Ergosterol extraction: a comparison of methodologies
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Ergosterol is a component of the cell membrane of mycorrhizal fungi and is frequently used to quantify their biomass. Arbuscular mycorrhizal (AM) fungi and ectomycorrhizal (ECM) fungi establish a symbiotic relationship with a respective host plant. Several methods are currently employed for quantification of ergosterol; however, these utilise a series of potentially hazardous chemicals with varying exposure times to the user. The present comparative study aims to ascertain the most reliable method to extract ergosterol whilst limiting hazard exposure to the user. Chloroform, cyclohexane, methanol and methanol hydroxide extraction protocols were applied to a total of 300 samples of root samples and a further 300 growth substrate samples across all protocols. Extracts were analysed via HPLC methodologies. Chromagraphic analysis showed chloroform-based extraction procedures produced a consistently higher concentration of ergosterol in both root and growth substrate samples. Methanol hydroxide, without the addition of cyclohexane, produced a very low concentration of ergosterol, with a reduction of quantified ergosterol of between 80 and 92 % compared to chloroform extractions. Hazard exposure was greatly reduced following the chloroform extraction protocol when compared with other extraction procedures.
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The author has adequately addressed both reviewers' comments and the manuscript can be accepted for publication.
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Comments to Author
The author has addressed all comments sufficiently for publication.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
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
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The reviewers have highlighted minor concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
The author has made substantial improvements to the manuscript and it is apparent chloroform is the most effective method for liberation and quantification of fungal biomass. However, the figures still need some review. In particular, presentation of statistical significance. Figure 1: You could combine the two left methods at the point they overlap i.e. after the sonication and then have two arrows running down to a single flow chart. Results: Line 209: You do not need to include all elements of the statistical test, the P value is sufficient. Is this section simply indicating all methods were capable of liberating fungal biomass, but chloroform was most efficient? Figure 2: I am unsure of your presentation of statistical significance. Usually * means p
Please rate the manuscript for …
Comments to Author
The author has made substantial improvements to the manuscript and it is apparent chloroform is the most effective method for liberation and quantification of fungal biomass. However, the figures still need some review. In particular, presentation of statistical significance. Figure 1: You could combine the two left methods at the point they overlap i.e. after the sonication and then have two arrows running down to a single flow chart. Results: Line 209: You do not need to include all elements of the statistical test, the P value is sufficient. Is this section simply indicating all methods were capable of liberating fungal biomass, but chloroform was most efficient? Figure 2: I am unsure of your presentation of statistical significance. Usually * means p
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
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
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Comments to Author
I am satistfied with the responses from the author in amending the manuscript. However, there are some minor concerns as the followings. Please keep using words/abbreviations consistently throughout the manuscript such as ml or mL, minutes or min, 1 minute or 60s. In the results section, please update/correct the Figure number mentioned in the texts. L107 It confused the readers about how it ended up with n=300. Please simply clarify how many replicates of plant and growth substrate were sampled in the manuscript. L136 Is the verb e.g., "was added" missing? L148 Please change to "Alekseyeva et al. (2021)". L209 Please explain briefly the details about multi-way ANOVA for overall results in the materials and methods to be related with the results here. It seems to be missing from the materials and …
Comments to Author
I am satistfied with the responses from the author in amending the manuscript. However, there are some minor concerns as the followings. Please keep using words/abbreviations consistently throughout the manuscript such as ml or mL, minutes or min, 1 minute or 60s. In the results section, please update/correct the Figure number mentioned in the texts. L107 It confused the readers about how it ended up with n=300. Please simply clarify how many replicates of plant and growth substrate were sampled in the manuscript. L136 Is the verb e.g., "was added" missing? L148 Please change to "Alekseyeva et al. (2021)". L209 Please explain briefly the details about multi-way ANOVA for overall results in the materials and methods to be related with the results here. It seems to be missing from the materials and methods. L316 "height" instead of "hight". Figure 1 Please capitalize the first letter in the flow chart boxes and correct "syringe" filter. Figure 2 Please check the number of samples. It should be n=300 instead of n=3,000 as running HPLC for 10 times per samples was not the real replicates. This is also concerned Table 1.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
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?
No: The manuscript does not involve any human and/or animal work.
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The reviewers have highlighted major concerns with the work presented. Please ensure that you address their comments.
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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data Generally, the author clearly described the experimental design and the details of extraction methods used in the manuscript. This would benefit for reproducibility for further studies. All methods used employed ultrasonication and heat treatment to disrupt fungal and root cells, however, their conditions varied among the methods. Together with the ratio between the amount of sample and that of the solvent, this may cause differentiation of extraction ability and applicability, and thus should be concerned when interpreting and comparing the results. The use of ultrasonic bath could also lead to unequally energy obtained among the samples within each batch. The underlying data were presented according to the statistical …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data Generally, the author clearly described the experimental design and the details of extraction methods used in the manuscript. This would benefit for reproducibility for further studies. All methods used employed ultrasonication and heat treatment to disrupt fungal and root cells, however, their conditions varied among the methods. Together with the ratio between the amount of sample and that of the solvent, this may cause differentiation of extraction ability and applicability, and thus should be concerned when interpreting and comparing the results. The use of ultrasonic bath could also lead to unequally energy obtained among the samples within each batch. The underlying data were presented according to the statistical analysis with a number of samples (n=320). Moreover, monitoring the volatile vapours released during the extraction operation in the current study is very beneficial regarding health risks to the users besides the ergosterol yields or extraction ability. 2. Presentation of results As the author performed ANOVA with post hoc test, would it be possible to include or present the statistic results on the result figure and table (such as on Figure 1 and in Table 1)? 3. How the style and organization of the paper communicates and represents key findings The texts were concise, and structure of the overall manuscript was easy to follow. Using diagrams or table or restructuring the texts when describe the extraction methods in detail would help in reducing the redundant texts and being more visualizable to the readers. For example, describe only once about sample preparation as it was the same for all extraction methods. Furthermore, the extraction steps of all methods used were very similar to each other: extraction with organic solvent in association with cell disruption by ultrasonication and heat treatment, followed by liquid-liquid phase separation, then pre-concentration and purification prior to HPLC measurement. However, the conditions or details in every step differed across the methods. Thus, these may be presented as a comparison table for being more visualizable. 4. Literature analysis or discussion The literature used in this manuscript sufficiently covered the underlying data and key findings. However, in accordance with Montgomery et al. (2000), the drawback of using fungal biomass estimation equation (Eq. 1) and its subsequent interpretation should be noted. 5. Any other relevant comments Could the author explain briefly about ability or suitability of using ergosterol as a biomarker for AMF? I usually find that ergosterol is used to determine saprotrophic and/or ectomycorrhizal fungi in soils. When or at which age were the samples collected? The materials and methods stated 6 months (L108) while the caption of Figure 1 was 9 months (L220). Please check the number of samples as it was confusing among 20 (L101-102), 320 (L108) and 3,200 (L218). When n=320, does it mean that 16 samples of roots were collected from each plant together with 16 samples of their respective growth substrate? As compost was applied during the experiment, could it affect the ergosterol yield (probably from the saprotrophic fungi) particularly in growth substrate? Furthermore, were there any sample preparation for plant root tissue samples to avoid e.g., contamination of saprotrophic fungi? Why the author used post hoc T testing and Bonferroni correction after ANOVA instead of other post hoc tests? Also, why performed (several) T test(s) among extraction protocols (4 extraction methods) within the same plant species instead of ANOVA? This may reduce the power of statistical test. What about using three-ways ANOVA? Which statistical tests were performed for the volatile matter measurement data? Please check the unit of the mean fungal biomass on Y-axis in Figure 1? Is it per g of roots or growth substrate?
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
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?
No: The manuscript does not involve any human and/or animal works.
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Comments to Author
The author presents an interesting manuscript detailing the efficacy of different methods for extraction of ergosterol, with compelling evidence that chloroform extraction liberates the greatest volume of fungal biomass. I have a few minor comments, whereby the methods section needs to review to ensure there is no repetition, figures should present significant differences between groups and the discussion needs reassessment. Similarly, all information discussing assessment of atmospheric VOCs should be removed. Title: Should be comparison? Abstract: Line 27: Review sentence, much better written in the introduction (line 60) Line 34: Extracts not extractions Methods: Be consistent with manufacturer details. I would recommend including brand and country of purchase, trademarks are also not required. …
Comments to Author
The author presents an interesting manuscript detailing the efficacy of different methods for extraction of ergosterol, with compelling evidence that chloroform extraction liberates the greatest volume of fungal biomass. I have a few minor comments, whereby the methods section needs to review to ensure there is no repetition, figures should present significant differences between groups and the discussion needs reassessment. Similarly, all information discussing assessment of atmospheric VOCs should be removed. Title: Should be comparison? Abstract: Line 27: Review sentence, much better written in the introduction (line 60) Line 34: Extracts not extractions Methods: Be consistent with manufacturer details. I would recommend including brand and country of purchase, trademarks are also not required. This includes for babybio and arthurs bowers compost. As the same the same procedure was exploited to pre-prepare samples and the same volume of substrate used in each protocol (A total of 2g sampled rhizosphere growth substrate was air dried at 25°C for 48 117 hours and 300mg root material without drying. Of both growth substrate and root samples, 300mg was weighed into 50mL centrifuge tubes), this should only be described once in the initial methods section. This section should have a sub-heading labelled "sample preparation for extraction." Similarly the sentence assessing atmospheric VOCs should only be discussed once. Results: Methods and statistical tests exploited should not be discussed in the results. Similarly, a p value is sufficient when discussing statistical differences. Significance between groups should be displayed on figures within discrete groups. For example, biomass liberated from oak. Please review publications and their presentation of significance. There are also a number of discussion points in the results which are not required. Table 1: Please ensure extraction methods are consistently labelled. Which extraction method do non-alkaline and alkaline refer to? Figures 2 - 3: Can you combine these figures and alter the scale so that it is consistent across the three figures? Discussion and conclusion The discussion require review and should be shortened. The main focus of this section should be on the efficacy of chloroform in fungal biomass extraction and perhaps how this could be applied environmentally. For example, routine assessment of fungal growth and characterisation of the symbiotic relationship between plants and fungi. Ensure referencing is consistent throughout, in the introduction Vancouver is used, but in the discussion the same style is not exploited. Leaf not leave I'm not sure information regarding atmospheric VOC is necessary as you couldn't monitor this due to the use of a fume hood, I would suggest removing these data from your manuscript.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
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
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