the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 20 Nov 2024
Promoted phosphorus transformation by increasing soil microbial diversity and network complexity – A case of long-term mixed-species plantations of Eucalyptus with N-fixing tree species
Abstract. Increased nitrogen (N) availability influences soil phosphorus (P) cycling through multiple pathways. Soil microorganisms are essential facilitating a wide range of ecosystem functions. However, the impact of mixed plantations of Eucalyptus and N-fixing tree species affect P transformation and microbiota interactions remains unknown. Therefore, we conducted a 17-year field experiment in pure Eucalyptus plantations (PPs) to assess the effects of soil P transformation in mixed plantations (MPs) of Eucalyptus and N-fixing trees species. The results showed that α-diversity indices for bacteria ACE and Chao1 as well as Shannon indices index for both bacteria and fungi were significantly higher in MPs than in PPs. Significantly higher relative abundances in MPs than in PPs were determined for the bacterial phyla Proteobacteria (0–10 cm soil layer only), Verrucomicrobia, and Rokubacteria and for the fungal phyla Mortierllomycota, Mucoromycota, and Rozellomycota. By contrast, those of the bacterial phyla Chloroflexi, Actinobacteria, and Planctomycetes and fungal phylum Ascomycota were significantly lower in MPs than in PPs. Gene copy numbers for 16S rRNA, internal transcribed spacer (ITS), N functional genes [nifH (0–10 cm soil layer only), AOB-amoA, narG, nirS, and nosZ (0–10 cm soil layer only)] and P functional genes [phoC, phoD (0–10 cm soil layer only), BPP, and pqqC] were also significantly higher in MPs than in PPs. The findings indicated that MPs can enhance soil microbial diversity, network complexity, and the relative abundance of functional genes associated with N and P cycling by optimizing soil nutrient levels and pH, thereby facilitating P transformation. Therefore, MPs of Eucalyptus and N-fixing tree species may represent a promising forest management strategy to improve ecosystem P benefits.
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Status: final response (author comments only)
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RC1: 'Comment on egusphere-2024-3456', Anonymous Referee #1, 19 Jan 2025
Jiyin Li and co-authors investigated the effects of monoculture plantation or mixed N-fixing tree species on soil phosphorus transformation in a eucalyptus plantation soil, they further tried to find explanatory biotic and abiotic factors for changes in transformation of phosphorus. I find this research potentially could contribute to our understanding of long-term (seventeen years) eucalyptus plantation soil restoration via introducing N-fixing tree species, particularly the interaction between two most important soil elements, i.e., N and P and related microbial diversity and network complexity. Overall, the topic was suitable for Biogeosciences, and would give some new points to the huge amount of soil nutrient restoration studies. But before it could be accepted for publishing, I have some questions and suggestions on the manuscript. First is a more detailed introduction on the experimental design, it would be ideal for providing detailed aspects of management (e.g., fertilizer amount and frequency, pesticide use) of the experimental plots. Second, I found the Materials and Methods was lack of relevant references. The authors should provide more references about the measuring methods of Soil properties and soil enzyme activity. Third, the organization and language of the paper need still need further modification. For example, in the discussion part, some contents belong to the repetition of the results, and the related and cited results can be summarized as supporting evidence without listing too many specific values (e.g., L404-406, L426-427, L487-490).
Specific comments:
1. L-26-L28: Please reorganize the sentence.
2. In this paper, your dissertation focuses on the introduction of N-fixing tree species to promote soil phosphorus transformation, but a description of the characteristics of N-fixing tree species is lacking in the introduction, please clarify.
3. L-93: There is a notation error here.
4. In the text, N-fixing tree should be changed to N-fixing tree species.
5. Line 135 “soil extracellular enzymes” should use the abbreviation.
6. In the text, you characterize the conversion of N and P in terms of soil enzyme activity, but you don't have a specific sentence in the text to describe it.
7. L-93: Wrong colon space on this line.
8. L262-263: You write “Significant (P < 0.05) increases in ……. were determined in both soil layers of the MPs and PPs ", I think it would be more precisely if you write “Significant (P < 0.05) higher of ……. were determined in both two investigated soil layers in MPs than those in PPs "
9. 9. L-374: Missing comma after “soil properties”.
10. L-439: Which specific result indicates that pH is the most crucial factor affecting microorganisms?
11. L441-L443: This sentence appears to have no correlation with the context and it is recommended to delete it.
12. L-449-450: It is recommended to restructure the sentence to indicate that Proteobacteria encompasses Rhizobia.
13. L-454-455: The sentence is repetitive in meaning.
14. The discussion indicated that these key microbial groups could increase the complexity of the network, but there were no corresponding results to support this view.
I highly value the large amount of work carried out by the authors. I hope my remarks will be valuable for the authors.Citation: https://doi.org/10.5194/egusphere-2024-3456-RC1 - AC1: 'Reply on RC1', X.M. Huang, 16 Mar 2025
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RC2: 'Comment on egusphere-2024-3456', Anonymous Referee #2, 24 Jan 2025
The authors examined key soil properties (e.g., pH, Nitrogen content, and Phosphorus content) and microbial diversity composition, comparing a Eucalyptus monoculture site with a Eucalyptus-Acacia mixed plantation site. They found significant differences in both soil chemical properties and microbial diversity composition between the two sites. This work and its topic align with the scope of Biogeosciences. However, the manuscript's organization and overall quality require significant improvement before it meets the publication standards of Biogeosciences.
General comments:
- The key message emphasized throughout the manuscript—that “increased soil microbial diversity and network complexity has resulted in enhanced Phosphorus transformation” —appears to be overstated for several reasons:
- While soil “P transformation” is repeatedly mentioned throughout the manuscript and included in data analyses (e.g., Figure 9), there is no clear definition or quantification methodology provided for this index. Phosphorus exists in soil in both organic and inorganic forms and undergoes continuous transformations through chemical, physical, and biological processes, making its transformation quantification complex. The authors only measured two P-related soil properties—total P (TP) and available P (AP), with AP notably absent from many follow-up analyses (Figure 8). It is unclear how the authors quantified “P-transformation” (which appears to be simply the Total P content) and drew conclusions about factors influencing this process.
- The authors appear to have conflated correlation with causation in their narrative. While the Eucalyptus plantations were established in 2004 (approximately 20 years ago), soil sampling and analysis were conducted in 2021, providing only a recent snapshot. While it is reasonable to describe the observed differences in key soil chemical properties and microbial diversity between the two types of Eucalyptus plantations, the causal claim that increased soil microbial diversity and network complexity resulted in enhanced Phosphorus transformation is not adequately supported by the presented data and results.
- Furthermore, according to the correlation matrix plot presented in Figure 8, Total P (TP) shows no significant correlations with either soil chemical properties or soil microbial diversity indices for most ofthe variable pairs. This lack of correlation directly contradicts the authors’ main argument about the relationship between microbial diversity and Phosphorus transformation.
- The “Introduction”section requires substantial revision. It contains excessive methodological descriptions, such as Microbiome co-occurrence networks analysis and Functional gene markers, while lacking crucial discussions of key questions, mechanisms, patterns, and processes. Methodology merely describes the work conducted rather than establishing research significance. The interesting aspects that should be emphasized include the relationship between N and P, the role of N-fixing plants in P transformation, the key players involved in these processes, and the main processes and influencing factors. Once these processes and key issues are clearly articulated, the methodological details would naturally fit into the Materials and Methods section. While the final paragraph includes hypotheses, these would be better integrated into the earlier parts of the Introduction.
- The Introduction should address whether findings from Eucalyptus plantations can be generalized to other plantation types globally. Given the wide variety of both monoculture and mixed-species plantations worldwide, the authors should discuss how their research on Eucalyptus plantations relates to or differs from other plantation systems, and clarify the broader applicability of their findings.
- All expressions of “significant (P < 0.05)”should be revised to include the appropriate test statistics. Throughout the manuscript, the authors need to add the corresponding test statistics (g., t or F values) alongside the P-values to comply with standard statistical reporting conventions. For t-tests, results should be reported as (t = XX, P < 0.05), and for ANOVA tests, results should be reported as (F = XX, P < 0.05).
- The manuscript contains numerous formatting errors in English text and symbols. For example:L2, Hyphens in title require spaces on both sides (e.g., "word - word" instead of "word-word"); redundant punctuation marks (e.g., double commas in L93); improper spacing in ratios (e.g., "C:N ratio" and "N:P ratio" should not have spaces around the colon); inconsistent hyphenation and capitalization in statistical terms (e.g., "z score" and "c score" should be "Z-score" and "C-score"). The authors should carefully review and correct all formatting issues throughout the manuscript, paying particular attention to: (1) proper use of hyphens and spaces; consistent capitalization; standard formatting of statistical terms; correct punctuation; proper ratio expressions
Specific comments:
L48-51, The opening statement about Phosphorus being an essential nutrient is too absolute and lacks proper context.
L79-81, The statement "... is crucial for developing forest management strategies aimed at enhancing soil fertility and optimizing ecosystem functionality" is an overreaching conclusion that lacks sufficient support. In particular, the concept of ecosystem functionality was never a focus of this study.
L236-237, the rationale for choosing these specific metrics (ACE, Chao1, and Shannon indices here in this study) over other available diversity measures for microbial community analysis should be explained.
L262-263, The description of results is unclear regarding which group showed an increase when compared to which group.
L245, There are inconsistent statements about the correlation analysis method used: L245 mentions Pearson correlation, L362 refers to Spearman correlation analysis, and Fig. 8 (L372) again states Pearson correlations. The authors need to clarify which correlation method was actually used and maintain consistency throughout the manuscript.
L372, Figure 8’s readability is poor due to the excessive number of correlated variables. With many variables showing covariation, it is difficult to identify meaningful relationships. The authors should justify the purpose of including so many variables in the correlation analysis and consider focusing on key variables that address their research questions.
L389-393, The meaning and purpose of Figure 9 are unclear. The figure caption only describes the visual elements but lacks explanation of what the figure aims to demonstrate or illustrate.
