Changes in Microbial Communities After Lettuce Cultivation in Sihwa Reclaimed Soils, Korea

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

the manuscript is very interesting and current because it is aimed at improving crops without pesticides, however the authors need to clarify some aspects:

1- The improvement of lettuce crops occurs at intervals of pH 5.0 - pH 7.0 however it is understood that it is not exclusively a question of pH values ​​otherwise it would be enough to use buffer mixtures in the soil, therefore there are other factors that need to be identified that facilitate the growth of lettuce (e.g. specific humidity values, salt composition, etc.);

2- It is indicated that mixed groups of Protobacteria, Actinobacteria and Acidobacteria improve the conditions of the rhizome and consequently the growth of lettuce however the authors need to clarify or formulate hypotheses in what way this consortium of bacteria can improve the growth of lettuce (e.g. Plant growth-promoting bacteria (PGPB) that release a variety of molecules that can significantly help plant growth and development).

Kind regards,

Author Response

Please see the attachment file.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

This manuscript describes an experimental protocol in which two contrasted soil types were tested using Lettuce and changes in Microbial community as measured variables. The authors provide information about the importance of soil microbial communities, many of which are irrelevant to the study's objective, while omitting crucial information necessary for a proper understanding of the experiment.

In the introduction section, the paragraph from lines 36-41 is better to introduce in Material and Method.

Lines 31-35 contain the same information as in lines 42-44

There is an association of bacterial diversity in the soybean rhizosphere, and then you talk about the Lettuce. It is not prudent to make comparisons between different crops.

In the Materials and Methods section, it is better to introduce a map of the study area (both zones). How many samples/area, repetitions? The experiment was a randomized block design? How many years, the period? This lack of information introduces considerable ambiguity. Similarly, the authors must refer to the sampling events, clarifying the precise timing of each, which undermines the reproducibility and clarity of the experimental design.

A critical omission is the lack of information regarding the Sihwa reclamation area before the implementation of the experimental protocol. This historical context is essential for interpreting the outcomes of the experiment. These shortcomings represent significant gaps in the description of the experimental protocol.

in the results section, there is no statistical interpretation of tables and figures.

In my opinion, this work lacks sufficient scientific value for proper publication in a journal. The experiment has a fairly short duration, but assessing physical and microbiological characteristics of soil normally needs long-term studies for significant results.

 

Many thanks,

Author Response

Please see the attachment file.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Please find my recommendation for "Physicochemical Properties of Soil in the Sihwa reclaimed land and Changes in Microbial community after lettuce cultivation"

The title should be checked for clarity !

Abstract:

1. L8-11: The objective/motivation of research should be clearly stated. In my opinion the first sentence would be more clear if is split in two, please reformulate
2. The abstract should contain representative numerical values as results; L16-18 clear data should be included

Introduction:

1. L36-41: The manuscript should better develop and present the current situation/ challenges faced by  Sihwa
2.  L42-46: The manuscript should provide clear exemplification about how soil microbiome responds to excess or lack of organic matter, salinity, etc.
3. In L47-55 the manuscript should connect "microorganisms secrete secondary metabolite" with identified/highlighted challenges from L36-41 and L42-46. In the current format there is no connectivity up to my opinion
4. L56-59; L59-61 - these statements should be exemplified
5. The knowledge gap is not clearly stated in this section, the reader cannot extract these information from the current form of the manuscript; In that way for the reader would be difficult to understand the necessity or this research;
6. Also the objectives (L63-70) should be clearly stated;
7. The authors should avoid to use statements as "this study represents the first case to elucidate" 

Methods:

1. L75: Please explain/justify the criteria for establishing the control samples location
2. The manuscript should present the number of plots established for study, the number of samples per plot, the number of real replicates, etc.
3. L78: Please clarify what means "20 kg of soil sampled from each site"; How many sites were considered - this is not clearly evidences (provide maps/GPS coordinates/description/representative characteristics)? What means 20 kg - one composite samples?
4. In my opinion the whole subsection "2.1 Sampling area" should be reconsidered by the authors
5. The authors report salinity but yet pore-water electrical conductivity conversion and temperature correction equations are absent. Please consider this 
6. In subsection "2.2 Analysis of soil samples" the used methods should be clearly cited at leas; for example "Lancaster method", "Tyrin’s method", etc.
7. ICP-OES method should be clearly presented
8. L94: Provide clear details about lettuce growing conditions (if is in controlled environment the manuscript should further clearly state the light cycle frequency, humidity, fertilizers if case, planting density, irrigation/watering salinity, etc... Such missing will imped the attribution of microbial shifts to agronomic factors
9. In my opinion the conventional-field controls differ in unknown soil texture, management history, and physicochemical status, violating comparability assumptions. This should be clarified in the manuscript
10. L127: Diversity indices are mentioned in the manuscript without clarifying rarefaction protocol or normalisation scheme. These details should be considered

Results:

1. In subsection "3.1 Physicochemical Characteristics of Sihwa Reclaimed Soil" the authors reiterate the numerical results from Table 1. This kind of approach should be totally avoided. Please consider this observation during whole manuscript where is the case
2. Table 1 should specify he number of samples from which the results comes (n=?); standard deviation or standard error should be included. Please consider this observation also through the whole manuscript where the case is 
3. Statistical metrics should be clearly provided/presented in the manuscript (p, F, etc.) - please consider for tables and figures also
4. Resolution of graphs should be seriously considered by authors; font size, color code, etc. Some of figures can not be see at all
5. The whole results section should consider to connect the presented results with corresponding statistical metrics for reliability

Discussions:

1. L 95: Such statement should be sustained either by the manuscript obtained research data either by literature - please consider this elsewhere also if the case
2. Several statements are made without longitudinal measurements or mechanistic evidence; correlation is treated as causation by authors which in my opinion is not correct scientifically
3. There are no interpretation of obtained data; the whole discussion section somehow reiterate the results section
4. Comparative and correlative analysis of obtained data with those available form literature are missing. The manuscript should integrate the available information from literature in their study
5. I recommend for authors to evidence their study limitations as well and also to highlight what are beyond the experimental data
6. Conclusion section would be benefic for manuscript
7. However, in my opinion the whole section of discussion should be seriously reconsidered by authors

Author Response

Please see the attachment file.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors I have read the improved manuscript with more attention and I consider it suitable for the publication on the Environments.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for considering my previous observation in "Physicochemical Properties of Soil in the Sihwa reclaimed land and Changes in Microbial community after lettuce cultivation" manuscript. Reading carefully the new version of  this manuscript I observed some parts that need to be clarified and improved by authors. Please find them below:

• L104: Please clarify which sampling design was applied by authors (completely randomized design/CRD or randomized block design/RBD)?
• L143: In my opinion there is an issue regarding the temporal resolution. In manuscript is stated that only a pre-cultivation (SR, NS) and post-harvest (SL, NL) snapshot were sequenced. Based on that the microbial succession is therefore interpolated, not observed; Therefore in my opinion conclusions about diversity increase during cultivation sound speculative. I recommend to authors to treat these carefully
• The discussion attributes reduced lettuce growth to salinity and pH alone, yet OM, Olsen-P, and possibly micronutrients also differ sharply. I recommend for authors to consider multivariate regression or path analysis to disentangle such drivers
• Statements that sustaine that Oscillatoriales improved photosynthetic carbon input into soil or that Eurotiales enhanced stress tolerance at the community level sound speculative up to my opinion as functional validation (metatranscriptomics, enzymatic assays) are absent. I recommend to authors to treat carefully such statements
• Please ensure clarity and accuracy for graphs. It is not recommended to use background color (gradient) especially if it do not have relevance

 

Author Response

we answered in word file

Author Response File: Author Response.docx