Chitosan-Loaded Copper Oxide Nanoparticles: A Promising Antifungal Nanocomposite against Fusarium Wilt Disease of Tomato Plants

Round 1

Reviewer 1 Report

1. In paragraph 2. Materials and methods. Description of the FTIR method presented in the draft 3.3.

2. Figure 2 Decorate the inscriptions. S-S, S=N…..

3. Table 1 Justification of periods 4 days, 6 days, 8 days. How does this relate to the development of the pathogen.

4. Page 10 Draw the diagram as a separate figure. Enter the name of the link in the text. Incorrect graphical data and visualization experiment in the same figure.

5. Figure 5. Draw the graphs in one scale, make the font of the drawing of all graphs the same size. It is desirable to make charts in color without "Fifty Shades of Grey"

6. In Conclusion 4 Give a quantitative assessment (%) of the experiments performed

and the effectiveness of methods to achieve results.

Author Response

Response to Reviewer 1

We greatly appreciate all the critiques and comments from you. Those comments are extremely helpful for us to improve our paper, and they provide valuable guidance for our future study. Additionally, the new version of the manuscript has been proof-read by a native English writer who is familiar with scientific and academic writing. We have edited the manuscript according to his advice. You could find the changes throughout the text. According to these comments, we have carefully improved our manuscript, and all the revisions are highlighted in yellow in the text. Please see below point-by-point responses to the comments:

Question 1: In paragraph 2. Materials and methods. Description of the FTIR method presented in the draft 3.3.

Response:  Many thanks for your kind comment, we have described the FT-IR method in Section Materials and methods (Highlighted: see lines: 156: 162)

Question 2: Figure 2 Decorate the inscriptions. S-S, S=N…..

Response: Many thanks for your kind comment, However I really tried to include additional functional groups  like S-S, S=N in the IR spectrum, but I could not find a clear signal band other than that what was included previously, this may be related to the that those bands are weak in the absorption….Thanks again.

Question 3: Table 1 Justification of periods 4 days, 6 days, 8 days. How does this relate to the development of the pathogen.

Many thanks for this important question: In table 1, although most similar experiments measure the average percent growth inhibition of nanoparticles against the fungal pathogen after only 8 days of the fungal growth. However, in our study we measured and followed this inhibition over a course of the incubation time (4, 6 and 8) days, in order to accurately ensuring the inhibition behavior of the  CH@CuO NPs against the target pathogen (with 3 isolates) throughout its continued growth and to figure out the potential activity of CH@CuO NPs from the beginning of the treatment or  after certain days of the fungal growth.

Question 4: Page 10 Draw the diagram as a separate figure. Enter the name of the link in the text. Incorrect graphical data and visualization experiment in the same figure.

Many thanks for your kind comment. I agree with you. I have corrected it.

Question 5: Figure 5. Draw the graphs in one scale; make the font of the drawing of all graphs the same size. It is desirable to make charts in color without "Fifty Shades of Grey"

Thanks again. All graphs are now with the same font of drawing.

Question 6: In Conclusion 4 Give a quantitative assessment (%) of the experiments performed and the effectiveness of methods to achieve results.

Response:  Many thanks for your kind comment, we provided the conclusion section with a quantitative assessment (%) for some experiments to be more brief and attractive for the journal readers ( Highlighted, See Lines:650-660 )

Reviewer 2 Report

1. There are extra dots in some sentences. Check the wording.

2. Species names, as well as the terms in vitro or in vivo, should appear in italics.

3. Capital letters are used unnecessarily.

4. Define abbreviations the first time they appear.

5. It would be necessary to discuss the modifications caused by the interaction between chitosan and CuO in the infrared spectra of the NPs.

6. There seem to be aggregates of NPs in figures 1A and 1B. They should be discussed in more detail.

7. The lines in figure 2 should be thinner to see the signals in the infrared spectrum in detail.

8. The legends of the statistical analysis should appear in Table 1 to identify treatment differences.

9. A full magnification and length mark should appear in figure 3 to identify details.

10. What was the criteria to choose the concentration of Kocide 2000 and to be able to compare it with the concentrations of CH@CuO NPs.

11. It remains to discuss the possible antifungal action of CH@CuO NPs. Please review: 10.5772/intechopen.106502; 10.1007/s10924-021-02296-y.

12. There is an error in figure 4A for the line that includes the treatments with the NPs.

13. The first part of the discussion does not have enough references.

14. It remains to discuss the beneficial effects of CuO mentioned in the conclusions.

Comments for author File: Comments.pdf

Author Response

Response to reviewers

Reviewer 2

Comments and Suggestions for Authors

1. There are extra dots in some sentences. Check the wording.
2. Species names, as well as the terms in vitro or in vivo, should appear in italics
3. Capital letters are used unnecessarily
4. Define abbreviations the first time they appear.
5. It would be necessary to discuss the modifications caused by the interaction between chitosan and CuO in the infrared spectra of the NPs.
6. There seem to be aggregates of NPs in figures 1A and 1B. They should be discussed in more detail.
7. The lines in figure 2 should be thinner to see the signals in the infrared spectrum in detail.
8. The legends of the statistical analysis should appear in Table 1 to identify treatment differences.
9. A full magnification and length mark should appear in figure 3 to identify details.
10. What was the criteria to choose the concentration of Kocide 2000 and to be able to compare it with the concentrations of CH@CuO NPs.
11. It remains to discuss the possible antifungal action of CH@CuO NPs. Please review: 10.5772/intechopen.106502; 10.1007/s10924-021-02296-y.
12. There is an error in figure 4A for the line that includes the treatments with the NPs.
13. The first part of the discussion does not have enough references.
14. It remains to discuss the beneficial effects of CuO mentioned in the conclusions.

Response to Reviewer 2

We greatly appreciate all the critiques and comments from you. Those comments are extremely helpful for us to improving our paper, and they provide valuable guidance for our future study. Additionally, the new version of the manuscript has been proof-read by a native English writer who is familiar with scientific and academic writing According to these comments, we have carefully improved our manuscript, and all the revisions are highlighted in yellow in the text. Please see below point-by-point responses to the comments:

Question 1: There are extra dots in some sentences. Check the wording

Response:  Many thanks for the critical revision, we checked all the manuscript and corrected all of them.

Question 2: Species names, as well as the terms in vitro or in vivo, should appear in italics

Response: Corrected

Question 3: Capital letters are used unnecessarily

Response:  Corrected

Question 4: Define abbreviations the first time they appear.

Response: Many thanks for your kind notification, we corrected all of them.

Question 5: It would be necessary to discuss the modifications caused by the interaction between chitosan and CuO in the infrared spectra of the NPs.

Response: I agree with you, a number of sentences were added to discuss this point (Highlighted: see Lines 294: 298). Thanks again for critical reviewing.

Question 6: There seem to be aggregates of NPs in figures 1A and 1B. They should be discussed in more detail.

Response:  Many thanks for your kind comment, we clarified this issue in the new version of the manuscript and provided our suggestion with a number of recent references related to this point. (Highlighted: See lines 278: 281)

Question 7: The lines in figure 2 should be thinner to see the signals in the infrared spectrum in detail.

Response: I totally agree with you, the IR spectrum was changed in the new version of the manuscript with to a more thinner line (See Figure 2). Thanks again.

Question 8: The legends of the statistical analysis should appear in Table 1 to identify treatment differences.

Response:  We thank the reviewer for his valuable comment. In the new version of the manuscript, we have presented data of Table 1 based on statistical view to simply identify the statistical difference between treatments (See Table 1).

Question 9: A full magnification and length mark should appear in figure 3 to identify details.

Response:   Many thanks for your kind comment, we add the scale bar on the images and the magnification was described in the figure legend (Highlighted: bar= 40 µm). Additionally, although, fluorescence microscope is used to determine the fungal cell viability after staining "red=dead cells or blue= live cells". However, I agree with you, that it will be more better to consider in the future work taking a high magnified images to describe any more details.  

Question 10: What was the criteria to choose the concentration of Kocide 2000 and to be able to compare it with the concentrations of CH@CuO NPs.

Response:  Many thanks for this question, we chosed Kocide 2000's concentration (2.5 g/l) based on following the recommended dosage of this fungicide in different published articles against different fungal pathogens including Fusarium oxysporum.

Question 11: It remains to discuss the possible antifungal action of CH@CuO NPs. Please review: 10.5772/intechopen.106502; 10.1007/s10924-021-02296-y.

Response: 

Done (Highlighted : See line 468:484)

Question 12: There is an error in figure 4A for the line that includes the treatments with the NPs.

Response:   Again, many thanks for your kind notice, it was corrected.

Question 13: The first part of the discussion does not have enough references

Response:  Done, we included different references in this issue (Highlighted: Ref. 34-46)

Question 14: It remains to discuss the beneficial effects of CuO mentioned in the conclusions

Response:

Done (Highlighted: See lines: 478:484)

Round 2

Reviewer 2 Report

1. Define FTIR on line 144.

2. An extra space appears on lines 155, 276, 277, 476, and 588.

3. The phrase "isolated Fusarium isolates" is redundant on line 241.

4. Figure 2 is not seen in the file. Therefore, the lines in Figure 2 cannot be seen as thinner to see the signals in the infrared spectrum in detail.

5. Figures 3, 4, 5, and 6 are not seen in the file.

6. The legend of Table 1 (a-f) indicating the statistical differences is missing.

7. The equivalence of the concentration of Kocide 2000 (2.5 g/l) with the concentrations of the CH@CuO NPs remains unclear.

8. Be consistent with the liter symbol (l or L).

Comments for author File: Comments.pdf

Author Response

Response to Reviewer 2

We greatly appreciate all the critiques and comments from you. Those comments are extremely helpful for us to improve our paper, and they provide valuable guidance for our future study. You could find the changes throughout the text. According to these comments, we have carefully improved our manuscript, and all the revisions are highlighted in yellow in the text. Please see below point-by-point responses to the comments:

Question 1: Define FTIR on line 144.

Response: Many thanks for your kind comment, we defined FTIR (See line 156, Highlighted)  

Question 2: An extra space appears on lines 155, 276, 277, 476, and 588.

Response: Corrected

Question 3: The phrase "isolated Fusarium isolates" is redundant on line 241.

Response: Corrected

Question 4: Figure 2 is not seen in the file. Therefore, the lines in Figure 2 cannot be seen as thinner to see the signals in the infrared spectrum in detail.

Response: Many thanks for your kind comment, we think that it was a technical system error; we have now fixed the figure to avoid their moving while PDF is generated.

Question 5: Figures 3, 4, 5, and 6 are not seen in the file.

Response: Many thanks for your kind comment, we have now fixed the figures to avoid their moving while PDF is generated.

Question 6: The legend of Table 1 (a-f) indicating the statistical differences is missing.

Response:  Many thanks for your kind comment, in below Table 1, we add a legend sentence indicated the statistical differences (See line 334, Highlighted).

Question 7: The equivalence of the concentration of Kocide 2000 (2.5 g/l) with the concentrations of the CH@CuO NPs remains unclear.

Response:  Many thanks for your kind comment, according to different published reports indicated that the dosage of Kocide 2000 in the range of 2-3 g/l is effective against different plant pathogens.  So, we selected 2.5 g/l as an effective/equivalent  dosage for the used fungicide in comparison with CH@CuO NPs. We also checked the product label of the manufacturing company. Kindly, see   references (Monir et al, 2021; ElAbeid et al. 2020; Bag et al. 2010; Prasad et al. 2008). See also (https://www.bartlett.ca/85256BB40074BCB1/52C92014929415F385257F86004D090C/0323CBE86C801F34852573F4006E4AFB/$file/Kocide+2000+Label+English.pdf.

Question 8: Be consistent with the liter symbol (l or L).

Response:  Many thanks for your kind comment, we corrected to "L" to be the same in all the manuscript including figures.

Again, many thanks for your critical reviewing the manuscript that significantly improved the manuscript, and they provide me with valuable guidance for the future study.