-->PONE-D-25-67322-->-->Biocontrol of rice blast by Pseudomonas mosselii PR5 through seed priming and foliar application reduces reliance on chemical pesticides-->-->PLOS One

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Reviewers' comments:

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-->Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Yes

Reviewer #2: Yes

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-->2. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

Reviewer #2: Yes

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-->3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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-->4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

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-->5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: This study addresses a relevant and important topic: the search for sustainable alternatives to chemical fungicides for controlling rice blast, a major threat to global food security. The experimental design is comprehensive, testing multiple application methods of the biocontrol agent Pseudomonas mosselii PR5 across three susceptible rice genotypes. The results demonstrate clear potential for PR5, particularly the combination treatments (SP+BCF and SeP+BCF), in reducing disease severity and promoting plant growth and yield. The manuscript is generally well-structured, but several critical flaws in methodology, data presentation, and interpretation must be addressed before it can be considered for publication.

The phrase "including an absolute control, a pathogen-inoculated control, a fungicide control, and five PR5 application modes" is not clear. It is not clear which control group receives the pathogen and which does not.

The term "seedling priming" (SeP) is introduced but is not a standard term in the literature. "Seed priming" is common

Bacterial culture filtrate (BCF)

Does it refer to a spray of the liquid growth medium containing bacterial metabolites but no live cells? Or is it a suspension of live bacteria?

The statement "SP+BCF achieved the highest yield and outperformed the fungicide in disease suppression" is main conclusion. However, the abstract does not provide the actual disease severity data (e.g., "SP+BCF reduced disease severity by X% compared to the pathogen control, compared to Y% for the fungicide").

For example, "SP+BCF produced the lowest AUPDC in V1 and V3" – how much lower? And crucially, how did it compare to the fungicide in those varieties? Was the difference statistically significant?

The final sentence of the results section ("SP+BCF achieved the highest yield and outperformed the fungicide in disease suppression") largely repeats the point made earlier about SP+BCF producing the lowest AUPDC and achieving the highest yield

The introduction should articulate the specific knowledge gap that this study aims to fill.

The introduction should set up the hypothesis that different application methods (priming vs. foliar) might have different effects, and that combining them could be synergistic. Currently, the introduction presents PR5 as a promising agent but doesn't foreshadow the central question of the paper: What is the best way to use it?

Cite some recent studies in the introduction https://doi.org/10.1016/j.bcab.2020.101729, https://doi.org/10.1002/aoc.5190, https://doi.org/10.1016/j.micpath.2024.107084

The treatment list in lines 106-109 is confusing and contains an error. It states: "T0, absolute control; T1, negative control; T2, positive control (commercial fungicide foliar application); ... T6, seed priming + BCF foliar; and 76, seedling priming + BCF foliar application." Later, in lines 128-132, it mentions a fungicide sprayed in "treatments T1". This is a major inconsistency. Which one is the fungicide treatment? A "negative control" typically receives no treatment and no pathogen, while a "positive control" receives a known effective treatment (like a fungicide) and the pathogen. The authors must clarify the treatment codes and ensure they are used consistently throughout the manuscript, including in all tables and figures.

To properly compare the biocontrol agent to the chemical standard, there must be a treatment that receives the pathogen and is sprayed with the fungicide but does not receive any PR5 treatment. As described, it's impossible to separate the effect of the fungicide from the potential confounding effects of PR5 if the fungicide was applied to the same plants as PR5 treatments.

The method describes the pathogen stock as a "paper disc" (Line 143). This is an unusual and non-standard method for long-term storage of M. oryzae. Typically, cultures are stored on agar slants, in sterile soil, or on filter paper in a dried state. The phrase "presented as a paper disc" is unclear.

Figure 1-3 and figure 9 the figures quality is poor these must be readable.

Reviewer #2: The manuscript investigates the biocontrol efficacy of the endophytic bacterium Pseudomonas mosselii PR5 against rice blast (Magnaporthe oryzae) across three susceptible rice genotypes. The research assesses different application techniques—such as seed priming, seedling priming, and bacterial culture filtrate (BCF) foliar spray—and contrasts their efficacy with that of a commercial fungicide. The findings demonstrate that combined applications, especially SP+BCF and SeP+BCF, significantly mitigate disease severity (PDI and AUDPC) while concurrently promoting plant growth and yield metrics, often surpassing the efficacy of chemical fungicides.

The study addresses an important issue in sustainable agriculture and plant pathology, fitting perfectly within the broad scope of PLOS ONE. The dual-action assessment of PR5 (growth promotion and disease suppression) is commendable. However, I have identified several critical methodological gaps, instances of confusing terminology, and a significant overreach in the Discussion section, in which prior findings are conflated with current data. Before considering the manuscript for publication, the authors should address these issues.

Section 2.2 details the pot preparation but completely omits the statistical design of the experiment. Was this a Completely Randomized Design (CRD) or a Randomized Complete Block Design (RCBD)? Furthermore, the authors note in the Discussion that pots were kept 25 cm apart in natural field conditions and that T0 (absolute control) pots were "physically separated by polyethylene sheets." This crucial methodological information should be included in Section 2.2, rather than being presented as an afterthought in the Discussion to clarify anomalous data. Please detail the exact spatial arrangement, post-inoculation environmental conditions, and randomization protocol.

In the Discussion, the authors state that PR5 produces IAA, solubilizes phosphorus, and produces siderophores and HCN. None of these biochemical assays were conducted or presented in the current manuscript. While these may be established traits of PR5 from the authors' previous works, the current phrasing makes it appear as though these mechanisms were proven in this specific study. The authors must strictly separate their current phenotypic/disease findings from previous in vitro characterizations. Please revise these sections.

In Lines 132-135, the authors mention centrifuging the bacterial solution (OD600 = 1) and mixing the supernatant with CMC for foliar application. Later, in line 225, the solution is referred to as a "bacterial cell-free (BCF)" application. Was this supernatant passed through a microbiological filter (e.g., 0.22 µm) to guarantee it was entirely cell-free? If no filtration was performed, viable cells likely remained in the supernatant, making the term "cell-free" scientifically inaccurate. Please clarify the exact sterilization/filtration protocol. If it was not filtered, the terminology must be corrected throughout the manuscript.

In Lines 124-125, T1 (pathogen only) is labeled the "negative control" and T2 (fungicide) is labeled the "positive control." In plant pathology, a pathogen-only treatment is typically considered a positive control for disease development. To avoid reader confusion, I recommend utilizing descriptive nomenclature throughout the manuscript.

The conclusion (Lines 368-377) reads too much like a second abstract, merely rehashing the results. A conclusion should synthesise the study's broader implications and practical applications in sustainable agriculture without repeating detailed data points.

Ensure that the meaning of the error bars is explicitly stated in the general methodology (Section 2.7), not just in the figure captions.

While generally readable, the manuscript contains minor grammatical inconsistencies and awkward phrasing (e.g., Line 121: "seedlings... are soaked", tense shifts). A final proofread is recommended to ensure consistent past-tense usage for methodologies and results.

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Reviewer #1: No

Reviewer #2: Yes:  HARUN BEKTAŞ

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Biocontrol of rice blast by Pseudomonas mosselii PR5 through seed priming and foliar application reduces reliance on chemical pesticides

PONE-D-25-67322R1

Dear Dr. Sultana,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Sofia Isabel Almeida Pereira

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

-->Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.-->

Reviewer #1: All comments have been addressed

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-->2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. -->

Reviewer #1: Yes

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-->3. Has the statistical analysis been performed appropriately and rigorously? -->

Reviewer #1: Yes

**********

-->4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

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-->5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.-->

Reviewer #1: Yes

**********

-->6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)-->

Reviewer #1: (No Response)

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-->7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review?  For information about this choice, including consent withdrawal, please see our Privacy Policy.-->

Reviewer #1: No

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