Review Reports
- Xiaoqing Qian1,2,*,
- Shifan Xie1 and
- Fulei Xu2,3
- et al.
Reviewer 1: Elaine Guedes Reviewer 2: Vellaichamy Mageshwaran Reviewer 3: Anonymous Reviewer 4: Anonymous Reviewer 5: Anonymous
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe research question is changes in microbial diversity as a function of N application to rice plants. This topic is very relevant to the world, as there is an excess of N in crops that not only causes environmental damage. The research shows that there is damage to the diversity of organisms in the soil.
The introduction needs to be backed up with more relevant research on the subject of soil microbial diversity.
, e.g. https://www.frontiersin.org/journals/environmental-science/articles/10.3389/fenvs.2022.1024610/full
The research employs a robust method of soil metagenomic analysis. The methodology is appropriate.
Further exploration of this subject in grain succession areas is necessary.
The last section of the conclusion needs to be rewritten to align with the research's aim. It is suggested to remove the paragraph regarding future research as it does not align with the research objective. Alternatively, consider revising the conclusion to align with the research objective. future research, as it is not in the research objective / and or add this objective to make sense with the conclusion.
Author Response
Reviewer #1：
The research question is changes in microbial diversity as a function of N application to rice plants. This topic is very relevant to the world, as there is an excess of N in crops that not only causes environmental damage. The research shows that there is damage to the diversity of organisms in the soil.
The introduction needs to be backed up with more relevant research on the subject of soil microbial diversity. , e.g. https://www.frontiersin.org/journals/environmental-science/articles/10.3389/fenvs.2022.1024610/full
The research employs a robust method of soil metagenomic analysis. The methodology is appropriate.
Further exploration of this subject in grain succession areas is necessary.
The last section of the conclusion needs to be rewritten to align with the research's aim. It is suggested to remove the paragraph regarding future research as it does not align with the research objective. Alternatively, consider revising the conclusion to align with the research objective. future research, as it is not in the research objective / and or add this objective to make sense with the conclusion.
Response: We have added the above mentioned literature to the introduction section. As to the grain succession area, we have provided the results of rice yield components (as suggested also by reviewers #4) and explained in the discussion.
We have rewritten the conclusion and removed the future research plans, while emphasized the objectives of this study.
Reviewer 2 Report
Comments and Suggestions for AuthorsIn order to achieve UN SDG of 12 to reduce the release of chemicals in soil, air and water, a collective action is required from various levels including scientific community. Considering this, the present work has importance and is aimed to study the soil bacterial community structure with response to the reduction of nitrogen fertilizer rate in rice field. The work will add value to the scientific community and the subject of interest.
Th following are the specific comments to the authors.
1. Title: The work is dealt with soil bacterial community then why the authors mentioned microbial community structure.
2. Page no. 3 line no. 120: Provide the manufacturer details of DNA isolation kit.
3. Page no. 4 line no. 167: Check the unit of 300 mg/kg N. I believe it is kg/ha.
4. Page no. 7 line no. 231: superscript of r2.
Author Response
Reviewer #2：
In order to achieve UN SDG of 12 to reduce the release of chemicals in soil, air and water, a collective action is required from various levels including scientific community. Considering this, the present work has importance and is aimed to study the soil bacterial community structure with response to the reduction of nitrogen fertilizer rate in rice field. The work will add value to the scientific community and the subject of interest.
The following are the specific comments to the authors.
- Title: The work is dealt with soil bacterial community then why the authors mentioned microbial community structure.
Response: It is not appropriate to mention microbial since we have only analyzed bacterial community. We have replaced “microbial” by “bacterial” in the title.
- Page no. 3 line no. 120: Provide the manufacturer details of DNA isolation kit.
Response: The company information of DNA isolation kit has been provided.
- Page no. 4 line no. 167: Check the unit of 300 mg/kg N. I believe it is kg/ha.
Response: “mg kg-1” has been revised as “kg ha-1”. Apologies for such a mistake.
- Page no. 7 line no. 231: superscript of r2.
Response: Revised.
Reviewer 3 Report
Comments and Suggestions for AuthorsOverall MS has targeted the good concept and did the fine experimental setup. Results are also according to the preer reviewed research.
Though, authors need to add following things in the MS to make it perfect:
1. Authors need to verify the indigenous culturable and predominant microbial communities in the experimental land.
2. Need to provide the experimental setups photograph in supplementary fig.
Author Response
Reviewer #3：
Overall MS has targeted the good concept and did the fine experimental setup. Results are also according to the preer reviewed research.
Though, authors need to add following things in the MS to make it perfect:
- Authors need to verify the indigenous culturable and predominant microbial communities in the experimental land.
Response: This paper is following up of the previous ones (Juanjuan Wang, Ruqing Xie , Nanan He , Wanlu Wang, Guiliang Wang, Yanju Yang, Qing Hu, Haitao Zhao, Xiaoqing Qian, Five years nitrogen reduction management shifted soil bacterial community structure and function in high-yielding ‘super ’ rice cultivation, Agriculture, Ecosystems and Environment, 2024, 10877), and we assume that the bacterial community structure in the previous papers represent the original bacterial community. We haven’t done cultivation on the soils and will include this work in the future research.
- Need to provide the experimental setups photograph in supplementary fig.
Response: A experiment setup is provided as Figure S1 in the supplementary file (S1).
Reviewer 4 Report
Comments and Suggestions for AuthorsThe methodology is thorough, however, consider adding the rice yield values obtained at different N fertilization rates, to provide more supporting evidence to strengthen your claim in line 284.
The article presents a detailed analysis of the soil microbial community at different rates of N fertilization in a short-term study. It might be useful to discuss the possible long-term adaptation changes in bacterial diversity and composition based on the literature.
Comments on the Quality of English LanguageLine 80: Wheat was the pre-crop
Line 244: Sodium potassium
Line 284: Gain grain development
Author Response
Reviewer #4：
The methodology is thorough, however, consider adding the rice yield values obtained at different N fertilization rates, to provide more supporting evidence to strengthen your claim in line 284.
Response: We have provided the rice yield in Table S1 in the supplementary document S1.
The article presents a detailed analysis of the soil microbial community at different rates of N fertilization in a short-term study. It might be useful to discuss the possible long-term adaptation changes in bacterial diversity and composition based on the literature.
Response: There are numerous papers dealing with long term nitrogen use on soil microbial community, while long term nitrogen fertilizer reduction is less studied. The authors have demonstrated previously that the top three major bacterial phyla shifted from Proteobacteria, Acidobacteria and Chloroflexi to Proteobacteria, Chloroflexi, and Acti nobacteria from first year to the fifth year after nitrogen fertilizer reduction, and the influential parameters are different (Juanjuan Wang, Ruqing Xie , Nanan He , Wanlu Wang, Guiliang Wang, Yanju Yang, Qing Hu, Haitao Zhao, Xiaoqing Qian, Five years nitrogen reduction management shifted soil bacterial community structure and function in high-yielding ‘super ’ rice cultivation, Agriculture, Ecosystems and Environment, 2024, 10877). We believe there are other factors, such as weather, are affecting the results, thus skip this discussion to avoid any confusion.
Comments on the Quality of English Language
Line 80: Wheat was the pre-crop
Response: Revised.
Line 244: Sodium potassium
Response: Revised.
Line 284: Gain grain development
Response: Revised.
Reviewer 5 Report
Comments and Suggestions for AuthorsThe paper is well-structured and has good logic. I have several comments below.
Line 68 What is NUE? Please give the full name.
Line 78-79 Rephrase this sentence
Line 85 What kind of N fertilizer did you use? Inorganic or organic?
Line 88 LN and HN are representing low and high nitrogen fertilizer?
Line 84 and line 89 The information is redundant.
Line 102 Any special equipment for “freeze-dried” ? How do you handle the contamination among every treatment during the sampling?
Line 113 to 116 Please provide the citation for every method you mentioned.
Line 120 Manufacture information for “PowerSoil DNA Isolation Kit” ?
Line 140 Provide a citation for the FAPROTAX method. What are the advantages compared to PICRUSt2?
Line 144 Is there any block effect that needs to be considered since the field is in a completely randomized block design?
Line 153 You mean the concentration of different form of N decreased?
