Endophytic Bacteria in Forest Protection: Pseudomonas silvicola Controls Pine Needle Blight in Masson Pine

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. The authors should conduct some biochemical test, which is produce by biocontrol such as chitinase, amylase etc.
2. authors should conduct at least 1 year field experiment to known efficacy of biocontrol agent
3. Mechanism of biocontrol for defense inside the plant should be evidence by defense ezymes or genes
4. In vitro should be italic

Author Response

Thanks for all your suggestions, we made a PDF file. Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The article includes extensive experimental material and, as a whole, represents a comprehensive study. However, a number of revisions/corrections need to be made to the manuscript before publication.

 

Title. “Antifungal endophytic bacteria” is a strange phrase.

 

Introduction.

Lines 36-37. Rephrase the sentence.

Lines 67-74. There should be a definition of what PSM-6 is. “Preliminary assessments of its antibacterial activity and inhibitory mechanisms were performed, providing valuable insight and strain resources for advancing biological control strategies for pine needle blight.” What are authors talking about here?

The main goal of the study should be stated more clearly.

 

Materials and methods.

Lines 131-134. Please clarify the information: centrifugation speed at which bacteria remained in the supernatant; what units of concentration (supernatant dilution?).

Lines 144-145. What is the target organism? What isolate (W1-W5) was use used in subsequent plant infection experiments and bacterium antagonism studies?

Lines 148-150. How was the number of bacterial cells controlled? Why wasn't the method of wells in agar, into which a suspension of bacteria of a certain concentration is added, used?

Line 205 and Lines 210-211. Bacterial suspensions in which concentration?

2.8. Effects of bacterial volatile organic compounds (VOCs). Explain the methodology.

Check lines 221-223.

Table S3 should be included in the main text of the article.

 

Results.

What about other pathogenic fungi, why haven't other pathogens been identified?

What were these 84 strains of bacteria? Did they all belong to the same species of bacteria?

I didn't find the figure S1.

“Crude extracts of PSM-6 secretions were prepared” Rephrase.

Line 374 “at a concentration of 5 μL”?

Figure 1. The parts of the figure should be aligned with each other. What is on the parts of the figure without letter designations? What is the difference between parts E and F? It is necessary to provide an explanation of all abbreviations in figure legend.

Figure 5. “Biocontrol mechanisms of PSM-6 against” This sounds strange, rephrase. The legend should specify what µl and dilutions correspond to. “(E) Inhibition of the dual-culture assay. (F) Inhibition of sterile supernatant. (G) Inhibition of the crude extract.” Inhibition of what and by what, specify the information.

Figure 6. Why do plants within the same group look different, including controls?

Figure 7. Clarify figure legend.

 

Discussion.

Given the abundance of results, their discussion should be expanded.

 

Conclusion.

The conclusion should be rewritten, building it logically and including a mention of all the main results obtained, as well as the prospects for further development of this work and/or practical use of the results obtained.

Author Response

Thanks for all your suggestions, we made a PDF file, please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors!

Thank you for submitting your article for review. It is devoted to the study of the influence of Pseudomonas silvicola bacteria on the resistance of P. massoniana to a fungal phytopathogen. The work is complex, since it studies the interaction of three living organisms: a plant, a fungus, and a bacterium. The work includes a large number of methods. The article is very well illustrated. I think that Figure 7 is the decoration of the article.

There are a number of comments and recommendations: 1. It is recommended that the authors indicate in the Introduction whether the fungus under study is a specific pathogen for P. massoniana or a non-specific pathogen. The strategy of interaction between the plant and the pathogen depends on this. 2. In Section 2.2, it is necessary to describe the composition and characteristics of the soil on which the seedlings were grown. 3. In the methodological part of the article (for example, Axio Imager M2 microscope (Zeiss, Germany)) it is necessary to indicate the city where the headquarters of the companies producing the equipment on which the authors worked is located. 4. In section 2.5, it is recommended to indicate what was positive and negative control in the study. 5. In the Methodological section, I was unable to see the titer of bacteria used in the experiments. It must be indicated. 6. Microorganisms Botrytis cinerea, Fusarium graminearum, F. oxysporum, N. camelliae-oleiferae, Pythium vexans, Diplodia pinea, Alternaria sp., Verticillium dahliae and Lecanosticta acicula must be briefly described, the strains, sources of receipt and cultivation conditions must be indicated. 7. In the paragraph devoted to statistical data processing, the authors indicated that they used Fisher's criterion and ANOVA analysis. It should be noted that such statistical processing is used only for data with a normal distribution. The authors must indicate how the primary data were checked for normality. 8. In Fig. 1 (A) no symptoms of damage are visible, this figure is proposed to be replaced with another one with more pronounced symptoms of damage. For Fig. 1 d, it is necessary to add explanations in the caption. 9. In Figure 2, the text needs to be made more readable. In its current form, the inscriptions are indistinguishable, they are very difficult to read. 10. The authors showed that the PSM-6 strain does not have cellulase degradation (Table 1). At the same time, this strain had the most pronounced fungicidal effect, in particular, damage to the cell wall of the fungus. It would be more logical if these bacteria had cellulalytic activity. Based on the available results, what do the authors associate with the pronounced antibacterial effect of this strain? Below, the authors write about inhibitory compounds - secondary metabolites, so what exactly could these compounds be? The authors write about this in the Discussion, but it would be good to isolate and identify them in the strain under study. 11. Figure 6 is uninformative and confuses the reader. It does not clearly express the desired effects. It is recommended to explain the abbreviations "dpi" in the caption to Figure 6. 12. It is recommended to add a concise sentence at the end of the Conclusions, summarizing all the data obtained. Respectfully Yours, reviewer. March 07, 2025

Author Response

Thanks for all your suggestions, we made a PDF file, please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Without conducting field experiments such study can't be validated. Therefore in my opinion it's not suitable for publication.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The general impression is that the authors did not have enough time to make the necessary changes to the text of the manuscript. Reveals carelessness in the way the authors worked with the manuscript text. Authors should check carefully the revised version of the manuscript, especially the new materials added to the text. The division of paragraphs into sentences, the use of capital letters, etc. require revision throughout the text. It is also worth noting that the added material is not inserted into the text logically and organically everywhere in the text. The English language of the material added by the authors also requires correction.

Some, but not all comments

In the Introduction and Conclusion sections the authors talk about “antibacterial activity against filamentous fungi” The authors do not see the difference between antibacterial and antifungal activity?

Line 117 “filling in the gaps regarding Pseudomonas aeruginosa” Where did this bacterium come from?

Line 415 “Inhibition of the dual-culture assay” The study of fungal growth inhibition by dual-culture assay?

Line 416 “Inhibition of sterile supernatant” Effects of sterile supernatant on fungal growth?

Line 417 “Inhibition of the crude extract”….

P. silvicola or Ps. silvicola; P. massoniana or Pi. massoniana Make it the same throughout the text.

2.11. Pot experiment for the disease control test. How many plants were in each group? How was the statistical significance of differences in the development of disease symptoms in groups 1-4 assessed? Given the differences in how plants appear in Figure 6 within one group, these data are especially important. If the experiment lasted 60 days, why does Figure 6 show plants after 49 days?

The legend to Figure 7 contains repetitions.

Comments on the Quality of English Language

The English language requires correction.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf