-->PONE-D-26-14713-->-->First Report of Corynespora cassiicola Causing Leaf Spot on Begonia fimbristipula in China-->-->PLOS One

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

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Reviewer #1: The manuscript titled "First Report of Corynespora cassiicola Causing Leaf Spot on Begonia fimbristipula in China" presents valuable findings, but several critical issues must be addressed before it can be considered for publication. My comments are structured as follows:

1. General Summary & Significance

This study investigates a severe leaf spot disease affecting Begonia fimbristipula, an important medicinal plant in southern China, resulting in significant yield losses. The core hypothesis is that an uncharacterized pathogen is responsible for this disease. The methodology employed field surveys to document disease incidence and symptoms, followed by isolation of the causative agent. Identification was achieved through a polyphasic approach combining detailed morphological characterization with multilocus phylogenetic analysis based on ITS, TEF1α, and RPB2 sequences. The pathogenicity of the identified organism was rigorously confirmed by fulfilling Koch's postulates through artificial inoculation and re-isolation. The findings indicate that the outbreaks observed in August 2024 resulted in over 60% disease incidence and >20% loss of fresh leaf yield. The pathogen was conclusively identified as Corynespora cassiicola, based on its distinctive morphological features and strong phylogenetic clustering with known reference strains. Artificial inoculation successfully reproduced the characteristic symptoms, and the pathogen was successfully re-isolated, thereby confirming its causal role.

This study holds significant value as it constitutes the first documented report of Corynespora cassiicola causing leaf spot on Begonia fimbristipula in China. This finding is original as it directly extends the known host range of this economically important and cosmopolitan fungal pathogen, providing valuable data for plant pathology and host-pathogen interaction studies. The scientific rigor of the study is commendable, particularly in its use of a robust polyphasic identification approach and the strict adherence to Koch's postulates for pathogenicity confirmation. The inclusion of multiple molecular markers (ITS, TEF1α, RPB2) strengthens the reliability of the molecular identification. The work provides a crucial foundational understanding for diagnosing and developing integrated management strategies for this newly emerging disease affecting a valuable medicinal plant.

2. Structure & Presentation

The title, "First Report of Corynespora cassiicola Causing Leaf Spot on Begonia fimbristipula in China," is adequate, appropriate, and accurately reflects the study's content. The abstract effectively covers all essential components: background, methods, results, and conclusion. It is comprehensive enough to stand alone for abstracting services.

The manuscript is generally well-written and the scientific information is communicated clearly. The language is understandable, although there are several instances of awkward phrasing, minor grammatical errors, and slightly repetitive sentences that detract from the overall polish expected for a high-impact journal.

Specific examples of awkward phrasing/grammar:

- In the Abstract's background, the sentence "Leaf spot disease has recently caused severe yield losses to B. fimbristipula in Guizhou and Hunan Provinces, but the causal pathogen remains unknown" could be rephrased for better flow, e.g., "...a significant leaf spot disease has recently emerged on B. fimbristipula in Guizhou and Hunan Provinces, causing severe yield losses, but its causal pathogen remains unidentified."

- In the Methodology section for PCR Amplification and Sequencing, the description of the PCR program for ITS contains a repetitive element: "...72°C extension for 1 min; and 72°C final extension for 10 min." This should be streamlined.

- The caption for Figure S1 (Fig S1 A1, A2, A3...) is grammatically fragmented and needs to be rewritten as complete, descriptive sentences.

A thorough revision to improve grammatical precision and eliminate minor redundancies would enhance conciseness. For example, the detailed descriptions of DNA extraction and PCR sequencing protocols are repeated almost verbatim in the abstract and the main Methods section, and are implicitly present in the Results. This should be streamlined, with full details appearing only once in the Methods section.

Visuals: The intent of the visuals (Figure S1 showing symptoms, Koch's postulates, colony, and conidia; Figure S2 showing a phylogenetic tree) is excellent and they are essential to support the findings. However, their presentation needs significant improvement:

Clarity and Quality: The embedded images (Figures S1 and S2) are low-resolution, making it difficult to discern important details (e.g., fungal structures, precise symptoms, and bootstrap values on the phylogenetic tree). High-resolution images are imperative for publication.

-The caption for Figure S1 is not understandable and requires complete rewriting to accurately and clearly describe each panel.

-A major inconsistency exists with Figure S2: the main text states phylogenetic analysis was performed using the "neighbor-joining (NJ) method," while the figure caption states "using Maximam Likelihood method." This is a critical contradiction that must be resolved. Ensure all details, particularly morphological features in S1 and bootstrap values/branching patterns in S2, are clearly legible. Completely rewrite the caption for Figure S1. Each panel (A1-A3, B1-B4, C, D) must be described clearly and concisely using full, grammatically correct sentences. Ensure the caption for Figure S2 is also clear and accurate, matching the text.

3. Section-Specific Comments

Introduction: The introduction is comprehensive and well-structured. It effectively establishes the economic and medicinal importance of B. fimbristipula, introduces the emerging disease problem, and adequately reviews existing literature on C. cassiicola's host range. The authors clearly delineate the knowledge gap that their study addresses, providing a strong rationale for the research.

