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PONE-D-19-25662

Novel bioassay to assess antibiotic effects of fungal endophytes on aphids

PLOS ONE

Dear Mr Collinson,

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.

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Both reviewers were encouraging about this work. However, they noted some elements that needed revision. In particular, I encourage you to revise the methods to meet those details that were noted as missing or unclear. I would also like to see the molecular results that both reviewers highlighted as missing. We look forward to seeing your revision. 

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We would appreciate receiving your revised manuscript by Dec 16 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.

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: N/A

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: Research is interesting. l re-read this paper numerous times looking for replication of the research and was unable to find it. Thus, I cannot appropriately comment on the science. There are numerous questions I have within the methodology sections (actual set up, sample size, infection methods) as well as the background and reasoning for choosing these insects. I feel that in order for this to be published there should be a replication of the life history events of these aphids like other life history related papers. Once replicated, I do believe this could only require minor revisions in elaborating in certain parts of this writing. Until then, my comments on that part are unnecessary.

Reviewer #2: The authours describe cup-based system suitable for bioassays involving two species of aphids with different feeding locations, seedlings and endophytes. The manuscript is well written, thanks, and the results are justified by the data. Several of the commercially available endophytes seem promising against aphids and I’m always in favour of biological pest control options when they are available. I am not requesting any major revisions but I’ve detailed suggestions, that could be considered minor revisions in the text below.

Keywords. I think the authours have the paper well covered with their keywords. Does PLOSOne have a keyword limit?

Abstract: The abstract is clear and mirrors the findings of the paper.

Introduction: The introduction clearly states the purpose and gives enough background information to make the objectives understandable.

Materials and Methods:

The creation of aphid clones (lines 119-214) was not very clear as written. For example, Line 194 suggests that all aphids used were raised on perennial rye grass…why mention the colony that was started on barely then? Was it your D. noxia aphid lines from barley that were then used as a control on barley in the endophyte experiments or did they come from the ryegrass colony? Lines 150-153 don’t really make it much clearer.

The materials and methods are also unclear at lines 194-200. How many adult aphids were went into plastic cups total? Then at 196, 24 and 48 nymphs were produced…were these per adult total or was it the grand total of nymphs produced? The bracketed set up of nymphs into treatments doesn’t make sense as is written if only 24 and 48 nymphs were produced total. Please make it clearer how you generated the 456 nymphs for the experiments is what I suppose I’m trying to say.

Lines 180-191. The molecular check of the seedlings for the presence of endophytes is not reported on in the results section or mentioned in the discussion. Could you please include a section in the results that details the molecular results please and then would it worthwhile to mention something about the ability of the seedlings in your novel bioassay to pick up the endophytes from the seed treatments and have them consistently expressed (ie. A percentage of infected plants in each treatment, to confirm that the seedlings had endophytes and that the bioassay allows for endophyte infiltration of seedlings?) If you are detecting fluorescence is there an option to report on the time to positive fluorescence as a semi-quantitative method of assessing the titer of endophytes in seedlings?

You’ve mentioned that seedlings were replaced every 7 days but have not described the methods of transferring aphids from one cup to another, or from one seedling to another. Could the handling of aphids have led to increased mortality? Ie. High mortality in nymphs of A. lentici WE control treatment.

Statistics: Fine.

Results:

Add molecular results please (as above)

Tables and Figures are all well done and necessary for data presentation. I liked the description of the shapes of the death curves in Figs 2 and 4.

In Fecundity tables where the mean number of offspring are shown, it would help to add in the standard error of the mean to show how much variability there was among aphids in each treatment. For example (Table 6 WE Total fecundity of 3.7 (±S.E.)

Line 321: That is high mortality of your nymphs on the Without Endophyte (WE) plants. Fig 4 helps to ease my concern because it seems most of them lived over 144 hours but that is a lot of aphids that did not make it to maturity and adulthood to reproduce. In the discussion at 446 – 451 you’ve given some good suggestions on why this bioassay might not be suitable for this species of root feeding aphid. It’s too bad that it wasn’t very suitable because there would be a great benefit to studying the life history of root feeding aphids outside of the soil. So, in light on the high mortality and potential unsuitability of the assay for root-feeding aphids, could you please add a caution to your abstract and conclusion to that affect. Readers might not pick up on the implications of the high nymphal mortality of A. lentisci in the WE treatment if they only read the abstract and skim the manuscript.

Discussion:

It might be useful to detail other aphid studies that might benefit from adoption of your cup bioassay to round off your discussion. For example, other insecticidal seed treatments perhaps, life history studies on other aphid species etc?

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Reviewer #1: No

Reviewer #2: No

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Novel bioassay to assess antibiotic effects of fungal endophytes on aphids

PONE-D-19-25662R1

Dear Dr. Collinson,

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

Additional Editor Comments (optional):

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: All comments have been addressed

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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

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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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

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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

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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: I apologize about the minimal comments submitted last time around. Not all of my comments copied and pasted into the boxes- that is entirely my fault and thus there is not much I can criticize here. Thank you for the replication clarification. I do believe moving forward for future experiments you should do more biological reps to account for variabilities in colony life history.

With that, reviewer 2 addressed most of my concerns that I had with the paper. I think the revisions that have been made are sufficient.

I am not sure if this is just a formatting thing with track changes but the spacing on table 4 is odd. The discussion is more robust now with the additions.

Reviewer #2: Thanks for addressing my comments so well. I'm happy with the changes that have been made.

References

It looks like you've made the references with citation manager software so I didn't take a close look at them.

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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

Reviewer #2: No