Root-Exudate-Mediated Modulation of the Rhizosphere Microbiome in Brassica juncea var. tumida During Plasmodiophora brassicae Infection

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript, titled: "Brassica juncea var. tumida regulate root exudates to alter the rhizosphere microbiome in response to Plasmodiophora brassicae infection", examines a specific form of competition in microbial communities in the soil influenced by the root exudates of the host plant. The pathogen (Plasmodiophora brassicae) isolated from symptomatic tumorous stem mustard roots was studied as well as the chemical structure of the compounds produced by the root network of a cruciferous host plant.

In the introduction chapter, it presents similar studies in sufficient detail and analyzes the open questions of the topic. The composition of the material and methods chapter is well structured and presents the applied research methods and statistical procedures in sufficient detail.

The chapter on presentation of results is well illustrated and allows an overview of the results achieved. In this manuscript, in the case of Figure 1, 2 and 4, the pictures are shifted to the left compared to the explanatory text. I suggest these be corrected. I also suggest a uniform, italicized spelling of scientific names throughout the text. This is especially true for the pathogen (P. brassicae) and the host plant (B. juncea var. tumida), which are mentioned in several places in the manuscript.

The discussion chapter is sufficiently detailed. It analyzes similar international results well and cites a sufficient number of references (45).

After making the minor corrections and additions suggested above, I recommend publishing the manuscript in the form of a scientific article.

Author Response

Comments 1: In the introduction chapter, it presents similar studies in sufficient detail and analyzes the open questions of the topic. The composition of the material and methods chapter is well structured and presents the applied research methods and statistical procedures in sufficient detail.The chapter on presentation of results is well illustrated and allows an overview of the results achieved. In this manuscript, in the case of Figure 1, 2 and 4, the pictures are shifted to the left compared to the explanatory text. I suggest these be corrected. I also suggest a uniform, italicized spelling of scientific names throughout the text. This is especially true for the pathogen (P. brassicae) and the host plant (B. juncea var. tumida), which are mentioned in several places in the manuscript.

Response1：We revised the scientific names. All revisions were highlighted during the editing process using track changes, with language enhancements implemented through DeepSeek software.

Reviewer 2 Report

Comments and Suggestions for Authors

The present article entitled “Brassica juncea var. tumida regulate root exudates to alter the rhizosphere microbiome in response to Plasmodiophora brassicae infection based on an interesting theme.

The experiment is well designed , However  article need some revision especially in English and presentation before acceptance; Here is some suggestion

-Line 97-98 “The pellet containing  high-purity resting spores was collected from the centrifuge tube bottom, re-suspended in sterile water”- it should be better to write  “The pellet containing  pure resting spores re-suspended in sterile water”

-Strains name should be in italic check  - line 206 and so on-

- Enhance the text size of all the figure in current front very hard to visulalize

- why authors have putted the alpha diversity  in supplementary  file ?, it should be better if present together with the beta diversity and put the value of significance  in the figure.

-Rephrase the  sentence  381-383 for clarity

 

Comments on the Quality of English Language

The present article entitled “Brassica juncea var. tumida regulate root exudates to alter the rhizosphere microbiome in response to Plasmodiophora brassicae infection based on an interesting theme.

The experiment is well designed , However  article need some revision especially in English and presentation before acceptance; Here is some suggestion

-Line 97-98 “The pellet containing  high-purity resting spores was collected from the centrifuge tube bottom, re-suspended in sterile water”- it should be better to write  “The pellet containing  pure resting spores re-suspended in sterile water”

-Strains name should be in italic check  - line 206 and so on-

- Enhance the text size of all the figure in current front very hard to visulalize

- why authors have putted the alpha diversity  in supplementary  file ?, it should be better if present together with the beta diversity and put the value of significance  in the figure.

-Rephrase the  sentence  381-383 for clarity

 

Author Response

Comments 1:-Line 97-98 “The pellet containing high-purity resting spores was collected from the centrifuge tube bottom, re-suspended in sterile water”- it should be better to write “The pellet containing pure resting spores re-suspended in sterile water” Response1： We revised the sentence. Comments 2:-Strains name should be in italic check - line 206 and so on- Response2： We revised them. Comments 3:- Enhance the text size of all the figure in current front very hard to visulalize Response3： The text size of figure was enlarged. Comments 4:- why authors have putted the alpha diversity in supplementary file ?, it should be better if present together with the beta diversity and put the value of significance in the figure. Response4： We agreed with reviewer. Rename 'Figure S2' as 'Figure 2,' and adjust all subsequent figure numbers sequentially. Comments 5:-Rephrase the sentence 381-383 for clarity Response 5： The sentences were revised (line 344-346). Our findings reveal that P. brassicae infection triggers significant metabolic reprogramming in tumorous stem mustard root exudates, characterized by the selective enrichment of defense-related compounds, particularly flavones and phenolic derivatives. Comments 6:The experiment is well designed , However article need some revision especially in English and presentation before acceptance; Here is some suggestion Response 6： All revisions were highlighted during the editing process using track changes, with language enhancements implemented through DeepSeek software.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Brassica juncea var. tumida regulate root exudates to alter the rhizosphere microbiome in response to Plasmodiophora brassicae infection” has been reviewed and in my opinion the findings of the manuscript are interesting and useful. Before further consideration of this manuscript, I have a few suggestions below:

-Authors need to rethink the title as the present topic is conveying the message of whole manuscript and it can be revised like “Root Exudate-Mediated Modulation of the Rhizosphere Microbiome in Brassica juncea var. tumida during Plasmodiophora brassicae Infection”. It

-The authors also need to revise the manuscript by stating explicitly the research question or hypothesis in the introduction.
-Please focus on the framing of the study around how B. juncea reprograms root exudation in response to P. brassicae, and the implications for rhizosphere microbiome assembly.

-The authors also requested to improve the flow of content in introduction, results and discussion by ensuring each section transitions logically from infection to exudate change -microbiome shift- functional implications.

-Please avoid overly dense or technical paragraphs; prioritize clarity and conciseness.

-In the result section, please avoid overinterpretation of correlation data (e.g., Spearman correlations should not imply causality without experimental validation).

-Please include heatmaps, network diagrams to link exudates and microbial taxa.

-In discussion section, please improve it by providing related findings to prior studies on clubroot or similar pathogen-host-microbiome systems. Discuss how specific metabolites may influence microbial recruitment mechanisms (e.g., signaling, antibiosis). Please also address limitations, e.g., lack of functional validation, pot vs. field conditions.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Comments 1:-Authors need to rethink the title as the present topic is conveying the message of whole manuscript and it can be revised like “Root Exudate-Mediated Modulation of the Rhizosphere Microbiome in Brassica juncea var. tumida during Plasmodiophora brassicae Infection”.

Response1：

Yes.  We agreed with you.

 

Comments 2: -The authors also need to revise the manuscript by stating explicitly the research question or hypothesis in the introduction.

Response 2：

 We revised the last paragraph of Introduction (line 48-60)

While current research has predominantly characterized microbial community responses to P. brassicae infection [19,20], critical knowledge gaps persist regarding the systematic manipulation of root exudates during pathogen-rhizosphere interactions. We hypothesize that P. brassicae infection induces metabolic reprogramming of root exudates in tumorous stem mustard, and the Pathogen-induced changes in root exudates selectively recruit beneficial microbes through chemotaxis, thereby remodeling rhizosphere microbiome structure and forming a disease-suppressive microbial community. To address these questions, we employed a dual-omics approach integrating 16S rRNA sequencing and untargeted metabolomics to characterize infection-induced metabolic shifts in tumorous stem mustard root exudates, to decipher correlated microbiome restructuring patterns, and to identify key exudate-microbiome networks associated with clubroot development. This work provides novel strategies for breeding resistant cultivars and engineering rhizosphere microbiomes, advancing sustainable disease management in cruciferous crops.

 

Comments 3:-Please focus on the framing of the study around how B. juncea reprograms root exudation in response to P. brassicae, and the implications for rhizosphere microbiome assembly.

Response3：

In the sections of materials and methods, result analysis and discussion, we have moved the content related to the changes in root exudates caused by Plasmodiophora brassicae to the beginning of the section on the changes in the root-associated microbial community.

 

 

Comments 4:-The authors also requested to improve the flow of content in introduction, results and discussion by ensuring each section transitions logically from infection to exudate change -microbiome shift- functional implications.

Response 4：

We revised the context in introduction, method, results and discussion and establish a clear cause-effect cascade: infection to exudate change -microbiome shift- functional implications.

​

Comments 5:-Please avoid overly dense or technical paragraphs; prioritize clarity and conciseness.

Response 5：

We revised the context and simplified language.

Comments 6:-In the result section, please avoid over interpretation of correlation data (e.g., Spearman correlations should not imply causality without experimental validation).

Response 6：

We revised the context (line 213-218)

Spearman's correlation analysis at the genus level revealed distinct microbial-metabolite co-occurrence patterns. In the CK group, Simkaniaceae showed strong positive correlations with 2,6-Dimethyl-7-octene-1,6-diol 8-O-glucoside, cibaric acid, and geniposidic acid (Figure 5a). In the P group, Paenibacillus was associated with multiple metabolites including 4-imidazolone-5-propionic acid and geniposidic acid (Figure 5b). While these associations suggest potential metabolic preferences, causal relationships require validation through gnotobiotic assays.

Comments 7:-Please include heatmaps, network diagrams to link exudates and microbial taxa.

Response 7：

Figure 5（heatmap） exhibited the relationship between exudates and microbial taxa at genus level.

Comments 8:-In discussion section, please improve it by providing related findings to prior studies on clubroot or similar pathogen-host-microbiome systems. Discuss how specific metabolites may influence microbial recruitment mechanisms (e.g., signaling, antibiosis). Please also address limitations, e.g., lack of functional validation, pot vs. field conditions.

Response 8：

We revised the discussion and added comparison between prior studies and our study (line 274-294).

We also discussed the mechanisms of metabolites influencing microbiome (line316-335).

Comments 9:Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Response9：

All revisions were highlighted during the editing process using track changes, with language enhancements implemented through DeepSeek software.

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

The present work brings valuable information on how host plants govern rhizomicrobiome. In order to improve your study some suggestions have been presented. As well, mandatory corrections are necessary. Here is my review:

• Title – Brassica juncea and Plasmodiophora brasicae must be italicized
• The obligatory use of italics for genera and species can be found in lines 12, 17, 18, 20, 25, 27, 31, 206, 207, 208-209, and 296.
• The word “species” – it sounds not accurate to use “species” following the genus or if you need to address number of species of a certain genus. Use “spp.” (case of line 303).
• The case of family citation, it is welcome to say “…within Brassicaceae, with notable…” in place of what is written in line 37. Avoid redundancy.
• Introduction: contextualize Brassicaceae in Chinese market, i.e., production, importance in diary diet, exportation…
• Introduction: first paragraph needs more citations (see lines 41-46). Reference #2 appears only in line 49.
• Lines 76-77: add references after the information of most studies focuses are responses of infection
• Section 2.1 – is it your methodology? Reference is absent.
• Section 2.2: same as section 2.1; in addition, greenhouse conditions for the specific plants of the study or in general?
• Line 163: please overwrite ®
• Section 2.5: use the correct IS for seconds (line 172) and minutes (lines 175 and 176)
• Line 307: remove the word “plants” after tomato
• Discussion:
  • Being a study of how host plants manipulate exudates to remodel microbiome, make comparison between the found microbiota and retrospective studies on microbial community responses in infections. What is the difference? Your dominant microbe is found in such conditions?
  • It was interesting to find salbutamol and there was no discussion on this and other metabolites.
  • During introduction the term “chemical language” is used. In the discussion section, there is one paragraph that this issue was brought again, however, the event has not been elaborated. Create a short period with references on the topic public goods and interspecies cheating.

 

Author Response

Comments 1:Title – Brassica juncea and Plasmodiophora brasicae must be italicized

Response 1：

We revised it.

 

Comments 2:The obligatory use of italics for genera and species can be found in lines 12, 17, 18, 20, 25, 27, 31, 206, 207, 208-209, and 296.

Response 2：

We revised them.

 

Comments 3:The word “species” – it sounds not accurate to use “species” following the genus or if you need to address number of species of a certain genus. Use “spp.” (case of line 303).

Response3：

We revised it.

 

Comments 4:The case of family citation, it is welcome to say “…within Brassicaceae, with notable…” in place of what is written in line 37. Avoid redundancy.

Response 4：

We revised it.

 

Comments 5:Introduction: contextualize Brassicaceae in Chinese market, i.e., production, importance in diary diet, exportation…

Response5：

We added one paragraph (line4-8 ) and added one reference.

China is the world's largest producer of Brassicaceae crops, with major cultivated varieties. As a core component of the Chinese diet, Brassicaceae vegetables account for over 30% of total vegetable intake, with per capita annual consumption exceeding 60 kg. China's Brassicaceae crop exports primarily consist of fresh products and processed goods, with key export commodities including fresh Chinese cabbage and dehydrated cabbage/cauliflower [1].

However, the sustainable production of these vital crops faces significant threats from clubroot disease.

National Development and Reform Commission/Ministry of Agriculture. National vegetable industry development plan. China Vegetables, 2012, 3:1-2

 

Comments 6:Introduction: first paragraph needs more citations (see lines 41-46). Reference #2 appears only in line 49.

Response 6：

We added one reference (line 15, NO. 2).

Xu, X.; Wu, C.; Zhang, F.; Yao, J.; Fan, L.; Liu, Z. Yao, Y. Comprehensive review of plasmodiophora brassicae: Pathogenesis, pathotype diversity, and integrated control methods. Front Microbiol. 2025, 16, 1531393.

 

Comments 7: Lines 76-77: add references after the information of most studies focuses are responses of infection

Response 7：

We added two references (line 49. NO. 19, 20).

Tian, X.; Wang, D.; Mao, Z.; Pan, L.; Liao, J. Cai, Z. Infection of plasmodiophora brassicae changes the fungal endophyte community of tumourous stem mustard roots as revealed by high-throughput sequencing and culture-dependent methods. PloS One. 2019, 14, e0214975. 470

Wang, D.; Sun, T.; Zhao, S.; Pan, L.; Liu, H. Tian, X. Physiological change alters endophytic bacterial community in clubroot of tumorous stem mustard infected by plasmodiophora brassicae. BMC Microbiol. 2020, 20, 244.

 

Comments 8: Section 2.1 – is it your methodology? Reference is absent.

Response：

We added one reference (line 65. NO. 21) about P. brassicae resting spore collection.

Wang, H.; Zhang, J.; Wang, Y.; Fang, B.; Ge, W.; Wang, X.; Zou, J. Ji, R. Transcriptome analysis of chinese cabbage infected with plasmodiophora brassicae in the primary stage. Sci Rep. 2024, 14, 26180.

 

Comments 9: Section 2.2: same as section 2.1; in addition, greenhouse conditions for the specific plants of the study or in general?

Response 9：

We added one reference (line 84. NO. 22)about P. brassicae resting spore inoculation.

Ji, R.; Zhao, L.; Xing, M.; Shen, X.; Bi, Q. Peng, S. Infection of plasmodiophora brassicae in chinese cabbage. Genetics and Molecular Research. 2014, 13, 10976-10982.

 

Comments 10:Line 163: please overwrite ®

Response 10：

We revised the sentence (line 94-96)

Ten biological replicates (individual plant roots) were subsequently immersed in 100 mL of sterile water within conical flasks (250 mL capacity).

 

Comments 11:Section 2.5: use the correct IS for seconds (line 172) and minutes (lines 175 and 176)

Response 11：

We revised it (line 103, 106, 107).

 

Comments 12:Line 307: remove the word “plants” after tomato

Response12：

We revised it.

 

 

Comments 13:Discussion:

Being a study of how host plants manipulate exudates to remodel microbiome, make comparison between the found microbiota and retrospective studies on microbial community responses in infections. What is the difference? Your dominant microbe is found in such conditions?

It was interesting to find salbutamol and there was no discussion on this and other metabolites.

During introduction the term “chemical language” is used. In the discussion section, there is one paragraph that this issue was brought again, however, the event has not been elaborated. Create a short period with references on the topic public goods and interspecies cheating.

Response13：

We analyzed the mechanism by which geniposidic acid and 4-imidazolone-5-propionic acid recruit Paenibacillus (line 316-324, 327-330), as well as the assembly pattern of public goods in microbial communities (line 332-335).

Salbutamol was excluded from detailed analysis because: No significant correlation with Paenibacillus populations, Potential environmental contamination source rather than plant biosynthesis.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have responded to all queries in the expected format and made the necessary revisions accordingly. I am happy to accept this manuscript in its current form.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

Include the abstract in the revised version.

regarding this part: "Salbutamol was excluded from detailed analysis because: No significant correlation with Paenibacillus populations, Potential environmental contamination source rather than plant biosynthesis." Was it declared? Please include.

Best regards.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf