Native Grasses Enhance Topsoil Organic Carbon and Nitrogen by Improving Soil Aggregates and Microbial Communities in Navel Orange Orchards in China

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General Assessment

This study is well-structured and comprehensive, addressing an important topic in sustainable agriculture. The long-term experimental design and the combination of soil physicochemical, microbial, and gene-level analyses provide strong evidence supporting the authors’ conclusions

 

The title is overly long and complex. Suggest simplifying to focus on the main contribution, e.g.,

“Native Grasses Improve Soil Aggregates and Microbial Communities in Navel Orange Orchards in China”.

 

References

Citations are given as numbers (e.g., “3”, “5”, etc.) but without the brackets “[ ]”. The result is not clearly distinguish the citation numbers from the text.

 

Methodological Issues

Lack of Baseline Data

The study began in 2019, but it doesn’t clearly state whether baseline soil conditions were measured prior to treatment application. Without baseline data, it’s difficult to determine how much of the change in SOC/TN/etc. is due to the treatment versus natural variability.

 

Sampling

line 126 Sampling was conducted using a “pipe drill and ring knife”, but specific depth, positioning (e.g., between trees or near roots), and soil moisture at sampling are vague. The soil aggregate structure and microbial communities are highly variable based on microenvironment.

 

Soil physicochemical analysis, nutrient content and enzyme activity

The measurements are not reproductive because the parameters of the analysis are missing. Which instrument was used.  Line 181 analyzed chemically How?

Line 191 „The particulate organic and mineral bonded organic contents were measured using the dichromate oxidation method as previously described.” It was not described…

 

DNA Extraction and Processing

The line 222 No mention of negative controls, extraction blanks, or mock communities during microbial DNA extraction and sequencing. The Contamination or PCR bias can affect community structure results.

 

qPCR

The line 232 The standard curve preparation mentions dilution but lacks details on replication, primer efficiency, and normalization (e.g., to 16S or total DNA). Please provide details on standard curve R² values, efficiency, and any normalization methods.

 

Microbial Analysis Interpretation Limitations

The authors use PICRUSt2, BugBase, FUNGuild, and FAPROTAX to predict functions from 16S/ITS data. These are predictive, not directly measured, functional data. The manuscript should emphasize that functional assignments are inferred, not confirmed. Write about the limitations in database coverage and resolution.

Author Response

Author's Reply to the Review Report (Reviewer 1)

 

General Assessment

This study is well-structured and comprehensive, addressing an important topic in sustainable agriculture. The long-term experimental design and the combination of soil physicochemical, microbial, and gene-level analyses provide strong evidence supporting the authors’ conclusions

Response: Thank you for your support and affirmation of this research.

 

The title is overly long and complex. Suggest simplifying to focus on the main contribution, e.g.,

“Native Grasses Improve Soil Aggregates and Microbial Communities in Navel Orange Orchards in China”.

Response: Thank you for your suggestion. The title is really too long, your suggestion is very good, but it may ignore the core content of improving soil organic carbon and nitrogen, so we optimized it on the basis of your suggestion, and we have reduced 31 words to 21 words in page 1 lines 2-4. Please see the revised version. The statement was list as follow: “Native Grasses Enhance Topsoil Organic Carbon and Nitrogen by Improving Soil Aggregates and Microbial Communities in Navel Orange Orchards in China”.

 

References

Citations are given as numbers (e.g., “3”, “5”, etc.) but without the brackets “[ ]”. The result is not clearly distinguish the citation numbers from the text.

Response: Thank you for your suggestion. The manuscript (word version) was submitted contains brackets, but the PDF does not. Maybe there was an error in the format conversion process. Therefore, We reconvert the manuscript into PDF format to ensure the content accuracy.

 

Methodological Issues

Lack of Baseline Data

The study began in 2019, but it doesn’t clearly state whether baseline soil conditions were measured prior to treatment application. Without baseline data, it’s difficult to determine how much of the change in SOC/TN/etc. is due to the treatment versus natural variability.

Response: Thank you for your suggestion. We have added the baseline soil conditions in page 3 lines 125-128. Please see the revised version. The statement was listed as follow: “Prior to treatment implementation, the baseline soil conditions were: pH 5.48, soil organic carbon (SOC) 7.16 g kg⁻¹, total nitrogen (TN) 0.18 g kg⁻¹, available phosphorus (Olsen-P) 12.54 mg kg⁻¹, and available potassium (AK) 137.48 mg kg⁻¹”.

 

Sampling

line 126 Sampling was conducted using a “pipe drill and ring knife”, but specific depth, positioning (e.g., between trees or near roots), and soil moisture at sampling are vague. The soil aggregate structure and microbial communities are highly variable based on microenvironment.

Response: Thank you for your suggestion. We have added the detailed introduction of soil collection (collection depth, location, etc.) in page 3-4 lines 137-144. Please see the revised version. The statement was listed as follow: “Three replicate plots (20 m ×20 m each) were established per treatment in a randomized block design. Within each plot, five sampling trees were selected according to the "S" shape, and soil samples were collected at the crown drip line (avoiding fertilization holes) with a soil moisture content of 25.3–27.5% during sampling. Prior to sampling, the layer of litter was removed from each selected representative sampling point. Then three independent sampling methods were performed: digging a 30-cm-deep soil profile, collecting soil cores with a pipe drill, and obtaining undisturbed samples using a ring knife”.

 

Soil physicochemical analysis, nutrient content and enzyme activity

The measurements are not reproductive because the parameters of the analysis are missing. Which instrument was used.

Response: Thank you for your suggestion. We have added the analysis parameters of the Soil physicochemical analysis, nutrient content and enzyme activity in page 4 lines 165-183. Please see the revised version. 

The statement was listed as follow: “Soil pH was measured using a pH metre (FE28-Standard, Mettler Toledo, Switzerland) in a 1:2.5 soil: water (w/v) mixture. The soil organic carbon (SOC) content was measured by the dichromate oxidation method as described by Bao [14]. The total nitrogen (TN) content was measured using an automatic Kjeldahl analyzer (Kjeltec 8400, FOSS Corporation, Denmark). Available phosphorus (Olsen-P) was measured using the molybdenum blue method with an ultraviolet spectrophotometer (UV-3600, Shimadzu Corporation, Japan). Available potassium (AK) was determined by ammonium acetate leaching-flame photometry with a flame photometer (FP6400A, Shanghai precision scientific instrument co., ltd, China). The soil bulk density and porosity were measured using a soil ring knife as previously described [15].

The activities of urease, catalase, sucrase, acid phosphatase, and amylase in the soil were assayed as described by Wang et al. [16]. Soil urease activity was measured by indophenol blue colorimetry with urea as the substrate. Soil catalase activity was determined volumetrically by measuring residual hydrogen peroxide through potassium permanganate (KMnO₄) titration in the presence of sulfuric acid (H₂SO₄). Soil sucrase activity was measured by 3, 5-dinitro salicylic acid colorimetry with sucrose as the substrate. Soil acid phosphatase activity was determined using the disodium phenyl phosphate colorimetric method with pH 5.0 acetate buffer. Soil α-amylase activity was measured by 3, 5-dinitro salicylic acid colorimetry using soluble starch as the substrate. ”

 

Line 181 analyzed chemically How?

Response: Thank you for your suggestion. We have rewritten this sentence in page 5 lines 214-216. Please see the revised version. The statement was listed as follow: “The TN and SOC content of the dried water stable aggregates were then determined by automatic Kjeldahl analyzer and the dichromate oxidation method respectively as previously described.”

 

Line 191 The particulate organic and mineral bonded organic contents were measured using the dichromate oxidation method as previously described.” It was not described…

Response: Thank you for your suggestion. We have rewritten this sentence in page 5 lines 225-226. Please see the revised version. The statement was listed as follow: “The POC and MAOC contents were measured using the dichromate oxidation method as described by Bao [14].”

 

 

 

DNA Extraction and Processing

The line 222 No mention of negative controls, extraction blanks, or mock communities during microbial DNA extraction and sequencing. The Contamination or PCR bias can affect community structure results.

Response: Thank you for your suggestion. We have added the information in page 6 lines 261-264. Please see the revised version. The statement was listed as follow: “Thereafter, primers were designed and PCR amplification was performed based on the primers. During the amplification process, sterile water was used as a negative control, and the absence of bands in the control indicated that there was no environmental pollution during the experiment.”

 

 

 

qPCR

The line 232 The standard curve preparation mentions dilution but lacks details on replication, primer efficiency, and normalization (e.g., to 16S or total DNA). Please provide details on standard curve R² values, efficiency, and any normalization methods.

Response: Thank you for your suggestion. We have added the information in page 7 lines 276-280. Please see the revised version. The statement was listed as follow: “repeated three times. Among them, the amplification efficiencies of cbbL, AOB, nirS, nirK, and nirH were 91.03%, 104.60%, 98.81%, 100.29%, 100.56%, respectively. The R² values were 0.9963, 0.9994, 0.9999, 0.9979, 0.9993, respectively. The slopes were -3.5573, -3.2165, -3.3507, -3.3151, -3.3085, respectively. The Y-inter values were 41.051, 37.557, 38.601, 37.957, and 38.634, respectively.”

 

Microbial Analysis Interpretation Limitations

The authors use PICRUSt2, BugBase, FUNGuild, and FAPROTAX to predict functions from 16S/ITS data. These are predictive, not directly measured, functional data. The manuscript should emphasize that functional assignments are inferred, not confirmed. Write about the limitations in database coverage and resolution.

Response: Thank you for your suggestion. We have added the information in page 7-8 lines 312-324 and in page 15 lines 539-544. Please see the revised version.

The statements were listed as follow: “It combines genomic data (NCBI RefSeq) with functional annotations (KEGG/COG/PFAM), but its 16S rRNA reliance limits strain-level phenotypic resolution.

It annotates prokaryotic functions (>80 categories; C/N/P/S cycling, pathogenesis) across >4,600 species, but its 16S rRNA dependency excludes metagenomic data and strainspecific metabolic distinctions.

It classifies fungal trophic modes (patho-/symbio-/saprotrophic) into ecological guilds, but lacks higher-taxon predictions and dynamic functional profiling.” 

“According to the prediction of BugBase; According to the prediction of FUNGuild.”

 

 

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I have reviewed the manuscript entitled "Improved topsoil organic carbon and nitrogen content by altering soil aggregates' physicochemical properties and their associated microbial communities under the management of native grasses in Gannan navel orange orchards in China." In my opinion, this work requires significant improvements for acceptance.

1. The title is too long; please summarize it and make sure the title is related to the results obtained.
2. In a background section of the introduction, the authors should provide more information on the effect of carbon on the physicochemical and biological properties of the soil. The information provided must be associated with grasses under medium- and long-term studies. The authors should highlight current research in relation to other studies.
3. The authors should provide more information on soil classifiers.
4. The authors should mention having performed normality and homoscedasticity tests prior to data analysis (ANOVA). I ​​suggest they mention the tests used.
5. They should standardize the units. In the text they mention g/L, mg/L, in the tables they write g.cm3, g.kg-1, mg.kg-1. Review the entire document.
6. In my opinion, the authors should improve the figures. I suggest increasing the font size on the X and Y axes. Also, increase the font size of the internal descriptions of the figures, literals, etc.
7. Also, standardize the color of the figures. I suggest all be presented in color.
8. In the discussion, the authors should use recent research background. While there is valuable underlying information, it should be discussed with updated information (2020-2025).
9. In lines 545-561, 571-582, 598-550, don't just compare your results with others. You need to discuss them in depth. The authors should expand the discussion. Provide information that discusses the chemical processes involved in changes in soil properties.
10. In the conclusions, mention the limitations of the research and future prospects.

Author Response

Author's Reply to the Review Report (Reviewer 2)

 

 

I have reviewed the manuscript entitled "Improved topsoil organic carbon and nitrogen content by altering soil aggregates' physicochemical properties and their associated microbial communities under the management of native grasses in Gannan navel orange orchards in China." In my opinion, this work requires significant improvements for acceptance.

 

1. The title is too long; please summarize it and make sure the title is related to the results obtained.

Response: Thank you for your suggestion. We have rewritten the title in page 1 lines 2-4. Please see the revised version. The statement was listed as follow: “Native Grasses Enhance Topsoil Organic Carbon and Nitrogen by Improving Soil Aggregates and Microbial Communities in Navel Orange Orchards in China”.

 

2. In a background section of the introduction, the authors should provide more information on the effect of carbon on the physicochemical and biological properties of the soil. The information provided must be associated with grasses under medium- and long-term studies. The authors should highlight current research in relation to other studies.

Response: Thank you for your suggestion. We have added the information in page 2 lines 61-66. Please see the revised version. The statement was listed as follow: “SOC significantly influenced soil properties and ecosystem functions. SOC can directly regulate soil nutrient cycling and biological activity [5]. Its content influenced soil productivity and degradation processes, thereby affecting both crop yield and quality. Therefore, SOC played a pivotal role in maintaining soil quality and agricultural ecosystem balance[6,7] Moreover, green manure can alleviate the degradation of organic matter, and enhance its versatility [8]. ”

 

3. The authors should provide more information on soil classifiers.

Response: Thank you for your suggestion. We have added the information on soil classifiers in page 3 lines 113-116. Please see the revised version. The statement was listed as follow: “According to the method of Classification and Retrieval of Soil Systems in China (Third Edition), the soil in this area is dominated by a red soil subclass of red loam, which belongs to moist iron-rich soil, showing moderate desilication and iron-rich aluminization.”

 

4. The authors should mention having performed normality and homoscedasticity tests prior to data analysis (ANOVA). Isuggest they mention the tests used.

Response: Thank you for your suggestion. We have added the information in page 8 lines 328-332. Please see the revised version. The statement was listed as follow: “Prior to analysis, the normality of data distribution was verified using Shapiro-Wilk tests, and homoscedasticity was confirmed by Levene's test. The results were analyzed using two-way ANOVAs. The mean values of each treatment were subjected to multiple comparisons using the least significant difference (LSD) test (p < 0.05).”

 

5. They should standardize the units. In the text they mention g/L, mg/L, in the tables they write g.cm3, g.kg^-1, mg.kg^-1. Review the entire document.

Response: Thank you for your suggestion. We have standardized the units of manuscripts. Please see the revised version.

 

6. In my opinion, the authors should improve the figures. I suggest increasing the font size on the X and Y axes. Also, increase the font size of the internal descriptions of the figures, literals, etc.

Response: Thank you for your suggestion. We have increased the font size of the figure 1, figure 2, and figure 3.

 

7. Also, standardize the color of the figures. I suggest all be presented in color.

Response: Thank you for your suggestion. We have modified figure 1, figure 2, and figure 3.

 

8. In the discussion, the authors should use recent research background. While there is valuable underlying information, it should be discussed with updated information (2020-2025).

Response: We have cited new research background in the supplementary content in page 18-20 lines 608-677. Please see the revised version.

 

9. In lines 545-561, 571-582, 598-550, don't just compare your results with others. You need to discuss them in depth. The authors should expand the discussion. Provide information that discusses the chemical processes involved in changes in soil properties.

Response: Thank you for your suggestion. We have added the information in page 18 lines 608-614, page 19 lines 637-640, page 19-20 lines 674-677. Please see the revised version.

The statement was listed as follow: “This may be due to biological disturbance caused by the growth of local grassroots, resulting in loose soil [35]. Moreover, grass cover can intercept rainwater and promote water infiltration, improving the soil's water retention capacity.

This was because grass cover introduces plant residues and root biomass into the soil, leading to an increase in organic matter content [45]. In addition, MAOC in soil aggregates can stabilize organic carbon in soil through isolation in pores, adsorption and physical sealing within aggregates [46]

Perhaps it was because the root exudates of native grass species affect the number of soil microbial communities, and soil moisture affects the respiration of microorganisms, thereby affecting the number of microbial communities [59].”

 

10. In the conclusions, mention the limitations of the research and future prospects.

Response: Thank you for your suggestion. We have added the limitations of the research and future prospects in page 21 lines 742-746. Please see the revised version. The statement was list as follow: “ While this study demonstrates the benefits of native grasses for soil nutrient sequestration and nutrient cycling, the findings are limited to the iron-aluminum enriched soils under specific management conditions. Future research should validate these results across diverse soil types and include economic assessments to facilitate farmer adoption.”

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript concerns the evaluation of the effect of 5-year treatment of navel orange orchard in China under the management of native grasses on the accumulation of soil organic carbon and nitrogen and the physicochemical properties and microbial communities of soil aggregates. The presented research results are important for improving soil fertility by increasing the content of soil aggregates, increasing the content of organic carbon in the soil and the number of beneficial microorganisms. The obtained results can be implemented into agricultural practice in organic orange cultivation as environmentally friendly solutions.

Remarks

1. The authors used two plant species as intercropping in the study: Centella asiatica (L.) Urban, that belongs to the family of Apiaceae and Stellaria media (L.) Cyr. that belongs to the family of Caryophyllaceae. Since grasses belong to the Poaceae (Gramineae) family, why do authors refer to these species as grasses?
2. The abstract sentence: 'Gannan navel orange (Citrus sinensis) orchards represent....' requires improvement (L. 15-18).
3. The sentence in lines 44-46 needs to be improved (changed to past tense).
4. The names of plant species should be given in a uniform manner, i.e. either both species should be given their full name or their abbreviated name (L. 80).
5. When describing the real-time quantitative polymerase chain reaction (qPCR) and high-throughput sequencing methodology, the kit used for DNA extraction from soil should be provided. The publications in which the primers used in these studies were developed should be provided, e.g. primers AmoA-1F, AmoA-2R: 'Rotthauwe, J. H., Witzel, K. P., & Liesack, W. 1997. The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and Environmental Microbiology, 63(12), 4704-4712)' (L. 223-226, 241-244).
6. Publications should be cited throughout the manuscript according to the journal's requirements.
7. The publication in References: ‘68. Frostegard et al. 2022’ is not cited in the manuscript.

Author Response

Author's Reply to the Review Report (Reviewer 3)

 

 

The manuscript concerns the evaluation of the effect of 5-year treatment of navel orange orchard in China under the management of native grasses on the accumulation of soil organic carbon and nitrogen and the physicochemical properties and microbial communities of soil aggregates. The presented research results are important for improving soil fertility by increasing the content of soil aggregates, increasing the content of organic carbon in the soil and the number of beneficial microorganisms. The obtained results can be implemented into agricultural practice in organic orange cultivation as environmentally friendly solutions.

Response: Thank you for your support and affirmation of this research.

 

Remarks

1. The authors used two plant species as intercropping in the study: Centella asiatica (L.) Urban, that belongs to the family of Apiaceae and Stellaria media (L.) Cyr. that belongs to the family of Caryophyllaceae. Since grasses belong to the Poaceae (Gramineae) family, why do authors refer to these species as grasses?

Response: Thank you for your suggestion. Indeed, from a taxonomic perspective, this term is not accurate enough. But in some ecological or agricultural studies, plants may be classified based on functional traits (such as growth habits, root types, etc.) rather than taxonomy. For example, Centella asiatica is a creeping herb that often forms a lawn like cover. Stellaria media is a common field weed with a slender and weak morphology resembling grass. If the research focuses on "surface cover" or "weed competition", the author may refer to these plants collectively as "grass like weeds" or "grasses" in the sense of functional groups. Perhaps due to specific expression habits in the research background, it is currently commonly referred to as native grass in the industry.

 

 

2. The abstract sentence: 'Gannan navel orange (Citrus sinensis) orchards represent....' requires improvement (L. 15-18).

Response: Thank you for your suggestion. We have improved that sentence in page 1 lines 14-16. Please see the revised version. The statement was listed as follow: “In Gannan navel orange (Citrus sinensis) orchards—a typical sloped farmland ecosystem—selected native grasses outperform conventional green manure due to their stronger ecological adaptability and lower management requirements.” 

 

3. The sentence in lines 44-46 needs to be improved (changed to past tense).

Response: Thank you for your suggestion. We have improved that sentence in page 2 lines 43-45. Please see the revised version. The statement was listed as follow: “By 2022, 126,000 ha would be planted to navel oranges in southern Jiangxi, with a yield of 1.586 million tons, which ranked first and third in the world, respectively. The navel orange industry had become a characteristic and leading agricultural industry in southern Jiangxi, China [2].” 

 

 

4. The names of plant species should be given in a uniform manner, i.e. either both species should be given their full name or their abbreviated name (L. 80).

Response: Thank you for your suggestion. We have given the full name of the native grasses in page 2 line 86. Please see the revised version. The statement was listed as follow: “Centella asiatica (L.) Urban and Stellaria media (L.) Cyr.”

 

 

5. When describing the real-time quantitative polymerase chain reaction (qPCR) and high-throughput sequencing methodology, the kit used for DNA extraction from soilshould be provided. The publications in which the primers used in these studies were developed should be provided, e.g. primers AmoA-1F, AmoA-2R: 'Rotthauwe, J. H., Witzel, K. P., & Liesack, W. 1997. The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and Environmental Microbiology, 63(12), 4704-4712)' (L. 223-226, 241-244).

Response: Thank you for your suggestion. We have given the kit used for DNA extraction in page 6 lines 259-260, page 7 lines 287-288. And the publication in which the primers used in page 6 line 265. Please see the revised version.

The statement was listed as follow: “The total DNA was extracted from the soil samples using the FastPure Soil DNA Isolation Kit (Shanghai Meiji Yuhua Biomedical Technology Co., Ltd., Shanghai, China).

The genomic DNA was extracted using the FastPure Soil DNA Isolation Kit (Shanghai Meiji Yuhua Biomedical Technology Co., Ltd., Shanghai, China).

[24].”

 

 

 

6. Publications should be cited throughout the manuscript according to the journal's requirements.

Response: Thank you for your suggestion. We have checked and confirmed each reference in the entire manuscript.

 

7. The publication in References: ‘68. Frostegard et al. 2022’ is not cited in the manuscript.

Response: Thank you for your suggestion. The manuscript (word version) was submitted contains the reference to 68, but the PDF does not. Maybe there was an error in the format conversion process. Therefore, We reconvert the manuscript into PDF format to ensure the content accuracy.

Author Response File: Author Response.pdf