Utilization of Rhodopseudomonas palustris in Crop Rotation Practice Boosts Rice Productivity and Soil Nutrient Dynamics

Round 1

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

Synergistic Effects of Crop Rotation and Rhodopseudomonas palustris Inoculation on Rice Productivity and Soil Nutrient Dynamics

This manuscript presents a two-season field study investigating the combined impact of purple non-sulfur bacteria (PNSB) inoculation on rice growth and yield. The authors aim to investigate the potential of these practices in enhancing rice growth and yield, and also exploring the underlying mechanisms.

From the experiment setup the rotation of Djulis is introduced in both treatment for -PNSB and +PNSB, in this case, the effects of rotation are not studied. I suggest the author to revised the manuscript accordingly, for title, abstract, ...

The experiment design is not clear. From Figure 1b, we understand that there were two treatments for Djulis (-PNSB and +PNSB), which is contradicted with the text. Please make these clear.

In figure 1a, please add necessary explanations for example which plants were grown between two rice field?

Abstract

Line 21-22, “Data on the growth and yield of rice were collected, statistically analyzed, and presented as mean ± standard error” should be present in materials and methods but not in abstract.

 

It would be beneficial to explore the economic implications of adopting these practices, including potential costs and benefits for farmers.

The manuscript could benefit from a broader discussion on the scalability of the findings and potential challenges in implementing the proposed practices in different agricultural settings.

I also suggest the authors focused on the analysis of rice growth, the inclusion of the data of Djulis would cause confusion to the reader.

 

Author Response

Answers to questions from reviewer 1

This manuscript presents a two-season field study investigating the combined impact of purple non-sulfur bacteria (PNSB) inoculation on rice growth and yield. The authors aim to investigate the potential of these practices in enhancing rice growth and yield, and also exploring the underlying mechanisms.

1. From the experiment setup the rotation of Djulis is introduced in both treatment for -PNSB and +PNSB, in this case, the effects of rotation are not studied. I suggest the author to revised the manuscript accordingly, for title, abstract, ...

We appreciate your suggestion to clarify the role of djulis rotation in our experiment. We understand that the current setup does not allow for the study of the effects of rotation, as it is introduced in both -PNSB and +PNSB treatments.

In response to your comments, we have made the following revisions:

• Title: We have revised the title to more accurately reflect the focus of our study, which does not include the effects of djulis rotation.
• Abstract: We have updated the abstract to clarify the experimental setup and the role of djulis rotation. This can be seen in Lines 16-18 and Lines 26-29.
• Introduction: We have made necessary changes in the introduction section to address your concerns. These changes can be found in Lines 76-77.

We hope these revisions address your concerns and make the focus of our study clearer.

2. The experiment design is not clear. From Figure 1b, we understand that there were two treatments for Djulis (-PNSB and +PNSB), which is contradicted with the text. Please make these clear.

To clarify, in our experiment, we indeed had two treatments for djulis: one with the addition of PNSB (+PNSB) and one without (-PNSB). We have now revised Lines 118-123 to better explain the experiment design and to resolve the contradiction. The text now clearly states the two different treatments for djulis and aligns with the representation in Figure 1b. We hope this clarification addresses your concern and enhances the understanding of our experiment design.

3. In figure 1a, please add necessary explanations for example which plants were grown between two rice field?

We understand that this information is crucial for a comprehensive understanding of our experimental setup. In response to your comment, we have revised Figure 1a and added necessary explanations in Lines 107-110 of the text. The revised figure now includes labels indicating the specific types of plants that were grown between the two rice fields. The corresponding text provides further details about these plants, their growth conditions, and their relevance to our study.

Abstract

4. Line 21-22, “Data on the growth and yield of rice were collected, statistically analyzed, and presented as mean ± standard error” should be present in materials and methods but not in abstract.

We understand your concern that the sentence “Data on the growth and yield of rice were collected, statistically analyzed, and presented as mean ± standard error” in Lines 21-22 is more appropriate for the Materials and Methods section rather than the Abstract. In response to your suggestion, we have removed this sentence from the Abstract.

5. It would be beneficial to explore the economic implications of adopting these practices, including potential costs and benefits for farmers.

We appreciate your insightful suggestion. While we acknowledge that this is indeed a valuable idea, it was not within the scope of our current study. However, realizing its potential significance, we have highlighted this aspect in our conclusion as a potential direction for future research (Lines 844-848). This will ensure that subsequent studies can build upon our work and explore this avenue further.

6. The manuscript could benefit from a broader discussion on the scalability of the findings and potential challenges in implementing the proposed practices in different agricultural settings.

We agree that discussing the scalability of our findings and the potential challenges of implementing the proposed practices in various agricultural settings is crucial. We have expanded our discussion on these topics in the conclusion section (Lines 836-842). Here, we delve into the practical implications of our research, potential obstacles in diverse farming contexts, and strategies for overcoming these challenges. We believe this comprehensive discussion will provide a more holistic understanding of our work and its applicability in the real world.

7. I also suggest the authors focused on the analysis of rice growth, the inclusion of the data of Djulis would cause confusion to the reader.

We understand that the inclusion of djulis data may lead to confusion for the readers. In response to your comment, we have revised our manuscript to focus primarily on the growth of rice. Where applicable, we have removed or minimized the data related to djulis to maintain clarity and focus. This change can be seen throughout the manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Comments on the revised version titled: “Synergistic Effects of Crop Rotation and Rhodopseudomonas palustris Inoculation on Rice Productivity and Soil Nutrient Dynamics” by Sundar, L.S., Yen, K.-S., Chang, Y.-T., Chao, Y.-Y. Manuscript ID: agriculture-2967589.

 

Comments:

1. The experimental design was not clear in the distribution of treatments. The field experiment was performed with two blocks of 9 m x 21 m, although the Materials and Methods did not explain whether the PNSB treatment was separated from the control un-inoculated. The authors mentioned that the PNSB inoculation was introduced into the rice field through running water. Were both treatments separated during trials? How can an inoculation be avoided in control plants during rice crop flooding?

2. Why the enzymatic activity (ROS) was performed exclusively in Trial 1 and not in Trial 2?

3. In Fig. 5, how do you explain that PNSB inoculation promoted high temperature and electrical conductivity in soil?

4. The authors mentioned that PNSB can fix N₂ and is one of the attributes of beneficial rhizobacteria. In this sense, the N quantification in soils or in plant biomass was not performed in both trials.

 

5. In the majority of results, there is a slight difference in the PNSB treatment compared to the control plants. Although the weather conditions in both trials were not determinants of the experiments, the authors did not provide evidence for any technique for detecting PNSB in roots or soil from inoculated rice treatments in both trials. The rhizobacteria detection is important to validate all results.

Author Response

Answers to questions from reviewer 2

1. The experimental design was not clear in the distribution of treatments. The field experiment was performed with two blocks of 9 m x 21 m, although the Materials and Methods did not explain whether the PNSB treatment was separated from the control un-inoculated. The authors mentioned that the PNSB inoculation was introduced into the rice field through running water. Were both treatments separated during trials? How can an inoculation be avoided in control plants during rice crop flooding?

Thank you for your question regarding the experimental design. We understand that the distribution of treatments and the separation of the PNSB treatment from the control were not clearly explained in the Materials and Methods section. Our field experiment was indeed conducted in two separate blocks, each measuring 9 m x 21 m. These blocks were separated by a ridge that was 1 meter wide to prevent any leakage or cross-contamination between the two areas. The fields were also arranged on a slightly slopy area, with the higher field designated as the control and the lower one as the treatment field. This arrangement ensured that any runoff from the control field would not contaminate the treatment field, thereby preventing any accidental inoculation of control plants during the flooding process. The PNSB inoculation was introduced into the rice field through running water, but only in the treatment field through its own water inlet pipe. The control field was not subject to this inoculation. We have now added these clarifications in Lines 91-97 and Lines 165-167 of our manuscript.

2. Why the enzymatic activity (ROS) was performed exclusively in Trial 1 and not in Trial 2?

Thank you for your question regarding the enzymatic activity analysis. In our study, the analysis of antioxidant enzymes was conducted exclusively in Trial 1 as a measure to ensure that no stress or bias was influencing our results. Upon finding no significant differences in this analysis, we decided not to repeat it in Trial 2. Instead, in Trial 2, we focused on analyzing the soil conditions, which we believed would provide additional valuable insights into our study. This decision was based on the results and learnings from Trial 1. We have now provided a more detailed explanation of this methodology in Lines 221-224 and Lines 252-258 of our manuscript.

