Effects of Different Manures in Combination with Fulvic Acid on the Abundance of N-Cycling Functional Genes in Greenhouse Soils

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

The suggestions for your review article are listed below.

1. Abstract

1.1 Keywords: I suggest you change “genes” to “nitrogen cycle functional genes”.

2. Materials and methods

2.1 Page 2 of 20, Experiment site: please inform “light intensity value” of the experimental plastic greenhouse.

2.2 Page 4 of 20, Determination of soil properties: Briefly describe the methodology of soil analyses. I suggest you add references to the methodology.

2.3 Page 5 of 20, Soil DNA extraction and determination of nitrogen cycle functional genes: Briefly describe how to determine the copy number of target genes.

2.4 Page 5 of 20, Table 4: please add the references of your primers.

2.5 Page 5 of 20, Data processing and statistical analyses: please add references for all statistical software.

3. Results

3.1 Explain the result of 16S rRNA gene analysis.

3.2 Page 9 of 20, Figure 2: I suggest you color the bar graphs of each target gene.

3.3 Page 10 of 20, Figure 3: All graphs need to be maximized.

4. General comments

4.1 I suggest you italicize and lowercase the statistical “P” symbols for all sections in your manuscript.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Response to Reviewer 1

Dear Reviewer/Editor,

We are grateful for the time and effort the reviewers have taken to help us improve the manuscript. Our responses to Reviewer #1 are marked in red in the revised manuscript.

Reviewer #1:

The suggestions for your review article are listed below.

1. Abstract

1.1 Keywords: I suggest you change “genes” to “nitrogen cycle functional genes”.

Reply: Done. Line 34.

2. Materials and methods

2.1 Page 2 of 20, Experiment site: please inform “light intensity value” of the experimental plastic greenhouse.

Reply: Given the greenhouse was covered with transparent plastic film, and the light intensity inside the greenhouse was close to the natural light, we did not determine “light intensity value”.

2.2 Page 4 of 20, Determination of soil properties: Briefly describe the methodology of soil analyses. I suggest you add references to the methodology.

Reply: We added more details of the methodology and the associated references. Since soil chemical properties were some common indicators, we mainly added more details about soil DNA extraction and determination of N-cycle functional genes. Line 163-195.

2.3 Page 5 of 20, Soil DNA extraction and determination of nitrogen cycle functional genes: Briefly describe how to determine the copy number of target genes.

Reply: We rewrote this section as you suggested. Line 163-195.

2.4 Page 5 of 20, Table 4: please add the references of your primers.

Reply: Done. Line 196

2.5 Page 5 of 20, Data processing and statistical analyses: please add references for all statistical software.

Reply: Done. Line 197-210.

3. Results

3.1 Explain the result of 16S rRNA gene analysis.

Reply: We have explained the result of 16S rRNA gene analysis. Line 226-234

3.2 Page 9 of 20, Figure 2: I suggest you color the bar graphs of each target gene.

Reply: Done as suggested.

3.3 Page 10 of 20, Figure 3: All graphs need to be maximized.

Reply: Done.

4. General comments 

4.1 I suggest you italicize and lowercase the statistical “P” symbols for all sections in your manuscript.

Reply: Done.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript investigated the “Effects of different manures in combination with fulvic acid on the abundance of functional N-cycling genes in greenhouse soils”. The topic is interesting, and the results have important implications for the improvement of nitrogen cycle and well as mitigation of environment concern. However, the manuscript was not very well prepared, several major issues need to be addressed before publication. The research results reported are too premature for publication. More work is needed to substantiate the abstract, introduction, and conclusions in your manuscript. In general, this study is limited and seems as a case study, which doesn't offer a large perspective that can be transferred to other contexts. The analysis did not well match the objectives proposed in the manuscript. Furthermore, there are many writing mistakes throughout the manuscript. Unfortunately, despite the respectable attempt provided by the authors, I cannot recommend publication in its present form.

Abstract. Comments is. It is better to state the value of the parameters in the best treatment in the abstract exhibited best performance within the treatments and year. (The results showed that fertilization treatments increased the abundance of functional genes for main nitrogen cycling in the soil, in particular, the abundance of nitrification and denitrification genes such as amoA-2, amoB, narG, nirK-1, nirS-1, nirS-2. And the response of adding sheep manure and pig manure treatments with nitrogen cycle function genes were higher than that of adding chicken manure; moreover, the same kind of manures with fulvic  acid had a tendency to reduce the nitrogen cycle function genes.)

Conclusion section.

The abbreviations should be defined at first place it mentioned, rather than in the Conclusion section in which the line numbers are also missed.

In addition, the language also needs substantial improvement.

Please refer to the comments in the attached text and answer them separately and correct them in the text.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

Response to Reviewer 2

Dear Reviewer/Editor,

We are grateful for the time and effort the reviewers have taken to help us improve the manuscript. Our responses to Reviewer #2 are marked in green in the revised manuscript.

Reviewer #2:

The manuscript investigated the “Effects of different manures in combination with fulvic acid on the abundance of functional N-cycling genes in greenhouse soils”. The topic is interesting, and the results have important implications for the improvement of nitrogen cycle and well as mitigation of environment concern. However, the manuscript was not very well prepared, several major issues need to be addressed before publication. The research results reported are too premature for publication. More work is needed to substantiate the abstract, introduction, and conclusions in your manuscript. In general, this study is limited and seems as a case study, which doesn't offer a large perspective that can be transferred to other contexts. The analysis did not well match the objectives proposed in the manuscript. Furthermore, there are many writing mistakes throughout the manuscript. Unfortunately, despite the respectable attempt provided by the authors, I cannot recommend publication in its present form.

Reply: Thank you for your comments. We rewrote the abstract, introduction, and conclusion of this manuscript and revised this manuscript according to your comments carefully.

Abstract. Comments is. It is better to state the value of the parameters in the best treatment in the abstract exhibited best performance within the treatments and year. (The results showed that fertilization treatments increased the abundance of functional genes for main nitrogen cycling in the soil, in particular, the abundance of nitrification and denitrification genes such as amoA-2, amoB, narG, nirK-1, nirS-1, nirS-2. And the response of adding sheep manure and pig manure treatments with nitrogen cycle function genes were higher than that of adding chicken manure; moreover, the same kind of manures with fulvic acid had a tendency to reduce the nitrogen cycle function genes.)

Reply: Thank you for pointing this out. We rewrote this section as you suggested. Line 30-32.

Conclusion section.

The abbreviations should be defined at first place it mentioned, rather than in the Conclusion section in which the line numbers are also missed.

Reply: Done.

In addition, the language also needs substantial improvement.

Reply: The language has been polished by a senior researcher in Rothamsted Research in UK.

Please refer to the comments in the attached text and answer them separately and correct them in the text.

1.The sentence in line 40，“show” should change“ showed”.

Reply: We used “proved” in the revised version. Line 41.

2.The sentence in lines 46-50, this unnecessary paragraph did not fit in manuscript.

Reply: We have deleted this paragraph.

3.The sentence in lines 61-64, how?

Reply: We rewrote this paragraph to make it clear. Line 59-62.

4.The sentence in line 97, the rewrite the sentence, the initial soil properties were following.

Reply: Done.

5.Why the use the ration 1:5 in pH and EC most of time used as 1:2 or 1:2.5.

Reply: The ratio 1:5 is commonly used for the pH determination of soil with high salt content (Lu Rukun. Analysis Method of Agricultural Chemistry in Soil. Beijing: Agriculture and Science Press, 1999. (in Chinese)). In this study, the soil salt content is high, so we used the ratio 1:5.

6.The sentence in lines 103-123, need a lot of improvement along with give a climax in sentences. What (94d Day?)

Reply: We rewrote this section. Because of the outbreak of the novel coronavirus, the first crop lasted 94 days from planting to harvesting.

7.The sentence in line 129, small letter.

Reply: Done.

8.The sentence in line 143 “Brassica chinensiswas”.

Reply: We revised this error.

9.The sentence in line 153 Table 2, detail add in the method and material along with add how to determination this element.

Reply: We added the details in “1.3.2. Determination of soil and manure properties”. The pH, EC, OM, TN, TP and TK was common parameters determined, and we used the associated methods described in “Analysis Method of Agricultural Chemistry in Soil” (Lu Rukun. Analysis Method of Agricultural Chemistry in Soil. Beijing: Agriculture and Science Press, 1999. (in Chinese)). Line 153-162.

10.The sentence in line 158, why it not took soil sample up to 0-20 cm whereas you are initial sample collected up to 0-20 cm.

Reply: Due to the shallow root system of Brassica chinensis, soil of 0-10 cm was sampled. We also changed the initial soil property of 0-20 cm to that of 0-10 cm.

11.The sentence in line 164, “1.3.2. Determination of soil properties”, the analysis methods should be presented more clearly.

Reply: We added more details of the methodology and the associated references. Since the chemical properties were some common indicators, we added more details about soil DNA extraction and determination of N-cycle functional genes. Line 163-195.

12.The sentence in lines 175-176, the DNA extraction in soil method should be presented more.

Reply: Done. Line 163-195.

13.The sentence in line 186, kindly add the full form each selection if used in first time.

Reply: Done.

14.The sentence in lines 197-200, not clear the result.

Reply: We rewrote this section. Line 214-220.

15. In table 5, add the above in row the initial soil properties, change the unit from % to g/kg (SOM).

Reply: Done. Line 221.

16. The sentence in line 235.

Reply: We revised this error.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The topic discussed in the paper is still relevant and presents an interesting approach to the subject of combined fertilization (here manure with fulvic acids) on N-cycling genes in soil. In general, the manuscript was prepared correctly, but the authors did not avoid some imperfections, which should be corrected and supplemented before publication. Below are my comments and suggestions.
1. In abstract, the sentence in lines 12-14 is too long and therefore incomprehensible. Needs to be reworded.
2. The abbreviations used AP should be explained - ? (line 25).
3. The key words completely repeat those contained in the title of the work - they should be changed.
4. The test contained in lines 97-101 will be more readable when we put it in a table. In addition, the taxonomy of the soil (according to WRB) used in the experiment should be provided
5. It should be explained why such doses of manure, FA and TN were assumed in the experimental scheme
6. It should be clearly indicated what plants were cultivated and not use the general term first, second and third crop (such a description may be included in Table 1), but it should be clearly indicated in the text and the systematic and Latin name of the plant should be included.
7. Table 1 - start the title with a capital letter, verify the unit for TN in the 6th column
8. In lines 162-163 there is no citation of authors.
9. In section 1.3.2, the authors/source of the used methods should be provided.
10. The text in lines 368 - 376 should be reworded, again the sentences are too long, which makes them illegible.
11. Text on lines 409 - 410; 419-424 - these are speculations because this aspect was not investigated in the study. It should be deleted or reworded.
12. The citations of old literature, over 10 years old, should be removed and replaced with newer literature.

Comments on the Quality of English Language

The quality of English language is generally good. However, I personally found some stylistic errors that should be consulted with the translator again.

Author Response

Response to Reviewer 3

Dear Reviewer/Editor,

We are grateful for the time and effort the reviewers have taken to help us improve the manuscript. Our responses to Reviewer #3 are marked in blue in the revised manuscript.

Reviewer #3:

The topic discussed in the paper is still relevant and presents an interesting approach to the subject of combined fertilization (here manure with fulvic acids) on N-cycling genes in soil. In general, the manuscript was prepared correctly, but the authors did not avoid some imperfections, which should be corrected and supplemented before publication. Below are my comments and suggestions.
1. In abstract, the sentence in lines 12-14 is too long and therefore incomprehensible. Needs to be reworded.

Reply: Done. Line 15-17.
2. The abbreviations used AP should be explained -? (line 25).

Reply: We changed AP to “available phosphorus”. Line 27.
3. The key words completely repeat those contained in the title of the work - they should be changed.

Reply: We have adjusted the keywords. Line 33.
4. The test contained in lines 97-101 will be more readable when we put it in a table. In addition, the taxonomy of the soil (according to WRB) used in the experiment should be provided

Reply: We have changed this as you suggested. Line 91-94.
5. It should be explained why such doses of manure, FA and TN were assumed in the experimental scheme

Reply: According to the nitrogen requirement for the growth of Brassica chinensis, an equal amount of nitrogen (150 kg per hectare) was applied for all treatments.
6. It should be clearly indicated what plants were cultivated and not use the general term first, second and third crop (such a description may be included in Table 1), but it should be clearly indicated in the text and the systematic and Latin name of the plant should be included.

Reply: We added the Latin name and the variety of the plants (Chinese cabbage) in the three growing seasons at the beginning of section 1.2. Line 98-99.
7. Table 1 - start the title with a capital letter, verify the unit for TN in the 6th column

Reply: Done. Table 2 Line 134.
8. In lines 162-163 there is no citation of authors.

Reply: We revised this error.
9. In section 1.3.2, the authors/source of the used methods should be provided.

Reply: Done. Line 153-157.
10. The text in lines 368 - 376 should be reworded, again the sentences are too long, which makes them illegible.

Reply: We rewrote these sentences as you suggested. Line 360-364.
11. Text on lines 409 - 410; 419-424 - these are speculations because this aspect was not investigated in the study. It should be deleted or reworded.

Reply: We deleted these sentences.
12. The citations of old literature, over 10 years old, should be removed and replaced with newer literature.

Reply: I have removed the old literature over 10 years old and replaced with newer literature, expect for some literatures of primers.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Author,

After I reviewed this revised manuscript, I found it to be better than the previous version. I am satisfied with the revision. All questions were answered.