Effects of Organic Fertilizer Substitution for Chemical Fertilizer on Grain Yield and 2-Acetyl-1-pyrroline (2-AP) of Fragrant Rice

Comments 1: [Introduction: Please add a research hypothesis.]

Response 1: Thank you for your valuable comments. We fully agree that there is a need to add a research hypothesis. The specific information is shown as (Page 2, Paragraph 4, Line 80-82) “Given this background, we hypothesized that 1) organic fertilizer substitution for chemical fertilizer can increase grain yield of fragrant rice; 2) organic fertilizer substitution for chemical fertilizer can improve 2-AP content of fragrant rice.”

Comments 2: [Materials and Methods: Subsection 2.1. What is the purpose of providing temperatures and precipitation? The experiment was conducted in a greenhouse facility.]

Response 2: We were really sorry for our careless mistake. Thank you for pointing out the problem. This potted experiment was conducted in the net-room of the practice base of South China Agricultural University. We mistakenly wrote it as greenhouse in the article. Now we have corrected it to net-room and added relevant description. Climate and precipitation data were provided to establish the environmental context of the experiment, thereby facilitating the evaluation of the applicability of the results and enhancing their credibility. The specific information is shown as (Page 3, Paragraph 5, Line 101-102) “The net-room is mainly composed of stainless steel wires with a diameter of 1 cm to prevent rats and birds from entering.”

Comments 3: [What organic fertilizer was used? Please provide the chemical composition of this fertilizer and the dose.]

Response 3: Thank you for pointing this out. We agree with the comment. Therefore, we have  included details regarding the type of organic fertilizer used and its relevant chemical composition in the manuscript. The specific information is shown as (Page 3, Paragraph 8, Line 128-129) “This kinds of organic fertilizer belonged to animal manure (43% organic matter, 2.31% nitrogen, 2.98% phosphorus and 3.82% potassium).”

Comments 4: [Provide the granulometric composition of the soil material in the pots and basic physicochemical properties.]

Response 4: Thank you for your constructive feedback. Below, we have provided further details regarding the granulometric composition and basic physicochemical properties of the soil material. The specific information is shown as (Page 3, Paragraph 5, Line 104-106) “The experimental soil was a sandy loam, with 21.44 organic C, 1.70 g kg-1 total N, 1.24 g kg-1 total P, 22.85 g kg-1 total K, 79.03 mg kg-1 available P, 108.30 available K, and pH=6.50.”

Comments 5: [According to what statistical method was the experiment established?]

Response 5: We sincerely appreciate the valuable comments. We have added the statistical method in this section according to the reviewer’s suggestion. The specific information is shown as (Page 5, Paragraph 16, Line 192-193) “Multiple comparisons were performed using the least significant difference (LSD) test.”

Comments 6: [For all analytical methods mentioned, the rules for performing these methods should be presented.]

Response 6: Thanks for your good advice. We have revised them according to your advice.

Comments 7: [Results: Table 1 - The units must be added (columns 3 and 4). Line 202 - Please standardize the spelling CK or T0? (note applies to the entire manuscript). The title of subsection 3.6 should be expanded.]

Response 7: Thanks for the comment. We have made the corrections as suggested by the reviewers.

Comments 8: [Discussion: From subsection 4.1, enzymatic activity should be separated as a separate subsection.]

Response 8: Thank you for your careful reading and constructive feedback. Following the reviewer’s suggestions, we have included a separate subsection to discuss enzyme activity. This modification improves readability. The specific information is shown as (Page15, Paragraph 31, 32, Line 479-511) “Nitrate reductase (NR) and glutamine synthetase (GS), key enzymes in nitrogen metabolism, directly regulate nitrogen uptake and assimilation efficiency [40]. This study revealed that substituting chemical fertilizers with organic fertilizers enhanced NR and GS enzyme activities in flag leaves during PI and HS stages. This enhancement may stem from the sustained nutrient supply provided by organic fertilizers, which offers energy support to maintain high enzyme activity. Elevated NR and GS activities serve as a critical foundation for improving nitrogen use efficiency. Accordingly, we infer that organic fertilizer substitution optimized nitrogen supply, providing sufficient nutrients during the vegetative growth stage to promote effective tiller formation, thereby increasing the number of effective panicles at the heading stage. This result is consistent with Moe et al. [41]. Additionally, peroxidase (POD) and catalase (CAT), as key antioxidant enzymes, scavenge reactive oxygen species (ROS) and protect cellular integrity [42, 43], while malondialdehyde (MDA) reflects oxidative stress levels [44]. The results showed that organic fertilizer substitution increased POD and CAT activities. At the same time, it reduced MDA content. This suggests that organic fertilizers alleviated oxidative stress in fragrant rice, reducing cell membrane damage and enhancing antioxidant capacity. This phenomenon indicates that organic fertilizer ap-plication effectively mitigated oxidative stress in rice, decreasing lipid peroxidation damage to cell membranes and thereby bolstering plant stress resistance, a finding consistent with previous studies [45,46]. Previous studies have confirmed that in-creased nitrogen levels positively promote the enhancement of antioxidant enzyme activities [47,48]. Thus, we hypothesize that the slow-release properties of organic fertilizers prevented drastic fluctuations in nutrient supply, reducing oxidative damage to plants caused by excessive ROS accumulation. Concurrently, the enhanced activity of key nitrogen metabolism enzymes further supported the high expression of antioxidant enzymes, ultimately manifested as increased POD and CAT activities and reduced MDA content.

In summary, the substitution of chemical fertilizers with organic fertilizers enhanced nitrogen use efficiency by increasing the activities of NR and GS, while simultaneously mitigating oxidative stress through an optimized antioxidant system (elevated POD and CAT activities and reduced MDA content). This dual regulatory mechanism supported the performance of fragrant rice during vegetative growth and stress adaptation. The experimental results ultimately manifested as improvements in both yield and aroma content.”

Comments 9: [Lines 424-425 Please refine this sentence.]

Response 9: Thanks for the comment. We have made the corrections as suggested by the reviewers. The specific information is shown as (Page14, Paragraph 28, Line 433-441) “This strategy provide a reference scientific basis for the efficient cultivation of fragrant rice. However, since this study was conducted under pot conditions, future research is recommended to further investigate the effects of 33.3% organic fertilizer substitution for urea and other substitution ratios under field conditions to confirm their applicability and effectiveness in practical agricultural production. Future studies should focus on optimizing fertilization ratios and evaluating performance under different environmental and management conditions to provide more comprehensive fertilization guidance, thereby promoting the cultivation of fragrant rice and the sustainable development of agriculture.”

Comments 10: [Conclusions: Lines 480-483 This sentence should be corrected. This cannot be concluded based on an experiment conducted in pots, under strictly controlled greenhouse conditions.]

Response 10: Thanks for the comment. We have made the corrections as suggested by the reviewers. The specific information is shown as (Page 16, Paragraph 33, Line 515-519)“The results of this study indicate that the strategy of substituting chemical fertilizers with organic fertilizers, particularly under the treatment with 33.3% organic fertilizer substitution for urea (T2), significantly improved the yield and aroma quality of fragrant rice. These findings provide a reference scientific basis for the effective enhancement of yield and aroma in fragrant rice.

Comments 11: [Line 348 - It should be: ...figure 7...]

Response 11: Thanks for the comment. We have made the corrections as suggested by the reviewers.

Comments 12: [Line 361 - Please improve the font size.]

Response 12: Thanks for the comment. We have made the corrections as suggested by the reviewers.

Comments 1: [This paper shows only substitution of N fertilizer (not other nutrients); so,this title is not in line with the treatments and the results of the experiment.]

Response 1: Thanks for your comments. In fact, the main components of organic fertilizer used in the experiment are 43% organic matter, 2.31% nitrogen, 2.98% phosphorus and 3.82% potassium.

Comments 2: [The contents of the Abstract may need to be revised after properly revising the data analyses and the presentation of the results.]

Response 2: Thanks for the comment. We have made the modification as suggested by the reviewers. The specific information is shown as (Page 1, Line 11) “improving grain qualities and economic benefits.”

Comments 3: [Normally organic fertilizer is high dose (voluminous) compared with inorganic fertilizer; so, application of organic fertilizers is normally less economical than inorganic fertilizer.]

Response 3: Applying organic fertilizer can improve grain qualities and price per unit, hence it will increase economic profits.

Comments 4: [Please explain bit more on the rationale of the positive effect of substituting N with organic fertilizer, as the main points of the urgency of this study so that those treatments were important to be examined.]

Response 4: Thank you for your valuable comments. We fully agree that there is a need to explain bit more on the rationale of the positive effect of substituting N with organic fertilizer. In the revised introduction, we have added the following in introduction part to clarify the rationale. The specific information is shown as (Page 2, Paragraph 3, Line 74-76) “Previous studies showed that organic fertilizer substitution for chemical could im-prove grain qualities, increasing nitrogen use efficiency, reducing the adverse effects of excessive nitrogen application on the environment [14,15].”

Comments 5: [Please mention the cultivar of rice tested to make it clear that the rice cultivars function as the second experimental factor making the experiment to be a factorial experiment, which necessitates the examination of the interaction effects, which also means different responses of both cultivars.]

Response 5: Thank you for your valuable comments. we have added a description of the cultivar of rice in the introduction section. The specific information is shown as (Page 2, Paragraph 4, Line 84-86) “and its physiological traits of fragrant rice (Qingxiangyou19 and Meixiangzhan2). Both Qingxiangyou19 and Meixiangzhan2 are widely planted in the area of South China because of their better grain qualities.”

Comments 6: [Please also explain more why those growth stages were important.]

Response 6: Thank you for your valuable comments. we have also added a description of those growth stage in the introduction section. The specific information is shown as (Page 2, Paragraph 4, Line 87-91) “(mid-tillering, panicle initiation and heading stages), yield and its components, 2-acetyl-1-pyrroline (2-AP) content, and substances related to 2-AP biosynthesis. Mid-tillering, panicle initiation, and heading stages are important period that deter-mine panicle number per ha, spikelets per panicle and 1000-grain-weight, respectively.”

Comments 7: [A good Method must be repeatable, i.e. everybody can easily repeat the experiment, and if repeated, similar results will be obtained. Therefore, all procedures used must be clearly and chronologically described in this section of a scientific paper. There are too many unclearly explained procedures of the experiment. Please also provide the entire photo of the entire pot experiment.]

Response 7: Thanks for your advice. We have revised the contents of “Material and Methods”, which make it more concise and clear. We are so sorry because some photos of pot experiment are missing.

Comments 8: [Is this the condition of the greenhouse or the climate of the land from which soil samples were taken? Since the experiment was conducted in a greenhouse then the climatic conditions of the greenhouse during the growth of the rice plants are the most important to be recorded.]

Response 8: The experiment was conducted in a net-room in 2024. The net-room is mainly composed of stainless steel wires with a diameter of 1 cm to prevent rats and birds from entering. The soil was taken from the paddy field in the Farm of SCAU. The authors have added the physical and chemical properties of soil. The specific information is shown as (Page 3, Paragraph 5, Line 99, 101-102) “The net-room is mainly composed of stainless steel wires with a diameter of 1 cm to prevent rats and birds from entering.”

Comments 9: [Please explicitly describe how many seedlings were transplanted to each treatment pot? If more than one, how the seedlings were arranged in each pot?]

Response 9: Thanks for your advice. We have added relevant descriptions of this aspect in the article. The specific information is shown as (Page 3, Paragraph 5, Line 115-116) “Four-hill seedlings was transplanted in a cross line, with four rice seedlings per each hill in every pot.”

Comments 10: [Diameter at top or bottom of the pots?]

Response 10: 26 cm is the diameter of bottom of the pot. Thanks. The specific information is shown as (Page 3, Paragraph 5, Line 114) “bottom diameter 26 cm, height 25 cm”

Comments 11: [At what moisture content?]

Response 11: The soils were taken from paddy field, dried in the sun, sieved. Then 10 kg dry soil was put into every pot.

Comments 12: [What was the soil condition at transplanting? Dry, flooded or saturated? If

flooded, was the soil pudled (become muddy) or not?]

Response 12: When the soil becomes muddy, then transplanting rice seedlings.

Comments 13: [Please explicitly describe how many replications were prepared for each

combination of fertilization treatment and varieties of rice, and which type of experimental design was used to arrange all the experimental units. According tothe principle of agricultural experimentation (see Gomez & Gomez (1984), Statistical procedures for agric research), the minimum number of replications is three (3x).]

Response 13: Every treatment was planted for 10 pots, and arranged in a split plot design. The specific information is shown as (Page 3, Paragraph 8, Line 127) “Each treatment was planted for 10 pots and arranged in split plot design.”

Comments 14: [100% of what amount?]

Response 14: All fertilizers were used as organic fertilizer, without applying area.

Comments 15: [Was this 33.3% of organic fertilizer to substitute 33.3% amount of Urea or substitute 33.3% amount of N in urea?]

Response 15: 33.3% of organic fertilizer were replaced 33.3% amount of N in urea. Thanks.

Comments 16: [% of what doses and what type of organic fertilizer?]

Response 16: Thank you for your valuable comments. We have added specific descriptions of the types and components of organic fertilizers in the article. The specific information is shown as (Page 3, Paragraph 8, Line 128-129) “This kinds of organic fertilizer belonged to animal manure (43% organic matter, 2.31% nitrogen, 2.98% phosphorus and 3.82% potassium).”

Comments 17: [Please describe what was the type of organic fertilizer, what was the recommended dose, and what types of nutrients contained in the fertilizer.]

Response 17: The organic fertilizer belongs to animal manure, the recommended does was 300 kg per ha. The major components of organic fertilizer used in the experiment are 43% organic matter, 2.31% nitrogen, 2.98% phosphorus and 3.82% potassium. The specific information is shown as (Page 3, Paragraph 8, Line 128-129) “This kinds of organic fertilizer belonged to animal manure (43% organic matter, 2.31% nitrogen, 2.98% phosphorus and 3.82% potassium).”

Comments 18: [Please describe what was actually sampled in this measurement, why it was only two replicates, and why was n=8 plants? What was represented by those 8 plants?]

Response 18: I am so sorry, there was a mistake. Actually three pots were randomly selected from each treatment for harvesting (n=12 hills). We have already corrected this mistake in the article. The specific information is shown as (Page 3, Paragraph 9, Line 132-133) “During the maturity stage, three pots were randomly selected from each treatment for harvesting (n = 12 hills).”

Comments 19: [Was it really 14% moisture content with air-drying? At what temperature and for how long?]

Response 19: Thanks for your advice. The grains were air-dried at room temperature for about thirty days after harvest. The moisture content of grain was measured by Cereal Crop Moisture Measure Instrument (TDS-1G), (developed by Zhejiang Tuopu Yunnong Science Technology Limited Company).

Comments 20: [what is tillering stage (MT)?]

Response 20: Tillering stage was the key growth stage in the production of rice, which determined panicle number of rice.

Comments 21: [What was represented by the 4 rice plants? Were they randomly selected from each treatment or each experimental unit? Please clearly describe, and they represent what, and why was only 4 rice plants?]

Response 21: Four rice plants were randomly selected from each treatment. They are taken from the same pot, i.e., 4 rice plants from the same treatment. Thanks.

Comments 22: [What is a standard leaf?]

Response 22: The meaning of the sentence was that normal leaf.

Comments 23: [Was the coefficient calculated from the actual leaf area or it was obtained from other's measurement? Please describe or referenced properly.]

Response 23: Thanks for your advice. It was obtained from other’s measurement. We have added relevant descriptions for it. The specific information is shown as (Page 4, Paragraph 10, Line 148-149) “Leaf area per plant was calculated by measuring the leaf area of standard leaves using the length-width coefficient method.”

Comments 24: [Of only 4 samples or per pot? Please be specific and explicit]

Response 25: There were four hills in every pot. And four rice seedling is for each hill when transplanting. So the total number of rice seedlings are for 16 rice seedlings when transplanting for per pot. Thanks.  

Comments 25: [Please clearly describe how these environmental conditions were maintained, and how they were maintained to the same conditions between treatments for a specific time period (explain for how long).]

Response 26: Photosynthetic characteristics of rice flag leaves were measured at the full heading stage at 9:00-11:00 on a sunny morning. The climate condition was relatively stable. Thanks a lot. The specific information is shown as (Page 4, Paragraph 11, Line 154) “at 9:00-11:00 on a sunny morning”

Comments 26: [Table 1 shows the highest average of panicle number per pot is 14 in T2V2, which means there were 14 flag leaves per pot; so, how can it be 25 randomly selected flag leaves?]

Response 27: I am so sorry, the sentence was not written properly. The number “14” in T2V2 was the average panicle number per hill for the treatment T2V2. Four hills were planted in every pot. Hence, the total number of flag leaves were 56 (14*4) for every pot.

Comments 27: [If they were randomly selected it means that there must be more than 25 flag leaves per pot. However, Table 1 show the highest average of panicle number per pot is 14 in T2V2; so, the 25 randomly selected flag leaves were taken from how many series of the pot experiment. This is not logical unless they were explicitly described especially sections 2.5, 2.6 and 2.8.]

Response 28: Thanks for your question. For T2V2, the average panicle number per hill was 14, four hills were for every pot. Hence, the total number of flag leaves were 56. Then 25 flag leaves were randomly selected.

Comments 28: [In this measurement at three different growth stages, 20 flag leaves were measured, and another 25 flag leaves measured in section 2.6; so, please explicitly describe from how many series of pot experiment they were all taken (a total of 45 flag leaves)?]

Response 29: In this experiment, ten pots were used for each treatment. For example, at maturity 48 flag leaves in one pot were for T2V2. The total number of flag leaves was about 480 flag leaves (48 *10).

Comments 29: [Please describe what kinds of preprocessed were done.]

Response 29: Thanks for your good advice. We have revised them according to your advice. The specific information is shown as (Page 5, Paragraph 16, Line 194-196) “Experimental data were subjected to outlier removal and data standardization using Microsoft Excel 2016 and SPSS 27.”

Comments 30: [Please be specific and please explicitly describe what you mean with ‘biological replicates’. In experimental research, data analysis and presentation of the analysis results must be based on the results of statistical analysis of the data obtained from at least three experimental replicates (at least three (3x) replicates of the entire experiment), not biological replicates. Please read Gomez & Gomez (1984). And, the technique used to analyzed those experimental data is in accordance with the experimental design used to arrange all the treatments and their replicaions (all experimental units) during the process of the experiment. Therefore, how all experimental units were arranged from the beginning of the experiment MUST be clearly explained in the Method. In addition, from previous sections, measurement variables were destructively measured at four growth stages (tillering, panicle initiation, full heading, and maturity), so please explicitly describe in the experimental design how many replications and experimental series were prepared during the experiment.]

Response 30: Thanks for your problems. I am so sorry because of my inaccurate sentence. In fact, there were ten replications for every treatment in this experiment. The total number of pots were 100.

Comments 31: [The experiment in this research paper was used to investigate the effects of two treatment factors, i.e. Fertilization (consisting of 5 levels: CK, T1, T2, T3, T4) and rice cultivars (Meixiangzhan-2 and Qingxiangyou-19), which means that it was a factorial experiment. The main focus of a factorial experiment is to find out whether the interaction effects are significant or not. If the interaction effect was significant on a measurement variable (for example grain yield), it means that the effects of the fertilization treatments are significantly different between the two cultivars; but if it was not significant, it means that both cultivars responded similarly. If the interaction effect was significant, a simultaneous mean comparison MUST be done between fertilization treatments in each cultivar, and between cultivars in each treatment of the fertilization; so, mean comparison MUST be done using a two-way contingency table (NOT using a table of mean values like Table 1 or Table 2, not using Fig.1 to Fig.6.). However, if the interaction effects were not significant, then only comparison of main effects can be done; so, Table 1, Table 2, and Gif. 1 to Fig.6 are not statistically valid mean comparison). Therefore, data analyses MUST be revised by first showing if the main effects were significant or not, and the interaction effects were significant or not, then proceed with statistically appropriate comparisons of mean values. In addition, data in Table 1 are not consistent. Let see panicle number of CK in cultivar #1 is 8 with 90 grains per panicle, which means there were 720 grains per pot. If the 1000-grain weight was 16.36 g, how can grain yield per pot became 36.28 g, while grain number was less than 1000? If 10 kg of soil in the pot can be assumed to make 25x20 cm growing area (because when multiplied with 20 cm height and bulk density is normally more than 1.0, which could be 1.2 kg/L), then without fertilization, grain yield of CK in cultivar #2 would be 8.5 ton/ha – is this right?]

Response 31: Thanks for your good advice. We have added the interaction effects between fertilizer and cultivars in Table 1-2 and Fig.1-6. The value in the third column of Table 1 was effective panicle per hill. Then four hills were for pot. The yield was 8*4*90*0.774*16.35/1000.

Comments 32: [I think this (conclusion on Fig.1) must be proven using an average growth rate (AGR) of the biomass accumulation, then compared them statistically if the AGR are significantly different between fertilization and between cultivars. If the differences are not statistically significant, then no conclusion about which treatment was stronger can be made, because if they are not significantly different it means that they are statistically the same (it means that there was no sufficient confidence to say that they are different). Scientific conclusion MUST be based on the results of statistical data analyses to make a CONFIDENT conclusion.]

Response 32: Thank you for your valuable comments. We have changed total aboveground biomass to crop growth rate. Please look at Fig. 1 in the manuscript. And we have added anova analysis in Fig.1. The specific information is shown as (Page 6, Paragraph 18, Line 225-239) “The average growth rate (AGR) of the two fragrant rice varieties, Meixiangzhan2 and Qingxiangyou19, under different treatment conditions is presented in Fig.1. The substitution of nitrogen fertilizer with organic fertilizer significantly affected the average growth rate of both varieties, with the T2 treatment exhibiting the most pronounced effect. Compared to the T1 treatment, the T2 treatment increased the AGR of Meixiangzhan2 by 16.84% and 90.65% during the MT-PI and PI-HS stages, respectively, with the most substantial increase observed during the PI-HS stage. During the PI-HS stage, the T3 treatment resulted in a significant increase of 74.97% in the AGR com-pared to T1. Similarly, for Qingxiangyou19, the T2 treatment elevated the AGR by 33.85% and 15.67% during the MT-PI and PI-HS stages, respectively, relative to T1, although these increases did not reach statistical significance. Among the treatments involving organic fertilizer substitution for nitrogen fertilizer, the T2 treatment exert-ed a stronger positive effect on the AGR than the T3 treatment, though the difference between the two was not statistically significant.”

Comments 33: [Similar comments for Fig.1 also apply to Fig.2]

Response 33: Thanks for your advice. We have added anova analysis in Fig.2.

Comments 34: [Similar comments for Fig.1 also apply to Fig.3]

Response 34: Thanks for your advice. We have added anova analysis in Fig.3.

Comments 35: [Similar comments for Fig.1 also apply to Fig.4]

Response 35: Thanks for your advice. We have added anova analysis in Fig.4.

Comments 36: [Similar comments for Fig.1 also apply to Fig.6]

Response 36: Thanks for your advice. We have added anova analysis in Fig.6.

Comments 37: [The Discussion may need to be revised after the authors properly revise the Results of data analyses and their presentation in the RESULTS.]

Response 37: Thanks for your advice. We have revised the results in the manuscript.

Comments 1: [Lines 79–85: Typically, the objective should be clearly separated from methodological or conclusive elements. These two sentences blend those elements, which may slightly affect clarity. However, this is a minor observation]

Response 1: Thanks for your good advice. We have revised these sentences according to you and other reviewer’s. (Page 2, Paragraph 4, Line 88-92) “(mid-tillering, panicle initiation and heading stages), yield and its components, 2-acetyl-1-pyrroline (2-AP) content, and substances related to 2-AP biosynthesis. Mid-tillering, panicle initiation, and heading stages are important period that determine panicle number per ha, spikelets per panicle and 1000-grain-weight, respectively.”

Comments 2: [Lines 92–93: As this is essentially a fertilization experiment, it would be useful to provide key soil properties such as pH, EC, CEC, and available nitrogen.]

