Optimizing the Incorporated Amount of Chinese Milk Vetch (Astragalus sinicus L.) to Improve Rice Productivity without Increasing CH₄ and N₂O Emissions

Round 1

Reviewer 1 Report

This is a well written and formulated manuscript related to GHG emissions in rice paddies to which Chinese milkvetch was added in various amounts to replace synthetic urea fertilizer. 

General comments: The “traditional amount” of CMV used on rice paddies, as well as the conclusion that 15 t CMV/ha should be applied rather than the “traditional amount,” requires further explanation. Is the traditional amount used across all soil types and environmental conditions in China, or elsewhere, for that matter? I suspect that the answer is no, and that this is really the traditional amount in that area or region. If I am correct, then I believe the authors need to discuss how their results may or may not apply to other regions, and perhaps to other cover crop species.

Abstract: I think it should be noted that mcrA and nosZ are PCR primers, as indicated on L130.

L32: 108 needs the 8 to be a superscript.

L44: change ‘substituting’ to ‘substitute for’

L86: What drives this traditional amount? Table 1 indicates that MV is 22.5 t/ha of CMV applied. Is this the traditional amount and how could it be so specific when CMV yields would likely vary from year to year?

L165 (Figure 1 title): CMV should be written out as well as any other abbreviations in figure titles or in the figure so that figures can stand alone. A reader should not have to refer to a table to get this information.

L169 (Table 2 title): All necessary information should be contained in each table. A reader should not have to go to another table to find abbreviation definitions.

Figures and Tables: See notes for Lines 165 and 169. Each table and figure should be able to stand alone without having to look at another table to see what abbreviations mean.

There are some minor grammatical errors that can be easily remedied with a quick review of the manuscript.

Author Response

Response to Reviewer 1 Comments

We are very grateful to the reviewer for spending her/his valuable time to review this manuscript and appreciate the constructive comments! We have carefully made revisions according to the comments. These comments have enabled us to provide a highly improved manuscript. The revised parts can be viewed in the new manuscript using the “Track Changes” function.

 

Point 1: The “traditional amount” of CMV used on rice paddies, as well as the conclusion that 15 t CMV/ha should be applied rather than the “traditional amount,” requires further explanation. Is the traditional amount used across all soil types and environmental conditions in China, or elsewhere, for that matter? I suspect that the answer is no, and that this is really the traditional amount in that area or region. If I am correct, then I believe the authors need to discuss how their results may or may not apply to other regions, and perhaps to other cover crop species.

Response 1: Thanks for the insightful comments! The reviewer's comment is very important because it relates to the application value of this study. The traditional application level of CMV (22.5 t ha⁻¹) is not only utilized in the study area but also in many other regions of China, such as Jiangxi Province [1], Henan Province [2], Zhejiang Province [3], Anhui Province [4], and Hunan Province [5]. Moreover, a similar application level (25.8 t ha⁻¹) is utilized in South Korea [6]. The present results may provide a reference for these regions. However, GHG emissions from paddies are affected by many factors, such as climate, soil properties, rice variety, fertilizer type, and water management [7,8]. Therefore, the optimal incorporation amount of CMV (15.0 t ha⁻¹) should be validated in these regions. For other species of cover crop, considering that their decomposition rates and products differ from those of CMV, further experiments are needed to confirm whether the subscription ratio affects GHG emissions.

According to the reviewer’s comments, we revised the manuscript (marked in green) as follows:

Line 32 in “Abstract” section, added “in the study area” to describe the conclusion more carefully: MV2/3, which involved partial substitution of synthetic N fertilizer with 15.0 t ha⁻¹ of CMV, resulted in improved rice productivity without increasing CH₄ and N₂O emissions, making it a recommended approach in the study area.

Lines 381-389 in “Discussion” section, added the description: The incorporation amount of CMV in the MV treatment (22.5 t ha⁻¹) is also utilized in many other regions in China. A similar incorporation level (25.8 t ha⁻¹) has been reported in South Korea. Therefore, the present results may provide a reference for these regions. However, GHG emissions from paddies are affected by many factors, such as climate, soil properties, rice variety, fertilizer type, and water management. Therefore, the optimal incorporation amount of CMV (15.0 t ha⁻¹) should be validated in these regions. Considering that the decomposition rate and products of other cover crops differ from those of CMV, further experiments are needed to confirm whether the substitution ratio affects GHG emissions.

Lines 475 in “Conclusions” section, added “in the study area” to describe the conclusion more carefully: Therefore, to improve rice productivity while mitigating GHG emissions in the study area, incorporating CMV at 15.0 t ha⁻¹ to substitute 77.1 kg ha⁻¹ of urea is a more desirable practice than the common approach of using only synthetic fertilizer or the traditional CMV application rate (22.5 t ha⁻¹).

 

References

1. Fan, Q.; Xu, C.; Zhang, L.; Xie, J.; Zhou, G.; Liu, J.; Hu, F.; Gao, S.; Cao, W. Application of milk vetch (Astragalus sinicus L.) with reduced chemical fertilizer improves rice yield and nitrogen, phosphorus, and potassium use efficiency in southern China. European Journal of Agronomy 2023, 144, 126762.
2. Zhang, J.S.; Zheng, C.F.; Zhang, L.; Zhang, C.L.; Lv, Y.H.; Nie, L.P.; Zhang, X.N.; Li, B.Y.; Cao, W.D.; Li, M., et al. Effects of Chinese milk vetch returning on soil properties, microbial community, and rice yield in paddy soil. Sustainability 2022, 14, 16065.
3. Wu, C.Y.; Chen, Y.; Wang, J.Y.; Wang, S.J. Estimation of turnover and equilibrium of soil organic matter using a mathematical approach. Pedosphere 2006, 16, 634-645.
4. Xu, P.; Wu, J.; Wang, H.; Tang, S.; Cheng, W.; Li, M.; Bu, R.; Han, S.; Geng, M. Combined application of chemical fertilizer with green manure increased the stabilization of organic carbon in the organo-mineral complexes of paddy soil. Environmental Science and Pollution Research 2023, 30, 2676-2684.
5. Zhou, X.; Lu, Y.H.; Liao, Y.L.; Zhu, Q.D.; Cheng, H.D.; Nie, X.; Cao, W.D.; Nie, J. Substitution of chemical fertilizer by Chinese milk vetch improves the sustainability of yield and accumulation of soil organic carbon in a double-rice cropping system. Journal of Integrative Agriculture 2019, 18, 2381-2392.
6. Kim, S.Y.; Gutierrez, J.; Kim, P.J. Considering winter cover crop selection as green manure to control methane emission during rice cultivation in paddy soil. Agric., Ecosyst. Environ. 2012, 161, 130-136.
7. Wang, W.; Sardans, J.; Wang, C.; Zeng, C.; Tong, C.; Asensio, D.; Peñuelas, J. Relationships between the potential production of the greenhouse gases CO₂, CH₄ and N₂O and soil concentrations of C, N and P across 26 paddy fields in southeastern China. Atmospheric Environment 2017, 164, 458-467.
8. Win, E.P.; Win, K.K.; Bellingrath-Kimura, S.D.; Oo, A.Z. Influence of rice varieties, organic manure and water management on greenhouse gas emissions from paddy rice soils. PLoS ONE 2021, 16 e0253755.

 

Point 2: Abstract: I think it should be noted that mcrA and nosZ are PCR primers, as indicated on L130.

Response 2: Thanks for the reviewer’s comment! Based on the reviewer’s comment and referred to other published literature [9,10], we added “gene” and revised the related parts throughout the manuscript to make the description more accurately.

 

References

9. Chen, D.; Zhou, Y.; Xu, C.; Lu, X.; Liu, Y.; Yu, S.; Feng, Y. Water-washed hydrochar in rice paddy soil reduces N₂O and CH₄ emissions: A whole growth period investigation, Environmental Pollution 2021, 274, 116573.
10. Li, H.; Meng, J.; Liu, Z.; Lan, Y.; Yang, X.; Huang, Y.; He, T.; Chen, W. Effects of biochar on N₂O emission in denitrification pathway from paddy soil: A drying incubation study. Science of the total environment 2021, 787, 147591.

 

Point 3: L32: 108 needs the 8 to be a superscript.

Response 3: Thanks for the reviewer’s comment! We corrected this error and checked those should be superscript or subscript throughout the manuscript.

 

Point 4: L44: change ‘substituting’ to ‘substitute for’

Response 4: Thanks for the reviewer’s comment! We corrected it and carefully checked the grammatical errors throughout the manuscript.

 

Point 5: L86: What drives this traditional amount? Table 1 indicates that MV is 22.5 t/ha of CMV applied. Is this the traditional amount and how could it be so specific when CMV yields would likely vary from year to year?

Response 5: Thanks for the reviewer’s comment! The traditional amount of CMV is 22.5 t/ha (the MV treatment). Farmers in many rice producing areas of China are used to applying CMV seeds with about 30 kg/ha. This sowing level corresponds to about 22.5 t/ha of CMV fresh weight in normal years. In this study, we set the different gradients of CMV by shifting the sowing level. For instance, 15.0 t/ha of CMV corresponds to 20 kg/ha seeds. To make the treatments accurately, before the incorporation, we collected CMV samples to test the actual fresh weight in each plot. When the fresh weight in a plot was deficient, we supplemented CMV from other fields. When it was superfluous, we removed parts of CMV.

According to the reviewer’s comments, we revised the “Experimental description” section. Detailed revisions marked in green are as follows:

Lines 116-117: There were five treatments with three replications in this experiment: urea only (CF), incorporating a traditional amount of CMV (sowing seeds with 30 kg ha⁻¹ and incorporating CMV with 22.5 t ha⁻¹ fresh weight) to partially substitute urea (MV), and incorporating 1/3 (MV1/3), 2/3 (MV2/3), and 4/3 (MV4/3) of MV to partially substitute urea. CF is a common management approach in paddy fields, and MV has been re-popularized in recent years. The other treatments are newly developed methods that need to be further explored. Before incorporation, CMV samples were collected to test the actual fresh weight in each plot. When the fresh weight in a plot was deficient, CMV was supplemented from other fields. When it was superfluous, some CMV was removed. The urea application amounts were designed based on the N inputs of CMV incorporation to ensure that the total N input was equal among treatments (Table 1).

 

Point 6: L165 (Figure 1 title): CMV should be written out as well as any other abbreviations in figure titles or in the figure so that figures can stand alone. A reader should not have to refer to a table to get this information.

Response 6: The reviewer’s comment makes sense! We have revised all the abbreviations in figure titles to make them more convenient to readers. Detailed revisions marked in green are as follows:

Figure 1. Rice yields affected by the substitution of synthetic N fertilizer with different amounts of Chinese milk vetch.

The vertical bars denote the standard errors of the means (n = 3). The different lowercase letters indicate significant differences among the means under different treatments in the same year (P < 0.05). CF, MV1/3, MV2/3, MV, and MV4/3 indicate incorporating 0, 7.5, 15.0, 22.5, and 30.0 t ha⁻¹ of Chinese milk vetch, respectively, to partially substitute urea.

Figure 2. CH₄ emission rate affected by the substitution of synthetic N fertilizer with different amounts of Chinese milk vetch.

The vertical bars denote the standard errors of the means (n = 3). CF, MV1/3, MV2/3, MV, and MV4/3 indicate incorporating 0, 7.5, 15.0, 22.5, and 30.0 t ha⁻¹ of Chinese milk vetch, respectively, to partially substitute urea.

Figure 3. N₂O emission rate affected by the substitution of synthetic N fertilizer with different amounts of Chinese milk vetch.

The vertical bars denote the standard errors of the means (n = 3). CF, MV1/3, MV2/3, MV, and MV4/3 indicate incorporating 0, 7.5, 15.0, 22.5, and 30.0 t ha⁻¹ of Chinese milk vetch, respectively, to partially substitute urea.

Figure 4. Cumulative emissions of CH₄ affected by the substitution of synthetic N fertilizer with different amounts of Chinese milk vetch.

The vertical bars denote the standard errors of the means (n = 3). Different lowercase letters indicate significant differences among the means under different treatments in the same year (P < 0.05). CF, MV1/3, MV2/3, MV, and MV4/3 indicate incorporating 0, 7.5, 15.0, 22.5, and 30.0 t ha⁻¹ of Chinese milk vetch, respectively, to partially substitute urea.

Figure 5. Cumulative emissions of N₂O affected by the substitution of synthetic N fertilizer with different amounts of Chinese milk vetch.

The vertical bars denote the standard errors of the means (n = 3). Different lowercase letters indicate significant differences among the means under different treatments in the same year (P < 0.05). CF, MV1/3, MV2/3, MV, and MV4/3 indicate incorporating 0, 7.5, 15.0, 22.5, and 30.0 t ha⁻¹ of Chinese milk vetch, respectively, to partially substitute urea.

Figure 6. Abundances of the functional genes affected by the substitution of synthetic N fertilizer with different amounts of Chinese milk vetch.

Samples were collected in 2021. The vertical bars denote the standard errors of the means (n = 3). Different lowercase letters indicate significant differences among the means under different treatments (P < 0.05). CF, MV1/3, MV2/3, MV, and MV4/3 indicate incorporating 0, 7.5, 15.0, 22.5, and 30.0 t ha⁻¹ of Chinese milk vetch, respectively, to partially substitute urea.

Figure 7. Directed graph of the partial least squares-path model.

MV:CF denotes the ratio of N from Chinese milk vetch to N from urea. SOC: soil organic carbon, DOC: dissolved organic carbon, TN: total nitrogen, AN: available nitrogen, AP: available phosphorus, AK: available potassium. Each box denotes an observed or latent variable. SOC, DOC, TN, AN, AP, and AK were loaded for the latent variable of soil nutrients. The numbers on the arrows and the arrow widths represent the path coefficients. The green and red arrows reflect positive and negative effects, respectively (P < 0.05). The dashed arrows indicate that the coefficients do not differ significantly from 0 (P > 0.05).

Figure S2. Pearson’s correlations between the abundances of functional genes and soil nutrients.

The blue and red ellipses denote significantly positive and negative correlations, respectively (n=15, P < 0.05). The color shades represent correlation coefficients (r). SOC: soil organic carbon, DOC: dissolved organic carbon, TN: total nitrogen, AN: available nitrogen, AP: available phosphorus, AK: available potassium.

 

Point 7: L169 (Table 2 title): All necessary information should be contained in each table. A reader should not have to go to another table to find abbreviation definitions.

Figures and Tables: See notes for Lines 165 and 169. Each table and figure should be able to stand alone without having to look at another table to see what abbreviations mean.

Response 7: The reviewer’s comment makes sense! We have revised all the abbreviations in table titles to make them more convenient to readers. Detailed revisions marked in green are as follows:

Table 1. Incorporation amounts of Chinese milk vetch and N inputs under different treatments.

CMV denotes Chinese milk vetch. MV: CF denotes the ratio of N from Chinese milk vetch to N from urea.

Table 2. Soil properties affected by the substitution of synthetic N fertilizer with different amounts of Chinese milk vetch.

Samples were collected in 2021. Values are reported as the means ± SEs. Different lowercase letters indicate significant differences (P < 0.05) in the variable means among treatments in the same year. ¹ SOC: soil organic carbon, DOC: dissolved organic carbon, TN: total nitrogen, AN: available nitrogen, AP: available phosphorus, AK: available potassium. CF, MV1/3, MV2/3, MV, and MV4/3 indicate incorporating 0, 7.5, 15.0, 22.5, and 30.0 t ha⁻¹ of Chinese milk vetch, respectively, to partially substitute urea.

Table 3. Global warming potential and greenhouse gas intensity affected by the substitution of synthetic N fertilizer with different amounts of Chinese milk vetch.

Different lowercase letters indicate significant differences in the variable means among treatments in the same year at P < 0.05. CF, MV1/3, MV2/3, MV, and MV4/3 indicate incorporating 0, 7.5, 15.0, 22.5, and 30.0 t ha⁻¹ of Chinese milk vetch, respectively, to partially substitute urea.

 

Point 8: There are some minor grammatical errors that can be easily remedied with a quick review of the manuscript.

Response 8: Thanks for the reviewer’s comment! According to reviewer’s comment, we carefully checked the grammatical errors and corrected them throughout the manuscript. Detailed revisions marked in green are as follows:

• Lines 14-32: We revised the “Abstract” section according to another reviewer’s comment.
• We replaced “GHGs emissions” with “GHG emissions” throughout the manuscript.
• Lines 56-58: Chinese milk vetch (Astragalus sinicus; CMV) is a leguminous green manure and winter cover crop that is widely grown in paddy fields in most Asian countries and is an important N source that can partially substitute for synthetic fertilizer.
• Lines 63-65: Some reported results have indicated that CMV incorporation does not significantly stimulate CH₄ emissions compared to treatments without residue amendment.
• Lines 80-82: Change “CMV has been typically considered to restrict N₂O emissions by partially replacing inorganic N fertilizer and due to its relatively slow mineralization rate” to “CMV has typically been considered to restrict N₂O emissions by partially replacing inorganic N fertilizer and due to its relatively slow mineralization rate”.
• Lines 103: Change “The experimental field plots were located in Yijiang Town, Wuhu City, China” to “The experimental field plots were located in Yijiang town, Wuhu city, China”.
• Table 1: Change “Applying rate” to “Application rate”.
• Lines 173: Change “At the maturity stage every year, rice plants were harvest in each plot.” to “At the mature stage every year, rice plants were harvested in each plot”.
• Lines 189: Change “The differences between the MV2/3 and MV treatments were insignificant” to “The differences between the MV2/3 and MV treatments were not statistically significant”.
• Lines 194: Change “The results indicated that partial substitution synthetic N fertilizer with a certain amount of CMV could increase the rice yield.” to “The results indicated that partial substitution of synthetic N fertilizer with a certain amount of CMV could increase the rice yield.”
• Change “Vertical bars denote the standard error of the mean” to “The vertical bars denote the standard errors of the means” in the captions of all figures.
• In the caption of table 2, change “Values are reported as the mean ± SE” to “Values are reported as the means ± SEs”.
• Lines 340-341: Change “The enhancement of rice yield may be attributed to the following reasons” to “Possible reasons for the enhancement of rice yield are as follows”.
• Lines 347-348: Change “promotes the accumulation of photosynthetic and its transportation to grain” to “promotes the accumulation of photosynthetic products and their transportation to grains”.
• Line 402: Change “activating unavailable K into available” to “converting unavailable K into available K”.
• Lines 409-410: Change “Another study also reports that the increasing pH in the soil under organic amendment” to “Another study also reported that the increasing pH in the soil under organic amendment”.
• Line 433: Change “probably because K plays a limiting factor in microorganisms growth” to “probably because K plays a limiting factor in microorganism growth”.
• Line 450: Change “the direct effects above were also observed in other studies” to “the direct effects reported above were also observed in other studies”.
• Lines 472-473: Change “which stimulated CH₄ and mitigated N₂O emissions” to “which stimulated CH₄ emissions and mitigated N₂O emissions”.
• Lines 476-477: Change “is a more desirable practice than the common practices of using only synthetic fertilizer” to “is a more desirable practice than the common approach of using only synthetic fertilizer”.

Moreover, we applied native English speaking editors to check the language of this manuscript:

 

We sincerely appreciate the reviewer’s work on this study. Best wishes!

Author Response File: Author Response.docx

Reviewer 2 Report

I have read the manuscript entitled “Optimizing the incorporated amount of Chinese milk vetch (Astragalus sinicus L.) to improve rice productivity without increasing CH₄ and N₂O emissions”. This is an excellent manuscript that conducted a three-year field experiment to investigate the emissions under different substitution ratios: urea only (CF), incorporating traditional amount of CMV (MV), and incorporating 1/3 (MV1/3), 2/3 (MV2/3), and 4/3 17 (MV4/3) of MV to partially substitute urea.. Although, I am satisfied with the overall presentation skills and writing of the manuscript. I am agree to highly recommend this paper for publication in the journal with minor spell check and errors in the manuscript.

Author Response

Response to Reviewer 2 Comments

 

We are very grateful to the reviewer for spending her/his valuable times to review this manuscript and appreciate the constructive comments! We have carefully made revisions according to the comments. These comments have enabled us to provide a highly improved manuscript. The revised parts can be viewed in the new manuscript using the “Track Changes” function.

 

Point 1: I have read the manuscript entitled “Optimizing the incorporated amount of Chinese milk vetch (Astragalus sinicus L.) to improve rice productivity without increasing CH₄ and N₂O emissions”. This is an excellent manuscript that conducted a three-year field experiment to investigate the emissions under different substitution ratios: urea only (CF), incorporating traditional amount of CMV (MV), and incorporating 1/3 (MV1/3), 2/3 (MV2/3), and 4/3 (MV4/3) of MV to partially substitute urea. Although, I am satisfied with the overall presentation skills and writing of the manuscript. I am agree to highly recommend this paper for publication in the journal with minor spell check and errors in the manuscript.

Response 1: Thanks for the comments! We carefully checked the grammatical errors and corrected them throughout the manuscript. Detailed revisions marked in green are as follows:

• Lines 14-32: We revised the “Abstract” section according to another reviewer’s comment.
• We replaced “GHGs emissions” with “GHG emissions” throughout the manuscript.
• Lines 56-58: Chinese milk vetch (Astragalus sinicus; CMV) is a leguminous green manure and winter cover crop that is widely grown in paddy fields in most Asian countries and is an important N source that can partially substitute for synthetic fertilizer.
• Lines 63-65: Some reported results have indicated that CMV incorporation does not significantly stimulate CH₄ emissions compared to treatments without residue amendment.
• Lines 80-82: Change “CMV has been typically considered to restrict N₂O emissions by partially replacing inorganic N fertilizer and due to its relatively slow mineralization rate” to “CMV has typically been considered to restrict N₂O emissions by partially replacing inorganic N fertilizer and due to its relatively slow mineralization rate”.
• Lines 103: Change “The experimental field plots were located in Yijiang Town, Wuhu City, China ” to “The experimental field plots were located in Yijiang town, Wuhu city, China ”.
• Table 1: Change “Applying rate” to “Application rate”.
• Lines 173: Change “At the maturity stage every year, rice plants were harvest in each plot.” to “At the mature stage every year, rice plants were harvested in each plot”.
• Lines 189: Change “The differences between the MV2/3 and MV treatments were insignificant” to “The differences between the MV2/3 and MV treatments were non-significant”.
• Lines 194: Change “The results indicated that partial substitution synthetic N fertilizer with a certain amount of CMV could increase the rice yield.” to “The results indicated that partial substitution of synthetic N fertilizer with a certain amount of CMV could increase the rice yield.”
• Change “Vertical bars denote the standard error of the mean” to “The vertical bars denote the standard errors of the means” in the captions of all figures.
• In the caption of table 2, change “Values are reported as the mean ± SE” to “Values are reported as the means ± SEs”.
• Lines 340-341: Change “The enhancement of rice yield may be attributed to the following reasons” to “ Possible reasons for the enhancement of rice yield are as follows”.
• Lines 347-348: Change “promotes the accumulation of photosynthetic and its transportation to grain” to “promotes the accumulation of photosynthetic products and their transportation to grains”.
• Line 402: Change “activating unavailable K into available” to “converting unavailable K into available K”.
• Lines 409-410: Change “Another study also reports that the increasing pH in the soil under organic amendment” to “Another study also reported that the increasing pH in the soil under organic amendment”.
• Line 433: Change “probably because K plays a limiting factor in microorganisms growth” to “probably because K plays a limiting factor in microorganism growth”.
• Line 450: Change “the direct effects above were also observed in other studies” to “the direct effects reported above were also observed in other studies”.
• Lines 472-473: Change “which stimulated CH₄ and mitigated N₂O emissions” to “which stimulated CH₄ emissions and mitigated N₂O emissions”.
• Lines 476-477: Change “is a more desirable practice than the common practices of using only synthetic fertilizer” to “is a more desirable practice than the common approach of using only synthetic fertilizer”.

Moreover, we applied native English speaking editors to check the language of this manuscript:

We sincerely appreciate the reviewer’s work on this study. Best wishes!

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript reports on the impact on N2O and CH4 of incorporating Chinese milk vetch to improve rice productivity

line 50 'Compared to the green manure with a high C/N ratio, such as ryegrass and oilseed rape, CMV shows a low C/N ratio'  - please add the C/N ratios for each.  does the glucosinolates in oilseed rape impact the function of methanogenic bacteria?

line 55 'N2O emissions are related to nitrification-denitrification processes and are directly influenced by the inorganic N applied during agricultural cultivation' - are also related to water filled pore space which shifts the balance between the 2 pathways which is important in rice production, need to expand this further.  temperature also has an impact.  the mineralisation of organic material will release N2O, need to add text about this.

line 61 'affected by the incorporation volume of green manure, which has a different influence on related functional microorganisms' - please explain why

Line 83 'field experiment was conducted from 2019 to 2021.' (10 m X 4 m) experimental plots 3 replicates for 5 treatments 15 in total.  The analysis is appropriate, the only reservation I have is the low number of replicates (n=3), I appreciate available resources may have dictated this.  Can an explanation of the design and why this number has been used be provided.

Discussion of of functional gene abundance is interesting, but is subject to the caveat that there are 3 replicates for each treatment.

Author Response

Response to Reviewer 3 Comments

 

We are very grateful to the reviewer for spending her/his valuable times to review this manuscript and appreciate the constructive comments! We have carefully made revisions according to the comments. These comments have enabled us to provide a highly improved manuscript. The revised parts can be viewed in the new manuscript using the “Track Changes” function.

 

Point 1: line 50 'Compared to the green manure with a high C/N ratio, such as ryegrass and oilseed rape, CMV shows a low C/N ratio'  - please add the C/N ratios for each.  does the glucosinolates in oilseed rape impact the function of methanogenic bacteria?

Response 1: Thanks for the insightful comments! According to the reviewer’s comments, we added the C/N ratios. Detailed revisions marked in green are as follows:

Lines 67-70: CMV shows a low C/N ratio (~15) and has a much weaker impact on CH₄ emissions than green manure with a high C/N ratio, such as ryegrass (~36) and oilseed rape (~25).

