Root Traits and Soil Bacterial Composition Explain the Rhizosphere Effects along a Chronosequence of Rubber Plantations
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript is well-redacted and may interest researchers, but the following key considerations must be addressed in the manuscript for publication:
1. In the descriptions of the results should be described in more detail, especially the biological explanation.
2. The figures are very small and cannot be seen in the manuscript. It is suggested that they be placed in a larger size and adjusted to the margin of the text.
3. Concerning statistical analyses, in the case of the letters (figure 1) placed on the bars, which post hoc test was used should be mentioned, and if the rhizosphere soils are being compared with bulk soils or between planting times.
4. Regarding format, care must be taken that the figure footer is below the figure, without going to the next page.
5. It is mentioned in lines 346-348: "However, some studies have suggested that the root morphological traits (such as diameter and SRL) are not clearly related to SOM stabilisation mechanisms, and their relationship remains to be investigated [56]". However, which studies are not mentioned, only one reference is cited, so it is suggested to add more bibliographic references.
6. In lines 379-381, it is mentioned, "In terms of ER on SOM decomposition, nitrogen fertilisers should be applied correctly in the process of managing rubber plantations." Add more specific information on how nitrogen fertilisers should be applied in the management process of rubber plantations.
7. It is suggested to write the calculated value of CO2-C and mention how it was calculated for SOM decomposition.
8. In the discussion, it is suggested to compare results with other authors.
Additional commentaries:
Line 109: Write the months of the seasons with lowercase letters
Line 111, 135, 136: Eliminate space between 28.5 and °C
Author Response
Response to Reviewer #1 Comments
General Comment: The manuscript is well-redacted and may interest researchers, but the following key considerations must be addressed in the manuscript for publication.
Response to general comment: We greatly appreciate the reviewer’s positive and encouraging comments. Necessary revisions have been made with respect to the comments. Below we listed the responses regarding each of the comments.
Comment 1: In the descriptions of the results should be described in more detail, especially the biological explanation.
Response: Detailed description of the results has been added, please see the Results section (Line 230-233, 256-258, 270-272, 278-284,).
Comment 2: The figures are very small and cannot be seen in the manuscript. It is suggested that they be placed in a larger size and adjusted to the margin of the text.
Response: We have enlarged the figures to see them clearly.
Comment 3: Concerning statistical analyses, in the case of the letters (figure 1) placed on the bars, which post hoc test was used should be mentioned, and if the rhizosphere soils are being compared with bulk soils or between planting times.
Response: Yes, we used Tukey's honest significant difference test as the post hoc test, and this information has been added in the Data analysis section (Line 203-205). We reanalyzed the data to know the differences of soil properties, microbial diversity, and SOM decomposition rates in bulk soil or rhizosphere among three age classes of rubber plantation using ANOVA with Tukey's honest significant difference test. t-test was used to compare the differences between bulk soil and rhizosphere. Then, the results of analysis were presented in the Results section and the Figure 1-3.
Comment 4: Regarding format, care must be taken that the figure footer is below the figure, without going to the next page.
Response: We have adjusted the format to ensure the figure footer is below the figure.
Comment 5: It is mentioned in lines 346-348: "However, some studies have suggested that the root morphological traits (such as diameter and SRL) are not clearly related to SOM stabilisation mechanisms, and their relationship remains to be investigated [56]". However, which studies are not mentioned, only one reference is cited, so it is suggested to add more bibliographic references.
Response: As suggested, two other references have been added. Please see Line 384.
Comment 6: In lines 379-381, it is mentioned, "In terms of ER on SOM decomposition, nitrogen fertilisers should be applied correctly in the process of managing rubber plantations." Add more specific information on how nitrogen fertilisers should be applied in the management process of rubber plantations.
Response: As suggested, more specific information about methods of nitrogen fertilisers application in the management process of rubber plantations have been added (Line 418-421).
Comment 7: It is suggested to write the calculated value of CO2-C and mention how it was calculated for SOM decomposition.
Response: We listed the formula for calculating value of CO2-C, and provided detailed explanation on how to calculate SOM decomposition rate based on this CO2-C (Line 155-158).
Comment 8: In the discussion, it is suggested to compare results with other authors.
Response: As suggested, we have compared our results to others at proper places (Line 331-332, 367-369, 376-378).
Comment 9: Line 109: Write the months of the seasons with lowercase letters
Response: We believe that the first letter of the months should be capitalized.
Comment 10: Line 111, 135, 136: Eliminate space between 28.5 and °C
Response: Done (Line 110, 138, 151).
Reviewer 2 Report
Comments and Suggestions for AuthorsMajor issues:
I’m concerned about the study design using three plantations, one of each age. As described it seems that you have n=1 for each plantation age with pseudo replication of plots within each, which would call into question the statistical analysis comparing differences by age.
It is also not clear that the soil properties at these plantations were identical prior to plantation establishment. Are supposed age effects actually due to age, or is it possible that initial pH/STN/etc at these sites differed? The geographic distribution of the sites (how close are they to each other?) could help, but even then, if there is not replication of plantations by age, how is it possible to identify age as the determining factor as opposed to some other environmental factor?
If I’m understanding the design correctly, there are major issues with this approach to analysis, but there may be a way to reframe the data without focusing on age that may be valid and valuable. This would likely require more than just a revision.
Minor issues:
Lines 69-71: Sentence is unclear
Lines 85-87: Which chemical traits?
Lines 140-141: Reference for STP and pH methods?
Line 142: Reference for this SOM method?
Line 144: How was water-holding capacity of soils determined?
Line 173: SRL has not yet been defined
Lines 222-223: These results need to be presented with more nuance, as there are not significant differences between SOC by age in bulk soil, nor STN in rhizosphere. Perhaps stating “rhizosphere or bulk soils” is meant to convey that it wasn’t both in all cases, but this should be clarified. It should also be clarified what you mean by “increasing age,” as these effects seem to be from young to middle-aged, but not increasing when progressing to old. Similar age pattern distinctions need to be examined throughout – increased with age vs increased from young to mid/old are somewhat different in their implications.
Line 263: ranged
Line 305-306: Incomplete sentence, unclear
Lines 382-388: This paragraph feels somewhat out of place as the final point in your discussion, and it is unclear why you conclude that the isotope method should be used even though you didn’t use it. How much difference is there in the accuracy of the methods?
Comments on the Quality of English LanguageIn general, some English language/grammatical editing is needed, though it is not highly distracting from the work.
Author Response
Response to Reviewer #2 Comments
General Comment: I’m concerned about the study design using three plantations, one of each age. As described it seems that you have n=1 for each plantation age with pseudo replication of plots within each, which would call into question the statistical analysis comparing differences by age.
It is also not clear that the soil properties at these plantations were identical prior to plantation establishment. Are supposed age effects actually due to age, or is it possible that initial pH/STN/etc at these sites differed? The geographic distribution of the sites (how close are they to each other?) could help, but even then, if there is not replication of plantations by age, how is it possible to identify age as the determining factor as opposed to some other environmental factor?
If I’m understanding the design correctly, there are major issues with this approach to analysis, but there may be a way to reframe the data without focusing on age that may be valid and valuable. This would likely require more than just a revision.
Response to General Comment: Sorry for the confusion caused by our vague description of the experimental design. Detailed description of sample plot selection has been added (Line 117-119). In fact, the five plots in each rubber plantation age were not adjacent to each other. In our study site, the rubber plantation was established after the logging of the secondary forest, and the area is about 10,000 hectares. The distances between the selected 15 plots (3 ages × 5 replicates) range from 100 m to 2,000 m apart from each other. The soil physicochemical properties before planting had a legacy effect on the soil microbiome of the rubber plantation. However, due to the time limitations of this study, we were unable to obtain data on the soil properties of the secondary forest several decades ago. Previous studies have also demonstrated that the impact of different tree planting durations on soil microbiota varies significantly (Han et al., 2007; Cao et al., 2010; Kang et al, 2018; Xu et al., 2020; Jin et al., 2022; Zhang et al., 2023). Therefore, in this study, we hypothesized that plantation age was the main factor influencing soil microbial composition. In addition, we also examined the correlation between microbial community structure and soil characteristics.
References:
Cao, Y., Fu, S., Zou, X., Cao, H., Shao, Y., & Zhou, L. (2010). Soil microbial community composition under Eucalyptus plantations of different age in subtropical China. European Journal of Soil Biology, 46(2), 128-135.
Han, W., Kemmitt, S. J., & Brookes, P. C. (2007). Soil microbial biomass and activity in Chinese tea gardens of varying stand age and productivity. Soil Biology and Biochemistry, 39(7), 1468-1478.
