New Strain of Bacillus amyloliquefaciens G1 as a Potential Downy Mildew Biocontrol Agent for Grape

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The study is interesting and provides useful information for controlling the grape's disease.  The article should be thoroughly revised for the clarity of the meanings.

A grammatical revision is needed throughout the manuscript. Some sentences are hard to understand. For example, lines 14-16, 21-22, 32, 53-54.

Scientific names should be italicized (line 21).

First, use the complete word, and then abbreviation (line 23). Check these points in the whole article and correct them.

Line 84. plate as a control Finally, the plates. Correct it.

Lines 89-92 check the font style.

2.2.2. Preparation of grape downy mildew sporangia. Why sporangia is underlined.

Table1. Follow the same style for writing the cultivar names in the whole article. In some places you, mentioned both (species and cultivar names) and in some places only cultivar.

Fruitage should be replaced with fruit.

Table 2. Provide proper legend for understanding. What is the meaning of letters with the numbers (75.8±6.5im).

Figure 1 and 2. Revise the figure legend.

 

Figure 8. High-resolution figure is required.  

Comments on the Quality of English Language

The manuscript requires careful language editing to enhance clarity and improve sentence readability. Several sentences are either overly complex or contain typographical errors which hinder comprehension. This will help to ensure that the scientific content is clearly conveyed. 

Author Response

1.A grammatical revision is needed throughout the manuscript. Some sentences are hard to understand. For example, lines 14-16, 21-22, 32, 53-54.

Thank you for your comments, I agree with you and have changed lines 14-16, 21-22, 32, 53-54 accordingly.

 

2.Scientific names should be italicized (line 21)

I have italicized the scientific names. line 21

 

3. First, use the complete word, and then abbreviation (line 23). Check these points in the whole article and correct them.

Thank you for your comments, I have checked the entire text and all words are complete words. Line 23

 

4. Line 84. plate as a control Finally, the plates. Correct it.

Thank you for your comments, I've made changes to it. line 81

 

5.Lines 89-92 check the font style.

Thank you for your comments, I've made changes to it. line 87-89.

 

6. 2.2.2. Preparation of grape downy mildew sporangia. Why sporangia is underlined.

Thank you for your comments, I've removed the underlining.

 

7. Table1. Follow the same style for writing the cultivar names in the whole article. In some places you, mentioned both (species and cultivar names) and in some places only cultivar.

Thank you for your comments, I've standardized on them.

 

8. Fruitage should be replaced with fruit

Thank you for your critique and correction, I've made changes to it

 

9.Table 2. Provide proper legend for understanding. What is the meaning of letters with the numbers (75.8±6.5im).

Thank you for your comments, These letters are meant to represent different groupings

 

10. Figure 1 and 2. Revise the figure legend.

Thanks for your criticism and correction, I have changed this

 

11. Figure 8. High-resolution figure is required

Thanks for your criticism and correction, I have changed this

 

Reviewer 2 Report

Comments and Suggestions for Authors

In general, the manuscript is interesting and relevant, because the problem of environmentally friendly control of crop diseases remains relevant for many years and each new biocontrol strain may have a potential to be used for development of biopreparations. However, I have some comments mainly concerning the proper description of the methods used as well as the description of the state-of-art in this field and the corresponding discussion (see below).

 Line 22: on the 7^th what? It seems the authors omitted a word.

Line 45: THEY have been used…

Lines 48-49: actually, the number of publications describing biocontrol agents to manage downy mildew is quite sufficient. For example, Google Scholar search for "downy mildew" AND grapewine AND "biological control" AND "endophytic bacteria" gives >300 references since 2020. Of course, not all of them are related to the subject of this study; nevertheless, I suppose there should be at least several dozens of papers within this theme. For example, already the first page of the search results contain 7 references, which can be associated with the theme of this manuscript. Therefore, I suppose it would be good to add more detailed information about such studies into the Introduction section to give readers a better understanding of the current state-of-art in this field.

Line 57: Please, indicate B. cinerea strain used in this study. It should have a number or any other designation. If this was not a strain, but infected leaves, then you should include information about the place and time of sample collection, host plant, and the procedure of a pathogen identification (how can you be sure this is P. viticola and not any other pathogen with similar traits?). I do not see such description in Subsection 2.2.2.

Line 58: please, indicate the origin of the listed media. Were there ready-to-use media, or you prepared them from the components? In the first case, you should indicate the manufacturer; in the second case, you should indicate their composition. Remember that the Materials and Methods section should contain detailed description of all materials, methods, and procedures used (or references to publications described the methods) to make it possible to reproduce your experiments by any reader.

Line 62: very unclear description of the process. What does it mean: clean the collected tissue? I suppose this phrase can be simply removed since you describe the processes in the further subsections.

Line 80: “were soaked in” is repeated twice.

Line 91: please, indicate the model of shaker, its manufacturer and location of its headwuarters. The same is for incubator (line 96).

Line 102: detached leaves? Or you arranged this experiment in planta?

Line 103: do you mean that you placed inoculated on each point for three times? Or do you mean the leaves were inoculated in three replications, i.e., each variant included 3 inoculated leaves? Please, explain.

Line 111: which cultivation media was used? Which equipment was used?

Lines 111-115. What 1000 times solution mean?? It is unclear how many experimental variants did you list here. I would also recommend to re-phrase the description of experimental variants to make them more clear for readers. Probably, you could formulate this as: the experimental variants included plants with the downy mildew manifestation (instead of “diseased”; by the way, please, explain what does it mean?? Naturally infected? Then at which stage of disease development did you do this experiment?) treated with:

(1) water (untreated control),

(2) suspension of tested bacteria (2´10⁸ CFU/mL),

(3) fermentation broth of the biocontrol strain CN181, and

(4) …fungicides (chemical control). Here, as I consider, you should indicate the final concentrations of active ingredients (mancozeb and dimetomorph) in the solution used to treat plants.

Line 115: what doest it mean - before and after 3 and 7 days of treatment? 3 days after treatment can be also called “before 7 days of the treatment”. Please, give more clear description. Maybe you mean that the measurement was carried out prior the treatment and 3 and 7 days after the treatment?

Line 117: what is the origin of this scale? Did you proposed it in this study? Can you add reference?

Line 120-121: give more detailed explanation of all indices used, since it is completely unclear how did you made these calculatuons, which parameters were used and how they were measured. Relative value of what? Total number of surveys - what are these surveys? Number of leaves at each level - which level? Condition index?

Line 149: what does it mean - reactivated?

Line 168: PDA is not a liquid medium. Please, check and correct. Which protein concentration did you use? Did 50 mL of bacillus solution mean the protein solution?? By the way, why did you make these test along with that on Petri plates described below?

Line 186: this is another method, so I would recommend to make a subsection for it.

Line 224: actually all three strains showed 100% inhibition, not only G1.

Fig. 1: what does it means: “the same as the same”? Please, indicate the year and location of field trials. Also, figure caption should include description of all abbreviations used on the figure, since every figure should be self-explanatory even being taken separately of the text. What are A and B parts of the figure?

Line 362: SpoVG is the main component of the antibacterial fraction of G1 metabolites, not the strain G1 itself.

Discussion and References: as I’ve already mentioned earlier, there is a number of papers studying biocontrol agents of downy mildew among endophytic bacteria including at least several dozens papers published since 2020. In the Discussion section, authors included information about various B. amyloliquefaciens strains to control various pathogens. I would recommend authors to add also information about existing biocontrol agents representing endophytic fungi and able to control the target disease on grape/grapewine and probably to compare the revealed antibacterial activities of those and your strain.

Comments on the Quality of English Language

The English level is fine, just some minor errors which, I suppose, will be corrected at the further stages.

Author Response

1. Line 22: on the 7^th what? It seems the authors omitted a word.

Thank you for your comments, I've made changes to it line 22.

2.Line 45: THEY have been used…

Thank you for your comments, I've made changes to it line 46

3.Lines 48-49: actually, the number of publications describing biocontrol agents to manage downy mildew is quite sufficient. For example, Google Scholar search for "downy mildew" AND grapewine AND "biological control" AND "endophytic bacteria" gives >300 references since 2020. Of course, not all of them are related to the subject of this study; nevertheless, I suppose there should be at least several dozens of papers within this theme. For example, already the first page of the search results contain 7 references, which can be associated with the theme of this manuscript. Therefore, I suppose it would be good to add more detailed information about such studies into the Introduction section to give readers a better understanding of the current state-of-art in this field.

Thank you for your criticism and correction, I have revised it and added relevant literature as appropriate

4.Line 57: Please, indicate B. cinerea strain used in this study. It should have a number or any other designation. If this was not a strain, but infected leaves, then you should include information about the place and time of sample collection, host plant, and the procedure of a pathogen identification (how can you be sure this is P. viticola and not any other pathogen with similar traits?). I do not see such description in Subsection 2.2.2.

Thank you for your criticism and correction, I have revised it

5.Line 58: please, indicate the origin of the listed media. Were there ready-to-use media, or you prepared them from the components? In the first case, you should indicate the manufacturer; in the second case, you should indicate their composition. Remember that the Materials and Methods section should contain detailed description of all materials, methods, and procedures used (or references to publications described the methods) to make it possible to reproduce your experiments by any reader.

I prepared them from the components，all materials were provided by the laboratory of Hebei Agricultural University

6.Line 62: very unclear description of the process. What does it mean: clean the collected tissue? I suppose this phrase can be simply removed since you describe the processes in the further subsections.

I have deleted it.

7.Line 80: “were soaked in” is repeated twice.

I have deleted it.

8.Line 91: please, indicate the model of shaker, its manufacturer and location of its headwuarters. The same is for incubator (line 96)

Thank you for your comments, I've made changes to it

9.Line 102: detached leaves? Or you arranged this experiment in planta?

yes detached leaves

10.Line 103: do you mean that you placed inoculated on each point for three times? Or do you mean the leaves were inoculated in three replications, i.e., each variant included 3 inoculated leaves? Please, explain.

I mean that each leaves were inoculated in three replications

11.which cultivation media was used? Which equipment was used?

NA media、shaker、incubator

12. What 1000 times solution mean?? It is unclear how many experimental variants did you list here. I would also recommend to re-phrase the description of experimental variants to make them more clear for readers. Probably, you could formulate this as: the experimental variants included plants with the downy mildew manifestation (instead of “diseased”; by the way, please, explain what does it mean?? Naturally infected? Then at which stage of disease development did you do this experiment?)

I mean diluting the solution to 100 times its original size and  spray the plants.

13.instead of “diseased”; by the way, please, explain what does it mean

I'm talking about spraying them from the start.

14.mancozeb and dimetomorph in the solution used to treat plants

80% Manganese Zinc Diclofenac diluted 1000 times
80% allylmorpholine diluted 2000 times

15.Line 115: what doest it mean - before and after 3 and 7 days of treatment? 3 days after treatment can be also called “before 7 days of the treatment”. Please, give more clear description. Maybe you mean that the measurement was carried out prior the treatment and 3 and 7 days after the treatment?

I mean the measurements were taken on the third and seventh day after treatment

16.Line 117: what is the origin of this scale? Did you proposed it in this study? Can you add reference?

I have cited the relevant literature

17.Line 149: what does it mean - reactivated?

yes

18.Line 168: PDA is not a liquid medium. Please, check and correct. Which protein concentration did you use? Did 50 mL of bacillus solution mean the protein solution?? By the way, why did you make these test along with that on Petri plates described below?

I have modified it.50 mL of bacillus solution mean the protein solution

19.Line 224: actually all three strains showed 100% inhibition, not only G1.

I have modified it

20.Fig. 1: what does it means: “the same as the same”? Please, indicate the year and location of field trials. Also, figure caption should include description of all abbreviations used on the figure, since every figure should be self-explanatory even being taken separately of the text. What are A and B parts of the figure?

I have modified it

21.Line 362: SpoVG is the main component of the antibacterial fraction of G1 metabolites, not the strain G1 itself.

I have modified it

Reviewer 3 Report

Comments and Suggestions for Authors

This paper gives significant contribution for the control of the one of the most important disease in vineyards P. viticola.

Hereby are comments for authors:

Introduction: 

34-37. give references to the statements

36 how the use of PPP can affect quality of the grape (also, instead fruits is recommended to use grape or berries depend on the means)  

37-38. why biological control is important, is there any importance of biological control in organic viticulture

46: add Latin names of diseases.

MM

55 for the cultivars kyoho and red globe use same approach, either capital for both or not and cross paretnt for all or for none (not signifficant for the paper).

63-86, Chapter 2.1.1- - 2.1.3. there are same part of the methods, list it for the stem, fruits and seeds and add particularities.

In the Results, authors compare the inhibition rate with fungicide mancozeb. So in the MM part, authors need to include the standard they used, application, dose or concentration for mancozeb, also add commercial name and formulation and amount of active ingredient (e.g. gram/ kg of mancozeb in the product) .

Results:

In 3.3., please refere more to the Fig 1 A and B in the text.

245-246. Move last sentence to the discussion part not in the results

262-263 - not well understandable

 

Author Response

 1.give references to the statements

I've listed relevant examples

2. 36 how the use of PPP can affect quality of the grape

because the long-term use of chemical pesticides is associated with a number of problems, such as, pesticide residue, and the development of resistance which in turn leads to a decreased quality of grape and wine.

3.also, instead fruits is recommended to use grape or berries depend on the means

I've made changes to it.

4. 37-38. why biological control is important, is there any importance of biological control in organic viticulture

Biological control precisely reduces pesticide residues and pesticide resistance in grapes. 

5. 46：add Latin names of diseases.

I've made changes to it.

6. 55 for the cultivars kyoho and red globe use same approach, either capital for both or not and cross paretnt for all or for none (not signifficant for the paper).

I've made changes to it.

7. 63-86, Chapter 2.1.1- - 2.1.3. there are same part of the methods, list it for the stem, fruits and seeds and add particularities.

I have given a brief overview of the same sections

8.In the Results, authors compare the inhibition rate with fungicide mancozeb. So in the MM part, authors need to include the standard they used, application, dose or concentration for mancozeb, also add commercial name and formulation and amount of active ingredient

I've labeled the concentrations of mancozeb and dimethylmorph

9. In 3.3., please refere more to the Fig 1 A and B in the text.

I've made changes to it.

10. 245-246. Move last sentence to the discussion part not in the results

I've made changes to it.

11.  262-263 - not well understandable

I mean that the colonies of G1 cultured on NA medium showed a certain degree of stickiness, had a foul odor, and did not produce any pigment