Evaluation of Bacillus subtilis Czk1 Metabolites by LC–MS/MS and Their Antifungal Potential against Pyrrhoderma noxium Causing Brow Rot Disease

Round 1

Reviewer 1 Report

Comments to the author Agriculture (2450406)

The manuscript entitled “Metabolites from Bacillus subtilis Czk1 evaluation by LC MS/MS and antifungal activity against the phytopathogen Phellinus noxius” is representing the interesting information and the author (s) performed a nice work. Overall, the manuscript is well structured; presenting novelty and authenticity of work. The results are reliable and manuscript is in accordance with the Journal’s scope. However for the improvements, some short comings in text are needed to be fixed for further improvement.

Suggested Title: Evaluation of Bacillus subtilis Czk1 Metabolites by LC‒2 MS/MS and their antifungal potential against Pyrrhoderma noxium causing brow rot disease

In abstract

Line 18: Whenever using the word “Phellinus noxius” use as Fungal pathogen/ fungi instead of phytopathogen/phytopathogenic  Phellinus noxius (need to check throughout the manuscript).

Need add some key and prominent metabolites along with ratio/quantity which showed strong inhibition towards pathogen.

Line 83-85: Shift these lines to conclusion section

Introduction

In the introduction, the author mostly discussed his own previous work about the strain. Need to improve introduction by citing some relevant work.

Material and Methods

Line 89:  Our laboratoryà add the detail of culture bank/lab/ university.

Line 106: QC?

Line 111: MS data à write down full form

Line 107: Add the detail of instrument, company model etc.

Line 112: Never start the sentence with abbreviation.

Line 125: Remove abbreviation from heading

Line 128: 12 h; the samples were rinsed thrice

Line 129: 10 min

Line 167: Add full from.

Table 3: highlight/bold the most prominent metabolite.

Discussion is very short wrt to present study. Need to add some comprehensive detailed information to validate the results of present study.

Line 318: Conclusion

In conclusion, add some future direction of work.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

What the source of the pathogen 

How you identified it 

How many replicate you used

All Figs need to improve and the legend should be explain everything in the fig 

This paper is suitable for this section "Crop protection, diseases, pests, and weeds.

 English language need to improve

I did not see that authors repeated his experiment or how many replicate they used

Most of figures need to improve even in most Fig did not mentioned what is (A, B,..) or what the meaning of bar or letter ………)

Discussion part need to improve here some new references maybe you can used for improve your discussion. If found useful, cite these recent references

Junior O.J.C.,Youssef K., Koyama R., Ahmed S., Dominguez A.R.,Mühlbeier D.T., Roberto S.R. 2019. Control of Gray Mold on Clamshell-Packaged ‘Benitaka’ Table Grapes Using Sulphur Dioxide Pads and Perforated Liners. Pathogens, 8, 271.https://doi.org/10.3390/pathogens8040271

Hadeel M. M. Khalil Bagy, Badawy F. M. Ibtesam, Eman A. A. Abou-Zaid, Badawy M. Sabah & Sallam, Nashwa, M. A (2021) Control of green mold disease using chitosan and its effect on orange properties during cold storage, Archives of Phytopathology and Plant Protection, 54 (11-12): 570-585   DOI: 10.1080/03235408.2020.1847568

Abo-Elyousr, K.A.M., Al-Qurashi, A.D. & Almasoudi, N.M. Evaluation of the synergy between Schwanniomyces vanrijiae and propolis in the control of Penicillium digitatum on lemons. Egypt J Biol Pest Control 31, 66 (2021). https://doi.org/10.1186/s41938-021-00415-4

Kashyap, A.S; Manzar, N.; Nebapure, S.M.; Rajawat, M.V.S.; Deo, M.M.; Singh, J.P.; Kesharwani, A.K.; Singh, R.P.; Dubey, S.C.; Singh, D. Unraveling Microbial Volatile Elicitors Using a Transparent Methodology for Induction of Systemic Resistance and Regulation of Antioxidant Genes at Expression Levels in Chili against Bacterial Wilt Disease. Antioxidants 2022, 11,404. https://doi.org/10.3390/antiox11020404

Mohamed I. Elsayed, Adel D. Al-Qurashi, Najeeb Marei Almasaudi and, Abo-Elyousr KAM 2022. Efficacy of some essential oils against gray mold of table grapes and their effect on fruit quality. South African Journal of Botany 146 (2022) 481_490 https://doi.org/10.1016/j.sajb.2021.11.046

Hadeel, MM. Kalil Bagy, Abo-Elyousr KAM, Abd El-Latif Hesham, Sallam, Nashwa, M. A. 2023. Development of antagonistic yeasts for controlling black mold disease of Onion. Egyptian Journal Biological Pest Control 33: 17: https://doi.org/10.1186/s41938-023-00664-5

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript "Metabolites from Bacillus subtilis Czk1 evaluation by LC‒MS/MS and antifungal activity against the phytopathogen Phellinus noxius" by Y. Liang et al. devoted to the study of novel biocontrol agent. The antifungal effect of B.subtilis strain on important agriculture pathogen was detected. The molecular basis of the effect was investigated using LC-MS metabolomics. The active antifungal compounds were identified in the culture extract. Despite the original findings and good experimental background the text is written poor and hard for perception. Guess, the manuscript could be acccepted for publication after minor revision (included extensive text editing).

Main points of criticism are marked below:

1. The antifungal action of the whole extract or culture broth could be lower or higher due to interaction of compounds in the complex mixture. Could the authors provide some evaluation of this fact. For example, if we will test the extract and mix of diacetyl and trans-2-octenoic acid (in the same concentrations like in the extract) - will the results be identical?

2. I guess the data analysis (PCA etc.) is not main and interesting for readers and could be tranferred into supplementary info.

3. Minor corrections needed:

lines 24-25: Here, the growth of P. noxium was inhibited after treatment with B. subtilis Czk1 culture 24 filtrate. - Please, rewrite. "The inhibition effect ... was detected" or something like this;

lines 27-28: Results of the LC–MS/MS analysis also confirmed that Czk1 can produce secondary metabolites - It's a strange and obvious conclusion, all bacterial strains produce a lot of secondary metabolites. Please, delete it;

line 67: with broad-spectrum antimicrobial activity - Thats not a characteristic for B. subtilis as a taxon. Please, re-formulte or delete this;

line 98: internal standard - What is the internal standard? I could not find the description anythere in the text;

line 97: eppendorf (EP)tube, - If you mean microcentrifuge tubes, please, rewrite this like "1.5 mL microcentrifuge tube". Now it's not clear what is used;

line 130: to tertiary butyl ethanol - The chemical name is incorrect;

line 139: area )were - area) were

line 318: 5. conclusion - Conclusion;

lines 326-328: The results can extend the knowledge on the use of B. subtilis Czk1 and its antifungal agents to control plant diseases. Furthermore, microorganisms and their metabolites can be used as an alternative method to reduce the field impact of biocides. - Please, rewrite for clarity.

Guess, the manuscript could be acccepted for publication after minor revision (included extensive text editing).

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 4 Report

I have some indication before accepting the paper for pubblication on Agriculture:

-Do not start a sentence with "And" (line 100, 136)

-Line 120 Please, indicate were did you buy metabolites for antifungal activities

- all over the text the binomial classification must be in Italicum

- paraghaph 3.3 Why did you tested only 2 metabolites for antifungal activity?

I found the discussion very poor, please integrate it with more examples in literature on antifungal molecules isolated by Bacillus species

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Discussion part need to improve here some new references maybe you can used for improve your discussion. If found useful, cite these recent references

Junior O.J.C.,Youssef K., Koyama R., Ahmed S., Dominguez A.R.,Mühlbeier D.T., Roberto S.R. 2019. Control of Gray Mold on Clamshell-Packaged ‘Benitaka’ Table Grapes Using Sulphur Dioxide Pads and Perforated Liners. Pathogens, 8, 271.https://doi.org/10.3390/pathogens8040271

Hadeel M. M. Khalil Bagy, Badawy F. M. Ibtesam, Eman A. A. Abou-Zaid, Badawy M. Sabah & Sallam, Nashwa, M. A (2021) Control of green mold disease using chitosan and its effect on orange properties during cold storage, Archives of Phytopathology and Plant Protection, 54 (11-12): 570-585   DOI: 10.1080/03235408.2020.1847568

Abo-Elyousr, K.A.M., Al-Qurashi, A.D. & Almasoudi, N.M. Evaluation of the synergy between Schwanniomyces vanrijiae and propolis in the control of Penicillium digitatum on lemons. Egypt J Biol Pest Control 31, 66 (2021). https://doi.org/10.1186/s41938-021-00415-4

Kashyap, A.S; Manzar, N.; Nebapure, S.M.; Rajawat, M.V.S.; Deo, M.M.; Singh, J.P.; Kesharwani, A.K.; Singh, R.P.; Dubey, S.C.; Singh, D. Unraveling Microbial Volatile Elicitors Using a Transparent Methodology for Induction of Systemic Resistance and Regulation of Antioxidant Genes at Expression Levels in Chili against Bacterial Wilt Disease. Antioxidants 2022, 11,404. https://doi.org/10.3390/antiox11020404

Mohamed I. Elsayed, Adel D. Al-Qurashi, Najeeb Marei Almasaudi and, Abo-Elyousr KAM 2022. Efficacy of some essential oils against gray mold of table grapes and their effect on fruit quality. South African Journal of Botany 146 (2022) 481_490 https://doi.org/10.1016/j.sajb.2021.11.046

Hadeel, MM. Kalil Bagy, Abo-Elyousr KAM, Abd El-Latif Hesham, Sallam, Nashwa, M. A. 2023. Development of antagonistic yeasts for controlling black mold disease of Onion. Egyptian Journal Biological Pest Control 33: 17: https://doi.org/10.1186/s41938-023-00664-5

minor types should be correct 

Author Response

Dear Reviewers:

Thank you for your comments concerning our manuscript entitled “Metabolites from Bacillus subtilis Czk1 evaluation by LC‒MS/MS and anti-fungal activity against the phytopathogen Pyrrhoderma noxium” (ID: 2450406). We have studied comment carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper.

Point1: Discussion part need to improve here some new references maybe you can used for improve your discussion. If found useful, cite these recent references

According to your suggestion, we have cited these references:

1.Kashyap, A.S.; Manzar, N.; Nebapure, S.M.; Rajawat, M.V.S.; Deo, M.M.; Singh, J.P.; Kesharwani, A.K.; Singh, R.P.; Dubey, S.C.; Singh, D. Unraveling Microbial Volatile Elicitors Using a Transparent Methodology for Induction of Systemic Resistance and Regulation of Antioxidant Genes at Expression Levels in Chili against Bacterial Wilt Disease. Antioxidants (Basel), 2022,11, 404. https://doi.org/10.3390/antiox11020404

2.Abo-Elyousr, K.A.M.; Al-Qurashi, A.D.; Almasoudi, N.M. Evaluation of the synergy between Schwanniomyces vanrijiae and propolis in the control of Penicillium digitatum on lemons. Egypt J Biol Pest Control, 2021,31, 66. https://doi.org/10.1186/s41938-021-00415-4

3.Bagy Hadeel M. M. K.; Abo-Elyousr K.A.M.; Hesham A. E. L.; Sallam Nashwa M. A. Development of antagonistic yeasts for controlling black mold disease of Onion. Egyptian Journal Biological Pest Control 2023,33,17. https://doi.org/10.1186/s41938-023-00664-5

Point2: minor types should be correct

According to your suggestion, we have revised this.

We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper.

Special thanks to you for your good comment.

Thank you and best regards.

Yours sincerely,

Yanqiong Liang

08 July 2023

Reviewer 4 Report

Accept in the present form

Author Response

Dear Reviewers:

   Thank you for your comments,We tried our best to improve the manuscript and made some changes in the manuscript. These changes will not influence the content and framework of the paper.

Special thanks to you for your good comment.

Thank you and best regards.

Yours sincerely,

Yanqiong Liang

08 July 2023