Antifungal Efficacy of Strawberry Leaf Extract and Its Effects on Conidia Cell Integrity of Postharvest Citrus Pathogens

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

This paper develops strawberry leaf water extract (SLE) as a biological control agent, which has both antibacterial and agricultural waste resource. The structure is complete and the diagram is clear. I have no problems from the experiment and hope the following suggestions will be helpful to the authors:

1. The "Statistical Analysis" section should include the number of repetitions for all experiments.
2. The medium of 4h PDS in Figure2 has some effects on the perception. Is there a picture of repeated experiments that can be used instead?
3. SLE shows a clear antibacterial effect, but as a reader, I might be more interested in knowing what exactly is responsible for the effect. I think we can use LC-MS or do a metabolomics analysis, or separate the crude extract, and then obtain specific substances, which may promote the subsequent small molecule protein interaction verification and other related experiments.
4. Strawberry leaves are rich in ellagic tannins, flavonoids, phenolic acids, etc., as well as the multi-target effects of SLE, which is the background of common knowledge. During the discussion, is there any data from other researchers' literatures to analyze and study specific natural products? I think that's more interesting to the reader.

Overall, this article is relatively complete and can be published with slight modifications. Good luck!

Author Response

Comments

This paper develops strawberry leaf water extract (SLE) as a biological control agent, which has both antibacterial and agricultural waste resource. The structure is complete and the diagram is clear. I have no problems from the experiment and hope the following suggestions will be helpful to the authors:

The "Statistical Analysis" section should include the number of repetitions for all experiments.

R: Thanks for the comments. The number of repetitions and trials is detailed at the end of each methodology paragraph. Explicitly in Line 136, 150 and 158 and 175.

Comments

The medium of 4h PDS in Figure2 has some effects on the perception. Is there a picture of repeated experiments that can be used instead?

R: We thank the reviewer for pointing this out. We agree that the panel for 4h PDS shows minor visual variations; however, this photograph remains the clearest and most accurate representation of the average experimental outcome. Unfortunately, due to technical constraints at this stage, we are unable to provide an alternative high-quality image.

Comments

SLE shows a clear antibacterial effect, but as a reader, I might be more interested in knowing what exactly is responsible for the effect. I think we can use LC-MS or do a metabolomics analysis, or separate the crude extract, and then obtain specific substances, which may promote the subsequent small molecule protein interaction verification and other related experiments.

R: Thanks for your comment. It would be very interesting in the future to undertake this type of experiment to elucidate more precisely what is responsible for the antifungal activity of the aqueous extract of strawberry leaves studied here.

Comments

Strawberry leaves are rich in ellagic tannins, flavonoids, phenolic acids, etc., as well as the multi-target effects of SLE, which is the background of common knowledge. During the discussion, is there any data from other researchers' literatures to analyze and study specific natural products? I think that's more interesting to the reader.

R.: Thanks to the comment. This topic it is describe in the lines 305 to 321.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

Manuscript horticulturae-4332277-v1

I have conducted a thorough and comprehensive evaluation of the manuscript entitled: “Antifungal efficacy and mode of action of strawberry leaf extract against fungicide-sensitive and resistant fungal pathogens on postharvest citrus.”

The article presents efficacy assays of an extract obtained by cold maceration of dried strawberry leaves. This filtrate is tested against three fungal species that are postharvest pathogens of citrus fruit. Its activity is evaluated in terms of inhibition of spore germination and mycelial growth.

The manuscript presents an interesting study with promising results; however, several aspects could be improved in order to enhance clarity and overall comprehension.

Comments to the authors:

TITLE

I suggest reconsidering the inclusion of “mode of action” in the title. While the manuscript provides convincing evidence of antifungal activity and associated cellular damage (e.g., membrane permeabilization and ultrastructural alterations), it does not elucidate a specific molecular or biochemical mechanism. Therefore, the term “mode of action” may be overstated. A more accurate wording would reflect the observed effects (e.g., antifungal activity or cellular damage) rather than a defined mode of action.

