Genetic and Cellular Basis of Resistance to Black Rot Caused by Xanthomonas campestris pv. campestris in Brassica rapa

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript titled "Inoculation tests, genetic studies and cytological analyses of resistance to black rot in Brassica rapa" presents a comprehensive study combining germplasm screening, genetic analysis, and cytological characterization to elucidate resistance mechanisms to Xanthomonas campestris pv. campestris (Xcc) in Brassica rapa. The topic is timely and relevant given the economic importance of Brassica crops and the challenge posed by black rot.

The current title is accurate and informative, but it could be more concise, specific, and engaging. While it lists the three major methodological components (inoculation, genetic analysis, cytology), it does not emphasize the novelty or key findings of the study—namely, the identification of resistant lines, the inheritance pattern, or the discovery of autophagy-associated responses. Suggested revised titles: “Resistance to Black Rot (Xanthomonas campestris) in Brassica rapa: Screening, Genetic Inheritance, and Cytological Insights” or “Genetic and Cellular Basis of Resistance to Xanthomonas campestris (black rot) in Brassica rapa”

In the Introduction (lines 50–58), several studies are cited regarding resistance to black rot in “cabbage.” However, the specific species (Brassica oleracea, Brassica rapa, or others) are not always clearly indicated. Since B. rapa is the focal species in this study and resistance mechanisms may differ across Brassica genomes (A, B, C), it is important to specify the species referenced in each case to avoid confusion. 

In line 90, the paragraph begins with the abbreviation "B. rapa." As a general rule in scientific writing, the genus name should never be abbreviated at the beginning of a new paragraph or sentence. Please write Brassica rapa in full here. Be sure to check for similar instances throughout the manuscript.

Lines 95–102, starting with “We identified 11 highly resistant accessions…”, include results (number of resistant accessions, cytological findings, etc.), which should not be in the Introduction. These lines should be moved to the Results or at least rephrased in the Discussion if intended to preview major findings.

The paragraph in lines 90–94 does attempt to state the objectives, but it could be made clearer and more direct. I recommend rewriting it to highlight the research gap and clearly list the threefold aim of the study: (1) screening for resistance sources in B. rapa, (2) genetic analysis, and (3) ultrastructural characterization of resistant and susceptible lines.

The subsection titles within the Materials and Methods section would benefit from greater consistency and clarity. I recommend renaming the sections currently titled “Observation using Scanning Electron Microscopy” and “Observation using Transmission Electron Microscopy” to follow the format of the other methodological subsections (e.g., “Scanning Electron Microscopy (SEM)” and “Transmission Electron Microscopy (TEM)”, respectively). This will make the structure more uniform and easier to navigate.

In addition, the description of the embedding and sectioning procedures for TEM is currently incomplete. Please specify:

• The embedding medium used (e.g., Spurr’s resin, Epon, etc.),
• The instrument or ultramicrotome used for sectioning and the thickness of ultrathin sections (typically 60–90 nm),
• Whether post-staining was performed (e.g., uranyl acetate and lead citrate),
• And the accelerating voltage and imaging mode used with the HITACHI Regulus 8100 TEM.

Providing this information will ensure the study can be fully replicated by other researchers and meet publication standards for ultrastructural studies.

The authors should clearly indicate the equipment used to obtain each panel—whether SEM, stereomicroscope, or TEM—in the figure legend. This applies to all figures throughout the manuscript to ensure transparency and reproducibility. Specifically for Fig. 2 panel (d), the imaging technique is unclear. It appears to be a surface view using a stereomicroscope or equivalent, but the image is not sharp enough to clearly identify stomata or lesions. Please clarify the equipment used and improving the image contrast or resolution to make the structures more discernible.

The Discussion is generally consistent with the Results and provides a solid interpretation of the findings. However, a few issues should be addressed:

Cellular Responses: The contrast between autophagy in ‘E5’ and necrosis in ‘E4’ is well interpreted, but speculative claims (e.g., induction of systemic acquired resistance) should be clearly framed as hypotheses or omitted, as they are not directly demonstrated.

Figure Consistency: The number of highly resistant lines is correctly stated, but avoid suggesting any were fully immune if not supported by the data.

Limitations: Please include a brief mention of limitations—e.g., use of a single pathogen race, and the need to specify population sizes for genetic analysis.

Clarity: Avoid repetition and clearly distinguish between results from this study and insights drawn from previous literature.

The Conclusions are mostly aligned with the main findings of the study and appropriately summarize the screening of resistant accessions, the inheritance model, and the ultrastructural observations. 

Author Response

Dear Editors and Reviewers,

We sincerely appreciate your letter and the reviewers' insightful comments regarding our manuscript entitled “Inoculation tests, genetic studies and cytological analyses of resistance to black rot in Brassica rapa” (Manuscript ID: horticulturae-3644670). The constructive feedback has been invaluable in enhancing both the quality and clarity of our work, while also providing important guidance for our ongoing research. We have carefully addressed each comment and implemented all suggested revisions, which we believe have significantly improved the manuscript. Below, we provide our point-by-point responses to the reviewers' comments (presented in italics), with corresponding revisions highlighted in the manuscript.

We sincerely thank the editor and all reviewers for their valuable feedback, which we have used to improve the quality of our manuscript. The reviewers' comments are presented below in italicized font, and specific concerns have been numbered. Our responses are given in regular font, and additions to the manuscript are highlighted in red text. Based on the editor’s and reviewers' comments, we have made extensive modifications to our manuscript and supplemented additional data to strengthen our results.

Thank you again for your constructive feedback and valuable suggestions, which have greatly improved the quality of our manuscript.

Comments 1: The current title is accurate and informative, but it could be more concise, specific, and engaging. While it lists the three major methodological components (inoculation, genetic analysis, cytology), it does not emphasize the novelty or key findings of the study—namely, the identification of resistant lines, the inheritance pattern, or the discovery of autophagy-associated responses. Suggested revised titles: “Resistance to Black Rot (Xanthomonas campestris) in Brassica rapa: Screening, Genetic Inheritance, and Cytological Insights” or “Genetic and Cellular Basis of Resistance to Xanthomonas campestris (black rot) in Brassica rapa”

Reply: Thank you for pointing this out. We appreciate your second suggestion, which has been valuable. Consequently, we have made a minor modification and adopted “Genetic and Cellular Basis of Resistance to Black rot Caused by Xanthomonas campestris pv. campestris in Brassica rapa” as the new title for this article.

 

Comments 2: In the Introduction (lines 50–58), several studies are cited regarding resistance to black rot in “cabbage.” However, the specific species (Brassica oleracea, Brassica rapa, or others) are not always clearly indicated. Since B. Rapa is the focal species in this study and resistance mechanisms may differ across Brassica genomes (A, B, C), it is important to specify the species referenced in each case to avoid confusion.

Reply: Thank you for pointing this out. We agree with this comment. Therefore, we have replaced the common English names of all species in this section with their corresponding Latin scientific names; for instance, "cabbage" is now referred to as "B. oleracea". Additionally, we have highlighted the modified content in red, as shown in lines 50-57.

 

Comments 3: In line 90, the paragraph begins with the abbreviation "B. rapa." As a general rule in scientific writing, the genus name should never be abbreviated at the beginning of a new paragraph or sentence. Please write Brassica rapa in full here. Be sure to check for similar instances throughout the manuscript.

Reply: This is a valid point. We agree with this comment. Therefore, have revised "B. rapa" in line 89 to "Brassica rapa" and thoroughly reviewed the entire text. The same error in line 110 has been rectified.

 

Comments 4: Lines 95–102, starting with “We identified 11 highly resistant accessions…”, include results (number of resistant accessions, cytological findings, etc.), which should not be in the Introduction. These lines should be moved to the Results or at least rephrased in the Discussion if intended to preview major findings.

Reply: We appreciate your question. These sentences have been removed from the introduction.

 

Comments 5: The paragraph in lines 90–94 does attempt to state the objectives, but it could be made clearer and more direct. I recommend rewriting it to highlight the research gap and clearly list the threefold aim of the study: (1) screening for resistance sources in B. rapa, (2) genetic analysis, and (3) ultrastructural characterization of resistant and susceptible lines.

