Impact of Land Use Types on Soil Physico-Chemical Properties, Microbial Communities, and Their Fungistatic Effects

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review for the article “Impact of land use intensity and soil microbiome on the fungi-2 stasis capacity against pathogenic and beneficial fungi” of Soil Systems, by Iacomino et al. 2024.

This manuscript presents an engaging study on soil fungistasis, examining how different land uses (ecosystems), physical and chemical soil factors and their microbial communities influence the soil’s ability to suppress saprophytic behaviors of pathogenic and beneficial fungi. By reviewing various ecosystems, which include agricultural and natural soils, the authors provide insights into how different management practices affect soil’s natural defenses. This work has meaningful implications for sustainable agriculture, offering practical data-backed recommendations on improving soil health through microbiome management with nutrient applications. The manuscript is well-written, introduced, justified, and methodologically well-studied. The results are significant and interesting, however, some points could be improved to strengthen the manuscript’s publication.

Strengths

The study presents a topic in sustainable agriculture: understanding soil’s natural defenses against pathogens. Given the current shift toward eco-friendly agriculture, this research is highly relevant, offering insights that could help reduce reliance on chemicals. Moreover, the scope of this study is well established, spanning multiple ecosystems with very different characteristics and management. Comparing both natural and agricultural soils, provides a well-rounded perspective on fungistasis, highlighting differences in natural suppression abilities and those affected by intensive land use. In addition, The researchers took an innovative approach by using varied glucose concentrations to examine the role of nutrients in fungistasis. This approach sheds light on how fungal growth is influenced by external carbon sources, supporting the idea that nutrient competition plays a major role in fungistasis than another hypothesis such as the inhibition components and antagonism. Additionally, the use of multiple statistical methods (PERMANOVA, ANOVA, PCA, Simper, etc) provides a solid foundation for analyzing the relationships between soil properties, microbiome composition, and fungistasis.

Weaknesses

The sample sizes vary across ecosystems, with some soils, such as greenhouse samples, overrepresented. This imbalance may impact statistical power and could balance the results, particularly in the PERMANOVA and PCA analyses. I recommend a more in-depth discussion of this limitation, including potential strategies like resampling or sensitivity analyses to account for the imbalance.

Regarding microbiome scope, the study only examines bacterial communities, even though fungal interactions are known to be relevant to fungistasis. Authors must explain why they are not included in ITS. Sequencing fungal communities would enrich the study’s findings, providing a more holistic view of microbial interactions impacting fungistasis. Without this, the study’s conclusions lack ecological depth. Maybe authors could suggest in future steps to investigate deeper on that.

While PERMANOVA and PCA are appropriate, using a DistML analysis, authors could reveal more nuanced variations in microbial community composition and soil physicochemical factors related to fungistasis. That is, adding DistML analysis could help to better understand how soil factors explain microbial communities and fungistatic variations among the different LUTs. The study doesn’t sufficiently address how pH, moisture, and temperature variations might influence microbial activity. DistML analysis could help with that.

Errors and Suggested Corrections

Inconsistent Terminology: Terms like “fungistasis relief” and “restoration of fungistasis” are used inconsistently, potentially confusing readers. Define these terms clearly in the introduction and maintain consistent language throughout the paper.

The results section omits some statistical details (e.g., F-values, p-values), particularly for ANOVA post-hoc tests. Including these specifics would increase transparency, allowing readers to better assess the strength of the findings.

Minor typos and inconsistent capitalization (e.g., “16s rRNA” instead of “16S rRNA”) slightly detract from the manuscript’s professionalism. Proofread and standardize formatting to improve readability and presentation.

Suggested Improvements

Sampling size: Balancing sample sizes across different soil types or using resampling techniques like bootstrapping would enhance the study’s robustness, allowing more reliable comparisons across ecosystems.

Discussing the lack of fungal community data (e.g., ITS sequencing) and the importance of studying it in the future would provide a fuller picture of microbial interactions affecting fungistasis, making the study’s conclusions more ecologically meaningful.

Incorporating DistML would improve the resolution of microbial community analysis, capturing finer compositional differences tied to fungistasis.

Expand the discussion on practical applications. An extended discussion on Trichoderma as a biocontrol agent, with specific recommendations on application rates and ideal soil conditions, would enhance the practical value for agriculture.

Author Response

Reviewer #1
Review for the article “Impact of land use intensity and soil microbiome on the fungistasis capacity against pathogenic and beneficial fungi” of Soil Systems, by Iacomino et al. 2024.

This manuscript presents an engaging study on soil fungistasis, examining how different land uses (ecosystems), physical and chemical soil factors and their microbial communities influence the soil’s ability to suppress saprophytic behaviors of pathogenic and beneficial fungi. By reviewing various ecosystems, which include agricultural and natural soils, the authors provide insights into how different management practices affect soil’s natural defenses. This work has meaningful implications for sustainable agriculture, offering practical data-backed recommendations on improving soil health through microbiome management with nutrient applications. The manuscript is well-written, introduced, justified, and methodologically well-studied. The results are significant and interesting, however, some points could be improved to strengthen the manuscript’s publication.

ANSWER: Thank you for your thoughtful and positive feedback on our manuscript. We are delighted to hear that you found our study engaging and appreciated the insights it provides into soil fungistasis across different ecosystems and land-use practices. We are also grateful for your constructive suggestions on areas that could be improved to strengthen the manuscript. We will carefully address these points to enhance the quality and clarity of our work.

2)Strengths
The study presents a topic in sustainable agriculture: understanding soil’s natural defenses against pathogens. Given the current shift toward eco-friendly agriculture, this research is highly relevant, offering insights that could help reduce reliance on chemicals. Moreover, the scope of this study is well established, spanning multiple ecosystems with very different characteristics and management. Comparing both natural and agricultural soils, provides a well-rounded perspective on fungistasis, highlighting differences in natural suppression abilities and those affected by intensive land use. In addition, The researchers took an innovative approach by using varied glucose concentrations to examine the role of nutrients in fungistasis. This approach sheds light on how fungal growth is influenced by external carbon sources, supporting the idea that nutrient competition plays a major role in fungistasis than another hypothesis such as the inhibition components and antagonism. Additionally, the use of multiple statistical methods (PERMANOVA, ANOVA, PCA, Simper, etc) provides a solid foundation for analyzing the relationships between soil properties, microbiome composition, and fungistasis.

ANSWER: Again, thank you for your positive and detailed feedback on our manuscript. The recognition of the robust statistical framework and the role of nutrient competition in fungal suppression is especially encouraging.

3)Weaknesses
The sample sizes vary across ecosystems, with some soils, such as greenhouse samples, overrepresented. This imbalance may impact statistical power and could balance the results, particularly in the PERMANOVA and PCA analyses. I recommend a more in-depth discussion of this limitation, including potential strategies like resampling or sensitivity analyses to account for the imbalance.

ANSWER: As noted in the Materials & Methods section, the number of samples collected varied across ecosystems due to logistical constraints, with the greenhouse ecosystem being overrepresented. It is important to note, however, that this imbalance does not affect the core experimental outcomes. After the soil samples were collected, soil extracts were prepared in equal numbers for each soil ecosystem to test their fungistasis capacity against the two fungi included in the study. These experiments were performed independently of the metagenomics analyses. Therefore, the variation in the number of replicates is relevant only to the metagenomics data and does not impact the experiments evaluating fungistasis.
In our study, the PERMANOVA analysis was employed to assess differences in bacterial community composition across ecosystems. Importantly, PERMANOVA does not inherently consider differences in the number of replicates between groups; it is robust to unequal sample sizes under most circumstances, as it is based on permutation testing rather than direct comparisons of group means. This minimizes the risk of skewed results due to unequal replication. For the PCA analysis, we emphasize that it was based on aggregated bacterial diversity indices and community composition. To ensure comparability across ecosystems, bacterial diversity and composition metrics were calculated for the same number of treatments, not replicates. This approach aligns with our broader analytical framework, as PCA was conducted jointly with chemical parameters and fungistasis capacity, focusing on ecosystem-level characteristics rather than replicate-level variation.

Regarding microbiome scope, the study only examines bacterial communities, even though fungal interactions are known to be relevant to fungistasis. Authors must explain why they are not included in ITS. Sequencing fungal communities would enrich the study’s findings, providing a more holistic view of microbial interactions impacting fungistasis. Without this, the study’s conclusions lack ecological depth. Maybe authors could suggest in future steps to investigate deeper on that.

ANSWER: We fully agree that fungal interactions play a critical role in soil ecosystems and can significantly impact fungistasis. While examining fungal communities through ITS sequencing would indeed provide a more holistic view of microbial interactions, our main hypothesis for this work was to explore the bacterial diversity and composition in relation to fungistasis, which has been less extensively studied compared to fungal dynamics.
This sentence was added to the Conclusions section: “Finally, future studies should incorporate fungal community analyses through ITS sequencing to provide a more comprehensive understanding of the microbial interactions driving soil fungistasis”, lines 473-476.

While PERMANOVA and PCA are appropriate, using a DistML analysis, authors could reveal more nuanced variations in microbial community composition and soil physicochemical factors related to fungistasis. That is, adding DistML analysis could help to better understand how soil factors explain microbial communities and fungistatic variations among the different LUTs. The study doesn’t sufficiently address how pH, moisture, and temperature variations might influence microbial activity. DistML analysis could help with that.

ANSWER: We appreciate the reviewer’s insightful suggestion regarding the use of DistML analysis to further explore microbial community composition and its relationship with soil physicochemical factors, including pH, moisture, and temperature. While we acknowledge the potential value of DistML in uncovering nuanced patterns, the scope of our current study is focused on leveraging PERMANOVA and PCA, which are well-suited to our dataset and research objectives. Expanding the analysis to include DistML would require additional computational resources, expertise, and time beyond the scope of this work. That said, we believe the current analyses sufficiently capture the relationships between microbial communities and soil factors within the context of fungistasis. The inclusion of pH, moisture, and temperature variations in our discussion reflects their importance, and we have emphasized their potential roles based on existing data. Future studies could indeed benefit from a deeper exploration using tools like DistML, and we will consider this approach in subsequent research to build upon our findings.

Errors and Suggested Corrections
Inconsistent Terminology: Terms like “fungistasis relief” and “restoration of fungistasis” are used inconsistently, potentially confusing readers. Define these terms clearly in the introduction and maintain consistent language throughout the paper.

ANSWER: We would like to clarify for the reviewer that, as defined in our abstract, fungistasis relief refers to a substantial increase in fungal growth, indicating a loss of the soil's inhibitory capacity. In contrast, fungistasis restoration refers to the opposite process, the recovery or return of fungistasis, which is the soil's capacity to inhibit fungal growth. We have revised the manuscript to ensure these terms are used consistently and accurately throughout the text: “Specifically, we found that, regardless of fungal species, a pulse application of glucose causes a temporary reduction in soil fungistasis (i.e., fungistasis relief), and the time required for fungistasis restoration (i.e., the recovery of soil's inhibitory capacity) varies with soil quality.” Lines 366-369.

The results section omits some statistical details (e.g., F-values, p-values), particularly for ANOVA post-hoc tests. Including these specifics would increase transparency, allowing readers to better assess the strength of the findings.

ANSWER: Done. The following sentences were adjusted in the manuscript: “The non-metric multidimensional scaling analysis (NMDS) of bacterial structure (Figure 2C) revealed clear separations among ecosystems (Fvalue= 14.25, Pvalue=0.001).” Lines 281-282.
“In the fungistasis bioassays, soil type, glucose application rate and incubation times had statistically significant effects on the growth of the two fungal species (three-way ANOVA, P < 0.01 for all three factors).” Lines 314-316.

Minor typos and inconsistent capitalization (e.g., “16s rRNA” instead of “16S rRNA”) slightly detract from the manuscript’s professionalism. Proofread and standardize formatting to improve readability and presentation.

ANSWER: We thank the reviewer for highlighting such inconsistencies. We dealt with them in order to improve the manuscript’ professionalism.

Suggested Improvements
Sampling size: Balancing sample sizes across different soil types or using resampling techniques like bootstrapping would enhance the study’s robustness, allowing more reliable comparisons across ecosystems.

ANSWER: As mentioned earlier, the sample size in our study is not a critical issue because we used soil extracts with the same number of replicates across all ecosystems for the fungistasis experiments. The metagenomics analysis was included primarily to provide an overview of how the structure of bacterial communities varies across ecosystems, rather than to serve as the primary focus of the study.

Discussing the lack of fungal community data (e.g., ITS sequencing) and the importance of studying it in the future would provide a fuller picture of microbial interactions affecting fungistasis, making the study’s conclusions more ecologically meaningful.

ANSWER: The limitation was mentioned in the Conclusions section as highlighted in the previous comment of the reviewer.

Incorporating DistML would improve the resolution of microbial community analysis, capturing finer compositional differences tied to fungistasis.

ANSWER: We already answered this point in the previous comments.

Expand the discussion on practical applications. An extended discussion on Trichoderma as a biocontrol agent, with specific recommendations on application rates and ideal soil conditions, would enhance the practical value for agriculture.

ANSWER: Done. We would like to inform the reviewer that the whole discussion section was adjusted as required: “This study, conducted with two fungal species and eight soil types spanning a broad range of physical, chemical, and microbiological traits, demonstrated that soil fungistasis responds qualitatively similarly but quantitatively differently depending on soil texture and microbiome composition. Specifically, we found that, regardless of fungal species, a pulse application of glucose causes a temporary reduction in soil fungistasis (i.e., fungistasis relief), and the time required for fungistasis restoration (i.e., the recovery of soil's inhibitory capacity) varies with soil quality. Our results further highlight that soil amendments with organic carbon, such as glucose, have concentration- and timescale-dependent effects on fungistasis: negative in the short term (e.g., hours) and positive in the medium term (e.g., days). A rapid but temporary relief of soil fungistasis following a pulse application of organic amendments has been documented in early studies [24, 25] and corroborated by more recent research [10, 26]. Furthermore, the intensity of fungistasis relief has been shown to depend on the biochemical properties of the applied organic amendments, with the fastest and most pronounced relief occurring when labile carbon-rich materials are used [5]. However, focusing solely on short-term responses provides only a partial view of the role of organic amendments. Indeed, organic amendments have been observed to exert positive effects on fungistasis in the medium and long term. For instance, Bonanomi et al. [27] demonstrated that repeated applications of organic amendments accelerate fungistasis restoration, a phenomenon linked to enhanced microbial activity that rapidly depletes the labile carbon stock in the soil.
The study underscores as well the practical implications of understanding fungi-stasis for sustainable agriculture. Organic amendments, such as labile carbon inputs, should be applied with careful consideration of their short- and long-term effects on soil fungistasis. For example, while glucose applications temporarily reduce fungi-stasis, they can enhance it over the medium term by stimulating microbial activity and depleting labile carbon stocks. An additional avenue for sustainable agriculture is the application of biocontrol agents, including both fungi and bacteria. In this context, Trichoderma, a well-known biocontrol agent, plays a dual role. As observed in our study, Trichoderma harzianum exhibited a slower fungistasis recovery in some soils compared to Botrytis cinerea, likely due to its greater saprophytic capacity. This suggests that Trichoderma's efficacy is closely tied to the availability of labile carbon and the composition of the soil microbiome. The findings of our study align with existing knowledge, confirming that the application of labile carbon, such as glucose, leads to a temporary disruption of fungistasis, which, however, is restored within a few days. This phenomenon appears to be generalizable, as it was observed across all studied soils and with both fungal species. Despite this general trend, our study revealed considerable variability among soils in terms of the intensity of fungistasis disruption and the time required for its recovery. For instance, in the case of Botrytis cinerea, the inten-sity of fungistasis disruption was significantly higher in soils from greenhouse, horti-cultural, and Quercus forest systems compared to those from grassland, shrubland, Pi-nus forest, and olive orchards. Similarly, for Trichoderma harzianum, the intensity of disruption varied widely among soils, with maximum levels in greenhouse and Fagus forest soils and minimum levels in grassland, shrubland, and olive orchard soils. The rate of fungistasis recovery also exhibited notable variability: it was rapid in soils from grassland, shrubland, and olive orchards but slower in those from greenhouse and Fagus forest systems. These observations underscore the critical role of soil type in shaping the dynamic response of fungistasis.
Our study, which analyzed eight soil types and 21 physical, chemical, and biochemical parameters, provides important insights into the factors influencing fungi-stasis. Notably, sandy soils, irrespective of their organic matter, nutrient, and microbial biomass content, were found to exhibit weaker fungistatic effects. Additionally, these soils required longer periods to re-establish fungistasis following glucose application. This contrasts with findings from a previous study conducted in Finland, which re-ported no significant differences in fungistasis among six soils toward Fusarium culmorum [28]. However, the Finnish study explored a narrower texture gradient com-pared to our investigation. The reduced fungistasis observed in sandy soils relative to silty or clayey soils could be attributed to differences in the resident microbiome or in organic molecule adsorption processes. Surprisingly, fungistasis did not positively correlate with overall enzymatic activity or microbial biomass in our study. Previous research on a limited number of soils [29] suggested that higher microbial and enzymatic activity facilitates the rapid recovery of fungistasis, as the microbiome efficient-ly metabolizes labile carbon, reinstating fungistatic conditions. The apparent contra-diction in our results may stem from differences in soil metabolic capacities. Agroecosystems and grasslands, which typically receive higher inputs of labile organic carbon through litterfall from lignin-poor tissues and root exudates of herbaceous species, appear more adept at rapidly restoring fungistasis. In contrast, Fagus forest soils, despite their high organic carbon content and microbial biomass, required the longest time to restore fungistasis toward T. harzianum. This discrepancy may be explained by the dominance of lignified, labile-carbon-poor organic inputs in Fagus forest ecosystems [30]. It is plausible that the fungal-dominated microbiome in these soils is less efficient at glucose catabolism compared to the microbiomes in grasslands or agroecosystems. Further studies are necessary to validate this hypothesis and elucidate the underlying mechanisms governing fungistasis dynamics in different soil types.
Notably, both fungal species studied (B. cinerea and T. harzianum) exhibited remarkably similar responses to the organic amendment. Specifically, a pronounced re-lief from fungistasis was observed following the glucose pulse, with restoration occur-ring within 168 hours for most soils. These findings support the nutrient-dependent nature of the selected fungal species, as their spores require an external carbon source to initiate germination [31]. Despite these overarching similarities, quantitative differences in the responses of the two fungi to the organic amendments were observed. B. cinerea showed a relatively shorter fungistasis restoration period compared to T. harzianum. This result is consistent with the known greater saprophytic capabilities of T. harzianum relative to the pathogenic B. cinerea [32]. Future research could extend this investigation by examining the fungistasis responses of fungal pathogens with high saprophytic capacities (e.g., Sclerotinia spp. or Fusarium spp.) compared to those with lower saprophytic tendencies (e.g., B. cinerea or Verticillium spp.). Additionally, as this study focused on nutrient-dependent fungal species, it remains unclear whether fungi-stasis is primarily driven by nutrient depletion or the presence of potential soil inhibitors [4]. Addressing this limitation in future studies would provide a more comprehensive understanding of the mechanisms underlying fungistasis.” Lines 364-452.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The paper is well written. Please align the paper to the technical requirements of the journal.

Best regards.

Author Response

Reviewer #2
Dear authors,
The paper is well written. Please align the paper to the technical requirements of the journal.
Best regards.

ANSWER: Thank you for your positive feedback on our manuscript. We have carefully reviewed the journal’s technical requirements and made the necessary adjustments to ensure alignment with the formatting and submission guidelines.

Reviewer 3 Report

Comments and Suggestions for Authors

The author explored the fungistasis capacity of soil on two fungi through extensive sampling and in-depth soil investigation, combined with indoor experiments. Although the author's workload is enormous and the data is abundant, the manuscript has not fully established the logical connections between the data, and its contribution to the manuscript topic is insufficient. The content that truly demonstrates the core of the paper - fungal fungistasis - is not sufficient and the evidence is not very solid. The entire manuscript gives me a feeling of incompleteness, and there are many errors in the details, which makes me fully concerned about whether the article can be published smoothly.

 

The detail comments as follows:

 

Title: Although there were significant differences in land use intensity among the 8 types of soils tested, the manuscript did not quantify and rank them. It only explored the differences in fungal fungistasis from the perspective of land use types and suggested replacing land use intensity. At the same time, the conversion of land use types drives the transformation of soil physicochemical properties and microorganisms. Why does the title only emphasize microbial communities without soil physicochemical properties?

 

Line15: agroecosystem

 

Line18: Delete “a spectrum of”.

 

Line34: “along the land use intensity gradient” Based on what? How to quantify?

 

Line38-39: landscapes

 

Line94: carried out

 

Line96: hypotheses

 

Line104: includes

 

Figure1: Why are there no photos of pinus forests? Even if there is no filming, an explanation should still be obtained.

 

Table1: geographical coordinate

 

Line195: chimaera

 

Line243-270: The names of all land use types must be consistent throughout the manuscript, with the first letter of 'Forest' in lowercase.

 

Table2: The decimal point in the table makes me very confused. Is it a bit symbol? In addition, the C/N ratio and pH of Olive orchard, as well as the FDA of Fagus Forest, did not indicate significant results. What does CEC mean? Suggest the author to add the full name of all abbreviations (like NMDS, B. cinerea, T. harzianum, P2O5 and et al) at the first occurrence of the main body, even at Abstract section, to improve readability. Wrong spell of “Limestione”.

 

Line277-278: The highest Shannon index is still in the olive orchard, and the expression should be rigorous.

 

Line279: NMDS

 

Figure2: What do the F and P values in the NMDS chart represent? What method is used for calculation? By the way, not all statistical methods are mentioned in the methods section. It is recommended to verify and supplement them.

 

Figure3: I think the legend does not indicate relative abundance (0%~4000%?), but more like the number of sequences. Olive orchard, not Olive_Orchad.

 

Line311-313: This result should correspond to a graph or table, at least included in the Supplementary Materials.

 

Line321: “statistically” I can't see any trace.

 

Figure4&5: The same “,” in the numerical values of the legend and plot title.

 

Line345: There are not just a few factors that are positively correlated, right?

 

Line349-354: How is microbial composition quantified?

 

Figue6A: What does CSC mean?

 

 

Insufficient citation of articles in the past three years.

Comments for author File: Comments.pdf

Author Response

Reviewer #3

The author explored the fungistasis capacity of soil on two fungi through extensive sampling and in-depth soil investigation, combined with indoor experiments. Although the author's workload is enormous and the data is abundant, the manuscript has not fully established the logical connections between the data, and its contribution to the manuscript topic is insufficient. The content that truly demonstrates the core of the paper - fungal fungistasis - is not sufficient and the evidence is not very solid. The entire manuscript gives me a feeling of incompleteness, and there are many errors in the details, which makes me fully concerned about whether the article can be published smoothly.

ANSWER: Thank you for your valuable feedback and for highlighting key areas for improvement in our manuscript. We understand your concerns regarding the need to strengthen the logical connections between the data presented and the central theme of fungal fungistasis. To ensure the manuscript’s rigor, we will thoroughly review and correct any errors in detail, ensuring clarity and consistency throughout.
 
The detail comments as follows:
 
Title: Although there were significant differences in land use intensity among the 8 types of soils tested, the manuscript did not quantify and rank them. It only explored the differences in fungal fungistasis from the perspective of land use types and suggested replacing land use intensity. At the same time, the conversion of land use types drives the transformation of soil physicochemical properties and microorganisms. Why does the title only emphasize microbial communities without soil physicochemical properties?

ANSWER: We have revised the title following the reviewer’s suggestion to: “Impact of Land Use types on Soil Physico-Chemical Properties, Microbial Communities, and Their Fungistatic Effects”.
The new title now emphasizes the exploration of fungal fungistasis in relation to land use types, as well as the transformation of soil physicochemical properties and microbial communities. 
 
Line15: agroecosystem

ANSWER: Done. 
 
Line18: Delete “a spectrum of”.

ANSWER: Done. 
 
Line34: “along the land use intensity gradient” Based on what? How to quantify?

ANSWER: We established this land-use intensity gradient by transitioning from natural ecosystems to agricultural ones, with grassland positioned as an intermediate category. Although grassland is considered a natural ecosystem, it was included in this intermediate position due to its exposure to mowing practices. This classification was based on the parameters outlined in Table 1.
 
Line38-39: landscapes

ANSWER: Done.
 
Line94: carried out
 
ANSWER: Done.

Line96: hypotheses
 
ANSWER: Done. 

Line104: includes

ANSWER: Done.

Figure1: Why are there no photos of pinus forests? Even if there is no filming, an explanation should still be obtained.
 
ANSWER: The figure was adjusted to include Pinus ecosystem; we apologize for the mistake.

Table1: geographical coordinate

ANSWER: Done.

Line195: chimaera

ANSWER: Done.

Line243-270: The names of all land use types must be consistent throughout the manuscript, with the first letter of 'Forest' in lowercase.

ANSWER: Done.

Table2: The decimal point in the table makes me very confused. Is it a bit symbol? In addition, the C/N ratio and pH of Olive orchard, as well as the FDA of Fagus Forest, did not indicate significant results. What does CEC mean? Suggest the author to add the full name of all abbreviations (like NMDS, B. cinerea, T. harzianum, P2O5 and et al) at the first occurrence of the main body, even at Abstract section, to improve readability. Wrong spell of “Limestione”.

ANSWER: Done. The decimals were presented by points instead of comma. Moreover, we have defined the abbreviation CEC as "Cation Exchange Capacity" and expanded all abbreviations, at their first appearance in both the Abstract and in the table to enhance readability. Finally, the spelling error of "Limestione" has been corrected.

Line277-278: The highest Shannon index is still in the olive orchard, and the expression should be rigorous.

ANSWER: Done. The sentence was adjusted as follows: “Species richness and the Shannon index for bacterial communities varied significantly among the ecosystems studied (Figure 2A, B). Bacterial diversity was lowest in horticultural and grassland soils for both metrics. On the other hand, both species richness and Shannon index were highest in Olive orchard soils.” Lines 275-278.

 
Line279: NMDS

ANSWER: Done.
 
Figure2: What do the F and P values in the NMDS chart represent? What method is used for calculation? By the way, not all statistical methods are mentioned in the methods section. It is recommended to verify and supplement them.

ANSWER: We would like to clarify to the reviewer that the statistical analysis presented in the nMDS figure was already described in the methods section, specifically within the statistical analysis subsection, as follows: “To assess the significance of the ecosystems diversity in terms of soil bacterial communities, Permutational Multivariate Analysis of Variance (PERMANOVA) was conducted with 999 permutations.” Lines 229-232. Consequently, the PERMANOVA test was employed for the analysis of metabarcoding data, which encompasses a large dataset including all identified taxa. Additionally, the figure caption has been revised to incorporate the reviewer’s comment, as follows: “In the nMDS chart, the P- and F-values represent the results of the PERMANOVA test conducted with 999 permutations on the bacterial data.”.
 
Figure3: I think the legend does not indicate relative abundance (0%~4000%?), but more like the number of sequences. Olive orchard, not Olive_Orchad.

ANSWER: We would like to inform the reviewer that the values presented (ranging from 0 to 4000) represent the number of sequences for each taxon shown in the heatmap. These values reflect the relative abundance, which can be expressed either as a percentage or as the actual number of sequences identified and assigned to a specific taxon. To clarify, relative abundance refers to the proportion or count of a given taxon relative to the total number of sequences in the dataset, providing a measure of its representation within the community. In contrast, absolute abundance refers to the actual number of individuals or copies of a taxon present in a given volume or weight of the sample, which would require additional normalization based on total biomass or sample size. In this context, we are referring only to relative abundance. Moreover, the olive orchard was adjusted as highlighted.
 
Line311-313: This result should correspond to a graph or table, at least included in the Supplementary Materials.

ANSWER: We believed it would be more effective to reduce the number of tables by incorporating the ANOVA results directly into the text. Since this represents a single analysis with limited data, we felt it was not extensive enough to warrant a standalone table.

Line321: “statistically” I can't see any trace.

ANSWER: The use of the word "statistically" to describe the decrease in the growth of the fungal species refers back to the first line, where we initially reported the results of the statistical test. 

 Figure4&5: The same “,” in the numerical values of the legend and plot title.

ANSWER: Done.
 
Line345: There are not just a few factors that are positively correlated, right?

ANSWER: We would like to inform the reviewer that we have highlighted the factors showing a strong positive correlation, as indicated by the length of the arrows. These factors include organic carbon content, total nitrogen (N), iron (Fe), magnesium (Mg), calcium (Ca), and sodium (Na).
 
Line349-354: How is microbial composition quantified?

ANSWER: This sentence was added to the statistical analysis section for more clarity: “For bacterial diversity, the Shannon index was used as a measure, while community composition was analyzed using the first axis of a PCoA based on the Bray-Curtis dis-similarity matrix.” Lines 237-240.

Figue6A: What does CSC mean?

ANSWER: We apologize for the mistake, we meant CEC, which is cation exchange capacity, the figure was adjusted.

 
Insufficient citation of articles in the past three years.

ANSWER: Done. We have added the following references:

-Zandyavari, N., Sulaiman, M. A., & Hassanzadeh, N. (2024). Molecular characterization and biocontrol potential of Trichoderma spp. against Fusarium oxysporum f. sp. dianthi in carnation. Egyptian Journal of Biological Pest Control, 34(1), 1.

-Majou, D. (2024). Effects of carbon dioxide on germination of Clostridium botulinum spores. International Journal of Food Microbiology, 110958.

-Lijiahong, G.; Yalun, F.; Xiaohua, P.; Zhengkun, Y.; Mengke, Z.; Zhiyu, S.; Ning, G.; Shuangchen, C.; Junliang, C.; Bing, B.; et al. Biocontrol potential of Trichoderma harzianum against Botrytis cinerea in tomato plants. Biol. Control 2022, 174, 105019.

-Ayaz, M.; Li, C.-H.; Ali, Q.; Zhao, W.; Chi, Y.-K.; Shafiq, M.; Ali, F.; Yu, X.-Y.; Yu, Q.; Zhao, J.-T.; et al. Bacterial and fungal biocontrol agents for plant disease protection: Journey from lab to field, current status, challenges, and global perspectives. Molecules 2023, 28, 6735. 

-Gokul, A.; Mabaso, J.; Henema, N.; Otomo, L.; Bakare, O.O.; Klein, A.; Daniel, A.I.; Omolola, A.; Niekerk, L.A.; Nkomo, M.; et al. Sustainable agriculture through the enhancement of microbial biocontrol agents: Current challenges and new perspectives. Appl. Sci. 2023, 13, 6507.

-Weinmann, M., Bradáčová, K., & Nikolic, M. (2023). Relationship between mineral nutrition, plant diseases, and pests. In Marschner's Mineral Nutrition of Plants (pp. 445-476). Academic Press.

 

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

I am glad that the author has taken into account my suggestions in revising the manuscript. The manuscript has made some improvements, except for the incorrect decimal points in Figures 4 and 5.

 Furthermore, forgive me for being a detail oriented person, as some sampling point names are still inconsistent throughout the entire text, such as Olive Orchard.

Author Response

I am glad that the author has taken into account my suggestions in revising the manuscript. The manuscript has made some improvements, except for the incorrect decimal points in Figures 4 and 5.
Furthermore, forgive me for being a detail oriented person, as some sampling point names are still inconsistent throughout the entire text, such as Olive Orchard.

ANSWER: We would like to show our gratitude to the reviewer for his constructive comments that have helped improving the quality of our manuscript. 
Both figures were adjusted according to the reviewer's comment. Moreover, the all sampling point names were made consistent all over the manuscript. For more details, please check the track-version manuscript.