Effects of Long-Term Straw Returning and Nitrogen Fertilizer Reduction on Soil Microbial Diversity in Black Soil in Northeast China
Round 1
Reviewer 1 Report
Title: Effects of long-term straw returning and nitrogen fertilizer reduction on soil microbial diversity in black soil in Northeast China.
Dear authors,
The topic of your paper is interesting. However, the quality of this paper is not adequate, there are some aspects which should be improved. Following are some recommendations to consider:
A.- Abstract, title and references:
The abstract is good. Please change some keywords, because they are already in the title.
Even though references are relevant and appropriate, they are mostly from Chinese authors. I understand that the topic of the work was about a Chinese soil, but they could be improved by searching some literature also from other authors.
B.- Introduction:
The research question is clearly outlined, enough arguing why this study may contribute with new knowledge.
C.- M&M:
Overall, methods need to be improved.
The experimental set-up could be better understandable by drawing a scheme.
Please explain what was the composition of the control soil you have chosen.
In the results section some analyses are presented, but there is not the method in this section. For example, how did you perform the inferred functionality of soil bacteria mentioned in the section 3.6?
It seems that pH of the soil was crucial in your study, please describe how you measured it.
Analysis of nitrogen, potassium etc have been performed by third parts? Otherwise, you should describe the methods as well.
Lines 138-141: what does this mean? This is not a method, and it seems just pasted from somewhere. Please describe in detail why you choose to use OTU clustering and how did you performed it.
Moreover, if you use OTU clustering instead of ASV, you should not compare your results with other works.
D.- Results:
Overall, this section is good. However, the figures should be improved. For example, the axis of Figure 2 could be improved.
Moreover, you should sort the microbial phyla, class and genus in alphabetical order.
E.- Discussion & Conclusion
The information in this section is quite good, however there are some key points lacking in my opinion. Which are the differences between section 4.1 and 4.2? It is so confusing reading one section and another, moreover they are in contrast each other. Is the straw returning a good strategy or not? The part of the functional analysis is totally missing in the discussion, but maybe is the most interesting?
Discussing about a shift in phyla composition (not present, for what I got), ignoring the data of class and genus is a huge disadvantage, in my opinion.
You should improve the discussion integrating both the data of class/genus with functional analysis and try to use those data to obtain a more concise conclusion, which are too much generic in my opinion.
The quality of English is appropriate.
Author Response
Dear Associate Editor Berry Song,
Dear Editor Dariusz Kadluczka,
Dear Reviewer 1,
Thank you very much for giving us the opportunity to revise our manuscript entitled “Effects of long-term straw returning and nitrogen fertilizer reduction on soil microbial diversity in black soil in Northeast China (agronomy-2481742)”. Thank you very much for your valuable comments on our MS. According to your comments and suggestions, we carefully revised throughout our MS. Below you will find our point-by-point response.
Reviewer 1
Title: Effects of long-term straw returning and nitrogen fertilizer reduction on soil microbial diversity in black soil in Northeast China.
Dear authors,
The topic of your paper is interesting. However, the quality of this paper is not adequate, there are some aspects which should be improved. Following are some recommendations to consider:
A.- Abstract, title and references:
Q1: The abstract is good. Please change some keywords, because they are already in the title.
Response:
Thank you for your constructive suggestions for our manuscript. According to your suggestion, we have changed our keywords as follow:
“Mollisols, fertilizer, community structure, high-throughput sequencing; bacterial diveristy”.
Please see the keywords in the revision manuscript.
Q2:Even though references are relevant and appropriate, they are mostly from Chinese authors. I understand that the topic of the work was about a Chinese soil, but they could be improved by searching some literature also from other authors.
Response:
Thank you for your constructive suggestions for our manuscript. We agree with your suggestion and we have updated our manuscript throughout whole manuscript. Please see the relevant parts and reference.
B.- Introduction:
The research question is clearly outlined, enough arguing why this study may contribute with new knowledge.
C.- M&M:
Overall, methods need to be improved.
Q3:The experimental set-up could be better understandable by drawing a scheme.
Response:
Thank you for your constructive suggestions for our manuscript. We have added a figure in here and please see the Fig. S1.
Q4:Please explain what was the composition of the control soil you have chosen.
Response:
Thank you for your constructive suggestions for our manuscript.
We have revised it as follow:
“Here, we examined the effects of four experimental treatments (Fig. S1): fertilizer (F), straw returning (S), FS (fertilizer × straw returning), and a control treatment (CK, without fertilizer addition and straw returning).”
Q5: In the results section some analyses are presented, but there is not the method in this section. For example, how did you perform the inferred functionality of soil bacteria mentioned in the section 3.6?
Response:
Thank you for your constructive suggestions for our manuscript.
We are sorry for our mistakes and we checked our material and method and revised it.
Please see them in the follows:
“The obtained operational taxonomic units (OTUs) were clustered by Uprase (Edgar, 2013) at similarity threshold of 97% (Li, 2015) and the taxonomy were annotated to the SILVA database (v138.1) (Quast et al., 2013). Before further analysis of Alpha diversity index (ACE, Chao1, Simpson, and Shannon) and Beta diversity (non-metric multi-dimensional scaling), the reads were normalized according to the lowest number of reads for a single soil sample. ACE, Chao1, Simpson, and Shannon indices were calculated on OTU level by “microeco” package R language (version 4.3.1). Non-metric multi-dimensional scaling (NMDS) was performed based on OTU level by “microeco” package R language (version 4.3.1). The indicator species of soil bacteria, i.e linear discriminant analysis (LDA) analysis were finished to identify bacterial indicator species on the basis of a normalized relative abundance matrix across group using the default parameters on the biocloud platform https://www.bioincloud.tech/. The soil bacterial functions based on OTU level were performed according to FAPROTAX function classifications on the biocloud platform https://www.bioincloud.tech/standalone-task-ui/faprotax.”
“The heatmap and cluster analyses based on Bray-Curtis dissimilarity was conducted on the biocloud platform https://www.bioincloud.tech/ using the most 20 relative abundance genera. All the figures were performed on the biocloud platform https://www.bioincloud.tech/. The Spearman’s rank correlation analysis for the relationships between the alpha diversity of soil bacteria and the soil physicochemical properties were finished via SPSS 12.0.”
Q6: It seems that pH of the soil was crucial in your study, please describe how you measured it.
Analysis of nitrogen, potassium etc have been performed by third parts? Otherwise, you should describe the methods as well.
Response:
Thank you for your constructive suggestions for our manuscript.
We have added the test method in the M&M. please see it in that and as follow:
“Soil pH was measured using a pH meter and soil to water ratio of 1:2.5 w/v. Total nitrogen (TN) was measured using an elemental analyzer (Elementar, Langenselbold, Germany). Available phosphor (AP), total potassium (TP), available nitrogen (AN), available phosphor (AP) and potassium (AK) were measured using an with a continuous flow analysis (SAN++, Skalar Analytical, Netherlands). Soil exchangeable calcium (Ca2+) and magnesium (Mg2+) were detected by ammonium acetate exchange-AAS method.”
Q7: Lines 138-141: what does this mean? This is not a method, and it seems just pasted from somewhere. Please describe in detail why you choose to use OTU clustering and how did you performed it.
Moreover, if you use OTU clustering instead of ASV, you should not compare your results with other works.
Response:
Thank you for your constructive suggestions for our manuscript.
According to your suggestion, we have revised our manuscript in M&M.
Please see them as follow:
The obtained operational taxonomic units (OTUs) were clustered by Uprase at similarity threshold of 97% (Li, 2015) and the taxonomy were annotated to the SILVA database (v138.1) (Quast et al., 2013). Before further analysis of Alpha diversity and Beta diversity (non-metric multi-dimensional scaling), the reads were normalized according to the lowest number of reads for a single soil sample.
For the second question, we admit that the QIIME2 now is the newest process for the sequences and the ASV may be suitable for current analysis. But now there are still some studies using the QIIME1 for the microbial ecology and we did this work in the 2013 and finished the sequence in 2018, so we used the QIIME1 as our process method and we compared our results to other studies also used QIIME1. Therefore, we only select QIIME1 as our sequence process and we thank you for your suggestion for our future work scheme.
D.- Results:
Q8: Overall, this section is good. However, the figures should be improved. For example, the axis of Figure 2 could be improved.
Moreover, you should sort the microbial phyla, class and genus in alphabetical order.
Response:
Thank you for your constructive suggestions for our manuscript.
We have remade our figure 2 according to your suggestion. Please see the figure 2 in the manuscript.
E.- Discussion & Conclusion
Q9: The information in this section is quite good, however there are some key points lacking in my opinion. Which are the differences between section 4.1 and 4.2? It is so confusing reading one section and another, moreover they are in contrast each other. Is the straw returning a good strategy or not? The part of the functional analysis is totally missing in the discussion, but maybe is the most interesting?
Response:
Dear reviewer,
Thank you for your constructive comments. According to you and reviewer2’s comments, we revised our discussion. Please see the discussion.
Discussing about a shift in phyla composition (not present, for what I got), ignoring the data of class and genus is a huge disadvantage, in my opinion.
You should improve the discussion integrating both the data of class/genus with functional analysis and try to use those data to obtain a more concise conclusion, which are too much generic in my opinion.
Response:
Thank you for your constructive suggestions for our manuscript. We thank you for your considerable suggestions.
We have revised our discussion according to your suggestion. Please see the discussion in the manuscript and as follow:
Dependent on the genus level, the dominant bacterial genus under straw returning and fertilizer application were Pseudarthrobacter, Sideroxydans, Sideroxydans, Candidatus_Udaeobacter and Anaerolinea (Fig. 2c and Table 4). Moreover, rice straw significant decreased the relative abundance of Pseudarthrobacter and Candidatus_Udaeobacter, but other genera relative abundance of fertilization and straw did not change compared to CK. This indicated that straw returning and fertilizer addition did not change most soil bacteria genera, but only changed the abundance of major degrading bacteria genera. This is consistent with previous studies (Bai et al. 2020; Wang et al. 2023). Pseudarthrobacter and Candidatus_Udaeobacter were common decomposers, which are primary decomposers with well-known cellulolytic activity (Zhan et al., 2018). Strangely, the increase of these bacteria was inhibited after adding straw, and the abundance of these degrading bacteria was increased by adding fertilizer. This may be that this experiment collected the soils after 5 years the straw added, and the rice straw may be more difficult to decompose after 5 years. Such bacteria cannot use the straw, but due to the impact of fertilizers, we guess these bacteria may be opportunist, because the presence of exogenous nutrient input, these bacteria genera could use exogenous nutrient. Therefore, further research is needed to explore the function of such bacteria genera.
4.3 Changes of soil bacterial functions between the experimental treatments
Analyzing the functions of bacterial community can help to determine the relationship between the fertilization and straw and a composite bacterial system. Our study showed that Chemoheterotrophy, aerobic_chemoheterotrophy, and nitrification were prominent in CK and F than in S and FS (Figure 7), but Chlorate_reducers were more prominent in F than in other treatments, and Plant_pathogen and dark_iron_oxidation were more prominent in S than in other treatments (Figure 7). This indicated that long-term fertilization and straw significantly change the soil bacterial functions, but the soil bacterial function had different response to fertilization and rice straw. We noted that the phyla Proteobacteria, Actinobacteria, Chloroflexi, and Bacteroidetes could effectively degrade lignocellulose, which significantly correlated with the functions of nitrogen fixation and Chemoheterotrophy. Our results indicated that not only is the degradation of straw directly influenced by the nitrogen fixation, but the fertilization utilization is also affected Chlorate_reducers, nitrification and fermentation.
Author Response File: 
Author Response.doc
Reviewer 2 Report
The manuscript (agronomy-2481742) should be accepted after minor revisions.
The authors described the effect of straw returning and nitrogen fertilizer reduction on soil microbial community structure and diversity in the long term and the correlations with soil chemical parameters. Overall the manuscript is well-written, and authors properly justify the study.
I found some issues in the "materials and methods" section regarding the analysis of soil in Table 1. I suppose that the parameter of "available phosphor" is referred to as total P. Moreover, it is not clear the methods used, for example, if some digestion or extraction is applied before the analysis. I suggest improving this section or reporting more references.
