Physiological, Biochemical, and Transcriptome Analyses Reveal the Potential Role of ABA in Dufulin-Induced Tomato Resistance to Tomato Brown Rugose Fruit Virus (ToBRFV)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General Assessment

The manuscript investigates the physiological, biochemical, and transcriptomic responses of tomato to Dufulin treatment during ToBRFV infection. The topic is timely, and the dataset is extensive; however, several issues related to citation accuracy, methodological completeness, data interpretation, and overextension of conclusions must be addressed before the work can be considered for publication. The manuscript has great potential, but substantial revisions are required to ensure scientific rigor, reproducibility, and alignment between data and interpretation.

Major Comments

1. Citation Format and Reference Accuracy

The Introduction contains inconsistent citation formatting. The sentence (line 40) referencing FAOSTAT mixes narrative citation, a URL, and numerical references, which is not compatible with MDPI’s numbered citation style. The FAOSTAT entry should be converted into a formal numbered reference and moved to the reference list.
Reference 15 is cited as “in press,” although it has already been published; this entry should be updated with complete publication details.
Reference 19 is not appropriate for supporting the statement about Dufulin’s toxicity and environmental behavior. This reference concerns synthesis and antitumor activity of aminophosphonates and does not address Dufulin’s ecological safety. A correct supporting reference should be provided.

The citations used to support WRKY–W-box binding specificity are inappropriate in the Discussion section (line 318). Reference 28 concerns bZIP/ABRE binding, and Reference 29 does not discuss WRKY transcription factors. These should be replaced with primary literature that specifically documents WRKY–W-box binding.

All citations in the text must be checked to avoid inaccuracies in the final version.

2. Excessive and Inconsistent Use of Abbreviations

The Abstract contains numerous abbreviations (SA, ABA, SOD, POD, CAT, etc.) that are not defined at first mention. Several abbreviations appear only once and do not benefit the reader.
The main text also contains inconsistencies, such as defining IAA only after its first use.
The manuscript should be revised to define only essential abbreviations at first mention, avoid abbreviations that appear only once, and apply consistent definitions throughout the text.

3. Insufficient Methodological Detail Affecting Reproducibility

The Materials and Methods section lacks critical detail necessary for replication. Essential information is missing from multiple subsections (Virus inoculation, Disease severity index, Biochemical and physiological assays, Hormone profiling, etc). Many of these procedures are cited from closed-access articles, which reviewers and readers cannot verify. All essential methodological details must be embedded directly in the manuscript.

4. Interpretation of Results Exceeds the Evidence Presented

Some interpretations in the Results and Discussion would benefit from greater caution. The suggestion that Dufulin enhances photosynthetic capacity is not yet supported by functional measurements; although pigment and transcript changes are informative, they do not directly demonstrate improved photosynthesis. Similarly, the conclusion that ABA is the primary pathway mediating resistance relies on correlative transcriptomic data. The notable increase in SA and the possibility that ABA elevations may reflect stress responses warrant further consideration. Finally, the proposed roles of SlABI5-like and SlWRKY4 are based on WGCNA correlations and promoter motif analyses. Without functional assays, these transcription factors are best described as potential or hypothesized regulators.

5. Need for Critical Experimental Controls

A major limitation of the study is the absence of a Dufulin-only control group. Without this control, it is impossible to determine whether the observed changes—including increases in pigments, antioxidant enzyme activities, and agronomic traits—reflect antiviral resistance or simply general growth promotion by Dufulin. Data presented in Supplementary Figures S1 and S2 further highlight this ambiguity. This omission substantially limits the mechanistic conclusions and should be addressed in the revision, at minimum through discussion and careful reinterpretation.

6. Statistical Analysis

The statistical analysis relies solely on pairwise Student’s t-tests across numerous traits and time points, without appropriate ANOVA models or correction for multiple comparisons. This greatly increases the risk of Type I statistical error and limits confidence in the reported physiological and hormonal effects. The statistical analysis should be strengthened.

Overall Recommendation

The manuscript contains valuable findings and a promising dataset, but several critical issues must be addressed before the work is publishable. A major revision is necessary to strengthen the manuscript’s scientific rigor, transparency, and clarity. With careful revision and a more cautious interpretation of the results, the study has the potential to make a meaningful contribution to understanding antiviral resistance mechanisms in tomato.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, below are my suggestions and comments:
1. Please note that in lines 51-52 you state: “To date, relatively few reports exist on chemical agents for ToBRFV management,” yet you only refer to studies conducted in China. Please add citations from other parts of the world.

2. Please refer in the introduction to other substances used to manage ToBRFV and explain the advantages of Dufulin over these substances.

3. Add molecular studies indicating the genes that have been implicated in the plant response to the presence of Dufulin, such as Harpin Binding Protein 1 (Hrbp1) and OsJAZ5.

4. Please add the RNA concentrations used in methodology 2.4 and 2.9.

5. I suggest that the discussion be integrated into a single section. In addition, I believe that an introductory paragraph is needed, as it goes straight into discussing SOD induction, leaving aside the discussion of the results presented in Figure 1.

