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Thanks for addressing the remaining comments!
[# PeerJ Staff Note - this decision was reviewed and approved by Sonia Oliveira, a PeerJ Section Editor covering this Section #]
Please address the last few comments.
**PeerJ Staff Note**: Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
The article entitled “A double agent? Unveiling the chemical profile of the pathogenic fungus Pyrrhoderma noxium as an endophyte in true mangroves” is well written after first revision. But for the improvement of quality of manuscript Add following suggestions
• Line 64 -65 Add the exact amount of economic losses caused by P. noxium in agriculture and forestry sector
• In results and discussion section: add sub heading p. noxium as double agent
• For improvement of manuscript add the LC-MS/MS chromatograph image for one sample analysis
• The data table with major classes of bioactive compounds, number of bioactive compounds identified per each class should be added.
The materials and methods section was improved allot after first revision but future improvement is reqgiured
• Lines 110 to 112: Add the accession numbers of all five plant samples
• Add the where the mangrove plant herbarium samples were deposited? Which improve plant authentication
• Add other databases also like pubmed
All underlying data in the present paper not provided; such as
• Add the sub heading P. noxium as double agent and add the results of your findings. It will explain the nature of P. noxium as endophyte and pathogenic fungus
• Add the relavent discussion points befor this to support p.noxium as double agent
• Add the LC-MS/MS chromatograph image for one fungal sample to improve the quality of the manuscript.
• The plant authentication of mangrove samples should be explained with deposition and accession number details
• The titles is mentioned as “A double agent? Unveiling the chemical profile of the pathogenic fungus Pyrrhoderma noxium as an endophyte in true mangroves” but the proper points to explain the P. noxium as double agent was not added
• As Fungal pathogen P. noxium will infect the mangrove plants and resides inside as mycelium for spreading the infection to entire plants, and shows symptoms in favorable conditions so it should be proved as pathogenic but authors isolated it from fresh mangrove plant samples which is already infected with P. noxium? If yes it not be described as endophyte. So have a better understanding on double agent : P, noxium.
• For the improvement of the manuscript better clarity is required on the content (data on favorable conditions to describe as endophyte/ sumbiont/parasitic)
The authors have significantly improved their manuscript and responded to all my comments. I do believe that original data from MS should be available, but it can be upon request. Therefore, based on my previous comments and the implemented corrections made by the authors, I recommend this manuscript to be accepted for publication.
No further comments from the previous round.
No further comments from the previous round.
I recommend adding original data to supplementary files or adding information that the data can be sent upon request.
Please address all reviewers' comments.
**PeerJ Staff Note:** Please ensure that all review, editorial, and staff comments are addressed in a response letter and that any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate.
**PeerJ Staff Note:** Your submission appears to have been at least partially authored or edited by a generative AI/large language model. When you submit your revision, please detail whether (and if so, how) AI was used in the construction of your manuscript in your response letter, AND mention it in the Acknowledgements section of your manuscript.
All basic reporting was well explained and self-understanding, but a few drawbacks were mentioned in the review file.
All methods and materials were included and explained very well, but a few data sets in experiments are missing, for example, the parameters for identification and confirmation of endophyte, and also plant sample identification. Bioactivity studies of the fungal isolate are also missing
Impact and novelty not assessed. Meaningful replication encouraged where rationale & benefit to the literature is clearly stated. Conclusions are well stated, linked to the original research question & limited to supporting results.
The conclusions should be elaborated.
The LC-MS graph is missing in the results section.
A few typographical mistakes were identified.
Repetition of the conclusion paragraph
Proof of identification of endophytic fungi from plants is missing.
Please consider an anatomical study of the host sample for the identification of the fungal isolate as an endophyte.
The authors provide an interesting study of the metabolite analysis produced by endophytes of mangroves belonging to the pathogenic fungal species Pyrrhoderma noxium. The manuscript's structure is correct, and the language is clear and comprehensive. All uploaded figures and tables are properly formatted and well described. The introduction is somewhat short but sufficient to provide all necessary background information. The references are properly selected. However, it seems that there are a lot of references cited in the supplementary file B that are not included in the main text. Please confirm with the editorial team if this is correct, or if the citation should be included after the main text with citations added in the Supplementary files section. The article addresses previously undiscovered aspects; however, some information on the direction of future studies should be included in the manuscript's conclusions. Most of the original data is included in the submission; however, I lack original files from LC-MS.
The presented research is properly planned and clearly presented. The used methods are adequately selected for the study. However, I do miss some minor aspects. Please indicate how the effectiveness of surface sterilization was confirmed, as well as whether chromatography calibration was performed. Since the methodology is qualitative, there is no need to calibrate quantitatively the extraction and separation of certain metabolites, but some physical parameters can influence, e.g., the retention time. Therefore, an internal calibrator (adding a compound of known retention time and concentration would confirm the proper data interpretation. Alternatively, external calibration could also be used. Additionally, I would recommend selecting one metabolite for purification or performing some simple activity test, e.g., against E. coli and/or Candida albicans, to confirm the biological activity of the obtained metabolites.
