Isolation and Characterization of Culturable Osmotolerant Microbiota in Hypersaline and Hypergypsic Soils as New Treatment for Osmotic Stress in Plants

Round 1

Reviewer 1 Report

This is a very good work and recommanded for publication with minor revrision.

In many regions across the globe, there exist soils known as saline and gypsic soils, posing challenges for the growth and prosperity of farms. These soils harbor high concentrations of sodium or calcium ions, severely limiting plant growth and crop production. As a result, these soils have traditionally been exploited for mineral extraction. However, in recent times, we have come to realize that these very soils can offer new biotechnological solutions to aid crops in coping with osmotic stress. Surprisingly, despite their low biodiversity, these environments house special microbiota that have adeptly adapted to the conditions and can interact with plants, helping them cope with diverse environmental stresses. These interactions hold the potential to revolutionize the creation of bioinoculants essential in managing osmotic stress.

 

The focus of this study was to delve into the diversity of cultivable populations in such environments and utilize them as regulators of soil nutrients and stress-relieving symbionts for plants experiencing osmotic stress. Among the carefully selected strains that encompass different scenarios, two strains, Stutzerimonas stutzeri A38 and Bacillus pumilus A49, emerged as capable of enhancing root growth under osmotic stress in Medicago sativa and Medicago polymorpha plants. Moreover, Peribacillus frigoritolerans A70 and Bacillus licheniformis A46 also demonstrated their potential in improving the performance of M. polymorpha, indicating their promising use in transforming wastelands into productive areas suitable for livestock feeding and other vital industries.

Minor editing of English language required

Author Response

Reviewer 1

This is a very good work and recommended for publication with minor revision.

In many regions across the globe, there exist soils known as saline and gypsic soils, posing challenges for the growth and prosperity of farms. These soils harbor high concentrations of sodium or calcium ions, severely limiting plant growth and crop production. As a result, these soils have traditionally been exploited for mineral extraction. However, in recent times, we have come to realize that these very soils can offer new biotechnological solutions to aid crops in coping with osmotic stress. Surprisingly, despite their low biodiversity, these environments house special microbiota that have adeptly adapted to the conditions and can interact with plants, helping them cope with diverse environmental stresses. These interactions hold the potential to revolutionize the creation of bioinoculants essential in managing osmotic stress.

The focus of this study was to delve into the diversity of cultivable populations in such environments and utilize them as regulators of soil nutrients and stress-relieving symbionts for plants experiencing osmotic stress. Among the carefully selected strains that encompass different scenarios, two strains, Stutzerimonas stutzeri A38 and Bacillus pumilus A49, emerged as capable of enhancing root growth under osmotic stress in Medicago sativa and Medicago polymorpha plants. Moreover, Peribacillus frigoritolerans A70 and Bacillus licheniformis A46 also demonstrated their potential in improving the performance of M. polymorpha, indicating their promising use in transforming wastelands into productive areas suitable for livestock feeding and other vital industries.

R: Thanks a lot for the comment! We are not very sure of the changes required, but we kindly incorporate some of your relaxions. We also amended the English with the help of a native colleague. Hope this, together with the rest of the revisions applied in order to cope with the other revisors is enough to accept the work. Thanks again!

Reviewer 2 Report

In this study, the authors determined the diversity of cultivable bacteria populations in stressed osmotic environments such as hypersaline and hypergypsic soils. The authors identified the isolates and characterized them in terms of some plant growth-promoting traits.  The authors also evaluated the effect of the best bacterial candidates (in terms of plant promoting traits) on in vitro colonization and germination of Medicago sativa. The authors also evaluated several growth parameters (root length, shoot length, dry weight) of the Medicago sativa and Medicago polymorpha in saline stress and in the presence of the best bacterial candidates. However, this manuscript has significant flaws in the writing and presentation of data in different sections of the manuscript that do not allow me to agree with the publication of this paper.

1. In this work, the authors only demonstrated that the presence of these bacterial isolates with growth promoting traits increased the stress resistance capacity of the tested plants. However, they do not offer any explanation or hypothesis of how these characteristics can enhance this higher resistance to saline stress.

2. Authors should be more careful with written English; there are several syntax and grammar errors throughout the manuscript that need to be proofread. And some scientific language needs to be revised. What does it means” plant growth-promoting skills performance”...

3. Authors should revise the entire introduction section to make the information more objective and concise. There is irrelevant information taking into account the purpose of the manuscript.

4. In the section Materials and Methods, authors should revise the text and remove “Here” in the beginning of sentences.

5. The authors reported that performed several Venn diagrams to better understand the diversity and distribution of each sample. Was this necessary? And did the authors really do that?

6. The authors describe the stress that plants have to face in saline and gypsic soils, however, all methodologies describe only saline stress conditions, and gypsic stress is disregarded. Even   in the section "2.5. In vitro germination and colonization tests under saline and gypsy stress conditions", only saline stress conditions are described.

7. The results section was sloppily organized and had a poor review by the authors. What did the authors want to present in Figure 5? The growth curves of the candidate's strains? If so, the colored graph represents the growth curves. The other graphs do not correspond to the figure caption.  

8. In Table 2, the authors present the name of the strains in one column and the place of isolation in another. However, it is impossible that the same strain (eg strain 14) could have been isolated from 3 different sampling sites 6, 7 and 8. The authors should clarify this. In the same Table 2, Yeast is not a species name.

9. The relative abundance of the different species or genus should be represented in a different type of graph, not in pie charts (Figure 3). The pie charts presented by the authors are very difficult to understand, the same colors for different sections of the pie chart, identifying the strain. The authors should merge the information from Table 2 and Figure 3 in a graph, with the relative abundance of the different taxonomic groups and identifying the isolation local.

10. The authors are very descriptive in the text of results, the figures in the manuscript contain most of the information and it is not necessary to describe them in detail in the text. In the case of the phenotyping quantification (Figure 8), the authors have several graphs designated by a, b, c,… each of them reporting a different phenotypic characteristic. However, in the text, the authors only refer to figure 8 and do not direct the reader to the individual figure 8a, 8b when describing the phenotypic characteristic.

 

Comments for author File: Comments.pdf

Author Response

In this study, the authors determined the diversity of cultivable bacteria populations in stressed osmotic environments such as hypersaline and hypergypsic soils. The authors identified the isolates and characterized them in terms of some plant growth-promoting traits.  The authors also evaluated the effect of the best bacterial candidates (in terms of plant promoting traits) on in vitro colonization and germination of Medicago sativa. The authors also evaluated several growth parameters (root length, shoot length, dry weight) of the Medicago sativa and Medicago polymorpha in saline stress and in the presence of the best bacterial candidates. However, this manuscript has significant flaws in the writing and presentation of data in different sections of the manuscript that do not allow me to agree with the publication of this paper.

1. In this work, the authors only demonstrated that the presence of these bacterial isolates with growth promoting traits increased the stress resistance capacity of the tested plants. However, they do not offer any explanation or hypothesis of how these characteristics can enhance this higher resistance to saline stress.

R: Thanks for the comment! We have made a new explanation about this in the text. In brief, one of the most intense restrictions that plants suffer under this stress is the acquisition of nutrients. Due to the osmotic pressure, the regular solubilization or capture of nutrients is even harder for plants, so the presence of microorganisms that can still carry out the process under these conditions is required to maintain reasonable levels of growth and development. On the other hand, the production of phytohormones, again under such stressing conditions (which used to be highly conditioned for non-adapted microbes), helps the plant to control ethylene level or develop a more efficient root architecture. Finally, some other skills as biofilm or antioxidants production, alleviate the stress in plants, allowing them to maintain their metabolic and developing processes. We have included these aspects both in the ‘Introduction’ and ‘Discussion’ section, with their respective literature citations. Hope this is ok now.

 

2. Authors should be more careful with written English; there are several syntax and grammar errors throughout the manuscript that need to be proofread. And some scientific language needs to be revised. What does it means” plant growth-promoting skills performance”...

R: Thanks a lot for the comment! Indeed, we are not natives, but we should revise more in deep this manuscript. Fortunately, we received some help from native colleagues to make those correction and we hope you can find it now in a correct shape. Any case, some concepts may have not a previous reference, so we tried to use the most correct ones in order to not cause more confusion. Thanks!

 

3. Authors should revise the entire introduction section to make the information more objective and concise. There is irrelevant information taking into account the purpose of the manuscript.

R: Thanks! We have completely revised the introduction, making it more concise and avoiding irrelevant information or reiterations. Thanks again!

 

4. In the section Materials and Methods, authors should revise the text and remove “Here” in the beginning of sentences.

R: Thanks a lot for the comment! We applied this change.

 

5. The authors reported that performed several Venn diagrams to better understand the diversity and distribution of each sample. Was this necessary? And did the authors really do that?

R: Thanks for the comment. Yes, we used to compare which strains compiled more skills and in which level. However, they were not showing a relevant result, so we agreed with you, and we quit this part of the text.

 

6. The authors describe the stress that plants have to face in saline and gypsic soils, however, all methodologies describe only saline stress conditions, and gypsic stress is disregarded. Even   in the section "2.5. In vitro germination and colonization tests under saline and gypsy stress conditions", only saline stress conditions are described.

R: Thanks a lot for the comment. Indeed, we didn’t use gypsum because the solubility in many tests was problematic, so we decided to apply the osmotic stress by NaCl to carry out the tests. After consulting with colleagues in the area, we conclude that salt-induced osmotic stress acts as a more intense osmotic stress than gypsum for the same molarities, so we decided to test the trait analyzed in this work with NaCl only. This is an operative decision, but we understand that the way we expressed in the previous version is not correct and may induce to confusion. In order to not cause this confusion, we have applied changes in the main texts, and we hope now is clearer and acceptable.

 

7. The results section was sloppily organized and had a poor review by the authors. What did the authors want to present in Figure 5? The growth curves of the candidate's strains? If so, the colored graph represents the growth curves. The other graphs do not correspond to the figure caption.  

R: So sorry, we didn’t include the last changes in this section (‘Results’) and many mistakes were included, as you mentioned. We have applied the clean version, and modified the legend in Fig.5, so we hope now is clear and tidy.

 

8. In Table 2, the authors present the name of the strains in one column and the place of isolation in another. However, it is impossible that the same strain (eg strain 14) could have been isolated from 3 different sampling sites 6, 7 and 8. The authors should clarify this. In the same Table 2, Yeast is not a species name.

R: Thanks a lot for the comment! Sorry for the confusion we caused here. Instead of strain, we should indicate the species isolated. We found several strains of same species in different sampling environments. We have clarified this in the text. Again, so sorry for this. About yeast, you’re completely right, so we have changed the labelling of the table in order to not make such a mistake. Thanks again!

 

9. The relative abundance of the different species or genus should be represented in a different type of graph, not in pie charts (Figure 3). The pie charts presented by the authors are very difficult to understand, the same colors for different sections of the pie chart, identifying the strain. The authors should merge the information from Table 2 and Figure 3 in a graph, with the relative abundance of the different taxonomic groups and identifying the isolation local.

            R: Thanks a lot for the comment! We understand it could be a little confuse, but we tried the version of merge both items as mentioned, and that was even more difficult to understand. About the pie chart, we consider is a simplified way to show the most resilient populations and their impact in the final population. We consulted this with colleagues because the stacked column graphs were not clear specially for the smaller populations. Moreover, as we are not comparing different treatment for the same area, or similar systems, the pie chart emerged as the best solution in order to have a fast picture of the different environments. In relation with the color of the sector, we joined those species that belong to similar taxa in order to simplify the visualization. Here we included some examples of publications that performed similarly. Hope this answer is ok for you. On the other hand, the legend is not that helping for it, so we decided to simplify and make some amendments.

Wan, X., Ruan, R., McLaughlin, R. W., Hao, Y., Zheng, J., & Wang, D. (2016). Fecal Bacterial Composition of the Endangered Yangtze Finless Porpoises Living Under Captive and Semi-natural Conditions. Current microbiology, 72(3), 306–314. https://doi.org/10.1007/s00284-015-0954-z

de Almeida, O. G. G., Capizzani, C. P. D. C., Tonani, L., Grizante Barião, P. H., da Cunha, A. F., De Martinis, E. C. P., Torres, L. A. G. M. M., & von Zeska Kress, M. R. (2020). The Lung Microbiome of Three Young Brazilian Patients With Cystic Fibrosis Colonized by Fungi. Frontiers in cellular and infection microbiology, 10, 598938. https://doi.org/10.3389/fcimb.2020.598938

Malli Mohan, G. B., Parker, C. W., Urbaniak, C., Singh, N. K., Hood, A., Minich, J. J., Knight, R., Rucker, M., & Venkateswaran, K. (2020). Microbiome and Metagenome Analyses of a Closed Habitat during Human Occupation. mSystems, 5(4), e00367-20. https://doi.org/10.1128/mSystems.00367-20

 

10. The authors are very descriptive in the text of results, the figures in the manuscript contain most of the information and it is not necessary to describe them in detail in the text. In the case of the phenotyping quantification (Figure 8), the authors have several graphs designated by a, b, c,… each of them reporting a different phenotypic characteristic. However, in the text, the authors only refer to figure 8 and do not direct the reader to the individual figure 8a, 8b when describing the phenotypic characteristic.

              R: Thanks for the comment. Indeed, the explanations were so extensive. We received an internal comment to expose everything in the main text, but we have as well the opinion that may be too much. We consider is more a style taste, but as we want to focus better the mains of the texts, we have applied several changes in order to point the most relevant results, leaving the rest to the self-explanatory figures. On the other hand, we have included the references of the figure 8 in the main text. Sorry for this mistake. Thanks again!

Reviewer 3 Report

The subject of the manuscript titled 'Isolation and characterization of culturable osmotolerant microbiota in hypersaline and hypergypsic soils as new treatment for osmotic stress in plants' fits the profile of the Soilsystems Journal. The work provides valuable data for our understanding of the microbiota of saline and gypsic soils, as well as the characteristics of the isolated strains. I believe your paper contributes significantly to our knowledge in this area. Furthermore, you have isolated some strains to assist Medicago plants against osmotic stress. However, the authors have made some shortcomings that should be corrected and/or revised before the publication of this work.

 1. The Introduction could be condensed.

2. 'per lite' should be corrected to 'per liter' in line 234.

3. There are some problems with the figure titles of Figure 5; please review them again.

The authors should review the manuscript for spelling errors and ensure accuracy in spelling.

Author Response

Reviewer 3

The subject of the manuscript titled 'Isolation and characterization of culturable osmotolerant microbiota in hypersaline and hypergypsic soils as new treatment for osmotic stress in plants' fits the profile of the Soilsystems Journal. The work provides valuable data for our understanding of the microbiota of saline and gypsic soils, as well as the characteristics of the isolated strains. I believe your paper contributes significantly to our knowledge in this area. Furthermore, you have isolated some strains to assist Medicago plants against osmotic stress. However, the authors have made some shortcomings that should be corrected and/or revised before the publication of this work.

1. The Introduction could be condensed.

R: Really appreciate your comment. It’s true, it’s being very dense and disperse, so we have applied changes and condense the material of this section.

 

2. 'per lite' should be corrected to 'per liter' in line 234.

R: Thanks a lot for the comment! It’s corrected now.

 

3. There are some problems with the figure titles of Figure 5; please review them again.

R: Thanks a lot for the comment! I’m so sorry, we didn’t include the last changes in this section (‘Results’) and many mistakes were included, as you mentioned. We have applied the clean version, and modified the legend in Fig.5, so we hope now is clear and tidy.

Reviewer 4 Report

The manuscript „Isolation and characterization of culturable osmotolerant micro-2 biota in hypersaline and hypergypsic soils as new treatment for 3 osmotic stress in plants” is an interesting approach to elucidate the function of single microbial strains in stressed environments. The authors analyses the culturable microorganisms which might cope with high amounts of salts in plant-soil systems. The effort on gypsum enriched soils were less but also the culturable community was analyzed. Due to focusing on different functions the ability of specialized strains was determined and the potential beneficial effect on Medicago species were estimated.

 

In general it is an interesting work which increases the understanding of beneficial microorganisms for improving salinized soil-plant system.

Nevertheless, there are some aspects needed to be revised before the manuscript can be published.

See detailed comments below.

 

INTRODUCTION

The introduction is very long. It shows mainly necessary information but might be shorten in some aspects.

 

MATERIAL AND METHODS

Lines 155-163: Especially the description of sampling sites is to short. Please give more information on that. What was the land use of the sites. Was there vegetation established? How was sampling conducted. You mentioned that you took 8 samples…and then you prepare a composite sample per site? Please give more information on that. Was there also a climatic gradient present. Do you have some climate data for the sites?

 

Lines 191-192: You mentioned that you calculated Shannon and Simpson indices and several others…please give the calculation for that indices.

 

RESULTS

Fig. 4: The red vectors are hard to read. Please increase the size of the letters.

Fig. 5: The description does not totally match the content. What is exactly shown here? In the description there is mentioned that you show the germination ratio but I think it is not shown here. Please check and clarify the description of that figure. Also, what means “mM” this is not defined. Also “OD550nm” (i-j).

Lines 548-574: Please refer to the respective figure concretely within the text.

 

Line 575: Delete “and” before “under…”

 

Line 603: Here you mention “label M stands for mock treatment” But there is no M – here you write “Mock” . Please check – also in Fig. 8.

 

DISCUSSION

The discussion contains superfluous information which are belonging rather to an introductory chapter. Delete lines 698-711.

Also, the paragraph from line 712-741 should be shortened.

Down from line 742 the real discussion starts.

Please avoid redundant information or overall introductory sentences.

But you should discuss more on the restriction of your finding especially regarding the culturable organisms from soil and the role of competing microorganisms which may mimic the beneficial effect of your strains. Are the strains you cultured representative for that environment? Please extend the discussion in that direction and give a more reserved conclusion.   

English quality is good but there are some typing errors which has to be checkt and corrected over the whole manuscript

Author Response

The manuscript „Isolation and characterization of culturable osmotolerant micro-2 biota in hypersaline and hypergypsic soils as new treatment for 3 osmotic stress in plants” is an interesting approach to elucidate the function of single microbial strains in stressed environments. The authors analyses the culturable microorganisms which might cope with high amounts of salts in plant-soil systems. The effort on gypsum enriched soils were less but also the culturable community was analyzed. Due to focusing on different functions the ability of specialized strains was determined and the potential beneficial effect on Medicago species were estimated. In general it is an interesting work which increases the understanding of beneficial microorganisms for improving salinized soil-plant system. Nevertheless, there are some aspects needed to be revised before the manuscript can be published.

See detailed comments below.

 

INTRODUCTION

The introduction is very long. It shows mainly necessary information but might be shorten in some aspects.

 R: Thanks a lot for the comment! We have applied a serious change in the section in order to focus it and avoid unnecessary information.

 

MATERIAL AND METHODS

Lines 155-163: Especially the description of sampling sites is to short. Please give more information on that. What was the land use of the sites. Was there vegetation established? How was sampling conducted. You mentioned that you took 8 samples…and then you prepare a composite sample per site? Please give more information on that. Was there also a climatic gradient present. Do you have some climate data for the sites?

 R: Thanks for the comment. We have included all data requested and some more in order to do this section more accurate and precise.

 

Lines 191-192: You mentioned that you calculated Shannon and Simpson indices and several others…please give the calculation for that indices.

 R: As the results were not significant, we didn’t include them in the text to not overload it even more. However, we indicated this situation in the correspondent section. However, the results obtained can be seen here:

  

 

 

RESULTS

Fig. 4: The red vectors are hard to read. Please increase the size of the letters.

R: I’m sorry for this, however the online software doesn’t allow us to make these changes

 

Fig. 5: The description does not totally match the content. What is exactly shown here? In the description there is mentioned that you show the germination ratio but I think it is not shown here. Please check and clarify the description of that figure. Also, what means “mM” this is not defined. Also “OD550nm” (i-j).

R: Thanks a lot for the comment! We are really sorry; this was belonging to other figure legend. Now, everything is well arranged. Hope you find good now. About the graph’s titles, we have described their meaning in the legend

 

Lines 548-574: Please refer to the respective figure concretely within the text.

R: Thanks! We have already referenced this correctly.

 

Line 575: Delete “and” before “under…”

R: Corrected!

 

Line 603: Here you mention “label M stands for mock treatment” But there is no M – here you write “Mock” . Please check – also in Fig. 8.

R: Thanks a lot for the comment! We have corrected these cases

 

DISCUSSION

The discussion contains superfluous information which are belonging rather to an introductory chapter. Delete lines 698-711.

R: We appreciate this comment. However, we consider relevant this introduction in order to refresh the topic and facilitate the understanding of the rest of the discussion. We consider this is more a style option and, if you don’t mind, we would like to maintain it here.

 

Also, the paragraph from line 712-741 should be shortened.

R: OK! We have applied strong changes and shortening to this section. Following your previous recommendation, we have reviewed all this section.

 

Down from line 742 the real discussion starts.

R: As mentioned before, we have applied strong changes and shortening to this section. Following your previous recommendation, we have reviewed all this section.

 

Please avoid redundant information or overall introductory sentences.

R: Thanks, we have applied this change

 

But you should discuss more on the restriction of your finding especially regarding the culturable organisms from soil and the role of competing microorganisms which may mimic the beneficial effect of your strains. Are the strains you cultured representative for that environment? Please extend the discussion in that direction and give a more reserved conclusion.   

R: Thanks a lot for the comment! We have applied changes in this sense.

Round 2

Reviewer 2 Report

1. The authors have greatly improved the manuscript, especially in English. The authors answered to the comments that were numbered in the text sent by the reviewer. However, the authors disregarded most of the comments and minor corrections that were made in the previously sent PDF document.

 

2. The authors improved Table 2 by changing the labels of the columns. However, they did not clarify the main issue: it is not possible to isolate the same strain from 3 different sampling sites. For example, strain 14 identified as belonging to the species Pseudoclavibacter helvolus could not be isolated at sites 6, 7, and 8. Each isolate has a strain designation, which means that the isolate at site 6 must have a designation (e.g. 14), and the isolate from locations 7 and 8 should have other names, respectively. Unless the authors designate strain 14 as the representative strain of isolates belonging to the species Pseudoclavibacter helvolus.

 

3. Although the authors include references to papers that use the pie chats to show the relative abundances. I still think that it is very difficult to understand the same color for different sections, identifying the strain.

1. The authors have greatly improved the manuscript, especially in English. The authors answered to the comments that were numbered in the text sent by the reviewer. However, the authors disregarded most of the comments and minor corrections that were made in the previously sent PDF document.

Author Response

Dear reviewer,

We really appreciate your comments. We are really sorry not to fully understand your meaning in some comments, but please, consider we have applied all your major and minor comments in a great effort to improve this manuscript. Your help is really appreciated because we feel the quality of the work improved and now is clearer and stronger. Considering your last comment, here we have tried to address them (and changes were carefully applied in the manuscript, consequently):

- Comment 1: The authors have greatly improved the manuscript, especially in English. The authors answered to the comments that were numbered in the text sent by the reviewer. However, the authors disregarded most of the comments and minor corrections that were made in the previously sent PDF document.

A: We hope you can reconsider the term 'disregarded', because we have been very incisive to change and adopt all the suggestions and comments, as well as change and resolve the mistakes indicated. If some precise one was not addressed, please tell us, because we were very carefully taking into the account your comments. 

- Comment 2: The authors improved Table 2 by changing the labels of the columns. However, they did not clarify the main issue: it is not possible to isolate the same strain from 3 different sampling sites. For example, strain 14 identified as belonging to the species Pseudoclavibacter helvolus could not be isolated at sites 6, 7, and 8. Each isolate has a strain designation, which means that the isolate at site 6 must have a designation (e.g. 14), and the isolate from locations 7 and 8 should have other names, respectively. Unless the authors designate strain 14 as the representative strain of isolates belonging to the species Pseudoclavibacter helvolus.

A: We understood now what you're meaning, and considered correct. Sometimes we are so focused on our history that we don't see these things. We have decided to rebuild it, simplifying and reordering in order to make it clearer. Thanks for the suggestion! 

- Comment 3: Although the authors include references to papers that use the pie chats to show the relative abundances. I still think that it is very difficult to understand the same color for different sections, identifying the strain. 

A: In this point, we consider it's just a matter of preferences. The pie charts with the organization we chose is based in functional groups of bacteria. May consider some other kind of organization, but this type of organization is valid for the type of description we are looking for. We understand that some readers may not find it clear at first, but many complex figures have different versions that could conversely be very understandable for a section of the readers and very difficult to interpret for the other. So we don't consider this a matter
for major review... We have consulted some colleagues in order to see how understandable, and in most of the cases they get our point. This organization aims to join close related strains, and in just one touch the audience can see where these 'clusters' were found along the sampling areas. We prefer to keep this option, hope you understand.

Said all this, we want to appreciate again all the help, time and support provided and hope to cope with your expectations, thanks!