Potential tradeoffs between effects of arbuscular mycorrhizal fungi inoculation, soil organic matter content and fertilizer application in raspberry production

Ke Chen; Jeroen Scheper; Thijs P. M. Fijen; David Kleijn

doi:10.1371/journal.pone.0269751

Peer Review History

Original SubmissionJanuary 28, 2022
16 Mar 2022 Decision Letter - Sergio Saia, Editor PONE-D-22-02818Potential tradeoffs between effects of arbuscular mycorrhizal fungi inoculation, soil organic matter content and fertilizer application in raspberry productionPLOS ONE Dear Dr. Chen, 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. Four reviewers assessed the ms and raised important concerns, especially the statistical analysis, that I warmly suggest you to address. Please submit your revised manuscript by Apr 28 2022 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, Sergio Saia, Ph.D. Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. 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 https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: (We thank Emiel van Riet for his assistance with the fieldwork. KC was funded by the China Scholarship Council (File No. 201706990023).) We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: (The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.) Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [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: Yes Reviewer #2: Yes Reviewer #3: No Reviewer #4: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No Reviewer #3: Yes Reviewer #4: I Don't Know ****** 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 Reviewer #3: Yes Reviewer #4: No ****** 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 Reviewer #3: Yes Reviewer #4: 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: It is interesting to study the interactive effects of SOM and AMF on the production of raspberry, the results showed that potentially beneficial effects of AMF and SOM can be offset by each other, probably driven by the dynamic relations between AMF and the host Plants. In materials and methods, pH value in soil, and other mineral nutrients such as Ca, Mg, Zn and Cu etc in soil needs to be supplemented. Additionally, the AMF-inoculated dosage, the spore number, and the mycorrhizal colonization determination method needs to be supplemented too. Reviewer #2: The manuscript aim is to study the interaction between soil organic matter content (SOM) and arbuscular mycorrhiza fungi (AMF) inoculation and how they are affected by mineral fertilization application in raspberry yield. The hypothesis of the work is there is a tradeoff between SOM and AMF. The objectives are interesting and novel because the authors studied the three factors interaction: SOM, AMF, and mineral fertilizer. The methodology is well written and honest because the authors explain that they have problems with plant survival and growth. However, the experimental design and data analysis have to be improved because the description of the blocks is not understandable. Looking at the excel data file of the supplementary material, the blocks hold different treatments and are not balanced. For example, blocks 1, 2, and 3 contain all the treatments (AMF, SOM, and fertilizer) for big plants. However, block 5 contain only inoculated, low SOM and 2 fertilizers for small and block 6 inoculated plants and low SOM but 1 plant of each fertilizer level. In my opinion, blocks have to contain all the treatments. In line 172-173, the authors say that “response variables were averaged per plant to avoid pseudoreplication”. But it is only possible in the single berry weight. The authors calculated the sum of all the values of the other variables, yield, flower, and fruit number per plant. The discussion part and the conclusions are well written. The reference 58, the journal is Acta Horticulturae, and in reference 69 the journal name is missed. In Figure 1, the graph only shows plant response to fertilizer level, however, in the caption effects of AMF and SOM are included. This caption need revision. Reviewer #3: The major limitation of this study is that AMF colonization in the roots of Rubus idaeus was not assessed. The authors conclude about the effects of AMF, however, without the data on AMF colonization there is no guaranty that AMF inoculation actually resulted in the formation of arbuscular mycorrhizas. Without mycorrhizas there can be no effects. Mycorrhization depends on multiple factors, such as quality of inoculum, target plant species, environmental factors, etc., so it cannot be taken for granted. AMF colonization assessment needs to be done. Reviewer #4: The manuscript reports data on a mixed experimental setup (plants of two different origins), without a detailed description of the experimental design: - the authors claim that they used a randomized complete block design, but no explanation of the variables on which blocks were designed and why is provided; - the data provided reveal that unbalanced block design is obtained, and many blocks lack most of the treatment. as my expertise in R is limited, I cannot check it by myself, thus I ask the authors if the linear mixed effects models take in account this uneven distribution of treatments - it seems to me that one single plant per treatment per block (when available) has been monitored: what does it mean that "Response variables were averaged per plant to avoid pseudoreplication", given that data originate from only one plant? please clarify - data provided in the excel file report only the variable "weight", assessed for each data point, although in the text other variables are analysed (flower number, fruit number, single berry weight (g/fruit) and total yield (g/plant)). As the journal requires authors to make all data underlying the findings described in their manuscript fully available without restriction, I think results fo all variables should be provided The absence of data and details make this manuscript difficult to be properly evaluated. ****** 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 Reviewer #3: No Reviewer #4: 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.] 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. https://doi.org/10.1371/journal.pone.0269751.r001
Revision 1
22 Apr 2022 Author Response Response to Reviewers Reviewer #1: It is interesting to study the interactive effects of SOM and AMF on the production of raspberry, the results showed that potentially beneficial effects of AMF and SOM can be offset by each other, probably driven by the dynamic relations between AMF and the host Plants. In materials and methods, pH value in soil, and other mineral nutrients such as Ca, Mg, Zn and Cu etc in soil needs to be supplemented. Additionally, the AMF-inoculated dosage, the spore number, and the mycorrhizal colonization determination method needs to be supplemented too. Reply: Thanks for the reviewer's suggestions. We have added pH values of the experimental soils in the revised manuscript in line 112 and 114. Unfortunately, we don’t have measures of Ca, Mg or Zn. As we used the same soils for the experiment, and we focus on the effect of artificial fertiliser (N, P and K), we think those nutrients have limited effects on the final results. We added the spore number of the AMF inoculum in line 115, and the dosage was added in line 124. In this study we have focussed on the management practice of AMF inoculation, rather than studying the effect of AMF on yield. In an earlier study we found that all plants were colonised similarly by AMF at the end of the experiment, likely because the plants were growing already for almost a year in non-sterilised soils (Chen et al 2022, DOI: 10.1016/j.agee.2021.107742). We nevertheless found (there and here) effects of AMF inoculation on yield, suggesting that the management practice of inoculation had an effect. Because we do find effects of AMF inoculation, and because we had limited access to the laboratory due to COVID-19, we decided not to measure root colonization for this study, and can therefore not add this information. However, we have now elaborated on this choice in the methods. Reviewer #2: The manuscript aim is to study the interaction between soil organic matter content (SOM) and arbuscular mycorrhiza fungi (AMF) inoculation and how they are affected by mineral fertilization application in raspberry yield. The hypothesis of th e work is there is a tradeoff between SOM and AMF. The objectives are interesting and novel because the authors studied the three factors interaction: SOM, AMF, and mineral fertilizer. The methodology is well written and honest because the authors explain that they have problems with plant survival and growth. However, the experimental design and data analysis have to be improved because the description of the blocks is not understandable. Looking at the excel data file of the supplementary material, the blocks hold different treatments and are not balanced. For example, blocks 1, 2, and 3 contain all the treatments (AMF, SOM, and fertilizer) for big plants. However, block 5 contain only inoculated, low SOM and 2 fertilizers for small and block 6 inoculated plants and low SOM but 1 plant of each fertilizer level. In my opinion, blocks have to contain all the treatments. Reply: Thanks for the reviewer's comments. We agree with the reviewer that it is unfortunate that we have incomplete treatment combinations due to relatively high mortality. The blocks were physical allocations of the pots to account for random gradients at the experimental site, and can therefore not be altered anymore. However, our used statistical methods (general linear mixed models) can cope with this unbalanced data quite well, by only looking at the available pairwise comparisons within block, and then generalising this pattern over all the blocks. But to make the unbalanced data more explicit, we added a supplementary table (line 159) in the revised manuscript, to indicate the exact number of replicated raspberry plants per treatment combination. In line 172-173, the authors say that "response variables were averaged per plant to avoid pseudoreplication". But it is only possible in the single berry weight. The authors calculated the sum of all the values of the other variables, yield, flower, and fruit number per plant. Reply: Thanks for the suggestion. We have clarified in lines 166-167 that only the single berry weight per plant was averaged. The discussion part and the conclusions are well written. The reference 58, the journal is Acta Horticulturae, and in reference 69 the journal name is missed. Reply: Thanks. We have added the journal names in both references. In the current version, the original reference 58 is now reference 51 (reference style is changed to meet requirements of the journal); and reference 69 is now reference 71. In Figure 1, the graph only shows plant response to fertilizer level, however, in the caption effects of AMF and SOM are included. This caption need revision. Reply: Thanks for the comments. We have revised the caption. Reviewer #3: The major limitation of this study is that AMF colonization in the roots of Rubus idaeus was not assessed. The authors conclude about the effects of AMF, however, without the data on AMF colonization there is no guaranty that AMF inoculation actually resulted in the formation of arbuscular mycorrhizas. Without mycorrhizas there can be no effects. Mycorrhization depends on multiple factors, such as quality of inoculum, target plant species, environmental factors, etc., so it cannot be taken for granted. AMF colonization assessment needs to be done. Reply: Thanks for the reviewer's comments. As also explained in a response to reviewer 1: In this study we have focussed on the management practice of AMF inoculation, rather than studying the effect of AMF on yield. Indeed, in an earlier study we found that all plants were colonised similarly by AMF at the end of the experiment, likely because the plants were growing already for almost a year in non-sterilised soils (Chen et al 2022, DOI: 10.1016/j.agee.2021.107742). We nevertheless found (there and here) effects of AMF inoculation on yield, suggesting that the inoculation had an effect. Because we do find effects of the management practice of AMF inoculation, and because we had limited access to the laboratory due to COVID-19, we decided not to measure root colonization for this study, and can therefore not add this information. However, we have now elaborated on this choice in the methods. Reviewer #4: The manuscript reports data on a mixed experimental setup (plants of two different origins), without a detailed description of the experimental design: - the authors claim that they used a randomized complete block design, but no explanation of the variables on which blocks were designed and why is provided; Reply: Thanks for the reviewer's comments. Each block in the randomized complete block design (RCBD) of the experiment was designed to consist of all treatment combinations. However, only a few of the plants survived the fruit production process or survived but did not produce fruits, and thus the final data were unbalanced. In the revised manuscript, we have clarified this, and we added a supplementary table (line 159) to indicate the number of replicated raspberry plants per treatment combination. - the data provided reveal that unbalanced block design is obtained, and many blocks lack most of the treatment. as my expertise in R is limited, I cannot check it by myself, thus I ask the authors if the linear mixed effects models take in account this uneven distribution of treatments Reply: Thanks for raising this. Yes, our statistical approach can deal well with these unbalanced data, because it first compares effects within block and then generalises effects over blocks (Zuur et al. 2009, ISBN:9780387874586). These methods have also been used in similar experiments with unbalanced data (e.g., Motzke et al. 2015, DOI: 10.1111/1365-2664.12357; Tamburini et al. 2015, DOI: 10.1007/s00442-015-3493-1 ) - it seems to me that one single plant per treatment per block (when available) has been monitored: what does it mean that "Response variables were averaged per plant to avoid pseudoreplication", given that data originate from only one plant? please clarify Reply: Thanks for spotting this mistake. We have clarified in lines 166-167 that it is the single berry weight per plant that was averaged. - data provided in the excel file report only the variable "weight", assessed for each data point, although in the text other variables are analyzed (flower number, fruit number, single berry weight (g/fruit) and total yield (g/plant)). As the journal requires authors to make all data underlying the findings described in their manuscript fully available without restriction, I think results fo all variables should be provided The absence of data and details make this manuscript difficult to be properly evaluated. Reply: Thanks for noticing this omision. We have added data of all variables in the revised excel file. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0269751.r002
27 May 2022 Decision Letter - Sergio Saia, Editor Potential tradeoffs between effects of arbuscular mycorrhizal fungi inoculation, soil organic matter content and fertilizer application in raspberry production PONE-D-22-02818R1 Dear Dr. Chen, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- 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. Kind regards, Sergio Saia, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0269751.r003
Formally Accepted
8 Jul 2022 Acceptance Letter - Sergio Saia, Editor PONE-D-22-02818R1 Potential tradeoffs between effects of arbuscular mycorrhizal fungi inoculation, soil organic matter content and fertilizer application in raspberry production Dear Dr. Chen: I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of prof Sergio Saia Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0269751.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .