Effects of Tillage Practices on Soil Organo-Mineral Complexes and Organic Carbon Distribution Under Continuous Maize Cropping in the Black Soil Region of Northeast China

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Review of the manuscript entitled: Effects of tillage practices on soil organo-mineral complexes  and organic carbon distribution under continuous maize cropping in the Black Soil Region of Northeast China.

 

Critical comments on the manuscript:

1. Line 33-40: You can extend your text with a concise paragraph emphasizing practical relevance, for example.
2. Line 54-67: The text aptly emphasizes the importance of mineral protection for SOC stability, but it is worth clarifying that this is not the only mechanism for stabilizing organic carbon in soil.
3. Line 79-97: The text accurately describes the role of calcium and Fe/Al oxides in SOC stabilization, but it should be supplemented and expanded with the information that their effect may be strongly dependent on specific soil conditions (e.g. type of clay minerals, organic matter content or moisture).
4. Line 169-198: The highlighted section lacks information about the research area, including the plot area. Information about the agrotechnical treatments conducted was completely omitted, and the amount of straw plowed into the soil was not provided.The doses used require a thorough explanation, i.e., the N:P:K ratios indicate the dominance of N fertilization. Why were such low doses of P and especially K used? I request a scientific explanation.The authors completely downplay the form of fertilization used.Simply specifying the dose is not a precise approach – what fertilizer was used, whether N fertilization was applied at one time or divided into doses, which ones, and at what times during plant development.What type of application was adopted?All of this requires a thorough explanation.
5. Provide a detailed calendar of treatments and plant growth and development.
6. Line 203-209: Describe in detail the procedure for collecting soil samples, what was used to collect them, how they were homogenized.
7. Line 2012-2013: “Kjeldahl method 212 was developed to determine total nitrogen (TN) - How was NO3- converted to NH4?. The determination of the total phosphorus (TP) was carried out using the HClO4-H2SO4 method.” - what method is this - what quantitative technique was used.
8. Line 252-269: The description of the extraction procedures for Fe and Al forms is correct and consistent with the commonly used methodology, however, it is worth noting that the methods used (DCB, ammonium oxalate, sodium pyrophosphate) do not completely separate all forms with different reactivity and stability.
9. Line 283-324: The interpretation of the results in this section is largely descriptive and, in places, oversimplified in explaining the mechanisms.In particular, attributing changes in fractions G0, G1, and G2 solely to the effects of tillage systems (e.g., no-tillage → increased aggregate stability) fails to account for the possible influence of other factors, such as variability in soil properties within the transect, differences in moisture content, or long-term chemical changes independent of tillage.Furthermore, the lack of broader statistical data (beyond individual p values ​​< 0.05) and the lack of analysis of factor interactions limits the strength of causal inferences.This section should be thoroughly revised.
10. Table 3: always provide 3 significant digits.
11. Line 360-377: The interpretation of the increase in organic carbon content in fractions G0 and G1 in the NT system is based on a rather simplistic assumption of the dominance of fresh organic matter and the role of Ca²⁺, without direct empirical confirmation in the presented results (e.g., there is a lack of data on the qualitative composition of SOM or humification dynamics).This fragment needs to be better described and documented in the literature, or the scope of chemical analyses should be broadened – absolutely necessary.
12. The Conclusions require extensive rewording and clarification.In particular, the statement that NT "most effectively improves SOC stability" and "inhibits Fe/Al redox cycles."This statement is mechanistic in nature, but has not been unequivocally confirmed by direct measurements of redox processes or stability dynamics over time. The authors attribute a key role to single factors, such as Ca²⁺, pH, and Fe/Al.This may oversimplify the multifactorial nature of soil processes, in which biological and structural interactions are also important.The authors believe that the authors should approach the issue of conclusions very carefully, paying particular attention to the complexity of the processes occurring in soil.

Author Response

Ref.: ISSN 2073-4395 (Agronomy)

Manuscript ID: agronomy-4295271

Title: Effects of tillage practices on soil organo-mineral complexes and organic carbon distribution under continuous maize cropping in the Black Soil Region of Northeast China

Dear Editor and Reviewer,

We sincerely express our gratitude to you, and the anonymous reviewers for the great work and time spent which helped us improve the quality of the manuscript.

In the revised manuscript, all the issues raised by the reviewers have been addressed accordingly. We have prepared the detailed, point-by-point responses, which are given in blue text after each of the comments from the reviewers. In our responses, we have also indicated line numbers that correspond to the text in the clean revised manuscript.

Once again, we appreciate the time and effort that you and the reviewer have dedicated to this manuscript.

With kind regards,

All authors

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

This manuscript addresses an important topic (organo-mineral complexes and SOC stabilization under tillage), with strong field data and mechanistic ambition. However, there are critical issues in alignment and logical flow across sections, particularly: (i) partial mismatch between stated objectives and presented results, (ii) abstract overclaims mechanistic insight, (iii) discussion introduces concepts not directly tested, and (iv) weak closure loop between objectives, results, and conclusion.

Objectives are not clearly reflected in the Abstract. The abstract emphasizes changes in organo-mineral fractions, NT superiority, and environmental drivers (Ca²⁺, Fe/Al, pH); but it does not explicitly reflect all three objectives, especially those of mechanistic interaction between SOM and minerals, and transformation pathways among G0, G1, G2.

Conclusion is not fully aligned with objectives as well. The manuscript lacks a clear, structured conclusion section, and instead conclusions are embedded in Discussion. Key claims are NT improves SOC stabilization, and Ca²⁺, Fe/Al, pH are drivers. However, objective 3 (transformation mechanisms among fractions) is not clearly concluded.

Overstatement of mechanistic understanding in Abstract. Abstract states “explore the mechanism…” and identifies key drivers but evidence is correlative (RDA, Pearson), not mechanistic.

Results strongly address Objective 1, partially Objective 2, weakly Objective 3. Objective 2 is partially addressed Ca²⁺, Fe/Al, pH analyzed but no direct process measurement (e.g., binding strength, turnover). Objective 3 is weak. IT only inferred via G0, G1, and G2 discussion like RDA relationships.

Discussion introduces concepts not directly supported by results. These are not measured or tested in the study.

Discussion lacks a clear narrative structure. Discussion is fragmented into subsections. It does not “tell a story”.

There is a weak linkage between statistical results and interpretations. RDA results are presented, but interpretation is descriptive, not process-based.

Inconsistency in interpretation of G2 fraction. Observations includes G2 is the highest OC per unit mass but lowest contribution per soil mass. Yet, discussion sometimes implies strong dominance.

Time comparison not fully integrated. Results repeatedly compare years but no synthesis of long-term trajectory.

Major Revision

Author Response

Ref.: ISSN 2073-4395 (Agronomy)

Manuscript ID: agronomy-4295271

Title: Effects of tillage practices on soil organo-mineral complexes and organic carbon distribution under continuous maize cropping in the Black Soil Region of Northeast China

Dear Editor and Reviewer,

We sincerely express our gratitude to you, and the anonymous reviewers for the great work and time spent which helped us improve the quality of the manuscript.

In the revised manuscript, all the issues raised by the reviewers have been addressed accordingly. We have prepared the detailed, point-by-point responses, which are given in blue text after each of the comments from the reviewers. In our responses, we have also indicated line numbers that correspond to the text in the clean revised manuscript.

Once again, we appreciate the time and effort that you and the reviewer have dedicated to this manuscript.

With kind regards,

All authors

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

Comment 6. This is not a precise description of the soil sampling procedure — please take this comment seriously.

Comment 7. In that case, can the nitrogen content really be described as total nitrogen content?

Comment 8. I do not accept the response to this comment.

Author Response

Ref.: ISSN 2073-4395 (Agronomy)Manuscript ID: agronomy-4295271

Title: Effects of tillage practices on soil organo-mineral complexes and organic carbon distribution under continuous maize cropping in the Black Soil Region of Northeast China

Dear Editor and Reviewer,

We sincerely express our gratitude to you, and the anonymous reviewers for the great work and time spent which helped us improve the quality of the manuscript.

In the revised manuscript, all the issues raised by the reviewers have been addressed accordingly. We have prepared the detailed, point-by-point responses, which are given in blue text after each of the comments from the reviewers.

Once again, we appreciate the time and effort that you and the reviewer have dedicated to this manuscript.

With kind regards,

All authors

 

 

 

 

 

Responses to comments from the Reviewer:

Reviewer 1

Comment 6. This is not a precise description of the soil sampling procedure — please take this comment seriously.

Response: Thanks for the correction.

Using a stainless steel shovel, take the soil sample and remove any stones, gravel, and residues of animal or plant origin. At each point, approximately 1 kg of soil was collected in the depth of 0-20 cm. Subsequently, all subsamples from a single plot were combined and thoroughly homogenized in a stainless steel pot by quartering to form a bulk sample. Then the sample was placed into a cloth bag and marked clearly.

Comment 7. In that case, can the nitrogen content really be described as total nitrogen content?

Response: We appreciate the reviewer's comment on NO₃⁻ to NH₄⁺.

To obtain total nitrogen inclusive of NO₃⁻, a salicylic acid-sodium thiosulfate pre-reduction step was implemented prior to standard Kjeldahl digestion (Bremner, 1965). In brief, the sample was incubated with a salicylic acid-sulfuric acid mixture at room temperature for 24 hours to immobilize NO₃⁻, followed by the addition of sodium thiosulfate and heating to effect reduction. Digestion, distillation, and titration were then carried out according to the conventional Kjeldahl protocol. This method consistently achieved a NO₃⁻ recovery rate exceeding 95%, thus guaranteeing the integrity of the total nitrogen measurements.

Bremner, J.M. Total Nitrogen. In: Norman, A.G., Ed., Methods of Soil Analysis: Part 2 Chemical and Microbiological Properties, 9.2, American Society of Agronomy, Wisconsin, 1965, 1149-1178.

Comment 8. I do not accept the response to this comment.

Response: Thank you for your important suggestions.

We fully agree that the DCB, ammonium oxalate and sodium pyrophosphate methods cannot completely separate all different reactivity and stability forms of iron and aluminum oxides. At present, the international soil chemistry community generally believes that no single chemical extraction method or combination can achieve the absolute separation of iron and aluminum forms. All methods provide components under the "operational definition" - that is, the extractable parts under specific reagent, temperature, time, pH and soil-liquid ratio conditions. There exists a phenomenon of coexistence of cross-extraction and incomplete extraction among different methods, which is a recognized technical limitation in this field.

Despite the above limitations, the sequential extraction or parallel extraction scheme of "sodium pyrophosphate →ammonium oxalate→DCB " adopted in this study remains a relatively reliable method for distinguishing complexed iron and aluminum, amorphous crystalline iron and aluminum, and total free iron and aluminum at present, and is widely used in the research of soil formation, environmental geochemistry and paleoclimate reconstruction. This combination can reveal the relative distribution trend of iron and aluminum forms, meeting the purpose of studying and comparing the differences between different treatments.

The separation procedures were carried out in strict accordance with standard protocols, thereby ensuring the repeatability and comparability of the obtained results.

In the method section, it is clearly pointed out that there may be situations where some amorphous iron and aluminum are extracted by DCB or some crystalline iron and aluminum are not completely dissolved by ammonium oxalate, and it is noted that this is an inherent methodological deviation.

 

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of the manuscript entitled: Effects of tillage practices on soil organo-mineral complexes and organic carbon distribution under continuous maize cropping in the Black Soil Region of Northeast China.

 

Detailed notes on the manuscript:

 

1. Line 10: „Organo-mineral complexes are intimately related to stabilize soil organic carbon and 10 maintain soil fertility.” very large generalization, the indicated complexes do not only concern carbon - write precisely.
2. Line 48-59: You describe the protective effect of soil minerals on SOC through the formation of organic-mineral complexes in a very general way.You don't specify which mineral soil components you're referring to, nor do you describe the mechanisms by which such complexes form. Their precise characteristics are lacking, such as their sorption or cation exchange capacities, or their activity in black soil.This information needs to be supplemented.
3. Line 74: If there is promotion of processes that favor the development of microorganisms, how does this relate to SOC sequestration - please elaborate on this issue.
4. No clearly defined research hypothesis?
5. Line 144: “Fertilization followed conventional local farming practices, and a locally adapted 144 corn cultivar was used.” - This is a scientific study, please describe the fertilization and other chemical treatments used, e.g. plant protection products used - be precise.
6. Table 2: Complete the results: if you determined available NPK forms, complete the table with the soluble carbon content.You should also determine the soil's electrical conductivity.Considering SOC stability, did you determine the non-hydrolyzable C content?– complete and explain.
7. Why didn't you discuss the results in Table 2?
8. Why didn't you provide the meteorological data?- please complete
9. Line 155: determine the sieve mesh diameter.
10. Line 155: Describe how pH, SOC, Ca were determined?
11. Line 22: I have a doubt about the results, namely why only samples from 2022 were analyzed.In my opinion, there is no reference point (e.g. to 2002).
12. Table 3 allows for comparison of cultivation methods only, I have no way of assessing the degree of change over time - why?

A single-year study cannot explain the mechanisms that determine the formation or degradation of organic-mineral bonds. The authors omitted a key factor: time and variability in meteorological conditions. I suggest supplementing the research results; the authors likely possess archived soil material from earlier years.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Editor,

I have been through following manuscript, agronomy-3994300-peer-review-v1, titled as “Effects of tillage practices on soil organo-mineral complexes and organic carbon distribution under continuous maize cropping in the Black Soil Region of Northeast China”. This manuscript addresses an important topic in soil science—how long-term tillage practices regulate organo-mineral complexes (G0–G2) and their associated soil organic carbon (SOC) in black soils of Northeast China. The study integrates mineral fractions, Ca associations, Fe/Al oxide forms, and SOC stabilization pathways using both chemical fractionation and multivariate analyses.

It is unclear whether the sites were independently replicated or pseudo-replicated.

• Each tillage measure appears to be represented by only3–4 sites, but these sites differ in location, soil history, and inherent properties. It is therefore problematic to treat tillage as a fixed effect without accounting for site variability.
• The authors should clarify whether the sites are true replicates or pseudo-replicates and if soil properties were comparable across sites before tillage treatments began in 2002.
• How the analysis accounted for spatial heterogeneity (mixed-effects models would be more appropriate).
• The paper relies heavily on the G0–G1–G2 gel-dispersion method, but the method has known limitations operational definitions do not always correspond to functional binding categories, and sodium chloride rinsing and ultrasonic dispersion can alter organo-mineral associations.
• Explain potential cross-contamination among fractions, selectivity of extraction steps, expected recovery and potential mass loss, and justification for using this method instead of density fractionation or mineral-associated organic matter (MAOM) protocols.
• The study does not directly measure redox potential, oxygen diffusion, or Fe(II)/Fe(III) ratios. Observed differences may reflect site variability, not tillage effect alone.
• The paper uses one-way ANOVA and LSD tests, but assumptions (normality, homoscedasticity) are not documented.
• LSD is not recommended with small sample sizes and unequal variances.
• Multivariate analyses (RDA) require clearer description of data scaling and variable selection.
• The discussion focuses heavily on classical colloid chemistry but should also reference newer mineral-associated organic matter (MAOM) frameworks.

