Mixed Chinese Fir Plantations Alter the C, N, and P Resource Limitations Influencing Microbial Metabolism in Soil Aggregates

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

 

Journal: Forests

Title: Mixed Chinese fir plantations alter the C, N and P resource limitation of microbial metabolism in soil aggregates

MS No. forests-2931581

 

 

Line 37: the soil quality scientifically reflects the rationalization…

The word “rationalization” seems a bit vague. Please be more specific.

 

Line 70: whole soil layer

The sudden appearance of these words here seems somewhat abrupt.

 

Lines 77-79: As…, and … Therefore, …

It was difficult to understand the connection between the sentences. Please pay attention to English, especially the conjunctions.

 

Lines 107-108: Mixed and pure stand with a mixture ratio of 3:1…

I couldn’t understand this sentence.

 

Line 115: different soil layers

How did you treat organic sol layer (O layer including fresh litter layer)?

 

Line 116: nonroot zone

I couldn’t imagine “nonroot” zone in the forest soil which contain more ore less contain fine roots.

 

Line 121: cut-ring method

Please explain this method.

 

Table 1: Aspect

The word “adret” is somewhat vague. Can you express it in terms of direction? (e.g., Southeast facing slope)

 

Line 174: Soil respiration (SR)

In this manuscript, instead of the term “soil respiration”, “microbial respiration” would be more appropriate.

 

Lines 184, 198, 221, 224: (1)~(4)

I think the heading for this section should be 2.5.1., 2.5.2….

 

Lines 193-194: equation (5) and (6)

It would be better to use a slash for the colon in the formula.

What is “ATAN”? Also, is the notation “Degree” correct as an expression in the formula? Please confirm.

 

Lines 242: tend to grow

Vague expressions

 

Lines 248-249:

Please be more specific.

 

Table 2 & Figure 2:

Please adjust the layout so that figures and tables including figure captions do not span pages.

 

Figures 2, 5, 6, 7:

These graphs are low resolution and unclear, and details are not clearly visible. Please prepare clearer graphs.

 

Figure 5:

I think panels A and D are especially important here. In contrast, the data in panels B, C, E, and F can be transferred (as simple tables) to the supplementary data. It would be more beneficial to enlarge panels A and D to make them clearer.

 

Line 332: axes

The "axis of the RDA" would be easier to understand.

 

Lines 420-421 and 422-424:

These sentences seem contradictory. C:N and C:` increased?, or relatively stable? Please check.

 

Lines 433-435:

For example, in soils lacking inorganic nitrogen, do they increase the secretion of enzymes to mineralize organic nitrogen to obtain inorganic nitrogen? If there is less nitrogen to be mineralized (i.e., organic nitrogen), wouldn't it be possible that the microorganisms would not benefit much from secreting more enzymes to mineralize this nitrogen, since less nitrogen would be obtained?

 

Line 454 and other many parts: “in our study [44]”

It is not clear whether the authors are discussing from the data obtained in this study or citing previous studies, although they are found in several places within this manuscript. It should clearly distinguish between the two.

Author Response

Dear reviewers, Thank you for offering us an opportunity to improve the quality of our submitted manuscript (forests-2931581). We appreciated very much the reviewers’ constructive and insightful comments. In this revision, we have addressed all of these comments/suggestions. We hope the revised manuscript has now met the publication standard of your journal. The revised portions are marked in blue ink in the paper. Below, our point-by-point responses to the issues raised by the reviewers are listed in blue text. Response to the comments of Reviewer #1 Journal: Forests Title: Mixed Chinese fir plantations alter the C, N and P resource limitation of microbial metabolism in soil aggregates MS No. forests-2931581 Line 37: the soil quality scientifically reflects the rationalization… The word “rationalization” seems a bit vague. Please be more specific. Response: Thanks to your constructive comments and suggestions, we have changed "rationalization" to "ecological sustainability" (Lines 36-37). Correction: Therefore, the soil quality sensitively reflects whether the above-ground vegetation is ecologically sustainable, especially in plantations. Line 70: whole soil layer The sudden appearance of these words here seems somewhat abrupt. Response: Thanks for your reasonable suggestion, we had revised it (Lines70-71). Correction: However, the microbial resource limitations in secondary soil structures are unclear but are necessary to explain soil microbial dynamics. Lines 77-79: As…, and … Therefore, … It was difficult to understand the connection between the sentences. Please pay attention to English, especially the conjunctions. Response: Thank you for your suggestion, we have revised it to make it easier to understand the connection of the sentences (Lines 78-80). Correction: In addition, aggregates are sites for soil microbial attachment and metabolic activities, and changes in aggregate C, N, and P resources may regulate the metabolism and reproduc-tion of soil microbes [32-34]. Lines 107-108: Mixed and pure stand with a mixture ratio of 3:1… I couldn’t understand this sentence. Response: We apologize for the unclear description and have revised it for clarity (Lines 108-112). Correction: Before afforestation, these were logging sites of pure Chinese fir forests and intense artificial disturbances (harvesting) have severely destroyed the vegetation [47]. In 1992, the mixed and pure Chinese fir stands were created with a row spacing of 2m × 3m. The mixed stands had a ratio of 3:1 of Chinese fir to mixed species and the density was ap-proximately 1,700 plants per hectare. Line 115: different soil layers How did you treat organic sol layer (O layer including fresh litter layer)? Response: Thank you for your valuable question. Before collecting the soil samples, we removed the litter layer from the soil surface, and we have revised this formulation in the text (Lines 120-123). Correction: The 5 point method was used to collect soil samples from each standard quadrat. Specifically, after removing soil surface litter, non-rhizosphere soil in different soil layers (0 ~ 10 cm and 10 ~ 20 cm) were collected in situ by a spade. The soils from 5 points were mixed in equal proportions to obtain mixed soil samples for each sample plot (Figure 1C). Line 116: nonroot zone I couldn’t imagine “nonroot” zone in the forest soil which contain more ore less contain fine roots. Response: I apologize for the mistake description, we have changed it to "non-rhizosphere soil" (Line 121-122). Correction: Specifically, after removing soil surface litter, non-rhizosphere soil in different soil layers (0 ~ 10 cm and 10 ~ 20 cm) were collected in situ by a spade. Line 121: cut-ring method. Please explain this method. Response: Thank you. We have added specific methods for determining soil physicochemical properties to the text (Lines 126-130). Correction: In addition, soil was collected in situ using a cutting ring for the determination of soil physico-chemical properties. Soil bulk density was calculated from the mass and volume of soil collected by each cutting ring; soil was dried at 65°C to constant weight to deter-mine soil water content (SWC). Table 1: Aspect The word “adret” is somewhat vague. Can you express it in terms of direction? (e.g., Southeast facing slope) Response: Thank you, we have revised Table 1 (Line 132). Line 174: Soil respiration (SR) In this manuscript, instead of the term “soil respiration”, “microbial respiration” would be more appropriate. Response: Thank you for your reasonable suggestions, which have been helpful in improving our manuscript. We have changed soil respiration to microbial respiration (MR) in the text and Figures / Tables of the manuscript. Lines 184, 198, 221, 224: (1) ~ (4) I think the heading for this section should be 2.5.1., 2.5.2…. Response: Thank you for your suggestion. We have adjusted the methodology section to make it more structured and easier to read, and we hope that this adjustment will meet with your approval (Lines 189-225). Lines 193-194: equation (5) and (6) It would be better to use a slash for the colon in the formula. What is “ATAN”? Also, is the notation “Degree” correct as an expression in the formula? Please confirm. Response: Thank you for your questions and suggestions regarding the equations in the manuscript: (1) We have changed the colon to a slash in equations (5) and (6); (2) "ATAN" indicates a function in Microsoft Excel (Office for windows 2021) that returns the inverse tangent of a number. To express the arctangent value in degrees, you need to multiply the result by 180/PI or use the DEGREES function. (3) "DEGREES" is the function that converts radians to degrees in Microsoft Excel (Office for windows 2021). We check and change "Degrees" to "DEGREES" in equation (6) (Lines 199). Correction: "VL=" [[("ln(BG) ∕ ln(URE)" )^"2" "+" ┤ 〖"(ln(BG) ∕ ln(ACP))" 〗^"2" ]^"1/2" (5) "VA=DEGREES" {"ATAN2" ["(ln(BG) ∕ ln(ACP)),(ln(BG) ∕ ln(URE))" ]} (6) Lines 242: tend to grow Vague expressions Response: Thanks, we specified these expressions (Lines 257-259). Correction: It is concerning that OC content increased significantly as aggregates particle size de-creased. Moreover, the contents of TN, ROC, AN, AP, and C:N were overall lower in the large aggregates than in the microaggregates in the three stands (Figure 2). Lines 248-249: Please be more specific. Response: Thank you, we specified these expressions (Lines 249-257). Correction: The C:P of 0-20 cm bulk soil and N:P of 0-10 cm bulk soil were significantly higher in SH stands than in pure stands. The C:N of 10-20 cm bulk soil in both mixed stands was significantly higher than that of the pure stand but the N:P was obviously lower than pure stand (Figure 2G-I). In addition, stand variations mainly led to significant differences in large aggregates and microaggregates in different soil layers. Specifically, C:N and C:P of large aggregates in 0-20 cm soil of SH stand and 0-10 cm soil of SM stand were significantly higher than those in SS stand (Figure 2G, H). And, C:N and N:P of microaggregates in SH stand were significantly different from those in SS stand (Figure 2G, I). Table 2 & Figure 2: Please adjust the layout so that figures and tables including figure captions do not span pages. Response: Thank you for your suggestions. We have adjusted the layout of Table 2 and Figure 2 to make it more reasonable. Figures 2, 5, 6, 7: These graphs are low resolution and unclear, and details are not clearly visible. Please prepare clearer graphs. Response: Thank you for your suggestions. We have improved the images to increase their sharpness (600 dpi), enlarged fonts and graphics, saved them in TIFF format, and uploaded them separately in the system. We hope our changes will improve your reading experience. Figure 5: I think panels A and D are especially important here. In contrast, the data in panels B, C, E, and F can be transferred (as simple tables) to the supplementary data. It would be more beneficial to enlarge panels A and D to make them clearer. Response: Thank you for your constructive suggestion, we have considered and adopted this opinion. We have enlarged panels A and D of Figure 5, and placed panels B, C, E, and F as supporting files in the supplementary files (Figure S1). Line 332: axes The "axis of the RDA" would be easier to understand. Response: Thanks, I've revised it (Line 325). Lines 420-421 and 422-424: These sentences seem contradictory. C:N and C:` increased?, or relatively stable? Please check. Response: Thank you. We have checked and revised the content here (Lines 403-419). Correction: In addition, the stoichiometric ratios reflect the relative dynamics of soil nutrients. In this study, the increase in the C:N ratio of the 10-20 cm bulk soil in the SH and SM stands was due to a significant increase in soil OC (Figure 2G). Although the SH stand significantly increased both soil OC and TP, the significant increase in soil C:P indicates that the SH stand is more conducive to soil carbon sequestration (Figure 2H). Related studies have shown that mixed tree species significantly alter soil carbon mineralization rates by changing soil microbial communities and structures [77]. Moreover, Zhang et al. found that mixed cultivation significantly enhanced soil microbial metabolic activity, where an increase in microbial carbon use efficiency was a key factor in promoting SOC accumulation [78]. The N:P is considered an index of the soil N and P nutrient limitation [79]. The impact of mixed plantations on soil N and P mineralization has been shown to be species-dependent and strongly regulated by nutrient release from leaf and root litter and in-put from root secretions [80, 81]. This explains the differences in soil N:P ratios among different forest stands in this study. However, this study indicates that the effect of mixed forests on N:P is regulated by soil layers (Figure 2I). Therefore, further studies on chemical elements in different soil layers may improve our understanding of organic carbon and nutrient cycling. Lines 433-435: For example, in soils lacking inorganic nitrogen, do they increase the secretion of enzymes to mineralize organic nitrogen to obtain inorganic nitrogen? If there is less nitrogen to be mineralized (i.e., organic nitrogen), wouldn't it be possible that the microorganisms would not benefit much from secreting more enzymes to mineralize this nitrogen, since less nitrogen would be obtained? Response: Thank you for your question. In soils deficient in inorganic nitrogen, microbes do increase the secretion of enzymes (e.g., urease) to mineralize organic nitrogen to obtain inorganic nitrogen [1]. Soil microbes regulate the production of extracellular enzymes to obtain nutrients and thus maximize their growth rate. However, the rationale behind the trade-off between enzyme production and growth is not fully understood. For example, microbes can regulate the rate of enzyme production per biomass to maximize biomass-specific growth rates [2]. In addition, microbes may also adapt by regulating their nitrogen use efficiency (NUE) when there is less mineralizable nitrogen in the soil. A high NUE means that microbes will retain more fixed organic nitrogen when nitrogen levels are limited, resulting in low nitrogen mineralization. This regulatory mechanism allows microorganisms to maintain growth and metabolism in nitrogen-limited environments [3]. In this study, the large mass of litter constitutes an important source of organic N mineralization available to soil microbes (Table 1); therefore, we did not consider altered microbial N balance due to organic N deficiency in this study. References： 1. Liu C.; Song Y.Y.; Dong X.F.; Wang X.W.; Ma X.Y.; Zhao G.Y.; Zang S.Y. Soil Enzyme Activities and Their Relationships With Soil C, N, and P in Peatlands From Different Types of Permafrost Regions, Northeast China. Front Env Sci-Switz 2021, 9. https://doi.org/10.3389/fenvs.2021.670769 2. Calabrese S.; Mohanty B.P.; Malik A.A. Soil microorganisms regulate extracellular enzyme production to maximize their growth rate. Biogeochemistry 2022, 158, 303-312. https://doi.org/10.1007/s10533-022-00899-8 3. Mooshammer M.; Wanek W.; Hammerle I.; Fuchslueger L.; Hofhansl F.; Knoltsch A.; Schnecker J.; Takriti M.; Watzka M.; Wild B. et al. Adjustment of microbial nitrogen use efficiency to carbon:nitrogen imbalances regulates soil nitrogen cycling. Nat Commun 2014, 5, 3694. https://doi.org/10.1038/ncomms4694 Line 454 and other many parts: “in our study [44]” It is not clear whether the authors are discussing from the data obtained in this study or citing previous studies, although they are found in several places within this manuscript. It should clearly distinguish between the two. Response: Thank you for your suggestion. We have revised it (Lines 444-445). In addition, other parts of the discussion section were checked and revised. Specifically, for the discussion made in response to the data from this study, we have added the source of the data (Figure or Table), and we hope that these revisions will clearly distinguish this study from others.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This study attempts to estimate changes in C, N, and P contents for microbial metabolism in soil aggregates. This article contains several methodological flaws and is not properly detailed.

 

General comments

The authors discovered that the type of forest planting alters the ratio of soil aggregates, which limits the amount of C, N, and P that soil microbes may use for volume metabolism. Unfortunately, this is only a statement of fact. No research has been conducted to determine the driving factors behind these changes. The authors have not researched any aspects of plant–soil interactions. No research has been conducted on tree plant root systems. The root systems of herbaceous plants have not been studied. Secretion of root exudates was not studied. The authors did not identify the taxonomic structure of the microorganisms (at the phylum level). There has been no research on soil fauna. The amount of litter in various tree stand types was reported by the authors, but no additional research was performed. The rate of litter decomposition and the rate at which organic matter entered the soil were not measured. This is not an exhaustive list of the factors that affect soil aggregate dispersion, and variations in organic matter intake and distribution. Therefore, all of the authors’ arguments have no factual evidence.

 

Specific issues

The authors must correctly specify the Latin names of the plants. You must provide an accepted biological name, not synonyms: Michelia macclurei - this name is a synonym of Magnolia macclurei (Dandy) Figlar (accepted Latin name).

Key woody plant characteristics were not included in this study. Average DBH? Average height? At the time of the research, what was the density of various forest stand types (number of trees per hectare)? At the time of the study, the tree planting scheme did not offer accurate information on stand density because weaker trees naturally die during growth.

Why did you calculate soil acidity (pH) if you did not use it in your study and even did not include it in the article?

Table 2. SWC? Perhaps it is the water content in the soil? You did not mention in the methodology that you defined this parameter. So, why were these data included in the table?

The information-presenting structure in Figure 2 is unfortunate. The figure is difficult to read.

Author Response

Dear reviewers,

Thank you for offering us an opportunity to improve the quality of our submitted manuscript (forests-2931581). We appreciated very much the reviewers’ constructive and insightful comments. In this revision, we have addressed all of these comments/suggestions. We hope the revised manuscript has now met the publication standard of your journal.

  The revised portions are marked in blue ink in the paper.

  Below, our point-by-point responses to the issues raised by the reviewers are listed in blue text.

 

Response to the comments of Reviewer #2

This study attempts to estimate changes in C, N, and P contents for microbial metabolism in soil aggregates. This article contains several methodological flaws and is not properly detailed.

 

General Comments

The authors discovered that the type of forest planting alters the ratio of soil aggregates, which limits the amount of C, N, and P that soil microbes may use for volume metabolism. Unfortunately, this is only a statement of fact. No research has been conducted to determine the driving factors behind these changes. The authors have not researched any aspects of plant–soil interactions. No research has been conducted on tree plant root systems. The root systems of herbaceous plants have not been studied. Secretion of root exudates was not studied. The authors did not identify the taxonomic structure of the microorganisms (at the phylum level). There has been no research on soil fauna. The amount of litter in various tree stand types was reported by the authors, but no additional research was performed. The rate of litter decomposition and the rate at which organic matter entered the soil were not measured. This is not an exhaustive list of the factors that affect soil aggregate dispersion, and variations in organic matter intake and distribution. Therefore, all of the authors’ arguments have no factual evidence.

Response: Thank you for your scientific questions and suggestions, which are very valuable for us to improve the quality of our manuscript. In this study, we mainly demonstrated that mixed stands affect the limitations of metabolic resources of soil microbes by altering soil aggregate structure (discussion 4.2) as well as soil C, N, P resources and their stoichiometry (discussion 4.3). Furthermore, we investigated the relationship between structural changes and soil aggregate stability, as well as the effects and mechanisms of mixed stands on soil nutrient stoichiometry, and found that mixed stands increased the proportion of soil large aggregates while decreasing the proportion of microaggregates, which directly led to an increase in soil aggregate stability (discussion 4.1). This study suggests that litter may be a significant factor in nutrient differences between stands and its impact on microbial metabolism. Future research should focus on the nutrient and decomposition processes of litter. Additionally, in-depth studies on microbial community structure and function may provide a better understanding of the resource-limited response of microbial metabolism to stand changes. Above are our next aims. We have revised the whole manuscript, including the Latin names of tree species, added some important data and modified the pictures, etc. We hope that the revised version of the manuscript will be approved by you.

 

Specific issues:

The authors must correctly specify the Latin names of the plants. You must provide an accepted biological name, not synonyms: Michelia macclurei - this name is a synonym of Magnolia macclurei (Dandy) Figlar (accepted Latin name).

Response: Thank you for your scientific advice. We have checked and corrected the Latin names of the two mixed species in this manuscript and defined them in the first occurrence (Lines 43-44).

 

Key woody plant characteristics were not included in this study. Average DBH? Average height? At the time of the research, what was the density of various forest stand types (number of trees per hectare)? At the time of the study, the tree planting scheme did not offer accurate information on stand density because weaker trees naturally die during growth.

Response: Thank you for your questions.

1) We have added the mean DBH and mean height of Chinese fir and mixed species in different stands in Table 1 (Line 132).

2) We have added the density of different stands in the Methods section (Line 132).

3) We have added the density of the stands at the time of stand construction in the Methods section of the manuscript (Line 112). We hope that these data will improve your understanding of this study.

 

Why did you calculate soil acidity (pH) if you did not use it in your study and even did not include it in the article?

Response: Thanks for the suggestion, we've removed it.

 

Table 2. SWC? Perhaps it is the water content in the soil? You did not mention in the methodology that you defined this parameter. So, why were these data included in the table?

Response:

Thank you for your question. In this study, SWC indicates soil water content and we have added its full name in Table 2 (Line 239). In addition, we have defined it in the method section as well as added the determination method (Lines 129-130).

 

The information-presenting structure in Figure 2 is unfortunate. The figure is difficult to read.

Response:

Thank you for your sensible suggestions. We have changed Figure 2 to a bar plot and changed the presentation structure. We hope that the revised picture will better visualize the differences of the indicator among different stands and aggregates (Line 260).

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Equation (6) (Line 200)

Describing functions on specific software such as Excel in formulas is not common and difficult for the reader to understand. It would be better to use a general mathematical expression.

Author Response

Dear reviewers,

Thank you for offering us an opportunity to improve the quality of our submitted manuscript (forests-2931581). We appreciated very much the editor and reviewers’ constructive and insightful comments. In this revision, we have addressed all of these comments/suggestions. Additionally, we have focused on and examined the manuscript for grammar and tense issues, as well as percent match. We also carefully sifted through the references to this manuscript. We hope the revised manuscript has now met the publication standard of your journal.

  The revised portions are marked in blue ink on the paper.

  Below, our point-by-point responses to the issues raised by the reviewers are listed in blue text.

 

Response to the comments of Reviewer #1

Equation (6) (Line 200)

Describing functions on specific software such as Excel in formulas is not common and difficult for the reader to understand. It would be better to use a general mathematical expression.

Response: Thank you for your comments. We have altered the formula to a general mathematical expression, which we hope will increase your understanding (Line 207).

Correction:

 

where atan2 is a mathematical function to compute the arctangent of a given point (x, y).

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors answered all the questions and comments. I am satisfied with the answers. The manuscript has been carefully revised. Thanks to the authors for their work.

Author Response

Dear reviewers,

Thank you for offering us an opportunity to improve the quality of our submitted manuscript (forests-2931581). We appreciated very much the editor and reviewers’ constructive and insightful comments. In this revision, we have addressed all of these comments/suggestions. Additionally, we have focused on and examined the manuscript for grammar and tense issues, as well as percent match. We also carefully sifted through the references to this manuscript. We hope the revised manuscript has now met the publication standard of your journal.

Author Response File: Author Response.pdf