Reasonable Nitrogen Regime in the Main Crop Increased Grain Yields in Both Main and Ratoon Rice
Round 1
Reviewer 1 Report
Applies to: 2.3.5. Statistical analysis:
1) The question of the minimum sample size has to be clarified (for each experiment).
2) The tested variables are certainly measurable but they must still be of a continuous variable nature (add to description).
3) The study shows that the authors used parametric analysis of variance (ANOVA) - it should be confirmed that the normality of the population distribution was examined (eg Kolmogorov-Smirnow test, or Shapiro-Wilk test, etc.) and the homogeneity of variance in the samples.
4) Why was LSD used as a post-hoc test (it has its drawbacks)? The disadvantage of this method is that it does not adjust the observed significance level for multiple comparisons. Explain in 2.3.5. Statistical analysis.
Other remarks:
1) Figure 1-7 - try to examine the quality of axis description.
2) Figure 4. Relationships between number .... You give R (correlation coefficient) and plots A, C, and E have a nonlinear trend (Pearson's correlation has the character of a linear relationship ...). Explain this in 3.4.2. Correlations between .... If you are using a trend line, also enter the coefficient of determination (R2). You can also provide equations that describe these trends (regression equation).
Author Response
Applies to: 2.3.5. Statistical analysis:
Point 1: The question of the minimum sample size has to be clarified (for each experiment).
Response 1: Four replicates were set for each treatment in this experiment. When root activity, NSC content, regenerated bud (length, fresh weight), regeneration rate and yield were invested, each replicate of these indicators was investigated. Therefore, the minimum sample size of each indicator in this experiment is 4. And we have clarified this question in 2.3.5.
Point 2: The tested variables are certainly measurable but they must still be of a continuous variable nature (add to description).
Response 2: The number, length and fresh weight of regenerated buds, regeneration rate, root activity and NSC content in stem are indicators that reflect the growth and development of rice, and these indicators will change as the external environment changes. For example, with the change of the N application rate, these indicators have corresponding values, and these values are continuously and separable, so they are continuous variable variables. Therefore, this experiment used Pearson's correlation and regression analysis to analyze their relationship. And we have added some sentence in 2.3.5 to clarify this question.
Point 3: The study shows that the authors used parametric analysis of variance (ANOVA) - it should be confirmed that the normality of the population distribution was examined (eg Kolmogorov-Smirnow test, or Shapiro-Wilk test, etc.) and the homogeneity of variance in the samples.
Response 3: We have checked the original data and find that the parameters of different treatments are of normal distribution and a common variance s2.
Point 4: Why was LSD used as a post-hoc test (it has its drawbacks)? The disadvantage of this method is that it does not adjust the observed significance level for multiple comparisons. Explain in 2.3.5. Statistical analysis.
Response 4: Yes, we are aware of the major drawback of a multiple-comparison procedure with a controlled per-comparison error rate. We also used Tukey’s multiple-comparison. Both of the analyses give the same results. For simplicity, we only present results of LSD method.
Other remarks:
Point 5: Figure 1-7 - try to examine the quality of axis description.
Response 5: We have improved the quality of axis description in Figure 1-7.
Point 6: Figure 4. Relationships between number .... You give R (correlation coefficient) and plots A, C, and E have a nonlinear trend (Pearson's correlation has the character of a linear relationship ...). Explain this in 3.4.2. Correlations between .... If you are using a trend line, also enter the coefficient of determination (R2). You can also provide equations that describe these trends (regression equation).
Response 6: We apologize for the mistake. It is inappropriate to use R to reflect the significance of nonlinear correlation. We have replaced R with R2, and provided regression equations to describe their correlation in Fig.4, Fig.6, Fig.7, Fig.8, Fig S5 and Fig S7.
Author Response File: 
Author Response.doc
Reviewer 2 Report
Manuscript Number: agriculture-1626902
Subject: Reasonable Nitrogen Regime in the Main Crop Increased Grain Yields in Both Main and Ratoon Rice
-The manuscript needs thorough and comprehensive corrections in all sections of the manuscript, from abstract to conclusions. The authors should put a tremendous effort into technically and linguistically shaping the contents of the manuscript.
