Antifungal and Toxicological Evaluation of Natural Compounds Such as Chitosan, Citral, and Hexanal Against Colletotrichum asianum

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General comment:

This manuscript (including the title, purpose of study, and performed experiments) represents a number of controversial intentions.

1. The title is: In vitro and in silico analysis of aldehydes and chitosan as antifungals against Colletotrichum asianum. According to this title, a reader expects that the manuscript will describe some experiments evaluating the effect of aldehydes (by the way, which aldehydes? It is too general even for the title) and chitosan against C. asianum as well as some in silico analysis of the same issues (say, some docking analysis to evaluate the potential of these compounds to bind with any essential targets, or something else).
2. Introduction. Introduction describes the problem of mango fruit storage associated with the infection with plant pathogens causing storage diseases (another one new point), the problems of application of chemical fungicides during fruit storage and need in alternative antifungals based on biopreparations. OK, the problem is quite real, and use of safe and nontoxic biopreparations to protect fruits from spoilage is a good and promising way. Then authors describe some published results of use of CH and aldehydes for the post-harvest antifungal treatments (OK, but I would want to see some numbers reflecting their efficiency, not only words. For example, % of disease suppression, or % of saved fruits, etc. I would also want to see a brief synopsis of such studies namely for mango fruits to understand the current research landscape in this area, the drawbacks of the existing solutions, and the novelty of the proposed study). Also, there is still no any information about the existing results of the use of in silico tools to evaluate antifungal effect of any tested compounds.
3. The purpose of the study is declared as: To find alternatives for the control of Colletotrichum asianum in mangoes, in vitro treatments based on chitosan and bioactive aldehydes (citral (CT) and hexanal (HX)) were evaluated. The tests focused on analyzing the capacity of these compounds to inhibit fungal development. Additionally, the cytotoxic effects of the treatments were investigated using tomato and cucumber seeds as biological models to evaluate germination and root development parameters. In addition, in silico analysis was performed to characterize absorption, distribution, metabolism, elimination, and toxicology (ADMET) properties. As a reader, I found that the antifungal effect on this culture was evaluated (OK), but then I found mentioning of cytotoxic effect evaluation on plants (Why? How does this agree with the title and Intro?) including assessment of their germination and root development (why? And how does it relate to cytotoxic effects and antifungal activity?) Finally, I read about in silico analysis of toxicological and other properties of these compounds in relation to their metabolism in human organism (Oh, another one quite different direction of studies, which was not reflected in the title, instead of the declared in silco analysis of antifungal properties).

The further text rises new questions. Actually, the study consists of three completely unrelated parts, from which only one corresponds to the title of the manuscript.

1. In vitro evaluation of the antifungal effect of three compounds. That is OK, it agrees to the title, and I have only minor comments to this part (see minor comments below). The only question is the novelty substantiation that requires some information (either in Intro, or Discussion sections) about the known results of the use of any agents to suppress the growth and development of this pathogen (if anybody did such experiments).
2. Evaluation of the phytotoxic effect of the tested compounds on tomato and cucumber seedlings (effect on germination and root development and histological study of root cross sections). Why? Why did authors evaluate phytotoxicity of these compounds? It is not important for the use of CH, CT, and HX for the treatment of stored fruits as declared in the introduction. It is also not important for evaluation of the toxicity of these compounds for humans (wrong choice of the model); moreover, at least chitosan toxicity for humans is already very well-studied (see my comments below).
3. In silico prediction of toxicological properties. First, the title declares the use of in silico tools for the evaluation of the antifungal effect against the chosen pathogen. Second, I have some doubts concerning the novelty of these results, since a number of similar in silico analyses have been already reported at least for chitosan and its derivatives.

Discussion. The general principle of the Discussion section is the discussion of results OBTAINED BY AUTHORS including their comparison with the data of other authors, explaining of any reasons providing author’s results, possible consequences, and how these results may be developed/applied further. In our case, almost a half of the Discussion section describes existing concepts of possible mechanisms of antifungal action of the tested compounds. However, authors did not study the mechanisms, so this part of discussion is not related with the obtained results and should be removed or significantly shortened. Thus, this section should be re-written. See also my comment below.

Taking into account all the above-mentioned, I consider the paper can not be accepted for publication. Authors should re-write the paper providing a strong logic in the formulating of the title, purpose of the study, experiments and discussion of results. Do not try to combine parts, which can not be combined in a logical manner. It can be a paper describing the antifungal effect plus phytotoxic effect to discuss the possibility to use CH and other compounds for antifungal treatment of mango plants. It can be a paper describing the antifungal effect of CH, CT, and HX against pathogens causing mango fruit spoilage during storage plus in silico analysis of their metabolism and toxicology in mammals in the case of use on mango fruits plus evaluation of their toxicity on animal models. It can also be a paper describing the antifungal effect of CH, CT, and HX against pathogens causing mango fruit spoilage during storage plus evaluation of their possible ecotoxicity in the case of environmental pollution with wastewater, fruit residues, etc. using the corresponding models for such evaluation (algae, small water invertebrates, etc.)

 

Other comments:

The introduction does not allow a reader to evaluate the novelty of the study. I would recommend authors to describe the current situation with the objects of their study. Which bioagents are used (or were reported to be used) for protective treatments of stored mango fruits? Are there any papers, which report the use of chitosan- and aldehyde-based preparations to protect mango fruits (it is important to evaluate the novelty of the study and its results)? Any other fruits (with more detailed information, say, pathogens to be controlled, percentage of infection and its reduction due to such treatments (with numbers - by 5%, by 40%, etc. - this would allow a reader to understand a possible economic efficiency of such treatments)? You mentioned something as [9,10], but almost without details. Note that the number of studies reported the antifungal effect of chitosan and its derivatives, as well as evaluated its toxicity or provided the ADMET analysis is quite large, so you should substantiate the novelty of your results. Say, you first made this study for mango/pathogen pair, or first evaluated the toxicity of any of tested compound (if any), etc.

 

In relation to the toxicity evaluation, the second question is the choice of a model organism. Authors soaked seeds of tomato and cucumber in solutions of the tested compounds to evaluate a potential harm to a mango fruit consumer. However, differences in physiology of plant seed germination process and processes occurring in humans are quite large. Evaluation of the root and seedling length in tomato and cucumber seedlings treated with tested compounds (phytotoxicity evaluation) is quite far from the evaluation of the physiological toxicity in humans who eat treated fruits. From this point of view, it seems that this part of the study poorly agrees with the declared aim, so I would recommend authors either exclude it from the study, or thoroughly substantiate why they use this model for toxicity evaluation (taking into account that at least CH toxicity has been already studied by a number of researchers). Alternatively, authors can declare this part as the phytotoxicity evaluation, but in this case they should substantiate its connection with the purpose of the whole study (evaluation of the possibility to use CH and aldehydes for mango fruit treatment). It would be more suitable to use animal model, say, to treat feed with the tested preparations and fed it to mice or rats, or even treat fruits with these compounds and fed them to animals.

