Foliar Biofortification of Maize (Zea mays L.) with Selenium: Effects of Compound Type, Application Rate, and Growth Stage

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

   The article addresses an interesting topic, presenting the possibility of introducing selenium into human nutrition, either directly or indirectly via meat consumption. However, the experiments were conducted in pots, which in my opinion are too small for maize cultivation. The authors conducted the experiment up to the grain harvest, using four plants per pot with a soil content of 5 kg. The Materials and Methods section lacks information on the number of days until the maize plants were harvested, as well as data on precipitation, temperature, and the size of the plants.

   Another issue is related to the statistical analyses. There are variables that showed no differences between the means, yet the authors state in the text that there were differences between treatments, which is not acceptable in scientific writing. Upon reviewing the means, it is clear that there is a large variation, but without statistically significant differences. When this occurs, it is usually due to a lack of normality in the data distribution or the presence of outliers. The Materials and Methods section does not mention that the authors performed a descriptive analysis of the data prior to conducting the analysis of variance.

   Another issue is with the statistical analysis of experiment 3, where the authors worked with doses. Since this is a quantitative factor of variation, a regression analysis should have been performed, rather than mean comparison tests. The conclusions need to be reviewed in light of the objectives. These general comments can be found in the annotations made in the PDF file of the article.

Comments for author File: Comments.pdf

Author Response

Question: Please add the name of the acronym in full.

Answer: Full explanation of acronym is in this book: Growth stages of mono-and dicotyledonous plants, Federal Biological Research Centre for Agriculture and Forestry, 2001.  In this book, the growth (phenological) phases for individual field crops are described in detail. Phenological stages have been added to the abstract and a reference to this book has been added in materials and methods. Line 19

Question: A photo of the experiment could be added. I think that pots of 5 L or 5 kg of soil are too small for growing corn, considering that the authors carried out the culture until grain production, with a total of 4 plants per pot.

Answer: The literature also presents 4 plants in one pot of similar size.

Nigussie, Ashenafi, et al. "Growth, nitrogen uptake of maize (Zea mays L.) and soil chemical properties, and responses to compost and nitrogen rates and their mixture on different textured soils: Pot experiment." Applied and Environmental Soil Science 2021.1 (2021): 9931763.;

Karim, Md Rezaul, et al. "Genotypic differences in zinc efficiency of Chinese maize evaluated in a pot experiment." Journal of the Science of Food and Agriculture 92.12 (2012): 2552-2559.

Question: Which system was used to classify the soil?

Answer: Classification was added and specified. Line 80

IUSS Working Group WRB. 2022. World Reference Base for Soil Resources. International soil classification system for naming soils and creating legends for soil maps. 4th edition. International Union of Soil Sciences (IUSS), Vienna, Austria

 

Question: What are the quantities of nutrients?

Answer: Quantity of nutrients was specified and which fertilizer was used. Line 91

Question: Better describe the phenological stage of the crop. Add, if possible, the number of days after seedling emergence.

Answer: Phenological phase of crop was specified and days after seedling emergence was added. Line 102

Question: If possible, add the number of days after seedling emergence.

Answer: Number of days after seedling emergence was added. 103

Question: It should be in the item: Selenium speciation analysis.

Table 3 shows the determination of SeCys2, which was not explained in the material and methods

Answer: It was moved and all species analyses were explained in Materials and Methods. Line 155

Question: All Se treatments presented higher averages than the control (without Se application) considering leaves, grains and straw. However, this was not verified for the Se content of the roots. Only the SeIV+Silwet and SeNPs+Silwet treatments showed higher levels of root Se when compared to the control.

The use of SeNPs resulted in the same leaf Se content when compared to the use of SeIV+Silwet.

Answer: It was modified and rewritten. Line 180

Question: This is true when SeNPs+Silwet was used. Note in figure 2 that the use of SeNPs does not present a difference in the Se contents of the grains when SeVI and SeVI+Silwet were used.

The use of SeNPs and SeNPs+Silwet was better when compared to SeIV and SeIV+Silwet treatments.

Answer: It was modified and rewritten. Line 187

Question: The results in Figure 2 do not support this statement. For Se content in grains, check that there is no difference in the means for SeIV and SeIV+Silwet. At the roots, there is no difference between treatments with and without Silwet. However, when we check the Se levels in the straw, we observe differences in the use of Silwet between the SeIV and SeIV+Silwet treatments and between SeNPs and SeNPS+Silwet.

