Isolation, identification and antibacterial activity of endophytic bacteria from the seeds of < Panax japonicus>

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National Natural Science Foundation of China (Grant NO.31660252).

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This work was supported by the four project grants:

Science and Technology Funded Project of Guizhou Provincial Science and Technology Department (Guizhou Science and Technology Foundation -ZK Grant NO.(2023) Key 002).

National Natural Science Foundation of China (Grant NO.31660252).

Guizhou Provincial Department of Education Youth Science and Technology Talent Development Project (Qian Jiao He KY character Grant NO.[2022]029).

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: N/A

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: This manuscript presents a study on the isolation, identification, and antibacterial activity of endophytic fungi and bacteria from Panax japonicus seeds. The topic is relevant and potentially significant for understanding seed biology and developing biocontrol agents. The study is generally well-structured, but there are several areas that require clarification and improvement before publication. Overall, the results are preliminary and lack strong evidence, but still valuable for further research.

Abstract (Lines 39-65):

• Line 40: Consider stating the specific aim of this study more clearly, such as: "The aim of this study was to isolate..."

• Line 48: It is confusing to read "Seven endophytic fungi strains were isolated" immediately followed by mentioning that "PjZ1, PjZ3, PjZ4 and PjZ5 were identified as Fusarium". This makes it appear that only these four were fungi and not all 7 strains. You should rephrase this to clarify that ALL 7 are fungal.

• Line 55: Instead of "Only PjZ4 had antibacterial activity", it may be more appropriate to say, "Among the isolates, only PjZ4 showed significant antibacterial activity...". This highlights that the other fungal isolates did have some inhibition on Bacillus subtilis, even if not significant.

• Line 58: Conclusion: It is advised to have a stronger conclusion. For instance, the authors could summarize the main findings, including the genus level of the identified strains, and highlight the significance of their study for future research.

• Line 63: The keywords could be improved by making them more specific. For example, replace "Antibacterial activity" with "Endophytic antibacterial activity".

Introduction (Lines 66-81):

• Line 68: The phrase "also known as Disporopsis fuscopicta Hance" is an unnecessary detail here as it is not the same genus.

• Line 70: Should add citations to support that "Pharmacological studies have shown that Panax japonicus..."

• Line 75: The statement regarding lack of reports on endophytic fungi/bacteria from seeds is good, but there needs to be something to indicate why this is valuable. For example, why are seeds and what importance is this to P. japonicus?

• Line 77-80: The end of the introduction, there should be something to emphasize the biological functions of the bacteria isolated.

Materials and Methods (Lines 82-162):

• Line 86: Provide the geographic coordinates or location details of the experimental site.

• Line 90: The composition of NA (Nutrient Agar) should be described better. The recipe is given as "Peptone 10g, Beef Paste 3g, Sodium Chloride 5g, Agar 20g, distilled water 1000mL, pH:7.2." It would be better practice to say "Beef Extract" instead of "Beef Paste", as well as identify if peptone is tryptone or another form.

• Line 103: Provide a citation or additional details to justify the use of this orthogonal testing method.

• Line 107-109: It is difficult to follow that section. Re-word and clarify that after disinfection, the seeds were plated to analyze contamination rate before proceeding with isolation.

• Line 115: Describe the sterile filter paper with more details, such as pore size and manufacturer.

• Line 117: Clarify why the seed is cut in half before plating.

• Line 119: What is meant by "seed preservation" here? It is better to say for "preservation of the isolated strains"

• Line 127-136: The morphological observation section lacks clarity. How many samples were observed per isolate? Were observations done in replicates? More detail on how results were "recorded" should be included.

• Line 137-139: The DNA extraction procedure needs to be described with more detail: What is the kit that was used? Also, it is better to use "according to the manufacture's instructions" rather than just stating "according to the instructions".

• Line 144: The reaction volume and composition is confusing. It would be better to say "a 50uL reaction volume containing..." instead.

• Line 151: The statement about the primer name is inconsistent, the previous section mentioned primer ITS1 and ITS4 but now are described as "primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGAC".

• Line 157: What percentage of agarose gel was used in gel electrophoresis?

• Line 166: Please add more detail about the selection of pure isolates. For instance, whether single colonies were used to prepare the pure isolates.

• Line 167: What does “adjusted to OD600=0.2 in 0.85% saline” means? Also, how was the 0.2 OD reading achieved?

• Line 174: It is good to mention the preparation of fungal cake, but describe the type of agar that was used.

• Line 178: Please mention which drug-sensitive paper was used.

Results (Lines 163-205):

• Line 215: The abbreviation ANOVA was already introduced, so no need to repeat it again.

• Line 222-229: It would be better to include a table of the Gram staining morphology results for better data presentation.

• Line 242: Avoid the use of the abbreviation for "16S ribosomal DNA", write as "16S rDNA" for consistency.

• Line 242-261: The method of identifying the endophytes is unclear. You should state the database that was used, such as NCBI (National Center for Biotechnology Information). Also, it would help to give more details regarding the process of phylogenetic tree construction.

• Line 276: Clarify that “Antagonistic experiments” are referring to the testing of antibacterial activity. Also, clarify that the bacteria used in the antagonistic experiments were pathogenic.

• Line 281-284: Describe the statistical significance of the differences observed.

• Line 298-307: The lack of activity should be presented in a table.

Discussion (Lines 308-376):

• Line 310: It is good that the authors note the potential of endophytes in productivity, but needs to be better linked to this study.

• Line 313: Clarify why the presence of Fusarium and Alternaria is important to discuss.

• Line 315: Is this significant? What do the non-inhibitory results suggest?

• Line 320: Again, why is it important to note the dominant fungi were not found?

• Line 330: Pseudocercospora variicola is a fungal species, but is treated as bacterial here. This section would benefit from being clarified, and better linked to the results, that this particular study may be considered a contaminant.

• Line 335: The authors should further discuss the importance of their finding in P. japonicus.

• Line 337-344: Needs a better transition to the importance of Pseudomonas and Enterobacteriaceae.

• Line 361-365: Reiterate more clearly. The authors should briefly list the identified species, but also highlight the dominant species in the study.

• Line 366-376: The conclusion is not comprehensive enough and should be revised to clearly state the main outcomes, limitations, and implications for future studies.

• Statistical Analysis: The manuscript briefly mentions statistical analysis (ANOVA) but more detailed information about the statistical methods used should be included. The data presentation could be enhanced by adding mean values, standard deviation values, and P values as appropriate.

• Language: The manuscript needs careful proofreading. There are several instances of grammatical errors and awkward phrasing that need to be revised.

Reviewer #2: The authors attempted to isolate and identify bacterial and fungal endophytes and studied the antibacterial activity of endophytic isolates from Panax japonicus. As a whole, the research findings may be helpful to other researchers. However, the scientific statements made in the MS are not clear, and the information flow is not up to the mark.

For example, the paper's title is “Isolation, identification and antibacterial activity of endophytic bacteria from the seeds of Panax japonicus." However, the MS reports both bacterial and fungal endophytes. For instance, under the abstract, the authors write, “Seven endophytic fungi strains were isolated from the seeds of Panax japonicus, named…………”

The conclusion is very weak and highlights only the isolation/identification of endophytic fungi. Innovation elements are missing from the MS. The conclusion before the references list is very weak from a scientific perspective, and many statements made don't fall under the conclusion.

The knowledge gap or the background provided in the Introduction is scientifically weak. Furthermore, the scientific arguments that are made under discussion are weak.

The authors have the results and data; however, the overall quality of the MS needs to be improved.

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Reviewer #1: No

Reviewer #2: No

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Dear Dr. Zhang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jun 28 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

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We look forward to receiving your revised manuscript.

Kind regards,

Raed Abduljabbar Haleem, Ph.D

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

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

Reviewer #3: No

Reviewer #4: Yes

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

Reviewer #3: No

Reviewer #4: Yes

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

The PLOS Data policy

Reviewer #3: No

Reviewer #4: Yes

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

Reviewer #3: No

Reviewer #4: Yes

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Reviewer #3: The title of your study sounds interesting. However, it appeared that this work is not as well planed scientifically as well as it should have been and the manuscript was so poorly written leading to very confusing story throughout the entire manuscript.

Let me give some examples as followed.

The title doesn't specify what group of microbial endophytes that the researchers are focusing on, but the introductory part of the abstract only mentions "endophytic bacteria" as the purpose of this study (L.26-29). However, the result part of the abstract suddenly focuses on "Seven endophytic fung[al] strains" L.34 and "Three strains of endophytic bacteria" L.38 at which sum up to be "11 endophyte species" L.50 in the conclusion part of the abstract. This bring up the question of where the number 11 come from. This is on top of the inconsistent issue with the correct way to write all scientific names both in the abstract and in the rest of the manuscript.

Introduction:

L.59 I[n]troduction

The beginning of the introduction is a bit confusing and should start with "The chemical composition of dried rhizome of Panax japonicus C.A. Mey. mainly includes ..." For the rest of the introduction, any scientific name that has already been written in full previously, you should abbreviate the genus name for the subsequent use in the manuscript.

Materials and methods:

Culture medium section is very confusing. Components of some media were described in details while some appears to be side-of-the-bottle instruction on how to prepare them while many only have the names of the media mentioned without any details. Also, one medium that was mentioned in the "Disinfection" sub-section was not included L.114.

The sub-section on "Screening of seed disinfection conditions" is even more confusing because you described the "disinfection time of 6min, 12min, 18min" L.105 which make me wonder where the "9min" in the rest of your manuscript comes from.

L.126 "the same as 1.6 Seed Disinfection Method" What does 1.6 refer to?

L.129 is very confusing. What is the "and line the purification, purify 2-3 times to keep the seeds."

L.135-136 (Three strains on each medium for each strain and three strains on each medium for observation and identification). ???

Molecular biology identification:

L.142 mentioned that "Endophytic fungal DNA was extracted" but then on the same paragraph L.146 why "DNA of each endophytic bacterium as a template"? Also, the PCR conditions were described inconsistently regarding the volume of each components and there was no mention of concentration of the primers used and no reference to them.

