PONE-D-19-35047

A diagnostic real-time PCR assay for the rapid identification of the tomato-potato psyllid, Bactericera cockerelli (Šulc, 1909) and development of a psyllid barcoding database.

PLOS ONE

Dear Dr. Sumner-Kalkun,

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.

This manuscript fell in a grey area between minor and major revisions. Three different reviewers examined the manuscript, and I also reviewed it. I agree with the 1st reviewer that you may be stretching a bit and could possibly focus some. This work will provide a useful tool. I think that alone makes it worth publication, and that opinion is shared by the reviewers. I also think that it is a complete and comprehensive piece of work. I, therefore, encourage you to focus on the comments form reviewer 1 and those about length etc. when preparing a resubmission.

We would appreciate receiving your revised manuscript by Feb 29 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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Sean Michael Prager, Ph.D.

Academic Editor

PLOS ONE

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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

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: No

Reviewer #2: No

Reviewer #3: No

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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

Reviewer #3: 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: This manuscript describes a qPCR assay to identify potato psyllid intercepted in shipments. The assay is paramount to Europe's ability to detect potential introductions of this psyllid, which would be harmful to agricultural production. The authors describe the assay and confirmed that it does not amplify the ITS gene of other psyllids.

My major concern for the manuscript is that it is overwritten and over-interpreted. The study is very simple - qPCR assay to detect potato psyllid - yet the text is over 50 pages long, includes unrelated information in the introduction, and includes an overly long discussion. The manuscript should be re-written to focus only on the assay and its use in trade commodities. Specific comments are provided in an attached document. I will apologize for my handwriting.

Reviewer #2: In this manuscript, the authors describe the design and validation of the first species-specific TaqMan probe-based real-time PCR assay, targeting the ITS2 gene region of Bactericera cockerelli, for robust and quick identification of the potato-tomato psyllid B. cockerelli, the main vector of ‘Candidatus Liberibacter solanacearum’ on potato and tomato crops in Central and Northern America and New Zealand. The authors examined false-positive rates in non-target psyllid species and false-negative rates in target species, including B. cockerelli at different life stages. The assay also compared amplification efficiency at different MgCl2 concentrations, primer concentrations, and annealing temperatures, and determined the detection limit at optimum conditions. The assay was designed and presented in a very robust way, however, I have some minor concerns the authors need to look into before the manuscript can be accepted.

Minor Concerns:

1. Data Availability: The authors need to add accession numbers for their sequence data.

2. Page 8 Line 163: What part of the body is used for micro-dissection to extract DNA? The authors should describe the micro-dissection procedure in more detail rather than only citing the papers.

3. Page 8 Line 172: “For amplification of ITS2 primers CA55p8sFcm-F and CA28sB1d-R [60] and for amplification of CO1 gene regions arthropod barcoding Primers LCO1490 and HCO2198 [61].” The authors should check the grammar here. It is not a complete sentence. It could be “For amplification of ITS2, primers CA55p8sFcm-F and CA28sB1d-R [60] were used, and for amplification of CO1 gene regions, arthropod barcoding Primers LCO1490 and HCO2198 [61] were used.”

4. Page 10 Line 204: “DNA was extracted as above using the non-destructive method, amplified and cloned into competent Escherichia coli cells using the TOPO TA cloning kit (Thermo-Fisher).” The authors should specify what genes (ITS2 or CO1?) they amplified for cloning, and what restriction enzyme (EcoRI?) they used to linearize the plasmid.

5. Page 10 Line 212: The authors need to list the real time PCR cycling conditions here, for example XX degrees for XX seconds.

6. Page 10 Line 223: “All reactions with non-target psyllid DNA were run in conjunction with a TaqMan Exogenous Internal Positive Control Reagent Kit (Applied Biosystems) to ensure false positives were not obtained due to inhibition within the reaction”. Here, “ensure” should be “rule out the possibility that”.

7. Page 11 Line 226: “DNA from all non-target psyllids was sequenced to ensure psyllid DNA was present in all reactions to rule out false negatives due to inefficient DNA extraction.” What DNA was sequenced? PCR product from ITS2 or CO1? The authors need to specify.

8. Page 11 Line 239: “6 subsequent dilutions were made. Stock DNA 10 ng/μl was linearised using EcoRI restrictions enzyme (New England Biolabs),” Here “6 subsequent dilutions” should be “8 subsequent dilutions”, according to the nine point 10-fold dilution series mentioned on Page 11 Line 236.

9. Page 12 Line 252: “A six point 1:10 dilution series starting at 10ng/μl was used with each dilution being performed in triplicate.” Here, “six point” should be “nine point” according to Page 11 Line 236.

10. Page 12 Line 263: “For each tested parameter, optimization was performed across an eight point 1:10 dilution series starting at 10ng DNA.” Here, “eight point” should be “nine point”, “10ng” should be “10ng/μl”.

11. In Supplementary table S1, green and red color coding should be explained in the text. What does TBC mean? Accession numbers should be given for all the sequences. Accession numbers in Table 3 should also be given and TBC should be explained.

12. Page 14 Line 289: “CO1 genes showed higher similarity and generally less conserved and variable regions compared to ITS2 regions.” Here “less conserved and variable” should be “less variable”.

13. Page 17 Line 310: “0.2 µ/mol” should be “0.2 µM”.

14. Page 18 Line 324: “The copy number calculator available at http://scienceprimer.com/copy-number-calculator-for-realtime-pcr was used.” Here a hyperlink should be created. According to the link and the formula given, 0.00001ng DNA equals 4.879×10000 copies, if length of gene region is considered 187bp (product length of ITS2 in real time PCR). However, the authors calculated that it equals to 200bp. Please double check the calculation.

15. Page 18 Line 337: “At primer concentration, 0.5 μM the assay was less sensitive only amplifying up to 0.001 ng DNA.” It should be “At primer concentration 0.5 μM, the assay was less sensitive only amplifying up to 0.001 ng DNA.”

16. Page 18 Line 338: “At higher primer concentrations (0.5 and 1.0) the assay showed higher sensitivity” Here “(0.5 and 1.0)” should be “(1.0 μM)”.

17. Page 19 Line 350: “The precision of the assay was lower at higher MgCl2 concentrations 6mM and 8mM (Supp Tab. S3).” Here “6mM and 8mM” should be “7.5mM and 9mM”.

18. Page 19 Line 354: “Reactions at 58 °C were 10 to 100-fold less sensitive than reactions at 58 °C.” Here it should be “Reactions at 58 °C were 10 to 100-fold less sensitive than reactions at 64 °C.”

19. Page 20 Line 367: “We have designed and validated the first species-specific, qualitative real-time PCR TaqMan assay for B. cockerelli by using the comparison of 73 non-target species to identify unique gene regions that were suitable for primer/probe design and species differentiation.” Here “qualitative” should be “quantitative”.

Reviewer #3: The manuscript presents a new real time assay that will make identification of the key pest commonly known as potato-tomato psyllid easier and faster. The assay has been rigorously developed, with appropriate controls, replication and sample size. The specificity of the assay is fairly assured by the inclusion, in its development and validation stage, of numerous non target species, including 9 congeneric species, representing about 30% of known European Bactericera taxa. The manuscript is well written, with thorough introduction and discussion, and methods and results clearly presented. There are only some minor issues that should be dealt with before the manuscript can be accepted for publication:

- Page 8 line 175: please replace amount of primers used with final concentration of primers (or add this)

- Page 10 line 213: please add cycling conditions of real time PCR, as done for CO1 and ITS2 amplification

- Table 1: should include also B. cockerelli, so to include fragment size of amplicons for this species. In alternative, fragment sizes can be added to the main text

Table 3: not clear what the "/" symbol in the CO1 column means

- Page 17 line 310: please check spelling of concentration

- Page 17 line 316: numbers seem not to add up: how many technical replicates were used per sample?

- Page 18 line 323: I have tried the formula myself using the concentration (0.00001 ng) and fragment size (187 bp) specified by the authors, but I get a quite different number of ITS2 copies (about 50,000 versus 200). Please double check, and add actual numbers to the formula.

Of some concern is the author's answer to the data accessibility question. Authors stated that they are not going to make all data available, with a generic "Some restrictions will apply". Please explain what data will not be made accessible and why.

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Reviewer #1: No

Reviewer #2: Yes: Penglin Sun

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 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: doc07744220200113085906.pdf

PONE-D-19-35047R1

A diagnostic real-time PCR assay for the rapid identification of the tomato-potato psyllid, Bactericera cockerelli (Šulc, 1909) and development of a psyllid barcoding database.

PLOS ONE

Dear Dr. Sumner-Kalkun,

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 appreciate the effort you have taken in both addressing the reviewer concerns and revising this work. In that context, I have rendered a decision of minor changes. This is primarily to give you the opportunity to make or address the additional reviewer comments.

==============================

We would appreciate receiving your revised manuscript by Apr 17 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,

Sean Michael Prager, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: (No Response)

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

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

Reviewer #1: Yes

**********

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

Reviewer #1: 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

**********

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

**********

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: The revised version of this manuscript is a substantial improvement from the original submission. I have only a handfull of minor suggestions in the attached PDF that the authors might consider.

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

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

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

Reviewer #1: 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.

Attachments

Attachment

Submitted filename: PONE-D-19-35047_R1_reviewer.pdf

A diagnostic real-time PCR assay for the rapid identification of the tomato-potato psyllid, Bactericera cockerelli (Šulc, 1909) and development of a psyllid barcoding database.

PONE-D-19-35047R2

Dear Dr. Sumner-Kalkun,

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,

Sean Michael Prager, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments: