Investigation into the Active Substance of Bacillus velezensis TRM82367 for Killing Aphis gossypii

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article "Investigation into the Active Substance of Bacillus velezensis TRM82367 for killing Aphis gossypii" discusses the use of a bacterium Bacillus velezensis to control the pest Aphid Aphis gossypii. The major concerns are the following. 

1. The authors have published a similar work [https://doi.org/10.3389/fmicb.2025.1559495]. They have used the same procedures and got similar results. Why was it not cited?

2. How does Bacillus australimaris TRM82479 published previously, compare with the current B. velezensis in controlling the aphid?

3. Add a space between number and their units throughout the manuscript.

4. Line 97 to 99, modify the sentence to use the past tense.

5. In equation (1), remove the words 'Deal with' in the denominator.

6. In equation (2), remove the words 'manage the'. Use the word 'observed' instead.

7. What was the need for partial 16S rRNA sequencing when you can get the entire 16S rRNA gene sequence from whole genome sequencing?

8. The protocol for library preparation for genome sequencing should be detailed in the methods section 2.5

9. Line 119, 'Based on the genomic prediction....', What procedure was followed for that? How many strains were sequenced?

10. Line 114, 'weighed 0.1 g'. What was weighed?

11. Line 160, change to the past tense.

12. Table 1 heading: Is it cotton strains or bacterial strains that were listed in the table?

13. Line 166- 167,  change '16Sr RNA' to '16S rRNA'.

14. What clustering algorithm was used within MEGA software to create the tree?

15. What do the authors mean by 'assembly effect'? The entire section 3.3.1 needs to be rewritten.

16. Line 183, check the correctness of '84 tRNA, 69 tRNA,....'.

17. Were the sequencing reads cleaned before assembling?

18. In Figure 2, mention the name of the bacteria and genome size in the center of the circos plot. 

19. What was the procedure followed for annotation? Was any software used for the annotation? Write a detailed procedure in mthe ethodology section.

20. The genome sequence of the strain Bacillus velezensis TRM82367 was not found in the NCBI database. The authors need to provide the NCBI accession ID. 

21. Line 260, the author Zhang Jun cited here is present in the list as reference number 20, not 19. Kindly check whether the in-text references are appropriate.

22. The words 'surfactin' and 'surfactant' were used interchangeably in this manuscript. But there is a difference between them. Authors should ensure correct usage throughout the manuscript.

23. There are only 23 in-text citations. But in the reference list, there are 25 references.

24. The mass spectrometry data can be deposited in a repository for further use by researchers.

25. The authors should have calculated ANI value using the genome sequence rather than the 16S rRNA sequence to conclude the species identity.

Minor corrections are:
1. In summary line 10, change 'have' to 'has'. Scientific names are considered as singular.
2. Line 25, change 'Aphis gossypiis' to 'Aphis gossypii'
3. Line 26, italicize 'Bacillus velezensis.'
4. Line 267, change 'its produce' to 'it produces'.
5. Delete the appendix section.

Overall, the manuscript was written in a simple and elegant way, but it misses much crucial information required to validate the experimental conclusions. The genome data should be made available in a public database. The manuscript can be revised, and it can go for one more round of review.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present a high active stain Bacillus velezensis TRM82367 isolated from Taklimakan Desert. Then tried deeply to investigate its characters on control of Aphis gossypii. The study is interesting, but there are many shortcomings need to be improved.

 

Specific Comments:

1. Simple Summary part: Line 10 to Line 14. The statement is too informal. The sentences in scientific research paper are not usually be described like that.
2. Materials and Methods part:

(1) I did not find the method(s) how the authors screened the strains.

(2) 2.5, I am afraid the authors did not conduct a genome sequencing.

(3) Statistics, Methods of data analysis did not provided.

3. Biological assay: I am afraid the surfactin homologues might not the main insecticidal active ingredient in the fermentation liquid. Because the authors just tested the activity of crude extract on Aphis gossypii while they did not separated the ingredients in the crude extract. They just analyzed the crude extract with LC-MS. The surfactin just the main ingredient in the crude extract. So I think the evidence is not sufficient.
4. Discussion part: This part is too simple. The authors did not compare their results with others’. And they did not summarize their academic contribution.

 

Minor Comments:

1. Line 25: There should add a space before “new”.
2. Line 79: There might missed a word.

Comments on the Quality of English Language

I strongly recommend the author to revise the manuscript with the help of a native English specialist before resubmit.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript provides useful information for readers. This manuscript can be considered for publication with major revisions. The authors should refer to the comments below and attached file for revisions and then I will give detailed comments.

(1) Line 26, 43, 263, 270, 315, 321, 339, 344, 363: Scientific names should be written in italics.

(2) What is LB medium? 

(3) Why did you screen the Bacillus and Streptomyces in 48h only? It was too short.

(4) The screening method needs mor detail. Remove "%" at the end of formulars 1 and 2.

(5) The discussion is too sketchy. You need to strengthen and rewrite the discussion strongly.

(6) Delete line 303-308 and line 310-312.

(7) There are 23 references in the text but 25 in the references section. The ref. #7 and #22 in the list need to be chec ked.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The article deals with a Bacillus velezensis isolate that shows insecticidal activity against Aphis gossypii. The study is interesting but requires substantial revisions regarding the introduction, methodology, results, and discussion.

Some recommendations:

Introduction

• The introduction should be improved, focusing specifically on the aphid Aphis gossypii and its economic importance;

• Regarding bacteria, discuss the Bacillus genus and mention that other species within this genus are used as bioinsecticides, such as Bacillus thuringiensis, and that Bacillus velezensis is gaining prominence as an entomopathogenic bacterium.

Objective

• Some results are mixed with the objective. I believe it would be better described as:
  “This study aimed to select and characterize bacterial isolates of Bacillus velezensis and Streptomyces sp. with insecticidal activity against A. gossypii.”

Materials and Methods

• Provide details on how the bacterial isolates were obtained, including the methodology and substrates used;

• Describe the experimental design. Was it completely randomized? How many insects were used per treatment? How many replicates? Was the experiment repeated at different times?

• Mortality was observed after 48 hours, but I suggest extending the observations to seven days after application;

• Mention that the antiSMASH website was used to search for genes related to pathogenicity;

• I strongly recommend the following article to check extraction conditions and assays with lipopeptides from Bacillus velezensis:
  https://doi.org/10.1016/j.biocontrol.2025.105888

• Detail how the lethal concentration was calculated. Was a Probit analysis used?

Results

• The statistical tests used were not mentioned in the Materials and Methods section.

Discussion

• The discussion should be expanded and improved. Several papers published this year have shown that B. velezensis has toxic effects against insects, and some of them identify the possible compounds involved in this activity.
  The authors should better explore their results and relate them to potential field applications.

Other suggestions

• The strain could be better characterized by providing morphological descriptions of the colonies and qualitative biochemical and physiological characteristics, which can be quickly obtained through laboratory tests;

• The bioassays with aphids should be better detailed, providing clearer statistical parameters and evaluating mortality over a longer period after bacterial application;

• The results are interesting and open avenues for exploring insecticidal compounds that are still uncommon in agriculture, but they need to be more thoroughly developed and described in greater detail.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,
I had asked you to submit the whole genome sequence of the bacteria to NCBI and provide the accession ID. Everybody knows very well that NCBI is currently frozen. So I asked you to provide the submission ID alone. But you have replied me with the accession ID of partial 16S rRNA sequence. Kindly amend the correction as mentioned in revision 1.

Author Response

Comment 1: I had asked you to submit the whole genome sequence of the bacteria to NCBI and provide the accession ID. Everybody knows very well that NCBI is currently frozen. So I asked you to provide the submission ID alone. But you have replied me with the accession ID of partial 16S rRNA sequence. Kindly amend the correction as mentioned in revision 1.

Response 1: In response to the reviewer's comment, the complete genome sequence has been deposited in the NCBI database under the accession number SUB15732899. This information has been added to the manuscript at lines 293-294.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors had revised the manuscript carefully. The study is interesting. All the comments had been improved. So I think it is ready to accept and publication.

Author Response

Dear Reviewer 2,     We are extremely grateful for your approval of our manuscript “Investigation into the Active Substance of Bacillus velezensis TRM82367 for killing Aphis gossypii” (Manuscript ID: biology-3939252). Your positive feedback is of great significance to us, and we would like to thank you for your careful review and recognition of our research.     We have double-checked the manuscript to ensure no minor errors exist, and the final version is ready for the journal’s subsequent processing.     Thank you again for your valuable time and support.                                                                                                                                                                                            Sincerely,                            Shiyu Wang 

Reviewer 3 Report

Comments and Suggestions for Authors

Base on explanation file that the authors uploaded, this manuscript is suitable for publication.

Best regards

Author Response

Dear Reviewer 3,     We are extremely grateful for your approval of our manuscript “Investigation into the Active Substance of Bacillus velezensis TRM82367 for killing Aphis gossypii” (Manuscript ID: biology-3939252). Your positive feedback is of great significance to us, and we would like to thank you for your careful review and recognition of our research.     We have double-checked the manuscript to ensure no minor errors exist, and the final version is ready for the journal’s subsequent processing.     Thank you again for your valuable time and support. Sincerely, Shiyu Wang 

Reviewer 4 Report

Comments and Suggestions for Authors

The article has been significantly improved, with more concrete and expanded results. In addition, the discussion has been enhanced and further developed. I only suggest a few adjustments regarding the text:

1. On line 102, correct Streptomyces

2. On line 401, italicize the scientific names mentioned

Author Response

Comment 1: On line 102, correct Streptomyces.

Response 1: Thank you for raising this question. We have made the revision at line 102 as suggested.

Comment 2:  On line 401, italicize the scientific names mentioned.

Response 2：Thank you for pointing this out. We have corrected the formatting of Fusarium, Escherichia coli, and Staphylococcus aureus to be consistent with standard microbiological nomenclature.