 

Abstract

Lines 17-18: please, specify: application for which purposes? Plant protection?

Line 18-19: Chitosan application in agriculture for plant protection is quite popular direction. I did some research activity in this area in recent years. See, for example, the Google Scholar search results for chitosan and plant protection: https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=chitosan+application+plant+protection&btnG=

Since 2021, such search gives almost 18000 results. Therefore, the phrase "little information is available on the application of these products" is completely incorrect in relation to chitosan (I did not check information for aldehydes). In relation to the toxicity, chitosan is known for its low toxicity and is widely used in biomedical applications and pharmaceutics (see, for example, https://link.springer.com/article/10.1007/s10965-022-03121-3, https://www.mdpi.com/1422-0067/23/18/10975, https://www.sciencedirect.com/science/article/pii/S000186861830277X?casa_token=bE64-OxPSP8AAAAA:p8Ai3vZNsrzEtyn5wSRVfD5WmBn0N-vpkJ1WI_U0PYrWBhnbn51B5kJ4CzYgE66_DrNk_nOOkA, etc.); therefore, one can not say there is little information about this (again, I did not look such information about aldehydes). Please, check and correct.

 

Introduction

Line 54: "The main success in its application..." - application of what? It is unclear.

Line 64-65: if you use plant model organisms, then this should be rather phytotoxicity (though it would look strange since you positioned CH and other two compounds as agents for treatment of harvested fruits). What was the purpose to evaluate cytotoxicity of the tested preparations on plant models? There are a number of studies reporting evaluation of the toxicity of CH-based preparations; it is considered to be low-toxic (see my comment to the Abstract section). In this case, what was the need to make toxicity studies? In addition, due to significant difference in physiology of animals/humans and plants, animal models would be much more suitable than plants if we talk about consumer safety. In relation to the in silico evaluation: also, there is a number of such studies for CH and its derivatives: https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=admet+chitosan&btnG=, so what was the need to make this work?

 

Materials and methods

Line 70: "...previously isolated mango fruit strain" - looks like you describe the strain of mango. I suggest re-phrase: ... Mycelial discs were obtained from the strain earlier isolated from mango fruits and identified as C. asianum". It would be also good to mention the strain ID.

Line 73: what type of equipment do you mention in this line? Please, add information.

Line 83-84: were the mentioned dilutions final? I mean, do 0.5-1.5% mentioned in these lines represent the final concentration in agarized medium, or there are concentrations prepared prior mixing with PDA (in this case, what are the final concentrations in the medium)? Please, explain this in the text.

 

Results

Fig. 1A: since the scale of the figure is insufficient to indicate all variants (only 5 out of 12 variants are visible), I would recommend to transform this figure into a table.

Fig. 2B: what does it mean? It seems that all variants demonstrated the same result; moreover, this result did not change with time.

Table 1: usually, when you present the results in the form of XX.XX±YY.YY, the number of symbols after the point should be equal (not XX.XX±YY.YYYY). Please, correct. Usually, the same number of symbols after the point is used for the whole table column or row.

Fig. 4A: please, check axis titles. What is “Relative seed elongation”? Probably, there should be “Relative radicle elongation”?

Table 2: please, specify measurement units for data shown in the table (%, mm, etc.). The note includes asterisks; in this case, these asterisks should also present in the table indicating to which data this note is attributed. Also, in this case, different notes should be indicated with different number of asterisks (in your case there is always one asterisk). Please, correct to avoid any confusing. “Randes from low toxicity…” - to which parameter this note is attributed?

Subsection 3.2.2. Here authors evaluated changes in plant cell development caused by the effect of the tested compounds. Again, this part of the study is completely unrelated to its purpose. How the development of cell wall of plant cells or radicle formation can be related to the toxicity for mammals? This section would agree with the purpose to evaluate phytotoxicity of CH, CT, and HX, but why such evaluation is needed for the concept of treatment of harvested and stored fruits?

Subsection 3.2.3: as I can see from the brief search, ADMET studies of chitosan and its derivatives have been already performed by other authors. See, for example: https://revistadechimie.ro/pdf/28%20GUMA.pdf. It would be good to explain here, why you decided to make such study in spite of the already existing reports. Probably, another form of chitosan, or the results made in other programs did not satisfy you for some reasons, or something else. Without such substantiation, it is unclear for me, as a reader, what new data you’ve obtained in this analysis and what was the purpose of such study.

Line 306-307: why did you use several platforms? Do they provide analysis of different toxicokinetic aspects? Please, explain in the text.

Line 308-309: chemical structures of … were different from the molecular weight, TPSA,…” of course, chemical structure and molecular weight are different things. Please, check the sentence and correct, since now it looks unclear.

 

Discussion

Lines 342-374 provide a detailed description of possible mechanisms of action of CH and aldehydes towards plant pathogenic fungi. However, the manuscript does not study this issue (I mean mechanisms), but rather evaluates the effect of these compounds on the growth and development of the pathogen chosen for this study. In this case, there is no need to describe the mechanisms in such a large text volume (this topic is not directly connected with the results obtained by authors); moreover, it is more logical to discuss the suppressive effect on this pathogen as compared with other antifungals, or the effect on this pathogen and other colletotrichum species, or other mango pathogens, etc.

Comments on the Quality of English Language

English is mainly OK, just some language editing is required.

Author Response

Reviewer #1

 

The title is: In vitro and in silico analysis of aldehydes and chitosan as antifungals against Colletotrichum asianum. According to this title, a reader expects that the manuscript will describe some experiments evaluating the effect of aldehydes (by the way, which aldehydes? It is too general even for the title) and chitosan against C. asianum as well as some in silico analysis of the same issues (say, some docking analysis to evaluate the potential of these compounds to bind with any essential targets, or something else).

The phrase “during post-harvest” was added to the title by suggestion to make it more understandable; the aldehydes evaluated are mentioned throughout the text (citral and hexanal), making the title short according to the journal's parameters.

 

Introduction. Introduction describes the problem of mango fruit storage associated with the infection with plant pathogens causing storage diseases (another one new point), the problems of application of chemical fungicides during fruit storage and need in alternative antifungals based on biopreparations. OK, the problem is quite real, and use of safe and nontoxic biopreparations to protect fruits from spoilage is a good and promising way. Then authors describe some published results of use of CH and aldehydes for the post-harvest antifungal treatments (OK, but I would want to see some numbers reflecting their efficiency, not only words. For example, % of disease suppression, or % of saved fruits, etc. I would also want to see a brief synopsis of such studies namely for mango fruits to understand the current research landscape in this area, the drawbacks of the existing solutions, and the novelty of the proposed study). Also, there is still no any information about the existing results of the use of in silico tools to evaluate antifungal effect of any tested compounds

Information regarding the percentages of fungal disease suppression recorded for chitosan, citral, and hexanal treatments was included, with an emphasis on tropical fruits. However, the potential effect has already been reported in a review article published by Rayón-Díaz et al. (2024). Furthermore, there is still no information on the results of using in silico tools to evaluate the antifungal effect of the tested compounds. These molecules have not yet been recorded in a molecular docking analysis that provides these answers, so this could be an interesting option for future tests.

 

The purpose of the study is declared as: To find alternatives for the control of Colletotrichum asianum in mangoes, in vitro treatments based on chitosan and bioactive aldehydes (citral (CT) and hexanal (HX)) were evaluated. The tests focused on analyzing the capacity of these compounds to inhibit fungal development. Additionally, the cytotoxic effects of the treatments were investigated using tomato and cucumber seeds as biological models to evaluate germination and root development parameters. In addition, in silico analysis was performed to characterize absorption, distribution, metabolism, elimination, and toxicology (ADMET) properties. As a reader, I found that the antifungal effect on this culture was evaluated (OK), but then I found mentioning of cytotoxic effect evaluation on plants (Why? How does this agree with the title and Intro?) including assessment of their germination and root development (why? And how does it relate to cytotoxic effects and antifungal activity?) Finally, I read about in silico analysis of toxicological and other properties of these compounds in relation to their metabolism in human organism (Oh, another one quite different direction of studies, which was not reflected in the title, instead of the declared in silco analysis of antifungal properties).

In vitro evaluation of the antifungal effect of three compounds. That is OK, it agrees to the title, and I have only minor comments to this part (see minor comments below). The only question is the novelty substantiation that requires some information (either in Intro, or Discussion sections) about the known results of the use of any agents to suppress the growth and development of this pathogen (if anybody did such experiments).

Evaluation of the phytotoxic effect of the tested compounds on tomato and cucumber seedlings (effect on germination and root development and histological study of root cross sections). Why? Why did authors evaluate phytotoxicity of these compounds? It is not important for the use of CH, CT, and HX for the treatment of stored fruits as declared in the introduction. It is also not important for evaluation of the toxicity of these compounds for humans (wrong choice of the model); moreover, at least chitosan toxicity for humans is already very well-studied (see my comments below).

High concentrations of aldehydes and essential oil compounds have been shown to cause phytotoxicity, so we rely on research using seed germination rate as a sensitive and efficient model, in addition, our commercial chitosan treatment does not report toxicity in plant cells, we use seeds as a model for several key reasons: 1) high sensitivity in early development, 2) Relevance for application in mango fruits because germination is a fundamental plant process and could give us answers to clues about the potential impact of the treatment and 3) simplicity and speed, which facilitates the generation of rapid results, in addition to ethical considerations compared to other models, being an alternative to these

 

In silico prediction of toxicological properties. First, the title declares the use of in silico tools for the evaluation of the antifungal effect against the chosen pathogen. Second, I have some doubts concerning the novelty of these results, since a number of similar in silico analyses have been already reported at least for chitosan and its derivatives

An adjustment was made to the article title, removing the word "in silico" and adding "toxicological prediction." The relevance of these tests helps us understand the chemical properties of the molecules studied, so the inclusion of these platforms provides a broader view.

 

Discussion. The general principle of the Discussion section is the discussion of results OBTAINED BY AUTHORS including their comparison with the data of other authors, explaining of any reasons providing author’s results, possible consequences, and how these results may be developed/applied further. In our case, almost a half of the Discussion section describes existing concepts of possible mechanisms of antifungal action of the tested compounds. However, authors did not study the mechanisms, so this part of discussion is not related with the obtained results and should be removed or significantly shortened. Thus, this section should be re-written. See also my comment below.

An adjustment was made to the focus of the article, discarding the word “in silico” and adding “toxicological prediction” and in vitro analysis.

 

The introduction does not allow a reader to evaluate the novelty of the study. I would recommend authors to describe the current situation with the objects of their study. Which bioagents are used (or were reported to be used) for protective treatments of stored mango fruits? Are there any papers, which report the use of chitosan- and aldehyde-based preparations to protect mango fruits (it is important to evaluate the novelty of the study and its results)? Any other fruits (with more detailed information, say, pathogens to be controlled, percentage of infection and its reduction due to such treatments (with numbers - by 5%, by 40%, etc. - this would allow a reader to understand a possible economic efficiency of such treatments)? You mentioned something as [9,10], but almost without details. Note that the number of studies reported the antifungal effect of chitosan and its derivatives, as well as evaluated its toxicity or provided the ADMET analysis is quite large, so you should substantiate the novelty of your results. Say, you first made this study for mango/pathogen pair, or first evaluated the toxicity of any of tested compound (if any), etc.

Figures were placed on the effectiveness of these treatments in inhibiting various phytopathogenic fungi upon introduction, In addition, the results of in vitro tests were complemented with toxicological predictions on the platforms and with the phytotoxicity test on seeds to determine the impact of the implemented treatments, since compounds such as citral and hexanal are not registered for purposes related to applications in fruits.

 

In relation to the toxicity evaluation, the second question is the choice of a model organism. Authors soaked seeds of tomato and cucumber in solutions of the tested compounds to evaluate a potential harm to a mango fruit consumer. However, differences in physiology of plant seed germination process and processes occurring in humans are quite large. Evaluation of the root and seedling length in tomato and cucumber seedlings treated with tested compounds (phytotoxicity evaluation) is quite far from the evaluation of the physiological toxicity in humans who eat treated fruits. From this point of view, it seems that this part of the study poorly agrees with the declared aim, so I would recommend authors either exclude it from the study, or thoroughly substantiate why they use this model for toxicity evaluation (taking into account that at least CH toxicity has been already studied by a number of researchers). Alternatively, authors can declare this part as the phytotoxicity evaluation, but in this case they should substantiate its connection with the purpose of the whole study (evaluation of the possibility to use CH and aldehydes for mango fruit treatment). It would be more suitable to use animal model, say, to treat feed with the tested preparations and fed it to mice or rats, or even treat fruits with these compounds and fed them to animals

We opted for a method that does not involve studies in mammals due to ethical restrictions and regulations regarding their handling. Therefore, we relied on reports by other authors using seeds as a study model and their effect on germination. These data can provide us with an answer related to morphological changes caused by the treatments, as well as adverse effects in these plant models (germination blockage). Furthermore, the notation of toxicity indices has been standardized by other research.

 

Abstract

Lines 17-18: please, specify: application for which purposes? Plant protection?

The text was modified to focus on the search for alternative treatments to synthetic fungicides.

 

Line 18-19: Chitosan application in agriculture for plant protection is quite popular direction. I did some research activity in this area in recent years. See, for example, the Google Scholar search results for chitosan and plant protection: https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=chitosan+application+plant+protection&btnG=. Since 2021, such search gives almost 18000 results. Therefore, the phrase "little information is available on the application of these products" is completely incorrect in relation to chitosan (I did not check information for aldehydes). In relation to the toxicity, chitosan is known for its low toxicity and is widely used in biomedical applications and pharmaceutics (see, for example, https://link.springer.com/article/10.1007/s10965-022-03121-3, https://www.mdpi.com/1422-0067/23/18/10975, https://www.sciencedirect.com/science/article/pii/S000186861830277X?casa_token=bE64-OxPSP8AAAAA:p8Ai3vZNsrzEtyn5wSRVfD5WmBn0N-vpkJ1WI_U0PYrWBhnbn51B5kJ4CzYgE66_DrNk_nOOkA, etc.); therefore, one can not say there is little information about this (again, I did not look such information about aldehydes). Please, check and correct.

Fixed summary focus.

 

Introduction

Line 54: "The main success in its application..." - application of what? It is unclear.

The reported treatment with the properties of generating a coating on the fruit was applied.

 

Line 64-65: if you use plant model organisms, then this should be rather phytotoxicity (though it would look strange since you positioned CH and other two compounds as agents for treatment of harvested fruits). What was the purpose to evaluate cytotoxicity of the tested preparations on plant models? There are a number of studies reporting evaluation of the toxicity of CH-based preparations; it is considered to be low-toxic (see my comment to the Abstract section). In this case, what was the need to make toxicity studies?

Because it is a formulation based on commercial grade chitosan, with a concentration of 4% and in its preparation different products than those reported (for chitosan) were used to acidify the dissolution medium, it was decided to perform this analysis since there was uncertainty about its effects.

 

In addition, due to significant difference in physiology of animals/humans and plants, animal models would be much more suitable than plants if we talk about consumer safety. In relation to the in silico evaluation: also, there is a number of such studies for CH and its derivatives: https://scholar.google.com/scholar?hl=en&as_sdt=0%2C5&q=admet+chitosan&btnG=, so what was the need to make this work?

As mentioned above, we opted for a method that does not involve studies in mammals due to ethical restrictions and regulations in their handling, so we relied on reports by other authors using seeds as a study model and the effect on their germination. These data can provide us with an answer related to morphological changes caused by the treatments, as well as adverse effects in these plant models (germination blockage). In addition, the notation of toxicity indices has been standardized by other research.

 

Materials and methods

Line 70: "...previously isolated mango fruit strain" - looks like you describe the strain of mango. I suggest re-phrase: ... Mycelial discs were obtained from the strain earlier isolated from mango fruits and identified as C. asianum". It would be also good to mention the strain ID.

The spelling correction was made and the GenBank accession numbers of the regions used were added.

 

Line 73: what type of equipment do you mention in this line? Please, add information

Team name added.

 

Line 83-84: were the mentioned dilutions final? I mean, do 0.5-1.5% mentioned in these lines represent the final concentration in agarized medium, or there are concentrations prepared prior mixing with PDA (in this case, what are the final concentrations in the medium)? Please, explain this in the text

The concentration of the PDA+CH dilution in the petri dish was corrected in the text.

 

Results

Fig. 1A: since the scale of the figure is insufficient to indicate all variants (only 5 out of 12 variants are visible), I would recommend to transform this figure into a table.

It was corrected to a table where all treatments are displayed.

 

Fig. 2B: what does it mean? It seems that all variants demonstrated the same result; moreover, this result did not change with time.

The intention is to make known the variables not visible in Fig. 1A, however the graphic figure was replaced by a table, according to the comments.

 

Table 1: usually, when you present the results in the form of XX.XX±YY.YY, the number of symbols after the point should be equal (not XX.XX±YY.YYYY). Please, correct. Usually, the same number of symbols after the point is used for the whole table column or row.

Fixed the number of symbols per column

 

Fig. 4A: please, check axis titles. What is “Relative seed elongation”? Probably, there should be “Relative radicle elongation”?

Yes, these are two variables, Relative Radicle Elongation (RRE) and another one, Relative Seed Germination (RSG). The abbreviation RSE was corrected to RSG because there was a spelling error.

 

Table 2: please, specify measurement units for data shown in the table (%, mm, etc.).

The note includes asterisks; in this case, these asterisks should also present in the table indicating to which data this note is attributed. Also, in this case, different notes should be indicated with different number of asterisks (in your case there is always one asterisk). Please, correct to avoid any confusing. “Randes from low toxicity…” - to which parameter this note is attributed?

The asterisks in the note were removed and replaced by the units of measurement of the data, in this case percentages (%), and the word “Normalized ranges” was added, with which the NREI and NRGI parameters were evaluated according to the index reported by Bagur-González et al. 2011.

 

Subsection 3.2.2. Here authors evaluated changes in plant cell development caused by the effect of the tested compounds. Again, this part of the study is completely unrelated to its purpose. How the development of cell wall of plant cells or radicle formation can be related to the toxicity for mammals? This section would agree with the purpose to evaluate phytotoxicity of CH, CT, and HX, but why such evaluation is needed for the concept of treatment of harvested and stored fruits?

As mentioned above, we opted for a method that does not involve studies in mammals due to ethical restrictions and regulations in their handling, so we relied on reports by other authors using seeds as a study model and the effect on their germination. These data can provide us with an answer related to morphological changes caused by the treatments, as well as adverse effects in these plant models (germination blockage). In addition, the notation of toxicity indices has been standardized by other research.

 

Subsection 3.2.3: as I can see from the brief search, ADMET studies of chitosan and its derivatives have been already performed by other authors. See, for example: https://revistadechimie.ro/pdf/28%20GUMA.pdf. It would be good to explain here, why you decided to make such study in spite of the already existing reports. Probably, another form of chitosan, or the results made in other programs did not satisfy you for some reasons, or something else. Without such substantiation, it is unclear for me, as a reader, what new data you’ve obtained in this analysis and what was the purpose of such study. Line 306-307: why did you use several platforms? Do they provide analysis of different toxicokinetic aspects? Please, explain in the text.

The complement of these analyses and the reasons why these platforms were used were included in the text.

 

Line 308-309: chemical structures of … were different from the molecular weight, TPSA,…” of course, chemical structure and molecular weight are different things. Please, check the sentence and correct, since now it looks unclear

The phrase was corrected to “chemical properties”

 

Discussion

Lines 342-374 provide a detailed description of possible mechanisms of action of CH and aldehydes towards plant pathogenic fungi. However, the manuscript does not study this issue (I mean mechanisms), but rather evaluates the effect of these compounds on the growth and development of the pathogen chosen for this study. In this case, there is no need to describe the mechanisms in such a large text volume (this topic is not directly connected with the results obtained by authors); moreover, it is more logical to discuss the suppressive effect on this pathogen as compared with other antifungals, or the effect on this pathogen and other colletotrichum species, or other mango pathogens, etc.

For us it is important to describe the effect of the treatments in inhibiting the development of the strain, so we decided to add reports on the application of these treatments and their mechanisms, which is why we decided to add this topic that is related to the effect of the agents evaluated.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript horticulturae-3524598, titled “In vitro and in silico analysis of aldehydes and chitosan as antifungals against Colletotrichum asianum”, addresses an important paper investigating the antifungal potential of chitosan, citral, and hexanal against C. asianum, the causative agent of mango anthracnose, and to assess their suitability as eco-friendly alternatives to synthetic fungicides. However, in my opinion this paper must be revised in a major manner for reasons of form and content.

Abstract

Could the authors elaborate on the study's importance in terms of food safety or sustainable agriculture more generally?

 Could the abstract be strengthened by defining the main findings regarding toxicity thresholds or inhibition percentages?

Introduction

The risks with synthetic fungicides are covered in the introduction; might the authors go into further detail on particular regulatory or consumer safety concerns?

 What are the differences between aldehydes and chitosan and other biological control agents used to combat C. asianum?

 Could the authors elaborate on the economic implications of mango losses brought on by fungal infections on a global scale?

Materials and Methods

For cytotoxicity testing, why were tomato and cucumber seeds chosen as biological models?

 Were controls incorporated to take into consideration possible inherent fluctuations in the rates of seed germination?

 Could the authors elaborate on whether the treatment fluids' pH was constant under various experimental setups?

Results

In comparison to conventional synthetic fungicides, were the reported antifungal effects of hexanal, citral, and chitosan statistically significant?

 Did these chemicals undergo individual testing or did the study investigate possible synergistic effects between them?

 Is it possible for the authors to shed additional light on the reasons why specific doses showed varying degrees of toxicity in cucumber versus tomato seeds?

Discussion

Could the authors elaborate on whether the mechanism mentioned in the discussion—plasma membrane disruption—was verified experimentally or deduced from earlier research?

 What is the difference between the results and earlier studies on fruit preservation coatings based on chitosan?

 Are there any possible restrictions or environmental issues related to the extensive use of chitosan and aldehydes that the authors could address?

Figures and Tables

Incorporating microscopic photos of fungal morphology following treatment would enhance clarity.

 Would it be simpler to interpret the results if a table gave the percentages of fungal inhibition for each tested concentration?

Conclusion

According to the conclusion, further omics research may shed further light. Do the authors have any particular genetic or metabolomic pathways in mind as potential contributors?

 How may mango growers use these findings to commercial post-harvest treatment plans?

 

 

Author Response

Reviewer #2

 

The tittle in my opinion could be more explicit and clearer. See just below my comments about this. For example, I would add “during post-harvest” after “Colletotrichum asianum”.

The sequence was added to the title.

 

The abstract is clear and embodies well the article, pointing out the main outcomes of the paper. But there are some genus names and species names that are not in italics and they should be

An improvement was made to the abstract, correcting the italicized names

 

Also, keywords were generally well chosen, but I wouldn’t repeat “Colletotrichum asianum” that is already in the tittle. I would replace this…

Replacing the word "Colletotrichum asianum" with "Mango disease" and "mango" with "alternative treatments."

 

The Introduction of the paper is in general well written and well organized, but for me the first two paragraphs are too summarized. They can be mor developed. For instance, in the second one, many other diseases and fungal diseases could be referred. I mean, introduction could be extended in this way. In addition, the first paragraph of the introduction could be rephrased and the language could be improved.

Text was added from line 37 to 40. Mentioning the factors that influence the development of phytopathogens and the most frequent fungal genera in post-harvest mango fruit

 

For example, in the second paragraph you mention 12 species of the genus that are problematic to mango fruit. You could provide a simple table with the species and correspondent reference point out the damages that those species can cause to the fruit during post-harvest, storage or marketing (in shelf for example).

It was decided to keep the mention, but adding the name of the species complexes related to mango

 

Moreover, I wonder if these species are also problematic to the plant itself or only for the fruit. Reading the tittle, we do not infer if the fungal species is only problematic to the fruit only or to the plant. I would clarify it. Please, see above my commnets in relation to the tittle

The title has been adjusted to reflect the fact that this fungus damages fruit postharvest, making it important to use disease control measures. However, various studies have linked the pathogenicity and virulence of the Colletotrichum genus to various parts of the plant tissue, including flowers, branches, stems, etc.

 

In lines 40 and 49-50 please put the names of fungal genera in italics.

Names were placed in italics

 

The Materials and Methods section is well organized; the methods are adequately described and with detail. Also, the authors employed adequate tools and also suitable statistics. Nevertheless, I think that statistics description needs some clarification. In the case of the tests for mycelial growth and sporulation, the number or replicates should be at least 3 as in the other tests.

Duplicates were used in the mycelial growth, sporulation, and fungal biomass tests due to the increased sample size (n=8); in the case of germination, triplicates were performed for each concentration. Additional information was added to the statistical analysis section, taking each analysis into account.

 

See lines #187 and 188: you mention here 3 replicates for all test and duplicates for mycelial growth and sporulation. But then in Results and also sometimes in the Materials and Methods (previous subsections of that one of Statistics) you mention n=8 (see for example Figure 1 and the correspondent methods or Table 1… or for example n=20 (see Figure 4). Please clarify this. Maybe, it should be better to address to each statistical tool and number of replicates in each subsection of the Methods, for each test done… It is just an idea… All these aspects must be clear! See also my comments below for the Results section on this matter.

Added analysis addressing each statistical tool with its number of replications in the section.

 

Moreover, I think that more information must be given about the strain used (Subsection 2.1). How old is it? When it was isolated, and from what geographical region, not simply giving the reference. Moreover, some information about the virulence of the strain could be given and information about the real damage in post-harvest and storage of the fruit. Also, why did you not test more than one strain? This is rather low for me. It would be nice to test more strains (even of the same species) but also within the genus, representing some of the 12 species that you mention in the second paragraph of the introduction

This strain was isolated from previous work by members of the lab. It was approximately 6 months to 1 year old at the time of its reactivation and evaluation in our experiments. It was isolated from Tommy Atkins mangos from the 5 de Mayo region of Nayarit, Mexico. Fruit damage caused by this strain has already been mentioned in a previous article.

 

In addition, when you reactivated the strain did you test that the strain was still virulent and capable of inducing damage to the fruit? Did you test that? For how long was it inactive?

The strain's inactivity lasted approximately 6 months to 1 year, so the Koch's postulate test was performed to determine its virulence status.

 

Please note that “In silico” in line 176 (title of subsection 2.5.2) must be in italics.

Corrected to italics

 

Another minor thing: in the tittle of subsection 2.1 I would not abbreviate Colletotrichum asianum to C. asianum.

Corrected to full name

 

Results are also well organised and properly described. Results are also illustrated with proper figures and tables. I would replace the patterns of the bars of figure 2. For example, using different colours instead of black, grey and white graphics

Changed to a color chart

 

Also, I would enlarge figures of Table 3. They are too small and we can barely see them…

Image sizes have been enlarged

 

In addition, I would enlarge also Figure 5. It is a very important part of the paper that must be with higher resolution, in my opinion.

Image sizes have been enlarged

 

Please note that is some figures captions you do not have the number of replicates (for example in Figure 2 and Table 2 and Table 3)… See my comments on this above!

The evaluated sample size was added

 

Lines 232 and 233. There are genera names not in italics. Please correct.

Fixed names in italics

 

Conclusions point out the main outcomes of the paper, point out also some future perspectives and directions. I liked them very much! Just put “in silico” in italics once more (like you did for “in vitro”).

It was corrected to italics as well as “C. asianum”

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Comments:

The submitted paper ” In vitro and in silico analysis of aldehydes and chitosan as antifungals against Colletotrichum asianum" by Edson Rayón-Díaz and colleagues is very interesting and it well constructed. It is a good contribution to the area of plant pathology, in particular for the field Plant Pathology and Disease Management and it is well placed within the SI about Fungal Diseases in Horticultural Crops.

The paper is clear, well written and well organised. But there are things to be improved.

The tittle in my opinion could be more explicit and clearer. See just below my comments about this. For example, I would add “during post-harvest” after “Colletotrichum asianum”.

The abstract is clear and embodies well the article, pointing out the main outcomes of the paper. But there are some genus names and species names that are not in italics and they should be.

Also, keywords were generally well chosen, but I wouldn’t repeat “Colletotrichum asianum” that is already in the tittle. I would replace this…

The Introduction of the paper is in general well written and well organized, but for me the first two paragraphs are too summarized. They can be mor developed. For instance, in the second one, many other diseases and fungal diseases could be referred. I mean, introduction could be extended in this way. In addition, the first paragraph of the introduction could be rephrased and the language could be improved.

For example, in the second paragraph you mention 12 species of the genus that are problematic to mango fruit. You could provide a simple table with the species and correspondent reference point out the damages that those species can cause to the fruit during post-harvest, storage or marketing (in shelf for example). Moreover, I wonder if these species are also problematic to the plant itself or only for the fruit. Reading the tittle, we do not infer if the fungal species is only problematic to the fruit only or to the plant. I would clarify it. Please, see above my commnets in relation to the tittle.

The objectives of the paper are clearly indicated at the end of introduction.

In lines 40 and 49-50 please put the names of fungal genera in italics.

The Materials and Methods section is well organized; the methods are adequately described and with detail. Also, the authors employed adequate tools and also suitable statistics. Nevertheless, I think that statistics description needs some clarification. In the case of the tests for mycelial growth and sporulation, the number or replicates should be at least 3 as in the other tests. See lines #187 and 188: you mention here 3 replicates for all test and duplicates for mycelial growth and sporulation. But then in Results and also sometimes in the Materials and Methods (previous subsections of that one of Statistics) you mention n=8 (see for example Figure 1 and the correspondent methods or Table 1… or for example n=20 (see Figure 4). Please clarify this. Maybe, it should be better to address to each statistical tool and number of replicates in each subsection of the Methods, for each test done… It is just an idea… All these aspects must be clear! See also my comments below for the Results section on this matter.

Moreover, I think that more information must be given about the strain used (Subsection 2.1). How old is it? When it was isolated, and from what geographical region, not simply giving the reference. Moreover, some information about the virulence of the strain could be given and information about the real damage in post-harvest and storage of the fruit.

Also, why did you not test more than one strain? This is rather low for me. It would be nice to test more strains (even of the same species) but also within the genus, representing some of the 12 species that you mention in the second paragraph of the introduction.

In addition, when you reactivated the strain did you test that the strain was still virulent and capable of inducing damage to the fruit? Did you test that? For how long was it inactive?

Please note that “In silico” in line 176 (title of subsection 2.5.2) must be in italics.

Another minor thing: in the tittle of subsection 2.1 I would not abbreviate Colletotrichum asianum to C. asianum.

Results are also well organised and properly described. Results are also illustrated with proper figures and tables.

I would replace the patterns of the bars of figure 2. For example, using different colours instead of black, grey and white graphics.

Also, I would enlarge figures of Table 3. They are too small and we can barely see them…

In addition, I would enlarge also Figure 5. It is a very important part of the paper that must be with higher resolution, in my opinion.

Please note that is some figures captions you do not have the number of replicates (for example in Figure 2 and Table 2 and Table 3)… See my comments on this above!

Lines 232 and 233. There are genera names not in italics. Please correct.

Discussion is well constructed and articulated, making the important comparisons with the literature.

Conclusions point out the main outcomes of the paper, point out also some future perspectives and directions. I liked them very much! Just put “in silico” in italics once more (like you did for “in vitro”).

The list of References is extensive and complete.

In summary, this is a fair work in my opinion that can deserve publication in Horticulturae journal after some improvements.

Kind regards.

 

Author Response

Reviewer #3

 

The tittle in my opinion could be more explicit and clearer. See just below my comments about this. For example, I would add “during post-harvest” after “Colletotrichum asianum”.

The sequence was added to the title.

 

The abstract is clear and embodies well the article, pointing out the main outcomes of the paper. But there are some genus names and species names that are not in italics and they should be

An improvement was made to the abstract, correcting the italicized names

 

Also, keywords were generally well chosen, but I wouldn’t repeat “Colletotrichum asianum” that is already in the tittle. I would replace this…

Replacing the word "Colletotrichum asianum" with "Mango disease" and "mango" with "alternative treatments."

 

The Introduction of the paper is in general well written and well organized, but for me the first two paragraphs are too summarized. They can be mor developed. For instance, in the second one, many other diseases and fungal diseases could be referred. I mean, introduction could be extended in this way. In addition, the first paragraph of the introduction could be rephrased and the language could be improved.

Text was added from line 37 to 40. Mentioning the factors that influence the development of phytopathogens and the most frequent fungal genera in post-harvest mango fruit

 

For example, in the second paragraph you mention 12 species of the genus that are problematic to mango fruit. You could provide a simple table with the species and correspondent reference point out the damages that those species can cause to the fruit during post-harvest, storage or marketing (in shelf for example).

It was decided to keep the mention, but adding the name of the species complexes related to mango

 

Moreover, I wonder if these species are also problematic to the plant itself or only for the fruit. Reading the tittle, we do not infer if the fungal species is only problematic to the fruit only or to the plant. I would clarify it. Please, see above my commnets in relation to the tittle

The title has been adjusted to reflect the fact that this fungus damages fruit postharvest, making it important to use disease control measures. However, various studies have linked the pathogenicity and virulence of the Colletotrichum genus to various parts of the plant tissue, including flowers, branches, stems, etc.

 

In lines 40 and 49-50 please put the names of fungal genera in italics.

Names were placed in italics

 

The Materials and Methods section is well organized; the methods are adequately described and with detail. Also, the authors employed adequate tools and also suitable statistics. Nevertheless, I think that statistics description needs some clarification. In the case of the tests for mycelial growth and sporulation, the number or replicates should be at least 3 as in the other tests.

Duplicates were used in the mycelial growth, sporulation, and fungal biomass tests due to the increased sample size (n=8); in the case of germination, triplicates were performed for each concentration. Additional information was added to the statistical analysis section, taking each analysis into account.

 

See lines #187 and 188: you mention here 3 replicates for all test and duplicates for mycelial growth and sporulation. But then in Results and also sometimes in the Materials and Methods (previous subsections of that one of Statistics) you mention n=8 (see for example Figure 1 and the correspondent methods or Table 1… or for example n=20 (see Figure 4). Please clarify this. Maybe, it should be better to address to each statistical tool and number of replicates in each subsection of the Methods, for each test done… It is just an idea… All these aspects must be clear! See also my comments below for the Results section on this matter.

Added analysis addressing each statistical tool with its number of replications in the section.

 

Moreover, I think that more information must be given about the strain used (Subsection 2.1). How old is it? When it was isolated, and from what geographical region, not simply giving the reference. Moreover, some information about the virulence of the strain could be given and information about the real damage in post-harvest and storage of the fruit. Also, why did you not test more than one strain? This is rather low for me. It would be nice to test more strains (even of the same species) but also within the genus, representing some of the 12 species that you mention in the second paragraph of the introduction

This strain was isolated from previous work by members of the lab. It was approximately 6 months to 1 year old at the time of its reactivation and evaluation in our experiments. It was isolated from Tommy Atkins mangos from the 5 de Mayo region of Nayarit, Mexico. Fruit damage caused by this strain has already been mentioned in a previous article.

 

In addition, when you reactivated the strain did you test that the strain was still virulent and capable of inducing damage to the fruit? Did you test that? For how long was it inactive?

The strain's inactivity lasted approximately 6 months to 1 year, so the Koch's postulate test was performed to determine its virulence status.

 

Please note that “In silico” in line 176 (title of subsection 2.5.2) must be in italics.

Corrected to italics

 

Another minor thing: in the tittle of subsection 2.1 I would not abbreviate Colletotrichum asianum to C. asianum.

Corrected to full name

 

Results are also well organised and properly described. Results are also illustrated with proper figures and tables. I would replace the patterns of the bars of figure 2. For example, using different colours instead of black, grey and white graphics

Changed to a color chart

 

Also, I would enlarge figures of Table 3. They are too small and we can barely see them…

Image sizes have been enlarged

 

In addition, I would enlarge also Figure 5. It is a very important part of the paper that must be with higher resolution, in my opinion.

Image sizes have been enlarged

 

Please note that is some figures captions you do not have the number of replicates (for example in Figure 2 and Table 2 and Table 3)… See my comments on this above!

The evaluated sample size was added

 

Lines 232 and 233. There are genera names not in italics. Please correct.

Fixed names in italics

 

Conclusions point out the main outcomes of the paper, point out also some future perspectives and directions. I liked them very much! Just put “in silico” in italics once more (like you did for “in vitro”).

It was corrected to italics as well as “C. asianum”

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

GENERAL COMMENTS:

 

I read authors replies to my comments. In some of them, they addressed my questions, but some of problems still remained at the same point.

 

Authors explained they work with toxicity evaluation on plants because of ethical concerns to avoid the work with animals. OK, but just one comment. There is another common way to make toxicological studies using human or animal cell cultures (see, for example, Refs. 29, 55). Such studies do not provide ethical concerns like in the case of use of live animals; at the same time they are much more suitable object of study due to the higher similarity of human and animal metabolism. Thus, I would recommend authors to think on the use of cell cultures in their future studies including such toxicological evaluations.

 

Title:

1) Phytotoxical, not fitotoxical; 2) “during postharvest” does not correspond to the performed study, since you did not work with real mango fruits (only in vitro analysis using fungal cultures) 3) no mention about in silico part of the study. 

Based on the manuscipt content, I would suggest authors to transform the title, say, into “Antifungal and toxicological evaluation of chitosan, citral, and hexanal as biocontrol agents against Colletotrichum asianum, a spoilage pathogen of mango fruits” (it is also possible to remove the last part of the title - “a spoilage pathogen…). In my opinion, such or similar title would provide the best correspondence to the manuscript content thus addressing a number of possible questions appearing for the reviewers/readers.

 

If you write “phytotoxical analysis”, this means you studied the effect of these compounds as phytotoxins on plants. In this case, it is incorrect to combine “phytotoxical analysis” with any analysis concerning the toxicology and pharmacokinetics of these compounds in mammals. That is why I suggest that you could call it in general as “toxicological study of CH, CT, and HX using plant models” (though it is still poorly combined with in silico analysis of their ADMET properties in mammals… but let it be so).

 

Introduction

It is required to show why authors decided to solve this problem (its significance) as well as to confirm the novelty of their approach or expected results. Authors substantiated the significance but still did not clearly emphasize the novelty of their study. They should clearly explain the presence of a gap in the existing information on the solved problem and which new knowledge and results they hoped to obtain by this study. Please, add this information into the Intro section.

Also, if authors write that they made toxicological studies for the three tested compounds, because they differ from similar compounds, which have already been tested in this field (see authors comment about commercial chitosan), they should clearly mention it in the text. In any case, it is very important to confirm the novelty of the study and its results and their significance, and it SHOULD be clearly mentioned/formulated in both Intro (it explains why we do such studies) and Discussion (we show which new results we obtained).

 

Discussion

There is no discussion of the obtained antifungal results (just description of possible antifungal mechanisms). At the same time, it is important part of your study. Please, supplement the Discussion section with this part. How good are these results compared to other antifungals against this pathogen (chemical or biological)? If there are no data on other antifungals against this pathogen, then it would confirm the novelty of your results and should be emphasized. I would also recommend to mention in the text why the ADMET analysis you made was necessary in view of a number of existing results of such analyses of Ch and probably aldehydes. This would also demonstrate that the results you obtained by such analysis are new.

 

Line 46: “regulate the regulation” can be changed to “control the regulation”. What is NOM-046-FITO-1995? Please, explain in the text.

 

Line 321: again: chemical properties were different from the molecular weight… this phrase looks strange, it is senseless to compare chemical properties and molecular weight or TPSA. Probably, authors mean that chemical properties of these compounds were different due to their difference in molecular weight and other characteristics?

Line 395-396: This phrase is related to the application of phytotoxic compounds on plants (not stored fruits). Early identification of phytotoxicity is important to avoid negative effects of the applied preparation on plants, which may result in the decrease in their productivity. It would be better to re-phrase it without an accentuation on phytotoxicity.

Author Response

Horticulturae

“Antifungal and toxicological evaluation of chitosan, citral, and hexanal as biocontrol agents against Colletotrichum asianum”

 

Manuscript ID:	Horticulturae-3524598
Full Title:	Antifungal and toxicological evaluation of chitosan, citral, and hexanal as biocontrol agents against Colletotrichum asianum
Article Type:	Original article
Keywords:	Mango disease; GRAS substances; cytotoxicology analyses; alternative treatments
Abstract:	The Colletotrichum genus is one of the ten most relevant pathogenic fungi in the post-harvest sector owing to its high infection rate in tropical fruits; however, the search for alternatives to synthetic fungicides is crucial because of their adverse effects on health and the environment. This study evaluated the efficacy of chitosan (CH), citral (CT), and hexanal (HX) against Colletotrichum asianum, as well as the toxicological potential of these treatments. In vitro tests, 1.0% CH, 0.03% CT, and 0.06% HX significantly inhibited fungal development in parameters of radial growth, sporulation, fungal biomass, and germination 78–100% (p < 0.05). Furthermore, the toxicity index was low to moderate for most concentrations using cucumber and tomato seed germination as a study model. Toxicokinetic predictions suggest that CH, CT, and HX molecules do not pose a danger to human consumption, suggesting that they promise alternatives to chemical fungicides for the control of phytopathogenic fungi
Response to Reviewers:	Reviewer #1   Authors explained they work with toxicity evaluation on plants because of ethical concerns to avoid the work with animals. OK, but just one comment. There is another common way to make toxicological studies using human or animal cell cultures (see, for example, Refs. 29, 55). Such studies do not provide ethical concerns like in the case of use of live animals; at the same time they are much more suitable object of study due to the higher similarity of human and animal metabolism. Thus, I would recommend authors to think about the use of cell cultures in their future studies, including such toxicological evaluations. The comment will be taken into account for future analysis and to complement our research   1) Phytotoxical, not fitotoxical; 2) “during postharvest” does not correspond to the performed study, since you did not work with real mango fruits (only in vitro analysis using fungal cultures) 3) no mention about in silico part of the study.  Based on the manuscipt content, I would suggest authors to transform the title, say, into “Antifungal and toxicological evaluation of chitosan, citral, and hexanal as biocontrol agents against Colletotrichum asianum, a spoilage pathogen of mango fruits” (it is also possible to remove the last part of the title - “a spoilage pathogen…). In my opinion, such or similar title would provide the best correspondence to the manuscript content thus addressing a number of possible questions appearing for the reviewers/readers. If you write “phytotoxical analysis”, this means you studied the effect of these compounds as phytotoxins on plants. In this case, it is incorrect to combine “phytotoxical analysis” with any analysis concerning the toxicology and pharmacokinetics of these compounds in mammals. That is why I suggest that you could call it in general as “toxicological study of CH, CT, and HX using plant models” (though it is still poorly combined with in silico analysis of their ADMET properties in mammals… but let it be so) The title of the article was rewritten based on the reviewer's suggestion Introduction It is required to show why authors decided to solve this problem (its significance) as well as to confirm the novelty of their approach or expected results. Authors substantiated the significance but still did not clearly emphasize the novelty of their study. They should clearly explain the presence of a gap in the existing information on the solved problem and which new knowledge and results they hoped to obtain by this study. Please, add this information into the Intro section. Also, if authors write that they made toxicological studies for the three tested compounds, because they differ from similar compounds, which have already been tested in this field (see authors comment about commercial chitosan), they should clearly mention it in the text. In any case, it is very important to confirm the novelty of the study and its results and their significance, and it SHOULD be clearly mentioned/formulated in both Intro (it explains why we do such studies) and Discussion (we show which new results we obtained). The purpose of the research was added on lines 79-82.   Discussion There is no discussion of the obtained antifungal results (just description of possible antifungal mechanisms). At the same time, it is important part of your study. Please, supplement the Discussion section with this part. How good are these results compared to other antifungals against this pathogen (chemical or biological)? If there are no data on other antifungals against this pathogen, then it would confirm the novelty of your results and should be emphasized. Added information related to the inhibitory effect in lines 358-372   I would also recommend to mention in the text why the ADMET analysis you made was necessary in view of a number of existing results of such analyses of Ch and probably aldehydes. This would also demonstrate that the results you obtained by such analysis are new. Information was added to lines 433-437, emphasizing the objective of the research.   Line 46: “regulate the regulation” can be changed to “control the regulation”. What is NOM-046-FITO-1995? Please, explain in the text The idea in the text was corrected, explaining what NOM-046 is about in Mexico.   Line 321: again: chemical properties were different from the molecular weight… this phrase looks strange, it is senseless to compare chemical properties and molecular weight or TPSA. Probably, authors mean that chemical properties of these compounds were different due to their difference in molecular weight and other characteristics? Properties in the chemical structure can provide us with greater insight into the benefits of these compounds, supporting the idea of ​​future combinations or their effects on controlling fungal diseases.   Line 395-396: This phrase is related to the application of phytotoxic compounds on plants (not stored fruits). Early identification of phytotoxicity is important to avoid negative effects of the applied preparation on plants, which may result in the decrease in their productivity. It would be better to re-phrase it without an accentuation on phytotoxicity. The text of lines 414-416 was reworded in relation to what was mentioned by the reviewer.

 

Reviewer 2 Report

Comments and Suggestions for Authors

I am writing to follow up on my review of manuscript horticulturae-3524598, entitled : In vitro and fitotoxical analysis of aldehydes and chitosan as antifungals against Colletotrichum asianum during postharvest. I'm pleased to inform you that the authors have addressed all my comments thoughtfully and respectfully.

Their revisions demonstrate a clear understanding of the points I raised, and the changes they have made significantly improve the quality of the manuscript. I believe the paper is now much stronger and ready for further consideration.

 

Author Response

Response to Reviewers:

Reviewer #2   I am writing to follow up on my review of manuscript horticulturae-3524598, entitled : In vitro and fitotoxical analysis of aldehydes and chitosan as antifungals against Colletotrichum asianum during postharvest. I'm pleased to inform you that the authors have addressed all my comments thoughtfully and respectfully.   Their revisions demonstrate a clear understanding of the points I raised, and the changes they have made significantly improve the quality of the manuscript. I believe the paper is now much stronger and ready for further consideration   No revisions/modifications

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Comments

I thank the authors for the improvements made and for the corrections. I believe now that the paper can be accepted for publication after only these minor changes being done, as follows:

Minor things

In the abstract, the “Colletotrichum” genus is not in italics and should be.

In introduction, lines 39-40, please delete “sp.” or “spp.” from the genera names. The genus is only “Colletotrichum” and not “Colletotrichum spp.” or “Colletotrichum sp.”. As you know, “sp.” means species (a species that is not identified) and “spp.” refers to several species within a genus.

In line 40, “Colletotrichum” is not in italics once more. Please correct.

In line 41 and 42, if the authors are referring to the species complexes, they should add Colletotrichum (or C.) before the name of each species. For example, it should be “C. gloeosporioides” and not only “gloeosporioides”. Once more, the names of the taxa must be in italics.

Author Response

Response to Reviewers:

Reviewer #3   In the abstract, the “Colletotrichum” genus is not in italics and should be. It has been corrected   In introduction, lines 39-40, please delete “sp.” or “spp.” from the genera names. The genus is only “Colletotrichum” and not “Colletotrichum spp.” or “Colletotrichum sp.”. As you know, “sp.” means species (a species that is not identified) and “spp.” refers to several species within a genus The words “sp.” and “spp.” were removed.   In line 40, “Colletotrichum” is not in italics once more. Please correct. Name corrected   In line 41 and 42, if the authors are referring to the species complexes, they should add Colletotrichum (or C.) before the name of each species. For example, it should be “C. gloeosporioides” and not only “gloeosporioides”. Once more, the names of the taxa must be in italics The “C” word was added abbreviating to the genus Colletotrichum and the taxa were placed in italics

 

Author Response File: Author Response.pdf