Reply: We appreciate your suggestions. This study outlines the problems to be addressed and proposes solutions to clarify the objectives and significance of the research. Modified the position to lines 89-99. Below, I will provide a brief revised version for your convenience in reviewing our modifications: “(1) comprehensive screening of resistant germplasm across diverse genetic resource is lacking; (2) the inheritance patterns governing Xcc resistance are unresolved; (3) subcellular defense mechanisms, particularly autophagy-mediated responses, in resistant genotypes are poorly understood.”

 

Comments 6: The subsection titles within the Materials and Methods section would benefit from greater consistency and clarity. I recommend renaming the sections currently titled “Observation using Scanning Electron Microscopy” and “Observation using Transmission Electron Microscopy” to follow the format of the other methodological subsections (e.g., “Scanning Electron Microscopy (SEM)” and “Transmission Electron Microscopy (TEM)”, respectively). This will make the structure more uniform and easier to navigate.

Reply: We acknowledge your feedback and have revised lines 150 and 160, highlighted in red for clarity.

 

Comments 7: In addition, the description of the embedding and sectioning procedures for TEM is currently incomplete. Please specify:

• The embedding medium used (e.g., Spurr’s resin, Epon, etc.),
• The instrument or ultramicrotome used for sectioning and the thickness of ultrathin sections (typically 60–90 nm),
• Whether post-staining was performed (e.g., uranyl acetate and lead citrate),
• And the accelerating voltage and imaging mode used with the HITACHI Regulus 8100 TEM.

Providing this information will ensure the study can be fully replicated by other researchers and meet publication standards for ultrastructural studies.

The authors should clearly indicate the equipment used to obtain each panel—whether SEM, stereomicroscope, or TEM—in the figure legend.

This applies to all figures throughout the manuscript to ensure transparency and reproducibility. Specifically for Fig. 2 panel (d), the imaging technique is unclear. It appears to be a surface view using a stereomicroscope or equivalent, but the image is not sharp enough to clearly identify stomata or lesions. Please clarify the equipment used and improving the image contrast or resolution to make the structures more discernible.

Reply: We appreciate your suggestions and fully concur with them. Our embedding medium consists of spurr’s resin and has a section thickness of 80nm. The ultramicrotome utilized is the Leica EMUZ7, with uranyl acetate and lead citrate for staining. Accordingly, we have elaborated on the procedures for scanning electron microscopy and transmission electron microscopy. These modifications are highlighted in red, located lines 150-170. Furthermore, figure 2d presents an image of a leaf captured with a standard camera, illustrating the lesions induced by the pathogen following its invasion through the stomata.

 

Comments 8: The Discussion is generally consistent with the Results and provides a solid interpretation of the findings. However, a few issues should be addressed:

Cellular Responses: The contrast between autophagy in ‘E5’ and necrosis in ‘E4’ is well interpreted, but speculative claims (e.g., induction of systemic acquired resistance) should be clearly framed as hypotheses or omitted, as they are not directly demonstrated.

Reply: Thank you for pointing this out. We appreciate your feedback. We have revised this sentence to clarify that the SAR arises from our speculation, rather than being a conclusion derived from our experiments. Please refer to lines 370-372 for this modification.

 

Comments 9: Figure Consistency: The number of highly resistant lines is correctly stated, but avoid suggesting any were fully immune if not supported by the data.

Reply: Thank you for pointing this out, but I think there may be a misunderstanding. Our description in lines 177-178 of the original text indicates that we did not identify materials that are completely immune.

 

Comments 10: Limitations: Please include a brief mention of limitations—e.g., use of a single pathogen race, and the need to specify population sizes for genetic analysis.

Reply: We appreciate your suggestions. Race1 is the predominant race worldwide. Therefore, the selected highly resistant materials are applicable to a wide range of cultivation areas. The genetic analysis of this study was based on phenotypic data from 18 P₁ plants, 17 P₂ plants, 23 F₁ plants, and 200 F₂ plants. Additionally, we have included Figure S2 to respond to your inquiries about population size.

 

Comments 11: Clarity: Avoid repetition and clearly distinguish between results from this study and insights drawn from previous literature.

Reply: I appreciate your thorough review of my manuscript. I have implemented revisions in specific sections to differentiate the findings of this study from those reported in the existing literature. The modifications are found in lines 361-363.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses resistance to black rot in Brassica rapa, analyzing inoculation tests, genetic studies and cellular mechanisms for the development of resistant varieties. The subject is relevant, and the study was well designed and presented. Therefore, I recommend the study for publication, with minor suggestions to improve the text.

Please check that the format of citations complies with the journal's standards.

Use short sentences, whenever possible.

Line 167: “analysis”

Materials and Methods

Please describe with more details how the genetic analysis was carried out.

Were the plants inoculated with a toothpick or a needle?

I suggest replacing the word “combat” to talk about the resistance response of the genotypes.

Figure 1: add “highly resistant” and “highly susceptible” to the top of the figure panel, so that these terms do not repeat in the figure title.

Line 293: please correct the sentence “cavities formed had formed”

The Discussion section is very well presented.

I congratulate the authors for their work and the editor for considering the manuscript for publication. It has become increasingly difficult to publish the results of studies on plant breeding for resistance to pests and pathogens, which often takes years to complete and even then is not adequately valued. Yet, plant resistance to pests remains the most sustainable pest control method, both environmentally and financially.

Comments on the Quality of English Language

Please review the text carefully to correct grammatical errors, typos and improve the flow of the text, using shorter sentences, whenever possible.

Author Response

Dear Editors and Reviewers,

We sincerely appreciate your letter and the reviewers' insightful comments regarding our manuscript entitled “Inoculation tests, genetic studies and cytological analyses of resistance to black rot in Brassica rapa” (Manuscript ID: horticulturae-3644670). The constructive feedback has been invaluable in enhancing both the quality and clarity of our work, while also providing important guidance for our ongoing research. We have carefully addressed each comment and implemented all suggested revisions, which we believe have significantly improved the manuscript. Below, we provide our point-by-point responses to the reviewers' comments (presented in italics), with corresponding revisions highlighted in the manuscript.

We sincerely thank the editor and all reviewers for their valuable feedback, which we have used to improve the quality of our manuscript. The reviewers' comments are presented below in italicized font, and specific concerns have been numbered. Our responses are given in regular font, and additions to the manuscript are highlighted in red text. Based on the editor’s and reviewers' comments, we have made extensive modifications to our manuscript and supplemented additional data to strengthen our results.

Thank you again for your constructive feedback and valuable suggestions, which have greatly improved the quality of our manuscript.

 

Comments 1: Please check that the format of citations complies with the journal's standards.

Reply: Thank you for your suggestions. We have rechecked the citation format of all references in the manuscript to ensure it complies with the journal's requirements.

 

Comments 2: Use short sentences, whenever possible.

Reply: We appreciate your suggestions. We have divided several long sentences into shorter ones, particularly in lines 45-48, and the revised content is highlighted in red.

 

Comments 3: Line 167: “analysis”

Reply: Thank you very much for your detailed review of my paper; this typo has been corrected, located in line 119.

 

Comments 4: Please describe with more details how the genetic analysis was carried out.

Reply: We appreciate your bringing this issue to our attention. The modifications are presented in lines 142-149. You will clearly know from the revised version how we select the most suitable model, what the minimum significant statistical value should be for the best model in the good-fit-test, and how to calculate genetic parameters.

Comments 5: Were the plants inoculated with a toothpick or a needle?

Reply: We appreciate your inquiry. Sterilized toothpicks were used for inoculation, and we have revised the sentence in line 176 to eliminate potential misunderstandings.

 

Comments 6: I suggest replacing the word “combat” to talk about the resistance response of the genotypes.

Reply: Thank you for your suggestion. We have used "combat" twice in the entire text, specifically in lines 176 and 195, and have replaced them with “to”.

 

Comments 7: Figure 1: add “highly resistant” and “highly susceptible” to the top of the figure panel, so that these terms do not repeat in the figure title.

Reply: We concur with your assessment. We marked “HR” and “HS” on highly resistant materials and highly susceptible materials in Figure 1, respectively, and explained in the title that they represent 'highly resistant' and “highly susceptible”.

Comments 8: Line 293: please correct the sentence “cavities formed had formed”

Reply: Thank you for pointing that out. We have corrected this sentence, and the modifications are located between lines 291 and 293. We used “accompanied by cavity formation” replace “cavities formed had formed”.

Author Response File: Author Response.pdf