Study on the Identification and Incidence Pattern of the Pathogen Causing Apple Scab in Wild Apple Forests of Ili, Xinjiang

Comments 1: The abstract does not clearly highlight the key aspects of the manuscript, such as t he problem statement, objectives, brief methodology, and main findings. In addition, the results and discussion are presented in a structure that is difficult to read. Please revise f or clarity and better flow.

Response 1: We thank the reviewer for this suggestion. We have completely rewritten the Abstract to follow a clear “problem–objective–methods–results–conclusion” structure. It now includes the research problem (apple scab in wild apples), objectives (pathogen identification and disease pattern), methods (field survey, isolation, pathogenicity), key results (symptom timing, peak severity, overwintering source), and conclusions.

Comments 2: The sentence is awkwardly structured and difficult to follow. Please consider revisin g for clarity..

Response 2: We appreciate this observation. We have rephrased the sentence in the Abstract to use active voice and improve clarity.

Comments 3: The description of symptom timing and disease progression is awkward.

Response 3: Thank you for pointing this out. We have broken down the long sentence in the Abstract into shorter ones and presented the timeline in a more logical sequence.

Comments 4: “Venturia inaequalis” should be in italics.

Response 4: We have corrected this oversight and italicized all instances of Venturia inaequalis throughout the text.

Comments 5: Recheck the citation format of “F. Yuan et al.”

Response 5: We thank the reviewer for highlighting this. We have corrected the citation to “Yuan F et al.” in the Introduction section in accordance with journal guidelines.

Comments 6: Check the citation format of “Yang Zhou.”

Response 6: We have corrected it to “Zhou Y” in the Introduction.

Comments 7: The use of “However” is unclear in the sentence about regional variation in disease patterns.

Response 7: We appreciate this suggestion. We have removed “However” and rephrased the sentence in the Introduction to improve logical flow.

Comments 8: Inconsistent spelling of “Huocheng.”

Response 8: We apologize for the inconsistency. We have standardized the spelling to “Huocheng” throughout the manuscript, including in Section 2.1 and Table 1.

Comments 9: Sampling details are unclear.

Response 9: Thank you for this comment. We have added specific information in Section 2.2.1: “A total of 150 leaves were collected from 30 trees, with 3 trees sampled from each of the 10 sample plots.”

Comments 10: Only one isolate was used for pathogenicity testing out of 29.

Response 10: We thank the reviewer for this note. We have clarified in Section 2.2.5 that strain XJAU-XY1-1 was selected based on its strong pathogenicity among the isolates.

Comments 11: The pathogenicity assay description is confusing regarding attached or detached fruits.

Response 11: We appreciate the reviewer’s attention to detail. We have clarified in Section 2.2.5 that the assay was performed on detached healthy fruits under laboratory conditions.

Comments 12: The developmental stage of fruits used in pathogenicity tests is unclear.

Response 12: We have specified in Section 2.2.5 that mature fruits collected in August were used.

Comments 13: Typo: “friut.”

Response 13: We have corrected this typo to “fruit” in Section 2.2.5.

Comments 14: The method for calculating disease incidence area proportion is not referenced.

Response 14: We have added a reference to the method described by Wang Xinru [39] in Section 2.3.

Comments 15: Images of inoculated and control fruits are missing.

Response 15: We have added a new figure (Figure 1) in Section 3.1.1 showing both inoculated and control fruits.

Comments 16: The number of measurements for fungal structures is not specified.

Response 16: We have added the following sentence in Section 3.1.2: “Measurements were based on 20 conidia and 20 conidiophores.”

Comments 17: Only one strain was used for molecular and pathogenicity tests, and no GenBank accession number was provided.

Response 17: We thank the reviewer for this important suggestion. We have expanded the molecular identification in Section 2.2.4 to include multiple gene loci (ITS, LSU, rpb2, tef1, tub2) and constructed a multi-locus phylogenetic tree. GenBank accession numbers are being processed and will be provided upon acceptance.

Comments 18: HC plot data are from only one year, limiting comparison reliability.

Response 18: We appreciate this observation. We have added an explanation in Section 2.1 that the HC plots were added in 2024 after initial surveys in 2023, and we have toned down comparative claims in Section 3.3.

Comments 19: Lack of environmental data weakens the comparison between XY and HC plots.

Response 19: We acknowledge this limitation. We have added a note in the Discussion that environmental factors will be analyzed in future studies.

Comments 20: It is unclear whether isolations were from conidia or ascospores.

Response 20: We have specified in Section 2.4 that isolations were made from pseudothecia and added molecular confirmation from overwintering structures in Section 3.5.

Comments 21: Figure captions for XY and HC plots are swapped.

Response 21: We apologize for the error. We have corrected the figure captions (now Figure 8 and Figure 9) in Section 3.5.

Comments 22: Same as above.

Response 22: Corrected as above.

Comments 23: The use of only one strain and one molecular marker (ITS) is insufficient for reliable species identification.

Response 23: We thank the reviewer for this critical comment. We have expanded the molecular analysis in Section 2.2.4 to include five gene loci and constructed a multi-locus phylogenetic tree to strengthen species identification.

Comments 24: The disease pattern described is already well known, and the study lacks novelty without environmental data.

Response 24: We have emphasized in the Introduction and Discussion that this is the first systematic report of apple scab on wild apples in the Ili region, highlighting its regional and host-specific significance. Environmental data have also been collected and will be studied later.

Comments 25: The discussion on ascospores or conidia is not well supported by the data, as the manuscript does not specify whether the isolations from various plant parts were derived from conidia, mycelium, or ascospores.

Response 25: We have revised the Discussion to clarify that the overwintering structures were molecularly identified as Venturia inaequalis and are likely pseudothecia, based on literature and morphological observations.

Comments 26: As previously mentioned, no environmental data were provided to support the authors’ claims. This weakens the comparison between the two plots, as the data only show differences in disease incidence without accounting for other confounding variables. In addition, only one year of data was included for the HC plot, and the lack of replication re duces the reliability of the findings.

Response 26: We have acknowledged this limitation in the Discussion and indicated that environmental data will be incorporated in future studies.

Comments 27: The conclusion is weak; more strains and molecular markers are needed.

Response 27: We have revised the Conclusion to highlight the preliminary nature of the findings and the need for multi-strain, multi-gene, and environmental data in future work.

4. Response to Comments on the Quality of English Language

Point 1:

Response 1: We have carefully addressed all the scientific and technical points raised by the reviewers in the revised manuscript and in the point-by-point response letter. However, due to time constraints, we were unable to perform a thorough language polish at this stage. In the later stage, we are willing to accept the editing service.

Comments 1: Line 29: I believe, 'apple scab', 'microscopy' and 'phylogenetic analysis' should be among the keywords

Response 1: We thank the reviewer for this helpful suggestion. We have updated the Keywords section to include: apple scab, microscopy, and phylogenetic analysis.

Comments 2: ....'inaequalis'

Response 2: We apologize for the typo. The spelling has been corrected to Venturia inaequalis.

Comments 3: Introduction. I believe the authors have to pay more attention on methods currently used for V. inaequalis identification.

Response 3: We appreciate this suggestion. We have expanded the Introduction to include a more detailed description of the current molecular and morphological methods used for the identification of V. inaequalis.

Comments 4:  I think the years (2023 or 2024) should be provided.

Response 4: Thank you for pointing this out. We have added a note in the caption of Table 1 specifying that the surveys in Xinyuan County (XY plots) were conducted from 2023 to 2024.

Comments 5: Lines 101-103: What mycroscope was used for morphological observation and what taxonomic keys were used for identification?

Response 5: We have added these details in Section 2.2.3 (Morphological Observation): "Images were captured using an Olympus BX51 microscope... Morphological identification was based on keys from [Fang, 1998] and [Úrbez-Torres et al., 2008]."

Comments 6: Line 106: Reference for ITS1 and ITS4 primers or primer sequences?

Response 6: We have added the reference for the ITS1/ITS4 primers (White et al., 1990) in Section 2.2.4.

Comments 7: Line 108: Profile of PCR amplification?

Response 7: We have provided the full PCR amplification profile in Section 2.2.4, including denaturation, annealing, and extension temperatures and times..

Comments 8: Line 109: 'Primers were synthesized' should be removed

Response 8: We appreciate this suggestion. This sentence has been removed from Section 2.2.4 as suggested.

Comments 9: Line 112: What algorithm was used for phylogenetic analysis? was bootstrapping conducted?

Response 9: We have specified in Section 2.2.4 that "Maximum Likelihood (ML) and Bayesian inference analyses were conducted," and that "ML bootstrap values (≥50%) are shown on the branches."

Comments 9: Line 113: The nucleotide sequence obtained in this study should be deposited in online database (such as GenBank NCBI) and corresponding accession numbers should be provided.

Response 9: We have expanded the molecular identification in Section 2.2.4 to include multiple gene loci (ITS, LSU, rpb2, tef1, tub2) and constructed a multi-locus phylogenetic tree. GenBank accession numbers are being processed and will be provided upon acceptance.

Comments 10: Figure 4: The authors should mention that C. variabile was used as an outgroup.

Response 10: We thank the reviewer for this note. We have updated the caption of Figure 4 (now Figure 3 in the revised manuscript) to state: "The tree is rooted with Coleroa robertiani (CBS458.64)."

Comments 11: Section 2.2.4 'Molecular identification'. In general if another DNA marker (e.g. ef1 alpha or beta-tubulin) would be used for molecular identification (optional).

Response 11: We appreciate the reviewer’s attention to detail. We have clarified in Section 2.2.5 that the assay was performed on detached healthy fruits under laboratory conditions.

Comments 12: Section 2.2.4 'Molecular identification'. In general, if another DNA marker (e.g., ef1 alpha or beta-tubulin) would be used for molecular identification (optional).

Response 12: We thank the reviewer for this valuable suggestion. Accordingly, we have expanded the molecular analysis. Section 2.2.4 now describes the use of five gene loci (ITS, LSU, rpb2, tef1, and tub2) for a robust multi-locus phylogenetic analysis.

Comments 13: Figure 5 and 6. I think the letter size of a legend in these graphs should be bigger (optional).Discussion. Probably, the authors should discuss how the results of this study can be used for applied purposes, for instance development of molecular identification systems for V.inaequalis or methods for control of the pathogen.

Response 13: We thank the reviewer for this note. Due to time constraints, we did not increase the font size of the legend in the charts (now Figures 6 and 7).

Comments 14: Discussion. Probably, the authors should discuss how the results of this study can be used for applied purposes, for instance, development of molecular identification systems for V. inaequalis or methods for control of the pathogen.

Response 14: We appreciate this insightful comment. We have added a new paragraph in the Discussion section exploring the applied implications of our findings, including the development of specific molecular detection tools and culturally relevant management strategies for apple scab in wild apple forests.