Answer: It was modified and rewritten. Line 192

Question: There was no difference between means (Figure 3). The time of Se application did not influence grain productivity.

Answer: It was modified and rewritten. Line 203

Question: The comparison of means does not allow such a statement (Figure 3). Verify that only the BBCH60 treatment presented greater amounts of straw when compared to only the control.  The other treatments did not show differences in relation to the control.

Answer: It was verified, modified and rewritten. Line 214

Question: The application of Se foliar at stages BBCH51, 60 70, 30+51 and 52+60 resulted in an accumulation of Se in the straw when compared to the control.

Answer: It was written. Line 219

Question: Table 2 should be subdivided.

Answer: Table 2 and also Table 3 were  subdivided and every Se compound was specified in Materials and Methods. Line 264

Question: Again, there was no difference between means. Such a statement cannot be realized. Check the frequency distribution of the data, whether the distribution is normal. Check for the presence of outliers. There is a problem with the data.

Answer: The distribution of data was normal. The sentences was rewritten.

Question: Again, there is no difference in averages. The authors cannot make such a claim.

Answer: It was supplied by explanation.

Question: In this comparison of means, the letter "a" represented the highest average observed. Check that for the SeVI variable the letter "b" represents the highest average. Authors need to standardize this across the article.

Answer: It was corrected.

Question: Experiment 3 – Perform regression analyses

Answer: Regression analyses of individual maize parts was performer. Line 333

Question: It is also not a conclusion, it could be included in the results. And the data presented in the article do not provide grounds for such a statement.

This has not been tested, should not be in the conclusion.

Answer: The recommendations was moved into discussion part. Line 442

 

Best regards,

 

Authors

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Biofortification is a process of increasing the content of nutrients in edible parts of plants, which has gained wide recognition as one of the methods of providing microelements and vitamins to the human body. Microelement deficiencies such as selenium, iodine, iron, zinc or magnesium affect from one third to one half of the world's human population. For this reason, the topic taken up in the reviewed article is very important and current.

The purpose and scope of the work have been correctly defined, and the adopted methodology of the work allows for achieving the intended goal. Nevertheless, the reviewer has several comments and suggestions for supplementing or explaining the issues raised by the authors.

The main objection, in the reviewer's opinion, concerns the lack of explanation of the compound and dose used in Experiment 2 and only the compound in Experiment 3. On what basis did the authors choose sodium selenate and a dose of 100 ug? Were any studies conducted earlier? Or was the selection based on literature data?

The second objection concerns the discussion of the results, which is very cursory. Please delve deeper into their analysis. Additionally, tables 2-4 lack the standard deviation. Moreover, regarding statistics (same-name groups), please standardize the lower-case letters in the results. Sometimes the letter "a" is assigned to the lowest result and sometimes to the highest.

 

And the last problem concerns the Conclusions chapter. I have the impression that part of the manuscript (lines 412-439) does not concern this work. In earlier chapters, including Results and Discussion, the authors did not mention cow's milk and feeding cows biofortified corn. It would be necessary to supplement the earlier parts of the manuscript to be able to present such conclusions.

Additionally, below are some suggestions that I hope will help improve this work.

1. Please standardize the notation of selenites(IV), selenites(VI) and selenides throughout the manuscript. For example, in lines 37-40 and 52-53 there are different notation systems

2. Please clarify the statement (lines 51-52) - (…) the Se uptake is impeded by the large selenium reservoir and the proportion of available Se in the soil (...)

3. Table 1 - please try to edit it so that the SD is in the line with the result and not below

4. Lines 83-84 - please clarify what exact NPK fertilizer was used

5. Line 91 - please provide the selenium valency in SeNPs

6. Lines 94, 126, 127 - mL and not ml

7. Lines 142-143 - please list what selenium speciation forms were marked

8. All Figures - I suggest adding axis titles (Yield or Total Se concentration). Additionally, the figure captions state: (…)Different lowercase letters in the columns indicate(…) – what columns are you referring to?

9. Line 191 – about 1 mg/kg

10. Line 217 – what previous experiments?

11. Line 226 – species name in italics

 

12. Lines 347-348 – definitely different forms of Se in nanoparticles? And not just one?

Kind regards,

Reviewer

 

Author Response

Question:

The main objection, in the reviewer's opinion, concerns the lack of explanation of the compound and dose used in Experiment 2 and only the compound in Experiment 3. On what basis did the authors choose sodium selenate and a dose of 100 ug? Were any studies conducted earlier? Or was the selection based on literature data?

 

Answer:

We chose the applied form according to the results of experiment 1 and further as the most used applied form of selenium due to the results of other authors dealing with this issue.

We chose the dose from previous studies and found 3 possible increases in the applied dose by our experiment.

 

The second objection concerns the discussion of the results, which is very cursory. Please delve deeper into their analysis. Additionally, tables 2-4 lack the standard deviation. Moreover, regarding statistics (same-name groups), please standardize the lower-case letters in the results. Sometimes the letter "a" is assigned to the lowest result and sometimes to the highest.

 

Answer:

We added standard deviation to all tables and standardized all lower-case letters in results.

 

Question:

And the last problem concerns the Conclusions chapter. I have the impression that part of the manuscript (lines 412-439) does not concern this work. In earlier chapters, including Results and Discussion, the authors did not mention cow's milk and feeding cows biofortified corn. It would be necessary to supplement the earlier parts of the manuscript to be able to present such conclusions.

Answer:

Our intention was to recommend the optimal application rate  to produce  maize sufficiently selenized. At the request of reviewer 1, we have moved this section to the discussion.

Additionally, below are some suggestions that I hope will help improve this work.

1. Please standardize the notation of selenites(IV), selenites(VI) and selenides throughout the manuscript. For example, in lines 37-40 and 52-53 there are different notation systems

Answer: It was corrected.

2. Please clarify the statement (lines 51-52) - (…) the Se uptake is impeded by the large selenium reservoir and the proportion of available Se in the soil (...)

Answer: Clarification of location of reservoirs was added. Line 56

3. Table 1 - please try to edit it so that the SD is in the line with the result and not below

Answer: It was edited.

4. Lines 83-84 - please clarify what exact NPK fertilizer was used

Answer: The concentrations of NPK fertilizer (solution) was added. Line 91

5. Line 91 - please provide the selenium valency in SeNPs

Answer: Valency of Se was added. Line 100

6. Lines 94, 126, 127 - mL and not ml

Answer: It was corrected.

7. Lines 142-143 - please list what selenium speciation forms were marked

Answer: It was added. Line 155

8. All Figures - I suggest adding axis titles (Yield or Total Se concentration). Additionally, the figure captions state: (…)Different lowercase letters in the columns indicate(…) – what columns are you referring to?

Answer: It was corrected.

9. Line 191 – about 1 mg/kg

Answer: It was added. Line 212

10. Line 217 – what previous experiments?

Answer: Previous experiments was specified. Line 240

11. Line 226 – species name in italics

 Answer: Species names was put into italics.

12. Lines 347-348 – definitely different forms of Se in nanoparticles? And not just one?

Answer: It was corrected. Line 392

Best regards,

 

Authors

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript should mention the interaction or relationship of experiments

1.      In my opinion, the current title is a bit clumsy. Please change it with a new one which should be smoother, more concise, and more direct.

2.      A background should be provided before the objective statement in the abstract, i.e. why were they study performed?

3.      The methodology is a bit ambiguous in the abstract. Please make it clearer by presenting the forms of Se, the stages of growth, and the specific concentrations of Se.

4.      In line 16, what is “BBCH”? please avoid the abbreviation for the first time

5.      Due to the lack of a clear methodology, the results in the abstract are confusing and they should be presented significant findings and numeric data.

6.      The abstract should be mentioned the data

7.      Because the three experiments have been stated to be separate, these factors may influence each other, while these interactions have not been raised. Thus, I concern about the conclusion in the abstract.

8.      Please modify the keywords,

9.      In the introduction, the first and second paragraphs should be merged.

10.  The statements in lines 58-62 should be cited.

11.  The novelty and necessity are not clear in the introduction. They should be stated right before the objective statement or hypotheses.

12.  The materials and methods should be separated into sub-sections and formatted according to the template of MDPI.

13.  Why were the three Se compounds chosen?

14.  “BBCH” is still undefined in the main text.

15.  After reading the materials and methods, I still concern about the interactions between the 3 three factors. For example, increasing the rate of Se compounds which include Na could increase the Na content as well. The excessive amount of Na content could affect the maize plants at other growth stage.

16.  There should be soil analytic methods because of the presence of Table 1.

17.  To avoid the title ‘experiment 1’ in line 154; please add the title of these experiment; it means that the main meaning of experiment should be added

18.  The Figure 1 should be reconsidered because some treatments show too large differences to be identical, especially in root and stover. Please check other figures as well.

19.  The Figure 2 should be reformatted due to a large gap between values in leaf compared to other parts. This makes the data in other plant parts difficult to read.

20.  Figures and Tables should be self-explainable. Please defined all of the abbreviations, symbols, and patterns in each Table or Figure.

21.   Scientific names should be italicized.

22.  The English writing of the manuscript should be checked as well.

23.  The conclusion should be rewritten. It is too wordy while it should have focused on the main findings and further applications. The irrelevant details can be moved to the discussion.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Question:

The main objection, in the reviewer's opinion, concerns the lack of explanation of the compound and dose used in Experiment 2 and only the compound in Experiment 3. On what basis did the authors choose sodium selenate and a dose of 100 ug? Were any studies conducted earlier? Or was the selection based on literature data?

 

Answer:

We chose the applied form according to the results of experiment 1 and further as the most used applied form of selenium due to the results of other authors dealing with this issue.

We chose the dose from previous studies and found 3 possible increases in the applied dose by our experiment.

 

The second objection concerns the discussion of the results, which is very cursory. Please delve deeper into their analysis. Additionally, tables 2-4 lack the standard deviation. Moreover, regarding statistics (same-name groups), please standardize the lower-case letters in the results. Sometimes the letter "a" is assigned to the lowest result and sometimes to the highest.

 

Answer:

We added standard deviation to all tables and standardized all lower-case letters in results.

 

Question:

And the last problem concerns the Conclusions chapter. I have the impression that part of the manuscript (lines 412-439) does not concern this work. In earlier chapters, including Results and Discussion, the authors did not mention cow's milk and feeding cows biofortified corn. It would be necessary to supplement the earlier parts of the manuscript to be able to present such conclusions.

Answer:

Our intention was to recommend the optimal application rate  to produce  maize sufficiently selenized. At the request of reviewer 1, we have moved this section to the discussion.

Additionally, below are some suggestions that I hope will help improve this work.

1. Please standardize the notation of selenites(IV), selenites(VI) and selenides throughout the manuscript. For example, in lines 37-40 and 52-53 there are different notation systems

Answer: It was corrected.

2. Please clarify the statement (lines 51-52) - (…) the Se uptake is impeded by the large selenium reservoir and the proportion of available Se in the soil (...)

Answer: Clarification of location of reservoirs was added. Line 56

3. Table 1 - please try to edit it so that the SD is in the line with the result and not below

Answer: It was edited.

4. Lines 83-84 - please clarify what exact NPK fertilizer was used

Answer: The concentrations of NPK fertilizer (solution) was added. Line 91

5. Line 91 - please provide the selenium valency in SeNPs

Answer: Valency of Se was added. Line 100

6. Lines 94, 126, 127 - mL and not ml

Answer: It was corrected.

7. Lines 142-143 - please list what selenium speciation forms were marked

Answer: It was added. Line 155

8. All Figures - I suggest adding axis titles (Yield or Total Se concentration). Additionally, the figure captions state: (…)Different lowercase letters in the columns indicate(…) – what columns are you referring to?

Answer: It was corrected.

9. Line 191 – about 1 mg/kg

Answer: It was added. Line 212

10. Line 217 – what previous experiments?

Answer: Previous experiments was specified. Line 240

11. Line 226 – species name in italics

 Answer: Species names was put into italics.

12. Lines 347-348 – definitely different forms of Se in nanoparticles? And not just one?

Answer: It was corrected. Line 392

Best regards,

 

Authors

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

thank you for explaining the inaccuracies and correcting the manuscript according to the proposed changes. In the opinion of the reviewer, the current version of the manuscript has been improved enough to be published in the Agriculture journal.

Kind regards,

Reviewer