Chitosan and Titanium Dioxide Are More Effective in Improving Seed Yield and Quality in Nanoparticle Compared to Non-Structured Form: A Case Study in Five Milk Thistle Ecotypes (Silybum marianum (L.) Gaertn.)
, Abdolhossein Rezaei Nejad 1, Hasan Mumivand 1, Karim Sorkheh 2, Nikolaos Nikoloudakis 3 and Dimitrios Fanourakis 4
Round 1
Reviewer 1 Report
The ms. “Chitosan and Titanium Dioxide in the Nanoparticle Form Are More Effective in Improving Seed Yield and Quality: A Case Study in Five Milk Thistle Ecotypes (Silybum marianum (L.) Gaertn.)” (Ms. Ref. No. agronomy-1788175-v1) presents original research regarding the efficiency assessment of chitosan or TiO2 treatments on the productivity of milk thistle. The study was conducted during two consecutive crop years. The experimental design was set up to investigate both the chitosan and TiO2 form (“bulk” particle and nanoparticles-NP), and various concentrations. Five milk thistle genotypes were considered to explore the effects across genetic variation.
There is a lot of work involved and the ms. has evident merit.
The topic falls within the aims and scopes of the Agronomy journal. Originality = fair.
The ms. is adequately structured and carefully written and may be considered for publication after proper revision. However, there are some issues that need to be addressed to improve the presentation of the results.
Major issues:
1. There are two main drawbacks of the study: 1) the authors did not assess the efficiency of the treatments on the quality of the crops (i.e., the influence of the treatments on the sylimarin content and on the fatty acids profiles of the seed oil – which are the main elements of interest of the species), and 2) I wonder if there really are any differences in the chitosan or TiO2 solutions, as used in “bulk” form or NP form (this form is important in the solid state, or if the two chemicals are not water soluble. The authors say they have used chitosan/TiO2 “solutions” to spray the plants. So, there is either a confusion regarding particles being dissolved or dispersed, which resulted in suspensions instead of solutions, or – if they have obtained solutions, then it has no importance whether the particles were “bulk” or NP prior to solubilization, the obtained solutions would be similar/identical. Please make it clear.
2. Please examine the influence of the treatments on the fatty acid profiles of the seed oil. It should not be difficult; you have the oil from the Soxhlet extractions. If it is not possible, you may find the fatty acid composition of the seed oil in https://doi.org/10.1016/j.lwt.2022.113319 and https://doi.org/10.3390/foods9111630. Please revise.
3. The title should be revised. In the current form, it contains a comparison element, without mentioning the reference (“are more effective…” than what?).
4. In the Abstract, lines 21-24: it is confusing, it seems that chitosan and TiO2 were both applied together. Please revise.
5. Line 26: What do you mean/point out by “besides Ti content”? Why is Ti important in the plant to be highlighted as an efficiency criterium?
6. In the Introduction: The authors should point out the originality/elements of novelty of their manuscript, as compared to the existing literature. In the current form, the state-of-the-art was not address, only two reviews were referred to, without mentioning anything about the specific results of similar studies, on other species. The authors should mention similar research carried out on other species to set up the grounds for the hypothesis in the current study. No hypothesis was formulated.
7. Also in the Introduction: Please motivate the choice for chitosan and TiO2. Also, what is the optimal stage to apply the treatments, according to literature?
8. Line 65: Positive effects, such as? Again, the Introduction is too general and vague, nothing specific neither related to the species, nor to the treatments.
9. Lines 49-51: The food applications should not be disregarded (for example, milk thistle seed cakes floor is a functional ingredient, see https://doi.org/10.3390/foods10112766.
10. In the M&M section: important information is missing here: climate, temperature/rainfall regime (can be given as a supplementary material), dimensions of each plot, days from seeding (lines 102-103), irrigation (total water amount, line 106). Please revise.
11. Why haven’t the authors investigated combined treatments chitosan-TiO2 (for example, 25/25 and 50/50 mg L-1, respectively)? This should have been best fitted here, not in a future study.
12. Section 2.2. What is described here seems to be a laboratory scale recipe to prepare chitosan NP. What quantity of chitosan NP did the authors obtain as described? Was it enough for the plant treatments? Please revise for the actual/real amounts.
13. Line 126: How was it purified?
14. Figure 1 is not commented at all. Please revise.
15. Regarding Figure 3, and in relationship with comment #1 (my concern that the solutions of either nano-, or “bulk” particles are practically the same thing): it would be good to show the micrographs for “bulk” treatments, to highlight the difference (if there is a difference). Please revise.
Minor issues:
1. Please write all the Latin words (including scientific names of the species or words such as per, in situ, etc.) with Italics. Please revise throughout the ms.
2. Line 57: Remove the second “also”.
3. Please revise the references (line by line) and ensure consistency with the journal’s guidelines. In the current form, there are several typing errors (e.g. line 581 – journal’s name).
Given the completed score sheet and the comments above, after careful evaluation, the paper “Chitosan and Titanium Dioxide in the Nanoparticle Form Are More Effective in Improving Seed Yield and Quality: A Case Study in Five Milk Thistle Ecotypes (Silybum marianum (L.) Gaertn.)” (Ms. Ref. No. agronomy-1788175-v1) needs Major Revision according to comments before being considered for publication in Agronomy journal.
Author Response
Khorramabad, 20 July 2022
Dear Editor,
Herewith we submit the revision of the manuscript entitled “Chitosan and titanium dioxide are more effective in improving seed yield and quality in nanoparticle compared to bulked form: A case study in five milk thistle ecotypes (Silybum marianum (L.) Gaertn.)” (ID: agronomy-1788175) by S Jafari, S Mousavi-Fard, A Rezaei Nejad, H Mumivand, K Sorkheh, N Nikoloudakis and D Fanourakis for publication in Agronomy.
We would like to thank you for the kind words and the valuable suggestions. Please find below a description on how we have dealt with the received comments. We hope that the manuscript can now be accepted for publication in your journal.
Yours sincerely,
Sadegh Mousavi-Fard
Reviewer #1
The ms. “Chitosan and Titanium Dioxide in the Nanoparticle Form Are More Effective in Improving Seed Yield and Quality: A Case Study in Five Milk Thistle Ecotypes (Silybum marianum (L.) Gaertn.)” (Ms. Ref. No. agronomy-1788175-v1) presents original research regarding the efficiency assessment of chitosan or TiO2 treatments on the productivity of milk thistle. The study was conducted during two consecutive crop years. The experimental design was set up to investigate both the chitosan and TiO2 form (“bulk” particle and nanoparticles-NP), and various concentrations. Five milk thistle genotypes were considered to explore the effects across genetic variation.
There is a lot of work involved and the ms. has evident merit.
The topic falls within the aims and scopes of the Agronomy journal. Originality = fair.
The ms. is adequately structured and carefully written and may be considered for publication after proper revision. However, there are some issues that need to be addressed to improve the presentation of the results.
Major issues:
There are two main drawbacks of the study: 1) the authors did not assess the efficiency of the treatments on the quality of the crops (i.e., the influence of the treatments on the sylimarin content and on the fatty acids profiles of the seed oil – which are the main elements of interest of the species), and 2) I wonder if there really are any differences in the chitosan or TiO2 solutions, as used in “bulk” form or NP form (this form is important in the solid state, or if the two chemicals are not water soluble. The authors say they have used chitosan/TiO2 “solutions” to spray the plants. So, there is either a confusion regarding particles being dissolved or dispersed, which resulted in suspensions instead of solutions, or – if they have obtained solutions, then it has no importance whether the particles were “bulk” or NP prior to solubilization, the obtained solutions would be similar/identical. Please make it clear.
Authors: (1) The present experiment included 45 treatments (5 ecotypes × 9 spray treatments), which were realized for two experimental years. The effects of chitosan and TiO2 on a wide range of agronomic traits were analyzed. It is currently not feasible to expand the evaluations to include seed oil composition. It is now mentioned that this feature ought to be treated in future studies (Lines 597–600).
(2) Since TiO2 does not dissolve in water, the term suspension is indeed more appropriate than solution (now corrected in Lines 149, 161 and 162). The particle size was determined by using field emission scanning electron microscopy. Under the same concentration, nanoparticles offer a much larger bioactive surface as compared to bulk form, which justifies the use of a discrete experimental unit. The obtained results validate that the employed substances were much more effective in promoting agronomic traits in the nanoparticle as compared to the bulk form.