L.178-185 were very confusing.

L.187 L.201 How did you measure the size of the ring of inhibition? Area? Circumference? Diameter?

L.210-211 A bit unclear writing.

L.215 What is 1.9.2?

Table 3 doesn't seem to has been clearly explained in text.

There are no mention to Fig.1 and Fig 2 in text.

L.282 What is "Streptomyces alternaria alternata"?

Fig.5 The phylogenetic tree reconstructions were improperly done such as lacking suitable outgroup sequences and limited reference sequences.

L.301-302 What is "endophytic fungal bacteria"? Which table or figures are the results of endophytic bacteria?

For the metabolites perhaps there is no need to tables 7 and simply report as no effect. Also, is table 8 the exact of table 7?

The discussion is misleading and not supported by your results. For example, what data from your study to support that the two genera of fungi were not the dominant species of endophytic fungi in the seeds when you found these 2 the most? What is the meaning to "dominant species"?

L.382 What is "salt tolerance after ping"?

The Conclusion is also very confusing with regard to "dominant species" and here we found "10 endophytic strains of endophytic fungi" L.407 and "10 strains of endophytic bacteria" L.409. Moreover, your study did not have any experiment on the effect of isolated microbial endophytes on the growth and development process of Panax

japonicus at all and thus appears to be over claiming.

There are so many other similar mistakes in this manuscript that I felt it would consume too much of my time to point them all out. Thus, I would suggest then you find a native English speaker to help you with proof reading it before any submission.

Reviewer #4: The study offers a useful sterilization protocol and a foundational catalog of endophytes from P.japonicus seeds, relevant to medicinal plant propagation and early-stage biocontrol exploration.

However, its scientific impact is limited by modest antibacterial results and the lack of functional validation of microbial roles. The manuscript requires structural and grammatical revisions, clearer scientific terminology, and more specific explanations to ensure clarity and relevance for a broader scientific audience. I have raised few questions and recommendation below based on overall reading/analyzing your paper.

The authors have not clearly or justifiably defined the purpose, context, and rationale of their study in a way that a broader audience, even an interdisciplinary scientific audience,can easily understand. They jump into procedural detail without first explaining why the work matters in a straightforward way. Ambiguities and missing explanation raise these question in introduction section: Why are the seeds being germinated in a lab? What problem is being solved? (The manuscript mentions contamination, but doesn't clearly connect it to seed failure, dormancy, and germination goals). Who benefits from this research? Is it for agricultural production, conservation, medicinal compound extraction? This is only vaguely implied. Why are E. coli, S. aureus, and B. subtilis used in tests? Not explained in ecological or applied terms.

Authors still did not report sterility checks (e.g., plating final rinse water), standard in seed disinfection protocols to confirm efficacy and rule out false negatives.

Why choose these test pathogens? These human pathogens are common in antagonism assays, but their relevance to P. japonicus seed health or soil microbiome context is unclear. Would plant pathogens (fro example Fusarium oxysporum, Pythium spp.) be more ecologically relevant?

The use of HgCl2 poses health and environmental risks. Recent protocols favor bleach (NaOCl) or H2O2 alternatives with comparable efficacy . Justify choice of HgCl₂ over safer agents, or test alternatives.

Antibacterial activity was only measured as inhibition zones from agar plugs and disc diffusion. For rigorous comparison,why minimal inhibitory concentrations (MICs) or broth microdilution assays were not performed?.

The discussion notes potential hormone-related inhibition by Fusarium spp., but offers no data on seed viability or germination trials post-sterilization. Incorporating a germination assay would solidify claims about contamination impact.

Have you considered testing endophyte re-inoculation on seed germination or seedling growth? Such functional assays are critical to demonstrate PGP potential and vave you deposited all sequence data in GenBank with accession numbers?

Instead of reporting what was done, the authors write Methods section as if they are giving instructions, like a lab manual.These kinds of descriptions r didactic, rather than declarative and scientific.For a research article, methods should be written in the past tense, reporting what was actually done, rather than describing steps as if in a lab manual.

The manuscript STILL REQUIRES careful revision for grammatical accuracy, sentence structure, and scientific terminology. Clearer sentence construction and restructuring of ambiguous or repetitive descriptions are recommended to improve clarity and precision. PLEASE go through the manuscript and check for grammatical/structural errors.

I CONCLUDE THESE MAJOR LIMITATION IN YOUR WORK: (Should be acknowledged in relevant section): The antibacterial activity results are modest,only one fungus showed weak inhibition, and none of the metabolites did. No functional testing was done (yet) to prove that these endophytes help or harm seed germination directly. It is more of a foundational study, setting the stage for future work (e.g., applying isolated strains to seeds and testing growth impact).

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Reviewer #3: No

Reviewer #4: Yes:  Saleem Ahmad

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Isolation, identification and antibacterial activity of endophytes from the seeds of Panax japonicus

PONE-D-24-46415R2

Dear Dr. Zhang,

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