Application of Biosynthesized Silver Nanoparticles from Oak Fruit Exudates against Pectobacterium carotovorum subsp. carotovorum Causing Postharvest Soft Rot Disease in Vegetables

Round 1

Reviewer 1 Report

Dear Authors,

The aim of this study is to synthetize silver nanoparticles using exudates form oak fruits against Pectobacterium carotovorum subsp. carotovorum, one of the most important causal agent of postharvest soft rot disease in vegetables.   The topic of this study is novel and attractive, but the style and the language should be improved, as well as used terminology. It is necessary to pay attention to consistency when using abbreviations. The formatting of the references should be redone in order to comply with the instructions for authors.

Language and grammar should be improved. Examples are included in the comments below.

Comments:

L57 - please change "Acacia cyanophylla" to "Acacia cyanophylla"

L58 - please change "to prepare stable SNPs, and SNPs had a strong" to "to prepare stable SNPs which had a strong"

L61 - please change "illnesses" to "diseases"

L63 - please change "Pectobacterium species" to "Pectobacterium species"

L68 - please change "deterioration [23,24] ." to "deterioration [23,24]."

L74 - please change "illnesses" to "diseases"

L82 - please change "antibacterial material" to "antibacterial agent"

L90-91 - please change "Fresh veggies from a vegetable 91 market in Kerman, Iran" to "Fresh vegetables from a market in Kerman, Iran"

L92 - please change "healthy veggies " to "healthy vegetables"

L98 - please change "little" to "small"

L149-150 - please change "cork oak" to "oak cork"

L155 - please change "Akhlaghi et al, [33]" to "Akhlaghi et al [33]"

L190 - please change "Pcc suspensions" to "Pcc suspensions"

L192 - in the title "Investigation on the curative 182 activity of SNPs against Pcc in vivo" what means 182?

L194 - please change "Hajian-Maleki et al, [34]" to "Hajian-Maleki et al [34]"

L227 - please change "Fig 3" to "Fig. 3"

L241 - please change "38°, 44°, 64° and 77°, respectively" to "38, 44, 64 and 77°, respectively"

L290 - please change "on" to "on"

L291 - please change "The in vitro growth index of Pcc" to "The in vitro growth index of Pcc"

L293 - please explain this "The growth of the animals". Which animals?

L307 - Fig. 10 please change "treatments. (vegetables" to "treatments (vegetables"

L321 - please change "soft rot illness in vivo" to "soft rot disease in vivo"

L340 - please change "caabilities" to "capabilities"

L340 - please change "were shown in vitro and in vivo" to "were shown in vitro and in vivo"

L346 - please change "growth.)." to "growth)."

Author Response

 Response to the comments, Reviewer 1

We have revised the manuscript according to reviewer comments and replied to the comments as following. All the changes addressed in the text as well.

• Language and grammar should be improved. Examples are included in the comments below.
• English quality improved throughout the entire text and introduction changed accordingly.

-           All Comments performed accordingly.

Comments:

L192 - in the title "Investigation on the curative 182 activity of SNPs against Pcc in vivo" what means 182?

Corrected accordingly.

L293 - please explain this "The growth of the animals". Which animals?

• Animals corrected to bacteria

                We hope that after considering all valuable comments provided by reviewers, the manuscript can be accepted for publication, although we are certainly willing to consider further changes if necessary.

Best regards,

Meysam Soltani Nejad

Reviewer 2 Report

Authors should check the 'Instruction for Authors' on the website of the Agronomy Journal and revise the use of SI units, Figures, and Tables in the text. For example, sometimes authors used ml, CFU/mL, sometimes used mL, CFU mL-1, ... Or sometimes used Fig.1 and sometimes used Figure,...

Where are originally the vegetables of the Iranian vegetable market in Kerman from? I think the authors should add this information in the material and method section.

Minor editing of English language required

Author Response

Response to the comments 

We have revised the manuscript according to reviewer comments and replied to the comments as following. All the changes addressed in the text as well.

Reviewer 2

-Authors should check the 'Instruction for Authors' on the website of the Agronomy Journal and revise the use of SI units, Figures, and Tables in the text. For example, sometimes authors used ml, CFU/mL, sometimes used mL, CFU mL-1, ... Or sometimes used Fig.1 and sometimes used Figure,...

- Corrected at full manuscript accordingly.

-Where are originally the vegetables of the Iranian vegetable market in Kerman from? I think the authors should add this information in the material and method section.

• Added accordingly

We hope that after considering all valuable comments provided by reviewers, the manuscript can be accepted for publication, although we are certainly willing to consider further changes if necessary.

Best regards

Meysam Soltani Nejad

Reviewer 3 Report

Manuscript ID: agronomy-2422650

Type of manuscript: Article

Title: Application of biosynthesized silver nanoparticles by oak fruits exudates against Pectobacterium carotovorum subsp. carotovorum causing postharvest soft rot disease in vegetables 

The paper presents the utilization of silver nanoparticles synthesized from AgNO₃ with the use of oak (Quercus brantii L) fruit exudates to reduce the pathogenic activity of bacterial soft rot in vegetables. It should be emphasized that the antibacterial properties of silver nanoparticles are known and used in many fields, so this side of the presented study is not very innovative. However, a certain novelty of the submitted work expanding knowledge in the discussed field is the use of oak fruit extract for the preparation of nanoparticles. In general, the topic discussed in the article is interesting, however, taking into account that nanoparticles may have the negative effects on the health of consumers, the possibility of its application raises some doubts. Therefore, it is advisable that the authors discuss the health context of consumers after the application of nanoparticles to vegetables during their post-harvest preservation. The manuscript is quite well organized, but some points particularly those associated with introduction, methodology and discussion need to be completed, explained or improved. Below, there are some comments and suggestions:

Introduction

Lines 46-47: The authors are writing that “Furthermore, nanobiotechnology has applications in all fields of food science, ...” - the use of nanoparticles arouse great interest in the food industry, nevertheless as due to the high reactivity of these compounds that can affect the health of humans and other organisms, they application in food processing is still limited.

Line 45:  The sentence: “…use of nanomaterials in research to produce novel insecticides and plant diseases [7]…” – shouldn’t it be e.g. “control plant diseases” instead “plant diseases”?

Lines 53-54: Shouldn’t it be „Many plants…” instead of „Many plans…”

Lines 57, 63: “Acacia cyanophylla”, ”Pectobacterium” - latin names of species should be written in italics.

Line 77-79: It is advisable to continue the thought from sentence: “We employed production of SNPs from oak (Quercus brantii L) fruit exudates in this work, which has been used in traditional Iranian medicine.” in the following ones, explaining to readers why the authors used oak extract (by briefly describing its properties).

Materials and Methods

Line 139: In the methodology, it would be better to first use the full name of the term "The zeta potential", and then introduce the abbreviation, e.g. the average value of the zeta potential (Z-average).

Line 144: The symbol of medium LB should be explained in this sentence, not

 under the Fig. 9. 

Lines 159, 162: Shouldn’t it be “ten microliters” instead of “ten liters”

Line 181-182: Were bacterial cultures really "centrifuged overnight"?

Line 187: The symbol “(DW)” would be better moved after the words "and weighted", as it describe “the decaying tissue weight”.

Line 192: There is a redundant number "182" in the title of subsection 2.10.

Line 227: It would be better to expand the "TEM" symbol in this sentence instead of under Fig. 3.

Figure 5, 10, 11 and Table 1:  The captions of the figures 5, 10-11 and table 1 should be changed, as they have the form of the text of the manuscript.

Figure 6:  Axis descriptions should be larger as they are illegible. Moreover, in the caption, the phrases “…PDI is 0.203 and other distribution at lower and up range from nanoparticle size shows that the synthesized particles are in lower and bigger range from particle size and the uniform distribution of silver nanoparticles...” and “…measurement of the silver nanoparticles (SNPs) suspension: the Mobility Mean 0.86 μm/s/V/cm; Standard Deviation: 0.57 ;Zeta Mean: ;-11.40 mV; Standard Deviation: 7.59“ seems redundant or need to be rewritten. 

Line 293: It should be “The growth of the bacterias…” instead of “The growth of the animals…”. In addition, to compare the growth of microorganisms, it is better to use predictive models, e.g. the Gompertz model, which allow estimating the parameters of growth kinetics, such as maximum growth rate or lag phase (stagnation phase), which can then be used to quantitatively compare the growth of microorganisms. The authors can find examples of the use of such model in papers https://doi.org/10.1007/s11947-020-02575-x; https://doi.org/10.1002/aocs.12365. 

Figure 11: There is not reference to Fig. 11 in the text of the manuscript.

Lines 339-340: It should be “antibacterial capabilities” instead of “antibacterial caabilities.”

Discussion

In the discussion, the authors should conduct a discourse on applicability of their results in the light of the fact that nanomaterials have direct or indirect ways of accumulating in biological bodies and used as food additives or as an antimicrobial agent have significant negative impacts on human health at an alarming rate (Sahani and Sharma, 2021, https://doi.org/10.1016/j.foodchem.2020.128318).

Language corrections are included in the review.

Author Response

Response to the comments, Reviewer 3

We have revised the manuscript according to reviewer comments and replied to the comments as following. All the changes addressed in the text as well.

Introduction

Lines 46-47: The authors are writing that “Furthermore, nanobiotechnology has applications in all fields of food science, ...” - the use of nanoparticles arouse great interest in the food industry, nevertheless as due to the high reactivity of these compounds that can affect the health of humans and other organisms, they application in food processing is still limited.

Dear reviewer, we agree with you and mentioned at lines 51-55 (introduction) that “ … food processing and due to their effects on the environment and human health, they should be consumed with caution. [11, 12]” and lines 443-445 and 453-455 (discussion).

Line 45:  The sentence: “…use of nanomaterials in research to produce novel insecticides and plant diseases [7]…” – shouldn’t it be e.g. “control plant diseases” instead “plant diseases”?

Corrected accordingly.

Lines 53-54: Shouldn’t it be „Many plants…” instead of „Many plans…”

Corrected accordingly.

Lines 57, 63: “Acacia cyanophylla”, ”Pectobacterium” - latin names of species should be written in italics.

Changed accordingly.

Line 77-79: It is advisable to continue the thought from sentence: “We employed production of SNPs from oak (Quercus brantii L) fruit exudates in this work, which has been used in traditional Iranian medicine.” in the following ones, explaining to readers why the authors used oak extract (by briefly describing its properties).

Mentioned at introduction, lines 88-90, “…which has been used in traditional Iranian medicine and this plant is traditionally applied for healthy problems,such as gastropathy, acute diarrhea inflammation, burns/cuts, and cancers”.

Materials and Methods

-Line 139: In the methodology, it would be better to first use the full name of the term "The zeta potential", and then introduce the abbreviation, e.g. the average value of the zeta potential (Z-average).

Changed accordingly.

-Line 144: The symbol of medium LB should be explained in this sentence, not

 under the Fig. 9.

Explained accordingly.

-Lines 159, 162: Shouldn’t it be “ten microliters” instead of “ten liters”

-Corrected accordingly.

-Line 181-182: Were bacterial cultures really "centrifuged overnight"?.

- Corrected accordingly.

-Line 187: The symbol “(DW)” would be better moved after the words "and weighted", as it describe “the decaying tissue weight”.

-Changed accordingly

Line 192: There is a redundant number "182" in the title of subsection 2.10.

-Removed accordingly.

Line 227: It would be better to expand the "TEM" symbol in this sentence instead of under Fig. 3.

-Corrected accordingly.

Figure 5, 10, 11 and Table 1:  The captions of the figures 5, 10-11 and table 1 should be changed, as they have the form of the text of the manuscript.

-Changed accordingly.

Figure 6:  Axis descriptions should be larger as they are illegible. Moreover, in the caption, the phrases “…PDI is 0.203 and other distribution at lower and up range from nanoparticle size shows that the synthesized particles are in lower and bigger range from particle size and the uniform distribution of silver nanoparticles...” and “…measurement of the silver nanoparticles (SNPs) suspension: the Mobility Mean 0.86 μm/s/V/cm; Standard Deviation: 0.57 ;Zeta Mean: ;-11.40 mV; Standard Deviation: 7.59“ seems redundant or need to be rewritten.

Changed accordingly.

-Line 293: It should be “The growth of the bacterias…” instead of “The growth of the animals…”. In addition, to compare the growth of microorganisms, it is better to use predictive models, e.g. the Gompertz model, which allow estimating the parameters of growth kinetics, such as maximum growth rate or lag phase (stagnation phase), which can then be used to quantitatively compare the growth of microorganisms. The authors can find examples of the use of such model in papers https://doi.org/10.1007/s11947-020-02575-x; https://doi.org/10.1002/aocs.12365. 

Dear reviewer, we used hajian malaki et al, methods. https://doi.org/10.1016/j.postharvbio.2019.06.002

Figure 11: There is not reference to Fig. 11 in the text of the manuscript.

Changed accordingly.

-Lines 339-340: It should be “antibacterial capabilities” instead of “antibacterial caabilities.”

