Priestia megaterium Inoculation Enhances the Stability of the Soil Bacterial Network and Promotes Cucumber Growth in a Newly Established Greenhouse

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear editor,

I have carefully reviewed the manuscript entitled “Bacillus megaterium inoculation enhances the stability of the soil bacterial network and promotes cucumber growth in a newly established greenhouse”. I have several comments and suggestions to improve the clarity and overall impact of the study, which I outline below.

The manuscript addresses a relevant and practically important topic by evaluating the effects of Bacillus megaterium on nutrient availability, the soil microbial community, and cucumber yield under greenhouse conditions. However, I consider that the experimental design presents several limitations that weaken the robustness of the inferences and the clear attribution of the observed effects to the bacterial inoculant. Below, I summarize the main points that, in my opinion, should be addressed or at least discussed in greater depth.

-The authors should expand the rationale for the study.

-The authors should select keywords that differ from the terms already used in the title.

-The authors should verify that all scientific names are written in italics and include the scientific names of all plant species cited throughout the manuscript.

-It is not clear why the study focuses specifically on assessing the effect of B. megaterium inoculation on K and P availability. This point should be clarified.

-The authors should specify which plant growth-promoting traits are known for the B. megaterium strain used in the study, indicate the origin of the strain, and describe how it was identified at the molecular level.

-The authors should explain why they decided to apply 100 mL of inoculant per plant. At what cell density was the inoculant applied? At which phenological stage of the cucumber crop was the inoculation carried out, and why was this stage chosen? Was any disinfection procedure implemented to ensure efficient colonization by B. megaterium? Why was sterile soil not used?

-The inoculant is formulated in a medium rich in organic and mineral components (yeast extract, bran, corn flour, brown sugar, salts, etc.), whereas the control treatment (CK) receives only irrigation water. This implies that any improvement in P and K availability, changes in the soil microbial community or increases in yield could be due, partially or entirely, to the effect of the carrier medium rather than to the activity of B. megaterium itself. A control treatment that receives the same carrier medium without bacteria is missing. Without this control, it is difficult to separate the microorganism effect from the substrate effect, which weakens the conclusion that the observed changes are specifically attributable to inoculation with B. megaterium.

-Both experiments are conducted with three replicates, which is the minimum acceptable number, especially considering the potential spatial heterogeneity within a greenhouse (gradients of moisture, temperature, light, etc.). With only three replicates per treatment, the statistical power to detect differences, particularly in soil and microbial community variables, is limited.

-In the first year, different doses of the inoculant are evaluated, whereas in the second year the timing of application is assessed using a single dose. This leads to two independent experiments rather than a factorial design (dose × timing) within the same cropping cycle. Consequently, it is not possible to robustly analyze the consistency of the results between years or the year × treatment interaction. Nor can the optimal combination of dose and application schedule be evaluated within a single year. In its current form, the study presents two distinct sets of treatments, which limits the ability to integrate the results into a more general agronomic recommendation.

-Across the different components of the study (colonization, nutrient dynamics, structure of the microbial community), different soil sampling depths are used. Although this may be justifiable given the nature of each assessment, the manuscript does not provide a sufficiently detailed explanation of this decision. This makes it difficult to directly compare colonization dynamics, nutrient variation and microbial community shifts, which are later interpreted jointly in the discussion.

Author Response

Dear Editor and Reviewers:

We thank the Editor and Reviewers for their constructive comments. All revisions have been implemented in the revised manuscript and highlighted in yellow for ease of inspection, as requested by the Editor.

Thank you for your comments concerning our manuscript entitled “Bacillus megaterium inoculation enhances the stability of the soil bacterial network and promotes cucumber growth in a newly established greenhouse” (Manuscript ID: agriculture-3970588). The comments are all valuable and very helpful for improving our article. We have revised the manuscript addressing all the comments by the reviewers and editors. We hope these revisions will meet with your approval. Below is a list of the responses to the reviewers’ comments.

 

Best regards,

Yours Sincerely,

Wenju Liu

Comments 1: The authors should expand the rationale for the study.

Revision: Thank you for pointing this out, we agree with this comment. Therefore, we expanded the rationale in the Introduction to emphasize why early-stage (newly established) greenhouse systems warrant targeted intervention and why nutrient bioavailability—particularly phosphorus (P) and potassium (K) in calcareous greenhouse soils—is a practical lever for sustainability. Please see L58-L61 (highlighted in yellow).

Comments 2: The authors should select keywords that differ from the terms already used in the title.

Revision: Thanks for your comment. We replaced the keyword list to avoid repeating the main title terms and to better reflect the methodological/functional focus of the study.

Comments 3: The authors should verify that all scientific names are written in italics and include the scientific names of all plant species cited throughout the manuscript.

Revision: Thanks for your comment. We formatted all scientific names in italics throughout the manuscript and ensured that plant species are provided with scientific names at first mention (e.g., cucumber, Cucumis sativus L.).

Comments 4: It is not clear why the study focuses specifically on assessing the effect of B. megaterium inoculation on K and P availability. This point should be clarified.

Revision: Thanks for your comment. We clarified that the study focuses on P and K because the inoculant is marketed/known as a P- and K-solubilizing PGPR (Please see L72-L73) and because calcareous greenhouse soils are prone to P and K immobilization and nutrient imbalance under intensive fertilization. Please see L58-L61 (highlighted in yellow).

Comments 5: The authors should specify which plant growth-promoting traits are known for the B. megaterium strain used in the study, indicate the origin of the strain, and describe how it was identified at the molecular level.

Revision: Thank you for this comment. The B. megaterium used in this study was applied as a commercial microbial inoculant product obtained from Run-Wo Biotechnology Co., Ltd. (Hebei Province, China). The focus of the present work was to evaluate the overall agronomic and microbiological performance of the commercial inoculant as a formulated product, rather than to characterize the origin or molecular identity of the individual strain contained within it.

The plant growth-promoting traits associated with this B. megaterium inoculant, including its capacity to enhance plant growth and mobilize soil phosphorus and potassium, have been documented in our previously published work. In the present study, we further examined functional traits directly relevant to the manuscript, including phytohormone production (IAA, GA₃, and ZR) under in vitro conditions, as well as soil colonization dynamics quantified by qPCR using B. megaterium-specific primers.

Comments 6: The authors should explain why they decided to apply 100 mL of inoculant per plant. At what cell density was the inoculant applied? At which phenological stage of the cucumber crop was the inoculation carried out, and why was this stage chosen? Was any disinfection procedure implemented to ensure efficient colonization by B. megaterium? Why was sterile soil not used?

Revision: We added a quantitative justification linking the per-plant application volume to the target field application rate, planting density, and product cfu concentration (approximately 2.1 mL product plant⁻¹ at 75 L ha⁻¹, equivalent to ~1.5 × 10⁹ cfu plant⁻¹). We also clarified that a per-plant irrigation volume of 100 mL was selected to ensure uniform delivery of the target dose to the root zone while avoiding runoff or waterlogging. In addition, the rationale for applying the inoculant at transplanting, to facilitate early root-zone establishment and microbial colonization, is now stated, and it is specified that soil was not sterilized or disinfected in order to preserve the indigenous microbial community and reflect farmer-relevant greenhouse management conditions. Please see these revisions were made in Methods Section 2.1 (Greenhouse experiment).

Comments 7: The inoculant is formulated in a medium rich in organic and mineral components (yeast extract, bran, corn flour, brown sugar, salts, etc.), whereas the control treatment (CK) receives only irrigation water. This implies that any improvement in P and K availability, changes in the soil microbial community or increases in yield could be due, partially or entirely, to the effect of the carrier medium rather than to the activity of B. megaterium itself. A control treatment that receives the same carrier medium without bacteria is missing. Without this control, it is difficult to separate the microorganism effect from the substrate effect, which weakens the conclusion that the observed changes are specifically attributable to inoculation with B. megaterium.

Revision: We thank the reviewer for raising this important point. In the present study, our primary objective was to evaluate the overall agronomic and microbiological effects of a commercially formulated B. megaterium inoculant as it is applied in practical greenhouse production, rather than to disentangle the individual contributions of the microbial cells and the carrier components. The inoculant used in this study consists of viable B. megaterium cells together with organic and inorganic constituents (e.g., yeast extract, bran, corn flour, brown sugar, and mineral salts), which are designed to function as an integrated formulation. Therefore, the comparisons in this study were intentionally framed between inoculant application and non-application, reflecting real-world farmer practice.

Similar approaches, focusing on the performance of commercial microbial products as integrated formulations without a carrier-only control, have been adopted in previous greenhouse and field studies (Moreno-Lora et al., 2023; Xiao et al., 2025). We appreciate the reviewer’s suggestion and fully agree that future work should employ a factorial experimental design (cells × carrier) to further disentangle formulation effects and microbial-specific mechanisms.

[1] Moreno-Lora, A., Velasco-Sánchez, Á., Delgado, A., 2023. Effects of microbial inoculants and organic amendments on wheat nutrition and development in a variety of soils. Journal of Soil Science and Plant Nutrition 23, 3329–3342.

[2] Xiao, M., Liu, X., Wang, X., Liu, W., Zhang, T., Yang, Y., 2025. Microbial inoculants drive disease suppression and rhizosphere modulation for effective management of pepper phytophthora blight. Applied Soil Ecology 208, 105971.

 

Comments 8: Both experiments are conducted with three replicates, which is the minimum acceptable number, especially considering the potential spatial heterogeneity within a greenhouse (gradients of moisture, temperature, light, etc.). With only three replicates per treatment, the statistical power to detect differences, particularly in soil and microbial community variables, is limited.

Revision: We thank the reviewer for this thoughtful comment regarding replication and statistical power. We acknowledge that three biological replicates represent the minimum commonly accepted level for greenhouse and soil microbiome experiments; however, this level of replication is widely used and considered sufficient to support statistical inference in similar controlled greenhouse studies, particularly when combined with randomized plot layout and consistent management practices.

In the present study, all treatments were arranged in a randomized block design within the greenhouse to minimize the influence of potential spatial heterogeneity (e.g., gradients of moisture, temperature, and light). Moreover, the main treatment effects were supported by consistent trends across plant growth, soil nutrient variables, and microbial community analyses. Similar replication levels (n = 3) have been adopted in recent greenhouse studies investigating soil properties and microbial community responses to microbial inoculants and fertilization practices (Lu et al., 2025).

[1] Lu, X., Li, Q., Li, B., Liu, F., Wang, Y., Ning, W., Liu, Y., Zhao, H., 2025. Bacillus megaterium controls melon Fusarium wilt disease through its effects on keystone soil taxa. Plant and Soil. https://doi.org/10.1007/s11104-025-07914-5

Comments 9: In the first year, different doses of the inoculant are evaluated, whereas in the second year the timing of application is assessed using a single dose. This leads to two independent experiments rather than a factorial design (dose × timing) within the same cropping cycle. Consequently, it is not possible to robustly analyze the consistency of the results between years or the year × treatment interaction. Nor can the optimal combination of dose and application schedule be evaluated within a single year. In its current form, the study presents two distinct sets of treatments, which limits the ability to integrate the results into a more general agronomic recommendation.

Revision: We appreciate the reviewer’s comment regarding the experimental structure. We would like to clarify that the study was conducted as two sequential greenhouse experiments completed over two consecutive years, both carried out in the same plastic-shed greenhouse and on the same soil beds. Apart from the specific inoculant treatments under investigation, all other experimental conditions—including soil type, greenhouse environment, crop cultivar, fertilization regime, irrigation management, and agronomic practices—were kept consistent between the two years.

The first-year experiment was designed to screen appropriate inoculant application rates, while the second-year experiment focused on optimizing application timing using the selected rate identified in year one. This sequential design allowed us to refine treatment selection under identical site conditions, thereby minimizing confounding effects related to spatial heterogeneity or inter-annual management differences. The experimental design has now been explicitly clarified in Methods Section 2.1. (highlighted in yellow).

Comments 10: Across the different components of the study (colonization, nutrient dynamics, structure of the microbial community), different soil sampling depths are used. Although this may be justifiable given the nature of each assessment, the manuscript does not provide a sufficiently detailed explanation of this decision. This makes it difficult to directly compare colonization dynamics, nutrient variation and microbial community shifts, which are later interpreted jointly in the discussion.

Revision: Thank you for this valuable comment. We have clarified the rationale for using different sampling depths by explaining their distinct and complementary purposes. Specifically, 0–5 cm root-affected soil was used to characterize short-term colonization dynamics at the root–soil interface, whereas 0–20 cm plough-layer soil was used to assess seasonal changes in soil nutrients and microbial community structure relevant to greenhouse management.

This clarification has now been incorporated into the Discussion (highlighted in yellow), where we explicitly explain how colonization dynamics, nutrient variation, and microbial community shifts were evaluated at appropriate spatial scales. In addition, the overall sampling scheme has been summarized in Table S1 and referenced in Methods Section 2.2. These revisions clarify that the two datasets provide complementary information and together support the study’s conclusions.

Reviewer 2 Report

Comments and Suggestions for Authors

The Submitted MS entitled : Bacillus megaterium inoculation enhances the stability of the 2 soil bacterial network and promotes cucumber growth in a newly established greenhouse" is of high interest. It focus on B. megaterium in vitro PGPR traits and effectiveness as growth promoter of cucumber while exactly determining inoculum characteristics and time for application.  Applying B. megaterium at 75 L ha-1 during cucumber seedlings transplantation, seems to be the best option in newly established greenhouse, system during two-year mono cropping. for growth, yield and nutrient availability. It also explain how B. megaterium  positively affects soil nutrients and cucumber plant nutrients (Phosphorus and potassium), enhancing their bioavailability and uptake. It also showed how soil microbial communities are effected by B. megaterium leading to an increased soil bacterial diversity. B. megaterium application also act by shaping a stable soil bacterial co-occurrence network,  enriching beneficial bacteria and suppressing the pathogen Xanthomonas.

I strongly recommend to publish this article after minor corrections:

Line 27: please to mention B. megaterium inoculum concentration .

Line 102-Line 105: Please to show clearly what is new? How your work coule be considered as original.

Line 122: What do you mean by "root affected soil"?

Line 130 please to write scientific names in italic "B. megaterium", please to check elsewhere.

Line 133: please to mention B. megaterium inoculum concentration.

Line 143-145: please to rewrite for clarity.

Line 202: plase to write as : "The supernatant was Collected and..."

Line 328-329 and Figure 3: it is not clear value of 4.6×104 cfu g-1 soil in "From 2 to 8 days after its application, the number of B. megaterium copies remained between 1.0×104 and 4.6×104 cfu g-1 soil", do you mean between 2 and 4 days.

Figure S1: needs statistics, please to show whether there is significant differences or no.

Author Response

Dear Editor and Reviewers:

We thank the Editor and Reviewers for their constructive comments. All revisions have been implemented in the revised manuscript and highlighted in yellow for ease of inspection, as requested by the Editor.

Thank you for your comments concerning our manuscript entitled “Bacillus megaterium inoculation enhances the stability of the soil bacterial network and promotes cucumber growth in a newly established greenhouse” (Manuscript ID: agriculture-3970588). The comments are all valuable and very helpful for improving our article. We have revised the manuscript addressing all the comments by the reviewers and editors. We hope these revisions will meet with your approval. Below is a list of the responses to the reviewers’ comments.

Best regards,

Yours Sincerely,

Wenju Liu

 

Comments 1: I strongly recommend to publish this article after minor corrections:

Response: We appreciate the positive evaluation and addressed all minor corrections requested below.

Comments 2: Line 27: please to mention B. megaterium inoculum concentration.

Revision: Thanks for your comment. Have done.

Comments 3: Line 102-Line 105: Please to show clearly what is new? How your work coule be considered as original.

Revision: Thanks for your comments. We have revised the Introduction to explicitly define the novelty of our work by identifying a specific knowledge gap: the lack of understanding of how microbial inoculants influence early-stage soil microbiome assembly and bacterial network stability in newly established greenhouse systems with short cultivation histories.

In particular, we now contrast this study with previous research that has predominantly focused on long-term monocropping greenhouses, including our own earlier work, and clearly state that the present study addresses the initial phase of greenhouse operation, when microbial communities are still forming and potentially more responsive to inoculation. Please see L104-L110 (highlighted in yellow).

Comments 4: Line 122: What do you mean by "root affected soil"?

Revision: We added a clear definition of ‘root-affected soil’ and specified the sampling distance from roots. Please see L176-L177 in Methods 2.2 first sentences (highlighted in yellow).

Comments 5: Line 130 please to write scientific names in italic "B. megaterium", please to check elsewhere.

Revision: All scientific names are now italicized throughout the manuscript, including in figure captions and supplementary material headings where applicable.

Comments 6: Line 133: please to mention B. megaterium inoculum concentration.

Revision: Have added the inoculum concentration and per-plant cfu calculation in Methods 2.1. Please see L146-L151 (highlighted in yellow).

Comments 7: Line 143-145: please to rewrite for clarity.

Revision: We rewrote the relevant sentences in Methods 2.1 for clarity. Please see L154-L160 (highlighted in yellow).

Comments 8: Line 202: plase to write as : "The supernatant was Collected and..."

Revision: Have corrected as suggested.

Comments 9: Line 328-329 and Figure 3: it is not clear value of 4.6×104 cfu g-1 soil in "From 2 to 8 days after its application, the number of B. megaterium copies remained between 1.0×104 and 4.6×104 cfu g-1 soil", do you mean between 2 and 4 days.

Revision: We thank the reviewer for noting the ambiguity in the original wording. The expression “from 2 to 8 days” has been revised to explicitly indicate the discrete sampling time points (2, 4, and 8 days after inoculation). We retain the range description because it represents the minimum and maximum values observed across all replicates at these sampling times, which reflects the relatively stable abundance of Bacillus megaterium during this period. The revised wording is now fully consistent with Figure 3. Please see L342 (highlighted in yellow).

Comments 10: Figure S1: needs statistics, please to show whether there is significant differences or no.

Revision: Thank you for the suggestion. We have ensured that Figure S1 clearly indicates statistical significance between treatments using asterisks (* p < 0.05, ** p < 0.01) and that the caption specifies the statistical test; non-asterisked comparisons are not significant.

Reviewer 3 Report

Comments and Suggestions for Authors

The article is good, but it needs to be revised before it can be accepted.

In particular, it is not clear to me whether the experiments were carried out in a greenhouse or in the field. In fact, greenhouse experiments usually refer to the use of pots, while field experiments refer to the use of randomized plots, which I believe is the case in this article. I therefore ask that this point be clarified, perhaps by showing images of the experimental setup.

What is the distance between the plants?

The concentration of B. megaterium used is also unclear: this can be found at the bottom of paragraph 2.1 and in the conclusions, but knowing how many bacteria are given in each treatment is fundamental to this article, so I ask that this point be specified more clearly.

The PCOA analysis shows the distribution based on sampling time. The times 60, 80, and 118 are much more comparable than the others. Could this be because the concentration of the harmless substance is very low at this point?

I also suggest including a table summarizing the different treatments carried out during the experiments, with an associated sampling scheme.

It is mentioned that the extent of colonization was evaluated, but these data are not described, nor is the procedure for this evaluation mentioned. I therefore ask for clarification on this point.

Half of the article contains the full words potassium and phosphorus, while the other half contains their abbreviations. I suggest using only the abbreviations throughout the article.

I suggest checking the quality of the figures and expanding the comments on them, as they are not always clear and comprehensive.

Not all names of microorganisms and plants are always written in italics. Please correct this.

p-value and not P-value.

 

 

Author Response

Dear Editor and Reviewers:

We thank the Editor and  Reviewers for their constructive comments. All revisions have been implemented in the revised manuscript and highlighted in yellow for ease of inspection, as requested by the Editor.

Thank you for your comments concerning our manuscript entitled “Bacillus megaterium inoculation enhances the stability of the soil bacterial network and promotes cucumber growth in a newly established greenhouse” (Manuscript ID: agriculture-3970588). The comments are all valuable and very helpful for improving our article. We have revised the manuscript addressing all the comments by the reviewers and editors. We hope these revisions will meet with your approval. Below is a list of the responses to the reviewers’ comments.

 

Best regards,

Yours Sincerely,

Wenju Liu

 Comments 1: In particular, it is not clear to me whether the experiments were carried out in a greenhouse or in the field. In fact, greenhouse experiments usually refer to the use of pots, while field experiments refer to the use of randomized plots, which I believe is the case in this article. I therefore ask that this point be clarified, perhaps by showing images of the experimental setup.

Revision: Thank you for your valuable comment. We have clarified that the study was conducted in a plastic-shed greenhouse, using randomized plots to simulate field conditions within the greenhouse, rather than using pots. Please see L128-130, L161. This photo shows the actual experimental site, which clearly demonstrates that it is a field experiment.

 

Comments 2: What is the distance between the plants?

Revision: We added plant density (3.56 plants m⁻²) derived from plot size and plant number, and provided the equivalent area per plant (~0.28 m²; ≈0.53 m × 0.53 m). Please see L163-L164.

 

Comments 3: The concentration of B. megaterium used is also unclear: this can be found at the bottom of paragraph 2.1 and in the conclusions, but knowing how many bacteria are given in each treatment is fundamental to this article, so I ask that this point be specified more clearly.

Revision: We specified the product concentration (7.0×10⁸ cfu mL^-1), and added an explicit per-plant cfu delivery calculation linked to the field rate and plant density. Please see Abstract and Methods 2.1 (highlighted in yellow).

 

Comments 4: The PCOA analysis shows the distribution based on sampling time. The times 60, 80, and 118 are much more comparable than the others. Could this be because the concentration of the harmless substance is very low at this point?

Response: Thank you for your observation. The temporal separation observed in the PCoA analysis primarily reflects the different plant growth stages, which are associated with shifts in root exudation and soil conditions. While the concentrations of harmless substances may play a role, the observed separation is more likely due to changes in the plant’s physiological development.

 

Comments 5: I also suggest including a table summarizing the different treatments carried out during the experiments, with an associated sampling scheme.

Revision: Thank you for the suggestion. We have added a new Table S1 summarizing the different treatments, inoculation timing, sampling depths, and time points. This table has been included in the Supplementary Material and referenced appropriately in the Methods section.

 

Comments 6: It is mentioned that the extent of colonization was evaluated, but these data are not described, nor is the procedure for this evaluation mentioned. I therefore ask for clarification on this point.

Revision: Thank you for pointing this out. We appreciate the opportunity to clarify this point. We have now explicitly stated that colonization was quantified using qPCR with Bacillus megaterium-specific primers. Additionally, we have ensured that Results Section 3.4 and Figure 3 provide a more detailed description of the colonization dynamics. This clarification has been included in Methods Section 2.5 and Results Section 3.4.

 

Comments 7: Half of the article contains the full words potassium and phosphorus, while the other half contains their abbreviations. I suggest using only the abbreviations throughout the article.

Revision: Thank you for your suggestion. We have standardized the terminology throughout the article by using the abbreviations P for phosphorus and K for potassium.

 

Comments 8: I suggest checking the quality of the figures and expanding the comments on them, as they are not always clear and comprehensive.

Revision: We reviewed all figures and figure captions for clarity and completeness, and expanded captions where needed to define treatments, sampling time points, and statistical tests.

 

Comments 9: Not all names of microorganisms and plants are always written in italics. Please correct this.

Revision: Thanks for your comments. Have corrected throughout (italic formatting).

 

Comments 10: p-value and not P-value.

Revision: Thanks for your comments. Have standardized to ‘p’ (e.g., p < 0.05) throughout.

