Tillage type and sentinel insect species affect the relative prevalence of the entomopathogenic fungus, Metarhizium robertsii, in soil

Shea A. W. Tillotson; Christina A. Voortman; John M. Wallace; Mary E. Barbercheck

doi:10.1371/journal.pone.0317149

Peer Review History

Original SubmissionSeptember 10, 2024
8 Oct 2024 Decision Letter - Rachid Bouharroud, Editor PONE-D-24-39841Tillage type and sentinel insect species affect the relative prevalence of the entomopathogenic fungus, Metarhizium robertsii, in soilPLOS ONE Dear Dr. Barbercheck, 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. ============================== Dear AuthorsPlease refer to reviewers comments especially those related to Metarhizium species identification. Please consider that as a vital piece of your feedback.Good luck ============================== Please submit your revised manuscript by Nov 22 2024 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, Rachid Bouharroud 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 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. When completing the data availability statement of the submission form, you indicated that you will make your data available on acceptance. We strongly recommend all authors decide on a data sharing plan before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire data will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption. Please be assured that, once you have provided your new statement, the assessment of your exemption will not hold up the peer review process. [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: Partly Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know Reviewer #3: 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: No Reviewer #3: 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 Reviewer #3: 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: Comments to the Authors The manuscript titled “Tillage type and sentinel insect species affect the relative prevalence of the entomopathogenic fungus, Metarhizium robertsii, in soil”. This research evaluated the in-season and legacy effects of soil management in four, three-year organic feed grain and forage production systems that varied in number and intensity of soil disturbances on the relative prevalence of the entomopathogenic fungus, Metarhizium robertsii. After carefully reviewing this research I can see some merit in this research but the current form not meet scientific standard so please carefully revise entire manuscript based on the following comments; 1. Line 111: The Tenebrio molitor and Galleria mellonella should be short form. The scientific name of fungi and insect name should be written as short form after first time mentioning carefully fixing these errors throughout the MS. 2. Line 138: check the font. 3. The introduction provides clear details about entomopathogens and their importance, otherwise the reader will be confused. 4. The methodological part should be short and informative, the current form hard to follow. 5. Line 607: which Metarhizium populations? Mention the species. 6. In this manuscript I can see several typographical errors so carefully correct it. Reviewer #2: The authors of the study performed interesting and long-term research, both in field and laboratory conditions. However, I have doubts about the correct designation of the species Metarhizium robertsii, which was the subject of the research. Doubts concern the lack of molecular tests to confirm species affiliation, which were performed only on the basis of morphological characteristics. Although the authors refer to their previous molecular research and 10 years of experience with the species M. robertsii, in my opinion this is not enough. Especially since, as Bischoff et al. reports in their 2009 study. it is not possible to distinquish taxa M. anisopliae, M. lepidiotae, M. pingshaense and M. robertsii, based on conidial morphology. Therefore, I suggest that the authors re-edit the manuscript and refer the obtained results to Metarhizium spp. I also have a question whether the larvae of both test species were infected only by fungi of the Metarhizium genus, or whether infections caused by e.g. Beauveria genus were also recorded? Reviewer #3: Dear Authors, Your manuscript on the effect of soil management on the prevalence of entomopathogenic fungus Metarhizium robertsii is interesting and brings original results with implications for improvement of biodiversity/ecological services of soil in both organic and conventional farming systems. The text of the manuscript is clear, methods are described in enough details, statistical analysis is sound and conclusions are based on obtained data. However, I believe there is some space for improvement as indicated in specific comments below. Numbers mean line numbers in manuscript. Title: it is fine, describes the study well. Abstract: I would recommend to add some values documenting the differences between treatments (it is not clear what is magnitude of observed differences, e.g. the range etc.). Introduction: I think it would be good to explain why the authors focused only on M. robertsii and did not look at other EPF species. 111 add order and family after species names 113-118 To me it could be reduced just to one hypothesis: "Greater frequency and intensity of soil disturbance has negative impact on relative prevalence of M. robertsii." 118-119 This could be the second hypothesis and authors can support it by citing recent findings on differences between those two bait species in the recoveries of different EPF species (e.g. Beauveria vs. Matarhizium). 243-244 Since these insect species were already mentioned in Introduction, here abbreviated version should be sufficient. 254-257 So there were no other EPF species (e.g. Cordyceps, Beaveria) found on this experimental plot? That is surprising. 494 Tillage has certainly positive role also in pest management of rodents and other pests 496 Perhaps other microogranisms might be mentioned as important, too. E.g. mycoparasitic fungi. 576-586 Some other technique of EPF isolation could be mentioned for comparison. E.g. in some studies EPF were isolated by culturing soil samples on selective media (Chase et al. 1986 , Correa et al. 2022 , Konopická et al. 2021 , 2022) so it might be useful for readers not familiar with these methods to discuss advantages of live insect-bait technique. 592-594 This species identity might be better placed earlier in Discussion (before discussion on system effect on prevalence of M. robertsii). 632 Better not to limit to insect pests, also mites and other invertebrates are important pets, not mentioning plant pathogens which can also be reduced by ecological services of beneficial microorganisms. ****** 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: Yes: Perumal Vivekanandhan Reviewer #2: No Reviewer #3: 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.0317149.r001
Revision 1
18 Dec 2024 Author Response Response to Reviewers PONE-D-24-39841 Tillage type and sentinel insect species affect the relative prevalence of the entomopathogenic fungus, Metarhizium robertsii, in soil We thank the reviewers for their comments and suggestions for improving the manuscript. Please find responses to the comments in BLUE CAPS below. Reviewer #1: Comments to the Authors The manuscript titled “Tillage type and sentinel insect species affect the relative prevalence of the entomopathogenic fungus, Metarhizium robertsii, in soil”. This research evaluated the in-season and legacy effects of soil management in four, three-year organic feed grain and forage production systems that varied in number and intensity of soil disturbances on the relative prevalence of the entomopathogenic fungus, Metarhizium robertsii. After carefully reviewing this research I can see some merit in this research but the current form not meet scientific standard so please carefully revise entire manuscript based on the following comments; 1. Line 111: The Tenebrio molitor and Galleria mellonella should be short form. The scientific name of fungi and insect name should be written as short form after first time mentioning carefully fixing these errors throughout the MS. REVISED AS SUGGESTED 2. Line 138: check the font. REVISED AS SUGGESTED 3. The introduction provides clear details about entomopathogens and their importance, otherwise the reader will be confused. WE BELIEVE THAT PARAGRAPH 2 OF THE INTRODUCTION DESCRIBES THE ROLE OF ENTOMOPATHOGENIC FUNGI AS A PEST SUPPRESSIVE ORGANISMS IN AGROECOSYSTEMS< AND WE CITE IMPORTANT REVIEW PAPERS ON THAT TOPIC. 4. The methodological part should be short and informative, the current form hard to follow. WE ACKNOWLEDGE THAT THE METHODS SECTION IS LONG AND DETAILED. THIS WAS A 3-YEAR SYSTEMS EXPERIMENT IN WHICH MANY MANAGEMENT VARIABLES DIFFERED AMONG SYSTEMS. WE HAVE REVISED TO SHORTEN AND IMPROVE CLARITY WHERE POSSIBLE, BUT CONSIDER THE AGRONOMIC DETAIL IMPORTANT TO UNDERSTANDIN THE EXPERIMENT. 5. Line 607: which Metarhizium populations? Mention the species. IN THE PAPER CITED, KEPLER ET AL. (2015) REFERRED TO MANIPULATION OF THE COMMUNITY OF METARHIZUM SPECIES AT A SITE IN GENERAL, RATHER THAN SPECIFIC SPECIES, HENCE OUR USE OF THE TERM ‘POPULATIONS’. 6. In this manuscript I can see several typographical errors so carefully correct it. WE HAVE CHECKED THE MS AND CORRECTED TYPOGRAPHICAL ERRORS Reviewer #2: The authors of the study performed interesting and long-term research, both in field and laboratory conditions. However, I have doubts about the correct designation of the species Metarhizium robertsii, which was the subject of the research. Doubts concern the lack of molecular tests to confirm species affiliation, which were performed only on the basis of morphological characteristics. Although the authors refer to their previous molecular research and 10 years of experience with the species M. robertsii, in my opinion this is not enough. Especially since, as Bischoff et al. reports in their 2009 study. it is not possible to distinquish taxa M. anisopliae, M. lepidiotae, M. pingshaense and M. robertsii, based on conidial morphology. Therefore, I suggest that the authors re-edit the manuscript and refer the obtained results to Metarhizium spp. I also have a question whether the larvae of both test species were infected only by fungi of the Metarhizium genus, or whether infections caused by e.g. Beauveria genus were also recorded? WE STORED SINGLE SPORE ISOLATES FROM OUR ASSAYS AND HAVE IDENTIFIED A REPRESENTATIVE SAMPLE OF THE ISOLATES BY MOLECULAR METHODS (ADDED TO THE MATERIALS AND METHODS AND SUPPLEMENTARY MATERIAL). ALL ISOLATES WERE IDENTIFIED AS METARHIZIUM ROBERTSII, WHICH IS CONSISTENT WITH OUR RESULTS FROM MORE THAN TEN YEARS OF RESEARCH AT OUR SITE. WE HAVE ISOLATED BEAUVERIA SP. AND ISARIA (CORDYCEPS) SP. EXTREMELY RARELY – ONLY A FEW TIMES DURING THE PAST FIFTEEN YEARS. THEREFORE, DETECTION OF BEAUVERIA WAS EXCLUDED FROM OUR ANALYSES. DURING THIS EXPERMENT< WE DID NOT DETECT ANY OTHER ENTOMOPATHOGENIC FUNGI WITH THE STANDARD, WIDELY PUBLISHED ASSAY METHOD THAT WE USED. THIS IS NOW INDICATED IN THE MS. Reviewer #3: Dear Authors, Your manuscript on the effect of soil management on the prevalence of entomopathogenic fungus Metarhizium robertsii is interesting and brings original results with implications for improvement of biodiversity/ecological services of soil in both organic and conventional farming systems. The text of the manuscript is clear, methods are described in enough details, statistical analysis is sound and conclusions are based on obtained data. However, I believe there is some space for improvement as indicated in specific comments below. Numbers mean line numbers in manuscript. Title: it is fine, describes the study well. Abstract: I would recommend to add some values documenting the differences between treatments (it is not clear what is magnitude of observed differences, e.g. the range etc.). EDITED AS SUGGESTED Introduction: I think it would be good to explain why the authors focused only on M. robertsii and did not look at other EPF species. EDITED AS SUGGESTED. M. ROBERTSII IS THE OVERWHELMINGLY DOMINANT SPECIES AT OUR RESEARCH SITE. WE HAVE INDICATED THIS IN THE MATERIALS AND METHODS SECTION UNDER THE SUBHEADING “RELATIVE PREVALENCE OF M. ROBERTSII” AND IN THE DISCUSSION. 111 add order and family after species names EDITED AS SUGGESTED. WE HAVE ADDED ORDER AND FAMILY NAMES OF TAXA UPON FIRST MENTION IN THE MAIN TEXT OF THE MS. 113-118 To me it could be reduced just to one hypothesis: "Greater frequency and intensity of soil disturbance has negative impact on relative prevalence of M. robertsii." EDITIED AS SUGGESTED. 118-119 This could be the second hypothesis and authors can support it by citing recent findings on differences between those two bait species in the recoveries of different EPF species (e.g. Beauveria vs. Matarhizium). WE KEPT THE HYPOTHESIS ABOUT DIFFERENCES DUE TO SENTINEL SPECIES USED IN ASSAYS AS A THIRD HYPOTHESIS. 243-244 Since these insect species were already mentioned in Introduction, here abbreviated version should be sufficient. EDITED AS SUGGESTED 254-257 So there were no other EPF species (e.g. Cordyceps, Beaveria) found on this experimental plot? That is surprising. CORRECT. WE ONLY EXTREMELY RARELY ISOLATE BEUAVERIA AND DID NOT DETECT CORDYCEPS IN THESE EXPERIMETAL PLOTS. THIS FINDING IS CONSISTENT WITH OTHER RESEARCH AT THE SITE SINCE THE SITE WAS FIRST MONITORED IN 2010. WE INDICATE THIS IN THE MATERIALS AND METHODS AND IN THE DISCUSSION. 494 Tillage has certainly positive role also in pest management of rodents and other pests YES, AGREED 496 Perhaps other microogranisms might be mentioned as important, too. E.g. mycoparasitic fungi. THAT IS CORRECT, BUT WE DID NOT ISOLATE FUNGI OTHER THAN M. ROBERTSII USING OUR METHODS AND DO NOT WANT TO SPECULATE ON BIOLOGICAL INTERACTIONS THAT MAY HAVE INFLUENCED OUR RESULTS. 576-586 Some other technique of EPF isolation could be mentioned for comparison. E.g. in some studies EPF were isolated by culturing soil samples on selective media (Chase et al. 1986 , Correa et al. 2022 , Konopická et al. 2021 , 2022) so it might be useful for readers not familiar with these methods to discuss advantages of live insect-bait technique. EDITED AS SUGGESTED IN DISCUSSION – WE HAVE ADDED TEXT AND CITATIONS FOR KONOPICKÁ 2022 2024; AND NISHI AND SATO 2018 IN THE DISCUSSION. THOSE AUTHORS, IN ADDITION TO CLIFTON ET AL, KEPLER ET AL, and STEINWENDER ET AL (ALREADY CITED IN THE TEXT) USED CULTURE-BASED AND MOLECULAR DETECTION METHODS. 592-594 This species identity might be better placed earlier in Discussion (before discussion on system effect on prevalence of M. robertsii). EDITED AS SUGGESTED 632 Better not to limit to insect pests, also mites and other invertebrates are important pets, not mentioning plant pathogens which can also be reduced by ecological services of beneficial microorganisms. EDITED AS SUGGESTED. EDITED TO REFLECT PESTS IN GENERAL THAT WOULD BE AFFECTED BY METARHIZIUM AS AN ENTOMOPATHOGEN AND AN DENDOPHYTE (ARTHROPODS AND PHYTOPATHOGENS) Attachments Attachment Submitted filename: Mr_Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0317149.r002
22 Dec 2024 Decision Letter - Rachid Bouharroud, Editor Tillage type and sentinel insect species affect the relative prevalence of the entomopathogenic fungus, Metarhizium robertsii, in soil PONE-D-24-39841R1 Dear Dr. Barbercheck, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Rachid Bouharroud Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0317149.r003
Formally Accepted
2 Jan 2025 Acceptance Letter - Rachid Bouharroud, Editor PONE-D-24-39841R1 PLOS ONE Dear Dr. Barbercheck, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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 Dr. Rachid Bouharroud Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0317149.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 .