Inoculation with Bradyrhizobium elkanii Reduces Nitrogen Fertilization Requirements for Pseudalbizzia niopoides, a Multipurpose Neotropical Legume Tree

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript effectively highlights the potential benefits of rhizobial inoculation in reducing nitrogen fertilization needs, which has significant implications for sustainable forestry and agroforestry practices. The methodologies are robust, featuring well-defined experimental designs and sound statistical analyses. However, several areas require additional clarification to strengthen the manuscript before it can be accepted.

1. The introduction effectively provides background on legume-rhizobia symbiosis, but it would benefit from a clearer statement of the novelty of this study. It is suggested to explicitly state the gap in knowledge regarding P. niopoides and its specific symbiotic relationship with B. elkanii.
2. The rationale for selecting B. elkanii SEMIA 6432 should be elaborated further. How does its symbiotic efficiency compare to other known rhizobial strains for related tree species?
3. The duration of 350 days in nursery cultivation is quite long. Were there any signs of nutrient depletion in the peat-based substrate, and could this have influenced results?
4. The interaction effects between nitrogen fertilization and inoculation need clearer discussion. Specifically, was there any evidence of a threshold beyond which additional nitrogen inhibited nodulation? 
5. The study does not assess how native soil microbial communities influence rhizobial establishment and function. Future research should examine competitive interactions between inoculated and native rhizobia. 
6. There should be more discussion on practical applications for forestry and nursery management. Are there specific recommendations for nitrogen application rates based on the results?
7. Line 60: Clarify the statement on P. niopoides being used in medicine. What aspects are being studied?
8. Line 100: Define “capillarity method” for nutrient solution application or provide a reference.

Author Response

Thank you for your time in reviewing our manuscript, and the effort to write these valuable insights for improving the comprehension of the text. We hope that the new version of the manuscript will clarify the issues expressed in the review. The attachment give the same questions.

1 - The introduction effectively provides background on legume-rhizobia symbiosis, but it would benefit from a clearer statement of the novelty of this study. It is suggested to explicitly state the gap in knowledge regarding P. niopoides and its specific symbiotic relationship with B. elkanii.

Response: Thank you for this comment. We have improved the Introduction with information about B. elkanii and P. niopoides within the context of the NFB symbiosis. This can be seen in lines 76-79; 82-86.

2 - The rationale for selecting B. elkanii SEMIA 6432 should be elaborated further. How does its symbiotic efficiency compare to other known rhizobial strains for related tree species?

Response: the motivation to use this strain was due to its already known ability to improve legume tree species and being recommended for its use. We added a clearer rationale in the lines 91-93. In addition, a new paragraph concerning the Bradyrhizobium genus in the introduction give a background to this choice (lines 54-60).

3 - The duration of 350 days in nursery cultivation is quite long. Were there any signs of nutrient depletion in the peat-based substrate, and could this have influenced results?

Response: Yes, this time was quite long when we compare it with other seedlings species. Notwithstanding, some native species have a lower growth rate during the nursery production, which seems to be the case during our experiment. No signs of nutrient depletion or toxicity in the peat substrate were noted, except the toxicity by higher N rates treatments, which was already expected.

4 - The interaction effects between nitrogen fertilization and inoculation need clearer discussion. Specifically, was there any evidence of a threshold beyond which additional nitrogen inhibited nodulation? 

Response: We included additional discussion about this topic in lines 297-300. Overall, the nodulation becomes inhibited from the 250 mg L^-1 N rate, and in a stronger way from 500 mgL^-1.

5 - The study does not assess how native soil microbial communities influence rhizobial establishment and function. Future research should examine competitive interactions between inoculated and native rhizobia.

Response: We agree with your comment, this is true particularly in the post-planting experiment, where we used natural soil collected from the region, which certainly contained unknown resident rhizobia population. We added a paragraph addressing this topic in lines 263-270.

6 - There should be more discussion on practical applications for forestry and nursery management. Are there specific recommendations for nitrogen application rates based on the results?

Response: In order to provide a conservative N fertilization approach, but not compromise the nodulation, a N rate up to 500 mg L^-1 in nutrient solution would be applicable (highlighted in line 330). We also made a discussion about the practical usefulness of the inoculation in the lines 335-339.