We searched the literature related to the effect of glucosinolates on CH₄ emissions on Web of Science utilizing the formula, “methane or CH₄” and “glucosinolates”. We found that the reported studies mainly focused on ruminants. The effect of glucosinolates from cruciferous green manure on CH₄ emissions is an interesting subject. We are very grateful to the reviewer for providing us with a promising research direction!

 

Point 2: line 55 'N₂O emissions are related to nitrification-denitrification processes and are directly influenced by the inorganic N applied during agricultural cultivation' - are also related to water filled pore space which shifts the balance between the 2 pathways which is important in rice production, need to expand this further.  temperature also has an impact.  the mineraliation of organic material will release N₂O, need to add text about this.

Response 2: Thanks for the insightful comments! The comments make the background of this study more comprehensive and complete. We added the description according to the reviewer’s suggestion. Detailed revisions marked in green are as follows:

Lines 76-80: N₂O emissions are related to nitrification-denitrification processes and are directly influenced by the inorganic N applied during agricultural cultivation [15]. Water management in paddy fields, such as flooding and drainage, will shift the balance between nitrification and denitrification and then affect N₂O emissions [16]. Soil temperature is another nonnegligible factor affecting N₂O emissions [17]. Moreover, the mineralization process of organic amendments produces N₂O [18]

 

Point 3: line 61 'affected by the incorporation volume of green manure, which has a different influence on related functional microorganisms' - please explain why

Response 3: Thanks for the insightful comments! We revised this part according to the reviewer’s suggestion. Detailed revisions marked in green are as follows:

Lines 88-91: In addition to the type of green manure, paddy soil properties are also directly affected by the incorporation volume of green manure. The incorporation volume regulates soil pH and SOC stocks, which elicit alterations in the composition and abundance of soil microorganisms, especially the functional genera related to C and N cycling [23,24].

 

Point 4: Line 83 'field experiment was conducted from 2019 to 2021.' (10 m X 4 m) experimental plots 3 replicates for 5 treatments 15 in total.  The analysis is appropriate, the only reservation I have is the low number of replicates (n=3), I appreciate available resources may have dictated this.  Can an explanation of the design and why this number has been used be provided.

Response 4: Thanks for the insightful comments! When designing the study, we referred to other studies on greenhouse gas emissions from paddy fields, especially for the related functional gene abundance. Many plot experiments conducted in a paddy filed utilize 3 replicates [1-11]. We are very grateful for the reviewer’s valuable comments and will set more replicates in the future research.

References:

1

Kim SY, Gutierrez J, Kim PJ. Considering winter cover crop selection as green manure to control methane emission during rice cultivation in paddy soil. Agric., Ecosyst. Environ. 2012, 161, 130-136.

2

Yuan J, Yuan Y, Zhu Y, et al. Effects of different fertilizers on methane emissions and methanogenic community structures in paddy rhizosphere soil, Science of The Total Environment. 2018, 627,770-781.

3

Qin H, Tang Y, Shen J, et al. Abundance of transcripts of functional gene reflects the inverse relationship between CH₄ and N₂O emissions during mid-season drainage in acidic paddy soil. Biology and Fertility of Soils. 2018, 54, 885-895.

4.  

Zhang W, Sheng R, Zhang M, et al. Effects of continuous manure application on methanogenic and methanotrophic communities and methane production potentials in rice paddy soil. Agriculture, Ecosystems & Environment. 2018, 258, 121-128.

5.  

Bertora C, Cucu MA, Lerda C, et al. Dissolved organic carbon cycling, methane emissions and related microbial populations in temperate rice paddies with contrasting straw and water management. Agriculture, Ecosystems & Environment. 2018, 265, 292-306.

6.  

Kim J, Yoo G, Kim D, et al. Combined application of biochar and slow-release fertilizer reduces methane emission but enhances rice yield by different mechanisms. Applied Soil Ecology. 2017, 117-118, 57-62.

7.  

Liu Y, Tang H, Muhammad A, et al. The effects of Chinese milk vetch returning with nitrogen fertilizer on rice yield and greenhouse gas emissions. Greenhouse Gases: Science and Technology. 2019, 9, 743-753.

8.  

Raheem A, Zhang J, Huang J, et al. Greenhouse gas emissions from a rice-rice-green manure cropping system in South China. Geoderma. 2019, 353, 331-339.

9.  

Zhou X, Lu Y H, Liao YL, et al. Substitution of chemical fertilizer by Chinese milk vetch improves the sustainability of yield and accumulation of soil organic carbon in a double-rice cropping system. Journal of Integrative Agriculture, 2019, 18, 2381-2392.

10.  

Lan T, Li M, Han Y, et al. How are annual CH₄, N₂O, and NO emissions from rice–wheat system affected by nitrogen fertilizer rate and type? Applied Soil Ecology. 2020, 150, 103469.

11.  

Dong D, Li J, Ying S, et al. Mitigation of methane emission in a rice paddy field amended with biochar-based slow-release fertilizer. Science of The Total Environment. 2021, 792, 148460.

 

Point 5: Discussion of of functional gene abundance is interesting, but is subject to the caveat that there are 3 replicates for each treatment.

Response 5: Thanks for the insightful comments! As mentioned above, many studies utilize 3 replicates to explore the effect of different fertilization on functional gene abundances in paddy fields. Moreover, we cited many references to support our conclusions in the manuscript:

• Chinese milk vetch → soil properties → functional gene abundances.

Lines 406-409: Methanogenic archaea are sensitive to increases in the pH of acidic paddy soil, and even a small increase from 6.3 to 6.6 will enhance the abundance of methanogens and stimulate CH₄ production [46,47], which was consistent with our results (Table 2, Figure 6a).

Lines 409-411: Another study also reports that the increasing pH in the soil under organic amendment positively regulates the gene abundance of mcrA [48].

Lines 412-413: A significantly positive direct effect of pH on the nirS gene was also observed in a structural equation model study [49].

Lines 421-422: As an important external source of DOC in paddies, organic residues positively regulate the dynamics of methanogens [51].

Lines 423-424: Other studies have similarly observed a significant relationship between DOC and methanotrophs [52,53].

Lines 426-428: Plant residue incorporation increases the assembly of nitrous oxide reductase (expression of the nosZ gene) by supplying labile carbon, an essential energy source for denitrifying bacteria [55].

• Functional gene abundances → GHGs emissions.

Lines 436-443: CH₄ is the final product of methanogens. The mcrA gene encodes the alpha subunit of the enzyme methyl coenzyme M reductase, which catalyzes the terminal step in biogenic methane production. Therefore, gene copies of mcrA are widely used to reflect the methanogen abundances and methanogenesis potentials [57]. Conversely, CH₄ can be utilized or oxidized by methanotrophs to produce organic tissue or CO₂. The pmoA gene encodes the subunit of the particulate methane monooxygenase that is found in all methanotrophs. Therefore, gene copies of pmoA are widely used to reflect the methanotroph abundances and CH₄ oxidation potentials [58].

Lines 450-452: Furthermore, the direct effects reported above were also observed in other studies [14,23], suggesting that the gene abundance of mcrA could be used as a predictor for CH₄ emissions in paddies with CMV incorporation.

Lines 455-459: Nitrite reduction is the critical and rate-limiting step in the denitrification process. Nitrite reductase is divided into soluble copper-containing enzymes and cytochrome enzymes, encoded by the nirK and nirS genes, respectively. Nitrous oxide reductase converts the greenhouse gas N₂O to harmless N₂ and determines the end product of denitrification, encoded by nosZ genes [59]. The nosZ/(nirK + nirS) ratio indicates the proportion of N₂O that is reduced to N₂ [61].

We sincerely hope that these explanations can allay reviewer’s concerns.

 

We are very grateful for the reviewer’s work on this manuscript. Best wishes!

Author Response File: Author Response.docx

Reviewer 4 Report

1.       The Introduction provides a clear overview of the research background the objectives. However, it would be helpful to provide more specific information on relevant studies investigating GHG emissions from paddy fields, discuss their similarities and differences, and articulate how this study can further advance our current understanding.

 

2.       It is important to clarify the controversy surrounding the impacts of Chinese milk vetch (CMV) incorporation on CH4 and N2O emissions. Provide a brief background on the conflicting results from previous studies to highlight the significance of this research.

 

3.       The article mentions different substitution ratios for CMV incorporation but does not provide clear information on how these ratios were determined or what they represent. Clarify how the substitution ratios were calculated and explain their practical relevance in agricultural settings.

 

4.       Regarding on the statement that "the mcrA and nosZ abundances indirectly regulated by the substitution ratios affected CH4 and N2O emissions”, elaborate on the mechanism or pathway through which these abundances influence emissions, providing relevant scientific background or references to support the claim.

 

5.       The recommendation for MV2/3 as a suitable substitution ratio is made based on its ability to improve rice productivity without increasing CH4 and N2O emissions. Provide more information on the factors that contribute to this favorable outcome and explain why MV2/3 is specifically recommended over other substitution ratios.

 

6.       Consider including limitations or potential biases of the study. Acknowledge any constraints in the research methodology or data interpretation that could affect the generalizability of the findings.

 

7.       Proofread the abstract for grammatical errors and ensure that the information is presented in a clear and concise manner.

The English language writing in the research article is generally clear and concise. The authors effectively communicate the main objectives, methods, and results of their study. However, there are a few areas where the language could be improved for better clarity and readability. Here are some suggestions using the “Abstract” as an example:

 

In the first sentence, consider rephrasing "that is widely grown in paddies" to "commonly cultivated in paddy fields" or a similar construction.

In sentence 2, it would be clearer to state "However, the impacts of incorporating CMV on CH4 and N2O emissions are still a subject of controversy."

In sentence 3, consider rephrasing "we conducted a three-year field experiment" to "we conducted a field experiment over three years" for smoother flow.

In sentence 4, instead of "incorporating 1/3 (MV1/3), 2/3 (MV2/3), and 4/3 (MV4/3) of MV to partially substitute urea," it would be clearer to say "with substitution ratios of 1/3 (MV1/3), 2/3 (MV2/3), and 4/3 (MV4/3) of MV for partial urea substitution."

In sentence 5, consider rephrasing "MV2/3, MV, and MV 4/3 showed a yield-increasing effect" to "MV2/3, MV, and MV4/3 resulted in increased yields."

In sentence 7, instead of "MV and MV4/3 decreased N2O emissions but promoted CH4 emissions," it would be clearer to say "MV and MV4/3 reduced N2O emissions but increased CH4 emissions."

In sentence 7, consider rephrasing "causing an increase in total global warming potential" to "resulting in an overall increase in global warming potential."

In sentence 8, instead of "MV2/3 showed a low greenhouse gas intensity value (0.46–0.47)," it would be clearer to say "MV2/3 exhibited a low greenhouse gas intensity value ranging from 0.46 to 0.47."

In sentence 9, consider rephrasing "the mcrA and nosZ abundances indirectly regulated by the substitution ratios affected CH4 and N2O emissions" to "CH4 and N2O emissions were influenced by the substitution ratios, which indirectly regulated the abundances of mcrA and nosZ."

In sentence 10, instead of "whether the CMV affected CH4 and N2O emissions was determined by the substitution ratios," it would be clearer to say "the impact of CMV on CH4 and N2O emissions was determined by the substitution ratios."

In sentence 11, instead of "MV2/3, which partially substituted synthetic N fertilizer with 15.0 t ha−1 of CMV, improved rice productivity without increasing CH4 and N2O emissions and is thus recommended," it would be clearer to say "MV2/3, which involved partial substitution of synthetic N fertilizer with 15.0 t ha−1 of CMV, resulted in improved rice productivity without increasing CH4 and N2O emissions, making it a recommended approach."

 

By incorporating these suggestions, the language in the research article will become more precise and easier to understand for readers. Please check carefully throughout the manuscript.

Author Response

Response to Reviewer 4 Comments

We are very grateful to the reviewer for spending her/his valuable times to review this manuscript and appreciate the constructive comments! We have carefully made revisions according to the comments. These comments have enabled us to provide a highly improved manuscript. The revised parts can be viewed in the new manuscript using the “Track Changes” function.

 

Point 1: The Introduction provides a clear overview of the research background the objectives. However, it would be helpful to provide more specific information on relevant studies investigating GHG emissions from paddy fields, discuss their similarities and differences, and articulate how this study can further advance our current understanding.

Response 1: Thanks for the insightful comments! According to the reviewer’s comments, we revised the “Introduction” section to provide more specific information, to indicate the insufficient of previous studies, and to articulate the value of this study. Detailed revisions marked in green are as follows:

To improve soil quality and increase rice productivity, the combined application of green manure and synthetic fertilizer in paddy fields is encouraged [3]. In agroecosystems, the production and consumption of CH₄ and N₂O are usually driven by microorganisms associated with the carbon (C) and nitrogen (N) cycles in farmlands, such as methanogenic archaea, methanotrophic bacteria, ammonia oxidizers and denitrifiers [4]. The combination of organic material and synthetic fertilizer affects the above-mentioned microorganisms by changing the substrate composition, soil nutrients, pH, and redox potential, which will likely alter CH₄ and N₂O emissions from paddy fields [5]. Therefore, confirming the specific impacts of the combined application on GHGs is significant for determining the application value of green manure.

