PONE-D-24-56644Mechanisms of growth promotion and resistance of PGPR to Elymus nutans Griseb at different temperaturesPLOS ONE

Dear Dr. wang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Massimiliano Cardinale, PhD

Academic Editor

PLOS ONE

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This work was financially supported by the Science and Technology Program of Tibet Autonomous Region (XZ202201ZY0005N), Sichuan Natural Science Foundation Project (2024NSFSC2074), National Natural Sciences Foundation of China (NO.41867013), China Forestry and Grassland Reform and Development Fund (GZFCG2023-17620), and Innovation Team Funds of China West Normal University (KCXTD2023-5). We also thank the anonymous reviewers for providing critical comments and suggestions that improved the manuscript.  

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Additional Editor Comments :

Considering the criticisms of Reviewer 3, which I agree with, I suggest ro temove the term "Mechanisms" from the title. You evaluated the effects of PGPR application, not the mechanisms.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: This is a valuable study as strains were obtained and characterized from the Tibetan Plateau, a unique and understudied ecosystem. The potential use of plant growth promoting bacteria in pasture production in this ecosystem is an interesting study area. However, it seems to me that the tested conditions (fertilizer scheme, substrate and temperature conditions) are not relevant to the application area, the Tibetan Plateau. Therefore, the results of this study are far less relevant and do not support the stated conclusions.

The results and discussion are very difficult to understand due to the use of many abbreviations, coded treatments and a lack of structure in the text. Therefore, I was also not able to verify the statistical analyses.

regarding availability of data:

- important information is lacking in Material and methods, e.g. NFM-agar, brief descriptions of methods, the preparation of inoculum (CFU counts etc)

- The manuscript does not disclose which five strains out of 273 strains were selected, and based on which criteria these were selected.

Reviewer #3: There is no experiment of mechanism of growth promotion and resistance of PGPR (eg. ROS, PO, PPO, PAL activity; amount of MDA, GSH, IAA, antioxidants, etc.) in this study. Here, title of this paper does not represent the results. This research is mainly focused on nutrient uptake efficiency of PGPR in peat soil based substrate with different temperature. In another words, authors should mention what and where is the PGPR mechanism in this study?

Reviewer #4: Comments for Authors

The abstract should be rewritten in a more systematic pattern to improve clarity and readability. Clarify the research aim, emphasizing the focus on temperature effects on PGPR-mediated plant growth and nutrient utilization. Specify the type of pot experiments conducted, including the temperature ranges used. Explain the significance of the treatments (T1, T2, T3, T4) briefly and consistently. Summarize the findings succinctly, focusing on the most significant results. The introduction section needs to incorporate more recent literature to provide a comprehensive background. In introduction section ([12] revealed that specific PGPR strains can impart cold resistance to rice plants, boosting their viability, fertility, and yield related traits and accelerating the progression of their reproductive cycle. Furthermore, variation in temperature tolerance exists among PGPR strains, affecting efficacy and plant health [13].), needs to rewrite. In methodology section the sub-heading “Identification of PGPR strains” is written well but its too lengthy, make it simple and short. In the result sectionIt is mention that percentage increases in plant traits like PT (plant height) and DW (dry weight). Could these increases be contextualized in terms of their biological or agricultural significance? What do these percentages mean for the practical application of PGPR in farming or plant growth?

Why were the specific temperature ranges (10 °C, 15 °C, 20 °C) chosen? Could the authors explain the relevance of these temperatures to the natural habitat or agricultural practices of Elymus nutans Griseb (EnG)?

There is a significant increase in Chl (chlorophyll content). What is the implication of increased chlorophyll content in terms of plant photosynthetic efficiency or overall health?

The root traits like RSA (root surface area) and RL (root length) are significantly affected by PGPR. Could the authors discuss how these changes in root architecture influence the overall nutrient uptake and drought resistance of EnG?

The paper mentions that RL was significantly higher at 15 °C than at 20 °C. What could be the physiological reasons behind this temperature-specific root growth pattern?

Why is there a significant increase in SS at 10 °C compared to 20 °C under the T3 treatment? Can the authors provide a rationale for this temperature-dependent response?

