Role of Humic Acid on Inducing Salt Tolerance of Ivy Geranium (Pelargonium peltatum L.) Plants

Round 1

Reviewer 1 Report

The manuscript title “Effect of humic acid on inducing salt tolerance of Ivy geranium plants” is conducted in a good manner and has scientific worth.

I have some minor comments for authors:

1-      Line 18: “as well as micro- and micronutrients levels,”. It seems to be micro- and macronutrients levels … Is it?

2-      Line 54: “Humic acid (HA) represents”. No need to repeat the full name again and again, just write HA is enough.

3-      2.4. Physiological growth characteristics:

“2.4.1.

2.4.2. Proline concentration in the plant shoot was estimated following the protocol of Bates et al. [36] using ninhydrin reagent.

2.4.3. The content of macro- and micro-elements (nitrogen, phosphorus, potassium, calcium, magnesium, copper, iron, manganese, zinc, and sodium) in the shoots of plants was assessed according to A.O.A.C. [37].”

“Please write detail methods of all aforementioned analysis”. Just giving their references is not enough.”

4.       Please add the figures of Ivy geranium plants with HA, saline water and control, to see the visual affects of different treatments Ivy geranium plants. Also, it will enhance the impact of the research.

English is fine

Author Response

Dear prospected reviewer 

We appreciate the time and effort you have dedicated to providing insightful feedback on ways to strengthen our manuscript. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We think that the manuscript has been greatly improved by these revisions and hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you have noted. The necessary corrections have been written in The red lines in the manuscript. To facilitate your evaluation, the following is a point-by-point response to the questions and comments

The manuscript title “Effect of humic acid on inducing salt tolerance of Ivy geranium plants” is conducted in a good manner and has scientific worth.

I have some minor comments for authors:

1-      Line 18: “as well as micro- and micronutrients levels,”. It seems to be micro- and macronutrients levels … Is it?

Thank you for your comments. We completely agree and corrected the word as indicated in the revised manuscript

2-      Line 54: “Humic acid (HA) represents”. No need to repeat the full name again and again, just write HA is enough.

 Thank you for your comments. All over the manuscript from the introduction, we add the full name in the first site and then used abbreviation

3-      2.4. Physiological growth characteristics:

“2.4.1.

2.4.2. Proline concentration in the plant shoot was estimated following the protocol of Bates et al. [36] using ninhydrin reagent.

2.4.3. The content of macro- and micro-elements (nitrogen, phosphorus, potassium, calcium, magnesium, copper, iron, manganese, zinc, and sodium) in the shoots of plants was assessed according to A.O.A.C. [37].”

“Please write detail methods of all aforementioned analysis”. Just giving their references is not enough.”

Thank you for your comments. As shown in the revised manuscript we add some details of analysis methods thank you again

4. Please add the figures of Ivy geranium plants with HA, saline water and control, to see the visual affects of different treatments Ivy geranium plants. Also, it will enhance the impact of the research.

Thank you for your comments. As indicated in the revised manuscript we put some of the pictures. In the future we will photograph any experiment to show the effect of treatment, thank you again

Once more, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We have worked hard to incorporate your feedback and hope that these revisions persuade you to accept our submission.

Thank you in advance for your time and attention.

Sincerely

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear Authors,

Congratulations on a well presented manuscript requiring very little to no additions. If possible could you also provide more information in your introduction regarding the composition of HA and also the advantages of foliar application over soil application since you chose foliar application as your method of choice for your current experiment.

Also note  line 21-23 in the abstract may need editing to make it clear what you mean

English is relatively fine

Author Response

Dear prospected reviewer 

We appreciate the time and effort you have dedicated to providing insightful feedback on ways to strengthen our manuscript. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We think that the manuscript has been greatly improved by these revisions and hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you have noted. The necessary corrections have been written in The red lines in the manuscript. To facilitate your evaluation, the following is a point-by-point response to the questions and comments

Dear Authors,

Congratulations on a well presented manuscript requiring very little to no additions. If possible could you also provide more information in your introduction regarding the composition of HA and also the advantages of foliar application over soil application since you chose foliar application as your method of choice for your current experiment.

Also note  line 21-23 in the abstract may need editing to make it clear what you mean

Thank you for your comments. As indicated in the revised manuscript, we add more information about the composition of humic acid and its application as foliar spraying. Additionally, we checked and rewrite the sentences in the Abstract to me more readable. Thank you again

Once more, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We have worked hard to incorporate your feedback and hope that these revisions persuade you to accept our submission.

Thank you in advance for your time and attention.

Sincerely

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors sought to evaluate the influence of humic acid on growth, flowering, prolines and other physiological parameters in geranium plants under saline stress. The manuscript presents important information and has potential for publication, however, some adjustments are necessary. Statistical analysis is not recommended for this work. Two quantitative factors were used, and a polynomial regression analysis is recommended for this study.

Tittle: Remove the word “effect”.

Abstract

The purpose of the study should be cited after the introductory part.

A general conclusion was lacking. Can HA be used as a mitigator of maximum saline stress in this study?

Introduction

Geranium is not a genus, the genus is Pelargonium sp. Review this information.

Reduce the target. There is no need to describe the analyzed variables. Substitute smaller terms such as “growth aspects, physiological aspects”... this is just an example.

Material and methods

Further detail the experimental design. It was unclear if it was DIC or DBC. How was it done?

The analyzed variables do not need to be described in subtopics. Write in plain text and provide more details on these analyses. It's just citing the variables without explaining how to make the measurements.

All analyzes should be more detailed.

The authors used Tukey's test to compare quantitative data. I suggest using regression analysis, and if there is interaction between the two factors, making response surface graphs.

Results

The results will be modified and presented differently with the new statistical analysis of the data.

Discussion

Same as the previous comment.

Conclusion

Same as the previous comment.

Author Response

Dear prospected reviewer 

We appreciate the time and effort you have dedicated to providing insightful feedback on ways to strengthen our manuscript. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We think that the manuscript has been greatly improved by these revisions and hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you have noted. The necessary corrections have been written in The red lines in the manuscript. To facilitate your evaluation, the following is a point-by-point response to the questions and comments

The authors sought to evaluate the influence of humic acid on growth, flowering, prolines and other physiological parameters in geranium plants under saline stress. The manuscript presents important information and has potential for publication, however, some adjustments are necessary. Statistical analysis is not recommended for this work. Two quantitative factors were used, and a polynomial regression analysis is recommended for this study.

Thank you so much for your comments. As indicated in the revised manuscript we use Two-way Anova to evaluate the sole effect of either irrigation with saline water or foliar application of humic acid as well as the interaction effects between two factors. As in papers like

https://doi.org/10.3390/horticulturae9080883

https://doi.org/10.3390/horticulturae9080945

Tittle: Remove the word “effect”.

Ok changed to Role

Abstract

The purpose of the study should be cited after the introductory part.

A general conclusion was lacking. Can HA be used as a mitigator of maximum saline stress in this study?

Thank you so much for your comments. As indicated in the revised manuscript we checked and revised the abstract to me more understandable

Introduction

Geranium is not a genus, the genus is Pelargonium sp. Review this information.

Thank you so much for your comments. We checked it and changed

Reduce the target. There is no need to describe the analyzed variables. Substitute smaller terms such as “growth aspects, physiological aspects”... this is just an example.

Thank you so much for your comments. We checked it and changed it as indicated in the revised manuscript

Material and methods

Further detail the experimental design. It was unclear if it was DIC or DBC. How was it done?

The analyzed variables do not need to be described in subtopics. Write in plain text and provide more details on these analyses. It's just citing the variables without explaining how to make the measurements.

All analyzes should be more detailed.

Thank you for your comments. As shown in the revised manuscript we add some details of analysis methods thank you again

The authors used Tukey's test to compare quantitative data. I suggest using regression analysis, and if there is interaction between the two factors, making response surface graphs.

