Optimizing Initial Nitrogen Application Rates to Improve Peanut (Arachis hypogaea L.) Biological Nitrogen Fixation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article entitled "Optimizing initial nitrogen application rates to improve peanut (Arachis hypogaea L.) biological nitrogen fixation” by Liu et al. explores the impact of different initial nitrogen fertilizer application rates on peanut nodule, root growth and biological nitrogen fixation (BNF). The research was conducted over two years using two peanut cultivars. It highlights that excessive basal nitrogen application inhibits nodule formation and BNF (as ARA)  in the early stages of peanut growth, but a split application of controlled release fertilization can mitigate this effect. Optimizing the balance between nitrogen fertilizer application and BNF can lead to higher yields and reduced environmental impact. The work addresses a topic of great interest and fits properly into the scope of Agronomy.

 

The main strength of the work is the article is well-organized, the methodology of the manuscript is clearly explained, and the article is easy to understand. However, there are serious deficiencies in the work that need to be corrected.

 

·         INTRO. Previous important works on peanut (Arachis hypogaea L.) and BNF closely related to the topic are omitted (neither discussed nor cited).

 

-       El-Sherbeny TMS, Mousa AM, Zhran MA. Response of peanut (Arachis hypogaea L.) plant to bio-fertilizer and plant residues in sandy soil. Environ Geochem Health. 2023 Feb;45(2):253-265. doi: 10.1007/s10653-022-01302-z.

-       Mondal, M.; Skalicky, M.; Garai, S.; Hossain, A.; Sarkar, S.; Banerjee, H.; Kundu, R.; Brestic, M.; Barutcular, C.; Erman, M.; et al. Supplementing Nitrogen in Combination with Rhizobium Inoculation and Soil Mulch in Peanut (Arachis hypogaea L.) Production System: Part II. Effect on Phenology, Growth, Yield Attributes, Pod Quality, Profitability and Nitrogen Use Efficiency. Agronomy 2020, 10, 1513. https://doi.org/10.3390/agronomy10101513.

 

 

·         P4 line 154: “nitrogen application significantly promoted the root growth of the peanut for two years 154 (Table 1)”. The parameters analyzed in Table 1 correspond to aerial parameters, not radicular ones. What conclusions do the authors draw from this table?

·         In relation to the way the data are presented, why do the authors present some data in table format and others as bar graphs? Given the complexity of all the parameters analyzed, a graphic representation of the results would be desirable, especially to visualize the impact of different amounts of Nitrogen on the aerial part.

·         P4 line 167: “The present results indicated that N45 treatment significantly enhanced root dry weight” . This statement is too categorical. It seems that it only happens (and not drastically) for HY22 and only during the year 2020.

·         P4 line 168: “while promoting water and nutrient absorption by the root system”. This conclusion is very categorical and not proven with these results.

·         The authors conclude that the N15 concentration increases the amount of nitrogen biologically fixed by the nodules. However, it does not seem that at this concentration ARA increases significantly (compared to higher concentrations).

·         What does “Root activity” mean?? Is it used as a synonym for acetylene reduction activity (ARA) of nodules? Is it other biochemical or physiological parameter? What do the units (ug.g-1FWh-1) mean? “There is no explanation in the text, or the legend. It is not clear, and the term is not intuitive. In that case (ARA) , What does “Root activity serves as a key indicator of root system development, with higher activity levels indicating that a plant is better equipped for nutrient absorption” mean? ARA is a measure of the biological nitrogen fixation (BNF) process in legumes and serves as an indicator of the activity of nitrogen-fixing enzymes in root nodules, providing insight into the plant's ability to fix atmospheric nitrogen into a usable form, but in any case, is an indicator of root system development.

Author Response

Dear Editors and Reviewers,

Firstly, we would like to express our sincere appreciation to reviewers for reviewing our manuscript. Thank you very much for spending your valuable time to provide these insightful and constructive comments on our manuscript. These comments can not only help us to revise and improve our manuscript, but also have important guiding significance for our future research. We have carefully revised our manuscript entitled “Optimizing initial nitrogen application rates to improve peanut (Arachis hypogaea L.) biological nitrogen fixation” (Manuscript ID: agronomy-2696735). We have tried our best to improve the quality of our manuscript and made the necessary corrections in the new version. The revised portions in the new version were all highlighted by using red-colored text. We also have provided our specific responses to the reviewers' professional and valuable comments. These responses are based on the results of the present study and our interpretations of the results. Point-by-point responses to the reviewers' comments are listed below this letter. We sincerely hope that our manuscript could be considered for publication in your journal.

Thank you for your kind consideration. We look forward to hearing from you.

With best wishes,

Ying Liu

Hunan Agricultural University, Changsha 410128, China

Email: [email protected]

Responses to the reviewers' comments

Reviewer #1:

Introduction

1. Previous important works on peanut (Arachis hypogaea L.) and BNF closely related to the topic are omitted (neither discussed nor cited).

       Ans: Thanks for your constructive suggestions. We have discussed and cited          according to your suggestion on line 48-53 in P2.

2. P4 line 154: “nitrogen application significantly promoted the root growth of the peanut for two years 154 (Table 1)”. The parameters analyzed in Table 1 correspond to aerial parameters, not radicular ones. What conclusions do the authors draw from this table?

Ans: We are sorry for this mistake. From this table 1, results showed that “nitrogen application significantly promoted the growth of the peanut for two years”. We have corrected it on lines 246 in P8.

3. In relation to the way the data are presented, why do the authors present some data in table format and others as bar graphs? Given the complexity of all the parameters analyzed, a graphic representation of the results would be desirable, especially to visualize the impact of different amounts of Nitrogen on the aerial part.

Ans: Thank you for your kind consideration, we have revised according to your suggestion on line 266-271 in P10-P11.

4. P4 line 167: “The present results indicated that N45 treatment significantly enhanced root dry weight”. This statement is too categorical. It seems that it only happens (and not drastically) for HY22 and only during the year 2020.
5. P4 line 168: “while promoting water and nutrient absorption by the root system”. This conclusion is very categorical and not proven with these results.

Ans: Thank you for your comments on our manuscript. We have revised according to your suggestion on line 259-262 in P9.

6. The authors conclude that the N15 concentration increases the amount of nitrogen biologically fixed by the nodules. However, it does not seem that at this concentration ARA increases significantly (compared to higher concentrations).

Ans: Thanks for your nice suggestions. There are many factors that influence ARA activity, and it is true that no significant increase was seen between the different nitrogen levels, but biologically fixed by the nodules was also related to the number of nodules and dry weight, and our study showed that the N15 significantly concentration increases the amount of number of nodules and dry weight, so N15 concentration may significantly increase the amount of nitrogen biologically fixed by the nodules. We also did the appropriate calculations. We found that BNF in HY22 and HY39 were significantly increased 43.79% and 42.55% under N15 treatments in 2020 compared to N60, and increased by 29.17% and 43.36% in 2021, However, ARA in HY22 and HY39 were significantly increased 106% and 135% under N15 treatments in 2020 compared to N60, and increased by 60.77% and 32.83% in 2021. It can be seen that although the increase of ARA is not significant, but BNF may still show significant differences.

7. What does “Root activity” mean?? Is it used as a synonym for acetylene reduction activity (ARA) of nodules? Is it other biochemical or physiological parameter? What do the units (ug. g^-1FWh^-1) mean? “There is no explanation in the text, or the legend. It is not clear, and the term is not intuitive. In that case (ARA), What does “Root activity serves as a key indicator of root system development, with higher activity levels indicating that a plant is better equipped for nutrient absorption” mean? ARA is a measure of the biological nitrogen fixation (BNF) process in legumes and serves as an indicator of the activity of nitrogen-fixing enzymes in root nodules, providing insight into the plant's ability to fix atmospheric nitrogen into a usable form, but in any case, is an indicator of root system development.

Ans: Roots are active absorption organs and synthetic organs. The growth and metabolic level of roots, that is, root activity, directly affect the growth and nutritional status of plant aboveground parts, and is one of the important physiological indicators of plant growth. Plants with strong root activity can better absorb water and nutrients in the soil and provide sufficient nutrient supply. The physiological parameter of root activity also appears in many references. For example,

88. Zhang, Huang Guo-qin, Bian Xin-min, et al. Effects of root interaction and nitrogen fertilization on the chlorophyll content, root activity, photosynthetic characteristics of intercropped soybean and microbial quantity in the rhizosphere. Plant Soil and Environment. 2013;59 (2):80-88. doi:10.17221/613/2012-pse

Liqun Xiu, Weiming Zhang, Di Wu, et al. Biochar can improve biological nitrogen fixation by altering the root growth strategy of soybean in Albic soil. Science of The Total Environment. 2021;773 (0):144564-144564. doi:10.1016/j.scitotenv.2020.144564

Root activity was determined using the triphenyl tetrazolium chloride (TTC) method. TTC is a redox pigment with a standard oxidation potential of 80 mV. When added to the roots, is reduced by the dehydrogenase (mainly succinate dehydrogenase) of the roots. Dehydrogenase activity can be used as an indicator of root activity. In brief,

Root tip samples of 0.5 g were placed in a 10 mL beaker, and an equal mixture of 0.4% TTC and phosphate buffer was added in 10 mL, the roots were fully submerged in the solution, and held in the dark at 37°C for 1~3 h. Thereafter, 1 mol/L sulfuric acid was added in 2 mL in order to stop the reaction. The root was dried with filter paper and then extracted with ethyl acetate. The absorbance of the extract at 485 nm was recorded. Root activity was expressed as TTC reduction intensity.

Root activity = amount of TTC reduction (µg)/fresh root weight(g) × time (h).

Note: FW, fresh root weight.

We have made changes on lines 132-139 in P3.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Topic:

The topic of the work and the publication itself are quite simple, but important for the needs of practice.

Abstract:

Line13-18 - No literature review needed here (1/3 of the chapter)

Line 23 - it is not 5 doses of nitrogen, but 4, because "N0" means lack of nitrogen - i.e. control

Introduction

The chapter is quite well written, but there are occasional shortcuts and frequent repetitions of the same thread

Line 39 – In my opinion, this is an unfortunate statement: “Nitrogen serves as a key element that limits crop growth and yields…..” Nitrogen increases……

Line 43 – The authors wrote: "these crops can obtain nitrogen" plants do not obtain nitrogen, only symbiotic bacteria

 Materials and methods

Line 94 - Is it a good statement: "the experimental field" since these are pot studies, not field experimental studies?

Statistical analysis - Duncan's test was used, which in my opinion is quite mild for such research.

 Results

Tables and figures quite well prepared.

However, it is quite difficult to read due to the lack of spaces between means ± standard deviation

In my opinion, there are so many comas’ places in table 2 unnecessarily: in the Root length and Root surface area columns. Real numbers would be enough: e.g. "1234" instead of "1234.12", and again Root average diameter - there should be 3 numbers after the decimal point "0.123" instead of "0.12"

Results

This chapter is generally correct, however:

The authors show significant differences, but this is not always statistically proven.

Line 152, 191: In my opinion, there should be no clear differences if you can't prove something. This is the case with the description of Figs 1 and 2 in the supplement. They point out differences, but these are visual differences - and we know that eyesight can fail.

Line 242-247 - Unnecessarily, the discussion of Results included elements that should have been included in the Chapter Discussion

 Line 333 - The unusual sound of "Fig. 7 shows that…..”

Conclusion

The first statement is too general and not very relevant to these results.

Lack of results is also a result - maybe some other conclusions?

Author Response

Dear Editors and Reviewers,

Firstly, we would like to express our sincere appreciation to reviewers for reviewing our manuscript. Thank you very much for spending your valuable time to provide these insightful and constructive comments on our manuscript. These comments can not only help us to revise and improve our manuscript, but also have important guiding significance for our future research. We have carefully revised our manuscript entitled “Optimizing initial nitrogen application rates to improve peanut (Arachis hypogaea L.) biological nitrogen fixation” (Manuscript ID: agronomy-2696735). We have tried our best to improve the quality of our manuscript and made the necessary corrections in the new version. The revised portions in the new version were all highlighted by using red-colored text. We also have provided our specific responses to the reviewers' professional and valuable comments. These responses are based on the results of the present study and our interpretations of the results. Point-by-point responses to the reviewers' comments are listed below this letter. We sincerely hope that our manuscript could be considered for publication in your journal.

Thank you for your kind consideration. We look forward to hearing from you.

With best wishes,

Ying Liu

Hunan Agricultural University, Changsha 410128, China

Email: [email protected]

Reviewer #2:

Abstract:

1. Line13-18- No literature review needed here (1/3 of the chapter)

Ans: Thank you for your comments on our manuscript. We have made changes according to your suggestions on lines 13-15 in P1.

2. Line 23 - it is not 5 doses of nitrogen, but 4, because "N0" means lack of nitrogen - i.e. control

Ans: We have revised according to your suggestion on line 21 in P1.

Introduction

3. The chapter is quite well written, but there are occasional shortcuts and frequent repetitions of the same thread

Ans: Thanks for your nice suggestions.

4. Line 39 – In my opinion, this is an unfortunate statement: “Nitrogen serves as a key element that limits crop growth and yields…..” Nitrogen increases……

Ans: Thank you for your comments on our manuscript. We have made changes according to your suggestions on line 36 in P1.

5. Line 43 – The authors wrote: "these crops can obtain nitrogen" plants do not obtain nitrogen, only symbiotic bacteria

Ans: Thank you for your comments on our manuscript. We have made changes according to your suggestions on line 40 in P1.

Materials and methods

6. Line 94 - Is it a good statement: "the experimental field" since these are pot studies, not field experimental studies?

Ans: We had revision and corrected on line 98 in P3.

Statistical analysis - Duncan's test was used, which in my opinion is quite mild for such research.

Ans: Thank you for your positive comments on our manuscript.

Results

7. Tables and figures quite well prepared.

However, it is quite difficult to read due to the lack of spaces between means ± standard deviation

In my opinion, there are so many comas’ places in table 2 unnecessarily: in the Root length and Root surface area columns. Real numbers would be enough: e.g. "1234" instead of "1234.12", and again Root average diameter - there should be 3 numbers after the decimal point "0.123" instead of "0.12"

Ans: Thanks for your nice suggestions. We have revised according to your suggestion on line 182 in P5.

8. This chapter is generally correct, however:

The authors show significant differences, but this is not always statistically proven.

Line 152, 191: In my opinion, there should be no clear differences if you can't prove something. This is the case with the description of Figs 1 and 2 in the supplement. They point out differences, but these are visual differences - and we know that eyesight can fail.

Ans: Thanks for your nice suggestions. We have deleted according to your suggestion on line 247, 167.

9. Line 242-247 Unnecessarily, the discussion of Results included elements that should have been included in the Chapter Discussion

Ans: Thanks for your nice suggestions. We have revised according to your suggestion on line 296 in P13.

10. Line 333 - The unusual sound of "Fig. 7 shows that…..”

Ans: Thanks for your nice suggestions. We have revised according to your suggestion on line 333 in P15.

Conclusion

11. The first statement is too general and not very relevant to these results.

Lack of results is also a result - maybe some other conclusions?

Ans: Thank you for your comments on our manuscript. We have made changes according to your suggestions on line 436-440 in P19.

