Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Authors of the manuscript proposed an interesting study of the intercropping effect on the rice blast occurrence, soil metabolites, and microbiome. The Introduction section is well-written and completely describes the current knowledge in this field. Materials and methods are described in details and correspond to the purpose of the study. Results are clearly presented. Discussion section is adequate.

The study has a high fundamental value. I consider the paper can be accepted for publication after some minor revisions (see below).

 

Line 26-27: please, check the sentence since it is unclear, whether the community assembly is significantly affected by the content of metabolites, or vice versa.

Line 30: “optimal control efficiency” - control of what?

Line 31: what is LTH?

Line 184: OTU or OUT?

Line 186: what is OTU? All abbreviations excepting very common ones (like DNA, HPLS, etc.) should be explained as they appear for the first time.

Line 397: “identical identified” - please, check the sentence and correct (remove “identical”.

Fig. 6: it would be better to increase the size of this picture.

Line 428: I suggest “optimal” should be instead of “optimist”.

Line 429-430. Unclear sentence. Please, check and correct.

Fig. 8: please, check the figure caption, since it seems you lots explanation for the E part (the current E-explanation does not correspond to the figure).

Comments on the Quality of English Language

English is quite fine, though some corrections is needed. The existing small errors can be corrected at the language editing stage prior proofreading.

Author Response

Comments 1: Line 26-27: please, check the sentence since it is unclear, whether the community assembly is significantly affected by the content of metabolites, or vice versa.

Response 1: Thank Reviewer 1 for your suggestion. We have revised and cleared the sentence now in line 28-33 to: “Soil microbiome analysis indicated that the bacterial genus Nocardioides, Marmoricola, Luedemannella, and Desulfomonile were significantly enriched in HKN after intercropped, while SY63 experienced a substantial accumulation of Ruminiclostridium and Cellulomonas after intercropping. Omics-based correlation analysis showed that the community assembly of Cellulomonas and Desulfomonile were significantly affected the content of the metabolites D-Sorbitol, D-Mannitol, Quinic Acid.”

Comments 2: Line 30: “optimal control efficiency" - control of what?

Response 2: Thank Reviewer 1 for your reminder. We have revised the “control efficiency” to “rice blast-control efficiency” or “control efficiency of rice blast disease” in line 31-32.

Comments 3: Line 31: what is LTH?

Response 3: LTH is the abbreviation of a rice susceptible variety “Lijiangxintuanheigu”, which is widely used in rice disease research (now in line 33).

Comments 4: Line 184: OTU or OUT?

Response 4: We have corrected the wrong words “OUT” to “OTU” (line 204), which is the abbreviation of “operational taxonomic units”. The corresponding errors in other parts of the manuscript has been corrected thoroughly.

Comments 5: Line 186: what is OTU? All abbreviations excepting very common ones (like DNA, HPLS, etc.) should be explained as they appear for the first time.

Response 5: Thank Reviewer 1 for your advice. OTU represents the abbreviation of operational taxonomic units, and we have added the complete sentence and abbreviation in line 201.

Comments 6: Line 397: “identical identified" - please, check the sentence and correct (remove “identical”

Response 6: Thank Reviewer 1 for the advice. We have removed "identical" from this sentence (now in line 418).

Comments 7: Fig. 6: it would be better to increase the size of this picture. Line 428: l suggest “optimal" should be instead of “optimist"

Response 7: Thank Reviewer 1 for your insightful suggestion. Fig 6 (now Figure 5) has been magnified and “optimist” has been replaced by “optimal” as recommended (Line 451).

Comments 8: Line 429-430. Unclear sentence, Please, check and correct.

Fig. 8: please, check the figure caption, since it seems you lots explanation for the E part (the current E-explanation does not correspond to the figure).

Response 8: Thanks for your suggestion. We have rephrased the sentence to “The disease indexes of rice blast of HKN after 50 mM/100 mM/ 200 mM D-Mannitol application were significantly decreased, with the optimal blast-control efficiency was 51.72% when the concentration of D-Mannitol was 100 mM” in line 451-452.

We have revised the figure caption of FIGURE 8 (now Figure 7) which correspond to the number list in the figure (line 467).

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

 

The manuscript “metabolites-3172537” by  Zhu et al., presents a study about the effectiveness of intercropping of resistance and susceptible rice varieties in the management of blast disease, caused by Magnaporthe grisea, via regulating soil metabolic and microbial recruiting. Although the idea is good and presents significant findings in the field of plant pathology, specifically regarding rice blast disease, however, I have several concerns about the methodology and experimental design, as well as the logical flow of ideas, and consistency of terminology.

Major concerns

1-    Throughout the manuscript, the authors did not mention the causal agent of rice blast disease (Magnaporthe grisea). Both anamorph and teleomorph of the pathogen MUST be mentioned within the introduction section and may be within the title also.

2-    The authors mentioned that Shanyou63 is a resistant variety and Huangkenuo is a susceptible variety, however, according to Figure 2, Shanyou63 recorded up to 7.5% DI which is very high for a resistant variety. Please explain??

3-    Throughout the manuscript, several abbreviations are NOT spelled out at their first appearance. For example, PBS (Line 121), OPLS-DA (Line 158), and many more. This issue Must be revised throughout the manuscript and ALL abbreviations MUST be spelled out at their first appearance

4-    The experimental design is fuggy and confusing. Although the authors had a separate subtitle “2.1 Filed experimental design”, they failed to mention the experimental design used in this study (CRD, Split-plot, … etc). Moreover, they did not mention the number of biological replicates or, the number of technical replicates throughout the manuscript.

5-     In the section “2.11 Correlation and statistical analysis”, the authors mentioned that “The statistical analysis of soil biochemical properties and rice blast occurrence degree were evaluated by independent sample T-test and Duncan's tests and P < 0.05 was regarded as significant statistically”, which I have several concerns about.

·         T-test is not the best option here, since the authors compare four different treatments (Mono-HKN, Mono-SY63, Inter-HKN, and Inter-SY63), I believe ANOVA fits better here.

·         Likewise, Duncan's test is not also the best option, It is well known that Duncan's test is more liberal and has a higher probability of committing Type I errors (false positives), especially when the number of comparisons is large. This is because it does not control the family-wise error rate as strictly as some other tests, however, some other post-hoc tests such as Tukey's HSD are more conservative and control the family-wise error rate, reducing the likelihood of Type I errors. This makes it more reliable when making multiple comparisons. In other words, compared to Tukey's HSD, Duncan's test is less conservative, meaning it might detect more significant differences, but this could come at the cost of increased false positives.

 

Consequently, this section must be rewritten and the following MUST be mentioned:-

·      The experimental design (CRD, split-plots, … etc.),

·      Statistical analysis used (one-way ANOVA, Two-way ANOVA, something else … etc).

·      Post-hoc analysis used (HSC, LSD, Bonferroni, Duncan, Dunnett, … etc)

·      Number of biological replicates and technical replicates per treatment.

I understand that some, NOT ALL, of this information is reported somewhere in the figure captions, however, it is poorly reported and still needs to be better described in section 2.11.

6-    The statistical analysis MUST be redone using Tukey's HSD as a post-hoc analysis for pair-wise comparisons. and accordingly, the significance letter might be changed which might affect the paper's conclusion.

7-    Finally, the manuscript should be thoroughly revised and improved in terms of comprehensibility and appropriateness of references. Overall, the manuscript should be carefully and deeply revised for grammar and English use, since many minor mistakes are found throughout the whole paper.

Other concerns

Title:

The title is clear and effectively conveys the core focus of the study, however, it could be slightly rephrased for better clarity. For example, the term “Rice intercropping” is very broad and could be understood as rice intercropping with other crops. Please clarify that it is an “intercropping of resistance and susceptible rice varieties …….”

Abstract:

The abstract effectively summarizes the study's aim, methodology, and key findings; however, it has a lot of redundancy (lines 17-20). Moreover,  some sentences are overly complex and could be simplified. It misses a clear statement of the research problem and the specific objective. Moreover, it requires summarizing results more succinctly to reduce redundancy and to better emphasize the novelty of the study in the concluding sentences

Introduction:

The introduction provides a solid background on rice blast disease and sets up the context for why the study is important. However, it contains some jargon that might be confusing to readers who are not experts in the field. Moreover, the research hypothesis must be stated more clearly at the end of the introduction instead of just listing a graphical abstract (Figure 1). Finally, It does NOT clearly state the objectives of the study and the novelty of the investigation.

·      I highly recommend restructuring the “Introduction” section to follow a more logical flow: general background, specific context, knowledge gaps, research objectives

·      Accordingly, the research gap and how this study addresses it, as well as the objectives of the study and/or the main research question(s) must be rewritten to better emphasize the aim of the work.

Materials and Methods:

The methodology is detailed, providing sufficient information for reproducibility, however, it is confusing and very poor in terms of logical flow. Moreover, some parts of the methodology are described in a verbose manner, which might obscure the key steps and procedures. For example:-

·         In section “2.1 Filed experimental design” - Lines 107-110, the authors mentioned that “THREE planting patterns were set: (1) intercropping of Huangkenuo and Shangyou63 (Inter-HKN-SY63), (2) monoculture of Shangyou63 (Mono-SY63), and (3) monoculture of Huangkenuo (Mono-HKN)”, which align with figure (1), however, throughout the study the presented data from FOUR treatments (Mono-HKN, Mono-SY63, Inter-HKN, and Inter-SY63) without any explanation of how they carried on the “Inter-SY63” plots. This issue MUST be explained better.

·         The authors presented some data about the disease index (DI) and incidence rate (IR) of rice blast, however, I can not see how they read for the disease incidence and how they calculated the disease index. Regardless that the disease severity is more important than disease incidence in evaluating for rice blast, ALL this information MUST be described more clearly and in a separate section/subtitle.  

Ensure consistency in terminology, e.g., consistently referring to the stages in the same order and using the same terms throughout the manuscript

Results:

Although the results are well-prepared in terms of figures and tables, they are poorly presented and most of them, if not all, are not readable. For example,

·         Figure 1 is useless and adds no significant data to the manuscript. I recommend removing it and submitting it as a graphical abstract.

·         Figure 2C-2G:- statistical analysis must be re-done using ANOVA instead of t-test. Moreover, ensure that all statistical results are accompanied by p-values to indicate significance levels.

·         Figure 3C and 3D: both figures are useless and do not add much to the data. Figure 3A (PCA scatter-plot of 4 treatments) is enough. Moreover, I recommend adding the PCA-associated loading plot which might be more useful for the study.

·         Figures 7B, 7C, and 7D:- all panels are not readable. Moreover, statistical analysis must be redone using ANOVA instead of t-test. Moreover, ensure that all statistical results are accompanied by p-values to indicate significance levels.

·         Figures 8B – 8H: all panels are not readable. Moreover, the statistical analysis must be redone using ANOVA instead of t-test. Moreover, ensure that all statistical results are accompanied by p-values to indicate significance levels.

Discussion:

The discussion provides a comprehensive analysis of the findings and places them within the broader context of existing research, however, like the introduction, it contains some redundancy and could be more focused. I highly recommend eliminating redundant statements. For example, if a point is made earlier in the discussion, avoid repeating it later unless it's to emphasize a new insight. Moreover, several references could be merged to improve the flow of the discussion. finally, the authors should discuss the limitations of the study and potential areas for future research.

Comments on the Quality of English Language

The manuscript should be thoroughly revised and improved in terms of comprehensibility and appropriateness of references. Overall, the manuscript should be carefully and deeply revised for grammar and English use, since many minor mistakes are found throughout the whole paper.

Author Response

Major concerns

Comments 1: Throughout the manuscript, the authors did not mention the causal agent of rice blast disease (Magnaporthe grisea). Both anamorph and teleomorph of the pathogen MUST be mentioned within the introduction section and may be within the title also.

Response 1: Thank Reviewer 2 for your suggestion. We have revised the sentence to “Rice blast, caused by the fungus Magnaporthe oryzae (anamorph: Pyricularia oryzae), is a highly prevalent and devastating disease that can lead to yield losses of up to 70–80%” in section Introduction in line 42. We also have added the Latin name of pathogen Magnaporthe oryzae in the Title.

Comments 2: The authors mentioned that Shanyou63 is a resistant variety and Huangkenuo is a susceptible variety, however, according to Figure 2, Shanyou63 recorded up to 7.5% DI which is very high for a resistant variety. Please explain??

Response 2: Thank Reviewer 3 for your concern. The disease index of Shanyou63 was 3.08 and the Disease incidence rate was 7.37% according to our investigation in the field trail. To our knowledge, Shanyou63 is a traditional resistant rice variety which was breed by the breeding scientists in our country at 1960s. We think Shanyou63 performed well in the resistant evaluation even after nearly 60 years promotion.

Comments 3: Throughout the manuscript, several abbreviations are NOT spelled out at their first appearance. For example, PBS (Line 121), OPLS-DA (Line 158), and many more. This issue Must be revised throughout the manuscript and ALL abbreviations MUST be spelled out at their first appearance

Response 3: Thank Reviewer 3 for your suggestion. We have added the full name of abbreviations at their first appearance throughout the manuscript carefully, like Phosphate Buffer Saline (PBS) in line 138, Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) in line 172-173, operational taxonomic units (OTUs) in line 201, and Potato Dextrose Agar (PDA) in line 242.

Comments 4: The experimental design is fuggy and confusing. Although the authors had a separate subtitle “2.1 Filed experimental design”, they failed to mention the experimental design used in this study (CRD, Split-plot, … etc). Moreover, they did not mention the number of biological replicates or, the number of technical replicates throughout the manuscript.

Response 4: Thank Reviewer 3 for your insightful suggestion. The experimental design used in this study was a randomized complete block design (RCBD). Four treatments (planting patterns) were set: (1) intercropping of Huangkenuo (Inter-HKN), (2) intercropping of Shangyou63 (Inter-SY63), (3) monoculture of Shangyou63 (Mono-SY63), and (4) monoculture of Huangkenuo (Mono-HKN), with three subplots per treatment as biological replicates.

We have now revised the subtitle “2.1 Field experimental design” to “2.1 Randomized Complete Block Design (RCBD)” and included a clearer description of the design in the revised manuscript.

Comments 5: In the section “2.11 Correlation and statistical analysis”, the authors mentioned that “The statistical analysis of soil biochemical properties and rice blast occurrence degree were evaluated by independent sample T-test and Duncan's tests and P < 0.05 was regarded as significant statistically”, which I have several concerns about.

    a. T-test is not the best option here, since the authors compare four different treatments (Mono-HKN, Mono-SY63, Inter-HKN, and Inter-SY63), I believe ANOVA fits better here.

    b. Likewise, Duncan's test is not also the best option, It is well known that Duncan's test is more liberal and has a higher probability of committing Type I errors (false positives), especially when the number of comparisons is large. This is because it does not control the family-wise error rate as strictly as some other tests, however, some other post-hoc tests such as Tukey's HSD are more conservative and control the family-wise error rate, reducing the likelihood of Type I errors. This makes it more reliable when making multiple comparisons. In other words, compared to Tukey's HSD, Duncan's test is less conservative, meaning it might detect more significant differences, but this could come at the cost of increased false positives.

Consequently, this section must be rewritten and the following MUST be mentioned:- • The experimental design (CRD, split-plots, … etc.), • Statistical analysis used (one-way ANOVA, Two-way ANOVA, something else … etc). • Post-hoc analysis used (HSC, LSD, Bonferroni, Duncan, Dunnett, … etc) • Number of biological replicates and technical replicates per treatment. I understand that some, NOT ALL, of this information is reported somewhere in the figure captions, however, it is poorly reported and still needs to be better described in section 2.11.

Response 5: Thank you for your valuable feedback on the statistical analysis section. We appreciate your concerns and would like to address them as follows:

    a. Regarding the use of the T-test, we understand your point that ANOVA might be more suitable when comparing four different treatments. However, in our study, we actually performed pairwise comparisons between the treatments (Mono-HKN&Inter-HKN, Mono-SY63&Inter-SY63) rather than comparing all four treatments simultaneously. Therefore, we believe that the T-test is appropriate for our analysis as it allows us to compare two groups at a time. We have now clarified this in the revised manuscript in the section 2.11.

    b. Regarding your concern about the use of Duncan's test, we understand your point about the potential for increased Type I errors. However, we chose Duncan's test based on its widespread use in our field and its ability to detect significant differences between pairwise treatments. We are aware of the alternative post-hoc tests available, such as Tukey's HSD, but we believe that Duncan's test in pairwise comparison has been able to demonstrate the significance between treatments, and the false positive rate is very low in our analysis.

The experimental design and statistical analysis were also revised in section 2.1 and 2.11.

Comments 6: The statistical analysis MUST be redone using Tukey's HSD as a post-hoc analysis for pair-wise comparisons. and accordingly, the significance letter might be changed which might affect the paper's conclusion

Response 6: Thank you for your valuable feedback. We understand the suggestion to use Tukey's HSD as a post-hoc analysis for pair-wise comparisons. However, we would like to respectfully explain that we have chosen Duncan's tests for our analysis based on specific considerations related to our data and research design. We believe that Duncan's tests are appropriate for our study and provide valuable insights. Nonetheless, we are willing to reevaluate our choice and consider the implications of using Tukey's HSD if necessary. We will carefully assess whether the change in statistical analysis and the potential alteration of significance letters would significantly impact the paper's conclusion.

Comments 7: Finally, the manuscript should be thoroughly revised and improved in terms of comprehensibility and appropriateness of references. Overall, the manuscript should be carefully and deeply revised for grammar and English use, since many minor mistakes are found throughout the whole paper.

Response 7: Thank you for your valuable feedback. We have thoroughly revised the manuscript to enhance its comprehensibility and ensure the appropriateness of the references. The entire manuscript has been carefully reviewed for clarity and coherence, with a focus on making sure the references are up-to-date and directly relevant to the context in which they are cited. Additionally, we have undertaken a comprehensive revision to correct any grammatical errors and improve the overall English usage, ensuring that the revised manuscript meets the required standards of academic writing.

Other concerns:
Comments 8: Title:

The title is clear and effectively conveys the core focus of the study, however, it could be slightly rephrased for better clarity. For example, the term “Rice intercropping” is very broad and could be understood as rice intercropping with other crops. Please clarify that it is an “intercropping of resistance and susceptible rice varieties …….”

Response 8: Thank Reviewer 2 for your suggestion. We have revised the title to “Rice varieties intercropping induced soil metabolic and microbial recruiting to enhance the rice blast (Magnaporthe oryzae) resistance”. We think the “rice varieties intercropping” could clarify the confused meaning of intercropping with other crops.

Comments 9: Abstract:

The abstract effectively summarizes the study's aim, methodology, and key findings; however, it has a lot of redundancy (lines 17-20). Moreover, some sentences are overly complex and could be simplified. It misses a clear statement of the research problem and the specific objective. Moreover, it requires summarizing results more succinctly to reduce redundancy and to better emphasize the novelty of the study in the concluding sentences

Response 9: Thank Reviewer 2 for your suggestion. We have thoroughly revised the abstract to reduce redundancy and better described.

Comments 10: Introduction:

The introduction provides a solid background on rice blast disease and sets up the context for why the study is important. However, it contains some jargon that might be confusing to readers who are not experts in the field. Moreover, the research hypothesis must be stated more clearly at the end of the introduction instead of just listing a graphical abstract (Figure 1). Finally, It does NOT clearly state the objectives of the study and the novelty of the investigation.

    a. I highly recommend restructuring the “Introduction” section to follow a more logical flow: general background, specific context, knowledge gaps, research objectives.

    b. Accordingly, the research gap and how this study addresses it, as well as the objectives of the study and/or the main research question(s) must be rewritten to better emphasize the aim of the work.

Response 10: Thank Reviewer 2 for your suggestion. We have revised the Introduction according to your recommendation.

Comments 11: Materials and Methods:

The methodology is detailed, providing sufficient information for reproducibility, however, it is confusing and very poor in terms of logical flow. Moreover, some parts of the methodology are described in a verbose manner, which might obscure the key steps and procedures. For example:

    a. In section “2.1 Filed experimental design” - Lines 107-110, the authors mentioned that “THREE planting patterns were set: (1) intercropping of Huangkenuo and Shangyou63 (Inter-HKN-SY63), (2) monoculture of Shangyou63 (Mono-SY63), and (3) monoculture of Huangkenuo (Mono-HKN)”, which align with figure (1),however, throughout the study the presented data from FOUR treatments (Mono-HKN, Mono-SY63, Inter-HKN, and Inter-SY63) without any explanation of how they carried on the “Inter-SY63” plots. This issue MUST be explained better.

    b. The authors presented some data about the disease index (DI) and incidence rate (IR) of rice blast, however, I can not see how they read for the disease incidence and how they calculated the disease index. Regardless that the disease severity is more important than disease incidence in evaluating for rice blast, ALL this information MUST be described more clearly and in a separate section/subtitle.

Ensure consistency in terminology, e.g., consistently referring to the stages in the same order and using the same terms throughout the manuscript

Response 11: Thank Reviewer 2 for your suggestion. We have revised the confused expression of experimental treatments in section 2.1 and added the disease investigation discrimination in section 2.2.

Comments 12: Results:

Although the results are well-prepared in terms of figures and tables, they are poorly presented and most of them, if not all, are not readable. For example,

1. Figure 1 is useless and adds no significant data to the manuscript. I recommend removing it and submitting it as a graphical abstract.
2. Figure 2C-2G: - statistical analysis must be re-done using ANOVA instead of t-test. Moreover, ensure that all statistical results are accompanied by p-values to indicate significance levels.
3. Figure 3C and 3D: both figures are useless and do not add much to the data. Figure 3A (PCA scatter-plot of 4 treatments) is enough. Moreover, I recommend adding the PCA-associated loading plot which might be more useful for the study.
4. Figures 7B, 7C, and 7D:- all panels are not readable. Moreover, statistical analysis must be redone using ANOVA instead of t-test. Moreover, ensure that all statistical results are accompanied by p-values to indicate significance levels.
5. Figures 8B – 8H: all panels are not readable. Moreover, the statistical analysis must be redone using ANOVA instead of t-test. Moreover, ensure that all statistical results are accompanied by p-values to indicate significance levels.

Response 12: Thank Reviewer 2 for your suggestion. We have removed Figure 1 in the manuscript and have submitted it as a graphical abstract and we have revised Figure 3 (now Figure 2), Figure 7 (now Figure 6) and Figure 8 (now Figure 7) as your recommendation.

Comments 13: Discussion:

The discussion provides a comprehensive analysis of the findings and places them within the broader context of existing research, however, like the introduction, it contains some redundancy and could be more focused. I highly recommend eliminating redundant statements. For example, if a point is made earlier in the discussion, avoid repeating it later unless it's to emphasize a new insight. Moreover, several references could be merged to improve the flow of the discussion. finally, the authors should discuss the limitations of the study and potential areas for future research

Response 13: Thank Reviewer 2 for your suggestion. We have carefully and thoroughly revised the Discussion section to reduce the redundancy, improve the fluency and to meet your requirements.

Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The authors performed interesting and important from the practical point of view experiments to identify possible mechanisms of increasing disease resistance in susceptible variety Huangkenuo under co-cultivation with resistant variety Shanyou63. The authors showed that protease production increases in susceptible variety Huangkenuo during intercropping. In addition, intercropping enriches the soil microbiome with Cellulomonas and Desulfomonile, which positively correlates with the increase of D-Sorbitol, D-Mannitol, Quinic Acid. It is shown that Quinic Acid inhibits the growth of mycelium of Magnaporthe oryzae. 

Abstract. Please provide the full species name of M. oryzae.

Line 92 "quenced by 16S rRNA High-throughput was detected to screen the significant bacterial"

It seems that "was detected" needs to be omitted to preserve the sense of the sentence, please check this.

Lines 123-125 "Soil enzymes urease (UE), nitrate reductase (NR), and protease (Pro) activities were determined by reductase kit instructions (Nanjing Mofan Biotechnology Co., Ltd.)". It appears that instructions specific to each of the 3 kits mentioned were used, not just from the reductase kit as can be understood from the text. Please correct it.

Please specify the activity of which group of proteases was measured?

Section 2.3 Extraction of soil metabolites. 

Did you use pre-filtration of the soil supernatant before mass spectrometric analysis to get rid of proteins, for example?

Section 2.4 Quantification of soil metabolites by LC-MS/MS

Please specify the versions of the programs used? Have you changed any parameters of the used programs or used default values?

Please specify more precisely the names of the public databases used and provide literature references where possible.

Lines 170-174.Please describe the process of sequencing metagenomic DNA in sequence and in more detail: first, the preparation of genomic libraries (also indicate which kits were used), and then describe on which instruments and where the libraries were sequenced.

Section 2.7 Data optimization of soil microbes

Please specify which programs (and which parameters were changed if applicable) were used to process metagenomic sequencing data.

Please increase the small font size in the figures to make it readable.

Please check the spelling of the section title "3.7 Beneficial microorganisms were significant related to the evolved soil metabolites"

Comments on the Quality of English Language

It is necessary to check the text for the quality of the English language, in a number of places this affects the perception of meaning. 

Author Response

Serious concerns
Comments 1: Abstract. Please provide the full species name of M. oryzae

Response 1: Thank Reviewer 3 for your notice. We have revised the full species name Magnaporthe oryzae of M. oryzae in the Abstract in line 30.

Comments 2: Line 92 "quenced by 16S rRNA High-throughput was detected to screen the significant bacterial' It seems that "was detected" needs to be omitted to preserve the sense of the sentence, please check this.

Response 2: Thank Reviewer 3 for the suggestion. We have removed “was detected” in Line 101 and the complete sentence is “Metabolome sequenced by LC-MS/MS and microbiome sequenced by 16S rRNA High-throughput to screen the significant bacterial communities and functional metabolites of rice rhizosphere soil in the intercropping system.”

Comments 3: Lines 123-125 "Soil enzymes urease (UE), nitrate reductase (NR), and protease (Pro) activities were determined by reductase kit instructions (Nanjing Mofan Biotechnology Co. Ltd.)". lt appears that instructions specific to each of the 3 kits mentioned were used, not just from the reductase kit as can be understood from the text, Please correct it.

Response 3: Thank you for the insightful suggestion. We have corrected the confused sentence and it has been revised to “Soil enzymes urease (UE), nitrate reductase (NR), and acid proteases (Acp) activities were determined by reductase kit (Nanjing Mofan Biotechnology Co., Ltd.) and the manipulation was referred to the kit instructions accordingly.” in line 142-143.

Comments 4: Please specify the activity of which group of proteases was measured?

Response 4: Thank Reviewer 3 for your notice. We detected the activity of acid proteases (Acp) and we have revised the “proteases (Pro)” to “acid proteases (Acp)” in line 135.

Comments 5: Section 2.3 Extraction of soil metabolites.

Did you use pre-filtration of the soil supernatant before mass spectrometric analysis to get rid of proteins, for example?

Response 5: We have not directly employed a specific method to remove proteins, However, we utilized a 70% methanol aqueous solution as the extractant, which is capable of dissolving or denaturing proteins to a certain extent. Coupled with two centrifugation steps (12,000 rpm, 4°C), this effectively removed insoluble macromolecules, including potential protein precipitates. Additionally, the entire sample handling procedure was conducted at low temperatures to minimize enzymatic reactions and protein denaturation.

We understand that the presence of proteins can potentially interfere with certain analyses, but based on our experimental design and the target analytes, these indirect removal measures have been sufficient to ensure the accuracy and reliability of our analytical results. Of course, if subsequent research necessitates stricter protein removal protocols, we will consider incorporating more specialized protein removal methods.

Comments 6: Section 2.4 Quantification of soil metabolites by LC-MS/MS

Please specify the versions of the programs used? Have you changed any parameters of the used programs or used default values?

Please specify more precisely the names of the public databases used and provide literature references where possible.

Response 6: Thank you for your thorough review. We used the LC-ESI-MS/MS system with UPLC ExionLC AD (UPLC, ExionLC AD, https://sciex.com.cn/) and QTRAP® System (MS, QTRAP® System, https://sciex.com). No parameters were altered from the manufacturer's recommended settings except for the solvent gradient program tailored for our specific analytes. The Waters ACQUITY UPLC HSS T3 C18 column (1.8 µm, 2.1 mm*100 mm) was employed under standard conditions. For public databases, we relied on the manufacturer's compound libraries and scientific literature for compound identification, without direct use of publicly accessible databases for this specific analytical step.

Comments 7: Lines 170-174. Please describe the process of sequencing metagenomic DNA in sequence and in more detail: first, the preparation of genomic libraries (also indicate which kits were used), and then describe on which instruments and where the libraries were sequenced.

Response 7: Thank you for the positive recommendation. The process of sequencing metagenomic DNA involved two main steps: preparation of genomic libraries and sequencing. For library preparation, total microbial genomic DNA was extracted from rhizosphere soil samples using the EZNA® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer's protocol. Subsequently, the V3-V4 region of the bacterial 16S rRNA gene was amplified with specific primer pairs (338F and 806R). The PCR products were then gel-extracted using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA), purified, and quantified with a Quantus™ Fluorometer (Promega, USA). These purified amplicons were pooled in equimolar ratios to construct the metagenomic libraries. Finally, the libraries were sequenced on the Illumina NovaSeq PE250 platform (Illumina, San Diego, USA) at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China), generating pair-end reads for downstream analysis. The detail discrimination was displayed in section 2.6 in line 176.

Comments 8: Section 2.7 Data optimization of soil microbes

Please specify which programs (and which parameters were changed if applicable) were used to process metagenomic sequencing data.

Response 8: Thanks for your suggestion. We have specified the programs discriminations of Section 2.7 in the manuscript.

Comments 9: Please increase the small font size in the figures to make it readable.

Response 9: Thank you for your recommendation. We have adjusted the figures to make it readable (now Figure 6 and 7).

Comments 10: Please check the spelling of the section title "3.7 Beneficial microorganisms were significant related to the evolved soil metabolites"

Response 10: Thank you for your reminder. We have revised the section 3.7 title as “Beneficial microorganisms were significantly related to the evolved soil metabolites” in line 415.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Unfortunately, the authors failed to satisfy most, if not all, of my previous concerns about the statistical analysis

Comments on the Quality of English Language

English is better in the revised version, but still needs for improvments

Author Response

Comments 1: Unfortunately, the authors failed to satisfy most, if not all, of my previous concerns about the statistical analysis.

Response 1: Thank you for your valuable feedback on the statistical analysis section. We have carefully considered all your suggestions and made the following revisions:

    a. Adhering to your recommendations, we have conducted a revised statistical analysis utilizing ANOVA and Tukey's HSD method, replacing the previously employed T-test and Duncan's test. This reevaluation, conducted at a significance level of P<0.05, encompassed the incidence of rice blast disease (Disease Index)as well as soil biochemical properties. Intriguingly, our findings indicate that the significance between the treatment pairs Mono-HKN & Inter-HKN and Mono-SY63 & Inter-SY63 remains unchanged, mirroring the outcomes from the initial statistical approach.

    b. Furthermore, we have revised the section “3.8 Correlated metabolites have significant control effects on rice blast” and conducted a thorough reanalysis. Gratifyingly, the significance results have remained consistent, unaltered from our previous assessment.

    The statistical analysis methods were revised in section 2.11 and we have redrawn Figure 2 (now Figure 1), Figure 7 (now Figure 6), Figure 8 (now Figure 7).

It's our earnest endeavor to ensure that the revisions made to the manuscript aptly address your concerns and contribute to enhancing the overall quality and clarity of our manuscript.

Comments 2: English is better in the revised version, but still needs for improvements.

Response 2: Thank Reviewer 2 for encouragements. We have thoroughly revised the manuscript to reduce redundancy and better described. We appreciate your continued support and guidance in this process.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors answered all questions thoroughly and corrected the manuscript significantly. I have no further comments. I suggest that the revised manuscript be accepted for publication in the journal.

Author Response

Thank you very much for your kind and comprehensive review of our manuscript. We are delighted to hear that we have been able to thoroughly address all of your questions and make significant improvements to the manuscript. 

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

Thanks for revisiting your statistical analysis. However, section 2.11 needs to be revised. the authors mentioned that "ANOVA was used to pairwise comparisons between the treatments Mono-HKN, Mono-SY63, Inter-HKN, and Inter-SY63 and Tukey's HSD as a post-hoc tests was used to analyze the significance between pairings".

ANOVA, or Analysis of Variance, is a test used to determine differences between treatments/groups, whereas Tukey's honestly significant difference test (Tukey's HSD) tests differences among sample means for significance. in other words, Tukey's HSD tests all pairwise differences.

please, Double check your statment.

 

Author Response

Comments: Thanks for revisiting your statistical analysis. However, section 2.11 needs to be revised. The authors mentioned that "ANOVA was used to pairwise comparisons between the treatments Mono-HKN, Mono-SY63, Inter-HKN, and Inter-SY63 and Tukey's HSD as a post-hoc tests was used to analyze the significance between pairings" ANOVA, or Analysis of Variance, is a test used to determine differences between treatments/groups, whereas Tukey's honestly significant difference test (Tukey's HSD) tests differences among sample means for significance. in other words, Tukey's HSD tests all pairwise differences. please. Double check your statement

Response: Thank you for your valuable feedback to our work. We have carefully made some minor revisions of section 2.11 in line 284-288 to clarify the statistical analysis of ANOVA and Tukey's HSD. We have also replaced Figure 1, 6, 7 to revise the figure caption and some wrong letters in the column, which were not affect the results. We have modified and added some descriptions of the results in Section 3.8. We are sincerely committed to ensuring that the revisions to our manuscript accurately address your concerns and contribute positively to elevating the overall quality of our work.