In-vitro assessment of Thyme oil (Thymus vulgaris) as antifungal agent against Phyllosticta citricarpa

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1. 

   Access Microbiology

   Aug 15, 2023

   The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.

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2. 

   Access Microbiology

   Aug 9, 2023

   Comments to Author

   The authors present an intriguing manuscript detailing the fungicidal properties of thyme oil against Phyllosticta citricarpa. The activity of which is hypothesised to inhibit mitochondrial activity. However, there remains some issues to address in terms of experimental design. In particular, use of XTT and the way it was used does not indicate thymus oil induced changes in mitochondrial activity. Similarly changes in morphology induced by thymus oil cannot be directly compared due to differences in magnification of SEM images presented. Collectively, the reviewer suggests presentation of additional SEM images at comparable magnifications, repeat of the XTT assay with standardised broths comprising either thyme oil or ethanol as a negative control. To confirm that thyme oil does indeed inhibit …

   Comments to Author

   The authors present an intriguing manuscript detailing the fungicidal properties of thyme oil against Phyllosticta citricarpa. The activity of which is hypothesised to inhibit mitochondrial activity. However, there remains some issues to address in terms of experimental design. In particular, use of XTT and the way it was used does not indicate thymus oil induced changes in mitochondrial activity. Similarly changes in morphology induced by thymus oil cannot be directly compared due to differences in magnification of SEM images presented. Collectively, the reviewer suggests presentation of additional SEM images at comparable magnifications, repeat of the XTT assay with standardised broths comprising either thyme oil or ethanol as a negative control. To confirm that thyme oil does indeed inhibit mitochondrial activity, additional rhodamine assays could be undertaken to provide evidence of diminished signal from treated P. citricarpa. The manuscript therefore requires a number of additional experiments to verify the hypothesis the author has outlined. Reviewer 2 comments Comment 1 (section 2.1.2) regarding Rhodamine fluorescence has not been addressed. The reviewers correctly suggests that excitation at specific wavelengths of the same compound (rhodamine) may not produce uniform levels of emission. Therefore emission from either cannot be compared to suggest one indicates high mitochondrial function and the other low. This can only be confirmed if a standard is exploited for verification. Similarly, the paper the author cites does not indicate that different excitation wavelengths can correspond to high or low mitochondrial activity. Rather, rhodamine is effective in visualisation of localization, distribution and movement of mitochondria. Comment 4 (section 2.1.4); just to confirm is the author suggesting Phyllosticta citricarpa doesn't produce characteristic black colonies due to the media it has been grown in? Simply because the strain was purchased from a culture collection, there may be a contaminant. Perhaps sequencing to verify would be worthwhile? Comment 5: The concentration the author has written does not make sense. Do you mean you make it up to 0.2% in 96% ethanol and then add 46ul to your media? Please utilise final % volume/volume to describe concentration. Figure 2: A negative control is exploited to ensure there are no contaminants that can impact the outcome of your experiment. For example, ethanol without fungal/microbial inoculum. "Asexual zone": perhaps simply refer to this zone as described in your response to the reviewers question. Reviewer 1 comments 3. Whilst the same methods as Ncango et al have been exploited in this study, it still does not explain why different temperatures have been utilised throughout this study. Please clarify. Page 7, line 76: please define asci and sporangia Page 7, Line 83: What do you mean by 'safety properties'? Perhaps remove? Page 8, Line 115 - 118: Still unsure how different excitation wavelengths denote high and low mitochondrial activity. Page 9, line 145 - 147: As discussed in comments please use standard nomenclature (i.e. % concentration thyme oil). Page 9, line 164 - 165: Denoting something as positive then indicating as a minus sign is perhaps misleading. Maybe indicate this by saying 'no fungal growth was denoted by '-'. Page 9, XTT assay: Perhaps a bit misleading as the activity of XTT is not purely based upon mitochondrial activity, but as a generalised 'metabolic activity'/cytotoxicity marker. https://pubmed.ncbi.nlm.nih.gov/792958/ Figure 1: I believe that simply using 'green fluorescence' is sufficient to provide evidence of higher mitochondrial activity in candida vs condidophores. Adding the red channel simply confuses things. Figure 2: Why is Phyloositica citricarpa black in A, yellow in B but white in C and D. Could this be contamination? Page 13; Broth microdilution: This can be shortened to simple highlight MIC of thyme oil compared to other substrates. Page 14; SEM: Again, thus section should be shortened to highlight only key results including changes in morphology. Ensure nomenclature is uniform across this section (italics). Figure 3: Representative images should be shown at both magnifications (2000x and 6000x) as otherwise we could assume differences are simply due to the place on the sample the image was acquired. Page 15, Line 376- 377: I am unsure why this assay has been undertaken in this manner. I would suggest growing P. citricarpa in broth and then exploiting thyme oil to assess metabolic activity following exposure to thyme oil vs ethanol. There is limited standardisation in the assay undertaken where relative levels of 'scraped material' could be lower in 'asexual' vs 'maximum growth' zones, meaning absorbance output would be lower. The author should also be careful when referring to XTT output as 'mitochondrial activity', as other non-mitochondrial enzymes metabolize XTT to formazan.

   Please rate the manuscript for methodological rigour

   Poor

   Please rate the quality of the presentation and structure of the manuscript

   Satisfactory

   To what extent are the conclusions supported by the data?

   Partially support

   Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

   No

   Is there a potential financial or other conflict of interest between yourself and the author(s)?

   No

   If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

   Yes

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3. 

   Access Microbiology

   Jul 4, 2023

   Comments to Author

   The manuscript by Magunga and Malebo investigates the effect of Thyme oil on the in-vitro growth of Phyllosticta citricarpa, the fungal pathogen causing Citrus black spot. The authors used microscopic analysis to study fungal structures and in-vitro growth assay to determine the effect of Thyme oil on fungal growth. In addition, they analyzed the composition of the used Thyme oil via GC/MS analysis. The manuscript addresses an interesting and highly topical field. However, there are many open questions that should be addressed, ideally experimentally, however if this is not possible at least in the discussion. Also, the writing style needs to be improved to increase readability. I would like to encourage the authors to work on the manscript. General points 1. There is no need to write Phyllosticta …

   Comments to Author

   The manuscript by Magunga and Malebo investigates the effect of Thyme oil on the in-vitro growth of Phyllosticta citricarpa, the fungal pathogen causing Citrus black spot. The authors used microscopic analysis to study fungal structures and in-vitro growth assay to determine the effect of Thyme oil on fungal growth. In addition, they analyzed the composition of the used Thyme oil via GC/MS analysis. The manuscript addresses an interesting and highly topical field. However, there are many open questions that should be addressed, ideally experimentally, however if this is not possible at least in the discussion. Also, the writing style needs to be improved to increase readability. I would like to encourage the authors to work on the manscript. General points 1. There is no need to write Phyllosticta citricarpa throughout the entire manuscript. This point was raised before by a previous reviewer and the authors claim to have amended it. In the method section alone, I find 8 examples of Phyllosticta citricarpa compared to 5 examples of P. citricarpa. 2. If the composition of Thyme oil is prone to differ dependent on cultivation area and the method used to extract the oil as mentioned by the authors, it would be good to understand which of the identified compounds cause(s) the observed inhibition of fungal growth. I suggest fractioning the essential oil and then testing the fractions for growth inhibition of P. citricarpa. 3. The authors study the in-vitro effect of Thyme oil. It seems to me that the project has ended, otherwise I would expect additional work based on the interesting findings. - Is there an established infection assay with P. citricarpa on citrus fruits? If so, the authors should test the effect of Thyme oil on fruits! This will massively increase the importance of the described observation that Thyme oil inhibits P. citricarpa growth and the claim that it could be an alternative for synthetic fungicides in CBS management. - How do the authors suggest Thyme oil should be applied if used in agriculture? In Their experiment, the fungus is in direct and constant contact with the compounds through the agar. I assume spraying would be the method of choice. However, is it realistic to extract Thyme oil and/or hydrosols in sufficient amounts and quality to work as an alternative for synthetic fungicide? How often would the oil have to be applied? Please discuss! - Line 253 says EO's can be antimicrobial. Is it a good idea to apply something with antimicrobial properties at a larger scale in agriculture? Would it affect the plant and soil microbiome? Please discuss! 4. Peel of citrus fruits is full of essential oils if I am not mistaken. Why is a fungus that is specialized on citrus fruits susceptible to EO's? Did the authors test EO's from a citrus plant as a control? 5. I am not a native English speaker myself and understand the challenges of writing in English very well. However, this article is very hard to read. Sometimes the authors repeat points throughout a section several times. Sometimes it is not clear at all why something is done in a certain way and, if explained at all, explanation only comes much later in the text. Also, I believe the current manuscript would greatly benefit from language editing. I'm not asking for anything fancy as I understand that such services are costly and therefore not affordable to everybody. Maybe the authors have a native speaker as a colleagues who can help? Line 1: No consistency of the word "in-vitro" throughout the manuscript (hyphenated vs unhyphenated, italic vs regular) Line 5: e-mail@e-mail.com ??? Lines 9/13/19: 3x furthermore in the abstract Line 16: Thyme (Thymus vulgaris) oil, also throughout the manuscript However, I don't think it is necessary to mention the botanical name again and again, I would just call it Thyme after the species was clarified. Line 18: I suggest including the word "analysis" after "GC-MS" here and throughout the manuscript. Lines 27/28: "This" should be followed by a noun, as in line 14 or line 35 (please check throughout the manuscript, I will not point out every example) Line 41: replace widespread with global, delete globally? Line 58: It is unclear if this amount accounts only for CBS treatment. Line 62: "resulting in critical losses of yield, quality, and profit" was explained extensively before. Line 72: - I am not entirely sure who is the target reader of this article. However, the first word a phytopathologist would use to describe SA would certainly not be anti-inflammatory. I suggest mentioning the fact that SA is one of the main phytohormones involved in plant immunity. - The authors continue to talk about anti-inflammatory activity. Why did they choose to do so instead of anti-fungal activity when it affects fungal growth? - In line 138 SA is suddenly an antifungal anti-mitochondrial compound Line 88: essential oils? Only Thyme oil was used? If other oils were tested this should be mentioned and shown. Lines 89/90: As far as I understand, the authors only tested Thyme oil and only tested it against P. citricarpa. The claim they make here is therefore not justified in my eyes. - "Thyme oil" instead of "EO's" - "as" instead of "can be"? - Delete "as well"? Line 100: Phyllosticta citricarpa strain, the collection must have a number for the strain, please mention it. Line 108: citricarpa instead of Citricarpa Line 110: 31 ul is not sufficient for reproduction, please mention the concentration of the compound! Line 149: "All plates were incubated at 25°C until different textured growth zones were observed." Does it mean that there is the possibility that with the oil it took 2 weeks more until the different zones appeared compared to the hydrosol or SA? Or was the time comparable? Was this followed? This point should be clarified. Line 204: C and CP should be introduced in this sentence. Lines 211/212: ascospores = sexual, conidia = asexual ?? Line 213: The abbreviation Rh 123 should be introduced before using (e.g. in line 199) Line 219, Figure 1: If possible, avoid green and red colors, especially on a grey background. The results will not be accessible for color blind readers. False colors could be used. Line 240: I don't think the country of origin of the study is of significance. If it was, - The location within Brazil should be mentioned as the country is (i) large and (ii) diverse in landscape and climate. - The same should be mentioned for the current study as well as the study with the results that differ dramatically from this manuscript. - What is the source and method of extraction of the oil in this study? Line 239: "not reflected on the table"? If the table shown is not the complete table, the missing compounds should be added to give the reader a complete overview. This point is of special importance to be able to compare the results to other studies analyzing Thyme oil composition. Line 266: … When Thyme oil, hydrosol and …? Line 311, Figure 2: The appearance of the fungus differs greatly between the plates (C & D white and fluffy, B yellow-brownish, A dark brown). Why is it happening and was it quantified. For many fungi it is known that production of melanin is a response to stress and correlates with aging. Was the incubation time of the plates the same or does the fungus differ in age? Line 363, Figure 3: I agree with the previous reviewer that the different magnifications are not the ideal way to present the results. I understand from the authors' response that it was done to improve the visibility of the conidia structures. I suggest including both, a picture of cells treated with Thyme oil at a lower magnification, comparable to the current picture of cells treated with ethanol. In addition, the current picture from the Thyme oil treatment should be shown to demonstrate the conidia structures alongside a picture from the ethanol treatment with a higher magnification. So, both treatments at lower and higher resolution please. Line 372: Do the authors test mitochondrial dehydrogenases, an indicator of metabolic activity. Would Thyme oil also affect the metabolic activity of the host plant cells? The peel of citrus fruits undergoes an extensive ripening process connected to metabolic changes. Since the compounds would be applied to both, the fungus, and the host, so an effect on the plant cells must be excluded (at least discussed). Lines 382-392, Figure 4: - The figure only shows the metabolic status of the fungal cells AFTER the treatment. As a control, the metabolic activity BEFORE the treatment must be included. - The authors test the asexual zone. In line 211 the authors say conidia are sexual structures, which I believe is wrong (see comment there). Since the effect of Thyme oil on the conidia is shown throughout the manuscript, I believe the asexual zone refers to the conidia. This connection is, however, entirely unclear and must be clarified in the text as well as the figure. For example, what is the connection the the results described in figure 1 (high mitochondrial activity in the conidia). - The authors included a statistical analysis of the results as requested by the reviewer. However, I don't think a detailed description of the bar chart itself is necessary. It is sufficient to describe the outcome. Also, I suggest including the results of the statistical analysis in the figure. - The axes of the figure are not labeled at all. Please change! Line 409: missing space between P. and citricarpa

   Please rate the manuscript for methodological rigour

   Poor

   Please rate the quality of the presentation and structure of the manuscript

   Satisfactory

   To what extent are the conclusions supported by the data?

   Partially support

   Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

   No

   Is there a potential financial or other conflict of interest between yourself and the author(s)?

   No

   If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

   Yes

   Read the original source
4. 

   Version published to 10.1099/acmi.0.000591.v2 on Access Microbiology

   Jun 14, 2023
5. 

   Access Microbiology

   May 11, 2023

   The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.

   Read the original source
6. 

   Access Microbiology

   May 11, 2023

   Comments to Author

   -In section 2.1.2 the authors used Rhodamine 123 (Rh123) to selectively image mitochondrial function (Δψm). They have stated that "For this dye, the high Δψm is indicated by yellow-green fluorescence (collected at 450 nm). On the other hand, low Δψm is indicated by red fluorescence at 625 nm". It is important to be noted that fluorochromes can give different color when collected at different emission wavelength, hence the red fluorescence observed in this study may not be related to low mitochondrial function. -The purpose of Mitochondrial mapping experiment is not clear in terms of how it is serving the objective of the present study -In section 2.1.4 The author stated that the agar diffusion method was done as described by Kock et al. (2007). However, this source is a review article that "interprets …

   Comments to Author

   -In section 2.1.2 the authors used Rhodamine 123 (Rh123) to selectively image mitochondrial function (Δψm). They have stated that "For this dye, the high Δψm is indicated by yellow-green fluorescence (collected at 450 nm). On the other hand, low Δψm is indicated by red fluorescence at 625 nm". It is important to be noted that fluorochromes can give different color when collected at different emission wavelength, hence the red fluorescence observed in this study may not be related to low mitochondrial function. -The purpose of Mitochondrial mapping experiment is not clear in terms of how it is serving the objective of the present study -In section 2.1.4 The author stated that the agar diffusion method was done as described by Kock et al. (2007). However, this source is a review article that "interprets a wide network of studies that reveal aspirin to be a novel antifungal". -In section 2.1.4 the fungal suspension preparation used in the agar diffusion method was not standardized and that could affect the results reliability. Moreover, according to the literature Phyllosticta citricarpa produce distinct black colonies. However, in the present study, the colonial morphology is different. Have the authors used a mutant strain? Additionally, the concentration of thyemol used in this section was not clearly specified. - In section 2.1.5 Broth microdilution assay: It is unclear which diluents was used for the serial dilution. Moreover, it is not clear if the negative control (ethanol) was also diluted to avoid any discrepancy of ethanol concentration between the test and negative control. Additionally, assessment of growth could be improved by using spectrophotometry analysis rather than visual, especially that the facility is available as indicated in 2.1.7. -In 2.1.7 Quantitative measurement of metabolic state: In order to determine the effect of the antimicrobial compounds on the activity of mitochondrion dehydrogenases, Phyllosticta citricarpa were scraped off from different textured zones on agar diffusion plates. However, it could have been better if the authors investigated the effect of the antimicrobial compounds on microbial metabolic activity by direct contact between the microbial cells and the test agent. Since the fungal suspension used in the agar diffusion assay was not standardised the results cannot be reliable and no correlation between the agent concentration and its effect on the metabolic activity of microbial cells can be generated from the current protocol. - The authors should have included a growth control (without treatment or ethanol), especially that they have concluded that "ethanol does not have any effect on growth of Phyllosticta citricarpa". However, such conclusion cannot be extrapolated without a comparison with a growth control. -Authors have not mentioned how many independent and technical replicate was performed for each experiment. -What is "asexual zone"? It was not clear how authors identified that zone. -It is understandable that statistical analysis is not applicable in some of the protocols. However, statistical significance should have been at least performed for section 2.1.7. -The paper could benefit from a clear separation between results and discussion sections as is challenging to follow at its current status. -No consistently in the scientific nomenclature of microorganism. After first mention, genus name should be abbreviated. -No accuracy in the references. For example, Halueendo, 2008 (line 66), Haleeondo, 2008 (line 344), these have no existence in the reference list. -Details of some the equipment is not mentioned. -It was not clear why authors used representative SEM images with different magnification to show the effect of the tested agent. It could have been more plausible to use the same magnification for a more reliable comparison as different magnification would show different structural details.

   Please rate the manuscript for methodological rigour

   Poor

   Please rate the quality of the presentation and structure of the manuscript

   Poor

   To what extent are the conclusions supported by the data?

   Not at all

   Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

   No

   Is there a potential financial or other conflict of interest between yourself and the author(s)?

   No

   If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

   Yes

   Read the original source
7. 

   Access Microbiology

   Mar 26, 2023

   Comments to Author

   Well-thought-out and well-done work. Much needed at the hour, where we are in need of harmless replacements for harmful synthetic components. I suggest this paper be accepted for publication. A few minor errors are to be corrected. 1. Line 103: 'The Phyllosticta citricarpa that was used in the study was supply by the national collection of fungi' needs to be changed to 'The Phyllosticta citricarpa that was used in the study was supplied by the national collection of fungi' 2. Line 109-110: 'Mitochondrial mapping was performed to determine which structure of P. citricarpa increased mitochondrial activity' to 'Mitochondrial mapping was performed to determine which structure of P. citricarpa has/has increased mitochondrial activity' 3. In 2.1.7, Quantitative measurement of metabolic state, Line 190, it …

   Comments to Author

   Well-thought-out and well-done work. Much needed at the hour, where we are in need of harmless replacements for harmful synthetic components. I suggest this paper be accepted for publication. A few minor errors are to be corrected. 1. Line 103: 'The Phyllosticta citricarpa that was used in the study was supply by the national collection of fungi' needs to be changed to 'The Phyllosticta citricarpa that was used in the study was supplied by the national collection of fungi' 2. Line 109-110: 'Mitochondrial mapping was performed to determine which structure of P. citricarpa increased mitochondrial activity' to 'Mitochondrial mapping was performed to determine which structure of P. citricarpa has/has increased mitochondrial activity' 3. In 2.1.7, Quantitative measurement of metabolic state, Line 190, it is mentioned that the cells were incubated at 37ºC for 3 hours. Kindly clarify why molds were incubated at 37ºC here alone, whereas, elsewhere it is incubated at 25ºC only. 4. Consider changing the heading 'Results' to ' Results and Discussion', as there is no separate section for discussion. 5. The organism name, Phyllosticta citricarpa is not italicized everywhere in the text (eg Line no. 300). 6. The organism name, is written as Phyllosticta citricarpa in some places (eg line no. 300) and as P. citricarpa in some places (eg line no.351). I suggest using 'Phyllosticta citricarpa' where it is first mentioned and 'P. citricarpa' everywhere else or as 'Phyllosticta citricarpa' it self throughout the article.

   Please rate the manuscript for methodological rigour

   Very good

   Please rate the quality of the presentation and structure of the manuscript

   Very good

   To what extent are the conclusions supported by the data?

   Strongly support

   Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?

   No

   Is there a potential financial or other conflict of interest between yourself and the author(s)?

   No

   If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?

   Yes

   Read the original source
8. 

   Version published to 10.1099/acmi.0.000591.v1 on Access Microbiology

   Mar 6, 2023