Corrected accordingly.

Discussion

In the discussion, the authors should conduct a discourse on applicability of their results in the light of the fact that nanomaterials have direct or indirect ways of accumulating in biological bodies and used as food additives or as an antimicrobial agent have significant negative impacts on human health at an alarming rate (Sahani and Sharma, 2021, https://doi.org/10.1016/j.foodchem.2020.128318).

-Added acordingly

-Comments on the Quality of English Language

-Language corrections are included in the review.

English quality improved throughout the entire text and introduction changed accordingly.

            We hope that after considering all valuable comments provided by reviewers, the manuscript can be accepted for publication, although we are certainly willing to consider further changes if necessary.

Best regards,

Meysam Soltani Nejad

Reviewer 4 Report

This study is aimed to investigate the application of biosynthesized silver nanoparticles by oak fruits exudates against Pectobacterium carotovorum subsp. carotovorum causing postharvest soft rot disease in vegetables. The design and the structure of the study are partially acceptable. The topic of the study is valuable but the study is negligently written. 

Examples for suggestions:

L77-84: Give a more exact objectives.

L79-81:  UV–vis spectrophotometer, transmission electron microscope (TEM), atomic force microscopy (AFM), and X-ray diffraction spectroscopy were used to analyze the synthesized SNPs (XRD). - This section is M and M and not Introduction.

L220: Figure 1 present no results. This figures should be deleted or moved to M and M.

L308: Figures 10 and 11 the same topic. Should be merged into one figure. 

L315: Fig. 11. An in vitro vegetable test of silver nanoparticles ...

Formating of References are inconsistent.

Writing style and English grammar should be improved.

Author Response

Response to the comments 

We have revised the manuscript according to reviewer comments and replied to the comments as following. All the changes addressed in the text as well.

Reviewer 4

Examples for suggestions:

L77-84: Give a more exact objectives.

L79-81:  UV–vis spectrophotometer, transmission electron microscope (TEM), atomic force microscopy (AFM), and X-ray diffraction spectroscopy were used to analyze the synthesized SNPs (XRD). - This section is M and M and not Introduction.

L220: Figure 1 present no results. This figures should be deleted or moved to M and M.

L308: Figures 10 and 11 the same topic. Should be merged into one figure. 

L315: Fig. 11. An in vitro vegetable test of silver nanoparticles ...

Formating of References are inconsistent.

• Reviewer suggestions performed accordingly

We hope that after considering all valuable comments provided by reviewers, the manuscript can be accepted for publication, although we are certainly willing to consider further changes if necessary.

Best regards

Meysam Soltani Nejad

Reviewer 5 Report

The manuscript presents an interesting topic, especially in the agriculture domain, but there are some uncertainties.

-          In the Abstract there specified different vegetable types such as potato, zucchini, carrot, and eggplant, but in Introduction, the accent is put on potatoes and the effects of Pectobacterium carotovorum on potatoes. I suggest rephrasing and write also the disease effects on other crops used in this study. Also, create a bond between the first and second paragraphs in the introduction.

-          In 2.5 paragraph:  laser intensity was 50 % ? (I = W/m^2 (Power/Aria)). I suppose that 50% represents the quantum efficiency; the wavelength was 657.00 ? What unit?

-          Write the equation in other lines.

-          Rephrase the sentences “The electro micrograph in the colloidal SNPs is spherical, hexagonal, and amorphous, 226 as shown in Fig 3 of a TEM electro micrograph of synthesized SNPs by oak fruit exudates. 227 Figures 3a, B show the TEM images that were taken.”

-          How were obtain the MIC and MBC? In section 3.7, specify the method that was used and how you arrived at those values on MIC and MBC.

-          The Discussions section is short, the results presented in the Results section are not discussed, the figures are not discussed.

-          The conclusions are too general.

Author Response

Response to the comments 

We have revised the manuscript according to reviewer comments and replied to the comments as following. All the changes addressed in the text as well.

Reviewer comments:

In the Abstract there specified different vegetable types such as potato, zucchini, carrot, and eggplant, but in Introduction, the accent is put on potatoes and the effects of Pectobacterium carotovorum on potatoes. I suggest rephrasing and write also the disease effects on other crops used in this study. Also, create a bond between the first and second paragraphs in the introduction.

Added accordingly.

-          In 2.5 paragraph:  laser intensity was 50 % ? (I = W/m^2 (Power/Aria)). I suppose that 50% represents the quantum efficiency; the wavelength was 657.00 ? What unit?