Reviewer 3 Report

Comments and Suggestions for Authors

 

The manuscript addresses an important topic related to soil carbon stabilization and organo-mineral complexes; however, it requires substantial improvement before it can be considered for publication. The major weaknesses include the lack of a proper experimental design, clear evidence of pseudoreplication, significant methodological gaps (particularly in the extraction and interpretation of the G0–G1–G2 fractions, analytical controls, and historical management information), and a statistical approach that is incompatible with the observational nature of the sampling. Additionally, the introduction presents conceptual imprecision, the description of soil and site characteristics is insufficient, and the methodological narrative does not allow reproducibility. These issues directly compromise the robustness and reliability of the conclusions, and they must be thoroughly addressed for the study to meet the expected scientific standards

Introduction:

• The text discusses the importance of organo-minerals, but does not provide a clear conceptual definition of fractions G0, G1, and G2 at the outset. The concept only appears later in the text, which impairs fluidity and makes it difficult for readers unfamiliar with the Chinese gel dispersion method to understand.
• The introduction mentions “severe degradation,” “thinning of the horizon,” and carbon loss, but does not present actual values (e.g., reduction in SOM, loss of A layer thickness, changes in CTC), which leaves the statements vague and unconvincing (L38-45).
• In the introduction, although mentioned, the binding mechanisms (calcium bridge, oxide complexation, specific sorption) are presented in an overly simplistic manner, without addressing: competition between cations, pH-dependent solubility, difference between crystalline and amorphous Fe/Al, recognized limitations in extraction methods (DCB, oxalate, pyrophosphate). In my opinion, this reduces the conceptual value of the introduction.
• The introduction discusses “carbon stability,” but does not clearly differentiate between POM and MAOM, nor how the G0–G1–G2 fractions relate to these categories widely accepted in the international literature. This compromises the study's inclusion in the current debate on C stability.
• The introduction does not justify why this “gel dispersion” method is suitable for the purpose. Discussions on recognised limitations are lacking; operational separation, not functional; possibility of mechanical rupture altering results; mixture of particulate materials and true complexes. This is critical, as the entire study depends on these fractions.
• Although the objectives are listed, they do not arise organically from the preceding text. The introduction should establish the scientific gap and then present the objectives, but here this is done abruptly.
• The review focuses almost exclusively on Chinese literature, failing to mention important works on MAOM (mineral-associated organic matter), the role of Al/Fe and pedogenesis in C sequestration, and the effects of management practices (NT, CT, RT) on oxides. This weakens the international credibility of the study.

Material and Methods:

• The paper states that areas in three latitudinal zones were selected, but does not present any experimental design (RCD, RCD, randomized blocks, fixed plots, etc.). This is a serious flaw; without a design, it is impossible to attribute differences to management rather than the environment.
• The methodology suggests that the areas “were selected” and not implemented as an experiment. This characterizes an observational study, but the authors treat it statistically as if it were a controlled experiment. The lack of control variables confuses interpretation.
• Each treatment appears in completely different locations, with different coordinates. This characterizes spatial pseudoreplication, not true experimental replication. The authors do not discuss this statistical problem.
• The points present differences in altitude, differences in local climate, differences in soil (not presented in the text). Without demonstrating homogeneity between points, comparisons between management practices become compromised.
• “Fertilization followed conventional practices”. (L144): This sentence is insufficient. It is not known; the dose of N, P, K, the chemical form (MAP? Urea?), whether liming was carried out over the years, whether organic fertilization was used. This is very serious because Ca²⁺ and Fe/Al are directly influenced by liming and P.
• (L152-154): The collection consists of five subsamples forming a composite sample. The problem is that each area receives only a single composite sample per treatment, which destroys any possibility of statistical inference. It is classic pseudo-replication.
• For a study focused on Fe/Al and organo-mineral complexes, it is mandatory to present; diffractometry (XRD), clay content, dominant mineral type (montmorillonite? kaolinite? vermiculite?). None of this appears.