My comments are listed as follows:
-The abstract is not properly structured and more results are appreciated.
-More recent papers that have been conducted for these purposes are advised to be included in the paper.
-The experimental program must be reconstructed. It is advised to include some photos of sample preparation for a better understanding of the test preparation and technical drawings of the test set up including all the instrumentation.
-The quality of figures is low. All Figures should be clear for the readers. Please improve their quality.
-More discussion should be made.
-Conclusion must be reconstructed & simplified into 5-6 main points.
Author Response
Point 1: The manuscript needs thorough and comprehensive corrections in all sections of the manuscript, from abstract to conclusions. The authors should put a tremendous effort into technically and linguistically shaping the contents of the manuscript.
Response 1: We have carefully revised and polished the manuscript according to your comments and suggestions.
My comments are listed as follows:
Point 2: The abstract is not properly structured and more results are appreciated.
Response 2: We have rewritten the abstract.
“Planting ratoon rice can realize one sowing and two harvests, which is of great significance for improving grain yield. However, the effects of nitrogen (N) regime in the main crop on the grain yield of ratoon rice and the associated physiological mechanisms are not clearly understood. The indica hybrid rice Liangyou 6326 was used, and three N fertilizer levels [100 kg ha-1 (low N, LN), 250 kg ha-1 (medium N, MN), and 400 kg ha-1 (high N, HN)]and four different ratios of basal tillering fertilizer to panicle fertilizer [7:3, 6:4, 5:5 and 4:6] applied to the main crop were designed to investigate their effects on the grain yields of the main and ratoon crops. The results showed that excessive N application rate and panicle N rate in main crop was not conducive to the improvement of yield and agronomic nitrogen use efficiency (ANUE) in both seasons. The increased yield in the ratoon crop was attributed to the increase in regeneration rate. Appropriate increasing the panicle N application rate was beneficial for increasing the ROA and NSC concentration in the main crop, resulting in increase in the number, length and fresh weight of regenerated buds, which caused an improvement in the regeneration rate. However, when excessive panicle N was applied in main crop, the excessive germination of regenerated buds decreased length and fresh weight of regenerated bud and resulted in a decrease in regeneration rate. These results suggest that in the production of ratoon rice, through reasonable N regime in the main crop could increase the yield and ANUE in both seasons.”
Point 3: More recent papers that have been conducted for these purposes are advised to be included in the paper.
Response 3: We have added some recent references in the introduction and discussion.
“Huang et al. (2022) reported that increasing N application rate in late growth stage of the main crop could improve the effective tillering percentage, leaf area index, canopy light interception rates and transport rate of stem and sheath in main crop, leading to an increase in yields of both seasons [17]. Properly postponed N application in the main crop was beneficial to increase the root activity and promote the growth of rice, resulting in increasing the yields of both seasons [18].”
“Wang et al. (2019) indicated that the N application rate in main crop had litter effect on the yield of ratoon crop [14]”
“Zhang et al. (2019) revealed that N application rate and N application method in main crop had a great effect on the yield of ratoon crop [12]. Some scholars reported that increasing N application rate in late growth stage of the main crop was beneficial to promote the growth and development of the aboveground and underground organs of rice, which could improve the yield of main and ratoon crop [17-19]. Nakano et al. (2009) indicated that N application rate of 22.5 g N m-2 in main crop had higher dry matter yield of ratoon crop than with 15.0 g N m-2, and more N was applied early in main crop could obtain higher dry matter yield [41].”
Point 4: The experimental program must be reconstructed. It is advised to include some photos of sample preparation for a better understanding of the test preparation and technical drawings of the test set up including all the instrumentation.
Response 4: We have added a technical drawing “Figure S2. Technical drawings for experimental program” and provided some photos (“Figure S3. The process of obtaining root samples from the field” and “Figure S4. Field investigation on the growth and development of regenerated buds”) to explain the experimental program in supplementary date.
Point 5: The quality of figures is low. All Figures should be clear for the readers. Please improve their quality.
Response 5: We have improved the quality of all figures.
Point 6: More discussion should be made.
Response 6: We have restructured and rewritten the discussion. And we discussed the results of the paper from the following four aspects:
4.1 Reasonable N regime in the main crop improved the growth and development of regenerated buds.
4.2 Reasonable N regime in the main crop improved the regeneration rate.
4.3 Effects of N regime in the main crop on the yield of the main and ratoon crops.
4.4 Effects of the N regime in the main crop on the N use efficiency of main and ratoon crops.
Point 7: Conclusion must be reconstructed & simplified into 5-6 main points.
Response 7: We have rewritten the conclusion.
“The rate and ratio of N application in the main crop had an important influence on the rice yield and ANUE of the main and ratoon crops. Excessive total and panicle N application rate in main crop was not conducive to improve the grain yield and ANUE in both seasons. Under different N application rate, appropriate increasing the panicle N application rate was beneficial for increasing the ROA and NSC concentration in stem and leaf in the main crop, resulting in improving the growth and development of regenerated buds, which caused an increase in the regeneration rate and yield of ratoon crop. A total N rate of 250 kg ha-1 and a ratio of basal tillering fertilizer to panicle fertilizer of 5:5 in the main crop could increase grain yields and ANUE in both seasons.”
Author Response File: Author Response.doc
Reviewer 3 Report
1. Effect of nitrogen regime on rice yield has been well studied and documented. Therefore authors need to justify how their work adds new knowledge to the field and what is the novelty of the work. 2. Authors need to justify the main conclusions with sufficient scientific evidences and in depth discussion of the reasons for such observations. Although authors have explained the results with sufficient depth the discussion lacks scientific merit. 3. Authors are advised to provide the nitrogen use efficiency at different rates of nitrogen applications. NUE is more scientific way of studying the nitrogen uptake by plants. 4. Nitrogen uptake rates need to be studied and discuss with more details. although more nitrogen is applied to soil the uptake patterns might be affected by the nutrient balance in the soil. Therefore it is important to report the nitrogen uptake rate in order to reach conclusions on the effect of nitrogen regime. 5. Authors have reported the initial soil nutrient levels. However, were the parameters equal for both years including the fields here high nitrogen amounts were used? It is also need to report the nutrient values in the yields and soil.Author Response
Point 1: Effect of nitrogen regime on rice yield has been well studied and documented. Therefore authors need to justify how their work adds new knowledge to the field and what is the novelty of the work.
Response 1: We have revised the introduction and discussion and added some sentence in introduction and discussion to explain the innovations of this article.
“However, there is a lack of indepth research on the effects of N regime in the main crop on the growth and development of regenerated buds and the regeneration rate. In addition, the N regime in the main crop on the yield of ratoon crop under different N application rate have not been reported so far. Since the N application rate vary greatly in different areas in the field production of ratoon rice, so it is of great significance to study the effects of N regime in main crop on the yield of ratoon rice under different N application rate.”
“There are few reports on the effect of N regime in main crop on the growth and development of regenerated buds. The growth and development of regenerated buds are usually reflected by their number, length and fresh weight.”
“However, there are few studies on the relationship between number of regenerated buds and regeneration rate. This research showed that the regeneration rate was non-linear correlated with the number of regenerated buds.”
The related references were also added in the revised manuscript.
Point 2: Authors need to justify the main conclusions with sufficient scientific evidences and indepth discussion of the reasons for such observations. Although authors have explained the results with sufficient depth the discussion lacks scientific merit.
Response 2: We have restructured and rewritten the discussion. And we discussed the results of the paper from the following four aspects:
4.1 Reasonable N regime in the main crop improved the growth and development of regenerated buds.
4.2 Reasonable N regime in the main crop improved the regeneration rate.
4.3 Effects of N regime in the main crop on the yield of the main and ratoon crops.
4.4 Effects of the N regime in the main crop on the N use efficiency of main and ratoon crops.
Point 3: Authors are advised to provide the nitrogen use efficiency at different rates of nitrogen applications. NUE is more scientific way of studying the nitrogen uptake by plants.
Response 3: Thank you for your valuable comments and we have provided effects of N regime in main crop on the nitrogen use efficiency of main and ratoon crop in 3.7 and discussed it in 4.4.
Point 4: Nitrogen uptake rates need to be studied and discuss with more details. although more nitrogen is applied to soil the uptake patterns might be affected by the nutrient balance in the soil. Therefore it is important to report the nitrogen uptake rate in order to reach conclusions on the effect of nitrogen regime.
Response 4: Due to workload, we did not study the N uptake rates. But we studied the effects of N regime in main crop on the agronomic nitrogen use efficiency of main and ratoon corp. And we have cited some literatures to discuss the changes of nitrogen uptake rates under different nitrogen fertilizer levels and the reasons for these changes in 4.4.
“N use efficiency (NUE) is also an important factor to determine whether the ap-plication of N fertilization is reasonable. N uptake rate and NUE are closely related to the N application rate. Xu et al. (2015) reported that appropriately increasing N application rate was beneficial to improve N uptake and NUE, leading an increase in rice yield [42]. However, under high N application rate, the N recovery efficiency de-creased, the ANUE first increased and then decreased, and rice yield did not increase. Zhang et al (2019). revealed that with the increase of N application rate, the N uptake rate and ANUE first increased and then decreased [43]. Our result showed that with the N application rate increased, ANUE decreased in main crop, no increasing trend in HN in ratoon crop, and first increased then decreased in both seasons (Table 6). Excessive N application reduces the number and physiological activity of roots [44], and reduces the absorption of inorganic N in paddy soil [43], resulting in less N absorbed by plants and lower N uptake rate and NUE.
The N application ratio also has a great influence on the ANUE. Because the seed-lings had less root system and less N was absorbed in the early stage of rice growth, appropriately reducing basal N and appropriately increasing panicle and tiller N could improve the NUE [45]. Our result is consistent with that (Table 6).”
Point 5: Authors have reported the initial soil nutrient levels. However, were the parameters equal for both years including the fields here high nitrogen amounts were used? It is also need to report the nutrient values in the yields and soil.
Response 5: The soil nutrient content in the article refers to the average annual soil nutrient content in the field. Due to the different N application rate will change soil nutrients fertility level, which could cause errors in the test results. The second year's experiment were conducted in different places on a same field. The purpose of this experiment was to study the effect of different N management methods on the grain yield of main and ratoon crop, so the nitrogen content in soil and rice yield were not studied. However, we have added some sentences in 4.4 to discuss the effect of different soil nutrient levels on rice yield and nitrogen use efficiency.
“In addition, Xu et al. (2011) reported that N uptake and N use efficiency were not only related to N regime, but also to soil characteristics [46]. Under high N supply of soil, reducing the N application rate in basal was beneficial to improve NUE and rice yield. However, under low N supply of soil, reducing the N application rate in basal would result in decrease in NUE and rice yield [47]. Liu et al. (2005) indicated that compared with low N supply of soil, the effect of N rate on rice yield was reduced, and NUE was also decreased under high N supply of soil [48]. In China, the yield of rice is usually 5-6 t ha-1 in N free areas of paddy soils [49]. In this experiment, the yield of N free area was 5.28 t ha-1 in the main crop and 8.43 t ha-1 in the total yield of two seasons, belonging to the area with medium and upper fertility. Therefore, during the ratoon rice production of the area, appropriately reducing the application rate of base tillering fertilizer and increasing the application rate of panicle fertilizer is beneficial to improve the NUE and the yield of both seasons.”
Author Response File: Author Response.doc
Round 2
Reviewer 2 Report
Manuscript Number: agriculture-1626902
Manuscript Subject: Reasonable Nitrogen Regime in the Main Crop Increased Grain Yields in Both Main and Ratoon Rice
The authors have made extensive modifications in the manuscript, adjusting it to the comments made by the reviewer.
Good luck
