Molecular Identification and Characterization of Peribacillus simplex LT4 Isolated from the Roots of Baby Maize (Zea mays L.)

Response to Reviewer 1 Comments

1. Summary

We sincerely thank the reviewers for their valuable time and constructive comments on our manuscript. Detailed, point-by-point responses are provided below, and all corresponding revisions have been incorporated and highlighted using track changes in the revised manuscript.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

A detailed, point-by-point response to each reviewer’s comment is provided below

Are all the cited references relevant to the research?

Yes/Can be improved/Must be improved/Not applicable

Yes

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

Yes

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

Yes

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Yes

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Research Overview: This study isolated the nitrogen-fixing bacterium Peribacillus simplex from the roots of maize seedlings. It further analyzes its growth characteristics and metabolic activities, with a specific focus on nitrogen fixation metabolism.

Critical Comments & Feedback:

Comments 1:

Significant Discrepancy in Results: There is a critical oversight regarding the primary subject of the study. While Table 1 identifies V4 as the optimal sample, the narrative text consistently refers to V3. This inconsistency must be corrected throughout the manuscript to ensure data integrity.

Response 1: Thank you for pointing this out. We agree with this comment. The discrepancy occurred due to an error during data entry in Table 1 (Burk agar position). This issue has now been corrected and highlighted in RED in the revised manuscript. Accordingly, the relevant text throughout the manuscript has been revised to ensure consistency, confirming that sample V3 is the optimal isolate.

Comments 2:

Terminology and Formatting: * Line 14: The acronym "RNFB" must be defined in full upon its first appearance.

Line 176 (Error Description): Please standardize the notation for Gram staining and reaction intensity. Suggested format: Note: Gram-negative (-); (-): no reaction; (+): positive reaction; (++): strong reaction.

Response 2: Agree. We have accordingly revised the manuscript to correct all errors identified by the reviewer throughout the text. In addition, the explanatory note for the biochemical reaction symbols has been modified to improve clarity. Specifically, the note “Gram-negative (−); (−): no reaction; (+): positive reaction; (++): strong reaction” has replaced the previous note in the manuscript. This revision can be found in the revised manuscript at line 176, and the change has been highlighted in RED for ease of reference.

“Gram-negative (−); (−): no reaction; (+): positive reaction; (++): strong reaction.”

Comments 3:

Qualitative Data Justification: The data is currently presented using qualitative descriptions (e.g., -, +, ++). What are the specific criteria used for these classifications? Additionally, please clarify if any statistical analysis was performed to support these qualitative observations.

Response 3:

Thank you for this valuable comment. We agree with the reviewer and have revised the manuscript to clarify how the qualitative observations were determined. Specifically, the symbols (−), (+), and (++) were assigned based on direct visual observation of bacterial reactions in the respective test media, including changes such as color development, halo formation, or other characteristic responses in the assay solutions. In this context, (−) indicates no visible reaction, (+) indicates a positive reaction, and (++) indicates a strong reaction. Because these assays were designed as qualitative screening tests and the results were recorded descriptively rather than quantitatively, no statistical analysis was performed for these data.

This clarification has been added to the revised manuscript (Section Materials and Methods, lines XX–XX) and highlighted in yellow for the reviewer’s convenience.

“(−) indicates no visible reaction; (+) indicates a positive reaction; and (++) indicates a strong reaction based on direct visual observation of bacterial responses in the respective assay media.”

Comments 4:

Phylogenetic Tree Analysis: It is recommended to include an outgroup in the phylogenetic tree to provide better evolutionary context. Furthermore, V3 should be positioned alongside the other two strains; even if the 16S rDNA sequences are identical, they may still represent distinct species.

Response 4:

Agree. We have accordingly redrawn and revised the phylogenetic tree following the reviewer’s suggestion to improve its clarity and presentation. The updated phylogenetic tree has been replaced in the revised manuscript as Figure 2 in the main text, and the change has been highlighted in RED for easy reference.

Comments 5

Metabolic Impact: I suggest providing a more in-depth discussion regarding the influence of nitrate reduction and citrate utilization on the efficiency and mechanisms of nitrogen-fixing bacteria.

Response 5:

Agree. We have accordingly revised the Discussion section in response to the reviewer’s suggestion to improve the interpretation and clarity of the results. The revised content has been incorporated into the Discussion section (lines 260–271) and highlighted in RED in the revised manuscript for the reviewer’s convenience.

Comments 6:

Experimental Design Rationale: The authors should justify the choice of maize seedlings for this research. Does the presence of this bacterium diminish in the roots of mature plants? Clarifying the temporal dynamics of the microbial population would strengthen the study.

Response 6 :

Thank you for this valuable comment. We agree with the reviewer and have clarified the rationale for the sampling time in the revised manuscript. In this study, root samples were collected 30 days after sowing, which corresponds to the mature vegetative stage of baby maize under our cultivation conditions. Since baby maize is typically harvested 40–45 days after sowing, plants at 30 days already possess a well-developed root system, allowing the isolation of bacteria that have established relatively stable colonization in the rhizosphere and root tissues. Therefore, this sampling stage is appropriate for capturing active root-associated nitrogen-fixing bacteria.

Response to Reviewer 2 Comments

1. Summary

We sincerely thank the reviewers for their valuable time and constructive comments on our manuscript. Detailed, point-by-point responses are provided below, and all corresponding revisions have been incorporated and highlighted using track changes in the revised manuscript.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes/Can be improved/Must be improved/Not applicable

A detailed, point-by-point response to each reviewer’s comment is provided below

Are all the cited references relevant to the research?

Yes/Can be improved/Must be improved/Not applicable

Yes

Is the research design appropriate?

Yes/Can be improved/Must be improved/Not applicable

Yes

Are the methods adequately described?

Yes/Can be improved/Must be improved/Not applicable

Yes

Are the results clearly presented?

Yes/Can be improved/Must be improved/Not applicable

Yes

Are the conclusions supported by the results?

Yes/Can be improved/Must be improved/Not applicable

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Research Overview: This study isolated the nitrogen-fixing bacterium Peribacillus simplex from the roots of maize seedlings. It further analyzes its growth characteristics and metabolic activities, with a specific focus on nitrogen fixation metabolism.

Critical Comments & Feedback:

Comments 1:

Brief Summary. Isolation, physiological characteristics and molecular identification of a bacterial strain named LT4 from baby maize roots based on 16S rRNA gene sequencing identified as Peribacillus simplex The aim of this study was to assess its nitrogen-fixing capability and environmental tolerance, proposing this strain as a candidate biofertilizer. The work has great strengths and contributions, namely: isolation of indigenous rhizosphere bacteria, integration of phenotypic and molecular characterization data with taxonomic analyses together with attempts to determine functional traits that could be used for agricultural applications (salinity, temperature and pH tolerance).

Comments 2:

General Comments on Scientific Content. The isolation and preliminary characterization of indigenous bacterial strains from maize roots is relevant, as well as the integration of morphological, biochemical, and molecular approaches are in principle quite adequate. Nonetheless, there are some conceptual and methodological limitations within the study that undermine the robustness of its conclusions, particularly in demonstrating nitrogen-fixing capacity and taxonomic identification precision

Comments 3:

A major conceptual worry stems from nitrogen fixation interpretation. Nitrogen ase activity estimation and nitrogen accumulation in culture medium are primarily used. Although these observations are consistent with nitrogen metabolism they do not independently establish atmospheric nitrogen fixation without the appropriate additional supporting evidence such as genetic proof of nitrogen fixing genes (for example, nifH) or complete nitrogen balance. The conclusion that strain LT4 is an effective nitrogen-fixing bacterium thus seems to rest on stronger grounds than can be supported by the data presented alone.

Response 3: Thank you for this insightful comment. In this study, the nitrogen-fixing ability of strain LT4 was evaluated through nitrogenase activity and nitrogen accumulation in nitrogen-free culture medium, which are commonly used methods for the preliminary screening of diazotrophic bacteria. The positive nitrogenase activity together with the increase in total nitrogen concentration in the culture medium suggests that strain LT4 possesses metabolic traits associated with nitrogen fixation.

However, additional verification such as those suggested (e.g., genetic confirmation) was not performed in the present study because subsequent experiments are planned, including reduced chemical nitrogen fertilization trials under greenhouse conditions and field experiments. These studies will further evaluate the nitrogen-fixing contribution of the strain before its potential development as a biofertilizer.

Comments 4:

The experimental design also suffers from a lack of sufficient methodological detail in certain key aspects. One of these includes the inadequately described phylogenetic analysis, as no information can at present be found details either the chosen method of tree construction or evolutionary model used, number of bootstrap replicates (none are mentioned), limiting both reproducibility criteria and evaluation of robustness for all taxonomic placements. Similarly, nitrogenase activity measurements are reported without statistical analysis or variability estimates and without clear indication of replication, impeding the assessment of reliability and reproducibility of trends among treatments.

Response 4:

Agree. We have revised and expanded the methodological description in the Materials and Methods section to address the reviewer’s comment and improve the transparency and reproducibility of the study.

First, the phylogenetic tree has been redrawn (Figure 2) and additional details on the tree construction method, evolutionary model, and number of bootstrap replicates have been included to clarify the robustness of the phylogenetic analysis. These revisions are described in the Materials and Methods section and highlighted in red in the revised manuscript.

Second, we have clarified the number of experimental replicates and the statistical analysis used for nitrogenase activity measurements, allowing better evaluation of variability among treatments and improving the reliability of the results.

These revisions strengthen the methodological rigor and clarity of the study.

Comments 5:

General Evaluation Questions. This manuscript is quite concise, clear and relevant to the area of agricultural microbiology and sustainable crop production. The organization is coherent and follows conventional scientific structure.

References are mostly recent and up-to-date, although some references seem relatively general and do not extremely directly support a specific methodological claim. The number of self-citations seems reasonable and not excessive.

More specific phenotypic evidence is required to assert organismal nitrogen fixation capability, as the experimental design only partially characterizes the strain in this regard. Further genetic, physiological or isotopic evidence would greatly augment the conclusions.

Phylogenetic analysis and measurements of nitrogenase activity lacked sufficient methodological detail to allow replication of results.

The figures and tables are relevant, mostly adequate. However, the absence of statistical indicators (standard deviations or error bars and number of replicates) prevents interpretation of the functional data.

The concluding remarks are generally in agreement with the experimental observations but phrased cautiously, acknowledging the preliminary nature of our evidence.

The statements on availability of data and conflict of interests are sufficient.

Response 5: Thank you for this comprehensive evaluation and constructive feedback. We appreciate the reviewer’s positive assessment of the manuscript’s clarity and organization. All comments have been carefully considered, and the manuscript has been revised accordingly, including improvements to methodological details, statistical reporting, and a more cautious interpretation of the conclusions.

Comments 6:

Specific Comments

i) According to the manuscript, nitrogen-fixing bacteria can convert atmospheric nitrogen into ammonium and nitrate. This sentence does not make sense since ammonia is directly made through biological nitrogen fixation while nitrate production requires subsequent nitrification processes. (Lines 42–44)

ii)Isolation was described using yeast extract mannitol agar. This medium is not selective for nitrogen-fixing bacteria as it contains organic nitrogen. This is a limitation that should be recognized, and the authors need to clearly explain how nitrogen-fixing bacteria were differentiated from other heterotrophic bacteria at this point.( Lines 72–83). The classification of the isolates as rhizosphere nitrogen-fixing bacteria on the basis of growth in nitrogen-free medium should be viewed with caution. These conditions on growth suggest potential nitrogen metabolism, but do not definitively indicate atmospheric nitrogen fixation (Lines 94–110)

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iii)In addition, there is insufficient methodological detail regarding phylogenetic identification of isolated strains using 16S rRNA gene sequencing. The manuscript should include details such as the phylogenetic method used (e.g., Neighbor-Joining, Maximum Likelihood), evolutionary model, alignment method and bootstrap replication number. (Figure 1 and Lines 117–128). The nitrogenase activity assay is described, but the manuscript cannot provide number of biological replicates, and show statistical variability. We need this information in order to produce result with the security level we desire. (Lines 149–159)

iv)Their categorization of all isolates as nitrogen-fixing bacteria was mainly based on physiological tests and should be interpreted more carefully. This conclusion could be strengthened by additional methods of confirmation. (Table 1 and Lines 182–191)

v)The trends in nitrogenase activity and nitrogen accumulation are interesting, but I would like to see some statistical indicators (error bars or confidence interval) on the figure. You should add these in for proper evaluation of variation and significance. (Figure 3 and Lines 227–236)

vi) The paper concludes that taxonomic identity is confirmed by 100% sequence similarity. Although this strongly supports identification, the authors should recognize that (repeat) 16S rRNA gene sequences may not resolve closely related species, and that additional genomic methodologies would lend further evidence to support identification. (Lines 271–277)

Response 6:

i) Agree. We have accordingly revised the sentence to clarify that biological nitrogen fixation directly produces ammonia/ammonium, while nitrate formation occurs through subsequent nitrification processes in soil. This modification was made to improve the scientific accuracy of the statement in accordance with the reviewer’s suggestion. The revised text can be found in lines 40–44 and has been highlighted in red in the revised manuscript.

ii) During the initial isolation step, yeast extract mannitol agar (YMA) was used as a general-purpose medium to recover a broad diversity of bacteria associated with baby maize roots, including both epiphytic and endophytic populations. Root fragments with adhering rhizosphere soil were homogenized in sterile distilled water to release microbial cells, and the resulting suspension was serially diluted and plated on YMA to obtain well-separated colonies. We acknowledge that YMA is not a selective medium for nitrogen-fixing bacteria because it contains organic nitrogen sources (yeast extract). Therefore, this step was intended only for the preliminary isolation and purification of bacterial colonies rather than for direct selection of nitrogen-fixing bacteria. After obtaining pure isolates, a secondary screening step was performed using nitrogen-free Burk medium, where only bacteria capable of growing in the absence of combined nitrogen were retained for further characterization. This sequential approach allowed us to differentiate potential nitrogen-fixing bacteria from other heterotrophic isolates, although we recognize that growth on nitrogen-free medium indicates potential nitrogen metabolism but does not definitively confirm atmospheric nitrogen fixation, which requires further physiological or molecular validation.

Agree. We have accordingly revised the description of the isolation procedure to acknowledge the limitation of using YMA medium and to clarify how potential nitrogen-fixing bacteria were differentiated from other heterotrophic isolates. Specifically, we clarified that YMA was used only for the initial recovery and purification of bacterial colonies, while subsequent screening on nitrogen-free Burk medium was performed to identify isolates with potential nitrogen-fixing capability. These revisions have been incorporated in lines 86–96 and highlighted in red in the revised manuscript to clearly address the reviewer’s concern.

iii) Agree. We have accordingly revised the phylogenetic analysis and redrawn the phylogenetic tree according to the reviewer’s suggestion to improve methodological clarity and presentation. The updated phylogenetic tree has been replaced as Figure 2 in the main text, and the corresponding methodological details have been clarified in the manuscript. These revisions have been highlighted in red in the revised manuscript for easy reference.

iv) Agree. We have accordingly revised the description and interpretation of the isolates to avoid definitive classification based solely on physiological tests. The text now clarifies that the isolates are considered potential nitrogen-fixing bacteria and that further confirmation using additional molecular or functional approaches would be required. These revisions have been incorporated in lines 178–189 and highlighted in red in the revised manuscript to clearly address the reviewer’s comment.

v) Thank you for pointing this out. We agree with this comment. Therefore, the original Figure 3 has been replaced with Table 4 to present the quantitative trends of nitrogenase activity and nitrogen concentration during the incubation period more clearly. The revised table now includes mean values ± standard deviation (SD) from three biological replicates, and statistical differences among incubation times are indicated by different letters based on the F-test (p < 0.01), which allows proper evaluation of data variability and significance. These revisions can be found in Table 4 (Lines 240–254) of the revised manuscript. In addition, the Discussion section has been expanded to better explain the observed trends in nitrogenase activity and nitrogen accumulation, and recent relevant references have been incorporated to support these findings. These revisions are highlighted in red in Lines 298–311 of the revised manuscript.

vi) Thank you for pointing this out. We agree with this comment. Therefore, we have revised the Discussion section to acknowledge the limitation of using 16S rRNA gene sequencing for resolving closely related species and to indicate that additional genomic approaches could provide stronger taxonomic evidence. This clarification has been incorporated in the revised manuscript, and the modifications are highlighted in red in the Discussion section