Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions

Márton Szoboszlay; Astrid Näther; Ewen Mullins; Christoph C. Tebbe

doi:10.1371/journal.pone.0222737

Peer Review History

Original SubmissionAugust 30, 2019
14 Oct 2019 Decision Letter - Luigimaria Borruso, Editor PONE-D-19-24482 Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions PLOS ONE Dear Dr. Christoph Tebbe, 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. We would appreciate receiving your revised manuscript by Nov 28 2019 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Luigimaria Borruso Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Additional Editor Comments (if provided): [Note: HTML markup is below. Please do not edit.] 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 #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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 #1: Yes Reviewer #2: Yes ****** 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 #1: Yes Reviewer #2: Yes ****** 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 #1: The paper "Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions" is interesting and intriguing paper, but my first impression is that, despite a large amount of data it should have been obtained from analyses, there is a is little in-depth analysis and enhancement of them. I think that it should be enhanced, by re-organizing it in a more linear structure. The Introduction is very clear and specific and it well focus on the subject of the work. Furthermore, it is also very smooth and pleasant to read. Materials and methods are very detailed but not very fluent, so I suggest to change the text to make it more pleasant, perhaps by eliminating repeating parts. The Results and discussion should be ordered, them are dispersive, it would be good to reorganize them in a more schematic way. Discussions are interesting and well focused on the results, but need to be reworked and more detailed. I provide some suggestions for the authors, as follows: 114-121: it would be good to insert the data of the soils in a table (or insert them in table 1). Moreover, have the chemical-physical data been collected every year? Or just once? It is not clear, please clarify it. 122-130: is the protocol already described in some work and/or modified by some other paper on the same topic? please specify it. 178-180: why authors used two libraries with different chemistries performed? Since the aim of the work is a comparative study of the microbiome, this could lead to an error in the comparison of the results. Did authors consider it? Please state this choice. 204: why was the 123 database chosen (release 2015), considering that the most recent version is 132 ( release 2018)? 322-329: I suggest to outline the community found in a table, with the percentage of phyla and genus retrieved in samples. I would also add a table with sequencing yields results reported in the text (easier to follow). 410-417: this sentence is very convoluted, it would be good to write the concept better. 430-432: the results are interesting, but why authors showed only the graph related to one of the sampling years? Authors should show them all in a single graph. 462-472: was the concentration of the Cry1Ab samples measured? If yes, authors should add it to material and methods as well as in the results. If not, all that period should be modified. Reviewer #2: Number: PONE-D-19-24482 Title: Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions General comments The research is original, following established scientific procedures and it is scientifically sound. The manuscript is well structured and the writing is concise and the English level allows a fluent lecture to understand the concepts and the authors’ intent. Therefore, please clarify a few things and finally, I would like to recommend the manuscript for the publication in PLOS ONE. Detailed comments Line 39: Instead of colon (:), I would rather prefer a point (.) after “decades”. Line 40: Please write Nowadays instead of “Today”. Line 68: Please use a comma (,) instead of “and” after “18S rRNA”. Line 71: Please write Among them instead of “E.g”. Line 73: Please add the abbreviation (N) after “nitrogen”. Line 117- 121: Please write “…total C and …total N contents”. Line 131: Please uniform the modus of reporting Throbäck et al. (2004) according the Journal’s requests. Line 251-253: Although the results in terms of abundance of 16S rRNA and nirS genes were not significant as a function of site and sampling year, I would like to see them in Table 2. Please add them. Line 370-372: I am wondering why the results of ITS diversity for the year 2014 are not shown in Figure 3. Please double-check. Figure 5: Why did you choose to perform the NMDS analysis of results concerning the year 2013 and not for the other two years (2012 and 2014)? References: Please check and uniform the references following the author's guidelines. Line 599: Please write “Bacillus thuringiensis” in italic font. ****** 6. 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 #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0222737.r001
Revision 1
24 Oct 2019 Author Response Dear Editor and Reviewers, we are grateful for your constructive comments that helped us improved the manuscript. Below we address the points and questions raised. Reviewer #1: R:The paper "Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions" is interesting and intriguing paper, but my first impression is that, despite a large amount of data it should have been obtained from analyses, there is a is little in-depth analysis and enhancement of them. I think that it should be enhanced, by re-organizing it in a more linear structure. The Introduction is very clear and specific and it well focus on the subject of the work. Furthermore, it is also very smooth and pleasant to read. Materials and methods are very detailed but not very fluent, so I suggest to change the text to make it more pleasant, perhaps by eliminating repeating parts. The Results and discussion should be ordered, them are dispersive, it would be good to reorganize them in a more schematic way. Discussions are interesting and well focused on the results, but need to be reworked and more detailed. A:This work was based on a large amount of data which, we agree, could be used to support the investigation of further questions. The aim of this manuscript, however, was not to present every bit of information that is possible to gain from the data but to fulfill the specific goals of the project as outlined in the introduction and present a focused study. The Results and Discussion sections are structured in a linear fashion along these goals. The materials and methods section was checked for redundancy. The data can be utilized to explore further questions in future manuscripts and is made available to the scientific community both as the raw sequencing output (European Nucleotide Archive) and a data matrices of the SV and TSV counts (Supplementary file 1). R:I provide some suggestions for the authors, as follows: 114-121: it would be good to insert the data of the soils in a table (or insert them in table 1). Moreover, have the chemical-physical data been collected every year? Or just once? It is not clear, please clarify it. A: The soil parameters were measured at every sampling time but had little variation. The goal of the project was not to study the effects of soil parameters on the rhizomicrobiome of maize, thus they are shown only as background information and were not used in the data analysis. Since the four sites were located in different biogeographical regions, they differed not just in soil parameters. It was more appropriate to use “site” in the analysis as a factor that combines the effects of soil type, climate etc. R: 122-130: is the protocol already described in some work and/or modified by some other paper on the same topic? please specify it. A: Obtaining samples of the rhizosphere by washing closely attached soil particles off the roots is a common practice. Yet the exact sample masses, volumes, and centrifugation parameters are optimized to each case and thus differ between studies. R: 178-180: why authors used two libraries with different chemistries performed? Since the aim of the work is a comparative study of the microbiome, this could lead to an error in the comparison of the results. Did authors consider it? Please state this choice. A: Sequencing of the bacterial and archaeal 16S rRNA amplicons from the samples from 2012 and 2013 took place before the sampling in 2014. At that time, the V3 chemistry was not available. Sequencing the 16S rRNA gene from the 2014 samples, and the nirK and ITS from all of the samples were performed later with the V3 chemistry. We agree that the potential bias of the different sequencing chemistries can add to or modify the differences in the 16S data between the 2014 samples and the samples from the other two years. Unfortunately, this effect can’t be separated from the year-to-year variation in this case. Being aware of this limitation, we did not quantify or test the significance of the differences in bacterial and archaeal community structure between 2014 and the other two years. The only exception is the Simpson diversity index, which we compared between the years. In this case, however, the only significant difference we found was driven by the high abundance of one SV in the Slovakian samples from 2013 (lines 356 – 361). We consider it extremely unlikely that this result is caused by the different sequencing chemistries. The analysis of the bacterial and archaeal community structure was focused on comparing the Bt to the non-Bt samples from the same site and year which were processed with the same sequencing chemistry in each case. A year-to-year variation in the bacterial and archaeal community structure was observed on the PCA plots (Figure 4 A-D). Potential biases of the sequencing chemistries can contribute to, but certainly not solely responsible for the distance of the 2014 samples from the other samples on the plots, especially considering the clear separation between the 2012 and 2013 samples. Accordingly, the presence of year-to-year variation was stated but it´s effect size was not quantified and responding SVs or taxa were not reported (lines 377 – 380). R: 204: why was the 123 database chosen (release 2015), considering that the most recent version is 132 (release 2018)? A: Version 123 was the most up-to-date release of the SILVA reference at the time of the analysis of the 16S sequencing data. R: 322-329: I suggest to outline the community found in a table, with the percentage of phyla and genus retrieved in samples. I would also add a table with sequencing yields results reported in the text (easier to follow). A: Such an outline of the community in a table format would result in two enormous tables, one for 16S the other for ITS, with 556 and 427 rows respectively. Furthermore, it would provide much information relevant to the objectives of the manuscript. Interested readers can easily generate these tables from the SV matrices provided in Supplementary file 1. R: 410-417: this sentence is very convoluted, it would be good to write the concept better. A: This section consists of short sentences with simple structure. They are not describing a concept just making simple statements. R: 430-432: the results are interesting, but why authors showed only the graph related to one of the sampling years? Authors should show them all in a single graph. A: The site in Denmark was not sampled in 2012 and the site in Spain in 2014 (Table 1 and also line 123). Therefore, the data from 2013 is