Corrected accordingly. “…Temperature was 25 °C, laser power was 50 %, DTC position was up, wavelength was 657.00 nm, and all measurements were done in triplicates.

-          Write the equation in other lines.

Added accordingly.

-          Rephrase the sentences “The electro micrograph in the colloidal SNPs is spherical, hexagonal, and amorphous, 226 as shown in Fig 3 of a TEM electro micrograph of synthesized SNPs by oak fruit exudates. 227 Figures 3a, B show the TEM images that were taken.”

-Changed accordingly.

-          How were obtain the MIC and MBC? In section 3.7, specify the method that was used and how you arrived at those values on MIC and MBC.

Dear reviewer, We applied “Akhlaghi et al” methods, previously mentioned in materials and methods.

“The Pcc was cultivated in LB medium overnight at 28 ± 1 °C to assess the MIC and MBC using the micro-dilution technique described by  Akhlaghi et al [33]. To achieve 500 μg /mL of SNPs in the first well of each row in 96-well microplates, 100 μ liters of SNPs were put to 100 μ liters of Mueller Hinton broth medium. SNPs were then serially 2X diluted in 96-well microplates with concentrations ranging from 500 to 0. 48 μg /mL in a total volume of 200 μL. After that, each well was infected with ten microliters of overnight bacterial culture (100 CFU/mL). At 28±1 °C, the plate was incubated for one day. MIC was calculated as the lowest concentration of SNPs at which there was no discernible growth (increase in turbidity). To measure MBC, ten microliters of each bacterial culture from the wells with greater concentrations than the MIC were cultivated on the NA for 24 hours at 28 °C. On the NA medium, MBC was defined as the lowest concentration with no bacterial growth. All of the tests were repeated three times.”

-          The Discussions section is short, the results presented in the Results section are not discussed, the figures are not discussed.

Added accordingly.

-          The conclusions are too general.

Changed accordingly. 

We hope that after considering all valuable comments provided by reviewers, the manuscript can be accepted for publication, although we are certainly willing to consider further changes if necessary.

Best regards

Meysam Soltani Nejad

Round 2

Reviewer 2 Report

Thank you for submitting the revised version. I have carefully reviewed your response to the reviewers' comments and the changes made in the revised manuscript better with the current format. But still, there are several issues that need to be discussed or addressed.

1. The introduction is well-written and provides a good background on the topic. However, it would be beneficial to include more recent studies to strengthen the relevance of the article. Especially, authors should provide more background information on the SNPs green synthesis.

2. The methodology section is thorough and well-explained. However, it would be helpful to provide more details on the methods. The authors should state where are originally the vegetables used in the study of the vegetable market from.

3. The results are presented clearly and concisely. However, the authors should provide more explanation for some of the findings, particularly those that are unexpected or contradict previous research.

4. In ‘3.7. MIC and MBC studies’ section, but the authors did not provide any data (table or figure) to support this result (I did not find any data in the manuscript!).

5. In this study, SNPs effectively decreased the postharvest soft rot disease in vegetables. The expected final result is an extension of the shelf life and an improvement in the quality of the vegetables. So, I think that the authors should provide data on the shelf life and quality of vegetables and the method of the evaluation of the vegetable shelf life in the result section as well as in the methodology section.

6. Figures and Tables:

 Fig. 10b: replace “rotting tissue (%)’ in Y-axis with “Rotting tissue incidence (%)”. Capitalizing the ‘potato’ on X-axis.

Fig. 10a: Please provide a scale bar for the figure.

The results in 3.8 and 3.9 sections, Fig. 9 and Table 1: Please provide the result of the T-test statistical analyses for the results between control and SNPs treatment. Without T-test statistical analyses, we cannot compared the results between Control and SNPs.

7. The discussion provides a good interpretation of the results and their implications. However, the authors should also compare the effectiveness of SNPs with Streptomycin.

Author Response

Dear Reviewer,

We have revised the manuscript according to reviewer comments and replied to the comments as following. All the changes addressed in the text as well.

Response to the comments:

1. The introduction is well-written and provides a good background on the topic. However, it would be beneficial to include more recent studies to strengthen the relevance of the article. Especially, authors should provide more background information on the SNPs green synthesis.

Added accordingly at introduction section.

2. The methodology section is thorough and well-explained. However, it would be helpful to provide more details on the methods. The authors should state where are originally the vegetables used in the study of the vegetable market from.

