Influence of Acacia mangium on Soil Fertility and Bacterial Community in Eucalyptus Plantations in the Congolese Coastal Plains
, Alessia Fiore 2, Silvia Tabacchioni 2, Giuseppe Aprea 2, Arthur Prudêncio de Araujo Pereira 3 and Annamaria Bevivino 2,*
Round 1
Reviewer 1 Report
Comments to authors
The manuscript entitled "Impact of Acacia mangium on soil fertility and microbiota composition
in Eucalyptus Plantations in the Congolese Coastal Plains" investigated the interactions between plant growth, nutrient cycling, and the microbial community structure in the Acacia and Eucalyptus plantations established on the nutrient-poor soils in the Congolese coastal plains.
The authors examined the soil bacterial community by 16S rRNA gene amplicon sequencing and evaluated the impacts of nitrogen-fixing trees (NFTs) on nutrient and microbiota dynamics. Their results showed that Actinobacteria, Proteobacteria, Firmicutes, and Acidobacteria were the dominant phyla and the bacterial community structure of the pure Eucalyptus was distinct from those containing Acacia or mixed species. The authors also suggested the importance of bacterial community to P cycling in this type of forest management. This study is valuable in revealing the roles of nitrogen-fixing trees in improving soil fertility and soil microbial diversity and sustaining Eucalyptus plantations established on the infertile and sandy soils. The research is within the scope of Sustainability. The manuscript was also written in a good manner.
Minor comments
1.Abstract:
“Overall, our findings highlight the benefits of introducing NFTs to improve soil fertility and soil microbial diversity and sustain Eucalyptus plantations established on the infertile and sandy soil of the Congolese coastal plains.” (Line 34-37)
Comment: This conclusion might not stand, because there were no significant differences in bacterial community diversity detected between samples as shown in the result part. If I understand correctly, this needs to be rephrased.
2. Introduction was well written, and it was enjoyable to read. I can also easily see the value of this study.
3. Materials and Methods:
I was wondering how the soil was classified and where the physicochemical properties came from. I saw the references, but it was not clear to me if the references provided the methods of measurement or the direct results. (Line 101-110)
“Resin available P was determined using two anion exchange resins strips (BDH#551642S) were used to determine available P (for more details see [33]).” Please rephrase this sentence. (Line 138-140)
4. Results
The sentence patterns in Line 213-218 were kind of repetitive making it not readable. You also mention higher or lower, but not mentioning compared with what samples. Please consider rephrase them.
For example, “found” and “observed” were repeatedly used. You could consider other ways of expression:
“Pure Acacia contained lower C concentration value (1.5%) compared with other samples, and the mixed-species nearby Eucalyptus 50A50EEu and nearby Acacia 50A50EAc stands had the highest (1.7%) C concentration.”
Line 225-226: “Figure show the relative abundance of soil bacterial community at the phylum level.”
Comment: “Figure shows the relative abundance of soil bacterial community at the phylum level.”
Question: How did you calculate the relative abundance of each phylum by sample or by bacterial taxa? Figure 1 does not seem robust to me. Please consider using another way to show the results of Figure 1.
The bacterial community composition was only shown in phylum level. It might be valuable to dig into the bacterial composition at class or genus level.
5. Discussion:
Generally, this is good discussion. I think the link between the structure of soil bacterial communities and soil fertility might be discussed more deeply.
Author Response
REPLY TO REVIEWER 1
We are thankful to the Reviewer for the positive comments to improve the manuscript. We have considered his valuable suggestions and modified the manuscript, accordingly.
Minor comments
Point 1: Abstract - “Overall, our findings highlight the benefits of introducing NFTs to improve soil fertility and soil microbial diversity and sustain Eucalyptus plantations established on the infertile and sandy soil of the Congolese coastal plains.” (Line 34-37).
Comment: This conclusion might not stand, because there were no significant differences in bacterial community diversity detected between samples as shown in the result part. If I understand correctly, this needs to be rephrased.
Response 1: We thank the Reviewer for this comment. On the basis of the results obtained during the review process, we modified the abstract (lines 37-39). Overall, our findings highlight the role of soil attributes, especially C, S, available P and C/N ratio at a lesser extent, in driving the soil bacterial community in mixed-species plantations and its potential to improve soil fertility and sustain Eucalyptus plantations.
Point 2: Introduction was well written, and it was enjoyable to read. I can also easily see the value of this study.
Response 2: We thank the Reviewer for the comment.
Point 3: Materials and Methods - I was wondering how the soil was classified and where the physicochemical properties came from. I saw the references, but it was not clear to me if the references provided the methods of measurement or the direct results. (Line 101-110)
Response 3: We thank the Reviewer for his/her suggestion. The soil was classified according previous findings in the studied area. Our previous publications provide both methods of measurements and results. In the current study, only data are presented and those of pH values, C and N have been added. Following the Reviewer’s suggestion, we added the values and the references in the sub-paragraph 2.1.1.
Point 4: “Resin available P was determined using two anion exchange resins strips (BDH#551642S) were used to determine available P (for more details see [33]).” Please rephrase this sentence. (Line 138-140)
Response 4: The sentence has been rephrased (see lines -158-163). More details have been given and a new reference added (38).
Point 5: Results - The sentence patterns in Line 213-218 were kind of repetitive making it not readable. You also mention higher or lower, but not mentioning compared with what samples. Please consider rephrase them. For example, “found” and “observed” were repeatedly used. You could consider other ways of expression:
“Pure Acacia contained lower C concentration value (1.5%) compared with other samples, and the mixed-species nearby Eucalyptus 50A50EEu and nearby Acacia 50A50EAc stands had the highest (1.7%) C concentration".
Response 5: We thank the Reviewer for the suggested modifications. Changes have been made accordingly. Please, see Lines 245-252.
Point 6: Line 225-226: “Figure show the relative abundance of soil bacterial community at the phylum level.”
Comment: “Figure shows the relative abundance of soil bacterial community at the phylum level.”
Response 6: We modified the result section (see paragraph 3.2) and this phrase has been removed.
Point 7: Question: How did you calculate the relative abundance of each phylum by sample or by bacterial taxa?
Response 7: The 16S rRNA sequences were assigned against SILVA database and each OTU was taxonomically classified. For this, we use QIIME scripts, specifically “summarize_taxa.py”, which provide summary information of the representation of taxonomic groups within each sample. It takes an OTU table that contains taxonomic information in each sample as input. To calculate the relative abundance, the script sums up all OTU counts and divide each count with the sum, same way that you would compute percentage.
Point 8: Figure 1 does not seem robust to me. Please consider using another way to show the results of Figure 1.
Response 8: We agree with the Reviewer. Figure 1 was modified.
Point 9: The bacterial community composition was only shown in phylum level. It might be valuable to dig into the bacterial composition at class or genus level.
Response 9: Done. We elaborated and discussed the results on the basis of the bacterial community composition at phylum, class, family, order and genus levels. We included Supplementary Figure S2, S3, S4 and S5 showing the relative abundances of classes, orders, families and genera, respectively.
Point 10: Discussion - Generally, this is good discussion. I think the link between the structure of soil bacterial communities and soil fertility might be discussed more deeply.
Response 10: We are grateful to the Reviewer for this comment. On the basis of the results presented in the new paragraph (3.4 Linkage between soil microbiota and soil parameters), the discussion section was modified. The link between the structure of soil bacterial communities and soil fertility was discussed more deeply (see Lines 530-554).
Reviewer 2 Report
Comments to the Author
The paper compared soil properties and the bacterial community structure under 100% A. mangium, 50% A. mangium+ 50% Eucalyptus, and 100% Eucalyptus plantations. Introducing nitrogen-fixing trees has benefits for enhancing soil C sequestration, improving N stock, stimulating microbial activity and P availability, and improving plant productivity. The topic is interesting and appropriate for Sustainability. Overall, the manuscript is well-written. However, revisions are needed for below aspects:
1. The author used “microbial community” and “microbiota composition” throughout the text. Did the author get any archaeal sequences from the 16S sequencing? If so, include the results to the manuscript. If not, the author may use “bacterial community” instead of “microbial community” in the title and text. For the same reason, be careful to draw any conclusions like Line 24 “Actinobacteria, Proteobacteria, Firmicutes, and Acidobacteria were the dominant phyla.”
