From Early Signals to Systemic Decline: Physiological Defense Landscape of Agave tequilana in the Fusarium oxysporum Pathosystem

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Early and late defence responses of Agave tequilana to three Fusarium oxysporum strains of varying pathogenicity are examined in this manuscript. The study is ambitious and integrative, addressing a significant knowledge gap in the pathogenesis of agave wilt, a topic with evident agricultural, industrial, and scientific importance.

Overall, the manuscript contains extensive experimental data, is well-structured, and scientifically relevant. However, several major issues with clarity, data interpretation, experimental presentation, and writing quality need to be addressed before publication. Many sections require much deeper integration of results and better contextualisation within modern plant pathology literature. Figures and quantification methods need to be clarified. The discussion would greatly benefit from a mechanistic synthesis and a more cautious interpretation of results.

Major Comments
The paper offers valuable comparative insights into compatible and incompatible F. oxysporum interactions in A. tequilana, which are underrepresented in the literature to date. The inclusion of three strains is a strength.
However, the emphasis on novelty could be sharper:
What does this study reveal that was previously unknown about agave-Fusarium interactions?
Experimental Design:
The overall design is suitable, but the following methodological details need to be clarified:
Sample size justification is missing for all experiments. For the enzymatic assays and biochemical analyses, clarify whether these were biological or technical replicates. How many independent plants were used per treatment? The inoculation method and infection pressure should be explained more thoroughly. Does a 50-µL drop artificially exaggerate local infection? How does this compare to natural infection under field conditions? The early phase (24–72 hours) and late phase (1–30 days) are distinct conceptual entities, but no explanation is provided for the chosen time frames.
Data Presentation:
Figures 2 and 3 are mainly qualitative microscopy images. Including quantitative data (for example, % area stained, relative fluorescence intensity, and number of colonised cells) would provide stronger evidence for claims and enable statistical comparisons.
Results Interpretation:
Some interpretations are exaggerated or not sufficiently supported:
The claim of "Absence of an effective HR" in FPA is based solely on ROS diffusion and PCD spread. HR is a genetically programmed and highly regulated process, and absence cannot be concluded without testing HR-associated gene expression or confirming whether PCD is controlled rather than catastrophic.
The conclusion that pathogenic strains detoxify saponins is not proven, even if it seems plausible.
Growth in a saponin-containing medium does not prove enzymatic detoxification; fungi may tolerate but not degrade them.
Phenylpropanoid pathway “redirection” toward lignification
Activation of PAL alone does not establish the pathway for metabolic redirection. It would need measurements of either lignin precursors or lignin quantification.
The proposed mechanistic model is interesting but speculative in several aspects. The authors should differentiate between data-driven conclusions and hypotheses.

Literature Integration
The manuscript occasionally references outdated literature, such as the HR conceptual framework. Recent discoveries about Fusarium effectors, host-specificity mechanisms, and plant immune signalling could deepen the interpretation, including effectoromics, small RNAs, and cell wall integrity sensing. This discussion could also be enhanced by comparing it with monocot systems such as banana, onion, and date palm.

The overall manuscript is clear, but it requires substantial editing:
Some sentences are either overly long or repetitive.
Results occasionally duplicate information already shown in figures.
Additionally, sections would benefit from more explicit topic sentences and smoother transitions.

Recommendation
Major Revision. This manuscript is valuable and innovative, but it lacks explicit data quantification, deeper interpretation, tighter writing, and a more comprehensive comparison with the current literature. Addressing these points will significantly enhance the manuscript's scientific impact and clarity.

Author Response

Dear reviewer, thank you for your critical observations and thoughtful comments, which have enabled us to improve the quality of our work. The comments were reviewed, and an effort was made to respond to all of the reviewer's comments and questions. A PDF file with detailed responses to your comments is included.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

With a short introduction, the authors introduce the reader to the specifications of their research, especially pointing out certain gaps in existing knowledge – this is valuable for the reader's interest.

The work is interesting, but it needs to be refined.

Below are the notes:

I would suggest you consider changing the abbreviations, especially "dpi". For an average person, it is an abbreviation referring to optics and dramatically hinders reading.

The descriptions below the figures also require that there be fewer abbreviations. Then they become more legible. The drawing with the description should be separable beyond the text and still understandable, and unfortunately this cannot be said in the current situation.

It is also not entirely understandable to write " for the ... strain" "in FPA", etc. . All processes taking place in the cells of the plant are caused by the presence of the strain. Thus, the way of writing should be modified throughout the text.

Another linguistic misunderstanding, introducing a slight confusion in the reader. The use of the phrase "for 7 days" suggests an addition stretched over time. And it seems that the differences in growth over time were simply shown.

 

Line 348 – what does it mean that "was added to each strain"? Even if it is known what the authors mean, the protocol for obtaining a suspension of fungal spores is not correctly formulated. And why as much as 15 days. For a PDA, a week is enough to obtain a suspension of the order of 10⁸.

And about the same experience. Why wasn't the mushroom growth control from the extract done without cooking?

The authors based the discussion on 3 publications. This is understandable due to the research profile, but it seems that one could be tempted to try to develop it.

Author Response

Dear reviewer, thank you for your critical observations and thoughtful comments, which have enabled us to improve the quality of our work. The comments were reviewed, and an effort was made to respond to all of the reviewer's comments and questions. A PDF file with detailed responses to your comments is included.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Review of the manuscript plants-4037569 From Early Signals to Systemic Decline: Physiological Defense Landscape of Agave tequilana in the Fusarium oxysporum Pathosystem

This work addresses a topic of regional importance: the interaction of Agave tequilana with Fusarium oxysporum, which, in addition to its practical significance, also has significant scientific value. The manuscript presents extensive research using classical biochemical methods. A comparative method was used for the study, using three Fusarium strains with varying pathogenicity, allowing the authors to draw conclusions and construct a preliminary model of effective and ineffective protection.

However, I have several comments to make to improve the article (three major comments throughout the article and minor comments within sections).

Important comments:

1. Article title. I suggest replacing Decline with Answer, and Landscape with Scenario (the authors themselves use this word in the Discussion section, Line 303). Furthermore, a pathosystem is two organisms, usually written like this: in the Agave tequilana-Fusarium oxysporum Pathosystem
2. I recommend describing the strains used in the work more carefully and in detail (in the Materials and Methods section) and achieving consistency in their mention in the text. This is important for understanding your entire work. Confusion arises when the authors describe the FOL and FNPA strains. In the abstract (line 31), the FOL strain is a pathogenic host-specific strain of F. oxysporum f. sp. lycopersici (FOL). In the Results (line 89), the FOL strain is a tomato-specific pathogenic strain (FOL). (line 30) non-pathogenic strain (FNPA), (line 93) Plants inoculated with the non-agave pathogenic strain (FNPA). The discussion reads: (lines 234-235) The comparative approach employed—contrasting a compatible interaction (FPA) with two incompatible or maladaptive ones (FNPA and FOL). Thus, by the time the reader reaches the Discussion section, they understand that the two strains, FNPA and FOL, are non-agave pathogenic and cause an incompatible interaction. Agave tequilana is not a host for these strains. I recommend emphasizing this in the Abstract and Results.
3. The article's writing style and English grammar need correction. Throughout the text (especially in the Results section), slang-like expressions are used that are unscientific and mislead the reader. For example, lines 114-115: "In FOL, PCD was early and dispersed at 24 hpi, with a progressive increase at 48–72 hpi in cell groups distributed throughout the root." I recommend writing not "In FOL," but "In agave roots, infected with the FOL strain, PCD was detected already after 24 hpi…” or when infected with the FOL strain. Further in the text, lines: 106-114, 117-122, 133-142, 167-169, 185, 189, 194-203, 205-209, 250, 254-260, 263-270, 274-285, 289-292. Also in the text there are sentences with an unclear meaning. For example: lines 35-36 – “Cell wall strengthening occurred with all strains, highest with FOL. The resistance mechanisms showed differential responses between strains.” Cell wall strengthening was found in agave roots, plants responded differentially to different strains. Further in the text, lines: 46, 55-56, 105-106, 138-139, 162-165, 188-189, 197 (unacceptable: "TPC did not differ from the control until," acceptable: "content of TPC..."), 222-223 (what does deficient mean), 245-247, 304-305 ("early PR inhibition," better written: early inhibition of activity or inhibition of PR protein accumulation).

Notes on sections:

Abstract: I recommend rewriting the abstract, removing unnecessary information (lines 26-29), and adding information on specific results, for example, from the discussion of lines 303-313.

Introduction. There is insufficient information on the pathogen Fusarium oxysporum and its pathogenesis (affects roots, causes wilting, penetrates blood vessels, etc.). In addition, the authors write (lines 231-233): "Since A. tequilana differs from classic models of interaction with Fusarium…” I recommend describing the classical and non-classical models of interaction with Fusarium in the introduction. This is also relevant to your work.

Results: In Figures 1-3, improve the photo quality (resolution). In Figure 1, specify 60 dpi (A, C, E, G) and 180 dpi (B, D, F, H). In Figures 2 and 3, indicate bars. In Figure 2, use arrows to indicate dead cells (B) and mycelium (C). In the captions to Figures 2 and 3, indicate the meaning of the arrows.

Lines 163 and 178: the text confuses references to Figure 4, A and Figure 4, B with chitinase and glucanase activity.

Discussion: Lines 245-247. Most likely, the authors wanted to indicate that strains FNPA and FOL did not contain Virulence factors against Agave tequilana. Sentence rewriting.

Lines 263-271: The discussion of the results on glucanase and chitinase activity is inconclusive and confusing. Rewrite it more clearly. Emphasize that the low glucanase and chitinase activity in agave roots infected with strains FNPA and FOL is associated with poor colonization of agave plants by these strains (FNPA and FOL).

Lines 303-313 repeat the conclusion; I recommend shortening it and moving some information to the abstract.

Materials and Methods: Lines 336-338: Explain abbreviations (FPC), (FAVR3). Explain the pathogenicity of strain FPA. Lines 393-397: Specify microscope settings, laser, and excitation/emission wavelengths. Sections 4.4.3.1, 4.4.3.2, and 4.4.3.3 do not specify the device used for measurements. Line 466: Explain abbreviations.

Conclusion: Based on the conclusion (lines 477-495), create a Figure-Schema representation of the compatible and incompatible interactions between Agave tequilana and Fusarium oxysporum.

All controversial points in the manuscript text are highlighted in yellow.

Comments for author File: Comments.pdf

Author Response

Dear reviewer, thank you for your critical observations and thoughtful comments, which have enabled us to improve the quality of our work. The comments were reviewed, and an effort was made to respond to all of the reviewer's comments and questions. A PDF file with detailed responses to your comments is included.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript has been improved and is now suitable for publication in its present form.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made the necessary scientific corrections. This version makes the work much more readable. I wish them continued success in their work.