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Mycorrhizal Inoculation Improves the Quality and Productivity of Essential Oil Distilled from Three Aromatic and Medicinal Plants: Thymus satureioides, Thymus pallidus, and Lavandula dentata

Agronomy 2022, 12(9), 2223; https://doi.org/10.3390/agronomy12092223

by Oumaima Akachoud^1,2,3

, Hafida Bouamama³, Natacha Facon¹, Frédéric Laruelle¹, Btissam Zoubi², Abderrazak Benkebboura², Cherki Ghoulam², Ahmed Qaddoury² and Anissa Lounès-Hadj Sahraoui^1,*

Reviewer 1:

Bouchra Soulaimani

Reviewer 2:

Mostafa Amani Machiani

Agronomy 2022, 12(9), 2223; https://doi.org/10.3390/agronomy12092223

Submission received: 31 August 2022 / Revised: 11 September 2022 / Accepted: 14 September 2022 / Published: 18 September 2022

(This article belongs to the Special Issue Plant–Microbial Interactions and Application of Microbial Products in Agricultural Ecosystems)

Round 1

Reviewer 1 Report

The paper « Mycorrhizal inoculation improves essential oil quality and productivity distilled from three aromatic and medicinal plants » reports interesting subject regarding the cultivation of medicinal plants. However, the paper needs to be deeply revised before acceptation for publication.

Here are some comments:

Title

I suggest to replace « Mycorrhizal inoculation improves essential oil quality and productivity distilled from three aromatic and medicinal plants » with « Mycorrhizal inoculation improves the quality and productivity of essential oil distilled from three aromatic and medicinal plants ».

Abstract

Line 18 : replace « …mass and essential oil yields in three AMP species endemic to Morocco and threatened with extinc tion, Thymus satureioides, Thymus pallidus, and Lavandula dentat » with «…mass and essential oil yields of three moroccan endemic AMP threatened with extinction, Thymus satureioides, Thymus pallidus, and Lavandula dentata » and verify if Lavandula dentata is a moroccan endemic plant !!!!

Line 23 : the sentence « Furthermore, the chemical composition of the essential oil showed increases in the proportions of major compounds such as oxygenated monoterpene » is not clear, please clarify if the increase is for the inoculated plants or the inthreared ones.

Line 23 : Replace « increases » with « an increase »

The conclusion needs to be improved.

Introduction

Line 47 : the statistics on the AMP export between 2002 and 2014 are very old, it is necessary to base on more recent statistics on the exports of thyme and lavender.

Line 51 : linalool and linalyl acetate are common components in lavander essential oils not in thyme !!! please verify.

9,10: are not the appropriate references. The authors should consult and cite articles and reviews on the essential oils of lavender and thyme. Here are some suggestions :

El Yaagoubi, M., Mechqoq, H., El Hamdaoui, A., Mukku, V. J., El Mousadik, A., Msanda, F., & El Aouad, N. (2021). A review on Moroccan Thymus species: Traditional uses, essential oils chemical composition and biological effects. Journal of Ethnopharmacology, 278, 114205.

Soulaimani, B., El Hidar, N., El Fakir, S. B., Mezrioui, N., Hassani, L., & Abbad, A. (2021). Combined antibacterial activity of essential oils extracted from Lavandula maroccana (Murb.), Thymus pallidus Batt. and Rosmarinus officinalis L. against antibiotic-resistant Gram-negative bacteria. European Journal of Integrative Medicine, 43, 101312.

Wagner, L. S., Sequin, C. J., Foti, N., & Campos-Soldini, M. P. (2021). Insecticidal, fungicidal, phytotoxic activity and chemical composition of Lavandula dentata essential oil. Biocatalysis and Agricultural Biotechnology, 35, 102092.

Soulaimani, B., Nafis, A., Kasrati, A., Rochdi, A., Mezrioui, N. E., Abbad, A., & Hassani, L. (2019). Chemical composition, antimicrobial activity and synergistic potential of essential oil from endemic Lavandula maroccana (Mill.). South African Journal of Botany, 125, 202-206.

Rathore, S., & Kumar, R. (2022). Essential Oil Content and Compositional Variability of Lavandula Species Cultivated in the Mid Hill Conditions of the Western Himalaya. Molecules, 27(11), 3391.

Line 56 : the reference 11 in not relevant

Materials and Methods

87-89 : who did the identification of the studied plants ?

Line 93 : add a reference for AMF species !!??

Line 151 : add the calculation of essential oil yield

Line 171 : add « s » to « essential oil » « essential oils »

Line 176 : « PDA » write the full name of the culture medium, then the abbreviation

Line 171 : Why did you use different methods for the antifungal test?

Results

Line 239-239 : scientific names in italics. Please check the whole document, especially the result section

Discussion

Line 432 : what do you mean by essential compounds (chlorophyll, soluble sugar, and proteins) ?????? !!!!!!!!!

Line 480 : with, not italic

The authors should discuss and compare the obtained results with another studies on the cultivation of AMP.

Conclusion

Line 521-523 : is not a conclusion

The authors should report the main conclusions of the study.

Author Response

Dear reviewer 1,

Thank you very much for your mail concerning our manuscript entitled "Mycorrhizal inoculation improves the quality and productivity of essential oil distilled from three aromatic and medicinal plants: Thymus satureioides, Thymus pallidus and Lavandula dentata." which was submitted to Agronomy and has been reviewed.

We carefully read the points you raised in your letter as well as the reviewers’ comments included at the bottom of it. The co-authors and I very much appreciated the encouraging, critical, and constructive comments on this manuscript by the reviewer. The comments have been very thorough and useful in improving the manuscript. We paid attention to all the comments and suggestions that were listed and did our best to improve the manuscript. We are submitting the corrected manuscript with the suggestion incorporated into the manuscript. In addition to the corrections performed in the manuscript, you will find below our responses to all the comments.

We thank you again for your valuable comments and suggestions that have improved the scientific value of the revised manuscript.

Responses to the comments:::

Title

Comment 1:

I suggest to replace « Mycorrhizal inoculation improves essential oil quality and productivity distilled from three aromatic and medicinal plants » with « Mycorrhizal inoculation improves the quality and productivity of essential oil distilled from three aromatic and medicinal plants ».

Response: The title was changed.

The title of the revised manuscript is now: “Mycorrhizal inoculation improves the quality and productivity of essential oil distilled from three aromatic and medicinal plants: Thymus satureioides, Thymus pallidus and Lavandula dentata”

Abstract

Comment 2 :

Line 18 : replace « …mass and essential oil yields in three AMP species endemic to Morocco and threatened with extinction, Thymus satureioides, Thymus pallidus, and Lavandula dentata » with «…mass and essential oil yields of three moroccan endemic AMP threatened with extinction, Thymus satureioides, Thymus pallidus, and Lavandula dentata » and verify if Lavandula dentata is a moroccan endemic plant !!!!

Response :

Replacement done: line 20-22.
Yes Lavandula dentata is not an endemic plant in Morocco. We have corrected the error in the lines 87-88.

Comment 3:

Response:

We have replaced the sentence by :

« The EOs of inoculated-AMP exhibited increased proportions of major compounds such as thymol (23.7%), carvacrol (23.36%) and borneol (18.7%) in T. satureioides, α-terpinene (32.6%), thymol (28.79%) and δ-terpinene (8.1%) in T. pallidus, and camphor (58.44%), isoborneol (8.8%) and fenchol (4.1%) in L. dentata. (lines 22-25).

Comment 4

Line 23 : Replace « increases » with « an increase »

Response: due to the improvement of abstract section , you will not found this correction in the revised article

Comment 5:

The conclusion needs to be improved.

Response: Now, in the revised version of the manuscript, the conclusion has been improved (lines 28-31.

Introduction

Comment 6

Line 47 : the statistics on the AMP export between 2002 and 2014 are very old, it is necessary to base on more recent statistics on the exports of thyme and lavender.

Response: You are right. Unfortunately, there is a lack of information on this topic. No recent statistics are available in the literature.

The only data found includes different species under the code: HS Code: 0910 - Ginger, saffron, turmeric, (Curcuma), thyme, bay leaves, curry, and other spices.

Comment 7:

Line 51 : linalool and linalyl acetate are common components in lavander essential oils not in thyme !!! please verify.

Response :

Linalyl acetate has been mentioned in the composition of two species of thyme: T. serpyllum and Thymus willdenowii Boiss (El yaagoubi et al., 2021), but as it is not a compound common to different species of thyme and exists in small quantities in lavender essential oils, we preferred not to mention it anymore.

For linalool, we have checked our references, and it has been identified in the majority of thyme species. This information has been confirmed and reported in several recent studies (El yaagoubi et al., 2021 ; El bakkal et al., 2020).

El-Bakkal, Salah eddine; Zeroual, Safia; Elouazkiti, Mohammad; Mansori, Mounir; Bouamama, Hafida; Zehhar, Naima; El-Kaoua, Mimoun (2020). Comparison of Yield Chemical Composition and Biological Activities of Essential Oils Obtained from Thymus pallidus; and Thymus satureioides Coss. Grown in Wild and Cultivated Conditions in Morocco. Journal of Essential Oil Bearing Plants, (), 1–14. doi:10.1080/0972060X.2019.1708216.

Comment 8:

9,10: are not the appropriate references. The authors should consult and cite articles and reviews on the essential oils of lavender and thyme. Here are some suggestions:

Rathore, S., & Kumar, R. (2022). Essential Oil Content and Compositional Variability of Lavandula Species Cultivated in the Mid Hill Conditions of the Western Himalaya. Molecules, 27(11), 3391.

Response: Now, in the revised version of the manuscript, all the references have been added under the numbers : 9, 10,11,12 and 13

Comment 9:

Line 56 : the reference 11 in not relevant

Response: We have removed the reference 11.

Materials and Methods

Comment 9:

87-89 : who did the identification of the studied plants ?

Response:

The identification of the plants studied was made at first by Prof. Hafida Bouamama, as well as by Dr. Salah Eddin El bakkal, who published an article in 2020 on the same plant species. A third confirmation has been made by Prof. Ouhammou Ahmed who is the head of the research of the Regional Herbarium "MARK" - UCA-FSSM at Cadi Ayyad University, Marrakech, Morocco.

Comment 10:

Line 93 : add a reference for AMF species !!??

Response: We have added the reference lines 104-105

Comment 11:

Line 151 : add the calculation of essential oil yield

Response: Done in line 175

Yield of the essential oil = (weight of the extracted essential oil /weight of the dried material) x (1/ essential oil density).

Comment 12:

Line 171 : add « s » to « essential oil » « essential oils »

Response: Done in line 196

Comment 13:

Line 176 : « PDA » write the full name of the culture medium, then the abbreviation

Response: PDA : Potato Dextrose Agar . Done in line 201

Comment 14:

Line 171 : Why did you use different methods for the antifungal test?

Response:

As we tested different fungal strains, namely F. culmorum in mycelian form, Z. tritici, and B. graminis in spore form, we choose an appropriate method for each fungus as reported in the following publications :

El-Alam I, Raveau R, Fontaine J, Verdin A, Laruelle F, Fourmentin S, et al. Antifungal and Phytotoxic Activities of Essential Oils: In Vitro Assays and Their Potential Use in Crop Protection. Agronomy. 2020;10(6):825. doi:10.3390/agronomy10060825.

Znini M, Cristofari G, Majidi L, El Harrak A, Paolini J, Costa J. In vitro antifungal activity and chemical composition of Warionia saharae essential oil against 3 apple phytopathogenic fungi. Food Sci Biotechnol. 2013;22(S1):113–119. doi:10.1007/s10068-013-0056-2.

Brent KJ, Hollomon DW. FUNGICIDE RESISTANCE: THE ASSESSMENT OF RISK [Internet]. 2 second, (revised) edition. Aimprint in the United Kingdom; 2007. Available from: https://www.frac.info/docs/default-source/publications/monographs/monograph-2.pdf

Results

Comment 15:

Line 239-239 : scientific names in italics. Please check the whole document, especially the result section

Response: Done.

Discussion

Comment 16:

Line 432 : what do you mean by essential compounds (chlorophyll, soluble sugar, and proteins) ?????? !!!!!!!!!

Response: There was a mistake in the formulation which is corrected now in the revised version of the manuscript.x(Thus, a clear positive correlation (Figure 15) was noticed between the mycorrhizal colonization and the accumulation of chlorophyll, soluble sugar, and proteins) line 485-486.

Comment 17:

Line 480 : with, not italic

Response: Done.

Comment 18:

The authors should discuss and compare the obtained results with another studies on the cultivation of AMP.

Response: Done (in track changes revised article).

Line 465-467 :”The beneficial effect of AMF on AMP growth has been reported in several other aromatic plant species such as Salvia officinalis L, Acacia gummifera and Ocimum basilicum L.^2–4”

Line: 471-474: “). These results are in agreement with previous reports on Coriandrum sativum L. inoculated with R. irregularis, which showed increased shoot concentration of nitrogen (44%), phosphorus (254%), and potassium (27%) ⁵”

Line 483-485: “accordingly to the research carried out by Yadav et al, which also shows an increase in those parameters in mycorrhizal Gloriosa superba L⁶.”

Line 501-502; “These findings are in line with several previous studies on inoculated AMP, Atractylodes lancea, Artemisia umbelliformis and Ocimum basilicum L. ^2,7”

Conclusion

Comment 18

Line 521-523 : is not a conclusion

The authors should report the main conclusions of the study.

Response: The conclusion has been rewritten in the revised version of the article (in track changes revised article).

Author Response File: Author Response.docx

Reviewer 2 Report

Tittle

The title is not clear. Please add the plant species in the title.

Abstract

- Underscore the scientific value-added to your paper in your abstract. Your abstract should clearly state the essence of the problem you are addressing, what you did and what you found and recommend. That will help a prospective reader of the abstract to decide if they wish to read the entire article.

- Please add the highest values of mentioned traits in abstract section.

- Please add the values and types of the main chemical constituents of each plants.

Introduction

- Please add the three medicinal and aromatic plants economic values and cultivation area in Morocco.

- In introduction, please add more information about the negative impacts of chemical inputs on the bioactive compounds of medicinal and aromatic plants. Use the manuscript:

Amani Machiani, M.; Javanmard, A.; Habibi Machiani, R.; Sadeghpour, A. Arbuscular mycorrhizal Fungi and Changes in Primary and Secondary Metabolites. Plants 2022, 11, 2183. https://doi.org/10.3390/plants11172183

Strzemski, M., Dzida, K., Dresler, S., Sowa, I., Kurzepa, J., Szymczak, G., Wójciak, M., 2021. Nitrogen fertilisation decreases the yield of bioactive compounds in Carlina acaulis L. grown in the field. Ind. Crops Prod. 170, 113698. https://doi.org/10.1016/j.indcrop.2021.113698.

- The novelty of study should be highlighted.

- The hypothesis should be added in introduction?

This Materials and methods section is good and the methods described clearly.

Lines 99-103: There was 9 treatments, no 12 treatments.

The method for measuring N content should be added in manuscript.

Please add the P and K content of three plant species.

Lines 242-249: How much percentage of dry matter increased by mycorrhiza application?

Lines 254-262: Please add the increasing or decreasing percentage, too.

Why the essential oil content of three plants was not reported in results section?

The p-cymene compound is one of the precursors compounds of thyme and lavender. Why this compound is not identified in the essential oil compounds of three medicinal and aromatic plants?

Please add the chromatogram of three plants essential oil constituents.

The conclusion section should be revised. This section repetitive. Please make sure your conclusions' section underscores the scientific value-added of your paper, and/or the applicability of your findings/results. Highlight the novelty of your study.

What suggestions do you have for future research in this field?

Author Response

Dear reviewer 2,

We thank you again for your valuable comments and suggestions that have improved the scientific value of the revised manuscript.

RESPONSES TO REVIEWER 2

Tittle

Comment 1:

The title is not clear. Please add the plant species in the title.

Response: Done “Mycorrhizal inoculation improves the quality and productivity of essential oil distilled from three aromatic and medicinal plants: Thymus satureioides, Thymus pallidus and Lavandula dentata.”

Abstract

Comment 2:

Underscore the scientific value-added to your paper in your abstract. Your abstract should clearly state the essence of the problem you are addressing, what you did and what you found and recommend. That will help a prospective reader of the abstract to decide if they wish to read the entire article.

Response: Done.

Background: In Morocco, extensive use, traditional practices, and climate change seriously impacted the productivity of aromatic and medicinal plants (AMP)./Line 8-9

Methods: To mitigate these adverse effects, this study aims at evaluating the potential of the arbuscular mycorrhizal fungi (AMF), Rhizophagus irregularis and Funneliformis mosseae, in improving biomass, essential oils (EOs), and biomolecules production in Thymus satureioides, T. pallidus, and Lavandula dentata /Line 16-19

Results: Compared to non-inoculated-AMP, AMF induced significant increases in biomass production by 37.1, 52.4, and 43.6%, and in EOs yield by 21, 74, and 88% in T. satureioides, T. pallidus and L. dentata respectively. The EOs of inoculated-AMP exhibited increased proportions of major compounds such as thymol (23.7%), carvacrol (23.36%) and borneol (18.7%) in T. satureioides, α-terpinene (32.6%), thymol (28.79%) and δ-terpinene (8.1%) in T. pallidus, and camphor (58.44%), isoborneol (8.8%) and fenchol (4.1%) in L. dentata.

Moreover, AMF significantly improved the anti-germinative and antifungal activities of the EOs. Indeed, IC50 values decreased by 1.8, 16.95, and 2.2 times against Blumerai graminis, Zymoseptoria tritici and Fusarium culmorum, respectively compared to non-inoculated-AMP. Line 20-28

Conclusion: This study highlights the performance of the symbiosis AMF-AMP in terms of high-quality of EOs production while respecting the environment. The associations F. mosseae-Thymus and R. irregularis-Lavandula are the most efficient/Line 28-31

Comment 3:

Please add the highest values of mentioned traits in abstract section.

Response: The highest values of the mentioned traits have been added (in track changes revised article)

Comment 4:

Please add the values and types of the main chemical constituents of each plants.

Response: the values and types of the main chemical constituents of each plants have been added in line 22-28

Introduction

Comment 5:

Please add the three medicinal and aromatic plants economic values and cultivation area in Morocco.

Response: Concerning the statistics on medicinal and aromatic plants, it is necessary to underline that there is a lack of data, and in the majority of the cases. We find only global information on several plants, and in our case, it is always the surfaces of wild plants which are quoted. It is not easy to have precise statistics on these three plants. Moreover, according to our research, no statistical information is available after 2014.

Comment 6:

In introduction, please add more information about the negative impacts of chemical inputs on the bioactive compounds of medicinal and aromatic plants. Use the manuscript:

Response: Done in line 64-70, (reference 16, 17)

Comment 7:

The novelty of study should be highlighted.

Response: This point has been highlighted in the revised version of our manuscript in the lines 83-89

Comment 8:

The hypothesis should be added in introduction?

Response: Done in line 90-94

Comment 9:

This Materials and methods section is good and the methods described clearly.

Response: Thanks a lot for the comment.

Comment 10:

Lines 99-103: There was 9 treatments, no 12 treatments.

Response: You are right. The correction is done in line 111.

Comment 11:

The method for measuring N content should be added in manuscript.

Response: The nitrogen method was already in the manuscript, please check line 151-156

Comment 12:

Please add the P and K content of three plant species.

Response:

We have added :

the P and K methods in the materials and methods section (line 157-167),
and also the results obtained (line 327-338),
as well as parameters in the PCA (Figure 15)
and the discussion section too (in track changes revised article).

Comment 13:

Lines 242-249: How much percentage of dry matter increased by mycorrhiza application?

Response: Done in line 272

“The biomass production expressed as shoot dry matter (SDM) per plant was significantly higher (P< 0.0001) in inoculated AMP (NI-AMP) than in non-inoculated ones (NI-AMP), with an improvement rate up to 37.1, 52.42 and 43.6% in T. satureioides, T. pallidus and L. dentata respectively,”

Comment 14:

Lines 254-262: Please add the increasing or decreasing percentage, too.

Response: Done in line 284-285

“Compared to NI-AMP, TCC increased by 38.5, 81.5 and 51.4% in inoculated T. satureioides, T. pallidus, and L. dentata, respectively”

Comment 15:

Why the essential oil content of three plants was not reported in results section?

Response:

The essential oil content of the three plants was already added, and the chemical composition of each plant was written on the up of the tables (T. satureioides: lines 361-372, T. pallidus: lines 382-390, and L. dentata: lines 398-405.

Comment 16:

The p-cymene compound is one of the precursors compounds of thyme and lavender. Why this compound is not identified in the essential oil compounds of three medicinal and aromatic plants?

Response:

Since p-cymene is an intermediate metabolite in the biosynthesis pathway of thymol, its concentration and consequently detection depends on several factors (such as harvesting season for example). Since the final compound (thymol) accumulates to a large extent, the p-cymene was below detection threshold in our conditions.

Comment 17:

Please add the chromatogram of three plants essential oil constituents.

Response: :as we have 9 chromatograms, the addition of these last ones will saturate the article, for this reason we put at your disposal an example of chromatograms obtained for T. pallidus

Comment 18:

Response: The conclusion has been rewritten.

Comment 19:

What suggestions do you have for future research in this field?

Response:

Our suggestions for future research in this field are to:

Develop a new fungicide based on the essential oils' main compounds, which have correlated positively to the antifungal activities, to be tested for the protection of plants during the treatment of seeds or storage.
Identification and multiplication of native AMF associated with each AMP, to be tested on a large scale under greenhouse conditions.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The manuscript has been improved and can be accepted for publication.

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Mycorrhizal Inoculation Improves the Quality and Productivity of Essential Oil Distilled from Three Aromatic and Medicinal Plants: Thymus satureioides, Thymus pallidus, and Lavandula dentata

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