Thank you so much for your comments. As indicated in the revised manuscript we use Two-way Anova to evaluate the sole effect of either irrigation with saline water or foliar application of humic acid as well as the interaction effects between two factors. As in papers like

https://doi.org/10.3390/horticulturae9080883

https://doi.org/10.3390/horticulturae9080945

Results

The results will be modified and presented differently with the new statistical analysis of the data.

Thank you so much for your kind comments. We revised and correct this section as possible as we can

Discussion

Same as the previous comment.

 Thank you so much for your kind comments. We revised and correct this section as possible as we can

Conclusion

Same as the previous comment.

 Thank you so much for your kind comments. We revised and correct this section as possible as we can

Once more, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We have worked hard to incorporate your feedback and hope that these revisions persuade you to accept our submission.

Thank you in advance for your time and attention.

Sincerely

Author Response File: Author Response.pdf

Reviewer 4 Report

The manuscript contains interesting data and may be published after substantial improvement in the style of presentation and correction of other more minor errors.

Abstract: L10-11 "Humic acid (HA) is a key signaling molecule for the induction of plant stress tolerance ...". It is hardly necessary to expand the list of plant signaling molecules by including humic acids abundant in soil. In any case, this is not a generally accepted view and therefore requires careful justification when mentioned.

L 24-25 "It is recommended to irrigate Ivy geranium with water of 4000 mg/l NaCl associated with the application of 1000 mg/l HA". Edit this sentence to clarify the recommendation to use saline water: it is unlikely that the authors meant to recommend specifically salinizing water to 4000 mg/l NaCl for irrigation.

L74 The authors write about the lack of data on the effect of humic acid on salt stress specifically in Ivy geranium plants. Nevertheless, the function of humic acid in the defense against stress, including the mitigation of salt injury, has been widely recognized in other plants. Such data should be cited in the Introduction to justify the purpose of the work.

L85 Please justify the choice of such salt concentrations. Provide data in the Introduction on salt content of marginal water resources that can be used for irrigation.

L91 Give the full Latin name of the experimental plant Ivy geranium with the unabbreviated generic name and authors.

L98 It is desirable to give the range or maximum salt content of tap water.

The beginning of the Methods section (L 87) states that the repetition is 6 plants (6 pots). At L102 it says that 6 plants were used, 2 per repetition, i.e. a repetition of 3. What is the repeatability for different parameters? How was material from 2 plants used if the repeatability is 3 (different plants for different tests or the average of the 2)?

What is the duration of the experiment? When was the material for the different tests selected?

Results. Reliability of differences and correctness of statistical analysis is not questionable. However, the presentation of ANOVA results is not standard. In my opinion, it should be indicated in the methodology or in the notes to the tables that a 2-factor ANOVA was performed, that the effect of both factors and their interaction is significant for almost all the parameters studied, and decipher how the averages in different rows of the tables were calculated.

In all tables of the manuscript, the averages are presented with excessive precision, which will make it very difficult to perceive and analyze the material. From the point of view of mathematics, 37.43±4.21 is meaningless, because already the number 7 is doubtful at such an error, we should write 37±4. From the aesthetic point of view I would recommend to leave one more sign: 37.4±4.2.

For numerical values in the text, the problem of extra characters is even more aggravated. Very few people will risk reading and even less analyzing the text "Stem length, stem diameter, leaf number plant -1, leaf area, shoot fresh and dry weights were significantly decreased by 51.03, 28.64, 17.80, 50.84, 14.87, and 57.23% as a result of irrigation with 2000 mg/l NaCl respectively and by 61.29, 35.24, 61.08, 61.32, 40.08, and 71.24% respectively under 4000 mg/l over non-salinized control plants" (L133-136). I would recommend that the authors drastically change the style of comments on the data in Tables 1 and 2. For any measurements it is better to write something like " irrigation with 2000 mg/l NaCl strongly reduced stem length, leaf area and shoot dry weight (50-57%) and had less effect on stem diameter, leaf number plant and shoot fresh weight (15-29%). When treated with 4000 mg/l ..... 61-71% and 35-61%, respectively" or better yet "Salt stress had a particularly strong effect on stem length, leaf area and shoot dry weight, reducing these parameters by a factor of 2 or more", and provide percentage data in tables for each mean.

Discussion. The section contains an excessive amount of poorly structured literature data, the number of which corresponds more to a large review article than to an experimental study. At the same time, the authors appear to address a rather narrow and specific question. A detailed explanation of stress mechanisms within the framework of this paper is unnecessary and impossible. Therefore, it is desirable to reduce the number of cited sources by 2 or more times. In my opinion, authors should choose what is the main goal of the paper. If the goal is to use Ivy geranium as another model plant for salt stress and humic acids research, then it should be more purposeful to write what turned out to be similar to other plants, and what new to stress research this new model object brought. If the main thing is the practical application of saline water for irrigation, then it is the ways of solving this problem in the literature that should be discussed.

Author Response

Dear prospected reviewer 

We appreciate the time and effort you have dedicated to providing insightful feedback on ways to strengthen our manuscript. We have incorporated changes that reflect the detailed suggestions you have graciously provided. We think that the manuscript has been greatly improved by these revisions and hope that our edits and the responses we provide below satisfactorily address all the issues and concerns you have noted. The necessary corrections have been written in The red lines in the manuscript. To facilitate your evaluation, the following is a point-by-point response to the questions and comments

The manuscript contains interesting data and may be published after substantial improvement in the style of presentation and correction of other more minor errors.

Thank you so much for your kind comment

Abstract: L10-11 "Humic acid (HA) is a key signaling molecule for the induction of plant stress tolerance ...". It is hardly necessary to expand the list of plant signaling molecules by including humic acids abundant in soil. In any case, this is not a generally accepted view and therefore requires careful justification when mentioned.

Thank you so much for your comments. As indicated in the revised manuscript we checked and revised the abstract to me more understandable

L 24-25 "It is recommended to irrigate Ivy geranium with water of 4000 mg/l NaCl associated with the application of 1000 mg/l HA". Edit this sentence to clarify the recommendation to use saline water: it is unlikely that the authors meant to recommend specifically salinizing water to 4000 mg/l NaCl for irrigation.

Thank you so much for your comments. As indicated in the revised manuscript we checked and revised it.

L74 The authors write about the lack of data on the effect of humic acid on salt stress specifically in Ivy geranium plants. Nevertheless, the function of humic acid in the defense against stress, including the mitigation of salt injury, has been widely recognized in other plants. Such data should be cited in the Introduction to justify the purpose of the work.

Thank you so much for your comments. As indicated in the revised manuscript we checked and revised it.

L85 Please justify the choice of such salt concentrations. Provide data in the Introduction on salt content of marginal water resources that can be used for irrigation.

We completely agree with your comment as indicated in the revised manuscript we add the brackish water in Saudi Arabia that ranged between 1000-6000 mg/l. so we selected the current experiment

L91 Give the full Latin name of the experimental plant Ivy geranium with the unabbreviated generic name and authors.

As indicated in the revised manuscript we add the full Latin name

L98 It is desirable to give the range or maximum salt content of tap water.

Thank you so much. As indicated in revised manuscript we add the salinity level of tap water as 230 mg/l

The beginning of the Methods section (L 87) states that the repetition is 6 plants (6 pots). At L102 it says that 6 plants were used, 2 per repetition, i.e. a repetition of 3. What is the repeatability for different parameters? How was material from 2 plants used if the repeatability is 3 (different plants for different tests or the average of the 2)?

We used each two plants’ averages as a replicate

What is the duration of the experiment? When was the material for the different tests selected?

Thank you. As indicated in the revised manuscript chlorophyll and proline were estimated in leaves, meanwhile, ions were estimated in the shoot

Results. Reliability of differences and correctness of statistical analysis is not questionable. However, the presentation of ANOVA results is not standard. In my opinion, it should be indicated in the methodology or in the notes to the tables that a 2-factor ANOVA was performed, that the effect of both factors and their interaction is significant for almost all the parameters studied, and decipher how the averages in different rows of the tables were calculated.

