Nitrogen Reduction with Bio-Organic Fertilizer Altered Soil Microorganisms, Improved Yield and Quality of Non-Heading Chinese Cabbage (Brassica campestris ssp. chinensis Makino)
Alimamy Fornah
Round 1
Reviewer 1 Report
Dear Authors,
I read your paper with interest and altogether, I think your submission might be suitable for publication in the journal after a major revision. On the other hand, there are several points that need to be addressed before I can suggest acceptance of the paper. These are detailed below. Please do recognize that these issues require restarting your bioinformatics workflow as the bare minimum. Additional errors might become obvious in a revised version.
Major points.
1., Rhizosphere. Based on the methodical details you provided, you did not specifically isolate rhizosphere soil, but bulk soil. This has to be corrected throughout the paper. Rhizosphere is about 1-3mm or <1cm close to roots and is isolated using specific techniques (e.g. 10.1007/s11104-020-04530-3, 10.1371/journal.pone.0262909). Therefore even large portions of soil that can be shaken off roots is not considered rhizosphere.
2., Sample preparation (2.3.1.). Enzymatic activity in 4 C stored soils is usure to remain stable. Did you do any preliminary assay to show that enzymatic stability does not change during your usual storage period? Do you have a reference to support this?
3., Sample preparation (2.3.2.). How did you homogenize your frozen soil samples prior to DNA extracion? Which steps of the work were done aseptically?
4., There is no mention of negative (process) controls for metagenomics assessment. A significant proportion of human-related microorganisms and other reads from ubiquitous indoor organisms can be there, making up a significant proportion of reads. These taxa are not meaningful to your experiment so they have to be discarded. Please see 10.1186/s12866-020-01839-y, 10.1111/jam.14919 or search results for "kitome" for details.
5., Changes. In L149, L176-181 and elsewhere: mentioned changes must be statistically significant, otherwise state that they are non-significant. Table S3 and S9 do not contain many statistically different changes vs controls (based on the post-hoc test compact letter display). Update your results and discussion accordingly.
6., Plant of study. When seeing the word Chinese cabbage in the title, I guess many associate to "napa cabbage" and not the plant you actually studied. Brassicas are an extremely variable. Please add botanical name to the abstract (perhaps title). In addition, make sure taxonomy is correct. The plant resembles Brassica rapa subsp. chinensis ("pak choi") to me, but I might be mixing varieties.
7., You apparently used a QIIME workflow. What is unclassified-k-Fungi? Are those fungal sequences for sure or did the algorithm default to the fungal kingdom when no meaningful results was found (and the same for other kingdoms)? What databases were used (name, version, citation)? A detailed workflow description is warranted. Also discard meaningless OTUs - your protists contain "Brassica" which is likely residual plant DNA.
8., Captions are not detailed enough. What is the difference between Fig 1a and Fig 1b?
9., Compositional data. Correlation study of read counts and other data is not meaningful, as these data negatively correlate with each other as the DNA fragments compete for a limited number of slots. To mathematically cope with the issue, the data have to be pre-treated before these procedures. See 10.3389/fmicb.2017.02224 for an excellent description.
10., False discovery rate. You did lots of statistical tests (by testing raw correlations between soil parameters and taxon abundances - which is very nice). Therefore, the chance of accidently finding an apparently "statistically significant", but actually random phenomenon is high, which has to be dealt with by using statistical correction of p-values. I suggest the sensitive Benjamini Hochberg procedure, which requires all p-values to be gathered. Alternatively, Bonferroni correction can be used which is simpler but will render more phenomena to be insignificant.
11., The tested soil seems to be quite "resistant" to your treatments. Please show compositional barplots in which there are changes (instead of Fig 2b, 3b, 4b). At these levels, not much has happened with the communities, though there are likely moving parts at, say, order level.
12., Was there any taxon abundance correlated with performance of cabbage? This could be the most significant finding (and by far most interesting part) of your experiment, yet, it is missing. Note point 9. - this has to be examined with logratio transformed numbers. Discussion should also be focused on expected changes in crop quality as a result of changes in the microbial community.
13., Your next experiment should contain a no crop control for bulk soils (with all the treatments).
Minor points.
Abstract:
- add nitrogen dose (kg N / ha) in T1.
- detail "fertilizer"
L62-63: This is not meaningful to readers outside of China. Provide approximate ha of crop area or produced tons/year.
L75: "acid soil" is not a texture type
L77: do you have data on P, and S?
L78: There are different versions of "superphosphate" and different potassium sulfates (K2SO4 . x H2O where x varies). Add exact chemical formulas or % element composition. Treatment main data should not only be available in Table S1.
L83: what is etc.? if it an undefined species, use "spp." otherwise add other taxa.
Fig S4 is an empty plot. Why?
L247: you cannot "increase chemical properties" of soils, please revise
Author Response
Cover Letter
Dear Editor and Reviewer,
Thank you very much for comments and constructive suggestions with regard to our manuscript titled “Reduced nitrogen with bio-organic fertilizer altered rhizo-sphere microorganisms improved yield and quality of non-heading Chinese cabbage (ID: agronomy-1743416). Those comments are very helpful for us to improve our manuscript. We have revised the manuscript thoroughly according to the reviewers’ constructive comments and useful suggestions. Please see the revised manuscript marked in red. We appreciate Editors/Reviewers’ warm work earnestly. Here are our point-to-point responses.
1., Rhizosphere. Based on the methodical details you provided, you did not specifically isolate rhizosphere soil, but bulk soil. This has to be corrected throughout the paper. Rhizosphere is about 1-3mm or <1cm close to roots and is isolated using specific techniques (e.g. 10.1007/s11104-020-04530-3, 10.1371/journal.pone.0262909). Therefore even large portions of soil that can be shaken off roots is not considered rhizosphere.
Response：Thanks for the comments. The sampling methods were not described in detail in this manuscript before. We have modified the method of soil sampling in the manuscript following reviewers’ suggestion, specifically, “Soil samples were collected from 5 to 10 cm soil layers around the plant root and passed through a sterilized 2-mm sieve to remove rocks, roots and organic residues. (Line112) ”
2., Sample preparation (2.3.1.). Enzymatic activity in 4 C stored soils is usure to remain stable. Did you do any preliminary assay to show that enzymatic stability does not change during your usual storage period? Do you have a reference to support this?
Response：We measured the enzyme activity according to the method descried by Yao et al (2021) and Wang et al (2020). The soils for enzymatic assay in their experiments were stored at 4℃ too. We measured the enzyme activity within three days.
Yao T, Zhang W, Gulaqa A , et al. Effects of Peanut Shell Biochar on Soil Nutrients, Soil Enzyme Activity, and Rice Yield in Heavily Saline-Sodic Paddy Field[J]. Journal of Soil Science and Plant Nutrition, 2021, 655–664.
Wang J , X Lu, Zhang J , et al. Regulating soil bacterial diversity, community structure and enzyme activity using residues from golden apple snails[J]. Scientific Reports, 2020, 10:16302.
3., Sample preparation (2.3.2.). How did you homogenize your frozen soil samples prior to DNA extracion? Which steps of the work were done aseptically?
Response：Soil samples were collected from 5 to 10 cm soil layers around the plant root with disposable gloves and sterilized bags. Then soil sample was passed through a sterilized 2-mm sieve to remove rocks, roots and organic residues. Subsequently, the powdery sample was collected in a new bag. In the whole process, we wearing a disposable medical mask. The sampling tools and containers containing soil samples were sterilized in advance to avoid interference from foreign substances.
4., There is no mention of negative (process) controls for metagenomics assessment. A significant proportion of human-related microorganisms and other reads from ubiquitous indoor organisms can be there, making up a significant proportion of reads. These taxa are not meaningful to your experiment so they have to be discarded. Please see 10.1186/s12866-020-01839-y, 10.1111/jam.14919 or search results for "kitome" for details.
Response：Thanks for the comments. As exogenous bio-organic fertilizer in this experiment made by animal feces, hair and plant residues, so human-related microorganisms and other reads were detected. We discarded some taxa are not meaningful following you suggestion. As our study focus on the microbial diversity of soil bacteria, fungi and protists, other microorganisms should not have much effect on them. We have been conducting an experiment about the continuous fertilization and the soil metagenomic function analysis to further analyze relevant information. When we do with soil metagenomic we would pay more attention to "kitome".
5., Changes. In L149, L176-181 and elsewhere: mentioned changes must be statistically significant, otherwise state that they are non-significant. Table S3 and S9 do not contain many statistically different changes vs controls (based on the post-hoc test compact letter display). Update your results and discussion accordingly.
Response：Thanks for the comments. We have updated results and discussion according to your comments. The changes were high light in the manuscript.
6., Plant of study. When seeing the word Chinese cabbage in the title, I guess many associate to "napa cabbage" and not the plant you actually studied. Brassicas are an extremely variable. Please add botanical name to the abstract (perhaps title). In addition, make sure taxonomy is correct. The plant resembles Brassica rapa subsp. chinensis ("pak choi") to me, but I might be mixing varieties.
Response：Thanks for the comments. We have added “non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino)” in the tittle following the article published on Horticulture Research.
Wang H, Li Z, Ren H, et al. Regulatory interaction of BcWRKY33A and BcHSFA4A promotes salt tolerance in non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. chinensis] Horticulture Research, uhac113, https://doi.org/10.1093/hr/uhac113
7., You apparently used a QIIME workflow. What is unclassified-k-Fungi? Are those fungal sequences for sure or did the algorithm default to the fungal kingdom when no meaningful results was found (and the same for other kingdoms)? What databases were used (name, version, citation)? A detailed workflow description is warranted. Also discard meaningless OTUs - your protists contain "Brassica" which is likely residual plant DNA.
Response：Thanks for the comments. Unclassified-k-Fungi can be found in the database, but has no classification information. Those fungal sequences were all refered to OUT, but most of them were meaningless OTUs, so we have discarded it as the same above. For the database, bacterial used RDP version 11.5 rRNA (http://rdp.cme.msu.edu/), Fungi and protist used UNITE version 8 (https://unite.ut.ee//, PR2 version 1 https://github.com/vaulot/pr2_database), we have added the information in “2.4 Statistical Analysis”(line152)
Some meaningless OTUs were discard and "Brassica" in the protist was discard in the OTUs.
8., Captions are not detailed enough. What is the difference between Fig 1a and Fig 1b?
Response：We have modified the captions following the reviewer’s suggestion. Added the note in Fig 1 as follows: “a: 20% reduction of total nitrogen with different bio-organic fertilizer, b: 30% reduction of total nitrogen with different bio-organic fertilizer. Different small letters represent significant difference at 0.05 level by Duncan’s test; the same as below”.
9., Compositional data. Correlation study of read counts and other data is not meaningful, as these data negatively correlate with each other as the DNA fragments compete for a limited number of slots. To mathematically cope with the issue, the data have to be pre-treated before these procedures. See 10.3389/fmicb.2017.02224 for an excellent description.
Response：The metagenome analysis in your suggestion is excellent. In this experiment, the data was also processed before correlation. Specifically, RDA analysis was carried out according to Cao et al (2019) and Cai et al (2017). We have carefully read the references you list, we will further optimize the data in the experiment with metagenome analysis, and eliminate the false-positive problem. Thank you very much.
Gao Z, Han M , Hu Y , et al. Effects of Continuous Cropping of Sweet Potato on the Fungal Community Structure in Rhizospheric Soil[J]. Frontiers in Microbiology, 2019, 10:2269
Cai F , Pang G , Li R X , et al. Bioorganic fertilizer maintains a more stable soil microbiome than chemical fertilizer for monocropping[J]. Biology & Fertility of Soils, 2017.
10., False discovery rate. You did lots of statistical tests (by testing raw correlations between soil parameters and taxon abundances - which is very nice). Therefore, the chance of accidently finding an apparently "statistically significant", but actually random phenomenon is high, which has to be dealt with by using statistical correction of p-values. I suggest the sensitive Benjamini Hochberg procedure, which requires all p-values to be gathered. Alternatively, Bonferroni correction can be used which is simpler but will render more phenomena to be insignificant.
Response: Bonferroni correction was used in the manuscript for the p-values as you suggestion. All the p-values listed in the manuscript can meet the conditions of bonferroni correction.
11., The tested soil seems to be quite "resistant" to your treatments. Please show compositional barplots in which there are changes (instead of Fig 2b, 3b, 4b). At these levels, not much has happened with the communities, though there are likely moving parts at, say, order level.
Response：Thanks for the comments. We added the compositional at barplots genus level Fig 2d, 3d, 4d. We kept compositional at barplots phylum level according to previous researches (Gao et al. 2021; Gao et al.2020)
Gao M, Yang J, et al. Effects of long-term biochar and biochar-based fertilizer application on brown earth soil bacterial communities - ScienceDirect[J]. Agriculture, Ecosystems & Environment, 2021,309.
Gao Z , Hu Y , Han M , et al. Effects of Continuous Cropping of Sweet Potatoes on the Bacterial Community Structure in Rhizospheric Soil. 2020. 21:102
12., Was there any taxon abundance correlated with performance of cabbage? This could be the most significant finding (and by far most interesting part) of your experiment, yet, it is missing. Note point 9. - this has to be examined with logratio transformed numbers. Discussion should also be focused on expected changes in crop quality as a result of changes in the microbial community.
Response：Thanks for the comments. We added the information about the taxon abundance correlated with yield and nitrate content of cabbage (Fig 2f, 3f, 4f).
Ding, H.; Fan, J.; Jia, C.; et al. Current situation and trend of vegetable seed industry development in China. China Vegetables, 2020, (09):1-8.
13., Your next experiment should contain a no crop control for bulk soils (with all the treatments).
Response：Thanks for the comments, we are doing the experiment about the Continuous fertilization (No.1) with non-heading Chinese cabbage about the soil metagenome, which contains a no crop control for bulk soils.
Minor points.
Abstract:
- add nitrogen dose (kg N / ha) in T1.
- detail "fertilizer"
Response：Thanks for the comments. We added the information in T1, "fertilizer" was mineral fertilizer (N, P, K), We added the information in Table 1and Table S1.
L62-63: This is not meaningful to readers outside of China. Provide approximate ha of crop area or produced tons/year.
Response：Thanks for the comments. We added the produced tons of2020 year in line62-63.
Ding, H.; Fan, J.; Jia, C.; Qin, C.; Yang, Y.; Zhang, H.; Zhang, F.; Wen, C.; Yu, Shuanchang.; Xu Y.; 2020. Current situation and trend of vegetable seed industry development in China. China Vegetables, (09):1-8.
L75: "acid soil" is not a texture type
Response：Thanks for the comments. We have corrected the information.
L77: do you have data on P, and S?
Response：We have the data of available P, K, which were relative stable in this experiment as we only changed the fertilizer application of N. So we do not measure the total P and S contents. Next time we will measure them, which will make the experiment more complete.
L78: There are different versions of "superphosphate" and different potassium sulfates (K2SO4 . x H2O where x varies). Add exact chemical formulas or % element composition. Treatment main data should not only be available in Table S1.
Response: We have added the information in the materials: 46% urea, 12% superphosphate and 52% Anhydrous potassium sulfate.
L83: what is etc.? if it an undefined species, use "spp." otherwise add other taxa.
Response: Thanks for the comments. We have corrected the information.
Fig S4 is an empty plot. Why?
Response: Please see Fig S4 below. It can’t appear because the format is not support. We have modify the format of the fig S4. Sorry about that.
L247: you cannot "increase chemical properties" of soils, please revise
Response: Thanks for the comments. We have revised this in the manuscript.
We would like to thank you again for giving the valuable comments. Hope the corrections will meet with approval.
Fangling Jiang
College of Horticulture, Nanjing Agricultural University / Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture, Nanjing 210095, P.R. China;
Tel: +86 2584396251, Fax: +86 2584396251,
Author Response File: 
Author Response.docx
Reviewer 2 Report
The introduction of this paper is great. The materials and methods could be improved. The result and conclusions are sound.
Author Response
Cover Letter
Dear Editor and Reviewer,
First of all, we would like to give thanks to the comments from academic editor and reviewers to our manuscript (ID: agronomy-1743416). We have highlighted the changes in the main text so that you can track the changes.
Thank you very much for your attention to our paper.
Sincerely yours,
We would like to thank you again for giving the valuable comments. We look forward to hearing good news from you.
Prof. Fangling Jiang
College of Horticulture, Nanjing Agricultural University / Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture, Nanjing 210095, P.R. China;
Tel: +86 2584396251, Fax: +86 2584396251,
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Dear Authors,
In my opinion, several points require your attention in my opinion, these are summarized below.
Major issues.
1., Despite claiming to use Bonferroni correction (L148), I do not see P values have changed (INV, UREA, etc.). Either write the values as punadj, or do the correction and write padj. Also, I'd not call a 0.00691 value extremely significant (L247).
2., Fig. 4d: not all bars have a sum of 1.
3., Discussion: restrict to statistically significant changes (versus CK, I think Burkholderia - Caballeronia - Parabulkholderia, Rhodanobacter, Ramlibacter, Dyella, C0119 and Bradyrhizobium might be worth a closer look for bacteria, while in Fungi, I'd check Mortierella, Olpidium, Humicola, Pseudaleuria and Alternaria).
4., Assignment of other kingdoms in community data. Your protists dataset contains lots of taxa that should be discarded. Given the contribution, you might consider discarding the whole protist dataset and discuss the fungi and bacteria only. The taxa to be discarded contain all your significantly correlating taxa (Fig 4e-f). Remove these as they're not protists: Chlorphyta are plants; Cryptomycota Mucoromycota and Ascomycota are fungi; Nematoda and Arthropoda are animals; etc. I'm unsure about the Streptophyta but they are also likely classified as plants.
5., L426 and elsewhere: the term "rhizobacterial community" should also be changed to "soil community" to match the title (also see my previous comment)
6., Your discussion does not discuss the background of the microbiome - feature correlations.
Minor / technical issues
Title: add a comma after "organisms"
L27-28: significantly decreased the abundance of the dominant phlya ..., L32-33 similarly
L29: genera
L41:in Europe
L51: TN not defined
L65: please either use m2, ha (10,000 m2) or km2
L415, 465, 499 (and possibly elsewhere): names of genera must be in italic
L431: we got no information about the "content" of Gemmatimonadates, but the "ratio" / "proportion"
L440: it correlated, not affected, your experiment does not allow one to draw conclusions on casual relationships
L447-453: these are raw results, not to be part of the discussion
Author Response
Cover Letter
Dear Editor and Reviewer,
Thank you very much for comments and constructive suggestions with regard to our manuscript titled “Reduced nitrogen with bio-organic fertilizer altered rhizo-sphere microorganisms improved yield and quality of non-heading Chinese cabbage (ID: agronomy-1743416). Those comments are very helpful for us to improve our manuscript. We have revised the manuscript thoroughly according to the reviewers’ constructive comments and useful suggestions. Please see the revised manuscript marked in red. We appreciate Editors/Reviewers’ warm work earnestly, and hope that the corrections will meet with approval. Here are our point-to-point responses.
Major issues.
1., Despite claiming to use Bonferroni correction (L148), I do not see P values have changed (INV, UREA, etc.). Either write the values as punadj, or do the correction and write padj. Also, I'd not call a 0.00691 value extremely significant (L247).
Response：Thanks for the comments. Bonferroni correction is used to eliminate false positives, which can be used in metagenomic analysis. Previously, we may have misunderstood the “Bonferroni correction”, we just make sure the Algorithm and formula was correct. We will do it in the metagenomic analysis. We write the values as punadj in L244-L255. And we changed “value extremely significant” in to “significant positively correlation with Rhodanobacte” when punadj =0.00691.
2., Fig. 4d: not all bars have a sum of 1.
Response：Thanks for the comments. We have discarded all the information of the protist according to 4 “Assignment of other kingdoms in community data….”
3., Discussion: restrict to statistically significant changes (versus CK, I think Burkholderia - Caballeronia - Parabulkholderia, Rhodanobacter, Ramlibacter, Dyella, C0119 and Bradyrhizobium might be worth a closer look for bacteria, while in Fungi, I'd check Mortierella, Olpidium, Humicola, Pseudaleuria and Alternaria).
Response：Thanks for the comments.
We give more attention on “Rhodanobacter, Dyella, Bradyrhizobium, Bacillus
Mortierella, Humicola, Fusarium” in the discussion.
4., Assignment of other kingdoms in community data. Your protists dataset contains lots of taxa that should be discarded. Given the contribution, you might consider discarding the whole protist dataset and discuss the fungi and bacteria only. The taxa to be discarded contain all your significantly correlating taxa (Fig 4e-f). Remove these as they're not protists: Chlorphyta are plants; Cryptomycota Mucoromycota and Ascomycota are fungi; Nematoda and Arthropoda are animals; etc. I'm unsure about the Streptophyta but they are also likely classified as plants.
Response：Thanks for the comments. We have discarded all the information of the protist, and discussed the fungi and bacteria only.
5., L426 and elsewhere: the term "rhizobacterial community" should also be changed to "soil community" to match the title (also see my previous comment)
Response：Thanks for the comments. We have changed "rhizobacterial community" to "soil community" throughout the manuscript.
6., Your discussion does not discuss the background of the microbiome - feature correlations.
Response：Thanks for the comments.We added the information in the discussion.
Dyella spp. was involved in Se biogeochemical cycle (Luo et al. 2022). Bradyrhizobium was a kind of nitrogen-fixing bacteria related to nitrogen fixation (Lajudie, 1994).
Mortierella spp dissolved insoluble phosphorus by releasing a variety of organic acids in soil, and promoted phosphorus cycling (Osorio & Habte, 2014). Mortierella spp promoted plant growth by improving soil phosphatase activity (Zhang et al., 2011). Humicola had a strong decomposition effect on organic matter (Malik &Sandhu,1973). Fusarium cause a variety of plant diseases, such as root rot, decreased the yield and quality of crops(Hu 1984).
Rhodanobacter was an essential clade involved in denitrification in acidic soils (Van et al., 2010).
Bacillus was a kind of probiotics, which could secrete plant hormones and promote plant growth (Bacon, et al. 2015).
Luo, X.; Wang, Y.; Li, Y.; An, j.; Wang, G.; Li, M.; Zheng, S.; 2022. Microbial oxidation of organic and elemental selenium to selenite. Sci. Total Environ. 0048-9697 https://doi.org/10.1016/j.scitotenv.2022.155203
Lajudie, P.; 1994. Polyphasic Taxonomy of Rhizobia: Emendation of the Genus Sinorhizobium and Description of Sinorhizobium meliloti comb. nov. Sinorhizobium saheli sp. nov. and Sinorhizobium teranga sp. nov.[J]. Int.j.syst.bacteriol, 44(4):715-733.
https://doi.org/10.1016/10.1099/00207713-44-4-715
Osorio N W and Habte M. 2014 Soil phosphate desorption induced by a phosphate-solubilizing fungus[J]. Commun Soil Sci Plant Anal, 45(4): 451～460. https://doi.org/10.1080/00103624.2013.870190
Zhang, H.; Wu, X.; Gang, L.; Qin, P.; 2011. Interactions between arbuscular mycorrhizal fungi and phosphate-solubilizing fungus ( Mortierella sp.) and their effects on Kostelelzkya virginica growth and enzyme activities of rhizosphere and bulk soils at different salinities. Biology and Fertility of Soils, 47(5):543-554. https://doi.org/10.1007/s00374-011-0563-3
Malik, K.; Sandhu,G.; 1973. Decomposition of organic matter by fungi in saline soils[J]. Mycopathologia et mycologia applicata, 50(4):339-347. https://doi.org/10.1007/BF02050034
Hu, G.; 1984. Discussion on the origin of sweet potato root rot in China. J. Henan Univ. Sci. Technol. (Agric. Ed.) 1, 10–13.
Van, R.; Van, E.; Jetten, M.; Hefting, M.; Kartal, B.; 2010. De-nitrification at pH 4 by a soil-derived Rhodanobacter-dominated community. Environ. Microbiol. 12, 3264–3271. https://doi.org/10.1111/j.1462-2920.2010.02301.x.
Bacon, C.; Palencia, E.; Hinton, D.; 2015. Abiotic and biotic plant stress-tolerant and beneficial secondary metabolites produced by endophytic Bacillus species. Springer India, 2015: 163-177.
https://doi.org/10.1007/978-81-322-2068-8_8
Minor / technical issues
Title: add a comma after "organisms"
Response：Thanks for the comments. We have added a comma after "organisms"
L27-28: significantly decreased the abundance of the dominant phlya ..., L32-33 similarly
Response：Thanks for the comments. We changed “significant” into “significantly” in line 32.
L29: genera
Response：Thanks for the comments. We changed “genus” into “genera”.
L41:in Europe
Response：Thanks for the comments. We changed “European” into “Europe”.
L51: TN not defined
Response：Thanks for the comments. We added the information “TN (total nitrogen content) in line 51.
L65: please either use m2, ha (10,000 m2) or km2
Response：Thanks for the comments. We changed “hm2” in to “ha”.
L415, 465, 499 (and possibly elsewhere): names of genera must be in italic
Response：Thanks for the comments. We have corrected the names of genera in italic.
L431: we got no information about the "content" of Gemmatimonadates, but the "ratio" / "proportion"
Response：Thanks for the comments. "content" in to "relative proportion" in L 392, 431
L440: it correlated, not affected, your experiment does not allow one to draw conclusions on casual relationships
Response：Thanks for the comments. We changed "affected" into "correlated with".
L447-453: these are raw results, not to be part of the discussion
Response：Thanks for the comments. We have discarded the results out of the discussion.
We would like to thank you again for giving the valuable comments. We look forward to hearing good news from you.
Fangling Jiang
College of Horticulture, Nanjing Agricultural University / Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture, Nanjing 210095, P.R. China;
Tel: +86 2584396251, Fax: +86 2584396251,
Author Response File: Author Response.docx
