Nitrogen Fixation, Carbohydrate Contents, and Bacterial Microbiota in Unelongated Stem of Manure Compost-Applied Rice at Panicle Initiation

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The manuscript is an improved version resubmitted to this Journal. The authors highlighted in red all the sentences that they changed or improved. From my point of view, the manuscript improved and addressed the changes suggested by the previous reviewers. The manuscript is well written and now has a more solid approach than its previous version. The topic it addresses and the data it offers are highly relevant both in scientific terms and applied to rice production. It uses molecular methods to approach the characterization of the microbial community linked to N2 fixation and is novel for understanding even the functional role of rice in N cycling.

However, I still find a few aspects that the authors could still improve. These are pointed out throughout the manuscript. Among these, my main criticisms are:

1. Adjust some key words, include some references in the introduction and if possible add the working hypothesis.

2. In general, it is necessary to complement the figure and table captions so that they are more informative and independent of the main text of the manuscript.

3. In methods there are still a few details to attend to, but the main thing is related to how the data were statistically analyzed. Here there are several doubts and the explanation of this section should be substantially improved.

4. The presentation of the results should be reinforced and improved by adding the parameters of the statistical analysis used in each case, either in the tables or in the text. Thus, the effect of the tested factors should be clearer beyond the aposteriori tests.

Once all these aspects have been improved or attended to, I consider that the manuscript will be ready for publication.

Comments for author File: Comments.pdf

Author Response

Reviewer 1

Thank you very much for your valuable time and efforts on revising the manuscript. Your comments must clarify interpretation for the data and results greatly. I have corrected all the points indicated in the PDF file. Our responses to your comments are listed below.

 

1. Adjust some key words, include some references in the introduction and if possible add the working hypothesis.

 

Some key words have been replaced, six citations have been added in Introduction and Materials and methods, six references have been added to References, and a working hypothesis has been added to the last paragraph of Introduction.

 

2. In general, it is necessary to complement the figure and table captions so that they are more informative and independent of the main text of the manuscript.

 

Figure legends and table notes have been corrected so that they become more informative independent of the main text.

 

3. In methods there are still a few details to attend to, but the main thing is related to how the data were statistically analyzed. Here there are several doubts and the explanation of this section should be substantially improved.

 

The materials and methods section has been corrected according to your indications, especially on descriptions for the methods of statistical analysis.

 

4. The presentation of the results should be reinforced and improved by adding the parameters of the statistical analysis used in each case, either in the tables or in the text. Thus, the effect of the tested factors should be clearer beyond the aposteriori tests.

 

The results section has been corrected according to your indications, especially on descriptions for the parameters of the statistical analysis.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Dear responsible author,

Thank you for your invitation to review this article.

Developing long-term environmental approaches to plant nutrition programs is very important for the future. In this context, it is necessary to investigate environmentally friendly practices for N2 fixation in plants. This symbiotic relationship, which is well known, especially in legume crops, should be studied in major crops such as rice, and new approaches should be developed. In this paper, the researchers have taken such an effect of the long-term application of matured compost to a new level from a previously detailed study. In this context, the detailed seasonal analysis of rice is very impressive. I also had the impression that many critical corrections had been made in the text of the article.

The main question the researchers tried to address in the study was to explain the seasonal variation of the effects of long-term matured compost and long-term chemical fertilizer application on nitrogenase activity, sucrose and glucose contents, and Bacterial microbiota in rice cultivation.

Although several studies examining N2 fixation of plants in symbiotic relationships have been published to date, this study is unique in that it explains the long-term effects of a previous large-scale study in a season-dependent manner. Therefore, the work is original.

Although changing the title in a work of this kind may seem troublesome for the authors, I think this title is insufficient for the content of the work.

Also, as a general rule, keywords should be written in alphabetical order.

The introduction is generally adequate. The basic information provided for understanding the subject is satisfactory. Although the purpose of the study is clearly stated, a hypothesis (H0 or H1) needs to be added to the scientific approach.

The methodology used in the study is appropriate for the research objectives.

In the study, the results are consistent with the evidence and arguments presented.  The results of the study showed that nitrogenase activity reached the highest activity in the US of MC-treated rice at panicle initiation (August). However, throughout the cultivation, sucrose content in EC increased after rooting regardless of the applied materials, while glucose content in US increased only in CF-treated plants. This observation is adequately discussed in the discussion section.

 

The references used in the study are appropriate in terms of both subject matter and relevance. However, the rule of italicizing Latin expressions in the titles of articles in the reference list was ignored.

Author Response

Reviewer 2

Thank you very much for your valuable time and efforts on revising the manuscript. Your comments must make readers of the manuscript more easily understand. Our responses to your comments are listed below.

 

> Although changing the title in a work of this kind may seem troublesome for the authors, I think this title is insufficient for the content of the work.

The title of manuscript has been changed so that the title includes all the contents of this work.

 

> Also, as a general rule, keywords should be written in alphabetical order.

 

Some key words have been replaced and shown in alphabetical order.

 

> The introduction is generally adequate. The basic information provided for understanding the subject is satisfactory. Although the purpose of the study is clearly stated, a hypothesis (H0 or H1) needs to be added to the scientific approach.

 

A hypothesis in this study has been added to the last paragraph of Introduction.

 

> In the study, the results are consistent with the evidence and arguments presented. The results of the study showed that nitrogenase activity reached the highest activity in the US of MC-treated rice at panicle initiation (August). However, throughout the cultivation, sucrose content in EC increased after rooting regardless of the applied materials, while glucose content in US increased only in CF-treated plants. This observation is adequately discussed in the discussion section.

 

The observation for that glucose content in US increased only in CF-treated plants has been added to Discussion.

 

> The references used in the study are appropriate in terms of both subject matter and relevance. However, the rule of italicizing Latin expressions in the titles of articles in the reference list was ignored.

 

Latin expressions and gene names in the titles of articles have been italicized in References.

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Manuscript Zhalaga Ao et al., “Nitrogen Fixation, Carbohydrate Contents, and Bacterial Microbiota in Unelongated Stem of Manure Compost-Applied Rice at Panicle Initiation”, Microbiology Research.

This paper describes seasonal changes of nitrogenase (acetylene reduction) activity, carbohydrate contents, and bacterial diversity in elongated culm (EC), unelongated stem (US), and crown root (CR) in manure compost (MC) and chemical fertilizer (CF) applied rice Oryza sativa L. japonica (cultivar Udai 21).

Therefore, the topic of this manuscript (Ms) is relevant for Microbiology Research.

However, I have several critical remarks to the Ms. Ms needs Major Revisions.

 

Major:

1) This manuscript (Ms) contains misprints, mistakes in English grammar and in the writing style. I recommend that the authors should use some help of a native English speaker or send the Ms to an English Editing Service that proofreads scientific writing.

 

2) Questions about manure compost and chemical fertilizer:

a) Authors should explain in detail the difference between manure compost (MC) and chemical fertilizer (CF) – what is not given enough is not clear.

b) Also, in Fig. 1 author should include photo of the rice parts growing on MC and CF treated fields.

c) In Fig. 1 author should include bar, e.g. bar 1 cm.

d) Has the diversity of bacteria been studied in the fields used where plants grew?

e) Can fungi in the soil somehow influence the nitrogen fixation and carbohydrate contents?

 

3) Authors should in “Materials and Methods” explain used nifH gene, also include the GeneBank accession number.

 

4) Authors should improve all legends for figures, e.g. explain all used abbreviations (e.g. OTUs), statistical treatment, etc. Also, e.g. I do not understand combination of the presentation of the statistical significance by using “*” and letters (like in Table 1).

 

5) Why do the authors study the content of glucose and sucrose only? Are there no other non-structural carbohydrates in rice?

 

6) Authors should improve presentation of the metagenomic data:

a) Authors should explain used operational taxonomic unit (OTUs) or amplicon sequence variants (ASVs), also include quantity of the obtained OTUs of ASVs in each sample. I do not understand Fig. 3.

b) Authors should present Shannon’s alpha diversity boxplot, Bray–Curtis beta diversity NMDS plot, Class-level taxonomical bar plots for the bacterial community composition, or Genus-level UpSet diagrams depicting overlapping taxa of bacteriological sow and next-generation sequencing. Like in Gamalero et al. 2021 or Aleynova et al. 2022.

- Gamalero E, Bona E, Novello G, Boatti L, Mignone F, Massa N, Cesaro P, Berta G, Lingua G. Discovering the bacteriome of Vitis vinifera cv. Pinot Noir in a conventionally managed vineyard. Sci Rep. 2020, 10, 6453.

- Aleynova OA, Nityagovsky NN, Dubrovina AS, Kiselev KV. The Biodiversity of Grapevine Bacterial Endophytes of Vitis amurensis Rupr. Plants 2022, 11, 1128.

 

Minor:

7) Why are there negative values (Fig. 4a,c,d)?

8) Line 341-342: “In addition, the ratio of the ratio of nifH gene copy number” correct to “In addition, the ratio of nifH gene copy number”.

Comments on the Quality of English Language

1) This manuscript (Ms) contains misprints, mistakes in English grammar and in the writing style. I recommend that the authors should use some help of a native English speaker or send the Ms to an English Editing Service that proofreads scientific writing.

Author Response

Reviewer 3

Thank you very much for your valuable time and efforts on revising the manuscript. Your comments have especially contributed to improvement of data analysis of bacterial microbiota and facilitated understanding to the contents. Our responses to your comments are listed below.

1) This manuscript (Ms) contains misprints, mistakes in English grammar and in the writing style.

Our response: More than 10 English mistakes have been corrected properly.

2) Questions about manure compost and chemical fertilizer:

a) Authors should explain in detail the difference between manure compost (MC) and chemical fertilizer (CF) – what is not given enough is not clear.

Our response: The difference between MC and CF has been explained in lines 87-91.

b) Also, in Fig. 1 author should include photo of the rice parts growing on MC and CF treated fields

Our response: Photographs of the rice parts growing on MC and CF have been added.

c) In Fig. 1 author should include bar, e.g. bar 1 cm.

Our response: Centimeter scale has been added.

d) Has the diversity of bacteria been studied in the fields used where plants grew?

Our response: A citation has been added in line 93.

e) Can fungi in the soil somehow influence the nitrogen fixation and carbohydrate contents?

Our response: I think it could influence. But even if I would add a sentence explaining this, it is only a guesswork. So I have not added the sentence.

3) Authors should in “Materials and Methods” explain used nifH gene, also include the GeneBank accession number.

Our response: The GeneBank accession number has been added in line 177.

4) Authors should improve all legends for figures, e.g. explain all used abbreviations (e.g. OTUs), statistical treatment, etc. Also, e.g. I do not understand combination of the presentation of the statistical significance by using “*” and letters (like in Table 1).

Our response: All abbreviations and statistical treatments have been explained in figures and tables. Asterisks in table have been deleted.

5) Why do the authors study the content of glucose and sucrose only? Are there no other non-structural carbohydrates in rice?

Our response: As written in lines323-330, in addition to sucrose and glucose, starch and fructose together with their oxidized forms need to be measured in the future study.

6) Authors should improve presentation of the metagenomic data:

a) Authors should explain used operational taxonomic unit (OTUs) or amplicon sequence variants (ASVs), also include quantity of the obtained OTUs of ASVs in each sample. I do not understand Fig. 3.

Our response: The used dissimilarity matrix (Bray–Curtis) has been explained in line 170. Unclassified bacterial sequences at phylum and class levels have been deleted before calculating the matrix. This procedure has improved the rarefaction curves. Figure 3 in the previous manuscript has been moved to Figure S1.

b) Authors should present Shannon’s alpha diversity boxplot, Bray–Curtis beta diversity NMDS plot, Class-level taxonomical bar plots for the bacterial community composition, or Genus-level UpSet diagrams depicting overlapping taxa of bacteriological sow and next-generation sequencing. Like in Gamalero et al. 2021 or Aleynova et al. 2022.

Our response: The alpha diversity boxplots have been added as Figure S2. Class-level taxonomical bar plots have been added as Figure 4A.

7) Why are there negative values (Fig. 4a,c,d)?

Our response: These negative values in Figure 4 have been deleted.

8) Line 341-342: “In addition, the ratio of the ratio of nifH gene copy number” correct to “In addition, the ratio of nifH gene copy number”.

Our response: The phrase has been corrected.

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

General comments

The authors investigated seasonal changes of nitrogenase activity and amount of sucrose and glucose in elongated culm (EC), unelongated stem (US), and crown root (CR) in manure compost (MC)- and chemical fertilizer (CF)-applied rice. Next, they evaluated bacterial microbiota in EC, US, and CR of MC-applied and CF-applied rice at panicle initiation. The authors discussed the relationship between the increase of nitrogenase activity in US of MC-applied rise at panicle initiation and other factors.

This study contains new findings regarding nitrogen fixation by endophyte in rice, but needs to be revised in order to be published as a scientific paper.

 

Major comments

1. There is no direct evidence for a link between glucose and nitrogenase activity, which is the main topic of this study. Therefore, the authors' conclusion of a causal relationship between glucose and increased nitrogenase activity is only a possibility. The authors need to present data or previous findings that clearly demonstrate the relationship. If this major consideration is a possibility, the value of this paper will be diminished.

 

2. The authors discussed the relationship between nitrogenase activity and alpha-proteobacteria. (L261-263, etc.) The authors should also present and discuss results for lower taxa (order, family, genus) for which nitrogen-fixing activity has been previously reported, such as Rhizobiales and Clostridiales (L257-260, L380-382).

 

3. It is not clear what has been discovered in this study. In particular, from the description in the first paragraph of discussion chapter, it appears that many of the authors' considerations are only possibilities. The authors should consider the results of this study and previous findings to come up with a convincing discussion, which should be the main focus of the discussion.

 

4. The significance of what was found in this study needs to be clarified.

 

Minor comments

Fig1: It is better to have a picture of the whole rice plant and a description of its parts.

L157: "as described previously" is unclear where it is mentioned.

L185-: This sentence is not important enough to be included in the results and could be deleted as it is mentioned in the methods (L127-128).

Table 1: For greater visual clarity, it is suggested to present the content in this table as a bar graph.

Table 1, L205-209: No significant differences (e.g. Nitrogenase, EC, MC rows) should be marked with the same letter. If "no letter" means "no significant difference", this should be stated.

L216-217: The authors need to clarify why they compared CR-MC and EC-CF. I did not understand what the authors intended, as the difference in the proportions of the two different experimental conditions was suddenly stated. It was not considered in Discussion chapter either.

 

L252-255(whereas-rice), L358-360: Talble2 shows no significant difference between US-MC (11.2+-3.3ab) and CR-MC (28.1+-3.8bc) for beta-Proteobacteria. Text and figures need to be matched: b in “28.1+-3.8bc” is wrong? Plus, “ab” in CRCF (14.6+-4.3ab) in alpha-Proteobacteria is correct?

Fig 4: Each measurement (Nitrogenase, nifH/16SrRNA gene, etc..) needs to be combined into one graph. It does not seem appropriate that multiple test results are listed within a single set of three graphs for Nitrogenase. The same applies to the others.

Was there really a significant difference between US-SF and US-MC for Nitrogenase and CR-CF and CR-MC for nifH/16S ratio by t-test? Sorry to be skeptical, but I don't think there is a significant difference based on the average value, SD and serial number.

L306-307 (Although-culm): The authors need to provide citations to support this sentence.

 

L308-310: The authors should state in more detail why they considered this.

L332: Need to clarify what "the knowledge from preceded studies" refers to or references.

L391: The authors should explain specifically how they were affected.

L397-399: The content of this sentence seems to be something that has not been discussed in Discussion chapter. The authors should summarize the content discussed within the Discussion chapter.

 

I hope my comments are helpful.

Author Response

Reviewer 4

Thank you very much for your valuable time and efforts on revising the manuscript. Your comments must clarify the content and emphasizing points of the manuscript. Our responses to your comments are listed below.

1. There is no direct evidence for a link between glucose and nitrogenase activity, which is the main topic of this study.

Our response: The data presentation and explanation have been totally reconstructed so that the importance of this study is clearly shown in the revised manuscript.

2. The authors discussed the relationship between nitrogenase activity and alpha-proteobacteria. (L261-263, etc.)

Our response: Bacterial microbiota data has been reanalyzed and the discussion has been focused on the class of Clostridiales while showing the result of lower taxa in Figure 4.

3. It is not clear what has been discovered in this study.

Our response: Discussion has been totally reconstructed according to the reviewers’ comments.

4. The significance of what was found in this study needs to be clarified.

Our response: The significance of the study has been emphasized in Abstract, Discussion, and Conclusions.

Fig1: It is better to have a picture of the whole rice plant and a description of its parts.

Our response: A picture of the whole rice plant and a description of its parts have been added.

L157: "as described previously" is unclear where it is mentioned.

Our response: The phrase has been deleted.

L185-: This sentence is not important enough to be included in the results and could be deleted as it is mentioned in the methods (L127-128).

Our response: The sentence has been deleted.

Table 1: For greater visual clarity, it is suggested to present the content in this table as a bar graph.

Our response: Information of Table 1 is too much to express as a bar graph and there is difficulty to express significant differences with multiple comparisons. So, the data of rice at panicle initiation has been shown as a bar graph as Figure 2.

Table 1, L205-209: No significant differences (e.g. Nitrogenase, EC, MC rows) should be marked with the same letter.

Our response: No significant differences have been marked with same latter in Table 1.

L216-217: The authors need to clarify why they compared CR-MC and EC-CF.

Our response: The sentence has been deleted and the focus on the data has been changed.

L252-255(whereas-rice), L358-360: Talble2

Our response: The data has been replaced with the reanalyzed data and explained carefully.

Fig 4: Each measurement (Nitrogenase, nifH/16SrRNA gene, etc..) needs to be combined into one graph.

Our response: The divided graphs have been combined together.

Was there really a significant difference between US-SF and US-MC for Nitrogenase and CR-CF and CR-MC for nifH/16S ratio by t-test?

Our response: p values have been checked again and these have been added in text.

L306-307 (Although-culm): The authors need to provide citations to support this sentence.

Our response: A citation has been inserted.

L308-310: The authors should state in more detail why they considered this.

Our response: The reason for it has been stated in more detail.

L332: Need to clarify what "the knowledge from preceded studies" refers to or references.

Our responses: Citations for it have been inserted.

L391: The authors should explain specifically how they were affected.

Our responses: The sentence has been deleted because it is only a guess.

L397-399: The content of this sentence seems to be something that has not been discussed in Discussion chapter.

Our responses: The sentence has been deleted and Conclusions have been focused on the significance of this study.

Round 2

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Major:

1) Fig. 1 – increase the size and quality.

 

2) “a) Authors should explain in detail the difference between manure compost (MC) and chemical fertilizer (CF) – what is not given enough is not clear.

Our response: The difference between MC and CF has been explained in lines 87-91.”

- It is necessary to include the data in the materials and methods.

 

3) “Has the diversity of bacteria been studied in the fields used where plants grew?

Our response: A citation has been added in line 93.”

- I did not find the information. Has a metagenomic analysis been performed for soils of the used fields?

 

4) Authors should improve presentation of the metagenomic data: include the Genebank accession numbers of the obtained metagenomic data, include information of the quantity of the all reeds and obtained OTUs or ASVs in each sample (e.g. as a table).

 

Minor:

7) Fig. 2c – explain “ND” in the legend for figure.

Comments on the Quality of English Language

Moderate editing of English language required

Author Response

Thank you very much for your important comments. I think that the quality of manuscript has increased much. Our responses to your comments are listed below.

1) Fig. 1 – increase the size and quality.

Our response: The size and quality of Fig. 1 have been increased and will submit this as a TIFF file.

2) “a) Authors should explain in detail the difference between manure compost (MC) and chemical fertilizer (CF) – what is not given enough is not clear.

- It is necessary to include the data in the materials and methods.

Our response: All the data that we analyzed in terms of MC and CF has been informed in lines 112-115.

3) “Has the diversity of bacteria been studied in the fields used where plants grew?

- I did not find the information. Has a metagenomic analysis been performed for soils of the used fields?

Our response: The result for metagenomic analysis in soil of the used fields can be found in reference 19 paper. So, I have added ‘as reported previously’ in front of the citation in line 93.

4) Authors should improve presentation of the metagenomic data: include the Genebank accession numbers of the obtained metagenomic data, include information of the quantity of the all reeds and obtained OTUs or ASVs in each sample (e.g. as a table).

Our response: DDBJ/EMBL/GenBank accession number for metagenomic data has been provided in line 407. The metagenomic data will be published soon. The information of all the OTUs in metagenome data with taxonomy has been provided as Table S1 as shown in lines 260-261 because it is too large to show in the manuscript.

7) Fig. 2c – explain “ND” in the legend for figure.

Our response: ‘ND’ in Fig. 2 has been explained in the legend.

Moderate editing of English language required

Our response: Several corrections in English have been completed.

Reviewer 4 Report (New Reviewer)

Comments and Suggestions for Authors

General comments

The authors responded appropriately to the reviewer’s comments and revised the manuscript. It is concluded that this manuscript should be accepted.

Author Response

Thank you very much for your valuable comments which have markedly improved the contents of manuscript. 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

It is an apparently original study, well justified and generally well written. However I detected several aspects that must be addressed before being accepted for publication, these aspects are indicated in the text file.

My main criticism of this version of the manuscript is that the methods are not complete, everything related to the statistical analyzes mentioned in the results is missing, as well as the calculation of diversity indices, for example. In addition, and perhaps as a consequence, not all results are supported by analytical statistics or robust statistical analysis. Thus, the significant differences or the higher or lower values that the authors mention and later discuss are not supported. This is striking because there is a well-executed multivariate statistical analysis for the bacterial populations, although it is not clear why this bacterial analysis was only done for August. Another point is regarding the seasonality of the N2 fixation activity, although it was mentioned in the objectives of the study, in the results and in the abstract, it is not explicitly discussed in the discussion and nothing is concluded about it either.

Comments for author File: Comments.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Symbiotic diazotrophic bacteria play a significant role in biological N fixation. The paper investigated the seasonal changes of nitrogenase activity and carbohydrate contents in various parts of rice in MC and CF applied rice paddy field at panicle initiation. The manuscript is well written, but the data is not sufficient to support the author's conclusions. In my view major revision is required. There are some suggestions:

1.     [Page 1, Line 19] Please give the abbreviation of “chemical fertilizer” in the abstract.

2.     [Page 2, Line 78] Please summarize the purpose/aim of the research in the last paragraph of introduction.

3.     [Materials and methods] Please provide a description of the methodology for the significance analysis.

4.     [Materials and methods] It’s necessary to illustrate the physicochemical properties of manure compost (MC) and chemical fertilizer (CF). Please modify.

5.     [Materials and methods] The dynamic of TN in different parts of rice should be tested to demonstrate the conclusion.

6.     [Figures] Please show the annotations in the pictures rather than description in the captions, including the meaning of the lines and dots.

7.     [Page 6, Line 193] It is inaccurate to express data in terms of ranges. Please revise to formation of “Mean ± Error bars”.

8.     [Page 6, Line 193] Descriptions of results should be in the past tense. Please harmonize the use of tenses throughout the manuscript.

9.     [Figure4] The figure title should be “Bacterial DNA reads/ Chloroplastic reads”.

10.  [Page 7, Line 217] Please show the results of significance analysis in the tables and figures. Use “*, **, ***” or “a, b, c” to indicate significance.

 

11.  [Page 9, Line 289] In order to better demonstrate the relationship between the N fixation ability and microbial community, correlation analysis is necessary.

Comments on the Quality of English Language

Moderate English changes required. English language and style are fine.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors investigated seasonal changes of nitrogenase (acetylene reduction) activity and carbohydrate contents in culm internode (IN), basal nodes (BN), and crown root (CR) of d in manure compost (MC)- and chemical fertilizer-applied rice. They found that nitrogenase activity increased after rooting and reached the highest activity in BN of MC-applied rice at panicle initiation. Bacterial and diazotrophic bacterial populations were most abundant in CR, and, alpha-Proteobacteria and Clostridia phyla were abundant in BN-specific bacterial microbiota at panicle initiation. These results were interesting and meaningful. However, the paper cannot be accepted in the status.

1. The contents involved in Title may not be sufficiently confirmed by the Results of text.

2. The article was lack of novelty and depth.

3. The logic of Introduction was a little confused.

Comments on the Quality of English Language

Can be better.

Reviewer 4 Report

Comments and Suggestions for Authors

The reviewed paper broadens both theoretical and practical knowledge related to the importance of biodiversity of microflora in connection with the type of soil, seasonality and the method of fertilization of agricultural fields.

 

Lines 78-85: It would be worthwhile to formulate hypotheses regarding the impact of soil microflora diversity, soil composition (soil type) and seasonal changes in enzyme activity, and the type of fertilization. Such a presentation of the research problem would be clearer.

In the titles of figures, it would be good to provide an explanation of the abbreviations used, it makes it easier to read without having to return to earlier fragments of the work, where they appear for the first time, this applies to the abbreviations IN, BN and CR MC, CF.

Line 219 – In the title of Table 1, it is worth explaining the given abbreviations

In the Conclusion part, it would be worth emphasizing the importance of the results obtained. This would be facilitated by the previously formulated hypotheses.

Comments for author File: Comments.pdf