European Grapevine Cultivars and Rootstocks Show Differential Resistance to Xylella fastidiosa Subsp. fastidiosa

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors conducted multi-year and multi-location experiments to assess grapevine cultivar and rootstock resistance to Xff, the causal agent of Pierce’s disease. The manuscript provides useful information, but requires satisfactory revisions to address three main shortcomings:

1) Important experimental details are missing from Materials & Methods. Specific examples are given below.

 

2) I disagree with the way the statistical analysis was conducted. In most cases, there were two factors in the experiment (e.g., cultivar/rootstock x isolate in Fig. 2, or cultivar x rootstock in Fig. 3), hence the data should be analyzed by two-way ANOVA. A two-way ANOVA has the major advantage of being able to test for interactions, e.g., between cultivar and isolate. For example, the following statement in the Discussion can be supported only when there are no significant cultivar x isolate interactions: “Thus, strain virulence does not appear to modify the susceptibility profiles.”

3) Given the largely inconclusive results from the water potential tests, the conclusions in the Discussion section about the potential usefulness of non-destructive water potential measurement should be removed.

 

Additional comments and suggestions:

 

The Abstract needs to describe better what was done. For example, the fact that the grape cultivars were assessed in different environments, including a climate-controlled greenhouse and open field conditions, needs to be mentioned. In addition, one of the important findings was that the results of resistance evaluations may differ between the field and the greenhouse; this should be mentioned in the Abstract.

 

Important experimental details are missing in Materials & Methods. In Section 2.2, start with a description of where the trials were done. Mention how many plants per cultivar (replicates) were evaluated each year. Were new potted plants started each year, or did you continue monitoring the same plants over multiple years? How were plants maintained, e.g., irrigation, fertilization, or pest control?

 

Section 2.3: when and how often was the disease severity index assessed?

 

Section 2.4: when and how often were plants sampled for Xf determination? Were all plants tested or only those showing symptoms? How many leaves per plant were sampled?

 

In Section 2.5: when and how often was water potential measured?

 

In Section 2.6, I do not understand why data were not analyzed by two-way ANOVA, as opposed to a one-way ANOVA separately for each Xff isolate. A two-way ANOVA would have the major advantage of being able to test for interactions between cultivars/rootstocks and isolates.

 

In line 236, what is “280 u²”? Shouldn’t the unit for AUDPC be “severity index-days”? Why use q instead of r for the correlation coefficient?

 

Lines 260-261: Materials & Methods should mention the cultivar x rootstock combinations evaluated, as well as the analysis conducted to evaluate rootstock combination effects. For each cultivar, this needs to be analyzed as a two-way ANOVA (rootstock x isolate).

 

Figure 1: This figure needs vertical axis titles.

 

Figure 2: Does this figure show only cultivars (as indicated in the legend) or cultivars and rootstocks (as indicated in the text)? This was only for 2019, correct? If so, that needs to be mentioned in the legend. I do not think Fig. 2(b) and the regression equation are needed; stating that there is a high and significant positive correlation between the two isolates is sufficient. It would be more important to test for significant cultivar x isolate interactions in the ANOVA. In this figure legend, and all others, acronyms need to be defined, such as Xff, WPI, rAUDPC, etc.

 

Table 1: I do not understand the numbers in this table. I assume 1/5 means that only a maximum score of 1 on the 5-point scale was attained. But why are some entries 1/3 or 2/2 for example? Shouldn’t the denominator be 5 in all cases since that was the maximum disease score?

 

Throughout the text: Cultivar and Rootstock should not be capitalized and italicized.

 

Additional editorial corrections are included in the attachment.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

A number of language corrections are included in the attached document.