Table 2 reports results without any unit of measure. Please, add them for all parameters. With regards to the community structure of soil bacteria, the relative abundances of the prominent bacterial OTUs is referred to a relative abundance >1%?
The Section 4.1. can be improved by discussing more the bacterial diversity found. The authors described the diversity related to the pH conditions, but the discussion about the other chemical parameters is poor, especially about phosphorus.
Author Response
Dear Associate Editor Berry Song,
Dear Editor Dariusz Kadluczka,
Dear Reviewer 2,
Thank you very much for giving us the opportunity to revise our manuscript entitled “Effects of long-term straw returning and nitrogen fertilizer reduction on soil microbial diversity in black soil in Northeast China (agronomy-2481742)”. Thank you very much for your valuable comments on our MS. According to your comments and suggestions, we carefully revised throughout our MS. Below you will find our point-by-point response.
Comments
Reviewer 2:
The manuscript (agronomy-2481742) should be accepted after minor revisions.
The authors described the effect of straw returning and nitrogen fertilizer reduction on soil microbial community structure and diversity in the long term and the correlations with soil chemical parameters. Overall the manuscript is well-written, and authors properly justify the study.
Q1: I found some issues in the "materials and methods" section regarding the analysis of soil in Table 1. I suppose that the parameter of "available phosphor" is referred to as total P. Moreover, it is not clear the methods used, for example, if some digestion or extraction is applied before the analysis. I suggest improving this section or reporting more references.
Response:
Dear reviewer,
Thank you for your considerable comments and we revised our manuscript according to your suggestion. Please see follow:
- We revised the “available phosphor” into “total phosphor”. Please see table 1 revised format.
- We revised the method for soil physicochemical parameters and added the references in the section of reference. See the line 126-134
“Soil pH was measured using a pH meter and soil to water ratio of 1:2.5 w/v. Soil organic carbon (SOC) was measured according to Yeomans and Bremner (1988). Total nitrogen (TN) was measured using an elemental analyzer (Elementar, Langenselbold, Germany). Available nitrogen (AN) was sequentially digested in H2SO4-HCLO4, 0.05 M NaHCO3, 2.0 M KCL. Available phosphorous (AP) was quantified using a colorimetric method upon extraction with 0.5 M NaCO3 (Zhu et al., 2021). Total phosphor (AP), total potassium (TK), available nitrogen (AN), available potassium (AK) were measured using a with a continuous flow analysis (SAN++, Skalar Analytical, Netherlands). Soil exchangeable calcium (Ca2+) and magnesium (Mg2+) were detected by ammonium acetate exchange-AAS method (Liu et al., 2009).”
Q2: Table 2 reports results without any unit of measure. Please, add them for all parameters. With regards to the community structure of soil bacteria, the relative abundances of the prominent bacterial OTUs is referred to a relative abundance >1%?
Response:
Dear reviewer,
Thank you for your considerable comments and we revised our manuscript according to your suggestion.
- Please see the table 2.
- We revised the part of community structure of soil bacteria. See the line 241-242 and 250-251 and 257-258:
“The relative abundance of the top 10 soil bacteria phyla (relative abundance > 1%) in all soil samples exhibited the following descending order:…. ”
“The relative abundance of the top 10 soil bacteria classes (relative abundance > 1%) in all soil samples exhibited the following descending order:….”
“Finally, the relative abundance of the top 10 soil bacteria genera (relative abundance > 1%) in all soil samples exhibited the following descending order:….”
Q3: The Section 4.1. can be improved by discussing more the bacterial diversity found. The authors described the diversity related to the pH conditions, but the discussion about the other chemical parameters is poor, especially about phosphorus.
Response:
Dear reviewer,
Thank you for your considerable comments and we revised this section, please see the section 4.1.
“We also found that soil nutrient characteristics (e.g. SOC and AN) were key environmental factors affecting the soil bacterial diversity (Table 4). Our result showed that fertilization and straw addition significantly changed the soil physicochemical properties, i.e. soil organic carbon decreased, whereas AN increased under F and S addition compared to CK (Table 2). Moreover, SOC positively correlated with OTU and AN positively correlated with Shannon. Previous studies reported that soil N application significantly changed the soil bacterial diversity (Hu et al., 2019).
In view of N fertilization, the increased availability of N, such as ammonium nitrogen and ammonium nitrogen, can satisfy the N requirements of microorganisms and directly increased the diazotrophic community abundance and richness (Grzyb et al., 2021). Similar, rice straw addition increased the carbon sources for soil bacteria and significantly increased the soil bacterial abundance. In contrast, TP positively but AP negtively correlated with soil bacterial richness (Chao1 and OTU) and Shannon (not significant), which is consistent with the results of Orchard et al., (2009). The abundance of soil bacteria decreases under low-P conditions, as P is an important component of nucleotides, energy compounds and cell membranes (Hu et al., 2019). However, some studies reported that demonstrated that soil P deficiency had a more important influence on soil bacteria than N, due to on diazotrophs inhabited by P content in barren soils (Wang et al., 2021). We guess black soil is a rich nutrient soil with high P content and therefore P did not show limitation on soil bacteria.”
Author Response File: Author Response.doc
Reviewer 3 Report
Comments and Suggestions for Authors
The manuscript “agronomy-24817428" Its publication should be reconsidered once the major errors have been corrected. This study investigates the impact on soil health and microbiological biodiversity of a black soil in China under addition of chemical fertilization and straw. The introductory section fulfills its purpose except for a comment on a paragraph that is recommended to be modified. The methodology section does not fulfill its purpose because it does not include references that support the use of these techniques, as well as some statistical tests that are not presented. In the results section, the authors seem to make many statistical and grouping analyses but do not establish the objective of these analyses. Comments and suggestions should be presented below:
- L32. The number does not have the correct format.
- L36-40. in the summary they mention acidification problems due to chemical fertilization processes, why not include them here as well.
- L44-45. This statement requires a reference.
- L51-59. It is necessary the existence of a paragraph that links and gives continuity to the theme of degradation with the biological part of the soil.
- L94. Why a general characterization of the straw was not done.
- L105-111. a diagram specifying the spatial distribution of the samples is required.
- L105-111. Why the methodologies used for soil physicochemical indicators are not established?
- L152-155. Some acronyms were not previously established.
- L163. I think it would be table 2. It is necessary to revise the numbering of the tables and their wording throughout the text.
- L167. These acronyms were not previously established.
- L175, 181. This technique is not established in the methodology section.
- L198. It is the only figure, there is no need to place a
- L199. Reorder table 4, it is too far away from the first mention.
- L208, 214. It is the next figure, isn't it?
- L239. This methodology was not established in its respective section.
- L241. At the beginning of the statement, it is mentioned that the authors are talking about figure 4, another mention is unnecessary.
- Figure 4, figure 6 and Figure 7. The meaning of the correlation and the colors is not clear in Figure 4, it is necessary to specify- Figure 5. Same question as the previous figure.
- L258. Figure 6 is never mentioned.
- L277. There is a space in the reference.
- L300. The C and N symbols had not been used for these compounds during the text.
- L322-326. Why?
- L343. Use acronyms.
none
Author Response
Dear Associate Editor Berry Song,
Dear Reviewer 2,
Thank you very much for giving us the opportunity to revise our manuscript entitled “Effects of long-term straw returning and nitrogen fertilizer reduction on soil microbial diversity in black soil in Northeast China (agronomy-2481742)”. Thank you very much for your valuable comments on our MS. According to your comments and suggestions, we carefully revised throughout our MS. Below you will find our point-by-point response.
Comments and Suggestions for Authors
Q1: The manuscript “agronomy-24817428" Its publication should be reconsidered once the major errors have been corrected. This study investigates the impact on soil health and microbiological biodiversity of a black soil in China under addition of chemical fertilization and straw. The introductory section fulfills its purpose except for a comment on a paragraph that is recommended to be modified. The methodology section does not fulfill its purpose because it does not include references that support the use of these techniques, as well as some statistical tests that are not presented. In the results section, the authors seem to make many statistical and grouping analyses but do not establish the objective of these analyses. Comments and suggestions should be presented below:
Response:
Dear editor,
Thank you for your considerable comments for our manuscript.
For this question: “The methodology section does not fulfill its purpose because it does not include references that support the use of these techniques, as well as some statistical tests that are not presented.”
We revised our material and method according to you and reviewer1’s comments. Please see the section of material and method and references as well as follow:
“According to the Overlap between PE reads for each soil sample, the paired-end sequence data obtained by Hiseq sequencing was spliced (Merge) into a sequence Tags, and the quality of Reads and the effect of Merge were filtered for quality control. There are mainly three steps as follows: 1) PE reads splicing: use FLASH v l.2.7 software (Magoč et al., 2011) to splice the reads of each sample through overlap, and the spliced sequence obtained is the original Tags data (Raw Tags). 2) Tags filtering: Use Trimmomatic v0.33 software (Bolger et al., 2014) to filter the spliced Raw Tags to obtain high-quality Tags data (CleanTags). 3) Removal of chimeras: Use UCHIME (version 4.2) software (Edgar et al., 2011) to identify and remove chimera sequences to obtain the final effective data (Effective Tags). The obtained operational taxonomic units (OTUs) were clustered by Uprase (Edgar, 2013) at similarity threshold of 97% (Li, 2015) and the taxonomy were annotated to the SILVA database (v138.1) (Quast et al., 2013). Before further analysis of Alpha diversity and Beta diversity (non-metric multi-dimensional scaling), the reads were normalized according to the lowest number of reads for a single soil sample. The indicator species of soil bacteria, i.e linear discriminant analysis (LDA) analysis were finished to identify bacterial indicator species on the basis of a normalized relative abundance matrix across group using the default parameters on the biocloud platform https://www.bioincloud.tech/. The soil bacterial functions based on OTU level were performed according to FAPROTAX function classifications on the biocloud platform https://www.bioincloud.tech/standalone-task-ui/faprotax. ”
Q2: - L32. The number does not have the correct format.
Response:
Dear editor,
Thank you for your constructive comments. We have revised it according to your suggestion.
Please see as follow:
“Black soil occupies a total area of 1030000 km2 in the Northeast region of the China Plain and is mainly distributed in the Hulunbeier Grassland, the Greater and Lesser Khingan Mountains, the Sanjiang Plain, the Songnen Plain, the Songliao Plain, and the Changbai Mountains (Ao et al. al., 2021).”
Q3: - L36-40. in the summary they mention acidification problems due to chemical fertilization processes, why not include them here as well.
Response:
Dear editor,
Thank you for your constructive comments. We have revised it according to your suggestion.
Please see as follow:
“However, due to unreasonable tillage practices (e.g. excessive fertilization utilization) and long-term straw removal from farmlands, soil acidification and erosion and degradation have become serious problems in this region (Wang et al., 2020).”
Q4: - L44-45. This statement requires a reference.
Response:
Dear editor,
Thank you for your constructive comments. We have revised it according to your suggestion.
Please see as follow:
“The application of chemical fertilizers can quickly and effectively supplement the nutrients required for optimal crop growth and significantly increases the content of key elements such as N, P, and K in black soil, all of which are known to increase agriculture yields (Rashid et al., 2021).”
Rashid, M., Hussain, Q., Khan, K. S., Alwabel, M. I., Hayat, R., Akmal, M., ... & Alvi, S. (2021). Carbon-based slow-release fertilizers for efficient nutrient management: synthesis, applications, and future research needs. Journal of Soil Science and Plant Nutrition, 21, 1144-1169.
Q5: - L51-59. It is necessary the existence of a paragraph that links and gives continuity to the theme of degradation with the biological part of the soil.
Response:
Dear editor,
Thank you for your constructive comments. We have revised it according to your suggestion.
“This strategy can also improve the physical properties of soil. For example, recent studies have demonstrated that straw returning increases soil porosity (Anning et al., 2021), reduces bulk density (Islam et al., 2022), improves soil permeability and water and fertilizer retention (Zheng et al. al., 2019), increases crop yields (Halko et al., 2023), and reduces the environmental pollution caused by straw burning.
