Inconsistent Responses of Rhizosphere Microbial Community Structure and Extracellular Enzyme Activity to Short-Term Nitrogen and Phosphorus Additions in Chinese Fir (Cunninghamia lanceolata) Plantations
Round 1
Reviewer 1 Report
The research is valuable both theoretically and practically. They broaden our understanding of the relationship between microbial communities, soil properties and plant communities. The results of the presented and future research will allow for the development of effective, but at the same time taking into account the proper functioning of forest ecosystems, methods of managing Chinese fir (Cunninghamia lanceolata) plantations.
Lines 112-121 –‘2.2 Soil sampling measurements’ It would be worth placing a schema of the experimental plots with an indication of the experimental variants.
Results:
Fig.S1 Please set in the figure title informations about abbreviastions CK, N1, N2, N3, N4, CK, P1, P2, P3, P4; The same in the case of Fig. S3
Conclusions: lines 387-394
It would be worth in this part of the text to refer to the hypotheses and clearly emphasize their truthfulness or not.
Author Response
- Lines 112-121 –‘2.2 Soil sampling measurements’ It would be worth placing a schema of the experimental plots with an indication of the experimental variants.
Response: According to your request, we have drawn a conceptual experimental design similar to a technical roadmap, including experimental treatments and indicators, as well as the relationship between the indicators to be explored.
- Fig.S1 Please set in the figure title informations about abbreviastions CK, N1, N2, N3, N4, CK, P1, P2, P3, P4; The same in the case of Fig. S3.
Response: We have refined the meaning of the abbreviations for experimental treatment in the titles of Figures S1 and S4.
- Conclusions: lines 387-394. It would be worth in this part of the text to refer to the hypotheses and clearly emphasize their truthfulness or not. lines 386-393:
Response: We rewrote the conclusion according to the hypothesis. The results verified the hypothesis that N addition reduced the ratio of fungi to bacteria and enhanced microbial C restriction, while P addition did not alleviate the microbial P restriction, which was inconsistent with the hypothesis.
Reviewer 2 Report
The article focuses on the important issue of N and P cycling in soil and rhizosphere as affected by shifts in EEAs activity and the structure of microbial community, in this case, under Chinese Firs trees. The objectives, the rationale for the problem, and the methodology of the study are unobjectionable. It is worth noting that the authors used a large body of to date literature sources on N- and P- cycling in rhizosphere of forest ecosystems.
Nevertheless, I believe that a number of improvements should be made to the text.
In addition to the linguistic problems, I would like to make the following remarks on the content of the article as such:
1. The authors use a large number of abbreviations, including some that are not always common. It would be good to add a list of abbreviations for ease of perception of this text.
2. It is great that the authors formulated a working hypothesis and included it into the end of the "Introduction" (L.90 – 97). However, it would be better if they clearly wrote in the text of the “Conclusion” whether the working hypothesis was confirmed by the results of the study (fully or partly) or not (L. 388 – 397).
3. It is necessary to give the official name of the soil in accordance with any generally accepted classification and its basic characteristics (either specifically for the experimental site, or at least averaged for this soil in the region of research).
4. L. 117. - Why did the authors use chloride as a nitrogen fertilizer and the most acidic form of phosphate as a phosphorus fertilizer? After all, this creates unfavorable conditions for acidity from the beginning of the experiment. It would be good to explain, at least briefly, the choice of these salts. Besides, atmospheric nitrogen deposition is represented by NO and NO2 dissolved in water, that is ultimately nitrite and nitrate, not ammonium. How about this? Please give a kind of explanation.
5. L. 254-256 – “The soil acidification by exogenous N input was not surprising, as it was often examined in forest soil, which was attributed to N addition saturating soil with N and NH4 + -N releasing more H+ into the rhizosphere by soil bacterial nitrification and root absorption [63,69]” – The main reason for acidification when applying ammonium chloride is the accumulation of chlorine anion in the soil due to the consumption of ammonium by plants and microorganisms. As for the nitrate anion, potassium nitrate, for example, is considered a physiologically neutral fertilizer - because of the consumption of both cation and anion. Probably both possible reasons for acidification should be mentioned.
6. L. 617 – 619 – The authors determined PLFA profiles, so, they have data on total PLFAs. I would recommend to make one more figure with total PLFA data, in order to find out whether there was a C deficiency or not.
7. L. 306 – “microbial C limitation in Chinese fir soil rhizosphere” – to me, sounds like an obvious oxymoron. I would reword it "changes in ....indices...that are commonly thought to be related to the C-limiting." Further, it would be good to discuss the data on the total PLFA content and how they relate to the dynamics of changes in these indices. The point is that the simple pattern "more nitrogen consumed - more C involved in cellular synthesis processes" seems not to work here. It works in a completely different way, namely: when additional amounts of nitrogen are introduced into the system, the ratio between the activities of a number of nitrogen cycle processes - autotrophic nitrification, heterotrophic nitrification, assimilatory and dissimilatory nitrаtereduction, nitrogen fixation, ammonification, etc. Many of these processes require readily available C both for the synthesis of the corresponding enzymes and intracellular substances. I highly recommend organizing a discussion around this complex interaction of carbon and nitrogen cycle processes.
Although the manuscript is well documented, it needs linguistic improvements by a native English speaker. Some parts of the text look incomprehensible/hardly understandable, or clearly need editing, for example:
L. 14-16 “This research aims to reveal impacts of short-term N and P inputs on microbial community structure and functions in rhizosphere soil, we determined soil physicochemical properties, phospholipid fatty acids, and seven hydrolytic enzyme activities in Chinese fir …” – this fragment evidently needs re-phrasing. Something like “….rhizosphere soil. To achieve these goals, the following experiments were conducted….etc”
L. 20 “…while showed a obviously unimodal pattern…” - an is missed.
L. 29 “Globally, atmospheric nitrogen (N) deposition.” – although it is clear that the authors mean chemical element N, the “atmospheric nitrogen” is molecular N2. This fragment needs rephrasing.
L. 38 “Soil microorganisms, play a critical role…” – no comma is needed.
L. 50 “sudies have reported…” – “t” is missed.
L. 54 “AMF in rhizosphere carrys” – please replace with carries
L.67 “to obtain N element” – should be rephrased.
L.113 – “Field addition experiments” – I would recommend “Field fertilization experiments”
L. 142 – “Soil available N was abstracted with 2M KCl solution” – was WHAT? “Maybe “extracted”?!
L. 198 – “were responsive to nutrient additions significant” – what does it mean, “significantly”?
L. 220 - “(Fig. 3, p < 0.000)” – looks weird.
L. 274 – 275 – “Ratios of rhizosphere C:N, C:P, and N:P are mainly decided by imbalance in rhizosphere C, N, 274 and P contents.” – what does it mean?
L. 281 – “Low P input maybe promot” – replace with ”may promote” or something like that.
L. 394 – “…rhizosphere N availability plays a essential role…” - …an essential role…
Therefore, the above-mentioned and similar fragments are to be re-written.
Note: the list of necessary corrections is not exhaustive. You need to check the entire text carefully.
Author Response
- The authors use a large number of abbreviations, including some that are not always common. It would be good to add a list of abbreviations for ease of perception of this text. L.618 – 619:
Response: According to your request, we have made a list of abbreviations for easy reading.
- It is great that the authors formulated a working hypothesis and included it into the end of the "Introduction" (L.90 – 97). However, it would be better if they clearly wrote in the text of the “Conclusion” whether the working hypothesis was confirmed by the results of the study (fully or partly) or not (L. 388 – 397). lines 386-393:
Response: We rewrote the conclusion according to the hypothesis. The results verified the hypothesis that N addition reduced the ratio of fungi to bacteria and enhanced microbial C restriction, while P addition did not alleviate the microbial P restriction, which was inconsistent with the hypothesis.
- It is necessary to give the official name of the soil in accordance with any generally accepted classification and its basic characteristics (either specifically for the experimental site, or at least averaged for this soil in the region of research). lines 110-111:
Response: According to the literature, we have given the official name of the soil in the sample plots, and the attached Table S2 shows the physical and chemical properties of 0-10cm soil layer at the sampling site.
- L. 117. - Why did the authors use chloride as a nitrogen fertilizer and the most acidic form of phosphate as a phosphorus fertilizer? After all, this creates unfavorable conditions for acidity from the beginning of the experiment. It would be good to explain, at least briefly, the choice of these salts. Besides, atmospheric nitrogen deposition is represented by NO and NO2 dissolved in water, that is ultimately nitrite and nitrate, not ammonium. How about this? Please give a kind of explanation. lines 117-118:
Response: N element was added to plots as NH4Cl, because the main input form of N deposition in China is ammonia nitrogen. P addition was selected as NaH2PO4 due to its good solubility and frequent use in agriculture.
- L. 254-256 – “The soil acidification by exogenous N input was not surprising, as it was often examined in forest soil, which was attributed to N addition saturating soil with N and NH4 + -N releasing more H+ into the rhizosphere by soil bacterial nitrification and root absorption [63,69]” – The main reason for acidification when applying ammonium chloride is the accumulation of chlorine anion in the soil due to the consumption of ammonium by plants and microorganisms. As for the nitrate anion, potassium nitrate, for example, is considered a physiologically neutral fertilizer - because of the consumption of both cation and anion. Probably both possible reasons for acidification should be mentioned. lines 256-258:
Response: We re-analyzed the reasons for soil acidification caused by exogenous nitrogen input. Plants and microorganisms absorb and use the ammonium ions imported into the soil to enter the cells, and in exchange release hydrogen ions into the environment, thus causing a decrease in pH.
- L. 617 – 619 – The authors determined PLFA profiles, so, they have data on total PLFAs. I would recommend to make one more figure with total PLFA data, in order to find out whether there was a C deficiency or not. lines 216, 238, 285-286, Table S3, Fig S3:
Response: We do a figure of total PLAFs and find that nitrogen and phosphorus additions had no significant effect on total PLFAs, that is, total microbial biomass did not reflect microbial carbon limitation in time in this study. This may be because the total PLFAs were controlled by soil moisture. However, changes in the ratio of microbial fungi to bacteria and vector length reflect the restriction of microbial nutrient metabolism.
- L. 306 – “microbial C limitation in Chinese fir soil rhizosphere” – to me, sounds like an obvious oxymoron. I would reword it "changes in ....indices...that are commonly thought to be related to the C-limiting." Further, it would be good to discuss the data on the total PLFA content and how they relate to the dynamics of changes in these indices. The point is that the simple pattern "more nitrogen consumed - more C involved in cellular synthesis processes" seems not to work here. It works in a completely different way, namely: when additional amounts of nitrogen are introduced into the system, the ratio between the activities of a number of nitrogen cycle processes - autotrophic nitrification, heterotrophic nitrification, assimilatory and dissimilatory nitrаtereduction, nitrogen fixation, ammonification, etc. Many of these processes require readily available C both for the synthesis of the corresponding enzymes and intracellular substances. I highly recommend organizing a discussion around this complex interaction of carbon and nitrogen cycle processes. lines 216, 285-286, 238, 294-300, 303, Table S3, Fig S3:
Response： For "microbial C limitation in Chinese fir soil", I reword it as " increasing vector length thought commonly to be related to the C-limiting." It was discussed in the paper that the total PLFAs were not significantly affected by nutrient additions, and the soil water content was significantly negatively correlated with total PLFAs, suggesting that the soil water content in this study may control the total PLFAs. In addition, there was no significant correlation between total PLFAs and extracellular enzyme activity, vector length, and enzyme ratio, so total PLFAs were not discussed much. This study focuses more on changes in the relative abundance of microbial taxa and their relationship to other indicators. We discuss the reasons for increased microbial carbon limitation. Because soil C and P cycles have complex coupling relationships, the introduction of additional nitrogen into forests can affect the interaction of microbially directed nitrogen cycles and carbon mineralization.We guessed that N addition delayed the mineralization of recalcitrant organic C by reducing the quality of soil organic matter and the oxidase activity, thus aggravating microbial carbon restriction. The decreases in relative abundances of fungi and AMF were likely linked to increases in microbial C limitation. Changes in rhizosphere microbial structure might be influenced by microbial nutrient restrictions.
- Although the manuscript is well documented, it needs linguistic improvements by a native English speaker. Some parts of the text look incomprehensible/hardly understandable, or clearly need editing. Thank you for your comments on the language problems in our paper.
Response： We have revised the spelling of words, single and plural forms, idioms, and the clear expression of sentence meaning in the paper. For example, lines 14-16, "To reveal the impacts....,....were measured in Chinese fir rhizosphere soil after one year of nutrient addition". lines 275, "Ratios of soil C:N, C:P, and N:P are mainly decided by nutrient contents condition".
