Mandarin Essential Oils as an Alternative Method of Controlling the Fungus Alternaria alternata (Fr.: Fr.) Keissler

Round 1

Reviewer 1 Report

Dear Authors

thank you very muhc for the paper.

Several suggestions are in the pdf file.

kind regards

Comments for author File: Comments.pdf

Author Response

Initially, we are grateful for the valuable suggestions and corrections made by the reviewer. Practically all of them were accepted and are highlighted in the text in yellow. Some points were kept, as the other 2 reviewers recommended so.

Best regards,

Correspondence author

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors,

You have written a good paper. I would just like to draw your attention to some technical details. Namely, from lines 97 to 110 including line 144, please move the SI units from the numbers (g, mm, ^oC).

Row 97 before PDA write first potato-dextrose-agar.

Line 31 and 149 write Alternaria in italics.

Row 152 and 256 delete (EO) or essential oils.

Table 1. instead of commas, put dots for decimals.

Author Response

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript contains valuable data related to the applicability of mandarin essential oil, but for better highlighting of the antifungal effect, it must also refer to solvent control (Tween 80). It is possible to influence the growth of the mycelium. In addition, the statistical analysis is very important to highlight the significance of the results compared to the controls.

Below are the specific suggestions:

1. I suggest to reformulate the sentence: "The inhibitory effect of different concentrations (1, 2, 4, 8, and 16 μL mL-1) of Fremont IAC 543 mandarin, IAC 2019Maria mandarin, Murcott IAC 221 22 tangor and Late IAC 855 willowleaf”? (lines 21-23)

2. Update the introduction by including recent references.

3. Include in the introduction details about the antimicrobial properties of mandarin EO, existing in the specialized literature. Include in the title of the manuscript the term “mandarin essential oil”, to avoid confusion

4. Do you mean optical microscope (lines 101-102? Please, include microscopic images. The equipment used must be written in detail throughout the manuscript.

5. Would you use any solvent to solubilize the EO (which is hydrophobic)? If it is Tween 80, did you make a solvent control?

6. For the percentage evaluation of the inhibition, you must refer to the mean diameter of the colony of the fungus in control (Pi=((dc-dt) x 100)/dc). Redo the calculations.

7. A statistical analysis of the results related to controls is necessary, in order to highlight the significance of the results.

8. Could you enter in Table 1 the percentages obtained for each class of compounds? The results should be written with mean ± SD in the whole manuscript, especially in Table 1.

9. How many leaves were studied for preventive and curative assays? Could you insert images with the appearance of treated and untreated leaves?

10. Can you insert images of the plates in the supplementary material to highlight the reduction of mycelia?

Author Response

Initially, we are grateful for the valuable suggestions and corrections made by the reviewer. All of them were accepted and are highlighted in the text in green.

below answers:

The manuscript contains valuable data related to the applicability of mandarin essential oil, but for better highlighting of the antifungal effect, it must also refer to solvent control (Tween 80). It is possible to influence the growth of the mycelium. In addition, the statistical analysis is very important to highlight the significance of the results compared to the controls.

Answer:

Tween® 80 was used as a surfactant in this study due to its low irritation, low toxicity, high stability, and small molecule that can minimize droplet size of the nanoemulsions. Thus, was observed by Muy-Rangel et al. (2018), there were no significant differences in the radial growth in treatments PDA and PDA-Tween® 80 after 120h on Alternaria spp.

Muy-Rangel MD, Osuna-Valle JR, García-Estrada RS, Martín-Hernández S, Quintana-Obregón EA. In vitro antifungal activity of garlic essential oil (Allium sativum L.) against Alternaria tenuissima. Rev Mexicana Fitopatol. (2018) 36:162–71.

1. Pongsumpun, S. Iwamoto, U. Siripatrawan. Response surface methodology for optimization of cinnamon essential oil nanoemulsion with improved stability and antifungal activity Ultrasonics Sonochemistry, 60 (2020), p. 104604
2. Ghosh, A. Mukherjee, N. Chandrasekaran, Eugenol – loaded antimicrobial nanoemulsion preserves fruit juice against microbial spoilage, Colloids Surf. B, Biointerfaces 114 (2014) 392–397
3. Goindi, A. Kaur, R. Kaur, A. Kalra, P. Chauhan, Nanoemulsions: an emerging technology in the food industry, in: A.M. Grumezescu (Ed.), Nanotechnology in the Agri-Food Industry, Academic Press, USA, 2016, pp. 651–688

Below are the specific suggestions:

1. I suggest to reformulate the sentence: "The inhibitory effect of different concentrations (1, 2, 4, 8, and 16 μL mL-1) of Fremont IAC 543 mandarin, IAC 2019Maria mandarin, Murcott IAC 221 22 tangor and Late IAC 855 willowleaf”? (lines 21-23)

The inhibitory effect of different concentrations (1, 2, 4, 8, and 16 μL mL-1) of EOs of Fremont IAC 543 mandarin, IAC 2019Maria mandarin, Murcott IAC 221 22 tangor and Late IAC 855 willowleaf

2. Update the introduction by including recent references.

References updated: 6, 8, 10 and 11

3. Include in the introduction details about the antimicrobial properties of mandarin EO, existing in the specialized literature. Include in the title of the manuscript the term “mandarin essential oil”, to avoid confusion

Lines 68-70

4. Do you mean optical microscope (lines 101-102? Please, include microscopic images. The equipment used must be written in detail throughout the manuscript.

Supplementary material – Conidia of Alternaria alternata observed in Leica DM750 light microscope (LM) (Leica Microsystems, Co., Wetzlar, Germany).

5. Would you use any solvent to solubilize the EO (which is hydrophobic)? If it is Tween 80, did you make a solvent control?

Tween® 80 was used as a surfactant in this study due to its low irritation, low toxicity, high stability, and small molecule that can minimize droplet size of the nanoemulsions. Thus, was observed by Muy-Rangel et al. (2018), there were no significant differences in the radial growth in treatments PDA and PDA-Tween® 80 after 120h on Alternaria spp.

Muy-Rangel MD, Osuna-Valle JR, García-Estrada RS, Martín-Hernández S, Quintana-Obregón EA. In vitro antifungal activity of garlic essential oil (Allium sativum L.) against Alternaria tenuissima. Rev Mexicana Fitopatol. (2018) 36:162–71.

6. For the percentage evaluation of the inhibition, you must refer to the mean diameter of the colony of the fungus in control (Pi=((dc-dt) x 100)/dc). Redo the calculations.

Pi = (dc - dt )x100 (1)

           dc

Where, Pi = Percent growth inhibition

dc = The mean diameter of the colony of the fungus in the control

dt = The mean diameter of the fungus colony in the treatment

7. A statistical analysis of the results related to controls is necessary, in order to highlight the significance of the results.

This was done when using the data of percentage evaluation of inhibition

8. Could you enter in Table 1 the percentages obtained for each class of compounds? The results should be written with mean ± SD in the whole manuscript, especially in Table 1.

The other reviewers accepted the data in percentage

9. How many leaves were studied for preventive and curative assays? Could you insert images with the appearance of treated and untreated leaves?

Lines 165-166 (three leaves each plate) and Insert supplementary material

10. Can you insert images of the plates in the supplementary material to highlight the reduction of mycelial

Insert supplementary material

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors

thank you very much for this new version.

However, I still do not see the controls used in the trial. 

The photos help a lot in easy interpretation; but again I don't see the controls.

Your graphs do not show the controls either.

kind regards.

Author Response

Dear reviewer,

We insert the control into the graphics and figures.

Best Regards

Authors

Author Response File: Author Response.docx

Reviewer 3 Report

I further argue that it is essential to introduce the standard deviation (mean±SD) for the GC analysis, especially since the authors mention that they worked in triplicate. Statistical analysis such as T-test, ANOVA, etc. highlights whether the differences between EOs are significant or not, compared to the controls as well as the comparison between them.

Author Response

Dear reviewer,

In fact, the GC evaluation was not performed in triplicate, so we do not present the SD. Sorry for this wrong information.

Tables with the results of the Tukey test (5%) were added to the supplementary material, where there is a comparison with the controls.

Best regards,

Authors

Author Response File: Author Response.docx