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Open AccessArticle
Peer-Review Record

Evaluation of Fungistatic Activity of Eight Selected Essential Oils on Four Heterogeneous Fusarium Isolates Obtained from Cereal Grains in Southern Poland

Molecules 2020, 25(2), 292; https://doi.org/10.3390/molecules25020292
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Molecules 2020, 25(2), 292; https://doi.org/10.3390/molecules25020292
Received: 17 December 2019 / Revised: 5 January 2020 / Accepted: 8 January 2020 / Published: 10 January 2020
(This article belongs to the Section Chemical Biology)

Round 1

Reviewer 1 Report

GENERAL COMMENTS

Although the theme and principal contents of the manuscript “Molecules-684254” is of interest and in accordance with the scope of “Molecules”, I think it could be accepted for publication only after a major revision, for specific reasons listed below. 

Generally, the overall writing is incomprehensible, which in some parts is difficult to understand or follow up. The experimental design and methodology is unclearly described. There are several mistakes in the “Results” section. Authors should give more details about the common findings of the related articles. They should also improve the content of “discussion”, being creative with the conclusions they have developed through their results.

The manuscript needs a thorough brush up in the English language. There are a lot of syntax and grammar errors. The authors would be advised to read through the manuscript carefully to eliminate these errors and to consult a microbiologist fluent in English.

SPECIFIC COMMENTS

Authors should provide the definitions of “fungistatic” and “fungicidal” activity (line 52). Are both terms (line 294) the same? Did their results demonstrate fungistatic (lines 71, 175 etc) or fungicidal activity (lines 312, 315)? It has been documented that antifungal activity of essential oils may be influenced due to the addition of Tween 80. Please provide any comments. While some essential oils and components may be considered less active, any data related to the mechanism of antifungal action could be added to the text. There is a major inconsistency between the results provided in the text and the results presented in tables 5, 6 and 7. There are different concentrations (text vs. table, e.g garlic 0.5% vs. 2%, cajeput 0.5% vs. 1%, Litsea 0.05% vs. 0.125%, lemongrass 0.05% vs. 0.125% and inhibition rates of grapefruit 71.03% vs. 12.35%). This is only for table 5. Please check the same for both tables 6 and 7 and rewrite correctly. Although not mandatory, the use of a reference strain (ATCC), could add clarity and reproducibility of the results. It should be also useful to check the drug resistant pattern of used isolates, since Fusarium species are among the most resistant fungi.

MORE (MINOR) COMMENTS

Line 52: Please cite a reference. Lines 72-76. Please rewrite the paragraph clearly. Line 100: “significantly lower”. Unnecessary use of significantly. Lines 125, 136: “Tables” instead of “Table”. Line 149: “Thyme oil (T)” instead of “Thyme oil”. Line 199: “In his case”. Please rewrite. Tables 5,6,7,8: Since you haven’t use any abbreviations for the used essential oils, you should do the same for Tea Tree oil. Also, in table 5, you have used “from tea tree”, and “from Litsea”. Please rewrite. Line 225: “0.125%” instead of “0,125%”. Please check if 2% of garlic is correct (table 9). Did garlic oil demonstrate medium (line 237) or weak (line 240) effect? Lines 269-270: “Hence …. EsO … They represent …” Please rewrite. Line 326: “aseptic”. Please rewrite. Line 345: “Segvic et al. believe”. Please rewrite. Please rewrite the reference 45 correctly. “Dimitra, Basil and Moschos are the first names of the authors”. You should write “Daferera, Ziogas and Polissiou”.

Author Response

Authors would like to thank the Reviewer for careful reading of this manuscript and for the thoughtful comments and considerable suggestions, which help to improve our manuscript. We have tried to do our best to respond to any comments and improve them. As indicated below, we have made changes accordingly to all the general and specific comments. Authors are specially thankfully for numerous important particular remarks and corrections, and tips for further research.

Authors should provide the definitions of “fungistatic” and “fungicidal” activity (line 52). - EsO may exhibit fungistatic activity characterized by partial inhibition of mycelial growth or fungicidal, causing complete inhibition of the development of a given species, comparable to those of synthetic fungicides [8]. The effect of the action depends on both the sensitivity of the fungus and the chemical composition and concentration of EsO. 

Are both terms (line 294) the same? - we corrected this

Did their results demonstrate fungistatic (lines 71, 175 etc) or fungicidal activity (lines 312, 315)? - we corrected this

It has been documented that antifungal activity of essential oils may be influenced due to the addition of Tween 80. Please provide any comments - Tween 80 is a nonionic surfactant and emulsifier derived from polyethoxylated sorbitan and oleic acid. Tween 80 (0.01%) was used as a control and did not affect the development of the tested fungi.

While some essential oils and components may be considered less active, any data related to the mechanism of antifungal action could be added to the text

We added to the introduction - Fungal growth disorders are caused by changes in the structure of fungi associated with the interaction of EsO on the enzymes responsible for cell wall synthesis. Inhibition of fungal growth by oils can be synonymous with changes in their ultrastructure. An important role in the degradation of the fungal cell membrane is attributed to lyophilic and polar compounds contained in oils. Lyophilic compounds disturb the structure of the cell membrane, inhibit its synthesis, as well as formation of spores and impede the respiration process. In contrast, polar compounds having active chemical groups participate in the degradation of the cell membrane. In sensitive species of fungi, there are changes in the structure of the cell membrane and in the composition of fatty acids and the formation of vacuoles in the cells of fungi. In parallel, the synthesis and regeneration of cell membrane components is inhibited, which inhibits cell growth and division as well as the production of spores [15,16].

We added to the discussion - EsO exhibited its antifungal activity  by  inhibit of hyphal growth,  the production and germination of conidia, a change in the morphology of the fungus, damage the cytoplasmic membrane, leading to the leakage of electrolytes and possibly lipid peroxidation induced by the increase in permeability and the reduction in ergosterol content (major component of the fungal membrane) and accumulation of massive lanosterol as well as an inhibition in wall formation [23,24]. Essential oils also have an inhibitory action on membrane ATPases and cytokine interactions and cell respiration, leading to rapid energy depletion and cell death [25].

There is a major inconsistency between the results provided in the text and the results presented in tables 5, 6 and 7. There are different concentrations (text vs. table, e.g garlic 0.5% vs. 2%, cajeput 0.5% vs. 1%, Litsea 0.05% vs. 0.125%, lemongrass 0.05% vs. 0.125% and inhibition rates of grapefruit 71.03% vs. 12.35%). This is only for table 5. Please check the same for both tables 6 and 7 and rewrite correctly. - Presence of such major inconsistency between the results provided in the text and the results presented in tables 5, 6 and 7 was caused by mistaken numbering and description of data putted inside mentioned tables. That “mixture” was our mistake(!) and Authors because of this fact are very sorry(!). Because of this mistake: - data from Table 5 de facto have to be in Table 7 (F. avenaceum); - data from Table 6 have to be in Table 5 (F. culmorum); and - data from Table 7 have to be in Table 6 (F. graminearum). First of all Authors remove this fatal mistake, and now data’s concern to the particular Fusarium species in Tables 5,6 and 7 inside main text are in the proper position. Automatically this inconsistency was removed. Tables 5,6 and 7 [and also Table 8,9,10] contains also some not necessary phrases, all such “addition” was removed. Authors formatted tables by consequent approximation of numbers include 0,1 and 100 (X.XXX for concentrations, Y.YY for % fungistatic activity, in table 10 approximation of correlation coefficient by format O.ZZ). Small additional corrections of text and in tables were putted to the main text and tables. So we corrected this.

Although not mandatory, the use of a reference strain (ATCC), could add clarity and reproducibility of the results. - We did not use strains from the ATTC collection. Our aim was control of chosen Fusarium species (isolates) from south Polish populations.

It should be also useful to check the drug resistant pattern of used isolates, since Fusarium species are among the most resistant fungi- Searching for the drug resistant patterns is very important from medicinal point of view. The phenomenon of drug resistant pattern is very similar to the phenomenon of fungicide resistant ability. Fusarium are really both drug and fungicide resistant - that is a reason to be dangerous and unpredictable infector of plants and animals, including humans. Authors decide to add to the chapter Discussion information from Danielewicz et al. (2013) paper concern to second phenomenon.

Danielewicz et al. [17] tested the sensitivity of six Fusarium species (F. avenaceum KZF-3, F. culmorum KZF-5, F. graminearum KZF-1, F. oxysporum KZF-4, F. langsethiae KZF-2 and F. equiseti KZF-6) to 6 fungicides from 4 chemical groups: azoxystrobin, prochloraz, thiophanate methyl, propiconazole, metconazole and tebuconazole. These studies showed that the strongest fungicidal effect on all tested Fusarium species was found for prochloraz, while thiophanate methyl and azoxystrobin showed only fungistatic activity.

The appearance of individuals (isolates) with reduced sensitivity to fungicides results in the emergence of forms resistant to these preparations. In addition, fungi often have a cross-resistance phenomenon with benzimidazole, dicarboximide and phenylamide fungicides. If a given phytopathogen species becomes resistant to one preparation, at the same time it becomes resistant to the whole group of chemical substances to which this preparation belonged.

The resistance of fungi to chemical compounds determines the specific structure of the cell wall (which protects the cell against external factors) and differences in the set of synthesized and extracellular secreted enzymes [19]. Some chemical fungicides, such as imazalil, prochloraz, and triflumizole, exhibited their antifungal mode by blocking the ergosterol biosynthesis, which can give rise to the disruption of cell structure and function, even to the death of cell [20].

Line 52: Please cite a reference. In terrestrial ecosystems, the effect of EsO on other organisms is called allelopathy, while in relations between plant and fungal organisms, their fungistatic or fungicidal properties are revealed.- The substances contained in the EsO of vascular plants exhibit interactions of a biochemical nature (both harmful and beneficial), in systems: plants - plants, microorganisms - microorganisms and plants - microorganisms. In 1937 Hans Molish introduced the concept of allelopathy [6,7].

Lines 72-76. Please rewrite the paragraph clearly - Present experiment was establish such a way that it was possible to identify variable spectrum of compounds and groups of compounds present in different EsO to estimate fungistatic activity. EsO and/or ingredients of EsO could potentially be useful in the formation of alternative fungicides. Synthetic fungicides are relatively high ecotoxic and resulting fungal resistance reactions.

Line 100: “significantly lower”. Unnecessary use of significantly. - we corrected this

Lines 125, 136: “Tables” instead of “Table”. - we corrected this

Line 149: “Thyme oil (T)” instead of “Thyme oil”- we corrected this

Line 199: “In his case”. Please rewrite. we corrected this

Tables 5,6,7,8: Since you haven’t use any abbreviations for the used essential oils, you should do the same for Tea Tree oil. Also, in table 5, you have used “from tea tree”, and “from Litsea”. Please rewrite.- Authors formatted tables by consequent approximation of numbers include N,0,1 and 100 (X.XXX for concentrations, Y.YY for % fungistatic activity and in table 10 approximation of correlation coefficient by format 0.ZZ). Small additional corrections of text and in tables were putted to the main text and tables, “addition” was removed - so we corrected this.

Line 225: “0.125%” instead of “0,125%”. - we corrected this

Please check if 2% of garlic is correct (table 9) - we corrected this

Did garlic oil demonstrate medium (line 237) or weak (line 240) effect?-  garlic oil demonstrate weak effect- we corrected this

Lines 269-270: “Hence …. EsO … They represent …” Please rewrite. - EsO due to the wide availability, limitation of the harmful effects of filamentous fungi and biodegradability are increasingly used in practice [21,22].

Line 326: “aseptic”. Please rewrite. Fungicidal activity of thyme oil is probably associated with high content of phenolic compounds (especially thymol).

Line 345: “Segvic et al. believe”. Please rewrite. Slightly different conclusions can be drawn from the work of Segvić et al. [39], in which it was shown that thymol had about three times stronger inhibition of pathogen growth than thyme oil. According to these observations, we can speculate that a strong antifungal activity of thyme oil can be attributed to thymol itself or, alternatively, speculate that such a strong fungistatic activity.

Please rewrite the reference 45 correctly. “Dimitra, Basil and Moschos are the first names of the authors”. You should write “Daferera, Ziogas and Polissiou” - we corrected this

Other many required corrections were included in the attached main manuscript.

Author Response File: Author Response.pdf

 

Reviewer 2 Report

This manuscript reports the fungistatic activity of essential oils on four heterogeneous Fusarium isolates. This is a valuable area of research, and the informed results are very interesting. However, numerous improvements should be incorporated into the manuscript. I think that the authors should add the suggested changes, and rewrite the manuscript.

General comments:

1) There are many typographical and grammatical errors throughout the manuscript. The manuscript could be improved by having someone with English as their native language read it.  

2) The method of essential oil identification of components has not been informed. Moreover, the authors did not report the retention index (or kovats index) of the identified compounds. The essential oils components should be identified by comparing their retention indices (determined on the basis of homologous n-alkane hydrocarbons) with those of pure authentic samples, peak enrichment on co-injection with authentic standards wherever possible and mass spectra with those of the mass spectral databases.

3) The authors describe several statistical analyzes in the materials and methods section, however, these statistical results were not reported in the results section. Hence, the discussion of these results could be questionable.

4) Monosporic cultures should be perfomed to each fungal strain.

5) Do essential oils form solutions in Tween 80/water?

6) I think that a plot of antifungal activity vs concentration (including all evaluated essential oils) to each fungal strain could simplify the results. These figures should be include statistic differences.

7) The antifungal activity of essential oils rich in phenolic compounds is something well known,  and lacks academic impact. Maybe, adding multivariate analysis (such as canonic correlation, Multiple regression Lineal, principal components, amnong others)  that relation the  antifungal activity with essential oil components could add novelty in the interpretation of the obtained results.

8) The discussion section is poorly developed. I think this section should be rewritten. Comparison with the antifungal activity of other essential oils should be incorporated. In addition to the deepening the discussion between the composition and the antifungal activity of the evaluated essential oils.

Author Response

We would like to thank the Reviewer for careful reading of this manuscript and for the thoughtful comments and considerable suggestions, which help to improve our manuscript. We have tried to do our best to respond to any comments and improve them. As indicated below, we have made changes accordingly to all the general and specific comments. And we are also gratefully for general questions, and tips for further research.

1) There are many typographical and grammatical errors throughout the manuscript. The manuscript could be improved by having someone with English as their native language read it. - The original text was translated and checked by native speaker (see the included certificate), but anyway, we are ready to order additional language checking and to cover costs of after revue language checking.

2) The method of essential oil identification of components has not been informed. Moreover, the authors did not report the retention index (or kovats index) of the identified compounds. The essential oils components should be identified by comparing their retention indices (determined on the basis of homologous n-alkane hydrocarbons) with those of pure authentic samples, peak enrichment on co-injection with authentic standards wherever possible and mass spectra with those of the mass spectral databases.- We added of the 4.3. Determination of the quantitative chemical composition of essential oils - The components were identified by comparison of their mass spectra with the spectrometer database of the NIST 11 Library (National Institute of Standards and Technology, Gaithersburg, MD, USA) and by comparison of their retention index calculated against n-alkanes (C9–C20). Each chromatographic analysis was repeated three times. The average value of the relative composition of the essential oil percentage was calculated from the peak areas (Cal). Literature values of Kovats retention indexes (L) based by Babushok et al. [18].

3) The authors describe several statistical analyzes in the materials and methods section, however, these statistical results were not reported in the results section. Hence, the discussion of these results could be questionable.- In section Results we add results of statistical analyses antifunginal activity eight essential oils for each isolates separately and four isolates together.

4) Monosporic cultures should be perfomed to each fungal strain.- The inoculum in the form of 10 mm diameter of media rings overgrown with Fusarium mycelium was used in the study. The spore suspension of tested Fusarium in a 0.01% sterile solution of Tween 80 (produced by BTL, Poland)—was obtained from a 10-day-old culture. The haemocytometer Thoma was used to obtain a spore suspension of 1⸱106 CFU⸱cm3.  Petri dishes (9 cm diameter) containing 20 cm3 PDA medium were inoculated in spore suspension and incubated at 25℃ for 10 days. Then, the discs (of 10 mm diameter) were cut out with a cork borer. Inoculum rings with a diameter of 10 mm overgrown by mycelium were obtained. Tested EsO were introduced into a PDA medium in the following concentrations: 0.125; 0.25; 0.5; 1.0; 2.0%. The positive control was the PDA medium with Funaben T (chemical seed mortar) in concentrations of 0.125, 0.25, and 0.5% and the negative control was the culture of the fungus on PDA medium (without oils) enriched with 0.01% Tween 80 with inoculum rings.

5) Do essential oils form solutions in Tween 80/water?- Yes, because Tween 80 is a nonionic surfactant and emulsifier derived from polyethoxylated sorbitan and oleic acid.

6) I think that a plot of antifungal activity vs concentration (including all evaluated essential oils) to each fungal strain could simplify the results. These figures should be include statistic differences. 7) The antifungal activity of essential oils rich in phenolic compounds is something well known, and lacks academic impact. Maybe, adding multivariate analysis (such as canonic correlation, Multiple regression Lineal, principal components, among others) that relation the antifungal activity with essential oil components could add novelty in the interpretation of the obtained results.

By suggestion of Reviewer we presents a plots of antifungal activity vs concentration including all evaluated essential oils to four Fusarium species (figure 1 and figure 2) and result of multiple regression of antifungal activity vs essential oil components (table2). All analyzed group of main chemical group in the oil component have a significant impact on antifungal activity.

The model describes only 60% of the variability of the antifungal activity, the remaining 40% variation of dependent variable is not included in the model. The presented model of multiple regression showed that the concentration of oils and their chemical composition significantly affect antifungal activity. The concentration of essential oil, monoterpenoids and sesquiterpenes are directly proportional to antifungal activity. Monoterpenes and sesquiterpenoids inhibit antifungal activity. However the value of the standard error of estimation is large and model is unacceptable to us in this form. The model requires detailed research. The model will be the theme of our next paper.

 

   

F. avenaceum

F. culmorum

   

F. graminearum

F. oxysporum

 

Figure 1. Plot antifungal activity vs concentration for four species Fusarium

Figure 2. Plot antifungal activity vs concentration for four species from Fusarium together

Table 1. Result of multiple regression

 

Regression Summary for Dependent Variable: Actifungall activity R= ,77694527 R2= ,60364395 Adjusted R2= ,60096285 F(6,887)=225,15 p<0,000 Std.Error of estimate: 21,775

b*

Std.Err.
of b*

b

Std.Err.
of b

t(887)

p-value

Intercept

   

61,601

2,1585

28,53925

0,000000

concentration

0,336381

0,021139

1740,112

109,3525

15,91288

0,000000

monoterpenes

-0,107375

0,026068

-0,233

0,0565

-4,11909

0,000042

monoterpenoids

0,257452

0,029329

0,309

0,0353

8,77820

0,000000

sesquiterpenes

0,196419

0,052579

1,595

0,4270

3,73566

0,000199

sesquiterpenoids

-0,466571

0,053122

-17,827

2,0298

-8,78297

0,000000

other chemical compounds

-0,218981

0,039955

-0,724

0,1321

-5,48065

0,000000

 

8) The discussion section is poorly developed. I think this section should be rewritten. Comparison with the antifungal activity of other essential oils should be incorporated. In addition to the deepening the discussion between the composition and the antifungal activity of the evaluated essential oils.- In the chapter discussion was added information about: mechanism of fungistatic activity, resistance ability to some chemical substance.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have answered all the questions and modified their manuscript according to the Reviewer's comments and suggestions properly. Thus, I suggest that this submission may be accepted in its current form for publication in “Molecules”

Author Response

The authors thanks for the review

Reviewer 2 Report

The manuscript has been greatly improved  and could be accepted form publication. However,  there are still suggestions that have not been incorporated into the manuscript. These suggestions could give add quality.

1) There are many typographical and grammatical errors throughout the manuscript. The manuscript could be improved by having someone with English as their native language read it. - The original text was translated and checked by native speaker (see the included certificate), but anyway, we are ready to order additional language checking and to cover costs of after revue language checking. 

R: ok

2) The method of essential oil identification of components has not been informed. Moreover, the authors did not report the retention index (or kovats index) of the identified compounds. The essential oils components should be identified by comparing their retention indices (determined on the basis of homologous n-alkane hydrocarbons) with those of pure authentic samples, peak enrichment on co-injection with authentic standards wherever possible and mass spectra with those of the mass spectral databases.- We added of the 4.3. Determination of the quantitative chemical composition of essential oils - The components were identified by comparison of their mass spectra with the spectrometer database of the NIST 11 Library (National Institute of Standards and Technology, Gaithersburg, MD, USA) and by comparison of their retention index calculated against n-alkanes (C9–C20). Each chromatographic analysis was repeated three times. The average value of the relative composition of the essential oil percentage was calculated from the peak areas (Cal). Literature values of Kovats retention indexes (L) based by Babushok et al. [18].

R: Please, the essential oil compounds should be reported in increasing order,  according to their kovats retention index.

3) The authors describe several statistical analyzes in the materials and methods section, however, these statistical results were not reported in the results section. Hence, the discussion of these results could be questionable.- In section Results we add results of statistical analyses antifunginal activity eight essential oils for each isolates separately and four isolates together.

R: OK

4) Monosporic cultures should be perfomed to each fungal strain.- The inoculum in the form of 10 mm diameter of media rings overgrown with Fusarium mycelium was used in the study. The spore suspension of tested Fusarium in a 0.01% sterile solution of Tween 80 (produced by BTL, Poland)—was obtained from a 10-day-old culture. The haemocytometer Thoma was used to obtain a spore suspension of 1⸱106 CFU⸱cm3.  Petri dishes (9 cm diameter) containing 20 cm3 PDA medium were inoculated in spore suspension and incubated at 25℃ for 10 days. Then, the discs (of 10 mm diameter) were cut out with a cork borer. Inoculum rings with a diameter of 10 mm overgrown by mycelium were obtained. Tested EsO were introduced into a PDA medium in the following concentrations: 0.125; 0.25; 0.5; 1.0; 2.0%. The positive control was the PDA medium with Funaben T (chemical seed mortar) in concentrations of 0.125, 0.25, and 0.5% and the negative control was the culture of the fungus on PDA medium (without oils) enriched with 0.01% Tween 80 with inoculum rings.

R: The authors did not respond to my comment. The culture used in the inoculum preparation (10 mm diameter of media rings) have to be initiated from an original monosporic culture. Monoconidial cultures are necessary to obtain pure fungal cultures.

5) Do essential oils form solutions in Tween 80/water?- Yes, because Tween 80 is a nonionic surfactant and emulsifier derived from polyethoxylated sorbitan and oleic acid.

R: These form solutions or homogeneous  suspensions. please check

6) I think that a plot of antifungal activity vs concentration (including all evaluated essential oils) to

each fungal strain could simplify the results. These figures should be include statistic differences. The antifungal activity of essential oils rich in phenolic compounds is something well known, and lacks academic impact. Maybe, adding multivariate analysis (such as canonic correlation, Multiple regression Lineal, principal components, among others) that relation the antifungal activity with essential oil components could add novelty in the interpretation of the obtained results.

By suggestion of Reviewer we presents a plots of antifungal activity vs concentration including all evaluated essential oils to four Fusarium species (figure 1 and figure 2) and result of multiple regression of antifungal activity vs essential oil components (table2). All analyzed group of mainchemical group in the oil component have a significant impact on antifungal activity.

R: Please, the plots of antifungal activity vs concentration, and multiple regression results should be add and discussed into the manuscript.

Author Response

Answers to questions of Reviewer II round 2

Authors would like to thank the Reviewer for careful reading of this manuscript and for the thoughtful comments and considerable suggestions, which help to improve our manuscript. We have tried to do our best to respond to any comments and improve them. Remarks and objections of were presented in 6 points - 2 points (1) and (3) were summarized as “OK”. To remaining 4 points: 2) and 4-6) Authors giving comments and answers:

Ad 2) [Field of results of chemical identification of chosen 8 EsO]

R: Please, the essential oil compounds should be reported in increasing order, according to their kovats retention index.

Authors answer:

Authors renew the Table 2, built new one, and new table is organized by the remark of Reviewer.

Ad 4) R: The authors did not respond to my comment [concern to the problem of sources of cultures]. The culture used in the inoculum preparation (10 mm diameter of media rings) have to be initiated from an original monosporic culture. Monoconidial cultures are necessary to obtain pure fungal cultures.

Comment of Authors: Yes, we use to our experiment monosporic cultures derived according to the procedure of Tousson and Nelson (1976) in the following 4 stages:

The spores were suspended in sterile water and diluted so that their number in the field of view of the microscope was from 1-10. The suspension was spread on the surface of petri dishes with 2% aqueous agar and incubated at 25°C for 18 hours. Periodically, the surface of the medium was checked using a microscope. Places were marked where there was only one germinating spore. The marked fragment was then transferred to PDA media tubes. Pure cultures were grown at a variable temperature of 25 ℃ during the day and 20 ℃ at night.

In line 513 we added: Tousson T.A., Nelson P.E. Fusarium: a pictorial guide to the identification of Fusarium species according to the taxonomic system of Snyder and Hansen. University Park: Pennsylvania State University Press, 1976.

Authors answer:

Authors corrected this gap shown by Reviwer: paper of Tousson and Nelson (1976) was cited with additional phrase monosporic (line 513)

Ad 5) Do essential oils form solutions in Tween 80/water?- Yes, because Tween 80 is a nonionic surfactant and emulsifier derived from polyethoxylated sorbitan and oleic acid.

R: These form solutions or homogeneous suspensions. please check

Comment of Authors: To describe the state of the EsO / Tween 80 / water “system” - we adopted the term "solution", which was corrected in the manuscript Essential oils disperse in sterile water with Tween 80, forming a heterogeneous mixture. However, the dispersion of the dispersed substance is so large that the mixture looks physically homogeneous. Such a system due to the small size of dissolved particles can be classified as colloidal solutions because it has common features with them - e.g. light scattering. Such a system does not create a real “classical” solution and is also not a typical suspension. The colloidal solution cannot be separated by filtration. In contrast, the suspension is a heterogeneous mixture, whose particles are larger than 100 nm (the suspension can be separated by filtration, it sediments). Term of solution a propos EsO / Tween 80 / water “system” was used also by Terzi (2007) “… solution containing 0,05% Tween and 0,5% TTO or 1% TTO …”. Terzi et al. In vitro antifungal activity of the tea tree (Melaleuca alternifolia) essential oil and its major components against plant pathogens. Letters Appl. Microbiol. 2007 44, 613–618.

Authors answer:

Authors corrected such inconsequence (=fuzzy situation) – solution versus suspension: in line 498 was added phrase “colloidal” – so we prefer term “colloidal solution”.

Ad 6) [Additive Tables and Figures describing statistic of the experiment have to be putted to the text of present paper] “Reviewer: I think that a plot of antifungal activity vs concentration (including all evaluated essential oils) to each fungal strain simplify the results. These figures should be include … of the obtained result”

Answer in Round I: By suggestion of Reviewer we presents a plots of antifungal activity vs concentration including all evaluated essential oils to four Fusarium species (figure 1 and figure 2) and result of multiple regression of antifungal activity vs essential oil components (table 2). All analyzed group of main chemical group in the oil component have a significant impact on antifungal activity.

R: Please, the plots of antifungal activity vs concentration, and multiple regression results should be add and discussed into the manuscript.

Authors comment: Authors included to the present paper new elements (by suggestions and remarks): figures – Figure 2 (new – old “Figure 2” was renumbered as Figure 7), Figure 3, Figure 4, Figure 5 and Figure 6 (these figures presenting plots antifungal activity vs concentration of oils) and tables – Table 11 and Table 12 (multiple regression results). Statistic problems are discussed in the text in section Results: 2.3., 2.4.

About F. culmorum.Grapefruit oil had the weakest effect and only at a concentration of 2.000% did it show fungistatic activity amounting on average to 71.03% (Table 5, Figure2). The result of Kruskal-Wallis test (H(9,n=230)=125.87, p=0.00) showed that the differences in fungistatic activity of the oils used were statistically significantly. In addition, the Kruskal-Wallis test (H(3,n=83)=4,69 p=0.20) showed that there are no statistically significant differences between Funaben T fungistatic activity and thyme, lemongras and Litsea cubeba oils. About F. graminearum. In this case, too, grapefruit oil had the weakest effect, and at a concentration of 2.000%, it showed lower fungistatic activity of 48.38% on average (Table 6, Figure 3).

          It was hypothesized that differences in fungistatic oil activity on the F. graminearum isolate are statistically significant. The result of Kruskal-Wallis test (H(9,n=231)=121.67, p=0.00) showed that the hypothesis was true. Differences in fungistatic activity of the eight oils were statistically significant. Also, the Kruskal-Wallis test (H(3,n=88)=4,96 p=0,17) confirms that there are no statistically significant differences between Funaben T fungistatic activity and thyme, lemongras and Litsea cubeba oils.

About F. avenaceum. In this case, too, grapefruit oil had the weakest effect; at the highest concentration applied (2.000 %), its average fungistatic activity was only 12.35%; garlic oil in the highest concentration – average activity 89.41% (Table 7, Figure 4). The Kruskal–Wallis tests (H(9,n=229)=133.13, p=0.00) indicated that differences in fungistatic activity of the oils in sensitivity of individual isolates with F. avenaceum were statistically significant. In addition, the U Mann-Whitney test showed that there are no differences between Funaben T and thyme oil (Z=0.00, p=1.00). About F. oxysporum. Grapefruit oil, however, had a stronger effect than against F. avenaceum, the highest concentration (2.000%) with an average fungistatic activity of 37.79%; garlic oil in the highest concentration with an average activity of 89.41% (Table 8, Figure 5).

The Kruskal-Wallis test ((H(9,n=231)=121,92, p=0,00) confirms that differences in fungistatic activity of the eight oils used were statistically significantly. In addition, small differences with thyme oil are visible to Litsea cubeba oils and lemongras. The Kruskal-Wallis test confirms that there are no statistically significant differences between Funaben T fungistatic activity and thyme, lemongrass, verben and Litsea cubeba (H(4, n=115)=1,89 p=0,76).

About 4 species of Fusarium (Additional statistical comments (combined analysis, Figure 6). The Kruskal-Wallis test showed that there are statistically significant differences between the fungistatic activity of the oils used for the four isolates tested (H (9,n=921)=486.05, p=0.00). Analysis of frame-to-gorchart showed that the greatest similarities to Funaben T's performance show thyme, lemongrass, verben and Litsea cubeba. Statistical analysis showed that only thyme oil does not have significant differences in fungistatic activity compared to Funaben T for all the fungi isolates at issue (Mann-Whitney Test Z=-0.401, p=0.69). Comments to Table 11 and Table 12. The presented model of multiple regression showed that the concentration of oils and their chemical composition significantly affect antifungistatic activity (Table 11). The concentration of essential oil, monoterpenoids and sesquitertenes are directly proportional to antifungal activity.The model describes only 60% of the variability of the antifungal activity (value of R2 in Table 12), the remaining 40% variation of dependent variable is not included in the model. However the value of the standard error of estimation is large (Table 12). The model requires detailed research.

Authors answer:

Authors add new figures (5), tables (2); statistical comments (6 points) were putted into text after suggestion of Reviewer, too.

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