A Biostimulant from Kappaphycus alvarezii Enhances the Growth and Development of Basil (Ocimum basilicum L.) Plants

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presented for the review describes the results of a single experiment with foliar treatment of basil plants with the extracts of Kappaphycus alvarezii. The topic undertaken is of significant importance with respect to the possibiloty of applying natural products as biostimulants.

I have some concerns regarding the work which are listed below:

1. Rephrase the title.
2. Introduction:
   1. please provide some details regarding the improvement in the performance of different plants species as affected by the marine algae extracts, why that approach seems promising and what are potential drawbacks or difficulties that needs to be tested etc.
   2. Please provide some justification for choosing basil plants as a subject of the study;
3. Material and methods:
   1. In my opinion some further evaluation of the physicochemical characterisitcs of collected algae and its extracts needs to be provided; you only mention the collection site and the colour; no information is given on the possible bioactive compounds present in the tested material, that would contribute to the effect on basil plants. Event though the very wide range of chemical analyses is performed, it cannot be concluded as to the mechanisms underlying the effects of these particular extracts.
   2. The times of foliar spraying should be specified, including plant growth stages
   3. I appreciate wide chemical analyses of plants
4. Results:
   1. In my opinion it is not quite correct to calculate, compare and discuss the “overall mean” for different treatments (Table 1 and further), please explain the approach. In my opinion it does not add any additional merit, as in fact you wanted to indicate the optimal dose of biostimulant. What is more, sometimes the differences between locations as measured by these averages were most likely affected by i.e. various control values for each block, as in the case of TPC or TS. In may opinion you should focus more on the effect of respective extracts within the subblock – it is mpre informative.
   2. Lines 360-365 – could be transferred to the introduction section
   3. Instead of States you should rather write: locations when comparing the results and in the escription of tables.
   4. I would suggest also to use the heat map for presenting the effect of various treatment not only for fatty acids and biogenic amines but also for other important characteristics of basil plants. I my opinion it would clearly indicate the choice of an optimal dose of respective extracts in terms of it effect on plant growth and chemical composition.
5. Some additional remarks are given in the attached pdf file.

To sum up, in my opinion after making some necesary corrections as indicated above the manuscript can be considered to be published.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The quality of English needs improvement, some but not all remarks are made in the attached file - pdf version of the manuscript with my comments

Author Response

Responses to the Reviewer

 

Reviewer #1

The manuscript presented for the review describes the results of a single experiment with foliar treatment of basil plants with the extracts of Kappaphycus alvarezii. The topic undertaken is of significant importance with respect to the possibiloty of applying natural products as biostimulants.

I have some concerns regarding the work which are listed below:

R: We appreciate the reviewer's comment and below we highlight the adjustments made to the manuscript following the suggestions.

 

Rephrase the title.

R: Taking into consideration the comments from Reviewer #1, as well as those from Reviewers #2, the title has been changed to “Biostimulant from Kappaphycus alvarezii enhance the growth and development of basil (Ocimum basilicum L.) plants”

 

Introduction:

please provide some details regarding the improvement in the performance of different plants species as affected by the marine algae extracts, why that approach seems promising and what are potential drawbacks or difficulties that needs to be tested etc.

R: Adjusted as requested, including a published work from the same research group, which shows various studies on biostimulants in plants: “In a review conducted by Munaro et al. (2022), it is noted that the various metabolites found in micro and macroalgae have significant effects on plants. These effects include increased germination rates, enhancement in length, weight, and growth of both aerial and root parts, as well as early flowering. Additionally, there is an intensification of pigments such as chlorophylls and carotenoids, along with primary and secondary metabolites. Thus, the use of algal-based biostimulants proves to be promising for promoting sustainable and productive agriculture”.

 

 

 

Please provide some justification for choosing basil plants as a subject of the study;

R: Added as requested: “Basil is not only a widely used culinary herb but also has significant medicinal properties, making it an important crop for both economic and health reasons”.

 

Material and methods:

In my opinion some further evaluation of the physicochemical characterisitcs of collected algae and its extracts needs to be provided; you only mention the collection site and the colour; no information is given on the possible bioactive compounds present in the tested material, that would contribute to the effect on basil plants. Event though the very wide range of chemical analyses is performed, it cannot be concluded as to the mechanisms underlying the effects of these particular extracts.

R: We appreciate the reviewer's consideration and understand the importance of this analysis. To address this, we have included supplementary material regarding the results obtained from biochemical analysis as well as the profile obtained from nuclear magnetic resonance (NMR).

 

The times of foliar spraying should be specified, including plant growth stages

I appreciate wide chemical analyses of plants

R: Adjusted as requested: “The application of the biostimulant of K. alvarezii was carried out weekly using a spray bottle, totaling five foliar applications. The times of foliar spraying were specified according to the plant growth stages to optimize absorption and effectiveness. The application volume was defined according to the plants' leaf growth; therefore, the following were carried out: 1st application – two sprays (1 mL per plant) during the early growth stage; 2nd application – three sprays (1.5 mL per plant) at the vegetative stage; 3rd application – five sprays (2.5 mL per plant) at the vegetative stage; 4th application – seven sprays (3.5 mL per plant) during the at the mid-growth stage; 5th application – twelve sprays (6 mL per plant) at the pre-flowering stage”.

 

 

 

 

Results:

In my opinion it is not quite correct to calculate, compare and discuss the “overall mean” for different treatments (Table 1 and further), please explain the approach. In my opinion it does not add any additional merit, as in fact you wanted to indicate the optimal dose of biostimulant. What is more, sometimes the differences between locations as measured by these averages were most likely affected by i.e. various control values for each block, as in the case of TPC or TS. In may opinion you should focus more on the effect of respective extracts within the subblock – it is mpre informative.

R: We understand the reviewer's consideration; however, we have added an additional paragraph in the “Materials and Methods” section to justify why we used the analysis in this format: “It is important to highlight that, due to the individualized experiments – i.e., with separate applications of Kal-SP and Kal-S – the statistical analysis was conducted considering the biostimulants separately, as both were cultivated with an individual control treatment. All data were analyzed by AgroEstat software (v. 1.1.0.712).”

 

Lines 360-365 – could be transferred to the introduction section

R: Added as requested

 

Instead of States you should rather write: locations when comparing the results and in the description of tables.

R: We appreciate the reviewer's comment. Indeed, this way it is much better. We have made adjustments throughout the manuscript.

 

I would suggest also to use the heat map for presenting the effect of various treatment not only for fatty acids and biogenic amines but also for other important characteristics of basil plants. I my opinion it would clearly indicate the choice of an optimal dose of respective extracts in terms of it effect on plant growth and chemical composition.

R: We understand that this indeed makes for easier visualization. We have adjusted all the tables as suggested by the reviewer.

 

 

 

Some additional remarks are given in the attached pdf file.

R: All points mentioned in the PDF have been corrected as requested. We emphasize that the manuscript, with all adjustments, is marked in the document.

 

To sum up, in my opinion after making some necessary corrections as indicated above the manuscript can be considered to be published.

R: We thank you for all the considerations made. All suggestions were very welcome and accepted, which improved the manuscript for potential publication.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The main question is:

Do you have any results about the chemical composition of the extracts (Kal-SP and Kal-SC)? Do you know any differences of them? To evaluate scientificly the effect of these two extracts as biofertilizer need some analyses about its physical and chemical properties: pH, phyto-chemicals, nutrients. Its necessary, because we are writing about the effects of something without knowing any background.

Anyway only some small corrections:

Not correct: Kappaphycus alvarezii is not a biofertilizer itself, its a algae (as the authors made clear later), thus the title is not correct. My suggestion is:

Biostimulant from Kappaphycus alvarezii enhance the growth and development of basil (Ocimum basilicum L.) plants

Formating problem with chemical formulas (subscripts).

Line 111 As I suggested before: biostimulants ’from’ not”of”.

Line 314 If you accept p=0.05 as significant result my suggestion is to correct p≤0.05.

Line 316 Not clear: „except for the leaf and dry root”.

Line 161 chlorophyll-a, chlorophyll-b, use hyphen everywhere in the manuscript

Author Response

Reviewer #2

Do you have any results about the chemical composition of the extracts (Kal-SP and Kal-SC)? Do you know any differences of them? To evaluate scientificly the effect of these two extracts as biofertilizer need some analyses about its physical and chemical properties: pH, phyto-chemicals, nutrients. Its necessary, because we are writing about the effects of something without knowing any background.

R: We agree with the point made by Reviewer 2, which was also mentioned by Reviewer 1. Therefore, we have included supplementary material regarding the results obtained from biochemical analysis as well as the profile obtained from nuclear magnetic resonance (NMR).

 

Anyway only some small corrections:

Not correct: Kappaphycus alvarezii is not a biofertilizer itself, its a algae (as the authors made clear later), thus the title is not correct. My suggestion is:

Biostimulant from Kappaphycus alvarezii enhance the growth and development of basil (Ocimum basilicum L.) plants

R: Title modified as suggested.

 

Formating problem with chemical formulas (subscripts).

R: We understand the point mentioned; however, based on biochemical literature, we have found that for fatty acids, the number of carbons and the position of unsaturation should be represented without subscripts, as it is not the minimum formula. Therefore, we adhere to the definitions provided by the International Union of Pure and Applied Chemistry (IUPAC – https://iupac.qmul.ac.uk/lipid/appABC.html#appA).

 

Line 111 As I suggested before: biostimulants ’from’ not”of”.

R: Adjusted as requested.

 

Line 314 If you accept p=0.05 as significant result my suggestion is to correct p≤0.05.

R: Adjusted as requested.

 

Line 316 Not clear: „except for the leaf and dry root”.

R: Adjusted as requested: “The results for the biochemical parameters were based on true triplicates, involving the analysis of three individual plants per treatment. Each plant was analyzed in triplicate, resulting in a total of nine repetitions per treatment for all analyses”.

 

Line 161 chlorophyll-a, chlorophyll-b, use hyphen everywhere in the manuscript.

R: Adjusted as requested.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript is devoted to the evaluation of the effect of Kappaphycus alvarezii extract on productivity indicators and a wide range of biochemical components in basil plants grown hydroponically. This work is of great scientific and practical interest, as it is aimed at creating natural plant growth stimulants and their use in protected ground conditions.

The study deserves a generally positive assessment; however, there are a number of points that require clarification.

 

1. The title of the article needs to be corrected in accordance with the rules of English grammar.
2. Additional clarifications are needed in the methodological section:
3. How was the extract solution (1-3-5-7%) obtained? By diluting the original extract in distilled water? Please specify.
4. Provide more detail on the basil cultivation conditions (Did you use a greenhouse? Specify the photoperiod, temperature, humidity, etc.).
5. What methods/instruments were used to measure the root length and height of basil plants?
6. Line 253 - should be standardized: one hundred = 100 mg (as elsewhere in the text).
7. Lines 253-254: correct, possibly remove unnecessary details (500 µl, 500 µl, 480 µl).
8. In line 270, the authors used the abbreviation FA - it is necessary to provide a breakdown, since this abbreviation has not been used before.
9. Section 3.
10. Table 1 - the font size should be increased. Please, here and in the tables below, indicate which indicators are given in the table. Mean value and mean error? In the table, the authors use the indicator "root size", but in reality, it is the length of the roots. These are different indicators, it is necessary to use the one that was actually determined.
11. The authors found an increase in number of nodes in the 3% and 5% Santa Catarina extract treatments. Was this due to an increase in shoot number? Leaves? Why did not the increase in number of nodes result in an increase in plant biomass?
12. I don't understand how the TCN data in the São Paulo treatments can be reliably different from each other? Especially: 1% 0.16 ± 0.01 b and 7% 0.16 ± 0.01 c? The reliability of the differences in other columns is also questionable based on the data presented in the table.
13. In lines 440-445 it is necessary to unify mL/L and mL.L−1.
14. Based on the results obtained, the differences in the response of basil to treatment with seaweed extracts from different regions are probably explained by differences in the biochemical composition of the extracts. Do the authors have data (including literature) to support this assumption? Or is there something else to the reason? This should be discussed in more detail.
15. The authors included only "phenolic components" in the keywords. In fact, there were many parameters under study. Therefore, I propose using the terms "productivity" and "biochemical composition" - they more accurately reflect the results of this study.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Minor editing of English is needed

Author Response

The manuscript is devoted to the evaluation of the effect of Kappaphycus alvarezii extract on productivity indicators and a wide range of biochemical components in basil plants grown hydroponically. This work is of great scientific and practical interest, as it is aimed at creating natural plant growth stimulants and their use in protected ground conditions.

The study deserves a generally positive assessment; however, there are a number of points that require clarification.

R: We appreciate the initial description of the evaluation and all the considerations made. Below, we respond point by point to the adjustments made in accordance with the reviewer's suggestions.

 

1. The title of the article needs to be corrected in accordance with the rules of English grammar.

R: In response to the considerations made by all reviewers (Reviewer #1, #2, and #3), the title has been changed to: “Biostimulant from Kappaphycus alvarezii enhance the growth and development of basil (Ocimum basilicum L.) plants”.

 

Additional clarifications are needed in the methodological section:

1. How was the extract solution (1-3-5-7%) obtained? By diluting the original extract in distilled water? Please specify.

R: We have adjusted the text as requested: “To obtain the concentrations, the pure biostimulant (100%) was diluted in distilled water”.

 

1. Provide more detail on the basil cultivation conditions (Did you use a greenhouse? Specify the photoperiod, temperature, humidity, etc.).

R: Indeed, this is very important information, and it has been included in the manuscript: “The experiment was conducted in a single-span arch greenhouse covered with a 0.1 mm transparent polyethylene film and consisted of a hydroponic system supported by an aluminum structure that provided a slope of 1.5% from the entrance to the exit. The pumps circulated the solution for 10 minutes in a 10 min cycle from 8 AM to 6 PM and for 10 min in a 1h cycle from 6 PM to 8 AM the next day.

During the cultivation period, from April 1 to May 9, 2024, the average maximum air temperature recorded was 30 ºC ± 3.5, and the minimum was 20.9 ºC ± 2.3. The average humidity varied from 90.7% ± 2.77 to 70.2% ± 11.1, and the photoperiod was 12/12 (light/dark)”.

 

1. What methods/instruments were used to measure the root length and height of basil plants?

R: Information added as suggested: “To measure the height of the aerial part and the length of the roots of the basil plants, a ruler was used for direct measurement. In addition, an analytical balance was used to weigh the plants”.

 

1. Line 253 - should be standardized: one hundred = 100 mg (as elsewhere in the text).

R: Adjusted as requested.

 

1. Lines 253-254: correct, possibly remove unnecessary details (500 µl, 500 µl, 480 µl).

R: Adjusted as requested.

 

1. In line 270, the authors used the abbreviation FA - it is necessary to provide a breakdown, since this abbreviation has not been used before.

R: Adjusted as requested.

 

Section 3.

1. Table 1 - the font size should be increased. Please, here and in the tables below, indicate which indicators are given in the table. Mean value and mean error? In the table, the authors use the indicator "root size", but in reality, it is the length of the roots. These are different indicators, it is necessary to use the one that was actually determined.

R: Adjusted as requested, including an increase in the font size of the tables, the indication that the values represent the mean and standard deviation, and the modification of "root size" to "length of the roots," which has been included in the text, figure and table captions.

 

1. The authors found an increase in number of nodes in the 3% and 5% Santa Catarina extract treatments. Was this due to an increase in shoot number? Leaves? Why did not the increase in number of nodes result in an increase in plant biomass?

R: We understand the highlighted point; however, the plants that exhibited an increase in the number of nodes did not show a corresponding increase in plant height, which would have contributed to biomass. Thus, this factor may be related solely to the development of additional branches rather than larger or broader leaves. Consequently, the statistical analysis did not reveal any significant differences among the treatments.

 

1. I don't understand how the TCN data in the São Paulo treatments can be reliably different from each other? Especially: 1% 0.16 ± 0.01 b and 7% 0.16 ± 0.01 c? The reliability of the differences in other columns is also questionable based on the data presented in the table.

R: The reviewer is correct in their observation, as we initially used only two decimal places, which could lead to rounding errors given the small differences. To avoid any confusion for the reader, we have included values rounded to four decimal places in this analysis.

 

1. In lines 440-445 it is necessary to unify mL/L and mL.L−1.

R: Adjusted as requested.

 

1. Based on the results obtained, the differences in the response of basil to treatment with seaweed extracts from different regions are probably explained by differences in the biochemical composition of the extracts. Do the authors have data (including literature) to support this assumption? Or is there something else to the reason? This should be discussed in more detail.

R: Indeed, this is a very important point. As requested by reviewers #1 and #2, we have included biochemical analyses and the profile obtained from nuclear magnetic resonance (NMR) in the supplementary material. Additionally, at the end of the manuscript, we have added a paragraph addressing the issue raised by the reviewer: “Additionally, it is important to note that there is a divergence, particularly for the 7% concentration treatment between Kal-SP and Kal-SC. This can be explained by the higher concentration leading to greater utilization of algal biomass. The composition of the biostimulant revealed differences that can be attributed to variations in total phenolic content, total flavonoid content, total carotenoids, total carbohydrates, total soluble sugars, total starch, and total protein. Furthermore, the metabolomic profile demonstrated significant differences in content, especially regarding amino acids and organic acids (see supplementary material – Table S1 and Table S2). Thus, the composition of the biostimulant can influence the morphological and biochemical outcomes in plants. However, it is important to highlight potential interference from higher concentrations, as there appears to be a consistent pattern for lower (1%) and medium (3% and 5%) concentrations”.

 

1. The authors included only "phenolic components" in the keywords. In fact, there were many parameters under study. Therefore, I propose using the terms "productivity" and "biochemical composition" - they more accurately reflect the results of this study.

R: We appreciate the feedback, and these adjustments have been made with the substitution of “phenolic components”.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I appreciate the changes that have been made to the manuscript. In fact most of the comments have been addresed as suggested. Regarding the “overall means” despite your explanation, I still think it is not an appropriate approach for that research, and they could or even should be easily removed from the results presentation and discussion without losing any significant merit.

After rereading and further consultation of the manuscript I have major doubts about the results of fatty acid analysis. The percentage share of capric acid (C10) in total fatty acids is far too high for most combinations. It is quite unlikely that a single fatty acid, or not even a major one, contributes over 90% of all fatty acids in plant material. Also behenic acid (C22) also is not as abundant in plants as your results suggest. The main fatty acids found in plants, including basil, are those with 16-20 carbon atoms, both saturated and unsaturated, including palmitic, linoleic, oleic acids etc. For references see:

• Vlaicu, P. A., Untea, A. E., Turcu, R. P., Saracila, M., Panaite, T. D., & Cornescu, G. M. (2022). Nutritional composition and bioactive compounds of basil, thyme and sage plant additives and their functionality on broiler thigh meat quality. Foods, 11(8), 1105;
• Tarchoune, I., Baâtour, O., Harrathi, J., Hamdaoui, G., Lachaâl, M., Ouerghi, Z., & Marzouk, B. (2013). Effects of two sodium salts on fatty acid and essential oil composition of basil (Ocimum basilicum L.) leaves. Acta physiologiae plantarum, 35, 2365-2372;

No valid explanation or even attention have been given to such unusual results . Moreover, one of the above mentioned references (Tarchoune et al. 2013) is cited in the manuscript as a source of similar results as presented in the work, which is simply not true. Therefore the whole section of fatty acid analysis should be deleted or the results recalculated if, hopefully, it is only  some kind of a mistake. 

Also units of the content of the respective compounds in the K. alvarezii material should be added into Tables S1 and S2 (supplementary material)

Additional comments are made in the pdf file.

Therefore in my opinion, the manuscript can be considered for publication but only after making additional improvements as mabove with a clear and valid statement or decision regarding the results of fatty acid analysis.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

In my opinion the manuscript should be proof-read by someone more fluent in English. 

Author Response

Dear Authors,

 

I appreciate the changes that have been made to the manuscript. In fact most of the comments have been addresed as suggested.

R: We appreciate all the adjustments that were previously discussed and those made in this second round. Your feedback has significantly contributed to the enhancement of the manuscript. Thank you for the time you dedicated to these analyses.

 

Regarding the “overall means” despite your explanation, I still think it is not an appropriate approach for that research, and they could or even should be easily removed from the results presentation and discussion without losing any significant merit.

R: We agree with the highlighted point and have removed it from the tables as well as from the text.

 

After rereading and further consultation of the manuscript I have major doubts about the results of fatty acid analysis. The percentage share of capric acid (C10) in total fatty acids is far too high for most combinations. It is quite unlikely that a single fatty acid, or not even a major one, contributes over 90% of all fatty acids in plant material. Also behenic acid (C22) also is not as abundant in plants as your results suggest. The main fatty acids found in plants, including basil, are those with 16-20 carbon atoms, both saturated and unsaturated, including palmitic, linoleic, oleic acids etc. For references see:

 

Vlaicu, P. A., Untea, A. E., Turcu, R. P., Saracila, M., Panaite, T. D., & Cornescu, G. M. (2022). Nutritional composition and bioactive compounds of basil, thyme and sage plant additives and their functionality on broiler thigh meat quality. Foods, 11(8), 1105;

Tarchoune, I., Baâtour, O., Harrathi, J., Hamdaoui, G., Lachaâl, M., Ouerghi, Z., & Marzouk, B. (2013). Effects of two sodium salts on fatty acid and essential oil composition of basil (Ocimum basilicum L.) leaves. Acta physiologiae plantarum, 35, 2365-2372;

 

No valid explanation or even attention have been given to such unusual results . Moreover, one of the above mentioned references (Tarchoune et al. 2013) is cited in the manuscript as a source of similar results as presented in the work, which is simply not true. Therefore the whole section of fatty acid analysis should be deleted or the results recalculated if, hopefully, it is only  some kind of a mistake.

R: We reanalyzed the data; however, we indeed obtained the results as shown. This may have been due to the column used. Therefore, as requested, we have removed all sections related to fatty acids from the manuscript. Thus, the PCA was redone with all data, without fatty acids.

 

Also units of the content of the respective compounds in the K. alvarezii material should be added into Tables S1 and S2 (supplementary material)

R: We apologize for the previous omission. The content has been appropriately adjusted.

 

Additional comments are made in the pdf file.

R: Adjusted as requested. The only issue that did not change is the comment 'Commercial name of the cultivar,' as no cultivar is highlighted, only the scientific name of the species.

 

Therefore in my opinion, the manuscript can be considered for publication but only after making additional improvements as mabove with a clear and valid statement or decision

We appreciate your feedback. Regarding the fatty acids, we have chosen to remove the results to prevent any confusion for the readers.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for accepting all of my suggestion.

Author Response

Thank you for accepting all of my suggestion.

R: We thank you once again for all the points raised by the reviewer, which greatly assist in improving the manuscript.

Author Response File: Author Response.pdf