Chinese milk vetch (Astragalus sinicus L.; CMV) is a leguminous green manure and winter cover crop that is widely grown in paddy fields in most Asian countries and is an important N source that can partially substitute for synthetic fertilizer [3]. Because CMV plays an important role in N management and yield increases in paddy fields, many studies have recently focused on the impacts of CMV incorporation on GHG emissions from paddy fields [6,7]. However, the current conclusions are ambiguous, limiting the promotion of CMV.

Some reported results have indicated that CMV incorporation does not significantly stimulate CH₄ emissions compared to treatments without residue amendment [8,9,10]. CMV shows a low C/N ratio (~15) and has a much weaker impact on CH₄ emissions than green manure with a high C/N ratio, such as ryegrass (~36) and oilseed rape (~25). The cumulative emissions under CMV treatments are 57.36%‒64.42% and 73.11%‒78.86% of those under ryegrass and rape treatments, respectively [10,11]. However, some studies have suggested that CMV incorporation remarkably increases CH₄ emissions by 24.66%‒508.68% compared with chemical fertilizer treatment, which may be due to the rich substrates provided by CMV and suitable environmental conditions for soil methanogenic archaea [12-14].

N₂O emissions are related to nitrification-denitrification processes and are directly influenced by the inorganic N applied during agricultural cultivation [15]. Water management in paddy fields, such as flooding and drainage, will shift the balance between nitrification and denitrification and then affect N₂O emissions [16]. Soil temperature is another nonnegligible factor affecting N₂O emissions [17]. Moreover, the mineralization process of organic amendments produces N₂O [18]. CMV has typically been considered to restrict N₂O emissions by partially replacing inorganic N fertilizer and due to its relatively slow mineralization rate [7,19]. However, other studies indicate that CMV incorporation does not affect N₂O emissions compared with the application of only chemical fertilizer, but the microprocesses related to N₂O emissions after green manure incorporation are still unclear [20-22].

In addition to the type of green manure, paddy soil properties are also directly affected by the incorporation volume of green manure. The incorporation volume regulates soil pH and SOC stocks, which elicit alterations in the composition and abundance of soil microorganisms, especially the functional genera related to C and N cycling [23,24]. Thus, the inconsistent conclusions above may have been caused by the single organic‒inorganic fertilizer substitution ratio utilized in most studies. The influence of different proportions of organic and inorganic fertilizers on CH₄ and N₂O emissions is meaningful for the application of CMV and needs to be further verified. The current study is expected (1) to assess the CH₄ and N₂O emissions from paddies with different incorporation amounts of CMV to substitute synthetic N fertilizer, (2) to reveal the microbial mechanisms that affect CH₄ and N₂O emissions, and (3) to explore a rational incorporated amount of CMV that is beneficial for improving rice productivity without increasing CH₄ and N₂O emissions.

 

Point 2: It is important to clarify the controversy surrounding the impacts of Chinese milk vetch (CMV) incorporation on CH₄ and N₂O emissions. Provide a brief background on the conflicting results from previous studies to highlight the significance of this research.

Response 2: Thanks for the insightful comments! According to the reviewer’s comments, we provided a brief background on the conflicting results from previous studies. Detailed revisions marked in green are as follows:

However, the current conclusions are ambiguous, limiting the promotion of CMV.

Some reported results have indicated that CMV incorporation does not significantly stimulate CH₄ emissions compared to treatments without residue amendment [8,9,10]. CMV shows a low C/N ratio (~15) and has a much weaker impact on CH₄ emissions than green manure with a high C/N ratio, such as ryegrass (~36) and oilseed rape (~25). The cumulative emissions under CMV treatments are 57.36%‒64.42% and 73.11%‒78.86% of those under ryegrass and rape treatments, respectively [10,11]. However, some studies have suggested that CMV incorporation remarkably increases CH₄ emissions by 24.66%‒508.68% compared with chemical fertilizer treatment, which may be due to the rich substrates provided by CMV and suitable environmental conditions for soil methanogenic archaea [12-14].

N₂O emissions are related to nitrification-denitrification processes and are directly influenced by the inorganic N applied during agricultural cultivation [15]. Water management in paddy fields, such as flooding and drainage, will shift the balance between nitrification and denitrification and then affect N₂O emissions [16]. Soil temperature is another nonnegligible factor affecting N₂O emissions [17]. Moreover, the mineralization process of organic amendments produces N₂O [18]. CMV has typically been considered to restrict N₂O emissions by partially replacing inorganic N fertilizer and due to its relatively slow mineralization rate [7,19]. However, other studies indicate that CMV incorporation does not affect N₂O emissions compared with the application of only chemical fertilizer, but the microprocesses related to N₂O emissions after green manure incorporation are still unclear [20-22].

 

Point 3: The article mentions different substitution ratios for CMV incorporation but does not provide clear information on how these ratios were determined or what they represent. Clarify how the substitution ratios were calculated and explain their practical relevance in agricultural settings.

Response 3: Thanks for the insightful comments! The original intention of CMV incorporation is to partially substitute N fertilizer. Therefore, the substitution ratios of synthetic N fertilizer with CMV in this study were represented by the ratios of N from CMV to N from urea. According to the reviewer’s comments, we remade Table 1 to show the specific substitution ratios and the amounts of urea substituted by CMV:

 

Table 1. Incorporation amounts of Chinese milk vetch and N inputs under different treatments.

Treatment		CMV			Urea				Total N (kg ha−1)		MV:CF (%)
		Application rate (t ha−1)	N input (kg ha−1)		Application rate (kg ha−1)	Substituted by CMV (kg ha−1)	N input (kg ha−1)
CF		0.0	0.0		480.0	0.0	220.8		220.8		0.0
MV25		7.5	17.7		441.4	38.6	203.1		220.8		8.7
MV50		15.0	35.5		402.9	77.1	185.3		220.8		19.1
MV75		22.5	53.2		364.3	115.7	167.6		220.8		31.7
MV100		30.0	70.9		325.8	154.2	149.9		220.8		47.3

CMV denotes Chinese milk vetch. MV:CF denotes the ratio of N from Chinese milk vetch to N from urea.

To provide practical relevance, we indicated the specific amount of urea substituted by CMV in the “Discussion” and “Conclusion” sections:

Lines 373-374 in the “Discussion” section: Therefore, incorporating CMV at 15.0 t ha⁻¹ as a substitute for 77.1 kg ha⁻¹ of urea (MV2/3) seems to be the optimal nutrient management strategy in the study area.

Lines 474-478 in the “Conclusion” section: Therefore, to improve rice productivity while mitigating GHG emissions in the study area, incorporating CMV at 15.0 t ha⁻¹ to substitute 77.1 kg ha⁻¹ of urea is a more desirable practice than the common approach of using only synthetic fertilizer or the traditional CMV application rate (22.5 t ha⁻¹).

 

Point 4: Regarding on the statement that "the mcrA and nosZ abundances indirectly regulated by the substitution ratios affected CH₄ and N₂O emissions”, elaborate on the mechanism or pathway through which these abundances influence emissions, providing relevant scientific background or references to support the claim.

Response 4: Thanks for the insightful comments! According to the reviewer’s comments, we added the influence mechanism of related gene abundances on emissions:

Lines 436-443: CH₄ is the final product of methanogens. The mcrA gene encodes the alpha subunit of the enzyme methyl coenzyme M reductase, which catalyzes the terminal step in biogenic methane production. Therefore, gene copies of mcrA are widely used to reflect the methanogen abundances and methanogenesis potentials [57]. Conversely, CH₄ can be utilized or oxidized by methanotrophs to produce organic tissue or CO₂. The pmoA gene encodes the subunit of the particulate methane monooxygenase that is found in all methanotrophs. Therefore, gene copies of pmoA are widely used to reflect the methanotroph abundances and CH₄ oxidation potentials [58].

Lines 455-459: Nitrite reduction is the critical and rate-limiting step in the denitrification process. Nitrite reductase is divided into soluble copper-containing enzymes and cytochrome enzymes, encoded by the nirK and nirS genes, respectively. Nitrous oxide reductase converts the greenhouse gas N₂O to harmless N₂ and determines the end product of denitrification, encoded by nosZ genes [59].

 

Point 5: The recommendation for MV2/3 as a suitable substitution ratio is made based on its ability to improve rice productivity without increasing CH₄ and N₂O emissions. Provide more information on the factors that contribute to this favorable outcome and explain why MV2/3 is specifically recommended over other substitution ratios.

Response 5: Thanks for the insightful comments! According to the reviewer’s comments, we revised the related parts to explain why MV2/3 is specifically recommended over other substitution ratios. Detailed revisions marked in green are as follows:

The GWP is considered an important indicator of the relative importance of CH₄ and N₂O. The CH₄-induced GWP accounted for more than 90% of the total GWP (Table 3), confirming that CH₄ is the dominant GHG emitted from paddies [37]. To further mitigate GHG emissions, future research should focus on reducing CH₄ emissions. Substitution of synthetic N fertilizer with a great amount of CMV increased the total GWP because the deleterious effects of increased CH₄ emissions exceeded the beneficial effects of decreased N₂O emissions (Table 3). Although MV and MV4/3 significantly increased rice yields compared with CF, the two treatments significantly increased CH₄ emissions and the total GWP (except for MV in 2021) (Figures 1 and 4, Table 3). MV1/3 did not significantly increase the total GWP, but its effect on rice yield was unsatisfactory compared with other CMV incorporation treatments. MV2/3 presented an excellent yield-increasing effect and did not increase GHG emissions, which consequently resulted in a low GHGI value ranging from 0.46 to 0.47. Therefore, incorporating CMV at 15.0 t ha⁻¹ as a substitute for 77.1 kg ha⁻¹ of urea (MV2/3) seems to be the optimal nutrient management strategy in the study area.

 

Point 6: Consider including limitations or potential biases of the study. Acknowledge any constraints in the research methodology or data interpretation that could affect the generalizability of the findings.

Response 6: Thanks for the insightful comments! According to the reviewer’s comments, we added two limitations of this study which needed to be explored in the future. Detailed revisions marked in green are as follows:

(1) We indicated that the optimal incorporation amount of CMV (15.0 t ha⁻¹) in other regions should be validated. And the optimal incorporation amount of other species of cover crop is needed to confirm:

Lines 381-389: The incorporation amount of CMV in the MV treatment (22.5 t ha⁻¹) is also utilized in many other regions in China. A similar incorporation level (25.8 t ha⁻¹) has been reported in South Korea [8,31]. Therefore, the present results may provide a reference for these regions. However, GHG emissions from paddies are affected by many factors, such as climate, soil properties, rice variety, fertilizer type, and water management [38,39]. Therefore, the optimal incorporation amount of CMV (15.0 t ha⁻¹) should be validated in these regions. Considering that the decomposition rate and products of other cover crops differ from those of CMV, further experiments are needed to confirm whether the substitution ratio affects GHG emissions.

• We indicated that the community composition of methanogens and methanotrophs may affect CH₄ emissions:

Lines 452-454: Moreover, in addition to the functional gene abundances, the community composition of methanogens and methanotrophs may affect CH₄ emissions, which needs to be further explored [14].

 

Point 7: Proofread the abstract for grammatical errors and ensure that the information is presented in a clear and concise manner.

However, there are a few areas where the language could be improved for better clarity and readability. Here are some suggestions using the “Abstract” as an example: Thanks for the insightful comments!

Response 7: We are very grateful for the reviewer's comments! We revised the “Abstract” section strictly following the reviewer's suggestions. And the revision makes the abstract smoother and easier to understand. Detailed revisions marked in green are as follows:

Chinese milk vetch (CMV) is a leguminous green manure that is commonly cultivated in paddy fields and can partially substitute synthetic nitrogen fertilizer. However, the impacts of incorporating CMV on CH₄ and N₂O emissions are still a subject of controversy. Therefore, we conducted a field experiment over three years to investigate the emissions under different substitution ratios: urea only (CF), incorporating traditional amount of CMV (MV), and with incorporation ratios of 1/3 (MV1/3), 2/3 (MV2/3), and 4/3 (MV4/3) of MV for partial urea substitution. Compared with CF, MV2/3, MV, and MV 4/3 resulted in increased yields. MV and MV4/3 reduced N₂O emissions but increased CH₄ emissions by 28.61% and 85.60% (2019), 32.38% and 103.19% (2020), and 28.86% and 102.98% (2021), respectively, resulting in an overall increase in total global warming potential (except for MV in 2021). MV2/3 exhibited a low greenhouse gas intensity value ranging from 0.46 to 0.47. The partial least squares-path model results showed that CH₄ and N₂O emissions were influenced by the substitution ratios, which indirectly regulated the gene abundances of mcrA and nosZ. Overall, the impact of CMV on CH₄ and N₂O emissions was determined by the substitution ratios. MV2/3, which involved partial substitution of synthetic N fertilizer with 15.0 t ha⁻¹ of CMV, resulted in improved rice productivity without increasing CH₄ and N₂O emissions, making it a recommended approach in the study area.

 

We sincerely appreciate the reviewer’s work on this study. Best wishes!

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

revisions complete

Reviewer 4 Report

My comments have been carefully and properly addressed. The current version can move forward for its publication process. Congratulations to the authors!