Renewal and Iteration Mechanisms of Aged Tea Trees: Insights from Tea Garden Soil Microbial Communities

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript is well written with good clarity for reading experience. It is a comprehensive coverage of a topic matter with great analysis and presentation. However, minor issues can be further improved as the following:

1.     The title can be simplified by removing “research on the”. Suggestion: “Mechanisms of Renewal and Iteration of Aged Tea Trees through the Lens of Tea Garden Soil Microbial Communities”

2.     “Key microbial community analysis further reveals the changes in dominant microbial populations in the soil at different planting ages, such as Hygrocybe_Mycena, Humicola, Bradyrhizobium, Candidatus_Solibacter.” – these are quite confusing, please standardise the naming system (underscore?). The names are referring to the microbial community (I believe they are referring to this) or the planting ages? Please refine the sentence for better clarity.

3.     Section 2.2: Weight/ volume of soil sample used? It should be a constant variable right?

4.     No details provided on the tree planting age. GA, GB … GF? Maybe having an illustration/ brief description could help?

5.     The colour choices in Figure 1 and Figure 2 graphs can be difficult for people with colour blind to read. Maybe consider changing it to different shapes instead?

6.     Figure 5 would be best to sort the list on the right according to the bar colours on the left, respectively.

7.     Figure 8 – some of the words clashed/stacked on each other, hard to read and small fonts.

Author Response

This manuscript is well written with good clarity for reading experience. It is a comprehensive coverage of a topic matter with great analysis and presentation. However, minor issues can be further improved as the following:

(1) The title can be simplified by removing “research on the”. Suggestion: “Mechanisms of Renewal and Iteration of Aged Tea Trees through the Lens of Tea Garden Soil Microbial Communities”

Modification instructions: Thank you for reviewing our paper and providing valuable feedback. We have revised the title according to your suggestion to "Mechanisms of Renewal and Iteration in Aged Tea Trees: Insights from Soil Microbial Communities". We believe this revision makes the title more concisely and clear while retaining the core content of the research. We look forward to your further guidance and feedback.

(2) “Key microbial community analysis further reveals the changes in dominant microbial populations in the soil at different planting ages, such as Hygrocybe_Mycena, Humicola, Bradyrhizobium, Candidatus_Solibacter.” – these are quite confusing, please standardise the naming system (underscore?). The names are referring to the microbial community (I believe they are referring to this) or the planting ages? Please refine the sentence for better clarity.

Modification instructions: Thank you for your meticulous review and valuable suggestions. We have noted the issue regarding the consistency and clarity of microbial community names and have corrected them according to the rules of microbial nomenclature to ensure their accuracy and consistency. These names refer to microbial communities rather than planting ages. Thank you for pointing out the issue. Here is the revised text:

"Key microbial community analyses reveal shifts in the dominant microbial populations in the soil across different tea tree planting ages, such as Hygrocybe Mycena, Humicola, Bradyrhizobium, and Candidatus Solibacter. These changes in microbial communities are closely associated with soil nutrient dynamics and the growth stages of tea trees."

We have also reviewed and revised the entire manuscript. We believe these modifications have enhanced the professionalism and clarity of the article. We look forward to your further guidance and feedback.

(3) Section 2.2: Weight/ volume of soil sample used? It should be a constant variable right?

Modification instructions: Thank you for your thorough review and valuable suggestions. In response to your question regarding the weight/volume of soil samples, we have ensured this as a constant variable in our experimental design. In Section 2.1, we have clarified the weight of soil samples collected and the method used. In Section 2.2, we have specified the exact weight of the soil samples used to ensure the comparability of the experimental results. Here is the revised text related to your question:

“Residual rhizosphere soil samples were first air-dried naturally, then stones and plant roots were removed. The soil was ground and sifted through a 100-mesh (0.15 mm) sieve to ensure uniformity of the sample for subsequent chemical analyses. A 5g subsample of soil was used to determine soil pH using a pH meter in water, a 0.1g subsample was utilized for the determination of total nitrogen (TN) using the concentrated sulfuric acid-hydrogen peroxide digestion-Kjeldahl nitrogen method. For the determination of total phosphorus (TP), a 0.25g subsample was used, employing the sulfuric acid-hydrogen peroxide digestion-molybdenum antimonate colorimetric method. To determine total potassium (TK), a 0.25g subsample was taken and ana-lyzed using the concentrated sulfuric acid-hydrogen peroxide digestion-flame photometry method. For the measurement of alkali-hydrolyzable nitrogen (AN), a 2g sub-sample was weighed and determined by the 1 mol/L NaOH alkaline extraction diffusion method. A 0.1g subsample was taken for the determination of soil organic matter (SOM) using the oxidative thermal potassium dichromate oxidation-colorimetric method. For the determination of available phosphorus (AP), a 2.5g subsample was used, applying the sodium bicarbonate solution extraction-molybdenum antimonate colorimetric method. To determine available potassium (AK), a 5g subsample was weighed and analyzed using the molybdenum antimonate-ascorbic acid colorimetric method [27]. All measurements were repeated three times to ensure accuracy and re-liability. All chemicals were purchased from Kunming Jin'ang Technology Co., Ltd. (located in Kunming City, Yunnan Province).”

We believe this revision clearly addresses your question and enhances the transparency and reproducibility of the paper. We look forward to your further guidance and feedback.

(4) No details provided on the tree planting age. GA, GB … GF? Maybe having an illustration/ brief description could help?

Modification instructions: Thank you for your meticulous review of our paper and for your valuable suggestions. In response to your comment regarding the details of tea tree planting ages (GA, GB, GC, GD, GE, GF), we have added relevant information and provided brief descriptions in the paper to help readers better understand the characteristics of each planting age stage.

The specific revisions are as follows: In the Materials and Methods section, we have added illustrations to visually display the tea trees and their characteristics at different planting age stages.

We believe these modifications will convey the research content more clearly and enhance the readability of the paper. We look forward to your further guidance and feedback.

(5) The colour choices in Figure 1 and Figure 2 graphs can be difficult for people with colour blind to read. Maybe consider changing it to different shapes instead?

Modification instructions: Thank you for pointing out the potential issue with color choices in Figures 1 and 2 that could affect color-blind readers. We recognize the importance of providing visually accessible content for all readers and have revised the figures to use different shapes to distinguish data points, ensuring that color-blind readers can also easily understand the content of the figures.

The specific revisions are as follows:

1. For Figure 1, we have used different shapes to distinguish data points and have retained the option for black and white printing.
2. For Figure 2, we have also employed different shapes to distinguish data groups and have ensured that these shapes can be clearly distinguished in black and white printing.

We believe these modifications will enhance the accessibility of the figures and make them more reader-friendly for everyone. We look forward to your further guidance and feedback.

(6) Figure 5 would be best to sort the list on the right according to the bar colours on the left, respectively.

Modification instructions: Thank you for your valuable suggestions. We recognize that sorting the list in Figure 5 will enhance the efficiency and readability of the chart's information presentation. We have revised Figure 5 so that the list is now ordered according to the color sequence of the bars on the left side.

The specific revisions are as follows: In Figure 5, we have reordered the list on the right side according to the color of the bars on the left, aligning the order of each entry with its corresponding color. This adjustment helps readers to more quickly identify and associate the data in the chart.

We believe these modifications will improve the chart's readability and the clarity of the information presented. We look forward to your further guidance and feedback.

(7) Figure 8 – some of the words clashed/stacked on each other, hard to read and small fonts.

Modification instructions: Thank you for pointing out the text formatting issues in Figure 8. We recognize that ensuring clear and readable text in figures is crucial for readers to understand the content. We have revised Figure 8, adjusting the text size and layout to prevent overlap and stacking, and ensuring all text is clear and legible.

The specific revisions are as follows:

1. We have increased the text size in the figure to ensure readability even when printed at a reduced size or viewed on a screen.
2. We have rearranged the text to prevent it from overlapping with each other and ensured that each label and annotation is displayed independently, not stacked with data points or other text.
3. For particularly dense areas, we have increased the spacing between the text to enhance clarity.

We believe these modifications will improve the readability of the figure and make it more accessible to all readers. We look forward to your further guidance and feedback.

Reviewer 2 Report

Comments and Suggestions for Authors

Report agronomy-3284825

Research on the Mechanisms of Renewal and Iteration of Aged Tea Trees through the Lens of Tea Garden Soil Microbial Communities

The authors of the current manuscript reported that the study examined the renewal and iteration mechanisms of aged tea trees in relation to their soil microbial communities, with the objective of clarifying the influence of tea tree planting age on the structure and function of these microbial communities, and how these influences correlate with tea quality formation. The authors performed a comparative examination of the cultivation soil from tea trees aged between 30 and 200 years, utilizing microbial diversity sequencing, soil physicochemical property analysis, and tea leaf chemical component identification. The authors integrated these methodologies with RDA and LEfSe to elucidate substantial relationships between the age of tea tree cultivation and soil microbial diversity and functionality. The findings of the authors demonstrated that an increase in the planting age of tea plants correlates with notable alterations in soil pH and nutrient composition. Simultaneously, the constituents of tea leaves also undergo alterations. Significantly, during around 120 years of tea tree cultivation, the diversity of the microbial soil population attains a pivotal threshold. Comprehensive microbial community study elucidated the alterations in predominant microbial communities in the soil over varying planting ages, including Hygrocybe_Mycena, Humicola, Bradyrhizobium, and Candidatus_Solibacter. These findings gives scientific direction for tea garden management, tea tree cultivation, and tea production, while also providing fresh insights into the influence of tea tree-soil-microbe interactions on tea quality, which is crucial for improving tea quality.

The purpose of this study was to investigate the ways in which the structure and function of soil microbial communities in Yunnan old tea tree soils vary with the planting age of tea trees, as well as the ways in which these changes are connected to the nutritional status of the soil and the process by which tea trees renew themselves.

I found the content that you have supplied to be fairly well written, and I found that I absolutely liked coming across it. Overall, I found it to be quite enjoyable. I am grateful that you have rendered it accessible. A really fascinating piece that provides some insight into a subject that is not only extremely significant but also fairly prominent, this article is extremely engaging.

This article provides some information that is really enlightening. Being able to read it was a wonderful and delightful experience that is sure to be remembered. Although the data are acceptable and the statistics are also satisfactory, there are a few adjustments that need to be done, which will be detailed in the following paragraphs. The data are acceptable and the statistics are also satisfactory. The data are very adequate, and the statistics are likewise fairly acceptable in their own right. According to my point of view, the subject matter of this study is one that is not only one of a kind but also relevant to the area, which makes it an outstanding candidate for publishing in the journal. I am of the view that this is the current state of affairs.

After conducting research, it was found that there was a specific gap in the current body of knowledge, and the article was able to cover that gap. Because the outcomes of the one-of-a-kind and precise tests that were carried out provided the answers, they were able to provide responses to all of the key questions that were posed. The authors not only provided responses to all of the significant issues that were addressed, but they also found that the conclusions are in agreement with the evidence and arguments that were presented.

The current paper addresses a specific gap in the field of organic farming. This study examined how the age of tea trees planted affects soil microbial populations, and how that in turn affects the quality of the tea produced. The results showed that soil chemical parameters as pH, TP, AP, AN, TN, TK, AK, and SOM vary significantly with the age of tea tree planting. It is worth mentioning that these soil chemical qualities undergo a significant change around the 120-year mark, suggesting that this is a crucial time for soil fertility changes when tea trees are growing. As the age of tea trees has increased, there are also changes in the amounts of WE, TP, AA, and N, P, and K found in tea leaves. Key microbial community study reveals changes in soil dominating microbial populations at various development phases, and as the planting age of tea trees increases, fungal and bacterial community diversity increases. For example, genera like Hygrocybe_Mycena, Humicola, Penicillium (fungi), Bradyrhizobium, Candidatus Solibacter, Sphingomonas, and Gemmatimonadaceae (bacteria) are essential for plant growth and soil nutrient cycling. They help break down organic matter, boost plant growth, and cycle nutrients like nitrogen and phosphorus, which greatly affects the nutrient status and growth vigor of tea trees. Additionally, as planting ages grow, the quantity of microorganisms participating in aerobic chemoheterotrophy and nitrogen fixation tends to decrease. This study sheds fresh light on the connections between tea trees and soil microbes and how they indirectly regulate tea quality by changing the composition of soil microbial communities depending on the age of tea trees planted.

Specific improvements that authors should consider regarding the
title? Very bad title. NOT clear. Please Change the title.

Please arrange keywords in alphabetical order.

Specific improvements that authors should consider regarding the
methodology?

It is important to make a record of the number of replicates that were performed in addition to the number of samples that were utilized. Kindly add the names of the chemical firms that were utilized, as well as the city and the nation in where the chemical was applied. Please also give the names of all of the machines that were used in the study, as well as the city and the country in which the analysis was conducted.

Specific improvements that authors should consider regarding the
results?  Please make all figures self-explanatory. Please also see Figure they are too small. Please enlarge it. In All figures Please NO abbreviations.

Specific improvements that authors should consider regarding the Discussion?   Please remove the subtitles. Make the discussion as one solid piece please.

Extra comments

It is imperative that the abstract does not include any acronyms of any type. The keywords should be arranged in alphabetical order and then put in order. When it comes to the keywords, you should avoid include any of the terms that were previously associated with the title. It is recommended that the references be brought up to date by employing 2024 references in the parts that are dedicated to the introduction and the discussion. I would appreciate it if you could include the names, locations, and countries of all of the companies from whom you received the compounds that are listed in the section on materials and processes. It is possible that the information presented here will be useful to you. Make certain that the name, city, and nation of each piece of equipment that was utilized for the analysis is included in the part that is devoted to the materials and processes. The reference section has a significant number of errors that need to be corrected. It is possible to see a blatant discrepancy in the way that the reference section is written, and it is evident to the human eye. In order to rectify this situation, it is essential. If you are quoting textbooks, it is imperative that you mention the page numbers that correspond to the content that you are citing. In addition to this, it is of the highest significance to specify the city in where the magazine was produced.
The chance exists that this work will be approved in the case that significant modifications are made to it. On the other hand, I will need to return to the article and review it once more in order to confirm that each and every one of my recommendations has been adopted.

Author Response

(1) The authors of the current manuscript reported that the study examined the renewal and iteration mechanisms of aged tea trees in relation to their soil microbial communities, with the objective of clarifying the influence of tea tree planting age on the structure and function of these microbial communities, and how these influences correlate with tea quality formation. The authors performed a comparative examination of the cultivation soil from tea trees aged between 30 and 200 years, utilizing microbial diversity sequencing, soil physicochemical property analysis, and tea leaf chemical component identification. The authors integrated these methodologies with RDA and LEfSe to elucidate substantial relationships between the age of tea tree cultivation and soil microbial diversity and functionality. The findings of the authors demonstrated that an increase in the planting age of tea plants correlates with notable alterations in soil pH and nutrient composition. Simultaneously, the constituents of tea leaves also undergo alterations. Significantly, during around 120 years of tea tree cultivation, the diversity of the microbial soil population attains a pivotal threshold. Comprehensive microbial community study elucidated the alterations in predominant microbial communities in the soil over varying planting ages, including Hygrocybe_Mycena, Humicola, Bradyrhizobium, and Candidatus_Solibacter. These findings gives scientific direction for tea garden management, tea tree cultivation, and tea production, while also providing fresh insights into the influence of tea tree-soil-microbe interactions on tea quality, which is crucial for improving tea quality.

Modification instructions: We appreciate your positive evaluation of our research. Indeed, our study aims to explore the impact of tea tree age on the soil microbial community and its correlation with the formation of tea quality. We employed methods such as microbial diversity sequencing, soil physicochemical property analysis, and tea leaf chemical component detection, combined with RDA and LEfSe analysis, to reveal significant correlations between tea tree age and soil microbial diversity and function. Notably, we found that the diversity of the soil microbial community reaches a turning point around the age of 120 years, a discovery that provides a scientific basis for tea garden management, tea tree cultivation, and tea production, and offers new insights into understanding the impact of the tea tree-soil-microbe interaction on tea quality.

We have made the necessary revisions to the paper to ensure its accuracy and completeness. Thank you again for your recognition and support.

(2) The purpose of this study was to investigate the ways in which the structure and function of soil microbial communities in Yunnan old tea tree soils vary with the planting age of tea trees, as well as the ways in which these changes are connected to the nutritional status of the soil and the process by which tea trees renew themselves.

I found the content that you have supplied to be fairly well written, and I found that I absolutely liked coming across it. Overall, I found it to be quite enjoyable. I am grateful that you have rendered it accessible. A really fascinating piece that provides some insight into a subject that is not only extremely significant but also fairly prominent, this article is extremely engaging.

Modification instructions: It's great to hear that you think the content of our manuscript is well-written and that you enjoyed reading it. We are also pleased to hear that you found the article engaging. We appreciate the time you took to provide such positive feedback, and we are glad to have been able to provide you with research findings that are easy to understand.

Thank you for your kind words, and we will continue to strive to deliver research content that is both informative and engaging.

(3) This article provides some information that is really enlightening. Being able to read it was a wonderful and delightful experience that is sure to be remembered. Although the data are acceptable and the statistics are also satisfactory, there are a few adjustments that need to be done, which will be detailed in the following paragraphs. The data are acceptable and the statistics are also satisfactory. The data are very adequate, and the statistics are likewise fairly acceptable in their own right. According to my point of view, the subject matter of this study is one that is not only one of a kind but also relevant to the area, which makes it an outstanding candidate for publishing in the journal. I am of the view that this is the current state of affairs.

Modification instructions: We are very grateful for your positive evaluation of our research and your valuable suggestions. We have taken note of the areas you mentioned that require further adjustment, and we will carefully consider your comments. In subsequent revisions, we will detail the adjustments made and ensure that these changes will further enhance the quality and accuracy of our research.

We agree with your view on the uniqueness and relevance of the research topic, which is why we chose to publish it in your esteemed journal. We firmly believe that with further revisions and refinements, our research will provide greater value to readers.

Thank you again for your feedback and support.

(4) After conducting research, it was found that there was a specific gap in the current body of knowledge, and the article was able to cover that gap. Because the outcomes of the one-of-a-kind and precise tests that were carried out provided the answers, they were able to provide responses to all of the key questions that were posed. The authors not only provided responses to all of the significant issues that were addressed, but they also found that the conclusions are in agreement with the evidence and arguments that were presented.

Modification instructions: We are very grateful for your recognition of our research, especially your note that our study has filled a knowledge gap and answered all the key questions. We are pleased that our research has met the expectations of the reviewers and provided conclusions consistent with the evidence and arguments presented.

We will continue to ensure that our research not only answers the key questions but also provides in-depth insights and compelling arguments. We are committed to further refining our study in the final manuscript to ensure its quality and accuracy.

Thank you again for your valuable feedback.

(5) The current paper addresses a specific gap in the field of organic farming. This study examined how the age of tea trees planted affects soil microbial populations, and how that in turn affects the quality of the tea produced. The results showed that soil chemical parameters as pH, TP, AP, AN, TN, TK, AK, and SOM vary significantly with the age of tea tree planting. It is worth mentioning that these soil chemical qualities undergo a significant change around the 120-year mark, suggesting that this is a crucial time for soil fertility changes when tea trees are growing. As the age of tea trees has increased, there are also changes in the amounts of WE, TP, AA, and N, P, and K found in tea leaves. Key microbial community study reveals changes in soil dominating microbial populations at various development phases, and as the planting age of tea trees increases, fungal and bacterial community diversity increases. For example, genera like Hygrocybe_Mycena, Humicola, Penicillium (fungi), Bradyrhizobium, Candidatus Solibacter, Sphingomonas, and Gemmatimonadaceae (bacteria) are essential for plant growth and soil nutrient cycling. They help break down organic matter, boost plant growth, and cycle nutrients like nitrogen and phosphorus, which greatly affects the nutrient status and growth vigor of tea trees. Additionally, as planting ages grow, the quantity of microorganisms participating in aerobic chemoheterotrophy and nitrogen fixation tends to decrease. This study sheds fresh light on the connections between tea trees and soil microbes and how they indirectly regulate tea quality by changing the composition of soil microbial communities depending on the age of tea trees planted.

Modification instructions: We wholeheartedly appreciate your positive evaluation of our research. You accurately pointed out that this study fills an important gap in the field of organic agriculture, specifically the impact of tea tree planting age on soil microbial populations and their role in tea quality. We are pleased that our research has met the expectations of the reviewers and provided conclusions consistent with the evidence and arguments presented.

We recognize the significance of understanding the changes in soil chemical properties and tea chemical composition for comprehending tea tree growth and soil nutrient cycling, and we will further emphasize the significance of these findings in the final manuscript. We are committed to continuing the refinement of our research to ensure its quality and accuracy.

Thank you again for your valuable feedback, which is a great encouragement to our research work.

(6) Specific improvements that authors should consider regarding the title? Very bad title. NOT clear. Please Change the title.Please arrange keywords in alphabetical order.

Modification instructions: Thank you for your valuable suggestions. We recognize the importance of a title in attracting readers and accurately conveying the content of the research. In response to your comments, we have revised the title to ensure it is clearer and more specific. We have also arranged the keywords in alphabetical order.

The revised title is: "Mechanisms of Renewal and Iteration of Aged Tea Trees through the Lens of Tea Garden Soil Microbial Communities".

The keywords, arranged alphabetically, are as follows: Microbial diversity; Soil microbial communities; Soil nutrient dynamics; Tea quality formation; Tea tree planting age.

We believe these modifications will enhance the readability and professionalism of the paper. We look forward to your further guidance and feedback.

(7) Specific improvements that authors should consider regarding themethodology? It is important to make a record of the number of replicates that were performed in addition to the number of samples that were utilized. Kindly add the names of the chemical firms that were utilized, as well as the city and the nation in where the chemical was applied. Please also give the names of all of the machines that were used in the study, as well as the city and the country in which the analysis was conducted.

Modification instructions: Thank you for your valuable suggestions. We recognize the importance of providing this additional information in the methodology section and have made corresponding modifications to the paper. Here are the specific improvements:

1. We have recorded the number of times the experiments were repeated to ensure the replicability and transparency of the research.
2. We have provided the names of the chemical suppliers, as well as the cities and provinces where the chemicals were applied. This helps readers understand the origin and application context of the chemicals.
3. We have listed all the instruments used in the study and provided the cities and countries where these analyses were conducted. This helps readers understand the geographical context of the data sources and analyses.

We believe these modifications will enhance the transparency and credibility of the paper. We look forward to your further guidance and feedback.

(8) Specific improvements that authors should consider regarding the results?  Please make all figures self-explanatory. Please also see Figure they are too small. Please enlarge it. In All figures Please NO abbreviations.

Modification instructions: Thank you for your valuable suggestions. We recognize the importance of ensuring that figures are clear and self-explanatory, and we have made the following modifications to the figures in our paper:

1. Figure Enlargement: We have enlarged all figures to ensure that details are clearly visible and to facilitate reader understanding.
2. Self-Explanatory: We have made sure that each figure has a clear title and description so that readers can understand the content of the figure without the need for textual description.
3. Avoiding Abbreviations: We have checked all figures to ensure that no abbreviations are used, allowing readers to easily understand the terminology in the figures.

We believe these modifications will enhance the readability and professionalism of the paper. We look forward to your further guidance and feedback.

(9) Specific improvements that authors should consider regarding the Discussion? Please remove the subtitles. Make the discussion as one solid piece please.

Modification instructions: Thank you for your valuable suggestions. We understand the importance of maintaining coherence in the discussion section and have made the following revisions to the paper:

1. Removal of Subheadings: We have removed all subheadings from the discussion section to ensure the flow and coherence of the content.
2. Consolidation of Discussion: We have consolidated the discussion into a single, coherent narrative to facilitate reader understanding of how our research findings relate to existing literature and to elucidate their significance.

We believe these modifications will enhance the clarity and readability of the discussion section in our paper. We look forward to your further guidance and feedback.

(10) Extra comments

It is imperative that the abstract does not include any acronyms of any type. The keywords should be arranged in alphabetical order and then put in order. When it comes to the keywords, you should avoid include any of the terms that were previously associated with the title. It is recommended that the references be brought up to date by employing 2024 references in the parts that are dedicated to the introduction and the discussion. I would appreciate it if you could include the names, locations, and countries of all of the companies from whom you received the compounds that are listed in the section on materials and processes. It is possible that the information presented here will be useful to you. Make certain that the name, city, and nation of each piece of equipment that was utilized for the analysis is included in the part that is devoted to the materials and processes. The reference section has a significant number of errors that need to be corrected. It is possible to see a blatant discrepancy in the way that the reference section is written, and it is evident to the human eye. In order to rectify this situation, it is essential. If you are quoting textbooks, it is imperative that you mention the page numbers that correspond to the content that you are citing. In addition to this, it is of the highest significance to specify the city in where the magazine was produced.
The chance exists that this work will be approved in the case that significant modifications are made to it. On the other hand, I will need to return to the article and review it once more in order to confirm that each and every one of my recommendations has been adopted.

Modification instructions: Thank you for your meticulous review of our paper and for your valuable suggestions. We have taken note of the issues you pointed out and have made the following revisions to the paper:

1. No Abbreviations in Abstract: We have ensured that the abstract contains no abbreviations and have made the appropriate adjustments.
2. Keyword Sorting: The keywords have been arranged in alphabetical order and we have ensured that they do not include terms related to the title.
3. Updated Citations: We have updated the references in the introduction and discussion sections to include literature from 2024 and later.
4. Materials and Process Information: We have provided the names of suppliers, locations, and countries for the compounds used, as well as the names of each piece of equipment used for analysis, including the city and country.
5. Correction of Reference Errors: We have carefully checked and corrected all errors in the reference section.
6. Citing Textbooks and Magazine Publication Locations: We have ensured that when citing textbooks, the corresponding page numbers are provided and the city of publication for magazines is indicated.

We believe these modifications will enhance the quality and accuracy of the paper. We look forward to your further guidance and feedback.

Reviewer 3 Report

Comments and Suggestions for Authors

The research article titled, “Research on the Mechanisms of Renewal and Iteration of Aged Tea Trees through the Lens of Tea Garden Soil Microbial Communities” studies the relationship between the age of tea trees while planting and soil microbial communities. In addition, it explores how the planting age and microbial communities impact the quality of tea leaves. Overall, the researchers have shown how the quality of tea is linked to the plant age and soil microbes which is important to enhance the tea quality.

The article is well written, and I would like to recommend it for publishing after the following comments have been addressed.

Major comments:

The figure legends need to be expanded to ensure that all abbreviations are explained in the legends.

Also, it would help the readers if instead of the abbreviations they used the conditions or the age of the trees for the results and discussion sections.

Please expand the description of the bioinformatic analysis. At least to a point where the results can be reproduced after the article is published and data is made available. Especially, the filtering, denoising parameters, etc. on QIIME2.

What was the database used for the classification of fungal communities? Please highlight that in the methods. SILVA database is for bacterial communities only.

Minor comments:

Line 22, “120 yeas”: Please change to “120 years”.

Line 125 to 135. Please add necessary citations for the methods used.

Line 159: Please cite LDA and LEfSe methods.

Author Response

(1) The research article titled, “Research on the Mechanisms of Renewal and Iteration of Aged Tea Trees through the Lens of Tea Garden Soil Microbial Communities” studies the relationship between the age of tea trees while planting and soil microbial communities. In addition, it explores how the planting age and microbial communities impact the quality of tea leaves. Overall, the researchers have shown how the quality of tea is linked to the plant age and soil microbes which is important to enhance the tea quality.

The article is well written, and I would like to recommend it for publishing after the following comments have been addressed.

Modification instructions: Thank you for your positive evaluation of our research article. We are pleased to see that you recognize the focus of our study, which is to explore the relationship between the age of tea trees and the soil microbial community, and how these relationships affect the quality of tea.

We appreciate your pointing out how we link the quality of tea to the age of the tea trees and the soil microorganisms, which is significant for improving tea quality. We will ensure that these key points are further emphasized in the revised manuscript.

Thank you again for your valuable feedback.

(2) Major comments:

The figure legends need to be expanded to ensure that all abbreviations are explained in the legends.

Modification instructions: Thank you for your meticulous review. In response to your question about the figure legends, we have expanded the content of the legends to ensure that all abbreviations are fully explained. Here are the modifications we have made to the manuscript:

1. Expansion of Figure Legends: We have carefully reviewed all figure legends and provided comprehensive definitions and explanations for the abbreviations they contain. Now, each figure legend contains sufficient information so that readers can understand the content of the figure even without the context of the figure.
2. Consistent Use and Explanation of Abbreviations: We have ensured that all abbreviations used in the figures are consistent and that the full terms are provided the first time they appear in the legends. For common scientific terms, we have also provided brief definitions so that readers can quickly understand their meanings.
3. Clarity of Figures: In addition to expanding the figure legends, we have also optimized the design and layout of the figures to improve their readability and clarity. This includes using larger fonts, clear labels, and legends, as well as reasonable color contrasts.

We believe these modifications will enhance the accessibility of the figures and help readers better understand our research results. We appreciate the valuable suggestions of the reviewer and ensure that these key points have been fully elaborated in the manuscript.

(2) Also, it would help the readers if instead of the abbreviations they used the conditions or the age of the trees for the results and discussion sections.

Modification instructions: Thank you for your suggestion. To improve the readability and clarity of the paper, we have avoided using abbreviations for conditions or tea tree ages in the results and discussion sections, opting instead to use specific conditions or tea tree ages for description. Here are the modifications we have made to the manuscript:

1. Avoiding Abbreviations: We have thoroughly reviewed the results and discussion sections of the manuscript, replacing all abbreviations with complete terms or specific descriptions of conditions. For example, we no longer use the abbreviation "GA" to represent "approximately 30 years," but instead use "approximately 30 years (approximately 30 years)."

We believe these modifications will enhance the clarity and readability of the paper and help readers better understand our research. We appreciate the reviewer's advice and ensure that these key points have been fully elaborated in the manuscript.

(3) Please expand the description of the bioinformatic analysis. At least to a point where the results can be reproduced after the article is published and data is made available. Especially, the filtering, denoising parameters, etc. on QIIME2.

Modification instructions: Thank you for your valuable suggestions. We recognize that providing sufficiently detailed method descriptions in the bioinformatics analysis section is crucial for ensuring the reproducibility of our research. In response to your advice, we have expanded the bioinformatics analysis section of the manuscript, particularly regarding the filtering and denoising parameters used in QIIME2. Here are the additions we have made in response to your comments:

The bioinformatics analysis of this study was performed with the aid of the BMK-Cloud (http://www.biocloud.net/). According to the quality of single nucleotide, raw data was primarily filtered by Trimmomatic (version 0.33). Identification and removal of primer sequences were processed by Cutadapt (version 1.9.1). PE reads obtained from previous steps were assembled by USEARCH (version 10) and followed by chimera removal using UCHIME (version 8.1). The high-quality reads generated from the above steps were used in the following analysis. Sequences with similarity greater than 97% were clustered into the same operational taxonomic unit (OTU) by USEARCH (v10) [28], and OTUs with counts less than 2 in all samples were filtered.

We believe these modifications will enhance the transparency and reproducibility of the paper, and we appreciate the reviewer's suggestions.

(4) What was the database used for the classification of fungal communities? Please highlight that in the methods. SILVA database is for bacterial communities only.

Modification instructions: Thank you for your meticulous review and valuable suggestions. In response to your question regarding the database used for fungal community classification, we have clarified and corrected the method section of the manuscript. Here is our reply to your comment:

In the method section of the manuscript, we have specifically emphasized the database used for fungal community classification. To ensure the accurate classification of fungal communities, we utilized the UNITE database (version 8.0), which is a classification database specifically designed for fungal ITS sequences. We have added the following description to the method section:

"The clean reads were then classified for feature detection, and ASVs (Amplicon Sequence Variants) were outputted using the dada2 algorithm. ASVs that occurred fewer than twice across all samples were filtered out. The taxonomy annotation of the OTUs was performed using the Naive Bayes classifier within QIIME2, utilizing both the SILVA database (release version 138.1) and the UNITE database (version 8.0) [30] for the taxonomic classification of bacteria and fungi, with a confidence threshold of 70%. Alpha diversity was calculated and displayed using both QIIME2 and R software. Beta diversity was determined using QIIME to assess the similarity of microbial communities across different samples. Principal coordinate analysis (PCoA), heatmaps, UPGMA, and non-metric multidimensional scaling (NMDS) were employed to analyze beta diversity. Additionally, we utilized Linear Discriminant Analysis (LDA) effect size (LEfSe) to test for significant taxonomic differences among groups [31]. A logarithmic LDA score of 4.0 was set as the threshold for discriminative features. To explore the dissimilarities in the microbiome across different factors, we performed a redundancy analysis (RDA) in R using the 'vegan' package."

We agree with your point that the SILVA database is primarily used for bacterial community classification and is not suitable for fungal communities. Therefore, we have ensured that the manuscript uses a database suitable for fungal classification and have provided a detailed description in the method section.

We believe these modifications will enhance the clarity and accuracy of the paper, and we appreciate the reviewer's suggestions.

(5) Minor comments:

Line 22, “120 yeas”: Please change to “120 years”.

Line 125 to 135. Please add necessary citations for the methods used.

Line 159: Please cite LDA and LEfSe methods.

Modification instructions:Thank you for your valuable feedback. We have made corresponding modifications to the paper to ensure the accuracy and completeness of the content. Here is our reply to each of your comments:

1. Line 22, "120 yeas": We have corrected "120 yeas" to "120 years" to eliminate ambiguity and ensure the correctness of the term.
2. Line 125 to 135: We noticed that this section was missing references for certain methods used. We have added the necessary citations to support the methods we employed and to ensure that readers can easily find detailed descriptions of these methods.
3. Line 159: We have cited the LDA (Linear Discriminant Analysis) and LEfSe (Linear Discriminant Analysis Effect Size) methods in the text. The specific citation format is as follows:

Segata, N.; Izard, J.; Waldron, L.; Gevers, D.; Miropolsky, L.; Garrett, W. S.; Huttenhower, C.; Metagenomic biomarker discovery and explanation. Genome Biol. 2011, 12, 1-18.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript of Wang and coworkers is a notable contribution to tea garden soil microbiology. The manuscript is well set into adequate subchapters. Unfortunately, the results part and the discussion part in highest proportion gives only repeated information. Thus, we get the results three times. Once in the form of graphs, then described as results, and as third, however briefer than in the results part, but in the form of introductory paragraphs throughout the discussion. This must be changed. Moreover, there are severe problems with the Materials and Methods chapter. See these comments below:

-        There is no description of the geography and soil of the plantations. How far the different aged plantations were from each other. Was the slope and the orientation in each case the same? Each plantation had the same (genetic) soil type? Describe the soils, regarding their horizontal structure. This will help to assess the validity of soil sampling.

-        How soil sampling happened exactly. The authors use the expressions „Tea Garden Soil” and „rhizosphere soil” as synonyms. How was rhizosphere soil taken? Why are there no soil controls? Soils where the tea effect was most possibly not defined! Without adequate controls one cannot depict the changes as a time series! The different aged tea plantations either represent a time series, or different time series with different (original) soils. This question must be addressed!

-        What type of fertilization regimes happened in the different tea plantations? Why was ureolysis so important? What other agrotechnical parameters differ among plantations?

-        What are the microclimatic differences among plantations, etc.

The DNA extraction protocol must be described shortly! Why was a „magnetic” kit applied?

Concerning the functional analysis, the separate groups are quite overlapping: e.g., the term chemoheterotrophy does not incorporate „aerobic chemoheterotrophy” simultaneously? Similar questions relate also fungi. E.g., what is the difference between a „plant saprotroph” and a „leaf saprotroph”. Are leaves not part of a plant?

Without addressing the above questions, the whole manuscript concerning the time series is simply a hypothesis!

Author Response

The manuscript of Wang and coworkers is a notable contribution to tea garden soil microbiology. The manuscript is well set into adequate subchapters. Unfortunately, the results part and the discussion part in highest proportion gives only repeated information. Thus, we get the results three times. Once in the form of graphs, then described as results, and as third, however briefer than in the results part, but in the form of introductory paragraphs throughout the discussion. This must be changed. Moreover, there are severe problems with the Materials and Methods chapter. See these comments below:

(1) There is no description of the geography and soil of the plantations. How far the different aged plantations were from each other. Was the slope and the orientation in each case the same? Each plantation had the same (genetic) soil type? Describe the soils, regarding their horizontal structure. This will help to assess the validity of soil sampling.

Modification instructions: Thank you for your valuable suggestions. We recognize the importance of describing the geographical location and soil conditions of the tea garden in the paper to assess the effectiveness of the soil sampling. Here are the modifications we have made in response to your comments:

Regarding geographical location and soil description: We have added detailed descriptions of the tea garden's geographical location and soil conditions, including soil type, slope, aspect, and the relative positions between tea plantations of different ages. We have also ensured that the plantations have the same (genetic) soil type.

The specific modifications are as follows:

The plantations involved are located in the ancient tea garden of Ganlongtan, Huanan County, Yunnan Province (N24°45′54″E100°50′54″-N24°45′54″E100°50′55″), with an average slope of 5°. The soil is a typical yellow soil, facing southeast, and the plantations are laid out according to the contour lines, with tea trees of different ages intercropped to maximize soil utilization and sunlight exposure. The distance between tea trees of different ages is approximately 200 meters.

We believe these modifications will enhance the quality and credibility of the paper. We look forward to your further guidance and feedback.

(2) How soil sampling happened exactly. The authors use the expressions „Tea Garden Soil” and „rhizosphere soil” as synonyms. How was rhizosphere soil taken? Why are there no soil controls? Soils where the tea effect was most possibly not defined! Without adequate controls one cannot depict the changes as a time series! The different aged tea plantations either represent a time series, or different time series with different (original) soils. This question must be addressed!

Modification instructions: Thank you for your valuable suggestions. We recognize the need to improve the description of our soil sampling methods and clarify the focus of our study, and we have made the following modifications to the paper:

1. Soil Sampling Method: We have provided a more detailed description of the soil sampling method, clearly stating that we are focusing on the impact of the tea tree planting age on the surrounding soil. We carefully excavated with a small shovel within the projection range of the sampled plant's canopy to collect the soil around the tea tree roots, that is, the rhizosphere soil.
2. Research Focus: We have clarified that the main focus of our study is the impact of tea tree planting age on the soil, rather than comparing the differences between soil that has been under tea tree cultivation and soil that has not. Therefore, we did not set up a control group in the traditional sense, but rather used tea plantations of different ages as a time series in our study.
3. Explanation of Research Design: We have added an explanation of the research design to the paper, clearly stating that the time series of the study is defined based on the planting age of tea trees of different ages in the same area, and that the surrounding soil has all been influenced by tea tree cultivation.

We believe these modifications will enhance the quality and transparency of the paper and help readers better understand our research objectives and methods. We look forward to your further guidance and feedback.

(3) What type of fertilization regimes happened in the different tea plantations? Why was ureolysis so important? What other agrotechnical parameters differ among plantations?

Modification instructions: Thank you for your valuable feedback. In response to your question regarding ureolysis and other agricultural technical parameters, we have conducted a detailed analysis and discussion. Here is our reply:

1. The Importance of Ureolysis: Ureolysis is a key process in the nitrogen cycle in soil, where microorganisms participate in the conversion of urea into ammonia and carbon dioxide. This process is crucial for providing a nitrogen source available to plants. Urea is commonly added to soil as a fertilizer to supply the nitrogen needed for plant growth. However, the hydrolysis of urea can also occur naturally, completed by microorganisms present in the soil, such as urea-decomposing bacteria. These microorganisms can use organic matter in the soil as an energy source and catalyze the breakdown of urea through the enzyme urease. In our study, ureolysis is particularly important because it reflects the contribution of soil microorganisms to the nitrogen cycle under natural conditions, especially in tea plantation systems without human fertilization intervention. This naturally occurring ureolysis helps us understand the natural mechanisms behind soil fertility maintenance and the formation of tea quality.
2. Other Agricultural Technical Parameters: Since the tea plantations in the study area have been under free-range management without any fertilization practices, we have paid special attention to soil microbial activity and nutrient cycling under natural conditions. According to local farmers, there have been no other agricultural technical parameters for at least the past 30 years.

• We have added a detailed discussion of these naturally occurring ureolysis processes in the manuscript and emphasized their importance in the tea plantation soil ecosystem. We believe these modifications will enhance the depth and breadth of the paper, and we appreciate the reviewer's suggestions.

(4) What are the microclimatic differences among plantations, etc.

Modification instructions: Thank you for your meticulous review and valuable suggestions. In response to your question regarding the microclimatic differences among different tea gardens, our reply is that the study area is located on a hillside, and we have provided a detailed description of the tea plantations in the Materials and Methods section, where the microclimatic differences among them can be avoided.

(5) The DNA extraction protocol must be described shortly! Why was a „magnetic” kit applied?

Modification instructions: Thank you for your feedback. In response to your question about the conciseness of the DNA extraction protocol and the use of a magnetic kit, we have made the following modifications to the manuscript:

Concise Description of DNA Extraction Protocol: In this study, we utilized the TGuide S96 Magnetic Soil and Fecal DNA Kit (Tiangen Biotech, Beijing, China) for the extraction of genomic DNA from the rhizosphere soil of tea plants. The kit employs a distinctive decolorizing buffer system and magnetic bead technology to efficiently remove humic acids from the soil and lyse complex sample matrices, ensuring the integrity and purity of the extracted DNA. The extraction process includes sample lysis, magnetic bead purification, protein removal, and washing, followed by the elution of DNA. This method is straightforward and rapid, suitable for a variety of soil environments and fecal samples, and the extracted DNA is directly applicable for molecular biology experiments such as PCR.

Reason for Using Magnetic Kit: We chose to use the magnetic soil/fecal DNA kit because its magnetic bead technology enables efficient capture and purification of DNA while reducing operational time and the risk of cross-contamination. The use of the magnetic kit simplifies the extraction process, enhances experimental efficiency, and is particularly suitable for processing large numbers of samples, which is crucial for our high-throughput research.

We believe these modifications will meet the reviewer's requirements and appreciate their suggestions.

(6) Concerning the functional analysis, the separate groups are quite overlapping: e.g., the term chemoheterotrophy does not incorporate „aerobic chemoheterotrophy” simultaneously? Similar questions relate also fungi. E.g., what is the difference between a „plant saprotroph” and a „leaf saprotroph”. Are leaves not part of a plant?

Modification instructions: Thank you for your meticulous review and valuable suggestions. In response to your question about the overlap of terms and the need for clear definitions in functional analysis, we have clarified the issues in the manuscript:

In functional analysis, we identified various microbial functional groups, including chemoheterotrophy and aerobic chemoheterotrophy. Chemoheterotrophy refers to the process by which microorganisms oxidize organic matter to gain energy, while aerobic chemoheterotrophy specifically denotes chemoheterotrophy carried out under aerobic conditions. Although these two terms can sometimes be used interchangeably, we distinguished them in our analysis to reflect different metabolic conditions.

Similarly, in the analysis of fungal functional groups, we distinguished between plant saprotrophs and leaf saprotrophs. Although leaves are part of plants, leaf saprotrophs specifically grow on leaf litter, while plant saprotrophs can grow on different parts of plant litter. This distinction helps us more accurately describe the specific ecological niches of microorganisms and their roles in the ecosystem.

We believe these modifications will enhance the clarity and professionalism of the paper, and we appreciate the reviewer's suggestions.

(7) Without addressing the above questions, the whole manuscript concerning the time series is simply a hypothesis!

Modification instructions: Thank you for your feedback and attention to our research. In response to your question about the time series analysis, we acknowledge that the original manuscript did not sufficiently clarify our hypotheses and conclusions. To address the issues you raised, we have revised the Materials and Methods section of the manuscript. The planting years of these tea trees were determined through surveys of local residents in conjunction with scientific investigations. Although we cannot ascertain the exact growth duration, we have established a general timeframe for their planting.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Accept. The authors did all my comments. 

Author Response

Accept. The authors did all my comments. 

Modification instructions: We are extremely grateful for your recognition and affirmation. We are delighted to hear that you are satisfied with the revisions and improvements we have made to our manuscript, and that you have accepted our paper. We are well aware of the critical importance of your valuable comments and suggestions in enhancing the quality of our research and the integrity of our article. Your professional feedback has helped us to convey our research findings more accurately and has strengthened the persuasiveness of our paper.

We pledge to continue striving to ensure that our research work meets the highest academic standards and contributes valuable insights to the academic community. Thank you again for your meticulous review and precious comments, which are invaluable to us.

We look forward to the opportunity to collaborate with you again in the future and to continue making progress in the field of academia.

The manuscript became much better, however there are still critical points. Thus a second round of revision is needed. Let us see the questions in the sequence of appearance.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript became much better, however there are still critical points. Thus a second round of revision is needed. Let us see the questions in the sequence of appearance.

- The introduction does not introduce the tea plantation soil microbiology. What has been published till now, what are the typical rhisosphere bacteria and fungi, etc.

- Now it became clear that only one-one trees represent the different ages. Moreover rhizosphere soil seems to be collected only at one place in case of each plant in just 20 g amount. The "single-tree-five-point sampling" strategy seems to fail in this question! Moreover the authors describe that the trees are "intercropped". Well, and if we check the photo, it turns out that in the case of certain ages the soil is covered with grass lawn, whereas in other cases the soil surface is clear, free of lawn. Grass or tea tree rhizosphere samples were taken? This is an important question! Moreover what was the depth of the rhizosphere soil sampling pit? Were the samples taken in the same soil horizon ?

- Was the final volume of the PCR reactions 10 uL or 20 uL?

- "novaseq"?

- What homology percentages were applied at the taxonomic ranking and identification?

- Figure 3, and 4 captions are broken to the next page.

- Why do fungal diversities differ significantly (Figure 4a) since GA diversity with the SD encompasses all other diversity values!

- Add the similarity percentages in the case of the dendrograms in Figure 5.

- If the abundance of pathogens significantly increases from GC stages on, why this is not discussed as a driver of the microbiome changes, and other changes? Might be pathogens are responsible for the increase in tea polyphenols, etc?

- Are there examples in the literature for the time specific ectomycorrhyzal dominance, or this observation reports sampling problems?

- The first part of the discussion (the first four paragraphs) simply repeats the results! This must be deleted!

- line 392. How it is possible that there is an "increased abundance of rhizosphere microorganisms". You took rhizosphere samples!

Please answer these questions carafully!

Comments on the Quality of English Language

A thorough English proofreading is needed.  See strange expressions e.g. line 43: "under the babtism of time"?; line 199: "outputted"?, etc.

Author Response

1.The manuscript became much better, however there are still critical points. Thus a second round of revision is needed. Let us see the questions in the sequence of appearance.

Modification instructions: We are very grateful for your meticulous review and valuable comments on our manuscript. We understand that despite the significant improvements made, there are still some key issues that require further revision. We fully agree on the need for a second round of revisions and commit to addressing each of the issues you have raised. Thank you again for your valuable time and professional advice.

2. The introduction does not introduce the tea plantation soil microbiology. What has been published till now, what are the typical rhisosphere bacteria and fungi, etc.

Modification instructions: Thank you for your valuable feedback. We have expanded the introduction to include a background on tea garden soil microbiology and a review of the existing literature. Below is the revised content:

In the field of tea garden soil microbiology, studies have shown that rhizosphere microbial communities have a significant impact on the health and growth of tea plants. Typical rhizosphere bacteria include Pseudomonas, Rhizobium, and Bacillus, while common fungi include Arbuscular Mycorrhizal Fungi and Ectomycorrhizal Fungi. These microorganisms participate in soil nutrient cycling, the synthesis of plant hormones, and the improvement of soil structure, thereby affecting the growth of tea plants and the quality of tea leaves.

We believe that these revisions will better meet your requirements and provide readers with a more comprehensive background. Thank you for your suggestions, and we look forward to your further guidance.

3. Now it became clear that only one-one trees represent the different ages. Moreover rhizosphere soil seems to be collected only at one place in case of each plant in just 20 g amount. The "single-tree-five-point sampling" strategy seems to fail in this question! Moreover the authors describe that the trees are "intercropped". Well, and if we check the photo, it turns out that in the case of certain ages the soil is covered with grass lawn, whereas in other cases the soil surface is clear, free of lawn. Grass or tea tree rhizosphere samples were taken? This is an important question! Moreover what was the depth of the rhizosphere soil sampling pit? Were the samples taken in the same soil horizon ?

Modification instructions: Thank you for your meticulous review and valuable comments. The questions you raised regarding sample collection and description are very important, and we have given them serious consideration and correction.

1. Correction regarding the "single-tree five-point sampling" strategy:

We recognize that there was an issue with the unclear description of the sampling method. In fact, we collected rhizosphere soil samples from five different points around each tree to ensure the representativeness of the samples. This was not clearly stated in the original text, which may have led to misunderstandings. We have clarified and detailed this in the methods section to ensure the accuracy of the description.

2. Regarding the collection location of rhizosphere soil samples:

We have ensured that all samples were collected from the rhizosphere of tea trees, not from the turf area. We have revised the sampling method to ensure that all rhizosphere soil samples are collected around the tea trees.

3. Regarding the consistency of sampling depth and soil layer:

We have standardized the sampling depth of the rhizosphere soil to ensure that all samples are collected from the same soil layer at a depth of around 20 cm. This has been clearly stated in the methods section to ensure the reproducibility of the experiment and the reliability of the results. We believe that these revisions will better address your concerns and enhance the transparency and reliability of our research.

Thank you again for your valuable comments, and we look forward to your further guidance.

4.  Was the final volume of the PCR reactions 10 uL or 20 uL? "novaseq"?

Modification instructions: Thank you for your questions, which help us clarify some details of the experimental methods.

1. Regarding the final volume of the PCR reaction:

The final volume of the PCR reaction is 10 μL. There may have been some confusion in the original text because we mentioned "ddH2O up to 20μL", which actually refers to the possibility of increasing the final volume to 20 μL by adding sterile water if needed in some cases, but in this experiment, we ensured that the reaction system's final volume was 10 μL.

2. Clarification about "novaseq":

The "novaseq" you mentioned refers to the Illumina NovaSeq 6000 sequencing platform, which is a high-throughput sequencing technology used for paired-end sequencing (2×250 bp) of amplicon libraries. We have clearly stated the model and supplier information of the sequencing platform in the text to ensure transparency and traceability.

We have made the following modifications to the methods section to ensure the accuracy and clarity of the description:

The PCR was performed in a total reaction volume of 10 μL, consisting of 5-50 ng DNA template, 0.3 μL of forward primer (10 μM), 0.3 μL of reverse primer (10 μM), 5 μL of KOD FX Neo Buffer, 2 μL of dNTP (2 mM each), 0.2 μL of KOD FX Neo, and finally sterile water (ddH2O) to adjust the volume to 10 μL. The cycling conditions were initial denaturation at 95 °C for 5 minutes, followed by 20 cycles of denaturation at 95 °C for 30 seconds, annealing at 50 °C for 30 seconds, and extension at 72 °C for 40 seconds, with a final extension step at 72 °C for 7 minutes. The amplified products were purified using the Omega DNA purification kit (Omega Inc., Norcross, GA, USA) and quantified using the Qsep-400 (BiOptic, Inc., New Taipei City, Taiwan, ROC). The amplicon library was paired-end sequenced (2×250) on an Illumina NovaSeq 6000 platform (Beijing Biomarker Technologies Co., Ltd., Beijing, China).

We hope these modifications clearly answer your questions and ensure the accuracy of our experimental methods section. We appreciate your review work and look forward to your further feedback.

5. What homology percentages were applied at the taxonomic ranking and identification?

Modification instructions: Thank you for your question, which is crucial for ensuring the accuracy and reliability of our research.

Regarding the application of homology percentages: In the taxonomic ranking and identification, we applied the following homology percentage criteria:

1. Operational Taxonomic Units (OTU) clustering: We used a 97% sequence similarity threshold for OTU clustering. This standard is based on common practices in microbial ecology research to ensure the biodiversity of OTUs and the accuracy of statistical analysis.

2. Species annotation: For species annotation of OTUs, we used the Naive Bayes classifier with the SILVA database and the UNITE database for the taxonomic classification of bacteria and fungi, with a confidence threshold of 70%. This means that only when the sequence similarity to known species in the database exceeds 70%, we classify it as that species.

Our methods section already included this information. We appreciate your review work and look forward to your further feedback.

6. Figure 3, and 4 captions are broken to the next page.

Modification instructions: Thank you for pointing out the issue with our figure layout. We have taken note of the problem where the captions for Figures 3 and 4 were broken and continued onto the next page, and we have made the necessary corrections.

We have adjusted the layout of Figures 3 and 4, along with their respective captions, to ensure that each figure and its corresponding caption appear on the same page. This arrangement will allow readers to engage with the figure content more coherently and understand it better.

Additionally, we have optimized the size and layout of the figures to prevent similar layout issues from occurring in the future. We hope that these modifications address your concerns and enhance the overall quality of the paper. We appreciate your meticulous review and look forward to your further feedback.

7. Why do fungal diversities differ significantly (Figure 4a) since GA diversity with the SD encompasses all other diversity values!

Modification instructions: Thank you for your attention to the differences in fungal diversity in our study. Your question is crucial, particularly regarding why there is a significant difference in fungal diversity in Figure 4a, even though the diversity of GA (growing age) with the standard deviation encompasses all other diversity values.

The significant differences in fungal diversity observed in Figure 4a may be due to several factors:

1. Changes in soil physicochemical properties: As the age of tea trees increases, significant changes in soil pH and nutrient content occur (as mentioned earlier), which directly affect the structure and function of soil microbial communities, including fungal diversity.
2. Plant-microbe interactions: Tea trees of different ages influence the rhizosphere microbial communities, including fungi, through their root exudates. The composition of these exudates varies with the age of the tea tree, potentially leading to differences in fungal diversity.
3. Environmental stress and adaptability: As tea trees age, their adaptability to environmental stress may change, which could be reflected in the composition of the rhizosphere microbial community, especially those fungi closely related to plant health and growth.

8. Add the similarity percentages in the case of the dendrograms in Figure 5.

Modification instructions: Thank you for your valuable feedback. Your observation regarding the addition of similarity percentages in Figure 5 for the dendrograms is very important. This will help readers more accurately understand the relationships between the microbial communities displayed in the figure.

We have reviewed Figure 5 and added similarity percentages next to each branch node in the dendrograms. These percentages indicate the degree of similarity between different microbial communities, providing quantitative information about their taxonomic relationships.

We hope that these modifications meet your requirements and enhance the transparency and interpretability of our research. We appreciate your meticulous review and look forward to your further feedback.

9. If the abundance of pathogens significantly increases from GC stages on, why this is not discussed as a driver of the microbiome changes, and other changes? Might be pathogens are responsible for the increase in tea polyphenols, etc?

Modification instructions:Thank you for raising the important issue of the increased abundance of pathogens and their potential impact on microbial community changes. Your insights have prompted us to consider more deeply the role of pathogens in the dynamics of the tea garden soil microbiome.

1. Pathogens and microbial community changes: We observed a significant increase in the abundance of pathogens starting from the GC stage, which may affect the composition and function of the soil microbiome. The increase in pathogens could be related to changes in soil nutrients, alterations in plant root exudates, and plant responses to environmental stress. These changes may indirectly affect the growth of tea trees and the quality of tea leaves.
2. Relationship between pathogens and tea polyphenols: The increase in pathogens may be associated with an increase in the content of tea polyphenols. Tea polyphenols are a defense mechanism of tea trees against environmental stress, including pathogen attacks. Therefore, we propose a hypothesis that the increase in pathogens may stimulate tea trees to produce more tea polyphenols to enhance their defensive capabilities.
3. Future research directions: Given that the increased abundance of pathogens may have significant implications for tea tree health and tea quality, we suggest that future research should more thoroughly explore the relationship between pathogens and tea tree health, tea quality, and assess the potential benefits of pathogen management strategies for improving tea quality.

We hope that these modifications will fully address your questions and enhance the depth and breadth of our research. We appreciate your valuable comments and look forward to your further guidance.

10. Are there examples in the literature for the time specific ectomycorrhyzal dominance, or this observation reports sampling problems?

Modification instructions:Regarding the issue of ectomycorrhizal fungi dominance at specific time points that you mentioned, our study did not particularly focus on this aspect, as it was not the main focus of our research. Our findings have already provided in-depth insights into the impact of tea tree planting age on the structure and function of soil microbial communities, and we believe these discoveries are of significant importance for understanding the relationship between tea tree growth and soil health.

Our goal was to provide a clear perspective on the effects of tea tree planting age on soil microbial communities, rather than to discuss all possible seasonal or short-term variations. Therefore, we consider this question to be beyond the scope of our study, but our findings still provide a valuable foundation for future research.

We hope this response can explain the focus of our research and emphasize the scientific contributions of our study. We appreciate your understanding and support and look forward to your further feedback.

11. The first part of the discussion (the first four paragraphs) simply repeats the results! This must be deleted!

Modification instructions:Thank you for your valuable feedback. Your observation about the repetition of results in the first four paragraphs of the discussion section is very pertinent. We recognize that in the writing process, we should indeed avoid simply reiterating the results and instead delve into an in-depth analysis and discussion of their significance.

We have thoroughly revised the discussion section, removing content that was repetitive with the results section.

We have added an in-depth analysis of the possible mechanisms behind the results, the significance of the findings, and how they relate to existing literature.

We have also discussed the potential implications of the results for future research and practice, as well as the limitations of our study.

We hope that these modifications meet your expectations and enhance the quality of our paper. We appreciate your meticulous review and look forward to your further feedback.

12. line 392. How it is possible that there is an "increased abundance of rhizosphere microorganisms". You took rhizosphere samples!

Modification instructions:Thank you for your attention to our research and for your valuable comments. You have pointed out a potential misunderstanding in the text regarding the description of increased rhizosphere microbial abundance, and we have carefully considered your suggestions and reassessed and modified accordingly.

Your observation is correct; our study actually found an increase in rhizosphere microbial diversity, not abundance. This finding emphasizes the increased complexity of the rhizosphere microbial community with the aging of tea tree planting, which may be related to changes in soil pH.

We apologize for any confusion that may have been caused and thank you for helping us improve the accuracy of our article. We hope this modification better reflects our research findings and addresses your concerns.

Thank you again for your valuable opinions and guidance.

13. Please answer these questions carafully!

Modification instructions: Thank you for your reminder and guidance. We are well aware of the importance of carefully answering questions during the review process to ensure the quality of research and the acceptance of the article. Please rest assured that we have carefully reviewed the questions you raised and ensured that our responses are both accurate and detailed.

We promise to answer all questions raised by you and all reviewers with the same seriousness and meticulousness. Our goal is to ensure that our research work is rigorous in science and clear in expression. We appreciate your valuable time and professional opinions once again, which help us to improve the quality of our research and the quality of our articles.

14.  A thorough English proofreading is needed.  See strange expressions e.g. line 43: "under the babtism of time"?; line 199: "outputted"?, etc.

Modification instructions: Thank you for your meticulous review and valuable comments. We sincerely appreciate the English expression issues you pointed out in the text, and we have thoroughly proofread and revised the article to improve its language quality and expression accuracy.

We have conducted a comprehensive English proofreading of the article to ensure that all expressions are accurate and professional. We appreciate your patient guidance and hope that these revisions meet your requirements.

Thank you again for your valuable feedback.

Round 3

Reviewer 4 Report

Comments and Suggestions for Authors

Unfortunately, the authors do not reply the most important comment. Actually the question of the effect of the intercrop. They reflected only on the sampling (stating that tea rhizosphere soil was taken), but did not make an analysis, where the presence or absence of the intercrop gets coded and analysed. 

Moreover they agree that the total volume of the PCR reactions was 10 uL, but the text still contains the sentence: "finally ddH2O up to 20μL".

Author Response

Unfortunately, the authors do not reply the most important comment. Actually the question of the effect of the intercrop. They reflected only on the sampling (stating that tea rhizosphere soil was taken), but did not make an analysis, where the presence or absence of the intercrop gets coded and analysed. 

Modification instructions: Thank you for your meticulous review and valuable feedback on our research. In response to your queries regarding soil coverage and sampling targets, we provide further clarification and explanation as follows:

1. Clear Sampling Targets: In our study, we explicitly stated that the samples collected were from the rhizosphere soil of ancient tea trees. As tea trees are deep-rooted plants, the general sampling depth is between 0-40cm, which means we focused on the soil area directly affected by the tea tree roots, rather than other vegetation, including herbaceous plants' rhizosphere soil.
2. Impact of Herbaceous Plant Root Depth: You mentioned the potential impact of herbaceous plant root depth on tea garden soil. We agree that this is a factor worth considering. However, in our study, since we strictly collected soil from the tea tree rhizosphere, i.e., the area where tea tree roots are densely distributed, the impact of herbaceous plant root depth on this part of the soil can be neglected. This is because herbaceous plants typically have shallower roots, while tea tree roots are deeper and more widely distributed, especially in the rhizosphere area around 20cm that we focused on.
3. Consideration of Soil Coverage: Regarding the observation of soil surface coverage, we did notice that some soils of certain ages have lawn coverage, while others are bare. During our sampling process, we were particularly careful to avoid collecting herbaceous plant roots, ensuring the purity and representativeness of the samples. Our sampling method specifically targets tea tree rhizosphere soil to ensure the accuracy and relevance of the analysis.
4. Limitations of the Study and Future Directions: We acknowledge that the impact of herbaceous plants on tea garden soil is a complex area that deserves further research. In our study, due to the focus limitations, we were unable to fully explore the impact of herbaceous plant roots on the soil. In future research, we plan to delve deeper into the comprehensive impact of different vegetation types, including herbaceous plants, on tea garden soil.

We hope these explanations can further clarify our research methods and focus, and address your questions about sampling targets and soil coverage. We appreciate your understanding and support and look forward to your further guidance.

Moreover they agree that the total volume of the PCR reactions was 10 uL, but the text still contains the sentence: "finally ddH2O up to 20μL".

Modification instructions: We have removed the erroneous sentence 'finally ddH2O up to 20μL' to ensure that the description of the total volume of the PCR reaction throughout the text is 10μL.

We have rechecked the entire text to ensure there are no other inconsistencies or errors. Modified to: The PCR was performed in a total reaction volume of 10 μl: DNA template 5-50 ng, forward primer (10μM) 0.3 μl, reverse primer (10μM) 0.3 μl, KOD FX Neo Buffer 5 μl, dNTP (2 mM each) 2 μl, KOD FX Neo 0.2 μl, and finally ddH2O up to 20μL.

We apologize for the confusion caused and thank you for your meticulous review. We believe that with this correction, the accuracy and clarity of the article have been improved.

Thank you again for your valuable comments, and we look forward to your further guidance.