A Polysaccharide-Based Integrated Nutrient Management System Enhances the Antioxidant Properties in Origanum dictamnus (Lamiaceae), a Valuable Local Endemic Plant of Crete

Round 1

Reviewer 1 Report

Dear authors,
the manuscript you have sent to me for evaluation addresses the very interesting issue of optimising the fertilisation process of the endemic Crete plant Origanum dictamnus L. Given the medicinal properties of this plant and the fact that it is an endangered species, it made sense for the authors to undertake this research. The manuscript has been prepared correctly for this type of work, but there are elements that need some revision. My proposed changes are set out below in bullet points.  
1. the title of the article in the typescript differs from that submitted to the MDPI system. This issue should be resolved. I suggest basing the title on the title that was included in the text/manuscript.
2. line 63 The acronym INM should be clarified here.
3. line 112 - "Chemicals" is not the correct term for the fertilisers that were used in the field trials. Please specify these combinations precisely e.g. "synthetic fertilisers" or "compounds derived from chemical synthesis" or "conventional fertilisers".
4.Row 281 - I suggest adding a citation.
5. line 290 - I suggest adding a citation.
6.I suggest explaining the abbreviations of the tested fertiliser combinations in table form. Then there will be no need to duplicate this information with each figure and with each table. This will increase the readability of the table and figure captions.
(7) Different fertilisation technologies were investigated in this study. For example, INM fertilisers were applied foliarly (INM-fa) and topdressing (INM-sa). The same was true for Chf fertilisers. So why was a mixture of plant extracts (MPE) only applied topically?
8 In the case of the control facility, I also see some inaccuracies. If fertilisers were applied foliarly and topdressing then there should also be two controls. 1) water applied topically and 2) water applied foliarly. Why was only one control included and how was it done?
9 Line 344 - please describe in detail the results shown in Figure 3. Where was the increase and where was the decrease in the trait analysed? A paragraph must not start and end with a single sentence, and this should be particularly avoided for a subsection in a manuscript.
10. subsections should be titled according to the results presented in it. I suggest avoiding formulating the title as a conclusion/summary. I therefore suggest changing the title of subsection 3.1 "The fertilisation scheme exerted limited effects on leaf colour" to "effect of fertilisation technology on leaf colour of Origanum dictamnus L.". This applies to the entire manuscript.
11 Please discuss in more detail the result shown in Figure 5A.
12 I suggest subsections 3.5 and 3.6 be combined and discussed in one paragraph. Then the title of subsection 3.5 could be as follows: "chlorophyll and antioxidant content of leaves of Origanum dictamnus L. depending on the fertilisation combination used".
13 Line 397 - please discuss in detail the results shown in Figure 6B. Similarly for Figure 6C and 6D.
14 Line 416 - as above.
15 Line 449 - we are not dealing with the x-axis in the table. I suggest correcting the tablei caption throughout the article.
16. line 609-621 - these are not conclusions. This part of the text is worth putting at the end of the "Discussion" section. The conclusions, on the other hand, should be based solely on the results presented in the manuscript. Conclusions = statement + justification based on the results obtained.

Author Response

REVIEWER 1 - Comments and Suggestions for Authors

Dear authors, the manuscript you have sent to me for evaluation addresses the very interesting issue of optimising the fertilisation process of the endemic Crete plant Origanum dictamnus L. Given the medicinal properties of this plant and the fact that it is an endangered species, it made sense for the authors to undertake this research. The manuscript has been prepared correctly for this type of work, but there are elements that need some revision.

Authors’ response: We would like to thank the reviewer for his/her favorable comments, suggesting our paper has been prepared correctly for publication after some revision.

My proposed changes are set out below in bullet points.

1. the title of the article in the typescript differs from that submitted to the MDPI system. This issue should be resolved. I suggest basing the title on the title that was included in the text/manuscript.

Authors’ response:Thank you for this comment. We have kept the title that is included in the text/manuscript. Please, see our answers to the reviewers’ suggestions, and also the amendments in the revised manuscript (see track changes).

2. line 63 The acronym INM should be clarified here.

Authors’ response: Ok. Thank you, this has been clarified in the revised manuscript (see track changes).

3. line 112 - "Chemicals" is not the correct term for the fertilisers that were used in the field trials. Please specify these combinations precisely e.g. "synthetic fertilisers" or "compounds derived from chemical synthesis" or "conventional fertilisers".

Authors’ response: Ok. The term “Chemicals” has been replaced by “compounds derived from chemical synthesis”, according to your suggestion (see track changes). Thank you.

4. Row 281 - I suggest adding a citation.

Authors’ response: Ok. The following citation was added: “Portable food diagnostic devices and methods: A review” (see track changes).

5. line 290 - I suggest adding a citation.

Authors’ response: Ok. The following citation was added: “DNA Barcoding and Fertilization Strategies in Sideritis syriaca subsp. syriaca, a Local Endemic Plant of Crete with High Medicinal Value” (see track changes).

6.I suggest explaining the abbreviations of the tested fertiliser combinations in table form. Then there will be no need to duplicate this information with each figure and with each table. This will increase the readability of the table and figure captions.

Authors’ response: The abbreviations of the tested fertilizers combinations are explained in “Supplementary material Methods S1”. However, many readers directly go to the tables and figures before reading the entire text and prefer that figures and tables are self-explanatory to facilitate readability. In this regard, we would prefer to keep unchanged this information with each figure and with each table unless the academic editor advises us differently.

(7) Different fertilisation technologies were investigated in this study. For example, INM fertilisers were applied foliarly (INM-fa) and topdressing (INM-sa). The same was true for Chf fertilisers. So why was a mixture of plant extracts (MPE) only applied topically?

Authors’ response: Indeed, the MPE was only soil-applied. However, since foliar and soil preliminary experiments in these plants generally did not reveal statistical differences (data not shown), we omitted the foliar application of MPE.

8 In the case of the control facility, I also see some inaccuracies. If fertilisers were applied foliarly and topdressing then there should also be two controls. 1) water applied topically and 2) water applied foliarly. Why was only one control included and how was it done?

Authors’ response: we would like to thank you for this comment. A drip irrigation system was permanently applied in all of the treatments. Furthermore, at the time that the INM and conventional foliar applications were performed (Schemes A and B), a foliar spraying with similar quantity of water was applied to the rest of the treatments (Schemes C, D, E and F). Similarly, at the time that the INM, conventional and biostimulant soil applications were performed (Schemes D, E and F), a soil application with similar quantity of water was applied to the rest of the treatments (Schemes A, B and C). So, the control was the same for both foliar and soil applications, as water was both foliarly and topically applied.

9 Line 344 - please describe in detail the results shown in Figure 3. Where was the increase and where was the decrease in the trait analysed? A paragraph must not start and end with a single sentence, and this should be particularly avoided for a subsection in a manuscript.

Authors’ response: This paragraph in the result subsection has now been considerably improved. Please, see the amendments shown with track changes.

10. subsections should be titled according to the results presented in it. I suggest avoiding formulating the title as a conclusion/summary. I therefore suggest changing the title of subsection 3.1 "The fertilisation scheme exerted limited effects on leaf colour" to "effect of fertilisation technology on leaf colour of Origanum dictamnus L.". This applies to the entire manuscript.

Authors’ response: In the results section, the titles of subdivisions are intentionally structured to depict in a clear way the most significant findings of this study. This widely used strategy was chosen to facilitate comprehension, readership, and results’ interpretation. In this regard, we would prefer to keep the results’ titles unchanged unless the academic editor advises us differently. Thank you.

11 Please discuss in more detail the result shown in Figure 5A.

Authors’ response: The result shown in Figure 5A has accordingly been given in more detail.

Please, see the amendments shown with track changes.

12 I suggest subsections 3.5 and 3.6 be combined and discussed in one paragraph. Then the title of subsection 3.5 could be as follows: "chlorophyll and antioxidant content of leaves of Origanum dictamnus L. depending on the fertilisation combination used".

Authors’ response: The subsections 3.5 and 3.6, according to the reviewer suggestion, have been combined and discussed in one paragraph and the title has been modified accordingly.

13 Line 397 - please discuss in detail the results shown in Figure 6B. Similarly for Figure 6C and 6D.

Authors’ response: According to the reviewer suggestions, Figure 6 has been better discussed in the revised3.5 section. Please, see the amendments shown with track changes.

14 Line 416 - as above.

Authors’ response: According to the reviewer suggestions, Figure 7 has been better discussed in the revised 3.6 section. Please, see the amendments shown with track changes.

15 Line 449 - we are not dealing with the x-axis in the table. I suggest correcting the tablei caption throughout the article.

Authors’ response: The tables have been accordingly improved throughout the article as suggested by the reviewer.  Please, see the amendments shown with track changes.

16. line 609-621 - these are not conclusions. This part of the text is worth putting at the end of the "Discussion" section. The conclusions, on the other hand, should be based solely on the results presented in the manuscript. Conclusions = statement + justification based on the results obtained.