3. In Fig. 5, how do you explain that PNSB inoculation promoted high temperature and electrical conductivity in soil?

We understand that the observed increase in soil temperature and EC following PNSB inoculation might suggest a direct effect of the PNSB treatment. However, this is not the case. The changes in soil temperature and EC were analyzed to ensure that the underground conditions were conducive for rice growth in both fields. This was an alternative approach to the antioxidant enzyme activity analysis conducted in Trial 1. The observed variations in soil temperature and EC were primarily due to environmental fluctuations and other treatments applied, rather than the PNSB inoculation itself. We have now clarified this point in Lines 253-258 of our manuscript. We appreciate your attention to detail, and we hope this addresses your query.

4. The authors mentioned that PNSB can fix N₂and is one of the attributes of beneficial rhizobacteria. In this sense, the N quantification in soils or in plant biomass was not performed in both trials.

We acknowledge that quantifying N in soils or plant biomass directly would have provided a more definitive measure of N fixation by PNSB. However, in our study, we opted to use the SPAD 502 leaf chlorophyll meter as a proxy for N content in plants. The SPAD meter measures the relative chlorophyll content, which has been widely recognized as an indirect indicator of plant N status. This is because chlorophyll molecules have N in their structure, and an increase in chlorophyll content often reflects an increase in N content. To further substantiate our approach, we have supplemented our manuscript with additional information and references (Lines 793-800) that support the use of SPAD readings as a reliable indicator of plant N content. We believe this method, while not direct, still provides valuable insights into the N-fixing capabilities of PNSB. We appreciate your suggestion and will consider direct N quantification in future studies to provide a more comprehensive understanding of the role of PNSB as beneficial rhizobacteria.

5. In the majority of results, there is a slight difference in the PNSB treatment compared to the control plants. Although the weather conditions in both trials were not determinants of the experiments, the authors did not provide evidence for any technique for detecting PNSB in roots or soil from inoculated rice treatments in both trials. The rhizobacteria detection is important to validate all results.

We acknowledge that direct detection of PNSB in roots or soil from inoculated rice treatments would have provided a more definitive validation of our results. However, in our study, we adopted an alternative approach. We measured the content of ALA in leaf tissues, as certain PNSB species such as R. palustris have been shown to enhance the production of ALA. When these bacteria are applied to plants, they can boost the ALA content in leaf tissues. This serves as an indirect indication of the presence and activity of PNSB. In addition, we measured the relative chlorophyll content, which is indicative of leaf N content. This measurement can also indirectly demonstrate the presence of PNSB through their N-fixing ability. We have added further clarifications and supporting references to these methods in Lines 260-261, Lines 407-410, and Lines 793-800 of our manuscript. However, we will certainly consider incorporating such techniques in our future research to further validate our findings.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (New Reviewer)

Comments and Suggestions for Authors

The authors have revised the 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

Synergistic Effects of Crop Rotation and Rhodopseudomonas palustris Inoculation on Rice Productivity and Soil Nutrient Dynamics

 In this paper, the authors have explored the utilization of Crop Rotation techniques and the application of Rhodopseudomonas palustris Inoculation as means to enhance Rice Productivity and Soil Nutrient Dynamics. However, upon reviewing the paper, several shortcomings have been identified:

 

 1. In the abstract, the authors state that rice grain yield increased by 104%, grains per hill by 103%, and so forth. Upon scrutinizing the data, it has become evident that these parameters have been miscalculated.

 

 2. The field experiment setup, design, degrees of freedom, and the relationship between Trial 1 and Trial 2 were not clearly elucidated. Additionally, the authors should incorporate initial soil data values for comprehensive understanding.

 

 3. Figure No. 2 appears to be superfluous to the paper's main objectives.

 

 4. In Figure 4, a comparison of sunshine hours between the two trials is presented, which seems unrelated to the research focus.

 

 5. Table 1 compares -PNSB and +PNSB treatments, raising questions about the maintenance of degrees of freedom in this type of analysis. Moreover, with only two treatments, the application of DMRT (Duncan's Multiple Range Test) warrants clarification.

 

 6. The data representation in Figure 5 is flawed, lacking X-axis legends. Furthermore, comparisons between trials conducted under different growing ecologies require homogeneity assessment. The labeling in the DMRT is also perplexing.

 

 7. The accuracy of percentage calculations for grain yield and other parameters needs verification.

 

 8. The manuscript overlooks the role of Djulis in rice crop cultivation, which should be addressed for a comprehensive understanding of the research findings.

Comments on the Quality of English Language

The article need extensive English Language improvement. I found several grammatical errors. 

Author Response

Response to comments from reviewer 1

Dear reviewer,

Thank you very much for your valuable comments and suggestions on our manuscript. We have made the changes to the manuscript based on your comments and suggestions. Here is a brief of what changes we have made.

Comments and Suggestions for Authors

1. In the abstract, the authors state that rice grain yield increased by 104%, grains per hill by 103%, and so forth. Upon scrutinizing the data, it has become evident that these parameters have been miscalculated.

The enhancement in rice grain yield and the number of grains per hill, as mentioned in the abstract, has been meticulously calculated. The formula used for this calculation is shown below with an example showing how we calculated percentage increase for grain yield. As outlined in Subsection 2.1 (Lines 106-109) and illustrated in Figure 1b, the "+PNSB" in Trial 2 refers to the combination of PNSB with crop rotation. This is compared with a control group, as highlighted in the abstract.                                                                    

% increase= (new value- original value / original value) x 100

Grain yield increase = 【(11.3-5.54) /5.54】x 100=104%

2. The field experiment setup, design, degrees of freedom, and the relationship between Trial 1 and Trial 2 were not clearly elucidated. Additionally, the authors should incorporate initial soil data values for comprehensive understanding.

In Subsection 2.1 (Lines 91-112), both the first and second paragraphs detail the experimental setup and design, including the degrees of freedom. This section also elucidates the relationship between Trial 1 and Trial 2. Complementing this, Figure 1b visually represents the setup and design of the current study. As for the initial soil information, it is mentioned in the first paragraph of Subsection 2.1 (Lines 91-94) that the field used for this study was divided into two distinct fields. One field was treated with PNSB, while the other was not. Given this division, the initial soil fertility would have been identical for both fields. Including initial soil information might lead to confusion among readers due to this uniformity. However, the soil results from Trial 1 without PNSB treatment can serve as a reference for initial soil conditions. This is because Trial 1 was a continuation of the existing monocropping practices for rice, thereby reflecting the initial state of the soil.

3. Figure No. 2 appears to be superfluous to the paper's main objectives.

Figure 2 has been omitted in response to the feedback received.

4. In Figure 4, a comparison of sunshine hours between the two trials is presented, which seems unrelated to the research focus.

In terms of comprehension and practical knowledge, the duration of sunlight plays a pivotal role in determining its influence on the growth of the rice crop, as opposed to the treatment (PNSB). The significance of sunlight duration has been underscored in Section 4.1 of our discussion (Lines 552-557). Furthermore, we have hypothesized a correlation between reduced plant height and sunlight duration in the second trial (Lines 685-687).

5. Table 1 compares -PNSB and +PNSB treatments, raising questions about the maintenance of degrees of freedom in this type of analysis. Moreover, with only two treatments, the application of DMRT (Duncan's Multiple Range Test) warrants clarification.

We, the authors, are deeply grateful for your insightful comments. We acknowledge that an error occurred during the typing process. Please be assured that this has been rectified in the updated version of the manuscript (Lines 309-310).

6. The data representation in Figure 5 is flawed, lacking X-axis legends.

The x-axis legend has been successfully added, as per your suggestion (Line 317). As for the y-axis, it represents multiple variables, each with distinct units. To maintain clarity, we have indicated the specific unit for each variable in parentheses directly on the y-axis.

7. Furthermore, comparisons between trials conducted under different growing ecologies require homogeneity assessment. The labeling in the DMRT is also perplexing

A homogeneity test between the trials is not feasible due to the distinct treatments within each trial, both with and without PNSB. Specifically, Trial 1 was conducted without a rotation effect, while Trial 2 incorporated this effect. Given that all four treatments are unique, we employed a one-way ANOVA for mean comparison, and utilized DMRT for mean separation.

8. The accuracy of percentage calculations for grain yield and other parameters needs verification.

To the best of our understanding, the calculation of percentage increase is performed using the following formula:

% increase= (new value- original value / original value) x 100

9. The manuscript overlooks the role of Djulis in rice crop cultivation, which should be addressed for a comprehensive understanding of the research findings.

The significance of Djulis as a rotational crop is elaborated upon in the fifth paragraph of the Introduction section (Line 70-78). Further discussion on its importance can be found in the first and second paragraphs of Subsection 2.1 (Line 102-105).

Comments on the Quality of English Language

The article needs extensive English Language improvement. I found several grammatical errors.

To enhance the linguistic quality of our manuscript, we have sought the expertise of a native English speaker. They have meticulously reviewed and refined the language used throughout the article.

We, the authors of this manuscript, express our gratitude for the insightful comments provided by the reviewer. We have diligently revised the article in response to these remarks, thereby improving both its quality and readability.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I read the manuscript carefully and found it to be interesting and in line with the theme of this journal. The design of the manuscript is also correct, for which I appreciate the experimental design diagrams as well as the mechanistic diagrams provided by the authors. I think the manuscript needs to be taken care of before it is accepted for publication:

1) purple non-sulfur bacteria (PNSB) needs to be given a clear species name (Rhodopseudomonas palustris), and whether to use purple non-sulfur bacteria or the photosynthetic bacterium Rhodopseudomonas palustris (L58) ? I think the authors need to think about. 

2) Why does an increase in ALA mean an enhanced photosynthetic rate? Although ALA is an intermediate in chlorophyll synthesis, the authors have actually measured the relative chlorophyll content (SPAD). If the relationship is consistent, it can only mean that an increase in ALA promotes chlorophyll formation, which implies an enhanced photosynthetic rate. The authors also need to expand a little on ALA in their discussion.

3) I think the authors need to pay attention to the principle of "one China". In many places, the author uses Taiwan (not a country) without adding China, which is obviously in violation of international rules. I consider the editors to check carefully. For example, L72, L91, L200, etc.  ROC shoudl correct to China or the People's Republic of China (PRC).

4) Figure 2 does not make any sense. Suggest deletion.

5) How do the authors determine soil fertility variables? It needs to be presented in Materials and Methods.

6) Reference citations need to comply with journal requirements, e.g. L219, L223, L226 corresponding to references 52, 53 and 54 should remove the year. Full text checking.

7) ppm in Figure 5 has been internationally banned and needs to be changed to mg/kg; 5-ALA was not shown in Figure 7 to be significantly different between the two treatments at any time, which does not seem to warrant the authors' assumption of an increase, which is statistically uncontroversial. The authors need to adjust the discussion.

8) Did the significant increase in plant height in the inoculated plants originate from improved soil fertility or auxin release? An analysis needs to be given in the Discussion.

9) In the conclusion, the authors need to give the limitations of this study, such as the fact that only one year of data was replicated, is a continuous multi-year observation needed?

Author Response

Response to comments from reviewer 2

Dear reviewer,

Thank you very much for your valuable comments and suggestions on our manuscript. We have made the changes to the manuscript based on your comments and suggestions. Here is a brief of what changes we have made.

Comments and Suggestions for Authors

 

1. Purple non-sulfur bacteria (PNSB) needs to be given a clear species name (Rhodopseudomonas palustris), and whether to use purple non-sulfur bacteria or the photosynthetic bacterium Rhodopseudomonas palustris (L58) ? I think the authors need to think about. 

In this revised manuscript, we have implemented significant improvements and consistently referred to the organism of interest as "purple non-sulfur bacteria" (Line 56), thereby providing a more precise description than the generic term "photosynthetic bacterium".

2. Why does an increase in ALA mean an enhanced photosynthetic rate? Although ALA is an intermediate in chlorophyll synthesis, the authors have actually measured the relative chlorophyll content (SPAD). If the relationship is consistent, it can only mean that an increase in ALA promotes chlorophyll formation, which implies an enhanced photosynthetic rate. The authors also need to expand a little on ALA in their discussion.

For your reference, the discussion section of the manuscript, specifically lines 644-657, offers an in-depth exploration of this subject matter.

3. I think the authors need to pay attention to the principle of "one China". In many places, the author uses Taiwan (not a country) without adding China, which is obviously in violation of international rules. I consider the editors to check carefully. For example, L72, L91, L200, etc.  ROC shoudl correct to China or the People's Republic of China (PRC).

We appreciate your suggestion to use “China” or “People’s Republic of China (PRC)” when referring to the ROC. While the People’s Republic of China (PRC) is commonly known as “China” and asserts the “One China” principle, the Republic of China (ROC), commonly known as “Taiwan.” Therefore, we have chosen to use the most specific and widely accepted terms to avoid ambiguity. The article here was also consulted regarding this: https://link.springer.com/article/10.1007/s11366-012-9224-0

4. Figure 2 does not make any sense. Suggest deletion.

In response to the feedback received, Figure 2 has been duly removed from the document.

5. How do the authors determine soil fertility variables? It needs to be presented in Materials and Methods.

This information has now been added to the material and method section of the article in the last paragraph of subsection 2.1 (Lines 135-138).

6. Reference citations need to comply with journal requirements, e.g. L219, L223, L226 corresponding to references 52, 53 and 54 should remove the year. Full text checking.

Both the in-text citations and the reference list throughout the manuscript have been meticulously checked, revised and enhanced in accordance with the feedback received.

7. Ppm in Figure 5 has been internationally banned and needs to be changed to mg/kg

In response to the feedback, the unit of measurement in Figure 5 (Line 317) has been meticulously updated, replacing ‘ppm’ with ‘mg/kg’.

8. 5-ALA was not shown in Figure 7 to be significantly different between the two treatments at any time, which does not seem to warrant the authors' assumption of an increase, which is statistically uncontroversial. The authors need to adjust the discussion.

While the increase in 5-ALA content in PNSB-treated plants was not statistically significant, it was nonetheless present. Consequently, we have chosen to use the term ‘increase’ rather than ‘significant increase’ or ‘statistically significant increase’ (Lines 369-370).

9. Did the significant increase in plant height in the inoculated plants originate from improved soil fertility or auxin release? An analysis needs to be given in the Discussion.

The augmentation in the height of the plants can be primarily attributed to the improvement in soil fertility, as stated in subsection 4.3 (Lines 683-685).

10. In the conclusion, the authors need to give the limitations of this study, such as the fact that only one year of data was replicated, is a continuous multi-year observation needed?

The conclusion section of this manuscript now includes a discussion on the limitations of our study, especially based on the comments provided here (Line 727-730).

The authors of this manuscript are grateful for the reviewer’s insightful comments. In response, we have made revisions to the article, thereby improving both its quality and readability.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript titled "Synergistic Effects of Crop Rotation and Rhodopseudomonas palustris Inoculation on Rice Productivity and Soil Nutrient Dynamics" contains interesting research results for science and agricultural practice. I appreciate that this is a two-year field study. The research is cognitively important, up-to-date and multi-faceted. However, the text of the manuscript requires improvement. I included detailed comments in the original text (see PDF).

General notes:

line 8, who represents this department... 2?
write how many repetitions there were in each year
how and when soil samples were collected for analysis
describe in detail the use of PNSB (in years)
describe in detail the fertilization for rice and djulis
results were reported selectively (describe in detail the results from the first year for rice, the results for djulis and the results for rice in the second year)
Figure 6 write what the asterisk * means
line 522 I understand that the grain quality was not analyzed
place figures and tables after their first mention in the text (e.g. Fig. 12)
line 653-657 correct literature references as required by the journal
in the summary (conclusion), write carefully the results from the first and second year
correct the reference list in accordance with the journal's requirements
correct the Latin names of species in the literature list

Other comments in the original text

I hope that my comments will help the authors improve the text of the article. Thank you for your cooperation and best regards

Comments for author File: Comments.pdf

Author Response

Response to comments from reviewer 3

Dear reviewer,

Thank you very much for your valuable comments and suggestions on our manuscript. We have made the changes to the manuscript based on your comments and suggestions. Here is a brief of what changes we have made.

Comments and Suggestions for Authors

1. Line 8, who represents this department... 2?

This issue has been fixed.

2. Write how many repetitions there were in each year

Each year, we maintained two distinct fields: one field was left without PNSB inoculation, while the other was treated with PNSB. The decision not to divide these fields into smaller plots was intentional. Our main objective was to observe the impact on plant growth and yield under conditions that closely mimic the real-world scenarios faced by farmers. This approach enhances the precision of our results.

3. How and when soil samples were collected for analysis

Comprehensive details regarding the methodology and timing of soil sample collection for analysis have been incorporated into the manuscript, specifically under subsection 2.1 (Line 127-138). This addition ensures a thorough understanding of the sampling process.

4. Describe in detail the use of PNSB (in years)

This information is conveyed in the concluding sentence of the final paragraph under subsection 2.2 (Line 157-160). However, for enhanced clarity, we have revised this sentence.

5. Describe in detail the fertilization for rice and djulis

The details pertaining to the quantity of fertilizer and the strategic approach for its application, specifically for both djulis and rice crops, have been comprehensively incorporated under Subsection 2.3 (Line 162-176).

6. Results were reported selectively (describe in detail the results from the first year for rice, the results for djulis and the results for rice in the second year)

We acknowledge the significance of the djulis results, but our primary objective was to augment rice productivity through the utilization of PNSB and crop rotation with djulis. Including the djulis data, while important, could potentially lead to confusion and overload the manuscript with excessive information. Therefore, we have chosen to concentrate solely on rice, as it was the principal crop under investigation in our study.

7. Figure 6 write what the asterisk * means

An asterisk (*) denotes a significant difference. This information has been incorporated into Figure 5 (previously Figure 6) caption (Line 353).

8. Line 522 I understand that the grain quality was not analyzed

Indeed, our study did not include an analysis of grain quality, and we have accordingly excised the term “grain quality” from our manuscript. While there is evidence to suggest that PNSB can enhance the quality of various fruits and vegetables, our research was solely concentrated on the aspects of growth and yield. We intend to broaden our focus in future studies to include the quality of grains, with a particular emphasis on rice, in relation to PNSB application. We have incorporated this intent into the conclusion section of our manuscript as a direction for future research (Line 727-729).

9. Place figures and tables after their first mention in the text (e.g. Fig. 12)

We have meticulously reviewed all Figures and Tables, ensuring they are positioned immediately following their initial reference within the text.

10. Line 653-657 correct literature references as required by the journal

The in-text references have been subjected to a thorough refinement process, ensuring their absolute compliance with the journal’s specific citation guidelines.

11. In the summary (conclusion), write carefully the results from the first and second year

The concluding section of this study has been meticulously updated to encapsulate the significant findings derived from our comprehensive research (Lines 717-732).

12. Correct the reference list in accordance with the journal's requirements

All citations have been meticulously verified and refined in accordance with the ‘Reference List and Citations Style Guide for MDPI Journals.’

13. Correct the Latin names of species in the literature list

The Latin names of the species have been appropriately rectified in accordance with the appropriate standards.

The authors of this manuscript express their gratitude for the reviewer’s insightful comments and have accordingly revised the article, thereby augmenting both its quality and readability.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

 I remain unconvinced by the experimental setup and assertions put forth by the authors. While efforts were made to enhance the manuscript, the data and rationale presented remain subject to scrutiny. Notably, the authors assert that rice production doubles with the addition of PNSB, a claim that warrants further investigation given its significant implications. Moreover, the suggestion that sunlight significantly influences yield variation, particularly within the same season, raises doubts about the validity of the findings. The reported rice yield of 11.3 t/ha in trial 2 appears improbable under the circumstances described. Consequently, I am unable to endorse the manuscript for publication in its current form.

Author Response

 

Response to 2^nd set of comments from reviewer 1

1. I remain unconvinced by the experimental setup and assertions put forth by the authors.

In this study, we utilized two fields, each measuring 9 m x 21 m, which were partitioned from a single larger field. The traditional practice of monocropping rice was previously employed, leading to a 11.6% decrease in grain yield compared to specified grain yield for this variety (Lines 703-704). To counteract this, we introduced the use of PNSB to stabilize the yield, in conjunction with crop rotation practices. In the first year, we maintained the monocropping practice in both fields. However, we supplemented one field with PNSB. The introduction of PNSB resulted in a significant increase in grain yield by approximately 44.5% from suggested yield of this variety. This increase could potentially be attributed to the enhanced tiller number induced by PNSB inoculation. For the second cultivation cycle, we transplanted djulis into the same field. In the PNSB-treated field, we continued the application of PNSB. Post-harvest, we crushed and incorporated the djulis stem into the soil of both fields and allowed the land to rest for a month. In the subsequent year, we transplanted rice again and continued the application of PNSB in the same field. This time, we observed a substantial increase in yield, which could again be due to a higher tiller number (Figure 9). Additionally, the data showed an increase in per plant grain yield, suggesting that PNSB inoculation also enhances per plant productivity (Table 3). This entire setup was designed to mimic the daily challenges faced by farmers and has been approved for use. It provides a practical and effective solution to enhance crop yield and productivity.

2. While efforts were made to enhance the manuscript, the data and rationale presented remain subject to scrutiny. Notably, the authors assert that rice production doubles with the addition of PNSB, a claim that warrants further investigation given its significant implications.

After meticulous verification of all data, we can confidently affirm the accuracy of the information presented in this study. We also sought assistances from experts within the same field and they also agreed. The compelling data from two rotation practices prompted us to highlight these findings. However, we are in the process of conducting more comprehensive field trials and a deeper analysis of PNSB traits to substantiate this study.

In addition to the ongoing field trials, we are also conducting further in vitro tests on the PNSB species used in this study. Preliminary results from one of our subsequent in vitro experiments have demonstrated the remarkable potential of this PNSB species. Not only does it fix and solubilize a majority of the nutrients (including those that were not studied before), but it also enhances the production of plant growth-promoting substances such as IAA, ALA, and siderophores. This has resulted in significant growth enhancement of rice seedlings, even under acidic conditions. The data from this promising experiment will be published shortly, providing further evidence of the potential of this PNSB species in sustainable agriculture. We remain committed to our research and will continue to conduct in-depth studies to validate and expand upon these findings.

3. Moreover, the suggestion that sunlight significantly influences yield variation, particularly within the same season, raises doubts about the validity of the findings.

Despite the consistency in the season, the years of cultivation varied. The weather data for the 1^st year indicated a decrease in sunshine hours, particularly during the early stages of plant growth, suggesting limited sunlight for photosynthesis. In this study, we did not present or discuss any yield changes associated with sunlight. Instead, we highlighted the potential influence of sunshine hours on plant height (Lines 684-686). The grain yield was either directly or indirectly linked to the treatments implemented in this study.

4. The reported rice yield of 11.3 t/ha in trial 2 appears improbable under the circumstances described.

After meticulous verification of all the data and consultation with experts in this field, we can confidently affirm the accuracy of all the data, both raw and presented. The near two-fold increase in rice yield may be attributed to the augmented plant growth, a result of the synergistic efforts of PNSB and crop rotation, with a notable impact on the number of tillers (Figure 9), especially on number of productive tillers (Table 3). Furthermore, there was a marked increase in the grain yield per plant in comparison to the yields from previous cultivation years (Table 3). However, it should be noted that we conducted our study on a 9 m x 21 m field, where all management practices, including weed and pest control, fertilizer application, and disease management, were carefully performed. This could have increased the performance of PNSB, thereby enhancing growth. Moreover, we minimized pesticide use and refrained from using herbicides, which are known to affect PNSB’s overall performance. Consequently, our restrained use of these chemicals may have boosted not only the growth of the rice but also that of the PNSB, further enhancing rice growth. Additionally, in a real-world scenario, such as a commercial field, the outcomes may vary slightly or show a marginal increase. Therefore, validating these results requires application and study in actual field conditions (stated in the conclusion as further research). The primary objective of this study was to demonstrate the efficacy of PNSB under field conditions and its synergy with crop rotation practice.