Cyclic Organic Peroxides as New Fungicides against Phytopathogenic Fungi
, Peter S. Radulov 1, Yulia Yu. Belyakova 1, Dmitrii I. Fomenkov 1, Vera A. Vil’ 1, Maria A. Kuznetsova 2, Valentina N. Demidova 2, Alexei P. Glinushkin 2 and Alexander O. Terent’ev 1,*
Round 1
Reviewer 1 Report
I feel that the manuscript is in the level for an article for Agrochemicals. Overall, the manuscript is well written. I recommend major revision of the manuscript as the results and the discussion should be divided and the discussion should be rewritten appropriately.
Some comments
Line 53: Add references
Line 233: Solanum tuberosum italics
Line 273: No need to write F.c., please correct all
Table 1: Write 0.0±0.0 and 100.0±0.0 to be consistent.
The drawback of this paper is the discussion, there is no discussion. Please separate results and discussion and rewrite the discussion.
Author Response
Reviewer 1
Comment: I feel that the manuscript is in the level for an article for Agrochemicals. Overall, the manuscript is well written. I recommend major revision of the manuscript as the results and the discussion should be divided and the discussion should be rewritten appropriately.
Response: Thank you for such a positive comment.
Some comments
- Line 53: Add references
Response: Since the fungicidal activity of cyclic organic peroxides was discovered fear years ago in group (ref. 32-33), the mechanism of the fungicidal action is still unknown. We have revised the text:
“The key feature of these compounds is that, due to the presence of the O-O bond, their mechanism of action can potentially be different from that of commercial fungicides.”
- Line 233: Solanum tuberosum italics
Response: Corrected
- Line 273: No need to write F.c., please correct all
Response: Corrected
- Table 1: Write 0.0±0.0 and 100.0±0.0 to be consistent.
Response: Corrected
- The drawback of this paper is the discussion, there is no discussion. Please separate results and discussion and rewrite the discussion.
Response: We ask the reviewer to leave it as it is. Due to the pioneering status of the study, it would be easier for readers to follow the material in this format. According to the rules of the journal, the "Discussion" section can be combined with the "Results" section.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The manuscript of Yaremenko and co-workers entitled “Cyclic Organic Peroxides as New Fungicides against Phytopathogenic Fungi” described the synthesis of peroxides with biological properties against species of phytopathogenic fungi. I consider it an important contribution to the field of research. However, it must be considered that this is an extension of other research carried out (Molecules 2020, 25, 1954 and Chem. Eur.J.2020,26,4734 –4751). I suggest to the authors that in the future they can explore other derivatives or other types of structures. For this manuscript, I recommend small modifications such as:
1. in line 34, inform the meaning of DMIs. Also for SDHIs in line 36.
2. Please, add the yield and the mass of products for each reaction in the data description.
3. Could the authors include some references among lines 271-277?
Author Response
Reviewer 2
Comment: The manuscript of Yaremenko and co-workers entitled “Cyclic Organic Peroxides as New Fungicides against Phytopathogenic Fungi” described the synthesis of peroxides with biological properties against species of phytopathogenic fungi. I consider it an important contribution to the field of research. However, it must be considered that this is an extension of other research carried out (Molecules 2020, 25, 1954 and Chem. Eur.J.2020,26,4734 –4751). I suggest to the authors that in the future they can explore other derivatives or other types of structures.
Response: Thank you for such a positive comment. We are going to continue with the study of the fungicidal activity of the cyclic organic peroxides.
Comment: For this manuscript, I recommend small modifications such as:
- in line 34, inform the meaning of DMIs. Also for SDHIs in line 36.
Response: Corrected
- Please, add the yield and the mass of products for each reaction in the data description.
Response: Corrected
- Could the authors include some references among lines 271-277?
Response: References added to part 2.6. Investigation of Fungicidal Activity in vitro
Author Response File: Author Response.pdf
Reviewer 3 Report
The authors synthesized organic peroxides derivates and tested their antifungal activity in vitro and in planta. A lot of information is missing from Materials and Methods (see attached pdf file). There is only results in Results and Discussion section. No paper was cited in this section. The authors need to add discussion: cite previous papers; and compare the efficacy of the compounds synthesized in this study with other previously reported compounds; how useful this new compounds relative to the existing chemical fungicide etc.
Azoxystrobin resistance is very common in many fungal species. The authors should verify that the fungal isolates used in this test are sensitive to azoxystrobin before the test.
The concentration of Azoxystrobin is not mentioned in the manuscript. How do you justify that the new compound is effective when comparing with Azoxystrobin at XX ug/mL vs. new compound at 30 ug/mL?
In planta test (Table 2), the new compound is inferior to the exiting fungicide (fluazinam). What's your thought about this? Discuss.
The data presented in this study doesn't support the L20-21: ...comparable or superior to the modern fungicides.
Some statistics can be done for Tables 1 and 2 to compare the mean values.
Comments for author File: Comments.pdf
Author Response
Reviewer 3
The authors synthesized organic peroxides derivates and tested their antifungal activity in vitro and in planta. A lot of information is missing from Materials and Methods (see attached pdf file). There is only results in Results and Discussion section. No paper was cited in this section. The authors need to add discussion: cite previous papers; and compare the efficacy of the compounds synthesized in this study with other previously reported compounds; how useful this new compounds relative to the existing chemical fungicide etc.
Response: Thank you for your detailed reading. We have corrected the manuscript according to your comments.
- Comment: Azoxystrobin resistance is very common in many fungal species. The authors should verify that the fungal isolates used in this test are sensitive to azoxystrobin before the test.
Response: Phytopathogenic fungi sensitivity to azoxystrobin was tested before the study. The information was added to section 2.6.
- Comment: The concentration of Azoxystrobin is not mentioned in the manuscript. How do you justify that the new compound is effective when comparing with Azoxystrobin at XX ug/mL vs. new compound at 30 ug/mL?
Response: We have added information about the concentration of Azoxystobin. The concentration of Azoxystrobin was 30 mg∙L−1.
- Comment: In planta test (Table 2), the new compound is inferior to the exiting fungicide (fluazinam). What's your thought about this? Discuss.
Response: The discussion was added to section 3.3.
- Comment: The data presented in this study doesn't support the L20-21: ...comparable or superior to the modern fungicides.
Response: The abstract was revised. The phrase "superior to modern fungicides (such as azoxystrobin)" was replaced by "superior to azoxystrobin". Indeed, the fungicidal activity of some peroxides is comparable or superior to that of azoxystrobin. It was shown in Table 1.
- Comment: Some statistics can be done for Tables 1 and 2 to compare the mean values.
Response: The information about statistical analysis was added to section 2.6 and 2.7.
- Comment: How many leaves (from how many plants) were used for each treatment? What's the experiment design (experiment run, replicate etc.)?
Response: One fully-expanded leave per plant were treated and in total 10 plants were used per one treatment. (section 2.7, line 323).
- Comment: What's LSD0.95?
Response: The least significant difference (LSD0.95) was used to compare the levels of a pathogen growth inhibition and disease severity between the tested compounds and to determine the significance of differences between results for different compounds at a 95% confidence level. The information was added to section 2.6.
Author Response File: Author Response.pdf
Reviewer 4 Report
Overall, there are many positives about this paper. The 6 fungi chose for study seem appropriate/relevant and the results with some of the bicyclic peroxides are quite interesting.
The introduction might be broadened to note that hydrogen peroxide, peracetic acid, Oxone and some other commercial oxidants have been explored for treatment of fungal infestations (see Copes and Ojiambo, Crop Protection, v 164, 2023, 106143) and an oxidative burst of hydrogen peroxide has been associated with plant response to disease (Levine,
Cell (1994), 79(4), 583-93). There are a number of reports describing activity of cyclic peroxides against yeast (e.g., Phillipson and Rinehart, J. Am. Chem. Soc. 1983, 7735 and probably many later examples) and a lead reference into this literature (perhaps already found in reference 12 or another of the reviews?) would provide the reader with a useful gateway into relevant literature.
It might be useful for the authors to speculate on a target for these drugs or to indicate if some follow-up research is in progress.
Given the focus of cyclic peroxides, it is unclear why a 1,1-dihydroperoxide (P1) is included.
Questions about Assays: Why were samples dissolved in acetone in some cases (section 2.5) and distilled water in others (section 2.6)? Section 2.5 is somewhat confusing. It is unclear why the authors did not include controls with and without acetone, especially since they didn’t seem to use acetone in section 2.6. A solution containing 1% acetone is likely to kill most fungi. Because the authors are adding the acetone-fungicide solution to the liquid potato-sugar agar at 50C, perhaps the acetone (bp 56 °C) has mainly evaporated prior to the agar solidifying so that the fungi can still grow. This should work, especially if the acetone concentration at the agar surface was less than that lower down. However, it would be nice in future work to compare acetone versus water to go with the acetone-fungicide versus pure acetone.
What is the composition of the “commercial adjuvant” described on line 231. It is probably proprietary, but it could influence things. It would not be unusual for such a commercial material to include a UV protectant, a detergent, and something that makes the composition sufficiently sticky and dense to be retained on the leaf.
The results of the translaminar experiments are interesting but can be confusing. Although a commercial fungicide is superior to P4 in terms of protection of potato leaves, both P4 and the commercial fungicide provide diminished protection when translaminar redistribution is required; this is not clear from the text. Is there an advantage to using the terms abaxial and adaxial (vs. upper and lower)?
Methods for the synthesis of the classes of peroxides represented by P1 -P13 have been previously reported. If experimental procedures and spectral characterization have been reported for any of the molecules in the paper, then procedures and characterization do not need to be included (but could be included in the SI if the presence would benefit readers).
Suggestions related to text:
Abstract: The discovery of the antimalarial activity of the peroxide natural product Artemisinin, an achievement that was recently recognized with the Nobel Prize, has brought…
This paper could be of interest to readers from multiple areas and it would be wise to include a short list/table of abbreviations: DMI, SDHI, HCP, PE, etc.
Author Response
Reviewer 4
Overall, there are many positives about this paper. The 6 fungi chose for study seem appropriate/relevant and the results with some of the bicyclic peroxides are quite interesting.
Response: Thank you for such a positive comment.
- Comment: The introduction might be broadened to note that hydrogen peroxide, peracetic acid, Oxone and some other commercial oxidants have been explored for treatment of fungal infestations (see Copes and Ojiambo, Crop Protection, v 164, 2023, 106143) and an oxidative burst of hydrogen peroxide has been associated with plant response to disease (Levine, Cell (1994), 79(4), 583-93). There are a number of reports describing activity of cyclic peroxides against yeast (e.g., Phillipson and Rinehart, J. Am. Chem. Soc. 1983, 7735 and probably many later examples) and a lead reference into this literature (perhaps already found in reference 12 or another of the reviews?) would provide the reader with a useful gateway into relevant literature.
Response: According to the reviewer’s suggestion, the introduction was revised.
- Comment: It might be useful for the authors to speculate on a target for these drugs or to indicate if some follow-up research is in progress.
Response: Thanks for the comment. We are afraid of misleading readers, so we ask the reviewer not to have us speculate about the target. At the moment we are continuing our research.
- Comment: Given the focus of cyclic peroxides, it is unclear why a 1,1-dihydroperoxide (P1) is included.
Response: We speculate that the superior activity of the cyclic peroxides P2-P8 compared to the acyclic peroxide P1 demonstrated the important role of the cyclic nature in the mechanism of action.
Comment: Questions about Assays: Why were samples dissolved in acetone in some cases (section 2.5) and distilled water in others (section 2.6)? Section 2.5 is somewhat confusing. It is unclear why the authors did not include controls with and without acetone, especially since they didn’t seem to use acetone in section 2.6. A solution containing 1% acetone is likely to kill most fungi. Because the authors are adding the acetone-fungicide solution to the liquid potato-sugar agar at 50C, perhaps the acetone (bp 56 °C) has mainly evaporated prior to the agar solidifying so that the fungi can still grow. This should work, especially if the acetone concentration at the agar surface was less than that lower down. However, it would be nice in future work to compare acetone versus water to go with the acetone-fungicide versus pure acetone.
Response: We used the medium with added acetone as a blank control. To avoid misunderstanding, section 2.8 has been rewritten. The efficacy of mycelial growth inhibition (Table 1) was estimated relative to the blank control.
- Comment: What is the composition of the “commercial adjuvant” described on line 231. It is probably proprietary, but it could influence things. It would not be unusual for such a commercial material to include a UV protectant, a detergent, and something that makes the composition sufficiently sticky and dense to be retained on the leaf.
Response: The used commercial adjuvant “Atomic™” was purchased from commercial sources and were used as is. We added available information about the content of this adjuvant to section 2.7.
- Comment: The results of the translaminar experiments are interesting but can be confusing. Although a commercial fungicide is superior to P4 in terms of protection of potato leaves, both P4 and the commercial fungicide provide diminished protection when translaminar redistribution is required; this is not clear from the text. Is there an advantage to using the terms abaxial and adaxial (vs. upper and lower)?
Response: The discussion of Table 2 was revised. The terms “abaxial” and “adaxial” are commonly used to describe such experiments in the literature.
- Comment: Methods for the synthesis of the classes of peroxides represented by P1 -P13 have been previously reported. If experimental procedures and spectral characterization have been reported for any of the molecules in the paper, then procedures and characterization do not need to be included (but could be included in the SI if the presence would benefit readers).
Response: Thanks for the comment. According to the journal’s rules we have included the synthetic methods and characterization data to the Materials and Methods section of the manuscript.
Suggestions related to text:
- Comment: Abstract: The discovery of the antimalarial activity of the peroxide natural product Artemisinin, an achievement that was recently recognized with the Nobel Prize, has brought…
Response: Corrected
- Comment: This paper could be of interest to readers from multiple areas and it would be wise to include a short list/table of abbreviations: DMI, SDHI, HCP, PE, etc.
Response: Full names for abbreviations were added in the text of the article.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Good to go.
Reviewer 3 Report
The authors addressed all of the reviewers' comments and suggestions. Mostly satisfactory, however I still can see only results in the Results and Discussion (except lines 276-277). The authors need to make the research outcome relevant to the previous studies by citing more papers. This manuscript lacks discussion.
