Biochar Application Alleviated Rice Salt Stress via Modifying Soil Properties and Regulating Soil Bacterial Abundance and Community Structure
, Yali Kong 1, Xiaochuang Cao 1, Lianfeng Zhu 1, Yongchun Zhang 2, Yunwang Ning 2, Wenhao Tian 1, Hui Zhang 2,*, Yijun Yu 3,* and Junhua Zhang 1,*Round 1
Reviewer 1 Report
This paper deals with as biochar application can alleviate salt stress in rice. A short trial was conducted in pots with or without NaCl application, using three years of biochar amended soils. Three year of soil amendment with biochar can be enough to modify the chemico-physical characteristics and microbial structure of the soil, so the data of this study can be very useful for new theoretical knowledge on the mechanism of salt stress mitigation by biochar.
General comments
Introduction is clear and cites the more recent literature in the field.
However, materials and methods lack the type and characteristics of the biochar used in the experiment, furthermore, was not explain as you performed the redundancy analysis.
The results are well presented but some corrections are needed because figures and tables do not match always with the text (look to the specific remarks). You do not present data on beta diversity that instead you had declared in the methods.
The discussion is sufficiently thorough, but do not deal with the shannon diversity index that decrease significantly with BS.
Conclusions are poor.
You must check citations to meet the style requirements of the journal
Specific remarks
Line 68: Why 667 m2?
Line 93: sulfuric acid-potassium dichromate oxidation method with external heating?
Line 175- 177: the values of SOM are corrected? Probably here are expressed in % and not in g/kg because are very lower compared to the values reported in the table 1.
Line 250-252: You write that the relative abundance of Chloroflexi decreased by 45.46% in the B treatment compared with the CK, but in fig. 4 it seems a lot less, while you write that Chloroflexi decreased by 16.12% in the BS treatment compared to the S treatment, but in fig. 4 it seems a lot more
Line 268: you did not applicate biochar under salt stress in this study but tested soil amended with biochar to salt stress
Line 272: maybe there is a punctuation error, check the semicolon
Line 291-292: check this sentence
Line 338 – 339: is repetitive
Author Response
> Reviewer 1
Comments and Suggestions for Authors
This paper deals with as biochar application can alleviate salt stress in rice. A short trial was conducted in pots with or without NaCl application, using three years of biochar amended soils. Three year of soil amendment with biochar can be enough to modify the chemico-physical characteristics and microbial structure of the soil, so the data of this study can be very useful for new theoretical knowledge on the mechanism of salt stress mitigation by biochar.
General comments
Introduction is clear and cites the more recent literature in the field.
- However, materials and methods lack the type and characteristics of the biochar used in the experiment, furthermore, was not explain as you performed the redundancy analysis.
Response 1: Thank you point it out. The information of biochar used in present study in list below: “Biochar is made from wood and calcined at 600℃. The characteristics of biochar were biochar yield 35.87 ± 0.51%, ash content 33.16 ± 0.75%, pH 8.83 ± 0.57, EC 1185 ± 117.07 μS/cm, CEC 33.5 ± 1.06 cmol/kg, and N 1.66 ± 0.02%”. For the detail, please see page 2, lines 76-78. Thanks. The purpose of the redundancy analysis is to analyze the effects of soil properties on microorganisms’ community structures, and biochar is considered as a treatment in our study which is not at the same level as soil properties, so we do not put it in the redundancy analysis.
- The results are well presented but some corrections are needed because figures and tables do not match always with the text (look to the specific remarks). You do not present data on beta diversity that instead you had declared in the methods.
Response 2: Thank you. We have revised it one by one according to the specific remarks. We are sorry for this negligence, and we have deleted the beta diversity in the methods.
- The discussion is sufficiently thorough, but do not deal with the shannon diversity index that decrease significantly with BS.
Response 3: Thank you. “The shannon diversity index decreased significantly with BS (Table 2), indicating that community diversity decreased significantly with BS. We speculated that the reason for this result was the emergence of relatively abundant dominant bacteria in BS treatment, such as Proteobacteria (Fig.4).” We added this discussion in the page 10, lines 368-371.
- Conclusions are poor.
Response 4: Thanks. We have improved our conclusion as following: “The application of biochar alleviates salt stress in rice via modifying soil properties and regulating soil bacterial abundance and community structure in the present study. The application of biochar significantly decreased the value of EC, soluble Na+ and Cl− contents, and increased CEC, SOM, HA, total nitrogen, and total phosphorus contents in the soil. In addition, the application of biochar In addition, the application of biochar in-creased the richness of soil microbial community and modified the bacterial community structure, such as increased Proteobacteria abundance and decreased Chloroflexi abundance, which were considered to be eutrophic bacteria and oligotrophic bacteria, respectively. The soil EC was the most significant indicator affecting the soil bacterial community structure.” We added this part to lines 409-428 of the revision.
- You must check citations to meet the style requirements of the journal
Response 5: Thank you. We have revised it one by one according to the style requirements of the journal.
- Specific remarks
(6.1) Line 68: Why 667 m2?
Response 6.1: Thanks. 667 m2 equal to 1/15 of hm2, and we have converted it into hm2 in the revision. Lines 86-87.
(6.2) Line 93: sulfuric acid-potassium dichromate oxidation method with external heating?
Response 6.2: Thanks, we have revised it according to your suggestion.
(6.3) Line 175- 177: the values of SOM are corrected? Probably here are expressed in % and not in g/kg because are very lower compared to the values reported in the table 1.
Response 6.3: We are sorry for this mistake, and we have corrected it. In addition, we have moved this information to the materials and methods section. Lines 78-83.
(6.4) Line 250-252: You write that the relative abundance of Chloroflexi decreased by 45.46% in the B treatment compared with the CK, but in fig. 4 it seems a lot less, while you write that Chloroflexi decreased by 16.12% in the BS treatment compared to the S treatment, but in fig. 4 it seems a lot more
Response 6.4: We are sorry for this mistake, and we have corrected it and checked our manuscript throughout to avoid the same mistake. The relative abundance of Chloroflexi in four treatments are as follows: CK (15.14%); B (12.70%); S (17.18%); BS (9.37%). The amendments are as follows: The relative abundance of Chloroflexi decreased by 16.12% in the B treatment compared with the CK treatment, and by 45.46% in the BS treatment compared to the S treatment. We have made corrections in lines 269-270 of the revision.
(6.5) Line 268: you did not applicate biochar under salt stress in this study but tested soil amended with biochar to salt stress
Response 6.5: Thank you very much for your correction. We have corrected this in Lines 285-287 in the revision. “In our study, we tested soil amended with biochar to salt stress, and the biomasses of rice aboveground tissue and panicles were both significantly increased (Fig. 1),”
(6.6) Line 272: maybe there is a punctuation error, check the semicolon
Response 6.6: Thank you very much. We have made corrections in line 291 of the revision. “Importantly, ionic toxicity and the osmotic effects induced by salt directly inhibit plant growth [1], and the decreases of the soluble salt content and EC are helpful to alleviate salt stress in plants.”
(6.7) Line 291-292: check this sentence
Response 6.7: Thank you very much for your advice. We have modified the sentence as follows: “The changes of soil nutrient status after biochar amendment can also affect rice growth under salt stress. Soil nutrient status includes the concentrations change of SOM, CEC, N, P, K and so on.” We have made corrections in lines 312-313 of the revision.
(6.8) Line 338 – 339: is repetitive
Response 6.8: We are very sorry for the repetition. We have deleted the latter sentence in the revision.
Reviewer 2 Report
The manuscript describes significant improvements in rice yields and soil quality and nutrient use in saline soils treated with biochar. One major effect of biochar amendment is reducing salt stress while enhancing nutrient availability. Considering the significant proportion of salt stressed rice areas, the findings can have potentially important impacts on maintaining or increasing rice yields without need for further land conversion. Nevertheless, similar observations as adequately discussed, have been published before.
Therefore one important aspect of this research that could be discussed and complemented by some experimental work is the potential impact of biochar amendment on the greenhouse gas balance of rice cultivation, which is of major global concern.
Biochar, mostly derived from easily available waste materials used as soil amendment leads to significant carbon sequestration into soil.
Similarly, as mentioned, biochar reduces the release of nitrous oxides, powerful greenhouse gasses.
Furthermore, the impact of biochar on methane release from rice fields might be discussed.
With agriculture the dominant sector in GHG release and rice one of its dominant contributors, any methodology preventing GHG release from rice cultivation could be of huge importance and should be quantified if possible, or discussed in detail to enhance the impact of this manuscripts.
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 3 Report
I find the manuscript interesting for the scientific community with a well oriented experimental design and a good discussion of the results. However, there is a lack, in terms of the results are based in a low number of samples (12), so information derived in this study could be biased by that aspect.
More specific comments below.
Figures and tables: Please, define the meaning of all CK, B, S, BS treatments. All of them must be self explanatory.
Abstract: Line 24. I don't see from the redundancy analysis that EC was the most significant factor affecting soil bacteria communities.
Introduction: Lines 57-64. I don't see the relationship of baterial inocula with this research.
Material and methods:
Line 74. What does organic substitution means?
Lines 107-129. No subsections (1.3.1 to 1.3.5) are required
Lines 114-115. Which of all the primers noted were actually used?
Lines 150. Which reference database was used, RDP or SILVA as indicated in line 215.
Results and analysis: I suggest to place figures and tables after mentioning them in the text, not before.
Lines 165-166. I would refer changes in porcentages. Also from line 184 to 188.
Line 1710 Replace The data ... with .. Data values. Check in other tables this change too.
Lines 174-178. This information is already in the table. No needed here.
Line 180. Replace rice harvest ... with .. the crop cycle.
Line 216. "significantly decreased" ... Is this based on the statistical analyses. If so, indicate with letters in Figure 3.B.
Line 217. "Dramatically" doesn't sound very scientific. Besides, an increase from 4548 to 1657 OTUs is not so dramatic.
Line 224. Table 2. Please, check decimal format in values.
Line 245-247. Phyla analysis is ok, but more information can be derived from the microbiological technique emplyed. For example, main changes of Proteobacteria and Chloroflexi families between treatments could be highligthed. This information will be very interesting for other researchers.
Lines 258-259. Analysing the figure 5, I don't understand how the statement "Soil bacterial communities were mainly affected by soil pH and EC" can be done.
Discussion: Lines 372-374. Again, I don't see that EC was the most signifcant factor affecting the soil bacteria community structure.
Conclusion: This section can be a little more developed using the results obtained.
Author Response
> Reviewer 3
I find the manuscript interesting for the scientific community with a well oriented experimental design and a good discussion of the results. However, there is a lack, in terms of the results are based in a low number of samples (12), so information derived in this study could be biased by that aspect.
Response: Thank you point it out. As we displayed in the material and method section, the soil used for the cultivation experiment in the present study was obtained from Jiangsu Academy of Agriculture in Jiangsu Province, China, and collected from a long-term positioning experiment with three years of organic substitution treatment and organic substitution + biochar treatment.
Where, the application of nitrogen fertilizer was 240 kg N/hm2, of which 25% was replaced by organic substitution according to pure nitrogen content, and the P and K fertilizers were 60 kg P2O5/hm2 and 90 kg K2O/hm2, respectively. The application of biochar was 45000 kg/hm2.
When we got the soil, we treated them with or without 3 g NaCl per kg soil and then transplant the rice cultivar Jinyuan 85. The rice rhizosphere soils were obtained after rice harvest for all the soil parameters’ measurement. Therefore, considering the soil are obtained from the long-term positioning experiment, our results are credible and the biases can be avoided.
More specific comments below.
- Figures and tables: Please, define the meaning of all CK, B, S, BS treatments. All of them must be self explanatory.
Response 1: Thank you point it out. CK: organic substitution; B: organic substitution + biochar; S: CK + NaCl; BS: B + NaCl. We have added this information in all figures and tables.
- Abstract: Line 24. I don't see from the redundancy analysis that EC was the most significant factor affecting soil bacteria communities.
Response 2: Thank you point it out. We are sorry for the missing of statistical analyses data for the redundancy analysis and we have provide this results as the supplementary material (Table S1). We also changed this sentence to: “Soil bacterial communities were mainly affected by soil pH and EC (P<0.05).”
- Introduction: Lines 57-64. I don't see the relationship of baterial inocula with this research.
Response 3: Thanks. The introduction of baterial inocula in the introduction section is try to explain the important and mechanism of bacteria to improve plant salt resistance ability stress. In the present study, we measured the bacterial abundance and community structure in the different treatments, and the changes of bacteria might involve in regulating rice to improve salt resistance ability. Therefore, we added these information in the introduction section.
