Soil Ecoenzymatic Stoichiometry Reveals Microbial Metabolic Limitations in Apple Orchards with Cover Crop and Organic Fertilizer Incorporation

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The manuscript entitled "Soil ecoenzymatic stoichiometry reveals microbial metabolic limitations in apple orchard with cover crop and organic fertilizer incorporation" sounds good and well presented. The results are well presented. I recommend it for publication, but there are few corrections that need to be done before final acceptance. I have my comments below:

1. Kindly cross-check the correspondence email ID.

2. Abstract: whta is TN? I think total nitrogen, please clarify and donot abbreviate.

3. Introduction: Please restructure the statement " The Loess Plateau..........Economic growth. Also indicate its importance.

4. Study area: Could you convert 2744 h sunshine in days for uniformity.

5. Kindly check the symbol of degree centigrade throughout.

6. What is 13.02 g Kg 1....I think topographical error. Please correct.

7. This statement does not make any sense.......more details of the experiment sites and design were reported by Zheng et al. (2019). ....Please report briefly the citation findings.

8. Some more local comparison studies in the discussion section will improve the quality of the manuscript.

Recommend it revision

 

 

Author Response

Responses to Reviewer Comments and Minor Revisions

(Responses in Blue Text)

Comments and Suggestions from Reviewer #1

The manuscript entitled "Soil ecoenzymatic stoichiometry reveals microbial metabolic limitations in apple orchard with cover crop and organic fertilizer incorporation" sounds good and well presented. The results are well presented. I recommend it for publication, but there are few corrections that need to be done before final acceptance. I have my comments below:

 

1. Kindly cross-check the correspondence email ID.

Re: Thanks for your comments. We have checked the correspondence email and provided the authors' email.

 

2. Abstract: whta is TN? I think total nitrogen, please clarify and donot abbreviate.

Re: We sincerely apologize for any inconvenience this may have caused. The measurement pertains to the total soil nitrogen content, and we will ensure this clarification is included in the revised manuscript.

 

3. Introduction: Please restructure the statement " The Loess Plateau..........Economic growth. Also indicate its importance.

Re: In response to the reviewer's request for restructuring the statement regarding the significance of the Loess Plateau in the apple industry, we have revised the introduction. The Loess Plateau is recognized as the primary apple-producing region in China, with its apple industry serving as a key economic driver and industrial pillar for local economic growth. It is crucial to highlight the paramount importance of the Loess Plateau in apple production, as it not only significantly contributes to the region's economy but also plays a vital role in supporting agricultural livelihoods and fostering regional development. Thank you for guiding us to enhance the clarity and emphasis of this key point.

 

4. Study area: Could you convert 2744 h sunshine in days for uniformity.

Re: In response to the reviewer's request for uniformity in the study area section, we have converted 2744 hours of sunshine into days. This amount corresponds to approximately 114 days of sunshine. Thank you for your attention to detail.

 

5. Kindly check the symbol of degree centigrade throughout.

Re: In response to the reviewer's comment regarding the symbol for degrees centigrade, we have ensured that the correct symbol "°C" is consistently used throughout the manuscript. Thank you for bringing this to our attention.

 

6. What is 13.02 g Kg 1....I think topographical error. Please correct.

Re: Thank you for your attention to the details in our article. The unit should be expressed as g·kg^-1. The labeling issue has been rectified in the revised manuscript.

 

7. This statement does not make any sense.......more details of the experiment sites and design were reported by Zheng et al. (2019). ....Please report briefly the citation findings.

Re: In response to the reviewer's comment on the unclear statement, we would like to clarify that our citation of the study by our senior colleagues from the same research group at the same experimental site aims to supplement and enrich the details of our experimental design. Thank you for guiding us in addressing this concern.

 

8. Some more local comparison studies in the discussion section will improve the quality of the manuscript.

Re: Including additional local comparison studies in the discussion section would enhance the quality of the manuscript. We will incorporate more local comparison studies to provide a more comprehensive analysis and strengthen the discussion section. Thank you for the suggestion to improve the quality of the manuscript.

 

 

Reviewer 2 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

RE: Soil ecoenzymatic stoichiometry reveals microbial metabolic limitations in apple orchard--

 

Thank you very much for re-evaluating this manuscript for possible publication in Agronomy Journal, which I previously reviewed. The Authors have incorporated most of my comments, but still there is a dire need to address a few more comments before it can get published Agronomy journal. Following these comments, I feel no hesitation in accepting this manuscript in Agronomy journal after moderate revision.

Comments

1.     The title contains eco-enzymatic -------------- but there is no data for enzyme is given in the results section.

2.     The procedure of cover crop i.e. positive, negative or insignificant is not given in the methodology section, this should be provided.

3.     The procedure for enzyme determination is given in the methodology, but the Enzyme data has been removed from the results, thus it is again suggested to add these data, to make the store clear. The title of the ms, the introduction and discussion all is enzyme centered, but the data which was given last time has been removed, this needs to be re-added to the results section along with the description.  

4.     Table 1, I suggest providing df, MS values along with ***/NS as superscript, the current version is not clear to show whether this is repeated analysis or not repeated, provision of degree of freedom along with MS values will make it clearer.

5.     Fig 1 and fig 2a,b, the legend is not clear, whether the different color belongs to treatment or effect of cover crop, this is much confusing, give pattern to various effect of cover like and color for the treatment, and also highlight where the effect of cover crop is given.

6.     Fig 2a,2b, the unit for MBC is missing this needs to be provided for understanding and clarity.

7.     Fig 2c, the various abbreviations should be spell out for clarity

8.     Figure 3. Enzymatic stoichiometry based on the relative proportions of C to N acquisition versus C to P acquisition. (a) Relationships of vector length and angle, with vector length representing soil C limitation for microbes and vector angle representing soil N/P limitation for microbes; The enzymes are represented by abbreviations: BG (β-1,4-glucosidase), NAG (β-1,4-N-acetylglucosaminidase), LAP (L-leucine aminopeptidase), AKP (alkaline phosphatase);----------------------- where are these enzyme presented in Fig 3, a, b or c or d,

9.     In conclusion, again there is 14 years, these results are from single years, or 14 years, this needs to be clear . Also “However, the organic matter left in the soil after cover crops are cut can aid orchards in surviving continuous droughts in winter and spring, while also increasing the microbial biomass carbon content” this is not clear, please revise.

 

 

Comments on the Quality of English Language

Minor grammer and functation problem 

Author Response

Responses to Reviewer Comments and Minor Revisions

(Responses in Blue Text)

Comments and Suggestions from Reviewer #2

RE: Soil ecoenzymatic stoichiometry reveals microbial metabolic limitations in apple orchard--

Thank you very much for re-evaluating this manuscript for possible publication in Agronomy Journal, which I previously reviewed. The Authors have incorporated most of my comments, but still there is a dire need to address a few more comments before it can get published Agronomy journal. Following these comments, I feel no hesitation in accepting this manuscript in Agronomy journal after moderate revision.

 

Comments

 

1. The title contains eco-enzymatic -------------- but there is no data for enzyme is given in the results section.

Re: Thank you for your attention to the consistency between our paper's title and its content. We completely understand your perspective. In our research, we adopted the approach of using vector angles and lengths to comprehensively represent the changes in enzymes related to the carbon, nitrogen, and phosphorus demands in soil. Therefore, we did not include specific enzyme activities in the main body of the text. However, we acknowledge that this might cause some confusion, and we appreciate your insight on this matter. Thus, in our revised version, we will add the specific enzyme activity data in the supplementary materials, enabling our readers to grasp our research approach and results in a more intuitive and detailed manner. Once again, we appreciate your feedback. We hope that this response clarifies your query, and we look forward to any further suggestions you might have concerning our adjustments.

 

 

2.The procedure of cover crop i.e. positive, negative or insignificant is not given in the methodology section, this should be provided.

Re: Thank you for bringing attention to the procedure regarding cover cropping in our methodology section. We apologize for any confusion this may have caused. In our study, we consider the use of cover crops as a positive measure which significantly contributes towards the overall methodology and results of our research. Upon constructive input, we understand that a detailed explanation of this should be present in the manuscript for better comprehension. In the revised version of this paper, we will make sure to include specific information about the procedure of cover cropping, emphasizing its positive implications, and detailing how it was implemented and influenced our findings within the context of the study.

 

3. The procedure for enzyme determination is given in the methodology, but the Enzyme data has been removed from the results, thus it is again suggested to add these data, to make the store clear. The title of the ms, the introduction and discussion all is enzyme centered, but the data which was given last time has been removed, this needs to be re-added to the results section along with the description.

Re: Thank you for your attention to the consistency between our paper's title and its content. We completely understand your perspective. In our research, we adopted the approach of utilizing vector angles and lengths to comprehensively represent the variations in enzyme activities related to the carbon, nitrogen, and phosphorus demands in soil. This methodology allowed us to capture a broader perspective on the enzyme-related changes in the soil environment.

We opted not to include specific enzyme activity measurements in the main body of the text to maintain focus on our unique analytical approach. However, we recognize that this decision may have created some ambiguity, and we value your insight on this matter.

To address this concern and provide a more detailed understanding of our research findings, we have decided to incorporate the specific enzyme activity data in the supplementary materials of our revised version. This addition will offer readers a more comprehensive view of our methodology and results, enhancing the clarity and interpretation of our study.

Once again, we appreciate your feedback and constructive suggestions. We trust that these adjustments will better align our paper with your expectations. We welcome any further recommendations you may have as we strive to enhance the quality and coherence of our research.

 

4. Table 1, I suggest providing df, MS values along with ***/NS as superscript, the current version is not clear to show whether this is repeated analysis or not repeated, provision of degree of freedom along with MS values will make it clearer.

Re: We appreciate your suggestion to enhance the clarity of the presentation by including degrees of freedom (df), mean squares (MS) values, and indicating significance levels using ***/NS superscript notations. This addition will provide a more detailed and informative interpretation of the data, distinguishing between repeated and non-repeated analyses.

By incorporating df and MS values along with the appropriate superscript indicators, we aim to offer a clearer representation of the statistical significance and variability in the results. We will implement these improvements in Table 1 to ensure better transparency and understanding of the analysis. Thank you for your valuable input, and we are committed to enhancing the comprehensibility and accuracy of our research data.

 

5. Fig 1 and fig 2a,b, the legend is not clear, whether the different color belongs to treatment or effect of cover crop, this is much confusing, give pattern to various effect of cover like and color for the treatment, and also highlight where the effect of cover crop is given.

Re: Thank you for your feedback regarding the clarity of the legends in Figure 1 and Figure 2a, b. We apologize for any confusion that these figures may have caused. In response to your comments, we will revise these figures to make the legends more clear and easier to interpret. We will assign patterns to various effects of cover crops and use specific colors for different treatments. Furthermore, we will highlight where the effects of cover crops are presented in the figures. We understand the importance of visual clarity in presenting our findings, and your feedback helps us improve the comprehensibility of our figures. We will make sure our revisions to the figures effectively address the confusions you have mentioned. Once again, thank you for your valuable insight. Your detailed feedback is greatly assisting us in improving our work.

 

 

6. Fig 2a,2b, the unit for MBC is missing this needs to be provided for understanding and clarity.

Re: Thank you for pointing out the omission in Figure 2a and 2b. We apologize for any inconvenience this may have caused. We acknowledge that the unit for MBC, which is mg/kg, was not included in these figures. We understand the importance of these details in interpreting and understanding the data accurately. In our revised version, we will make sure to include the unit (mg/kg) for MBC in Figure 2a and 2b. This will provide much needed clarity and assist in proper understanding of our results. Again, we deeply appreciate your valid observation and thank you for helping us enhance our paper.

 

7. Fig 2c, the various abbreviations should be spell out for clarity

Re: Thank you for your valuable comment on Figure 2c. We realize that the abbreviations used in the figure may not have been adequately explained, leading to possible confusion.In the revised version of our paper, we will make sure to spell out all abbreviations in the figure to improve its clarity and ease of understanding. We fully agree that clarity is important in enabling the reader comprehend and interpret our findings better.

 

8. Figure 3. Enzymatic stoichiometry based on the relative proportions of C to N acquisition versus C to P acquisition. (a) Relationships of vector length and angle, with vector length representing soil C limitation for microbes and vector angle representing soil N/P limitation for microbes; The enzymes are represented by abbreviations: BG (β-1,4-glucosidase), NAG (β-1,4-N-acetylglucosaminidase), LAP (L-leucine aminopeptidase), AKP (alkaline phosphatase);----------------------- where are these enzyme presented in Fig 3, a, b or c or d,

Re: We apologize for the confusion stemming from incorrectly labeled legends in Figure 3. We appreciate your patience and vigilance in pointing out this oversight. For clarity, the phosphorus-related enzyme should be represented as ln(BG)/ln(BG+AKP) and the nitrogen-related enzyme as ln(BG)/ln(BG+LAP+NAG) in the figure. In the revised version of our manuscript, we assure you that these discrepancies will be addressed, and we will make the necessary adjustments to the figure to correctly represent these enzymes. Thank you once again for your observation and feedback, which are invaluable in improving the accuracy and clarity of our research findings.

 

9. In conclusion, again there is 14 years, these results are from single years, or 14 years, this needs to be clear . Also “However, the organic matter left in the soil after cover crops are cut can aid orchards in surviving continuous droughts in winter and spring, while also increasing the microbial biomass carbon content” this is not clear, please revise.

Re: Thank you for your attention to the details in our conclusion. We acknowledge that there may have been some confusion regarding the time span of the data used in our study. To clarify, our research is based on a long-term study that began in 2005. However, the data we used and presented in this particular manuscript originate from the soil samples collected exclusively in 2019. In our revised manuscript, we will ensure this is clearly stated to avoid any misunderstandings. In regard to your second point, we apologize for any ambiguity in our description of the impact of organic matter left in the soil after cover crops are cut. In the revised version, we will focus on enhancing the clarity of this explanation.

 

 

 

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

Although I find the methods and results in this research adequate, my main objection lies with the significance of results: the authors cannot claim that the soil treatments applied for 14 consecutive years had the effects on soil microbial C, N, and enzymatic activity found out of only 4 sample dates only in the last year of the experiment: this represents just a snapshot of soil properties at the time taken. To prove the persistence and significance of these soil treatments, the same analysis should have been done and the same effects should have been observed repeatedly over several years.

Some specific comments, to improve the manuscript: 

In section 2.1, the phrase "Agriculture in this region is completely dependent on natural precipitation" is repeated twice.

Section 2.3. Please elaborate more on the chloroform fumigation method: how does it differentiate between microbial C and N compared to total C and N (including manure inputs for example). What were the controls?

Figure 1. Authors mentioned in the Materials and Methods section that irrigation of the plot depended solely on natural precipitation. No irrigation treatments were described. Please explain where the irrigation results described in the caption originated from.

Section 3.2. Please rephrase: Changes in soil microbial carbon and nitrogen. "Soil microbial amount" is an incorrect term.

Figure 3: Are the claims of negative/insignificant/positive effect of soil treatments meaningful, when vector angle values are essentially identical (although with statistical differences)?

Supplemental figures (e.g. S1) mentioned in the manuscript were not provided.

All this research was set up to improve soil fertility for an apple tree crop. Did it work for the crop? Were harvest data collected each year? Tree physiology data? Nothing relevant to the crop is presented.

Comments on the Quality of English Language

Use of the English language in the manuscript is at a good level. Authors and the proofreader should pay attention to spaces between words (missing in several cases).

Author Response

Responses to Reviewer Comments and Minor Revisions

(Responses in Blue Text)

Comments and Suggestions from Reviewer #3

Although I find the methods and results in this research adequate, my main objection lies with the significance of results: the authors cannot claim that the soil treatments applied for 14 consecutive years had the effects on soil microbial C, N, and enzymatic activity found out of only 4 sample dates only in the last year of the experiment: this represents just a snapshot of soil properties at the time taken. To prove the persistence and significance of these soil treatments, the same analysis should have been done and the same effects should have been observed repeatedly over several years.

Some specific comments, to improve the manuscript: 

In section 2.1, the phrase "Agriculture in this region is completely dependent on natural precipitation" is repeated twice.

Re: Thank you for highlighting this duplication in section 2.1. Indeed, the phrase "Agriculture in this region is completely dependent on natural precipitation" was repeated by mistake. We appreciate your keen observation which allowed us to correct this error.

 

Section 2.3. Please elaborate more on the chloroform fumigation method: how does it differentiate between microbial C and N compared to total C and N (including manure inputs for example). What were the controls?

Re: Thank you for your comments on Section 2.3 and the request for more details concerning the chloroform fumigation method utilized in our paper. Chloroform fumigation is an analytical method used to estimate microbial biomass in soil by differentiating between microbial carbon and nitrogen and total carbon and nitrogen.

The method essentially works by subjecting a soil sample to chloroform fumigation which leads to the lysis or disruption of microbial cells releasing microbial Carbon and Nitrogen. This is followed by measuring the increased extractable amounts of C and N after fumigation, which represents the microbial C and N.

To differentiate microbial C and N from total soil C and N including other organic inputs like manure, we used non-fumigated soil samples as our control. Here, extraction allows us to measure the initial soil C and N before fumigation. By comparing the resultant values (after fumigation-extraction and control extraction), we can estimate the part of C and N in the soil that was part of microbial biomass.

Controls used in our study were indeed non-fumigated soil samples of the same type and treatment.

In the revised manuscript, we will incorporate this detail to avoid any ambiguity regarding the methodology used in this section. We appreciate your input to help us improve this aspect of our work.

 

Figure 1. Authors mentioned in the Materials and Methods section that irrigation of the plot depended solely on natural precipitation. No irrigation treatments were described. Please explain where the irrigation results described in the caption originated from.

Re: Thank you for your question regarding the irrigation results stated in the caption for Figure 1. There seems to be a misunderstanding. As mentioned in the Materials and Methods section, irrigation of the plot relied completely on natural precipitation, indicating no additional man-made irrigation treatments were performed. Therefore, the "irrigation results" in the diagram should actually be interpreted as "precipitation results". I see that our wording may have caused confusion, and we greatly appreciate you pointing this out.

Adding on, we believe that the differences in soil moisture content that are reflected in the irrigation results arise primarily due to the varying absorptive capacities of the soil, resulting predominantly from long-term fertilising effects between fruit trees and cover crops.

 

Section 3.2. Please rephrase: Changes in soil microbial carbon and nitrogen. "Soil microbial amount" is an incorrect term.

Re: Thank you for pointing out our error, we will correct this statement in the revised manuscript

 

Figure 3: Are the claims of negative/insignificant/positive effect of soil treatments meaningful, when vector angle values are essentially identical (although with statistical differences)?

Re: Your insightful comment is appreciated. We concur that the vector angle values are largely indistinguishable, which could potentially lead to misinterpretations. Our assertions of negative, insignificant, and positive effects are primarily based on the observed statistical differences. Our aim is to depict trends or potential pathways from the results, instead of absolute disparities. While acknowledging that despite statistical variance, the practical or biological implications might not be substantial due to the almost identical vector angles, we remain keen on examining the correlation between vector angle and length, alongside other soil nutrients, under various growth stages and fertilization treatments. This approach aims to further unravel potential pathways that cover fertilization has on soil carbon, nitrogen, and phosphorus limitations.

 

Supplemental figures (e.g. S1) mentioned in the manuscript were not provided.

Re: My sincere apologies for this oversight. We indeed have supplemental figures that support the findings of our study. There seems to have been an oversight while submission, causing supplemental data to be left out. In the revised manuscript, we will ensure to include all the supplemental figures that are referenced in the text. Once again, I apologize for the confusion and appreciate your understanding.

 

 

All this research was set up to improve soil fertility for an apple tree crop. Did it work for the crop? Were harvest data collected each year? Tree physiology data? Nothing relevant to the crop is presented.

Re: We appreciate your astute observation on the direct impact of our study on the apple tree crop. Our apologies, should we have fallen short in fully accentuating this facet in the manuscript. Indeed, our fundamental objective was to enhance soil fertility, thereby raising the apple crop yield. Whilst a major focus was attributed to the alterations in soil characteristics, it is indubitable that the utmost indication of success would be the amplification in the crop's output. Therefore, your inquiry pertaining to variations in the harvest data and tree physiology bears great relevance. We regret any confusion, but our expertise in plant physiology is admittedly limited. While we have compiled data pertaining to the fruit tree yields, our inability to execute a comprehensive comparison across each time period has precluded their integration in this study.

Round 2

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

In my first review I had mentioned right from the beginning:

"Although I find the methods and results in this research adequate, my main objection lies with the significance of results: the authors cannot claim that the soil treatments applied for 14 consecutive years had the effects on soil microbial C, N, and enzymatic activity found out of only 4 sample dates only in the last year of the experiment: this represents just a snapshot of soil properties at the time taken. To prove the persistence and significance of these soil treatments, the same analysis should have been done and the same effects should have been observed repeatedly over several years".

The authors did make changes to their manuscript according to my comments, but the main argument above was not answered at all. Obviously, the authors do not have data from other years to support their conclusions. I found the body of data provided very insufficient to conclude that 14 years of standard agronomic practices result in soil conditions observed in only 1 year.

One more issue, the supplemental figures mentioned in the manuscript, still are nowhere to be found.

I cannot accept this manuscript for publication in Agronomy.

Author Response

Responses to Reviewer Comments and Suggestions

Comments and Suggestions for Authors

In my first review I had mentioned right from the beginning:

 

"Although I find the methods and results in this research adequate, my main objection lies with the significance of results: the authors cannot claim that the soil treatments applied for 14 consecutive years had the effects on soil microbial C, N, and enzymatic activity found out of only 4 sample dates only in the last year of the experiment: this represents just a snapshot of soil properties at the time taken. To prove the persistence and significance of these soil treatments, the same analysis should have been done and the same effects should have been observed repeatedly over several years".

The authors did make changes to their manuscript according to my comments, but the main argument above was not answered at all. Obviously, the authors do not have data from other years to support their conclusions. I found the body of data provided very insufficient to conclude that 14 years of standard agronomic practices result in soil conditions observed in only 1 year.

Re: Your insightful comment is appreciated.We appreciate your astute observation on this long-term fertilization trial. For long-term fertilization experiment, researchers pay more attention to the final effects of fertilization tests on soil properties, enzyme activities and microbial community^[1-5]. We focusing on the differences between long-term fertilization treatments, rather than the differences between years. Our study investigated the activities of soil enzymes associated with Carbon, Nitrogen, and Phosphorus acquisition under different fertilizer management and cover crop schemes during crucial growth stages of apple trees.

 

The experiment was established in 2008, this long-term positioning trial data is measured annually, and our colleagues have exhibited the outcomes of prolonged experimental endeavors across diverse research domains^[6-14]. The study aimed to scrutinize the dynamic changes in soil nutrients following the integration of cover cropping with fertilization, and analyze the attributes of microbial metabolic limitations under cover crop and fertilization practices.

 

One more issue, the supplemental figures mentioned in the manuscript, still are nowhere to be found.

Re: My sincere apologies for this oversight. We indeed have supplemental figures that support the findings of our study. There seems to have been an oversight while submission, causing Supplemental files to be left out. In the revised manuscript, we will ensure to include all the supplemental figures that are referenced in the text. Once again, I apologize for the confusion and appreciate your understanding.

 

 

 

 

 

Reference

1. Lin, Y., Ye, G., Luo, J., Di, H. J., Liu, D., Fan, J., & Ding, W. (2018). Nitrosospira cluster 8a plays a predominant role in the nitrification process of a subtropical Ultisol under long-term inorganic and organic fertilization. Applied and Environmental Microbiology, 84(18). https://doi.org/10.1128/AEM.01031-18
2. Liu, B., Xia, H., Jiang, C., Riaz, M., Yang, L., Chen, Y., Fan, X., & Xia, X. (2022). 14 year applications of chemical fertilizers and crop straw effects on soil labile organic carbon fractions, enzyme activities and microbial community in rice-wheat rotation of middle China. Science of the Total Environment, 841(June), 156608. https://doi.org/10.1016/j.scitotenv.2022.156608
3. Jones, J., Savin, M. C., Rom, C. R., & Gbur, E. (2017). Denitrifier community response to seven years of ground cover and nutrient management in an organic fruit tree orchard soil. Applied Soil Ecology, 112, 60–70. https://doi.org/10.1016/j.apsoil.2016.12.009
4. Ji, L., Wu, Z., You, Z., Yi, X., Ni, K., Guo, S., & Ruan, J. (2018). Effects of organic substitution for synthetic N fertilizer on soil bacterial diversity and community composition: A 10-year field trial in a tea plantation. Agriculture, Ecosystems and Environment, 268(September), 124–132. https://doi.org/10.1016/j.agee.2018.09.008
5. Liu, J., Shu, A., Song, W., Shi, W., Li, M., Zhang, W., Li, Z., Liu, G., Yuan, F., Zhang, S., Liu, Z., & Gao, Z. (2021). Long-term organic fertilizer substitution increases rice yield by improving soil properties and regulating soil bacteria. Geoderma, 404(June), 115287. https://doi.org/10.1016/j.geoderma.2021.115287
6. Zheng, W., Zhao, Z., Lv, F., Wang, R., Wang, Z., Zhao, Z., Li, Z., & Zhai, B. (2021). Assembly of abundant and rare bacterial and fungal sub-communities in different soil aggregate sizes in an apple orchard treated with cover crop and fertilizer. Soil Biology and Biochemistry, 156(3), 108222. https://doi.org/10.1016/j.soilbio.2021.108222
7. Zhao, Z., Ma, Y., Feng, T., Kong, X., Wang, Z., Zheng, W., & Zhai, B. (2022). Assembly processes of abundant and rare microbial communities in orchard soil under a cover crop at different periods. Geoderma, 406(3), 115543. https://doi.org/10.1016/j.geoderma.2021.115543
8. Lv, F. lian, Zheng, W., Zhai, B. nian, & Li, Z. yan. (2022). Cover cropping and chemical fertilizer seasonally mediate microbial carbon and phosphorus metabolisms in an apple orchard: Evidence from the enzymatic stoichiometry method. Applied Soil Ecology, 178(3), 104579. https://doi.org/10.1016/j.apsoil.2022.104579
9. Sompouviset, T., Ma, Y., Zhao, Z., Zhen, Z., Zheng, W., Li, Z., & Zhai, B. (2023). Combined Application of Organic and Inorganic Fertilizers Effects on the Global Warming Potential and Greenhouse Gas Emission in Apple Orchard in Loess Plateau Region of China. Forests, 14(2). https://doi.org/10.3390/f14020337
10. Zheng, W., Zhao, Z., Lv, F., Yin, Y., Wang, Z., Zhao, Z., Li, Z., & Zhai, B. (2021). Fungal alpha diversity influences stochasticity of bacterial and fungal community assemblies in soil aggregates in an apple orchard. Applied Soil Ecology, 162(3), 103878. https://doi.org/10.1016/j.apsoil.2020.103878
11. Zheng, W., Zhao, Z., Lv, F., Wang, R., Gong, Q., Zhai, B., Wang, Z., Zhao, Z., & Li, Z. (2019). Metagenomic exploration of the interactions between N and P cycling and SOM turnover in an apple orchard with a cover crop fertilized for 9 years. Biology and Fertility of Soils, 365–381. https://doi.org/10.1007/s00374-019-01356-9
12. Zhao, Z., Ma, Y., Zhang, A., Chen, Y., Zheng, Z., Zheng, W., & Zhai, B. (2023). Response of apple orchard bacteria co-occurrence network pattern to long-term organic fertilizer input. Applied Soil Ecology, 191(3), 105035. https://doi.org/10.1016/j.apsoil.2023.105035
13. Zheng, W., Zhao, Z., Gong, Q., Zhai, B., & Li, Z. (2018). Responses of fungal–bacterial community and network to organic inputs vary among different spatial habitats in soil. Soil Biology and Biochemistry, 125(July), 54–63. https://doi.org/10.1016/j.soilbio.2018.06.029
14. Zheng, W., Gong, Q., Lv, F., Yin, Y., Li, Z., & Zhai, B. (2020). Tree-scale spatial responses of extracellular enzyme activities and stoichiometry to different types of fertilization and cover crop in an apple orchard. European Journal of Soil Biology, 99(3), 103207. https://doi.org/10.1016/j.ejsobi.2020.103207

 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript discusses how the soil ecoenzymatic stoichiometry reveals microbial metabolic limitations in apple orchard with cover crop and organic fertilizer incorporation.

 

The text is generally not well-written; various language issues exist.

Some points are raised below.

 

1.           Abstract: Provide full name for CMNPK.

2.           P. 2: “There are more than 100 enzymes present…”

3.           P. 2: “Ecoenzymatic stoichiometric theory integrating the metabolic and stoi-chiometric theory of ecology, revealing the relationships between microbial metabolism and soil nutrients availability…” Language revision.

4.           P.3: “Basen on the above…”

5.           P.4: “The total applied rates of chemical fertilizer in NPK were 192 kg N ha−1 year−1, 108 kg P2O5 ha−1 year−1 and 168 kg K2O ha−1 year−1, in MNPK were 96 kg N ha−1 year−1, 54 kg P2O5 ha−1 year−1 and 84 kg K2O ha−1 year−1, respectively.” On what ground were these fertilizer doses determined? Why weren’t exactly the same?

6.           P. 4: References are needed regarding the following determinations: SOC, TP, AP, AN, DOC, DON, MBC, MBN.

7.           P.4: “…specific to each enzyme…” More details are needed.

8.           P.4: “The negative control well received 200 μL buffer and 50 μL substrate.” Not clear.

9.           P.5: Table S2 to be removed to main text.

10.        Discussion: “The soil had higher soil moisture, could improve the availability of substrate for mi-crobial and ultimately stimulated the activity of BG at the setting and maturing periods…” Language revision.

11.        P.11: “It has been suggested that the soil enzyme activities were linearly increasing after cover crop termination because the decomposition of cover crop residue (Nevins et al., 2018). Besides, organic fertilizers were adopted in last year and soil samples were collected in next year, would have been decomposed and transformed into soil organic matter (Lorenz et al., 2007). Improved soil organic content by organic fertilizer, which impacts microbial activity and could increase the rates of cover crop residue decomposition, all of which could contribute to greater value of soil enzyme activity in CM and CMNPK treatments at the maturing period” Language revision.

12.        P. 11: “…and was likely due to the biotic factors are directly involved in the decomposition of soil organic matter…”Language revision.

13.        P.11: “   to adapt for environmental variation…” Language revision.

 

 

Comments on the Quality of English Language

Moderate revision needed.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript by Shibiao and Colleague's works on “Soil eco enzymatic stoichiometry reveals microbial metabolic limitations in an apple orchard with cover crop and organic fertilizer incorporation” was a great field of research, and I found it suitable for the journal “ Agronomy”.. They provide significant data to explain how soil stoichiometry and associated microbial metabolic affect apple orchards. The data presentation in writing is somehow Ok, and the authors nicely discussed the finding with a range of literature and a well-made conclusion. Although this is well-prepared manuscript, Shibiao and Colleagues can accept my suggestion below:

1. Figures and tables are completely missing in the manuscript, although these are cited in the running text of the manuscript. Therefore, the authors are requested to incorporate the same in the revised version kindly.

2. Photographs of the experimental plots should improve the quality of the manuscript. Therefore authors are advised to kindly provide at least one photograph of the apple orchard where experiments were carried out.

3. Time frame of the experiments is required to be clearly mentioned.

4. Abstract and conclusion need slight improvement pertaining to results.

5. Latest references, say 2022, or 2023, pertaining to this manuscript must be included.

6. Introduction should include the hypothesis behind the experimentation work and can clearly cite the expected outcomes and their importance in apple orchards.

7. There are certain topographical errors in the abstract and in the text which need to be corrected. Examples: The word “in” is used twice in the last sentence of the abstract. The word should be “regulated by”, not “regulatedby” in page no. 8, article 4.2.

  I suggest a major improvement of the manuscript before acceptance.

Reviewer 3 Report

Comments and Suggestions for Authors

Re: Soil ecoenzymatic stoichiometry reveals

Based on my evaluation, I recommend major changes to the manuscript for possible publication in the Agronomy Journal due to the following comments.

The quantity and quality of data is sufficient for publication, but it is not clear that this the data from one year or 14 years, this should be clear first.

The topic of the title, and most of the data presented in figures, except Fig 8 and Fig  S3, do not support each other, I suggest to change the topic as per data and conclusion been derived

In all figures, the bars number are much in number, and thus it is very difficult to conclude from this, why not to take two groups of four treatments each for Cover crop and No cover crop with in each period, at-least that type of arrangement will be a bit more clear, also provide all these bar with different pattern and color for easy understanding

The results section lack, treatment comparison in term of quantitative way and the in discussion, no mechanistic approach is been used that why cover crop with MNPK had decreased microbial C and P limitations, the main conclusion of the manuscript

Figure 1., Please cross check the MBC contents, it this not much more higher than ordinary soil, or you can cross check the unit. Secondly, this is not clear that this the average of 14 years data or only one year, if one year which one i.e. 2019, then need to be written clearly. Also what does the different letter indicates, and whether this is within the growth period or across the growth period, this should also be mention, I would suggest to remove the figure c, one can easily calculate the C/N from fig a & b, if needed.

Fig 2. sizes represent Spearman correlations coefficients (r)=---------------------- for color the scale is provided, but what about the size, this should be explain, also please cross check the data, and analysis how the AN show negative correlation with MBC/MBN and SWC, please cross check

What is the difference between fig 4 and Fig 7, can you please elaborate

Fig S2, please give appropriate symbol to the treatments, and also show the color of the legends for periods, this is not clear in the figures, due to different color management. The figure caption is also not in line with the information provided on the y-axis. Also, the treatment notation should be make clear, it will be better to present the CC with Green color, with different symbols and no-CC with any other color, and legend for periods may be based on color or symbols.

FigS3, %IncMSE, may please be define in the figure caption, also if we re-arrange the soil properties, the trend will be change, so what information, we can get from this figure.

Table S2, give the DF number in the parenthesis in front of each factors, along with MSE, to show which design was used for analysis and which was the subject for repeated measurement design, otherwise, the ANOVA section should be deleted, and of no use.

Minor comments (No line number, make the review processing very difficult, but I just take the start of the statement from the relevant section)

 

Abstract

measures of organic amendment-------------------- what do you mean by this terminology here

this amendment---------------- which amendment, you have not yet defined it before

cover crop (C) and no cover crop with four sub-plots comprising no fertilizer (CK), organic fertilizer (M), chemical fertilizer (NPK) and partial substitution of chemical fertilizers by organic fertilizer (MNPK=---------------- No information about the rate both for CC and Fertilization is provided, this should be provided to know how much was added

CMNPK--------------- where this treatment came from, it should be defined before that C was added for Cover Crop, I should suggest removing C, and write the C in full like MNPK under cover crop---------

guidance in in---------------- remove one in from here

Introduction

Consequently, cover cropping could decrease the us-age of fertilizer, which is another way to save chemical fertilizer-------------------- need proper reference to claim

Yet, the increases in soil nutrients by organic amendment treatments may suppress soil enzyme activities. For instance, the lit-erature has demonstrated the activity of P-acquiring enzyme activity was decreased with soil P content increasing (Sinsabaugh et al., 2008; Zeng et al., 2022).------------------------ these statement do not support each other

Besides, data from several studies suggest that the soil water content under cover crop------------------ why it will decrease, does this not prevent much more evaporation from bare soil, in case of no cover crop

The objective/aims of the study is not clear, please make it more robust

Methodology

Study site and treatments---------- we also need to provide information about the max/min temperature, and soil nutrients contents at-least NPK

cover crop (C),---------------- which was the cover crop, how much was the CC addition per year, how this was management, what was the amount of residue leftover after cover crop,

the residues were plowed into the soil.----------- how much was the residue

The sowing rate was 7.5 kg ha−1 year−1.----------------------- this is not clear to  me, please rephrase

decomposed goat----------- how much decomposed for how long the decomposition was done and how was done

according the method described by Jones------------------add ‘to’ after according

chloroform fumigation-extraction method.--------------------- give reference as well as Kec values, for this conversion

To determine the all treatments effects within the different growth periods, one-way ANOVA was performed,-------------------- awkward statement, please revise

Result

The results section, lack any quantitative determination of the observed variable and seems to be stretched as compared to the number of tables/illustration and amount of data. This section should be improved by provision of some quantitative description of the treatment involved, to make it clear, whether cover crop and which soil amendment is better and how much it is better

Discussion

This should be framed based on the set hypothesis/objective in the introduction section. Framing based on the results heading, present this section is a repetition of the result