Soil microorganisms are the most abundant life form in soil and play a crucial role in maintaining ecological functions such as biogeochemical cycles (Abs Sui et al, 2023; Garaycochea et al., 2023), litter decomposition, and plant growth (Bonilla et al., 2012). Moreover, due to their sensitivity to environmental changes, shifts in microbial communities can reflect changes in ecological functions (Yu et al., 2016). The decomposition and transformation process of straw returning to the soil is carried out under the impetus of soil microorganisms. Straw returning to the soil provides sufficient carbon sources for the reproduction and growth of soil microorganisms, increasing the content and types of carbon sources (Hao et al., 2019; Su et al., 2020). Li et al. (2017) reported that straw returning changed the bacterial community structure in the north plain of China, and increased the abundance of bacteria related to the degradation of complex organic matter. Zhao et al. (2019) also found that returning straw significantly changed the structure and abundance and diversity of soil bacterial community. Compared with the single application of chemical fertilizers, straw returning combined with chemical fertilizers significantly improved soil fertility, increased soil enzyme activity and bacterial abundance, and changed the bacterial community structure (Chen et al., 2017). Therefore, studying the effect of long-term straw returning on the soil microbial community has important implications for evaluating its impact on soil ecology, as well as the identification of environmentally friendly agricultural practices.”
Q6: - L94. Why a general characterization of the straw was not done.
Response:
Dear editor,
Thank you for your constructive comments. We have revised it according to your suggestion.
Please see follow:
“The rice straw was crushed into 8~10 cm, and the content of total nitrogen (TN), total phosphors (TP) and total potassium (TK) were 0.098, 0.026 and 2.1 g.kg-1, respectively.”
Q7: - L105-111. a diagram specifying the spatial distribution of the samples is required.
Response:
Dear editor,
Thank you for you and reviewer1’s constructive comments. We have added a scheme figure. Please see the figure S1 in the manuscript.
Q8: - L105-111. Why the methodologies used for soil physicochemical indicators are not established?
Response:
Dear editor,
Thank you for you and reviewer1’s constructive comments. We have revised here in the manuscript and also see as follow:
“Soil pH was measured using a pH meter and soil to water ratio of 1:2.5 w/v. Total nitrogen (TN) was measured using an elemental analyzer (Elementar, Langenselbold, Germany). Available phosphor (AP), total potassium (TP), available nitrogen (AN), available phosphor (AP) and potassium (AK) were measured using an with a continuous flow analysis (SAN++, Skalar Analytical, Netherlands). Soil exchangeable calcium (Ca2+) and magnesium (Mg2+) were detected by ammonium acetate exchange-AAS method.”
Q9: - L152-155. Some acronyms were not previously established.
Response:
Dear editor,
Thank you for your constructive comments. We have revised here in the manuscript and also see Q8.
Q10:- L163. I think it would be table 2. It is necessary to revise the numbering of the tables and their wording throughout the text.
Response:
Dear editor,
Thank you for your constructive comments. We revised the numbering of the table and wording throughout the text. Please see in the manuscript.
Q11: - L167. These acronyms were not previously established.
Response:
Dear editor,
Thank you for your constructive comments. We have revised them in the manuscript. Please see them in the manuscript and also as follow:
“ACE, Chao1, Simpson, and Shannon indices were calculated on OTU level by “microeco” package R language (version 4.3.1).”
Q12: - L175, 181. This technique is not established in the methodology section.
Response:
Dear editor,
Thank you for your constructive comments. We revised them in the material and method.
“The Spearman’s rank correlation analysis for the relationships between the alpha diversity of soil bacteria and the soil physicochemical properties were finished via SPSS 12.0. ”
“Non-metric multi-dimensional scaling (NMDS) was performed based on OTU level by “microeco” package R language (version 4.3.1).”
Q13: - L198. It is the only figure, there is no need to place a
Response:
Dear editor,
Thank you for your constructive comments. We are sorry for our mistake and we have renumbering our figure throughout the manuscript. Please see them in the manuscript
Q14: - L199. Reorder table 4, it is too far away from the first mention.
Response:
Dear editor,
Thank you for your constructive comments. We adjust the position of table 4 in the manuscript.
Q15: - L208, 214. It is the next figure, isn't it?
Response:
Dear editor,
We are sorry for our mistake. We revised the numbering of figure and table in the manuscript.
Q16: - L239. This methodology was not established in its respective section.
Response:
Dear editor,
Thank you for your constructive comments.
We added the method in the material and method. Please see them in the manuscript and as follow:
“The heatmap and cluster analyses based on Bray-Curtis dissimilarity was conducted on the biocloud platform https://www.bioincloud.tech/ using the most 20 relative abundance genera.”
Q17: - L241. At the beginning of the statement, it is mentioned that the authors are talking about figure 4, another mention is unnecessary.
Response:
Dear editor,
Thank you for your constructive comments.
We transfer the follow sentence into the composition of genera. Please see the manuscript.
Q17- Figure 4, figure 6 and Figure 7. The meaning of the correlation and the colors is not clear in Figure 4, it is necessary to specify- Figure 5. Same question as the previous figure.
Response:
Dear editor,
Thank you for your constructive comments.
We add the meaning for these figures.
“The color gradient (red, whit, blue) represents the relative abundance of the soil genera from high to low in the different treatments.”
Q18: - L258. Figure 6 is never mentioned.
Response:
Dear editor,
Thank you for your constructive comments.
We revised relevant parts in the manuscript and also renumbering our figures and tables.
Q19: - L277. There is a space in the reference.
Response:
Dear editor,
We revised it and also check similar problem throughout manuscript.
Q20: - L300. The C and N symbols had not been used for these compounds during the text.
Response:
Dear editor,
Thank you for your considerable comments. We revised this in the manuscript and as follow:
“we found that the increase in soil AN, TK, TN and AP content caused…..”
Q21: - L322-326. Why?
Response:
Dear editor,
Thank you for your considerable comments.
We agree with your comments and we revised our result 3.5.
Please see as follow:
“Redundancy analysis indicated that the soil microbial community structure was significantly correlated with the soil physicochemical properties (Figure 5). At the OTU level, TP, and AK were significantly associated with CK and F (Figure 5, P<0.05), and TK was significantly associated with FS and S (Figure 5, P<0.05), whereas other soil parameters were not significantly associated with the community structure of soil bacteria (Figure 5, P>0.05).”
Q22: - L343. Use acronyms.
Response:
Dear editor,
Thank you for your considerable comments.
We revised it according to your suggestion.
“..under S returning and F application were..”.
Author Response File: Author Response.doc
Round 2
Reviewer 3 Report
Comments and Suggestions for Authors
The manuscript “agronomy-24817428" Its publication should be reconsidered once the major errors have been corrected. This study investigates the impact on soil health and microbiological biodiversity of a black soil in China under addition of chemical fertilization and straw. The introductory section fulfills its purpose. The methodology section, although improved, does not fulfill its purpose because it does not include references that support the use of these techniques. In general, the text presents many errors in the use of acronyms and symbols of elements that should be homogenized throughout the text. Comments and new suggestions are presented below:
Modifications made.
- L32. The number does not have the correct format.
- modification was made.
- L36-40. in the summary they mention acidification problems due to chemical fertilization processes, why not include them here as well.
- modification was made.
- L44-45. This statement requires a reference.
- modification was made.
- L51-59. It is necessary the existence of a paragraph that links and gives continuity to the theme of degradation with the biological part of the soil.
- modification was made.
- L94. Why a general characterization of the straw was not done.
- modification was made.
- L105-111. a diagram specifying the spatial distribution of the samples is required.
- modification was made.
- L105-111. Why the methodologies used for soil physicochemical indicators are not established?
- modification was made.
- L152-155. Some acronyms were not previously established.
- modification was made.
- L163. I think it would be table 2. It is necessary to revise the numbering of the tables and their wording throughout the text.
- modification was made.
- L167. These acronyms were not previously established.
- modification was made.
- L175, 181. This technique is not established in the methodology section.
- modification was made.
- L198. It is the only figure, there is no need to place a
- modification was made.
- L199. Reorder table 4, it is too far away from the first mention.
- modification was made.
- L208, 214. It is the next figure, isn't it?
- modification was made.
- L239. This methodology was not established in its respective section.
- modification was made.
- Figure 4, figure 6 and Figure 7. The meaning of the correlation and the colors is not clear in Figure 4, it is necessary to specify- Figure 5. Same question as the previous figure.
- modification was made.
- L258. Figure 6 is never mentioned.
- modification was made.
- L277. There is a space in the reference.
- modification was made.
- L322-326. Why?
Modifications not made.
- L241. At the beginning of the statement, it is mentioned that the authors are talking about figure 4, another mention is unnecessary.
- L300. The C and N symbols had not been used for these compounds during the text.
- L343. Use acronyms.
New modifications
- L32. I don't understand how we went from 1.09x106 to 1030000, is that correct?
- L38. excessive fertilization is a tilling practice?
- L38-40. This paragraph is not well written, please rewrite it.
- L40-42. Why?
-L44. The first mention of any symbol or acronym requires establishing the meaning before the first mention.
-L56. There is still no line of writing that smoothly links the degradation of the soil with the biological part, the jump is very abrupt.
-L92. N was used to specify nitrogen, but was subsequently dropped, homogenizing throughout the text. In the same way for the other elements.
- L123. The acronym for total nitrogen (TN) was already established, why not continue using it? homogenize for this and the other acronyms throughout the text.
-L123. Why were no references placed?
- Table 1. Use acronyms.
- Under what level of significance were the statistical analyses performed and following what procedures, in addition to placing the references.
- The soil organic carbon technique was not established in the methodology.
- For the reader, the difference between the ACE, Chao1, Simpson, and Shannon indices in the methodology is not very clear.
- “Moreover, soil Ca had an obvious positive correlation with OTU”, It is not so obvious, specify
- It is necessary to put the percentages in Figures 2a, b and c.
- “The relative abundance of Chloroflexi in the FS treatment differed significantly compared to the CK (Table 5). The relative abundances of Bacteroidetes and Acidobacteria in the S and FS treatments differed significantly compared to the CK, respectively (Table 45)”. Under what level of significance?
- “Based on a linear discriminant analysis (LDA) effect size (LEfSe) score > 2, nine groups of soil bacterial community were found to be significantly different between the F, S, and FS treatments (Figure 3)”. Under what level of significance?
- “As illustrated in Figure 4, heatmap and cluster analyses based on Bray-Curtis dissimilarity demonstrated that the experimental treatments had a substantial effect on the relative abundance of the different members of the soil bacterial community (Figure 4)”. It had already been mentioned that it referred to figure 4.
- Figures 4 and 6 are very complex, however, there is a very poor analysis of them.
- “The effect of long-term fertilization on the bacterial community”. It is not clear what type of fertilization the authors are referring to.
- “Our results demonstrated that rice straw returning increased soil N, P, and K, with the FS treatment exhibiting the highest levels of these nutrients compared to the other treatments”. Were these variations significant?
- The difference between bacterial community structure and bacterial community composition is not very clear during the reading of the text.
-
Comments for author File: 
Comments.pdf
NONE
Author Response
Dear Associate Editor Berry Song,
Dear Editor Dariusz Kadluczka,
Dear Reviewer 2,
Thank you very much for giving us the opportunity to revise our manuscript entitled “Effects of long-term straw returning and nitrogen fertilizer reduction on soil microbial diversity in black soil in Northeast China (agronomy-2481742)”. Thank you very much for your valuable comments on our MS. According to your comments and suggestions, we carefully revised throughout our MS. Below you will find our point-by-point response.
Comments and Suggestions for Authors
The manuscript “agronomy-24817428" Its publication should be reconsidered once the major errors have been corrected. This study investigates the impact on soil health and microbiological biodiversity of a black soil in China under addition of chemical fertilization and straw. The introductory section fulfills its purpose. The methodology section, although improved, does not fulfill its purpose because it does not include references that support the use of these techniques. In general, the text presents many errors in the use of acronyms and symbols of elements that should be homogenized throughout the text. Comments and new suggestions are presented below:
Response:
Dear reviewer,
Thank you for your constructive comments for our manuscript. We have revised our manuscript according to your comments. Please see the line 126-134 and 155-178:
- We revised the material and method carefully and also added the references in the methods.
“Soil pH was measured using a pH meter and soil to water ratio of 1:2.5 w/v. Total nitrogen (TN) was measured using an elemental analyzer (Elementar, Langenselbold, Germany). Available nitrogen was sequentially digested in H2SO4-HCLO4, 0.05 M NaHCO3, 2.0 M KCL. Available phosphorous was quantified using a colorimetric method upon extraction with 0.5 M NaCO3 (Zhu et al., 2021). Total phosphor (AP), total potassium (TP), available nitrogen (AN), available potassium (AK) were measured using a with a continuous flow analysis (SAN++, Skalar Analytical, Netherlands). Soil exchangeable calcium (Ca2+) and magnesium (Mg2+) were detected by ammonium acetate exchange-AAS method (Liu et al., 2009). ”
“According to the Overlap between PE reads for each soil sample, the paired-end sequence data obtained by Hiseq sequencing was spliced (Merge) into a sequence Tags, and the quality of Reads and the effect of Merge were filtered for quality control. There are mainly three steps as follows: 1) PE reads splicing: use FLASH v l.2.7 software (Magoč et al., 2011) to splice the reads of each sample through overlap, and the spliced sequence obtained is the original Tags data (Raw Tags). 2) Tags filtering: Use Trimmomatic v0.33 software (Bolger et al., 2014) to filter the spliced Raw Tags to obtain high-quality Tags data (CleanTags). 3) Removal of chimeras: Use UCHIME (version 4.2) software (Edgar et al., 2011) to identify and remove chimera sequences to obtain the final effective data (Effective Tags). The obtained operational taxonomic units (OTUs) were clustered by Uprase (Edgar, 2013) at similarity threshold of 97% (Li, 2015) and the taxonomy were annotated to the SILVA database (v138.1) (Quast et al., 2013). Before further analysis of Alpha diversity index (ACE, Chao1, Simpson, and Shannon) and Beta diversity (non-metric multi-dimensional scaling), the reads were normalized according to the lowest number of reads for a single soil sample. ACE, Chao1, Simpson, and Shannon indices were calculated on OTU level by “microeco” package R language (version 4.3.1) (R Core Team, 2022). Non-metric multi-dimensional scaling (NMDS) was performed based on OTU level by “microeco” package R language (version 4.3.1) (R Core Team, 2022). The indicator species of soil bacteria, i.e linear discriminant analysis (LDA) analysis were finished to identify bacterial indicator species on the basis of a normalized relative abundance matrix across group using the default parameters on the biocloud platform https://www.bioincloud.tech/. The soil bacterial functions based on OTU level were performed according to FAPROTAX function classifications on the biocloud platform https://www.bioincloud.tech/standalone-task-ui/faprotax. ”
Comments:
Revierew3,
Modifications made.
- L32. The number does not have the correct format.
- modification was made.
- L36-40. in the summary they mention acidification problems due to chemical fertilization processes, why not include them here as well.
- modification was made.
- L44-45. This statement requires a reference.
- modification was made.
- L51-59. It is necessary the existence of a paragraph that links and gives continuity to the theme of degradation with the biological part of the soil.
- modification was made.
- L94. Why a general characterization of the straw was not done.
- modification was made.
- L105-111. a diagram specifying the spatial distribution of the samples is required.
- modification was made.
- L105-111. Why the methodologies used for soil physicochemical indicators are not established?
- modification was made.
- L152-155. Some acronyms were not previously established.
- modification was made.
- L163. I think it would be table 2. It is necessary to revise the numbering of the tables and their wording throughout the text.
- modification was made.
- L167. These acronyms were not previously established.
- modification was made.
- L175, 181. This technique is not established in the methodology section.
- modification was made.
- L198. It is the only figure, there is no need to place a
- modification was made.
- L199. Reorder table 4, it is too far away from the first mention.
- modification was made.
- L208, 214. It is the next figure, isn't it?
- modification was made.
- L239. This methodology was not established in its respective section.
- modification was made.
- Figure 4, figure 6 and Figure 7. The meaning of the correlation and the colors is not clear in Figure 4, it is necessary to specify- Figure 5. Same question as the previous figure.
- modification was made.
- L258. Figure 6 is never mentioned.
- modification was made.
- L277. There is a space in the reference.
- modification was made.
Q1: - L322-326. Why?
Response:
Dear editor,
We are sorry for that mistake. We revised this sentence carefully according to your comment. Please see line 309-405:
“The rice straw is composed of complex material components such as hemicellulose, lignin, cellulose, etc. (Sun et al., 2014), and the soil requires a large number of enzymes, a variety of microorganisms and the combined action of soil bacteria and fungi in order to adequately degrade the straw material applied to the soil, thus promoting the nutrient cycle in the soil. After decomposition, the rice straw can provide plants with nutrients necessary for growth, and straw can also increase the effective nutrient content and nitrogen content, improve soil fertility. Therefore, we guess the rice straw input significantly changed the soil nutrients (i.e SOC and TK), but for fertilization treatment, the only fertilization changed the soil TP and TK, but the SOC did not input and thus give a different response on soil bacterial community composition.”.
Modifications not made.
Q1: - L241. At the beginning of the statement, it is mentioned that the authors are talking about figure 4, another mention is unnecessary.
Response:
Dear editor,
Thank you for your considerable comments. We agree with your comments and delete another mention. Please see the line 304-306:
“As illustrated in Figure 4, heatmap and cluster analyses based on Bray-Curtis dissimilarity demonstrated that the experimental treatments had a substantial effect on the relative abundance of the different members of the soil bacterial community.”.
Q2:- L300. The C and N symbols had not been used for these compounds during the text.
Response:
Dear editor,
Thank you for your considerable comments. We revised all the symbols throughout the MS.
Q3:- L343. Use acronyms.
Response:
Dear editor,
Thank you for your considerable comments.
We revised this part using acronyms. Moreover, we also revised the whole MS for acronyms.
New modifications
Q4: - L32. I don't understand how we went from 1.09x106 to 1030000, is that correct?
Response:
Dear editor,
We are sorry for our mistake and we correct the format “1, 090, 000 km2” in the MS.
Q5: - L38. excessive fertilization is a tilling practice?
