Consecutive Fertilization-Promoted Soil Nutrient Availability and Altered Rhizosphere Bacterial and Bulk Fungal Community Composition
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn the manuscript by Want et al., the authors describe a study of soil nutrients, enzymes, and microbial communities from a larch plantation that experienced fertilization for three years. Fungal and bacterial communities from plant roots, rhizosphere soil, and bulk soil were sampled over time. In addition, soil nutrients and soil enzyme activities were measured in both rhizosphere and bulk soil over time. The inclusion of both bulk soil and rhizosphere soil for all measurements a highlight of this study for me. The time points measured were at 0 days, 5 days, 5 days, 30 days, and a final time point that the authors call “long-term.” However, it is unclear to me after reading this manuscript how long exactly after the fertilization started the “long-term” samples were collected. What is clear is that the fertilization was done for three years, which in my opinion, is not a long enough time to consider this a long-term effect. Thus, I recommend the authors re-phrase their manuscript to reflect this. This is one of several suggestions I have for the authors, which are detailed below.
1. In my experience, long-term fertilization experiments are much longer than three years. Even the references used by the authors describe fertilization studies of 22 years (reference 12), 30 years (reference 51), and over 100 years (reference 34). Soil organisms do respond quickly to fertilization, so I think it is valid to study these organisms within this time frame. But, even after three years, the effects that the authors report on soil bacteria and fungi, to me, are still not a long-term change. In order to report a long-term change and new equilibrium for these communities, multiple years would need to be recorded and I’m not sure how the “long-term” soils were sampled. In the Abstract, it seems like samples were perhaps collected in “three consecutive years of fertilization” (lines 18-19), but the figures only show one “long-term” sample, which makes this confusing to understand what exactly was done. I suggest the authors rephrase this throughout their manuscript.
2. Line 110 mentions sampling in “unfertilized and long-term fertilized areas”. However, the results do not show a comparison of fertilized and unfertilized treatments. Thus, my understanding is that any comparison between the fertilized soils and unfertilized soils were comparing the samples at 5 days, 15 days, 30 days, and “long-term” to the day 0 sample. A proper study design for comparing fertilized and unfertilized treatments would be to have samples from unfertilized plots sampled at the same time as the fertilized plots. Thus, statements about comparing unfertilized and fertilized treatments are very misleading (see for example, lines 214-216, lines 234-236, and lines 409-413). These all need to be rephrased, as there is not a true unfertilized treatment.
3. Line 112 mentions sampling three replicate fertilization plots. More information is needed here. How large were these plots? How large was the circular ditch for fertilization. I think this is typically done around one tree. Does this mean only three separate trees were sampled? In addition, if all three trees were at the same plantation, which I think they were, then this is pseudo replicated. Mainly, though, there was not enough information provided to adequately tell if this study had adequate replication.
4. It is unclear to me how the p-values for the community data were calculated (lines 246 and 258). A univariate ANOVA, as described in the methods would not be appropriate for these multivariate data. Multivariate analyses, like PERMANOVA and PERMDISP, should be used here. These could help with some of the conclusions of the NMDS data. For example, it was unclear to me as to why rhizosphere soil were analyzed separately for bacterial communities and bulk soil were analyzed separately for fungal communities. PERMANOVA could help to determine which groups to separate out; if there are significant interactions, for example.
Minor comments:
Line 119 – More detail is needed on how rhizosphere soil and bulk soil were separated. In addition, were roots washed before storage?
Lines 163-166 – Obtaining splice variants is not appropriate for this study. The Illumina reads for this study amplified the rRNA gene (16S for bacteria and ITS1 for fungi). Because these were not mRNA, there should be no splice variants.
Line 184 – I have not ever seen scientists report a finding as “extremely significant”. I just see p-values reported and the reader can determine the level of significance. Despite this, the authors do not use this term in the results, so I think it can be removed here.
Line 193 – R was used, so not all statistics were performed in SPSS, as stated on line 182.
Line 497-498 – Mortierellales is fungal, not bacterial.
Lines 501-521 – I would be cautious on any interpretation you make regarding the endophyte guild. Figure 9 does not have endophyte alone as a guild. The fungal guild that increased in the long-term fertilization samples is listed as “Endophyte-Litter Saprotroph-Soil Saprotroph-Undefined Saprotroph.” Fungal taxa can fall into multiple guilds, thus whether these endophytes are actively associating with the larch trees is uncertain, especially given that these trees are ECM-associated.