Methodology: The methodologies employed are scientifically sound, valid, and based on established plant pathology techniques for pathogen isolation, identification, and pathogenicity testing. Refer to https://doi.org/10.3390/v11070588 as justification for using specific Phylogenetic Analysis as a more robust method for examining the evolutionary distance between C. cassiicola isolates. Ensure that the method stated in the main text (e.g., "neighbor-joining (NJ) method") perfectly matches the method specified in the Figure S2 caption (currently " Maximum Likelihood method). Provide a brief justification for the chosen method.

The DNA extraction kit, PCR primers, concentrations, and programs are outlined. GenBank accession numbers are provided, which is excellent for data transparency. However, the critical contradiction regarding the phylogenetic method between the text and Figure S2 caption must be resolved to ensure reproducibility and credibility. Clarification is also needed on whether all 10 isolates underwent molecular identification or if ZBTK-1 was exclusively characterized, with justification if the latter. Explicitly state whether molecular identification was performed on all 10 representative isolates (ZBTK1-ZBTK10) or solely on isolate ZBTK-1. If only one isolate was fully sequenced, provide a clear scientific justification for this approach (e.g., genetic redundancy, resource limitations, or ZBTK-1 being representative after preliminary screening of all 10).

For the control, specifying "sterile PDA plugs without mycelia would add clarity.

Results: The results are credible and logically presented, supported by the analytical methods described. Descriptions of symptoms are clear. The reported incidence (60%) and yield loss (>20%) are impactful, though a brief explanation of how these figures were estimated (even if observational) would enhance their validity.

In the "Field Disease Symptoms and Incidence" subsection, briefly describe the methodology used to estimate the "over 20% fresh leaf yield loss." Even if qualitative, outlining the observational criteria would add credibility (e.g., visual assessment scale, sampled areas, etc.).

Consider Quantitative Pathogenicity Data: If quantitative data on disease severity (e.g., lesion diameter measurements, disease index scores) were collected during the pathogenicity assays, incorporate them into the Results section. It would significantly strengthen the evidence for pathogenicity. If not collected, state that qualitative visual assessments were deemed sufficient.

Discussion: The discussion is logical, comprehensive, and effectively connects the findings to their broader implications. It goes beyond merely restating the results by contextualizing them within existing literature, emphasizing the expanded host range of C. cassiicola, and discussing its potential impact on B. fimbristipula cultivation and regional agriculture. The proposed integrated management strategies (cultural, chemical, resistance breeding) are relevant and practical. However, I suggest expanding the study's limitations to include the complex biochemical interactions—such as how the pathogen might manipulate host cell signaling or phytohormone pathways to overcome plant immunity (refer to https://doi.org/10.15414/jmbfs.3422)—that remain to be elucidated, beyond identifying the causal agent.

Conclusion: The conclusion is well-written and succinctly summarizes the key takeaway.

Reviewer #2: Comments

First Report of Corynespora cassiicola Causing Leaf Spot on Begonia fimbristipula in China

� The sentence provides useful context, but it is slightly descriptive for an abstract. It could be more concise while retaining key relevance.

� “severe yield losses” is vague and could be strengthened with brief quantitative indication.

� Brief mention of scale or scope would improve the methodological clarity.

� This is a strong and informative result statement, effectively conveying the scale and impact of the disease

� It could be made more concise by avoiding repetition of procedural details.

� The statement is slightly overstated, as management aspects are not directly addressed within the abstract.

� slightly lengthy. It could be streamlined for better readability while retaining key information.

� Including examples of diseases or brief statistics would strengthen the argument.

� This provides clear context and significance; however, quantitative data on yield loss would improve precision.

� listing multiple hosts may slightly reduce conciseness.

� The claim regarding disease control is somewhat broad without direct supporting evidence.

� The survey duration and frequency are not described.

� The parameters are relevant, but methods for measuring incidence and yield loss are not explained.

� Justification for sterilization duration is not provided

� “±0°C” appears unrealistic and may indicate a reporting error.

� It lacks detail on how single spores were isolated and confirmed.

� criteria for choosing representative isolates are not specified.

� The measurement approach is appropriate, but increasing sample size could improve statistical reliability.

� Reliance solely on comparison may introduce subjectivity without clear criteria.

� justification for selecting only one isolate is lacking.

� kit-based methods may limit methodological transparency.

� Thermal cycling conditions are clearly described, though minor redundancy could be reduced for conciseness

� This is a standard validation step, but lacks details such as voltage and run time.

� Providing accession numbers enhances transparency and data availability.

� More robust approaches (e.g., ML or Bayesian) could strengthen analysis.

� no justification for model selection is provided.

� plant selection criteria is not described.

� Wounding may artificially enhance infection and should be justified or discussed.

� Inclusion of a control is appropriate and strengthens experimental validity

� statistical analysis of results is not mentioned.

� Criteria for symptom assessment are not specified.

� The approach is methodologically sound and essential for confirming causality.

� The use of a single representative isolate simplifies analysis but may not capture genetic variability among isolates.

� Reliance on commercial kits limits methodological transparency.

� Minor redundancy could be reduced.

� This is a routine step, but lacks details such as electrophoresis conditions.

� No justification for concatenation strategy is provided.

� It lacks justification for plant age selection.

� The absence of wounding increases realism, though infection efficiency may vary.

� statistical analysis is not mentioned.

� Observation frequency is appropriate, but scoring criteria are not defined.

� Quantitative data effectively demonstrate disease severity and impact.

� Symptom description is clear and detailed, aiding disease identification.

� The conclusion is appropriate but relies on descriptive comparison.

� This validates the experimental setup and strengthens conclusions.

� The statement is valid but somewhat general.

� This is a reasonable inference, though not experimentally tested.

� The statement is justified but slightly overstated without applied data.

� Suggestions are relevant but remain hypothetical.

� The authors appropriately acknowledge key limitations.

� The statement is relevant but slightly broad.

Reviewer #3: This manuscript reports the identification of Corynespora cassiicola as the causal agent of leaf spot disease on Begonia fimbristipula, based on field observations, morphological characterization, multilocus phylogenetic analysis (ITS, TEF1α, and RPB2), and pathogenicity assays fulfilling Koch’s postulates. The study addresses a relevant phytopathological issue, particularly considering the economic and medicinal importance of B. fimbristipula in southern China, and contributes to documenting disease occurrence in this host.

Overall, the experimental framework adopted by the authors follows a generally accepted approach for pathogen identification, combining classical mycological methods with molecular phylogenetics and pathogenicity testing. The successful fulfillment of Koch’s postulates and the multilocus identification provide support for the conclusion that C. cassiicola is associated with the observed disease symptoms. However, despite this technically valid foundation, the manuscript in its current form presents several limitations that need to be addressed before it can be considered for publication.

A central issue is that the study remains largely descriptive, with limited quantitative analysis to support the conclusions. In particular, the pathogenicity assays are described qualitatively, with visual confirmation of symptom development, but without quantitative measurement of disease severity (e.g., lesion size, lesion number per unit area, or severity indices) and without statistical analysis. The inclusion of quantitative disease assessments and appropriate statistical comparisons would substantially strengthen the robustness and reproducibility of the findings.

Another important limitation concerns the phylogenetic analysis. Although multiple isolates were obtained from infected tissues, only a single representative isolate appears to have been used for multilocus sequencing and phylogenetic reconstruction. This restricts the ability to assess intra-population variability and limits the generalization of the identification. The authors should clarify the rationale for selecting a single isolate and, if possible, include additional isolates in the phylogenetic analysis or explicitly acknowledge this limitation in the discussion.

The manuscript also presents structural issues that affect clarity. Notably, sections of the Methods describing molecular identification appear to be duplicated, which disrupts the logical flow of the manuscript and should be corrected. In general, the organization of the Methods section would benefit from careful revision to improve coherence and avoid redundancy.

Regarding statistical analysis, the manuscript includes only minimal quantitative treatment of the data. While descriptive statistics are provided for morphological measurements, there is no statistical evaluation of pathogenicity results or comparison among isolates. As such, the statistical analysis cannot be considered rigorous in its current form. The authors should either incorporate appropriate statistical analyses where relevant or clearly justify the descriptive nature of the study.

Data availability is another aspect that requires clarification. While sequence data appear to be deposited in public databases, it is not fully clear whether all data underlying the findings (e.g., raw measurements, experimental observations, or additional datasets) are available, as required by PLOS ONE data policies. The Data Availability statement should be revised to explicitly indicate where all supporting data can be accessed.

The interpretation of the results, particularly in the Discussion and Conclusion sections, should also be moderated. Some statements extend beyond the data presented, especially those related to broader implications for disease management, crop protection, and resistance breeding. While these perspectives are relevant, they are not directly supported by experimental evidence in this study and should be presented more cautiously as potential implications rather than demonstrated outcomes.

In addition, the manuscript requires substantial improvement in English language quality. Numerous grammatical errors, awkward sentence constructions, and redundancies affect readability and clarity. Careful language editing by a fluent English speaker or professional editing service is strongly recommended to meet the journal’s standards.

Despite these limitations, the study has several strengths. It addresses an underreported host-pathogen interaction, applies a combination of classical and molecular identification approaches, and provides baseline information that may be useful for future epidemiological and phytopathological studies. With improved data presentation, clearer methodological description, and more cautious interpretation of results, the manuscript could represent a useful contribution.

In summary, the study is technically sound in its core approach but only partially supports its conclusions in its current form due to limitations in quantitative analysis, statistical rigor, and presentation. I therefore recommend major revision, with particular emphasis on strengthening data analysis, improving methodological clarity, and refining the interpretation and language of the manuscript.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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First Report of Corynespora cassiicola Causing Leaf Spot on Begonia fimbristipula in China

PONE-D-26-14713R1

Dear Dr. Lu,

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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Reviewers' comments: