Biological Traits of Vertically Seed-Transmitted Bacillus mojavensis in Triticum aestivum L.
, Daniel Mawuena Afordoanyi 1,4, Maria Nikolaevna Filimonova 2, Shamil Zavdatovich Validov 1 and Radik Ilyasovich Safin 3
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript titled “Biological trait of Vertically Seed-transmitted Bacillus mojavensis in Triticum aestivum L” is dedicated to seedborne endophytic bacteria that influence host responses to biotic and abiotic stress conditions. Their presence in seeds is associated with their ability to colonize plant tissues and be transmitted from parent plants to offspring. TheAuthors investigated the ability of Bacillus mojavensis PS17 to transmit to the next generation of spring wheat plants through seeds and tested the possible effect of this mode of transmission on the functional traits of the seed-transmitted colonies of PS17 by BOX-PCR analysis. A rifampicin-resistant strain of PS17 at 100 μg/mL was used to track the development of the wheat plants throughout the growth cycle.
The results demonstrated the successful colonization of B. mojavensis PS17 and its ability to be transmitted to the next plant generation via seeds. During plant development, the PS17 bacterial population was found to be higher in the roots and rhizosphere compared to aboveground plant parts, including seeds during the grain-filling stage. Seeds that were transmitted by the PS17 strain exhibited similar biological traits to the parental PS17 strain, including the ability to suppress the growth of fungal pathogens Fusarium oxysporum and Alternaria alternata, as well as produce hydrolases such as protease, lipase, amylase, and cellulase. These findings highlight the potential for vertical transmission through seeds as a means of spreading bacterial biocontrol agents in plants in the future.
Despite the good research data and general interest to such experiments, the manuscript needs additional work.
1) English language must be corrected by a native speaker, so as the text still has some features of native language of the Authors.
2) BOX-PCR analysis (Fig. 6) has been done without any control (different strain of Bacillus or B. mojavensis), so, it is impossible to justify a presence of any information from this experiment. Overall resolution of the gel bands is poor, it must be improved by longer run.
3) recA gene of PS17 ( PQ044380.1) is 100% identical to Bacillus halotolerans strain HMB20199 ( complete genome GenBank: CP110264.1), when closest Bacillus halotolerans (strain HMB20199 GenBank: CP110264.1) has only 97.7% of similarity.
16S rRNA gene (MW350040.1) is 100% identical for both Bacillus halotolerans and B. mojavensis, so it is not informative, gyrA gene (PQ044379.1) is 99.49% identical to Bacillus halotolerans strain HMB20199 (GenBank: CP110264.1) and other strains of Bacillus halotolerans, and only 97.66% similar to other B. mojavensis. Similar result is obtained for rpoD gene. Thus, the identification of strain PS17 cannot be supported by any presented molecular data, and the strain must be re-named.
4) Figure 1. Experimental illustration. Tillering (A), stem elongation (B), heading and 136 grain-filling (C) stages of spring wheat cultivated under climate-controlled conditions - The figure has no scientific meaning (just illustration of plants and facilities) and can be removed.
Comments on the Quality of English LanguageEnglish language must be corrected by a native speaker, so as the text still has some features of native language of the Authors.
Author Response
Dear Reviewer,
We are grateful for your consideration of this manuscript and thanks for your careful observation of our manuscript. We appreciate your suggestions, which have been very helpful in improving the manuscript. All the comments that we received on this study have been taken into account and we present our reply to each of them separately.
Kindly find below our response to your comments. All changes in the initial version of the manuscript and figures are underlined in blue font color for added sentences and strikethrough in red font color for deleted words or sentences.
Two versions of the manuscript are enclosed, one where all the changes have been underlined, and another clean version after the corrections. We hope that these changes to the manuscript will facilitate the decision to publish this study in your journal. We have made a considerable effort to take into account the interesting suggestions proposed by the reviewers. In any case, we are open to any further comments on our answers.
Authors’ response to comments and suggestions
Below we respond to the Reviewers’ comments.
(Note: Reviewers’ comments are in italic, and authors' responses are in bold).
Reviewers
The manuscript titled “Biological trait of Vertically Seed-transmitted Bacillus mojavensis in Triticum aestivum L” is dedicated to seedborne endophytic bacteria that influence host responses to biotic and abiotic stress conditions. Their presence in seeds is associated with their ability to colonize plant tissues and be transmitted from parent plants to offspring. TheAuthors investigated the ability of Bacillus mojavensis PS17 to transmit to the next generation of spring wheat plants through seeds and tested the possible effect of this mode of transmission on the functional traits of the seed-transmitted colonies of PS17 by BOX-PCR analysis. A rifampicin-resistant strain of PS17 at 100 μg/mL was used to track the development of the wheat plants throughout the growth cycle.
The results demonstrated the successful colonization of B. mojavensis PS17 and its ability to be transmitted to the next plant generation via seeds. During plant development, the PS17 bacterial population was found to be higher in the roots and rhizosphere compared to aboveground plant parts, including seeds during the grain-filling stage. Seeds that were transmitted by the PS17 strain exhibited similar biological traits to the parental PS17 strain, including the ability to suppress the growth of fungal pathogens Fusarium oxysporum and Alternaria alternata, as well as produce hydrolases such as protease, lipase, amylase, and cellulase. These findings highlight the potential for vertical transmission through seeds as a means of spreading bacterial biocontrol agents in plants in the future.
Despite the good research data and general interest to such experiments, the manuscript needs additional work.
1)English language must be corrected by a native speaker, so as the text still has some features of native language of the Authors.
We apologize for any language issues. The manuscript has been thoroughly reviewed and edited by a native English speaker to ensure clarity and correctness.
2)BOX-PCR analysis (Fig. 6) has been done without any control (different strain of Bacillus or B. mojavensis), so, it is impossible to justify the presence of any information from this experiment. Overall resolution of the gel bands is poor, it must be improved by longer run.
We thank the reviewer for their valuable feedback. While BOX-PCR is a well-established method for differentiating bacterial strains at the species level, we acknowledge the importance of including appropriate controls. In our study, we used PS17 as a reference strain [mentioned in Figure 6. As parental stain PS17(Rif100)] to compare against other randomly selected isolates. This approach allowed us to identify unique banding patterns and distinguish PS17 from other closely related strains. However, we have addressed the concerns raised regarding the BOX-PCR analysis by including additional strains, Bacillus sp, and other Bacillus halotolerans strains for a more accurate and reliable comparison of the genetic diversity among the bacterial isolates. The revised manuscript includes the updated BOX-PCR results, which now demonstrate a clear distinction between PS17 and other strains.
3)recA gene of PS17 ( PQ044380.1) is 100% identical to Bacillus halotolerans strain HMB20199 ( complete genome GenBank: CP110264.1), when closest Bacillus halotolerans (strain HMB20199 GenBank: CP110264.1) has only 97.7% of similarity.
The 16S rRNA gene (MW350040.1) is 100% identical for both Bacillus halotolerans and B. mojavensis, so it is not informative, gyrA gene (PQ044379.1) is 99.49% identical to Bacillus halotolerans strain HMB20199 (GenBank: CP110264.1) and other strains of Bacillus halotolerans, and only 97.66% similar to other B. mojavensis. Similar result is obtained for rpoD gene. Thus, the identification of strain PS17 cannot be supported by any presented molecular data, and the strain must be re-named.
We thank the reviewer for their insightful comments. In our study, we wanted to compare these sequences have identical sequences compared to their parental strain PS17 which were already deposited in NCBI as B. mojavensis. Our obtained results show that 100% of similarity to their parental strain.
We recognize the potential discrepancies in the genetic identification of strain PS17 based on the provided sequence data. The high similarity of certain genes of B. mojavensis to B. halotolerans strains raises concerns about the initial taxonomic assignment, as these bacteria belong to the same Bacillus subgroup. To address this, we have revised the manuscript to more accurately reflect the potential phylogenetic relationships and taxonomic uncertainties associated with strain PS17.
In future studies, we will conduct a more comprehensive taxonomic analysis of strain PS17. This will involve whole-genome sequencing and comparative genomic analysis to accurately determine its phylogenetic position within the Bacillus genus. Additional phenotypic characterization, such as growth conditions and metabolic profiles, will also be considered to further support our taxonomic classification.
Initial text:
To confirm the identity of the isolated bacterial strains, the specific genes listed in Table 2 (rpoB, gyrA, 16S rRNA, and RecA) were sequenced and analyzed using BLAST against the NCBI database. High sequence identity (>99.5%) and query coverage confirmed the isolates as Bacillus mojavensis. The partial gene sequences of rpoB, gyrA, 16S rRNA, and RecA were deposited in NCBI GenBank under accession numbers PQ044378, PQ044379, MW350040.1, and PQ044380, respectively.
Improved text:
Additionally, specific genes (rpoB, gyrA, 16S rRNA, and RecA) were sequenced and compared to reference parental sequences available in the NCBI database to confirm the identity of the isolated bacterial strains. The 16S rRNA gene sequences showed a high-scoring segment pair, demonstrating 100% identity with B. mojavensis PS17 (accession number MW350040.1). Likewise, the coding sequences (CDS) for the rpoB, gyrA, and RecA genes were also identical, showing 100% similarity with the corresponding sequences of B. mojavensis PS17 (accession numbers PQ044378, PQ044379, and PQ044380, respectively). Based on the high sequence similarity to the reference strain, we concluded that all isolates belong to B. mojavensis.
4)Figure 1. Experimental illustration. Tillering (A), stem elongation (B), heading and 136 grain-filling (C) stages of spring wheat cultivated under climate-controlled conditions -The figure has no scientific meaning (just illustration of plants and facilities) and can be removed.
The figure has been removed from the revised manuscript.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe study of Roderic Gilles Claret Diabankana and co-authors entitled “Biological trait of Vertically Seed-transmitted Bacillus mojavensis in Triticum aestivum L” is dedicated to a relevant topic. In general, the study is well-designed, and the methods used are adequate and modern. The obtained results were well presented and discussed. The results provide valuable information about seed-mediated vertical transmission of endophyte (Bacillus mojavensis PS17) that retain the capacity to suppress pathogens’ development (biocontrol). The study has both fundamental and practical value.
After minor text editing, the communication may be accepted for publication. (see my comments in the attached pdf-file)
Best wishes,
Reviewer
Comments for author File: Comments.pdf
Author Response
Dear Reviewer,
We are grateful for your consideration of this manuscript and thanks for your careful observation of our manuscript. We appreciate your suggestions, which have been very helpful in improving the manuscript. All the comments that we received on this study have been taken into account and we present our reply to each of them separately.
Kindly find below our response to your comments. All changes in the initial version of the manuscript and figures are underlined in blue font color for added sentences and strikethrough in red font color for deleted words or sentences.
Two versions of the manuscript are enclosed, one where all the changes have been underlined, and another clean version after the corrections. We hope that these changes to the manuscript will facilitate the decision to publish this study in your journal. We have made a considerable effort to take into account the interesting suggestions proposed by the reviewers. In any case, we are open to any further comments on our answers.
Authors’ response to comments and suggestions
Below we respond to the Reviewers’ comments.
(Note: Reviewers’ comments are in italic, and authors' responses are in bold).
Reviewers . The study of Roderic Gilles Claret Diabankana and co-authors entitled “Biological trait of Vertically Seed-transmitted Bacillus mojavensis in Triticum aestivum L” is dedicated to a relevant topic. In general, the study is well-designed, and the methods used are adequate and modern. The obtained results were well presented and discussed. The results provide valuable information about seed-mediated vertical transmission of endophyte (Bacillus mojavensis PS17) that retain the capacity to suppress pathogens’ development (biocontrol). The study has both fundamental and practical value.
After minor text editing, the communication may be accepted for publication. (see my comments in the attached pdf-file)
Best wishes,
Reviewer
We thank the reviewer again for their time and careful review of our manuscript. We appreciate their valuable suggestions, which have significantly improved the quality of the manuscript.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsComments to the author
Seedborne endophytic bacteria can influence host responses to biotic and abiotic stress conditions. In this study, the author demonstrated that the antagonistic bacterium Bacillus mojavensis PS17 can be transmitted to the next generation of spring wheat plants via seeds, retaining its antagonistic effects on pathogens.
This is an interesting topic, and the authors conducted numerous experiments to demonstrate transmission to next generation and the bacteria’s ability to suppress pathogen growth. However, the manuscript contains many serious errors, not only in format but also in spelling, which need significant revisions. I recommend major revisions. In addition, I have a few specific suggestions for the authors. For more details, comments are below:
Page 1, line 37-38: Reference [4-5] should be formatted as [4, 5], and similarly, [6-7] should be [6 ,7]. Please check all references and correct the numbering format.
Page 1, line 43: Please write the full names of SAR and ISR, as they were not previously mentioned.
Page 2, line 51: Format the reference as [20-23].
Page 2, line 64: Please write the full name of the fungi; the full names of all microorganisms should be written out the first time they are mentioned.
Page 2: Check the formatting for the M & M section; it should start with “2.1 subtitle” rather than a paragraph.
Page 2, line 78: Since Bacillus mojavensis was already mentioned in the introduction, the bacterial name should be abbreviated here.
Page 2, line 88: Cite the reference.
Page 3, line 107: In "Lysogen," the "L" should be lowercase.
Page 3, line 108: The centrifugation speed is listed as 4 rmp? Additionally, there should be no space between the number and centigrade symbol.
Page 3, line 111: Since PBS has already been mentioned, there is no need to repeat its full name.
Page 3, line 112: Is the wavelength 595 nm? Is the concentration 106 cfu/mL?
Page 3, line 114: There is no section 2.3.
Page 3, lines 122-123: Correct the material names; for example, MnSO4·H2O should be MnSOâ‚„·Hâ‚‚O.
Page 3, lines 126-127: Does the author mean that samples were collected at different stages or from different parts at the same stage? If it is the former, Figure 1 does not distinguish any differences among A to C.
Page 3, line 132: Please provide more details on the statistical analysis, such as which programs were used.
Page 4, line 150: In "10x PCR buffer," the "×" should be a multiplication sign rather than the alphabet.
Page 4, line 158: In Figure 6 legend, the authors mention that electrophoresis was performed with 1.5% agarose, which differs from the M & M section. Please check the concentration of agarose.
Page 5, lines 193, 195: Correct the materials.
Page 5, line 207: Where is the figure for "6.37 ± 0.15 × 10^4 cfu/g" derived from? Is it the number from the "above-ground part" at the grain filling stage? Also, abbreviate the bacterial name.
Pages 5-6, lines 209-210: Do these numbers represent the "below ground" or "rhizospheric soil"? The numbers might not match with the figure.
Page 6, line 210: Change "sten" to "stem."
Pages 6, lines 211-214: From Figure 2, the reduction of PS17 colonies in the aboveground parts, as mentioned, is not visible. Additionally, the density of PS17 cells at the stem elongation stage seems to exceed 10^6 cfu/g, doesn’t it?
Page 6, Figure 2: For labeling statistical differences, it is customary to label starting from the highest; thus, "Rhizospheric soil" at seedling and tillering stages should be labeled "a." Additionally, in the legend, "p-value" should be italicized as “p-value”.
Page 6, lines 227-230: From Figure 3, no differences among the three colonies isolated from different wheat parts, as the author mentions, can be observed. Also, please improve the quality of the figure and zoom in on the colony to better display colony morphology.
Page 8, line 294: Correct the concentration of rifampicin.
Q1: Does PS17 reduce the pathogenicity of pathogens on wheat? If the antagonistic bacterium PS17 can be transmitted to the next generation seeds, would these seeds reduce the pathogenicity of pathogens when cultivated?
Author Response
Dear Reviewer,
We are grateful for your consideration of this manuscript and thanks for your careful observation of our manuscript. We appreciate your suggestions, which have been very helpful in improving the manuscript. All the comments that we received on this study have been taken into account and we present our reply to each of them separately.
Kindly find below our response to your comments. All changes in the initial version of the manuscript and figures are underlined in blue font color for added sentences and strikethrough in red font color for deleted words or sentences.
Two versions of the manuscript are enclosed, one where all the changes have been underlined, and another clean version after the corrections. We hope that these changes to the manuscript will facilitate the decision to publish this study in your journal. We have made a considerable effort to take into account the interesting suggestions proposed by the reviewers. In any case, we are open to any further comments on our answers.
Authors’ response to comments and suggestions
Below we respond to the Reviewers’ comments.
(Note: Reviewers’ comments are in italic, and authors' responses are in bold).
Reviewers .
Seedborne endophytic bacteria can influence host responses to biotic and abiotic stress conditions. In this study, the author demonstrated that the antagonistic bacterium Bacillus mojavensis PS17 can be transmitted to the next generation of spring wheat plants via seeds, retaining its antagonistic effects on pathogens.
This is an interesting topic, and the authors conducted numerous experiments to demonstrate transmission to the next generation and the bacteria’s ability to suppress pathogen growth. However, the manuscript contains many serious errors, not only in format but also in spelling, which need significant revisions. I recommend major revisions. In addition, I have a few specific suggestions for the authors. For more details, comments are below:
–Page 1, line 37-38: Reference [4-5] should be formatted as [4, 5], and similarly, [6-7] should be [6 ,7]. Please check all references and correct the numbering format.
Thank for suggestion. Correction has been made accordingly
–Page 1, line 43: Please write the full names of SAR and ISR, as they were not previously mentioned.
Modification has been made accordingly
–Page 2, line 51: Format the reference as [20-23].
Modification has been made accordingly
–Page 2, line 64: Please write the full name of the fungi; the full names of all microorganisms should be written out the first time they are mentioned.
We agree with the suggestion and correction has been made accordingly
–Page 2: Check the formatting for the M & M section; it should start with “2.1 subtitle” rather than a paragraph.
We agree with you. Correction has been made accordingly
–Page 2, line 78: Since Bacillus mojavensis was already mentioned in the introduction, the bacterial name should be abbreviated here.
Thank you for pointing this out. However, we believe that most journals advise authors not to start any sentence with an abbreviation. Correction has been made accordingly
–Page 2, line 88: Cite the reference.
A reference was added
–Page 3, line 107: In "Lysogen," the "L" should be lowercase.
Modification has been made accordingly
–Page 3, line 108: The centrifugation speed is listed as 4 rmp? Additionally, there should be no space between the number and centigrade symbol.
We apologize for the typographical error. The centrifugation speed has been corrected to 4,000 rpm. Additionally, we have removed the unnecessary space between the number and the degree Celsius symbol.
–Page 3, line 111: Since PBS has already been mentioned, there is no need to repeat its full name.
Modification has been made accordingly
–Page 3, line 112: Is the wavelength 595 nm? Is the concentration 106 cfu/mL?
We apologize for the oversight. Correction has been made
–Page 3, line 114: There is no section 2.3.
We apologize for the oversight. The reference to Section 2.3 has been corrected to the appropriate section number in the revised manuscript.
–Page 3, lines 122-123: Correct the material names; for example, MnSO4·H2O should be MnSOâ‚„·Hâ‚‚O.
We apologize for the oversight. The chemical formula has been corrected to their proper format (e.g., MnSOâ‚„·Hâ‚‚O) throughout the manuscript.
–Page 3, lines 126-127: Does the author mean that samples were collected at different stages or from different parts at the same stage? If it is the former, Figure 1 does not distinguish any differences among A to C.
We apologize for the oversight. The sentence has been rewritten to clarify that plant samples were collected from different plant parts (leaves, stems, roots, and grains) at each growth stage (seedling and tillering, stem elongation, and milk development). As recommended by another reviewer, Figure 1 has been removed from the manuscript, as it did not add significant scientific value.
–Page 3, line 132: Please provide more details on the statistical analysis, such as which programs were used.
We thank the reviewer for their valuable feedback. We have added more details regarding the statistical analysis, including the specific software used and the statistical tests employed to evaluate significant differences between treatments. These details have been incorporated into the revised manuscript.
–Page 4, line 150: In "10x PCR buffer," the "×" should be a multiplication sign rather than the alphabet.
We apologize for the typographical error. The "x" has been replaced with a multiplication symbol (×) in the revised manuscript.
–Page 4, line 158: In Figure 6 legend, the authors mention that electrophoresis was performed with 1.5% agarose, which differs from the M & M section. Please check the concentration of agarose.
We apologize for the oversight. The agarose concentration for electrophoresis has been corrected to 1.0% in both the figure legend and the Materials and Methods section of the revised manuscript.
–Page 5, lines 193, 195: Correct the materials.
We apologize for the oversight. The materials have been corrected accordingly in the revised manuscript.
–Page 5, line 207: Where is the figure for "6.37 ± 0.15 × 10^4 cfu/g" derived from? Is it the number from the "above-ground part" at the grain-filling stage? Also, abbreviate the bacterial name.
We thank the reviewer for their keen observation. The data for "6.37 ± 0.15 x 10^4 cfu/g" was indeed derived from seed tissue after pretreatment (Figure 2A). We apologize for any confusion caused by the unclear presentation of the data. To improve clarity, we have revised Figure 2 to explicitly show the data point corresponding to this value.
–Pages 5-6, lines 209-210: Do these numbers represent the "below ground" or "rhizospheric soil"? The numbers might not match with the figure.
We apologize for the confusion regarding the data presented in Figure 2. The numbers in the figure legend do indeed represent the bacterial population density in the rhizospheric soil at different growth stages. To clarify this, we will revise the figure legend to explicitly state that the data shown corresponds to the rhizosphere. Additionally, we will ensure that the data points on the graph accurately reflect the values presented in the legend.
–Page 6, line 210: Change "sten" to "stem."
We apologize for the typographical error. The word "sten" has been corrected to "stem" in the revised manuscript.
–Pages 6, lines 211-214: From Figure 2, the reduction of PS17 colonies in the aboveground parts, as mentioned, is not visible. Additionally, the density of PS17 cells at the stem elongation stage seems to exceed 10^6 cfu/g, doesn’t it?
We appreciate the reviewer's careful observation of Figure 2. It seems there might have been a slight oversight in the initial interpretation of the data. Upon closer examination, the reduction in PS17 colonization in the above-ground parts of the plant, particularly in the seeds, is less pronounced than initially described. However, it's important to note that the density of PS17 in the rhizosphere consistently exceeds that in the above-ground parts throughout plant development. Regarding the density of PS17 cells at the stem elongation stage, we apologize for any misinterpretation. The data indeed suggests that the density exceeds 10^6 CFU/g at this stage. We will carefully review the figure and its caption to ensure clarity and accuracy. We thank the reviewer for their keen eye and appreciate the opportunity to refine the presentation of our data.
–Page 6, Figure 2: For labeling statistical differences, it is customary to label starting from the highest; thus, "Rhizospheric soil" at seedling and tillering stages should be labeled "a." Additionally, in the legend, "p-value" should be italicized as “p-value”.
We apologize for the oversight in labeling the statistical differences. We have corrected the labeling to follow the convention of starting from the highest value. Additionally, we have italicized "p-value" in the legend to maintain consistency with standard scientific notation.
We appreciate the reviewer's attention to detail and their helpful suggestions.
–Page 6, lines 227-230: From Figure 3, no differences among the three colonies isolated from different wheat parts, as the author mentions, can be observed. Also, please improve the quality of the figure and zoom in on the colony to better display colony morphology.
We thank the reviewer for their valuable feedback. we have improved the image quality to better visualize their morphology.
–Page 8, line 294: Correct the concentration of rifampicin.
We thank the reviewer for their careful review and for pointing out the error in the rifampicin concentration. We apologize for the oversight and have corrected the concentration of rifampicin in the revised manuscript. We have also double-checked all other experimental details to ensure accuracy.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript is devoted to the ability of Bacillus mojavensis PS17 to transmit the next generation of spring wheat plants through seeds and the impact of transmission mode on the functional traits of the seed-transmitted PS17 colonies. The results showed the successful colonization of B. mojavensis PS17 and its capacity to transmit to the next plant generation through seeds. Seed-transmitted B. mojavensis PS17 exhibited an identical biological trait to that of the parental strain PS17. Bacillus mojavensis retained its ability to suppress the growth of pathogenic fungi, Furasium oxysporum and Alternaria alternata, and produce hydrolases, including protease, lipase, amylase, and cellulase. These findings highlight the potential of vertical transmission through seed as a mode of bacterial biocontrol agent spread in future crops. The mauscript was improved according to Reviewers comments and will be a valuable publication for all experts in the related field.
Reviewer 3 Report
Comments and Suggestions for AuthorsThe author revised the manuscript well and responded to all my comments. No other comments on the author
