Fertigation of Ajwain (Trachyspermum ammi L.) with Fe-Glutamate Confers Better Plant Performance and Drought Tolerance in Comparison with FeSO₄

Round 1

Reviewer 1 Report

Authors have presented their research work in the manuscript entitled 'Fertigation of water-stressed ajwain plants with environment-friendly Fe-chelated glutamate confers a better induction for drought tolerance in comparison with FeSO4', is an interesting piece of work and good for reacders. However, there are below concers:

1) No page numbers found so could not provide very specific comments.

2) The title is too long, can authors shorten it?

3) Abstract can be improved in light of language such as: what do you mean by better crop production? So, other chemicals which are used and are approved by Government organisations to use are not producing better crops compared to no-chemical application crops? Change of language is suggested. The use of word foliary doesnt suit the scientific publications, please use foliar application instead. 

4) What would be the cost of application of Fe-glu and FeSO4 in compared to readily available alternates in the market? IS the cost significant to recommend this to a grower?

5) Moreover, Ajwain is a cold loving crop and is mainly grown during July to March depending upon the season you would like to grow. So, my question is how stressed can plants be when grown at the arrival of rainy season or in winter?

6) Introduction section: Too much use of words like However, Furthermore, Moreover, etc. Please use alternate words or rephrase your sentences.

7) Materials and methods section: Provide the coordinates for the field trial. Experimental design is not clear. Were there four rows per treatment? Why there is no estimation of Fe in soil? How many seasons the field trials were performed. Was the canal water quality and nutrient composition tested? Supplier information of FeSO4 and Fe-glu is not provided.

8) Results: Data presented in for. (what are authors trying to say?). Improved English is required, can authors get this edited by an English speaker. Reason, use of words like significantly adversely (together!), Comparatively and then comparison in same sentence, WHY? These mistakes are quite repetitive and annoying to the reader.

9) Discussion: Better production? Again use of this word throughout the manuscript is not appropriate, not a word used in scientific publications. Inadequate number of citation to support the research outcomes. 

10) Conclusions: In this paragraph, first 6 lines, authors have used better word 6 times. This is just too much better. If Authors are recommending this to be used by farmers, can they please provide the rate of application and estimated cost? 

To make such a claim; the exogenous use of Fe-glu will be favored due to its environment-friendly nature in comparison with FeSO4, did authors perform any surveys or research in this area? Pleas make claims which can be supported by sound data available.

11) Figures: Please provide figures (1 & 2) with proper labels. The labels are not visible. Also, how four row of each treatment are adjacent to each other for each treatment? Did authors not say in M&M that the four rows of equal size were one replication. Clarification required. 
Figures 3&4 have LSD 5% value for FAA and GSG graphs on the border, please adjust them same as others.

Author Response

Response to Reviewer 1

Comment: Authors have presented their research work in the manuscript entitled 'Fertigation of water-stressed ajwain plants with environment-friendly Fe-chelated glutamate confers a better induction for drought tolerance in comparison with FeSO4', is an interesting piece of work and good for readers.

Response: We are thankful to the worthy reviewer for admiring the idea and work done

Comment: No page numbers found so could not provide very specific comments

Response: Now the page numbers has been inserted at bottom of the page 

Comment:  The title is too long, can authors shorten it?

Response: Suggestion followed and title has shorten possibly

Comment: Abstract can be improved in light of language such as: what do you mean by better crop production? So, other chemicals which are used and are approved by Government organisations to use are not producing better crops compared to no-chemical application crops?

Response: Abstract has been improved in English language and the required information has included in the text in red text

Comment: Change of language is suggested.

Response: Suggestion followed and manuscript has improved extensively in English language

Comment: The use of word foliary doesnt suit the scientific publications, please use foliar application instead.

Response: Suggestion followed

Comment: What would be the cost of application of Fe-glu and FeSO4 in compared to readily available alternates in the market? IS the cost significant to recommend this to a grower?

Response: The demanded information has been incorporated in conclusion section highlighted red 

Comment: Moreover, Ajwain is a cold loving crop and is mainly grown during July to March depending upon the season you would like to grow. So, my question is how stressed can plants be when grown at the arrival of rainy season or in winter?

Response: Information has incorporated in the text in Materials and Methods section

Comment:  Introduction section: Too much use of words like However, Furthermore, Moreover, etc. Please use alternate words or rephrase your sentences.

Response: Manuscript has been revised extensively following the comments

Comment:  Materials and methods section: Provide the coordinates for the field trial.

Response: Required information has been incorporated in materials and Methods section

Comment: Experimental design is not clear. Were there four rows per treatment?

Response: Detail regarding the experimental plan along with a flow chart diagram showing the research layout has been provided as supplementary material

Comment: Why there is no estimation of Fe in soil?

Response: Required information has been incorporated in materials and Methods section

Comment: How many seasons the field trials were performed.

Response: Required information has been incorporated in materials and Methods section

Comment: Was the canal water quality and nutrient composition tested?

Response: Information regarding the quality and nutrient composition has been incoporated in red text

Comment: Supplier information of FeSO4 and Fe-glu is not provided.

Response: Required information has been incorporated in materials and Methods section

Comment: Results: Data presented in for. (what are authors trying to say?). Improved English is required, can authors get this edited by an English speaker. Reason, use of words like significantly adversely (together!), Comparatively and then comparison in same sentence, WHY? These mistakes are quite repetitive and annoying to the reader.

Response: Manuscript has been extensively improved in English language

Comment: Discussion: Better production? Again use of this word throughout the manuscript is not appropriate, not a word used in scientific publications. Inadequate number of citation to support the research outcomes.

Response: suggestion followed. Correction has incorporated in the manuscript by replacing the word better production. New citations has included to support the data.

Comment: Conclusions: In this paragraph, first 6 lines, authors have used better word 6 times. This is just too much better.

Response: Sentences have been improved with alternate words

Comment: If Authors are recommending this to be used by farmers, can they please provide the rate of application and estimated cost?

Response: Required Information has incorporated in text in conclusion section highlighted in red

Comment: To make such a claim; the exogenous use of Fe-glu will be favored due to its environment-friendly nature in comparison with FeSO4, did authors perform any surveys or research in this area? Please make claims which can be supported by sound data available.

Response: The detailed supportive information to claim the exogenous use of Fe-glu as environment-friendly has been incorporated in Introduction section.

Comment:  Figures: Please provide figures (1 & 2) with proper labels. The labels are not visible.

Response: Labels of figures has incorporated in clear way that are visible to the readers

Comment: Also, how four row of each treatment are adjacent to each other for each treatment? Did authors not say in M&M that the four rows of equal size were one replication. Clarification required.

Response: Correction has incorporated in text as well as a flow chart diagram has added for the clarification.

Comment: Figures 3&4 have LSD 5% value for FAA and GSG graphs on the border, please adjust them same as others.

Response: Corrected

Reviewer 2 Report

This manuscript describes a comprehensive analysis of water-stressed ajwain plants after foliar spraying of Fe-chelated glutamate in comparison with FeSO4 and concludes that Fe-chelated glutamate confers better drought tolerance in the plants. 

Overall, the submission is of good quality with conclusions well supported and of general interests. 

 There are needs for improvements before publication, as detailed below: 

1. Experiment and design: 1a) As described in section 2, two main plots were used for normal irrigation and water-stressed plants, each main plot has six subplots each corresponding to one treatment. It is not clear to the reviewer how many rows were used for each replicate and importantly, how individual plants were selected for various analysis and how many plants were analyzed for each replicate; 1b) seeds for this project were "bought from local market" with no subspecies or variety name. Authors need to provide such information and ensure that seed-derived plants are reasonably uniform and suitable for this research project. 1c) for foliar spraying, there was no dosage information; 1d) section 2.17, how was the seed yield per plant "estimated manually"? 
2. Ajwain is a herb and spice, production quantity does not represent quality. It would be good to include in your analysis some traits relative to quality such as thymol content. 
3. In conclusion part: 3a) While applying Fe-glutamate helped water stress tolerance, it is also noted that it's foliar spraying also helped plants with no water stress. These results also need to be mentioned and discussed. 
4. Since Fe-glutamate preparation also needs FeSO4, the chemical that is not so environmentally friendly. The reviewer does not see any convincing support to the claim that the use of Fe-glutamate is "environment-friendly".
5. The manuscript is readable but of a low level of English literacy. e.g. punctuation marks are missing in several places, many typos and inappropriate use of certain terms such as "better production per capita". It is recommended that you have some native speaker proofread your manuscript. 

Author Response

Response to Reviewer 2

Comment: Overall, the submission is of good quality with conclusions well supported and of general interests.

 Response: We are thankful to the worthy reviewer for admiring the idea of the research quality and conclusions

Comment: Experiment and design: 1a) As described in section 2, two main plots were used for normal irrigation and water-stressed plants, each main plot has six subplots each corresponding to one treatment. It is not clear to the reviewer how many rows were used for each replicate and importantly, how individual plants were selected for various analysis and how many plants were analyzed for each replicate;

Response: Detail regarding the experimental plan along with a flow chart diagram showing the research layout has been provided as supplementary material

Comment: 1b) seeds for this project were "bought from local market" with no subspecies or variety name. Authors need to provide such information and ensure that seed-derived plants are reasonably uniform and suitable for this research project.

Response: The required information has incorporated in red text

Comment: 1c) for foliar spraying, there was no dosage information;

Response: The detail regarding the dosage of the foliar spray has incorporated in detailed

Comment: 1d) section 2.17, how was the seed yield per plant "estimated manually"? 

Response: detailed information regarding the estimation of yield manually has added in the text

Comment: Ajwain is a herb and spice, production quantity does not represent quality. It would be good to include in your analysis some traits relative to quality such as thymol content. 

Response: Nice comment and we admire the worthy reviewer for the suggestion. The suggestion can be included in future studies

Comment: In conclusion part: 3a) While applying Fe-glutamate helped water stress tolerance, it is also noted that it's foliar spraying also helped plants with no water stress. These results also need to be mentioned and discussed.

 Response: Information is included in Results section in yield section and the additional supportive data to the results has incorporated in discussion part

Comment: Since Fe-glutamate preparation also needs FeSO4, the chemical that is not so environmentally friendly. The reviewer does not see any convincing support to the claim that the use of Fe-glutamate is "environment-friendly"

Response: the detailed convincing information has added in the introduction part in red text.

Comment: The manuscript is readable but of a low level of English literacy. e.g. punctuation marks are missing in several places, many typos and inappropriate use of certain terms such as "better production per capita". It is recommended that you have some native speaker proofread your manuscript. 

Response: Manuscript has been extensively improved in English language and all the incorporated improvements are highlighted in red

Reviewer 3 Report

The paper is interesting and full of data; anyway is not well written, there are several repetitions and many parts are unclear. I suggest an extense revision of the manuscript, mainly for the english language.

Just some comments:

Scientific name of the plant should be entered in the title. Anyway in the text it looks totally missing, except in the table description.

There are several typing errors. Please, Improve the formatting of tables.

mat e met

Try to explain better how the fertigation experiments were performed, i would suggest to do a graphical scheme of the experimental design in field. The experimental plan si described in a not clear way. I understand that is not easy to describe this experiments, due to the number of the steps, but some graph or table could help to improve understanding.

English language is to greatly improve especially for certain paragraphs (i.e. 2.1, 2.2….) Also in description of results, english is very poor and it is difficult to understand the text.

Fig 2: the names in the tags are bad formatted. Maybe during  PDF creation.

There is No correspondence between text and figure… authors miss to refer to fig 1 when they show the FTIR analysis. Moreover is appropriate to specify FTIR as Fourier Transform Infrared Spectroscopy, at least when it is cited for the first time

The analysis pattern for this study is large and well planned, but it should be more accurate: in many paragraphs, several information are missing, i.e. TBA (how much the concentraton), or DNS assay , but also for the another protocols, or you choose to describe well the procedure, or choose to totally refer to the bibliographic note

Paragraph 2.15.3, specify what is IR…

Figure 2 is not totally clear. I can't see difference between stressed and no-stressed plants. I mean, the pictures don't allow to appreciate the difference.

all the tables need to be more clear, please add the meaning of abbreviations in the footnote. Rename control plants. What is LSD%...? Tables must be more clear reading the numbers; It should be avoided to search in the text the abbreviations meaning. For ex. In table 4 please specify in the footnote which kind of nutrients you are talking about. And also for other tables.

Finally, try to comment and explain much better the observed results, which are interesting but they can be improved with more comments . The correlation analysis is good.

Anyway, even the article is quite original and interesting, is well known that GLU is an endogenous low molecular weight Fe chelator, and, since it is able to chelate more ions, the reduction of free iron could decrease the oxidative effect and the consequent damage , due to the Fenton reaction, to the different cellular structure.

Author Response

Response to Reviewer 3

Comment: The paper is interesting and full of data; anyway is not well written, there are several repetitions and many parts are unclear. I suggest an extense revision of the manuscript, mainly for the english language.

Response: Authors are thankful to the worthy reviewer for appreciating the idea and work done. Now the manuscript has been extensively improved in English language and all the incorporated improvements are highlighted in red

Comment: Scientific name of the plant should be entered in the title. Anyway in the text it looks totally missing, except in the table description.

Response: Suggestion followed

Comment: There are several typing errors. Please, Improve the formatting of tables.

mat e met

Response: All the typing and formatting errors has been corrected

Comment: Try to explain better how the fertigation experiments were performed, i would suggest to do a graphical scheme of the experimental design in field. The experimental plan si described in a not clear way. I understand that is not easy to describe this experiments, due to the number of the steps, but some graph or table could help to improve understanding.

 Response: Detail regarding the experimental plan along with a flow chart diagram showing the research layout has been provided as supplementary material

Comment: English language is to greatly improve especially for certain paragraphs (i.e. 2.1, 2.2….) Also in description of results, english is very poor and it is difficult to understand the text.

 Response: Manuscript has been extensively improved in English language and all the incorporated improvements are highlighted in red

Comment: Fig 2: the names in the tags are bad formatted. Maybe during  PDF creation.

 Response: Correction has incorporated in the tags

Comment: There is No correspondence between text and figure… authors miss to refer to fig 1 when they show the FTIR analysis.

Response: Corrected

Comment: Moreover is appropriate to specify FTIR as Fourier Transform Infrared Spectroscopy, at least when it is cited for the first time

 Response: suggestion followed

Comment: The analysis pattern for this study is large and well planned, but it should be more accurate: in many paragraphs, several information are missing, i.e. TBA (how much the concentraton), or DNS assay , but also for the another protocols, or you choose to describe well the procedure, or choose to totally refer to the bibliographic note

Response: thanks for the worthy reviewer for well planned analytical work. Now the missing information has incorporated in all sections of materials and methods in red text. 

Comment: Paragraph 2.15.3, specify what is IR…

Response: It was the typing error and now it is corrected

Comment: Figure 2 is not totally clear. I can't see difference between stressed and no-stressed plants. I mean, the pictures don't allow to appreciate the difference.

Response: The Pictures might be of low quality

Comment: all the tables need to be more clear, please add the meaning of abbreviations in the footnote. Rename control plants.

Response: suggestion followed. Now the tables are presented in a clear way. Hope the worthy reviewer will be satisfied with the presentation of tables. A list of abbreviation has incorporated at the end of Results section with Figure 5

Comment: What is LSD%...?

Response: Information incorporated in Materials and methods. Please see section 2.19.

Comment: Tables must be more clear reading the numbers; It should be avoided to search in the text the abbreviations meaning. For ex. In table 4 please specify in the footnote which kind of nutrients you are talking about. And also for other tables.

Response: A list of abbreviation has incorporated at the end of Results section with Figure 5

Comment: Finally, try to comment and explain much better the observed results, which are interesting but they can be improved with more comments.

Response: suggestion followed and improved following comments

Comment: The correlation analysis is good.

Response: We are thankful to worthy reviewer for appreciation

Reviewer 4 Report

Sustainability Review:  “Fertigation of water-stressed ajwain plants with environment-friendly Fe-chelated glutamate confers a better induction for drought tolerance in comparison with FeSO4”  Qasim Ali1, Sumreena Shahid et al.

This manuscript reports a comparison between foliar spray of either Fe-glutamate or Fe sulphate on ajwain plants under irrigation or water stress. It is indicated that there is some benefit in terms of yield under water stress, with Fe-glu having a greater beneficial effect than FeSO4, and there are suggestions this may be due to improved Fe use and improved tolerance to oxidative stress.

While the data represents a substantial amount of work and may show some significant indications of the benefit of Fe-glu over Fe SO4, in my view, it is generally difficult to fully appreciate the results and conclusions, due to the poor presentation. While I sympathise with authors for whom English may not be a first language, I found the English presentation to be poor with frequent clumsy, verbose, or meaningless sentences. In addition, the Tables and Figures have inadequate legends. In view of these general comments I could have many minor specific comments, and would recommend that the manuscript be improved editorially to improve presentation before being resubmitted to a journal. A few more specific comments are listed below.

Tables 1,2,3  no legend outlining the abbreviations; results presented to too many decimal places (it is not possible to measure with confidence all these parameters to 2 or 3 decimal places); the level of significance (1% or 5% ?) is not clearly indicated; some differences between treatments which apparently show significant differences (by superscripts) are well within the LSD values, which suggests they may not actually be significantly different.

Table 2. Chl a/b ratio appears to be decreased in some cases under water stress, in contrast to comments (p13).

Table 5 would be better included as an appendix.

Changes in Chl concentrations do not necessarily represent changes in photosynthesis, as suggested in discussion.

Author Response

Response to Reviewer 4

Comment: While the data represents a substantial amount of work and may show some significant indications of the benefit of Fe-glu over Fe SO4,

Response: Authors are thankful to the worthy reviewer for appreciating the outcomes.

Comment: in my view, it is generally difficult to fully appreciate the results and conclusions, due to the poor presentation. While I sympathise with authors for whom English may not be a first language, I found the English presentation to be poor with frequent clumsy, verbose, or meaningless sentences.

Response: Manuscript has been extensively improved in English language with help of an expert and all the incorporated improvements are highlighted in red

Comment: In addition, the Tables and Figures have inadequate legends. In view of these general comments I could have many minor specific comments, and would recommend that the manuscript be improved editorially to improve presentation before being resubmitted to a journal. A few more specific comments are listed below.

Response: Now the manuscript has been improve extensively in language, and presentation

Comment: Tables 1,2,3  no legend outlining the abbreviations; results presented to too many decimal places (it is not possible to measure with confidence all these parameters to 2 or 3 decimal places);

Response: A list of abbreviation has incorporated at the end of Results section with Figure 5

Comment: the level of significance (1% or 5% ?) is not clearly indicated; some differences between treatments which apparently show significant differences (by superscripts) are well within the LSD values, which suggests they may not actually be significantly different.

Response: corrections ahs been incorporated in tables and results section in red text

Comment: Table 2. Chl a/b ratio appears to be decreased in some cases under water stress, in contrast to comments (p13).

Response: Correction has incorporated in text and tables

Comment: Table 5 would be better included as an appendix.

Response: Suggestion is followed and now the Table 5 has given as supplementary material

Comment: Changes in Chl concentrations do not necessarily represent changes in photosynthesis, as suggested in discussion.

Response: Discussion has improved following the comments in red text

Round 2

Reviewer 1 Report

Authors have incorporated most of the comments, thank you.

Line 107: The new reference to a weblink is just inserted in the text, can authors use this in citation manager and properly cite it. 

line 177: Bring figure legend to justify alignment.

Line 194: Symbol for degree has underline, please remove

Line 242: Space between 0.5 and g

Lines 285-286: Insert spaces between the quantities and units as through out the test

Line 391: Figure 2: I really don't see the difference between the water stressed plants when treated with FeSO4 or Fe-Glu.

Figure 3: First graph on right hand is missing LSD value

Figure 5: Move the legend for this figure under the figure. Same as all other figures.

Discussion still requires adequate number of references to be added. Example: sentences in 4th paragraph have no references at all. And all the paragraphs thereafter also lack references. Add as appropriately.

Conclusions: The claims made in these conclusions solely rely on one years of field trial data. The presents a serious risk for farmers. To be statistically significant for a field application, this work should have been carried out at different locations and during at least 3 years.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Sustainability: Second Review:  “Fertigation of water-stressed ajwain plants with environment-friendly Fe-chelated glutamate confers a better induction for drought tolerance in comparison with FeSO4”  Qasim Ali1, Sumreena Shahid et al.

In addition to my original comments (see below), I believe the results overall , and conclusions are complicated by the fact that the control plants must be under a degree of Fe deficiency, as spraying with Fe solution of either chelate form leads to an increase in yield and other parameters. This makes it difficult to assess how much of the responses to Fe spray for the stressed plants is due to just Fe addition, or to some kind of induction of water stress tolerance, as suggested.  Making the assumption that the response is due to the latter would be incorrect, and the authors need  to take care in making specific claims on this.

My reviewer’s comments for my second review of the revised manuscript are appended to each point below (indicated by number as first or second review).

(Authors’) Responses to Reviewer 4

Comment (1): While the data represents a substantial amount of work and may show some significant indications of the benefit of Fe-glu over Fe SO4,

Response: Authors are thankful to the worthy reviewer for appreciating the outcomes.

Comment (1): in my view, it is generally difficult to fully appreciate the results and conclusions, due to the poor presentation. While I sympathise with authors for whom English may not be a first language, I found the English presentation to be poor with frequent clumsy, verbose, or meaningless sentences.

Response: Manuscript has been extensively improved in English language with help of an expert and all the incorporated improvements are highlighted in red

Comment (2): In my view the manuscript still can be significantly improved in English and made more succinct.

Comment (1): In addition, the Tables and Figures have inadequate legends. In view of these general comments I could have many minor specific comments, and would recommend that the manuscript be improved editorially to improve presentation before being resubmitted to a journal. A few more specific comments are listed below.

Response: Now the manuscript has been improve extensively in language, and presentation

Comment (2): in my view, the legends are still inadequate. I believe that Tables and Figures should be explained in the legend (including  abbreviations at the first time of use) to the extent that it is not necessary to read the text elsewhere in order to understand the details.

Comment (1): Tables 1,2,3  no legend outlining the abbreviations; results presented to too many decimal places (it is not possible to measure with confidence all these parameters to 2 or 3 decimal places);

Response: A list of abbreviation has incorporated at the end of Results section with Figure 5

Comment (2) See above. In some cases I still believe that there are too many decimal places.

Comment (1): the level of significance (1% or 5% ?) is not clearly indicated; some differences between treatments which apparently show significant differences (by superscripts) are well within the LSD values, which suggests they may not actually be significantly different.

Response: corrections ahs been incorporated in tables and results section in red text

Comment (2):  5% significance LSD has now been added. Are LSD values only applying to the non-stressed results, or combined for both the non-stresssed and stressed results ?  This is not clear.

Comment (!): Table 2. Chl a/b ratio appears to be decreased in some cases under water stress, in contrast to comments (p13).

Response: Correction has incorporated in text and tables

Comment (2) Earlier comment has been addressed.

Comment (1): Table 5 would be better included as an appendix.

Response: Suggestion is followed and now the Table 5 has given as supplementary material

Comment (2) Earlier comment has been addressed.

Comment (1): Changes in Chl concentrations do not necessarily represent changes in photosynthesis, as suggested in discussion.

Response: Discussion has improved following the comments in red text

Comment (2) Earlier comment has been addressed.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf