TIDRE: An Efficient Tool for Isolating Antagonistic Endophytes and Screening Resistant Plants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled, “TIDRE: An Efficient Tool for Isolating Antagonistic Endophytes and Screening Resistant Plants” presents a novel and practically relevant methodology (TIDRE) for targeted isolation of disease-resistant endophytes and screening resistant plant materials. The study is comprehensive, methodologically sound, and supported by relevant examples (Panax notoginseng and grapevine). However, following comments must be addressed before acceptance of the MS.

• The TIDRE approach is presented as innovative, however, it does not sufficiently distinguish it from existing tissue-based antagonism screening or duel-culture assays. Add a concise comparison with established methods e.g., tissue imprinting, enrichment-based isolation in introduction to strengthen the claim.
• In materials and methods: add details of Type of statistical tests applied, Data normality checks, Post-hoc tests used for multiple comparisons etc.
• The correlation between plant health and antagonistic activity is overstated. Authors have reported differences between healthy and diseased leaves but no formal correlation or regression analysis is presented. The conclusion about plant health and antagonistic potential must be either statistically supported or rephrased.
• The use of three plants per health category (pseudo-ginseng) may not adequately reveal biological variability. This limitation must be mentioned in the discussion section.   
• This study relied on in vitro antagonism assays. If possible add at least, preliminary in planta validation to strengthen the practical applicability of TIDRE approach.
• The statements suggesting direct application of TIDRE in resistance breeding and PEM technology are speculative. It must be toned down or stated as future potential rather than demonstrated outcomes.
• Several sentences require grammatical correction and improved clarity (e.g., “Furtherly, we identified…” should be replaced with “Furthermore, we identified”). A thorough language edit is recommended.
• Terms such as “disease-resistant endophytes,” “antagonistic endophytes,” and “resistant endophytes” are used interchangeably. Standardizing terminology throughout the manuscript will improve clarity.
• Some figure legends (e.g., Figures 2 and 4) lack sufficient methodological details to be understood independently. Legends should clearly define experimental conditions, sample size, and statistical indicators.
• Sections 4.1 and 4.3 partially repeat known concepts of endophyte–plant–pathogen interactions. These sections could be streamlined to focus more on insights specific to TIDRE outcomes rather than general background.
• References should be formatted consistently according to the journal’s guidelines.
• The antagonism values in Table 3 appear to be ratios rather than percentages, but this is not clearly stated. Units and calculation methods should be clarified in the table caption.

 

Comments on the Quality of English Language

• Several sentences require grammatical correction and improved clarity (e.g., “Furtherly, we identified…” should be replaced with “Furthermore, we identified”). A thorough language edit is recommended.

Author Response

Dear editor and reviewers,

The authors are greatly appreciated for those valuable comments, which were all helpful to improve our manuscript. The present version has been revised according to comments, and the point to point response to comments was attached as followed:

Response to Reviewer 1’ Comments

Comments 1: Comments and Suggestions for Authors

The manuscript entitled, “TIDRE: An Efficient Tool for Isolating Antagonistic Endophytes and Screening Resistant Plants” presents a novel and practically relevant methodology (TIDRE) for targeted isolation of disease-resistant endophytes and screening resistant plant materials. The study is comprehensive, methodologically sound, and supported by relevant examples (Panax notoginseng and grapevine). However, following comments must be addressed before acceptance of the MS.

Response 1: Thank you for your thoughtful and constructive comments on our manuscript. We have carefully considered your suggestions and will address each point in detail in the revised version of the manuscript.

We appreciate your positive assessment of the work and the opportunity to improve it. All specific responses to your comments will be clearly outlined in the revised manuscript and the point-by-point response letter.

Comments 2: The TIDRE approach is presented as innovative, however, it does not sufficiently distinguish it from existing tissue-based antagonism screening or duel-culture assays. Add a concise comparison with established methods e.g., tissue imprinting, enrichment-based isolation in introduction to strengthen the claim.

Response 2: We thank the reviewer for the valuable suggestion. To better highlight the novelty of TIDRE, we have added a concise comparison with established methods (e.g., tissue imprinting and dual-culture assays) in the Introduction. The revisions clarify that, unlike conventional sequential workflows, TIDRE integrates pathogen-specific selection directly into the initial isolation step, enabling targeted enrichment of antagonistic endophytes. We believe this strengthens the claim regarding the method's innovation.

Comments 3: In materials and methods: add details of Type of statistical tests applied, Data normality checks, Post-hoc tests used for multiple comparisons etc.

Response 3: We have revised the “Materials and Methods” section (now lines 167-173) to include the requested details on the specific statistical tests applied, data normality checks, and post-hoc tests used for multiple comparisons. Thank you!

