PONE-D-23-39732Early assessment of fungal and oomycete pathogens in greenhouse irrigation water using Oxford nanopore amplicon sequencingPLOS ONE

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Reviewers' comments:

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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: No

Reviewer #2: Yes

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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 manuscript reads very well and I had an easy time getting through it. I just have a few minor comments for authors (see below).

Page 6 and 7 under Data processing and analysis, I suggest authors cite filling, porechop, guppy, minimap2, or at least provide a link to where software could be downloaded.

Page 9. Add the species, so consider changing "...the oomycete Phytophthora (accession number:

EF126351.1) and Pythium (accession number: MW366735.1)..." To "...the oomycete Phytophthora infestans (accession number: EF126351.1) and Pythium sp. (accession number: MW366735.1)"

Pythium intermedium was transferred to Globisporangium intermedium. I suggest authors use the new name (see Uzuhashi et al. 2010 "Phylogeny of the genus Pythium and description of new genera" and Nguyen et al. 2022 "Whole genome sequencing and phylogenomic analysis show support for the splitting of genus Pythium"

I suggest italicizing the genus names in the figures, if not too much of a hassle.

I suggest authors deposit raw data generated into a repository like NCBI SRA, and state the accession number in the manuscript.

Reviewer #2: This manuscript presents early assessment potential of used of MinION amplicon sequencing for environmental monitoring in irrigation water. They use spike material in irrigation water and evaluate the tool for identification of Fungi and Oomycete pathogens. I think this is valuable work and information that is need to evaluate the tools, however I think they can go more far in the analysis of it and state more about the finding of non spike material. They evaluate some potential but not too clear what the best dilution or way to do it more far, they did not made the analysis to the species level but only to the genera level.

Specific comments:

Abstract: I think they can add more information in abstract about type of samples, numbers more details on results. Region of amplicon used...

P3L69, should rewrite this sentence, a verb I think is missing.

P5 Greenhouse samples and material should be presented in a table to get more clear.

L150 need # for the supplement info in.

L157 any info about the DNA conc. used.

L204, info on Miracloth

L246, please check new litterature about Pythium intermedium, now Globisporangium and could be a complex species.

L258, the spike-in preparation not always to clear to follow, the plasmid and not plasmid prep ...

L299 Concentration of DNA, not always clear?

L304-305 on water or plasmid material, the 1.56ng/ul?

L325, seems results and discussion are put together in what I seen in https://journals.plos.org/plosone/s/submission-guidelines seems they are supposed to be distinct sections?

L376 and Figure 2 are very low correlation.

L410- seem you only have genera information, why and it is possible to get to the species level, would have been great information on the use of the method and limit. I believe information need to be add and also part of your limitation in your bioinformatic pipeline, should be part of discussion. Figure 3B do we see Phytophthora?

L449 I believe you want to say Phytophthora fragaria and not Pythium intermedium here in parenthesis. L461 more info about the amplification bias and/or references.

L461, interesting but I believe more work need to be done in analysis, info about the species level, any other info from the water samples, important species find, concern any info about the other genera found, any concern, any problem they could cause...Return from L347... Peronospora...

Figure 1 the names of plant hosts and Fungi and Oomycete do not necessary align with the samples position? Recheck it.

Recommendation on concentration what type of spiking, info sequences of water without spike material only?

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Reviewer #1: No

Reviewer #2: No

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Early assessment of fungal and oomycete pathogens in greenhouse irrigation water using Oxford nanopore amplicon sequencing

PONE-D-23-39732R1

Dear Dr. Kudjordjie,

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.

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Kind regards,

Jaime E. Blair, PhD

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: N/A

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

**********

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: Thank you for addressing my comments.

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Reviewer #2: They have respond to my comments. L189-205 information on software version should be add mainly for porechop and reference material.

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Reviewer #1: No

Reviewer #2: No

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