Evaluating the Effect on Cultivation of Replacing Soil with Typical Soilless Growing Media: A Microbial Perspective

Round 1

Reviewer 1 Report

This is a well-written paper with a good structure to evaluate the missing microbial part of soilless substrate study, they provided great amount of information with traditional microbial composition analysis with additional functional analysis, I think this is a good paper to publish in Agronomy.

Just a minor suggestion, it would be great to discuss more about the organic matter relationships with microbes, can the microbes use the high OM in substrates (peat and coir)? What’s the differences between substrate OM and soil OM? In addition, since peat use is not sustainable, is there any benefits to replace peat with coir in microbial perspectives?

Author Response

Dear Reviewer,

Thank you for your comments concerning our manuscript entitled “Evaluating the effect of replacing soil with typical soilless growing media: a microbial perspective” (agronomy-1995237). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied comments carefully and have made correction which we hope meet with approval. In the revised version, the deleted parts were marked with blue font and strikeout, while the added parts were marked with red font and underline. The main corrections in the paper and the responds to the reviewer’s comments are as following: Replies to the reviewer’s comments:

(Just a minor suggestion, it would be great to discuss more about the organic matter relationships with microbes, can the microbes use the high OM in substrates (peat and coir)? What’s the differences between substrate OM and soil OM? In addition, since peat use is not sustainable, is there any benefits to replace peat with coir in microbial perspectives?)

Response: We would like to express our sincere thanks to the reviewers for the constructive and positive comments. In this study, OM had a major impact on the bacterial and fungal communities. Unfortunately, limited indicators do not allow us to further analyze how OM affects these microbial processes. However, the substrate OM is quite different from soil OM and has fewer nutrients that are easily available directly. In addition, the study indicated that the peat based substrate and coir substrate can basically perform the common soil functions, but coir substrate has salt accumulation problem. In the future, it is hoped that more research can focus on SGM and find better methods or materials to completely replace peat. Thank you very much for your comments and suggestions again.

Author Response File: Author Response.docx

Reviewer 2 Report

All comments are in pdf.

I recommend major revision, but I am not sure if it is possible to improve it. Also, I am not English native speaker, but the language must be improved.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you for your comments concerning our manuscript entitled “Evaluating the effect of replacing soil with typical soilless growing media: a microbial perspective” (agronomy-1995237). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied comments carefully and have made correction which we hope meet with approval. In the revised version, the deleted parts were marked with blue font and strikeout, while the added parts were marked with red font and underline. The main corrections in the paper and the responds to the reviewer’s comments are as following: Replies to the reviewer’s comments:

1. (I recommend major revision, but I am not sure if it is possible to improve it. Also, I am not English native speaker, but the language must be improved.)

Response: We would like to express our sincere thanks to the reviewers for the constructive and positive comments. We have improved the grammar and expression, and tried our best to avoid the language problem with the help of coauthor.

2. (Please indicate these problems.)

Response: Thank you very much for this comment. We have added these problems “, numerous fungi of unknown function and a shortage of available nutrients and microbiomes,”

3. (animals)

Response: Thank you for pointing out this. The “animal” has been replaced by the “animals” in the revised manuscript.

4. (living in soil)

Response: Thank you for pointing out this. The “in” has been added between “living” and “soil”.

5. (Reformulate this sentence not to star with the same word as the previous one.)

Response: Thank you very much for this comment. The two sentences have been combined into one sentence as “They, especially rhizosphere microbes, can manage the soil fertility by regulating ab-sorption and release of main elements in soil”

6. (What was dimension of the growth chamber?)

Response: Thank you very much for this suggestion. We have added dimension of the growth chamber as “(12.5×5.0×12.8 L)”.

7. (Please describe containers in which the plants were incubated in the greenhouse. What was dimension of the greenhouse?)

Response: Thank you very much for this suggestion. We have added the container as “a U-shaped slot” and dimension of the greenhouse as “60×40 m²”.

8. (Was there any leftover of the SGM on the roots. How much? Approximately.)

Response: Thank you very much for this comment. We are sorry that we only took enough samples of SGM and did not evaluate the leftover of SGM.

9. (Please explain this phrase: "measured extracted". I do not understand what you did.)

Response: We are sorry for that we used a wrong word. The “measured extracted” has been changed to “extracted directly”.

10. (Please reformulate first sentence)

Response: Thank you for pointing out this. This sentence has been changed to “Part of fresh SGM samples was used to measure mass water content”.

11. (Delete space)

Response: We apologize for our carelessness. The “space” has been deleted in the revised manuscript.

12. (You mean the rizosphere samples from different soilless substrates. Please correct.)

Response: We are sorry to make it unclear. This sentence has been changed to “Total microbial genomic DNA of the different SGM samples was extracted”.

13. (if mothur is a name of the software it should be written with the upper capital letter.)

Response: Thank you for pointing out this. The “mothur” is a software and it has been changed to “MOTHUR”

14. (was performed)

Response: Thank you very much for this comment. The “was also analyzed” has been replaced by “was performed”.

15. (was also performed)

Response: Thank you very much for this comment. The “was performed” has been replaced by “was also performed”.

16. (Please delete: "were responsible for the microbial structure", and instead say something like this: "best explained variability within microbial community structure". RDA as well as correlation analyses does not imply causation. Please be careful regarding this.)

Response: Thank you very much for this suggestion. We will be careful regarding this issue in the future. The “were responsible for the microbial structure” has been replaced by “best explained variability within microbial community structure”.

17. (predicted)

Response: We are sorry for this mistake. The “prediction” has been modified to “predicted”.

18. (Why not use PERMANOVA instead of two-way ANOVAs?)

Response: Thank you very much for this comment. The ANOVAs were used to assess the difference in physicochemical characteristics of SGM rather than in microbial characteristics of SGM. Therefore, we did not use PERMANOVA.

19. (Please increase size of the titles of the x and y axes.)

Response: Thank you very much for this suggestion. We have increased the size of numbers and letters of all figures in the revised manuscript.

20. (Please place + sign as the superscript of the letter H. The same with the - sign in the NO3 where it should be placed as superscript of the letter O and not of the number 3 (or number 4 in ammonia). Please correct here and elsewhere in the manuscript.)

Response: Thank you very much for this comment. The + and - signs were the superscripts, but they did not look like due to the font in this manuscript.

21. (If hierarchically clustered heatmap is more detailed maybe the circus could be deleted. Why there are two representations of the same?)

Response: We apologize for this poor choice of words. The hierarchically clustered heatmap showed the relationship on bacteria and fungi among the four treatments, whereas the circus showed the relative abundance for bacteria and fungi at phylum level. Therefore, the “for more details” has been changed to “In addition”.

22. (Line up marking of the treatment in the same order. In Figure 2 there is DL TL DY TY for bacteria and DL DY TL TY for funghi as in figure S2. Marking of the treatments MUST be equal throughout the manuscript (in text, figures or tables).)

Response: Thank you very much for this comment. This problem you mentioned appeared in Figure 2C. This is because clustering is involved in Figure 2 C and D, thus we cannot sort the four treatments in the same order.

23. (That is odd as in the soil this relationship is one most important in structuring microbial communities. How do you explain that?)

Response: Thank you very much for this comment. The expression of this sentence was wrong. In fact, pH was significantly correlated with both bacteria and fungi (Fig. 3 C and D). However, the correlation with bacteria was at p<0.05 level rather than at p<0.01 level. Therefore, this sentence has been changed to “while pH was no observed this phenomenon.”

24. (Increase the size of the text on all four parts of this figure.

Response: Thank you very much for this suggestion. We have increased the size of numbers and letters of all figures in the revised manuscript.

25. (Why not run RDA against microbial community structure instead of your treatments.)

Response: Thank you very much for this comment. The microbial community has been analyzed a lot. However, we also want to observe the difference between different treatments, thus the information of microbial community was no displayed in here.

26. (Nevertheless, You should incorporated real soil sample like control to which SGM and differences within would be compared to.)

Response: Thank you very much for this comment. Our aims are to evaluate the microbial communities of typical soilless growing media under common irrigation patterns in soilless culture systems. In general, soil can be used as a control, whereas soil microbiome varies in different ecosystems and has been studied well. Therefore, we did not use soil as a control, but the soil cited from previous studies was an important comparison factor in the discussion.

27. (In abstract you stated that the effect of irrigation was minor. What is correct?)

Response: Thank you very much for this comment. The irrigation pattern played a minor role relative to the SGM type. Therefore, there is no contradiction between the two sentences.    

28. (It is vital... to what? This sentence should be rewritten. It seems unfinished.)

Response: Thank you very much for this comment. This sentence has been rewritten as “the SGM type and irrigation pattern are very important for tomato production process in SCS”.

29. (than in common)

Response: We are sorry for this mistake. The “than common soil ecosystems” has been changed to “than those in common soil ecosystems”

30. (What is "original" SGM?)

Response: Thank you for pointing out this. The original SGM is raw SGM without watering and fertilization. The original SGM has been modified to “those in raw SGM”.

31. (This sentence is not correct. Supplementary figure S2 show no difference between treatments consisted of different irrigation patterns and soilless media. Hence unidentified fungi were not important. Please delete the sentence.)

Response: Thank you very much for this suggestion. This sentence has been deleted in the revised manuscript.

32. (There)

Response: Thank you for pointing out this mistake. The “there” has been replaced by “There”.

33. (pH does not preferre anything. Please reformulate this sentence.)

Response: Thank you very much for this comment. This expression is wrong, and we have reformulated this sentence as “pH had a more significant effect on the fungal community based on this network analysis”.

34. (in this study.)

Response: Thank you very much for this suggestion. The “as this study” has been modified to “in this study”.

35. (they were also)

Response: Thank you for pointing out this mistake. The “are” has been added between “they” and “also”.

36. (Four lines above you stated that in this study salt accumulation occurred (marked in yellow). Here you state that it might be some salinization. Please decide whether was salinization or not.)

Response: Thank you very much for this comment. We are sorry to make it unclear. Here we stated has been modified as “However, high EC values were observed only in coir substrate (Fig. 1B). Therefore, we speculate that the salinization problems are more likely to occur in coir substrate.”

37. (This antagonism could not be explained by N and P limitation because if both nutrients were limiting both enzymes would be equaly (both positively or both negatively) related to microbes as both enzymes increase availability of those nutrients. Please try to find better explanation why those activities were in antagonistic relation. Also it is hard to believe in N-limitation in experiments in which minimum nitrate concentration was 1 g/kg!)

Response: Thank you very much for your constructive comments and suggestions. We are sorry to make it inaccurate. This sentence has been changed to “In addition, we found that urease and phosphatase had obvious antagonistic effect on these bacterial and fungal genera, which may be that the lack of available carbon sources leads to microbial competition for energy in N and P utilization”.

38. (compost)

Response: Thank you for pointing out this problem. The “composting” has been changed to “compost”.

39. (in peat based substrate)

Response: We are sorry for this mistake. The “in based peat substrate” has been changed to “in peat based substrate”.

40. (Why conduct microbiome research if only simple questions would be asked. This conclusion could also be drawn from much simpler set of data i.e. figure S2. There was no difference for the tomato yield regardles of the SGM or irrigation used. Why were you interested in microbiome research of SGM? Does tomato grow better or worse in SGM compared to soil? And there was no soil in this research to which the results could be compared to. Why? By my opinion the discussion should be written about microbial community structure peculiarities related to different treatments (SGMs/irrigations). But again, everything seems pointless if there was no difference in tomato growth.)

Response: Thank you very much for your comments. The points you mentioned are very good. The main aim of this study is to evaluate the microbial community of typical soilless growing media under common irrigation patterns in soilless culture systems. Meanwhile, we want to assess the effect of different treatments on tomato yield. The results showed that there are no significant differences on microbial community function as well as tomato yield between different treatments. In addition, we found that the SGM have similar microbial function with soil. In fact, there are no significant differences on the tomato yield between the SGM and soil, which should be related to the similar microbial function between the SGM and soil. Therefore, we study indicated that peat based substrate and coir substrate are very good SGM from a microbial perspective and can be used to replace the soil. However, this is a preliminary evaluation study and there must be many shortcomings. We are very sorry for that and would appreciate your more advice and help.

41. (Major and minor role in what?)

Response: Thank you very much for this comment. This sentence has been changed to “SGM type played a major role and irrigation pattern played a minor role in the impact of environmental parameters and microbial communities”.

42. (That is not correct. You did only analysis of the SGMs and not of the soil. So, you could not state that.)

Response: Thank you for pointing out this problem. This statement has been deleted and the sentence has been changed to “We also found that the common soil functions can be basically performed by the two SGM.”

43. (In conlusions there is no space for assumptions. You shoul stic to your data and what they had tell you. No more or less than that. So what would be your major conclusion that come out from your results? For instance, I noted that bacterial and fungal community structures were different between treatments. How that could have reflected on microbial functions?)

Response: Thank you very much for your constructive comments and suggestions. The bacterial and fungal community structures were different between treatments, but there were no significances on the microbial function between different treatments. So instead of focusing on the differences between treatments, we focused on their role in replacing soil. The results indicated that peat based substrate and coir substrate can perform most of the functions of soil. This also explains why the SGM can be used more and more widely in alternative soil from a microbial perspective. In addition, we have replaced some speculative conclusions with speculative ones. We are very sorry for the unsolved problems. Thank you very much for your guidance and help. We will continue to improve the future research under your comments and suggestions. We also look forward to your further guidance.

Author Response File: Author Response.docx

Round 2

Reviewer 2 Report

Comments are in the pdf file.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

Thank you for your comments concerning our manuscript entitled “Evaluating the effect of replacing soil with typical soilless growing media: a microbial perspective” (agronomy-1995237). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our research. We have studied comments carefully and have made correction which we hope meet with approval. In the revised version, the deleted parts were marked with blue font and strikeout, while the added parts were marked with red font and underline. The main corrections in the paper and the responds to the reviewer’s comments are as following: Response to the reviewer letter.

Author Response File: Author Response.docx