the best to demonstrate the point raised in this section as it includes all the sites. R: 462-472: was the concentration of the Cry1Ab samples measured? If yes, authors should add it to material and methods as well as in the results. If not, all that period should be modified. A: The concentration of the Cry1Ab protein was not measured in the rhizosphere samples. The fact that MON810 releases it in its rhizosphere has been demonstrated previously in several studies (see references in the section). Reviewer #2: General comments The research is original, following established scientific procedures and it is scientifically sound. The manuscript is well structured and the writing is concise and the English level allows a fluent lecture to understand the concepts and the authors’ intent. Therefore, please clarify a few things and finally, I would like to recommend the manuscript for the publication in PLOS ONE. Detailed comments R: Line 39: Instead of colon (:), I would rather prefer a point (.) after “decades”. - A: Corrected. R: Line 40: Please write Nowadays instead of “Today”. A:Corrected. R: Line 68: Please use a comma (,) instead of “and” after “18S rRNA”. A:“And” is appropriate. R: Line 71: Please write Among them instead of “E.g”. A: Corrected. R: Line 73: Please add the abbreviation (N) after “nitrogen”. A: We don’t consider it necessary to indicate the chemical symbol of nitrogen. It’s known by the readers. R: Line 117- 121: Please write “…total C and …total N contents”. A: Corrected. R: Line 169: Please uniform the modus of reporting Throbäck et al. (2004) according the Journal’s requests. A:The references were updated. R: Line 251-253: Although the results in terms of abundance of 16S rRNA and nirS genes were not significant as a function of site and sampling year, I would like to see them in Table 2. Please add them. A: Differences between sites and sampling years are shown in Figure 1. R: Line 370-372: I am wondering why the results of ITS diversity for the year 2014 are not shown in Figure 3. Please double-check. A: The samples from 2014 were not included in the fungal ITS sequencing due to financial constraints. We apologize that a clear statement on this was missing from the method section. We added it to lines 181 and 341. R: Figure 5: Why did you choose to perform the NMDS analysis of results concerning the year 2013 and not for the other two years (2012 and 2014)? A: The NMDS plots in Figure 5 serve to demonstrate that regarding differences between the sites, a similar pattern was found in the 16S, nirK, and ITS datasets approximating the geographical distances between the sites. The site in Denmark was not sampled in 2012 and the site in Spain in 2014 (Table 1 and also line 123). Therefore, the data from 2013 is the best to demonstrate this as it includes all the sites. R: References: Please check and uniform the references following the author's guidelines. A: The references were updated. R: Line 599: Please write “Bacillus thuringiensis” in italic font. A: Corrected. Attachments Attachment Submitted filename: Response to the reviewers.docx https://doi.org/10.1371/journal.pone.0222737.r002
26 Nov 2019 Decision Letter - Luigimaria Borruso, Editor PONE-D-19-24482R1 Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions PLOS ONE Dear Dear Prof. Dr. Christoph C Tebbe, 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. ============================== I will be delighted to accept personally your manuscript after you have responded to the last few requirements of Reviewer 2. ============================== We would appreciate receiving your revised manuscript by Jan 10 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Luigimaria Borruso Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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 #1: All comments have been addressed Reviewer #2: (No Response) ******** 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 #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 #1: Yes Reviewer #2: 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 #1: Yes Reviewer #2: 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 #1: All comments indicated in the review have been addressed in a clear and categorical way. I am fully satisfied. Reviewer #2: Number: PONE-D-19-24482R1 Title: Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions General comments The quality of the revised manuscript has been improved. Thanks to the authors that have followed the reviewers’ comments. Therefore, please clarify a few things and finally, I would like to recommend the revised manuscript for the publication in PLOS ONE. Detailed comments Line 122: Please remove a bracket. Line 379: “did not” instead of “do not”. Line 384: “showed” instead of “show. Line 386: “did not” instead of “do not”. ****** 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 #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0222737.r003
Revision 2
26 Nov 2019 Author Response All four modifications suggested for the 2nd revision have been accepted and implemented Attachments Attachment Submitted filename: Response to the 2ndreviewerscomments.docx https://doi.org/10.1371/journal.pone.0222737.r004
2 Dec 2019 Decision Letter - Luigimaria Borruso, Editor Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions PONE-D-19-24482R2 Dear Dr. Christoph C Tebbe, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Luigimaria Borruso Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0222737.r005
Formally Accepted
6 Dec 2019 Acceptance Letter - Luigimaria Borruso, Editor PONE-D-19-24482R2 Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions Dear Dr. Tebbe: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Luigimaria Borruso Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0222737.r006

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