Seed Surface Sterilization Can Alter Root Microbiomes, Increase Endophyte Diversity and Enhance Plant Growth
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsLine 117-130: Did you follow any previous described protocol or you established your own procedure? Please insert the reference or write this is our own adapted procedure for sample processing.
Line 135-151: Why did you use this PCR protocol? Is it previously used in similar studies or is your own PCR protocol? If used from previous studies, please insert the reference or write if it is your own protocol.
Line 156: How did you draw phylogenetic tree? Please describe it.
Line 167-187: Is this procedure taken from references 21 and 22 or it is your own protocol? Please describe it better within the text.
Paragraph 2.6 and 2.7: Please describe if it is your adapted procedure or you used previous studies. Describe it within both paragraphs because methodology without references is not well prepared.
This study has revealed interesting data, pictures are well prepared, please make them bigger to read better data inside graphs.
You have discussed your results with previous studies which is very good methodology. Although you have made a comprehensive concluding remarks at the end.
Author Response
Comment: Line 117-130: Did you follow any previous described protocol or you established your own procedure? Please insert the reference or write this is our own adapted procedure for sample processing.
Response: I appreciate this request and have attempted to clarify the methodology with references. On line 117 I changed the sentence to read, "The collected plants were cleaned and surface sterilized following a protocol somewhat similar to previously established guidelines [19]. " and the text that follows outlines the adapted protocol. On line 123, I altered the sentence and inserted a reference to a patent which contained the protocol we used for inspiration, "Adapting a protocol used for isolating endophytes from seeds [20], the surface sterilized plant tissue samples were placed in 15 mL conical tubes with three banded tungsten carbide beads and 3 mL of sterile water." I hope you agree it is now clearer.
Comment: Line 135-151: Why did you use this PCR protocol? Is it previously used in similar studies or is your own PCR protocol? If used from previous studies, please insert the reference or write if it is your own protocol.
Response: I understand your confusion and have tried to clarify the PCR protocol which had two steps. On line 136 I changed the sentence to say, "A PCR protocol similar to one we used previously [8] was carried out with a Mastercycler Nexus Thermal Cycler...''. Then on line 144 I changed the sentence explaining the second PCR reaction, "The resulting PCR product was carried forward into a semi-nested PCR of our own design consisting of 30 cycles using the 27-F and 1387-R primers (5'-GCCCGGGAACGTATTCACCG-3') and the same conditions as the previous reaction.". I hope you find these changes clarify the PCR methodology.
Comment: Line 156: How did you draw phylogenetic tree? Please describe it.
Response: The phylogenetic tree is a coarse representation of the established taxonomic levels present in the plant samples. I drew it in Powerpoint and have indicated that on line 156, "A phylogenetic tree of plants sampled was drawn to the level of genus with the software Powerpoint (Microsoft, USA) based on established plant taxonomy.".
Comment: Line 167-187: Is this procedure taken from references 21 and 22 or it is your own protocol? Please describe it better within the text.
Response: We had already indicated that this turfgrass bioassay was similar to that in the reference, but have rewritten the sentence so that may be easier to understand. Here it is (from line 162): "To screen purified endophytes for potential benefit to maize growth, we decided to adapt the turfgrass bioassay methodology suggested by others [18]." From there we continue to outline the methods we used, since there were some differences to that outlined in reference [18]. On line 174, we continued to outline our method as we adapted it from [18] and have not changed what was written.
Comment: Paragraph 2.6 and 2.7: Please describe if it is your adapted procedure or you used previous studies. Describe it within both paragraphs because methodology without references is not well prepared.
Response: In paragraph 2.6 the methodology has been written out in detail and has no reference indicated, so it is clearly the methodology we developed ourselves for this experiment. In paragraph 2.7 however, thank you for asking for a reference because we forgot to mention one. On line 213 I inserted the sentence with reference, "For TRFLP, we employed a protocol we’d previously developed [24]."
Comment: This study has revealed interesting data, pictures are well prepared, please make them bigger to read better data inside graphs.
Response: Thank you for your comment; we agree that the data is interesting. We have made all the text in all the figures as large and legible as possible; do you mean that some figure in particular is illegible? In order to change the text in figures like figure 3, the entire graphic would have to be overhauled and perhaps split into multiple smaller figures; I'd rather not have to do that.
Comment: You have discussed your results with previous studies which is very good methodology. Although you have made a comprehensive concluding remarks at the end.
Response: Thank you for your positive comments. We did our best to review the literature and contextualize our results.
Reviewer 2 Report
Comments and Suggestions for AuthorsPlease read the attached file.
Comments for author File: 
Comments.pdf
Author Response
Comments: The presented work is relevant and necessary...This is a significant and useful contribution for geobotanists and ecologists.
Response: Thank you for your support. I hope that we can continue to do this type of work which is necessary to preserve and utilize the immense biodiversity present in Colombia.
Comments: Consider how the keywords section of the manuscript indicates the application of the perennial ryegrass biotest in vitro.
Response: I'm a little confused about this comment. I think it means that they keywords should be changed to better reflect the use of the perennial ryegrass biotest? To this end I've put the keywords "plant bioassay, ryegrass" into the list. Hope that you agree this makes it more accessible in the keyword section.
Comment: In the Introduction, the authors provide extensive information about corn, its global and national economic importance, and the ecological rationale for using beneficial bacteria to enhance corn stability and productivity. This is well presented. However, the purpose of the study should be clearly formulated in this section.
Response: Thank you for this comment. I see that I presented background information but I could've said more about our purpose. To help remedy this, on line 51 I inserted the sentence, "To be clear, the main purpose of our research presented in this manuscript was to bioprospect in Colombian plants for endophytes that could be used to develop microbial inoculants for improving maize production." I also added on line 109 the sentence, "Although our search for growth promoting endophytes did not yield significant results, our inclusion of uninoculated and unsterilized controls allowed us to observe that the seed surface is an important part of microbiome transmission from seed to plant. The implications of the seed surface being involved in vertical transmission has potentially important implications for experimental science and plant microbiome engineering, which we will discuss.". I think that with these added statements that the study's purpose becomes a bit clearer?
Comment: Line 196: Please clarify what "0.2" refers to.
Response: Do you refer to this sentence? "These inoculums then had there OD600 adjusted to 0.2." I've changed the sentence to, "In preparation for inoculation, broth in each test tube had its optical density adjusted to OD600=0.2".
Comment: The Materials and Methods section is sufficiently described. + The Results section aligns with the submitted figures, photos, and supplementary materials.
Response: Good to hear you approve. :-)
Comment: Lines 523–526: This content was already discussed in the Introduction. Consider removing it from here and integrating the result directly into the Discussion section.
Response: Thank you for this suggestion - I was repeating the taxonomic results rather than writing a discussion. To improve this flow of the section, I removed some of the taxonomic results and incorporated that paragraph into the next. Here's how I changed that bit, "It was not surprising to isolate the majority of the endophytes from root tissues, as this is generally the most heavily colonized part of the plant [32]. It was likewise expected to see that Proteobacteria would be the most common phylum of endophytes, as its been observed to makeup the core of the seed transmitted plant microbiome [25]. There are many reports of the genera Bacillus, Pseudomonas, Microbacterium, Enterobacter and Pantoea containing examples of plant growth promoters [33]. "
Comment: The manuscript discusses spermopheres but does not study them. Is this keyword appropriate?
Response: I guess you're right. I tend to want to use spermosphere as a word to define the "seed surface microbiome", but that is incorrect. I have removed it from the keywords and left "seed microbiome" and "seed surface sterilization" instead.
Comment: The Conclusion section largely repeats the Abstract. Please revise it to provide a more evolutionarily significant conclusion and include potential applications.
Response: Thank you for this valuable suggestion. I have rewritten the conclusion and it is now different from the abstract, but also I think it better communicates future steps and evolutionary implications of our results. Here is the new conclusion, "This study uncovers a bit of the hidden diversity of endophytic bacteria in wild plants from underexplored ecosystems in Colombia. While our search for growth-promoting potential among our endophyte isolates proved ultimately inconclusive, we have identified some candidates which warrant study in field trials; we also observed the ability of select strains and microbial consortia to reshape maize root microbiomes. Most importantly, we have also shown that seed surface sterilization has a significant effect on root microbiome composition and plant phenotype—probably through the removal of seed-borne pathogens and increasing the evenness of endophytic bacterial species.
These results challenge conventional methodologies in plant microbiome research, suggesting that routine sterilization practices may inadvertently distort natural microbial assemblages and plant responses, potentially explaining discrepancies between laboratory and field outcomes. From an evolutionary perspective, the seed surface microbiome may be a resilient and adaptable subset of the mother plant’s own microbiome that has contributed to plant health and reproduction over hundreds of millions of years. As most plant microbial inoculants are intended for deposition on seeds, the members of this surface microbiome may already be preadapted for this type of agricultural application and warranty further study. Testing our endophytes, as well future isolates from seed surface microbiomes, is an important endeavor for future field trials with maize under realistic agronomic settings. Likewise, the impact of seed surface sterilization on plant health, performance and microbiome should be studied under field conditions, where we can better understand the seed surface microbiome’s importance in vertical transmission and potential as a leverage point in plant microbiome engineering."