7 - Line 60: Clarify the statement on P. niopoides being used in medicine. What aspects are being studied?

Response: Clarified in the reviewed version, lines 71-73. The leave’s extract was studied against cancer cells, the results being positive.

8 - Line 100: Define “capillarity method” for nutrient solution application or provide a reference.

Response: We have added a detailed, concise description of the method, lines 115-117. The capillarity method consisted in submerging the base of the tubes in the nutrient solution, allowing capillarity action to transport moisture up to the substrate surface.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study provides valuable insights into the potential of Bradyrhizobium elkanii inoculation to reduce nitrogen fertilization requirements in Pseudalbizzia niopoides seedlings, contributing to sustainable agroforestry and reforestation efforts. The research is well-structured, featuring a robust experimental design, and clear presentation of results. Notably, the findings demonstrate enhanced root proliferation and plant growth, particularly at lower nitrogen rates, underscoring the benefits of biological nitrogen fixation.

After minor revisions, I recommend this article for publication.

The following suggestions are provided to enhance the manuscript:

1. The figure legend (Figure 1) should be more self-explanatory to assist readers. It is advisable to improve them.
2. The text in Figure 2 is difficult to read, and the overall resolution is too low for effective visualization.
3. In Figure 4b - Example of root nodules in an inoculated plant, you can highlight the root nodules for better understanding.
4. In the methods section, include more details about the statistical analysis, specifying the tests employed and the significance thresholds set.
5. There is inconsistency in font type and size across all figures. This can be improved for better uniformity.
6. In Figures 2 and 3, it is unclear whether the data is presented as Mean ± SD or Mean ± SEM.
7. While the study discusses future research, a more precise articulation of its limitations would enhance understanding.

Author Response

Dear reviewer, thank you for your time in review our manuscript and the efforts to make these relevant points in order to improve the work. We hope the new version of the manuscript will clarify the issues expressed in the review. Attached you find the pdf version of the responses.

1 - The figure legend (Figure 1) should be more self-explanatory to assist readers. It is advisable to improve them.

Response: We made a new figure layout, in order to leave the sequence of experiments more clear.

2 - The text in Figure 2 is difficult to read, and the overall resolution is too low for effective visualization.

Response: The resolution of the figure was good when we made the manuscript, maybe it could lose quality during the upload process within the .doc file. Anyway, we have edited the figure, with font standardization and a better display in the graph space.

3 - In Figure 4b - Example of root nodules in an inoculated plant, you can highlight the root nodules for better understanding.

Response: Thanks for this very good idea, especially because during the review we made a mention of the nodules in the discussion. The nodules were highlighted with arrows in the figure.

4 - In the methods section, include more details about the statistical analysis, specifying the tests employed and the significance thresholds set.

Response: We added the method of estimation used for the generalized linear models (maximum likelihood) and the significance threshold for all comparisons (α = 0.05), lines 165-168.

5 - There is inconsistency in font type and size across all figures. This can be improved for better uniformity.

Response: The figures were reviewed and edited using the journal’s font type.

6 - In Figures 2 and 3, it is unclear whether the data is presented as Mean ± SD or Mean ± SEM.

Response: Unfortunately, we missed to inform this relevant information. The vertical bars indicate the standard error of the mean (SEM). We have highlighted in the figure legends.

7 - While the study discusses future research, a more precise articulation of its limitations would enhance understanding.

Response: We agree with your comment. We made some discussion about the study’s limitations, concerning the use of non-sterile environment (lines 263-270) and a topic about the economic viability of the inoculation for seedling production (lines 335-339).

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear responsible author,

Thank you for your invitation to review this article.

Nowadays, all biologically based approaches to reducing conventional fertilizers are valuable. In this context, the contribution of this study to the scientific world is considered important. However, making the following improvements will increase the effectiveness of the study:

The title of your article promises a satisfying scientific work. However, using a dot as the final character in titles is not common. The journal editor's opinion on this matter is also important.

Nitrogen doses were mentioned in the abstract, but the inoculation method of Bradyrhizobium elkanii was not mentioned. It is a shortcoming that the abstract is too generalized without mentioning more objective conclusions.

Keywords should be given in alphabetical order.

