PONE-D-19-33958

In Situ Processing and Efficient Environmental Detection (iSPEED) of pests and pathogens of trees using point-of-use real-time pcr

PLOS ONE

Dear Dr Hamelin,

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.

Broadly, I think many readers will recognize the utility of the iSPEED platform. Rapid, DNA-based field detection of insects and pathogens is sorely needed by the forest health community. However, it is important to get the details right. The manuscript is not yet suitable for publication, but can be improved sufficiently by addressing the reviewers’ comments. In particular, you must address the following points:

1) Both reviewers noted that some of the citations are formatted improperly, and as a result, some works are missing from the references list. Please correct.

2) I agree with Reviewer 1 that a fair amount of material in the Results section should be moved to the Introduction or Discussion.

3) I also agree with Reviewer 1 that additional details are required about the simplified DNA extraction methods, qPCR assay setup, and any cross-reactivity tests that were performed.

4) There are some typographical and grammatical errors scattered throughout the manuscript (e.g., “…AGM as a broader host range than EGM…”, 1st paragraph, p. 12; “…significant effect age effect…”, 1st paragraph, p.15). Reviewer 1 has provided a marked-up copy that identifies other minor errors. Please check the manuscript carefully and revise as necessary.

We would appreciate receiving your revised manuscript by Feb 16 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.

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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,

Frank H. Koch, PhD

Academic Editor

PLOS ONE

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1. 

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2.  Thank you for stating the following in the Acknowledgments Section of your manuscript:

This work was funded by Genome Canada, Genome British Columbia, Genome Quebec, the

Canadian Forest Service and the Canadian Food Inspection Agency, through a Genomics

Applications Partnership Program (GAPP 6102; Genome Canada) grant. Also this work is funded

by Genome Canada, Genome British Columbia, Genome Quebec, the Canadian Forest Service,

the Canadian Food Inspection Agency and FP Innovations through a Large Scale Applied Research

Project (LSARP 10106; Genome Canada) grant.

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:

RCH was funded by information Genome Canada Large Scale Applied Research Program #10106 and by a Genome Canada Genomics Application Partnership Program # 6102.

3.  We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

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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 #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: 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 #1: Yes

Reviewer #2: 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 #1: The authors present a simplified DNA extraction procedure and a mobile real-time PCR platform for detecting DNA of selected fungal pathogens and an invasive insect for in-field testing. The authors present novel qPCR assays for detection of the rust pathogens Cronartium ribicola and C. comandrae, and also adapted published assays for Phytophthora ramorum, Septoria musiva, and Lymantria spp. to the mobile qPCR platform. A marked up copy of the manuscript is attached.

Reviewer's Specific Comments:

1. The Results section contains a substantial amount of background information/discussion about the various diseases/pests that would be more appropriate for the Introduction and/or Discussion sections.

2. The description of the simplified DNA extraction method and the field-ready qPCR assay setup are both missing crucial details, (e.g., the amount of tissue that was put into the extraction, the pH of the extraction buffer, the model of the mobile thermocycler [found elsewhere in manuscript], the number of biological/technical replicates for the qPCR reactions, how the threshold fluorescence value was determined, statistical analysis details).

3. It is unclear if any testing on cross-reactivity of the Cronartium spp. assays was tested. Given that the polymorphism targeted for the specific probes is just 2 of 14 nucleotides within the probe, and the primers are common, it is likely that the two assays may cross react on the non-target template DNA of the opposite species. As one purpose for the test stated in the manuscript is to identify hybrids between the two species, the specificity of the assays must be tested and the data presented.

4. The discussion of the iSPEED platform vs. isothermal detection methods (i.e. LAMP) should be more thoroughly developed and expanded. The reviewer acknowledges the authors' points regarding the applicability of existing qPCR assays with the mobile qPCR platform, but the authors do not specifically discuss the challenges/limitations of LAMP assay development.

5. The authors need to define the abbreviation "NA" used in the tables in the supplemental data. The reviewer assumes this designation indicates the target was not detected. The authors also need to specify which qPCR probes were labeled with FAM and which were labeled with CY5.

Reviewer #2: The article is original, has good technical quality and large general interest. Many forest ecosystems suffer from the presence of pests and pathogens, very often unpredictable in changing climate conditions. International and national phytosanitary regulations also stipulate measures aimed at managing the risk of introducing harmful organisms, often following pest-risk analysis based on scientific evidence. The large number of plants traded and the inconspicuousness of many pests, in particular microbial pathogens, highlight the need to mitigate the risk associated also with the intercontinental trade pathways (Eschen et al. 2018). Real-time PCR assays based on the TaqMan system have been recently developed for the identification of many pests and pathogens e.g. Phytophthora sp., including P. cactorum, P. megasperma, P. plurivora, P. pseudosyringae and P. quercina that cause significant damage to forest ecosystems (Nowakowska et al. 2016). The development of molecular methods has become powerful tool which facilitates diagnosis, i.e. PCR methods aiding species identification, making the process more fast and accurate. The paper describes very useful method of rapid and reliable environmental detection of tree pests and pathogens thanks to the real-time PCR. The prevention measures can then be undertaken in order to mitigate the development of the harmful organisms in forest stands.

About the content:

The title, abstract and keywords clearly reflect paper's content.

Introduction presents the problem clearly.

Experimental methods are adequate. The markers were appropriately chosen for the species detection and the sensitivity of each primer + probe set was tested. The idea of lyophilization of the PCR reaction mixtures ensures the stability of the reagents in the field conditions. The applied appropriate concentration of trehalose into the PCR mixtures ensured the long term stability (more than 260 days) of the reagent kit at room temperature.

Discussion and results are justified.

References are adequate but incomplete. Please check the bibliography citation in the text (on p. 4, 5, 8, 9, 11 and 17) because some of them are missing in the literature at the end of the MS. All citations should have numbers in the MS.

About Presentation:

Length is commensurate with the paper's content.

Quality of tables is adequate but quality of figures 4 & 5 could be improved.

Some data could be improved:

- Check the spelling of the Latin names, e.g. “Clostridium perfrigens” should be “Clostridium perfringens” on p. 17, or “C. comandrae” in Supplementary Table S7

- “thedetection” on p. 17,

About Scientific evaluation:

The general scientific approach is properly stated and well explained. A good quality tool (iSPEED protocol) has been developed and its usefulness for the chosen insects and pathogen detection in situ has been statistically proven. Of course, the field tests will not replace laboratory tests, but instead, provide an effective screening which may speed up prevention measures undertaken in a forest stand. Thanks to such techniques, it is also possible to monitor the biodiversity of the investigated species. The increasing number of new alien pests and pathogens of woody plants in many parts of the world will certainly promote the development of fast, reliable and costless techniques of the harmful organism detection in the field.

Therefore, I recommend publishing the article after minor corrections.

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Reviewer #1: No

Reviewer #2: Yes: Justyna Nowakowska

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Attachments

Attachment

Submitted filename: PONE-D-19-33958_reviewer.pdf

PONE-D-19-33958R1

In Situ Processing and Efficient Environmental Detection (iSPEED) of pests and pathogens of trees using point-of-use real-time pcr

PLOS ONE

Dear Dr Hamelin,

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.

**From the Academic Editor: I appreciate the many changes you made in response to my comments and those of the reviewers. At this point, the manuscript is nearly suitable for publication. I went through the revised version (and the supporting information) carefully, and identified a small number of further edits I would like you to make. They are all minor, and rather than list them here, I've attached track-change versions of both the main text and supporting information documents as guidance.

I have one specific request: Please go through the reference list and ensure that all capitalization, italics and formatting are correct. If at all possible, use standard journal name abbreviations. As you probably know, PLOS ONE doesn't perform further editing once a manuscript is accepted. **

We would appreciate receiving your revised manuscript by Apr 16 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 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'.
• A rebuttal letter will not be necessary.

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,

Frank H. Koch, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

[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.

Attachments

Attachment

Submitted filename: Manuscript_AE_tracked_changes.docx

Attachment

Submitted filename: Supporting Information_AE_tracked_changes.docx

In Situ Processing and Efficient Environmental Detection (iSPEED) of tree pests and pathogens using point-of-use real-time PCR

PONE-D-19-33958R2

Dear Dr. Hamelin,

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.

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With kind regards,

Frank H. Koch, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Thank you for completing one more round of edits. I believe the manuscript is now suitable for publication. However, if you're given the chance to revisit the manuscript, I noticed a few very minor things you should consider fixing (they're cosmetic, but if I was an author I'd want to know about them).

P. 14, first paragraph -- "...yielded a positive real-time PCR results..." (singular-plural disagreement, either delete "a" or change "results" to "result")

P. 20, last line -- "homogeneized" should be "homogenized"; insert "the" before "kit"

References 43 and 44 -- "Phytophthora ramorum" should be italicized

S6 Table caption -- insert "using" after "antennae"

Reviewers' comments: