DURING SEEDS DEVELOPMENT BETWEEN TWO SOYBEAN CULTIVARS
Review round: 1
Reviewer: 3

Output:
Basic reporting: This manuscript is clear and readable. It has acceptable literature references and background/context provided.The manuscript has professional article structure, and figures. However, it is not really hypothesis driven.
Experimental design: The experimental design is appropriate for this experiment. There lacks a fundamental research questions besides the mention that the genetic mechanisms for the differences in seed composition has not be revealed. Methods are described with sufficient detail & information to replicate.
Validity of the findings: The authors have done a lot of work on this research yet I fail to see the impact and novelty of this research among the many already published in this particular research question. Data analysis has been provided. Conclusions could be improved. Right now they read as the conclusion for results and not addressed to the research community.
Additional comments: General Comments:
The authors of the study “Comparative transcriptome analysis during seeds development between two soybean cultivars set out to identify the genetic mechanisms for the differences in seed composition between two soybean cultivars with distinct protein and oil content using RNA-Seq technology. Overall, this manuscript addresses a major problem in soybean breeding which is the negative association between protein and oil. It is well written, and the authors have done a lot of work to identify the dynamic expression for the seed development process in soybean by evaluating DEG’s for protein vs oil in soybean.

Specific comments:
Line 48-: I believe your introduction has a lot of background and literature but does not demonstrate how this study justifies the novelty and the gap being filled. I believe there have been other studies that have addressed the genetic mechanisms for the differences in seed composition.
Lines 145: I thank the authors for their detailed explanation for the PCA analysis. Where any R statistical packages used to run the PCA. Please include any additional details if any were used.
I believe the authors of this study have demonstrated a lot of work to understand the genetic mechanisms for the differences in seed composition over time. But I would recommend improving on this study is novel from other studies and can be useful for other soybean researchers or breeders.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSIS DURING SEEDS DEVELOPMENT BETWEEN TWO SOYBEAN CULTIVARS
Review round: 2
Reviewer: 1

Output:
Basic reporting: no comment
Experimental design: no comment
Validity of the findings: no comment
Additional comments: The authors have addressed nearly all my concerns.

However, in the rebuttal letter, the author mentioned that there are three replicates at each time point. There are three time points compared in the manuscript. The data deposited in the NCBI SRA only have 6 samples (method section). The authors need to address this discrepancy.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARATIVE TRANSCRIPTOME ANALYSIS DURING SEEDS DEVELOPMENT BETWEEN TWO SOYBEAN CULTIVARS
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript is well structured, the methods is sufficient. The figures and tables are in well readable. It has acceptable literature references and background.
Experimental design: The experimental design is appropriate for this experiment.
Validity of the findings: The authors have done a lot of work on this research.the results are reliable.
Additional comments: The author has  answered my questions, I don't have any other questions. In my opinion, it is suitable for publication.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TEMPERATE SOUTHERN AUSTRALIAN COASTAL WATERS ARE CHARACTERISED BY SURPRISINGLY HIGH RATES OF NITROGEN FIXATION AND DIVERSITY OF DIAZOTROPHS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The writing is clear. The introduction is setting the stage for the study sufficiently.
Literature cited is extensive although a few key ones seem to be missing (see below).
Figure 1 seems low resolution. Some revision would improve it (see below).
The N2 fixation raw and calculated data should be included.
The sequences need to be submitted to the appropriate sequence database. qPCR assay limits of detection and quantification should be reported.
The manuscript is self-contained.
Experimental design: There is a continuous interest in understanding distributions of diazotrophs in the world’s oceans. If the authors can resolve the methodological issues detailed below, this study is adding to the global database of N2 fixation rates and parallel diazotroph communities, providing data from yet another area from which these measurements have not yet been made. The research is essentially a monitoring study. The time of day should be reported for sample collection. If the time varied substantially across samples, the expression data are not going to be comparable (because UCYN-A nifH expression has a diel cycle).
Technical methods for sequencing and qPCR seem ok, although a few additional details should be added.
For the N2 fixation methods, a few other details need to be provided. Importantly, the batch of 15N2 used is the same one used in a previous study demonstrating 15N-DIN contamination in the gas. The authors need to be able to show gas contamination is not influencing their results.
Validity of the findings: The data reported here primarily have value when exploring the importance of coastal areas in general as contributors to N2 fixation and global distributions of UCYNA lineages. Explanations of these patterns with a few environmental snapshot measurements is correlational. n=1 for each environmental datapoint. Multiparameter analyses could further elucidate the patterns explained in the text.

The discussion feels too long and speculative given the scope of study. I wish the focus was on providing more new information and less of a literature review.
Additional comments: L89 The diazotrophs that are not cyanobacteria are not necessarily only heterotrophic. By assuming that all nifH sequences that are not falling with cyanobacteria are from heterotrophic bacteria, you extrapolate from the nifH genes to metabolisms which are unknown. This is a common fallacy in the diazotroph literature.

L104 Need references for the fact that UCYN-B and C are free-living.

L130 You have mentioned UCYN-A several times by now. Define with the full name when mentioning it for the first time.

L131 Include appropriate references for Gamma A. Did you find it here? Should come back to it in the discussion if you did.

L173-174 Were the dissolved nutrient samples filtered for particulates only after freezing, not filtered prior to freezing? If so, can you show this approach did not bias your data?

L197 The same lot of Sigma 15N2 gas has previously been shown to contain 15N-DIN contaminants (Dabundo et al. 2014). This is a major concern. Did you make any attempts to check the gas contamination?

L198 Were any measurements made on actual gas enrichment? What was the final calculated gas enrichment?

L204 At what time of the day did you conduct the incubations? How much did the initiation and termination times vary across stations and sampling times?  If this was highly variable, it could also bias comparisons among sites/seasons.

L300-301 Why were PCR conditions different for the A1 and A1?

L357 But did your gas contain 15N-DIN contaminants?

L348-439 I don’t agree with these statements. Salinity and silica could equally well be indicators of the different water masses in which multiple other factors may have been different, correlating with salinity and silica, and played greater roles. You have no way of knowing these two that you happened to measure were the drivers for the changing communities.

L449 Amplicon based approaches should not be interpreted quantitatively. They are always relative abundances. Given the fact that the rest of the community may be changing drastically, same proportion of an OTU in the community based on amplicon sequencing analyses may mean the actual abundances increased, decreased, or remained stable. Therefore comparisons between amplicon sequencing and qPCR should be worded differently.

L458-459 You don’t discuss the time of day for measurements of nifH expression. At what time did you collect the RNA samples? Was the time of day identical across samples? UCYN-A nifH expression has a diel pattern, therefore the results are only comparable across sites and seasons if the collection time was the same.

L474 Why is change in salinity at these ranges potentially driving the communities?

L 481 Also see Mulholland et al. 2012.

L486-488 These rates should only be reported if you can demonstrate your batch of 15N was not contaminated with 15N-DIN. The raw 15N2 data should be included, with data from blanks shown. Did you include any controls incubated without tracer to check if background shifted during incubation? Was the background delta 15N measured for T0 for each sample? Data for any of such controls should be reported.

L546 Physico-chemical data being based on n=1 per site per sampling occasion and fairly limited.

L675-768 This part is essentially literature review, and very long speculation with your small dataset. The literature review seems fine, but overextended. All of your data are correlative - it is difficult for me to get excited about this part.

L712-713 Mills et al. 2020, ISME J provides insights into DIN and UCYNA. Their findings are important for your conclusions (as they appear to contradict them).

L760-763 The statement about influence of salinity on freshwater diazotrophic cyanobacteria here seems like a stretch. You studied completely different species in a marine environment. The link to your study seems weak.

L765  State as a conclusion from your data, not as a hypothesis at this late stage of the narrative.

Figures

Figure 1.The different nutrients should be separated into different graphs or shown in a table, as they require different scales to see any changes.
You measured particulate C and N also, where are the data?

Figure 3. Please separate the UCYNA1 and A2 OTUs.

Figure 4. Did you check assay cross-hybridization for UCYN-A1 and A2 under your assay conditions?

Figure 5. What time of the day were cDNA collected? The data are comparable only if the sampling time was identical. Different colors for different symbols would help here.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TEMPERATE SOUTHERN AUSTRALIAN COASTAL WATERS ARE CHARACTERISED BY SURPRISINGLY HIGH RATES OF NITROGEN FIXATION AND DIVERSITY OF DIAZOTROPHS
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript by Messer et al. report nitrogen fixation rates, diazotroph community composition and quantification of UCYN-A (A1 + A2/A3 sublineages) in an inverse estuary. The findings add on to the literature on the significance of nitrogen fixation also in coastal temperate waters. I think that this is a very nice area to study shifts in diazotroph communities and activity. The manuscript is very well written and the data is clearly presented.
Experimental design: My main criticism concerns the presentation of data related to sublineages of UCYN-A. In the current study the authors have used an OTU clustering approach of 97% similarity. The cluster is then identified by the most abundant sequence type in the cluster which the authors use to identify the cluster. The problem with this method is that UCYN-A sublineages are >97% similar on the amino acid level and the UCYN-A diversity is therefore “hidden” in the clustering. This needs to be revised or discussed more thoroughly, especially since different UCYN-A sublineages are targeted in the qPCR analysis which cannot be identified on the OTU level in the amplicon libraries. To use the full potential of the data, analysis of amplicon sequence variants would have been a better choice.

Further, in literature it is well documented that the UCYN-A2 qPCR assay by Thompson et al. 2014 also amplifies the UCYN-A sublineage UCYN-A3 which frequently co-occurs with UCYN-A1 (see Turk-Kubo et al. 2017 and Farnelid et al 2016 for evaluation of qPCR primers targeting UCYN-A). In the current study, with lack of support from the amplicon libraries it is not possible to conclude whether UCYN-A2 and/or UCYN-A3 is co-occurring with UCYN-A1.

It is also not clear if Gamma A sequences were present in the study? This would be interesting to know since it is one of the most well documented and widely distributed HBN’s.
Validity of the findings: The discussion on correlations between SiO4 concentrations and diazotroph community structure (Lines 731-746) is highly speculative as SiO4 may be linked to abiotic factors rather than the occurrence of diatoms and neither have been explored in this study. The authors draw the conclusion that cyanobacterial diazotrophs associated with diatoms were not important constituents of the diazotroph communities (Lines 737-739). It should be noted that the primers used in this study have been shown to favor amplification of gammaproteobacterial taxa while systematically underrepresenting Richelia and Calothrix (Turk-Kubo et al. 2015). Thus in order to draw conclusions about the occurrence of symbionts of diatoms analyses need to be coupled to microscopy observations or complemented by qPCR analyses specific to these groups.
Additional comments: Farnelid, H., Turk-Kubo, K., Muñoz-Marín, M., and Zehr, J. (2016) New insights into the ecology of the globally significant uncultured nitrogen-fixing symbiont UCYN-A. Aquat Microb Ecol 77: 125–138.
Turk-Kubo, K.A., Farnelid, H.M., Shilova, I.N., Henke, B., and Zehr, J.P. (2017) Distinct ecological niches of marine symbiotic N2-fixing cyanobacterium Candidatus Atelocyanobacterium thalassa sublineages. J Phycol 53: 451–461.
Turk-Kubo, K.A., Frank, I.E., Hogan, M.E., Desnues, A., Bonnet, S., and Zehr, J.P. (2015) Diazotroph community succession during the VAHINE mesocosm experiment (New Caledonia lagoon). Biogeosciences 12: 7435–7452.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TEMPERATE SOUTHERN AUSTRALIAN COASTAL WATERS ARE CHARACTERISED BY SURPRISINGLY HIGH RATES OF NITROGEN FIXATION AND DIVERSITY OF DIAZOTROPHS
Review round: 2
Reviewer: 1

Output:
Basic reporting: Thank you for the revised manuscript. This study is interesting and an important contribution to the growing litterature on the activity and distribution of unicellular and non-cyanobacterial diazotrophs. The manuscript is generally well written.

In the revised manuscript, multiple punctuation errors such as missing brackets, double full stops, and spelling mistakes (e.g Proteobateria) are present which need to be corrected prior to publication
Experimental design: no comments
Validity of the findings: Similar to my comment in the previous review, I still find the section in the discussion on SiO4 concentration in relation to increased abundances of non-cyanobacterial diazotrophs highly speculative. A concentration of 1.1 uM SiO4, as observed in the waters of the Gulf should not be described as "rich" as similar concentrations represent very low post diatom bloom levels of SiO4 in oligotrophic waters (e.g. Sargasso Sea). Considering the overall low concentrations of SiO4 in the Gulf, I encourage a revision or removal of this section.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: TEMPERATE SOUTHERN AUSTRALIAN COASTAL WATERS ARE CHARACTERISED BY SURPRISINGLY HIGH RATES OF NITROGEN FIXATION AND DIVERSITY OF DIAZOTROPHS
Review round: 2
Reviewer: 2

Output:
Basic reporting: This manuscript presents interesting findings of N2 fixation rates and diazotrophic community structure in an inverse estuary in Southern Australia. The authors’ findings of relatively high N2 fixation rates adds to the mounting evidence that N2 fixation may be more important in coastal environments than was previously recognized. The manuscript is very well-written and clear.
Experimental design: My only concern about the experimental design is the potential for 15N contaminants in the Sigma batch of 15N2 gas used, which could have inflated N2 fixation rate estimates (as discussed by Reviewer #1 in the previous round of review). The authors now discuss this issue in lines 589-602 of the current manuscript. I feel that a slightly longer description of the calculations/assumptions used to estimate the potential inflated N2 fixation rates (referencing Table 2 of Dabundo et al. (2014)), would help convince the reader that contamination was not an issue.
Validity of the findings: The manuscript conclusions are well-stated and supported by the data.
Additional comments: There are a few instances of small grammatical/formatting errors (L205, 217, 486-488, 523-526)

L486-497: Were UCYN-A abundances significantly correlated with N2 fixation rates?

The authors may wish to discuss the fact that UCYN-A qPCR abundances were relatively low while N2 fixation rates were quite high. I suspect that a quick calculation multiplying maximum UCYN-A cell-specific N2 fixation rates from the literature by the observed qPCR abundances would indicate that UCYN-A likely accounts for a small fraction of the observed N2 fixation rates. However, this method has huge caveats, including that nifH gene abundances are not equivalent to cell concentrations; it is possible that nifH underestimates cells due to incomplete DNA extraction or other methodological issues. Whether or not this should be discussed can be at the discretion of the authors.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ESTIMATING THE NATIONAL AND REGIONAL PREVALENCE OF DRINKING OR EATING MORE THAN USUAL DURING CHILDHOOD DIARRHEA IN MALAWI USING THE BIVARIATE SAMPLE SELECTION COPULA REGRESSION
Review round: 1
Reviewer: 1

Output:
Basic reporting: this is a very good applied work that uses an advanced method to solve an important applied problem. I do not see any major issue here apart from the fact that the authors need to reference properly the methods used, specifically they have to cite Marra et al JASA 2017 and the GJRM package which the authors used for the analysis
Experimental design: It looks fine as far as I can see
Validity of the findings: They look robust as far as I can see
Additional comments: very good work, please fix the references


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ESTIMATING THE NATIONAL AND REGIONAL PREVALENCE OF DRINKING OR EATING MORE THAN USUAL DURING CHILDHOOD DIARRHEA IN MALAWI USING THE BIVARIATE SAMPLE SELECTION COPULA REGRESSION
Review round: 1
Reviewer: 2

Output:
Basic reporting: 27-28 It is known that conventional methods may be biased. Please elaborate more about the conventional methods and why it is biased. By simply reading the background part, the readers may have more questions about how the samples are selected in conventional methods in detail and the impact of the results.

34 Please write this sentence clearer and more concise.

39 Please comment more on the maps of prevalences

44 The weakly association of diarrhea and drinking or eating is an impressive finding

47 Readers may be interested in knowing more about what kind of strategies

59-63 I appreciate your investigation of diarrhea data, however, the structure of these sentences is monotonous. Please try to use another way to rephrase these sentences.

67 2015 to 2016 would be a better wording compared to 2015/2016

70 Readers would like to see more about how DHS data was selected. Please use more citations instead of assuming “ that is, selection into the sample is based on whether the child has diarrhea or not”

72-78 I appreciate your citations for these examples, however, I suggest that confidence interval or P-value be included from these studies to show a statistically significant result. For eg, Asfaha et al 2018, saying households with ≥3 number of children under-five [AOR = 4.05, 95% CI (1.91, 8.60)] was associated with childhood diarrhea

76-78 A good hypothesis

79-81 A bit ambiguous of the logic behind it. Please rephrase the sentence to make it clearer. Do you mean prevalence would be underestimated due to diarrhea status when children were selected for the study?

85 Why does the flexibility in marginal distribution benefit your study? Please comment more on this part.

152 I suggest using the same ‘diarrhea’ consistently throughout the article

155 Please elaborate on the alpha level of the confidence interval that you use

159 13.1 is a percentage

193 Please explain more about how nonrandomness was corrected.

Data and tables look good. Here are some minor issues. A lot of sentences are written in the passive voice. I suggest that rephrase these sentences and make them in an active voice. For eg. Change 'something was collected' to 'we collected something'. Besides, there are some minor spelling issues. Sentences should be written more scientifically, avoiding using words 'say XXXX', 'let xxx be', etc.
Experimental design: The research goal meets the aims and scope of the journal. The hypothesis is creative and the results are impressive. However, there is still space for improvement. For example, in the beginning, the article should convey more explicitly how the conventional method was biased in detail, and make it clearer how this flaw led to your hypothesis.
Validity of the findings: 214 Please follow the order, conclusion, and then discussion

In the discussion part, I would suggest adding more insights on future changes/policy-making/advocates/solutions as mentioned in the beginning.
Additional comments: Overall, the article is well written. Hope to see Scientific English Language improvement in the future.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ESTIMATING THE NATIONAL AND REGIONAL PREVALENCE OF DRINKING OR EATING MORE THAN USUAL DURING CHILDHOOD DIARRHEA IN MALAWI USING THE BIVARIATE SAMPLE SELECTION COPULA REGRESSION
Review round: 1
Reviewer: 3

Output:
Basic reporting: 1) The author needs to cite more recent published papers. Currently, there's no cited paper published after 2018.

2) The English language is a big problem. The author should make a careful reversion to ensure that an international audience can clearly understand the text.
For example:
L28, "due to selection procedure." -> "due to the selection procedure."
L30, "non randomness of sample" -> "non-randomness of the sample"
L60, "Diarrhea is considered as the second" -> "Diarrhea is considered the second"
L60, “under five” -> “under-five”
L64, “minimise” -> “minimize”
L72, "house hold" -> "household"
L75, "with large number" -> "with a large number"
L77, "there is negative association" -> "there is a negative association"
L166, "diarrhoea" ->"diarrhea"
…
Similar problems exist in the remaining paper. The author needs to fix all of them.
Experimental design: The author needs to explain the formula in L125. The meaning of those symbols is not very clear.
Validity of the findings: 1) Fig. 1 (c) keeps the first five digits after the decimal point. At the meanwhile, Fig. 2 (c) holds the first two digits after the decimal point. The numbers in Fig. 1 (c) are very close to each other, which contrasts the claim in L166 "... copula parameter for the bivariate model of diarrhoea and drinking more fluids was -1.23 with confidence interval (-1.53,-1.09)."

2) In the caption, "Dark (high) and white (low)." However, Fig. 1 (c) and Fig. 2 (c) do not follow it.

3) The color of the figure is not illustrative. The author needs to make the color more contrast when the range of the number is wider.

4) It would be better if the author could use numbers to evaluate the prevalence in different regions in addition to figures.
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ESTIMATING THE NATIONAL AND REGIONAL PREVALENCE OF DRINKING OR EATING MORE THAN USUAL DURING CHILDHOOD DIARRHEA IN MALAWI USING THE BIVARIATE SAMPLE SELECTION COPULA REGRESSION
Review round: 2
Reviewer: 1

Output:
Basic reporting: I would suggest using "diarrhea" instead of "diarrhoea" in table 1 to keep it consistent.
Experimental design: well written
Validity of the findings: well written
Additional comments: English writing has improved since last time. The whole article looks fluent and consistent and easy to read. However, some minor issues need to be fixed.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: ESTIMATING THE NATIONAL AND REGIONAL PREVALENCE OF DRINKING OR EATING MORE THAN USUAL DURING CHILDHOOD DIARRHEA IN MALAWI USING THE BIVARIATE SAMPLE SELECTION COPULA REGRESSION
Review round: 2
Reviewer: 2

Output:
Basic reporting: Acceptable
Experimental design: Acceptable
Validity of the findings: Acceptable
Additional comments: No additional comments


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF GUT MICROBIOTA BETWEEN ADULTS WITH AUTISM SPECTRUM DISORDER AND OBESE ADULTS
Review round: 1
Reviewer: 1

Output:
Basic reporting: Good.
Experimental design: 1. Please describe the inclusion criteria and exclusion criteria in detail;
2. Please specify the time and frequency of fecal collection. Please note that the time taken and dietary conditions could affect the structure of feces;
3. Please pay special attention to the effect of antibiotics before enrolled in the study;
4. Please indicate whether the enrolled autistic patients have gastrointestinal symptoms;
Validity of the findings: this study is a small sample single center study, so please give the conclusion carefully.
Additional comments: This study compared the difference of intestinal flora between autistic and obese patients, which is of great significance for clinical improvement of comprehensive treatment of autism and obesity. However, in order to obtain clearer information, the authors are requested to supplement the following information:
1. Please describe the inclusion criteria and exclusion criteria in detail;
2. Please specify the time and frequency of fecal collection. Please note that the time taken and dietary conditions could affect the structure of feces;
3. Please pay special attention to the effect of antibiotics before enrolled in the study;
4. Please indicate whether the enrolled autistic patients have gastrointestinal symptoms;
In addition, this study is a small sample single center study, so please give the conclusion carefully.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF GUT MICROBIOTA BETWEEN ADULTS WITH AUTISM SPECTRUM DISORDER AND OBESE ADULTS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Overall, the text reads rather well but English language still need significant revisions in terms of grammar and spelling mistakes. Introduction may want to include the changes to autism in adults, as they transit from childhood including metabolic co-morbidity like obesity. Any studies reporting gut microbiota changes in autistic obese child and obese adults – any differences? How does obesity affect autism in adults? In general, the structure of the paper conforms to the PeerJ requirements. Likewise, figures are relevant and of sufficient qualities, although larger texts for figure 6 are preferable. Raw data is provided as per journal policy.
Experimental design: The study recruited ASD with normal BMI but I note some were obese (max BMI was 31.9). While I like the general idea of this study but I am not entirely clear on the intention of experimental design. Does the researcher hope to differentiate obese ASD from obesity, if this is the case, then researcher should recruit obese ASD. If the intention is to differentiate ASD from obesity, then there shouldn’t be any obese ASD adults or to have different groups of ASD i.e. non-obese ASD and obese ASD with obesity as controls. Again, the grouping depends on the research hypothesis which I am not clear of. The psychological status of ASD adults has not been provided for e.g. the autism spectrum quotient. Likewise the medical history and metabolic status of ASD and obese adults are not provided.
Validity of the findings: The study findings do provide a couple of interesting observations. Limitations of the study are not provided in the discussion. What is the significance of higher biodiversity in ASD vs. obese? Due to grouping design as described above, the impact of results somehow become limited since the findings become somewhat expected, e.g., fusobacteria is enriched in obesity and contradictory, e.g., enriched fusobacteria in children but reduced in adults.
Additional comments: While the idea is good, but study design somehow limits the usefulness of findings.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF GUT MICROBIOTA BETWEEN ADULTS WITH AUTISM SPECTRUM DISORDER AND OBESE ADULTS
Review round: 1
Reviewer: 3

Output:
Basic reporting: No comment
Experimental design: I have a few comments on the methodology, which can be seen in the general comments for the author section.
Validity of the findings: No comment.
Additional comments: This is a well-made study investigating the gut microbiota of adults with ASD, as compared to obese adults. The authors rightfully conclude that certain bacteria differs between these two groups, and thus future experiments on gut microbiota in ASD need to take the BMI or growth curves into account. The article is well written, and thus the information are easily accessible.
However, I do have a couple of comments, mainly on the methodology. These issues does not affect the conclusion of the article, but need to be mentioned, possibly in a limitation section.

L. 61-62: Since the manuscript focuses on adults, I will recommend deleting the definition of obesity in children.

L. 80. “… which can lead to the ASD-like behaviors, language development deficits, and so on”. This comment needs a reference.

L. 97: “…comparison of intestinal microbiota characteristics between patients with ASD and obese patients are rare”. Does any study exists investigating this? If not, the authors should write that they are not aware of studies investigating this or something like that. “rare” suggest that other such studies exist.

L. 104. I have a couple of questions regarding the study population with ASD.
- How was the ASD diagnosed in these patients? K-SADS? ADOS?
- The authors have included 21 patients diagnosed with ASD and 21 obese persons. It would have been beneficial to have included a third group, that was obese and had ASD, when trying to discuss the effects of obesity versus ASD. This is not critical to the study, but would be beneficial for future studies.
- BMI of ASD cases. You have a BMI range going from underweight (15.9) to obese (31.9) amongst your patients with ASD. Could these extremes have different gut microbiota? E.g. did the obese patient(s?) with ASD have a gut microbiota more like the obese case group?

L. 109: The authors included obese adults from a gym. Are these obese adults in the process of losing weight, since they were recruited at the gym? In that case, were they currently undergoing a diet? The exercise as well as introduction of low calory diets, may have impacted their gut microbiota.

L. 110: I would like more information on how the samples were collected? At the research lab, or in the home of study participants? Were samples immediately (how quick?) transferred to -80 °C, or were they transported to the lab on cold ice?

L. 117. The authors mention that they used the QIAamp DNA stool mini kit. Gram-positive bacteria are notoriously difficult to extract DNA from, and often require additional steps to process (see pmid: 26182345, as well as the new preprint on bioRxiv: https://www.biorxiv.org/content/10.1101/2020.06.15.151753v1 for details). Often this include bead-beating while others utilize extended chemical lysis steps. This is not critical to the conclusions of this study, since all samples were processed similarly, but it might have caused gram-positive bacteria to be underrepresented. This should be mentioned in a limitation section.
If the authors prefers QIAGEN as provider, they have released a PowerFecal kit. This is a good alternative to QIAamp that can be automated on the same machinery, but which does include bead-beating.

Figure 4+5+6 – what test do the authors use to compare the differences in mean abundance between ASD and obesity?

L225-227. “At the phylum-> Figure 1).” Since the authors does not use these information in the subsequent discussion, and has already mentioned it in the results, I think these three lines can be deleted from the discussion.

L. 258. “As all know…”. May I suggest using another phrase?

L. 266-268. That the reason Megamonas was more abundant amongst obese patients, was due to its ability to produce substrates for lipogenesis, is interesting, and probably not wrong. However, it is stated as a fact in  the section. It should be more clear, that it is a speculation by the authors.

L. 281-282: Yes, leaky gut has been suggested to be involved in ASD. But since leaky gut has not been mentioned previously in the manuscript, and is not mentioned later, the speculated relationship between increased Ruminococcus and leaky gut, does not really fit into the rest of the article.

L. 346-351: The authors mentioned that they observed enrichment with P. copri in both obese patients and patients with ASD. Conversely Kang et al. reported a reduced relative abundance of this bacteria. The authors uses this to conclude that children and adults with ASD may have different microbiota compositions. However, gut microbiota can be altered in a lot of ways, meaning that the differences in gut microbiota between this study and the study by Kang et al., may be a result of differences in study population, lifestyle, methodology or just normal variations in gut microbiota. Unless the authors compares the gut microbiota in children and adults with ASD in parallel, I would hesitate to make that conclusion with confidence. Furthermore, the ASD of children are not the focus on this study, and thus I am unsure why the authors wish to comment on differences between adults and children.

While the study seems well designed and the article is well written, it would benefit from a limitation section. This should include, but not necessarily be limited to:
-	The wide range in BMI amongst the included patients with ASD.
-	The risk of behavioral/dietary modifications that may arise from recruiting patients at a gym.
-	The potential underrepresentation of gram-positive bacteria due to lack of mechanical disruption.

A couple of minor typos
- L. 91. “..are positive correlated with obese..” – this should be replaced with “are positively correlated with obesity…”
- L. 95: “…studies have shown that…” replace “have shown” with either reporting/documenting/showing.
- L. 218. “common” – does the authors means “similarities”?
- L. 334. “reched”. I am unsure what the authors mean with this word. It is probably just a typo.
- L. 366. “specie” – replaced with “species”. While “specie” is correct for singular form in the original Latin version, the meanings have diverged and currently, “species” is usually as both singular and plural.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF GUT MICROBIOTA BETWEEN ADULTS WITH AUTISM SPECTRUM DISORDER AND OBESE ADULTS
Review round: 2
Reviewer: 1

Output:
Basic reporting: The authors have revised accordingly to my comments.
Experimental design: The authors have revised accordingly and provided limitations as suggested.
Validity of the findings: The authors have revised accordingly and provided additional analysis, which is acceptable in its current form.
Additional comments: The current version is much improved and is suitable for publication in its current form.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF GUT MICROBIOTA BETWEEN ADULTS WITH AUTISM SPECTRUM DISORDER AND OBESE ADULTS
Review round: 2
Reviewer: 2

Output:
Basic reporting: I observed a potential error with the reference "Association, 2013"
I can see that the authors utilize reference management software – which is highly recommended. However, it seems that an error has occurred when entering the data for the American Psychiatric Association. The software has understood it as a name, and thus only used the last part of the name. I do not know what software the authors use, although I know that it is a known problem in Mendeley – here, it can be solved when entering the name, by clicking the “institution” option in the dropdown menu below. I am not sufficiently familiar with other reference management softwares to guide in these.

There are some minor typos and and grammatical errors, listed below:
L. 65: “…were obesity”. This should be “…were obese”.

L. 69: “Recent decades”. This should be “In recent decades”.

L. 75: “though regulating”. This should be “through regulation”.

L. 81. “Additionally, other study”. This should be “Additionally, another study”

L. 82 + l. 100: Replace “mirobiota” with “microbiota”.

L. 91: Replace “Though” with “Through”

L. 109 “With a mean ages years (ranging…” Something is wrong with this sentence. It seems like the () has been misplaced.

L. 115: “in one month prior” this should be “for one month prior”.

L. 117-119: “Twenty-one obese adults (…) …neurophychiatric diseases,”. Rephrase this sentence. This could be like this: “Twenty-one gender and age matched obese adults (…) that did not suffer from ASD, other neurodevelopmental disorders or neuropsychiatric diseases”.

L. 243-245: “only the phylum Fusobacteria… enriched in obese group”. When you see a decrease amongst ASD, it makes sense that you see an enrichment in the obese groups, since you are comparing those two groups. I would recommend that you just write that the relative abundance of phylum Fusobacteria were lower amongst your ASD group compared to the obese group.

L. 248-251: “Furthermore, Koliada… BMI(Kolliada et al., 2017).” This sentence is a bit unclear.
Experimental design: L. 192-196. I appreciate the information on the weight distribution of the ASD group. However, I do not think, that no significant difference in AMOVA are a sufficiently strong argument to group all ASDs. Despite no overall difference in microbiota composition, you might still see differences among the less dominant bacteria. Especially considering the fact that the individual subgroups was small when performing the AMOVA. I would recommend trying to perform the analyses again (alpha diversity, PCoA, AMOVA, and potentially the Picrust), but only for the group consisting of normal weight ASDs. Then see if the results are still the same as what you observed for all ASDs combined. The results of this subanalysis can then be either placed in the supplementary, or, if the results were indeed similar, just mention that you perform this subanalysis, and that it provided no differences.
Validity of the findings: No Comment
Additional comments: L. 247: Kostic et al, 2012 only reports on the species Fusobacterium nucleatum, that belongs to the Fusobacteria phylum. I would not suggest extrapolating characteristics from a single species to the whole phylum. Thus, I would suggest either finding a source that states that most members of the phylum can induce host inflammatory responses, or scale down the comment. E.g. “members of the Fusobacteria phylum can induce”. Later (346-361) you comment on the reduced relative abundance of the genus Fusobacterium (to which F. nucleatum belongs). I think you should just discuss this here, and avoid mentioning Kostic et al when commenting on the phyla.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: COMPARISON OF GUT MICROBIOTA BETWEEN ADULTS WITH AUTISM SPECTRUM DISORDER AND OBESE ADULTS
Review round: 3
Reviewer: 1

Output:
Basic reporting: No comment
Experimental design: No comment
Validity of the findings: No comment
Additional comments: I believe that the manuscript has ended up being very good, with well presented and interesting results. I highly recommend accepting the manuscript as it now stands.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DISCOVERING POTENTIAL CANDIDATES OF RNAI-BASED THERAPY FOR COVID-19 USING COMPUTATIONAL METHODS
Review round: 1
Reviewer: 1

Output:
Basic reporting: The manuscript is well motivated, introduced in a broadly understandable manner, and self-contained. Literature and background discussion is extensive and precise in detail.

Artwork and figures are very good if not perfect, except that tables like Fig. 3 could possibly be transmitted better, but that is a point the PeerJ technical office may work out together with the authors.

Data is shared and documented / linked.
Experimental design: Covid19 therapies is an area of immediate pressure, so of urgency and importance. siRNA/miRNA suppression therapies are among the promising approaches. Here the authors combine several established modern bioinformatics tool approaches are combined into a candidate-predictive workflow, as clearly summarized in Fig. 1.

The study is worked out and described in thorough detail.

There are no apparent ethical issues within this work visible.

As the authors narrow down to two suggested miRNA / siRNA any subsequent experimental studies would come up with a small number of experiments.
Validity of the findings: The study appears to be on high level and well worked out.

Predicting only 2 candidates might indicate that the acceptance thresholds in each step were quite tight. While I would wish the authors the success that 1 or 2 of them materialize into a working thereapy, I would encourage, if not, to extend the study to predict the next 20 candidates which might still be a manageable number to be tested.
Additional comments: As specified in the 3 evaluation areas (not forwarded to authors), this study appears to be well worked out and written conclusively.
The authors have set up an innovative prediction pipeline for candidates for RNAi based therapy.

As a footnote:
Predicting only 2 candidates might indicate that the acceptance thresholds in each step were quite tight. While I would wish the authors the success that 1 or 2 of them materialize into a working thereapy, I would encourage, if not, to extend the study to predict the next 20 candidates which might still be a manageable number to be tested.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: DISCOVERING POTENTIAL CANDIDATES OF RNAI-BASED THERAPY FOR COVID-19 USING COMPUTATIONAL METHODS
Review round: 1
Reviewer: 2

Output:
Basic reporting: Line 32: CoV instead of COV.
Line 33&34: pls specify the date at which these numbers were taken.
Line 78 and 86: M Witkos et al., 2011).== pls check.
Line 94: You might mean Rous sarcoma?
Line 124: the human == delete the article "the".
Line 151: fref et al., 2016) == was that correct?
Line 162: Analyses instead of Analysis.
Line 163: These two procedure is === These two procedures are.
Line 164:overall workflow is shown in 1. pls clarify!!
Line 167: The conserved regions obtained by (Rangan et al., 2020). == Grammatical error. and here in this case, the Year is inside the parenthesis.
 Line 214 &215: leave a space between the value and its identifies, I mean the Celsius degree.
Line 260: clash should be CLASH.
Line 262: you need a "," after GO.
Line 308: we conducted the analysis the same as the previous section: the sentence needs revision.
Line 311: mention the red flour beetle and put the scientific name in parenthesis.
Line 316: Also indicate the common names and put the scientific name in parenthesis.

--Did you mention Fig.1 and Fig. 2 anywhere in the text before their appearance?
--In the ligand of Fig. 2 4, and 6 the word location is mentioned three times>> did you mean location??
Experimental design: This article fall under the umbrella of Bioinformatics. it tries to establish an miRNA-based CoV targeting. the core question is reliable and well articulated. The analyses were done on scientific merit and the results showed good conclusion that could be utilized in future.
Validity of the findings: The findings of this article is of great importance as it approached molecular targeting of SARS-CoV-2 in reliable and efficient manner.
Additional comments: Dear Colleague,
Nice work and well-witten article.
however, some the results section needs more polishing especially for punctuations


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SOIL PROPERTIES AND ROOT TRAITS JOINTLY SHAPE FINE-SCALE SPATIAL PATTERNS OF BACTERIAL COMMUNITY AND METABOLIC FUNCTIONS WITHIN A KOREAN PINE FOREST
Review round: 1
Reviewer: 1

Output:
Basic reporting: See below section.
Experimental design: See below section.
Validity of the findings: See below section.
Additional comments: Dear Author:
The manuscript “Soil properties and root traits jointly shape fine-scale spatial patterns of bacterial community and metabolic functions within a Korean pine forest”  is quite interesting and informative. The methods and experimental design seem reasonable and sound. However, there are few concerns with this manuscript which need proper consideration. Such as:

1.	Why you took samples from 0-10cm ? usually the surface sampling is done from 0-15cm.
2.	Line 122: Based on which classification system the soil is classified as Haplic Andosols?
3.	Line 136: How the temperature in the color box was kept  4 °C?
4.	How many samples were collected per plot (30 m × 40 m)?
5.	Line 142: Rephrase the sentence “Those for soil organic matter and particulate organic matter analyses were air dried”.
6.	Line 145: The air dried sample passed…. should be replaced by “ the air dried samples were passed through 2 mm sieve” .
7.	Line 145: The word  “measured for” should be replaced by “analyzed for”.
8.	Line 144-154; never start the sentence with abbreviation.
9.	 Line 202: what temperature was used for oven drying of roots?
10.	Why pH and nutrient contributed to 63% of variance of metabolic functions ? Add the pH and nutrients data for all samples as a table which can explain such a huge variation?
11.	What could be the possible reasons for the largest variance in metabolic functions due to soil pH? Explain in discussion section.
12.	What are the possible reasons for such a significant difference in soil microbial composition even at a small distance?
13.	The literature used is up to 2016. Try to add latest references in discussion section.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SOIL PROPERTIES AND ROOT TRAITS JOINTLY SHAPE FINE-SCALE SPATIAL PATTERNS OF BACTERIAL COMMUNITY AND METABOLIC FUNCTIONS WITHIN A KOREAN PINE FOREST
Review round: 1
Reviewer: 2

Output:
Basic reporting: The manuscript reports the spatial shift in the bacterial community in response to the soil properties and root traits. The authors reported after rigorous analysis of Illumina 16S rRNA amplicon sequencing that soil organic matter and roots biomass are the main drivers of bacterial community composition in tested samples. Furthermore, after the analysis of metabolic functions through Biolog plates, it was found that pH is the main factor responsible for the variation of metabolic properties of bacterial communities. Overall, the manuscript seems to be well-composed and may be accepted after minor revisions.
Experimental design: The experimental design is fine.
Validity of the findings: Good
Additional comments: 1) In the introduction, authors should specify the introduction and background to bacterial communities and not microbial communities since they tested only bacterial communities
2) The metabolic variation (functional variation) on the basis of Biolog Eco plates was measured with soil samples probably having a complex mixture of microorganisms; however, the genetic variation was based only on the 16S rRNA gene representing the bacterial portion of complex communities, how authors could correlate the findings of both functional and genetic variation?
3) Authors should specify the term microbial community in case of metabolic measurements (Biolog plates) and bacterial communities in case of genetic variation at a fine-scale level
4) The discussion is supported by old references at some places, these references may be replaced with updated literature


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SOIL PROPERTIES AND ROOT TRAITS JOINTLY SHAPE FINE-SCALE SPATIAL PATTERNS OF BACTERIAL COMMUNITY AND METABOLIC FUNCTIONS WITHIN A KOREAN PINE FOREST
Review round: 1
Reviewer: 3

Output:
Basic reporting: Teng et al manuscript reports interesting findings about the role of root traits and soil properties in structuring soil microbes across different geographic distances. Overall, manuscripts reports very convincing and interesting results, and below are my suggestions that may improve the current work.
Major: Novelty: many studies have reported the role of soil pH in microbial communication? How you results are different from previous studies?
Minor
Ln 33. “environmental variables” which environmental variable you thing is prominent in determining microbial communities?
At some level manuscript is too wordy and sentences are full of redundant phrases (e.g. ln 43-44, first portion of ln 47). Authors may want to check and address this issues.
Ln 58/ln94. For very descriptive and established facts, authors have unnecessarily cited bulk of references, which are even longer than the contents of actual sentences, authors may want to check n correct citation.
Ln 81. I appreciate that the reviewers have acknowledged that “Much less is known, however, about the role of roots in regulating the microbial communities”. BUT again, the cited references have little to nothing to do with the contents of this particular notion. Rather authors may want to support their root traits aspects with relevant literature (e.g. Rhizosphere 16 (2020): 100249).
Ln 106. You may want to clarify which “metabolic functions” are you going to study?
Ln 227. What does “with an average of 6.28” this mean? What is the unit?

Ln 275. Did you test, what was the relationship between SOC and root biomass? Some recent studies showed a positive relationship between SOC and root biomass(Rhizosphere 16 (2020): 100248.). Please discuss this aspect, while linking root traits with microbial communities.

Discussion: you may want to link root traits with microbial communities using relevant literature. You discussion is full of general sentences, which are loaded with references(not necessarily relavent).

Figures are blurred and their quality is not good.

Overall, it is a very well-performed study.
Experimental design: Teng et al manuscript reports interesting findings about the role of root traits and soil properties in structuring soil microbes across different geographic distances. Overall, manuscripts reports very convincing and interesting results, and below are my suggestions that may improve the current work.
Major: Novelty: many studies have reported the role of soil pH in microbial communication? How you results are different from previous studies?
Minor
Ln 33. “environmental variables” which environmental variable you thing is prominent in determining microbial communities?
At some level manuscript is too wordy and sentences are full of redundant phrases (e.g. ln 43-44, first portion of ln 47). Authors may want to check and address this issues.
Ln 58/ln94. For very descriptive and established facts, authors have unnecessarily cited bulk of references, which are even longer than the contents of actual sentences, authors may want to check n correct citation.
Ln 81. I appreciate that the reviewers have acknowledged that “Much less is known, however, about the role of roots in regulating the microbial communities”. BUT again, the cited references have little to nothing to do with the contents of this particular notion. Rather authors may want to support their root traits aspects with relevant literature (e.g. Rhizosphere 16 (2020): 100249).
Ln 106. You may want to clarify which “metabolic functions” are you going to study?
Ln 227. What does “with an average of 6.28” this mean? What is the unit?

Ln 275. Did you test, what was the relationship between SOC and root biomass? Some recent studies showed a positive relationship between SOC and root biomass(Rhizosphere 16 (2020): 100248.). Please discuss this aspect, while linking root traits with microbial communities.

Discussion: you may want to link root traits with microbial communities using relevant literature. You discussion is full of general sentences, which are loaded with references(not necessarily relavent).

Figures are blurred and their quality is not good.

Overall, it is a very well-performed study.
Validity of the findings: Teng et al manuscript reports interesting findings about the role of root traits and soil properties in structuring soil microbes across different geographic distances. Overall, manuscripts reports very convincing and interesting results, and below are my suggestions that may improve the current work.
Major: Novelty: many studies have reported the role of soil pH in microbial communication? How you results are different from previous studies?
Minor
Ln 33. “environmental variables” which environmental variable you thing is prominent in determining microbial communities?
At some level manuscript is too wordy and sentences are full of redundant phrases (e.g. ln 43-44, first portion of ln 47). Authors may want to check and address this issues.
Ln 58/ln94. For very descriptive and established facts, authors have unnecessarily cited bulk of references, which are even longer than the contents of actual sentences, authors may want to check n correct citation.
Ln 81. I appreciate that the reviewers have acknowledged that “Much less is known, however, about the role of roots in regulating the microbial communities”. BUT again, the cited references have little to nothing to do with the contents of this particular notion. Rather authors may want to support their root traits aspects with relevant literature (e.g. Rhizosphere 16 (2020): 100249).
Ln 106. You may want to clarify which “metabolic functions” are you going to study?
Ln 227. What does “with an average of 6.28” this mean? What is the unit?

Ln 275. Did you test, what was the relationship between SOC and root biomass? Some recent studies showed a positive relationship between SOC and root biomass(Rhizosphere 16 (2020): 100248.). Please discuss this aspect, while linking root traits with microbial communities.

Discussion: you may want to link root traits with microbial communities using relevant literature. You discussion is full of general sentences, which are loaded with references(not necessarily relavent).

Figures are blurred and their quality is not good.

Overall, it is a very well-performed study.
Additional comments: Teng et al manuscript reports interesting findings about the role of root traits and soil properties in structuring soil microbes across different geographic distances. Overall, manuscripts reports very convincing and interesting results, and below are my suggestions that may improve the current work.
Major: Novelty: many studies have reported the role of soil pH in microbial communication? How you results are different from previous studies?
Minor
Ln 33. “environmental variables” which environmental variable you thing is prominent in determining microbial communities?
At some level manuscript is too wordy and sentences are full of redundant phrases (e.g. ln 43-44, first portion of ln 47). Authors may want to check and address this issues.
Ln 58/ln94. For very descriptive and established facts, authors have unnecessarily cited bulk of references, which are even longer than the contents of actual sentences, authors may want to check n correct citation.
Ln 81. I appreciate that the reviewers have acknowledged that “Much less is known, however, about the role of roots in regulating the microbial communities”. BUT again, the cited references have little to nothing to do with the contents of this particular notion. Rather authors may want to support their root traits aspects with relevant literature (e.g. Rhizosphere 16 (2020): 100249).
Ln 106. You may want to clarify which “metabolic functions” are you going to study?
Ln 227. What does “with an average of 6.28” this mean? What is the unit?

Ln 275. Did you test, what was the relationship between SOC and root biomass? Some recent studies showed a positive relationship between SOC and root biomass(Rhizosphere 16 (2020): 100248.). Please discuss this aspect, while linking root traits with microbial communities.

Discussion: you may want to link root traits with microbial communities using relevant literature. You discussion is full of general sentences, which are loaded with references(not necessarily relavent).

Figures are blurred and their quality is not good.

Overall, it is a very well-performed study.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SOIL PROPERTIES AND ROOT TRAITS JOINTLY SHAPE FINE-SCALE SPATIAL PATTERNS OF BACTERIAL COMMUNITY AND METABOLIC FUNCTIONS WITHIN A KOREAN PINE FOREST
Review round: 2
Reviewer: 1

Output:
Basic reporting: I have already reviewed the manuscript “Soil properties and root traits jointly shape fine-scale spatial patterns of bacterial community and metabolic functions within a Korean pine forest” on October 19, 2020 and my decision was major revision. I am again of the opinion that the manuscript is well written and explores some new knowledge. I am impressed an in favor of publishing it in your esteem journal. However, there are some suggestions/ comments/ changes which must be adopted before its final publication.
Experimental design: The design of the study is well appropriate and there is no need for its further improvement.
Validity of the findings: The findings are valid and well supported by literature.
Additional comments: I have already reviewed the manuscript “Soil properties and root traits jointly shape fine-scale spatial patterns of bacterial community and metabolic functions within a Korean pine forest” on dated October 19, 2020 and my decision was major revision. I am again of the opinion that the manuscript is well written and explores some new knowledge. I am impressed an in favor of publishing it in your esteem journal. However, there are some suggestions/ comments/ changes which must be adopted before its final publication. My comments are appended below:

•	The abstract has some repetitions which needs to be avoided like “Root biomass explained the second largest variance in soil bacterial community composition after SOC”
•	Introduction is well written and supports the study but there is a need of English language editing as well as new literature.
•	  Why soil depth was kept 0-10cm? Mostly we take surface soil up to 15cm.

My detail comments have already been submitted to you October 19, 2020. In addition to the above comments, those submitted earlier must be properly adopted before making final decision.


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SOIL PROPERTIES AND ROOT TRAITS JOINTLY SHAPE FINE-SCALE SPATIAL PATTERNS OF BACTERIAL COMMUNITY AND METABOLIC FUNCTIONS WITHIN A KOREAN PINE FOREST
Review round: 2
Reviewer: 2

Output:
Basic reporting: The manuscript reports the spatial shift in the bacterial community in response to the soil properties and root traits. The authors reported after rigorous analysis of Illumina 16S rRNA amplicon sequencing that soil organic matter and roots biomass are the main drivers of bacterial community composition in tested samples. Furthermore, after the analysis of metabolic functions through Biolog plates, it was found that pH is the main factor responsible for the variation of metabolic properties of bacterial communities. Overall, the manuscript seems to be well-composed and may be accepted after minor revisions.
Experimental design: The experimental design is fine
Validity of the findings: Good
Additional comments: The authors have addressed my comments and the MS is now ready for publication


Instruction: You are one of the reviewers, your task is to write a review for the article. You will be given the title of the article, the number of the round in which the article is located, and your order among the reviewers.

Input:
Title: SOIL PROPERTIES AND ROOT TRAITS JOINTLY SHAPE FINE-SCALE SPATIAL PATTERNS OF BACTERIAL COMMUNITY AND METABOLIC FUNCTIONS WITHIN A KOREAN PINE FOREST
Review round: 2
Reviewer: 3

Output:
Basic reporting: Authors have sufficiently addressed the raised concerns and manuscript can be considered for publication.
Experimental design: Authors have sufficiently addressed the raised concerns and manuscript can be considered for publication.
Validity of the findings: Authors have sufficiently addressed the raised concerns and manuscript can be considered for publication.
Additional comments: Authors have sufficiently addressed the raised concerns and manuscript can be considered for publication.