Thank you so much for your kind response. As indicated in the MM Statistical section we used Two-Way ANOVA to evaluate the sole effect of HA or saline water as well as their interaction of Ivy geranium plants

In all tables of the manuscript, the averages are presented with excessive precision, which will make it very difficult to perceive and analyze the material. From the point of view of mathematics, 37.43±4.21 is meaningless, because already the number 7 is doubtful at such an error, we should write 37±4. From the aesthetic point of view I would recommend to leave one more sign: 37.4±4.2.

For numerical values in the text, the problem of extra characters is even more aggravated. Very few people will risk reading and even less analyzing the text "Stem length, stem diameter, leaf number plant -1, leaf area, shoot fresh and dry weights were significantly decreased by 51.03, 28.64, 17.80, 50.84, 14.87, and 57.23% as a result of irrigation with 2000 mg/l NaCl respectively and by 61.29, 35.24, 61.08, 61.32, 40.08, and 71.24% respectively under 4000 mg/l over non-salinized control plants" (L133-136). I would recommend that the authors drastically change the style of comments on the data in Tables 1 and 2. For any measurements it is better to write something like " irrigation with 2000 mg/l NaCl strongly reduced stem length, leaf area and shoot dry weight (50-57%) and had less effect on stem diameter, leaf number plant and shoot fresh weight (15-29%). When treated with 4000 mg/l ..... 61-71% and 35-61%, respectively" or better yet "Salt stress had a particularly strong effect on stem length, leaf area and shoot dry weight, reducing these parameters by a factor of 2 or more", and provide percentage data in tables for each mean.

Thank you so much for your kind comments. As indicated in the revised manuscript we checked and change the style of writing in some sites in the manuscript. Thank you again

Discussion. The section contains an excessive amount of poorly structured literature data, the number of which corresponds more to a large review article than to an experimental study. At the same time, the authors appear to address a rather narrow and specific question. A detailed explanation of stress mechanisms within the framework of this paper is unnecessary and impossible. Therefore, it is desirable to reduce the number of cited sources by 2 or more times. In my opinion, authors should choose what is the main goal of the paper. If the goal is to use Ivy geranium as another model plant for salt stress and humic acids research, then it should be more purposeful to write what turned out to be similar to other plants, and what new to stress research this new model object brought. If the main thing is the practical application of saline water for irrigation, then it is the ways of solving this problem in the literature that should be discussed.

Thank you so much for your kind comments. As indicated in the revised manuscript we checked and change the style of writing in some sites in the manuscript. Thank you again

Once more, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We have worked hard to incorporate your feedback and hope that these revisions persuade you to accept our submission.

Thank you in advance for your time and attention.

Sincerely

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

The manuscript has been improved according to suggestions.

Author Response

Dear prospective prof

Thank you so much for your kind comments that increased the overall value of our manuscript. thank you again

Yours, truly

Author Response File: Author Response.pdf

Reviewer 4 Report

Please reduce the number of decimal points in data in the text expressed as % (e.g. 98.6 instead of 98.59) (L159-160, L188, L192, L200-201, L219, L230, L246).

L133 Check the Cooper (1977) citation and put it in References if necessary.

L379 Check the Sahar et al. [2009] citation and put it in References if necessary.

Author Response

Dear prospective prof

Thank you so much for your kind comments that increased the overall value of our manuscript.

1- Please reduce the number of decimal points in data in the text expressed as % (e.g. 98.6 instead of 98.59) (L159-160, L188, L192, L200-201, L219, L230, L246).

As indicated in revised manuscript we do all changes suggests by you in a red line

2- L133 Check the Cooper (1977) citation and put it in References if necessary.

L379 Check the Sahar et al. [2009] citation and put it in References if necessary.

Thank you so much for your valuable recommendation we add the two reference in the references section in red line

thank you again

Yours, truly

Author Response File: Author Response.pdf