In the introduction, the authors have mentioned topics that will enable readers to acquire certain essential knowledge.  However, the links between these topics are missing. For example, the concept of nitrogen-fixing bacteria is an old and widely known phenomenon. The authors should describe Bradyrhizobium elkanii more comprehensively within “nitrogen-fixing bacteria.” Furthermore, although some basic information about the plant material of the study, Pseudalbizzia niopoides, is provided, it is still unclear why this species was chosen and its economic importance. Also, some information on nitrogen applications in the conventional cultivation of this species should be added. In addition, the introduction should include references to previous studies on the two main topics of the article (Bradyrhizobium elkanii and Pseudalbizzia niopoides). 

The material method section should indicate the variety name, if any, of P. niopoides used in the study. If there is no registered variety, this should be indicated. There are many sources of nitrogen in agricultural applications. Why urea was used in this study should be explained. How were nitrogen doses determined? It would be useful to provide at least one standard dose of nitrogen for this plant species as a reference to conclude that the bacteria used reduced the nitrogen requirement.

The results and discussion section were written by the general plan of the article and shared clearly and understandably.

The study's references are pertinent in terms of their topic matter.

 

Author Response

Dear reviewer, we thank you for your valuable time in reading and review our manuscript. In fact, your considerations were very relevant to promote improvements on our work. We have edited your comments in form of questions and answers. We hope the new version of the manuscript will clarify the issues expressed in your review. Attached you find a pdf file of our responses, which is colourized, to facilitate the comprehension.

 

1 - The title of your article promises a satisfying scientific work. However, using a dot as the final character in titles is not common. The journal editor's opinion on this matter is also important.

Response: Thanks for remember us of this style convenience, we have removed the dot from the title.

2 - Nitrogen doses were mentioned in the abstract, but the inoculation method of Bradyrhizobium elkanii was not mentioned. It is a shortcoming that the abstract is too generalized without mentioning more objective conclusions.

Response: We added some main conclusions in the abstract (lines 27-30), in order to make it more clear about the research findings.

3 - Keywords should be given in alphabetical order.

Response: corrected in the reviewed version.

4 - In the introduction, the authors have mentioned topics that will enable readers to acquire certain essential knowledge.  However, the links between these topics are missing. For example, the concept of nitrogen-fixing bacteria is an old and widely known phenomenon. The authors should describe Bradyrhizobium elkanii more comprehensively within “nitrogen-fixing bacteria.” Furthermore, although some basic information about the plant material of the study, Pseudalbizzia niopoides, is provided, it is still unclear why this species was chosen and its economic importance.

Response: We have included a paragraph in the Introduction section addressing the genus Bradyrhizobium, going deeper to the rationale of its choice for this experiment (lines 54-60). Also, in lines 82-86 we have made a clearer statement on the choice of P. niopoides.

5 - Also, some information on nitrogen applications in the conventional cultivation of this species should be added. In addition, the introduction should include references to previous studies on the two main topics of the article (Bradyrhizobium elkanii and Pseudalbizzia niopoides).

Response: nitrogen application is done in native seedlings to ensure a proper raising of the seedlings. We added some lines (76-79) about this topic in the Introduction. Also, a paragraph about Bradyrhizobium elkanii was added (lines 54-60) and more information about P. niopoides (lines 82-86).

5 - The material method section should indicate the variety name, if any, of P. niopoides used in the study. If there is no registered variety, this should be indicated.

Response: Added in the MM section, lines 98-99. There is no commercial variety for this species. The seeds were collected from a healthy tree growing spontaneously in pasture.

6 - There are many sources of nitrogen in agricultural applications. Why urea was used in this study should be explained. How were nitrogen doses determined? It would be useful to provide at least one standard dose of nitrogen for this plant species as a reference to conclude that the bacteria used reduced the nitrogen requirement.

Response: Urea was chosen because it does not contain other nutrients than nitrogen, in order to not influence the seedling nutrition. We place the increasing N rates based on mean values of N concentration used in experimentation with other native tree species. This information was added in lines 121-124.

 

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author has thoroughly addressed all the points raised in my initial review. The enhancements in clarity and depth significantly improve both the scientific rigor and the readability of the manuscript. I commend your responsiveness to feedback and recommend that this manuscript be considered for publication.

Author Response

Thank you again for taking the time to review our changes to the manuscript. Your comments were certainly helpful in better understanding the work.