Effects of Continuous Straw Return on Soil Nutrients and Microbial Community Structure of Paddy Fields in Northeast China

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is of particular interest to specialists. The manuscript pays sufficient attention to microbial studies, however, soil studies are practically not described. If the experiment took 8 years, how many times were soil samples taken? Where is the soil data for 8 years? The authors stated the name "Effects of continuous straw returning on soil nutrients and microbial community structure of Paddy Field in Northeast China", therefore, it is necessary to add the characteristics of the studied sites before and during the experiment as a separate section. The activity of microorganisms depends on such important soil parameters as humidity, temperature, pH of the medium, and such data are not available. It is also important to describe the general climatic conditions for the entire study period, since this was a field experiment. I hope that the expressed wishes will be able to significantly improve the manuscript.

Author Response

Revised manuscript agronomy-3207042 " Effects of continuous straw returning on soil nutrients and microbial community structure of Paddy Field in Northeast China"

Dear Holly Luan Editor and reviewers:

On behalf of my co-authors, thank you for giving us the opportunity to revise our manuscript.

The manuscript has already been corrected and is attached for further evaluation.

We would like to express our great appreciation to you and the reviewers for your comments on our paper. We look forward to hearing from you.

Thank you and best regards.

Yours sincerely,

Qiuju Wang

Heilongjiang Academy of Agricultural Sciences

368 Xuefu Road, Harbin, P.R.China, 150086

 

 

 

 

Responses to Reviewer

 

Reviewer 1:

The manuscript is of particular interest to specialists.

Thank you for your constructive feedback and valuable insights on our manuscript.

(1) The manuscript pays sufficient attention to microbial studies, however, soil studies are practically not described. If the experiment took 8 years, how many times were soil samples taken? Where is the soil data for 8 years?

RESPONSE：

We only collected a soil sample once. Eight years is an experimental process.

This research specifically focuses on nutrient-deficient albic soils, a prevalent but agriculturally challenging soil type in Northeast China. To address soil fertility limitations, we conducted a long-term field experiments (8-years continuous straw returned) to investigate whether continuous straw returning could enhance soil nutrient availability, consequently, improve crop productivity. Improving the fertility of white slurry soils is a cumulative process. Based on previous pre-test experiences, we chose an eight-year trial period. The study focused on the analysis of soil nutrient changes and soil microbial communities during the eighth year of straw return, aiming to investigate the effects of straw return. The study counted the growth of plants in each year, and three areas in the experimental field were selected each year to test the crop yield per unit area and related indexes, as shown in Figure 2, which shows the distribution of all the monitoring indexes in the eight years of the study.

I hope you can understand that if we had tested soil microorganisms 5 years ago, we should have published a paper earlier. We are just maintaining such a valuable soil sample and will persevere.

Line324: We added “2023”.

Line 172: We changed “during the experimental process” to “at 2023”.

 

(2) The authors stated the name "Effects of continuous straw returning on soil nutrients and microbial community structure of Paddy Field in Northeast China", therefore, it is necessary to add the characteristics of the studied sites before and during the experiment as a separate section. The activity of microorganisms depends on such important soil parameters as humidity, temperature, pH of the medium, and such data are not available. It is also important to describe the general climatic conditions for the entire study period, since this was a field experiment. I hope that the expressed wishes will be able to significantly improve the manuscript.

RESPONSE：

This is a good question, we didn't express it clearly.

Line 551: We added “The activity of microorganisms depends on such important soil parameters as humidity, temperature, pH of the medium. The experimental site is situated in the Northeastern region of China, characterized by a cold temperate climate zone. As elaborated in Section 2.1 of the manuscript, the site’s key climatic parameters, including mean annual temperature, annual precipitation, frost-free period, and annual active temperature, are based on 8-year averaged data (2016–2023). This approach accounts for interannual climatic variability and ensures a robust characterization of the experimental environment. The albic soils at this location inherently possess low levels of organic matter, nitrogen, and phosphorus, making them representative of the region's typical low-fertility soils. Consequently, while changes in soil nutrient composition over the eight-year period were not individually detailed in the study, they were assessed in terms of the cumulative effects resulting from successive straw returns.”

 

Thank you for your suggestions, which have improved the logical coherence of the paper.

 

Reviewer 2 Report

Comments and Suggestions for Authors

The work does not introduce new information. The positive effects of straw have been known for a long time. The paper is written quite correctly. Incomplete description of the methodology for determining the content of components in soil. This needs to be supplemented. The authors use methods described half a century ago. Such an old thing. There are a lot of colourful figures in the text and not very readable. Well-edited tables would make more sense. In the text, the authors only state how much what has increased or decreased. There is absolutely no indication of the range of values of the labelled quantities. The discussion lacks comparison with other data. There are only numbers from references. This is not enough. Twenty-six observations are highlighted in the text. Data from the supplement should be in the text.

Comments for author File: Comments.pdf

Author Response

Revised manuscript agronomy-3207042 " Effects of continuous straw returning on soil nutrients and microbial community structure of Paddy Field in Northeast China"

Dear Holly Luan Editor and reviewers:

On behalf of my co-authors, thank you for giving us the opportunity to revise our manuscript.

The manuscript has already been corrected and is attached for further evaluation.

We would like to express our great appreciation to you and the reviewers for your comments on our paper. We look forward to hearing from you.

Thank you and best regards.

Yours sincerely,

Qiuju Wang

Heilongjiang Academy of Agricultural Sciences

368 Xuefu Road, Harbin, P.R.China, 150086

 

 

Responses to Reviewer

 

Reviewer 2:

The work does not introduce new information. The positive effects of straw have been known for a long time. The paper is written quite correctly. Incomplete description of the methodology for determining the content of components in soil. This needs to be supplemented.

RESPONSE：

Thank you for your constructive feedback and valuable insights on our manuscript. We have revised the manuscript in accordance with the suggestions in the Appendix. Details of the modifications are described as follows:

• We have supplemented the detailed description of the method used for determination soil nutrients in Chapter 2.3.

Line 131: We added “For the assessment of alkali-hydrolyzable nitrogen, the method utilizes principles of alkaline hydrolysis, employing sodium hydroxide (NaOH) under high-temperature sealed conditions to hydrolyze soil samples. This process converts organic nitrogen into ammonium nitrogen. The resulting ammonia is subsequently collected through diffusion or distillation, absorbed by boric acid, and quantified using standard acid titration techniques. This method effectively indicates the soil's short-term mineralizable nitrogen potential. The determination of available phosphorus is conducted using Olsen's classical sodium bicarbonate extraction method (0.5 mol/L NaHCO₃, pH 8.5), which utilizes competitive adsorption mechanisms to extract active phosphorus fractions. The extract is then quantified spectrophotometrically using the molybdenum-antimony ascorbic acid colorimetric method, providing precise characterization of phosphorus availability in neutral-to-alkaline soils. The analysis of available potassium is conducted through extraction using neutral ammonium acetate (1 mol/L NH₄OAc), where cation exchange mechanisms facilitate the release of exchangeable potassium from soil colloids. The filtered extract is subsequently measured via flame photometry, a method that ensures simplicity and minimizes interference. For the determination of total organic carbon (TOC), the modified Walkley-Black wet oxidation method, as adapted by Douglas, is employed. This involves the oxidation of organic carbon to CO₂ using potassium dichromate-sulfuric acid under heated conditions. The residual oxidant is back-titrated with ferrous sulfate to quantify the carbon content.”

 

(2) The authors use methods described half a century ago. Such an old thing. There are a lot of colourful figures in the text and not very readable. Well-edited tables would make more sense.

RESPONSE：

To more effectively illustrate the impact of straw return on soil nutrient composition, we have replaced the original bar chart with a table;

We deleted figure 1

Line 255: We added Table 1.

Line 411: We added Table 2.

Line 455: We added Table 3.

 

(3) In the text, the authors only state how much what has increased or decreased. There is absolutely no indication of the range of values of the labelled quantities.

RESPONSE：

We have proofread and revised the units throughout the manuscript. We changed “increase ***” to “increase from *** to ***”.

 

(4) The discussion lacks comparison with other data. There are only numbers from references. This is not enough.

RESPONSE：

According to the recommendations, we have revised the discussion section.

Line 468: We added “In alignment with existing research, the application of straw return has been shown to significantly increase total organic carbon content (Figure 1d), thereby providing essential organic carbon and altering the soil C/N ratio, which enhances soil fertility and supports sustainable agricultural systems [31-33]. A meta-analysis of 446 datasets from various studies in China indicated that straw return increased soil organic carbon (SOC) content by an average of 13.97% [31]. Furthermore, the duration of straw return is critical, with optimal outcomes observed within 6-9 years of continuous application, after which the benefits may decline [31]. This is corroborated by a global synthesis reporting an average SOC increase of 3.68 Mg C ha^-1 due to straw return, with a corresponding carbon efficiency of 20.51% [32]. Additionally, straw return has been demonstrated to promote the accumulation of soil organic carbon over extended periods. In China, for example, increased straw return has resulted in significant SOC accumulation in croplands over the past 40 years (1980-2020), with an average national carbon sequestration rate of 27 Tg C yr^-1 [33]. This long-term accumulation is crucial for enhancing soil health and mitigating climate change. Consistent with the findings, straw returning is an effective management practice for regulating soil nutrients and reducing the loss of organic carbon, alkali-hydrolyzed nitrogen, available phosphorus, and available potassium from agricultural soils.”

Line 493: We added “The nutrient composition of soil fluctuates with the planting period, in this study (Table 1), compared to the sowing stage, the content of detection indexes in all samples with a noteworthy declining tendency though a long-time treatment, that might be attributed to the consumption of nutrients during the physiological process of plant growth [37-39], including the development of floral buds, the production of shoot and root biomass.”

 

We have revised the conclusion section by eliminating redundant descriptions and streamlining the overall presentation.

Line 563: We added “In the rice planting field, experimental results concerning soil nutrient content indicated that the straw returning treatment (SR) significantly enhances the accumulation of soil organic carbon, available phosphorus, alkali-hydrolyzed nitrogen, and available potassium, compared to native soil groups. In comparison to the CK treatment, the successive application of square blocks of rice straw to the field positively influenced spikelets per panicle and grain filling, thereby contributing to an overall increase in rice yield. The significant alterations in nutrient composition within Albic soil represent another major factor contributing to the substantial fluctuations observed in crop yield-related indices. The results from soil microbial community analyses indicated that straw incorporation prior to crop planting exerts varying effects on soil microbial diversity and community structure in Albic soil, enhancing the proliferation of bacteria or fungi in specific soils. For example, relative to CK, the SR treatment demonstrated greater efficacy in increasing the relative abundance of Bacteroidales in the deeper soil layers of samples collected at both sowing and maturity stages. Conversely, the concentrations of the other two predominant species, Anaerolineales and Micrococcales, demonstrated a declining trend following the return of straw. In terms of fungal communities, Thelebolales, which represented the second highest mean abundance among total microorganisms, exhibited a significantly increasing trend with straw incorporation. In contrast, the variation trend of Mortierellales was opposite to that of Thelebolales during the straw return at the maturity period. Collectively, these findings suggest that straw incorporation may positively influence the enhancement of nutrient composition in Albic soil and offer potential environmental benefits.”

 

We have revised and corrected the formatting of all cited references.

Line 624: We added “2.   Xiu, L.Q.; Zhang, W.M.; Sun, Y.Y..; Wu, D.; Meng, J.; Chen, W.F. Effects of biochar and straw returning on the key cultivation limitations of Albic soil and soybean growth over 2 years. Catena 2019, 173, 481–493. 3.       Liu, Z.J.; Zhou, W.; Shen, J.B.; Li, S.T.; He, P.; Liang, G.Q. Soil quality assessment of Albic soils with different productivities for eastern China, Soil Till Res 2014, 140, 74–81.”

Line 630: We added “5.   Liu, T.; He, G.J.; Lau, A. K. H. Statistical evidence on the impact of agricultural straw burning on urban air quality in China. Sci Total Environ. 2020, 711, 134633. 6.   Singh, G.; Gupta, M.K.; Chaurasiya, S.; Sharma, V.S.; Pimenov, D.Y. Rice straw burning: a review on its global prevalence and the sustainable alternatives for its effective mitigation. Environ Sci Pollut Res Int. 2021, 28, 32125–32155. 7.       Shi, T.T.; Liu, Y.Q.; Zhang, L.B.; Hao, L.; Gao, Z.L. Burning in agricultural landscapes: an emerging natural and human issue in China. Landscape Ecol 2014, 29, 1785–1798.”

Line 674: We added “25. Noveriza, R.; Rahajoeningsih, S.; Harni, R.; Miftakhurohmah. Molecular identification of white root fungal pathogens and in vitro effect of nanopesticide. IOP Conf Ser: Earth Environ Sci 2020, 418, 012085. 26.      Chen, S.F.; Zhou, Y.Q.; Chen, Y.R.; Gu, J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics, 2018, 34, i884–890.”

 

(5)Twenty-six observations are highlighted in the text. Data from the supplement should be in the text.

RESPONSE：

We have incorporated the test results regarding the influence of straw return on fungal incidence into the manuscript.

Line275: We added “figure 1”.

• Figure 2 presents the statistical results of crop yield-related indicators sampled across the continuous eight years using box-and-line diagrams. This approach is intended to demonstrate trends in variations and facilitate statistical analysis. We believe this revision more accurately depicts the changes in yield, thereby enabling a comprehensive assessment of the impact of straw return;
• We have added the Tables 2 and 3 in the manuscript to facilitate a deeper understanding of the alterations in soil bacterial and fungal communities at the order level following straw incorporation;

Thank you for your suggestions, which have improved the logical coherence of the paper.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors should take into account the following:
1. the abstract states that the research was conducted in pots, however the methodology and results mention plots.
2. why did the authors use such a high dose of straw to assess its effect on soil properties, is this a realistic amount to be obtained in agricultural practice in the region of the research conducted?
3. was the rice crop grown in rotation or in monoculture, where was the straw used in the plots with permanent addition of straw obtained from?
4. what was the soil tillage system?
5. what was the nutrient balance over the 8-year period, and what was the C:N ratio of the introduced straw?
6. what can explain the decrease in assimilable potassium during the growing season?
7. why was it decided to test the soil in the 0-10 and 10-20 cm layers since the straw was mixed with the soil to a depth of 20 cm?
8.Are the data presented in the figures average values over a period of 8 years?, it would be useful to show how the parameters changed during the first years after the establishment of the experiment to be able to evaluate the dynamic impact assessment.
9. figure 5 , 6, 7 are not very clear.

Author Response

Revised manuscript agronomy-3207042 " Effects of continuous straw returning on soil nutrients and microbial community structure of Paddy Field in Northeast China"

Dear Holly Luan Editor and reviewers:

On behalf of my co-authors, thank you for giving us the opportunity to revise our manuscript.

The manuscript has already been corrected and is attached for further evaluation.

We would like to express our great appreciation to you and the reviewers for your comments on our paper. We look forward to hearing from you.

Thank you and best regards.

Yours sincerely,

Qiuju Wang

Heilongjiang Academy of Agricultural Sciences

368 Xuefu Road, Harbin, P.R.China, 150086

 

 

Responses to Reviewer

 

Reviewer 3:

The authors should take into account the following:

1. The abstract states that the research was conducted in pots, however the methodology and results mention plots.

RESPONSE：

Line 11: We changed “pot” to “field”.

 

2. Why did the authors use such a high dose of straw to assess its effect on soil properties, is this a realistic amount to be obtained in agricultural practice in the region of the research conducted?
3. Was the rice crop grown in rotation or in monoculture, where was the straw used in the plots with permanent addition of straw obtained from?

 

RESPONSE：

Sorry, this was a mistake. Thank you very much for discovering it.

Line 116: We changed “80g/kg” to “7500 kg/ha”.

The experimental field is situated in northeastern China, where a single crop is cultivated annually. Rice is grown in a monoculture system from May to October, while the soil remains frozen from November to April. Upon reaching maturity, the rice is harvested, and the straw residue is left in the field, subsequently being crushed and incorporated into the soil through tilling.

 

4. What was the soil tillage system?

RESPONSE：

Line111: We added “Following the autumn rice harvest each year, the straw is processed in this manner. After the winter period, when the soil temperature surpasses 5 °C, water is introduced to level the field surface. The water level is maintained at 3-5 centimeters, and once the soil has settled, rice transplanting is conducted.”

 

5. What was the nutrient balance over the 8-year period, and what was the C:N ratio of the introduced straw?

RESPONSE：

Line131: We added “The straw in the experimental field was returned to the field on site, with an average organic carbon content of 34.65%, total nitrogen content of 0.542%, and a carbon to nitrogen ratio of 64:1. The experiment did not take into account the nutrient balance over the 8-year period, as the carbon and nitrogen deposition from paddy fields cannot be estimated as they enter the soil and evaporate into the hydrosphere and atmosphere.”

 

6. What can explain the decrease in assimilable potassium during the growing season?

RESPONSE：

Line501: We added “The consistent application of straw to agricultural fields is a primary factor contributing to the enhancement of soil's available potassium content. During the plant growth cycle, particularly at the rapid growth stage, there is a substantial demand for potassium to facilitate various physiological processes, which primarily accounts for the reduction in soil's available potassium content. Furthermore, in Northeast China, the plant growth period predominantly spans from May to October. During this period, increased precipitation and irrigation practices significantly contribute to the depletion of available potassium in the soil.”

 

7. Why was it decided to test the soil in the 0-10 and 10-20 cm layers since the straw was mixed with the soil to a depth of 20 cm?

RESPONSE：

In most plants, particularly in juvenile stages, the root system predominantly resides within the uppermost soil stratum, which serves as the principal source for nutrient absorption. Consequently, examining the nutrient composition within the 0-10 cm soil layer provides a direct indication of the primary nutrients accessible to plants. As plants mature, their root systems extend downward, penetrating deeper into the subsurface soil. Therefore, analyzing the nutrient content of the 10-20 cm soil layer offers insights into the nutrient availability during the intermediate and later stages of plant growth, thereby contributing significantly to the assessment of overall soil fertility. Furthermore, conducting stratified soil analyses prevents the obscuring of variability between distinct soil layers, which might occur if the entire soil profile were examined collectively. Considering that many references design experiments in this way, we did not provide a specific description.

Of course, if you feel it is necessary to annotate, we will add some content in the discussion section.

 

8. Are the data presented in the figures average values over a period of 8 years?, it would be useful to show how the parameters changed during the first years after the establishment of the experiment to be able to evaluate the dynamic impact assessment.

RESPONSE：

We only collected a soil sample once. Eight years is an experimental process. We are just maintaining such a valuable soil sample and will persevere.

Line324: We added “2023”.

Line 172: We changed “during the experimental process” to “at 2023”.

 

9. figure 5 , 6, 7 are not very clear.

We have revised. Stacked graphs illustrate the variations in species communities (Figure 5 and Figure 6). To more accurately capture the impact of straw return on the relative abundance of microorganisms, the study supplemented an analysis of variations of bacterial and fungal at order level across various treatments and detection depths (see Table 2 and Table 3).

 

Thank you for your suggestions, which have improved the logical coherence of the paper.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for considering the reviewer's wishes.

Author Response

Thanks！

Reviewer 2 Report

Comments and Suggestions for Authors

The work has only been partially improved. The methodology has been improved. There are many figures in the text but the words describing the figures are too small. Some figures have been correctly replaced by tables. The notation of units has still not been corrected. Data from the supplement should be in the text. 15 comments are marked in the text.

Comments for author File: Comments.pdf

Author Response

Responses to Reviewer

 

Reviewer 2:

The work has only been partially improved. The methodology has been improved. There are many figures in the text but the words describing the figures are too small. Some figures have been correctly replaced by tables. The notation of units has still not been corrected. Data from the supplement should be in the text. 15 comments are marked in the text.

RESPONSE：

Thank you for your constructive feedback and valuable insights on our manuscript.

All figures have been submitted as 300DPI files. You may contact the editor to download them.

Comment 1

E-mail addresses of the authors are missing.

RESPONSE：

Line 6: We deleted “; e-mail@e-mail.com”

 

Comment 2

Please write the units correctly. Correct everywhere.

RESPONSE：

Line 320: We changed “hm²” to “ha”.

We checked “/ha”, “kg/”and “g/”.

 

Comment 3

Increase the letters in the figures.

RESPONSE：

Using letters may look more aesthetically pleasing.

In the paper, we used "*" to indicate whether there are significant differences. We believe it is not appropriate to add letters, as different letters can only indicate whether there are significant differences (p < 0.05). "***" can express extremely significant differences (p < 0.001). However, if you insist on the modification, we are also agreeable to changing it to letters.

 

Comment 4

Figure S2 ?

All data from the supplement must be included in the text of the article.

Comment 5

Figure S3 ?

Comment 6

Increase the letters in the figures.

Comment 7

Figure S4a?

Comment 8

Figure S4c ?

Comment 9

Figure S3 ?

Comment 10

Figure S1a ?

RESPONSE：

This was our mistake, thank you very much for discovering it.

In the last revision, Figure S5 was changed to Table 2,3.

In the last revision, Figure 1 was changed to Table 1, and Figure S1 was changed to Figure 1, but we did not sequentially change all Figure labels.

Line 309, 607: We changed “(Figure S2)” to “(Figure S1)”.

Line 312, 609: We changed “(Figure S3)” to “(Figure S2)”.

Line 610: We changed “(Figure S4)” to “(Figure S3)”.

Line 371: We changed “(Figure 3a and Figure 3c)” to “(Figure 4a and Figure 4c)”.

Line 375: We changed “(Figure 3b and Figure 3d)” to “(Figure 4b and Figure 4d)”.

Line 378: We changed “(Figure S4a)” to “(Figure S3a)”.

Line 385: We changed “(Figure S4b and Figure S4d)” to “(Figure S3b and Figure S3d)”.

Line 381: We changed “(Figure S4c)” to “(Figure S3c)”.

Line 530: We changed “(Figure S3)” to “(Figure S1)”.

Line 559: We changed “(Figure S1a)” to “(Figure 1a)”.

Line 480: We changed “(Figure 1d)” to “(Table 1)”.

Line 549: We changed “(Figure 5c)” to “(Table 2)”.

Line 551: We changed “(Figure 5e)” to “(Table 2)”.

Line 553: We changed “(Figure 5e)” to “(Table 2)”.

Line 553: We changed “(Figure 7d)” to “(Table 3)”.

Line 607: We deleted “Figure S1. Disease Index Evaluation of rice leaf blast (a), panicle neck blast (b), sheath rot disease (c), rice sheath blight (d), and rice fales smut (e). Note: CK: no rice straw returning; SR: rice straw returning. * p < 0.05, ** p < 0.01, and *** p < 0.001.”.

Line 614: We deleted “Figure S5. A phylogenetic tree elucidating the microbial community differences between the control and treatment groups based on the representative sequences of fungal OTUs using the Neighbor-Joining method. (a). Samples taken at sowing stage; (b). Samples taken at maturity stage. The color of the separate leaves indicates the different classes of the microorganisms. The outer circle indicates the distribution of the order-level taxonomy of OTUs. Numbers at nodes indicate bootstrap values based on 1000 replicates. Bar, 0.03 substitutions per nucleotide position.”.

 

 

 

Comment 11

This sentence here is not necessary.

RESPONSE：

Line 603: We deleted “6. Patents

This section is not mandatory but may be added if there are patents resulting from the work reported in this manuscript.”.

 

Comment 12

At this address, we get this notation.

  File not found

RESPONSE：

Line 603: We deleted “The following supporting information can be downloaded at: www.mdpi.com/xxx/s1,”.

 

Comment 13

Y.Y..;

correct

RESPONSE：

Line 634: We changed “Y.Y..” to “Y.Y.”.

 

Comment 14

No space.

A.K.H.

RESPONSE：

Line 640: We changed “A. K. H” to “A.K.H”.

 

Comment 15

Correct the spelling of the names and the title of the journal.

 

RESPONSE：

Line 721: We changed “J. Chen, J.” to “Chen, J.”.

Line 735: We changed “Liu, Y.H. Zhang, S.H” to “Liu, Y.H.; Zhang, S.H”.

Line 689: We changed “Journal of Environmental Management” to “J Environ Manage”.

 

Thank you for your suggestions, which have improved the logical coherence of the paper.