Saccorhiza polyschides Extract as Biostimulant for Reducing Salt Stress Effect in Common Bean Crops

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The number of keywords needs to be reduced.

There are many reports on the impact of salt stress on Common beans. Please introduce the key conclusions in the review, rather than discussing the effects of salt stress on plants in general.

In the introduction, it is necessary to use original papers as much as possible instead of citing reviews.

What is the basis for setting the concentration of the extraction solution?

Why choose seeds based on color and size?

What is the concentration basis for simulating salt stress treatment?

Please provide a detailed description of the sampling process for the determination of physiological and biochemical indicators. It should include sampling time, blade position and weight, etc.

The method description suggests merging similar indicators and reducing the number of subheadings to make the writing more concise.

The annotations of tables and figures should be placed separately below, rather than in the same position as the title.

Delete Table 1.

The discussion needs to be reduced.

The conclusion needs to provide key quantitative information.

Author Response

The number of keywords needs to be reduced. Done

There are many reports on the impact of salt stress on Common beans. Please introduce the key conclusions in the review, rather than discussing the effects of salt stress on plants in general.

We have included general aspects that are affected by salinity in plants because they are some of those we have tested during the work such as chlorophyll content, oxidative stress or antioxidant activities. In this way we intended to offer the reader an explanation of the aspects evaluated rather than a review of salinity. Nevertheless, we have included some more references concerning common bean as suggested by the reviewer.

 

In the introduction, it is necessary to use original papers as much as possible instead of citing reviews. Done

What is the basis for setting the concentration of the extraction solution?

Previous work in our laboratory tested different concentrations for different algae (Abdelhamid ennoury et al., 2022) and the range of concentrations selected for this work has proven to be the most effective. The methodology used to estimate the concentration has been included in M&M.

https://doi.org/10.1016/j.sajb.2022.07.035

Why choose seeds based on color and size?

The selection of seeds based on shape and color was made because within each lot there is always some variability, and we tried to make them as similar as possible to each other.

 

What is the concentration basis for simulating salt stress treatment?

The salt concentration was selected from previous studies of our group (Nhaala et al., 2024 underreview; Latique et al., 2021),where the concentration of 68.4 mM of NaCl showed impactfully negative effects on common beans.

https://doi.org/10.3390/agronomy11020265

Please provide a detailed description of the sampling process for the determination of physiological and biochemical indicators. It should include sampling time, blade position and weight, etc. Done

The method description suggests merging similar indicators and reducing the number of subheadings to make the writing more concise. Done

The annotations of tables and figures should be placed separately below, rather than in the same position as the title. Done

Delete Table 1. The table has been deleted and the information is mentioned in the discussion section.

The discussion needs to be reduced. Although this section can be a little extensive, we believe the discussion is complete and all relevant aspects of the work are discussed. In addition, other reviewers have even suggested including some other aspects. In this sense, it would help us to know what information you think could be summarized or reduced.

The conclusion needs to provide key quantitative information. Done

Reviewer 2 Report

Comments and Suggestions for Authors

General comments

The manuscript titled “Saccorhiza polyschides extract as biostimulant for reducing salt 2 stress effect in common bean crops” has been submitted to agronomy. In this study, authors have tested the efficacy of Saccorhiza polyschides extract at tree concentrations (1%, 2%, and 5%)  in enhancing the salinity tolerance of common bean crops. Two different application methods were used: spraying the 24 aerial parts of the plant or by irrigation together with the watering solution. They finally concluded that the concentrations of 2% and 5% SPE applied  by spraying best improve the growth and development of common bean plants under saline conditions. In my opinion, the research results are relevant and of considerable interest to researchers. The manuscript describes well the research work and results. However, I have listed some comments that help authors improve the manuscript.

Specific comments

Keywords

-           Please put it in alphabetical order. In addition, keywords should not repeat words found in the title of the manuscript

Introduction

-          If possible add a brief overview of factors that support increased saline levels in agricultural lands, and some few effects (symptoms) of salt stress in Phaseolus vulgaris.

-          it is important to add some publications of specific work on Saccorhiza polyschides, which have been used either to alleviate salt stress in plants or on Phaseolus vulgaris. If this is your first work, it is worth mentioning.

M&M

-          - I appreciate the graphical experience plan (fig. 1).

-          The origin of the brown alga Saccorhiza polyschides used is not mentioned in the m/s

-          on what basis do you use the 68.4 mM of NaCl to apply salt stress on common bean. The authors need to provide more details

-          The physico-chemical characteristics of soil used in the experiment are missing and should be given.

-          Plants were sprayed every 5 days with different SPE concentrations (1%, 2%, and 5%) and were watered every 4 days with fresh water (NSS), with NaCl solution (68.4 mM for SS), or with NaCl solution mixed with the SPE in the irrigation method. Then, 8 treatments were applied: NSS, SS, SPE+SS+Irrigation and SPE+SS+Spray. I think SPE+NSS Irrigation or spray treatments are missing in this experiment. How the authors could clarify that?

-          It is not clear how salt stress was introduced. The author should revise his article to clarify whether he applied salt stress with a gradual increase in NaCl concentrations to avoid osmotic shock until the final concentrations were reached.

-          How were the growth parameters measured especially the leaf diameter, authors need to more detail.

-          Sections in the methods section should be arranged as in the results section.: growth parameters, physiological then biochemical parameters.

-          Why not reanalyze the soil for salt level after the experiment?

-. Why not analyze the plants for nutrient contents to justify the treatment effects?

-          L 203: review the form of the '' extinction coefficient 6.2 mM- 1 cm-1)''. The same for all the m/s

-          Please indicate the type of ANOVA you used.

-In the discussion section, the authors are asked to compare their results with other works that have used specific algae, Saccorhiza polyschides

Conclusion

Please indicate the relevance of your results to soil fertilization management in sustainable agriculture

References

- Add the missing DOI and check all references citation in the text and list (volume number and pages).

 

 

Author Response

Keywords

           Please put it in alphabetical order. In addition, keywords should not repeat words found in the title of the manuscript. Done

Introduction

-          If possible add a brief overview of factors that support increased saline levels in agricultural lands, and some few effects (symptoms) of salt stress in Phaseolus vulgaris. Done

-          it is important to add some publications of specific work on Saccorhiza polyschides, which have been used either to alleviate salt stress in plants or on Phaseolus vulgaris. If this is your first work, it is worth mentioning.

As we mentioned in discussion section as far as we know, no experiments were made using SPE to improve the resilience and productivity of common bean plants under salt stress conditions. However, we compared our results with others done with Ascophyllum nodosum extracts, whose application is the most studied under salt stress conditions in Lactuca sativa, Arabidopsis thaliana, and Solanum lycoperssicum among others, as we also mentioned in the discussion.

 

M&M

-          - I appreciate the graphical experience plan (fig. 1). Thank you

-          The origin of the brown alga Saccorhiza polyschides used is not mentioned in the m/s

This information has been included in the introduction and in M&M section

-          on what basis do you use the 68.4 mM of NaCl to apply salt stress on common bean. The authors need to provide more details

-          The physico-chemical characteristics of soil used in the experiment are missing and should be given.

 

We have used the same commercial substrate for all conditions (this information has been included in M&M), so we assume that if the physicochemical characteristics of the substrate are affecting, they will do so to the same extent in all of them, which is why they have not been determined in this work.

-          Plants were sprayed every 5 days with different SPE concentrations (1%, 2%, and 5%) and were watered every 4 days with fresh water (NSS), with NaCl solution (68.4 mM for SS), or with NaCl solution mixed with the SPE in the irrigation method. Then, 8 treatments were applied: NSS, SS, SPE+SS+Irrigation and SPE+SS+Spray. I think SPE+NSS Irrigation or spray treatments are missing in this experiment. How the authors could clarify that?

Thank you for your insightful feedback. We appreciate your suggestion regarding the inclusion of algal extract + no salt stress. In response to your concern, we would like to clarify that we have conducted previous studies focusing on the effects of algal extracts used alone without salt stress. These studies have demonstrated the biostimulant properties of the extracts under normal conditions. However the primary objective of the current study was to investigate whether the algal extract provide protective and stimulating effects specifically under salt stress conditions. Our aim was to understand how the SPE can mitigate the adverse effects of salt stress and promote plant growth and health in such challenging environments. Therefore our experimental design intentionally focused on comparing the effects of the algal extract under salt stress versus untreated similar stress conditions. Thank you once again for your valuable suggestion, and question. We hope this clarification addresses your concern and provides context for our experimental design choices.

 

-          It is not clear how salt stress was introduced. The author should revise his article to clarify whether he applied salt stress with a gradual increase in NaCl concentrations to avoid osmotic shock until the final concentrations were reached.

The salt stress was applied by irrigation as specified in the M&M section. However, we did not determine if there was an accumulation of salt in the soil after the experiment which might produce osmotic shock. The same stress applications were used across both experimental conditions: Salt stress alone and Salt stress + biostimulation, which produce the same effect on all the specimens.

Although it would have been interesting to separate the double negative effect the NaCl has on the plant (drought stress and osmotic stress), the objective of the present work was to determine whether the extract had a positive mitigating effect on saline stress in general, which it did, in comparison with control plants.

-          How were the growth parameters measured especially the leaf diameter, authors need to more detail. Done

-          Sections in the methods section should be arranged as in the results section.: growth parameters, physiological then biochemical parameters. Done

-          Why not reanalyze the soil for salt level after the experiment?

The objective of our work is to evaluate the effect of SPE application on common bean plants under salt stress, not in the soil. Although the reviewer's suggestion is interesting, we do not understand how this could contribute to the objective of our work.

-. Why not analyze the plants for nutrient contents to justify the treatment effects?

The reviewer's suggestion is another interesting approach that we will keep in mind for future work in which we will also measure the yield of the crops once we find out the most adequate SPE concentration and the most effective application method.

-          L 203: review the form of the '' extinction coefficient 6.2 mM- 1 cm-1)''. The same for all the m/s Done

-          Please indicate the type of ANOVA you used. Done

-In the discussion section, the authors are asked to compare their results with other works that have used specific algae, Saccorhiza polyschides.

As we mentioned in discussion section as far as we know, no experiments were made using SPE to improve the resilience and productivity of common bean plants under salt stress conditions. However we compared our results with others done with Ascophyllum nodosum extracts, which application is the most studied under salt stress conditions in Lactuca sativa, Arabidopsis thaliana, and Solanum lycoperssicum among others, as we also mentioned in the discussion.

 

Conclusion

Please indicate the relevance of your results to soil fertilization management in sustainable agriculture

 

Since the effect on the soil was not analyzed in this work, as it was not the objective of the experiment, we cannot provide such information. It is true that SPE have a biofertilizing effect (Mamede et al.2024). However, we have not analyzed this aspect as this, again, was not the primary goal of the study. We only tested the biostimulant effect on the plant.

References

- Add the missing DOI and check all references citation in the text and list (volume number and pages). Done

 

Reviewer 3 Report

Comments and Suggestions for Authors

The paper addresses the issue of the counteracting phenomenon of soil salinisation. The proposed method of using Sacchorhiza polyschides extract seems to be an interesting solution. Unfortunately, doubts remain regarding the extraction of the material and its analysis for contaminants, e.g. heavy metals, hrmons, herbicides, etc. Such analysis is extremely important because, on the one hand, we know whether the extracted extract has other contaminants and whether we are thus contributing to the spread of contaminants. On the other hand, organisms such as Sacchorhiza polyschides can often be bioindicators of environmental contamination. The results obtained are intriguing and somewhat surprising, as it is the spraying method of dosing that is more effective compared to irrigation. It would be worthwhile to supplement the manuscript with such a discussion.

 

Also the method section should be completed with the following information: 

Section 2.2 lacks information on the drying conditions of the SP.

There is also no information on at what temperature the extract was prepared. Why the SP was first soaked for 24 h and only then heated.

What method of filtration was used (if gravity filtration, the type of filter should be given). Why the authors decided on this method of obtaining the extract. What is the reproducibility of the composition of the extract obtained? Is the extract obtained stable. How long can it be stored, under what conditions, etc.? 

Line 122 - the authors mention that an analysis of the composition of the extract obtained was performed. Unfortunately, they do not state the content of which substances was determined and what method was used. In addition, the work should be carefully analysed and the spelling of the sum formulas of chemical compounds and the Latin names of plants should be corrected.

Author Response

The paper addresses the issue of the counteracting phenomenon of soil salinisation. The proposed method of using Sacchorhiza polyschides extract seems to be an interesting solution. Unfortunately, doubts remain regarding the extraction of the material and its analysis for contaminants, e.g. heavy metals, hrmons, herbicides, etc. Such analysis is extremely important because, on the one hand, we know whether the extracted extract has other contaminants and whether we are thus contributing to the spread of contaminants. On the other hand, organisms such as Sacchorhiza polyschides can often be bioindicators of environmental contamination. The results obtained are intriguing and somewhat surprising, as it is the spraying method of dosing that is more effective compared to irrigation. It would be worthwhile to supplement the manuscript with such a discussion.

The approach proposed by the reviewer on the possibility that algae may contain contaminants is very interesting, although we believe that this type of study is not the objective of the present work. If the algae had a high content of contaminants, we do not believe that there would be a stimulatory effect on plant growth. In our opinion, such an approach would be necessary if the application of the extract had a negative effect on growth to try to explain this response, but this is not the case. However, we agree that in the future it would be interesting to conduct a more in-depth study on the composition of these extracts.

Regarding the application method, although we do not know the reason why the spraying method had better results than irrigation, we have included a hypothesis in the discussion section about this aspect. However, further research in this sense is necessary.

Also the method section should be completed with the following information: 

Section 2.2 lacks information on the drying conditions of the SP. Done

There is also no information on at what temperature the extract was prepared. Why the SP was first soaked for 24 h and only then heated. Done

What method of filtration was used (if gravity filtration, the type of filter should be given). Why the authors decided on this method of obtaining the extract. What is the reproducibility of the composition of the extract obtained? Is the extract obtained stable. How long can it be stored, under what conditions, etc.? 

Gravity filtration was used because it is straightforward, inexpensive, and does not require specialized equipment. Also, Whatman No.1 filter paper is suitable for routine filtration applications and effectively separates larger particles and debris from the algal solution. This information was already included in Mat and Met section.

The storage is at low temperature of 4°C, to help prolong its stability. The extract should also be stored in a dark container to protect it from light that can degrade sensitive compounds, and sealed to prevent contamination.

Typically, algal extracts can be stable for several weeks to months, under optimal conditions. The shelf life of the extract would need to be determined experimentally, but an estimate would be: 1 to 3 months when its refrigerated at 4°C. However, we always prepared on fresh for these experiments.

The stability of the composition is undetermined at present. However, it raises interesting questions regarding a possible future marketing of the extract. Further studies would be necessary in this sense.

 

Line 122 - the authors mention that an analysis of the composition of the extract obtained was performed. Unfortunately, they do not state the content of which substances was determined and what method was used. We removed the table of composition because of the comment of reviewer 1.

 In addition, the work should be carefully analysed and the spelling of the sum formulas of chemical compounds and the Latin names of plants should be corrected. Done

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

Thank you for your responses to my comments. However, the quality of the photos currently supplied is insufficient. Higher resolution and better quality images are needed to ensure the clarity and accuracy of the illustrations in your manuscript.

 In addition, I would like to highlight a few suggestions which I believe will improve the clarity and quality of your documents for next time:

1. it would be useful to include the page number and corresponding line for each comment addressed in the answer sheet.  This will make it easier to reread and check the changes made.

2. To facilitate the revision process, I recommend that you use Microsoft Word's "Track Changes" feature. This feature allows all changes to be visible, which considerably simplifies the editing process for reviewers and authors. 

Author Response

Thank you for your responses to my comments. However, the quality of the photos currently supplied is insufficient. Higher resolution and better quality images are needed to ensure the clarity and accuracy of the illustrations in your manuscript.

 In addition, I would like to highlight a few suggestions which I believe will improve the clarity and quality of your documents for next time:

1. it would be useful to include the page number and corresponding line for each comment addressed in the answer sheet.  This will make it easier to reread and check the changes made.
2. To facilitate the revision process, I recommend that you use Microsoft Word's "Track Changes" feature. This feature allows all changes to be visible, which considerably simplifies the editing process for reviewers and authors. 

Thank you for your comments, we believe they improve the quality of our paper.

Following your advice, higher resolution images have been included to substitute the previous ones.

On the other hand, we will consider your suggestions for facilitating the correction of our future manuscripts.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for responding to the comments and making changes to the manuscript. 

Regarding the presence of contaminants in their effect on plant growth, it is not so obvious that their presence can only have a toxic effect. Some contaminants can stimulate growth/development particularly at the germination or initial plant development stage.

Author Response

Comments and Suggestions for Authors

Thank you for responding to the comments and making changes to the manuscript.

Regarding the presence of contaminants in their effect on plant growth, it is not so obvious that their presence can only have a toxic effect. Some contaminants can stimulate growth/development particularly at the germination or initial plant development stage.

Thank you for this input. We agree that further studies on the composition of the extract should be carried out in order to deepen our knowledge on what specific components stimulate plant growth and defense. Although an initial composition characterization was carried out, the present work only focused on whether the extract could have any effect at all against salt stress conditions. Once our hypothesis has been confirmed, new experiments in this sense will be designed in the near future which will be included in  a future manuscript.