For example:

“Antifungal efficacy of strawberry leaf extract against fungal pathogens on postharvest citrus” or “Antifungal efficacy of strawberry leaf extract and its effects on fungal cell integrity in postharvest citrus pathogens”

 

ABSTRACTS

Línea 29, cambiar 0.1 g mL-1 y poner antes 8 and 24 hour

INTRODUCTION

The introduction is generally well structured and provides relevant background on postharvest diseases and plant-derived antifungal compounds. However, it would benefit from improved focus and clarity. Currently, a significant portion of the text is devoted to strawberry metabolites and previous work by the authors, which, although relevant, is presented in excessive detail and detracts from the main research question.

Importantly, the knowledge gap is not clearly articulated. The authors should explicitly state what remains unknown regarding the antifungal potential of strawberry leaf extract, particularly in relation to citrus postharvest pathogens and fungicide-resistant strains. Clarifying this gap would strengthen the justification and novelty of the study.

Finally, the connection between strawberry-derived compounds and their application to citrus postharvest disease control could be better emphasized to improve coherence.

 

MATERIAL AND METHODS

The Materials and Methods section is generally well organized and covers in vitro, cellular, and in vivo assays. However, several aspects limit the reproducibility and robustness of the study.

First, the characterization of the strawberry leaf extract (SLE) is insufficient. Although some general compositional parameters are provided, no detailed chemical profiling is included, and key components such as SAGs are indirectly estimated. This makes it difficult to compare results with other studies or reproduce the extract.

Second, the concentrations of SLE used throughout the experiments are not clearly defined in terms of extract equivalence, which may lead to ambiguity.

Third, the selection and characterization of fungal isolates require further detail, particularly regarding the level and confirmation of fungicide resistance.

Additionally, the in vivo assay is limited to a single pathogen and does not include resistant strains, which weakens the applicability of the conclusions.

Finally, the methods used to explore the mode of action are mostly descriptive, lacking quantitative or biochemical analyses, which should be taken into account when interpreting the results.

The section “2.1 Chemicals”

Appears unnecessary and could be removed to improve the organization and readability of the manuscript. The listed reagents (e.g., Tween 80, SYTOX™ Green, imazalil) are better described within the specific experimental sections where they are actually used (e.g., conidial preparation, membrane integrity assays, in situ trials). This would enhance clarity and avoid redundancy. Additionally, fungicides such as TBZ and PYR are mentioned later but not introduced consistently, which should be corrected.

The section “2.2 SLE extract preparation”.

The description of the SLE preparation requires further clarification and improvement to ensure reproducibility. Although the extraction procedure is outlined, the chemical characterization of the extract is limited and lacks detailed profiling of its bioactive components. In particular, the quantification of SAGs through indirect antifungal activity assays is not sufficiently robust and should be reconsidered or clearly justified.

Additionally, the definition of extract concentrations used in subsequent experiments is not entirely clear and should be explicitly stated (e.g., dry weight equivalent basis and dilution steps). Further methodological details, such as extraction conditions, number of independent extract preparations, and storage conditions, should also be included.

The description of the strawberry leaf extract (SLE) preparation would benefit from further clarification and additional detail to ensure reproducibility and scientific rigor. Although the general extraction procedure is described (aqueous extraction at a 1:10 ratio for 24 h at 25 °C), several aspects remain insufficiently defined.

First, the chemical characterization of the extract is limited. While some general parameters are reported (dry extract content, total phenolics, and SAGs), no detailed analytical characterization (e.g., chromatographic profiling) is provided. In particular, the quantification of SAGs is based on indirect estimation through antifungal activity, which is not a standard or robust analytical method. This point should be clarified and, if possible, supported by a more appropriate analytical approach.

Second, the definition of the extract concentrations used throughout the study is not entirely clear. The manuscript refers to an initial extract concentration of 0.1 g dry weight mL⁻¹, but subsequent assays use values such as 0.05 and 0.1 g·mL⁻¹ without explicitly explaining how these relate to the original extract (e.g., whether they correspond to dilution steps or reconstituted dry extract equivalents). This should be explicitly clarified to avoid ambiguity.

Additionally, important methodological details are missing, including extraction conditions such as agitation, the number of independent extract preparations, and storage conditions prior to use. Providing this information would improve the reproducibility of the study.

Finally, the manuscript would benefit from acknowledging the limitations associated with the use of a crude extract with limited chemical characterization, particularly given that the biological activity is attributed to this complex mixture.

2.3 Fungal isolates and conidial suspensions preparation.

The description of the fungal isolates and conidial suspension preparation is generally clear; however, several aspects should be improved to strengthen the robustness and reproducibility of the study.

First, the selection of fungal isolates requires further justification and detail. The study includes both fungicide-sensitive and fungicide-resistant isolates; however, no information is provided regarding the level of resistance, how this resistance was previously characterized, or whether it was confirmed under the conditions of the present study. Including this information would be essential to support the claims related to effectiveness against resistant strains.

In addition, only a single isolate per species and resistance category appears to have been used. This limits the representativeness of the results and should be acknowledged as a limitation, as intraspecific variability may significantly influence antifungal responses.

Second, although the preparation of conidial suspensions is described, some methodological details are missing. For instance, it is not specified whether the conidial suspensions were freshly prepared for each experiment or stored prior to use, and for how long. This is relevant because storage conditions can affect conidial viability and experimental outcomes.

Furthermore, the filtration step using cheesecloth is mentioned, but additional clarification on its purpose (e.g., removal of hyphal fragments) and consistency across replicates would improve clarity.

Finally, while the conidial concentration is adjusted using a Neubauer chamber, it would be helpful to indicate whether viability was assessed (e.g., germination rate prior to treatment), as this could influence the interpretation of antifungal effects.

2.4 Fruit.

The section “2.4 Fruit” appears unnecessary as a standalone subsection and does not add significant value in its current form. The information provided (fruit origin, disinfection, and pre-treatment handling) is directly related to the in situ assay described later and would be more appropriately integrated into Section 2.8.

Merging this information into the corresponding experimental section would improve the overall organization and readability of the Materials and Methods, allowing the reader to follow each experimental procedure more cohesively without the need to refer to separate sections.

2.5 Evaluation of SLE effect on mycelial growth, conidia germination and minimum killing conidia time.

The section describing the evaluation of the antifungal effect of SLE on mycelial growth, conidia germination, and minimum killing time is generally understandable; however, several aspects should be clarified and improved to enhance methodological transparency and reproducibility.

First, the rationale behind the selection of SLE concentrations (0.05 and 0.1 g·mL⁻¹) is not provided. It would be helpful to indicate whether these concentrations were based on preliminary experiments, previous studies, or represent physiologically or practically relevant levels.

Second, although the methodology for assessing mycelial growth inhibition is described, additional detail is needed. For example, it should be clarified whether the 5 µL drops of conidial suspension were placed centrally or in multiple points per plate, and how many technical replicates per plate were used. This information is necessary for accurate reproducibility.

Regarding conidia germination assays, the criteria used to define germination (e.g., germ tube length in relation to conidium size) are not specified and should be clearly stated. In addition, it is not indicated how many conidia were counted per sample, which is essential to assess the robustness of the data.

In the minimum killing time assay, the methodology would benefit from further clarification. The use of growth recovery on PDA after exposure is appropriate; however, it should be specified whether the assessment was qualitative (presence/absence of growth) or quantitative (e.g., colony diameter or CFU counts). Moreover, the detection limit of this approach should be discussed, as low levels of surviving conidia might not be detected.

Finally, the section would benefit from a clearer connection to later analyses, particularly those related to cellular damage, to improve the overall coherence of the methodological framework.

Erase gaps in line 140

2.6 Evaluation of conidial membrane integrity.

The membrane integrity assay using SYTOX™ Green is appropriate to evaluate changes in cell permeability; however, the current implementation is largely qualitative and lacks sufficient detail and quantification to support strong mechanistic conclusions.

