Characterizing the Endophytic Microbiome and Microbial Functional Assemblages Associated with Fengtang Plum (Prunus salicina Lindl.) Development and Resistance

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript presents an in-depth analysis of the stage-specific microbial community structure and functional profiles in Fengtang plum fruits. The research addresses an important gap in understanding endophytic microbiota in stone fruits and has the potential to contribute significantly to horticultural microbiology and fruit quality science. However, several issues related to terminology, methodology, interpretation, and language must be addressed before the manuscript can be considered for publication.

Title:

• "Microbat" should be corrected to "Microbial".
• "Endphytic" should be "Endophytic".

Abstract:

• The sentence “With notably no reported brown rot caused by Monilinia spp..” is grammatically incomplete and disrupts the logical flow.

Keywords:

• The keyword “microbat function” is inappropriate and unrelated to the content of the study. It should be removed or replaced.

Introduction:

• The term “resistance” is vague. It is unclear whether it refers specifically to Monilinia spp. or to general pathogen suppression.
• The relationship between microbiome and fruit aroma is mentioned but lacks explanation. The chemical nature of these compounds and microbial contributions should be briefly discussed.
• While terroir variables like diurnal temperature shifts and low relative humidity are introduced, their impact on microbial dynamics remains underexplored.
• The introduction is too brief to establish a strong rationale for the study:"The brevity of the introduction limits the reader's ability to fully grasp the scientific context, and it falls short of establishing a compelling rationale for the investigation."
• Typographical Issues:
• Line 41: “bits” → should be “exhibits”
• Line 42: “friut” → “fruit”
• Line 44: “harbors considerable” → “harbor a considerable”
• Line 60: “significant economically” → “economically significant”

Materials and Methods:

• The study focuses solely on fruit tissue. To support claims about terroir-specific microbiota, it would have been valuable to include additional sample types (e.g., leaves, soil).
• Surface sterilization protocols are critical in endophyte research and are missing.
• PCR conditions for 16S and ITS amplification (temperatures, cycles) are not described, limiting reproducibility.
• Statistical analysis details are insufficient. Methods used for beta diversity, alpha diversity comparisons, and functional differential analysis should be stated clearly.
• Typographical Issues:
• Line 64: “during April to June 2024” → “from April to June 2024”
• Line 92: “OUT” → should be “OTU”

Results:

• Lines 150–158 include long taxonomic lists that may reduce readability:"Several sections over-rely on extensive taxonomic listings without integrating ecological interpretation, which may reduce readability and scientific coherence."
• Data on OTU percentages, diversity indices, or abundance shifts would benefit from additional tables rather than being embedded only in the text.

Discussion:

• Lines 270–276: The opening paragraph is too generic. It should clearly summarize the key findings and implications.
• Lines 277–297: The discussion frequently restates results instead of offering deeper interpretation or connecting findings to broader literature.
• Lines 360–370: The role of Ralstonia is inadequately discussed:
• "The discussion around Ralstonia lacks depth; its functional role in asymptomatic fruits is posed as a question but not sufficiently examined or hypothesized." Potential explanations such as latent colonization, non-pathogenic strains, or competitive inhibition by Pseudomonas/Trichoderma could be explored.
• Line 311: It is recommended to add the following sentence to further emphasize the role of environmental factors in shaping cultivar-specific traits:
  “In addition to microbial shifts, environmental factors such as temperature, relative humidity, and solar radiation are also known to influence plant morphological and phytochemical traits, which may indirectly contribute to cultivar-specific characteristics (Aydın and Coskun, 2013).” 

  Aydın, D., Coskun, O.F. Comparison of Edta-Enhanced Phytoextraction Strategies with Nasturtıum officinale (Watercress) On An Artificially Arsenic Contaminated Water. Pak. J. Bot., 45(4): 1423-1429, 2013.

Conclusion:

• The conclusions summarize the study well but should more strongly emphasize the scientific novelty, practical implications, and limitations.
• A brief mention of the analytical approach (multi-stage sampling + functional prediction tools) would also enhance this section.

Author Response

Title:

Comments 1: "Microbat" should be corrected to "Microbial".

Response 1:  Thank you for pointing this out. We agree with this comment. Therefore, we have corrected "Microbat" to "Microbial".

Comments 2: "Endphytic" should be "Endophytic".

Response 2:  Thank you for pointing this out. We agree with this comment. Therefore, we have corrected "Endphytic"to  "Endophytic".

Abstract:

Comments 3: The sentence “With notably no reported brown rot caused by Monilinia spp..” is grammatically incomplete and disrupts the logical flow.

Response 3:  Thank you for pointing this out. We agree with this comment. Therefore, we have corrected the sentenceto  "and notable resistance to brown rot caused by Monilinia spp."

Keywords:

Comments 4: The keyword “microbat function” is inappropriate and unrelated to the content of the study. It should be removed or replaced.

Response 4:  Thank you for pointing this out. We agree with this comment. Therefore, we have removed the keyword “microbat function”.

Introduction:

Comments 5: The term “resistance” is vague. It is unclear whether it refers specifically to Monilinia spp. or to general pathogen suppression.

Response 5:  Thank you for pointing this out. We have pointed that " Fengtang plum exhibits atypical resistance to brown rot fungi Monilinia spp. compared to other stone fruit" in line 82. I have restructured the entire Introduction section by relocating the original paragraphs describing plums and Honey Plum (Prunus salicina 'Fengtangli') to lines 75-82. This adjustment may help present the botanical description in a more logical sequence and enhance the clarity of species-specific characterization.

Comments 6: The relationship between microbiome and fruit aroma is mentioned but lacks explanation. The chemical nature of these compounds and microbial contributions should be briefly discussed.

Response 6:  Thank you for pointing this out. We have briefly discussed the chemical nature of these compounds and microbial contributions in coffee and tomato in line68-74

 

 Comments 6: While terroir variables like diurnal temperature shifts and low relative humidity are introduced, their impact on microbial dynamics remains underexplored.

Response 7:   Thank you for pointing this out. In lines 42-51, we have included examples demonstrating how seasonal variations, different plant varieties, and tissue-specific differences shape microbial community structures. Specifically, we illustrated that seasonal shifts significantly alter rhizosphere microbiota composition, distinct cultivars harbor genotype-specific endophytes, and specialized microbial consortia colonize specific anatomical structures like xylem versus phloem tissues. These empirical cases systematically validate our proposed framework regarding spatiotemporal and host factors governing microbiome assembly.

Comments 8: The introduction is too brief to establish a strong rationale for the study:"The brevity of the introduction limits the reader's ability to fully grasp the scientific context, and it falls short of establishing a compelling rationale for the investigation."

Response 8:   Thank you for pointing this out.We have improved the Introduction section by supplementing the following content:

1. In Lines 42-55, we added explanations about how tissue types, fruit varieties, and geographical environments influence microbial communities.
2. In Lines 58-66, we elaborated on the relationship between microbial community dynamics and fruit resistance.
3. Lines 68-74 briefly address the correlation between microorganisms and specific aroma compounds.
4. Lines 83-99 provide a concise overview of microbial community characteristics in plums (Prunus salicina) across different developmental stages and cultivars.

Comments 9: 

• Line 41: “bits” → should be “exhibits”
• Line 42: “friut” → “fruit”
• Line 44: “harbors considerable” → “harbor a considerable”
• Line 60: “significant economically” → “economically significant”

Response 9:   Thank you for pointing this out. We have  corrected all typographical issues.

Materials and Methods:

Comments 10: The study focuses solely on fruit tissue. To support claims about terroir-specific microbiota, it would have been valuable to include additional sample types (e.g., leaves, soil).

Response 10:   Thank you for pointing this out. We aimed to investigate how the microbial community of Fengtang plum diversifies throughout its developmental stages, and whether it contributes to the fruit's resistance against brown rot disease or volatile compound emissions. Therefore, we have not included additional sample types in the current study. However, your suggestion is particularly insightful. We plan to investigate microbial communities of other sample types in future studies to identify terroir-specific microbiota.

Comments 11: Surface sterilization protocols are critical in endophyte research and are missing.

Response 11: Thank you for pointing this out. We have supplemented the surface sterilization protocol in the Materials and Methods section.

Comments 12: PCR conditions for 16S and ITS amplification (temperatures, cycles) are not described, limiting reproducibility.