Author Response

For research article:

Title: European Grapevine Cultivars and Rootstock Show Differential

Resistance to Xylella fastidiosa Subsp. fastidiosa

Authors: Sara Martínez, Maite Lacuesta, Juan Bautista Relloso, Ana

Aragonés, Ana Herran, Amaya Ortiz-Barredo *

Manuscript ID: horticulturae-2679554, Received: 9 Oct 2023

Submitted to section: Viticulture (Genetic Resources for Viticulture)

 

Response to Reviewer-1 X Comments
1. Summary
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections changes in the re-submitted files.
2. Questions for General Evaluation	Reviewer’s Evaluation	Author´s Response and Revisions
·        Does the introduction provide sufficient background and include all relevant references?	Yes
·        Are all the cited references relevant to the research?	Yes
·        Is the research design appropriate?	Must be improved	We agree with your right suggestion, please, kindly see our corresponding responses in the point-by-point responses bellow
·        Are the methods adequately described?	Must be improved
·        Are the results clearly presented?	Can be improved
·        Are the conclusions supported by the results?	Can be improved

 

 

3. Point-by-point response to Comments and Suggestions for Authors

Comment 1: The authors conducted multi-year and multi-location experiments to assess grapevine cultivar and rootstock resistance to Xff, the causal agent of Pierce’s disease. The manuscript provides useful information, but requires satisfactory revisions to address three main shortcomings: 1) Important experimental details are missing from Materials & Methods. Specific examples are given below. Response: Thank you for pointing this out. We agree with this comment. Therefore, several details have been added in the Materials & Methods section for the new version of the manuscript. In addition, a general overview of the trials has been included in Materials & Methods section. 2) I disagree with the way the statistical analysis was conducted. In most cases, there were two factors in the experiment (e.g., cultivar/rootstock x isolate in Fig. 2, or cultivar x rootstock in Fig. 3), hence the data should be analyzed by two-way ANOVA. A two-way ANOVA has the major advantage of being able to test for interactions, e.g., between cultivar and isolate. For example, the following statement in the Discussion can be supported only when there are no significant cultivar x isolate interactions: “Thus, strain virulence does not appear to modify the susceptibility profiles.” Response: We agree with your right suggestion and the new manuscript has been modified. In the new version, the ANOVA analyses are presented in detail (2.6 Statistical Analyses section) as well as the statistical parameters that justify the significant interactions (sections 3.1 and 3.2.). The statistical results obtained in this way are conveniently discussed in the discussion section. 3) Given the largely inconclusive results from the water potential tests, the conclusions in the Discussion section about the potential usefulness of non-destructive water potential measurement should be removed. Response: We agree with your opinion. The results from the water potential tests are not conclusive enough to consider the potential usefulness of non-destructive water potential measurement as an early detection technology for Xf infection as it was already included in the discussion (line 503). In addition, the results obtained show differences among cultivars, but it was not possible to establish a significant relationship between infection and potential water in all cultivars. Bearing in mind these results, the authors agree with your opinion. This alternative technology would be discarded from the discussion as an alternative diagnostic measure unless further research is carried out to discriminate its potential use for different cultivars, evaluation times and agroclimatic conditions. It has been included in the Discussion section. Comment 2: Additional comments and suggestions: The Abstract needs to describe better what was done. For example, the fact that the grape cultivars were assessed in different environments, including a climate-controlled greenhouse and open field conditions, needs to be mentioned. In addition, one of the important findings was that the results of resistance evaluations may differ between the field and the greenhouse; this should be mentioned in the Abstract. Response: We agree and have modified the abstract as you have properly suggested. Important experimental details are missing in Materials & Methods. In Section 2.2, start with a description of where the trials were done. Mention how many plants per cultivar (replicates) were evaluated each year. Were new potted plants started each year, or did you continue monitoring the same plants over multiple years? How were plants maintained, e.g., irrigation, fertilization, or pest control? Response: We agree with your comment, especially with regards to monitoring the plants over one or multiple years as this is an important factor in discussing the results obtained. We have modified the section 2.2. to clarify all your properly suggested issues. Section 2.3: when and how often was the disease severity index assessed? Response: We agree and have modified section 2.3. to clarify all your suggested issues. Section 2.4: when and how often were plants sampled for Xf determination? Were all plants tested or only those showing symptoms? How many leaves per plant were sampled? Response: We agree and have modified section 2.4. to clarify all your suggested issues. In Section 2.5: when and how often was water potential measured? Response: We agree and have modified section 2.5. to clarify all your suggested issues. In Section 2.6, I do not understand why data were not analyzed by two-way ANOVA, as opposed to a one-way ANOVA separately for each Xff isolate. A two-way ANOVA would have the major advantage of being able to test for interactions between cultivars/rootstocks and isolates. Response: We have modified section 2.6. to describe the programs, ANOVA analysis and the transformation of the rAUDPC data prior to assess the normality of the data and the homogeneity of variance by Shapiro–Wilk´s and Levene´s tests. In line 236, what is “280 u2”? Shouldn’t the unit for AUDPC be “severity index-days”? Why use q instead of r for the correlation coefficient? Response: Line 236. The AUDPC units, as indicators of resistance or susceptibility, are not easily interpretable. In an effort to standardize the AUDPC, same researchers often use the relative AUDPC (rAUDPC) (Jeder et al., 2001*). The rAUDPC is calculated by dividing the AUDPC by the “maximum potential AUDPC”. The maximum potential AUDPC is simply the AUDPC a cutivar would have if it had had 100% infection all days during the reading period. Considering that the period in which AUDPC was evaluated in our experiments was 56 days (from 8 to 16 WPI) and the maximum Severity Index (SI) value in the reference scale is 5, the maximum value of AUDPC would be 280 square units. *Additional Reference: Jeger, M. J., and Viljanen-Rollinson, S. L. H. 2001. The use of the area under the disease progress curve (AUDPC) to assess quantitative disease resistance in crop cultivars. Theoretical and Applied Genetics 102:32–36     Use of q instead of r for the correlation coefficient. Response: We totally agree. We calculated the Pearson coefficient of a sample: r. It has been modified in new version of the manuscript. Lines 260-261: Materials & Methods should mention the cultivar x rootstock combinations evaluated, as well as the analysis conducted to evaluate rootstock combination effects. For each cultivar, this needs to be analyzed as a two-way ANOVA (rootstock x isolate). Response: We agree. In fact, the analysis carried out to evaluate rootstock combination effects have been conducted for each variety (Tempranillo and Garnacha Tintorera).  It has also been modified in the new version of the manuscript. Figure 1: This figure needs vertical axis titles. Response: We agree. The figure has been modified for the vertical axis and series (“Av. Relative Humidity” instead of “Relative Humidity”) Figure 2: Does this figure show only cultivars (as indicated in the legend) or cultivars and rootstocks (as indicated in the text)? This was only for 2019, correct? If so, that needs to be mentioned in the legend. I do not think Fig. 2(b) and the regression equation are needed; stating that there is a high and significant positive correlation between the two isolates is sufficient. It would be more important to test for significant cultivar x isolate interactions in the ANOVA. In this figure legend, and all others, acronyms need to be defined, such as Xff, WPI, rAUDPC, etc. Response: We agree. The new manuscript specifies and presents statistical analyses showing that there is no significant interaction between Cultivars (according to the terminology indicated in the text) and strains. Thus, the correlation between both strains could be established. The descriptive parameters of this correlation are detailed in section 3.1. and we have considered removing the graph in figure 2 as it does not provide additional information to the text. The year and acronyms have been mentioned and defined in the figure legends.  Table 1: I do not understand the numbers in this table. I assume 1/5 means that only a maximum score of 1 on the 5-point scale was attained. But why are some entries 1/3 or 2/2 for example? Shouldn’t the denominator be 5 in all cases since that was the maximum disease score? Response: The authors tried to reflect in Table 1 the maximum Severity Index (SI) values recorded in any of the individual inoculated plants in the experiments. We have considered interesting to show this index in the table as it depicts two different data readings: 2 and 4 months after inoculation (8WPI and 16 WPI) as an independent reference data of the AUDPC and rAUDPC values. Some other authors also reflect this in their scientific studies on the assessment of varietal susceptibility to Xf, References among others: Rashed et al., 2013 [reference nº 63] and Moralejo et al., 2019, [reference nº 39]. Throughout the text: Cultivar and Rootstock should not be capitalized and italicized. Response: We agree with this suggestion. In fact, the term cultivar should not be capitalized nor italicized. However, in the latest scientific articles of the vituculture section of Horticulturare (these serve as an example of others not mentioned) the term cultivar and variety are used indistinctly for the denomination of genotypes independently (or omitting) of the rootstock on which they have been grafted. The section 2.1. Terminology, was added to the manuscript to specify that the term Cultivar (capitalized and italicized) includes the study of varieties grafted on certain rootstocks. In our opinion it helps the reader to understand that this factor could potentially be a conditioning factor of varietal susceptibility and as a frame for further studies. 5. Additional clarifications Additional editorial corrections are included in the attachment. Author´s clarifications: The authors are extremely grateful for your suggestions, and we acknowledge the effort made to allow us to resubmit this manuscript for your consideration for publication in the viticulture section. We truly consider it very appropriate to present a work that depicts, for the first time, the comparative susceptibility of many European cultivars and that, with no doubt, establishes a framework to be able to compare varietal selections, either local or new commercial varieties. The breeding activity in grapevine is continuous and intense. To reinforce this statement, please review the latest articles published in the viticulture section, where both new and local varieties are described and characterized in detail. The presence of Xf in Europe suggests a future scenario in which the varietal selection will be based, among other factors, on resistance to this dangerous pathogen.    PLEASE SEE THE ATTACHMENT

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The article is very interesting, and the experimental procedure done in the correct way. Disease progression and disease severity are the most important parameters taken in consideration to assess plant susceptibility. However, what is missing is the infection level of the pathogen during the test, in association with disease progress, especially for those resistant. Have you any data about that? If yes, I suggest to insert them.

The article does not need any English revision and can be accepted with minor revisions.

Author Response

For research article:

Title: European Grapevine Cultivars and Rootstock Show Differential

Resistance to Xylella fastidiosa Subsp. fastidiosa

Authors: Sara Martínez, Maite Lacuesta, Juan Bautista Relloso, Ana

Aragonés, Ana Herran, Amaya Ortiz-Barredo *

Manuscript ID: horticulturae-2679554, Received: 9 Oct 2023

Submitted to section: Viticulture (Genetic Resources for Viticulture)

 

Response to Reviewer-2 X Comments
1. Summary
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections changes in the re-submitted files.
2. Questions for General Evaluation	Reviewer’s Evaluation	Author´s Response and Revisions
·        Does the introduction provide sufficient background and include all relevant references?	Yes
·        Are all the cited references relevant to the research?	Yes
·        Is the research design appropriate?	Can be improved	We agree with your right suggestion, please, kindly see our corresponding responses in the point-by-point responses bellow
·        Are the methods adequately described?	Yes
·        Are the results clearly presented?	Yes
·        Are the conclusions supported by the results?	Yes

 

 

3. Point-by-point response to Comments and Suggestions for Authors   Comment 1: Dear authors, The article is very interesting, and the experimental procedure done in the correct way. Disease progression and disease severity are the most important parameters taken in consideration to assess plant susceptibility. However, what is missing is the infection level of the pathogen during the test, in association with disease progress, especially for those resistant. Have you any data about that? If yes, I suggest to insert them. The article does not need any English revision and can be accepted with minor revisions. Response: We agree with this suggestion. The infection level in association with disease progress was not recorded in the first two field trials carried out exclusively under field conditions, the bacteria detection was recorded only at the end of the symptomatology period (16WPI). The bacterial detection progress (but not quantification) was recorded in the last two trials carried out with a low number of cultivars under field and greenhouse conditions and they are showed in Fig. 8-C. The record suggested would have been interesting to guide future trials on disease progression in each of the varieties to explain the cultivar resistance. We consider, after the results obtained, that not only this parameter should be included but also the quantification of tyloses and gums production by the plants involved in the symptomatology and resistance, as it was related in the introduction section (line 59, reference Pérez-Donoso, 2016). We thank you for your suggestion and include this guidance (orientación) in the discussion section. PLEASE SEE THE ATTACHMENT

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Review comments

 The manuscript studies the important diseases of European grapes, providing a good theoretical basis for production prevention and control. However, the manuscript still has the following problems:

 1. Framework issue: The article feels that it is a 3-year patchwork of data, which has not been systematically studied on the same grape cultivars and strains. Therefore, it only draws some simple conclusions and does not propose any meaningful viewpoints.

 2. The disease studied in the manuscript mainly occurs in the xylem, and the final symptoms of the disease are reflected on the leaves. Therefore, only detecting the disease area of the leaves does not have enough data. Why not perform some testing and observation on the xylem, which is more meaningful than detecting the water potential.

 3. The description of the test material is not clear. Table 1 should be listed in the materials and methods, and indicate which strains were tested for those cultivars in which years.

 4. Figure 1 lacks a horizontal coordinate. Is relative humidity an average value?

Author Response

For research article:

Title: European Grapevine Cultivars and Rootstock Show Differential

Resistance to Xylella fastidiosa Subsp. fastidiosa

Authors: Sara Martínez, Maite Lacuesta, Juan Bautista Relloso, Ana

Aragonés, Ana Herran, Amaya Ortiz-Barredo *

Manuscript ID: horticulturae-2679554, Received: 9 Oct 2023

Submitted to section: Viticulture (Genetic Resources for Viticulture)

 

Response to Reviewer-3 X Comments
1. Summary
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections changes in the re-submitted files.
2. Questions for General Evaluation	Reviewer’s Evaluation	Author´s Response and Revisions
·        Does the introduction provide sufficient background and include all relevant references?	Must be improved	We agree with your right suggestion, please, kindly see our corresponding responses in the point-by-point responses bellow
·        Are all the cited references relevant to the research?	Can be improved
·        Is the research design appropriate?	Not applicable
·        Are the methods adequately described?	Must be improved
·        Are the results clearly presented?	Must be improved
·        Are the conclusions supported by the results?	Must be improved

 

 

 

 

3. Point-by-point response to Comments and Suggestions for Authors

 

The manuscript studies the important diseases of European grapes, providing a good theoretical basis for production prevention and control. However, the manuscript still has the following problems:

Comment 1.    Framework issue: The article feels that it is a 3-year patchwork of data, which has not been systematically studied on the same grape cultivars and strains. Therefore, it only draws some simple conclusions and does not propose any meaningful viewpoints.

Response 1: We agree with your valuable comment.

The pathogen and handling conditions are worth to be considered as set out in the current European regulations. Xf is in the category of “Priority quarantine pathogen” (Regulation (EU) 2016/2031 and Commission Delegated Regulation (EU) 2019/829), this means that we are dealing with one of the 20 most dangerous pathogens for Euro-pean wine-growing areas as well as for other agricultural areas with major crops. Consequently, its handling, including for scientific purposes, has been extremely re-stricted even under regional rules (limited to areas where the disease has been widely detected or in accredited facilities (such as high biosafety greenhouses) to avoid any risk of pathogen spread.

The varietal selection and the way the trials were conducted correspond to the following chronology: The first trial in 2019 was carried out in Mallorca-Balearic Is-lands, under field conditions. The area for testing had to be restricted and carried out with local Xf isolated strains (XYL 2055/17 and XYL 2177/18). Until then (at the time the trials started) there was no reference to the strains´ virulence in different cultivars or rootstocks. A total of 28 Cultivars and Rootstocks were evaluated. That amount (28) was selected due to their vast degree of establishment (approx. 75%) in the most im-portant wine-growing areas of Spain. Once the results of the first trial were analised, and according to the space availability restrictions, a second trial was carried out the following year (2020). It was conducted during the vegetative period of the grapevine, in the same location and under the same operational conditions. In this year, due to above mentioned space availability restrictions, the number of cultivars to be evaluated was reduced to 22 in total vs 28. Due to the results obtained in the previous year re-garding the strain virulence, the susceptibility was assessed just against the most viru-lent strain (XYL 2055/17). Considering the results obtained in these two trials carried out under field conditions (2019 and 2020), it was decided to carry out a new trial during a new growing season in 2021. In this case, the objective of this third trial was evaluating the influence of climate conditions on disease progression. This is the reason of the restricted number of plants in this trial (5 Cultivars and 1 Rootstock). The assay was carried out under field conditions (the same location in the Balearic Islands) and also in a greenhouse located in the Basque Country. This particular study under greenhouse (in the Basque Country) was located in a free disease area of Spain and in a facility accredited for handling infected plant material with this harmful pathogen. The 2021 trials were carried out with the only authorized to import strain for this type of experimental trials (IVIA 5770). The cultivars evaluated each year as well as the in-oculated strains are detailed in Table 1, included in the results section.

We are aware that the current disease scenario as well as the operational restrictions in the trials have greatly conditioned the conduction of the trials and therefore the results that could be extracted from them. However, from our point of view, they are of great value as:

• We have conducted a multi-year and multi-location experiments to assess grapevine cultivar and rootstock resistance to Xff, the causal agent of Pierce’s disease.
• it is the first time that the different susceptibility (with a large number of European grapevine cultivars) is revealed,
• the first reference of the virulence of different Xf strains isolated in European grapevine, and
• confirms the influence of agro-climatic parameters in the disease development and, consequently, in the cultivar susceptibility.

The authors selected the section of Viticulture (Special Issue "Genetic Resources for Viticulture") in Horticulturae Journal because it offers an appropriate space for the assessment of the results by pathologists but, especially, by researchers involved in grapevine breeding. The last 8 scientific articles published in this special issue refer to the varietal description of new minority or local varieties by classical descriptive parameters. Judging by the current scenario in America in relation to this disease (more than 50 years after its detection) and the progression of the incidence of this emerging disease in Europe, the authors believe that in a very near future, the varietal structure in European wine-growing areas could be modified and studies, such as this one presented here, set the first main reference for this.

 

Your perception on the above listed outcomes of the research is of great value as it offers valuable insights that should be adequately considered in the manuscript to enrich the discussion of the results obtained. In addition, a general overview of the trials has been included in Materials & Methods section.

 

Comment 2.    The disease studied in the manuscript mainly occurs in the xylem, and the final symptoms of the disease are reflected on the leaves. Therefore, only detecting the disease area of the leaves does not have enough data. Why not perform some testing and observation on the xylem, which is more meaningful than detecting the water potential.

 

Response 2: We thank you for your comment on this. As it is described in the introduction section, the main factors causing PD symptoms appear to be related to water stress and the blockage of xylem vessels by the bacterial biofilm, as well as the resulting production of tyloses and gums by the plant, which cause hydraulic dysfunctions leading to desiccation and plant death within a few years [references 10,11]. Subsequently, characteristic leaf symptoms such as registered in the leaves for this work become apparent. However, the development of symptoms may not be entirely due to the vessel’s occlusion. Instead, the pathogenesis may be more complex and occur starting from the earliest stages of infection, before the colonization of vessels [12 ,13]. Considering the conditions for handling Xf infected material, mentioned above, and the large number of cultivars to be evaluated, the authors chose to select the leaf symptoms as an indicator of varietal resistance. Indicators of the cultivar susceptibility and the severity of the disease caused by Xf has been evaluated in other scientific reference works based on the leaf symptom scale, first described by Sue in 2013 (As reported in materials and methods). This scale has also been recently used by other authors for the same purposes, both on grapevine (Rashed et al., 2013 [Reference nº 63] and Moralejo et al., 2019 [Reference nº 39]) and on other major Xf hosts (Olive by Baró et al., 2021, Reference nº 43).

 

Based on the Severity Index data, it was possible to calculate the disease progression expressed as the absolute area under the disease progression curve (AUDPC) and its transformed rAUDPC to assess quantitative disease resistance in grapevine (Jeder et al., 2001*).

 

*Jeger, M. J., and Viljanen-Rollinson, S. L. H. 2001. The use of the area under the disease progress curve (AUDPC) to assess quantitative disease resistance in crop cultivars. Theoretical and Applied Genetics 102:32–36

 

Your suggested records will be of great interest to guide future trials on disease progression in each of the varieties to explain the cultivar resistance. We consider, after the results obtained based on just the leaves symptomatology and water potential, that not only these parameters should be included but also alterations on xylem vessels of the plants involved in the symptomatology and resistance and the quantification of plant secondary metabolites involved in the symptomatology and resistance. We thank you for your suggestion and it will be for sure included in the discussion section.

 

Comment 3.    The description of the test material is not clear. Table 1 should be listed in the materials and methods, and indicate which strains were tested for those cultivars in which years.

Response 3: The authors agree with your suggestion and so it has been modified in the new manuscript.

In addition, a general overview of the trials has been included in Materials & Methods section.

 

Comment 4.    Figure 1 lacks a horizontal coordinate. Is relative humidity an average value?

Response 4: We agree. The figure 1 has been modified in the new version of the manuscript.

PLEASE SEE THE ATTACHMENT

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

It can be seen that the author's research difficulty is significant. Finally, a point of view is that the introduction section describes too much and can be slightly reduced.

Author Response

Thank you for your suggestions for improving the manuscript
I have shortened the introduction, eliminating redundant parts or previously well known information. The number of references has not been reduced. 
I enclose the modified document, showing in red the modifications made.

Author Response File: Author Response.docx