Review Reports - Effects of Combined Application of Different Nitrogen Forms on Substrate Nutrient Utilization, Root Microenvironment, and Tomato Yield

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Mainly all corrections to be made and comments are marked in paper;
Some typos in paper (incomprehensible use of punctuation marks where they should not be and their incorrect application, and thus the sentences become unclear) have been noticed but they are easy to correct;
In "Materials and Methods" the literary source (standard method or previous author's work) for pH and EC is missing; there is a lack of information on the weight of the substrate per pot; there is ambiguity in the use of the terms "substrate" and "soil" (they are not the same in terms of where the plants are grown), so if the substrate is studied, please do not refer to them as soil in paper, but substrate samples and not soil samples; Table 1 should be completely revised to be clearer;
Table 4 should be arranged as in Table 1;
Please write references in complete form and not only „Author et al., since I believe that all the authors who contributed to the writing of the paper cited here should be listed.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

English is generally good, but could be improved.

Author Response

Dear Editors and Reviewers：

Thank you for your letter and the reviewers' comments on our manuscript entitled "Effects of Combined Application of Different Nitrogen Forms on Substrate Nutrient Utilization, Root Microenvironment, and Tomato Yield" (microorganisms-4010377). These comments have been valuable and helpful in revising and improving our paper, and have important guiding significance for our research. We have carefully studied the comments and made revisions, and hope to receive approval. The editor's and reviewers' comments are shown in bold below, with specific issues numbered. Our response is given in red, and the revisions to the manuscript are highlighted in yellow.

Reviewer 1

Some typos in paper (incomprehensible use of punctuation marks where they should not be and their incorrect application, and thus the sentences become unclear) have been noticed but they are easy to correct.

Response:

Thank you very much for your careful review of our manuscript. In response to the issue of "a large number of misspellings (misuse or misplacement of punctuation marks)", we have checked each sentence and made unified corrections: unify the punctuation marks in both Chinese and English, and delete redundant Spaces and repeated symbols; Standardize the spacing before and after all reference parentheses, unit abbreviations and numbers. Split long sentences or add commas to ensure clarity. All the changes have been highlighted in yellow in the revised draft for your easy verification. Thank you again for your patient guidance!

In "Materials and Methods" the literary source (standard method or previous author's work) for pH and EC is missing; there is a lack of information on the weight of the substrate per pot; there is ambiguity in the use of the terms "substrate" and "soil" (they are not the same in terms of where the plants are grown), so if the substrate is studied, please do not refer to them as soil in paper, but substrate samples and not soil samples; Table 1 should be completely revised to be clearer.

Response:

Thank you for your detailed review and valuable suggestions on our manuscript. Regarding your comments on the "Materials and Methods" section, we have reviewed and revised them one by one, adding the standard methods and original literature for pH and EC determination (Line 160-162). The following are the added references:"[28] Sparks, D. L. (Ed.). Methods of Soil Analysis. Part 3: Chemical Methods; Soil Science Society of America: Madison, WI, USA, 1996.[29] Mu, W.; Han, N.; Qu, Z.; Zheng, M.; Shan, Y.; Guo, X.; Mu, Y. ECWS: soil salinity measurement method based on electrical conductivity and moisture content. Agronomy 2024, 14, 1345."

The revised manuscript clarifies that each pot should be filled with 2.5 kg (±50 g) of air-dried substrate (Line 102). The misuse of "soil" and "substrate" has been corrected. Table 1 (Line 106) has been completely rewritten using the three-line table format. The specific modifications are as follows:

Table 1. Physical and chemical properties of the substrate

Substrate

A N

(mg kg^-1)

A K

(g kg^-1)

A P

(g kg^-1)

T N

(g kg^-1)

T K

(g kg^-1)

T P

(g kg^-1)

(mS cm^-1)

Bulk Density

(g cm^-3)

substrate:peat:vermiculite=2:1:1

429.77

0.36

0.90

1.96

0.90

0.64

1.56

0.54

6.49

Note: AN:Available N AK:Available K AP:Available P TN:Total N TK:Total K TP:Total P The substrate was purchased from Gansu Lv neng Ruiqi Agricultural Technology Co., Ltd. (Gansu, China).

3.Table 4 should be arranged as in Table 1.

Response:

Thank you for your careful review and valuable suggestions. We have revised Table 4. The revised Table 4 is as follows:

Table 4. Effects of different treatments on nutrient content in the rhizosphere substrate of tomato.

Treatments	T N (g kg^-1)	T P (g kg^-1)	T K (g kg^-1)	Ca (g kg^-1)	Mg (g kg^-1)	A N (mg kg^-1)	A P (mg kg^-1)	A K (mg kg^-1)	EC (mS cm^-1)	pH
CK	3.78±0.03 d	1.71±0.09 a	10.08±0.20 b	2.01±0.06 a	2.01±0.07 a	278.10±13.45 d	0.64±0.02 ab	3.08±0.07 a	1.07±0.04 c	6.44±0.06 a
T1	5.63±0.22 c	1.55±0.01 b	11.39±0.18 a	1.90±0.01 a	1.92±0.01 a	702.77±14.15 bc	0.64±0.01 a	2.75±0.08 b	1.22±0.04 b	5.78±0.12 b
T2	4.48±0.20 d	1.36±0.03 c	11.92±0.19 a	1.68±0.01 b	1.92±0.004 a	644.43±27.29 c	0.60±0.01 b	2.31±0.09 c	1.07±0.05 c	6.28±0.02 a
T3	7.97±0.39 b	1.39±0.03 c	10.15±0.24 b	1.94±0.02 a	1.97±0.001 a	712.10±24.36 b	0.54±0.01 c	2.72±0.09 b	1.55±0.03 a	5.22±0.10 cd
T4	7.54±0.33 b	1.25±0.05 cd	10.17±0.22 b	1.93±0.01 a	1.94±0.01 a	784.43±17.19 a	0.56±0.02 c	2.74±0.13 b	1.31±0.03 b	5.06±0.03 d
T5	9.52±0.17 a	1.21±0.03 d	10.48±0.27 b	1.91±0.05 a	1.93±0.001 a	819.43±18.78 a	0.53±0.01 c	2.68±0.11 b	1.5±0.03 a	5.42±0.07 c

Notes: AN:Available N AK:Available K AP:Available P TN:Total N TK:Total K TP:Total PData in the table are presented as the mean ± standard error (n=3). Within a column, different letters indicate significant differences at the 0.05 level.

4.Please write references in complete form and not only „Author et al., since I believe that all the authors who contributed to the writing of the paper cited here should be listed

Response:

Thank you for your careful review and guidance. I have completed each reference according to MDPI requirements, listing all authors (no longer using "et al."), and verified the year, title, journal, volume, issue, page number, and DOI. These have been highlighted in yellow in the revised manuscript for your convenience.

Change "compared with" to "compared to". This applies to all of the following such cases.

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. We have checked each one and changed "compared with" to "compared to" in the revised draft. All the revised contents have been highlighted in yellow in the revised draft for your convenience to check.

6.Delete per plant and retain yield

Response:

Thank you for your careful review and valuable suggestions. We have removed "per plant" and retained "yiegld" as requested, which is highlighted in yellow in the revised manuscript (Line 31) for your convenience.

"experimental material" is changed to "experimental plant material".

Response:

Thank you for your careful review and valuable suggestions. We have changed "experimental material" to "experimental plant material" as required. This is highlighted in yellow (Line 95) in the revised manuscript for your convenience.

The full name for this abbreviation should be written, e.g. operational taxonomic unit (OTU), because it appears for the first time in the text.

Response:

Thank you for your careful review and valuable suggestions. We have provided the full name of the OTU (Operational Taxonomic Unit) in the revised manuscript (Line 434) and added a "Abbreviations" section at the end of the revised manuscript for readers' convenience.

Reviewer 2 Report

Comments and Suggestions for Authors

The organization and concept of the study are fine. However, it needs tobe improved significantly. Comments can be found in the attachment.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Rigorous English editing is required.

Author Response

Dear Editors and Reviewers：

Reviewer 2

1.There is a mistake in authors and affiliation. Have a look at them and correct.

Response:

Thank you for pointing out the errors in the author list and affiliations. After checking each item, we have corrected the order of authors and the designation of the corresponding authors as follows:

Shuyan Jiang ¹, Jianhong Sun ¹, Ning Jin ¹, Shuya Wang ², Shuchao Huang ², Zhaozhuang Li ¹, Jihua Yu ^1,2, Jian Lyu ^1,2^* and Li Jin²^*

College of Horticulture, Gansu Agricultural University, Lanzhou 730070, China.
State Key Laboratory of Aridland Crop Science (Gansu Agricultural University), Lanzhou, Gansu Province 730070, China

* Correspondence: lvjiangs@126.com; jinli0124@163.com

2.Too long sentence and hard to understand. Additionally, before starting the material and methods add background and need of the current study.

Response:

Thank you for your careful review and valuable suggestions. We have shortened the sentences and highlighted the key points, and added a section on "Research Background and Necessity." The revised version is highlighted in yellow (Line 12-15) for your convenience in checking. Specific revisions are as follows: In facility tomato production, the excessive application ratio of ammonium nitrogen (NH₄⁺-N) often leads to root acidification and calcium-magnesium antagonism. Although amide nitrogen (urea-N) has better buffering properties, it needs to be hydrolyzed before utilization, resulting in a lag effect. Previous studies have mostly focused on a single nitrogen source or a fixed proportion, and there is still a lack of systematic evidence on the nitrogen supply effects of different nitrogen application combinations at different growth stages of tomatoes.

3.write what do you mean by 0% and 100% and so on.

Response:

Thank you for your careful review and valuable suggestions. We have explained the meaning of % in the revised manuscript, highlighting it in yellow (Line 20-22). The specific revisions are as follows: Six AN–UN ratio treatments were designed: CK (0% AN, 0% UN), T1 (100% AN, 0% UN), T2 (0% AN, 100% UN), T3 (25% AN, 75% UN), T4 (50% AN, 50% UN), and T5 (75% AN, 25% UN).

4.How much increment? use some figures like this % increase.

Response:

Thank you for your careful review and valuable suggestions. We have used percentages (%) to represent increments, which are highlighted in yellow in the revised manuscript (Lines 22-23). Specific revisions are as follows: T3 (25% NH₄ + 75% urea) increased the yield per plant by 64.04% and 5.10% compared to T1 and T2, respectively, and increased the total N, P, K, and Mg accumulations by 29.0% and 20.7%, respectively.

5.Are you sure 5% is that much difference?

Response:

Thank you for your careful review and valuable suggestions. After re-verifying the data, we confirmed that the yield of the T3 treatment was 5% higher than that of the T2 treatment. This is likely because the T3 treatment (25% ammonium nitrogen and 75% amide nitrogen) allows the ammonium nitrogen to be directly absorbed, which is beneficial for rapid seedling growth. Amide nitrogen, on the other hand, needs to be slowly hydrolyzed by urease, continuously releasing NH₄⁺, providing a stable nitrogen source for the mid-to-late growth stages. The T3 treatment may have avoided temporary nitrogen deficiency, leading to the significant difference in tomato yield.

6.You need to provide a reference point, such as "By how much higher than the control/other treatments", and clearly state the specific value of the increase.

Response:

Thank you for your careful review and valuable suggestions. I have provided reference points in the revised manuscript, clearly stating the specific values that exceed them. These are marked in yellow in the revised manuscript (Line 22-24). The specific modifications are as follows: In addition, compared with T1 and T2 the nitrogen fertilizer uptake rate of the T3 treatment in-creased by 17.00% and 24.90%, respectively and the EC increased by 27.04% and 44.84%, respectively.

7.always use full name at first appearence along with its acronymum and then use short name in the following.

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. We have provided the full names of all Abbreviations when they first appear and added a section "Abbreviations" at the end of the revised draft to facilitate readers' reference.

8.Positively but not significantly?

Response:

Thank you for your careful review and valuable suggestions regarding our manuscript. RDA analysis can only determine whether environmental factors are correlated with the treatment samples; a positive correlation is indicated when the arrow segment forms an acute angle with the treatment sample point. In our manuscript, we found that the RDA analysis showed that enzyme activities, total nitrogen (N), and electrical conductivity were positively linked to microbial communities in T3 and T4, whereas communities in CK, T1, T2, and T5 correlated with nutrients and pH.

9.Too hard to said this because pots study findings cannot be compared with the field experiments with larger variations.

Response:

Thank you for your careful review and valuable suggestions. Our wording in the original manuscript was inadequate, and we have now revised the sentences as requested. These are highlighted in yellow in the revised manuscript (Line 28-30). The specific revisions are as follows: Under controlled pot conditions, T3 optimizes the rhizosphere microenvironment, enhances microbial abundance and nutrient uptake, and provides a theoretical basis for precise nitrogen management in tomato.

10.Rearrange the keywords and avoid to use the words that are already available in the title of the manuscript.

Response:

Thank you for your careful review and valuable suggestions. We have re-selected the vocabulary. These are highlighted in yellow in the revised manuscript (Line 31-32). Specific revisions are as follows:

Keywords: forms of nitrogen; tomato yield; plant mineral elements; substrate nutrients; substrate microorganisms

11.Instead of this use economic importance of this crop. Worldwidely area under tomato cultivationa and how much revenue is generated by tomato farming. Additionally highest producing countries etc

Response:

Thank you for your careful review and valuable suggestions. We have made the required revisions, which are highlighted in yellow in the revised manuscript (Line 37-41).The specific modifications are as follows: Tomato is the third most important vegetable crop worldwide, with an annual planting area of 4.73-5.00 million hectares and a global output of 186 million t (fresh weight) averaging 3.7-3.8 kg m⁻². Based on international wholesale prices in 2022 the world tomato industry generates more than USD 120 billion per year, with China being the largest producer[2].

12.Follow the MDPI Citation policies because the citation number should be before the full stop.

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. I have changed the full text citation numbers to before the period as required by MDPI. Update in the revised draft and highlight it in yellow.

13.Try to put some studies on tomato.

Response:

Thank you for your careful review and valuable suggestions. I have tried to focus some of my research on tomatoes. This is highlighted in yellow in the revised manuscript (Line 55-59). The specific modifications are as follows: In tomato, raising the NH₄⁺ proportion in the nutrient solution from 0 % to 50 % markedly shortened primary-root length, decreased lateral-root number and reduced root surface area, with 10 mmol L⁻¹ NH₄⁺ almost completely suppressing lateral-root formation. By contrast, a NO₃⁻/NH₄⁺ ratio of 75:25 produced the highest photosynthetic rate, largest leaf area and greatest single-plant yield, outperforming the ammonium-only treatment by more than 18% [12].

14.After the objectives of your study, put the hypothesis you want to test during the current investigation.

Response:

Thank you for your careful review and valuable suggestions. I have presented the hypotheses I wish to test following my research objectives. These are highlighted in yellow in the revised manuscript (Line 87-90). The specific modifications are as follows: Our hypotheses are as follows: (i) Compared to a single nitrogen source, the combined application of ammonium and amide nitrogen will increase tomato yield and nutrient accumulation. (ii) Compared to a single nitrogen source, the combined application of ammonium and amide nitrogen will alter substrate enzyme activity and the microbial community.

15.Put coordinates of your experimental location.

Response:

Thank you for your careful review and valuable suggestions. I have added the coordinates of the experimental site. They are highlighted in yellow in the revised manuscript (Line 93-94). The specific modifications are as follows: The experiment was conducted in a glass greenhouse located at the College of Horticulture, Youdaoplaceholder0 Agricultural University, Lanzhou, China (36°05 '39.86 "N, 103°42' 31.09" E).

16.From where you got the seeds?

Response:

Thank you for your careful review and valuable suggestions. I have added the seed source. It is highlighted in yellow in the revised manuscript (Line 96-97). The specific modifications are as follows: The tomato seeds (' Jinfan 502 ') are provided by the Gansu Academy of Agricultural Sciences, China.

17.You didn't do surface sterlization of the seeds?

Response:

Thank you for your careful review and valuable suggestions. I sterilized the tomato seeds and added relevant information. These are highlighted in yellow in the revised manuscript (Line 96-97). The specific modifications are as follows: First, soak the seeds in a 10% sodium hypochlorite solution for 5 minutes, and then in hot water at 50-55℃ for 25-30 minutes for disinfection.

How much substrate is present in it?

Response:

Thank you for your careful review and valuable suggestions. I have added the substrate weight for each pot. It is highlighted in yellow in the revised manuscript (Line101-102). The specific modification is as follows :Fill each basin with 2.5 kg (±50 g) of air-dried substrate.

19.Put the table in opposite direction for better visulaization.

Response:

Thank you for your careful review and valuable suggestions. I have revised Table 1 as requested. The specific revisions are as follows:

Table 1. Physical and chemical properties of the substrate

Substrate

A N (mg kg^-1)

A K (g kg^-1)

A P

(g kg^-1)

T N

(g kg^-1)

T K

(g kg^-1)

T P

(g kg^-1)

(mS cm^-1)

Bulk Density

(g cm^-3)

substrate:peat:vermiculite=2:1:1

429.77

0.36

0.90

1.96

0.90

0.64

1.56

0.54

6.49

Note: AN:Available N AK:Available N AP:Available P TN:Total N TK:Total K TP:Total P The substrate was purchased from Gansu Lv neng Ruiqi Agricultural Technology Co., Ltd. (Gansu, China).

20.How you set this frquencey? do you have any intervention limits by which you restore the watering?

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. The three-day irrigation interval was set based on the initial experiment. Under our greenhouse conditions (30±2℃/20±2℃ day and night, VPD 1.2 kPa), when 1000 mL of nutrient solution was applied, the relative humidity of the substrate decreased from 75% to approximately 60% within 72 hours. The relative humidity of the substrate was maintained within the range of 60-70% throughout the entire experimental process, which could prevent drought and waterlogging stress. In this experimental environment, the relative humidity of the substrate should be maintained at 60-70%, and 1000 mL of nutrient solution should be applied every three days.

21.Along with the refernce you also need to mention here the experimental design and treatment arrangments.

Response:

Thank you for your careful review and valuable suggestions. I have revised the experimental design and treatment arrangements. These are highlighted in yellow in the revised sections (Line 111-120). The specific revisions are as follows: Our experimental design follows a previous study reported by our group [23], which set up 12 treatments (T1-T12) with three nitrogen forms applied in combination and a control (CK) without nitrogen fertilizer. The nutrient solution used in the experiment was prepared based on Hoagland nutrient solution, with the nitrogen forms in the Hoagland nutrient solution formula adjusted, while the contents of other elements remained unchanged. This experiment reduced the number of treatments by 7, focusing on the ratio of ammonium nitrogen to amide nitrogen. The nitrogen forms in the Hoagland nutrient solution formula were adjusted to ammonium nitrogen and amide nitrogen, while the contents of other elements remained unchanged. One week after planting, the plants were irrigated with nutrient solution every three days, with 1000 mL of nutrient solution each time. In this experiment, the nitrogen-free treatment (CK) was used as the control, and different nitrogen form ratios (T1-T5) were set under the same conditions of N (15 mM), P (1 mM), K (6 mM), Mg (2 mM) and Ca (5 mM).

The word "configure d" is incorrect

Response:

Thank you for your careful review and valuable suggestions. I have changed the word "configured" to "supplied". It is highlighted in yellow in the revised manuscript (Line125).

23.are these the standard protocols? put reference of them.

Response:

Thank you for your careful review and valuable suggestions. I have added appropriate references, which are highlighted in yellow in the revised manuscript (Line 130). Specific references：[24] Jones, J. B., Jr. Tomato Plant Culture: In the Field, Greenhouse, and Home Garden, 2nd ed. CRC Press: Boca Raton, FL, USA, 2007. https://doi.org/10.1201/9781420007398.

24.You didn't differentiate between marketable and non marketable yields?

Response:

Thank you for your careful review and valuable suggestions. In this study, we assessed the overall tomato yield by removing unsaleable deformed and rotten fruits. Thank you again for your detailed review and guidance.

25.size of sieve?

Response:

Thank you for your careful review and valuable suggestions. I have added the sieve size, which is highlighted in yellow in the revised manuscript (Line 147-149). The specific modifications are as follows: The samples were ground separately, passed through a 0.25 mm sieve and stored in self-sealing bags for the determination of mineral element contents.

26.How to calculate them. Place these method references here

Response:

Thank you for your careful review and valuable suggestions. I have added appropriate references, which are highlighted in yellow in the revised manuscript (Lines 170-172). Specific references：[30] Cassman, K.G. Ecological intensification of cereal production systems: yield potential, soil quality, and precision agriculture. Proc. Natl. Acad. Sci. USA 1999, 96, The 5952-5959. https://doi.org/10.1073/pnas.96.11.5952

[31] Cassman, K.G.; Dobermann, A. Nitrogen and the future of agriculture: 20 years on: This article belongs to Ambio's 50th Anniversary Collection. Theme: Solutions - oriented research. Ambio 2022, 51, 17-24. https://doi.org/10.1111/amb.13144

27.How you design the primers?

Response:

Thank you for your careful review and valuable suggestions. I have added the relevant content, which is highlighted in yellow in the revised manuscript (Line 219-226). DetailsThe specific modifications are as follows: Taking the DNA extracted above as the template, The V3-V4 variable region of the 16S rRNA gene was amplified by PCR using the upstream primers s338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTW TCTAAT-3’)carrying Barcode sequences. The ITS2 region of fungi was amplified using primers ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA 3’) and ITS2R (5’-GCTGCGTTCTTCATCGATGC-3’). These primers have been widely used in high-throughput sequencing research and are regarded as standard primer pairs for analyzing bacterial and fungal communities in matrix and rhizosphere samples.

28.Have you performed normality and homogenity test on your collected data before proceeding for statistical analysis.

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. Before conducting statistical analysis, I carried out normality and homogeneity of variance tests on all the collected data.

29.Is this yield include both marketable and non marketable yield? because in some studies it is reported that increase in total yield doesn't corresponde to the increase in marketable yield

Response:

Thank you for your careful review and valuable suggestions regarding our manuscript. In this study, "yield" refers to the total fresh weight of mature fruit harvested per plant (excluding unsaleable rotten or deformed fruit).

Put the p values throughout the result section where you are describing your results in the form of statistics. You can also put the stars instead of p values like *: p< 0.01, **: p< 0.01, ***: p< 0.001

Response:

Thank you for your careful review and valuable suggestions. I have placed the p-value in the results section, which describes the results in statistical data form, and highlighted it in yellow in the revised manuscript (Line 259-267). The specific modifications are as follows: The nitrogen fertilizer uptake rate, partial factor productivity from applied N, and agronomic efficiency of applied N differed significantly among nitrogen treatments (p < 0.05) ranging from 13.10% to 18.07%, 166.90 to 473.49 kg·kg⁻¹, and 70.41 to 377.00 kg·kg⁻¹ respectively.Among all treatments, T3 showed significantly higher nitrogen fertilizer uptake rate, partial factor productivity of nitrogen, and agronomic efficiency of nitrogen than the other treatments (p < 0.05). Compared with T1 T3 significantly increased the nitrogen fertilizer uptake rate, partial factor productivity of nitrogen, and agronomic efficiency of nitrogen by 17.03%, 64.04%, and 96.20%, respectively (p < 0.05). Compared with T2 T3 also significantly increased the nitrogen fertilizer uptake rate, partial factor productivity of nitrogen, and agronomic efficiency of nitrogen by 24.87%, 5.10%, and 6.40%, respectively (p < 0.05)

This also have a bit higher standard error.

Response:

Thank you for your careful review and valuable suggestions. Regarding your concern about the large standard error, we found no errors in data processing after checking the data. The large standard error is likely due to the fact that the partial factor productivity from applied N is a ratio that varies with yield, which could further amplify differences between repeated trials. Although this difference exists, there are still significant differences between treatments. Therefore, the observed standard error does not affect the statistical significance or conclusions of this study.

32.Use a different color scheme to have a better visulation and clear difference among the N concentration in different organs. also correct the y axis title. is it correct? mg.piant?

Response:

Thank you for your careful review and valuable suggestions. I have corrected the y-axis title "mg plant⁻¹". We have considered the overall color scheme for suitability and aesthetics; please allow us to use the current color scheme.

33.Also describe the meaning of dots in the circles

Response:

Thank you for your careful review and valuable suggestions. The dots within each circle represent the individual replicate values for that treatment. These are highlighted in yellow in the revised manuscript (Line 470-472). The specific revision is as follows: The dots within each circle represent the individual replicate values for that treatment, showing the spread and distribution of the data.

34.It describes the meaning of the arrow length

Response:

Thank you for your careful review and valuable suggestions. The longer the arrow, the stronger the correlation. This is highlighted in yellow in the revised manuscript (Line 489-491). The specific revision is as follows: The longer the angle between the environmental factor arrow and the sample point (acute angle indicates positive correlation, obtuse angle indicates negative correlation), the stronger the correlation.

35.The figure is not clearly visible. Update it with a clear one that can recognize clearly what is written inside the picture.

Response:

Thank you for your careful review and valuable suggestions. We have updated the revised manuscript (Line 507). The specific revisions are as follows:

36.This section is unnecessarily long and less focused towards the outcomes of the study. Make is shorter, put more attention towards the outcomes of your study, and make your result justifiable using previous study results along with the reason of obtaining such results.

Response:

Thank you for your careful review and valuable suggestions. We have rewritten it as requested. It is marked in yellow in the revised manuscript (Line 511). The specific revisions are as follows: The specific modifications are as follows: The results demonstrate that combined application of ammonium-N (AN) and amide-N (UN) significantly improved tomato yield, nitrogen-use efficiency and substrate properties, with T3 (25 % AN + 75 % UN) showing the best overall performance. This indicates that an appropriate AN : UN ratio can better match the N demand of tomato at different growth stages. For growth and N-utilization, T3 markedly increased single-plant yield and agronomic efficiency at the late harvest stage (120 d). The synergistic AN–UN supply mechanism is likely responsible: AN can be absorbed directly, favouring rapid seedling growth [32], whereas UN is slowly hydrolysed by urease and continuously releases NH₄⁺ [33], providing a steady N source for mid- and late-season development and avoiding temporary deficiency or loss. Anderson J [34] reported that moderate UN enhances crop dry-matter accumulation and N-use efficiency, consistent with our T3 results. Regarding substrate nutrients, T3, T4 and T5 increased total-N and alkali-hydrolysable N com-pared with CK, T1 and T2. This is related to the transformation characteristics of different N sources: AN is readily adsorbed by colloids and less prone to leaching [35], while UN hydrolysis continuously replenishes NH₄⁺, raising the N-supply capacity of the substrate [36]. Part of the NH₄⁺ released from UN is nitrified, releasing H⁺ and thus lowering substrate pH [37, 38]. The de-creases in total-P, available P, available K, Ca and Mg under N fertilization relative to CK are probably due to large nutrient uptake by the plants and/or rhizosphere acidification. Acidifica-tion enhances Fe³⁺ and Al³⁺ activity, forming sparingly soluble phosphates (AlPO₄, FePO₄) and reducing P availability [39, 40]. NH₄⁺ competes with K⁺ for adsorption sites because of similar charge and ionic radius, decreasing K content [41], and occupies divalent cation sites, causing Ca²⁺ and Mg²⁺ to remain in solution and migrate with water [40]. Enzyme activities were signifi-cantly increased by UN co-application, especially under T3. UN hydrolysis depends on urease [42], and UN application enhances urease, nitrate reductase and nitrite reductase activities [43]. Tong. [44] also found that different N combinations markedly increased catalase and sucrase in substrate, consistent with our results.

In terms of mineral element uptake, T3 significantly increased the accumulation of N, P, K, Ca and Mg in tomato plants compared with single-N treatments, and these elements were gradu-ally translocated to the fruits during late growth. The synergistic supply of the two N forms in T3 modulates carbon–nitrogen metabolism balance and may enhance the activity of N-assimilation enzymes such as GS/GOGAT [45], while the slow release of amide-N avoids proton toxicity from excessive NH₄⁺ and provides more ATP and carbon skeletons for mineral absorption [45]. Fang [46] also reported that an adequate N supply can activate AMT2 gene expression and synergisti-cally improve mineral uptake efficiency, consistent with our results. Regarding microbial com-munity structure, PCoA showed clear separation among N treatments, with T3 and T4 forming a distinct cluster. RDA further indicated that microbial communities under T3 and T4 were posi-tively correlated with TN, electrical conductivity and various enzyme activities. Yin [47] and Fierer [48] demonstrated that combined N fertilization can alter substrate physico-chemical properties and enzyme activities, thereby driving shifts in microbial community structure. LEfSe analysis revealed that T3 enriched bacterial groups involved in C and N cycling and the poten-tial plant-growth-promoting fungus g_Penicillium. Hassine, M. [49] showed that such microor-ganisms can enhance nutrient transformation and improve the rhizosphere environment, ulti-mately promoting tomato growth and yield formation.

Therefore, our results demonstrate that balanced co-application of AN and UN, especially the T3 ratio, simultaneously improves substrate nutrient status, enzyme activities and microbial community structure, leading to higher tomato yield and N-use efficiency. These findings cor-roborate previous studies and provide a reliable basis for optimizing N form and ratio in sub-strate-grown greenhouse tomatoes.

37.Also mention the future directions that arises after the findings of current investigations.

Response:

Thank you for your careful review and valuable suggestions. We have added future directions, which are highlighted in yellow in the revised manuscript (Line 584-586). The specific revisions are as follows: Future research will focus more on the regulatory effect of the T3 treatment on tomato quality and validate these conclusions under different varieties and long-term field trials.

38.use the standard template of the Author contribution.

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. The standard template contributed by the author as you requested has been used. I have revised it in the revised Author Contributions (Line 587-592) and highlighted it in yellow.Specific changes are as follows: the Author Contributions: Conceptualization, Jian Lyu and Li Jin; methodology, Jihua Yu ; software,Shuya Wang ; validation, Shuchao Huang ; formal analysis, Zhaozhuang Li; investigation, Ning Jin; resources, Jianhong Sun; data curation, Jianhong Sun; writing—original draft preparation, Shuyan Jiang; writing—review and editing,Jian Lyu and Li Jin; visualization,Zhaozhuang Li; supervision, Shuchao Huang; project administration, Shuya Wang; funding acquisition, Jian Lyu and Li Jin; All authors have read and agreed to the published version of the manuscript.

Reviewer 3 Report

Comments and Suggestions for Authors

I have reviewed the manuscript entitled "Effects of Combined Application of Different Nitrogen Forms on Substrate Nutrient Utilization, Root Microenvironment, and Tomato Yield." It is an interesting paper; however, I offer comments and suggestions that may improve the manuscript before its possible acceptance.

1. I suggest including the statement of the hypothesis or hypotheses before mentioning the general objectives. Furthermore, the hypothesis should be discussed in the discussion section.

2. One of the components of the introduction is the background section. I suggest you mention at least three studies similar to or related to those proposed by the authors.

3. In the section "Determination of Substrate Enzyme Activities," I suggest you mention the reagents used, including the reagent grade and brand.

4. In the statistical analysis, I suggest that the authors mention the tests used to verify compliance with the criteria for normality and homoscedasticity. Were the assumptions confirmed?

5. I suggest that, in the conclusion section, you highlight the contribution of your research compared to other similar studies. Also, mention the limitations of the study.

Author Response

Dear Editors and Reviewers：

Reviewer 3

I suggest including the statement of the hypothesis or hypotheses before mentioning the general objectives. Furthermore, the hypothesis should be discussed in the discussion section.

Response:

Thank you for your careful review and valuable suggestions. We have added the hypotheses we wish to test, which are highlighted in yellow in the revised manuscript (Line 87-90). Our responses to these hypotheses are highlighted in yellow in the revised manuscript (Line 512-528). The specific revisions are as follows:

Our hypotheses are as follows: (i) Compared to a single nitrogen source, the combined application of ammonium and amide nitrogen will increase tomato yield and nutrient accumulation.(ii) Compared to a single nitrogen source, the combined application of ammonium and amide nitrogen will alter substrate enzyme activity and the microbial community. (Line 87-90)

The results showed that the combined application of ammonium nitrogen (AN) and amide nitrogen (UN) significantly improved tomato yield, nitrogen fertilizer utilization and substrate properties, with the T3 treatment (25% AN + 75% UN) showing the best overall performance. This indicates that a suitable AN:UN ratio can better meet the nitrogen requirements of tomatoes at different growth stages. In terms of growth and nitrogen fertilizer absorption, the T3 treatment significantly improved the yield per plant and nitrogen fertilizer absorption in the later harvest period (120 days). This may be due to the synergistic nitrogen supply mechanism of AN and UN: AN can be directly absorbed, which is beneficial to the rapid growth of seedlings [32]; while UN is slowly hydrolyzed by urease, continuously releasing NH₄⁺ [33], providing a stable nitrogen source for the middle and late growth stages and avoiding temporary nitrogen deficiency or loss. Anderson J [34] reported that an appropriate amount of UN can improve crop dry matter accumulation and nitrogen fertilizer utilization, which is consistent with our T3 treatment results. Regarding substrate nutrients, the total nitrogen and alkaline hydrolyzable nitrogen contents of treatments T3, T4, and T5 were all increased compared to CK, T1, and T2. This is related to the conversion characteristics of different nitrogen sources: AN is easily adsorbed by colloids and is not easily leached [35], while UN hydrolysis continuously replenishes NH₄⁺, improving the nitrogen supply capacity of the substrate [36]. (Line 521-528)

One of the components of the introduction is the background section. I suggest you mention at least three studies similar to or related to those proposed by the authors.

Response:

Thank you for your careful review and valuable suggestions. We have added the relevant content to the background section as requested. It is highlighted in yellow in the revised manuscript (Line 54-59). The specific modifications are as follows: The combined application of different nitrogen forms can produce different physiological effects on plant growth[9, 10]. For example, when NH₄⁺ and NO₃⁻ are applied in combination, tea plants often grow better and have higher bio-mass[9]. Chen et al.[11] found that a 1:1 ratio of ammonium nitrogen to nitrate nitrogen can op-timize the growth and some quality indices of tobacco. In tomato, raising the NH₄⁺ proportion in the nutrient solution from 0 % to 50 % markedly shortened primary-root length, decreased lat-eral-root number and reduced root surface area, with 10 mmol L⁻¹ NH₄⁺ almost completely sup-pressing lateral-root formation. By contrast, a NO₃⁻/NH₄⁺ ratio of 75:25 produced the highest photosynthetic rate, largest leaf area and greatest single-plant yield, outperforming the ammo-nium-only treatment by more than 18% [12].

In the section "Determination of Substrate Enzyme Activities," I suggest you mention the reagents used, including the reagent grade and brand.

Response:

Thank you for your careful review and valuable suggestions. We have added the relevant content as requested. It is highlighted in yellow in the revised manuscript (Line 185-195). The specific modifications are as follows: Urease kit (indophenol blue method, GRS-UE-1-M, lot 20230812): phenol ≥ 99% (AR, Sinopharm), sodium hypochlorite solution 5% (CP, Xilong Science), Tris-HCl buffer pH 9.0 (≥ 99%) Amresco). Sucrase kit (DNS method, GRS-SC-1-M, lot 20230815): 3,5-dinitrosalicylic ac-id ≥ 98% (AR, Aladdin), NaOH pellets ≥ 97% (GR, Sinopharm), sucrose ≥ 99.5% (HPLC) Sigma). Nitrate-reductase kit (sulfanilic acid–naphthylamine method, GRS-NR-1-M, lot 20230810): sulfanilic acid ≥ 99 % (AR, Macklin), naphthylamine ≥ 99 % (AR, Aladdin), KNO₃ ≥ 99 % (GR, Sinopharm). Nitrite-reductase kit (Griess method, GRS-NiR-1-M, lot 20230811): sulfanilic acid ≥ 99% (AR, Titan), α-naphthylamine ≥ 98% (AR, Aladdin), NaNO₂ ≥ 99% (GR) Sinopharm). Catalase kit (high-sensitivity probe method, GRS-CAT-1-M, lot 20230809): chromogenic probe 2,4-dichlorophenol ≥ 98% (HPLC, TCI), H₂O₂ solution 30% (CP, Xilong Science).

4.In the statistical analysis, I suggest that the authors mention the tests used to verify compliance with the criteria for normality and homoscedasticity. Were the assumptions confirmed?

Response:

Thank you for your careful review and valuable suggestions. We have added the relevant content, which is highlighted in yellow in the revised manuscript (Line 239-244).The specific modifications are as follows: All experimental data were analyzed using Excel 2021 and SPSS 26.0 and figures were generated with Origin 2021. Prior to analysis of variance, the normality of residuals was assessed using the Shapiro–Wilk test, and the homogeneity of variances was evaluated using Levene’s test. The results indicated that the data satisfied the assumptions of normality and homoscedasticity. Subsequently, one-way analysis of variance (ANOVA) was performed, followed by Duncan's multiple range test to determine significant differences among treatments at P < 0.05.

I suggest that, in the conclusion section, you highlight the contribution of your research compared to other similar studies. Also, mention the limitations of the study.

Response:

Thank you for your careful review and valuable suggestions. We have added relevant content, which is highlighted in yellow in the revised manuscript (Line 577-586). The specific modification is as follows: This study utilized the characteristic that amide nitrogen can slowly decompose into nitrate nitrogen, and investigated the effects of mixed application of ammonium and amide nitrogen. It was found that the mixed application of ammonium and amide nitrogen improved substrate nutrients and altered the substrate microbial environment, thereby increasing tomato yield. This may provide a practical solution for optimizing nitrogen fertilizer forms in greenhouse tomato cultivation. Currently, the results of this study are based only on a single variety and a single pot substrate; this fertilization scheme has not yet been validated in different varieties and long-term field trials. Future research will focus more on the regulatory effect of the T3 treatment on tomato quality and validate these conclusions in different varieties and long-term field trials.

Reviewer 4 Report

Comments and Suggestions for Authors

I think this is an outstanding research work showing that a combined application of N as AN-Urea mixture improved substrate properties, increased microbial counts, enhanced tomato mineral absorption and yield, and is recommended for efficient tomato cultivation. I think the paper should be published with very few minor corrections.

General questions:

In agricultural production, ammonium nitrogen (NH₄⁺), nitrate nitrogen (NO₃⁻), and urea-N (urea) are the main nitrogen sources. This experiment investigated the effects of different ratios of ammonium nitrogen and urea-N. The experimental design of this study was based on the previous work of the authors research,[sic LL105-106, ref 22; Sun et al., Plants (2023. 12(24): p. 4175. https://doi.org/10.3390/plants12244175)]. Ift he study follows the same experimental design as the previous work by the authors why does not consider nitrate nitrogen.?

The tables need formatting to be readable.

Maybe you should include a section for Abbreviatures to include the many in the text. See: TP, AP, AK, TK, SUC, CAT, AIKN, NiR, NR, URE, TN, EC, RDA, OTU, PCoA, LEfSe, LDA,

Specific questions

L10 delete “These authors contributed equally to this work”

LL14-16: Needs re-writing. There are 6 treatments. A control 0–0 (T0 better than CK?) receiving 0mM N, and 5 treatments receiving 15mM N with ammonium-N (AN) and amide-N (UN) combined at six ratios: 100 %–0 (T1), 0–100 % (T2), 25 %–75 % (T3), 50 %–50 % (T4), 15 and 75 %–25 % (T5).

L28 Introduction. All the cite numbers are after full stop. Correct.

LL131-133: Why 105℃.? The standard is 80℃.

L137: Need adding digestion method prior to colorimetric P and AAS analysis.

L149: Need adding digestion method prior to colorimetric P and AAS analysis.

Fig 6. Y-axis: Ca instead of Ga

L441: PcoA?

L451 OUT?

L467 Define LEfSe

Figure 10 is impossible to see anything. What do the bars mean? Looks like you need to separate in different figures or supplementary material.

Author Response

Dear Editors and Reviewers：

Reviewer 4

In agricultural production, ammonium nitrogen (NH₄⁺), nitrate nitrogen (NO₃⁻), and urea-N (urea) are the main nitrogen sources. This experiment investigated the effects of different ratios of ammonium nitrogen and urea-N. The experimental design of this study was based on the previous work of the authors research,[sic LL105-106, ref 22; Sun et al., Plants (2023. 12(24): p. 4175. https://doi.org/10.3390/plants12244175)]. Ift he study follows the same experimental design as the previous work by the authors why does not consider nitrate nitrogen?

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. We fully recognize that NH₄⁺, NO₃⁻ and amide nitrogen are all major nitrogen sources in tomato production. Our previous pot research included NH₄⁺, NO₃⁻ and amide nitrogen. In our early research, we found that the combined application of ammonium nitrogen and amide nitrogen has a very good effect. Compared with single fertilization, the mixed application of ammonium nitrogen and amide nitrogen can increase tomato yield and nitrogen utilization efficiency. At present, research on different forms of nitrogen mainly focuses on the ratio of nitrate nitrogen to ammonium nitrogen, with little attention paid to the effects of the ratio of ammonium nitrogen to amide nitrogen on tomato yield, substrate nutrient utilization, and substrate microbial communities. Therefore, we chose to focus on the effects of the mixed application of ammonium nitrogen and amide nitrogen on tomato yield and substrate. Thank you again for your comprehensive review and guidance.

2.The tables need formatting to be readable.

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. We have thoroughly reorganized all the tables: we have uniformly adopted three-line tables and removed vertical and redundant horizontal lines. Column headings and units are placed on separate lines, with saliency letters in lowercase. Table titles are placed above the tables, and table notes are moved below the tables to explain the statistical methods and the meaning of saliency annotations. At the same time, wide tables are appropriately rotated and reduced to ensure complete display within one page. Thank you again for your comprehensive review and guidance.

3.Maybe you should include a section for Abbreviatures to include the many in the text. See: TP, AP, AK, TK, SUC, CAT, NiR, NR, URE, TN, EC, RDA, OTU, PCoA, LEfSe, LDA.

Response:

Thank you for your careful review and valuable suggestions. We have added the full name at its first appearance in the text and added a "Abbreviations" section at the end of the revised manuscript for readers' convenience. The specific revisions are as follows:

The following abbreviations are used in this manuscript:

AK–available potassium	TK–total potassium
AP–available phosphatase	TP–total phosphorus
AN–available nitrogen	TN–available nitrogen
URE–urease	NR–nitrate reductase
NiR–nitrite reductase	CAT–catalase
SUC–sucrase	EC–electrical conductivity
OTU–operational taxonomic unit	PCoA–principal coordinate analysis
RDA–redundancy analysis	LDA–linear discriminant analysis
LEfSe–linear discriminant analysis effect size

L10 delete “These authors contributed equally to this work”

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. Line 10 "These authors contributed equally to this work" has been deleted as you requested. Thank you again for your patient review and guidance.

5.LL14-16: Needs re-writing.

Response:

Thank you for your careful review and valuable suggestions. It has been rewritten as requested, and is highlighted in yellow in the revised manuscript (Line 17-22). The specific modifications are as follows: Therefore, in this experiment, tomato cultivar ‘Jingfan 502’ was used. All treatments received the same total nitrogen concentration (15 mM), but the nitrogen was supplied as different combinations of ammonium nitrogen (AN) and amide nitrogen (UN). Six AN–UN ratio treatments were designed: CK (0% AN, 0% UN), T1 (100% AN, 0% UN), T2 (0% AN, 100% UN), T3 (25% AN, 75% UN), T4 (50% AN, 50% UN), and T5 (75% AN, 25% UN).

Introduction. All the cite numbers are after full stop. Correct.

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. We have reviewed the entire text one by one as you requested. All citation numbers have been adjusted to the end of the period to ensure compliance with the MDPI reference format norms.

LL131-133: Why 105℃.? The standard is 80℃.

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. Samples were briefly heated at 105°C to inactivate enzymatic activity, and then immediately transferred to an oven at 80°C for drying to constant weight, a temperature commonly used in plant tissue analysis to ensure complete dehydration while minimizing thermal damage to tissue components. Thank you again for your patient review and guidance.

Need adding digestion method prior to colorimetric P and AAS analysis.

Response:

Thank you for your careful review and valuable suggestions. The digestion methods have been added and are highlighted in yellow in the revised manuscript (Line 162-167). The specific modifications are as follows: A 0.5 g sample of air-dried substrate, sieved to 0.25 mm, was placed in a 100 mL digestion tube pre-soaked over-night with 5 mL of concentrated HNO₃, and 2 mL of HClO₄ were added the next day. The tube was then heated in a graphite block digester (ED36, LabTech) using the following temperature ramp: 120°C for 1 h, 160°C for 2 h, and 190°C until white fumes ceased. After cooling, the digest was diluted to 50 mL with deionized water and used for subsequent analyses.

Fig 6. Y-axis: Ca instead of Ga- L149：

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. The Y-axis label in Figure 6 has been corrected from "Ga" to "Ca", and the high-resolution image has been re-exported. Thank you again for your patient review and guidance. The specific modifications are as follows:

PcoA? - OUT?

Response:

Thank you for your careful review and valuable suggestions. The full names of PCoA and OTU have been added and are highlighted in yellow in the revised manuscript (Line 459-464). The specific changes are as follows: OTU (Operational Taxonomic Units) PCoA (Principal Coordinates Analysis)

11.L467 Define LEfSe-

Response:

Thank you for your careful review and valuable suggestions. LEfSe has been defined in the revised manuscript and is highlighted in yellow (Line 492-496). The specific modifications are as follows: LEfSe (Linear Discriminant Analysis Effect Size) is a statistical method for identifying mi-crobial (or other) taxa that are significantly different and possess a sufficiently large effect size among multiple groups.

12.Figure 10 is impossible to see anything. What do the bars mean? Looks like you need to separate in different figures or supplementary material.

Response:

Thank you for your careful review and valuable suggestions. We have updated the images and explained the meaning of the bars in the revised manuscript (Line 507). The specific revisions are as follows:

Figure 10. LefSe diagrams of bacterial (A) and fungal (B) communities under different treatments, and LDA discrimination results of bacterial (C) and fungal (D) communities. The LDA bar chart represents the matrix microbial groups that show significant differences under different treatments.

Reviewer 5 Report

Comments and Suggestions for Authors

The manuscript shows a detailed work about the effects of some ratios of ammoniun and ureid nitrogen on the growth and yield of tomato. The authors carried out a deep study on several response variables in soil and plant. A great contribution is the analysis of the effects of nitrogen forms on the soil microbiota.
Overall the manuscript describes a study well planned, conducted and analyzed.
Some suggestions are:
1. Include the description of the experimental design.
2. Make the tables more readable.
3. Specify the groups of means in the Figures 3, 4, 5, and 6.
4. Argue why the authors consider to include ACoP where the two major components contribute only with around 50% of the variance.

Comments for author File: Comments.pdf

Author Response

Dear Editors and Reviewers：

Reviewer 5

1.It would good to have here the statistical experiment design

Response:

Thank you for your careful review and valuable suggestions. I have revised the experimental design and treatment arrangements, which are highlighted in yellow in the revised manuscript (Line 110-121). Specific modifications are as follows :Our experimental layout followed the protocol previously reported by our group [23] in which twelve nitrogen-form combinations (T1–T12) plus a nitrogen-free control (CK) were originally tested. The present study retained the same nutrient-solution preparation and randomized com-plete-block arrangement, but reduced the number of treatments to the six most informative ratios (CK, T1, T2, T3, T4, T5) while maintaining the identical total-N concentration (15 mM) and ir-rigation schedule as described in the cited work.

2.Make sure that the name of the variable is the same in Table 3

Response:

Thank you for your careful review and valuable suggestions. We have ensured that the variable names are the same as in Table 3. The specific modifications are as follows:

Table 3. Effects of different treatments on nitrogen fertilizer use efficiency in tomato.

Treatments

nitrogen fertilizer uptake rate (%)

Partial factor productivity from applied N

(kg kg⁻¹)

Agronomic efficiency of applied N

(kg kg⁻¹)

15.44±0.21 b

14.47±0.34 c

18.07±0.34 a

13.10±0.18 d

15.74±0.16 b

288.64±1.95 c

450.49±2.36 b

473.49±11.98 a

235.34±8.43 d

166.90 ±6.10 e

192.15±1.95 c

354.00±2.36 b

377.00±11.98 a

138.85±8.43 d

70.41±6.10 e

Notes: Data are presented as mean ± standard error (n = 5). Different lowercase letters within the same column indicate significant differences among treatments at p < 0.05 (one-way ANOVA followed by post hoc test).

3.The editor requested that The short vertical lines on the bars indicate the mean ± standard error (n=3) in the caption of Figure 3-6 be marked. and different letters indicate sig-nificant differences at the 0.05 level. The short vertical lines on the column are changed to represent the mean ± standard error (n=3), and different letters indicate significant differences within organs at the 0.05 level.

Response:

Thank you for your careful review and valuable suggestions. We have revised the caption in Figure 3-6 as required, changing "The short vertical lines on the bars indicate the mean ± standard error (n=3), and different letters indicate significant differences at the 0.05 level" to "The short vertical lines on the bars represent the mean ± standard error (n=3), and different letters indicate that the differences between different organs are significant at the 0.05 level." This is highlighted in yellow in the revised manuscript (Line 334-426).

Argue why the authors consider to include ACoP where the two major components contribute only with around 50% of the variance.

Response:

Thank you for your careful review and valuable suggestions. In our study, the first two principal axes explained approximately 45% to 51% of the total variance in the PCoA analysis, which is not particularly high. However, we believe that including PCoA analysis was appropriate and valuable for the following reasons:1. PCoA is a commonly used exploratory tool in microbial ecology, suitable for high-dimensional and complex community data. In such datasets, the first two principal axes typically explain approximately 30% to 60% of the variance, while an explanation rate close to 50% is widely accepted and reported in studies of bacterial and fungal community structure.2. Although the explained variance is not high, PCoA analysis revealed clear and consistent separation patterns. In this study, both bacterial and fungal communities showed significant separation of treatments T3 and T4 from other treatments on the PC1 axis, and replicates within each treatment were tightly clustered. This indicates good reproducibility of the experimental results and suggests that nitrogen speciation has a significant and directional impact on rhizosphere microbial community structure.

PCoA results need to be interpreted in conjunction with other analytical methods (including RDA and LEfSe), which further support the conclusion that nitrogen speciation significantly alters microbial community structure. Therefore, PCoA is more suitable as an auxiliary visualization tool than as an independent quantitative indicator.

Thank you again for your patient review and guidance.

5.Make the tables more readable.

Response:

Specify the groups of means in the Figures 3, 4, 5, and 6

Response:

Thank you for your meticulous review of our manuscript and your valuable suggestions. We made modifications to two plans based on our own understanding. Option One: Processing groups have been added in Figures 3, 4, 5 and 6. Option Two: The average value graphs of mineral elements in four periods were respectively added to Figures 3, 4, 5 and 6. Thank you again for your comprehensive review and guidance. The specific modifications are as follows:

3.5.1. Nitrogen Uptake and Allocation

3.5.2. Potassium Uptake and Allocation

3.5.3. Phosphorus Uptake and Allocation

3.5.4. Calcium and Magnesium Uptake and Allocation