Line 155 What is meant by nitrogen reduction? Is it the process of converting nitrogen (N₂) into ammonia (NH₃), or does it refer to decreasing the use of nitrogen fertilizer? Please clarify this for the entire paper.
Figure 3A I would suggest use divergent bar plot, it would be more clear.
Figure 3B Be careful the format of the label.
Table 3 Did you adjust your p-value after the correlation analysis? I am concerned about the false discovery rate.
Discussion: How reliable is it to use 16S data to predict function without collecting RNA or protein information? Please discuss the limitations of FAPROTAX analysis.
Comments on the Quality of English LanguageGood, can be improved
Author Response
Reviewer #5：
The paper is well-structured and has good logic. I have several comments below.
Line 68 What is NUE? Please give the full name.
Response: It should be nitrogen use efficiency (NUE). Revised.
Line 78-79 Rephrase this sentence
Response: Revised as “This site belongs to the warm temperate monsoon climate zone, with an average annual temperature of 14.1 ℃. The average annual precipitation is 892.3 millimeters.”
Line 85 What kind of N fertilizer did you use? Inorganic or organic?
Response: (in the form of urea) has been added.
Line 88 LN and HN are representing low and high nitrogen fertilizer?
Response: Yes. The explanation has been provided, in line 89.
Line 84 and line 89 The information is redundant.
Response: The sentence in Line 89 has been deleted.
Line 102 Any special equipment for “freeze-dried” ? How do you handle the contamination among every treatment during the sampling?
Response: The relevant information has been provided. Please refer to the changes in LINE 103-107
Line 113 to 116 Please provide the citation for every method you mentioned.
Response: Five extra references [27-31] have been provided to the specific method.
Line 120 Manufacture information for “PowerSoil DNA Isolation Kit” ?
Response: Provided.
Line 140 Provide a citation for the FAPROTAX method. What are the advantages compared to PICRUSt2?
Response: A citation has been provided. As indicated in the literature, FAPROTAX is more suitable for predicting the ecological functions of environmental samples, such as biogeochemical cycle process (especially the cycle of carbon, hydrogen, nitrogen, phosphorus, sulfur and other elements). FAPROTAX is a manually constructed database based on published literature, which corresponds the functions of classification and metabolism of prokaryotic microorganisms. Because it is based on the published and verified culturable bacteria literature, its prediction accuracy is good, but the coverage of prediction may be reduced compared with PICRUSt2.
Line 144 Is there any block effect that needs to be considered since the field is in a completely randomized block design?
Response: The block effect could be neglected as the whole field was treated evenly before the experimental plot was built.
Line 153 You mean the concentration of different form of N decreased?
Response: Yes. We have revised the sentence to make it more readable.
Line 155 What is meant by nitrogen reduction? Is it the process of converting nitrogen (N₂) into ammonia (NH₃), or does it refer to decreasing the use of nitrogen fertilizer? Please clarify this for the entire paper.
Response: We changed “nitrogen reduction” into ‘nitrogen fertilizer reduction” throughout the manuscript.
Figure 3A I would suggest use divergent bar plot, it would be more clear.
Response: As it is a standard output from LEfSe analysis, we still haven’t figured out how to converse this figure into a divergent bar plot.
Figure 3B Be careful the format of the label.
Response: The font style of the label has been unified.
Table 3 Did you adjust your p-value after the correlation analysis? I am concerned about the false discovery rate.
Response: No, we have not adjusted the P-values after the analysis. The coefficient values and p values were the results of Spearman correlation analysis.
Discussion: How reliable is it to use 16S data to predict function without collecting RNA or protein information? Please discuss the limitations of FAPROTAX analysis.
Response: The method of FAPROTAX has been widely accepted after its publication (Stilianos Louca, Laura Wegener Parfrey, Michael Doebeli Decoupling function and taxonomy in the global ocean microbiome. Science, 2016, 353:1272-1277). It includes over 80 functional classifications, including carbon, nitrogen, phosphorus, sulfur cycling, animal and plant pathogens, methane generation, fermentation, etc., covering more than 4600 different prokaryotic species. It has also flexible database (uses SILVA or Greengene).
The limitation of this analysis is the coverage. We might under estimate the functional abundance to some degree, with should be compensated by other methods such as RNA and protein analyses.