2. Major contributions/new findings: In lines 19-21 & 80, the author stated that this research topic “has never been evaluated in the Congolese coastal plains”, thus it is of significance to conduct the research in this area. Based on this study, did the author get any new findings compared to previous publications on the same topic? How does this work expand what we have known from previous publications? The author may need to add literature on the same topic in the introduction and highlight the major contributions of this work in the discussion and conclusion.
3. About phosphorus (P) data: In sections 2.2 & 3.1, it is essential to add phosphorus (P) analyses in the title and text. One of the major findings of this study is “Only P showed a positive correlation with bacterial community structure” (Abstract & Fig 4), thus I would suggest the author highlight P results in tables, figure, and text.
Also, the author can dig deeper about P to strengthen the manuscript:
The author can do a correlation analysis between P contents in the 36 composite samples and the relative abundance (%) of individual bacterial groups to see which specific groups are sensitive to P changes. Add the results to discussion.
Another thing I noticed is that P content is highest in 100E when compared to 100A and 50A50E in R1YI and R2Y2 rotations (Table 1). Since the author is attempting to present the benefit of introducing A. mangium to Eucalyptus plantations, a good explanation is needed for this data.
4. Statistical analysis for relative abundance in result 3.3 is needed.
5. The author presented that 50A50E had the highest C and N contents compared to 100A and 100E in Table 1. However, 50A50E did not have the greatest microbial richness and diversity indices based on Table 3. The author needs to explain why a soil with “better soil fertility” does not support a more diverse bacterial community. This is important, since the author included “soil fertility’ in the manuscript title.
7. Logic and structure of the results: Describe the overall bacterial community structure first (a portion of section 3.2, and sections 3.4 and 3.5), then take a closer look at each individual group (a portion of section 3.2, and section 3.3). Last, you can present any correlations or links between microbial results and soil properties.
Material and method:
1. Lines 104-105 “Soils contain less than 1.5 % of iron oxides content [29] and their pH values are low as well as C and N content [5].” Avoid vague description like “low”. Present PH, C, and N values.
2. Line 159: gaps are missing.
3. Line 170-171: Delete “For more details see the Illumina MiSeq 170 protocol https://web.uri.edu/gsc/files/16s-metagenomic-library-prep-guide-15044223-b.pdf.”
Results:
1. Table 1 errors: double check the significance sign “a” and “b” for C% and N% in 100A and 50A50E.
2. Table 2 errors: double check the significance sign “a” and “b” for N% in 100A and 50A50E.
3. Figure 1 y-axis: since the author already has “percentage (%)” as the y-axis title, it is no necessary to use % for the data. For example, use 90 instead of 90%. The percentage is a general description, the author can use “relative abundance (%)” to keep consistent with literature.
4. Table 3 and Figure 2 are redundant. Keep one of them.
5. Figure 3: Any specific reason to keep both weighted and unweighted UniFrac metrics?
Author Response
Dear Reviewer,
we are very grateful for taking the time to provide your valuable comments and suggestions. The comments encouraged us to carefully revise and refine our manuscript and helped us to improve the quality of the article. Below we describe our responses point-by-point to your comments.
Main comment
The paper compared soil properties and the bacterial community structure under 100% A. mangium, 50% A. mangium+ 50% Eucalyptus, and 100% Eucalyptus plantations. Introducing nitrogen-fixing trees has benefits for enhancing soil C sequestration, improving N stock, stimulating microbial activity and P availability, and improving plant productivity. The topic is interesting and appropriate for Sustainability. Overall, the manuscript is well-written. However, revisions are needed for below aspects:
Point 1: The author used “microbial community” and “microbiota composition” throughout the text.
Response 1: ‘Microbial community’ has been changed into ‘bacterial community’ throughout the text.
Point 2: Did the author get any archaeal sequences from the 16S sequencing? If so, include the results to the manuscript. If not, the author may use “bacterial community” instead of “microbial community” in the title and text.
Response 2: Yes, but a very insignificant number of archaeal sequences were obtained as we did not use specific primers for this purpose. For this reason, we decided to remove the archaeal sequences to avoid bias in our analyses.
Point 3: For the same reason, be careful to draw any conclusions like Line 24 “Actinobacteria, Proteobacteria, Firmicutes, and Acidobacteria were the dominant phyla.”
Response 3: The sentence has been changed into “At the phylum level, the soil bacterial community was dominated by Actinobacteria, followed by Proteobacteria, Firmicutes, and Acidobacteria”
Point 4: Major contributions/new findings: In lines 19-21 & 80, the author stated that this research topic “has never been evaluated in the Congolese coastal plains”, thus it is of significance to conduct the research in this area. Based on this study, did the author get any new findings compared to previous publications on the same topic? How does this work expand what we have known from previous publications? The author may need to add literature on the same topic in the introduction and highlight the major contributions of this work in the discussion and conclusion.
Response 4: We thank the Reviewer for this comment. Accordingly, we modified the Introduction (Lines 82-94). Also, these recommendations were highlighted in the Discussion and Conclusion sections.
Point 5: About phosphorus (P) data: In sections 2.2 & 3.1, it is essential to add phosphorus (P) analyses in the title and text. One of the major findings of this study is “Only P showed a positive correlation with bacterial community structure” (Abstract & Fig 4), thus I would suggest the author highlight P results in tables, figure, and text.
Response 5: Phosphorus has been added in sections 2.2 and 3.1 and also in Tables 1 and 2 and figure captions as requested. Furthermore, due to the additional Spearman ranking coefficient test, a more in-depth analysis of correlation between soil parameters and bacterial community structure was performed (see 3.4 and Discussion section, lines 531-555).
Point 6: Also, the author can dig deeper about P to strengthen the manuscript: The author can do a correlation analysis between P contents in the 36 composite samples and the relative abundance (%) of individual bacterial groups to see which specific groups are sensitive to P changes. Add the results to discussion.
Response 6: This has been already argued in the current discussion section. This may explain the correlation between bacterial structure and available P that was observed in this study. Soil available P decreased in stands containing Acacia relative to Eucalyptus [5],[11] because of its requirement by NFTs to sustain symbiotic root nodules and atmospheric N2 fixation processes [9],[10], even though its status improved in all planted stands compared to savannas [6]. This also is shown by the high extractable P in the forest floor of the Acacia stands [13] resulting from important inputs of P in organic residues [11], and P amounts in Acacia litterfall [11] relative to Eucalyptus, probably due to its ability to retranslocate P [10].
In addition, broaden analyses have been made. Spearman test revealed the correlation between P and bacterial community from order through family to genus. This have been added and discussed. In conclusion, sentences on P have been added throughout the manuscript.
Point 7: Another thing I noticed is that P content is highest in 100E when compared to 100A and 50A50E in R1YI and R2Y2 rotations (Table 1). Since the author is attempting to present the benefit of introducing A. mangium to Eucalyptus plantations, a good explanation is needed for this data.
Response 7: We thank the reviewer for the remark. Soil available P concentration was higher beneath 100E when compared to 100A and 50A50E in R1Y7 and R2Y2 rotations (Table 1) because of P requirement by NFTs to sustain symbiotic root nodules and atmospheric N2 fixation processes. Although P is lower in NFTs stands, it is still higher than in the natural savanna. Therefore, introducing Acacia will not threaten the ecosystems. This is explained in the Discussion section as follow (lines 567-574) “In previous studies a decrease in soil available P has been reported in stands containing Acacia relative to Eucalyptus established in the Congolese coastal plains [5],[11] because of well-known requirement of NFTs to sustain symbiotic root nodules and atmospheric N2 fixation processes [9],[10],[68]. However, its status improved in all planted stands with Eucalyptus and/or Acacia compared to savannas [6]. Its significance is also shown through the great amount of extractable P in the forest floor of the Acacia stands [13]resulting from important inputs of P in organic residues and litterfall [11] relative to Eucalyptus, probably due to Acaciaability to retranslocate P [10]”.
Point 8: Statistical analysis for relative abundance in result 3.3 is needed.
Response 8: We performed the ANOVA analysis and modified the entire paragraph by adding the P values (see the revised 3.2).
Point 9: The author presented that 50A50E had the highest C and N contents compared to 100A and 100E in Table 1. However, 50A50E did not have the greatest microbial richness and diversity indices based on Table 3. The author needs to explain why a soil with “better soil fertility” does not support a more diverse bacterial community. This is important, since the author included “soil fertility’ in the manuscript title.
Response 9: We improved the Discussion section (see lines 515-528): Despite the highest N content and the lowest C/N ratio, the lowest microbial richness and diversity indices were found in mixed-species plantations nearby Acacia even though there were no significant differences among stands. Li et al. [28] reported a significant decline in bacterial species richness and diversity and a substantial shift of bacterial community composition after N addition in a subtropical deciduous oak mixed forest in China. We suggest that the loss of one or more species does not dramatically affect the functioning of the ecosystem, probably due to the high functional redundancy of soil microorganisms [73]. As suggested by Dukunde et al. [31], although soil characteristics have been frequently reported as strong drivers of microbial diversity [74], tree species have been shown to exhibit a stronger impact on community structure than the soil environment. Due to the long-term of Acacia and Eucalyptus rotation, repeated soil sampling over forest development will permit a more-in depth investigation of changes in soil parameters and microbial diversity in the Congolese coastal plains.
Point 10: Logic and structure of the results: Describe the overall bacterial community structure first (a portion of section 3.2, and sections 3.4 and 3.5), then take a closer look at each individual group (a portion of section 3.2, and section 3.3). Last, you can present any correlations or links between microbial results and soil properties.
Response 10: We thank the Reviewer for this comment. Accordingly, we changed the logic and structure of the soil microbiota results and presented the results in the following paragraphs:
3.2. Sequencing data and overall composition of bacterial community along the field sites
3.3. Bacterial Alpha and Beta Diversity
3.4 Relationship between soil microbiota and soil characteristics
Point 11: Material and method - Lines 104-105 “Soils contain less than 1.5 % of iron oxides content [29] and their pH values are low as well as C and N content [5].” Avoid vague description like “low”. Present PH, C, and N values.
Response 11: Values have been added.
Point 12: Line 159: gaps are missing.
Response 12: Done.
Point 13: Line 170-171: Delete “For more details see the Illumina MiSeq 170 protocol https://web.uri.edu/gsc/files/16s-metagenomic-library-prep-guide-15044223-b.pdf.”
Response 13: Done.
Point 14: Results - Table 1 errors: double check the significance sign “a” and “b” for C% and N% in 100A and 50A50E.
Response 14: Done.
Point 15: Table 2 errors: double check the significance sign “a” and “b” for N% in 100A and 50A50E.
Response 15: Done.
Point 16: Figure 1 y-axis: since the author already has “percentage (%)” as the y-axis title, it is no necessary to use % for the data. For example, use 90 instead of 90%. The percentage is a general description, the author can use “relative abundance (%)” to keep consistent with literature.
Response 16: We modified the Figure 1.
Point 17: Table 3 and Figure 2 are redundant. Keep one of them.
Response 17: We removed Table 3.
Point 18: Figure 3: Any specific reason to keep both weighted and unweighted UniFrac metrics?
Response 18: As you know, weighted UniFrac is useful for examining differences in community structure, while unweighted UniFrac is sensitive to differences in low-abundance groups. So we agreed to show both the UniFrac metrics together because there are cases where both, only one, or neither may yield significant differences between treatments.
