Co-Inoculation of Rhizobia and a Multifunctional Microbial Consortium Is Associated with Improved Soybean Performance and Bacterial Community Reassembly in Soybean Fields

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The presentation of the manuscript has improved substantially compared to the previous version, and the figures and tables have been appropriately incorporated. However, some minor editorial issues still require attention. For example, in the captions of the supplementary figures (Supplementary Figures 1 and 2), the term "Supplementary" should be used consistently.

For the first heading in Supplementary Tables 1 and 2, the authors may wish to harmonize the terminology by using either "rhizobial strain" or "rhizobium" consistently across both tables.

I also recommend including the standard deviation values for the data presented in Supplementary Tables 1, 2, and 3, as well as in Tables 1 and 2 of the main text. Providing measures of variability would facilitate a more complete interpretation of the results.

The revised results are now consistent with the stated objectives, and the conclusions are adequately supported by the data presented.

 

Comments on the Quality of English Language

No comments

Author Response

Response to Reviewer 1

We sincerely thank the reviewer for the positive assessment of the revised manuscript and for the careful editorial suggestions. We have addressed all comments as detailed below.

Comment 1

In the captions of Supplementary Figures 1 and 2, the term “Supplementary” should be used consistently.

Response

We fully agree with this comment and thank the reviewer for identifying this inconsistency. We have systematically checked all figure citations and figure captions throughout the main text and supplementary materials. The term “Supplementary Figure” is now used uniformly for all supplementary figures, including in-text citations, figure captions, and the supplementary material file. No abbreviated or inconsistent forms remain in the revised manuscript.

Comment 2

For the first heading in Supplementary Tables 1 and 2, the authors may wish to harmonize the terminology by using either “rhizobial strain” or “rhizobium” consistently.

Response

We fully agree with the reviewer. Since HH103 and TY3-5-1 refer to specific bacterial strains, we have standardized the first column heading in both Supplementary Table 1 and Supplementary Table 2 as Rhizobial strain to ensure terminological consistency across all supplementary tables.

Comment 3

The standard deviation values should be included for the data presented in Supplementary Tables 1, 2, and 3, as well as in Tables 1 and 2 of the main text.

Response

We agree that measures of variability are necessary for a complete interpretation of the results. We have recalculated and reformatted Tables 1 and 2 and Supplementary Tables 1–3 so that all quantitative data are now presented as mean ± standard deviation. The numbers of biological replicates have also been specified in the corresponding table footnotes. Statistical grouping letters remain included where applicable.

Author Response File: Author Response.docx

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The article text and tables and drawings has been improved.  

Soybean cultivar has not been sufficiently described - we only know that it is commonly grown in the area of field study. 

Weather condition during soybean vegetation are presented in extremly short form - a better form would be more detiled data in the form of a table or graph, showing rainfalls and temperatures every decade. 

Information of height of first pod setting - I cannot see it. 

 

 

Author Response

Response to Reviewer 2

We thank the reviewer for the constructive comments regarding the experimental materials, meteorological conditions, and agronomic measurements.

Comment 1

The soybean cultivar has not been sufficiently described.

Response

We fully agree that the previous description was insufficient. We have expanded the description of soybean cultivar ‘Heihe 43’ in Section 2.1 Experimental Site and Materials. The revised text now covers its breeding institution, growth habit, growth duration, regional adaptability, and the rationale for its selection in this study.

Comment 2

Weather conditions during soybean vegetation were presented in an extremely short form. More detailed rainfall and temperature data should be provided for every ten-day period.

Response

We fully agree with this suggestion. The original manuscript only reported seasonal mean temperature and total precipitation, which was insufficient to characterize the meteorological conditions during soybean growth.

We have added detailed dekad-scale (10-day interval) meteorological data from sowing to harvest as Supplementary Table 4, which includes mean, minimum, and maximum air temperature, as well as cumulative precipitation for each 10-day period from May to October 2025. The data source (Tongjiang National Meteorological Station, approximately 35 km from the experimental site) has also been specified.

A brief summary of seasonal meteorological conditions remains in Section 2.2.2 Field Plot Experiment, while full time-series data are provided in the supplementary material.

Comment 3

Information on the height of first pod setting cannot be found.

Response

We fully agree and appreciate the reviewer for identifying this missing information. We acknowledge that the measurement protocol for first pod height was documented in the original manuscript, yet the corresponding dataset was not included. Unfortunately, the field experiment was conducted last year, and we no longer have access to the raw measurement records of first pod height to supplement valid, complete data for Table 1 and the result section. Given the unavailability of original experimental data, we are unable to add this indicator to the manuscript. We have noted this limitation clearly in the discussion section to inform readers of the missing metric.

 

 

Author Response File: Author Response.docx

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

This study investigated the degradation, acidification, and lack of available phosphorus and nitrogen in the black soil of Northeast China due to long-term continuous cropping, and found that the nitrogen fixation effect of rhizobia alone was unstable in the field. The study employed a combined inoculum of rhizobia and multifunctional Bacillus to inoculate soybeans in black soil, focusing on green nitrogen reduction and rhizosphere microbial regulation. This approach has both theoretical value and practical application guidance, aligning with the journal's focus on soil sustainability, farmland microorganisms, and green crop production. However, the following revisions are needed before formal publication:

Q1: Key information regarding experimental materials and methods is missing, and the logic needs improvement.

1. Descriptions of compound microbial agents, rhizobium application dosages, and application methods are vague; variables are not standardized.
2. A blank control (no fertilizer application) is missing, making it impossible to distinguish between the "basic fertilizer effect" and the "microbial agent synergistic effect."
3. Details of molecular experiments are missing; sequencing quality control and OTU clustering threshold annotations are incomplete.

Q2: Some data interpretations in the results are not rigorous, and key mechanisms lack direct evidence.

1.The entire text only observes increased alkaline nitrogen solubility, increased available phosphorus, and increased enzyme activity after CRF treatment, but it does not measure rhizobium nitrogen fixation efficiency, root phosphorus and potassium activation, root exudates, or the number of microbial agents colonizing the rhizosphere. Therefore, it cannot confirm that "rhizobium nitrogen fixation + Bacillus phosphorus and potassium solubilization synergy" is the core mechanism for yield increase; it can only describe the correlation, and many expressions in the text easily confuse "correlation" with "causation."

2.CRF treatments showed increased Chao1 abundance and decreased Shannon evenness, described only as "community recombination," without analysis of whether this was due to targeted enrichment of functional microorganisms or suppression of indigenous weaker microorganisms leading to the decreased evenness. LEfSe was used to screen for CRF biomarker genera, but no correlation analysis was performed considering microbial functions (nitrogen fixation, phosphorus solubilization, organic matter decomposition).

3. During the maturity stage, CRF levels of sucrase, urease, and acid phosphatase were significantly higher than in the F stage, but catalase showed no difference across treatments, mentioned only briefly without discussion of catalase function (soil antioxidant capacity, stress response) or the background of soil acidification.

Q3: The discussion section lacks depth, failing to differentiate between existing research and the innovative aspects of this study in terms of comparative analysis and mechanism speculation.

1. Weak comparison with relevant literature on soybean inoculation in Northeast China's black soil: Numerous domestic studies have investigated the co-inoculation of Northeast China soybeans with Bacillus and Rhizobium. However, this paper only cites a few similar domestic black soil experiments, without comparing the yield increase and soil improvement effects of this study with previous findings, and without explaining the sources of the synergistic effect (strain combination, regional soil type).
2. The synergistic mechanism of co-inoculation is only listed as hypotheses, without stratified verification: The paper proposes three synergistic pathways (balanced nutrient supply, metabolite-induced nodulation, and improved soil microenvironment), but fails to corroborate each one with existing data from this study: for example, whether increased pH promotes nodule formation, and whether increased organic matter improves inoculant survival; these are only discussed in general terms.
3. Insufficient emphasis on the limitations of the experiment, and overly generalized conclusions from short-term experiments. The experiment was conducted in a single season of field trials. The effects of soil organic matter, acidification improvement, and changes in the microbial community were all short-term and transient. However, the conclusions were presented in a way that could lead readers to believe that long-term black soil remediation could be achieved. The experiment did not discuss the interference of interannual climate and differences in indigenous rhizobium communities on the effectiveness of the inoculant.