Response:
Dear editor,
We revised this part according to your suggestion. See line 37-40:
“However, black soil in this region suffered serious problems, such as soil acidification, soil erosion and degradation due to unreasonable fertilization utilization and long-term straw removal from farmlands (Wang et al., 2020).”.
Q6: - L38-40. This paragraph is not well written, please rewrite it.
Response:
Dear reviewer,
We revised this sentence according to your comment. This is same with the Question 5 , please see the 37-40:
“However, black soil in this region suffered serious problems, such as soil acidification, soil erosion and degradation due to unreasonable fertilization utilization and long-term straw removal from farmlands (Wang et al., 2020).”.
Q7: - L40-42. Why?
Response:
We revised this sentence to make it clearly.
“This irrational application of fertilizers and the straw removal of black soil in Northeast China poses a serious threat not only to national food security but also to the environment.”.
Q8: -L44. The first mention of any symbol or acronym requires establishing the meaning before the first mention.
Response:
We revised this according to your suggestion.
“..elements such as nitrogen (N), phosphorus (P), and potassium (K) in black soil..”
Q9: -L56. There is still no line of writing that smoothly links the degradation of the soil with the biological part, the jump is very abrupt.
Response:
We revised this according to your suggestion. Please see line 45-48.
“However, excessive application of chemical fertilizers can lead to serious ecological impacts such as environmental pollution and soil degradation due to soil salinization and acidification, leading to serious damage to the soil structure, thus causing the occurrence of soil crusting (Horrigan et al., 2002).”
Q10: -L92. N was used to specify nitrogen, but was subsequently dropped, homogenizing throughout the text. In the same way for the other elements.
Response:
We revised this according to your suggestion throughout the manuscript.
Q11: - L123. The acronym for total nitrogen (TN) was already established, why not continue using it? homogenize for this and the other acronyms throughout the text.
Response:
We revised this according to your suggestion throughout the manuscript.
Q12: -L123. Why were no references placed?
Response:
We revised this according to your suggestion. Please see the line 126-133.
Q13: - Table 1. Use acronyms.
Response:
We revised this according to your suggestion. Please see the line 134-136.
Q14: - Under what level of significance were the statistical analyses performed and following what procedures, in addition to placing the references.
Response:
We revised this according to your suggestion. Please see the line 181.
Q15: - The soil organic carbon technique was not established in the methodology.
Response:
We revised this according to your suggestion. Please see the line 126-127.
“Soil organic carbon (SOC) was measured according to Yeomans and Bremner (1988).”
Q16: - For the reader, the difference between the ACE, Chao1, Simpson, and Shannon indices in the methodology is not very clear.
Response:
Thank you for your considerable comments for our manuscript. We revised this according to your comments. Please see the line 165-167.
“Four indices were used to evaluate alpha diversity, including community richness (Chao1 and ACE), community diversity (Shannon and Simpson).”.
Q17: - “Moreover, soil Ca had an obvious positive correlation with OTU”, It is not so obvious, specify
Response:
Thank you for your considerable comments for our manuscript.
We deleted this sentence in the manuscript.
Q18: - It is necessary to put the percentages in Figures 2a, b and c.
Response:
We revised the figure 2a, b and c according to your suggestion.
Q19: - “The relative abundance of Chloroflexi in the FS treatment differed significantly compared to the CK (Table 5). The relative abundances of Bacteroidetes and Acidobacteria in the S and FS treatments differed significantly compared to the CK, respectively (Table 45)”. Under what level of significance?
Response:
Thank you for your comment. We revised it as follow and see line 246-248:
“The relative abundance of Chloroflexi in the FS treatment differed significantly compared to the CK (Table 5, Duncan test and P<0.05). The relative abundances of Bacteroidetes and Acidobacteria in the S and FS treatments differed significantly compared to the CK, respectively (Table 5, Duncan test and P<0.05)”.
Q20: - “Based on a linear discriminant analysis (LDA) effect size (LEfSe) score > 2, nine groups of soil bacterial community were found to be significantly different between the F, S, and FS treatments (Figure 3)”. Under what level of significance?
Response:
Thank you for your constructive comment.
We revised it as follow and see line 290-292.
“Based on a linear discriminant analysis (LDA) effect size (LEfSe) score > 2, nine groups of soil bacterial community were found to be significantly different between the F, S, and FS treatments (Figure 3, P<0.05).”.
Q21:- “As illustrated in Figure 4, heatmap and cluster analyses based on Bray-Curtis dissimilarity demonstrated that the experimental treatments had a substantial effect on the relative abundance of the different members of the soil bacterial community (Figure 4)”. It had already been mentioned that it referred to figure 4.
Response:
Thank you for your constructive comment.
We deleted the figure 4 and please see the line 303 and follow:
“As illustrated in Figure 4, heatmap and cluster analyses based on Bray-Curtis dissimilarity demonstrated that the experimental treatments had a substantial effect on the relative abundance of the different members of the soil bacterial community.”.
Q22: - Figures 4 and 6 are very complex, however, there is a very poor analysis of them.
Response:
Thank you for your considerable comments.
We revised it as follow and please see the line 301-306 and
Q23: - “The effect of long-term fertilization on the bacterial community”. It is not clear what type of fertilization the authors are referring to.
Response:
We revised it as follow and please see the line 345-348:
“The effect of long-term fertilization combined with straw return on the bacterial community was similar to that of a single application of chemical fertilizers and did not significantly influence the recovery of the bacterial community structure and diversity.”.
Q24: - “Our results demonstrated that rice straw returning increased soil N, P, and K, with the FS treatment exhibiting the highest levels of these nutrients compared to the other treatments”. Were these variations significant?
Response:
Thank you for your constructive suggestion.
We revised this sentence according to your suggestion.
See follow and the line 406-408.
“Our results demonstrated that rice straw returning significantly increased soil N, P, and K, with the FS treatment exhibiting the highest levels of these nutrients compared to the other treatments.”.
Q25: - The difference between bacterial community structure and bacterial community composition is not very clear during the reading of the text.
Response:
Thank you for your constructive suggestion.
We revised the relevant paragraph according to your suggestion. Please see the line 245-240 and line 263-273.
Author Response File: Author Response.doc
