Description, Identification, and Growth of Ectomycorrhizae in Tuber sinense-Mycorrhized Castanea mollissima Seedlings

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study presents a detailed investigation into the ectomycorrhizal relationship between Tuber sinense (truffle) and Castanea mollissima (chestnut) seedlings. The authors successfully established a controlled greenhouse system to observe and characterize the morphological changes during ectomycorrhizal development over an eight-month period. The integration of resin sectioning with confocal microscopy is a notable strength, providing a detailed view of the symbiotic structures. The study contributes valuable insights into truffle ectomycorrhizae development and offers a methodological foundation for future research in similar plant-fungal systems.

1. The authors acknowledge the limitations of using ITS sequences for truffle identification, particularly the controversy surrounding Tuber sinense and Tuber indicum. They suggest the use of additional molecular markers like LSU and rpb2 to improve reliability. While this is a valid point, the study could be strengthened by including data or at least a more detailed discussion on why these additional markers were not used in this study. Was it due to resource constraints, availability of samples, or a specific focus on ITS?
2. The variability in mycorrhizal colonization rates, even within the same time points (e.g., T3 and T8 in Table 1), is relatively high. The authors should discuss potential reasons for this variability. Could it be due to inconsistencies in the inoculation process, genetic differences among seedlings, or microenvironmental variations within the growth chamber? Addressing this would enhance the robustness of the results.
3. The description of the mycorrhizal model construction using Icy and ggPlantmap is brief. More details on the modeling process would be beneficial. For example, what specific parameters were included in the model? How was the model validated? Including this information would improve the transparency and reproducibility of this aspect of the study.
4. Some of the figure legends (e.g., Figure 2 and 3) contain abbreviations that are explained in the text but could be defined directly in the legend for clarity.
5. The description of the statistical analysis mentions Student's t-test and one-way ANOVA, but it lacks specifics. It would be helpful to specify what data sets were analyzed with each test and what post-hoc tests were used, if any.
6. The terms "mycorrhization rate" and "colonization rate" are used interchangeably. While the meaning is clear, maintaining consistency in terminology would improve the clarity of the manuscript.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

In the manuscript(agriculture-3543584) entitled ‘Description, identification, and growth of ectomycorrhiza during Tuber sinense mycorrhized Castanea mollissima seedlings’ by Wang et al., this manuscript established an exogenous mycorrhizal synthesis system for Chinese chestnut (Castanea mollissima) seedlings by inoculating spore suspension of Chinese tuber fungus (Tuber sinense), and systematically observed the dynamic process of mycorrhizal formation. Combined with the techniques of resin sectioning and laser confocal microscopy, the stages of mycorrhizal morphology development were revealed. The study aims to elucidate the developmental mechanism of truffle mycorrhiza and provide theoretical support for mycorrhizalized seedling cultivation and sustainable cultivation. However, I have some comments that should be addressed by the Authors prior to the publication.

Comments:

1. Introduction, Comparison of literatures on mycorrhizal developmental differences between T. sinense and other Tuber species.
2. It is recommended to add a description of the steps for the preparation of spore suspensions. Secondly, improve the PCR reaction conditions. The sample size should be unspecified in the statistical analysis.
3. In the discussion section, possible reasons for higher colonization rates in this study (e.g., improved inoculation methods) should be addressed.
4. The discussion section should be supplemented with potential mechanisms for differences in mycorrhizal colonization rates .
5. In the conclusion section, the limitations of the study  and future research directions should be clarified.

Author Response

Please see the attachment.

Author Response File: Author Response.docx