6. Verify that scientific names are in italics.

7. I believe that the discussion needs to be restructured and integrated. As it stands, it is very difficult to follow the results obtained and view them as a whole. I also recommend improving the conclusions, as they were written in a very simple manner.

 

Comments on the Quality of English Language

It needs improvement.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors report their investigation into Dufulin-Induced Tomato Re- 3
sistance to Tomato Brown Rugose Fruit Virus (ToBRFV). The Results read pretty clearly and overall the work looks good and will be beneficial to the community. But, I recommend some changes before publication because there are some weak areas:

(1) Methods too sparse: the authors need to ensure experimental reproducibility and spatial-temporal clarity. So, the exact "chain of custody" for the virus—moving from the initial Agrobacterium infiltration to the mechanical sap inoculation—and providing specific metrics for chemical treatments, such as the exact volume of Dufulin sprayed per plant needs to be done. The sampling protocol must explicitly link the specific leaf position (e.g., the third true leaf) and the exact time points (1, 3, or 7 dpi) to each analytical platform, ensuring readers know exactly which tissues were used for the transcriptome versus the hormone profiling.

Additionally, the technical descriptions must describe the fundamental laboratory parameters. This includes reporting the precise amount of fresh tissue weight used for extractions, the specific spectrophotometric wavelengths (nm) used for enzyme assays, and the exact hardware specifications for high-throughput sequencing, such as the Illumina platform model and read length.

(2) Figures blurry/text too small. There are a lot of 'big data' figures that are not well-presented. Ensure only high-resolution graphics with legible text are used.

Comments on the Quality of English Language

There are a large number of typos, such as missed spaces between sentences. Also check supplementary writing.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript addresses a relevant and timely topic by examining Dufulin-induced resistance to Tomato brown rugose fruit virus using a combination of physiological, biochemical, hormonal, and transcriptomic approaches. The dataset is extensive, and the revised version demonstrates that the authors have made a genuine effort to improve the manuscript.
The revision has improved methodological transparency, corrected citation and formatting issues, and strengthened the statistical analysis. These changes enhance the technical quality and reproducibility of the study. The transcriptomic data and WGCNA analysis are competently executed and provide a useful resource for future investigations.
Nevertheless, some key limitations remain. Most notably, the absence of a Dufulin-only control restricts the interpretation of many observed effects and prevents clear separation of antiviral resistance from general growth-promoting responses. In addition, conclusions regarding the central role of ABA signaling and the regulatory functions of SlABI5-like and SlWRKY4 remain largely correlative and should be interpreted with appropriate caution. While these limitations do not invalidate the study, they require that the mechanistic claims remain conservative and clearly framed as hypotheses rather than demonstrated pathways. Further minor refinement of language and consistency in cautious interpretation would strengthen the final presentation.

Comments on the Quality of English Language

The English is generally understandable, but the manuscript requires language polishing, particularly to reduce overstatement, improve idiomatic phrasing, and ensure consistent, cautious interpretation. 

Author Response

Comments 1: 

The manuscript addresses a relevant and timely topic by examining Dufulin-induced resistance to Tomato brown rugose fruit virus using a combination of physiological, biochemical, hormonal, and transcriptomic approaches. The dataset is extensive, and the revised version demonstrates that the authors have made a genuine effort to improve the manuscript. The revision has improved methodological transparency, corrected citation and formatting issues, and strengthened the statistical analysis. These changes enhance the technical quality and reproducibility of the study. The transcriptomic data and WGCNA analysis are competently executed and provide a useful resource for future investigations. Nevertheless, some key limitations remain. Most notably, the absence of a Dufulin-only control restricts the interpretation of many observed effects and prevents clear separation of antiviral resistance from general growth-promoting responses. In addition, conclusions regarding the central role of ABA signaling and the regulatory functions of SlABI5-like and SlWRKY4 remain largely correlative and should be interpreted with appropriate caution. While these limitations do not invalidate the study, they require that the mechanistic claims remain conservative and clearly framed as hypotheses rather than demonstrated pathways. Further minor refinement of language and consistency in cautious interpretation would strengthen the final presentation.

Response: Many thanks for your great comments. According to your suggestions, the results we obtained in the absence of Dufulin-only control were described and summarized more carefully to make our study more rigorous. In addition, the role of ABA signaling and the functions of SlABI5-like and SlWRKY4 have been interpreted with appropriate caution in this revised manuscript (lines 24-34; lines 204-210; lines 308-313; lines 504-510).

Comments 2: 

The English is generally understandable, but the manuscript requires language polishing, particularly to reduce overstatement, improve idiomatic phrasing, and ensure consistent, cautious interpretation.

Response: Many thanks for your comments. We have improved the language of this manuscript again (highlighted in yellow).

Greatly appreciate again!

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for addressing the suggestions. For my part, your manuscript is accepted. I only ask that you check that all gene names are in italics.

Comments on the Quality of English Language

Ok

Author Response

Comments 1: Comments and Suggestions for Authors

Dear authors,

Thank you for addressing the suggestions. For my part, your manuscript is accepted. I only ask that you check that all gene names are in italics.

Response: Many thanks for your comments. According to your comments, we have checked that all gene names are in italics.

Comments 2: Comments on the Quality of English Language: Ok.

Response: Thank you very much for your comments.