Soil Bacteriome Resilience and Reduced Nitrogen Toxicity in Tomato by Controlled Release Nitrogen Fertilizer Compared to Urea

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The article describes the effect of fertilizers on plant biomass, soil nitrogen levels, and soil microbial properties; two forms of nitrogen fertilizers were compared. This research on trending topic is valuable and scientifically sound. However, there is room for improvement. The experimental design is good but not described clearly. The used methods should also be described better. Conclusions should be corrected according to the results. Please check the comments:

Major remarks:

11)       Article name does not reflect the content. It is not fully clear what was studied and what was the influencing factor. Also, plant toxicity was reduced compared to what? Authors mention microbiome, however, only the gene library analysis is mentioned in methods. So probably it’s better to say “bacterial community” instead of “microbiome”.

2)   Experimental design. There were 160 samples in total: planted/unplanted (2) * sterilized/not (2) * quick/controlled release (2) * high/low rate (2) *10 replicates = 160. But what about unfertilized control? How many samples were not fertilized? This should be added to the total number.

3.       Line 15 “soils with either low or high initial microbial N competitor populations” – but it was sterilized (not low, but absent) and unsterilized (not high, but present population) soil, wasn’t it?

4.       Please add information how many samples were used for DNA extraction (160+(unfertilized)*4 sampled cores from each pot)?

5.       Library preparation: how many samples were sequenced in the one run? How many samples were added to the same library?

6.       In the introduction and discussion, the competition between bacteria and plants for available N was mentioned. Please add references

7.       Lines 371-373 – are there any studies regarding the nitrogen losses in gaseous forms after ESN application?

8.       Please provide some information about the nitrogen release speed from ESN. It could be important for the context of results comparison in Lines 386-395

9.       Conclusion should be rewritten. Some results show that ESN, on the contrary, is still somewhat toxic if applied in high doses.

Other minor remarks:

Line 13 and 66 why tomato sort is mentioned only in 1 place                  

Line 77 – is seed sterilization before planting common for growing tomatoes?

Line 81 –The information about soil sterilization should be somewhere here

Line 209,211,213,228,245 – check the links

Figure 2 – why such a big difference was observed for the autoclaved and non-autoclaved soil? Unfertilized and autoclaved went below 0? Please check carefully

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study is focused on analyzing the effects of applying toxic levels of nitrogen using quick-release and controlled-release fertilizers. Research on soil microbiome responses to different agricultural practices, such as fertilizer application, tillage techniques, and crop rotation, has been extensively documented. However, there appears to be a lack of consensus in the results reported over time. As such, it remains pertinent to further contribute to the publication of results in this domain. Nevertheless, I recommend conducting major revisions before publishing the study.

One of the noteworthy aspects is how the study documents that the effects of nitrogen toxicity on soil microbiota have been previously reported (lines 35, 63, and 330). However, these results do not align with the findings in this study (line 353). While differences may be attributed to variations in crops or application doses, it is likely that a pronounced effect such as soil autoclaving can significantly alter the results.

I recommend performing analyses exclusively on non-autoclaved samples to determine whether removing this substantial effect reveals differences between the treatments. While autoclaving the samples is justified to reduce nitrifying bacterial populations, it's essential to remember that autoclaving is a generic sterilization method that affects not only the study group but the entire microbial diversity, potentially leading to significant result alterations.

In addition to these major issues, the following minor revisions are suggested:

-        The title refers to "soil microbiome resilience," but it appears to be associated with fertilizer use. It is unclear. Consider changing the title of the study.

-        Line 11: The phrase " However, it is unknown if similarly toxic rates of controlled-release fertilizers also have toxic effects on the soil microbiome" seems to suggest that toxic effects occur with quick-release fertilizers. The study results demonstrate that this is not always the case.

-        In many parts of the text, italics are not used when referring to species and genes (lines 13, 516, 170, 171, 180, 181, 287, 288, 290, 291, or 298).

-        Line 15: Specify whether the soils mentioned have been sterilized or not.

-        Lines 51 to 62: Rewrite this paragraph as it appears redundant.

-        Line 78: "distilled."

-        Line 95: "controlled"

-        Line 106: “Fertilizer was applied to the pots one week after trans-106 plantation at high rates [2 g urea (0.92 g N), 2.05 g ESN (0.90 g N)] and low rates [0.5 g urea 107 (0.23 g N), 0.55 g ESN (0.24 g N)] (Table 1).”

-        Line 117: “The shoots and roots were cleaned immediately following harvest”.

-        Line 136: Indicate the primer concentrations used.

-        In the Results section, remove the recurring "(Error! Reference source not found.)" notation.

-        Sections 3.1 and 3.2: Clarify that the results are independent of the presence or absence of plants.

-        Line 254: The text "Figure 3" appears duplicated.

-      Line 336: Mention other observed symptoms in addition to "reduced growth," such as visual effects on the leaves, etc.

Comments for author File: Comments.pdf

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Editors and Authors,

Review:

“Soil microbiome resilience and reduced plant toxicity by control-release nitrogen fertilizer”

Carley R. Rohrbaugh, Mary M. Dixon, Jorge A. Delgado, Daniel K. Manter *, Jorge M. Vivanco *

This paper is well written and describes an experiment designed to investigate the physiological response of tomato plants and the soil microbial biome to no Nitrogen, a small amount of urea, high amounts of urea, a small amount of slow-release nitrogen and high amounts of slow-release nitrogen. Plant responses were measured by dry weight measurements of roots and shoots. The microbiome was investigated by extracting soil DNA and amplifying he 16 S rDNA component. Thorough analyses of the resulting DNA sequences were undertaken.

Introduction

The introduction is well written.

Materials and Methods

Plant response

Why did the authors not measure tomatoes as an indicator / measure of the plant’s response to the varying N treatments. What interests a farmer is the crop. This cannot be changed now but is something that might be considered in the future and briefly discussed.

Microbiome section needs improvement.

The experimental design is not clear. 160 soil samples are mentioned but we do not know if DNA was extracted from all 160?  Figure 3 reports on that microbiome data in but I do not see 160 data points, nor is there an (n) in the legend.  Were sampled pooled, if so, then how?

The authors cite that Meta Genome Analysis (shot gun sequencing of raw no PCR amplified DNA) is the gold standard to measure functional gene in an MGA and yet they chose a PCR method. They use PICRUSt2 Douglas et al 2020 to quantify and compare functional genes in the microbiome. I would suggest without proper reference to the limitations of the approach and 16S copy number and intragenomic variations.  Inferences based on this approach require the necessary caveats which are missing in the results and discussion.

Results

Generally, the results are well presented but some minor changes required.

L263:  Change the text to did not significantly change the overall community.

L266: change to six bacterial species.

L272:  change bacteria to bacterial

1.      The standout result in this study was a quantification of how autoclaving the soil changed the microbiomes. Not a surprise that it would change them but interesting to see how and on such a scale and across all treatments.

2.      I agree with the authors findings that the Nitrogen treatments applied in this study did not have a large impact on the overall community structure and biodiversity and that this can be described as resilience.

3.      I was pleased to see that some key nitrogen associated species did change and the name of the species were reported. More importantly Table 3 shows that “nitrogen” functional genes were responsive.      

Discussion and conclusion

1.      Although not the subject of the study – the autoclave results deserve more discussion. Clearly autoclaving a soil does not sterilize it, but it does have impact that is long lasting and was abundantly clear after the 57 days of the experiment.  We do we learn from this??  The re is a method note/paper that could be written. Which phylogroups are impacted and a recommendation that if we want to conclude anything that can be vaguely correlated to what happens in a farmer’s field then it is a method that should be abandoned.

2.      While the microbiomes showed general resilience in terms of total community structure – I would suggest this is to be expected and now you have empirically proven it for tomatoes under the experimental conditions. Where you use resilient, I would suggest there is massive redundancy both in terms of species richness and functionality.    

3.      In terms of the bacterial biodiversity of species associated with the Nitrogen Cycle, herein lies the new and most relevant results for the question you asked. Likewise with e fold increases in nitrogen associated genes.

That said you should explain the limits of how PICRUSt2 works and that we are looking at a “snapshot”, a photograph of one moment in time.

The paper is well written, the analyses are thorough even if the NGS method chosen wasn’t the best approach.  Caveats need to be included in the discussion so that more can be made of the results.

I recommend the paper be published with the adjustments I have suggested.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, thank you for taking my comments into consideration. The manuscript was improved significantly.

I discovered minor error in Line 364 - "urea" twice mentioned in the sentence.

Author Response

Thanks for your comments, "urea" issue was addressed. 

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have made the requested changes.

Author Response

Thanks for your review.