- Material and methods:
4.1 Line 74. What does organic substitution means?
Response 4.1: We are sorry that we didn't explain clearly here. Organic substitution is one treatment of Jiangsu Academy of Agriculture long-term positioning experiment with three years. Where, the application of nitrogen fertilizer was 240 kg N/hm2, of which 25% was replaced by organic substitution according to pure nitrogen content, and the P and K fertilizers were 60 kg P2O5/hm2 and 90 kg K2O/hm2, respectively.
4.2 Lines 107-129. No subsections (1.3.1 to 1.3.5) are required
Response 4.2: Thank you very much. We have deleted it.
4.3 Lines 114-115. Which of all the primers noted were actually used?
Response 4.3: the primer of 16S V4: 515F-806R was used in present study. We have revised it.
4.4 Lines 150. Which reference database was used, RDP or SILVA as indicated in line 215.
Response 4.4: Thank you point it out. SILVA was used and we have revised it.
- Results and analysis: I suggest to place figures and tables after mentioning them in the text, not before.
Response 5: Thank you. We revised it according to your nice suggestion.
5.1 Lines 165-166. I would refer changes in percentages. Also from line 184 to 188.
Response 5.1: Thanks, we have revised it according to your suggestions.
5.2 Line 171 Replace The data ... with .. Data values. Check in other tables this change too.
Response 5.2: Thank you. We have revised it and checked throughout our manuscript.
5.3 Lines 174-178. This information is already in the table. No needed here.
Response 5.3: Thank you very much to point it out. This is the information of original soil we got from the long-term positioning experiment before we added the salt. We put this information in the wrong place. In the revised manuscript, we have moved them to the materials and methods.
5.4 Line 180. Replace rice harvest ... with .. the crop cycle.
Response 5.4: Thank you very much for your advice. We have replaced it.
5.5 Line 216. "significantly decreased" ... Is this based on the statistical analyses. If so, indicate with letters in Figure 3.B.
Response 5.5: We have deleted “significantly”.
5.6 Line 217. "Dramatically" doesn't sound very scientific. Besides, an increase from 4548 to 1657 OTUs is not so dramatic.
Response 5.6: Thanks. We have deleted “dramatically”.
5.7 Line 224. Table 2. Please, check decimal format in values.
Response 5.7: Thanks. We have revised it.
5.8 Line 245-247. Phyla analysis is ok, but more information can be derived from the microbiological technique emplyed. For example, main changes of Proteobacteria and Chloroflexi families between treatments could be highligthed. This information will be very interesting for other researchers.
Response 5.8: Thank you very much for your suggestions. We have made a brief analysis on lines 429-440.
5.9 Lines 258-259. Analysing the figure 5, I don't understand how the statement "Soil bacterial communities were mainly affected by soil pH and EC" can be done.
Response 5.9: We are sorry for the missing of P-values when we do the redundancy analysis and we have provided it in the supplementary table 1. From the statistical analysis, only soil pH and EC are significant at the level of P < 0.05.
- Discussion: Lines 372-374. Again, I don't see that EC was the most signifcant factor affecting the soil bacteria community structure.
Response 6: We are sorry for the missing of statistical analyses data for the redundancy analysis and we have provided it in the supplementary table 1. From the statistical analysis, only soil pH and EC are significant at the level of P < 0.05.
- Conclusion: This section can be a little more developed using the results obtained.
Response 7: Thanks. We have improved our conclusion as following: “The application of biochar alleviates salt stress in rice via modifying soil properties and regulating soil bacterial abundance and community structure in the present study. The application of biochar significantly decreased the value of EC, soluble Na+ and Cl− contents, and increased CEC, SOM, HA, total nitrogen, and total phosphorus contents in the soil. In addition, the application of biochar in-creased the richness of soil microbial community and modified the bacterial community structure, such as increased Proteobacteria abundance and decreased Chloroflexi abundance, which were considered to be eutrophic bacteria and oligotrophic bacteria, respectively. Soil bacterial communities were mainly affected by soil pH and EC (P<0.05). This study provides a theoretical basis for alleviating saline-alkali paddy soil in the future.”
Reviewer 4 Report
The paper is devoted to the study of the effect of biochar on various chemical properties of the soil, as well as the effect on the bacterial communities of the soil under conditions of salt stress.
The title of the paper corresponds to the content.
The abstract reflects the main content of the paper. Greater emphasis is placed on changing the bacterial community of the soil. The authors should note the effect of biochar on plants under salt stress.
Research methods require additional explanation. It is necessary to describe the methods of microbiological studies carried out.
Lines 82-83: Add a description of the rice variety used (development phases and ripening times, average plant height).
Authors should pay more attention to describing the results obtained.
It is necessary to improve the quality of Figures 1 and 2.
Lines 273-276: how is the leaching of salts from the soil under the conditions of the model experiment, if a closed drainage system was used. If the drainage system was open, was the drainage run examined?
Author Response
Please see the attachment.
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
Nearly all my comments have been considered and/or replied. However, there is one I don't find in the text:
5.8 Line 245-247. Phyla analysis is ok, but more information can be derived from the microbiological technique emplyed. For example, main changes of Proteobacteria and Chloroflexi families between treatments could be highligthed. This information will be very interesting for other researchers.
Response 5.8: Thank you very much for your suggestions. We have made a brief analysis on lines 429-440.
I don't find in the file the supplied the Table1. Supplementary material.
Just solving this two issues I find the manuscript ready for publication.
Author Response
Nearly all my comments have been considered and/or replied. However, there is one I don't find in the text:
5.8 Line 245-247. Phyla analysis is ok, but more information can be derived from the microbiological technique emplyed. For example, main changes of Proteobacteria and Chloroflexi families between treatments could be highligthed. This information will be very interesting for other researchers.
Response 5.8: Thank you very much for your suggestions. We have made a brief analysis on lines 429-440.
Response: We are sorry for the misleading of your meanings when we answer this question at first time. Now we explored the changes of microorganisms between different treatments at family level in Fig. S1 and described it in our revised manuscript.
At the family level, the common dominant bacterial groups (relative abundance top 10) in the soil under all the treatments are: Gallionellaceae, Pedosphaeraceae, Prolixibacteraceae, Spirochaetaceae, Anaerolineaceae, Solibacteraceae, Sulfurimonadaceae, SC-I-84, Rhodocyclaceae, and Bryobacteraceae (Fig. S1). The Gallionellaceae family are the most abundance microorganism which belong to Proteobacteria phyla and displayed a higher abundance in BS treatment (15.79%) than other treatment (Fig. S1). The change trends of Anaerolineaceae, which belong to Chloroflexi, was same as that of phyla. The application biochar significantly decreased the abundance of Anaerolineaceae with or without salt supplication.
I don't find in the file the supplied the Table1. Supplementary material.
Response: Thanks. We are sorry for the misleading. We put the Table S1 in the main manuscript before the Reference section. The same to the Fig. S1.
Just solving this two issues I find the manuscript ready for publication.
Reviewer 4 Report
The authors answered questions from reviewers, but despite the positive aspects of this study, there are the following remarks:
82-86 – write notation
95 – delete ()
153 – Microbiological
218 – after 136.4% write «respectively»
231-232 – recalculate %, especially for SOM
573 – There is no CEC in Figure 5. Why do the soil pH, CEC, and SON content affect the structure of the soil microbial community?
574 – may be better to refer to Table S1
594 – round values in Table S1
594 – it is better to rename Table S1 as a one-way analysis of variance since it is one-way on line 178
Author Response
82-86 – write notation
Response: Thank you, we have revised it according to your suggestion.
95 – delete ()
Response: Thank you, we have deleted it.
153 – Microbiological
Response: Thank you, we have revised it.
218 – after 136.4% write «respectively»
Response: Thanks, we have added “respectively” in line 166.
231-232 – recalculate %, especially for SOM
Response: Thank you very much to point it out. We have recalculated and revised it.
573 – There is no CEC in Figure 5. Why do the soil pH, CEC, and SON content affect the structure of the soil microbial community?
Response: Thanks. We are sorry for the discrepancy between the picture and text in the manuscript and we have deleted this sentence.
574 – may be better to refer to Table S1
Response: Thanks. We have added reference of Table S1.
594 – round values in Table S1
Response: Thanks. We have revised it and retained three decimal for the values in the Table S1.
594 – it is better to rename Table S1 as a one-way analysis of variance since it is one-way on line 178
Response: Thanks. We have renamed the title of Table S1.