Author Response

Response to Reviewer 3

We sincerely thank the reviewer for the detailed and constructive evaluation. We agree that several descriptions in the previous version were incomplete and that some mechanistic interpretations exceeded the direct evidence provided by the experiment. We have revised the Materials and Methods, Results, Discussion, and Conclusions accordingly.

Q1.1

Descriptions of the compound microbial inoculant, rhizobial dosage, and application method are vague, and variables are not standardized.

Response

We fully agree with this comment. We have substantially expanded the description of inoculants and their application protocols in Section 2.1 of line 101-106 and Section 2.2.2 in line 120-125. The revised manuscript now clearly specifies:

1. Full taxonomic identity of each bacterial strain;

2. Viable cell concentration of the rhizobial suspension and the granular microbial consortium;

3. 1:1:1 mixing ratio of the three Bacillus strains based on viable cell counts;

4. Standardized application rates per pot (pot experiment) and per hectare (field experiment) in SI units;

5. Application methods: rhizobial inoculant via seed coating, and microbial consortium via soil incorporation with basal fertilizer;

6. Exact timing and depth of field application;

7. Peat as the carrier material for the granular consortium inoculant.

We have also cross-checked and standardized the definitions of F, CF, RF, and CRF throughout the main text, tables, figure legends, and supplementary materials, and corrected the inconsistent treatment definitions in the original Table 2 footnote in Line191-192.

Q1.2

A blank control without fertilizer is missing, making it impossible to distinguish the basic fertilizer effect from the microbial-agent synergistic effect.

Response

We fully agree that a full factorial design including an unfertilized–uninoculated control is required to quantify the absolute contribution of basal fertilizer and test fertilizer × inoculant interactions.

The present field experiment was designed specifically to compare different inoculation strategies under a uniform conventional fertilization background, rather than to quantify the independent effect of fertilization. Therefore, all field treatments received the same basal fertilizer rate, and treatment F served as the fertilization-only reference.

We have clarified this experimental objective in Section 2.2.2 line132-134 and Future studies will adopt a complete factorial design to disentangle fertilization and inoculation effects.

Q1.3

Details of the molecular experiments, sequencing quality control, and OTU clustering threshold are incomplete.

Response

We fully agree, and we have thoroughly expanded the bacterial community analysis protocol in Section 2.3.4. The revised manuscript now reports full experimental and bioinformatics details, including:DNA purity and concentration verification (agarose gel electrophoresis + spectrophotometry);Complete PCR reaction system and thermal cycling program;Sequencing library preparation procedures;Full bioinformatics workflow: demultiplexing, paired-end read merging (FLASH v1.2.11), quality filtering (Trimmomatic v0.39, Q20 threshold), chimera removal (UCHIME v4.2);OTU clustering at 97% sequence similarity using UPARSE v7.0.1090;Taxonomic assignment against the SILVA 138.1 database with a 70% confidence threshold;Rarefaction procedure for diversity analysis;Exact software names and versions for all analyses.

Q2.1

The study did not directly measure nitrogen fixation, nutrient activation, root exudates, or inoculant colonization; therefore, the proposed synergistic mechanism cannot be confirmed.

Response

We fully agree with this important comment. Our experiment measured nodulation traits, soil nutrient indicators, extracellular enzyme activities, soybean performance, and bacterial community composition, but did not directly quantify nitrogenase activity, isotopic biological nitrogen fixation, plant nutrient uptake, root exudate profiles, or the colonization and persistence of inoculated strains.

Accordingly, we have systematically revised all causal statements throughout the entire manuscript (Introduction, Results, Discussion, and Conclusions). Deterministic expressions such as “promoted nitrogen fixation” and “enhanced phosphorus and potassium solubilization” have been replaced with evidence-based descriptions such as “was associated with”, “was consistent with”, and “may reflect”. All proposed mechanistic pathways are now explicitly presented as testable hypotheses that require further validation via physiological, isotopic, metabolomic, and strain-tracking approaches.

Q2.2

The increase in Chao1 and decrease in Shannon diversity were described only as community reassembly, without examining targeted enrichment or suppression of indigenous taxa. LEfSe biomarkers were not related to microbial functions.

Response

We fully agree with this comment. First, we have revised the alpha diversity interpretation in Section 3.4 to explicitly distinguish richness and evenness. Higher Chao1 and ACE indices indicate increased bacterial richness, while lower Shannon and Simpson indices reflect reduced community evenness (i.e., selective enrichment of dominant taxa) rather than a uniform increase in overall diversity. To support this interpretation, we have added Pielou’s evenness index to the alpha diversity analysis (Supplementary Table 3).

Consistent with the limitations of 16S rRNA amplicon sequencing, we have avoided assigning confirmed metabolic functions to individual biomarker taxa solely based on taxonomic identity. Any functional discussion in the manuscript is now described as putative and supported by previous literature, rather than experimentally verified in this study

Q2.3

Catalase showed no significant difference, but its function and relationship with soil acidification were not discussed.

Response

We fully agree, and we have expanded the relevant discussion in Section 4.2. Catalase decomposes hydrogen peroxide and is widely recognized as an indicator of microbial oxidative stress regulation and soil biological resilience. The absence of a significant treatment effect indicates that the inoculation treatments did not measurably alter this component of soil oxidative stress response under the conditions of this one-season field experiment. This result also demonstrates that the enhancement of sucrase, urease, and acid phosphatase cannot be generalized to all soil enzyme systems.

We further note that the modest pH differences observed within a single growing season do not support a direct causal link between soil acidification and catalase activity in this study.agree and have expanded this part of the Discussion. Catalase decomposes hydrogen peroxide and is commonly associated with microbial oxidative-stress regulation and soil biological resilience. The absence of a significant treatment effect suggests that the inoculation treatments did not measurably alter this component of soil oxidative-stress response under the conditions of the present experiment. This result also indicates that the responses of sucrase, urease, and acid phosphatase should not be generalized to all soil enzyme systems. We further note that the modest differences in rhizosphere pH observed during one season do not establish a direct causal relationship between soil acidification and catalase activity.

Q3.1(未

Comparison with previous studies of soybean co-inoculation in Northeast China is insufficient.

Response

We agree. We have expanded the Discussion to compare the magnitude and pattern of soybean yield, nodulation, nutrient, and microbial-community responses in our study with previous co-inoculation studies conducted in Northeast China and other black-soil or cool-climate soybean systems. We now discuss both similarities and differences, with particular attention to soil type, initial pH and nutrient status, soybean cultivar, rhizobial strain identity, Bacillus composition, fertilization background, and climatic conditions.

We have also clarified that the distinctive aspect of the present study is not simply the use of Rhizobium–Bacillus co-inoculation, but the joint evaluation of stage-resolved rhizosphere soil indicators, extracellular enzyme activities, soybean performance, and bacterial community responses under a Sanjiang Plain field environment.

Q3.2

The three proposed synergistic pathways are not supported by stratified evidence from the study.

Response

We agree. We have reorganized the mechanistic discussion into three evidence levels.

First, directly observed responses include nodulation traits, soybean agronomic performance, rhizosphere nutrient indicators, enzyme activities, and bacterial community composition. Second, associations supported by the present dataset include relationships among selected soil variables, microbial taxa, and plant performance. Third, untested mechanisms include enhanced nitrogen-fixation efficiency, root-exudate-mediated interactions, direct phosphorus or potassium solubilization by the inoculated strains, and improved inoculant survival.

The latter mechanisms are now clearly identified as hypotheses rather than conclusions. We have also removed statements implying that the measured increase in pH directly promoted nodulation or that higher soil organic matter directly improved inoculant survival, because these causal links were not tested.

Q3.3

The limitations of the single-season experiment are insufficiently emphasized, and the conclusions are overly generalized.

Response

We agree and have added a dedicated limitations paragraph. The revised manuscript now states that the experiment was conducted at one site, with one soybean cultivar, during one growing season, and under one fertilization regime. The field design did not include an unfertilized control, and the study did not directly measure biological nitrogen fixation, nutrient-transformation rates, root exudates, inoculant colonization, or the composition and competitiveness of indigenous rhizobial populations.

We have therefore restricted the conclusions to short-term, treatment-associated responses observed during the 2025 soybean growing season. Statements implying long-term remediation of black soil, persistent reversal of soil acidification, or stable restructuring of the indigenous microbiome have been removed. We now emphasize that multi-year, multi-site experiments are required to determine persistence, climate dependence, and interactions with indigenous rhizobial communities.

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

This manuscript can be published in its current form.

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

The introduction provides sufficient and up‑to‑date background information to support the research topic. The writing in the Materials and Methods section is clear, and the experimental design appears appropriate for the study’s objectives.

However, the Results section contains several critical omissions that prevent a proper evaluation of the manuscript:

• Tables 1, 2, and 3, which are cited in the text, are missing.

• Figures 1, 2, and 3 are also absent.

• The text refers to “Figure 2a" implying the existence of additional subfigures (e.g., 2b, 2c, 2d), yet these are neither included nor mentioned elsewhere.

• It is essential that all referenced figures and tables be provided within the Results section.

Additionally, the supplementary materials require correction:

• In Supplementary Figures 1 and 2, please correct the spelling of “supplementary” and “treatment.”

• In Supplementary Figure 3, correct the term “connectwork.”

• In Supplementary Tables 1, 2, and 3–6, the legends currently appear in Chinese and should be translated into English.

Given the extent of missing essential content, the manuscript cannot be adequately assessed in its current form.

Reviewer 2 Report

Comments and Suggestions for Authors

Manusript Land-4335696 Co-Inoculation of Rhizobia and a Multifunctional Microbial Consortium Improves Soil Fertility and Reshapes Bacterial Community Assembly in Soybean Fields, by Hou et al. The manuscript deals with inoculation of two rhizobial strains and co-inoculation with three Bacillus species in a pot and in a field trial carried out only one season. After careful review on the manuscript, despite scientifically sound, I do not consider it approptiate for publidation. The main reason is that some interpretation and statements can not be supported by data. It is not reasonable that soil pH and soil organic matter changes in a single crop season due to co-inocultion with plant-growth promiting microorganisms. Some misinterpretation apparently occurred here. In addition, the main tables (Tables 1 and 2) and Figures (Fig. 1 and 2) are missing in the manuscript. I could see only the supplemental tables and figures, but part of the tables are in Chinese, and I could not read. Supplementary table 3 seems to have formatation problems. Further comments:

Abstract would benefit from some numerical results.

Introduction must be amended for language. Some sentences ae confusing and repetitive.

Material and Methods need deep reformulation in terms of clearnes, sequence and supporting references on the methods. Units must follow the International Sistem. I could not understand the unit of area "mu". Lines 114-117. I don't know if these amounts are high or low, because I do not know the unit represented by "mu".

Results: main tables and figures are missing. Only supplemental could be seen.

L184. An increase in 37.5% in SOM can not be expected from inoculation. 

Discussion: some statements are speculative, such as in lines 261, 264, 267-270, 285, 292.

Further comments were done in the attached pdf file.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Some parts are confusign and need to be improved. Please check the attached pdf file.

Reviewer 3 Report

Comments and Suggestions for Authors

In General „Introduction” is a well written chapter of the article, however, the fragment between lines L 44 and L 50 and L 51 and L 58 contains repeated content.

In chapter 2 “Materials and Method” L 98 only soybean cultivar name “Heihe43” is mentioned – please add some basic characteristic. Please explain why this particular soybean cultivar was chosen?

L 101-L 103: it is mentioned that conventional compound fertilizer was used – what about its rate.  

L 106 – L112 “Pot experiment” – no information on plants watering.

L 115 - L 116: 20 kg /mu compound fertilizer and compound inoculant used at a rate of 4 kg / mu: please explain what is mu? Use SI system.

“Field Plot Experiment” - no information on weather conditions (temperatures, precipitation) during soybean vegetation.

“Soybean Agronomic Traits and Yield”- no information on the height of the first pod setting in soybeans.

L 127 : 100-seed weight – why not 1 000 seed weight?  

The text of the article is rather short. Why are the figures and tables placed not directly in the text of the article but in the Supplementary Materials? This makes it difficult to follow the essence of the article. Furthermore, it contradicts the recommendations for authors, which clearly state that figures and tables should be placed directly at the point where they are first cited.

The tables are not clearly labeled. The table headings and footnotes are written in Chinese. Tables 2 and 3 lack explanations of the abbreviations used.

The drawings are not clearly labeled. For example, Figure 1 does not even specify which experiment it refers to (pot or plot experiment).