Comments 4: The correlation between plant health and antagonistic activity is overstated. Authors have reported differences between healthy and diseased leaves but no formal correlation or regression analysis is presented. The conclusion about plant health and antagonistic potential must be either statistically supported or rephrased.

Response 4: Upon careful reconsideration, we agree that the original wording could overstate the relationship between plant health and antagonistic activity in the absence of formal correlational analysis. Accordingly, we have revised the relevant passages (e.g., lines 188 and 309 in the revised manuscript) to present the observations in a more cautious and descriptive manner, avoiding causal or strongly correlative language.

Comments 5: The use of three plants per health category (pseudo-ginseng) may not adequately reveal biological variability. This limitation must be mentioned in the discussion section.

Response 5: Thank you very much for this suggestion. We acknowledge that the preliminary comparison based on three plants per health category may not fully encompass the natural biological variability within P. notoginseng populations. In response to your suggestion, we have added a discussion of this limitation in the revised manuscript, noting that future studies with larger sample sizes are warranted to confirm and generalize the observed trends. We appreciate this comment, which has helped us present a more balanced and critical perspective on our findings.

Comments 6: This study relied on in vitro antagonism assays. If possible add at least, preliminary in planta validation to strengthen the practical applicability of TIDRE approach.

Response 6: This is indeed a limitation of our research; the current state cannot provide validation results at the plant level. However, our previously published results (Guo et al., 2024) may provide indirect validation. In that study, grape cuttings containing pathogen-antagonistic endophytic bacteria were found to exhibit significantly enhanced disease resistance at both the in vitro leaf and grapevine plant levels. We have included the relevant discussions in this revision. Thank you!

Comments 7: The statements suggesting direct application of TIDRE in resistance breeding and PEM technology are speculative. It must be toned down or stated as future potential rather than demonstrated outcomes.

Response 7: Thank you for this constructive suggestion. We agree that the claims regarding the direct application of TIDRE should be presented more cautiously. Accordingly, we have revised the relevant statements throughout the manuscript (e.g., in the revised lines 78, 162, 240, 361, and 390) to tone down definitive assertions and reframe them in terms of future potential and methodological promise, rather than as demonstrated outcomes.

We believe these edits have improved the objectivity and rigor of the manuscript.

Comments 8: Several sentences require grammatical correction and improved clarity (e.g., “Furtherly, we identified…” should be replaced with “Furthermore, we identified”). A thorough language edit is recommended.

Response 8: We thank the reviewer for highlighting the need for language improvements. We have conducted a thorough edit of the manuscript to correct grammatical errors, enhance clarity, and ensure consistent academic phrasing. Specific issues, such as the use of "Furtherly," have been corrected (e.g., to "Furthermore"). We believe the revised text is now clearer and more professionally presented.

Comments 9: Terms such as “disease-resistant endophytes,” “antagonistic endophytes,” and “resistant endophytes” are used interchangeably. Standardizing terminology throughout the manuscript will improve clarity.

Response 9: We thank the reviewer for raising this important point regarding terminology. We agree that consistent and precise terminology is crucial for clarity. In the revised manuscript, we have carefully standardized the use of these terms to accurately reflect their distinct meanings. Specifically, the term "disease-resistant endophytes" is now used to describe the functional outcome for the host plant (i.e., conferring disease resistance), "antagonistic endophytes" specifies those with a direct inhibitory mechanism against pathogens, and the ambiguous use of "resistant endophytes" has been corrected accordingly.

Comments 10: Some figure legends (e.g., Figures 2 and 4) lack sufficient methodological details to be understood independently. Legends should clearly define experimental conditions, sample size, and statistical indicators.

Response 10: Thank you for pointing out the need for greater detail in the figure legends. In response to your comment, we have thoroughly revised the legends for Figures 2 and 4 by adding essential methodological information, including specific experimental conditions, sample sizes (replicates), and clear descriptions of statistical indicators.

These additions ensure that each figure can now be understood independently, significantly improving the clarity of the presented data. The updated legends are provided in the revised manuscript.

Comments 11: Sections 4.1 and 4.3 partially repeat known concepts of endophyte–plant–pathogen interactions. These sections could be streamlined to focus more on insights specific to TIDRE outcomes rather than general background.

Response 11: Thank you for your comment. We have reorganized, condensed, and revised the Discussion section accordingly to focus more on insights specific to the TIDRE outcomes.

Comments 12: References should be formatted consistently according to the journal’s guidelines.

Response 12: Thank you for your reminder. We have carefully checked and revised the reference list throughout the manuscript to ensure that all citations now conform consistently to the journal’s formatting guidelines.

Comments 13: The antagonism values in Table 3 appear to be ratios rather than percentages, but this is not clearly stated. Units and calculation methods should be clarified in the table caption.