Jin, Q., Zhang, Y., Ma, Y., Sun, H., Guan, Y., Liu, Z., ... & Wang, Q. (2022). The composition and function of the soil microbial community and its driving factors before and after cultivation of Panax ginseng in farmland of different ages. Ecological Indicators, 145, 109748.
Kang, H., Gao, H., Yu, W., Yi, Y., Wang, Y., & Ning, M. (2018). Changes in soil microbial community structure and function after afforestation depend on species and age: case study in a subtropical alluvial island. Science of the Total Environment, 625, 1423-1432.
Xu, J., Liu, B., Qu, Z. L., Ma, Y., & Sun, H. (2020). Age and species of Eucalyptus plantations affect soil microbial biomass and enzymatic activities. Microorganisms, 8(6), 811.
Zhang, G., Yang, W., Hu, J., Liu, J., Ding, W., & Huang, J. (2023). Effects of tea planting age on soil microbial biomass C: N: P stoichiometry and microbial quotient. Plant, Soil and Environment, 69(5), 221-229.
Comment 1: Lines 69-71: Sentence is unclear
Response: We rewrote the sentence to make it clear (Line 69-70).
Comment 2: Lines 85-87: Which chemical traits?
Response: The detailed root chemical traits, N content and C:N ratio, have been added (Line 85).
Comment 3: Lines 140-141: Reference for STP and pH methods?
Response: The references have been added (Line 144).
Comment 4: Line 142: Reference for this SOM method?
Response: The reference has been added (Line 146).
Comment 5: Line 144: How was water-holding capacity of soils determined?
Response: The method we used to determine the water-holding capacity of soil is as follows: soil samples were collected using an auger (100 m3). To prevent soil particles from scattering, a piece of filter paper was placed underneath the auger. Then we put augers in a dish and poured water into the dish until it covered the filter paper. After the soil reach saturation point, the soil was removed from the auger and weighted. After oven drying at 105 ℃ for 24 h, the soil weighted again. Finally, the water-holding capacity of soil was calculated by these weight value.
Comment 6: Line 173: SRL has not yet been defined
Response: SRL has defined in the Introduction section (Line 64-65).
Comment 7: Lines 222-223: These results need to be presented with more nuance, as there are not significant differences between SOC by age in bulk soil, nor STN in rhizosphere. Perhaps stating “rhizosphere or bulk soils” is meant to convey that it wasn’t both in all cases, but this should be clarified. It should also be clarified what you mean by “increasing age,” as these effects seem to be from young to middle-aged, but not increasing when progressing to old. Similar age pattern distinctions need to be examined throughout – increased with age vs increased from young to mid/old are somewhat different in their implications.
Response: According to the comments, we further analyzed and described the results to make them clear (Line 230-233). As suggested, more precise expression was used to describe the differences among the three ages classes throughout the manuscript.
Comment 8: Line 263: ranged
Response: Done (Line 286).
Comment 9: Line 305-306: Incomplete sentence, unclear
Response: The sentence was replaced by “These results Consistent were consistent with previous studies study that which indicated that the rate of soil C decomposition is positively facilitated by REs” (Line 331).
Comment 10: Lines 382-388: This paragraph feels somewhat out of place as the final point in your discussion, and it is unclear why you conclude that the isotope method should be used even though you didn’t use it. How much difference is there in the accuracy of the methods?
Response: In fact, we listed our study limitation in this paragraph. Based on the C isotope labelling method, we can distinguish native SOM- and root-derived CO2 in intact plant-soil systems, in other word, we can know the rate of SOM decomposition caused by the root existed, and this is defined as the rhizosphere priming effect. However, we just studied the rhizosphere effect on SOM decomposition using the incubation of rhizosphere soil that was collected by the adhering soil method. Therefore, we can further investigate the rhizosphere processes using isotope labeling in situ in future study. Both methods are widely used.
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors of the Manuscript review - Root characteristics and soil bacterial composition explain rhizosphere effects in a chronosequence of rubber plantations.
First of all - I would like to congratulate you on the article, it is clear that it was a lot of work. However, I think it's necessary that with all the effort you've put into it for the benefit of science, it should have been better discussed, so as not to lose interesting data, although some of it is quite logical in relation to the life cycle of tree species.
Secondly - I need to know how you defined the best strategy to determine the bacterial communities of the rhizospheric soil and the soil, after all, there are new concepts and with greater precision when compared to the one adopted in the present work.
I made some comments and suggestions.
Thank you again for correcting this work.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer #3 Comments
General Comment: First of all - I would like to congratulate you on the article, it is clear that it was a lot of work. However, I think it's necessary that with all the effort you've put into it for the benefit of science, it should have been better discussed, so as not to lose interesting data, although some of it is quite logical in relation to the life cycle of tree species.
Secondly - I need to know how you defined the best strategy to determine the bacterial communities of the rhizospheric soil and the soil, after all, there are new concepts and with greater precision when compared to the one adopted in the present work.
Response to General Comment: Firstly, we are grateful to the reviewer for his positive and encouraging comments. Further discussion of the results has been added according to the comments.
Secondly, our study chose OTU approach to determine the bacterial communities of the rhizospheric soil and the bulk soil. The ASV approach has several mature bioinformatic applications for analysis with their own advantages. The evidence is mounting that the future of targeted sequencing lies with the ASV approach. In fact, OTU and ASV have been developed to minimize the effects of targeted sequencing error, each with their own pros, cons, and idiosyncrasies. Despite a significant risk of reference bias and loss of novel sequences, OTUs are computationally fast and easy for both generation and comparison between samples (refer to https://zymoresearch.eu/blogs/blog/microbiome-informatics-otu-vs-asv). On the one hand, it would be a tremendously extensive work to reanalyze using ASV approach. On the other hand, in this study, we primarily focused on the differences in community structure between samples rather than identifying specific species. Therefore, OTU approach was sufficient to support the scientific hypothesis and the results are reliable.
Comment 1: Line 11 - I suggest change - "Rubber tree plantations (Hevea brasiliensis) are expanding into the tropical regions of southwest China to ensure production to meet the growing demand for latex. The effects of long-term plantations on soil carbon processes are still unclear. Also, the effects of the plant's rhizosphere on the decomposition of soil organic matter (SOM) play a crucial role in predicting soil carbon dynamics. The rhizosphere and soils corresponding to a chronosequence of ages (4, 15 and 30 years) of rubber plantations were collected and incubated to determine the effect of the rhizosphere (RE) on SOM decomposition."
Make this change because the first sentence will hook the reader. I also suggest breaking it up to make it more striking, thus creating an impactful second sentence.
Response: Thanks for your suggestion, we have revised this part according to your suggestion (Line 11-16).
Comment 2: Line 19 - Explain further the sentence "REs in SOM decomposition varied significantly in different age classes of rubber plantations. This limited the accumulation of organic carbon in the soil in older plantations.", or rewrite it.
Response: The sentence has been replaced by “The REs on SOM decomposition varied significantly in different age classes of rubber plantation, and the higher REs on SOM decomposition in older plantations might limit the accumulation of organic carbon in the soil.” (Line 20-21).
Comment 3: Line 33 - Has agricultural expansion moved to the tropics only because of population growth? Or because of geopolitical and structural issues in the developed countries, almost all of which are in the more temperate climates of the Northern Hemisphere? With less capacity for large-scale production? Or do the subtropical regions where these countries are located not have the soil and climate characteristics that make safe production possible?
In other words, this sentence causes a great deal of disagreement, I suggest changing it.
Response: To avoid disagreement, we merged the first two sentences as show in Line 34-36.
Comment 4: Line 48 - Has agricultural expansion moved to the tropics only because of population growth? Or because of geopolitical and structural issues in the developed countries, almost all of which are in the more temperate climates of the Northern Hemisphere? With less capacity for large-scale production? Or do the subtropical regions where these countries are located not have the soil and climate characteristics that make safe production possible?
In other words, this sentence causes a great deal of disagreement, I suggest changing it.
Response: The sentence in Line 48 is unconcerned with agricultural expansion. We think you would like to make some other comments for Line 48.
Comment 5: Line 85 - Much is known about the communities or microbial ecology of agricultural and fruit species, but little is known about forest species. However, the influence of communities and even certain types of plant-microorganism associations have already been seen in some studies involving different planting ages. Example: The process of biological nitrogen fixation in mixed and monoculture planting systems has been well evaluated, and this may provide evidence that could corroborate your work.
I suggest checking out the review article by Monteiro et al. (2019) on the influence of BNF on N and C cycles, consequently in production systems with Acacia mearnsii.
Response: Indeed, we overlooked the importance of nitrogen-fixing bacterial communities in the discussion. In the revised version, we have added this literature in the Discussion section (Line 410).
Monteiro, P. H. R., Kaschuk, G., Winagraski, E., Auer, C. G., & Higa, A. R. (2019). Rhizobial inoculation in black wattle plantation (Acacia mearnsii De Wild.) in production systems of southern Brazil. Brazilian Journal of Microbiology, 50, 989−998.
Comment 6: Line 106 - Is there any information about the species of undergrowth regarding root architecture, microbial groups that work with these species or even the influence of interaction with the plants or the soil where the Hevea brasiliense plantations are located, that could explain why they are in this text? If you would like to participate in the discussion, please withdraw.
Response: Due to the management and operation of the plantations, there is few undergrowth in the rubber plantation. Although the undergrowth had some effects on soil environments, in this study, we focused on micro-scale (rhizosphere) changes. The effects of undergrowth can be disregarded. Certainly, additional information has been included within the Discussion section (Line 433-434).
Comment 7: Are all the planting areas under the same type of soil?
Response: Yes, the planting areas we studied possess the same type of soil. After the logging of the secondary forest, the rubber plantation was established in the study site, with a total area of 10,000 hectares. The 15 selected sampling plots were characterized by a flat terrain and consisted of laterite (Oxisols) soil.
Comment 8: Line 120 - Is this figure correct? Have the plants reduced in size from 15 to 30 years? Latex extraction can reduce plant growth, but to such an extent?
Or is this a reflection of the area planted, is all the soil homogeneous as described above? I think the influence of this soil is greater, do you have data on soil classification, or do you have any idea what might have influenced this reduction between the ages/sample plots?
Response: This figure is correct. Although 15-year-old rubber plantation has a higher plant height value than 30-year-old rubber plantation, there was no significant difference based on one-way ANOVA analysis. The selected sampling sites were consisted of laterite (Oxisols) soil. The different height value between 15-year-old and 35-year-old rubber plantation may be attributed to several environmental factors, such as wind, soil nutrition, and cutting practices.
Comment 9: Line 130 - Processing time between collection and testing? Longer or shorter than 72 hours?
Response: Root samples and soil samples used to incubation were tested within 48 hours, and soil samples used to microbiological sequencing were stored at −80 ℃ until were determined (might longer than 72 hours, but shorter than one month). The freezing conditions at −80 ℃ can preserve the activity and stability of microbes for an extended period. The detailed information has been added (Line 138-139).
Comment 10: Line 157 - Primers for the ITS region? But then were the fungal community analyzed? And why isn't it in the paper?
Response: It is true that we did not analyze fungal community. This is our fault. We deleted “and ITS2 genes” (Line 163-164).
Comment 11: Line 162 - This separation is no longer used, there are new algorithms such as DADA2 and even the concept of ASV - Amplicon sequence variant - is any of the inferred unique DNA sequences recovered from a high-throughput analysis of marker genes.
These new algorithms separate species by 1 bp, as opposed to the 97% in this work. After all, this is somewhat arbitrary, there were species that showed greater similarity than 97%, but were different species, just as there were identical species with similarities of less than 97%.
This is the part that most compromises the work, as it changes the structure of the microbial community too much. The way it is presented in the paper is not wrong, but it is no longer applied in studies of bacterial communities. In other words, it loses its innovative character to use this tool.
Response: We strongly agree with the opinions of the reviewer. The ASV approach has several mature bioinformatic applications for analysis with their own advantages. The evidence is mounting that the future of targeted sequencing lies with the ASV approach. In fact, OTU and ASV have been developed to minimize the effects of targeted sequencing error, each with their own pros, cons, and idiosyncrasies. Despite a significant risk of reference bias and loss of novel sequences, OTUs are computationally fast and easy for both generation and comparison between samples (refer to https://zymoresearch.eu/blogs/blog/microbiome-informatics-otu-vs-asv). On the one hand, it would be a tremendously extensive work to reanalyze using ASV approach. On the other hand, in this study, we primarily focused on the differences in community structure between samples rather than identifying specific species. Therefore, OTU approach was sufficient to support the scientific hypothesis and the results are reliable.
Comment 12: Line 303 – Why?
Response: The RE on SOM decomposition was calculated by dividing the rate of SOM decomposition in rhizosphere by the rate of SOM decomposition in bulk soil. In addition, the rates of SOM decomposition in bulk soil had no significantly different among three age class of rubber plantations. So, the higher RE on SOM decomposition in 15-year- and 30-year-old rubber plantation mainly attributed to the higher rhizosphere SOM decomposition rate.
Comment 13: Line 307 - Explain it better! Why can roots contribute more to COS? What differs from the woody material in the upper part? That's a good discussion.
Response: Detailed explanation has been added in this part, please see Line 336-344. Root C inputs from plants to soil are comprised of root turnover, root-associated fungal turnover, and rhizodeposition (Keller et al., 2021). More active components in root-derived C can increase SOM turnover via priming effects, and can promote slow-cycling C formation given their effects on microbial efficiency (Sokol and Bradford, 2019). It has been proven that root biomass and rhizodeposition input have higher SOC fractions formation efficiency than aboveground inputs (Villarino et al., 2021).
References:
Keller A B, Brzostek E R, Craig M E, et al. (2021). Root-derived inputs are major contributors to soil carbon in temperate forests, but vary by mycorrhizal type. Ecology letters, 24(4): 626-635.
Sokol N W, & Bradford M A. (2019). Microbial formation of stable soil carbon is more efficient from belowground than aboveground input. Nature Geoscience, 12(1): 46-53.
Villarino S H, Pinto P, Jackson R B, et al. (2021) Plant rhizodeposition: A key factor for soil organic matter formation in stable fractions. Science Advances, 7(16): eabd3176.
Comment 14: Line 309 – Why?
Response: Root biomass production was not measured in three age classes of the rubber plantation in the study, so we think that this sentence is inappropriately. We rewrote the sentence, and replaced by “In the present study, higher root exudation rate in the MR and OR plantations (Table 1) might conducive to the higher RE on SOM decomposition by increasing the activity of microbes, thereby resulting in a decreased rhizosphere SOC content”.
Comment 15: Line 312 - Could it be that the accumulation of carbon in the root exudates has increased because the plant is using less of the carbon that was used for growth and metabolic activity for the development of the plant as a whole?
Response: We agree with your opinion. This inference was added in this part (Line 345-348).
Comment 16: Line 361 - Firmicutes are not described in your essay, I know it's in the supplementary material, but explain their behavior, as well as the Phylum Gemmatimonadetes.
Response: The reason we did not describe Firmicutes and Gemmatimonadetes is that the relative abundances of these two phyla were lower than other phyla and they had no significantly difference among the three age classes of rubber plantation. As suggested, the describe of Firmicutes and Gemmatimonadetes was added in the Result part (Line 270-272, 278-280).
Reviewer 4 Report
Comments and Suggestions for AuthorsQ.1: In line 104 – 106 authors are mentioned about different sites but didn’t give any information about site parameters and vegetation characteristics for rubber tree plantations.
Q.2: Throughout the manuscript diversity of the soil bacterial community in Rubber Tree Plantations as well as their classification also not explained.
Q.3: Conclusion part also need to more explanatory.
Q.4: How the age of chronosequence (4, 15, and 30 years) of rubber plants was confirmed
Q.5. What could be probable reason for the observation reported in line no. 226-227 “SRL, RTD, RN content, and root exudation exhibited opposite trends”
Q.6: Define the criteria of soil sample selection
Q.7. Author should highlight the limitations of the study.
The whole manuscript should be checked carefully for spelling and stylistic errors. Some long sentences, misspellings, etc., are still noticeable throughout the text. Please carefully proofread the manuscript to ensure that it is free from any errors.
Comments on the Quality of English LanguageAcceptable
Author Response
Response to Reviewer #4 Comments
General Comment: The present research seems scientifically important and interesting. However, it is necessary to make some changes or corrections to the initial manuscript.
Response to general comment: We greatly appreciate the reviewer’s positive and encouraging comments. Necessary revisions have been made with respect to the comments of the reviewer.
Comment 1: In line 104 – 106 authors are mentioned about different sites but didn’t give any information about site parameters and vegetation characteristics for rubber tree plantations.
Response: In fact, we mentioned only one site where we conducted studies. Sorry for the vague expression, and we rewrote this sentence. It has been replaced by “The study was conducted at the experimental farm of Chinese Academy of Tropical Agricultural Sciences (19°28′ N, 109°29′ E) located in Danzhou, Hainan Province, China. This site was established after clearing natural tropical forest.” (Line 103-105).
Comment 2: Throughout the manuscript diversity of the soil bacterial community in Rubber Tree Plantations as well as their classification also not explained.
Response: In this study, we focused on the differences in community structure between samples and their effect on the rhizosphere effects on SOM decomposition. According to our results, only bacterial community composition had effect on the rhizosphere effects on SOM decomposition (Figure 4). Therefore, we just discussed bacterial community composition in the Discussion section. As suggested, in the revision, we have further added some descriptions about the classification of soil bacterial communities in the Discussion section (354-359).
Comment 3: Conclusion part also need to more explanatory.
Response: We have made more explanatory in the Conclusion part (Line 442-444).
Comment 4: How the age of chronosequence (4, 15, and 30 years) of rubber plants was confirmed.
Response: We confirmed the age chronosequence of rubber plants by the following methods. Generally, rubber plantations start tapping rubber in the 7th or 8th year, then we estimate the age of rubber trees by the cut marks on the rubber trunk firstly. Then we consulted the local farmers to confirm it.
Comment 5: What could be probable reason for the observation reported in line no. 226-227 “SRL, RTD, RN content, and root exudation exhibited opposite trends”
Response: The results showed that SRL, RTD, RN content, and root exudation increased with increasing plantation age. As rubber trees mature, the soil nutrients become increasingly depleted, resulting in more extensive and complex root systems. Higher RTD, RTD, and RN content are often associated to the mechanical support and resource acquisition of plants (Greinwald et al., 2021; Yan et al., 2022). Root exudation tends to increase as tree mature, which may attribute that the enhancement of plant-microbe interactions may stimulate rhizosphere secretions (Nguyen et al., 2020). In addition, the strength increases in cutting glue may also stimulate changes in the root systems (Comas et al., 2000).
References:
Comas, L. H., Eissenstat, D. M., Lakso, A. N. (2000). Assessing root death and root system dynamics in a study of grape canopy pruning. The New Phytologist, 147(1), 171-178.
Greinwald, K., Dieckmann, L. A., Schipplick, C., et al. (2021). Vertical root distribution and biomass allocation along proglacial chronosequences in Central Switzerland. Arctic, Antarctic, and Alpine Research, 53(1), 20−34.
Nguyen, T. T., Do, T. T., Harper, R., et al. (2020). Soil health impacts of rubber farming: The implication of conversion of degraded natural forests into monoculture plantations. Agriculture, 10(8), 357.
Yan, H., Freschet, G. T., Wang, H., et al. (2022). Mycorrhizal symbiosis pathway and edaphic fertility frame root economics space among tree species. New phytologist, 234(5), 1639−1653.
Comment 6: Define the criteria of soil sample selection.
Response: According to the reviewer’s suggestion, we have incorporated additional details pertaining to the criteria employed for the selection of soil samples to ensure precise analysis. These encompass random sampling techniques, determination of sampling depth, utilization of appropriate sampling tools, and the implementation of measures to prevent contamination (Line 124-139).
Comment 7: Author should highlight the limitations of the study.
Response: In fact, the last paragraph in Discussion section was described as the limitations of our study. According to the comment, we further summarized the limitations of the study, please see Line 423-434.
Comment 8: The whole manuscript should be checked carefully for spelling and stylistic errors. Some long sentences, misspellings, etc., are still noticeable throughout the text. Please carefully proofread the manuscript to ensure that it is free from any errors.
Response: We have re-checked our manuscript carefully again, some grammar mistakes have been revised, such as Line 37, 216, 255, 291, 345-346, and 356.
Round 2
Reviewer 3 Report
Comments and Suggestions for AuthorsDear authors,
I would like to congratulate your work again because I believe that this microbiological approach to forest species is necessary, after all, microorganisms present in soil are often neglected by the forestry sector. This is why it is so difficult to see the use and appreciation of the ecosystem services of microorganisms present in forests and even the application of biologicals aimed at the productivity and quality of forest species.
Thus, as we begin to understand which microbial groups are present and their functions, we will be able to positively interfere in future forest production, using their bioprospecting.