Reviewer 4 Report

Comments and Suggestions for Authors

The manuscript presents the results of a greenhouse experiment investigating the effect of a bacterial strain identified by the authors as Bacillus megaterium on cucumber growth. Please verify the correct taxonomic designation of the strain. Since 2020, Bacillus megaterium has been reclassified as Priestia megaterium (see: doi:10.1007/s00253-021-11424-6). I strongly recommend using the current and accepted nomenclature throughout the manuscript.

The experimental design appears appropriate, and control treatments were included. The results are clearly presented and, in most cases, adequately interpreted. However, several issues require attention, as detailed below:

1. Please correct the citation formatting throughout the manuscript by adding spaces before square brackets (e.g., line 48: “sustainability [1, 2]”).
2. Please clarify what is meant by “some common measures” (line 55).
3. Ensure that all Latin names of bacterial strains are italicized consistently throughout the manuscript (e.g., lines 70, 79, 130–133, 457).
4. I do not agree with the statement: “It is still unclear how B. megaterium may affect soil P and K bioavailability” (line 83). These mechanisms have been thoroughly investigated and described in numerous previous studies.
5. Please clearly explain why both natural and chemical fertilization were applied (lines 148–151). Was fertilization applied to the experimental treatments as well, or only to the control plots?
6. My major concern relates to the characterization of the applied bacterial strain. Although phytohormone production (IAA, ZR, GA₃) was measured, it remains unclear whether the strain is capable of solubilizing phosphorus or potassium.
• Were in vitro tests conducted prior to application to confirm P and K solubilization ability?
• Was the bacterial cell concentration verified experimentally, or does the value reported in line 159 originate from the manufacturer’s specification?
• Please note that inoculation likely introduced not only bacterial cells but also accompanying nutrients (possibly containing P and K). Was this accounted for in the interpretation of the results?
• Additionally, please clarify whether the value presented in the Conclusions (line 550) was directly measured or only calculated.
7. Please avoid colloquial expressions such as “we wondered” (line 165).
8. The phrase “three triplicates” (line 200) is unclear. Please specify whether three or nine replicates were used.
9. In lines 203–204, please clarify that the precipitated residue was dissolved in methanol after vacuum drying, rather than the organic solvent itself.
10. In the Results section, multiple results are often discussed simultaneously (e.g., lines 259 and 291), which makes it difficult to associate specific values with individual treatments.
11. Please discuss why increases were observed in the 75BM and 300BM treatments but not in the 150BM treatment (Section 3.2, lines 217–287). Although differences were observed, they were not statistically significant.
12. The statement “This indicated that the B. megaterium strain could produce these phytohormones for cucumber plant growth” (lines 318–319) requires clarification.
• Were the detected hormone levels high compared to other PGPM strains?
• Rather than being cucumber-specific, such hormone production may be generally beneficial for plant growth and should be discussed as such.
13. Regarding qPCR quantification, I have concerns about expressing the results as CFU g⁻¹ soil. In practice, qPCR quantifies gene copy numbers rather than viable cell counts.
• Were culture-based (media-dependent) methods used for validation?
• Why was a lower abundance observed in the 300BM treatment compared to 75BM (Figure 3), despite the higher inoculation dose?
• Furthermore, please justify whether a population level of approximately 10⁴ CFU g⁻¹ soil is sufficient to claim successful colonization, considering that total bacterial abundance in soil can reach 10¹⁰–10¹¹ CFU g⁻¹.
14. Please ensure that correct and consistent units are used throughout the manuscript (e.g., replace “ml” with “cm³”). SI units should be applied consistently.
15. Figures 4 and 8 are overly complex and difficult to interpret, particularly Figure 8. Simplification is strongly recommended.
16. In Table S1, please verify why available phosphorus (AP) values are significantly higher than total phosphorus (TP), as AP should logically represent only a fraction of TP. The same concern applies to potassium measurements.

17. The Discussion section requires revision. At present, it mainly reiterates the results rather than critically discussing them in the context of existing literature. Although approximately 30 publications are cited in this section, only a few are directly compared with the results of the present study. This limits the reader’s ability to assess the novelty and scientific contribution of the findings.

Author Response

Dear Editor and Reviewers:

We thank the Editor and Reviewers for their constructive comments. All revisions have been implemented in the revised manuscript and highlighted in yellow for ease of inspection, as requested by the Editor.

Thank you for your comments concerning our manuscript entitled “Bacillus megaterium inoculation enhances the stability of the soil bacterial network and promotes cucumber growth in a newly established greenhouse” (Manuscript ID: agriculture-3970588). The comments are all valuable and very helpful for improving our article. We have revised the manuscript addressing all the comments by the reviewers and editors. We hope these revisions will meet with your approval. Below is a list of the responses to the reviewers’ comments.

Best regards,

Yours Sincerely,

Wenju Liu

 The manuscript presents the results of a greenhouse experiment investigating the effect of a bacterial strain identified by the authors as Bacillus megaterium on cucumber growth. Please verify the correct taxonomic designation of the strain. Since 2020, Bacillus megaterium has been reclassified as Priestia megaterium (see: doi:10.1007/s00253-021-11424-6). I strongly recommend using the current and accepted nomenclature throughout the manuscript.

Response: We thank the reviewer for bringing this important taxonomic update to our attention and acknowledge that Bacillus megaterium has recently been reclassified as Priestia megaterium. For consistency with previous studies and with the commercial product labeling used in this work, we retain the name Bacillus megaterium throughout the manuscript while explicitly acknowledging the updated nomenclature.

The experimental design appears appropriate, and control treatments were included. The results are clearly presented and, in most cases, adequately interpreted. However, several issues require attention, as detailed below:

1.Please correct the citation formatting throughout the manuscript by adding spaces before square brackets (e.g., line 48: “sustainability [1, 2]”).

Response: Thank you for noting this formatting issue. We have corrected the citation formatting.

2.Please clarify what is meant by “some common measures” (line 55).

Response: Thank you for the comment. We have clarified what “some common measures” refers to by specifying representative management practices used to alleviate monocropping obstacles in greenhouse vegetable systems. Please see L56-57 (highlighted in yellow).

3.Ensure that all Latin names of bacterial strains are italicized consistently throughout the manuscript (e.g., lines 70, 79, 130–133, 457).

Response: Thank you for the suggestion. We have checked the entire manuscript and ensured that all Latin names (genus/species) are italicized consistently.

4.I do not agree with the statement: “It is still unclear how B. megaterium may affect soil P and K bioavailability” (line 83). These mechanisms have been thoroughly investigated and described in numerous previous studies.

Response: We appreciate this important point and agree that the biochemical mechanisms of phosphorus and potassium solubilization by Bacillus megaterium have been well documented. We have therefore revised the text to clarify that the remaining knowledge gap does not concern the solubilization mechanisms per se, but rather the functional performance and ecological impacts of B. megaterium inoculation in newly established greenhouse systems with short planting histories, where soil microbial interaction networks are still developing. Please see L83-88.

5.Please clearly explain why both natural and chemical fertilization were applied (lines 148–151). Was fertilization applied to the experimental treatments as well, or only to the control plots?

Response: Thank you for this comment. The combined application of organic manure and chemical fertilizers reflects the standard fertilization practice commonly adopted by local greenhouse cucumber growers. All treatments, including the control (CK), received the same fertilization regime, and the microbial inoculant was the only experimental variable. This experimental design allowed us to evaluate the effects of Bacillus megaterium inoculation within the context of farmer-relevant conventional fertilization practices, rather than under nutrient-limited or artificial conditions.

6.My major concern relates to the characterization of the applied bacterial strain. Although phytohormone production (IAA, ZR, GA₃) was measured, it remains unclear whether the strain is capable of solubilizing phosphorus or potassium.

Were in vitro tests conducted prior to application to confirm P and K solubilization ability?

Response: Thank you for this question. Yes, the phosphorus- and potassium-solubilizing capacities of Bacillus megaterium have been confirmed in our previous foundational studies using standard in vitro assays. Those results demonstrated that this strain is capable of mobilizing insoluble P and K sources.

In the present study, our objective was not to re-verify these established traits under laboratory conditions, but rather to evaluate the functional performance of the commercial inoculant under greenhouse conditions, as reflected by changes in soil available P and K and their accumulation in cucumber plants.

Was the bacterial cell concentration verified experimentally, or does the value reported in line 159 originate from the manufacturer’s specification?

Response: Thank you for the clarification request. The reported viable cell concentration was determined experimentally by us prior to application and was not solely based on the manufacturer’s specification.

Please note that inoculation likely introduced not only bacterial cells but also accompanying nutrients (possibly containing P and K). Was this accounted for in the interpretation of the results?

Response: Thank you for raising this important point. We acknowledge that, as a commercial microbial inoculant, the formulation may introduce trace amounts of accompanying nutrients together with bacterial cells. However, the quantities of these nutrients are negligible relative to the background soil nutrient pools and the conventional fertilization applied uniformly across all treatments, and they are primarily intended to support bacterial viability rather than to directly contribute to plant nutrition.

Importantly, the objective of this study was to evaluate the agronomic and microbiological effects of the commercial inoculant as a formulated product, as it is applied in practice, rather than to isolate the effects of individual components. Therefore, the observed responses are interpreted as the outcome of inoculation with the commercial product as a whole (including viable cells and formulation components).

Additionally, please clarify whether the value presented in the Conclusions (line 550) was directly measured or only calculated.

Response: Thanks for your comment. the value presented in the Conclusions (5.25 × 10¹³ cfu ha⁻¹) was calculated from the labeled concentration (7.0 × 10⁸ CFU mL⁻¹) and the field application rate (75 L ha⁻¹).

7.Please avoid colloquial expressions such as “we wondered” (line 165).

Response: Thanks for your comment. We have revised the wording to a more formal scientific style.

8.The phrase “three triplicates” (line 200) is unclear. Please specify whether three or nine replicates were used.

Response: Thank you for catching this ambiguity. We clarified the replication statement to specify the exact number of biological replicates collected at each time point. Please see L 219 (highlighted in yellow).

9.In lines 203–204, please clarify that the precipitated residue was dissolved in methanol after vacuum drying, rather than the organic solvent itself.

Response: Thank you for the correction. We revised the sentence to clarify that the dried residue was re-dissolved in methanol after vacuum drying. Please see L 223 (highlighted in yellow).

10.In the Results section, multiple results are often discussed simultaneously (e.g., lines 259 and 291), which makes it difficult to associate specific values with individual treatments.

Response: Thank you for this helpful comment. The Results section has been revised to clearly associate specific values with individual treatments and plant compartments.

11.Please discuss why increases were observed in the 75BM and 300BM treatments but not in the 150BM treatment (Section 3.2, lines 217–287). Although differences were observed, they were not statistically significant.

Response: Thank you for this insightful question. The non-significant response at 150BM, increases at 75BM and 300BM, suggests that PGPR responses can be non-linear and dose-dependent, and that intermediate rates may not necessarily yield monotonic effects due to interactions with indigenous microbiota, resource competition, or variability in establishment under field-like greenhouse conditions (Compant et al., 2019).

Compant, S., Samad, A., Faist, H., Sessitsch, A., 2019.A review on the plant microbiome: Ecology, functions, and emerging trends in microbial application. Applied Microbiology and Biotechnology 103, 8045–8061.

12.The statement “This indicated that the B. megaterium strain could produce these phytohormones for cucumber plant growth” (lines 318–319) requires clarification.

Were the detected hormone levels high compared to other PGPM strains?

Response: Thank you for these insightful comments. In this study, the detected levels of IAA, GA₃, and ZR produced by Bacillus megaterium fall within the range commonly reported for plant growth-promoting microorganisms under comparable in vitro culture conditions, rather than being exceptionally high. Our intention was not to rank this strain against other PGPMs based on absolute hormone production, but to confirm that the commercial inoculant possesses representative phytohormone-producing capability that is consistent with its growth-promoting function.

Rather than being cucumber-specific, such hormone production may be generally beneficial for plant growth and should be discussed as such.

Response: Thank you for these insightful comments. We fully agree that phytohormone production by PGPMs is not crop-specific. Accordingly, we revised the relevant text to discuss hormone production as a general plant growth-promoting trait, which can contribute to root development, nutrient uptake, and plant performance across a wide range of crops. Any cucumber-specific wording has been removed, and the interpretation has been generalized in the revised manuscript.

13.Regarding qPCR quantification, I have concerns about expressing the results as CFU g⁻¹ soil. In practice, qPCR quantifies gene copy numbers rather than viable cell counts.

Were culture-based (media-dependent) methods used for validation?

Response: We appreciate these important methodological concerns. We revised the manuscript to report qPCR results as gene copy numbers (copies g⁻¹ soil) rather than CFU and clarified that qPCR quantifies DNA copies rather than viable cells. Culture-based enumeration was not performed in this study.

Why was a lower abundance observed in the 300BM treatment compared to 75BM (Figure 3), despite the higher inoculation dose?

Response: The lower abundance observed in 300BM relative to 75BM may reflect variability in establishment and density-dependent interactions with the resident microbiota under soil conditions.

Furthermore, please justify whether a population level of approximately 10⁴ CFU g⁻¹ soil is sufficient to claim successful colonization, considering that total bacterial abundance in soil can reach 10¹⁰–10¹¹ CFU g⁻¹.

Response: Thank you for this important and technically relevant question. We agree that a population size of 10⁴ copies g⁻¹ soil is several orders of magnitude lower than the total bacterial abundance typically reported for bulk soils. However, successful colonization by plant growth-promoting microorganisms should be interpreted in a functional and ecological context, rather than by direct comparison with total microbial biomass.

First, PGPMs primarily exert their effects at the root–soil interface, where localized populations interacting with plant roots can influence nutrient mobilization, hormone signaling, and microbial interactions, even when their absolute abundance is low relative to the total soil microbiome. Second, previous studies have shown that PGPM populations on the order of 10³–10⁵ copies g⁻¹ soil can be sufficient to induce measurable plant and soil responses, particularly when persistence over time is observed rather than transient detection.

In the present study, B. megaterium was consistently detected across multiple sampling points following inoculation, indicating establishment and persistence rather than short-term survival. We therefore interpret colonization success in terms of detectable persistence and associated functional responses (changes in soil P and K availability, plant nutrient accumulation, and microbial community structure), rather than relative abundance alone.

 

14.Please ensure that correct and consistent units are used throughout the manuscript (e.g., replace “ml” with “cm³”). SI units should be applied consistently.

Response: Thank you for your comment. We standardized units throughout the manuscript, using consistent SI notation. Where volume units are reported, we use “mL” (SI-accepted; equivalent to cm³) consistently and avoid mixed forms such as “ml”.

15.Figures 4 and 8 are overly complex and difficult to interpret, particularly Figure 8. Simplification is strongly recommended.

Response: We thank the reviewer for the constructive suggestion regarding figure complexity. We carefully re-evaluated Figures 4 and 8 and acknowledge that they contain multiple layers of information. However, in this study, these figures were intentionally designed to present the multivariate relationships and network properties of soil bacterial communities, which are central to the ecological interpretation of our results.

Similar levels of complexity are commonly adopted in published studies focusing on microbial community correlations and co-occurrence networks, where simplifying the figures may risk obscuring key structural patterns and interactions. After careful consideration, we therefore retained the current presentation to preserve the completeness and interpretability of the results, while ensuring that all figure legends clearly guide the reader through the main messages conveyed by each panel.

 

16.In Table S1, please verify why available phosphorus (AP) values are significantly higher than total phosphorus (TP), as AP should logically represent only a fraction of TP. The same concern applies to potassium measurements.

Response: Thank you for identifying this inconsistency. We re-checked the nutrient units and found that TP and TK were incorrectly displayed with “mg kg⁻¹” in the table; they should be reported as “g kg⁻¹”. We corrected the units (and any related text) accordingly, which resolves the apparent contradiction that AP exceeds TP.

17.The Discussion section requires revision. At present, it mainly reiterates the results rather than critically discussing them in the context of existing literature. Although approximately 30 publications are cited in this section, only a few are directly compared with the results of the present study. This limits the reader’s ability to assess the novelty and scientific contribution of the findings.

Response: Thank you for this constructive suggestion. We revised the Discussion to move beyond reiteration of results and to more explicitly position our findings in the context of existing literature. (highlighted in yellow)

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Editor,

I consider that the revised version of the manuscript entitled “Bacillus megaterium inoculation enhances the stability of the soil bacterial network and promotes cucumber growth in a newly established greenhouse” is now ready for publication in Agriculture.

Comments on the Quality of English Language

Dear Editor,

I consider that the revised version of the manuscript entitled “Bacillus megaterium inoculation enhances the stability of the soil bacterial network and promotes cucumber growth in a newly established greenhouse” is now ready for publication in Agriculture.

Author Response

Dear Editor and Reviewers:

Thank you very much for your positive feedback and for recognizing the improvements made in the revised manuscript. We appreciate your constructive comments throughout the review process, which have greatly helped enhance the clarity and quality of the work. We are glad to hear that the manuscript is now ready for publication in Agriculture.

Best regards,

Yours Sincerely,

Wenju Liu

Reviewer 4 Report

Comments and Suggestions for Authors

The revised version of the manuscript has been significantly improved, and most of the previously raised comments have been adequately addressed or convincingly justified. The overall quality of the manuscript has clearly increased.

Nevertheless, I remain concerned about the continued use of outdated taxonomic nomenclature for the bacterial strain investigated in this study. According to the LPSN (List of Prokaryotic Names with Standing in Nomenclature) database, Priestia megaterium is currently the valid and recommended name for this species in scientific publications. While I acknowledge that the former name, Bacillus megaterium, is still frequently used due to historical precedence and commercial recognition, the application of the currently accepted nomenclature is strongly recommended to ensure scientific accuracy, consistency with contemporary literature, and long-term citability of the manuscript. It is worth noting that the use of the updated name Priestia megaterium in high-quality, PubMed-indexed publications has increased markedly since 2025, with approximately 30–40% of recent studies already adopting this nomenclature either as the primary or equivalent species name. A commonly accepted and recommended approach in microbiological and genomic literature is the use of the formulation: Priestia megaterium (formerly Bacillus megaterium). I strongly encourage the Authors to adopt this convention. At minimum, the currently valid name should be explicitly mentioned in the manuscript text when describing the microbiological material.

Furthermore, if the Authors have thoroughly characterized the strain used in the experiment, this information should be clearly described in the Materials and Methods section. In its current form, the description of the applied bacterial strain is insufficiently detailed and does not fully support the interpretation of the presented results.

 

In addition, several minor issues remain and require the Authors’ attention:

1. Ensure that all Latin names of bacterial strains are consistently italicized throughout the manuscript (e.g., lines 69, 611, 797). In line 698, “megaterium” should not be capitalized.
2. Please unify the formatting of temperature units—there should be no space before the degree Celsius symbol (°C).
3. In Figure 3, the Y-axis description contains an error (“copes”) and should read “gene copies.” Moreover, throughout the manuscript, expressions such as “Bacillus megaterium copies” or “population copies” are used when referring to gene copy numbers. These should be corrected and replaced with appropriate and precise terminology and units.
4. Line 633: “Institutional Review Board Statement: ‘Not applicable’ for studies not involving humans or animals.” The wording should be standardized to “not applicable,” in accordance with the Journal’s guidelines.

Author Response

Dear Editor and Reviewers:

 

We thank the Editor and Reviewers for their constructive comments. All revisions have been implemented in the revised manuscript and highlighted in yellow for ease of inspection, as requested by the Editor.

Thank you for your comments concerning our manuscript entitled “Bacillus megaterium inoculation enhances the stability of the soil bacterial network and promotes cucumber growth in a newly established greenhouse” (Manuscript ID: agriculture-3970588). The comments are all valuable and very helpful for improving our article. We have revised the manuscript addressing all the comments by the reviewers and editors. We hope these revisions will meet with your approval. Below is a list of the responses to the reviewers’ comments.

 

Best regards,

Yours Sincerely,

Wenju Liu

 

Nevertheless, I remain concerned about the continued use of outdated taxonomic nomenclature for the bacterial strain investigated in this study. According to the LPSN (List of Prokaryotic Names with Standing in Nomenclature) database, Priestia megaterium is currently the valid and recommended name for this species in scientific publications. While I acknowledge that the former name, Bacillus megaterium, is still frequently used due to historical precedence and commercial recognition, the application of the currently accepted nomenclature is strongly recommended to ensure scientific accuracy, consistency with contemporary literature, and long-term citability of the manuscript. It is worth noting that the use of the updated name Priestia megaterium in high-quality, PubMed-indexed publications has increased markedly since 2025, with approximately 30–40% of recent studies already adopting this nomenclature either as the primary or equivalent species name. A commonly accepted and recommended approach in microbiological and genomic literature is the use of the formulation: Priestia megaterium (formerly Bacillus megaterium). I strongly encourage the Authors to adopt this convention. At minimum, the currently valid name should be explicitly mentioned in the manuscript text when describing the microbiological material.

Response: Thank you for your thoughtful and constructive comment regarding the bacterial strain nomenclature. We fully acknowledge the importance of using accurate and updated taxonomic nomenclature to ensure scientific consistency and clarity.

As you correctly pointed out, Priestia megaterium is the currently accepted name for this species, and we appreciate your recommendation to adopt the updated nomenclature. In response to your comment, we have now replaced Bacillus megaterium with Priestia megaterium throughout the manuscript. We have added the previous name Bacillus megaterium (now Priestia megaterium) in parentheses at the first mention of the strain.

Furthermore, if the Authors have thoroughly characterized the strain used in the experiment, this information should be clearly described in the Materials and Methods section. In its current form, the description of the applied bacterial strain is insufficiently detailed and does not fully support the interpretation of the presented results.

Response: Thank you for your valuable suggestion. In response, we have revised the Materials and Methods section to provide a more detailed description of the Priestia megaterium strain used in our study. We have clarified that the strain was originally classified as Bacillus megaterium and has since been reclassified to Priestia megaterium. Additionally, we have included information about the strain's whole-genome sequencing, which has been completed and confirms its genomic characteristics. This revision should address your concern regarding the strain’s characterization and support the interpretation of our results. Please see L177-180.

In addition, several minor issues remain and require the Authors’ attention:

1. Ensure that all Latin names of bacterial strains are consistently italicized throughout the manuscript (e.g., lines 69, 611, 797). In line 698, “megaterium” should not be capitalized.

Response: Thank you for pointing this out. We have carefully reviewed the manuscript and ensured that all Latin names of bacterial strains, including Priestia megaterium, are consistently italicized throughout the manuscript.

2. Please unify the formatting of temperature units—there should be no space before the degree Celsius symbol (°C).

Response: We appreciate your careful attention to detail. We have unified the formatting of temperature units throughout the manuscript and removed the space before the degree Celsius symbol (°C) in accordance with the journal's guidelines.

3. In Figure 3, the Y-axis description contains an error (“copes”) and should read “gene copies.” Moreover, throughout the manuscript, expressions such as “Bacillus megaterium copies” or “population copies” are used when referring to gene copy numbers. These should be corrected and replaced with appropriate and precise terminology and units.

Response: Thank you for pointing out this inconsistency. We have corrected the error in Figure 3, where “copes” has been replaced with “gene copies” on the Y-axis. Additionally, we have reviewed the manuscript and replaced expressions with the appropriate terminology, such as “gene copy numbers,” throughout the manuscript to ensure accuracy and consistency.

4. Line 633: “Institutional Review Board Statement: ‘Not applicable’ for studies not involving humans or animals.” The wording should be standardized to “not applicable,” in accordance with the Journal’s guidelines.

Response: Thank you for your suggestion. We have revised the Institutional Review Board statement in line 633 to use the standardized wording, “not applicable,” in accordance with the journal’s guidelines.