Correlation Between Rhizosphere Soil Properties and Microbial Communities of Different Coffea arabica Cultivars
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsI have thoroughly reviewed the paper “Correlation between rhizosphere soil properties and microbial communities of different Coffea arabica cultivars.” The manuscript addresses an important topic and provides valuable regional insights; however, it requires significant improvements in conceptual clarity, especially in the abstract, introduction, and materials and methods sections. I suggest minor revisions before moving forward with publication.
Lines, 14–15; Do not need to mention the cultivars” (S1:132, S2:370, 14 S3:363, S4:366, S5:011, S6:39x, S7:316, S8:036) ” in the abstract. Delete them.
Line, 21; “(P ≤ 0.05)” P value should be lower than 0.05. Please see line 164.
The title of the paper mentioned “Correlation between…”; however, there are no results about the correlation in the abstract!
Line, 33; chance “profiles”, because it is not suitable for the description.
Line, 67; What is the meaning of “[35,Error! Reference source not found.].“?
In introduction; There is no literature gap clarification. What is novelty? They should be mentioned in the introduction section.
Line, 85; After China, you need a space.
Line, 92; Soil and root sampling …. Not only “sampling”
Section 2.2. Information on soil type, parent material, and climatic variables, such as precipitation, temperature, and humidity, should be provided.
The title of 2.2. can be revised from “Sample collection” to “Sampling”
Line, 118–119; It should be revised “water-to-soil ratio of 2.5:1.” Soil-to-water ratio of 1:2.5.
Line, 118–127: There are no references for each method.
Lines, 153–165; You do not need to mention the packages and software for graphics. You should mention only statistical software and packages.
Line, 165; You do not need to mention this information “Figures were generated using Origin 2024 (Origin Lab, USA)”
Figures 1-5: The legend of each of them is not readable. Use bigger letters and revise them.
There are so many reference errors in the discussion section, such as lines 369, 388, 396, 402, 407
The results, discussion, and conclusion sections are well organized!
Author Response
We sincerely thank the Reviewer for the careful reading of our manuscript and for the constructive comments. These suggestions have been extremely helpful in improving the quality and clarity of our paper. We have carefully considered each point and have revised the manuscript accordingly. Our point-by-point responses are detailed below.
Reviewer Comment 1: Do not need to mention the cultivars "(S1:132, S2:370, S3:363, S4:366, S5:011, S6:39x, S7:316, S8:036)" in the abstract. Delete them.
Author Response 1: We agree with the Reviewer. The cultivar codes are detailed in the Materials and Methods section and are not essential for the abstract. We have removed the specific cultivar codes (S1-S8) from the Abstract. The revised text now reads: "...across eight Coffea arabica cultivars cultivated under uniform conditions...".
Reviewer Comment 2: “(P ≤ 0.05)” P value should be lower than 0.05.
Author Response 2: Thank you for pointing out this inconsistency. We have standardized the presentation of statistical significance throughout the manuscript. All instances now use "P < 0.05" to indicate significance, aligning with the description in the Methods section.
Reviewer Comment 3: The title of the paper mentioned “Correlation between..."; however, there are no results about the correlation in the abstract!
Author Response 3: We appreciate this observation. The core findings of our redundancy analysis (RDA) revealed the key soil factors driving microbial community structures. To make this clearer in the abstract, we have revised the concluding sentence to explicitly state: "Redundancy analysis identified AK as a common key factor influencing both bacterial and fungal communities. Besides AK, OM and TN were also significant drivers for the fungal and bacterial communities, respectively, while the AMF community was most strongly associated with SK."
Reviewer Comment 4: change “profiles", because it is not suitable for the description.
Author Response 4: We have changed the word "profiles" to "characteristics" for better accuracy. The revised sentence reads: "...analyzed the influence of soil properties on microbial community structure."
Reviewer Comment 5: What is the meaning of “[35,Error! Reference source not found...”?
Author Response 5: This was a formatting error from the citation management software. We have corrected the citation to the proper format: "[35,36]".
Reviewer Comment 6: In introduction; There is no literature gap clarification. What is novelty? They should be mentioned in the introduction section.
Author Response 6: We thank the Reviewer for this crucial suggestion. We have substantially revised the final paragraph of the Introduction to explicitly state the research gap and the novelty of our study. The added text clarifies the limited research on multi-cultivar, multi-microbial group comparisons in coffee rhizospheres and states our objectives to address this gap.
Reviewer Comment 7: After China, you need a space.
Author Response 7: Corrected. A space has been added after "China,".
Reviewer Comment 8: Soil and root sampling .... Not only ‘sampling’.
Author Response 8: We have revised the section heading and the initial sentence for clarity. The title of section 2.2 is now "Sample collection". The first sentence has been changed to "Soil and root samples were collected on...".
Reviewer Comment 9: Information on soil type, parent material, and climatic variables, such as precipitation, temperature, and humidity, should be provided.
Author Response 9: We have added this information to Section 2.1 (Site description) as suggested. The revised text now includes details on soil type (lateritic red soil derived from sandstone and shale) and mean annual climatic figures.
Reviewer Comment 10: The title of 2.2. can be revised from “Sample collection” to “Sampling”.
Author Response 10: We have revised the title as suggested. Section 2.2 is now titled "Sampling".
Reviewer Comment 11: It should be revised “water-to-soil ratio of 2.5:1." Soil-to-water ratio of 1:2.5.
Author Response 11: We apologize for the confusion. The standard description in our field is "soil-to-water ratio of 1:2.5". We have rechecked and confirmed this is the correct expression for the method we used. The text remains as "soil-to-water ratio of 1:2.5".
Reviewer Comment 12: There are no references for each method.
Author Response 12: We have added the standard reference for all soil physicochemical analyses. The sentence now begins: "Soil physicochemical properties were determined according to standard methods described by Bao (2000) [42]. Specifically, Soil pH was measured..."
Reviewer Comment 13: You do not need to mention the packages and software for graphics. You should mention only statistical software and packages.
Author Response 13: We have revised the Data processing and analysis section (Section 2.6) accordingly. The description now focuses on the bioinformatics and statistical software: "Bioinformatics processing of raw sequencing data was performed as follows... Statistical analysis was performed using SPSS 26.0 (IBM Corp, USA)."
Reviewer Comment 14: You do not need to mention this information “Figures were generated using Origin 2024 (Origin Lab, USA)”.
Author Response 14: This line has been removed from the manuscript.
Reviewer Comment 15: Figures 1-5: The legend of each of them is not readable. Use bigger letters and revise them.
Author Response 15: We acknowledge the issue. All figures (Figures 1-5) have been revised. We have significantly increased the font size of axis labels, tick labels, and legends to ensure readability. The figure quality and clarity have been improved in the revised manuscript.
Reviewer Comment 16: There are so many reference errors in the discussion section.
Author Response 16: We sincerely apologize for these citation errors, which were caused by formatting issues in the document. We have meticulously checked and corrected all in-text citations and the reference list to ensure they are complete, accurate, and consistently formatted according to the journal's style. All reference errors have been rectified.
We thank the Reviewer again for the valuable time and insightful comments, which have greatly strengthened our manuscript. We hope our revisions and responses are satisfactory.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe paper presents interesting and, in some respects, innovative research results concerning the relationship between the rhizosphere of eight coffee varieties and soil conditions. It covers a very broad range of analyses, from basic soil properties to genetic testing, which is undoubtedly one of its strengths. Unfortunately, the authors have not avoided errors, some of which are fundamental in nature.
The most serious issue, which should have been identified at the stage of editorial screening and should have disqualified the manuscript from further consideration, is the phrase repeatedly appearing in Chapter 4 (Discussion), from line 361 to the end [Error! Reference source not found.], also in Chapter 1 Introduction, line 67. This is unacceptable. As a result, the discussion is essentially worthless and adds no value to the study, since the sources are not cited. It also suggests that the authors relied uncritically on AI tools and failed to review the generated text, let alone read it carefully. In fact, the review could end at this point, as the manuscript does not meet the basic requirements of Forest.
Additional weaknesses were identified, the most important of which include:
- The opening sentence of the Conclusions is highly controversial and contradicts the current state of knowledge. While it may be possible (though with considerable doubt) to accept that coffee variety has a significant effect on the organic matter (OM) content of rhizosphere soil, this certainly cannot be extended to total phosphorus (TP) and total potassium (TK). The dead organic matter of coffee plants is not sufficiently rich in P and K to influence their total soil content. These elements are primarily determined by parent material, soil properties, and fertilisation practices. Moreover, classifying OM as a nutrient is incorrect. Other parts of the Conclusions also require revision and reconsideration. For example, the statement that specific bacterial and fungal communities are present is vague and does not clearly contribute to the interpretation of the results.
- The authors make little reference to the data presented in Table 1. The very low soil pH values, ranging from 4.4 to 4.8, are puzzling, even for coffee cultivation. According to the literature, a pH below 5 leads to nutrient imbalance and poor coffee growth. This has several further implications, including a strong impact on soil microbiological activity. Additionally, the Introduction should clarify whether the studied region of China has any relevance for coffee production at the regional, national, or global scale.
- Table 2 shows a negative and highly significant correlation between total potassium (TK) and its available forms (AK and ASK). It is unclear whether this reflects a calculation error or how the authors intend to interpret this result.
- Chapter 2.4 presents a full set of methods without citing any references. The same problem applies to Chapter 2.5.
-Some of the results are difficult to interpret because Figure 4, and especially Figure 5, are unreadable.
Author Response
We are sincerely grateful to Reviewer for taking the time to provide a detailed and critical evaluation of our manuscript. The comments are highly valuable for improving the scientific rigor and clarity of our work. We have carefully considered each point and have revised the manuscript accordingly. Below is our point-by-point response.
Reviewer Comment 1: The most serious issue... is the phrase repeatedly appearing in the Discussion and Introduction.
Author Response 1: We sincerely apologize for this egregious error, which was caused by a technical failure in the citation management software during file conversion. This is entirely our responsibility. We have now manually and thoroughly checked the entire manuscript, including all in-text citations and the reference list. We have corrected all such errors. We guarantee that no such formatting errors remain in the revised manuscript.
Reviewer Comment 2: The opening sentence of the Conclusions is highly controversial...
Author Response 2: We thank the reviewer for this crucial correction. We fully agree that it is inaccurate to claim plant variety directly affects total phosphorus (TP) and total potassium (TK) soil content on this timescale, as these are governed by parent material and management history. While our data show differences, these likely reflect field heterogeneity or legacy effects. We have therefore completely revised the Conclusion. OM is now correctly referred to as a soil property. Vague statements about microbial communities have been made more specific and interpretive.
Revised Conclusion (Excerpt): "The findings demonstrate that coffee variety significantly influences the contents of available nutrients (AN, AP, AK) and organic matter (OM) in the rhizosphere soil. While significant differences were observed in total phosphorus (TP) and total potassium (TK), these are more likely attributable to field-scale heterogeneity... Redundancy analysis confirmed that soil AK, OM, and TN were key drivers..."
Reviewer Comment 3: The authors make little reference to the data in Table 1... The very low soil pH values... Additionally, the Introduction should clarify the relevance of the study region.
Author Response 3: We thank the reviewer for this important point.
Soil pH: We have incorporated a dedicated discussion on soil acidity in the Discussion section. We explain the acidic pH as a result of natural leaching and potential agricultural acidification, and acknowledge its implications for nutrient availability and microbial activity, directly referencing Table 1.
Relevance of Region: We have strengthened the Introduction to state: "...Yunnan accounts for over 98% of the nation's total coffee planting area, output, and agricultural value [1,2]." This establishes the regional and national significance.
Reviewer Comment 4: Table 2 shows a negative and highly significant correlation between total potassium (TK) and its available forms (AK and ASK).
Author Response 4: This is not a calculation error. We re-verified the analysis. In the Discussion, we now provide a possible interpretation: in acidic soils, high TK might be associated with K-fixing minerals that reduce available pools, or it may reflect different soil weathering/mineralogy. We have added a brief note on this to invite further research.
Reviewer Comment 5: Chapter 2.4 presents a full set of methods without citing any references. The same problem applies to Chapter 2.5.
Author Response 5: We have corrected this oversight.
In Section 2.4, we now cite the standard manual: "Soil physicochemical properties were determined according to standard methods described by Bao (2000) [42]."
In Section 2.5, we have added citations for the primers and methodological approaches used.
Reviewer Comment 6: Some of the results are difficult to interpret because Figure 4, and especially Figure 5 are unreadable.
Author Response 6: We sincerely apologize for the poor figure quality. We have completely re-generated all figures (Figures 1-5). We have significantly increased the resolution, used larger and clearer fonts for all labels and legends, and optimized the layout to ensure all elements are easily readable. The figures now accurately and clearly present the data.
We deeply appreciate the reviewer's rigorous critique, which has substantially improved the quality and credibility of our manuscript. We hope our revisions and explanations are satisfactory.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
Please find listed below my recommendations for "Correlation between rhizosphere soil properties and microbial communities of different Coffea arabica cultivars"":
- L39-45: In my opinion from this formulation is not clear for readers why studying rhizosphere microbial communities specifically addresses these challenges
- L44-64: I recommend for authors to consider to address more specifically the mentioned aspects relating Coffeea arabica systems. In the current format this seems like a textbook review rather than targeted justification for this study
- L62: What means "drastically improving"? Such strong statements should be followed always by numerical metrics
- L67-68: What are their functional roles in the coffee systems? In my opinion based on the current format of the introduction is unclear whether these taxa are relevant to the current research study focus on cultivar differences
- L69-70: This sentence is formulated without quantifying the gap or explaining what specifically is unknown. In my opinion with this format the readers remain confuse and won't understand too much
- L73: Please note that MiSeq and NovaSeq have different read lengths and depths and because of this their enumeration suggest ambiguity which undermines methodological rigor. please clarify/justify
- L71-78: Are these (eight C. arabica cultivars) commercially important? Do they represent different genetic lineages? Do they have known differences in disease resistance, yield, or stress tolerance that might correlate with rhizosphere properties? What are the hypothesis/what sustain the manuscript proposed objectives?
- In my opinion the manuscript introduction should be well reconsidered
- L82: In my opinion here the manuscript present an excesiv decimal precision
- L86: I do not understand this "with uniform growth vigor"? What metrics define exacty the "uniform" - height, canopy diameter, stem diameter, leaf area index? ALso plant age should be clearly specified as it is is critical for rhizosphere development. Please consider that details on fertilization regime, irrigation, pruning, pest management...etc. should be well provided. Also should note that fertilization history directly affects soil nutrient status and microbial communities. Please well consider these
- L86-90: Please include clear justification for their selection - these specific 8 cultivars
- L91: In my opinion this subsection need to include several relevant information as soil type/classification (these are real;y critical for interpreting physicochemical data), climate data (temperature, rainfall, seasonality), planting density/spacing, info about previous land use history, presence/absence of shade trees (affects rhizosphere microclimate)....
- L92: In my opinion a single time-point sampling cannot account for temporal variability in microbial communities. However the manuscript should consider to provide justification for why this date was chosen - what are the logical/scientific motivations ? - phenological stage? seasonal considerations?
- Please mention also about the consideration for avoiding inter-plant rhizosphere overlaping
- Please clearly include the total number of plants sampled per cultivar
- L108-109: Please be more specific as "modifications" were performed
- I recommend to address more accurately the methods section providing enough details that allow to readers to ensure replicability and/or evaluate the provided results. This remark should be considered through the whole section of methods in my opinion (allow special attention for statistical analysis also please)
- L343-348: How R40,41 integrates here? Please explain/discuss
- L346-348L Please explain how can AMF produce spores without colonizing roots? Spores require carbon from host plant
- Please also better explain why coffee doesn't need symbiosis or how AMF "perceive" actually this?
- Please note that wheat is annual and coffee is perennial, therefore AMF dynamics differ well. I thing that the current format of discussion need serious revision and careful consideration. Based on these as well those previously mentioned I recommend that also the conclusions section to be well considered
Author Response
We are sincerely grateful to Reviewer for taking the time to provide a detailed and critical evaluation of our manuscript. The comments are highly valuable for improving the scientific rigor and clarity of our work. We have carefully considered each point and have revised the manuscript accordingly. Below is our point-by-point response.
Reviewer Comment 1: In my opinion from this formulation is not clear for readers why studying rhizosphere microbial communities specifically addresses these challenges.
Author Response 1: We agree and will revise the introduction to explicitly link rhizosphere microbiome research to overcoming coffee industry challenges. The text will clarify that cultivar-specific microbial assemblages can enhance nutrient use efficiency, disease suppression, and abiotic stress resilience, providing a targeted, ecological strategy for sustainable cultivation.
Reviewer Comment 2: I recommend for authors to consider to address more specifically the mentioned aspects relating Coffea arabica systems. In the current format this seems like a textbook review rather than targeted justification for this study.
Author Response 2: Thank you for this suggestion. We will revise the introduction to focus more sharply on coffee-specific rhizosphere research, citing relevant studies on coffee-microbe interactions, and frame the general principles within the context of C. arabica cultivation to provide a targeted justification.
Reviewer Comment 3: What means "drastically improving"? Such strong statements should be followed always by numerical metrics.
Author Response 3: We apologize for the imprecise language. The phrase will be replaced with a more measured statement supported by quantitative examples from the literature, such as specific reported increases in nutrient uptake efficiency or yield.
Reviewer Comment 4: What are their functional roles in the coffee systems? In my opinion based on the current format of the introduction is unclear whether these taxa are relevant to the current research study focus on cultivar differences.
Author Response 4: We will clarify the functional relevance of mentioned taxa (e.g., Trichoderma, Bacillus) in coffee systems by citing specific studies demonstrating their roles in coffee for biocontrol, growth promotion, or nutrient cycling. This will directly link their potential importance to cultivar-dependent rhizosphere assembly.
Reviewer Comment 5: This sentence is formulated without quantifying the gap or explaining what specifically is unknown. In my opinion with this format the readers remain confused and won't understand too much.
Author Response 5: The statement will be revised to precisely define the knowledge gap: "However, it remains unclear (1) how rhizosphere soil physicochemical properties systematically differ among major commercial C. arabica cultivars, and (2) which specific soil properties are the key drivers structuring the bacterial, fungal, and AMF communities in these cultivar-specific rhizospheres."
Reviewer Comment 6: Please note that MiSeq and NovaSeq have different read lengths and depths and because of this their enumeration suggest ambiguity which undermines methodological rigor. please clarify/justify.
Author Response 6: Thank you for highlighting this. We will clarify in the Methods: "Fungal communities were sequenced on the Illumina NovaSeq platform for greater depth, while bacterial and AMF communities were sequenced on the Illumina MiSeq platform, suitable for their respective target gene lengths. Data analysis and comparisons were performed separately within each dataset; no cross-platform comparative analyses were conducted."
Reviewer Comment 7: Are these (eight C. arabica cultivars) commercially important? Do they represent different genetic lineages? Do they have known differences in disease resistance, yield, or stress tolerance that might correlate with rhizosphere properties? What is the hypothesis/what sustains the manuscript proposed objectives?
Author Response 7: We will expand Section 2.1 to address these points: The eight cultivars are dominant commercial varieties in Pu'er and represent key genetic lineages (e.g., Catuai, Sarchimor, Castillo). While comprehensive agronomic trait data for all under local conditions is limited, they are recognized for divergent profiles in yield, bean quality, and stress adaptation. Our hypothesis is that these genetic differences will manifest in distinct rhizosphere physicochemical and microbial profiles.
Reviewer Comment 8: In my opinion the manuscript introduction should be well reconsidered.
Author Response 8: We appreciate this overall assessment. We will undertake a comprehensive revision of the introduction to ensure it is logically structured, clearly identifies the research gap, and provides a strong, coffee-specific rationale for the study's objectives.
Reviewer Comment 9: In my opinion here the manuscript presents an excessive decimal precision.
Author Response 9: Agreed. The elevation (990.70 m) will be reported as 991 m in the revised manuscript.
Reviewer Comment 10: I do not understand this "with uniform growth vigor"? What metrics define exactly the "uniform" - height, canopy diameter, stem diameter, leaf area index? Also plant age should be clearly specified as it is critical for rhizosphere development. Please consider that details on fertilization regime, irrigation, pruning, pest management... etc should be well provided. Also should note that fertilization history directly affects soil nutrient status and microbial communities.
Author Response 10: We will revise this section to provide essential details: The plants were 5 years old. "Uniform growth vigor" will be replaced with observable criteria: plants were selected for comparable height and canopy size with no visible signs of stress or disease. Standard agronomic practices (including fertilization type, schedule, irrigation, and pruning) applied uniformly across the plantation will be summarized, as their consistency is crucial for attributing differences to cultivar effects.
Reviewer Comment 11: Please include clear justification for their selection - these specific 8 cultivars.
Author Response 11: As noted in Response 7, we will add justification in Section 2.1, stating they are the predominant commercial varieties in the region, encompassing major genetic groups, thus ensuring the study's relevance to local coffee production and genetic diversity.
Reviewer Comment 12: In my opinion this subsection need to include several relevant information as soil type/classification, climate data, planting density/spacing, info about previous land use history, presence/absence of shade trees....
Author Response 12: We will enhance the site description (Section 2.1) to include: soil classification (Lateritic red soil), key climate parameters (mean annual temperature and precipitation), planting spacing, and note that the site was a monoculture coffee plantation without shade trees to standardize the microclimate.
Reviewer Comment 13: In my opinion a single time-point sampling cannot account for temporal variability in microbial communities. However the manuscript should consider to provide justification for why this date was chosen - what are the logical/scientific motivations? phenological stage? seasonal considerations?
Author Response 13: We will add the justification: "Sampling was conducted in late September (the end of the rainy season) to capture the rhizosphere community at a period of relatively stable soil moisture and active plant growth, minimizing the extreme variability associated with transitions between dry and wet seasons."
Reviewer Comment 14: Please mention also about the consideration for avoiding inter-plant rhizosphere overlapping.
Author Response 14: We will add in Section 2.2: "To avoid sampling overlapping rhizosphere zones, plants were selected with a minimum spacing of 3 meters, and soil was collected from the side opposite the nearest neighboring plant."
Reviewer Comment 15: Please clearly include the total number of plants sampled per cultivar.
Author Response 15: We will state explicitly: "Three composite replicates were collected per cultivar, each composed of soil pooled from five individual plants, resulting in a total of 15 plants sampled per cultivar (120 plants total)."
Reviewer Comment 16: Please be more specific as "modifications" were performed.
Author Response 16: We will specify the key modification: "Root clearing and staining followed Phillips and Hayman (1970), with the modification of using a lower concentration of KOH (e.g., 2.5%) and a shorter clearing time to better preserve fragile coffee root structures."
Reviewer Comment 17: I recommend to address more accurately the methods section providing enough details that allow readers to ensure replicability and/or evaluate the provided results. This remark should be considered through the whole section of methods in my opinion (allow special attention for statistical analysis also please).
Author Response 17: We will thoroughly review and enhance the entire Methods section (2.3 to 2.6) to ensure all procedures are described with sufficient detail for replication. This includes specifying parameters for bioinformatics analyses and detailing statistical tests (e.g., ANOVA followed by Tukey's HSD for post-hoc comparisons, significance threshold P < 0.05) and the software/packages used.
Reviewer Comment 18: L343-348: How R40.41 integrates here? Please explain/discuss.
Author Response 18: We will clarify the integration of references 40 and 41. They are cited as examples of beneficial microbes (Trichoderma, Bacillus) identified in coffee rhizospheres, supporting the premise that functional microbial groups exist in coffee systems and their abundance may be genotype-dependent, which our study investigates across cultivars.
Reviewer Comment 19: L346-348: Please explain how can AMF produce spores without colonizing roots? Spores require carbon from host plant.
Author Response 19: We will refine the discussion to address this. The high spore density likely represents a legacy bank from previous vegetation or past colonization events. Under high soil P conditions (as found), AMF symbiosis is suppressed. The carbon for maintaining or slowly turning over this spore bank could come from residual carbon in the soil organic matter or from very low-level, transient/aborted colonization attempts insufficient for root staining detection.
Reviewer Comment 20: Please also better explain why coffee doesn't need symbiosis or how AMF "perceive" actually this?
Author Response 20: We will revise the text to avoid implying coffee "doesn't need" symbiosis. Instead, we will state: "Under conditions of high phosphorus and potassium availability, the cost-benefit balance of the AMF symbiosis may shift, reducing the plant's carbon allocation to and dependency on AMF, and/or leading the fungus to perceive a less favorable environment for successful colonization, thus prioritizing spore dormancy."
Reviewer Comment 21: Please note that wheat is annual and coffee is perennial, therefore AMF dynamics differ well. I think that the current format of discussion need serious revision and careful consideration. Based on these as well those previously mentioned I recommend that also the conclusions section to be well considered.
Author Response 21: We thank the reviewer for this critical point. We will revise the discussion to remove the inappropriate analogy with annual wheat systems and instead frame the observed AMF dynamics (high spore bank, low colonization) within the context of perennial crop ecology, long-term soil feedbacks, and nutrient management. The conclusions will also be carefully reviewed to ensure they accurately reflect the study's findings and their implications for perennial coffee cultivation.
We deeply appreciate the reviewer's rigorous critique, which has substantially improved the quality and credibility of our manuscript. We hope our revisions and explanations are satisfactory
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsI would like to thank the authors for their thorough and careful consideration of my comments. I am pleased to see that they have been fully addressed; therefore, I have no further comments in this regard.
Upon rereading the revised manuscript, I noticed one minor technical inaccuracy. In the Abstract (lines 15–20), when listing the forms P, N, and K, I suggest first presenting their total forms, followed by the available ones.
Author Response
Comment 1: Upon rereading the revised manuscript, I noticed one minor technical inaccuracy. In the Abstract (lines 15-20), when listing the forms P, N, and K, I suggest first presenting their total forms, followed by the available ones.
Response 1: Thank you for catching this inaccuracy and for the constructive suggestion. We have revised the sentence in the Abstract accordingly. The list of soil chemical properties now presents the total forms of nitrogen, phosphorus, and potassium first, followed by their available forms, as recommended.
Revised text (L17-20): “analyzed soil chemical properties—including pH, organic matter (OM), total nitrogen (TN), total phosphorus (TP), total potassium (TK), alkali-hydrolyzable nitrogen (AN), available phosphorus (AP), available potassium (AK), and slowly available potassium (SK)”
Author Response File: 
Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsDear Authors,
Thank you very much for considering to improve the "Correlation between rhizosphere soil properties and microbial communities of different Coffea arabica cultivars" manuscript. Reading carefully the new version of this manuscript I observed few aspects that in my opinion could be considered. Please find them listed as follows:
- L44-47: This is unclear for me at least, "fundamentally shift" is vague and lacks specificity. What specific shift is being proposed? From what to what? Please provide clarification and better formulate
- L79-86:n Here in my opinion the manuscript should provide specific citations showing what has been done and explicitly state what has not been investigated. The statement from L80-82 needs citation. However, the current form does not cite specific studies to demonstrate the gap, it does not explain why this gap exists - is because of methodological limitations? recent technology?, also it does not quantify "limited" - how many studies exist actually or were considered? These needs consideration in my opinion
- L87-96: Here in my opinion the manuscript should clearly present the addressed objectives/hypothesis and do not mix applied methodologies
- L116-118: Why 15 cm? Coffee root architecture extends beyond this. Why 10-30 cm depth variation within the same sample? Please consider to include justification
- L400-401: Please consider to address why no colonization occurred and acknowledge potential methodological limitations like detecting low colonization rates
- Please consider that RDA shows correlation, it doesn't account for co-correlation among variables - I recommend better formulation
- L506-509: This sounds as recommendation but in that case it should take into account also the yield data. Please careful consider the practical recommendations
Author Response
Comment 1: L44-47: This is unclear for me at least, “fundamentally shift” is vague and lacks specificity. What specific shift is being proposed? From what to what? Please provide clarification and better formulate.
Response 1: Thank you for this comment. We have revised the sentence to clarify the proposed shift in approach. The text now explicitly states a shift “from a management model dependent on chemical inputs toward a systemic management strategy based on the regulation of rhizosphere microbial ecology,” thereby providing the specificity requested.
Revised text (L45-50): “To address these challenges, it is essential to shift from a management model dependent on chemical inputs toward a systemic management strategy based on the regulation of rhizosphere microbial ecology. This necessitates a focused attention on the rhizosphere—a critical interface for interactions among plant roots, soil, and microorganisms, which plays a central role in plant health and ecosystem functioning.”
Comment 2: L79-86: Here in my opinion the manuscript should provide specific citations showing what has been done and explicitly state what has not been investigated. The statement from L80-82 needs citation. However, the current form does not cite specific studies to demonstrate the gap, it does not explain why this gap exists – is because of methodological limitations? recent technology?. also it does not quantify “limited” – how many studies exist actually or were considered? These needs consideration in my opinion.
Response 2: We thank the reviewer for this detailed suggestion. The entire paragraph has been rewritten to address all points raised: 1) Specific citations for beneficial microbes in coffee rhizosphere have been added; 2) The gap is explicitly defined as the lack of systematic, multi-cultivar studies integrating all microbial groups and soil properties, supported by citations of studies with a narrower focus; 3) The reason for the gap is attributed to methodological evolution from culture-dependent methods to recent high-throughput sequencing; 4) The term “limited” is quantified by stating that studies adhering to the required rigorous design are “notably scarce.”
Revised text (L77-96): “Furthermore, beneficial microorganisms such as Trichoderma [39], Bacillus [40], and Actinomycetes [41] have been identified in the coffee rhizosphere. However, current research on coffee remains constrained within specific paradigms. Relevant studies typically focus on a single microbial group—for example, analyzing arbuscular mycorrhizal fungi and associated fungal communities under different management practices [42], or isolating specific biocontrol agents such as Trichoderma [39] or Actinomycetes [41]—or on a single environmental driver, such as assessing the response of bacterial communities to altitudinal gradients [43]. Consequently, there is a clear lack of systematic research that simultaneously investigates how multiple commercially important Coffea arabica cultivars shape the physicochemical environment of the rhizosphere and the integrated structure of bacterial, fungal, and arbuscular mycorrhizal fungal communities. This gap can be attributed to methodological evolution. Early culture-dependent methods [40,41] possess inherent limitations in capturing microbial community complexity, while the application of high-throughput sequencing technology for holistic microbiome analysis in coffee rhizosphere research has only recently been developed [42,43]. To elucidate the correlations and differences in rhizosphere soil physicochemical properties and microbial communities among different Coffea arabica cultivars, it is essential to conduct multi-cultivar, replicated field comparisons under uniform conditions. Nevertheless, studies adhering to such a rigorous design remain notably scarce.”
Comment 3: L87-96: Here in my opinion the manuscript should clearly present the addressed objectives/hypothesis and do not mix applied methodologies.
Response 3: We agree and have revised this section. The study objectives are now clearly and separately listed first. The methodology employed to achieve them is stated in a subsequent sentence.
Revised text (L97-103): “To address this knowledge gap, the objectives of this study were as follows: (1) to examine the differences in arbuscular mycorrhizal fungi (AMF) colonization and spore density among different cultivars; (2) to assess the variations in rhizosphere soil properties and microbial diversity; and (3) to analyze the influence of soil properties on microbial community structure. To achieve these objectives, soil chemical analysis and high-throughput sequencing techniques were integrated within a uniformly designed field experiment.”
Comment 4: L116-118: Why 15 cm? Coffee root architecture extends beyond this. Why 10-30 cm depth variation within the same sample? Please consider to include justification.
Response 4: Thank you for pointing this out. We have added justifications for the chosen sampling distance and depth. We also clarified the measures taken to avoid interference from adjacent plants.
Revised text (L123-140): “Sampling was conducted on September 24, 2023, during the late rainy season to capture a relatively stable rhizosphere microbial community. Soil and root samples were collected at a distance of 15 cm from the coffee plant base—targeting the rhizosphere zone with higher fine root density—and at a depth of 10–30 cm to encompass the main soil volume occupied by absorbing roots. To avoid overlap between the rhizosphere zones of adjacent plants, sampled coffee plants were spaced at least 3 m apart both within and between rows. Sampling points (15 cm from the plant base) were oriented away from neighboring plants to ensure that the collected rhizosphere soil originated exclusively from the target cultivar. A five-point sampling method was applied per cultivar. Samples from the five points were thoroughly mixed to form one composite replicate. Three biological replicates were prepared for each of the eight coffee cultivars, resulting in a total of 120 sampled plants (8 cultivars × 5 plants per composite sample × 3 replicates), i.e., 15 individual plants per cultivar. Before sampling, surface debris was removed. Soil within the target depth was then excavated using a small shovel. Roots were gently shaken to dislodge adhering soil, which was defined as the rhizosphere soil. Both the rhizosphere soil and the corresponding roots were separately placed into pre-labeled zip-lock bags. All samples were immediately stored in an icebox and transported to the laboratory.”
Comment 5: L400-401: Please consider to address why no colonization occurred and acknowledge potential methodological limitations like detecting low colonization rates.
Response 5: We have added explanations in the discussion section to address both points raised by the reviewer: 1) the inhibitory role of high soil available phosphorus, supported by a citation, and 2) the acknowledgment of the potential technical limitation of the staining method used.
Revised text (L418-420 & L431-433): “Since high phosphorus availability is a well-documented suppressor of AMF colonization, the elevated soil available phosphorus levels measured in this study (Table 1) likely constitute a major factor inhibiting colonization[49]... Additionally, it is important to acknowledge the methodological limitation of the trypan blue staining technique used, which may fail to detect early or low-intensity colonization events.”
Comment 6: Please consider that RDA shows correlation, it doesn’t account for co-correlation among variables – recommend better formulation.
Response 6: We thank the reviewer for this important methodological note. A statement clarifying the nature of the RDA results has been inserted at the beginning of the relevant paragraph. Furthermore, the terminology used to describe the relationships has been adjusted from “influenced/affected” to “correlated with / associated with” to better reflect the correlative nature of the analysis.
Revised text (L474-496): “In recent years, growing evidence has highlighted the close relationship between coffee rhizosphere soil factors and microbial communities. It should be noted that the redundancy analysis (RDA) conducted in this study reveals statistical correlations between soil variables and microbial community composition, but does not account for potential co-correlation among the explanatory variables. Our analysis identified that OM, TN, AK, ASK, and AN were significantly correlated with the fungal community; TP, AP, AK, and ASK showed significant correlations with the bacterial community; and TK was strongly correlated with the AMF community. In contrast, pH exhibited only a weak correlation with all communities. This pattern can be interpreted through the mechanism of coffee roots maintaining pH balance during growth by increasing NO₃⁻ uptake and HCO₃⁻ release or regulating the NH₄⁺/NO₃⁻ uptake ratio [43]. Supporting this, studies on coffee rhizosphere soils at different altitudes found that OM content was significantly positively correlated with fungal diversity at high altitudes (1400 m-1600 m), while TN at low altitudes (800 m-1000 m) was associated with shifts in community composition, likely through promoting the proliferation of saprotrophic fungi (e.g., Trichoderma). ASK was linked to indirect effects on fungal community structure in acidic soils (pH 4.8-5.2) via regulating the activity of sulfur-oxidizing bacteria [61]. Similarly, research on rhizosphere soils of C. canephora and C. arabica in Malang, East Java, Indonesia, demonstrated that TP and AP were significantly correlated with bacterial community structure, potentially through promoting the proliferation of phosphate-solubilizing bacteria (e.g., Pseudomonas putida). In alkaline soils (pH 6.8-7.2), ASK was associated with enhanced bacterial mineralization of organic phosphorus, possibly by regulating the activity of sulfur-reducing bacteria [62].”
Comment 7: L506-509: This sounds as recommendation but in that case it should take into account also the yield data. Please careful consider the practical recommendations.
Response 7: We appreciate the reviewer's caution. The concluding sentence has been modified to frame the recommendation within the context of future research that integrates rhizosphere ecology with yield evaluation, rather than as a direct practical recommendation based solely on the current data.
Revised text (L540-543): “Thus, we recommend that these two varieties be prioritized as core subjects in future breeding programs that integrate rhizosphere ecology with yield evaluation, thereby exploring pathways toward the dual objectives of productivity and sustainability.”
Author Response File: Author Response.pdf