The increases in NAE (nitrogen agronomic efficiency), PAE (phosphorus agronomic efficiency), and KAE (potassium agronomic efficiency) are significant. Could the authors elaborate on the potential long-term benefits of these increases for sustainable agriculture?

The interaction between temperature and PGPR significantly affects RL. Could the authors discuss the possible mechanisms or environmental factors that might drive this interaction?

Discussion is written well however for the clarification of the above question modify this section accordingly.

Reviewer #5: The paper (PONE-D-24-56644) is a nice demonstration of growth promotion and resistance enhancement by PGPR to Elymus nutans Griseb at variable temperatures.

I have a few minor/major comments that authors should address before the manuscript can be accepted for publication.

Abstract is overly dense and uses unexplained abbreviations (e.g., "SS and SP") which may confuse non-expert readers.

The broader ecological and economic implications of focusing on Elymus nutans in the introduction are not sufficiently emphasized.

Put clear distinction between biological and technical replicates.

There is a lack of details on how environmental factors like light intensity and humidity were controlled across treatments.

Statistical methods (e.g., SPSS) are mentioned but without sufficient elaboration on key assumptions (e.g., normality checks).

Results:

Limited explanation for unexpected trends, such as the decrease in physiological efficiency of nitrogen (NPE) at higher temperatures in T3 treatment.

You mentioned p-values but could not discuss the biological significance of findings.

Table 2 presents complex statistical results without adequate textual interpretation for non-specialists.

For figures, legends are overly technical and could be simplified for clarity.

The color coding in heatmaps (Figure 7a) is inconsistent, making interpretation less intuitive. Also, text is not legible.

In Fig. 2, measuring scales are not legible. Better to use a common scale for all and that should be clearly visible.

Improve discussion: there are repetitive points about the benefits of PGPR, leading to redundancy.

Alternative mechanisms for PGPR effects (e.g., microbial community shifts) are not discussed.

Limited exploration of variability among the five PGPR strains tested.

Discuss limitations of the study in the conclusion, such as the exclusion of field trials or the potential for long-term ecological impacts. Give practical recommendations for scaling PGPR applications are underdeveloped.

Below are some papers for authors for scientific discussion and for probable citation:

doi.org/10.1007/s00344-023-11119

https://doi.org/10.1016/B978-0-323-91595-3.00011-2

Plant Stress 11, 100397

Plant Growth Regulation 99 (3), 449-464

https://doi.org/10.3389/fpls.2021.746780

https://doi.org/10.3390/microorganisms9122451

Environmental and Experimental Botany 200, 104911

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Reviewer #2: Yes:  Marie Legein

Reviewer #3: No

Reviewer #4: Yes:  Arshad Iqbal

Reviewer #5: No

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Submitted filename: PONE-D-24-56644.pdf

PONE-D-24-56644R1Effect of PGPR on growth and nutrient utilization of Elymus nutans Griseb at different temperaturesPLOS ONE

Dear Dr. wang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Apr 19 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Massimiliano Cardinale, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

Reviewer #4: All comments have been addressed

Reviewer #5: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #3: Partly

Reviewer #4: Yes

Reviewer #5: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Thank you for revising your manuscript thoroughly. Some comments remain, most importantly a comment raised by another reviewer concerning biological and technical replicates:

- In response to this comment, you have made adjustments in section 2.1, regarding the isolation of bacterial strains. However, it is in the experimental part of the manuscript i.e. in the incubation experiment (2.5) that biological and technical replicates are needed. Can you provide more detail here, i.e. how many replicates per condition, how many seeds per pot, how many measurements per parameter, did you repeat it at different timepoints, with freshly made microbial mixtures? Secondly, the plants were incubated in three incubators, were the conditions randomized between these incubators?

Other comments are minor:

- Abstract: The study started at an earlier stage then the demonstration of growth promoting effects. I suggest including more context: i.e. rhizosphere bacteria were isolated (at this stage it is still unknown if these are PGPR), selected based on known PGPR traits which were assessed in vitro, and finally tested in plants. This context underlines the novelty of the research: novel PGPR were selected specifically for Eng on Tibetan plateau.

- M&M: 2.6.3: briefly explain the methods used to determine these plant nutrient use efficiency, instead of including a reference.

- M&M: Change “excellent” strains to e.g. “selected” strains, to remain objective

- Results: Table 1: change in caption > 5 strains. Please include identification of the strains, preferably also in abstract and discussion. Are these species known for their PGPR effects?

- readability of the results and discussion remains difficult. Below a few recommendations to improve:

o describe the different treatments instead of uniquely referring to T1, T2 etc

o guide the reader to better understand figure 7; i.e. how was this figure made, what does it show, and how should it be interpreted?

o explain the differences between physiological efficiency (PE) and apparent recovery efficiency (RE) and how this is relevant for this research.

- Discussion: This phrase is misleading and should be nuanced: “Consistent with these findings, the culture substrate containing PGPR elicited IAA production (Table 1)” > The PGPR produced IAA on certain media, but PGPR eliciting IAA production in plants was not studied. Secondly, it is not sure that IAA was also produced in the conditions of the incubation experiment.

Reviewer #3: The title of this paper was different from now. So, some additional data and corrections are necessary to accept this manuscript as full length paper (comments are given as attached file). Hope, the author will response accordingly.

Reviewer #4: (No Response)

Reviewer #5: Authors have addressed my comments. The manuscript can now be accepted. I have not looked into comments from other reviewers.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #2: Yes:  Marie Legein

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

**********

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: PONE-D-24-56644_R1.pdf

Attachment

Submitted filename: Comments on PLOS.docx

PONE-D-24-56644R2Effect of PGPR on growth and nutrient utilization of Elymus nutans Griseb at different temperaturesPLOS ONE

Dear Dr. wang,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by May 14 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Massimiliano Cardinale, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Academic Editor:

Both reviewers accepted the manuscript but pointed out that the abstract is neither fluent nor clear. Therefore, I read it carefully and I found that the language is below the minimal scientific standard. For example:

- "In order to show the growth-promoting effects of selected PGPR isolated from the rhizosphere soil of Elymus nutans Griseb on the Tibetan Plateau." cannot be a stand-alone sentence: main clause is missed;

- "By isolating and purifying the rhizosphere bacteria from the rhizosphere soil of Elymus nutans Griseb (EnG) in the Qinghai-Tibet Plateau, use the selective culture medium to determine whether the strains have plant growth-promoting ability and measure the magnitude of their plant growth-promoting ability". This sound as a protocol where someone is giving you instructions.

I read the rest of the manuscript and I found further parts where the language is poor. Just as a few examples:

- "Five selected strains (S1, S2, S3, S4 and S5) with high growth promoting ability were screened by the determination of growth-promoting ability". Really confusing and repetitive.

- "Inoculate the purified strains onto Nitrogen free medium (NFM)..." This sound as a protocol where someone is instructing you to inoculate the strain on different media. In the article, you must write "Strains were inculated onto NFM..." etc.

Therefore, it is necessary that the manuscript is revised by a native english speaker before it can be accepted and published.

Another important aspect to be clarified concerns the statistical analysis: it is stated that both least significant difference (LSD) and Tukey test (HSD) were used. However, in the graphs where post-hoc letters are reported, it is not indicated which post-hoc method was used. Considering that these two post-hoc tests are very different (LSD the most permissive, HSD the most stringent one), it is really necessary that this is clearly indicated: significant differences assessed by HSD have a much greater value that those assessed by LSD!!

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: No

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

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Reviewer #2: Authors have addressed my comments. The added text in the abstract could be written more fluently, but the manuscript can be accepted.

Reviewer #3: See the attached file. In abstract, there is also a comment. It is suggested to solve this query and correct the word.

hope you will formulate this PGPR in future for commercial use.

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Reviewer #2: Yes:  Marie Legein

Reviewer #3: Yes:  Prof. Dr. Mohammad Delwar Hossain

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Attachments

Attachment

Submitted filename: Comments on PLOS.docx

Effect of PGPR on growth and nutrient utilization of Elymus nutans Griseb at different temperatures

PONE-D-24-56644R3

Dear Dr. wang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Massimiliano Cardinale, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for revising the manuscript according to my concerns.

Please, at the proofs correction step, remember to correct one sentence in the paragraph 2.3. "Identification of PGPR strains" as follows: "The 16S rRNA gene was amplified by polymerase chain reaction (PCR) ... ..."

Reviewers' comments: