Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is devoted to the identification of pathogenic fungi infecting fruit trees. The authors performed morphological and molecular genetic analyses to identify the pathogen. Some minor corrections should be addressed to further improve the manuscript.

Line 21, 66 and throughout the maniscript: please, check the genus name " Monilia" (this is a former name)

Table 1: since GenBank accession numbers could be considered part of the study results they may be removed from the Table in Materials and Methods section

Line 143: please, provide a reference for "CTAB method" or brief description.

Line 149: please, specify the manufacturer of "Taq Mix" and concentration of primers (not the volume)

Line 157: it is preferable to use the term  "similar nucleotide sequences" rather than "homologous DNA sequences"

Line 271, 273, 277:  it is preferable to use "similarity" rather than "homology"

Line 287: please, provide information about the instrument/software used for sequence concatenation in Materials and Methods section.

Figure 7. Please, provide Accession numbers for the strains in the dendrogram; increase branch length clearer presentation

Lines 359-360: please, clarify the statement "the distribution of Monilinia species depends on geography rather than the hosts" since the  above statement was "host or geographical location did not play a key role in the evolution of M. yunnanensis". In addition, using such concerved sequences as ITS, tub2 and laccase gene is not preferable for evolutionary studies within a single species.

Author Response

Dear reviewers and editors

    Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files

 

Author's Reply to the Review Report (Reviewer 1)

The manuscript is devoted to the identification of pathogenic fungi infecting fruit trees. The authors performed morphological and molecular genetic analyses to identify the pathogen. Some minor corrections should be addressed to further improve the manuscript.

Comments 1: Line 21, 66 and throughout the manuscript: please, check the genus name " Monilia" (this is a former name)

Response 1: we have revised “Monilinia” throughout the manuscript.

Comments 2: Table 1: since GenBank accession numbers could be considered part of the study results they may be removed from the Table in Materials and Methods section

Response 2: Table 1 was removed to the results part, and revised as Table 2.

Comments 3: Line 143: please, provide a reference for "CTAB method" or brief description.

Response 3: we have provided a reference for this.

Comments 4: Line 149: please, specify the manufacturer of "Taq Mix" and concentration of primers (not the volume)

Response 4: we have specified the manufacturer of "Taq Mix" and concentration of primers.

Comments 5: Line 157: it is preferable to use the term "similar nucleotide sequences" rather than "homologous DNA sequences"

Response 5: we have revised homologous to similar.

Comments 6: Line 271, 273, 277:  it is preferable to use "similarity" rather than "homology"

Response 6: we have revised "similarity" to "homology".

 

Comments 7: Line 287: please, provide information about the instrument/software used for sequence concatenation in Materials and Methods section.

Response 7: we have provided information about the software used for sequence concatenation.

Comments 8: Figure 7. Please, provide Accession numbers for the strains in the dendrogram; increase branch length clearer presentation

Response 8: we have revised the phylogenetic tree, then marked the isolates in this study and other ex-type isolates. we refer to most of the literature, and most of the strains in the phylogenetic tree use the combination of strain number and species name.

Comments 9: Lines 359-360: please, clarify the statement "the distribution of Monilinia species depends on geography rather than the hosts" since the above statement was "host or geographical location did not play a key role in the evolution of M. yunnanensis". In addition, using such concerved sequences as ITS, tub2 and laccase gene is not preferable for evolutionary studies within a single species.

Response 9: here we refer to published literature, ITS, tub2 and laccase gene combine could solve species identification on M. yunnanensis. Due to these Conservative marker gene could not preferable for evolutionary studies, we revised these statement in discussion and conclusion parts.

Reviewer 2 Report

Comments and Suggestions for Authors

Manuscript horticulturae-4399796: Characterization and Pathogenicity of Monilia yunnanensis Causing Malus Jinxiu Brown Rot Disease in Shaanxi, China.
Overall, the manuscript addresses an important phytopathological problem and provides a potentially valuable first report of Monilia yunnanensis associated with brown rot of Malus ‘Jinxiu’. The work combines field observations, morphology, pathogenicity assays, and multi-locus phylogenetic analyses, which represents a solid framework for pathogen identification. However, some conceptual and methodological issues should be addressed before publication. Please find below my comments and suggestions:
Major comments
1.    The scope of sampling and the scope of the conclusions do not match. The authors explicitly state that samples were collected from only three orchards (Lines 90-91). However, these three localities are all located in the same region and do not represent the full diversity of apple production systems throughout Shaanxi Province. Therefore, this statement in the Abstract "The results showed that the dominant causal agent of Malus ‘Jinxiu’ brown rot in Shaanxi Province is Monilia yunnanensis." (Lines 20-21) is somehow problematic because no orchards outside Yan'an were sampled. Could other Monilinia species occur in those regions non-sampled? Because no surveys were conducted in other major apple-producing regions of Shaanxi, these conclusions should be tempered and restricted to the surveyed orchards or northern Shaanxi region. The authors themselves acknowledge that the three orchards were geographically close and that broader surveys are needed (Lines 344-348), further highlighting the limitation of the current sampling design. Please revise this issue.
2.    Pathogenicity experiments need stronger statistical and experimental support. The pathogenicity assays successfully satisfy Koch's postulates and provide evidence that the isolates are pathogenic on apple fruit. However, several aspects of experimental design and statistical analysis require clarification. The replication structure is ambiguous (Lines 125-127), and the manuscript should clearly identify the experimental unit used for statistical analyses. Because all inoculations were performed on wounded fruits (Lines 121-122), the experiments demonstrate pathogenic capability rather than natural infection potential, and this limitation should be acknowledged in the Discussion. Furthermore, lesion expansion rate was the only disease parameter evaluated (Lines 132-133), whereas additional metrics such as final lesion size, disease incidence, or sporulation would provide a more comprehensive assessment of virulence. Finally, the statistical methods section (Lines 168-170) does not specify the post hoc test used following ANOVA, nor does it describe how assumptions of normality and homogeneity of variances were assessed. These issues should be clarified and, where possible, strengthened.
3.    Phylogenetic analysis could be Improved. The multilocus approach is appropriate for species identification; however, the phylogenetic framework remains relatively limited because it relies on only three loci (ITS, tub2, and lcc2). Inclusion of additional informative loci commonly used in fungal phylogenetics (for example, TEF1-α, GAPDH, ACT) would provide stronger resolution of relationships among closely related Monilinia species. Most importantly, the phylogeny supports species identification but does not justify broader conclusions regarding the influence of host or geographic origin on the evolution of M. yunnanensis (Lines 338-340). These statements should be moderated accordingly.
4.    The Discussion largely repeats the Results section. The Discussion would benefit from greater analytical depth. Much of the section currently reiterates findings already presented in the Results (Lines 293-299) without sufficiently exploring their biological or epidemiological significance. The authors should more thoroughly compare their findings with previous reports of M. yunnanensis on apple hosts in China, particularly regarding pathogen prevalence and disease incidence. The cross-infection experiments between ‘Jinxiu’ and ‘Fuji’ cultivars (Lines 295-297) are potentially one of the most important findings of the study, yet their implications for pathogen movement and disease epidemiology in mixed-cultivar orchards are not discussed. Finally, the Discussion should address the remarkably high disease incidence observed in surveyed orchards (Lines 174-175) and elaborate on the practical implications of cultivar susceptibility and pathogen spread for apple production systems in northern Shaanxi.

Author Response

Dear reviewers and editors

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted/in track changes in the re-submitted files

Author's Reply to the Review Report (Reviewer 2)

Comments 1: The scope of sampling and the scope of the conclusions do not match. The authors explicitly state that samples were collected from only three orchards (Lines 90-91). However, these three localities are all located in the same region and do not represent the full diversity of apple production systems throughout Shaanxi Province. Therefore, this statement in the Abstract "The results showed that the dominant causal agent of Malus ‘Jinxiu’ brown rot in Shaanxi Province is Monilia yunnanensis." (Lines 20-21) is somehow problematic because no orchards outside Yan'an were sampled. Could other Monilinia species occur in those regions non-sampled? Because no surveys were conducted in other major apple-producing regions of Shaanxi, these conclusions should be tempered and restricted to the surveyed orchards or northern Shaanxi region. The authors themselves acknowledge that the three orchards were geographically close and that broader surveys are needed (Lines 344-348), further highlighting the limitation of the current sampling design. Please revise this issue.Response 1: we have revised “Monilinia” throughout the manuscript.

Response 1: As a newly introduced apple variety, Malus ‘Jinxiu’ is only cultivated in the northern part of Shaanxi Province in recent years. Due to the small cultivation area and the fact that brown rot did not occur in some orchards in our field survey in 2025, we revised these statements in the abstract, results and discussion parts. In future studies, we will expand the scope of investigation and sampling, and in the same time we should pay attention to other stone fruit tree related diseases.

Comments 2: Pathogenicity experiments need stronger statistical and experimental support. The pathogenicity assays successfully satisfy Koch's postulates and provide evidence that the isolates are pathogenic on apple fruit. However, several aspects of experimental design and statistical analysis require clarification. The replication structure is ambiguous (Lines 125-127), and the manuscript should clearly identify the experimental unit used for statistical analyses. Because all inoculations were performed on wounded fruits (Lines 121-122), the experiments demonstrate pathogenic capability rather than natural infection potential, and this limitation should be acknowledged in the Discussion. Furthermore, lesion expansion rate was the only disease parameter evaluated (Lines 132-133), whereas additional metrics such as final lesion size, disease incidence, or sporulation would provide a more comprehensive assessment of virulence. Finally, the statistical methods section (Lines 168-170) does not specify the post hoc test used following ANOVA, nor does it describe how assumptions of normality and homogeneity of variances were assessed. These issues should be clarified and, where possible, strengthened.

Response 2: we have clarified statistical analysis in the materials and methods part. Due to the lack of natural infection potential experiment, so this limitation have been k acknowledged in the discussion part. We refer to similar publications, and found that the rate of fruit lesion expansion is often a pathogenicity level of pathogenicity. Because fruits size usually different among different apple varieties, we did not choose final lesion as a pathogenicity level. We found that disease incidence, or sporulation between different isolates did not showed any significant difference in the pathogenicity assays. Assumptions of normality and homogeneity of variances were clarified in the materials and methods part.

Comments 3: Phylogenetic analysis could be Improved. The multilocus approach is appropriate for species identification; however, the phylogenetic framework remains relatively limited because it relies on only three loci (ITS, tub2, and lcc2). Inclusion of additional informative loci commonly used in fungal phylogenetics (for example, TEF1-α, GAPDH, ACT) would provide stronger resolution of relationships among closely related Monilinia species. Most importantly, the phylogeny supports species identification but does not justify broader conclusions regarding the influence of host or geographic origin on the evolution of M. yunnanensis (Lines 338-340). These statements should be moderated accordingly.

Response 3: we have revised the phylogenetic analysis appropriately. Here we refer to published literature, ITS, tub2 and laccase gene combine could solve species identification on M. yunnanensis. We used GAPDH gene sequencing in the previous experiment and obtained the same results. Because multiple genes of the published strains were needed for multi-gene joint construction, we chose ITS, tub2, and lcc2 to be more suitable for our study. Due to these Conservative marker gene could not preferable for evolutionary studies, we revised these statement in discussion and conclusion parts.

Comments 4: The Discussion largely repeats the Results section. The Discussion would benefit from greater analytical depth. Much of the section currently reiterates findings already presented in the Results (Lines 293-299) without sufficiently exploring their biological or epidemiological significance. The authors should more thoroughly compare their findings with previous reports of M. yunnanensis on apple hosts in China, particularly regarding pathogen prevalence and disease incidence. The cross-infection experiments between ‘Jinxiu’ and ‘Fuji’ cultivars (Lines 295-297) are potentially one of the most important findings of the study, yet their implications for pathogen movement and disease epidemiology in mixed-cultivar orchards are not discussed. Finally, the Discussion should address the remarkably high disease incidence observed in surveyed orchards (Lines 174-175) and elaborate on the practical implications of cultivar susceptibility and pathogen spread for apple production systems in northern Shaanxi.

Response 4: we have revised the discussion part appropriately. We have added a brief paragraph discussing the importance of M. yunnanensis spread among different apple cultivar in the orchards, also including the fungicide resistance profiles, disease cycle, or epidemiology.