L406-408 There is a logical inconsistency in the manuscript's core arguments. While L406-408 emphasizes how soil properties influence microbial community composition ("Soil properties are key in influencing the composition of microbial communities..."), the main thesis appears to argue that differences in microbial community diversity lead to variations in soil P transformation.
L465-468, the text here is redundant as similar sentences appear in the Introduction. Moreover, this background information belongs in the Introduction section rather than the Discussion, where the focus should be on interpreting results and their implications.
L508-L512 This is for sure. The introduced trees are N-fixing trees.
Citation: https://doi.org/10.5194/egusphere-2024-3456-RC2 - AC2: 'Reply on RC2', X.M. Huang, 16 Mar 2025
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RC3: 'Comment on egusphere-2024-3456', Anonymous Referee #3, 13 Feb 2025
This study quantified soil fungal and bacterial communities, genes, and networks for both pure Eucalyptus (PP) and mixed Eucalyptus-Acacia (MP) plantations. The plantations have been growing for 17 years, allowing authors to report long-term differences caused by co-planting Eucalyptus with a nitrogen-fixing tree species. The results are interesting, consisting of many differences between the plantation types in the composition and function of the microbial communities. Although I cannot address many of molecular methods, as they are outside of the scope of my expertise, I hope my comments below help improve the manuscript. Once they are addressed, I believe it will be a good fit for Biogeosciences.
The hypotheses presented in the last paragraph of the introduction are unclear. For (1), it is stated that diversity and composition of soil microorganisms will change with mixed planting. How will they change? For (2), “mixed plantations intensify the response to the beneficial impacts of N-fixing tree” is unclear and should be reworded. For (3), this hypothesis seems to overlap with hypothesis (1) (both mention diversity), but is more specific, suggesting that there will be higher diversity in mixed plantations.
The rationale for making measurements at the two depths (0-10 and 10-20cm) are unclear. Please provide an explanation for why these two depths were chosen.
The rationale for the different alpha index analyses (ACE, Chao1, Shannon) should be mentioned. That is, why are all three used and in what ways do insights from them differ?
It would be helpful to mention the perceived function of the different genes that were measured. For example, in the paragraph at L198 and in Figs. 5-7.
I think that there should be a discussion of why there was higher TP in PPs than MPs and whether trees in MPs and PPs might differ in whether they are limited by N vs. P.
The introduction and discussion would benefit from discussing mixed plantations between N-fixing and non-fixing trees in general. How representative are Eucalyptus-Acacia plantations of mixed plantations elsewhere?
The direction of causality is unclear. Throughout the manuscript, the authors argue that microbial diversity, structure, complexity promote P transformation. However, sentences such as that on L68-70 suggest causality is in the other direction.
The manuscript should be checked for typos and grammar. There are many instances of minor mistakes.
Specific comments:
Title: I would change to: “Soil microbial diversity and network complexity promote phosphorus transformation: A case of long-term mixed-species plantations of Eucalyptus with a nitrogen-fixing tree species”
L24-26: Clarify that the study was in both PPs and MPs. The sentence makes it sound like the study was just done in PPs.
L30: The two soil layers tested should probably be mentioned before reporting specific results for one of them.
L63: “soil health” is a vague statement. Be more specific.
L95: This sentence states that N content influences soil pH. Typically, the direction is one where an increase in N content lowers soil pH. The results show that pH however increased, which I found surprising. Although the discussion has a few lines on why, it may be good to address the hypothesized direction of change somewhere in the introduction.
L99: Change the part of the sentence that follows the comma to “thereby accelerating nutrient cycling and improving soil fertility”
L106: It is unclear what is meant by “soil nutrient effectiveness”.
L111: Replace “fewer” with “less or no”
L117: This might be a good time to mention the N-fixing tree species that is used in the MPs.
L125-126: I am unsure of what is meant by “along with genes associated with N and P cycling”.
L262-263: Clarify that the increase was in going from PPs to MPs.
L305: Can you explain by what metric pH is the most important regulator? It is not immediately clear from looking at Figure 3b.
L376: Please provide a number for the “high goodness of fit.”
L450-451: Having actinobacteria in this sentence is misleading. Actinorhizal plants form N-fixing symbioses with Frankia, which are actinobacteria. However, Acacia is not an actinorhizal N fixer. Instead, Acacia forms N-fixing symbioses with Rhizobia, which are Proteobacteria.
Table 1: Clarify whether the +/- refers to the standard deviation or the standard error.
Table 2: Bacteria is misspelled.
Figure 1: In the caption mention the threshold p value (my guess is p < 0.05) that determines whether differences between treatments are significant or not.
Figure 4: The Zi-Pi plots have the connectors (high among module connectivity) and module hubs (high within module connectivity) switched in the legend. Also, it is not clear what is meant by “node color node size” in the caption.
Figure 9: The caption appears to explain 9a, but not 9b.
Citation: https://doi.org/10.5194/egusphere-2024-3456-RC3 - AC3: 'Reply on RC3', X.M. Huang, 16 Mar 2025
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