Please see attachment for the new manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

Some comments:

1. In my opinion is better in Keywords to use words that are different from the Title.

2. Check the style of references

3. The Conclusions need to be more detailed

4. The Material and Methods section is well written

5. The sections Results and Discussion are described in detail.

Author Response

Dear Editors and Reviewers,

Firstly, we would like to express our sincere appreciation to reviewers for reviewing our manuscript. Thank you very much for spending your valuable time to provide these insightful and constructive comments on our manuscript. These comments can not only help us to revise and improve our manuscript, but also have important guiding significance for our future research. We have carefully revised our manuscript entitled “Optimizing initial nitrogen application rates to improve peanut (Arachis hypogaea L.) biological nitrogen fixation” (Manuscript ID: agronomy-2696735). We have tried our best to improve the quality of our manuscript and made the necessary corrections in the new version. The revised portions in the new version were all highlighted by using red-colored text. We also have provided our specific responses to the reviewers' professional and valuable comments. These responses are based on the results of the present study and our interpretations of the results. Point-by-point responses to the reviewers' comments are listed below this letter. We sincerely hope that our manuscript could be considered for publication in your journal.

Thank you for your kind consideration. We look forward to hearing from you.

With best wishes,

Ying Liu

Hunan Agricultural University, Changsha 410128, China

Email: [email protected]

Reviewer #3:

Some comments:

1. In my opinion is better in Keywords to use words that are different from the Title.
2. Check the style of references
3. The Conclusions need to be more detailed
4. The Material and Methods section is well written
5. The sections Results and Discussion are described in detail.

Ans: Thank you for your comments on our manuscript. We have modified the keywords and checked the style of references. The conclusions revised portions in the new version were all highlighted by using red-colored text.

Please see attachment for the new manuscript.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

None

Comments for author File: Comments.pdf

Author Response

Dear Editors and Reviewers,

Firstly, we would like to express our sincere appreciation to reviewers for reviewing our manuscript. Thank you very much for spending your valuable time to provide these insightful and constructive comments on our manuscript. These comments can not only help us to revise and improve our manuscript, but also have important guiding significance for our future research. We have carefully revised our manuscript entitled “Optimizing initial nitrogen application rates to improve peanut (Arachis hypogaea L.) biological nitrogen fixation” (Manuscript ID: agronomy-2696735). We have tried our best to improve the quality of our manuscript and made the necessary corrections in the new version. The revised portions in the new version were all highlighted by using red-colored text. We also have provided our specific responses to the reviewers' professional and valuable comments. These responses are based on the results of the present study and our interpretations of the results. Point-by-point responses to the reviewers' comments are listed below this letter. We sincerely hope that our manuscript could be considered for publication in your journal.

Thank you for your kind consideration. We look forward to hearing from you.

With best wishes,

Ying Liu

Hunan Agricultural University, Changsha 410128, China

Email: [email protected]

Reviewer #4:

1. What is the main question addressed by the research?

The inoculation in peanuts is a relatively new technique that is been adopted by farmers around the word.

2. Do you consider the topic original or relevant in the field? Does it address a specific gap in the field?

Yes, there is many doubts about inoculation in peanuts still.

3. What does it add to the subject area compared with other published material?

Despite being a vase study, the methodologies used enriched the quality of the manuscript.

4. What specific improvements should the authors consider regarding the

methodology? What further controls should be considered?

As I had commented to the editor in my review, in my opinion this type of study should be performed on the field, as there is a great interaction between the soil and nitrogen -fixing bacteria.

5. Are the conclusions consistent with the evidence and arguments presented

and do they address the main question posed?

Yes.

6. Are the references appropriate?

Yes.

7. Please include any additional comments on the tables and figures.

The quality of both is adequate

Ans: Thank you for your comments on our manuscript. Please see attachment for the new manuscript.

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

The submitted article addresses the interesting and important issue of optimizing nitrogen management in legumes. Increasing the possibility of using biological pathways for the plant to obtain this nutrient will help reduce the amount of N contributed in mineral fertilizers, which is one of the goals of sustainable agriculture.

The research conducted by the Authors has produced valuable material and I strongly recommend that it be published. However, in my opinion, the potential of the data has not been adequately exploited in the current version of the paper. The manuscript should be substantially revised to fully reflect the quality of the results obtained.

The baseline experiment was, in my opinion, well planned and conducted, and the methodological description needs few additions (these I point out directly in the manuscript). My biggest concerns are with the presentation of the results of the study, as it does not make full use of the results of the statistical analysis (analysis of variance), while it contains too many generalizations that are not grounded in this analysis. 

My suggestion is to first present the results of the analysis of variance, highlighting which of the tested sources of variation (factors and their interactions) influenced the dependent variables studied, and then to present in tables or graphs and discuss the resulting differences between the means. The results of the analysis of variance are shown for the variables in Tables 1, 2 and 3, but this information is already missing for the variables presented in Figures 1-5. The results of the analysis of variance are shown for the variables in Tables 1, 2 and 3, but this information is already missing for the variables presented in Figures 1-5. However, the data presented in the tables are difficult to interpret (it is difficult to guess what interaction of factors they represent). Many times, the trends described in the text do not correspond to the difference labels in the tables and figures. I would suggest showing the effects of the main factors in the tables, even alongside the ANOVA results, and presenting only significant interactions in the figures. I think this would make it easier to fully interpret the results.  The current version neglects to explain emerging differences between years (e.g., in root traits, nitrogenase activity) and between varieties, as well as interesting interactions. 

In my opinion, when interpreting the effect of N fertilization rates, N0 should be taken as a control treatment, and then the effects of increasing doses of N should be observed. Comparing lower doses to higher doses seems illogical to me.

I also suggest that you do not add to the presentation of changes in percentage terms each time. This only obscures the results of the statistical analysis.

In addition to the above comments, I also suggest rethinking the order in which variables are presented. In my opinion, the following order would be more logical: peanut root architecture, peanut root activity, nodulation and BNF activity, plant agronomic above-ground morphological characteristics, peanut root dry weight and aboveground dry weight, root-to-shoot ratio, peanut nitrogen content (nitrogen derived from fertilizer (Ndff), nitrogen accumulation from different nitrogen sources), correlations.

Once the interpretation of the results is properly improved, the conclusions can be expanded and refined.

Other detailed suggestions are also included directly in the manuscript.

I hope my suggestions will help Authors improve the article in terms of content and form.

Comments for author File: Comments.pdf

Author Response

Dear Editors and Reviewers,

Firstly, we would like to express our sincere appreciation to reviewers for reviewing our manuscript. Thank you very much for spending your valuable time to provide these insightful and constructive comments on our manuscript. These comments can not only help us to revise and improve our manuscript, but also have important guiding significance for our future research. We have carefully revised our manuscript entitled “Optimizing initial nitrogen application rates to improve peanut (Arachis hypogaea L.) biological nitrogen fixation” (Manuscript ID: agronomy-2696735). We have tried our best to improve the quality of our manuscript and made the necessary corrections in the new version. The revised portions in the new version were all highlighted by using red-colored text. We also have provided our specific responses to the reviewers' professional and valuable comments. These responses are based on the results of the present study and our interpretations of the results. Point-by-point responses to the reviewers' comments are listed below this letter. We sincerely hope that our manuscript could be considered for publication in your journal.

Thank you for your kind consideration. We look forward to hearing from you.

With best wishes,

Ying Liu

Hunan Agricultural University, Changsha 410128, China

Email: [email protected]

Reviewer #5:

Introduction

1. Line 48, Please see the previous sentence started with 'however' and revise one of them.

Ans: We have revised on line 42.

2. Line 92-98, Please add information on what parameters were taken as measures of root growth and biological nitrogen fixation, and that two different genotypes were evaluated.

Ans: Thanks for your nice suggestions. We have revised on line 89-93.

Materials and Methods

3. Line 98, under the experimental field pot conditions at Shandong Academy of Agricultural Sciences Station.
4. Line 101, the information on N content is needed here.
5. Line 106, for one year?
6. line 111, grammatical issue “is”?
7. line 120, please add information on plant irrigation.
8. line 122, maybe rather 'morphological' (?)
9. Line 131-132, that is, when? How many days after planting or emergence? If it took place earlier than 30 days after planting (which I think it did), it should also be described earlier.
10. Line 148, before these formulas, a proper introduction to them is necessary.
11. Line 149, please correct this subtitle.
12. Line 156, please add the number for this formula.
13. Line 158-159, please verify the linguistic correctness of these sentences.

Ans: Thanks for your nice suggestions. We have tried our best to revise and made the necessary corrections in the new version. Our manuscript and made the necessary corrections in the new version The revised portions in the new version were all highlighted by using red-colored text.

Results

14. In addition to the above comments, I also suggest rethinking the order in which variables are presented. In my opinion, the following order would be more logical: peanut root architecture, peanut root activity, nodulation and BNF activity, plant agronomic above-ground morphological characteristics, peanut root dry weight and aboveground dry weight, root-to-shoot ratio, peanut nitrogen content (nitrogen derived from fertilizer (Ndff), nitrogen accumulation from different nitrogen sources), correlations.

Ans: Thanks for your nice suggestions. We have tried our best to revise and made the necessary corrections in the new version.

15. Please see the general comments. In addition, throughout the manuscript, the analysis of significant differences should be revised because in many places the authors overgeneralize the description by writing about an increase or decrease in the values of variables when the data in the tables indicate that there are no differences between the means.

Ans: Thanks for your nice suggestions. We have tried our best to revise the analysis of significant differences and made the necessary corrections in the new version. Our manuscript and made the necessary corrections in the new version The revised portions in the new version were all highlighted by using red-colored text.

16. As it does not make full use of the results of the statistical analysis (analysis of variance), while it contains too many generalizations that are not grounded in this analysis.

Ans: Thanks for your nice suggestions. We have tried our best to revise and made the necessary corrections in the new version.

17. I would suggest showing the effects of the main factors in the tables, even alongside the ANOVA results, and presenting only significant interactions in the figures. I think this would make it easier to fully interpret the results. The current version neglects to explain emerging differences between years (e.g., in root traits, nitrogenase activity) and between varieties, as well as interesting interactions.

Ans: Thanks for your nice suggestions. We have tried our best to revise and made the necessary corrections in the new version.

Discussion

18. A large part of the work is an analysis of morphological features of aboveground plant parts, which were omitted from the Discussion.

Ans: Thanks for your nice suggestions. We have revised and made the necessary corrections on line 382-386.

19. Line 424-426, It is just that the differences between the varieties were not shown in the paper.

Ans: Thanks for your nice suggestions. We have revised and made the necessary corrections on line 416-419.

Please see attachment for the new manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

-

Author Response

Dear Editors and Reviewers,

Thank you for your comments on our manuscript. Please see attachment for the new manuscript.

 

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

Thank you for the substantial revision of the manuscript. I accept the corrections made. However, several were overlooked, and since they are important, I think they should be completed. I have marked them in the manuscript, which I am enclosing.

Comments for author File: Comments.pdf

Author Response

Dear Editors and Reviewers,

Thank you for your comments on our manuscript. Please see attachment for the new manuscript.

1. Line 92, This term must be defined. Do you mean respiratory activity of roots?

Ans: Thanks for your nice suggestions. We have revised on line 198-201.

2. Line 134, Please proofread for typos.

Line 136, Please provide first the full name.

Line 140, I suggest writing it down as equation (1).

Line 143, I suggest using the full name in the title, not the abbreviation.

Line 149, Please provide a full name first.

Line 150, Please improve the formula. It should be: Ndff = (15N atom% excess in plant sample / 15N atom% excess in fertilizer) × 100 or Ndff = 15N atom% excess in plant sample × 100/ 15N atom% excess in fertilizer.

Ans: Thanks for your nice suggestions. The revised portions in the new version were all highlighted by using purple-colored text.

3. Line 159, Please provide more information about the ANOVA conducted (what model was used, what sources of variation were examined for their effect on the dependent variables, what tests were used to verify the basic assumptions of the ANOVA). What post hoc test was used to assess differences between means? Please add also HERE the information on how the relationships (correlations) between variables were expressed. Please proofread for language accuracy.

Ans: Thanks for your nice suggestions. The revised portions in the new version were all highlighted by using purple-colored text.

Author Response File: Author Response.pdf