PONE-D-26-00871Genome-wide analysis of the invasive fungal pathogen Neopestalotiopsis unveils high genomic diversity, effector repertoires, and the assessment of fungicide resistance risks.PLOS One

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North Carolina Department of Agriculture and Consumer Services (NCDA & CS).

Grant contract number 25-025-4018

https://www.ncagr.gov/].

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North Carolina Department of Agriculture and Consumer Services (NCDA & CS).

Grant contract number 25-025-4018

https://www.ncagr.gov/]

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Additional Editor Comments:

The paper has now been evaluated, and based on the reviewers’ comments and my own assessment, minor revision is recommended. While the study appears scientifically sound and suitable for publication, several minor changes are suggested to improve scientific clarity, coherence, and overall presentation.

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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: 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: This study presents a timely and comprehensive genomic investigation of Neopestalotiopsis species associated with strawberry diseases in North Carolina, addressing an important gap in genomic resources for this emerging pathogen complex. By sequencing and analyzing 50 strains from major production regions, the authors provide valuable insights into population structure, pathogenicity-related gene content, and fungicide resistance mechanisms.

The paper reads well and could be considered for publication, although addressing several areas for improvement would strengthen it. One such area is the phenotypic validation of fungicide resistance, which would improve the interpretation of resistance-associated mutations, particularly for cyp51 variants linked to DMI resistance. These mutations are more complex than the single, well-established point mutations that govern resistance to benzimidazole and QoI fungicides. Another concern is the reliance on short-read Illumina sequencing without complementary long-read (PacBio or Oxford Nanopore) data, which may limit the resolution of repeat-rich or accessory genomic regions. In addition, the taxonomic status of Clade C warrants clearer resolution, although this may reasonably fall outside the primary scope of the present study. Similarly, the predicted effector repertoire is based solely on in silico analyses and remains hypothetical in the absence of functional or expression-based validation.

Nevertheless, as a pioneering large-scale genomic resource for Neopestalotiopsis, the findings in this paper provide a valuable foundation for future work.

Given the breadth of genome data generated, performing additional analyses such as exploring recombination or horizontal gene flow among lineages could further enhance understanding of population dynamics and the spread of fungicide resistance.

Reviewer #2: Neopestalotiopsis spp. is among the most important emerging strawberry pathogens worldwide. In 2017, this pathogen began to draw attention because of its high impact on crops, as it can attack different plant organs. This paper is a significant contribution that sheds light on various aspects of the pathogen's genomics, explaining why it is aggressive and difficult to control. It provides basic information and links it to an applied solution to manage fungicide resistance.

The paper is very well written. During the review, it called my attention to the fact that the authors no longer use the term "Aggressive and non-aggressive" as used by Baggio et al. (2021. Different groups in the USA have reported the aggressive Neopestalotiops sp., including Canada. Also, recently, Moparthi et al. (2026) reported the identification of different species within the N. rosae complex, including N. hispanica (a controversial report for some groups in the USA). In this paper, they also do not refer to the term "aggressive isolates," as it was called in the last years, for the widespread variant (as it has been called).

I believe the paper should include, in the introduction, a discussion of this information, contrasting how " Aggresive" isolates differed from the rest of N. rosae.

Is clade C, the "aggressive" Neopestalotiopsis? (referred to as N. hispanica? in Moparth et al.) I believe this part needs clarification, given that many reports have emphasized this term.

I strongly suggest that the authors clearly state whether the isolates analyzed fall into the so-called aggressive category or, if they decide that the term is no longer needed based on their results, indicate this in the results and discussion sections.

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Reviewer #1: No

Reviewer #2: No

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Genome-wide analysis of the invasive fungal pathogen Neopestalotiopsis unveils high genomic diversity, effector repertoires, and the assessment of fungicide resistance risks

PONE-D-26-00871R1

Dear Dr. Adhikari,

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,

Pramod Prasad, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

The major concerns are effectively addressed in the revised version. The manuscript is now clear, rigorous, and impactful. The article can be accepted for publication.

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 #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 #2: Yes

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

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 #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 #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 #2: The findings reported in this paper will contribute to a better understanding of why N. rosae is so aggressive toward strawberries, and why is it so difficult to manage? The point mutations reported herein also explain and complement the findings of other groups currently working on this pathogen.

No more comments. Just correct a minor typo in the abstract: " Neopestalotiopisrosae". Row 14

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Reviewer #2: No

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