Added at materials and methods” …Fresh vegetables prepare from Baft city vegetable farms, Kerman Province, Iran.”

3. The results are presented clearly and concisely. However, the authors should provide more explanation for some of the findings, particularly those that are unexpected or contradict previous research.

Dear reviewer, We mentioned at conclusion section that “…based on our literature review there was not such record mediating fruit exudates of cork oak (Quercus brantii L.) in order to biosynthesize silver nanoparticles and applying against Pcc as a postharvest plant pathogen.”

4. In ‘3.7. MIC and MBC studies’ section, but the authors did not provide any data (table or figure) to support this result (I did not find any data in the manuscript!).

Table 1 Added accordingly in Results section.

5. In this study, SNPs effectively decreased the postharvest soft rot disease in vegetables. The expected final result is an extension of the shelf life and an improvement in the quality of the vegetables. So, I think that the authors should provide data on the shelf life and quality of vegetables and the method of the evaluation of the vegetable shelf life in the result section as well as in the methodology section.

Dear reviewer, Our next investigation is affects SNPs on the shelf life and quality of vegetables and know we have not any data about it.

6. Figures and Tables:

 Fig. 10b: replace “rotting tissue (%)’ in Y-axis with “Rotting tissue incidence (%)”. Capitalizing the ‘potato’ on X-axis.

Changed accordingly.

Fig. 10a: Please provide a scale bar for the figure.

The scale bar added.

The results in 3.8 and 3.9 sections, Fig. 9 and Table 1: Please provide the result of the T-test statistical analyses for the results between control and SNPs treatment. Without T-test statistical analyses, we cannot compared the results between Control and SNPs.

In table 2 added accordingly.

7. The discussion provides a good interpretation of the results and their implications. However, the authors should also compare the effectiveness of SNPs with Streptomycin.

This sentence “According to MIC and MBC test, the SNPs antibacterial activities against Pcc are less than streptomycin.” added. 

We hope that after considering all valuable comments provided by reviewers, the manuscript can be accepted for publication, although we are certainly willing to consider further changes if necessary.

Best regards

Meysam Soltani Nejad

Reviewer 5 Report

The authors made the changes according to the observations, the only inconsistency remains in paragraph 2.5. Physicochemical properties of SNPs (TEM, AFM, XRD, DLS and FTIR):

-          Line 171 - “(I = W/m^2 (Power/Aria)” this was an example of the laser intensity formula, not something that can be introduced into the main text. I am interested in passing the value of the laser intensity in the text, which is not a percentage.

The laser power measurement unit is “W”, not in percent, perhaps the laser power transmission is 50% - line 170.

Author Response

Dear Reviewer

Thanks for providing your valuable comments. We have revised the manuscript and replied to the comments as following. All the changes addressed in the text as well.

Comments and Suggestions for Authors

The authors made the changes according to the observations, the only inconsistency remains in paragraph 2.5. Physicochemical properties of SNPs (TEM, AFM, XRD, DLS and FTIR):

-          Line 171 - “(I = W/m^2 (Power/Aria)” this was an example of the laser intensity formula, not something that can be introduced into the main text. I am interested in passing the value of the laser intensity in the text, which is not a percentage.

 -The formula removed from manuscript text.

The laser power measurement unit is “W”, not in percent, perhaps the laser power transmission is 50% - line 170.

-Dear reviewer, added and corrected to “the laser power transmission”.

               We hope that after considering all valuable comments provided by reviewers, the manuscript can be accepted for publication.

 Best regards

Meysam Soltani Nejad

Round 3

Reviewer 2 Report

Thank you for submitting the revised version. I have carefully reviewed your response to the reviewers' comments and the changes made in the revised manuscript better with the current format. But still, there are no statistical analyses in Fig. 9. So please add the result of the statistical analyses between control and SNPs. It will strongly support your obtained result.

Author Response

Dear Reviewer

Thanks for providing your valuable comments. We have revised the manuscript and replied to the comments as following. All the changes addressed in the text as well.

Response to the comments:

Thank you for submitting the revised version. I have carefully reviewed your response to the reviewers' comments and the changes made in the revised manuscript better with the current format. But still, there are no statistical analyses in Fig. 9. So please add the result of the statistical analyses between control and SNPs. It will strongly support your obtained result.

Dear reviewer, added accordingly.

We hope that after considering all valuable comments provided by reviewers, the manuscript can be accepted for publication.

 Best regards

Meysam Soltani Nejad