Microbial Biostimulants and Seaweed Extract Synergistically Influence Seedling Growth and Morphology of Three Onion Cultivars

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please address the following:

- In Figure 1: add standard deviation

 - Why at 17 DAS the germination was measured is there an explanation? why not at 1 week? or 3, 4 weeks?

- Add in Table 1 a column indicated the abbreviation of products which are used in the following tables.

- “Spectrum” and “Spectrum DS” are the same? see Table 1.

- Discussion should be improved regarding the following topis:

-        -  The germination differences between cultivars in other onion varieties or crops in the literature.

-     -     Which components in the seaweed product could be important for the better results obtained?

-     -     Which physiological response are affected in the plants due to the application of the different biostimulants

Comments on the Quality of English Language

I think english could be improved. For instance, line 198, not start with "As shown in Table 2...", the table could be refered at the end of the senetce in brackets: "Onion germinations was not affected....seaweed extract (Table 2)."

Author Response

Responses to Reviewer 1.

We are very grateful for your insightful comments and suggestions to improve our manuscript. We revised the manuscript according to your comments. Please see the following responses to your valuable comments (all the line numbers refer to the revised manuscript with track-change enabled).

Comments and Suggestions for Authors

- In Figure 1: add standard deviation

Response: Thank you for your suggestion. We included the standard error bars at the top of each bar. The standard error of germination rates ranged from 0.44 to 0.60, while the standard error of relative germination rates ranged from 0.0044 to 0.0070. Therefore, it might be difficult to discern error bars in the figure. Based on your suggestion, we supplemented detailed descriptions of the statistical analyses to the Figure 1 caption to enhance the clarity.

 - Why at 17 DAS the germination was measured is there an explanation? why not at 1 week? or 3, 4 weeks?

Response: Thanks for your question. Due to the substantial number of onion seedlings in this study (6720 plant cells need to be investigated for germination data collection), we only collected germination data once. As no new seedling emergence was observed after a few days, data collection was finalized at 17 days after sowing (DAS). We acknowledge that collecting germination data at 2 or 3 weeks after sowing might enhance comparability with other studies and will incorporate this into our future research methods.

- Add in Table 1 a column indicated the abbreviation of products which are used in the following tables.

Response: Thanks for your valuable advice. We have added a column indicating the abbreviation of products to Table 1 as suggested. We believe these changes will improve the manuscript's readability.

- “Spectrum” and “Spectrum DS” are the same? see Table 1.

Response: Thanks for your question. No, they are different products produced by the same manufacturer. “Spectrum DS” is especially designed for drought-stressed and high salinity soils. We have supplemented this information into Table 1.

- Discussion should be improved regarding the following topics:

- The germination differences between cultivars in other onion varieties or crops in the literature.

Response: Thanks for your valuable advice. We have added relevant discussion to lines 233-240.

- Which components in the seaweed product could be important for the better results obtained?

Response: Thank you for your question. Seaweed extracts are known to contain plant hormones, potassium, and bacteriostatic polysaccharides, which may contribute to their beneficial effects on plant growth. We have supplemented studies on this product (Kelpak) on tomato seedling and onion to make comparisons with our study. Besides, we discussed the plant growth-promoting components contained in this product (Lines 362-370).

- Which physiological response are affected in the plants due to the application of the different biostimulants.

Response: Thanks for your suggestion. We found plant height, leaf area, shoot DW, root DW, root/shoot, and root length of onion seedlings were improved by different biostimulants. We added a summary of the parameters that were significantly affected by the application of different biostimulants in lines 457-461. Besides, we discussed further about the synergistic effects of microbial biostimulants and seaweed extract Kelpak (Lines 462-471).

 

Comments on the Quality of English Language

I think english could be improved. For instance, line 198, not start with "As shown in Table 2...", the table could be refered at the end of the senetce in brackets: "Onion germinations was not affected....seaweed extract (Table 2)."

Response: Thank you for your suggestion. We have revised lines 228-229 accordingly. Additionally, we have revised other places with similar issues (lines 260-263, 313-318).

Reviewer 2 Report

Comments and Suggestions for Authors

Recommendations for authors

 1.      The discussion was somewhat limited, specifically in explaining how the physiological responses were influenced by the treatments. On the other hand, no data were generated to make inferences, but I consider it important that some documented effects in similar experiments can be exposed.

 

2.      The authors suggest a long-term effect; however, the experiment is carried out in a single agricultural cycle (October to December 2022). This does not imply that the information generated is not relevant, but rather that the projection of the experiment does not reveal the influence of the long-term treatments, as indicated in the manuscript.

 

3.      The interpretation of the tables is somewhat difficult to understand, even more so with so many levels of significance (0.05, 0.01 and 0.001), so a schematic table where the values ​​that mark the statistical differences can be clearly seen would be very helpful. and more graphics.

 

4.      I agree that any effect that an agricultural input may have been important, however, the effect of using these commercial products did not make a difference in relation to root growth and biomass. For example, in Figure 1, a germination graph is presented between the three varieties, but it does not indicate the effect of any stimulant, nor of the positive control.

 

5.      Tables 3 to 8 present the results in terms of number of leaves, plant height, stem diameter and leaf area, however, it is not clear to see the effect of the controls and the interaction between the treatments.

 

6.      It seems to me that the information in the tables is a little confused in the way it is presented, in fact, since the results come from the same treatments, it is advisable to present a composite table where the most important and relevant data are compiled. Moreover, consider that number of leaves is a characteristic marked by effect of genetic, rather than by the influence an external stimulant, at least in the first phenological stages.

 

7.      If any synergistic effect occurs, it is necessary to better document it and interpret it from a statistical and biological point of view. Particularly, because if there is a synergism, therefore, I consider it important to discuss this point further.

 

8.      The Ithenticate analyses (19%) It seems a little high to me, I recommend working on the document a little more to lower those similarity values.

Author Response

Responses to Reviewer 2

We appreciate your time and efforts in providing valuable comments on this manuscript. We have revised the manuscript accordingly. Below are our point-by-point responses to your comments (all the line numbers refer to the revised manuscript with track-change enabled):

1. The discussion was somewhat limited, specifically in explaining how the physiological responses were influenced by the treatments. On the other hand, no data were generated to make inferences, but I consider it important that some documented effects in similar experiments can be exposed.

Response: Thanks for your valuable suggestion and pointing out the areas to be improved. Due to a lack of equipment for measuring physiological responses in onion leaves, we did not have any data on their physiological responses. We have improved the discussion session by comparing our results with similar studies (lines 233-240, lines 360-370), and summarizing the physiological responses that were affected by the biostimulants treatments (lines 457-461). Besides, we added a discussion about the synergistic effects of microbial biostimulants and seaweed extract Kelpak to lines 462-471 as suggested.

2. The authors suggest a long-term effect; however, the experiment is carried out in a single agricultural cycle (October to December 2022). This does not imply that the information generated is not relevant, but rather that the projection of the experiment does not reveal the influence of the long-term treatments, as indicated in the manuscript.

Response: Thanks for your valuable feedback. We understand your concern. We suggest a long-term study is necessary or as subsequent/follow-up research in our conclusion /abstract. We simply indicate the limitation of our results. In fact, we did conduct a follow-up study and the results are available. We are in the process of developing the manuscript.

3. The interpretation of the tables is somewhat difficult to understand, even more so with so many levels of significance (0.05, 0.01 and 0.001), so a schematic table where the values ​​that mark the statistical differences can be clearly seen would be very helpful. and more graphics.

Response: Thanks for your insightful comment. Different levels of significance are only presented in Table 2, significant differences in other tables and figures are indicated by Tukey’s HSD test at p<0.05. The reason we present Table 2 is to show the significance level of main effects and their interactions. To enhance clarity and simplify the presentation, we removed the F-values in Table 2 as significance levels are more informative. We understand graphics marked with statistical differences would be more informative to audiences. However, this study was designed to screen a wide range of biostimulants products (9 microbial biostimulants + 1 seaweed extract) on 3 different onion cultivars, the significant interactions (as shown in Table 2) between experimental factors made it difficult to present data in graphics.

4. I agree that any effect that an agricultural input may have been important, however, the effect of using these commercial products did not make a difference in relation to root growth and biomass. For example, in Figure 1, a germination graph is presented between the three varieties, but it does not indicate the effect of any stimulant, nor of the positive control.

Response: Thank you for your insightful comment. As shown in columns 3 and 4 (counting from left) of Table 2, both microbial biostimulants and the seaweed extract Kelpak significantly influenced seedling biomass and root morphology. However, neither microbial biostimulants nor Kelpak affected germination (indicated by 'NS' in Table 2). Therefore, germination data were pooled across biostimulant treatments and analyzed solely by cultivar for Figure 1. We added the explanation in Lines 231-232 to enhance clarity.

5. Tables 3 to 8 present the results in terms of number of leaves, plant height, stem diameter and leaf area, however, it is not clear to see the effect of the controls and the interaction between the treatments.

Response: Thank you for your feedback. The significant levels of experimental factor interactions are presented in Table 2. The effect of controls or the difference between biostimulants and related controls are detailed in Tables 3 to 8. You might want to emphasize the effect of biostimulants comparing to related controls is more important than the main effects, which we agree. Therefore, we discussed the difference between microbial biostimulants and related controls in Results and Discussion sessions for each Table. For example, in Lines 270-272, we compared the Spectrum DS to related control, and found that the plant height was increased by 8%. Similar comparisons can be found in L272-273, 279-280, 281-283, 326-327, etc.

6. It seems to me that the information in the tables is a little confused in the way it is presented, in fact, since the results come from the same treatments, it is advisable to present a composite table where the most important and relevant data are compiled. Moreover, consider that number of leaves is a characteristic marked by effect of genetic, rather than by the influence an external stimulant, at least in the first phenological stages.

Response: We appreciate your feedback and valuable advice. We tried our best to tell the story smoothly through introducing the Three-way ANOVA results in Table 2 first; due to the significant interactions across experimental factors, listing microbial biostimulants’ effects under different onion cultivars, with or without seaweed extract applications, respectively; and wrap up the results in PCA biplots. The PCA biplots can present some correlations between parameters. In addition, we added one paragraph summarizing and explaining the synergistic effects of microbial biostimulants and seaweed extract Kelpak. To improve the readability of our manuscript, we supplemented more discussion explaining our stories and the reasons why we gave these conclusions.

From our results, as shown in Table 2 and Figure 4, microbial biostimulants affected the leaf number significantly. Leaf number can reflect the plant growth rate of onion seedlings since they will produce new leaves earlier than others if they grow faster.

7. If any synergistic effect occurs, it is necessary to better document it and interpret it from a statistical and biological point of view. Particularly, because if there is a synergism, therefore, I consider it important to discuss this point further.

Response: Thanks for pointing out this. We have supplemented related discussion in lines 456-471. We summarized the parameters that were significantly affected by the biostimulant treatments, explained the synergistic effect in a statistical way (Table 2, lines 462-463) and biological point of view (combined application resulted in more positive effects on the parameters collected in this study, lines 457-461).

8. The Ithenticate analyses (19%) It seems a little high to me, I recommend working on the document a little more to lower those similarity values.

Response: Thanks for your feedback. We obtained the Ithenticate report from the Editor. The Similarity Index was 15%. According to your recommendation, we have carefully revised the whole manuscript based on the Ithenticate report to lower the similarity index.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors, it is interesting work and it is good to know that you have done huge work. As my purpose is to review your work, I have some questions and suggestions for you.

First of all, when ever you speak about biostimulants, please be ready to explain them. In all text it is missing.

You will see my suggestions or/and questions, please answer them, or apply, when ever I ask for, information.

Introduction needs more information. 

MM chapter needs more explanations, as you will se by my suggestions.

Your best parts are the last three figures and their explanations. Please simplify your results and discussion regarding the tables. They are so confused.

Conclusion is not conclusion. You need to show some criticism regarding your work. What was missing? Why it was done by this way and not on another? What should be changed in the future?

Comments for author File: Comments.pdf

Author Response

Responses to Reviewer 3.

General comments

Dear authors, it is interesting work and it is good to know that you have done huge work. As my purpose is to review your work, I have some questions and suggestions for you.

Response: We sincerely appreciate your time and valuable feedback on our manuscript. Your comments have significantly contributed to the improvement of our manuscript. We have carefully revised the manuscript based on your questions and suggestions. Please find our point-to-point response in the following contents. Please note that the line numbers mentioned in our responses refer to the revised manuscript with tracked changes.

First of all, when ever you speak about biostimulants, please be ready to explain them. In all text it is missing.

Response: We have provided a detailed explanation of “biostimulants” in lines 52-61.

You will see my suggestions or/and questions, please answer them, or apply, when ever I ask for, information.

Response: A point-by-point response to each of your comments, with corresponding line numbers in the revised tracked-change manuscript, is provided below.

Introduction needs more information.

MM chapter needs more explanations, as you will se by my suggestions.

Response: Thanks for pointing this out. We have improved the Introduction and MM sessions according to your suggestions and questions, as shown in our point-to-point response below.

Your best parts are the last three figures and their explanations. Please simplify your results and discussion regarding the tables. They are so confused.

Response: Thanks for your acknowledgement. While PCA biplots can visualize the data we collected, PC1 and PC2 can only represent a total of 56% of the variance. Therefore, we believe the presentation of the tables and other figures are necessary. According to your suggestion, we have simplified our results and discussions to enhance the clarity of our manuscript.

Conclusion is not conclusion. You need to show some criticism regarding your work. What was missing? Why it was done by this way and not on another? What should be changed in the future?

Response: Thanks for your valuable questions. To comprehensively assess the effects of a wider range of biostimulant products and explore potential interactions between microbial biostimulants, seaweed extracts, and onion cultivars, this study was limited to evaluating morphological and biomass parameters. Subsequent research should investigate the long-term impacts of biostimulants on yield and quality, as well as delve deeper into plant physiological responses. Given the superior performance of LALRISE Mycorrhizae, Mighty Mycorrhizae, Spectrum DS, Spectrum Myco, Spectrum, Tribus Continuum, and Kelpak in enhancing onion seedling growth compared to other tested biostimulants, future studies could narrow their focus to these products to further elucidate their mechanisms of action. We have incorporated these contents into the Conclusions, as shown in lines 549-555.

Detailed comments

1. ‘a major vegetable crop’ It is not. Please correct the statement.

Response: We have removed the ‘major’ for clarity (line 9).

2. English please.

Response: Thanks for pointing it out. We have revised accordingly (line 11).

3. Are these commercial? Please mention it if so. Or which one is?
4. This is commercial? Please mention if so.

Response: All the biostimulant products used in this study are commercial. We have revised the related contents (lines 14, 16, 123).

5. Please describe what are biostimulants in general. Then explain what kind of materials/substances/ are they? Then describe where these substances can be found, and what types are present in bacteria, fungi and seaweeds.

Response: Thanks for your valuable feedback. In general, biostimulants are either microorganisms or non-microbial substances applied to plants or their surrounding rhizosphere, which can benefit plant growth or crop quality by enhancing nutrient availability and uptake. Microbial biostimulants, such as arbuscular mycorrhizal fungi (AMF) and plant growth-promoting bacteria (PGPB), benefit plant growth through forming symbiotic relationships with plants and producing growth-stimulating compounds. While non-microbial biostimulant seaweed extracts are rich in phytohormones, minerals, and polysaccharides that promote plant growth. We have supplemented related contents to lines 56-60.

6. Please explain in what way?

Response: Thanks for your question. The germination and seedling stages are the most vulnerable to various stress factors due to their reliance on seed reserves and underdeveloped root systems. Young plants are highly susceptible to drought, salinity, extreme temperatures, nutrient deficiencies, and other stresses. Besides, young shoots are delicate and can be easily damaged by wind. We have explained this in lines 72-76.

7. What you mean with "early"? Please explain.

Response: Thanks for your question. Early inoculation refers to applying microbial biostimulants during the germination or seedling stages. Since microbes can multiply independently over time, smaller initial doses are sufficient when applied earlier compared to later stages of plant growth. We have revised lines 78-79 in accordance with your advice.

8. Explain please "latter stages".

Response: Thank you for your advice. We have revised the content as ‘Previous studies have shown that microbial biostimulants inoculated in 4- or 5-leaf seedling stage improved lettuce leaf health compared to both untreated controls and treatments where biostimulants were applied later, including one week post-transplant or at the head formation stage (lines 82-84).

9. Can you please describe which biostimulants are important for plant growth and development, mainly for onion. Characterize them please.

Response: Thanks for your question. Both microbial and non-microbial biostimulants can benefit plant growth and development. We have expanded on the effects of specific biostimulants such as arbuscular mycorrhizal fungi (AMF), plant growth-promoting bacteria (PGPB), and seaweed extract, which are related to this study, within lines 56-60 and 65-66.

10. Origin of the seed please?

Response: Thanks for your question. We have supplemented the origin of seeds in lines 103-107.

11. Are these microbial products were commercial? Please mention it. Which manufacturer? Products were not evaluated. Please define clearly what was evaluated.

Response: Thanks for your questions. They are commercially available. The manufacturer information is presented in Table 1. We have revised the contents in lines 123-125 accordingly.

12. All were mixed (bacteria vs fungi) ? Please explain better.

Response: Thanks for pointing this out. Not all microbial biostimulants were mixed products. We have deleted ‘mixed’ in line 126 to make it clear.

13. Where to? Substrate? How you did it?

Response: Yes, they were applied to the substrate. The application methods were explained in lines 130-133.

14. In some products you are mentioning the bacteria/fungi present and in other, not. Please uniformize it and write all MO present in each product that you were using per experiment.

Response: Thanks for your question. Due to the limited space, we cannot list all microbial species in Table 1. To address your concern, we revised the ‘major ingredients’ column in Table 1 to make it uniformed. In addition, we added a Supplementary Table S1 to list all microbial species name and their contents in biostimulant products.

15. These were so close to the harvesting date. It can not function. I want to see your discussion related with these dates….

Response: Thank you for your feedback. The Tribus series products were applied every two weeks as recommended by the manufacturer. Seedlings were harvested at 58 days after sowing (DAS), which corresponds to 14 days after the final application (at 44 DAS), aligning with the recommended application frequency. Therefore, the last application had sufficient time to exert its effects.

16. Or done?

Response: Thanks for pointing this out. We have replaced ‘collected’ with ‘recorded’ to enhance clarity (line 148).

17. If you have applied 28 and 44 days some of them, this number of days is incorrect. Please adjust.

Response: Thank you for your valuable feedback. We acknowledge the possibility of delayed germination beyond 17 DAS. However, these later-emerging seedlings are typically smaller and would not be included in subsequent plant morphology and biomass assessments. Therefore, the timing of germination data collection (17 DAS) aligns with the current experimental design and subsequent measurements. We appreciate your suggestion to collect germination data after all treatments are complete. We will incorporate this method into our future studies.

18. Where from this formula originated? Please cite the author
19. Where from this formula originated? Please cite the author

Response: Thanks for your suggestion. We have cited the authors of these equations.

19. Please explain clearly what was control in your experiment. You are mentioning control group (what it means?) and after with or without Kelpak. Is it with or without? Define only one. Please be clear. As it is, there is a confusion.

Response: Thank you for your feedback. The description of relative germination rate (RGR) calculations has been revised to improve clarity (lines 152-159).

To calculate RGR, we need to understand there are three experimental factors: microbial biostimulant, onion cultivar, and seaweed extract. The RGR for a specific treatment is determined by comparing its germination rate to a corresponding control group. This control shares the same onion cultivar and seaweed treatment but lacks the treatment of any microbial biostimulants. For example, to calculate the RGR for yellow onion treated with Spectrum without seaweed extract, we compare its germination rate to that of yellow onion without any microbial biostimulant and without seaweed extract. However, if we are calculating the RGR for yellow onion treated with Spectrum and seaweed extract, the control group becomes yellow onion without a microbial biostimulant but with seaweed extract.

Further explanations regarding our experimental design are provided in our response to comment No. 25.

21. No repetitions?pn

Response: Thanks for your question. There were 2 repetitions from different trays. For each experimental unit, there were 112 plants (subsamples).

22. Which plant parameters were analysed and why each of these methods was used? Need to explain, please.

Response: Thanks for your questions. Shoot morphology, biomass, and root morphology parameters were analyzed by Shapiro-Wilk and Levene’s test to test their normality and homogeneity. These tests were required because there are three primary assumptions in ANOVA: the responses for each factor level have a normal population distribution; the variance of the data in each group is approximately equal; the observations in each group are independent of each other. Therefore, we need to confirm the normality and the homogeneity of variance before we run ANOVA. PCA was performed to visualize the variance in different parameters across different experimental factors. We have revised line 199-200 to enhance clarity.

23. You are comparing Kelpak with a group of 9 MO treatments. Why? Can you please explain somewhere what is the importance of this comparison.
24. Explain this, please. It is not clear what you want to say.
25. What is a level? Explain it, please.

Response: Thanks for your valuable questions. We did the compassion because the two-way and three-way interactions are significant, we have to present the results of each treatment combination separately. We chose to present with or without seaweed extract applications in separate tables based on two reasons: Seaweed extract only had significant impact on 7 parameters, comparing to 13 parameters by microbial biostimulants, and 14 parameters by cultivar; Seaweed extract only have two different conditions (with application or without).

In experimental design, one ‘level’ represents a specific condition within the experimental factor. For example, in our study, we have three levels of onion cultivar, white, yellow and red; we have two different levels of seaweed extract application, with, and without seaweed extract application; we also have ten different levels of microbial biostimulants (table 1). As a result, we have 3x2x10=60 treatment combinations. We have supplemented explanation in lines 193-195.

26. Is this present in your treatments? Which one?
27. Which one? It os not just to say seaweed extract, you need to say its name and genera.

Response: Thanks for your question. Bacillus subtilis is present in Spectrum DS, Spectrum Myco, Spectrum, Tribus Original, and Tribus Continuum products.

One of the seaweed extracts used in the cited study is made from the same genus but different species (Ecklonia arborea vs Ecklonia maxima in our study).

We have revised the contents in lines 246-254 accordingly.

28. This is not seaweed. Explain please why you have compared your results with such algae.

Response: We compared our results with microalgae because it shared similarities with seaweed extract as both are rich in polysaccharides and carbons. However, we decided to delete this comparison to simplify our results and discussions.

29. What you mean with this? Some MO or seaweed included or not? Please be clear.

Response: Thanks for your feedback. We have improved the logic flow in lines 290-294 We discussed one possible reason resulting in the ‘biostimulants effects on leaf number and pseudostem diameter were not significant’:

Prolonged biostimulant application is often a promising approach to enhance plant growth benefits [35]. Therefore, onions may require a longer time to respond with changes in these two parameters.

30. Why this happened? Please discuss it and explain what should be a reason to happen.

Response: Thanks for your insightful question. We have discussed the reason in lines 344-351. In short, the competition for nutrients between mycorrhizal fungi and plant might have caused the tip-burn in the onion seedlings.

31. As yours MO? Do you have common MO with these studies? First of all, tomato and cucumber are from different groups and not monocots. Then they are fruit crops. Of course their metabolites are different than in onion. What you really want to say comparing your results with these two crops? Be precise please.

Response: Thanks for your feedback. Yes, both our study and the cited study combined the application of mycorrhizal fungi and seaweed extract. We agree with you that tomato and cucumber are dicots while onion is monocot. However, improving nutrient availability, alleviating the stress met by the plants, and enhancing water and nutrient uptake, all these effects caused by biostimulants will benefit both monocot and dicot plant growth. One of the potential reasons for the synergistic effects is the polysaccharide contained in seaweed extracts can serve as nutrient source for microbial biostimulants. We have revised the discussion about the synergistic effects of microbial biostimulants and seaweed extract accordingly (lines 456-471).

32. Has different root system. Can not compare it with onion.

Response: Thanks for your feedback. We have deleted this discussion to enhance clarity.

33. Explain why control plants have longer roots, please.

Response: Thanks for your suggestion. The reduction in root length microbial biostimulants treated groups could potentially be attributed to competition for nutrients between the onion seedlings and the establishing microbial communities (lines 397-399).

34. What are specifically PC1 and PC2? Please explain.

Response: Thanks for your question. Principal components PC1 and PC2 are not individual variables, they are created/calculated to represent the variation within the dataset. However, the correlations of each variable with a PC indicate the strength and direction (positive or negative) of its association with that PC. We have interpreted the biological meaning of PC1 and PC2 in lines 479-487. Briefly, PC1 strongly negatively correlated with biomass variables, indicating a decrease in plant biomass along the x-axis; PC2 positively correlated with root growth variables while negatively correlated with shoot growth parameters.

35. In fact they are commercial products and not biostimulants, as you have not any note about their chemical/biological constitution (in main part of them).

Response: Thanks for pointing this out. We have added Table S1 to list the biological constitutions in the biostimulant products used in this study. To make it clear, we replaced ‘biostimulants’ with ‘biostimulant products’.

36. This is part of results/discussion. Here you do not need to repeat these facts,

Response: Thanks for your suggestion. We have revised the conclusions to give a more concise summary of the results. Additionally, the revised section now incorporates the limitations of our study and explores potential future research directions.

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Can be accepted but after some corrections and answers

Comments for author File: Comments.pdf

Author Response

Responses to Reviewer 3 – Review Round 2

General comments

Can be accepted but after some corrections and answers.

Response: Thank you again for your insightful comments. We have revised the manuscript based on your comments raised in review round 2. To ensure clarity, all changes from review round 1 have been implemented. In the current manuscript, we have tracked the changes made specifically to address your comments from review round 2. Please find our point-to-point response to your review comments follows below.

Eliminou: b

Response: Corrected (line 65).

Comentado [PT1]: Can not use "other stresses". It means they are a lot. Or omit or define which ones.

Response: Thanks for pointing this out. We have revised this sentence accordingly (lines 75-76).

Comentado [PT2]: Please explain the water quantity per seed. It was the same for all? Should be uniform. If not, this is a variable to consider.

Response: Thanks for your question. Yes, all cells received the same initial water application. Following the first biostimulant application at 0 DAS, subirrigation was used to saturate the substrate in each cell. Subsequent biostimulant applications employed subirrigation, ensuring uniform distribution of both biostimulants and water to each seedling. We have supplemented related contents in lines 114-115.

Comentado [PT3]: Onion is not germinating. Please use correct word.

Response: Thank you. We have revised the contents in line 220 accordingly.

Comentado [PT4]: What are optimal? Please define clearly.

Response: Thanks for your question. 'optimal' refers to ideal conditions without drought, salinity, or high temperature. We have revised this sentence to enhance clarity (lines 228-231).

Comentado [PT5]: Define ideal conditions, please. Why it may not have a significant impact? Please explain

Response: Thanks for your question. We have defined ideal conditions as conditions in the absence of stresses such as drought, high salinity or high temperatures. From lines 224-233, our results initially showed that germination rates in this study were higher compared to previous research. We hypothesized that for seeds with such a high baseline germination rate, biostimulants might have a limited effect on further improvement. For instance, raising germination from 95% to 96% might be difficult to achieve. Conversely, if stress factors such as drought, high salinity, or high temperature were introduced and germination rates dropped (e.g., to 40%), biostimulants could potentially have a much more significant impact, raising germination to 80%. We have revised lines 231-234 accordingly.

Comentado [PT6]: Please rewrite it. What other crops? Which stressful conditions? Please be clear.

Response: Thanks for your feedback. We have revised this sentence to improve clarity (lines 246-247).

Eliminou: [31]

Response: Author’s name was cited.

Eliminou: out

Eliminou: the application of

Response: Corrected (lines 253-254).

Comentado [PT7]: Tip burn can result from excess soil salts or soil borne pathogens. Did you sterilize the soil? Did you check soil pH or EC? Please explain why you did not analyse the EC or pH.

Response: Thanks for your question. We measured the EC and pH during the experiment, both were within the normal range. Therefore, we discussed other reasons that might cause the tip burn on onion seedlings. The seed germination mix (BM2) used in the experiment should not contain any soil borne pathogens. We have supplemented related discussion in lines 323-326.

Comentado [PT8]: English please

Response: Thanks for your feedback. We have revised the English language and logic flow in lines 512-528.