Identification and Functional Analysis of Endophytic Bacteria Bacillus cereus in Sphagnum palustre

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Identification and functional analysis of endophytic bacteria Bacillus cereus in Sphagnum palustre” by Wang et al. demonstrates a plant growth-promoting potential of newly isolated endophytic bacterium Bacillus cereus strain 11. This isolate originates from bryophyte Sphagnum palustre, which has garnered increased attention due to its multifunctional characteristics. The reason why this plant was chosen for bacterial isolation is well explained in the introduction, as well as previous research on endophytes originating from this plant species. However, in the introduction it is also necessary to emphasize the agricultural and ecological importance of species of the genus Bacillus.

 

In the Materials and Methods, authors should briefly describe the method for seed sterilization. It is unclear why the maize germination test lasted two days and what method was used.

 

 

Some sections, especially the discussion, list other research without appropriate flow and link with this study. In figures, name of the species along the strain code should be included.

 

The conclusion should be separated from the discussion and expanded by focusing on general points which they inferred from this study and the importance of future studies in field conditions should be emphasized, given that these studies are limited to testing the effect of B. cereus on maize seeds and seedlings in controlled experiments.

There are also multiple typing and other textual errors in the manuscript, so authors should carefully edit the manuscript.

 

Overall, the manuscript is topical and of significant interest to the journal's readership. I recommend this manuscript for publication after considering above-mentioned suggestions that could further improve its quality.

Comments on the Quality of English Language

There are multiple textual errors in the manuscript, so authors should carefully edit the manuscript.

Author Response

1. The reason why this plant was chosen for bacterial isolation is well explained in the introduction, as well as previous research on endophytes originating from this plant species.However, in the introduction it is also necessary to emphasize the agricultural and ecological importance of species of the genus Bacillus.

Answer：We sincerely appreciate your constructive comments, which have significantly improved the quality of our manuscript. The suggested revisions have been carefully incorporated into the revised version. For full details, please refer to the marked changes in the manuscript.

2. In the Materials and Methods, authors should briefly describe the method for seed sterilization. It is unclear why the maize germination test lasted two days and what method was used.

Answer：Maize seeds were surface-sterilized using a stepwise chemical disinfection protocol (75% ethanol for 5 min, followed by 2% sodium hypochlorite for 10 min), as described by Peng Xue and Luo Yanju [52-54]. To enhance germination efficiency, seeds were soaked for 8 h prior to planting, which both improved germination rates and reduced the germination cycle from the typical 3–7 days to 48 h. Germination was defined as the emergence of the embryo through the seed coat, consistent with the criteria established by Zhao Baorong [55], Xiong Chunmei [56], and Du Feng [57]. These references have been incorporated into the manuscript.

3. Some sections, especially the discussion, list other research without appropriate flow and link with this study.

Answer：In accordance with your recommendations, we have carefully reviewed the entire manuscript and implemented the necessary revisions.

4. In figures, name of the species along the strain code should be included.

Answer：In accordance with your suggestions, we have carefully revised and clearly marked the modifications in Figure 2.

5. The conclusion should be separated from the discussion and expanded by focusing on general points which they inferred from this study and the importance of future studies in field conditions should be emphasized, given that these studies are limited to testing the effect of cereus on maize seeds and seedlings in controlled experiments.

Answer：In response to your valuable suggestions, we have revised both the Discussion and Conclusions sections to highlight the significance of field experiments in our study.

6. There are also multiple typing and other textual errors in the manuscript, so authors should carefully edit the manuscript.

Answer：We sincerely appreciate your valuable suggestions and have accordingly conducted a thorough revision of the entire manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

thank you for your Manuscript.

The presented manuscript is a high-quality scientific work using a comprehensive systems approach to studying the object, which is endophytic bacteria isolated for the first time from moss Sphagnum palustre L., identified as spore-forming Bacillus cereus using a complex of molecular-biological, morpho-physiological and biochemical methods. The morphological, physiological and biochemical features of isolated strains bacteria, their ability to utilize various substances, to increase the appearance of water-soluble phosphorus, to fix the nitrogen, to produce the protease, IAA, siderophore and, NH₃ are shown. The observed effects of the influence of the isolated strains bacteria on seeds and growing plants of the test crop – corn was by authors with the bacteria characterized physiological abilities are logically connected and explained.

The work is read easily and with interest. The results obtained are original, correspond to the goals and objectives set in the manuscript, and can be the basis for the development of highly effective microbial biofertilizers with a range of useful functions for agricultural plants.

Despite the fact that the authors very clearly and reasonably explain in the results section all the identified properties of the studied n J11 strain's bacteria and their effects on the test plant culture, combining the discussion and conclusion is not a very good solution. I suggest moving some of the identified effects explanations from the results section and from the conclusion to the discussion section, making the latter a separate section. And the conclusion should be shorter.

Comments on the sections of the manuscript:

1. Title of manuscript

1) Authors. Lines 2-3: Identification and Functional Analysis of Endophytic Bacteria 2 Bacillus cereus in Sphagnum palustre

Reviewer: Still, at the first mention it is better to indicate the scientist who described the plant, in this case - Sphagnum palustre L.

2. Materials and Methods

2) Authors. 150-154: The sequences obtained were input into DNAMAN software for sequence assembly and BLAST comparison in the NCBI database (www.ncbi.nlm.nih.gov/BLAST, December 15, 2024 ). Finally, the phylogenetic tree of  strain J11 was constructed using MEGA 11.0 software, employing the Neighbor-Joining  algorithm with a bootstrap value set to 1,000 and other values at the default settings.

Reviewer: It is necessary to indicate the developer or his location for all software mentioned and used in the work.

3) Authors. 167-168: The absorbance (OD) at a wavelength of 600 nm was measured, and the growth curve of the strain under these conditions were plotted.

Reviewer: It is necessary to indicate the name of the device, brand, company and country of manufacture.

 4) Authors. 185-187: The soluble phosphorus content in the supernatant was determined using the molybdenum antimony colorimetric  method.

Reviewer: It is necessary to provide a literary reference for the method.

5) Authors. Lines 238-239: Seed Sterilization: 20 plump, uniformly sized Huawan 267 maize seeds were selected, and sterilized the maize seeds following the method outlined by Peng Xue et al. [39-41].

Reviewer: Why are only 20 plump seeds used - is there a standard or methodology to refer to?

6) Authors. Line 242: The seeds were immersed at room temperature for 8 hours.

Reviewer: Please justify why the seeds are immersed at room temperature for 8 hours. It would be better to provide a literary reference to the method.

7) Authors. Lines 245-246: Cultivation: 20 seeds, after soaking, were placed in a germination box (12.0×12.0×5.0 245 cm³) lined with a layer of filter paper, with 5 seeds per row across 4 rows

Reviewer: It is also necessary to clarify, 20 seeds is a repetition in each variant of the experiment, including the control?

8) Authors. Line 262: Peat moss and perlite were mixed in a 1:1 ratio and sterilized at 121°C for 30 mins.

Reviewer: It is necessary to provide a link to a publication in which this ratio is substantiated.

9) Authors. Line 263: The soil was wetted with 200 mL of sterile water…

Reviewer: Before this line in the manuscript, a substrate based on a mixture of peat moss and perlite was designated for growing plants. This line indicates soil - is this, apparently, a typo? If not, then it is necessary to indicate the type of soil, its main agrochemical characteristics. And, in the text of the manuscript, it is then necessary to correct - the plants were grown in a substrate or in soil.

10) Authors. 264-265: …and one germinated seed was planted per pot at a depth of approximately 2 cm.

Reviewer: How many plants are there in one variant of plant treatment? Please clarify.

11) Authors. 266-270: The control group was watered daily with 5 mL of sterile water, while the experimental group was watered daily with 5 mL of the diluted (16 times ) bacterial suspension from section 2.0.1, for a continuous period of 7 days followed by an additional period of 3 days where both groups were watered with only sterile water ( 5 mL each). Each treatment was replicated three times.

Reviewer: Please clarify and justify the time periods between three plant treatments over a period of seven days followed by a three-day period of watering with sterile water.

3. Results and Analysis

12) Authors. 327-328: However, the duration of each phase is influenced by the growth environment and the medium composition [45].

Reviewer: …as well as genetically determined physiological characteristics of a specific strain of microorganism.

13) Reviewer: Figure 5,7,9 - Please indicate what is shown on the graphs as the range of each mean value - the error of the mean or the confidence interval or the standard deviation?

14) Reviewer: On the graphs of Figure 5: Ad; Cb and Cc - there are no deviations from the average; they should be provided.

15) Reviewer: 376-377: The explanation under figure 5 - "D: Iron-producing carrier; E: Nitrogen fixation; F: Produces ammonium" is not entirely successful, it would be better, for example, - a test for iron production, a test for the ability of microorganisms to fix nitrogen, a test for the ability of the studied microorganisms to produce ammonium…

16) Authors. 422-423: suggesting that J11 bacterial strain has no significant impact on the germination rate, germination index, and secondary root number of maize plants

Reviewer: It would be better to add an explanation that this is on the second day from soaking the seeds.

For example: suggesting that J11 bacterial strain has no significant impact on the germination  rate, germination index, and secondary root number of maize plants on the 2 Day after soaking the seeds.

17) Authors. 423-425: This implies that under consistent environmental conditions, moss plant spore germination mainly relies on stored nutrients [53].

Reviewer: To logically connect with the previous paragraph, I suggest adding the following:

This implies that under consistent environmental conditions, plants seeds and also moss plant spore germination mainly relies on stored nutrients

18) Reviewer: In Figure 7, I suggest that for a more pronounced demonstration of the differences between the variants and the control, the ordinate axis should start not from the zero mark, but, for example, in graphs a and b from 50%; c from 20CI; d and e – from 2 pcs and cm respectively; f – from 4 mm.

19) Authors. 439-444: The differences in the root and shoot lengths of the experimental groups compared to the control groups were significantly pronounced ( P <0,05) under various dilutions, as shown in Figures 7e and 7f. The root length increased by 9.37%, 13.87%, 20.08%, 33.92%, 39.28%, and 25.00% respectively, with each successive dilution, indicating a gradual in- crease in root length that peaked at a 16-fold dilution, reaching a maximum of 3.12±0.30 cm, before decreasing subsequently.

Reviewer: Since, judging by the graph 7 e and f, the increase in root length by 9.37%, as well as shoot length by 4.58%, 5.85%, is not significant, it would be better to indicate that not only the significant effect, but also in the form of a trend.

For example: The differences in the root and shoot lengths of the experimental groups compared to the control groups were in the form of a trend or significantly pronounced (P <0,05)…

4. Discussion

20) Reviewer: It would be better to make the Discussion a separate section and move some of the explanations to this section from the Results and Conclusions section.

5. Conclusion

21) Reviewer: The conclusion will be shortened to the required size after moving part of the text to the Discussion section.

 Despite the comments made, the manuscript can be recommended for publication after minor revision.

Author Response

1. Lines 2-3: Identification and Functional Analysis of Endophytic Bacteria 2 Bacillus cereusin Sphagnum palustre

Reviewer: Still, at the first mention it is better to indicate the scientist who described the plant, in this case - Sphagnum palustre L.

Answer：We sincerely appreciate your positive decision and valuable feedback on our manuscript. In response to your constructive comments, we have carefully revised the manuscript accordingly, with all modifications highlighted in blue for your convenience.

2. 150-154: The sequences obtained were input into DNAMAN software for sequence assembly and BLAST comparison in the NCBI database (www.ncbi.nlm.nih.gov/BLAST, December 15, 2024). Finally, the phylogenetic tree of strain J11 was constructed using MEGA 11.0 software, employing the Neighbor-Joining algorithm with a bootstrap value set to 1,000 and other values at the default settings.

Reviewer: It is necessary to indicate the developer or his location for all software mentioned and used in the work.

Answer：Following your insightful comments, we have thoroughly revised the manuscript and incorporated the suggested modifications.

3. 167-168: The absorbance (OD) at a wavelength of 600 nm was measured, and the growth curve of the strain under these conditions were plotted.

Reviewer: It is necessary to indicate the name of the device, brand, company and country of manufacture.

Answer：Following your insightful comments, we have thoroughly revised the manuscript and incorporated the suggested modifications.

4. 185-187: The soluble phosphorus content in the supernatant was determined using the molybdenum antimony colorimetric method.

Reviewer: It is necessary to provide a literary reference for the method.

Answer：Following your insightful comments, we have supplemented the corresponding references in the manuscript.

5. Lines 238-239: Seed Sterilization: 20 plump, uniformly sized Huawan 267 maize seeds were selected, and sterilized the maize seeds following the method outlined by Peng Xue et al.[39-41].

Reviewer: Why are only 20 plump seeds used - is there a standard or methodology to refer to?

Answer：For the germination assay, we employed 12×12×5 cm³ germination boxes containing 20 seeds per box, which was determined to be the optimal quantity without compromising experimental outcomes. Each experimental setup included three biological replicates, with 20 seeds constituting one technical replicate. These methodological details were established based on the protocol described by Xiong Chunmei [56], as referenced in the revised manuscript.

6. Line 242: The seeds were immersed at room temperature for 8 hours.

Reviewer: Please justify why the seeds are immersed at room temperature for 8 hours. It would be better to provide a literary reference to the method.

Answer：The 8-hour room temperature seed soaking treatment was implemented to effectively break seed dormancy and accelerate germination. This optimal soaking duration was determined through our preliminary experiments, which demonstrated its efficacy in shortening the germination cycle while maintaining seed viability. Our methodology was developed with reference to the protocol established by Zhao et al. [55], as duly cited in the manuscript.

7. Lines 245-246: Cultivation: 20 seeds, after soaking, were placed in a germination box (12.0×12.0×5.0 245 cm³) lined with a layer of filter paper, with 5 seeds per row across 4 rows

Reviewer: It is also necessary to clarify, 20 seeds is a repetition in each variant of the experiment, including the control?

Answer：We appreciate your insightful comment regarding the methodological description. In response, we have revised the manuscript to clarify the experimental design. Specifically, both the experimental and control groups consisted of three biological replicates (n=3), with each replicate comprising one germination box containing 20 seeds (technical replicates). This design resulted in a total of three boxes (60 seeds) per treatment group and three boxes (60 seeds) for the control group. The modifications have been carefully implemented in the relevant sections of the manuscript.

8. Line 262: Peat moss and perlite were mixed in a 1:1 ratio and sterilized at 121°C for 30 mins.

Reviewer: It is necessary to provide a link to a publication in which this ratio is substantiated.

Answer：In accordance with your recommendation, we have incorporated the relevant references into the manuscript

9. Line 263: The soil was wetted with 200 mL of sterile water…

Reviewer: Before this line in the manuscript, a substrate based on a mixture of peat moss and perlite was designated for growing plants. This line indicates soil - is this, apparently, a typo? If not, then it is necessary to indicate the type of soil, its main agrochemical characteristics. And, in the text of the manuscript, it is then necessary to correct - the plants were grown in a substrate or in soil.

Answer：We appreciate your keen observation. Upon careful review, we have identified and corrected these errors in the manuscript.

10. 264-265: …and one germinated seed was planted per pot at a depth of approximately 2 cm.

Reviewer: How many plants are there in one variant of plant treatment? Please clarify.

Answer：We acknowledge that our original description of this methodology was unclear, and we have carefully revised the relevant sections in the manuscript to provide a more precise explanation of the experimental setup.

11. 266-270: The control group was watered daily with 5 mL of sterile water, while the experimental group was watered daily with 5 mL of the diluted (16 times) bacterial suspension from section 2.0.1, for a continuous period of 7 days followed by an additional period of 3 days where both groups were watered with only sterile water (5 mL each). Each treatment was replicated three times.

Reviewer: Please clarify and justify the time periods between three plant treatments over a period of seven days followed by a three-day period of watering with sterile water.

Answer：The irrigation protocol was implemented as follows: From days 1-7, the experimental group received daily applications of 5 mL bacterial suspension at 6:00 pm, while the control group was treated with an equal volume of sterile water. During days 8-9, both groups were irrigated with 5 mL sterile water daily at 6:00 pm to maintain adequate hydration for plant growth. We have supplemented the specific temporal details of these treatments in the revised manuscript.

12. 327-328: However, the duration of each phase is influenced by the growth environment and the medium composition[45].

Reviewer: as well as genetically determined physiological characteristics of a specific strain of microorganism.

Answer：We sincerely appreciate your valuable suggestion, which has significantly improved our manuscript. We have carefully incorporated this recommendation and made the corresponding revisions throughout the text.

13. Reviewer: Figure 5,7,9 - Please indicate what is shown on the graphs as the range of each mean value - the error of the mean or the confidence interval or the standard deviation?

Answer：We sincerely appreciate your valuable suggestion, which has significantly improved our manuscript. We have carefully incorporated this recommendation and made the corresponding revisions in the text.

14. Reviewer: On the graphs of Figure 5: Ad; Cb and Cc - there are no deviations from the average; they should be provided.

Answer：In response to your valuable suggestion, we have revised Figure 5 accordingly

15. Reviewer: 376-377: The explanation under figure 5 - "D: Iron-producing carrier; E: Nitrogen fixation; F: Produces ammonium" is not entirely successful, it would be better, for example, - a test for iron production, a test for the ability of microorganisms to fix nitrogen, a test for the ability of the studied microorganisms to produce ammonium…

Answer：In response to your valuable suggestion, we have revised Figure 5 accordingly

16. 422-423: suggesting that J11 bacterial strain has no significant impact on the germination rate, germination index, and secondary root number of maize plants

Reviewer: It would be better to add an explanation that this is on the second day from soaking the seeds. For example: suggesting that J11 bacterial strain has no significant impact on the germination rate, germination index, and secondary root number of maize plants on the 2 Day after soaking the seeds.

Answer：We have carefully incorporated this recommendation and made the corresponding revisions in the text.

17. 423-425: This implies that under consistent environmental conditions, moss plant spore germination mainly relies on stored nutrients[53].

Reviewer: To logically connect with the previous paragraph, I suggest adding the following:

This implies that under consistent environmental conditions, plants seeds and also moss plant spore germination mainly relies on stored nutrients

Answer：We have carefully incorporated this recommendation and made the corresponding revisions in the text.

18. Reviewer: In Figure 7, I suggest that for a more pronounced demonstration of the differences between the variants and the control, the ordinate axis should start not from the zero mark, but, for example, in graphs a and b from 50%; c from 20CI; d and e – from 2 pcs and cm respectively; f – from 4 mm.

Answer：In response to your valuable suggestion, we have revised Figure 7 accordingly.

19. 439-444: The differences in the root and shoot lengths of the experimental groups compared to the control groups were significantly pronounced (P <0,05) under various dilutions, as shown in Figures 7e and 7f. The root length increased by 9.37%, 13.87%, 20.08%, 33.92%, 39.28%, and 25.00% respectively, with each successive dilution, indicating a gradual in- crease in root length that peaked at a 16-fold dilution, reaching a maximum of 3.12±0.30 cm, before decreasing subsequently.

Reviewer: Since, judging by the graph 7e and f, the increase in root length by 9.37%, as well as shoot length by 4.58%, 5.85%, is not significant, it would be better to indicate that not only the significant effect, but also in the form of a trend.

For example: The differences in the root and shoot lengths of the experimental groups compared to the control groups were in the form of a trend or significantly pronounced (P <0,05)…

Answer：We have carefully incorporated this recommendation and made the corresponding revisions in the text.

20. Reviewer: It would be better to make the Discussion a separate section and move some of the explanations to this section from the Results and Conclusions section.

Answer：Following your recommendation, we have reorganized the manuscript by separating the Conclusion section from the Discussion. The results and partial conclusions have been incorporated into the Discussion section, which has been substantially revised to improve clarity and logical flow.

21. Reviewer: The conclusion will be shortened to the required size after moving part of the text to the Discussion section.

Answer：We have carefully incorporated this recommendation and made the corresponding revisions in the text.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript plants-3565617 describes the isolation of strain J11 from Sphagnum palustre. The authors have done a great job, but I have questions and comments that need to be clarified before the manuscript is published.

1. The authors isolated a spore-forming bacillus from sphagnum plants. The sterilization method used by the authors does not kill the Bacillus spores. The authors did not prove in any way that the strain they isolated is an endophyte.
2. The authors identified strain J11 as Bacillus cereus, for which nitrogen-fixing activity has not previously been demonstrated. Growth on Ashby medium is not unambiguous evidence for classifying it as a nitrogen fixer. Without additional evidence, the authors cannot claim that strain J11 is capable of fixing nitrogen.
3. The Bacillus cereus species is pathogenic. Strain J11 has hemolytic and proteolytic properties. Before recommending strain J11 as a biofertilizer, the authors must prove its safety for animals and humans.

Minor remarks:

Lines 32-34: The ecological importance of Sphagnum palustre is stated here, but the references are weak. Support the information with stronger references.

Lines 41-42: The information provided does not match the references. Sheridan studied another Sphagnum species and did not show nitrogen fixation for this plant.

Section Materials and Methods: Why were different temperatures used to describe the growth and biochemical activity of bacteria.

Lines 186-187: Provide a reference to the method.

Line 299: Provide the 16S rRNA gene sequence number for strain J11.

Line 302: What does the bootstrap value indicate by "high reliability"?

Figure 2b: What was the principle used to select the strains for sequence comparison? Why weren't only the type strains for their species selected?

Line 472: Replace "com" with "maize".

Author Response

1. The authors isolated a spore-forming bacillusfrom sphagnum The sterilization method used by the authors does not kill the Bacillus spores. The authors did not prove in any way that the strain they isolated is an endophyte.

Answer：We sincerely appreciate your thorough and insightful review of our manuscript. Regarding your question about endophytes, we would like to clarify that endophytes are defined as microorganisms (including bacteria, fungi, or archaea) that colonize the intracellular or intercellular spaces of healthy plant tissues without causing disease, and their presence may vary across developmental stages or plant organs.

As detailed in Materials and Methods (Section 2.3, Lines 110–115), we implemented a rigorous surface-sterilization protocol for Sphagnum samples to eliminate epiphytic microorganisms. This included [Sphagnum was rinsed with sterile water for 3 times, soaked in 75 % ethanol for 30 s, soaked in hydrogen peroxide solution for 6 min, and finally rinsed with sterile water for 3 times until the last rinsed sterile water was cultured on NA medium for 3 days without microbial growth, indicating that the surface disinfection of Sphagnum was completed.]. Thus, the isolated Bacillus cereus strain could only originate from the inner tissues of Sphagnum.

We fully acknowledge that methodological improvements are always valuable. If you could recommend alternative sterilization approaches or procedural refinements, we would be grateful for the opportunity to incorporate your expertise in future work.

2. The authors identified strain J11 as Bacillus cereus, for which nitrogen-fixing activity has not previously been demonstrated. Growth on Ashby medium is not unambiguous evidence for classifying it as a nitrogen fixer. Without additional evidence, the authors cannot claim that strain J11 is capable of fixing nitrogen.

Answer：.We sincerely appreciate your constructive suggestion, which undoubtedly enhances the scientific rigor of our study. In this study, strain J11 could grow in Ashby medium, indicating that it has the potential to grow in a nitrogen-free environment, rather than nitrogen fixation. We have revised it in the manuscript.

3. The Bacillus cereusspecies is pathogenic. Strain J11 has hemolytic and proteolytic properties. Before recommending strain J11 as a biofertilizer, the authors must prove its safety for animals and humans.

Answer：We sincerely appreciate your constructive suggestion, which undoubtedly enhances the scientific rigor of our study. While current time constraints and laboratory limitations prevent us from implementing this experiment immediately, we fully acknowledge its importance and plan to address it in future research.

4. Lines 32-34: The ecological importance of Sphagnum palustreis stated here, but the references are weak. Support the information with stronger references.

Answer：In response to your valuable suggestion, we have incorporated relevant references to strengthen this section of the manuscript.

5. Lines 41-42: The information provided does not match the references. Sheridan studied another Sphagnumspecies and did not show nitrogen fixation for this plant.

Answer：We have carefully incorporated this recommendation and made the corresponding revisions in the text.

6. Section Materials and Methods: Why were different temperatures used to describe the growth and biochemical activity of bacteria.

Answer：We have carefully incorporated this recommendation and made the corresponding revisions in the text.

7. Lines 186-187: Provide a reference to the method.

Answer：We have carefully incorporated this recommendation and made the corresponding revisions in the text.

8. Line 299: Provide the 16S rRNA gene sequence number for strain J11.

Answer：Following your recommendation, we have deposited the gene sequence of this strain in the NCBI database (Accession number: [SUB15242777]). The corresponding accession number has been incorporated into the manuscript to ensure full transparency and reproducibility.

9. Line 302: What does the bootstrap value indicate by "high reliability"?

Answer：The bootstrap value represents a statistical measure of branch reliability in phylogenetic analysis, obtained through resampling methods. Following established phylogenetic standards (Felsenstein, 1985), nodes with bootstrap support ≥70% are considered statistically significant. In our analysis, the relevant branch demonstrates maximal bootstrap support (100%), indicating particularly robust phylogenetic confidence.

10. Figure 2b: What was the principle used to select the strains for sequence comparison? Why weren't only the type strains for their species selected?

Answer：In phylogenetic sequence analysis, strain selection must adhere to two key principles: (1) sequence similarity (>97–99% for genus/species-level discrimination) and (2) taxonomic representativeness (complete gene region coverage). We acknowledge that our initial selection in Figure 2b did not fully meet these criteria. In response, we have replaced all reference strains with validated type strains from public culture collections (e.g., DSMZ, ATCC), ensuring both high sequence similarity (>99%) and comprehensive genomic coverage. This revision significantly strengthens the phylogenetic reliability of our analysis.

11.Line 472: Replace "com" with "maize".

Answer：We sincerely appreciate your meticulous review. Following your suggestion, we have standardized the terminology by replacing "com" with the scientifically accurate term "maize" throughout the manuscript to ensure consistency with botanical nomenclature.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The revised manuscript contains fewer errors, and the authors have partially made the necessary corrections. However, I have several questions and comments regarding the revised version of the manuscript:

1. I agree that the method of sterilization of the plant surface used by the authors kills vegetative cells, but Bacillus cereus forms resistant spores. The authors could have studied the resistance of spores (or cell culture with spores) to the sterilization method they used as a control. But, as far as I know, the only reliable confirmation of the endophytic nature of bacteria to date is confocal microscopy with 3D reconstruction of the plant object. Thus, I have two doubts about the validity of using the term "endophytic bacterium" for strain J11:
i) There is no evidence that the isolate was initially found in the internal tissues of sphagnum and not on the surface of the plant in the form of resistant spores;
ii) The authors do not provide any evidence that J11 cells are able to penetrate into plants upon inoculation.

2. The authors should carefully match the references in the text of the manuscript with the References section. For example, line 42, "Sheridan et al.'s [16]," but [16] Markham, 2009 and this article doesn't contain information about S. palustre. Also, line 44, "Turetsky et al.'s [17, 18] research", but [17] Turetsky, 2003 and [18] Gao, 2024.
Overall, the correctness and use of references are very weak.

3. Instead of the GenBank accession number of the 16S rRNA gene sequence, the authors provided a submission number that does not lead to the sequence.
The genus Bacillus consists of a huge number of species, and therefore the taxonomic identification of strains of this genus based on the 16S rRNA gene sequence is not informative. For example, the species B. sanguinis, B. proteolyticus, B. albus and others are very close to the species Bacillus cereus. The authors do not draw these species in Figure 2b, which makes it impossible to assess the correctness of classifying strain J11 as a Bacillus cereus species.

4. What do the authors mean by the phrase "with a bootstrap value of 100%, indicating high reliability"? This sentence implies that the sequences of strains J11 and ATCC 14579 differ. How different are they? What is the percentage of identity between them?

Author Response

Response to the Reviewer

1. I agree that the method of sterilization of the plant surface used by the authors kills vegetative cells, but Bacillus cereusforms resistant spores. The authors could have studied the resistance of spores (or cell culture with spores) to the sterilization method they used as a control. But, as far as I know, the only reliable confirmation of the endophytic nature of bacteria to date is confocal microscopy with 3D reconstruction of the plant object. Thus, I have two doubts about the validity of using the term "endophytic bacterium" for strain J11:
2. i) There is no evidence that the isolate was initially found in the internal tissues of sphagnumand not on the surface of the plant in the form of resistant spores;
3. ii) The authors do not provide any evidence that J11 cells are able to penetrate into plants upon inoculation.

Answer：First and foremost, we sincerely appreciate your constructive feedback, which has significantly enhanced the quality of our manuscript.

Secondly, regarding your question about endophytes, we would like to clarify the methodology used in this study. Following Gao Xin's method[38], we surface-sterilized the Sphagnum to eliminate any potential contamination from epiphytic microorganisms. To further ensure that the obtained strains were indeed endophytic, we removed the epidermis of the Sphagnum and extracted tissue from the inner section. Subsequently, we performed grinding and isolation for cultivation, referencing Huo Tianqi's method [45], which led to the acquisition of strain J11. These procedures have been highlighted in the text.

As for your suggestion on using confocal microscopy and 3D reconstruction of plant tissues, we agree that it is an excellent approach. In fact, we previously employed this method in our published paper, "The Colonization and Effect of Isaria cateinannulata on Buckwheat Sprouts." However, since the current study primarily focuses on demonstrating the growth-promoting effects of strain J11, we did not emphasize its colonization after inoculation. The colonization-related findings are being prepared for submission to another journal. If you are interested in this aspect, we would be delighted to discuss it further with you in the future.

2. The authors should carefully match the references in the text of the manuscript with the References section. For example, line 42, "Sheridan et al.'s [16]," but [16] Markham, 2009 and this article doesn't contain information about S. palustre. Also, line 44, "Turetsky et al.'s [17, 18] research", but [17] Turetsky, 2003 and [18] Gao, 2024.

Overall, the correctness and use of references are very weak.

Answer：We sincerely thank you for your valuable comments. We have carefully reviewed and checked the references to the full text, and corrected them in the manuscript.

3. Instead of the GenBank accession number of the 16S rRNA gene sequence, the authors provided a submission number that does not lead to the sequence.

The genus Bacillus consists of a huge number of species, and therefore the taxonomic identification of strains of this genus based on the 16S rRNA gene sequence is not informative. For example, the species B. sanguinis, B. proteolyticus, B. albus and others are very close to the species Bacillus cereus. The authors do not draw these species in Figure 2b, which makes it impossible to assess the correctness of classifying strain J11 as a Bacillus cereus species.

Answer：First, we have carefully reviewed the manuscript and corrected the gene sequence accession number of strain J11. Additionally, in accordance with your suggestion, we have expanded the phylogenetic tree to include the B. sanguinis, B. proteolyticus, and B. albus strains for a more comprehensive analysis.

4. What do the authors mean by the phrase "with a bootstrap value of 100%, indicating high reliability"? This sentence implies that the sequences of strains J11 and ATCC 14579 differ. How different are they? What is the percentage of identity between them?

Answer：In phylogenetic analysis, the Bootstrap value (resampling probability) serves as a statistical measure to evaluate the reliability of tree branches. Branches with Bootstrap support ≥ 75% are generally regarded as highly credible, indicating robust evolutionary relationships. In response to your previous suggestion, we have revised the phylogenetic tree accordingly and updated the relevant description in the manuscript.

 We have carefully addressed all comments and implemented corresponding revisions to improve the manuscript. These modifications, which are highlighted in the revised manuscript for easy reference, primarily involve textual refinements and do not affect the core findings or overall structure of the study.

We sincerely appreciate the Editors and Reviewers for their time and constructive feedback, which have significantly strengthened our work. We hope the revised version meets your expectations.

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

The authors described the method of isolating strain J11 in more detail. Now the manuscript contains a more substantiated assumption about the endophytic origin of the strain.

The additional information provided by the authors ambiguously indicates that strain J11 belongs to the species Bacillus sereus, but increases the probability of this. The PV465233 sequence has 99.86% identity with the type strains of the species B. sanguinis and B. proteolyticus. When discussing these results, the authors could indicate the presumptive nature of the assignment of strain J11 to the species Bacillus sereus, since subsequent molecular genetic studies can confirm or refute the proposed taxonomic definition.

Author Response

1.The authors described the method of isolating strain J11 in more detail. Now the manuscript contains a more substantiated assumption about the endophytic origin of the strain.

The additional information provided by the authors ambiguously indicates that strain J11 belongs to the species Bacillus cereus, but increases the probability of this. The PV465233 sequence has 99.86% identity with the type strains of the species B. sanguinis and B. proteolyticus. When discussing these results, the authors could indicate the presumptive nature of the assignment of strain J11 to the species Bacillus cereus, since subsequent molecular genetic studies can confirm or refute the proposed taxonomic definition.

Answer：We sincerely appreciate your valuable feedback and constructive comments, which have significantly enhanced the quality of our manuscript.

Your insightful suggestion has been carefully addressed in the revised manuscript, with the corresponding changes highlighted in blue for your convenience.

Once again, we are deeply grateful for your thorough review and expert recommendations. We are committed to further refining this paper to meet the highest publication standards and to honor the effort and expectations you have kindly invested in our work.