Response 13: Thank you for your comment. The values in the original Table 3 represent the inhibition rates (%) of the target antagonistic strains against the pathogens. In response, we have revised the captions for both Table 2 and Table 3 to explicitly state that the values are presented as inhibition rates, thereby clarifying the units and avoiding potential misunderstanding.

Reviewer 2 Report

Comments and Suggestions for Authors

Please see comments (attached docs)

Comments for author File: Comments.pdf

Author Response

Dear editor and reviewers,

The authors are greatly appreciated for those valuable comments, which were all helpful to improve our manuscript. The present version has been revised according to comments, and the point to point response to comments was attached as followed:

Response to Reviewer 2’ Comments

Comments 1: Section 2.1

Line 85: Any NCBI coded for these isolates?

Response 1: The fungal isolates used in this study were all conventional pathogenic strains isolated, purified, and identified by our laboratory. They were not specifically assigned NCBI (e.g., GenBank) accession numbers during the initial phase of this work. However, these strains are available from the corresponding author upon reasonable request. We appreciate your comment, which helps improve the clarity of our manuscript.

Comments 2: Section 2.2

Line 94: Include also parts where leaf samples were collected

Line 98: Days old of plants sampled and what specific plant parts where leaves were sampled?

Response 2: We sincerely thank you for these constructive suggestions aimed at improving the methodological clarity of our manuscript. In response to your comments, we have revised Section 2.2 accordingly. Specifically, at line 94, we have clarified the Panax notoginseng leaves were randomly sampled. Furthermore, at line 99, we have added the grapevine cuttings age and a detailed description of the specific leaf parts sampled. We believe these additions enhance the precision and reproducibility of the methods described.

Comments 3: Section 2.3

Line 105: From what specific parts

Line 113: Describe properly  Any reference if there are?

Line 118: Any reference as a form of validation on these methods?

Response 3: Thank you for your valuable comments regarding the clarity and rigor of the methodology in Section 2.3. We have revised the manuscript accordingly to address each point raised:

Regarding the specific plant parts sampled (Line 105), this detail has been comprehensively described in the revised Section 2.2.

Concerning the sterilization procedure (Line 113), we have expanded the description to provide a complete and precise account of the steps. Furthermore, we have added Reference [21] to cite the established protocol upon which our method was based.

To validate the methods in question (Line 118), we have included Reference [22] in the revised manuscript to support the methodological approach.

We believe these revisions have significantly enhanced the clarity, reproducibility, and scholarly foundation of the methods section. Thank you for the constructive feedback.

Comments 4: Section 2.4

Line 132: How? Any technical method to describe this?

Line 134: Provide reference for this?

Response 4: We appreciate the reviewer's attention to methodological rigor. Regarding the queries, we wish to clarify that the techniques mentioned (aseptic isolation and antagonism co-culture) are fundamental and widely adopted procedures in this field. In response to the request for a reference, we have added a citation (Reference [25]) to a standard protocol that details these conventional steps.

Comments 5: Section 2.5

Reference, after genomic extraction prior to BLAST analysis, include details for clarity?

Response 5: Thank you for this constructive suggestion. As suggested, we have added detailed descriptions of the steps between genomic extraction and BLAST analysis. Specifically, we have included the procedures for PCR product verification (gel electrophoresis), purification, quantification, as well as post-sequencing sequence assembly, trimming, and quality control. These additions (highlighted in red in the revised manuscript) ensure the clarity and completeness of the methodology.

Comments 6: Section 2.6

Is this with the endophytes? How about if there are more than two endophytes present which is more potential compared to only 1 endophytes, if there are cases in your observation?

How many treatments then- using bacterial endophytes?

Response 6: Thank you for your question. We appreciate the opportunity to clarify our experimental design. In this stage of the study focusing on screening disease-resistant grapevine cuttings, we employed three common pathogenic fungi (Botrytis cinerea, Fusarium solani, and Fusarium graminearum) which had been previously isolated and identified in our laboratory. The primary objective was to evaluate the inherent disease resistance of different individual plant accessions by performing antagonistic co-culture assays between their leaf tissue segments and these three pathogens. This initial screening phase was designed to assess plant-level resistance and therefore did not involve the specific evaluation of effects attributable to single or multiple endophytes. We hope this clarification is helpful.

Comments 7: Figure 2: ok

Response 7: Thank you.

Comments 8:

Line 196: Describe properly in which how about if there are more than 1 bacterial endophytes in a certain plant samples, then how could affect the experimental research- let say 1 endophytes was used only?

Line 197: Explain in detail in the methodology how rigid and accurate in determining antagonistic effect? In real situation how about if a combine presence of multiple bacterial endophytes present and what happens to the resistance ability against fungal pathogens?

Figure 4: Provide specific treatments with known and identified bacterial endophytes with desired treatment combination (how about in some cases isolated bacterial endophytes present is more than 1 endophytes? Hence additional experiment with combined effect of isolated bacterial endophytes

Discussion 4.1-4.3: The effects of combined bacterial endophyte isolates against fungal pathogens exhibit different resistance responses compared to the use of a single isolate. Therefore, additional experiments are needed to validate whether using more than one bacterial endophyte can provide stronger evidence or a reliable benchmark demonstrating that bacterial endophyte consortia can enhance antifungal activity and potentially reduce the development of fungal resistance.

Response 8: We sincerely thank the reviewer for raising this important point regarding the potential effects of single versus multiple endophytes, which is indeed a valuable aspect we had not focused on initially.

In this study, when multiple bacterial morphotypes were recovered from an antagonistic tissue segment, our protocol was to purify each and conduct individual confirmatory bioassays to identify the active strain(s). We have clarified this step in the revised manuscript.

The reviewer's suggestion to investigate combined effects and synergies within endophyte consortia is an excellent direction for future research. While incorporating such a study was beyond the scope of the current work, we have acknowledged this limitation and highlighted the investigation of multi-strain interactions as a key future perspective in the revised Discussion section.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors present a time-efficient method for isolating fungal-antagonist endophytes from plant tissues. Essentially the method skips the process of isolating and generating pure cultures of endophytes which then go into antifungal bioassays. The method is a good concept.

I request that the authors remove the use of TIDRE 'technology'. No new technology or tools are presented. Rather, the authors have improved a method. I notice the authors do interchange the use of 'technology', 'method', 'protocol' and 'approach'. I suggest the authors use TIDRE method or TIDRE protocol.

 

Minor points-

• line 55-56 of the introduction. The authors use homogenisation and surface sterilization as examples of inefficient and lengthy processes, but the authors use both of these steps in their TIDRE method. Suggest the authors provided alternate examples such as purifying endophytes and generating isolates/strains with or without antifungal activity.
• Figure 2 panels e and f. Select one of panel e and f. I don't see what is different about them. Then the authors can enlarge one of these panels to make clearer to the reader the level of antifungal suppression.
• the Discussion is rather long for a methods paper. Suggest removing section 4.2 and condensing section 4.3.

Author Response

Dear editor and reviewers,

The authors are greatly appreciated for those valuable comments, which were all helpful to improve our manuscript. The present version has been revised according to comments, and the point to point response to comments was attached as followed:

Response to Reviewer 3’ Comments

Comments 1: The authors present a time-efficient method for isolating fungal-antagonist endophytes from plant tissues. Essentially the method skips the process of isolating and generating pure cultures of endophytes which then go into antifungal bioassays. The method is a good concept. I request that the authors remove the use of TIDRE 'technology'. No new technology or tools are presented. Rather, the authors have improved a method. I notice the authors do interchange the use of 'technology', 'method', 'protocol' and 'approach'. I suggest the authors use TIDRE method or TIDRE protocol.

Response 1: Thank you for your positive feedback and constructive suggestion regarding terminology. As recommended, we have replaced “TIDRE technology” throughout the manuscript with “TIDRE method” or “TIDRE protocol” and have ensured consistent use of these terms. This revision more accurately describes our work as an improved methodological procedure. We appreciate your helpful comment.

Minor points-

Comments 2: line 55-56 of the introduction. The authors use homogenisation and surface sterilization as examples of inefficient and lengthy processes, but the authors use both of these steps in their TIDRE method. Suggest the authors provided alternate examples such as purifying endophytes and generating isolates/strains with or without antifungal activity.

Response 2: The reviewer correctly points out the inconsistency in our original phrasing, which arose from imprecise wording. We have revised the sentence as suggested to more accurately reflect our intended meaning.

Comments 3: Figure 2 panels e and f. Select one of panel e and f. I don't see what is different about them. Then the authors can enlarge one of these panels to make clearer to the reader the level of antifungal suppression.

Response 3: We sincerely thank the reviewer for this valuable suggestion. In accordance with the comment, we have merged subpanels e and f in Figure 2 and have accordingly updated the figure legend to reflect the revised manuscript.

Comments 4: the Discussion is rather long for a methods paper. Suggest removing section 4.2 and condensing section 4.3.

Response 4: Thank you for your suggestion. We have revised and shortened the Discussion section accordingly.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors have addressed all the comments. The MS can be accepted for the publication.

Reviewer 2 Report

Comments and Suggestions for Authors

none- satisfied with the integration of suggestions

Comments for author File: Comments.pdf