The 2′,4′-Dichloro-chalcone Inhibits the In Vitro Growth and Pathogenicity of Fusarium tricinctum and Trichothecium roseum by Activating Cyanide-Resistant Respiration

Round 1

Reviewer 1 Report

Authors described the antifungal activity of known and commercially available 2’,4’-dichlorochalcone against two pathogenic strains Fusarium tricinctum and Trichothecium roseum.

The introduction is sufficient background for the rest of the manuscript.

The manuscript is an interesting work, which showed that 2’,4’-dichlorochalcone in concentration of 100 µM reduced growth of  F. tricinctum and T. roseum in potato and apple, respectively.

I have a few comments:

1. In whole manuscript sometimes „in vivo”  and „in vitro” is italic, sometimes not, please unify in accordance with MDPI requirements.

2. Line 79 and 82: “3-Hydroxy-4-methoxy” is not a full name of compound, is like an abbreviation. Furthermore, it is not a proper name of compound, which is described  by Sivakumar et al [23].  It should be “3’-hydroxy-4-methoxychalcone”, please improve it.

3. Line 96: “The F. tricinctum and T. roseum tested were isolated from potato tubers and apple…” – please improve the sentence. It doesn’t make sense using “tested”.

4. Lines 129-130: “A stock solution of 0.1 M 2ʹ,4ʹ-dichloro-chalcone, was prepared by dissolving 0.27 g in 500 μL of DMSO, 100 μL Tween 80 and 400 μL of anhydrous ethanol (Figure 2).” Based on MW of 2’,4’-dichlorochalcone (MW=277.15 g/mol) using 0.27g of chalcone and dissolving it in 1mL (500µl of DMSO, 100 µl Tween80 and 400 µl of anhydrous ethanol; the final volume was 1mL) the stock solution is not 0.1M but 0.001M or 1 mM. Please explain it and correct it.

5. Figure 2. The preparation of stock solution does not match the description in lines 129-130. Figure 2 have no information about dissolving chalcone in 500 µl of DMSO. Please improve it. Figure 2 also possess wrong information about stock solution 0.1M.

6. Please describe also in a few sentences how to prepare subsequent dilutions, as illustrated in Figure 2.

7. In materials and methods section, provide a number representing the volume, e.g. 20 μL instead of “Twenty μL”.

8. Line 183: “containing x mM -mercepto-ethanol and 2% (w/v) SDS” please improve the name β-mercaptoethanol and the concentration.

9. Figures 4, 6, 7B, 7D, 8, 9C and 9F: the concentration of chalcone on axis x should be from 0 to 100 µM, not from 100 to 0 µM.

10. Line 354-355: “Candida glabrata” should be italic

Moderate editing of English language required

Author Response

The following is point-to-point response to the editor and reviewers’ comments.

Response to Reviewer 1 Comments

Point 1：In whole manuscript sometimes “in vivo” and “in vitro” is italic, sometimes not, please unify in accordance with MDPI requirements.

Response 1:We are sincerely grateful for your thoughtful suggestions.We have revised all the words "in vivo" and "in vitro" in the revised draft according to the requirements of the MDPI.

Point 2：Line 79 and 82: “3-Hydroxy-4-methoxy” is not a full name of compound, is like an abbreviation. Furthermore, it is not a proper name of compound, which is described by Sivakumar et al [23].  It should be “3’-hydroxy-4-methoxychalcone”, please improve it.

Response 2: We must thank you for your kind suggestions. Meanwhile, we are very sorry for our negligence in many details of our manuscript. We have revised of the mistakes in the article content.

Point 3：“The F. tricinctum and T. roseum tested were isolated from potato tubers and apple…” – please improve the sentence. It doesn’t make sense using “tested”.

Response 3: Thank you very much for your helpful and insightful comments and suggestions about our manuscript. According to your suggestion, we have revised and the mistakes.

Point 4：Lines 129-130: “A stock solution of 0.1 M 2ʹ,4ʹ-dichloro-chalcone, was prepared by dissolving 0.27 g in 500 μL of DMSO, 100 μL Tween 80 and 400 μL of anhydrous ethanol (Figure 2).” Based on MW of 2’,4’-dichlorochalcone (MW=277.15 g/mol) using 0.27g of chalcone and dissolving it in 1mL (500µl of DMSO, 100 µl Tween80 and 400 µl of anhydrous ethanol; the final volume was 1mL) the stock solution is not 0.1M but 0.001M or 1 mM. Please explain it and correct it.

Response 4: We must thank you for your kind suggestions. Meanwhile, we are very sorry for our negligence in details of our manuscript. “A stock solution of 0.1 M 2ʹ,4ʹ-dichloro-chalcone, was prepared by dissolving 0.27 g in 500 μL of DMSO, 100 μL Tween 80 and 400 μL of anhydrous ethanol (Figure 2).” Based on MW of 2’, 4’-dichlorochalcone (MW=277.15 g/mol) using 0.27g of chalcone and dissolving it in 1mL (500µl of DMSO, 100 µl Tween80 and 400 µl of anhydrous ethanol; the concentration of stock solution is 0.001M or 1 mM. We are sincerely grateful to you.

m=0.27g; M=277.15 g/mol

Point 5：The preparation of stock solution does not match the description in lines 129-130. Figure 2 have no information about dissolving chalcone in 500 µl of DMSO. Please improve it. Figure 2 also possess wrong information about stock solution 0.1M.

Response 5: Thank you for your kind suggestion. According to your suggestion, we have revised the inaccurate concentration units about stock solution in lines 129-130, and improved the model diagram in Figure 2).

Point 6：Please describe also in a few sentences how to prepare subsequent dilutions, as illustrated in Figure 2.

Response 6: Thank you very much for your helpful and insightful comments and suggestions about our manuscript. According to your suggestion, we have improved some important information about how to prepare subsequent dilutions. The revised texts in lines 118-120 are as follows:“After filtration with 0.22µm Polytetrafluoroethylene, chalcone solution of 0.01 µM, 0.1 µM and 1 µM were prepared by multiple dilution method(1:100000; 1:10000; 1:1000).”

Point 7：In materials and methods section, provide a number representing the volume, e.g. 20 μL instead of “Twenty μL”.

Response 7: We must thank you for your kind suggestions. Meanwhile, we are very sorry for our negligence in many details of our manuscript. We have corrected the mistakes about method of number representing in all text.

Point 8：Line 183: “containing x mM -mercepto-ethanol and 2% (w/v) SDS” please improve the name β-mercaptoethanol and the concentration.

Response 8: Thank you very much for your helpful and insightful comments and suggestions about our manuscript. According to your suggestion, we have revised the section.

Point 9：Figures 4, 6, 7B, 7D, 8, 9C and 9F: the concentration of chalcone on axis x should be from 0 to 100 µM, not from 100 to 0 µM.

Response 9: Thank you very much for your suggestions and your patience. Meanwhile, we must apologize for our irregular writing. We have revised the order of sample in Figures 4, 6, 7B, 7D, 8, 9C and 9F.

Point 10：Line 354-355: “Candida glabrata” should be italic.

Response 10: Meanwhile, we are very sorry for our negligence in many details of our manuscript. We have revised the italic form of “Candida glabrata” in revised manuscript .

Point 11：Moderate editing of English language required.

Response 11：Thank you very much for your suggestions and your patience. Meanwhile, we must apologize for my irregular writing. We have thoroughly checked sentence by sentence and have revised the manuscript by International Science Editing Service (www.internationalscienceediting.com) and the certification of language editing for our manuscript.

Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript “The 2ʹ,4ʹ-dichloro-chalcone Inhibits the in vitro Growth and Pathogenicity of Fusarium tricinctum and Trichothecium roseum by Activating Cyanide-Resistant Respiration and Disrupting Respiration” investigates the potential of 2ʹ,4ʹ-dichloro-chalcone as an antifungal agent against two significant postharvest crop pathogens, Fusarium tricinctum and Trichothecium roseum. The compound was found to significantly inhibit fungal growth and pathogenicity both in vitro and in vivo. It disrupts fungal cell membranes, triggers ROS production, and activates the alternative respiratory pathway, specifically AOX, leading to respiratory stress. The research presents a promising eco-friendly alternative to chemical fungicides for postharvest disease control.

Overall, the study is well-structured and provides valuable insights into the antifungal properties of 2ʹ,4ʹ-dichloro-chalcone. It addresses an important issue in agriculture by exploring eco-friendly alternatives to chemical pesticides.

Here are my comments and required corrections:

Title:

Consider simplifying the title for better readability.

Abstract:

·         Reduce the number of words to 200 as reported in the authors' guidelines.

·         Clarify the main findings in the abstract. Mention the significant inhibitory effect of 2ʹ,4ʹ-dichloro-chalcone on fungal growth and pathogenicity.

·         State the implications of your results more explicitly. How could this research impact the field of postharvest disease control?

·         Mention the need for further research briefly. For instance, explore potential applications of this compound in agricultural practices.

Introduction:

·         Start with a concise background of the issues posed by F. tricinctum and T. roseum in agriculture. Explain the current challenges in controlling these pathogens.

·         Clearly state the research objective early in the introduction. Let readers know that this study aims to investigate the antifungal properties of 2ʹ,4ʹ-dichloro-chalcone.

·         Provide a brief rationale for choosing this compound. Why is it relevant or interesting in the context of postharvest disease control?

·         You may consider mentioning the importance of finding eco-friendly alternatives to chemical fungicides as a motivator for this research.

·         Discussion:

·         Organize the discussion section into subsections for clarity. For example, sections on "Inhibitory Mechanisms" and "Implications for Postharvest Preservation."

·         Elaborate on the potential practical applications of your findings. How might this research be used in real-world scenarios for crop protection?

·         Discuss any limitations of the study. Are there any factors that might affect the compound's efficacy in the field?

·         Address the broader implications of your research for sustainable agriculture and food safety. How could the use of compounds like 2ʹ,4ʹ-dichloro-chalcone contribute to these goals?

Conclusions:

·         Summarize the key findings in a concise manner.

·         Clearly state the practical implications of your research. What are the takeaways for postharvest disease control?

·         Highlight the novelty and significance of your study. What makes it stand out in the field of agricultural research?

·         Suggest directions for future research, especially regarding the molecular mechanisms underlying the compound's action and its potential for practical application in crop protection.

In summary, the manuscript addresses an important research topic, and the findings have the potential to contribute to the field. However, some revisions and additions are necessary to improve the clarity and completeness of the manuscript. I recommend addressing the specific comments mentioned above to enhance the quality of the manuscript.

The manuscript's English language quality is generally good but could benefit from improvements in sentence structure, verb tense consistency, and clarity.

Author Response

The following is point-to-point response to the editor andreviewers’ comments.

Response to Reviewer 2 Comments

Title:

Point 1：Consider simplifying the title for better readability.

Response 1: Special thanks for the Reviewer’s good comments. This title is really too long, and we have revised a few words and the title was changed to “The 2ʹ,4ʹ-dichloro-chalcone Inhibits the in vitro Growth and Pathogenicity of Fusarium tricinctum and Trichotheciumroseum by Activating Cyanide-Resistant Respiration ”

Abstract:

Point 1：Reduce the number of words to 200 as reported in the authors' guidelines.

Response 1: We are sincerely grateful for your thoughtful suggestions. According to your suggestion, we have reduced the number of words in Abstract section.

Point 2：Clarify the main findings in the abstract. Mention the significant inhibitory effect of 2ʹ,4ʹ-dichloro-chalcone on fungal growth and pathogenicity.

Response 2: Thank you very much for your helpful and insightful comments and suggestions about our manuscript. According to your suggestion, we have revised the section.

Point 3：State the implications of your results more explicitly. How could this research impact the field of postharvest disease control?

Response 3: We gratefully appreciate your time, patience, and helpful comments regarding our manuscript. To state the implications of our results more explicitly, we have revised the section for “Abstract”. The revised text in lines 27-28 are as follows:

“2ʹ,4ʹ-dichloro-chalcone have potential use as alternative safety method in the control of postharvest diseases by F. tricinctum and T. roseum in agricultural practices.”

Point 4：Mention the need for further research briefly. For instance, explore potential applications of this compound in agricultural practices.

Response 4: We are sincerely grateful for your thoughtful suggestions. According to your suggestion, we have rewrited the“Abstract” section, descriptions about contents were added in the last paragraph.

Introduction:

Point 1：Start with a concise background of the issues posed by F. tricinctum and T. roseum in agriculture. Explain the current challenges in controlling these pathogens.

Response 1: We must thank you for your kind suggestions. According to your suggestion, we have revised the section and the revised texts are as follows:

“Fusarium tricinctum and Trichotheciumroseum are important fungal pathogens of various plant diseases worldwide. F. tricinctum infests many cereal crops, vegetables, and traditional Chinese medicinal plants including rice, wheat, barley, maize, potato, lily and licorice. [1-6]. T. roseum infests a wide range of fruits and vegetables such as apples, peaches, melons and tomatoes [7-10], causing severe preharvest and postharvest rots. Infection by F. tricinctum and T. roseum not only leads to a reduction in product quality, but also to increased food safety risks through their production of toxic and carcinogenic mycotoxins. Currently, chemical fungicides are commonly used in the postharvest control of these two pathogens, which can have negative impacts on the development of fungal resistance and environmental pollution.”

Point 2：Clearly state the research objective early in the introduction. Let readers know that this study aims to investigate the antifungal properties of 2ʹ,4ʹ-dichloro-chalcone.

Response 2: Thank you very much for your helpful and insightful comments and suggestions about our manuscript. Yes, we also think that state the research objective early in the introduction is good. However, in the introduction section, when we expand on the advantages of chalcone, 2 ʹ, 4 ʹ- dichloro chalcone was mentioned in the following text, so we still insist on choosing option that the research objective of 2 ʹ, 4 ʹ- dichloro chalcone need be placed in the last paragraph of the introduction.

Point 3：Provide a brief rationale for choosing this compound. Why is it relevant or interesting in the context of postharvest disease control?

Response 3: We are sincerely grateful for your thoughtful suggestions. According to your suggestion, we have revised the section and the revised texts in lines 74-76 are as follows:

“Some studies have reported that chalcone derivatives with halogen substituents were highly fungicidal [25-26]. However, the inhibitory effects of chlorinated chalcones on pathogenic fungi have been less characterized.”

Point 4：You may consider mentioning the importance of finding eco-friendly alternatives to chemical fungicides as a motivator for this research.

Response 4: We must thank you for your kind suggestions.According to your suggestion, we have revised the section and the revised texts in lines 42-46 are as follows:

“Currently, chemical fungicides are commonly used in the postharvest control of these two pathogens, which can have negative impacts on the development of fungal resistance and environmental pollution [12]. Therefore, there is a need for the development of alternative and environmentally safe means for postharvest disease prevention during storage of important crop products.”

Discussion:

Point 1：Organize the discussion section into subsections for clarity. For example, sections on "Inhibitory Mechanisms" and "Implications for Postharvest Preservation."

Response 1: We are sincerely grateful for your thoughtful suggestions. According to your suggestion.To state the implications of our results more explicitly, we have revised the section. Three subsections were divided in revised manuscript, and the secondary title respectively was 4.1 Antifungial activity of 2ʹ,4ʹ - dichloro-chalcone in vitro, 4.2 The possible antifungial mechanism of 2ʹ,4ʹ- dichloro-chalcone against F. tricinctum and T. roseum, and 4.3 2ʹ,4ʹ-dichloro-chalcone treatment significantly inhibited the pathogenicities of F. tricinctum and T. roseum in vivo.

Point 2：Elaborate on the potential practical applications of your findings. How might this research be used in real-world scenarios for crop protection?

Response 2: We do appreciate you for giving us comments and pointing out this issue. According to your suggestion, we have revised the section and the revised texts in lines 345-350 are as follows:

“Chalcones are members of the flavonoid family, are precursors for the synthesis of flavonoids and isoflavones in plants. Due to their content of active α and β unsaturated carbonyl functional groups, they are chemically very reactive, it can effectively inhibit the growth of bacteria, mold and yeast, and prolong the shelf life of foods. In postharvest preservation, chalcones compounds can be used as film forming materials be applied in vegetables and fruits to protect them from microorganisms.”

Point 3：Discuss any limitations of the study. Are there any factors that might affect the compound's efficacy in the field?

Response 3: We gratefully appreciate for your kindly suggestion. According to your suggestion, we have added some information of limitation in revised manuscript. The revised texts in line 429-431 are as follows:

“Although chalcones compounds have very good antifungial activity，it easy to decompose at high temperature and the antimicrobial effect will also decline.”

Point 4：Address the broader implications of your research for sustainable agriculture and food safety. How could the use of compounds like 2ʹ,4ʹ-dichloro-chalcone contribute to these goals?

Response 4: Thank you very much for your suggestions and your patience. According to your suggestion, we have added some information for sustainable agriculture and food safety in revised manuscript in lines 347-350. The added information is as following:

“It can effectively inhibit the growth of bacteria, mold and yeast, and prolong the shelf life of foods. In postharvest preservation, chalcones compounds can be used as film forming materials be applied in vegetables and fruits to protect them from microorganisms.”

Conclusions:

Point 1：Summarize the key findings in a concise manner.

Response 1: We thank you very much for giving us an opportunity to revise our manuscript. According to your suggestion, we have summarized the key findings in conclusion section. The revised information is as following:

“Our results showed that 1 µM of 2ʹ,4ʹ-dichloro-chalcone strongly inhibited mycelial growth and conidial production of F. tricinctum (32.3%) and T. roseum (65.2%) in vitro. In addition, this treatment also significantly inhibited the expansion of potato dry rot from F. tricinctum (48.6%) and apple rot spot from T. roseum (36.2%).”

Point 2：Clearly state the practical implications of your research. What are the takeaways for postharvest disease control?

Response 2: We sincerely thank you for your insightful and helpful comments, corrections, and suggestions on our manuscript, as well as the important guiding significance to our researches. According to your suggestion, we have summarized the section in a concise manner. The revised texts in line 438-441 are as follows:

“The present investigation might be helpful to explore the application of 2ʹ,4ʹ-dichloro-chalcone as an effective antifungal agent in treating postharvest infections caused by F. tricinctum and T. roseum.”

Point 3：Highlight the novelty and significance of your study. What makes it stand out in the field of agricultural research?

Response 3: Thank you very much for your helpful and insightful comments and suggestions about our manuscript. According to your suggestion, we have revised the section. The revised texts in lines are as follows:

“As an environmentally friendly, green and efficient fungicide, 2ʹ,4ʹ-dichloro-chalcone will be widely used in agricultural practices.”

Point 4：Suggest directions for future research, especially regarding the molecular mechanisms underlying the compound's action and its potential for practical application in crop protection.

Response 4: Special thanks for the Reviewer’s good comments. According to your suggestion, we have revised the section.The revised text are as follows:

“Characteristics of film formation of 2ʹ,4ʹ-dichloro-chalcone and other chlorinated chalcones molecular should be further investigated as alternative film-forming materials for the postharvest preservation of fruits and vegetables, sustainable agriculture and food security.”

Once again, thank you very much for your comments and suggestions.

Author Response File: Author Response.docx