Authors’ response: Following the reviewer’s suggestion, the lines 609-621  have been transferred to the end of the “Discussion” section. We thank the reviewer for the valuable suggestions.

Reviewer 2 Report

The concept of this research is interesting and useful to the researchers who are working in this biomedical field. The methods used in this study need to be appropriate and should provide detailed methodology. The result interpretation needs improvement and discussion section can be improved further to enhance the quality of the manuscript. The following comments can improve the manuscript: quality.

Comments

The background information behind this topic selection in introduction section needs to be improved to support the reason and significance of this study.

What are the major biomolecules present in Origanum dictamnus?

Strengthen the novelty statement and improve the statements that describe the significance of this study.

Why did the author select the Origanum dictamnus?

How many times have the studies been performed or repeated?

Some references are old, so author should update with recent references

What is the continuation of this study that should be mentioned in the conclusion section?

Check the table format

Correct the figure legends and check the x and y axes. axis content

The manuscript has some typo errors and improves the language of this manuscript.

The manuscript has some typo errors and improves the language of this manuscript.

Author Response

REVIEWER 2 - Comments and Suggestions for Authors

The concept of this research is interesting and useful to the researchers who are working in this biomedical field. The methods used in this study need to be appropriate and should provide detailed methodology. The result interpretation needs improvement and discussion section can be improved further to enhance the quality of the manuscript.

Authors’ response: We would like to thank the reviewer for his/her favorable comments.

The following comments can improve the manuscript quality.

Comments

The background information behind this topic selection in introduction section needs to be improved to support the reason and significance of this study.

Authors’ response: According to the Reviewer suggestions, the background information behind this topic selection in the introduction section has been improved in the revised manuscript, thus offering stronger support regarding the reason and significance of this study. Please, see our answers to the reviewers’ suggestions, and also the amendments in the revised manuscript (see track changes).

What are the major biomolecules present in Origanum dictamnus?

Authors’ response: As suggested by the reviewer, we have further improved the introduction section by adding a few citations concerning the biomolecules in O. dictamnus, taken from the study “The O. dictamnus L. extracts possess high scavenging capacities, high phenolic contents and are rich in flavonoids” and “Identification of Bioactive Compounds in Plant Extracts of Greek Flora and Their Antimicrobial and Antioxidant Activity”.

Strengthen the novelty statement and improve the statements that describe the significance of this study.

Authors’ response: As suggested by the reviewer, the novelty statement that describes the significance of this study has been considerably improved in the introduction section to support the significance of this study.

Why did the author select the Origanum dictamnus?

Authors’ response: O. dictamnus L. is traditionally cultivated only at local scale in Crete as one of the most popular and therapeutic aromatic-medicinal plants; otherwise, it is directly sourced from the wild since ancient times and is strongly associated with the Cretan cultural heritage. Therefore, it represents a traditional herbal medicine with approved use by the European Medicines Agency. Furthermore, its unique structure in polysaccharide-containing subcuticular compartments strengthens its use in polysaccharide-INM fertilization strategies.

Please, see our answers to the reviewers’ suggestions, and also the amendments in the revised manuscript (see track changes).

How many times have the studies been performed or repeated?

Authors’ response: Each experimental design was performed in completely randomized blocks of 10 plants of O. dictamnus per block, three blocks per treatment, which were randomly located in different rows. Although the present study focused only on one growth cycle in Crete, it offered for the first time reference data for growth/yield aspects and several herbal quality aspects in O. dictamnus, related to polysaccharide-INM fertilization strategies.

Please, see our answers to the reviewers’ suggestions, and also the amendments in the revised manuscript (see track changes).

Some references are old, so author should update with recent references

Authors’ response: Several recent references have been added in the revised version of the manuscript.

What is the continuation of this study that should be mentioned in the conclusion section?

Authors’ response: As suggested by the reviewer, the continuation of this study has been added at the end of the “Conclusion and Prospects” section.

Check the table format

Authors’ response: Ok. Corrected.

Correct the figure legends and check the x and y axes. axis content

Authors’ response: Ok. Corrected.

The manuscript has some typo errors and improves the language of this manuscript.

Authors’ response: Ok. Corrected.

Round 2

Reviewer 1 Report

The authors have followed most of my comments and suggestions for change. Consequently, I believe that the article in this form can be published in the journal Polysaccharides.

Reviewer 2 Report

Manuscript quality is considerably improved

Minor language proof correction need to done before publishing this work