Response 12: Thank you for pointing this out. We have supplemented PCR conditions for 16S and ITS amplification in the Materials and Methods section.（line140-144）

Comments 13: Statistical analysis details are insufficient. Methods used for beta diversity, alpha diversity comparisons, and functional differential analysis should be stated clearly.

Response 13: Thank you for pointing this out. We have supplemented methods used for beta diversity, alpha diversity comparisons, and functional differential analysis in the Materials and Methods section.（line162-169）

Comments 14:

Line 64: “during April to June 2024” → “from April to June 2024”

Line 92: “OUT” → should be “OTU”

Response 14: Thank you for pointing this out. We have corrected these typographical issues.

Results:

Comments 15: Lines 150–158 include long taxonomic lists that may reduce readability:"Several sections over-rely on extensive taxonomic listings without integrating ecological interpretation, which may reduce readability and scientific coherence."

Response 15: Thank you for pointing this out. To address the readability concern while maintaining scientific rigor, we have reorganized the description of the heatmap (Lines 207-231). We deleted the simple taxonomic listings and conducted trend descriptions of microbial community changes across different periods. Biological function predictions linking microbial dynamics with fruit physiological changes were added (such as involvement in early defense system establishment, sugar accumulation, and tissue degradation). The differences in bacterial-fungal community interactions were emphasized by highlighting their 'distinct temporal characteristics compared to bacteria'.

Comments 16: Data on OTU percentages, diversity indices, or abundance shifts would benefit from additional tables rather than being embedded only in the text

Response 16: We sincerely appreciate the reviewer's thoughtful suggestion regarding data presentation. We have modified Figure 2 by adding trend lines to better visualize the changes in microbial abundance, and included a microbial abundance table (Table S2) in the supplementary files.

Discussion:

Comments 17: Lines 270–276: The opening paragraph is too generic. It should clearly summarize the key findings and implications.

Response 17: Thank you for pointing this out.In the revised manuscript (Lines 339-348-288), we have described the stage-dependent microbial assembly as  key findings summarize.

Comments 18: Lines 277–297: The discussion frequently restates results instead of offering deeper interpretation or connecting findings to broader literature.

Response 18: Thank you for pointing this out. In the revised manuscript (Lines 358-367), we have offered a deeper interpretation about “bacterial community follows a distinct successional sequence，whereas the fungal enrichment process demonstrates independent temporal specificity" instead of restates results.

Comments 19: Lines 360–370: The role of Ralstonia is inadequately discussed:"The discussion around Ralstonia lacks depth; its functional role in asymptomatic fruits is posed as a question but not sufficiently examined or hypothesized." Potential explanations such as latent colonization, non-pathogenic strains, or competitive inhibition by Pseudomonas/Trichoderma could be explored.

Response 19: Thank you for pointing this out.This advice is very enlightening. In the revised manuscript (Lines 445-457), based on a review of relevant literature and our research findings, we have discussed two key aspects: the carriage of non-pathogenic strains of Ralstonia, and the bioantagonistic effects of non-pathogenic strains/Trichoderma harzianum against Ralstonia.

Comments 20: Line 311: It is recommended to add the following sentence to further emphasize the role of environmental factors in shaping cultivar-specific traits:
“In addition to microbial shifts, environmental factors such as temperature, relative humidity, and solar radiation are also known to influence plant morphological and phytochemical traits, which may indirectly contribute to cultivar-specific characteristics (Aydın and Coskun, 2013).” 

Response 20: Thank you for pointing this out. In the revised manuscript (Lines 377-380), we have added the sentence to  further emphasize the role of environmental factors in shaping cultivar-specific traits.

Conclusion:

Comments 21: The conclusions summarize the study well but should more strongly emphasize the scientific novelty, practical implications, and limitations. A brief mention of the analytical approach (multi-stage sampling + functional prediction tools) would also enhance this section.

Response 21: Thank you for pointing this out. This advice is very enlightening. In the revised manuscript (Lines 484-498), we have explicited states the study's originality in microbiome characterization and ecological transition discovery, highlightsed analytical approach (sampling + prediction tools), linked specific taxa/functions to agricultural applications and acknowledgesd prediction method constraints.

 

 

 

 

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

The general proposal of this manuscript submitted to Horticulturae encompasses the characterization of the diversity, composition, and functions of endophytic microbial communities (bacterial and fungal) at different stages of fruit development of Fengtang plum (Prunus salicina Lindl.), among other aspects related to quality development and disease resistance. I also provide the following considerations:

I wonder if the authors actually meant to write microbat in the title and other parts of the text. The definition of this in English is: “Microbats are small bats belonging to the  suborder Microchiroptera…” Authors should be careful with every word written in a manuscript, especially because this reflects the level of attention given to the main text and to the details of the research.

In my opinion, the methodology section could be enriched with more biochemical, physiological, and in planta phenotypic parameters to justify a full article instead of a short communication to be published.

The abstract could be more critical by pointing out the study's limitations—for example, how relying on functional predictions from sequencing data rather than direct metagenomic data is a disadvantage. The association of Lactobacillus and Trichoderma with aromatic compounds and resistance to fungal pathogens could be presented in a more cautious and well-grounded way.

The introduction could benefit from a more in-depth review of the existing literature on the microbiome of other plum varieties or related fruits during developmental stages.

The methodological section could be improved with more details about the selection criteria of the independent orchards and information about the specific environmental conditions during sampling at each stage. The sequencing depth achieved and the criteria for determining microbial sampling sufficiency could be more explicitly clarified and explained.

Regarding the presentation of functional prediction results (Figures 6 and 7) as well as other related findings, a formal statistical analysis of functional differences between the stages could strengthen the conclusions about metabolic changes and other associated biochemical shifts.

The discussion of results often, regarding several aspects, remains limited to the description of figures and tables.

Some statements lack deeper critical insight in the discussion, including aspects of the dominance of Ralstonia in the later stages—the discussion about the absence of symptoms in Fengtang plum and the possible role of this genus could be more exploratory. The section could benefit from a more detailed discussion on possible interactions between the bacteria and fungi identified at each stage.

Comments on the Quality of English Language

See comments for authors

Author Response

Comments 1：I wonder if the authors actually meant to write microbat in the title and other parts of the text. The definition of this in English is: “Microbats are small bats belonging to the  suborder Microchiroptera…” Authors should be careful with every word written in a manuscript, especially because this reflects the level of attention given to the main text and to the details of the research.

Response 1:  Thank you for pointing this out. We agree with this comment. Therefore, we have corrected "Microbat" to "Microbial".

Comments 2：In my opinion, the methodology section could be enriched with more biochemical, physiological, and in planta phenotypic parameters to justify a full article instead of a short communication to be published.

Response 2:  We sincerely appreciate the reviewer's constructive suggestion, which would undoubtedly enhance the comprehensiveness of our study. We fully agree that incorporating additional biochemical, physiological, and phenotypic parameters could provide more comprehensive dimensions to the research. However, we regret to note that there is currently very limited research on microbial community changes during different developmental stages of plum fruits, and no reported studies specifically on the microbial community dynamics during the development of Fenghuang plums. Consequently, we were unable to predetermine specific physiological or biochemical indicators that might correlate with microbial community shifts. At this stage, our study primarily focuses on establishing fundamental data regarding the changes in microbial communities across different developmental stages of Fenghuangli plums. Building upon these current findings, we plan to conduct follow-up studies integrating metabolomics and metagenomics approaches to correlate these microbial community changes with relevant physiological, biochemical, and phenotypic parameters. This will enable us to elucidate the specific mechanisms by which microbial community dynamics influence fruit quality formation during the maturation process.

Comments 3：The abstract could be more critical by pointing out the study's limitations—for example, how relying on functional predictions from sequencing data rather than direct metagenomic data is a disadvantage. The association of Lactobacillus and Trichoderma with aromatic compounds and resistance to fungal pathogens could be presented in a more cautious and well-grounded way.

Response 3:  Thank you for pointing this out. We agree with this comment. Therefore, we have revised the abstract to supplement the description of limitations regarding functional predictions derived from 16S/ITS sequencing data rather than metagenomic analysis. Additionally, we adopted more cautious language when speculating about the relationships between lactic acid bacteria and Trichoderma with aromatic compounds, as well as their potential resistance to fungal pathogens.(line27-35)

Comments 4：The introduction could benefit from a more in-depth review of the existing literature on the microbiome of other plum varieties or related fruits during developmental stages.

Response 4:  Thank you for pointing this out. We have added content in Lines 83-99 to introduce recent research findings regarding the significant dynamic changes observed in the microbial communities of plum fruits (Prunus salicina) across different developmental stages and varieties.

Comments 5：The methodological section could be improved with more details about the selection criteria of the independent orchards and information about the specific environmental conditions during sampling at each stage. The sequencing depth achieved and the criteria for determining microbial sampling sufficiency could be more explicitly clarified and explained.

Response 5:  Thank you for pointing this out. In the revised manuscript (Lines 118-125), we have supplemented details about the selection criteria of the independent orchards and information about the specific environmental conditions during sampling at each stage.The detailed clarifications and explanations regarding sequencing depth and the criteria for determining the sufficiency of microbial sampling have been provided in the Supplementary Materials.

Comments 6：Regarding the presentation of functional prediction results (Figures 6 and 7) as well as other related findings, a formal statistical analysis of functional differences between the stages could strengthen the conclusions about metabolic changes and other associated biochemical shifts.

Response 6: Thank you for pointing this out. This is very inspiring. We have conducted statistical analysis and added error bar charts to the "functional prediction results"(fig.6-7). To better present the experimental results, the top 15 functional group bar charts and PCA plots were placed in the supplementary files (Fig. S2).

Comments 7：The discussion of results often, regarding several aspects, remains limited to the description of figures and tables.

Response 7: Thank you for pointing this out. We appreciate the reviewers' attention to the discussion section. In the first two paragraphs of section 4.1 of the discussion, we described the microbial composition of different developmental stages of the honey plum to enable readers to better understand whether the microbial community composition and succession patterns at different developmental stages of the honey plum are consistent or similar to those in other studies, and to attempt to explore the reasons for any differences.

Comments 8：Some statements lack deeper critical insight in the discussion, including aspects of the dominance of Ralstonia in the later stages—the discussion about the absence of symptoms in Fengtang plum and the possible role of this genus could be more exploratory. The section could benefit from a more detailed discussion on possible interactions between the bacteria and fungi identified at each stage.

Response 8: Thank you for pointing this out. This advice is very enlightening. In the revised manuscript (Lines 381-395), based on a review of relevant literature and our research findings, we have discussed two key aspects: the carriage of non-pathogenic strains of Ralstonia, and the bioantagonistic effects of non-pathogenic strains/Trichoderma harzianum against Ralstonia.(line445-457).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I would like to thank the authors for carefully revising the manuscript. It is evident that the majority of the suggestions raised in the previous round have been addressed appropriately, and the overall quality of the manuscript has significantly improved. Nevertheless, I believe that a few additional clarifications and minor refinements would further enhance the clarity and completeness of the study. These points are listed below and can be addressed with minimal effort:

Although the sample collection process is given in detail, information on how many replicates were made in each phenological stage should be added.

Alpha diversity analyses were performed correctly and meaningfully. However, p-values ​​for the results of the statistical test were not clearly given.

It was not stated why the LDA score threshold (LDA > 4) was chosen, I recommend that information be provided.

Author Response

Coments1：Although the sample collection process is given in detail, information on how many replicates were made in each phenological stage should be added.

Response1:Thank you for point this out. We previously described the sampling principles for each phenological period from line 115 to line 118. However, perhaps my language description was not precise enough, causing ambiguity. Now, I have made a few minor changes to this paragraph to make the language description more accurate. We have highlighted it with blue.

Coments2：Although the sample collection process is given in detail, information on how many replicates were made in each phenological stage should be added.

Response2：Thank you for point this out. I have added explicit p-values at line 177 and line 179.

Comments 3: It was not stated why the LDA score threshold (LDA > 4) was chosen, I recommend that information be provided.

Response3：Thank you for point this out. According to past statistical analysis method research, it is believed that through Linear discriminant analysis, when the LDA value is greater than 4, features (genes, pathways or taxa) that represent the differences between two or more biological conditions (or categories) can be identified. These features have statistical significance, biological consistency and effect correlation. I have added this explanation to lines 269-271 and included the reference.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript has been improved following some of my previous recommendations and thus I have no more comments.

Author Response

Comments：The manuscript has been improved following some of my previous recommendations and thus I have no more comments.

Response： We are pleased that the previous revisions align with your suggestions and requirements. Thank you for time and professional guidance.