Response 2: Thanks a lot. We have added the data about physical and chemical traits of the soil. The specific information is shown as (Page 3, Paragraph 5, Line 105-107) “The experimental soil was a sandy loam, with 21.44 organic C, 1.70 g kg-1 total N, 1.24 g kg-1 total P, 22.85 g kg-1 total K, 79.03 mg kg-1 available P, 108.30 available K, and pH=6.50.”

Comments 3: [Lines 105–106: Please clarify whether T1 is considered the main control treatment (in the context of substitution and objective), and whether CK serves as an absolute control.]

Response 3: Thanks for your questions. We completely agreed with you. The treatment CK was used to calculated fertilizer use efficiency. Regarding the content of the manuscript, these data were not used in the manuscript.

Comments 4: [Lines 112–113: Indicate the main characteristics of the organic fertilizer used (e.g., source/feedstock, pH, N content).]

Response 4: Thanks for your good advice. We have added organic fertilizer components  according to you and other reviewer’s. The specific information is shown as (Page 3, Paragraph 8, Line 120-132) “This kinds of organic fertilizer belonged to animal manure (43% organic matter, 2.31% nitrogen, 2.98% phosphorus and 3.82% potassium).”

Comments 5: [Line 114: Clarify whether the nitrogen rate of 150 kg/ha is based on rice nutrient requirements and available soil nitrogen. Also, was the dose of organic fertilizer calculated based on its nitrogen and organic matter content (w/w basis)?]

Response 5: Thanks for your advice. The nitrogen rate (150 kg N/ha) is decided by the local agricultural department recommendation. The dose of organic fertilizer is also based on recommendation rate.

Comments 6: [Lines 173–174: Was ANOVA performed prior to conducting LSD multiple comparisons?]

Response 6: Yes. Thank you very much.

Comments 7: [Lines 174–176: This information may be better placed in table footnotes.]

Response 7: Yes. Thanks for your good advice. We have revised it according to your advice.

Comments 8: [Lines 176–177: This sentence appears redundant and could be removed for clarity.]

Response 8: OK. Thanks for your good advice. We have removed it according to your advice.

Comments 9: [Lines 183–184, 197–198: These sentences read more like discussion content and could be relocated to the discussion section.]

Response 9: Thanks for your good advice. We have revised them according to your advice. The specific information is shown as (Page 13, 14, Paragraph 27, Line 409-410, 419-420) “The increase in yield was primarily attributed to the synergistic enhancement of effective panicles and grains per panicle” and ” These findings suggest that the ratio of organic to inorganic fertilizer application may exhibit a threshold effect on yield formation.”

Comments 10: [Lines 332, 340: Consider relocating the label “A” from line 332 to line 340 for better continuity.]

Response 10: Thanks for your good advice. We have revised them according to your advice.

Comments 11: [Line 348: It appears that “Figure 6” should actually be labeled “Figure 7.”]

Response 11: Thanks for your good advice. We have revised it.

Comments 12: [Lines 359–360: For completeness, consider adding a note that the substitution occurs at different ratios or combinations in the sentence “…organic fertilizer instead of nitrogen fertilizer…”]

Response 12: Thanks for your good advice. We have revised it according to your advice. The specific information is shown as (Page 13, Figure 7, Line 393-394) “Correlation among detected parameters of fragrant rice under different ratios of organic fertilizer instead of nitrogen fertilizer.”

Comments 13: [Lines 372–373, 421–423: For clarification, the term “optimization” usually refers to results obtained through a regression model or a formal optimization method. Since the study identifies a "best treatment" rather than an optimized value, consider rephrasing.]

Response 13: Thanks for your good advice. We have revised them according to your advice. The specific information is shown as (Page 13, 14, Paragraph 27, 28, Line 409-410, 424-426) “with the T2 treatment achieving the best value.” and “adopting 33.3% organic fertilizer substitution for chemical fertilizer method to meet the rhythm of nitrogen supply”

Comments 14: [Lines 421–424, 480–483: Recommending a specific ratio for field adoption based solely on a controlled experiment may be premature. Also, it would be appropriate to suggest further investigation of application rates around the T2 treatment to confirm its effects on yield and aroma under field conditions.]

Response 14: Thanks for your good advice. We have revised them according to your advice. The specific information is shown as (Page 14, Paragraph 28, Line 438-447) “This strategy provide a reference scientific basis for the efficient cultivation of fragrant rice. However, since this study was conducted under pot conditions, future research is recommended to further investigate the effects of 33.3% organic fertilizer substitution for urea and other substitution ratios under field conditions to confirm their applicability and effectiveness in practical agricultural production. Future studies should focus on optimizing fertilization ratios and evaluating performance under different environmental and management conditions to provide more comprehensive fertilization guidance, thereby promoting the cultivation of fragrant rice and the sustainable development of agriculture.” and (Page 16, Paragraph 33, Line 521-526)“The results of this study indicate that the strategy of substituting chemical fertilizers with organic fertilizers, particularly under the treatment with 33.3% organic fertilizer substitution for urea (T2), significantly improved the yield and aroma quality of fragrant rice. These findings provide a reference scientific basis for the effective enhancement of yield and aroma in fragrant rice.

Comments 15: [Figures: Include label letters (A, B, C, D, etc.) in each figure caption for clarity. Also, indicate that error bars represent standard error. Improve spacing in the figure legend between treatments.]

Response 15: Thanks for your good advice. We have revised them according to your advice.

Comments 16: [Tables: Adjust column formatting to ensure consistency in some headers and content. If applicable, indicate that +/– SE indicate standard error.]

Response 16: Thanks for your good advice. We have revised them according to your advice. The specific information is shown as (Page 6, 8, Table1, 2, Line 215-216, 275-276) “Data are presented as means ± standard error (Mean ± SE).”

Comments 17: [Methodology: Consider merging some subsections that are closely related, as seen in the discussion and results sections.]

Response 17: Thanks for your good advice. We have revised them according to your advice. The specific information is shown as (Page15, Paragraph 31, 32, Line 480-512) “Nitrate reductase (NR) and glutamine synthetase (GS), key enzymes in nitrogen metabolism, directly regulate nitrogen uptake and assimilation efficiency [40]. This study revealed that substituting chemical fertilizers with organic fertilizers enhanced NR and GS enzyme activities in flag leaves during PI and HS stages. This enhancement may stem from the sustained nutrient supply provided by organic fertilizers, which offers energy support to maintain high enzyme activity. Elevated NR and GS activities serve as a critical foundation for improving nitrogen use efficiency. Accordingly, we infer that organic fertilizer substitution optimized nitrogen supply, providing sufficient nutrients during the vegetative growth stage to promote effective tiller formation, thereby increasing the number of effective panicles at the heading stage. This result is consistent with Moe et al. [41]. Additionally, peroxidase (POD) and catalase (CAT), as key antioxidant enzymes, scavenge reactive oxygen species (ROS) and protect cellular integrity [42, 43], while malondialdehyde (MDA) reflects oxidative stress levels [44]. The results showed that organic fertilizer substitution increased POD and CAT activities. At the same time, it reduced MDA content. This suggests that organic fertilizers alleviated oxidative stress in fragrant rice, reducing cell membrane damage and enhancing antioxidant capacity. This phenomenon indicates that organic fertilizer ap-plication effectively mitigated oxidative stress in rice, decreasing lipid peroxidation damage to cell membranes and thereby bolstering plant stress resistance, a finding consistent with previous studies [45,46]. Previous studies have confirmed that in-creased nitrogen levels positively promote the enhancement of antioxidant enzyme activities [47,48]. Thus, we hypothesize that the slow-release properties of organic fertilizers prevented drastic fluctuations in nutrient supply, reducing oxidative damage to plants caused by excessive ROS accumulation. Concurrently, the enhanced activity of key nitrogen metabolism enzymes further supported the high expression of antioxidant enzymes, ultimately manifested as increased POD and CAT activities and reduced MDA content.

In summary, the substitution of chemical fertilizers with organic fertilizers enhanced nitrogen use efficiency by increasing the activities of NR and GS, while simultaneously mitigating oxidative stress through an optimized antioxidant system (elevated POD and CAT activities and reduced MDA content). This dual regulatory mechanism supported the performance of fragrant rice during vegetative growth and stress adaptation. The experimental results ultimately manifested as improvements in both yield and aroma content.”

Comments 1: [The content of this paper is only substitution of N or urea only, and not other types of fertilizers, so the title of this paper must be revised. It can be Effects of organic fertilizer substitution for Urea Fertilizer (or N Fertilizer) …. Or, Effects of Substituting N Urea with Organic Fertilizer …. It depends on what was substituted: N-Urea or Urea amount (which is not clearly explicit in the Methods.]

Response 1: We think this is an excellent suggestion. Thank you for a critically focused title! We have changed the title of the article, which has been highlighted in red in the revised version. The specific information is shown as (Page 1, Line 2) “Effects of Substituting N-Urea with Organic Fertilizer”

Comments 2: [The contents of the Abstract must also be revised after properly revising the presentation of data analysis results, their proper interpretation, the Discussion, and the Conclusion.]

Response 2: Thank you for your valuable suggestions. We fully agree that it is necessary to describe more experimental details in the abstract and present the results of data analysis more clearly. Therefore, we have made the following revisions in the abstract to enable readers to have a clearer understanding of the experimental details and results. The specific information is shown as (Page 1, Line 20, 29-34) “Nitrogen application rates for all treatments were standardized at 150 kg N/ha.” and “Similarly, compared to the T1 treatment, the T2 treatment significantly increased the dry matter accumulation, leaf area, and photosynthetic rate of Meixiangzhan2 and Qingxiangyou19, while also significantly enhancing the activities of nitrate reductase and glutamine synthetase. Furthermore, the activities of peroxidase and catalase in the flag leaves were significantly elevated, whereas the malondialdehyde content was significantly decreased.”

Comments 3: [Was 33.3% dose of Urea substituted with the same amount of organic fertilizer, or was 33.3% of Urea-N substituted with organic fertilizer with the same amount of N. Please revised here and in the Methods to make it explicit and repeatable.]

Response 3: We appreciate your attention to methodological clarity. Your suggestions have been helpful in enhancing the clarity and reproducibility of the article. We have revised the abstract content based on your suggestions. The specific information is shown as (Page 1, Line 17-20) “33.3% of Urea-N substituted with organic fertilizer with the same amount of N (T2), 66.6% of Urea-N substituted with organic fertilizer with the same amount of N (T3), all organic fertilizer (T4), and no any fertilizer as a control (CK).”

Comments 4: [The experiment in this paper is a factorial pot experiment. The treatments applied to each replicate pot is called treatment combination in which there are 10 treatment combinations in the experiment (because each variety in each pot must be applied one of the fertilization treatments). So, to make it explicit and clear, please do not say per treatment, which is unclear. Please talk about measurement results per pot OR per replicate of treatment combination.]

Response 4: Thank you for your valuable feedback. We have revised them according to your advice.

Comments 5: [There are many problems of the design of the experiment. Although I have commented since the previous version of this paper, no fully relevant revisions have been done in this revised version. All procedures and the design of the experiment of a scientific paper MUST be repeatable, i.e. everybody can easily repeat the experiment.

So, please explicitly describe how many replications were prepared for this two-factors experiment having 10 treatment combinations; and how many experiment series for measuring yield attributes and the other 3 times destructive measurements (tillering stage (MT), panicle initiation stage (PI), and full heading stage (HS)); then, how can only 10 pots were prepared per treatment. In a pot experiment, 1 pot functions as 1 replicate of a treatment or treatment combination.

The experiment in this paper has 10 treatment combinations, each consisting of combination of 2 varieties and 5 fertilization treatments. Each treatment combination must be replicated in 3 pots, which is a minimum number of replications.]

Response 5: Thank you for your valuable suggestions. We are very sorry for not following your previous suggestion to make completely relevant revisions. Firstly, we would like to clarify the rationale behind planting only ten pots. Each treatment combination was replicated ten times by planting ten pots, which provides adequate statistical power for the measurement of the relevant indicators in this study. Secondly, in response to your feedback, we have amended the "Materials and Methods" section to explicitly state the number of replicates for each treatment combination and to detail the specific experimental conditions under which each replicate was utilized for indicator measurements. The specific information is shown as (Page 3, Paragraph 8, Line 132-136) “Each treatment combination was planted in ten pots, that is to say, each treatment combination was repeated ten times. When sampling, three replicates were randomly selected from each treatment combination to determine the average growth rate and leaf area, three replicates to determine enzyme activity, and four replicates to determine yield.”

Comments 6: [What was the unit?]

Response 6: We were really sorry for our careless mistake. Thank you for pointing out the problem. We have made the corrections as suggested by the reviewers. The specific information is shown as (Page 3, Paragraph 5, Line 110) “21.4 g kg^-1 organic C”

Comments 7: [What was the unit?]

Response 7: We were really sorry for our careless mistake. Thank you for pointing out the problem. We have made the corrections as suggested by the reviewers. The specific information is shown as (Page 3, Paragraph 5, Line 111-112) “108.3 mg kg⁻¹ available K”

Comments 8: [Please explicitly mention how many replications and/or how many series of the experiment.]

Response 8: Thank you for your valuable feedback; we sincerely apologize for not clearly describing the experimental methods. We have revised them according to your advice. The specific information is shown as (Page 3, Paragraph 8, Line 132-133) “Each treatment combination was planted in ten pots, that is to say, each treatment combination was repeated ten times.”

Comments 9: [What about application of other nutrients, and how much. However, if there was no application of other nutrients, please explicitly mention here.]

Response 9: Thank you for your critical suggestions. No additional nutrients were added in the experimental methods, and this has been explicitly stated in the Materials and Methods section as per your suggestion. The specific information is shown as (Page 3, Paragraph 8, Line 141) “No other nutrients were added.”

Comments 10: [This is actually not a split plot design (please read a principle of experimental design, for example: Gomez & Gomez (1984), Statistical Procedures for Agric. Research.]

Response 10: We were really sorry for our careless mistake. Thank you for pointing out the problem. We have revised them according to your advice.

Comments 11: [How can it be 12 hills? Line 117 says 5 hills per pot???]

Response 11: I am so sorry, there was a mistake. In practice, four hill seedings were transplanted into each pot. Consequently, we randomly selected three pots from each treatment combination for harvesting, resulting in the collection of 12 hills. We have revised the errors in the description within the Materials and Methods section. The specific information is shown as (Page 3, Paragraph 6, Line 121-122) “Four hill seedlings was transplanted in a cross line in each pot.”

Comments 12: [Once again, rice plants grow in clump; please explicitly explain that there were 5 hills of rice plants; then how these 4 plants were selected and from how many hills in each pot?]

Response 12: We sincerely apologize for repeating the same error as above. In fact, four hill seedings were transplanted into each pot. In the experimental methods, four hills were randomly selected from each treatment, all of which were taken from the same pot, representing four rice plants from the same treatment combination. We have revised the errors in the description within the Materials and Methods section. The specific information is shown as (Page 3, Paragraph 6, Line 121-122) “Four hill seedlings was transplanted in a cross line in each pot.”

Comments 13: [Please explicitly explain, were the 25 flag leaves from each treatment or from each replication? If they are from each treatment combination, how they were separated which are from replication #1, replication #2, etc. If they are from each replication, then this is impossible. Table 1 shows the highest average of panicle number was 14 which means that the highest number of flag leaves was 14, then how it become 25 flag leaves??? In Experimental Design, scientists do not talk about 25 flag leaves per treatment, which is really uncertain for the readers (not repeatable); please explicitly mention how many flag leaves were measured per replication of the experiment and how many replications.]

Response 13: Thank you for your valuable feedback. We sincerely apologize for the misunderstanding caused by our lack of clarity. The 25 flag leaves were randomly selected from four replicates of each treatment combination. Taking T2V2 as an example, the average number of panicles per hill is 14, with four hills per pot. Consequently, the total number of flag leaves is 56, which exceeds the required 25, ensuring sufficient availability for sampling. We have revised them according to your advice. The specific information is shown as (Page 4, Paragraph 12, Line 179-180) “The 25 flag leaves were randomly selected from three replicates of each treatment combination.”

Comments 14: [These are the other 25 flag leaves samples again??? They are from how many replications??? The above comments also apply to this Line 169.]

Response 14: Thank you for highlighting this issue. We sincerely apologize for the misunderstanding caused by our lack of clarity. The same sample of 25 flag leaves was used; consequently, we have consolidated the two experimental methods to enhance the clarity and reproducibility of the methodology. The specific information is shown as (Page 4, Paragraph 12, Line 175-189) “Determination of Nitrogen Metabolizing Enzyme Activity, Anti-Stress Enzyme Activity and MDA Content in Leaves

Samples were collected at key rice growth stages (tillering, panicle initiation, and full heading stages). From each treatment combination, 25 flag leaves were randomly selected and combined the 25 flag leaves for processing. The 25 flag leaves were randomly selected from three replicates of each treatment combination. Then stored in an ultra-low temperature freezer at −80 °C for subsequent analysis of antioxidant response parameters and nitrogen metabolism enzyme activities in leaves. The extraction and activity levels of nitrate reductase (NR) and glutamine synthetase (GS) were determined following the methods described by Mo et al. (2018) [9]. Absorbance was measured at 540 nm, with NR activity expressed as mg·min−1·g−1 FW and GS activity expressed as U·min−1·g−1 FW. The activities of peroxidase (POD) and catalase (CAT), as well as the malondialdehyde (MDA) content, were determined following the methods described by Mostofa et al. [15]. POD and CAT activities were expressed as U·min−1·g−1 FW (FW, fresh weight), while MDA content was expressed as μmol·g−1 FW.”

Comments 15: [These are the other 20 flag leaves, so where did they sampled from???]

Response 15: Thank you for highlighting this issue. I am so sorry, there was a mistake. In fact, the same sample of 25 flag leaves was used. Taking T2V2 as an example, the average number of panicles per hill is 14, with four hills per pot. Consequently, the total number of flag leaves is 56, which exceeds the required 25, ensuring sufficient availability for sampling.

Comments 16: [There are may scientific errors in Table 1. Table 1 does not compare mean values between fertilization treatments BUT between fertilization treatments in each variety. This can only be done if the interaction effect was significant.

The interaction effect was significant only on 1000 grain weight, but mean comparison must be done between fertilization treatments in each variety and between varieties in each fertilization treatment. Mean value of V1 in CK CANNOT be compared with that of V2 in T1 for example, because they are not statistically comparable, because they are different varieties fertilized with different fertilization treatments, how can they be compared? Were they different because of different varieties OR were they different because of different fertilization? If the interaction effects are not significant such as in effective panicles, grain number, GF%, and yield/pot, mean comparison can only be compared between mean values of fertilization treatments averaged across varieties if F was significant, and between mean values of varieties each avegared across those 5 fertilization treatments.

Please also show the LSD value for each mean comparison (that’s the principle of experimental design).]

Response 16: Thank you for your valuable feedback. We have revised them according to your advice.

Comments 17: [How can yield is per pot while effective panicles are per hill. In a pot experiment, 1 pot is 1 experimental unit, how can it be different between effective panicle and yield? This is not consistent with the principle of agricultural experimentation and the principle of experimental design.]

Response 17: Thank you for raising this question. Please allow me to clarify that the values in the third column of Table 1 represent the number of effective panicles per hill. As described earlier, four hills were transplanted into each pot. Taking CKV1 as an example, the yield per pot is calculated as (8 × 4 × 90 × 0.774 × 16.35) / 1000.

Comments 18: [There are scientific errors in Fig.1. First, Fig.1A, there are two types of AGR (MT-PI & PI-HS), why there is only one ANOVA result? Similar comment to Fig.1B. Secondly, both ANOVA tables show non-significant V*F interaction, how can mean comparison be done separately in each variety? These are really out of statistical procedures. Thirdly, looking at each figure (A or B), it seems that types of AGR were used as an independent variable of research (which should dependent variables) BUT how can the sources of variation of the ANOVA consist of V (variety), F (Fertilization), and V*F interaction??? Similar statistical analysis errors also occur in Fig.2, Fig.3, Fig.4 & Fig.6.]

Response 18: Thank you for pointing out our error, as this has been instrumental in enhancing the quality of our manuscript. We have followed your suggestion and have re-conducted the one-way analysis of variance (ANOVA) on the data.

Comments 19: [Similar comments to Fig.1 also apply to Fig.2.]

Response 19: Thank you for pointing out our error, as this has been instrumental in enhancing the quality of our manuscript. We have revised them according to your advice.

Comments 20: [Similar comments to Table 1 also apply to this Table 2.]

Response 20: Thank you for pointing out our error, as this has been instrumental in enhancing the quality of our manuscript. We have revised them according to your advice.

Comments 21: [Similar comments to Fig.1 also apply to Fig.3 although some show significant interaction effects.]

Response 21: Thank you for pointing out our error, as this has been instrumental in enhancing the quality of our manuscript. We have revised them according to your advice.

Comments 22: [Similar comments to Fig.1 also apply to Fig.4.]

Response 22: Thank you for pointing out our error, as this has been instrumental in enhancing the quality of our manuscript. We have revised them according to your advice.

Comments 23: [ANOVA table in Fig.5 shows non-significant interaction effect; which means that the response of both varieties to fertilization treatments is statistically similar. Therefore, mean comparison between fertilization treatments cannot be done in each variety. Instead, it must be done between the mean values averaged across both varieties. In addition, because main effect of variety is significant then mean values between varieties averaged across fertilization treatments must also be done.]

Response 23: Thank you for pointing out our error, as this has been instrumental in enhancing the quality of our manuscript. In accordance with your request, we have restructured the configuration of Figure 5 and reconducted a one-way analysis of variance (ANOVA) on the data.

Comments 24: [Similar comments to Fig.1 also apply to Fig.6.]

Response 24: Thank you for pointing out our error, as this has been instrumental in enhancing the quality of our manuscript.

Comments 25: [The contents of Discussion may need to be revised after properly revising the presentation of the analysis results and their interpretation.]

Response 25: Thank you for your valuable suggestions. We have revised the manuscript in accordance with your recommendations, incorporating additional content to strengthen its logical coherence. The specific information is shown as (Page 14, Paragraph 28, Line 458-459) “This phenomenon was even more pronounced under the T4 treatment, leading to a de-crease in yield compared to T1.”

Comments 26: [The contents of the Conclusion may also need to be revised after properly revising the presentation of the analysis results, their interpretation, and the Discussion. Interpretation of significant effects of different fertilization treatments can be done only for the measurement variables showing significant interaction effects.]

Response 26: Thank you for your valuable feedback. We have revised them according to your advice.

Comments 27: [It is also not clearly explicit in the Method about this fertilization treatment. In Line 132, N application was 150 kg/ha, which means it was around 333 kg/ha Urea. Was substituting 33.3% of urea means substituting 111 kg/ha with 111 kg/ha organic fertilizer, OR was it means that 50 kg/ha N was substituted with 2164.5 kg/ha organic fertilizer??? To make a repeatable nature of the Methods, please make clearly explicit description about the treatments in the Methods, so that an explicit implication of the research results can be made.]

Response 27: We appreciate your attention to methodological clarity. Your suggestions have been helpful in enhancing the clarity and reproducibility of the article. In response to your suggestions, we have explicitly described this fertilization treatment in the Abstract, Materials and Methods, and Conclusion sections. The specific information is shown as (Page 1, Line 17-20) “33.3% of Urea-N substituted with organic fertilizer with the same amount of N (T2), 66.6% of Urea-N substituted with organic fertilizer with the same amount of N (T3), all organic fertilizer (T4), and no any fertilizer as a control (CK).” and (Page 4, Paragraph 8, Line 141-144) “In the T2 treatment, the application of organic fertilizer was designed to substitute for 33.3% of the nitrogen content that would have been provided by urea. For the T3 treatment, the amount of organic fertilizer was calculated in the same manner.” and (Page 17, Paragraph 31, Line 542-543) “33.3% of Urea-N substituted with organic fertilizer with the same amount of N”