Innovative Approaches for Engineering the Seed Microbiome to Enhance Crop Performance

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1. line no- 14-15: Consider breaking this sentence into two for better readability and to emphasize the key aspects of seed science.

2. Line 25-It would be helpful to briefly define what these innovative approaches entail to provide better clarity to the reader.

3. Line 33-35: You may want to specify how these findings can be practically applied in sustainable agriculture for a stronger conclusion. How can these findings be practically applied in sustainable agriculture to strengthen the conclusion? describe in review...

4. While the introduction provides a broad overview of seed microbiomes, it does not sufficiently highlight the engineering aspect, such as genetic modifications, synthetic biology approaches, or precision microbiome editing.

5. The introduction primarily emphasizes the benefits of microbial applications but does not discuss potential challenges, such as microbial stability, competition with native microbiota, regulatory constraints, or reproducibility of introduced microbes under field conditions.

6. Limited Coverage of Host-Microbe Interactions at the Genetic Level.

7. Specific Microbial Strains and Their Effects: The section mentions that Bacillus subtilis is less effective than Trichoderma species for cucumber seed germination, but does not specify the conditions or context of the comparison.  While the effectiveness of microbial strains is well-documented, it would be useful to briefly explain how these microbes inhibit pathogens or promote growth. 

8. Mechanisms of Action: The section could be structured more clearly with subheadings such as: Direct growth promotion (hormone production, nutrient solubilization), Stress tolerance enhancement (osmolytes, antioxidant enzymes, ethylene modulation), Disease suppression (antibiotics, competition, induced systemic resistance)...

9. Some concepts, such as Enterobacter spp. and heavy metal chelation, appear abruptly without sufficient background. A brief introduction to the role of microbes in phytoremediation would improve coherence.

Author Response

We sincerely thank Reviewer 1 for his/her positive comments and constructive criticism that have greatly improved our manuscript. For detailed responses to each point raised, please see below.

 

Comments 1: [line no- 14-15: Consider breaking this sentence into two for better readability and to emphasize the key aspects of seed science.]

Response 1: [Thank you for the suggestion. We have split the sentence to improve readability and highlight key aspects of seed science. Further details are provided in the revised manuscript.]

 

Comments 2: [Line 25-It would be helpful to briefly define what these innovative approaches entail to provide better clarity to the reader.]

Response 2: [Thank you for the suggestion. We have defined the innovative approaches in the main text to keep the abstract concise. Further details are included in the revised manuscript.]

 

Comments 3: [Line 33-35: You may want to specify how these findings can be practically applied in sustainable agriculture for a stronger conclusion. How can these findings be practically applied in sustainable agriculture to strengthen the conclusion? describe in review...]

Response 3: [Thank you for the good suggestion. We have further specified how those findings can be practically applied in sustainable agriculture in the “8. Conclusions and future directions” session. Further details are included in the revised manuscript.]

 

Comments 4: [While the introduction provides a broad overview of seed microbiomes, it does not sufficiently highlight the engineering aspect, such as genetic modifications, synthetic biology approaches, or precision microbiome editing.]

Response 4: [Thank you for the good suggestion. We have improved the introduction by further highlighting the engineering aspects including genetic modifications, synthetic biology approaches and precision microbiome editing. Further details are included in the revised manuscript.]

 

Comments 5: [The introduction primarily emphasizes the benefits of microbial applications but does not discuss potential challenges, such as microbial stability, competition with native microbiota, regulatory constraints, or reproducibility of introduced microbes under field conditions.]

Response 5: [Thank you for this insightful suggestion. We agree that addressing potential challenges associated with microbial applications would enrich the manuscript. Therefore, we have expanded the introduction to briefly discuss these important issues, including microbial stability, competition with native microbiota, regulatory constraints, and reproducibility of introduced microbes under field conditions.]

 

Comments 6: [Limited Coverage of Host-Microbe Interactions at the Genetic Level. ]

Response 6: [Thank you for pointing this out. We have now included additional content discussing host-microbe interactions at the genetic level, including insights from genome-wide association studies (GWAS) and plant genetic loci that influence seed microbiome composition. These revisions are reflected in the updated sections of the manuscript.]

 

Comments 7: [Specific Microbial Strains and Their Effects: The section mentions that Bacillus subtilis is less effective than Trichoderma species for cucumber seed germination, but does not specify the conditions or context of the comparison.  While the effectiveness of microbial strains is well-documented, it would be useful to briefly explain how these microbes inhibit pathogens or promote growth.]

Response 7: [Thank you for your helpful feedback. We have clarified the context of the comparative effectiveness between Bacillus subtilis and Trichoderma species in cucumber seed germination. We also expanded the section to include brief descriptions of the mechanisms by which these microbes inhibit pathogens and promote plant growth.]

 

Comments 8: [Mechanisms of Action: The section could be structured more clearly with subheadings such as: Direct growth promotion (hormone production, nutrient solubilization), Stress tolerance enhancement (osmolytes, antioxidant enzymes, ethylene modulation), Disease suppression (antibiotics, competition, induced systemic resistance)... ]

Response 8: [Thank you for the constructive suggestion. We have revised Section 7 to improve clarity and readability by introducing subheadings that categorize the major mechanisms of action: (1) direct growth promotion, (2) stress tolerance enhancement, and (3) disease suppression. These changes aim to better organize the content and highlight key microbial functions.]

 

Comments 9: [Some concepts, such as Enterobacter spp. and heavy metal chelation, appear abruptly without sufficient background. A brief introduction to the role of microbes in phytoremediation would improve coherence.]

Response 9: [Thank you for pointing this out. We agree that a brief introduction to microbial roles in phytoremediation would improve coherence. We have revised the section to provide appropriate background before discussing specific microbial examples such as Enterobacter spp. and heavy metal chelation.]

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is well-structured and comprehensive, covering multiple key aspects of the seed microbiome, including its composition, functions, engineering approaches, biopriming, and future directions. The content is thorough and demonstrates the authors' in-depth understanding of the field.

My main suggestions and comments are as follows:

1. The title in the submission system does not match the one in the manuscript.

2. In a review paper, phrases such as "we examined ****" should be avoided in the abstract.

3. The introduction is somewhat lengthy and could be streamlined to highlight the research background and significance, avoiding excessive detail.

4. Repetition should be minimized. For example, the advantages of biopriming are mentioned multiple times across different sections and could be consolidated.

5. The manuscript lacks a critical analysis of the current limitations in the field, which should be appropriately addressed.

6. The conclusion section could be more concise, focusing on the core findings and future research priorities, while avoiding redundancy with the main text.

7. Some references are incomplete, such as references 15 and 61.

8. The title needs further refinement to make it more specific and substantive, rather than being overly general.

Author Response

We sincerely thank Reviewer 2 for his/her positive comments and constructive criticism that have greatly improved our manuscript. For detailed responses to each point raised, please see below.

 

Comments 1: [The title in the submission system does not match the one in the manuscript.]

Response 1: [Thank you for pointing that out. The academic editor recommended the current title, but the submission system still displays our original one.]

 

Comments 2: [In a review paper, phrases such as "we examined ****" should be avoided in the abstract.]

Response 2: [Thank you for the helpful comment. We have revised the abstract to remove first-person phrases such as “we examined” and rephrased the content to maintain an objective, review-style tone.]

 

Comments 3: [The introduction is somewhat lengthy and could be streamlined to highlight the research background and significance, avoiding excessive detail.]

Response 3: [Thank you for the suggestion. We have revised the introduction to improve focus and readability by condensing some of the detailed explanations. The revised version emphasizes the research background and significance of seed microbiomes, while reserving specific mechanisms and examples for later sections.]

 

Comments 4: [Repetition should be minimized. For example, the advantages of biopriming are mentioned multiple times across different sections and could be consolidated.]

Response 4: [Thank you for your thoughtful feedback. We have reviewed the manuscript for repetitive content and consolidated overlapping discussions of seed biopriming. This has improved the flow and clarity of the text while maintaining the necessary emphasis on its significance.]

 

Comments 5: [The manuscript lacks a critical analysis of the current limitations in the field, which should be appropriately addressed.]

Response 5: [Thank you for this important observation. We have added a brief critical analysis outlining key limitations in seed microbiome engineering, including knowledge gaps, technical challenges, and translational barriers. This addition provides a more balanced perspective on the current state of the field.]

 

Comments 6: [The conclusion section could be more concise, focusing on the core findings and future research priorities, while avoiding redundancy with the main text.

Response 6: [Thank you for the helpful suggestion. We have revised the conclusion to improve conciseness and clarity by focusing on the key findings of the review and streamlining the discussion of future research priorities. Redundant elements previously covered in the main text have been removed for better flow.]

 

Comments 7: [Some references are incomplete, such as references 15 and 61.]

Response 7: [Thank you for pointing this out. We have carefully reviewed the reference list and corrected incomplete entries, including references 15 and 61. Full citation details have now been provided in accordance with the journal's formatting guidelines.]

 

Comments 8: [The title needs further refinement to make it more specific and substantive, rather than being overly general.]

Response 8: [Thank you for your valuable feedback. In response, we have refined the title to better reflect the scope and specificity of the review. We aimed to highlight both the innovative methodologies and the applied relevance of seed microbiome engineering in crop improvement.]

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript of Yang and coworkers is a strange review. It is strange first because the starting five pages bear no information, simply runs the sentence „Seed associated beneficial microbes have been shown to support seed germination, enhance seedling vigor, increase nutrient uptake, and improve plant resilience to various stresses” into the ground. Second the only information bearing part reports on seed biopriming, though we get no information what this expression means, how is it made. Third, throughout the text seed and vegetative plant microbiota are mixed up. I.e., seed-related changes are not treated, only consequences in the vegetative plant. Fourth, the future directions simply shortly repeat the former chapters focusing on vegetative plant associated microbes.

            Why don’t we get information on the composition of the seed microbiota? Which anatomical portion of the seed contains microbes, and what is the typical composition? What is the difference between the endophytic seed microbiota and the periphytic seed microbiota? What are the challenges of seed microbiota manipulation, which microbes are easily passed to the germ line, why, which microbes survive the dormancy, can be dormancy manipulated with microbes, and thousands of other such questions can be formulated.

            The authors emphasize the importance of „seed science”. But where are the important findings? What is typically seed and not plant related?

            There are, moreover, some minor mistakes or wording issues. Let us see some of them.

• line 45. Cyanobacteria are bacteria! Why are they separately listed (specified)?
• line 73. „chemical fungicides”. Why only fungicides?
• line 100. „priority effects” What is that?
• line 119. „microbial inheritance” Strange technical term.
• line 129 and on. Italicization of species or generic names appears haphazardly.
• Figure 2. is strange. The information content is zero.
• line 256. „mycoflora” Are fungi plants?

Summing up, the manuscript must be rejected in its present form.

Author Response

We sincerely thank Reviewer 3 for his/her constructive criticism that have greatly improved our manuscript. For detailed responses to each point raised, please see below.

 

Comments 1: [The manuscript of Yang and coworkers is a strange review. It is strange first because the starting five pages bear no information, simply runs the sentence „Seed associated beneficial microbes have been shown to support seed germination, enhance seedling vigor, increase nutrient uptake, and improve plant resilience to various stresses” into the ground. Second the only information bearing part reports on seed biopriming, though we get no information what this expression means, how is it made. Third, throughout the text seed and vegetative plant microbiota are mixed up. I.e., seed-related changes are not treated, only consequences in the vegetative plant. Fourth, the future directions simply shortly repeat the former chapters focusing on vegetative plant associated microbes.]

Response 1: [Thank you for your thoughtful feedback. We have significantly revised the introduction to provide clearer and more concise background information. We now define “seed biopriming” and briefly explain the process to improve reader understanding. We have also carefully distinguished between seed-associated microbiota and vegetative plant microbiota throughout the text, ensuring that seed-specific aspects are clearly highlighted. Additionally, we revised the future directions section to avoid repetition and to more clearly focus on seed-related microbial research. More details can be found in the revised manuscript.]

 

Comments 2: [Why don’t we get information on the composition of the seed microbiota? Which anatomical portion of the seed contains microbes, and what is the typical composition? What is the difference between the endophytic seed microbiota and the periphytic seed microbiota? What are the challenges of seed microbiota manipulation, which microbes are easily passed to the germ line, why, which microbes survive the dormancy, can be dormancy manipulated with microbes, and thousands of other such questions can be formulated.]

Response 2: [Thank you for raising these important points. While a comprehensive discussion of all related questions is beyond the scope of this review, we now provide a clearer overview and references to guide interested readers toward more detailed studies. We also briefly address key challenges in seed microbiota manipulation, such as microbial transmission to the next generation, survival during dormancy, and potential for microbiome-based modulation of dormancy. More details can be found in the revised manuscript.]

 

Comments 3: [The authors emphasize the importance of „seed science”. But where are the important findings? What is typically seed and not plant related?]

Response 3: [Thank you for this valuable comment. We have revised the manuscript to better highlight key seed-specific findings, such as the unique composition and transmission of seed microbiota, seed-based microbial inheritance, and the role of microbes in seed germination and early seedling development. We also clarified how seed science differs from general plant microbiome studies by emphasizing microbial interactions that occur specifically during the seed stage. More details can be found in the revised manuscript.]

 

Comments 4: [some minor mistakes or wording issues]

Response 4: [Thank you for the helpful comments. We have clarified the classification of cyanobacteria, broadened the term “chemical fungicides,” and added explanations for “priority effects” and “microbial inheritance.” Scientific names have been consistently italicized. Additionally, we replaced the outdated term “mycoflora” with “fungal communities” to reflect accurate terminology. More details can be found in the revised manuscript.]

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

All corrections have been incorporated by Author.