Multifunctional Endophytic Fungi from Ginger (Zingiber officinale) with Antimicrobial, Enzymatic, and Antioxidant Potential

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this work, the authors isolated endophytic fungi from ginger (Zingiber officinale). The antifungal and antibacterial activities of these fungi were analyzed. Also the production of hydrolytic enzymes and antioxidant properties were evaluated. It is an interesting research work, but is not novel. There are several works reporting endophytic fungi from ginger with different biological activities.

 

General comments

The manuscript needs improvement in terms of the description of the methodology, presentation, and analysis of the results. The number of replicates for each experiment should be mentioned. In antifungal experiments, a number of replicates of two was done, which is insufficient. The discussion should be focus on its results; there is much irrelevant information.

 

Specific comments 

1. The title should be modified so that it is more related to the research work carried out.
2. The name of microorganisms must be in cursive
3. Nomenclature of nine endophytes (J34, J35, etc) is not clear, it could be eliminated
4. Table 2, antifungal activity, Are the showed results the mean of two replicates and the standard deviation?.
5. The results shown in Table 3 are not understood, table 3 must be eliminated as well the methodology and result description related with this experiment.
6. Table 6: concentration of the extracts must be indicated
7. The discussion should be rewritten, without adding irrelevant information and ambiguous phrases as “This suggests that the mechanisms behind fungal inhibition may be more effective or diverse against cinerea, possibly due to the differences in pathogenicity and resistance 336 profiles between these fungi” (lines 335-336)

Author Response

We sincerely thank Reviewer 1 for the time and effort dedicated to evaluating our manuscript and for providing constructive and insightful comments that have helped us improve its quality. We have carefully addressed all the points raised, revising the text accordingly to enhance its clarity, precision, and scientific rigor. We truly appreciate the reviewer’s contribution to refining our work. We hope that this revised version, which also incorporates the modifications suggested by the other reviewers, will now meet the expectations of both the reviewer and the editorial board, and that it will be favourably considered for publication in Agronomy.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript has written well. Authors have clearly described the research content. However, the manuscript needs revision for better clarity

1.Revise abstract. Abstract should reveal findings of the study with clear results 

2.Introduction part should  be revised. Add the importance of microbes and microbial metabolites associated with diverse sources for various applications in a brief manner 

cite the following

 https://doi.org/10.1007/s00284-025-04379-8 

 https://doi.org/10.1007/s13205-025-04396-1 

 https://doi.org/10.1016/j.bbrc.2025.152730 

 https://doi.org/10.1016/j.bcab.2024.103034 

 https://doi.org/10.1016/j.bcab.2025.103714 

What are the standards authors used in the bioassays

Results should be described with suitable recent findings

Conclusion part should reveal challenges and future prospects 

Author Response

We sincerely thank Reviewer 2 for the careful evaluation of our manuscript and for the thoughtful and constructive comments provided. We have carefully considered each of the reviewer’s suggestions and revised the manuscript accordingly to improve its clarity, coherence, and scientific depth. The introduction has been updated with recent and relevant references, the results and conclusions have been refined to better highlight the study’s significance, and additional clarifications have been incorporated where necessary. We greatly appreciate the reviewer’s contribution to strengthening our work and believe that the revised version now presents a clearer and more comprehensive manuscript that meets the standards of Agronomy.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript being reviewed explores the isolation and identification of endophytic fungi with potential biocontrol activity. Although the content of the paper fits within the size of the journal, the research, in its present form, is insufficient to be published. The study merely describes isolation, identification, and the initial in vitro biocontrol tests. These measures constitute a preliminary screening and are not novel enough, or deep enough, to be published in this journal.

No statistics or illustrations have been given to support the endophyte biocontrol activity. The reported findings need to be ascertained by the figure images. Necessary tests, including disc diffusion tests and in vivo (pot or greenhouse) experiments, are missing. In the absence of these, the biocontrol agent functional validation of the isolates is not complete. These antagonistic endophytes have been described in detail at the time of isolation and identification.

This article does not offer any new understanding or innovative input to the field.

 

Comments on the Quality of English Language

Dear Editor

The manuscript being reviewed explores the isolation and identification of endophytic fungi with potential biocontrol activity. Although the content of the paper fits within the size of the journal, the research, in its present form, is insufficient to be published. The study merely describes isolation, identification, and the initial in vitro biocontrol tests. These measures constitute a preliminary screening and are not novel enough, or deep enough, to be published in this journal. No statistics or images have been given to support the endophyte biocontrol activity. Necessary tests, including disc diffusion tests and in vivo (pot or greenhouse) experiments, are missing. In the absence of these, the biocontrol agent functional validation of the isolates is not complete. These antagonistic endophytes have been described in detail at the time of isolation and identification. This article does not offer any new understanding or innovative input to the field. The paper requires significant improvement in scientific writing, structure, and clarity. It is below the standard of the journal in its present form.

Author Response

We sincerely thank Reviewer 3 for the time and effort devoted to evaluating our manuscript and for the constructive feedback provided. We appreciate the reviewer’s critical assessment and have carefully considered all the points raised. In this revised version, we have improved the structure, strengthened the discussion, clarified the novelty and scope of the study, and incorporated additional materials (including raw experimental data in .xlsx format and a new figure illustrating antifungal activity assays). We hope that these improvements address the reviewer’s concerns and demonstrate the scientific relevance and robustness of our work.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

This is a well-structured and comprehensive study that explores the biotechnological potential of endophytic fungi isolated from ginger tubers. The authors successfully isolate, identify, and characterize nine fungal strains, evaluating their antimicrobial, enzymatic, and antioxidant activities. The work is relevant to the fields of sustainable agriculture, biocontrol, and industrial biotechnology. 

Major Comments

1. Fungal Identification and Phylogenetic Analysis

• The use of both morphological and molecular methods (ITS and 28S rRNA) for identification is appropriate and strengthens the taxonomic assignments. However, the phylogenetic trees (Figure 1) are difficult to interpret due to poor resolution and labeling. The inclusion of bootstrap values is essential to support the clade's credibility.

2. Antagonistic Assays

• The dual-culture assay for antifungal activity is standard and well-executed. The high inhibition of B. cinerea by P. melinii (J47) is noteworthy. However, the antibacterial assay on bacterial lawns is qualitative and lacks quantitative rigor. The use of terms like “++” and “+” is subjective and not standardized.

3. Enzymatic Activity

• The use of the Enzyme Index (EI) is appropriate, but the authors should clarify why an EI > 2 was considered “significant,” as this threshold is not universally accepted. The enzymatic profiles are well-documented, but the ecological or functional implications of these activities in the ginger host are not discussed.

4. Antioxidant and Antibacterial Assays with Extracts

• The DPPH assay is standard, and the results are clearly presented. The strong antioxidant activity of P. melinii and P. cucumerina is a highlight. The antibacterial assay using crude extracts is well-performed, but the lack of activity against K. pneumoniae is not sufficiently explored.

Minor Comments

1. Introduction: The introduction is thorough but could be more focused. The historical background on endophytes is lengthy; consider condensing and emphasizing the novelty of studying ginger-associated endophytes.
2. The discussion is comprehensive but somewhat repetitive. Consider reorganizing to highlight the most novel findings (e.g., the antioxidant activity of P. cucumerina). The comparison with previous studies is good, but more emphasis should be placed on the unique contributions of this work.
3. Methods: The incubation temperature for antibacterial assays (25°C) is low for bacterial growth. Justify this choice or repeat at 37°C for clinical relevance.
4. The manuscript is generally well-written, but there are occasional grammatical errors and awkward phrasings (e.g., “fungal candidates suitable for using it”).

Author Response

We sincerely thank Reviewer 4 for the time and effort devoted to evaluating our manuscript and for providing detailed and constructive comments that have significantly contributed to improving its quality. We have carefully addressed each of the points raised, revising the text to enhance its scientific accuracy, methodological clarity, and linguistic precision. We deeply appreciate the reviewer’s insightful feedback, which has helped us refine the manuscript and strengthen the presentation of our results.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All the comments to improve the manuscript were incorporated by the authors

Author Response

We sincerely thank the reviewer for their positive evaluation and kind appreciation of our work. We are pleased that the revisions and improvements incorporated in the manuscript have met their expectations. We truly value their time and consideration in reviewing our study.

Reviewer 2 Report

Comments and Suggestions for Authors

Revised Manuscript has been accepted for publication as the authors revised the manuscript according to all the comments 

Author Response

We sincerely thank the reviewer for their positive assessment and for acknowledging the improvements made to the manuscript. We are very grateful for their time and constructive feedback throughout the review process, and we are pleased that the revised version meets their approval for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors did not perform greenhouse or other functional validation of the isolates, though they could revise this. The article does not add anything new or innovative to the field.

Author Response

We sincerely thank the reviewer for taking the time to re-evaluate our revised manuscript and for sharing their additional observations. We fully understand the concern regarding the absence of greenhouse or other in vivo validation experiments. As previously stated, this work was conceived as an initial screening and characterization study, aimed at identifying promising endophytic fungal isolates with potential biotechnological applications. The subsequent functional validation of selected strains under greenhouse or field conditions constitutes an important next step, which we have explicitly acknowledged as part of our future research objectives in the Conclusions section.

Regarding the reviewer’s remark on novelty, we respectfully emphasize that the revised version now clearly outlines the innovative aspects of our study, including (i) the unique geographic origin of the isolates (southern Europe, where ginger endophytes have not been previously reported), (ii) the identification of uncommon fungal taxa such as Plectosphaerella cucumerina and Pseudogymnoascus pannorum as ginger endophytes, and (iii) the integrated evaluation of multiple bioactivities (antifungal, antibacterial, enzymatic, and antioxidant) from the same set of isolates. We believe these features together provide a meaningful contribution to the current understanding of ginger-associated endophytes and their multifunctional potential.

We appreciate the reviewer’s continued attention and trust that the clarified scope and demonstrated improvements will help convey the scientific relevance and originality of this work.