First, it is not specified whether the fluorescence signal was quantified or if the analysis was based solely on visual observation. The description suggests a qualitative assessment based on microscopy images, which limits the ability to objectively compare treatments. Including quantitative measurements (e.g., percentage of fluorescent conidia) would significantly strengthen the analysis.

Second, the number of conidia evaluated per sample and the criteria used to classify cells as “fluorescent” or “non-fluorescent” are not indicated. These details are essential to ensure reproducibility and to assess the robustness of the results.

Additionally, while the use of positive and negative controls is appropriate, it would be beneficial to clarify whether these controls were included in all experimental replicates and analyzed under identical conditions.

Finally, although the assay demonstrates increased membrane permeability in treated conidia, this type of evidence should be interpreted cautiously, as it reflects a general cellular effect rather than a specific mode of action. Therefore, the results should be presented as indicative of membrane damage rather than definitive mechanistic insight.

2.7 Evaluation of conidial ultrastructure

The use of transmission electron microscopy (TEM) to evaluate ultrastructural changes in conidia is appropriate and provides valuable visual evidence of cellular damage. However, the methodology and its description would benefit from additional detail and a more cautious interpretation.

First, the analysis appears to be entirely qualitative. While representative micrographs are mentioned, no criteria are provided to define or quantify the observed alterations (e.g., proportion of damaged conidia, types of structural damage, or frequency of specific features such as membrane disruption or vesicle formation). Including some level of quantitative or semi-quantitative assessment would strengthen the reliability of the observations.

Second, the number of conidia examined per sample and the number of independent observations are not clearly specified. This information is important to assess the robustness and reproducibility of the ultrastructural analysis.

Additionally, although control samples are included, it should be clarified whether image acquisition and selection were performed under identical conditions and based on predefined criteria, in order to avoid potential bias in representative image selection.

Finally, while TEM observations clearly indicate severe cellular damage after SLE treatment, these types of alterations should be interpreted as evidence of general cytotoxic or structural effects rather than a specific mode of action. Therefore, the conclusions drawn from this analysis should be appropriately framed to avoid overinterpretation.

2.8 Application of SLE on lemon in situ

The in situ assay on lemons is a valuable component of the study, as it provides applied relevance to the antifungal activity observed in vitro. However, several aspects of the experimental design and description require clarification and improvement.

First, the assay is limited to a single pathogen (P. digitatum, sensitive isolate), despite the broader scope of the study, which includes multiple species and fungicide-resistant strains in vitro. This inconsistency weakens the overall conclusions regarding the effectiveness of SLE against a broader spectrum of postharvest pathogens. The authors should justify this choice or consider expanding the in situ analysis to include additional isolates, particularly resistant ones.

Second, additional methodological details would improve reproducibility. For example, it is not clearly specified how many fruits were used per treatment in each replicate, whether the wounds were treated independently or averaged per fruit, and how variability between fruits was managed.

Third, the experimental conditions (e.g., storage at 24 °C and 95% relative humidity for 5 days) are described but not justified. It would be helpful to indicate whether these conditions simulate commercial postharvest environments.

Additionally, the choice of applying the extract directly into artificial wounds represents a preventive/curative scenario under controlled conditions, which may not fully reflect practical application methods. This limitation should be acknowledged.

Finally, while disease incidence is reported, no additional parameters (e.g., lesion diameter or severity) are included. Incorporating more detailed disease assessments would provide a more comprehensive evaluation of the antifungal effect.

2.9 Statistic analysis

The statistical analysis is generally appropriate (ANOVA followed by Tukey’s test); however, the description lacks sufficient detail to ensure transparency and reproducibility.

First, it would be helpful to clearly specify the experimental unit for each assay (e.g., plate, conidial suspension, or individual fruit), as this directly affects the validity of the statistical analysis. This is particularly important for the in situ experiments, where it is not clear whether each wound, each fruit, or each replicate was considered as an independent observation.

Second, although replication is mentioned, the structure of the replicates (biological vs. technical) is not consistently defined across experiments. Clarifying this distinction would improve the robustness and interpretation of the results.

Additionally, there is a typographical error (“tree repetitions” instead of “three repetitions”) that should be corrected.

Finally, it would be beneficial to indicate whether the assumptions of ANOVA (normality and homoscedasticity) were verified prior to analysis, or at least to state the criteria used for applying parametric tests.

RESULTS

3.1 Antifungal action of SLE on mycelial growth, conidia germination and time of death

The results presented in this section clearly show that SLE exerts a strong inhibitory effect on mycelial growth, conidial germination, and viability across the tested fungal pathogens. The inclusion of both fungicide-sensitive and resistant isolates is a strength of the study. However, several aspects of data presentation and interpretation should be reconsidered to improve clarity and avoid overstatement.

First, while complete or near-complete inhibition is reported for several conditions, the results are mainly presented descriptively, with limited quantitative detail beyond colony diameter measurements. In particular, the germination and killing time assays would benefit from more explicit quantification (e.g., percentage of germinated conidia, survival rates), rather than relying primarily on representative images.

Second, the determination of the “minimum killing time” appears to be based on the absence of visible growth after re-plating. While this is a common approach, it should be interpreted cautiously, as it does not exclude the presence of a small number of surviving conidia below the detection limit. This limitation should be acknowledged in the text.

Additionally, the variability between isolates is only briefly described. Although differences in sensitivity are mentioned (e.g., the higher tolerance of the PDR isolate), these observations are not explored in depth or supported by statistical comparisons across isolates, which would strengthen the conclusions.

Finally, some of the statements in this section tend to overgeneralize the results. For example, reporting “total inhibition” or “complete loss of viability” may be too strong given the methodological limitations of the assays used. It would be more appropriate to frame these results in terms of strong inhibitory effects under the specific experimental conditions tested.

 

In Table 1, the descriptive text currently placed below the table appears to function as a header or caption rather than as a footnote. Its current positioning disrupts the flow of the text and may suggest that the table has been inserted into the manuscript without proper formatting.

I recommend moving this text above the table as a proper caption or integrating it into the table title, in accordance with standard formatting guidelines. This would improve clarity and presentation.

 

3.2 Effect of SLE on conidial membrane integrity

The use of SYTOX™ Green staining provides useful evidence of increased membrane permeability in conidia treated with SLE. The inclusion of both positive and negative controls is appropriate and supports the validity of the observations. However, several aspects of data presentation and interpretation should be improved.

First, the results appear to be based solely on qualitative observations from fluorescence microscopy images. No quantitative analysis (e.g., percentage of fluorescent conidia) is provided, which limits the robustness of the conclusions and prevents objective comparison between treatments and isolates. Including quantitative data would significantly strengthen this section.

Second, the number of conidia analyzed and the criteria used to classify cells as permeabilized are not specified. These methodological details are essential to assess the reliability and reproducibility of the results.

Additionally, while the fluorescence signal clearly indicates increased membrane permeability, this type of assay reflects a general indicator of loss of membrane integrity rather than a specific cellular target. Therefore, the interpretation of these results should be moderated. The data support the occurrence of membrane damage but do not allow the identification of a specific mode of action.

Finally, some statements in this section may overinterpret the findings by implying mechanistic insight beyond what the data can support. It would be more appropriate to describe these observations as evidence of cellular damage associated with SLE treatment.

3.3 Internal structure conidial after SLE treatment

The ultrastructural analysis by transmission electron microscopy (TEM) provides clear visual evidence of cellular alterations in conidia treated with SLE, including cytoplasmic disorganization, vesicle formation, and structural damage to the cell membrane and wall. These observations are relevant and support the antifungal effect described in previous sections. However, several aspects of data presentation and interpretation should be improved.

First, the analysis is entirely qualitative, based on representative micrographs. No quantitative or semi-quantitative evaluation of the observed damage is provided (e.g., proportion of affected conidia or frequency of specific structural alterations), which limits the robustness of the conclusions. Including such information would strengthen the reliability of the findings.

Second, the number of conidia analyzed and the criteria used to select representative images are not specified. This raises concerns about potential bias in image selection and makes it difficult to assess the reproducibility of the observations. These aspects should be clarified.

Additionally, while the observed cellular damage is consistent with strong antifungal activity, it reflects general structural disruption rather than a specific targeted mechanism. Therefore, the interpretation of these results should be moderated. The data support the occurrence of severe cellular damage following SLE treatment but do not provide sufficient evidence to define a specific mode of action.

Finally, some statements in this section tend to overinterpret the ultrastructural observations by implying mechanistic conclusions beyond the scope of the data. A more cautious wording is recommended, emphasizing descriptive cellular effects rather than mechanistic insights.

3.4 Effect of SLE in situ application over green mold incidence on lemons

The in situ assay on lemons provides valuable information on the potential applicability of SLE under postharvest conditions. The reduction in disease incidence compared to the untreated control indicates that SLE has a measurable protective effect. However, several aspects of the experimental scope and data interpretation should be reconsidered.

First, the experiment is limited to a single pathogen (P. digitatum, sensitive isolate), whereas the in vitro section includes multiple pathogens and fungicide-resistant strains. This discrepancy restricts the extent to which the in situ results can support the broader claims of efficacy against different fungal species and resistant isolates. The conclusions should therefore be adjusted accordingly, or the limitation should be explicitly acknowledged.

Second, the level of disease control achieved by SLE appears to be moderate (approximately 50% reduction in incidence), which is substantially lower than that of the synthetic fungicide (IMZ), which provided complete control. This difference should be more critically discussed, particularly in terms of practical applicability and comparison with existing treatments.

Additionally, the evaluation is limited to disease incidence, without considering other relevant parameters such as lesion size or severity. Including such measurements would provide a more comprehensive assessment of treatment efficacy.

Furthermore, the experimental setup (application in artificial wounds under controlled conditions) represents a simplified system that may not fully reflect commercial postharvest practices. This limitation should be acknowledged and discussed, especially when suggesting potential applications of SLE.

Finally, some of the statements in this section and the associated discussion may overstate the practical potential of SLE based on these results alone. A more cautious interpretation is recommended, emphasizing that while SLE shows promising activity, further optimization and validation are required under more realistic conditions.

 

DISCUSSION

The Discussion appropriately summarizes the main findings of the study and places them within the context of existing literature on plant-derived antifungal compounds. The manuscript effectively highlights the potential of strawberry leaf extract (SLE) as a sustainable alternative for postharvest disease control. However, several aspects of interpretation should be reconsidered to improve accuracy and avoid overstatement.

First, the discussion tends to overinterpret the mechanistic insights derived from the study. While the results clearly demonstrate antifungal activity and associated cellular damage (e.g., membrane permeabilization and ultrastructural alterations), these observations are primarily descriptive and do not provide sufficient evidence to establish a specific mode of action. Therefore, statements referring to a defined mechanism should be moderated and reframed in terms of general cellular effects.

Second, the conclusions regarding the broad-spectrum efficacy of SLE, particularly against fungicide-resistant strains, are mainly based on in vitro assays. However, the in situ validation is limited to a single pathogen and a sensitive isolate. This discrepancy should be more clearly acknowledged, and the scope of the conclusions should be adjusted accordingly.

Additionally, the comparison with synthetic fungicides could be presented more critically. Although SLE shows promising activity, its efficacy in vivo is clearly lower than that of imazalil under the tested conditions. This difference should be discussed in terms of practical implications and the need for further optimization (e.g., formulation, dosage, or application methods).

Furthermore, while the discussion emphasizes the advantages of plant-derived extracts and multi-target effects in reducing resistance development, these claims remain speculative in the absence of detailed mechanistic evidence. A more cautious and evidence-based interpretation is recommended.

Finally, the manuscript would benefit from a clearer acknowledgment of its main limitations, including the use of a single isolate per condition, the limited chemical characterization of the extract, and the qualitative nature of several analyses. Explicitly addressing these limitations would strengthen the scientific rigor and credibility of the study.

 

Comments for author File: Comments.pdf

Author Response

In the atached file are list the reply to the Reviewer 2

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

I would like to thank the authors for responding to all the comments. I have nothing further to add.

Author Response

Thanks you for yours comments.  

We are sending a marked version changing some similarities in wording and sentence structure that were detected by Editor office.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The goal of the manuscript is interesting, however it needs a deeply revision and introduce much more information (specially data and statistical analysis results) before being published to support the conclusions obtained. Additionally, I think that there are deep failures in methodology that affect to most of the experiments. Authors can see more comments about it in the attached file.

Comments for author File: Comments.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor and Authors,
I am writing in reference to manuscript entitled " Antifungal efficacy and mode of action of strawberry leaf extract against fungicide sensitive and resistant fungal pathogens on postharvest citrus" by Pia Di Peto, Gabriela Michavila, Mario A. Debes, Bjorn V. Welin, Nadia R. Chafoun, Sabrina I. Volentini and Luciana Cerioni
The paper written in this form is not acceptable for publication and I am suggesting a revision of the manuscript before it is published.

Comments for author File: Comments.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This study evaluated the in vitro and in vivo antifungal activities of the aqueous extract of strawberry leaves, PSP2, against the main post-harvest pathogenic fungi of citrus and its mechanism of action. The topic of this paper has clear practical significance and application prospects, and is in line with the current research trend of seeking sustainable alternatives to synthetic fungicides. The experimental design is relatively complete, covering aspects such as inhibition of mycelial growth and spore germination, observation of cell membrane integrity and ultrastructure, and in vivo experiments in fruits. The overall writing of the paper is standardized, but some parts need to be strengthened in terms of the accuracy of experimental method description, the completeness of result data presentation, and the depth of discussion to enhance the rigor and scientific value of the research:

1. The method section clearly describes the preparation process of PSP2 extract, but lacks basic chemical characterization of the extract, which is particularly important. As a study on the biological activity of plant extracts, it is necessary to supplement preliminary qualitative/quantitative data on the yield of the extract, main active component categories (such as total phenols, flavonoid content) or characteristic components (such as the quantitative determination of ellagic acid tannins and strawberry acyl sugar glycosides by LC/GC-MS). This will help readers understand the material basis of its biological activity and provide key information for subsequent studies on its stability, standardized production, and comparison with other extracts.
2. In Section 2.9, it is mentioned that "three complete sets of experiments were conducted, with three repetitions for each condition", but only representative pictures are selected in the figures (Figure 1, Figure 2, etc.). It is recommended to supplement a table after the figures to show the inhibition rates of the three plate experiments and conduct statistical analysis.
3. As mentioned in the previous point, in Section 3.1 when describing the inhibitory effect of PSP2 on mycelial growth and spore germination, it is strongly recommended to provide specific quantitative data in the main text or tables.
4. Line 236, "achieving nearly 50%", the expression is not precise. Here, specific, statistically analyzed incidence rates should be given.
5. The discussion section has compared some studies on plant extracts, but it can be more indepth. For example, when discussing the in vivo efficacy of PSP2 (about 50%), in addition to comparing with Cynara cardunculus extract (about 10%), it can briefly mention the efficacy ranges of other reported plant extracts or biological preparations in other literature to more objectively position the effect level of PSP2 in the current research field.
6.  The study mentions that PSP2 comes from agricultural waste of strawberries and is sustainable. It is recommended to briefly outline the key issues that need to be solved for its transition from the laboratory to practical application at the end of the discussion or conclusion section, such as: scale-up of extraction process, cost assessment, compatibility with existing post-harvest processing procedures, and requirements for formulation development. This can significantly enhance the significance and value of the research, as mentioned in the first point.
7. The reference list format should follow the requirements of the journal's website. Some references are not standardized. Additionally, many of the references are from a long time ago. It is recommended to include more recent papers.

In summary, the article is innovative, but many experimental data need to be quantified, rather than just descriptions of pictures, especially avoiding the use of "Nearly" as a reader would prefer to see precise numbers. I hope my suggestions can be helpful to the authors and good luck to you!