Overall, this study presents meaningful results that align with current trends in research on novel biologically active compounds in endophytic microorganisms, highlighting a possible dual ecological role. This dual ecological function of Pyrrhoderma noxium is a promising aspect that should be further investigated both for basic science and for its application potential. I do have to verify some additional confirmation of the obtained results, as mentioned before. Firstly, to confirm the presence of the detected metabolites, e.g., by providing calibration results, or additional experimental confirmation of the obtained metabolite structure, e.g., NMR. Secondly, to confirm the metabolites' activity by performing an antimicrobial test using whole and/or separated extracts.
In conclusion, I recommend that this article be accepted for publication in the PeerJ journal after the addition of supporting information regarding LC-MS calibration, the original LC-MS data, and the performance of antimicrobial tests using the obtained extracts.
In-text comments:
Line 120: Please explain how Surface sterilization effectiveness was confirmed.
Line 139: Please use rcf instead of rpms
Line 155: Please do not use the whole URL address; https://blast.ncbi.nlm.nih.gov is enough, but please add the access date
Line 172: Did you perform calibration, and if yes, please indicate how.
Line 303: Suggest rearranging supplementary tables B1-6 to begin with the compound name (indicate which isolates are confirmed producers, and later proposed properties, as it better reflects the content of this manuscript, as the compounds were detected, and the literature search was for possible application, not vice versa.
329: Has this been evaluated? Generally, there are few studies dedicated to evaluating chemicals against plant beneficial microorganisms. If a study is based solely on a literature review, the likelihood of finding such activity is low.
The manuscript is generally well-structured and follows a logical scientific format. The introduction effectively sets the stage by providing relevant background on mangrove ecosystems, endophytic fungi, and P. noxium as a pathogen.
Suggestions for Improvement:
1. Line 291 states: "Bioactive compounds produced by EFs have attracted significant attraction" which should read "attracted significant attention."
2. Figure 1 (colony morphology) would benefit from higher resolution images. While your descriptions of morphological differences (smooth transitions vs. concentric circles, brown dot sizes) are detailed in the text, the current resolution makes it difficult for readers to visually verify these observations. Clearer images would significantly strengthen the morphological evidence supporting your conclusions.
3. It would be helpful to briefly explain why five mangrove species were sampled but only three P. noxium isolates were selected for detailed analysis. What criteria guided your selection of these particular isolates from presumably more initial endophytic fungi?
The research question is clearly defined and addresses an important knowledge gap—whether P. noxium, a well-documented pathogen, could exist as an endophyte in mangrove ecosystems and what chemical profile it might exhibit in this context. The methods you've employed (DNA barcoding, phylogenetic analysis, and LC-MS/MS) are appropriate and rigorous for addressing this question. I particularly appreciate your deposition of sequences in GenBank (PQ821345, PQ821348, PQ821349), which enhances transparency.
Suggestions for Improvement:
1. The authors mention using Hamzah et al. (2018) with "minor modifications" for surface sterilization—could the authors please specify what these modifications were? This detail is crucial for evaluating the effectiveness of your endophyte isolation protocol.
2. It would be helpful to clarify whether your LC-MS/MS analyses were performed with biological and/or technical replicates, and if so, how many. If replicates weren't used, a brief acknowledgment of this limitation would be appropriate.
3. Could the authors briefly describe the confidence level applied for compound identification (e.g., Level 2 [library spectrum match] for entries matched against mzCloud)? This would help readers assess the reliability of your chemical profiling.
The conclusions are generally well-supported by the data presented.
Suggestions for Improvement:
1. The identification of compounds with potential therapeutic applications is one of the most exciting aspects of the study. To ensure absolute clarity and prevent any potential misinterpretation, please reframe statements about bioactivities to emphasize that these are literature-based predictions rather than experimentally validated activities within this specific study. For example, instead of "identified compounds from the P. noxium isolates may possess," consider phrases like "literature suggests compounds identified in P. noxium may possess" or "compounds identified in P. noxium have been previously reported to possess."
2. In the discussion of biological control implications (lines 329-331), the statement that "P. noxium does not appear to produce bioactive compounds that inhibit the growth of Trichoderma sp. and Streptomyces sp." could be phrased more precisely as "Our chemical profiling did not identify compounds known to target Trichoderma or Streptomyces..." This would more accurately reflect the scope of the methodology.
3. For the Jaccard Similarity Index in Table 3, please clarify whether this was calculated from presence/absence data or abundance-weighted data. If no replicates were used in this calculation, a single sentence acknowledging that this provides a preliminary similarity measure would be sufficient and intellectually honest.
4. The speculation about geographical clustering in the phylogeny (lines 253-255) is interesting. Consider softening the language here (e.g., "may suggest," "could be influenced by") to reflect the preliminary nature of this observation based on a limited number of sequences.
5. A brief explanation of why AA2AA and SA2AA show greater similarity despite coming from different host species (beyond just geographic proximity) would strengthen the interpretation of the results.
This work represents a genuinely novel contribution to fungal ecology that challenges our understanding of P. noxium as solely a pathogen. The authors' discovery of its endophytic lifestyle in mangroves opens exciting new research directions with potential implications for both natural product discovery and pathogen management strategies.
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