Comments on the Quality of English LanguageI don't have any specific comments about the quality of the english language. Some minor editing is needed, though.
Author Response
Thank you very much for your revision advices. We have revised the entire manuscript according to your feedback. Now we have replaced “long-term fertilization” with “consecutive fertilization” in the whole manuscript. Please find the detail point-by-point response in the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsForests-2841732
The authors of the manuscript “Long-term fertilization promoted soil nutrient availability and altered rhizosphere bacterial and bulk fungal community composition ” are contributing to filling the knowledge gaps regarding long-term nutrient availability and changes in microbial community structure under different management practices regarding the fertilization time frame.
I found this paper interesting to read as it has a well-guided study and discussion. I recommend this manuscript for publication after minor revisions.
INTRODUCTION
55 tempus: “are important”
54-67 this section should be shortened, as there are many basic information that can be condensed (regarding N, P and K).
83 Please add some more information about the importance of rhizosphere soil in nutrient cycling and some key differences compared to the bulks soil. As this paper focuses on rhizosphere and bulk soil, this should be addressed in the introduction. Also add information about the inclusion of the root itself when the paper addresses the differences between rhizosphere and bulk soil? What was the purpose of including plant material in this study?
MATERIALS AND METHODS
102-103 Grammar “ranging from … and …”
103-104 Repetitive use of “belongs”
109 What is the soil type and texture? The color is less relevant.
123 How did you samples the rhizosphere soil? Please add information about the sampling method (e.g., shaking https://doi.org/10.1016/j.geoderma.2021.115589 or washing https://doi.org/10.1007/s13213-012-0491-y)
138 NH3-N: 3 needs to be subscript
137-143 please rewrite and try to use comprehensive units -> Enzymes released g-1 soil d-1 mg -1 glucose for example.
137-143 Grammar: The activity units of XY were defined as […] was one enzyme activity. Please check for grammar and phrasing (one enzyme activity).
RESULTS
295/304 delete “including bulk soil and rhizosphere soil” You only sampled these two types
DISCUSSION
466 It would be helpful to get a plot which shows the absolute abundance of the investigated organisms for root, rhizosphere and bulk soil so we can get an impression of the differences.
CONCLUSION
The conclusion should be shortened a bit and be more focused on the key findings without repeating specific results (“The NMDS analysis results showed…”, “The RDA and co-occurrence network analyses indicated”)
FIGURES
Figure 4, 5, 6 and 9: Please use a consistent order in the legend (either alphabetical or by specific groups). “Other” and “Unknown” should come last. “unclassified_p_” should come after the specific name -> Fungi (unclassified)
Figure 8: Please increase the label size. It is very hard to read.
Author Response
Thank you very much for your advices. We have made comprehensive revisions to the manuscript according to your suggestions.Please find the detail point-by-point response in the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsIn general, a professional English-style correction is needed. Several grammar errors, incorrect terms, colloquial jargon, and inconsistencies were found. Please use active voice.
Abstract:
Line 16 and further. Please substitute the common name “larch” with the scientific name in all manuscript sections.
Line 22. NMDS is an ordination analysis, not a hypothesis test analysis.
Line 25. Please describe first the causes (fertilization) and then the consequences (microbial shifts).
Introduction:
Line 49. Please mention the results and cite those “rarely reported” (few) works.
Line 59. Please include data from forest or perennial crops instead of annual crops (reference 11 is based mostly on annual crops).
Materials and Methods
Line 104. Please add “a.s.l” after “meters”
Line 109. Please mention the soil type according to any international nomenclature system and cite the source.
Line 109. Please cite the work determining the soil thickness of “50 meters”???
Line 110. Please describe the exact sampling site locations, or (better) add a map.
Line 111. Please describe the tree density and age at each sampling site.
Line 111. Please state the fertilization regime followed for the long-term group (composition, dose, and frequency of fertilizer, including seasonal variations, if any)
Line 116. Please provide the fertilizer brand and formula. Please include trace elements. Please report the operational doses administered (weight/Ha and weight/tree).
Line 120. Please change as: “… samples were sent for sequencing (see section 2.4).”
Line 123. Please state the total number of collected samples and replicates and cases where samples were pooled as a composite sample (if any).
Line 125, please use N for nitrogen, C for carbon, and P for phosphorous.
Line 126. Please cite correctly the instrument used (Catalog number, brand, city, and country of company’s headquarters).
Line 127. Please cite correctly the method used and the instrument (2400â…¡, USA).
Line 128, 132. Please cite correctly the used instrument.
Line 136. Please cite the used kit correctly (Catalog number, brand, city, and country of company’s headquarters). References 22 and 23 do not refer to the kit.
Line 137. Please remove “The determination methods are detailed in the manual” and briefly describe the method and operational adaptations (if any).
Line 138. Please use subindex for Formulas like NH3 (NH3)
Lines 138 to 143. Please cite.
Line 146. Please cite the used kit correctly (Catalog number, brand, city, and country of company’s headquarters). References 22 and 23 do not refer to the kit.
Line 184. Please state the specific cases where normality criteria were not met.
Line 195. Please state which samples were used in each case.
Line 199. Please do not add spaces between numbers and the “<” symbol as in line 184.
Results:
Line 204, 208, 210 (and so). Please use N for nitrogen, C for carbon, and P for phosphorous.
Line 205, 207, 227 (and so). Please use consistently “p<0.05” as in line 184.
Figure 1 and 2. Please mention the number of repetitions as (n=X) in the footnote. Fuse these two figures in the new Figure 1.
Line 245, 252. NMDS is not a hypothesis test. Please include additionally a PERMANOVA analysis.
Figure 3. The use of a color palette is inadequate. Please use a different symbol for rhizosphere R (circles), RS: Rhizosphere soil (squares); BS: Bulk soil (triangles); 262 RSLF (diamonds). Further, to ease comparisons, use the same color for the samples obtained in the same event (same color for samples R0, RS0, BS0). Use a consistent color palette in all figures.
Figures 4 and 5. Please change the taxa order in the legend to match the order in the bars. Please clarify if the top 1% abundance is by sample or average. Consider substitute bars with heatmaps showing similarity distances by taxa abundance and by sample; it would be more informative. Fuse these two figures in the new Figure 3.
Line 321. Please explain how taxa were subsampled based on NMDS analysis. I would strongly recommend filtering after a SIMPER test. Otherwise, please remove Figure 6.
Figure 6. Please change the taxa order in the legend to match the order in the bars. Send this figure to Supplementary files.
Figure 7. Please explain what the different colors in the arrows refer to. Please use the same symbol and color palette recommended for Figure 3.
Figure 8. Please change the arrows from green to blue to meet the standard visualization. Increase font size in annotations. Please explain the meaning of circle size in nodes and arrow thick on edges. Please include a table with the network’s properties as supplementary material. I strongly recommend including a global network considering all samples, all soil nutrient content, and all enzymes’ activities. Please eliminate “Abbreviation instructions refer to the annotations 387 below Fig. 7.” and add the corresponding explanation.
Line 391. Please add the word “predicted” before the word “proportions”
Line 396. Please add the word “predicted” before the word “fungal”
Figure 9. Please change the taxa order in the legend to match the order in the bars. Send this figure to Supplementary files.
Discussion.
Line 425, 441. NMDS is not a hypothesis test analysis.
Lines 426 to 456. No probable causation is provided or supported by data. This section must be rewritten. Argumentation, including physiologic, genetic, biochemical, and ecological mechanisms of specific taxa explaining microbial community shifts, should be included. Direct reference to close research groups in China should be restrained and reserved only for critical data.
Line 503, 508. Please add the word “predicted” before the word “proportions”
Conclusions.
Line 531. NMDS is not a hypothesis test analysis.
Comments on the Quality of English LanguageIn general, a professional English-style correction is needed. Several grammar errors, incorrect terms, colloquial jargon, and inconsistencies were found. Please use active voice.
Author Response
Thank you very much for your advices. We have revised grammar errors and expression in the manuscript according to the reviewers’ advices. We have made comprehensive revisions to the manuscript according to your suggestions. Please find the detail point-by-point response in the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsThe authors have addressed all observations
Comments on the Quality of English LanguageMinor typos were found, the typesetting process will correct them.
