Seed Treatment with Calcium Carbonate Containing Bacillus amyloliquefaciens PMB05 Powder Is an Efficient Way to Control Black Rot Disease of Cabbage

Round 1

Reviewer 1 Report

The paper entitled " Seed treatment with calcium carbonate containing Bacillus amyloliquefaciens PMB05 powder is an efficient way to control black rot disease of cabbage ", has important information about  black rot disease (BRD) caused by Xanthomonas campestris pv. campestris (Xcc), which often occurs in an environment with high humidity and temperature. Also, this ms gives response to that the seed treatment with calcium carbonate containing Bacillus amyloliquefaciens PMB05 powder is efficient to control black rot disease of cabbage.

After careful reading of the ms I found it suitable for publication, since the authors used suitable and standard methods and analysis. This is a good work. The abstract abides by all the editing instructions and presents the objectives of the study. The introduction is supported by well selected bibliographic data. Also discussion is rich, since many important scientific references have been taken into consideration. I suggest to publish the ms after minor revision, since I suggest to improve the ms as follows:

Abstract

Introduction

p. 2, line 87: a period is missing at the end of the sentence

Materials and Methods

Discussion

Conclusions

- Expand part of the conclusion with one to two more sentences.  

References

Comments for author File: Comments.pdf

Author Response

Reviewer 1

The paper entitled " Seed treatment with calcium carbonate containing Bacillus amyloliquefaciens PMB05 powder is an efficient way to control black rot disease of cabbage ", has important information about  black rot disease (BRD) caused by Xanthomonas campestris pv. campestris (Xcc), which often occurs in an environment with high humidity and temperature. Also, this ms gives response to that the seed treatment with calcium carbonate containing Bacillus amyloliquefaciens PMB05 powder is efficient to control black rot disease of cabbage.

 After careful reading of the ms I found it suitable for publication, since the authors used suitable and standard methods and analysis. This is a good work. The abstract abides by all the editing instructions and presents the objectives of the study. The introduction is supported by well selected bibliographic data. Also discussion is rich, since many important scientific references have been taken into consideration. I suggest to publish the ms after minor revision, since I suggest to improve the ms as follows:

Introduction

 line 87: a period is missing at the end of the sentence

 Conclusions

- Expand part of the conclusion with one to two more sentences.

Response:

Thank you for the reviewer’s comments. The conclusion has been revised in the revised manuscript accordingly.

Reviewer 2 Report

The manuscript ID agriculture-2318612 entitled " Seed treatment with calcium carbonate containing Bacillus amyloliquefaciens PMB05 powder is an efficient way to control black rot disease of cabbage", is an interesting study. But  my  suggest it needs to  a major revision to be published in this journal. I do have some comments about the manuscript and data interpretation/discussion that could improve the overall quality of the manuscript:

1-      The large number of abbreviations made it very difficult to understand the scientific content, so that the reader in each sentence first searched for the abbreviation in order to understand the meaning of this sentence. Therefore, I suggest minimizing these abbreviations as much as possible and inserting a section for abbreviations.

2-      In this study, an artificial fertilizer containing calcium was used as a control. This is good and useful for the study to a large extent, but it was necessary to use an antibiotic as well as a control to compare the results of the study with the results of the antibiotics used. So that this title can be mentioned, and the authors described it as an effective way, how is that, and it was not compared to existing market pesticides?

3-      The authors studied the effect of the materials used in vivo only, but why was it not used in vitro until it was consistent with the title of the manuscript, and they mentioned that it controls and is not plant resistance, or is it possible to change the title so that it is consistent with the study on plants and becomes directed to resistance?

4-      Abstract: Although carefully written, it needs to reduce abbreviations as much as possible.

5-      Introduction:

 line 35: please change "Taiwan[1] ." to Taiwan[1].

Line 62: rewrite "  [26,27 ] [28] " as [26-28].

6-      Materials and Methods:

Line 100: Please revise and check this sentence " nutrient broth agar (NA) plate".

Lines 112:114: I think it has its rightful place in the discussion section.

Line 164: What do these symbols mean? And also enter the appropriate reference for this method.

Lines 172:176: Please explain the method used in detail.

7-      Results:

Revise the numbers written in Table 1.

Figure 4 needs more clearing and high resolution.

In Figure 6A, please enter an indication of the x-axis.

8-      Discussion: The discussion section needs to be supported by recent citations, as this part contains only 11 references that are already present in the introduction section.

9-      Reference:

Self-citation is very high, reaching 17.5%, and needs to be reduced.

in this manuscript,  the last five years have only 9 references, however the manuscript should be supplemented with more recent references. It is suggested that the manuscript be supplemented with such references;

doi 10.5943/ppq/9/1/3

doi.org/10.1007/s13399-023-03949-9

doi.org/10.3390/jof8080775.

Author Response

The manuscript ID agriculture-2318612 entitled " Seed treatment with calcium carbonate containing Bacillus amyloliquefaciens PMB05 powder is an efficient way to control black rot disease of cabbage", is an interesting study. But  my  suggest it needs to  a major revision to be published in this journal. I do have some comments about the manuscript and data interpretation/discussion that could improve the overall quality of the manuscript:

• The large number of abbreviations made it very difficult to understand the scientific content, so that the reader in each sentence first searched for the abbreviation in order to understand the meaning of this sentence. Therefore, I suggest minimizing these abbreviations as much as possible and inserting a section for abbreviations.

Response:

Thank you for this comment. This is good advice. After thinking about it, we have removed the abbreviation of BRD, hoping the revised manuscript will easy to read for readers.

• In this study, an artificial fertilizer containing calcium was used as a control. This is good and useful for the study to a large extent, but it was necessary to use an antibiotic as well as a control to compare the results of the study with the results of the antibiotics used. So that this title can be mentioned, and the authors described it as an effective way, how is that, and it was not compared to existing market pesticides?

Response:

Thanks for this comment. The main issue of this manuscript is to explore whether plant immunity intensifying microorganisms (Bacillus amyloliquefaciens PMB05) that can strengthen plant immune responses in calcium carbonate-added environments. At the same time, it is also explored whether making it into a powder and treating the seeds can effectively reduce the occurrence of cabbage black rot in both the greenhouse and the field. Under this framework, we therefore did not perform comparisons of antibiotic treatments. This is good advice. We will remember this point for future experiments.

As for the "it is an effective way" we mentioned, it means that using powder to treat seeds can indeed effectively reduce the occurrence of diseases, and it is also a simple way to apply on seeds.

3-      The authors studied the effect of the materials used in vivo only, but why was it not used in vitro until it was consistent with the title of the manuscript, and they mentioned that it controls and is not plant resistance, or is it possible to change the title so that it is consistent with the study on plants and becomes directed to resistance?

Response:

Thanks for this suggestion. The goal in this manuscript is to analyze the effect of calcium carbonate on the B. amyloliquefaciens PMB05 to enhance plant immunity. Plant immunity must occur in living plant cells, so in vitro analysis cannot be used in this experiment.

Regarding the relationship between disease resistance and disease control, our interpretation is that B.amyloliquefaciens PMB05 can increase the strength of immune signals in the process of induced-plant immunity, which is the “increase of plant disease resistance”. The increase of plant disease resistance can bring the reduction of subsequent disease occurrence. Therefore, the overall use of PMB05 to reduce the occurrence of diseases is the main reason we use "control".

• Abstract:Although carefully written, it needs to reduce abbreviations as much as possible.

Response:

Thanks for this good comment. We have corrected it in the revised manuscript.

5-      Introduction:

 line 35: please change "Taiwan[1] ." to Taiwan[1].

Line 62: rewrite "  [26,27 ] [28] " as [26-28].

Response:

Thanks. We have corrected the same errors in the new manuscript as well as in the full text.

6-      Materials and Methods:

Line 100: Please revise and check this sentence " nutrient broth agar (NA) plate".   nutrient agar

Lines 112:114: I think it has its rightful place in the discussion section.

Line 164: What do these symbols mean? And also enter the appropriate reference for this method.

Response:

Thanks for the reminders. We have corrected in the manuscript and added the original reference in the manuscript.

Lines 172:176: Please explain the method used in detail.

Response:

Thank you! More detail information on preparing the powder was described in the materials and methods.

7-      Results:

Revise the numbers written in Table 1.

Figure 4 needs more clearing and high resolution.

In Figure 6A, please enter an indication of the x-axis.

Response:

Thanks! These points were corrected as suggested in the revised manuscript.

8-      Discussion: The discussion section needs to be supported by recent citations, as this part contains only 11 references that are already present in the introduction section.

Response:

Thanks. This is a good suggestion we must heed. We added 7 references in the Introduction and Discussion.

9-      Reference:

Self-citation is very high, reaching 17.5%, and needs to be reduced.

in this manuscript,  the last five years have only 9 references, however the manuscript should be supplemented with more recent references. It is suggested that the manuscript be supplemented with such references;

doi 10.5943/ppq/9/1/3

doi.org/10.1007/s13399-023-03949-9

doi.org/10.3390/jof8080775.

Response:

Thanks for this comment. This manuscript is based on the topic of improving plant immune response by microorganisms. Indeed, there are few current studies, and most of the references are focus on the relevant mechanisms of plant immune response. Thank you for this kind reminder and for providing our excellent literature. These references have now been included in the revised manuscript.

Reviewer 3 Report

I thoroughly enjoyed reading the manuscript regarding the efficient use of calcium carbonate containing Bacillus amyloliquefaciens PMB05 powder in controlling black rot disease of cabbage. However, I have some concerns that require further attention.

1. The manuscript needs a more detailed introduction explaining the rationale for selecting calcium carbonate and PMB05. It would be beneficial to provide a more comprehensive explanation of how the addition of calcium carbonate in the formulation for preparing PMB05 enhances plant immune responses against black rot disease.

1. The author used fluorescence microscopy for callose deposition, and the microscopic images require a scale bar. Furthermore, a more detailed description of the methodology used would improve the manuscript.

3. The hot water method is a common and effective strategy widely used in seed disinfection in production. Soaking seeds in 45-55°C hot water can economically and effectively kill microbes that are latent or stuck to the inside and outside of seeds. Therefore, it is recommended to use hot water-treated seed as a control for this experiment.

Author Response

I thoroughly enjoyed reading the manuscript regarding the efficient use of calcium carbonate containing Bacillus amyloliquefaciens PMB05 powder in controlling black rot disease of cabbage. However, I have some concerns that require further attention.

1. The manuscript needs a more detailed introduction explaining the rationale for selecting calcium carbonate and PMB05. It would be beneficial to provide a more comprehensive explanation of how the addition of calcium carbonate in the formulation for preparing PMB05 enhances plant immune responses against black rot disease.

Response:

Thanks for the good comment. We have added the description of the importance of the calcium involved in the plant immune system. And, the point on calcium carbonate as a calcium fertilizer commonly used in the field was also added in the revised manuscript.

2. The author used fluorescence microscopy for callose deposition, and the microscopic images require a scale bar. Furthermore, a more detailed description of the methodology used would improve the manuscript.

Response:

Thanks for this reminder. We have included scale bars in the microscope pictures. At the same time, some methods are also described in more detail in the revised manuscript.

3. The hot water method is a common and effective strategy widely used in seed disinfection in production. Soaking seeds in 45-55°C hot water can economically and effectively kill microbes that are latent or stuck to the inside and outside of seeds. Therefore, it is recommended to use hot water-treated seed as a control for this experiment.

Response:

Thanks! This is a great topic. The reason we did not use hot water treatment is mainly because in many experiments we also used the bacterial suspension of Xcc as inoculum. In such cases, even hot water treatment of the seeds will have no visible control effect. Therefore, we did not design a hot water treatment in each trial.

Round 2

Reviewer 2 Report

I thank you for amending the manuscript and it remains only to avoid exaggerated self-citation

Reviewer 3 Report

no further comments