Please examine the influence of the treatments on the fatty acid profiles of the seed oil. It should not be difficult; you have the oil from the Soxhlet extractions. If it is not possible, you may find the fatty acid composition of the seed oil in https://doi.org/10.1016/j.lwt.2022.113319 and https://doi.org/10.3390/foods9111630. Please revise.
Authors: The present experiment included 45 treatments (5 ecotypes × 9 spray treatments), which were realized for two experimental years. The effects of chitosan and TiO2 on a wide range of agronomic traits were analyzed. It is currently not feasible to expand the evaluations to include seed oil composition. It is now mentioned that this feature ought to be treated in future studies (Lines 597–600).
The title should be revised. In the current form, it contains a comparison element, without mentioning the reference (“are more effective…” than what?).
Authors: As suggested, the title of the manuscript was adjusted accordingly (Lines 4 and 5).
In the Abstract, lines 21–24: it is confusing, it seems that chitosan and TiO2 were both applied together. Please revise.
Authors: Done (Line 27).
Line 26: What do you mean/point out by “besides Ti content”? Why is Ti important in the plant to be highlighted as an efficiency criterium?
Authors: The reference to Ti content was removed from that sentence (Line 32).
In the Introduction: The authors should point out the originality/elements of novelty of their manuscript, as compared to the existing literature. In the current form, the state-of-the-art was not address, only two reviews were referred to, without mentioning anything about the specific results of similar studies, on other species. The authors should mention similar research carried out on other species to set up the grounds for the hypothesis in the current study. No hypothesis was formulated.
Authors: As suggested, this is now dealt in the introduction (Lines 71–76, 79–81).
Also in the Introduction: Please motivate the choice for chitosan and TiO2. Also, what is the optimal stage to apply the treatments, according to literature?
Authors: As suggested, this is now dealt in the introduction (Lines 71–76, 79–81).
Line 65: Positive effects, such as? Again, the Introduction is too general and vague, nothing specific neither related to the species, nor to the treatments.
Authors: As suggested, this is now dealt in the introduction (Lines 71–76, 79–81).
Lines 49-51: The food applications should not be disregarded (for example, milk thistle seed cakes floor is a functional ingredient, see https://doi.org/10.3390/foods10112766.
Authors: Since silymarin content and constituents were not investigated in the current study, it seems better not to highlight these aspects. Instead, it is now mentioned that these features ought to be treated in future investigations (Lines 597–600).
In the M&M section: important information is missing here: climate, temperature/rainfall regime (can be given as a supplementary material), dimensions of each plot, days from seeding (lines 102-103), irrigation (total water amount, line 106). Please revise.
Authors: As suggested, meteorological data are now provided (Lines 140–142, and new Table S1).
Why haven’t the authors investigated combined treatments chitosan-TiO2 (for example, 25/25 and 50/50 mg L-1, respectively)? This should have been best fitted here, not in a future study.
Authors: Considering that 5 genotypes with a high genetic distance were selected, a total of 45 treatments were obtained for this experiment. Hence, it is difficult in terms of labor involved to further add experimental units.
Section 2.2. What is described here seems to be a laboratory scale recipe to prepare chitosan NP. What quantity of chitosan NP did the authors obtain as described? Was it enough for the plant treatments? Please revise for the actual/real amounts.
Authors: This is mentioned in the materials and methods (Lines 132–136).
Line 126: How was it purified?
Authors: This is now mentioned in the materials and methods (Lines 161 and 162).
Figure 1 is not commented at all. Please revise.
Authors: Done (Line 193–196).
Regarding Figure 3, and in relationship with comment #1 (my concern that the solutions of either nano-, or “bulk” particles are practically the same thing): it would be good to show the micrographs for “bulk” treatments, to highlight the difference (if there is a difference). Please revise.
Authors: This is now treated in the materials and methods (Lines 161, 162 and 193–196).
Please write all the Latin words (including scientific names of the species or words such as per, in situ, etc.) with Italics. Please revise throughout the ms.
Authors: Done (Line 4).
Line 57: Remove the second “also”.
Authors: Done (Line 77).
Please revise the references (line by line) and ensure consistency with the journal’s guidelines. In the current form, there are several typing errors (e.g. line 581 – journal’s name).
Authors: References were checked and adjusted to the appropriate format.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Article titled "Chitosan and titanium dioxide in the nanoparticle form are more effective in improving seed yield and quality: A case study in five milk thistle ecotypes (Silybum marianum (L.) Gaertn.)" is an appropriate work by the authors.
Nanoparticles have been used in agriculture to enhance plant productivity. Nanoparticles have advanced or unique features due to their small size and high surface-to-volume ratio. This paper focuses on the investigation of chitosan on various morphological, physicochemical, yield and quality parameters of Silybum marianum L.
The scientific structure and overall theme of the manuscript are solid and acceptable. The nanoparticle treatment in the experiment showed positive effects on the measured parameters characterizing plant growth, photosynthesis, and enzyme activity.
In my opinion, the overall concept is interesting and important. The paper is acceptably written and will be a suitable contribution that will be of interest to readers of Agronomy.
The manuscript needs major revisions to make it acceptable for publication, I have a few suggestions that I believe will improve the manuscript.
My suggestions regarding this MS are to accept after revision with the following points:
1. The authors have provided a very good introduction, but could explain more about the molecular mechanisms of plant tolerance to nanoparticles and potential mitigation from chitosan toxicity.
2. The authors could have included new aspects - hormonal regulation of nanoparticle tolerance (interactions with SA, CYT, etc.), including new references - in the introduction and discussion section.
3. Add more perspectives on photosynthetic parameters/discuss which types of parameters are more sensitive and why.
4. Add some recent references in MS. It is important to discuss the regulatory mechanisms of plants.
5. Improve the quality of the figure and simplify them.
6. Statistically significant differences need to be better highlighted - use SE and Tukey or another HSD test...
The paper brings many new aspects and the novelty of the paper is fine, but I would encourage the authors to discuss more ecophysiological aspects as well using new references: DOI: 10.1111/jfpp.16491; 10.1007/s11738-021-03264-8
FINAL COMMENTS
- The manuscript is useful and innovative and contains original data.
- The study presents relevant issues in greater depth than some other related publications, so I recommend its publication after MAJOR revisions.
Author Response
Khorramabad, 20 July 2022
Dear Editor,
Herewith we submit the revision of the manuscript entitled “Chitosan and titanium dioxide are more effective in improving seed yield and quality in nanoparticle compared to bulked form: A case study in five milk thistle ecotypes (Silybum marianum (L.) Gaertn.)” (ID: agronomy-1788175) by S Jafari, S Mousavi-Fard, A Rezaei Nejad, H Mumivand, K Sorkheh, N Nikoloudakis and D Fanourakis for publication in Agronomy.
We would like to thank you for the kind words and the valuable suggestions. Please find below a description on how we have dealt with the received comments. We hope that the manuscript can now be accepted for publication in your journal.
Yours sincerely,
Sadegh Mousavi-Fard
Reviewer #2
Given the completed score sheet and the comments above, after careful evaluation, the paper “Chitosan and Titanium Dioxide in the Nanoparticle Form Are More Effective in Improving Seed Yield and Quality: A Case Study in Five Milk Thistle Ecotypes (Silybum marianum (L.) Gaertn.)” (Ms. Ref. No. agronomy-1788175-v1) needs Major Revision according to comments before being considered for publication in Agronomy journal.
Article titled "Chitosan and titanium dioxide in the nanoparticle form are more effective in improving seed yield and quality: A case study in five milk thistle ecotypes (Silybum marianum (L.) Gaertn.)" is an appropriate work by the authors.
Nanoparticles have been used in agriculture to enhance plant productivity. Nanoparticles have advanced or unique features due to their small size and high surface-to-volume ratio. This paper focuses on the investigation of chitosan on various morphological, physicochemical, yield and quality parameters of Silybum marianum L.
The scientific structure and overall theme of the manuscript are solid and acceptable. The nanoparticle treatment in the experiment showed positive effects on the measured parameters characterizing plant growth, photosynthesis, and enzyme activity.
In my opinion, the overall concept is interesting and important. The paper is acceptably written and will be a suitable contribution that will be of interest to readers of Agronomy.
The manuscript needs major revisions to make it acceptable for publication, I have a few suggestions that I believe will improve the manuscript.
My suggestions regarding this MS are to accept after revision with the following points:
The authors have provided a very good introduction, but could explain more about the molecular mechanisms of plant tolerance to nanoparticles and potential mitigation from chitosan toxicity.
Authors: In our experiments, plants were cultivated under control (non-stress) conditions. The suitable concentration range was selected on the basis of both a comprehensive literature survey, and a pre-experiment (Lines 131–136). Symptoms of toxicity were not apparent in any of the 45 treatments. Additionally, molecular data were not obtained. Discussing toxicity tolerance mechanisms or molecular aspects falls out of the scope of the obtained data.
The authors could have included new aspects - hormonal regulation of nanoparticle tolerance (interactions with SA, CYT, etc.), including new references - in the introduction and discussion section.
Authors: The suitable concentration range was selected on the basis of both a comprehensive literature survey, and a pre-experiment (Lines 131–136). Symptoms of toxicity were not apparent in any of the 45 treatments. Additionally, hormone concentration data were not obtained. Discussing toxicity tolerance mechanisms or hormonal regulation falls out of the scope of the obtained data.
Add more perspectives on photosynthetic parameters/discuss which types of parameters are more sensitive and why.
Authors: Spray treatments promoted net photosynthesis. Our data indicate that this improvement is related to both increased (1) stomatal conductance, and (2) photosynthetic efficiency (Lines 613–615, 617 and618). The increased stomatal conductance was associated with enhanced leaf hydration status (Lines 622–625).
Add some recent references in MS. It is important to discuss the regulatory mechanisms of plants.
Authors: Done (Lines 73–76, 79–81, 135, 196, 633).
Improve the quality of the figure and simplify them.
Authors: During the manuscript file conversion to pdf, a figure quality loss is apparent. All figures will be separately provided to editorial office upon request.
Statistically significant differences need to be better highlighted - use SE and Tukey or another HSD test...
Authors: The statistics of the present investigation consider a three-way ANOVA (ecotype × treatment × year). By using Fisher’s LSD, we can lower our chances of a Type II error (false negative). In our case, this means that by using Fisher’s LSD we can lessen our chances of not finding a significant difference between two experimental units. Also, there is one other possible advantage of Fisher’s LSD. If the sample standard deviation for each group (of a pairwise comparison) is equal to it’s population standard deviation, then our pooled SD will be more accurate and we gain more degrees of freedom. We can acknowledge that Tukey and Duncan tests are more ‘famous’ but we feel that in our three-way experimental design, data fit best under the Fisher’s LSD test.
The paper brings many new aspects and the novelty of the paper is fine, but I would encourage the authors to discuss more ecophysiological aspects as well using new references: DOI: 10.1111/jfpp.16491; 10.1007/s11738-021-03264-8
Authors: The former reference deals with silybin production by using cell cultures. The latter reference was included as suggested (Line 633).
FINAL COMMENTS
- The manuscript is useful and innovative and contains original data.
- The study presents relevant issues in greater depth than some other related publications, so I recommend its publication after MAJOR revisions.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors of the revised ms. “Chitosan and titanium dioxide are more effective in improving seed yield and quality in nanoparticle compared to bulk form: A case study in five milk thistle ecotypes (Silybum marianum (L.) Gaertn.” (Ms. Ref. No. agronomy-1788175-v2) have addressed the reviewers’ comments. Proper changes have been made in the ms. according to suggestions and consequently, the ms. was improved compared to its initial submission. However, I do not agree with the rebuttals provided with respect to some of my comments from the first review round and the authors still need to work on their ms.
1. I understand that it is difficult from the experimental design viewpoint to assess the combined chitosan-TiO2 treatments, and I accept their rebuttal. However, I cannot accept the authors’ rebuttal concerning the suggested chemical analyses (seed fatty acid profile [FAP] and silymarin content), with the standard phrase that this will be treated in another study (see comment #2 from the first review round). Frankly, this is a very poor rebuttal. Moreover, the fact that the authors completely disregarded the other suggestions on papers dealing reporting these valuable products, choosing to avoid any mention of silymarin or discussion about the FAP only because they refuse to perform these analyses denotes superficiality and carelessness. Avoiding discussion about the main compounds of the plant only because you did not investigate them does not improve the quality of the paper, on the contrary, it is just like hiding it. I can understand there may be difficult for the authors to perform the indicated analyses and that the paper focuses on the agronomic yield components. However, it is worth mentioning (in the Introduction) and discussing in the (R&D section) both the silymarin and FAP, even if it is based on previously published results. For this, I have indicated very relevant papers (in the first review round), which will certainly improve the quality of the manuscript. Please revise.
2. The same comments apply for the rebuttal to my comment #9 from the first review round (again, the standard dismissing 0 value phrase in the manuscript and in the rebuttal that “these features ought to be treated in future investigations”). Refusing to discuss/mention (hiding) evident applications only because you did not address some aspects hoping that this would pass unnoticed is a very wrong approach, with negative impact on the quality of the manuscript. My suggestions were meant to strengthen the importance of the studied crop and point out the relevance of your study. Please revise.
3. I would suggest rephrasing as “non-structured form” instead of “bulk form”. In my opinion, it sounds better. If you accept my suggestion, please revise throughout the whole ms.
Therefore, after careful examination, my recommendation term = Major revision.
Author Response
Reviewer #1
The authors of the revised ms. “Chitosan and titanium dioxide are more effective in improving seed yield and quality in nanoparticle compared to bulk form: A case study in five milk thistle ecotypes (Silybum marianum (L.) Gaertn.” (Ms. Ref. No. agronomy-1788175-v2) have addressed the reviewers’ comments. Proper changes have been made in the ms. according to suggestions and consequently, the ms. was improved compared to its initial submission. However, I do not agree with the rebuttals provided with respect to some of my comments from the first review round and the authors still need to work on their ms.
I understand that it is difficult from the experimental design viewpoint to assess the combined chitosan-TiO2 treatments, and I accept their rebuttal. However, I cannot accept the authors’ rebuttal concerning the suggested chemical analyses (seed fatty acid profile [FAP] and silymarin content), with the standard phrase that this will be treated in another study (see comment #2 from the first review round). Frankly, this is a very poor rebuttal. Moreover, the fact that the authors completely disregarded the other suggestions on papers dealing reporting these valuable products, choosing to avoid any mention of silymarin or discussion about the FAP only because they refuse to perform these analyses denotes superficiality and carelessness. Avoiding discussion about the main compounds of the plant only because you did not investigate them does not improve the quality of the paper, on the contrary, it is just like hiding it. I can understand there may be difficult for the authors to perform the indicated analyses and that the paper focuses on the agronomic yield components. However, it is worth mentioning (in the Introduction) and discussing in the (R&D section) both the silymarin and FAP, even if it is based on previously published results. For this, I have indicated very relevant papers (in the first review round), which will certainly improve the quality of the manuscript. Please revise.
Authors: As indicated, the denoted information is now provided in both Introduction (Lines 60–62, 65–74) and Discussion (Lines 630–635, 674–686) sections. All the references, which were earlier suggested (round 1 of comments), were now included in the manuscript (highlighted in the reference list).
The same comments apply for the rebuttal to my comment #9 from the first review round (again, the standard dismissing 0 value phrase in the manuscript and in the rebuttal that “these features ought to be treated in future investigations”). Refusing to discuss/mention (hiding) evident applications only because you did not address some aspects hoping that this would pass unnoticed is a very wrong approach, with negative impact on the quality of the manuscript. My suggestions were meant to strengthen the importance of the studied crop and point out the relevance of your study. Please revise.
Authors: As suggested, this information is now added in the Introduction (Lines 60–62, 65–74). All the references, which were earlier suggested (round 1 of comments), were now included in the manuscript (highlighted in the reference list).
I would suggest rephrasing as “non-structured form” instead of “bulk form”. In my opinion, it sounds better. If you accept my suggestion, please revise throughout the whole ms.
Authors: As suggested, this is now clarified in the manuscript tilte (Line 5) as well as in several passages over the text (Lines 26, 107, 206, 589, 702).
Author Response File: Author Response.pdf
Reviewer 2 Report
The authors significantly improved the manuscript and addressed all comments. Still, the manuscript needs to amend a lot of typos, improve figs etc... For the citation potential of this paper, I again recommend adding more up-to-date papers to the discussion.
Author Response
Reviewer #2
Comments and Suggestions for Authors
The authors significantly improved the manuscript and addressed all comments. Still, the manuscript needs to amend a lot of typos, improve figs etc... For the citation potential of this paper, I again recommend adding more up-to-date papers to the discussion.
Authors: (1) As suggested, this information is now added in the Disscution along with recent references (Lines 630–635, 674–686); (2) All the figures will be provided in the editorial office in separate files to assure appropriate quality; (3) The manuscript was cross-checked, and typos were corrected throughout the text; and (4) All the references, which were earlier suggested (round 1 of comments), were now included in the manuscript (highlighted in the reference list).
Author Response File: Author Response.pdf
