Physical and Chemical Properties Evaluation of Liquid Organic Fertilizers (LOFs) and Their Effects on Promoting Rice Growth

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study presents the potential of polyamine-enriched liquid organic fertilizers to enhance rice growth and mitigate salt stress, aligning with global priorities in sustainable agriculture. The comparative analysis of LOFs derived from brassica, mushroom, and soybean materials offers insights into nutrient dynamics and stress resilience mechanisms. In sum, the following revisions are essential:

• Specify the rationale for selecting the 1:75 dilution ratio for foliar application. Provide dose-response data or literature evidence supporting this choice, particularly regarding phytotoxicity thresholds and nutrient bioavailability.
• Reconcile the paradox in Figure 6 where LOFs inhibited root growth but enhanced shoot growth. Does this reflect hormonal crosstalk (e.g., PA-IAA antagonism) or osmotic imbalances?
• Address scalability: Discuss energy inputs for fermentation (e.g., molasses sourcing, 60-day process) versus conventional fertilizer production. A life-cycle assessment (LCA) would strengthen sustainability claims.
• Elaborate on the specific roles of polyamines (PAs) versus other LOF components (IAA, GA, nutrients) in observed outcomes. For instance, did PA-enriched LOFs uniquely upregulate ion transporters (e.g., SOS1, NHX1) or antioxidant genes (SOD, CAT)? Consider including qPCR or enzymatic activity data to disentangle PA-specific effects from general nutrient benefits.
• Include PA quantification via LC-MS/MS alongside HPLC to confirm dansylation-based measurements, as dansyl derivatives may exhibit matrix-dependent recovery variations.
• Justify the 100 mM NaCl treatment duration (7–14 days) in relation to rice growth stages and real-world salinity exposure scenarios. Include ECe (soil salinity) equivalents for field relevance.
• Discuss the metabolic fate of exogenous PAs: Are absorbed PAs directly modulating stress responses, or do they stimulate endogenous PA biosynthesis? Isotopic labeling (e.g., 15N-Spd) could resolve this.
• Contrast PA accumulation patterns (Figure 11) with prior studies: Why did Put dominate over Spd/Spm in salt-stressed controls, contrary to typical rice PA metabolism under salinity?
• Provide full ANOVA results (F-values, p-values) and post-hoc tests for all figures. For example, Figure 7’s growth data requires clarity on whether SOF-CF equivalence is statistically validated (p>0.05).
• Compare LOF performance against commercial biofertilizers (e.g., seaweed extracts, vermicompost teas) to benchmark efficacy. Currently, the CF-only comparison limits practical relevance.

Author Response

This study presents the potential of polyamine-enriched liquid organic fertilizers to enhance rice growth and mitigate salt stress, aligning with global priorities in sustainable agriculture. The comparative analysis of LOFs derived from brassica, mushroom, and soybean materials offers insights into nutrient dynamics and stress resilience mechanisms. In sum, the following revisions are essential:

1. Specify the rationale for selecting the 1:75 dilution ratio for foliar application. Provide dose-response data or literature evidence supporting this choice, particularly regarding phytotoxicity thresholds and nutrient bioavailability.

Author response: The 1:75 dilution was selected based on the phytotoxicity of LOFs through seed germination and available nutrients. The 1:50 dilution is quite dense and has a high EC. The 1:100 may be too dilute contained low nutrients. The rationale for using 1:75 dilution is described on Page 11, at the end of section 3.4.

2. Reconcile the paradox in Figure 6 where LOFs inhibited root growth but enhanced shoot growth. Does this reflect hormonal crosstalk (e.g., PA-IAA antagonism) or osmotic imbalances?

Author response: Thank you very much for bringing up this important point. I looked at some literature which stated that the roles of PA in root and shoot growth greatly varied depending on plant species, concentration of applied PA, endogenous PA, and ‘hormonal crosstalk’ as per your suggestions. I have added discussion concerning root growth inhibition on Page 19, Lines 454 – 465.

3. Address scalability: Discuss energy inputs for fermentation (e.g., molasses sourcing, 60-day process) versus conventional fertilizer production. A life-cycle assessment (LCA) would strengthen sustainability claims.

Author response: Molasses Sourcing: Molasses, a byproduct of sugar production, is often used as a substrate for fermentation. The energy required for sourcing and transporting molasses can vary depending on the distance and method of transportation. However, molasses can be easily obtained and bought in the Northest Thailand as cheap byproduct from sugar factories. Combining with the main ingredients for fermentation process are plant-based materials which can be bought from fresh markets. About 60-Day Process: The fermentation process typically spans around 60 days. This involves maintaining optimal conditions for microbial growth, which requires energy for temperature control, aeration, and agitation. This process could be conducted on a small scale by farmers. Compared with chemical fertilizers, production of organic fertilizers does not require expensive machinery, less energy consumption for processing, and almost no waste and pollution are generated. This advantage of producing LOFs is addressed in the final paragraph of Discussion.

4. Elaborate on the specific roles of polyamines (PAs) versus other LOF components (IAA, GA, nutrients) in observed outcomes. For instance, did PA-enriched LOFs uniquely upregulate ion transporters (e.g., SOS1, NHX1) or antioxidant genes (SOD, CAT)? Consider including qPCR or enzymatic activity data to disentangle PA-specific effects from general nutrient benefits.

Author response: Thank you so much for your valuable comments. This article focuses on the effects of LOFs on growth and physiology with a view to providing basic information for future applications at the field experiment level. So, the authors did not collect materials for molecular studies. Your suggestion on evaluation of the effects of LOFs on up-regulation of gene expression is highly constructive and should be investigated in future experiments.

5. Include PA quantification via LC-MS/MS alongside HPLC to confirm dansylation-based measurements, as dansyl derivatives may exhibit matrix-dependent recovery variations.

Author response: We highly appreciate your concern about the quantification of PAs using HPLC of dansyl derivatives. We agree that using LC-MS/MS will give a more accurate quantification of PAs. However, we thought PA quantification based on HPLC was sufficient for our purposes. We compared our results with other studies in plants, all of which employed HPLC and found that the range of the amount of PAs we obtained under salinity was reasonably comparable with previous reports although the content varies with plant species, growth stage, level of stress etc. For example, Quinet et al. (2010), in rice, reported Put (~0.2 – 0.7 umol/g FW); Spd (~0.05 – 0.3 umol/g FW); Spm (~0.06-0.13 umol/g FW), Puyang et al. (2015) reported, in Kentucky bluegrass, Put (~0.5 – 2 umol/g FW); Spd (~0.05 – 0.7 umol/g FW); Spm (~0.4 – 2 umol/g FW), Raziq et al. (2022), in tomato, Put (~0.01 – 0.4 umol/g FW); Spd (~0.02 – 0.8 umol/g FW); Spm (~0.01 – 0.6 umol/g FW). Do et al. (2014), in rice, Put (<0.1 – 4 umol/g FW); Spd (~0.1 – 1.2 umol/g FW); Spm (~0.1 – 0.5 umol/g FW)

References: Quinet M, Ndayiragije A, Lefèvre I, Lambillotte B, Dupont-Gillain CC, Lutts S. Putrescine differently influences the effect of salt stress on polyamine metabolism and ethylene synthesis in rice cultivars differing in salt resistance. J Exp Bot. 2010 Jun;61(10):2719-33. doi: 10.1093/jxb/erq118. Epub 2010 May 14. PMID: 20472577; PMCID: PMC2882274.

Puyang, X., An, M., Han, L., Zhang, X. 2015.Protective effect of spermidine on salt stress induced oxidative damage in two Kentucky bluegrass (Poa pratensis L.) cultivars. Ecotoxicology and Environmental Safety 117,96-106, https://doi.org/10.1016/j.ecoenv.2015.03.023.

Raziq, A., Ud Din, A.M., Anwar, S., Wang, Y., Jahan, M.S., He, M., Ling, C.G., Sun, J., Shu, S., Guo, S. 2022. Exogenous spermidine modulates polyamine metabolism and improves stress responsive mechanisms to protect tomato seedlings against salt stress, Plant Physiology and Biochemistry,187,1-10  https://doi.org/10.1016/j.plaphy.2022.07.005.

Do PT, Drechsel O, Heyer AG, Hincha DK, Zuther E. Changes in free polyamine levels, expression of polyamine biosynthesis genes, and performance of rice cultivars under salt stress: a comparison with responses to drought. Front Plant Sci. 2014 May 8;5:182. doi: 10.3389/fpls.2014.00182. PMID: 24847340; PMCID: PMC4021140.

6. Justify the 100 mM NaCl treatment duration (7–14 days) in relation to rice growth stages and real-world salinity exposure scenarios. Include ECe (soil salinity) equivalents for field relevance.

Author response: The use of 100 mM NaCl for salt stress treatment of rice was based on our prior experience working with this rice cultivar (KDML105) which is highly sensitive at young seedling stage (14 days after germination), but less sensitive at the growth stage used in this study (30 days after germination). At lower concentrations (eg. 50-75 mM NaCl) the plants were quite tolerant while 150 mM would be too strong.  The concentration used also depend on the number of days the plants are treated before the tissues are collected for physiological studies. In this study, plant tissues were inspected after 7 and 14 days of salt stress, so 100 mM NaCl was used. The sign of stress was monitored non-destructively by the reduction in net photosynthesis rate after 7 days of salt stress. In addition, this 100 mM concentration in pot experiments resulted in soil EC of approximately 6 – 8 dS/m corresponding to salinity level commonly occurs in rice field around this region (moderately saline).

7. Discuss the metabolic fate of exogenous PAs: Are absorbed PAs directly modulating stress responses, or do they stimulate endogenous PA biosynthesis? Isotopic labeling (e.g., 15N-Spd) could resolve this.

Author response: Thank you for your constructive comments. I agree with you that using isotopically labelled exogenous PA would elucidate the questions regarding the fate of applied PAs in plants whether they directly modulate stress response or indirectly stimulate endogenous PA biosynthesis. According to several published papers and review articles, I have not been able to find any reports on tracking isotopically labelled PAs after application to plants. However, many papers reported that exogenous PAs led to modulation of PA metabolism and increased levels of endogenous PAs. For example: (1) Spraying Spd to rice panicles after pollination led to increased content of Spd and Spm in developing and mature rice grains (Fu et al., 2019). (2) Spd application enhanced biosynthesis of endogenous Spd and Spm in tomato seedlings (Raziq 2022) (3) Exogenous Put led to an increase in endogenous Put in rice seedlings under salt stress especially in salt-tolerant variety (Quinet 2010) (4) Exogenous Spd increased endogenous Spd and Spm, but decreased Put in Kentucky blue grass under salt stress (Puyang et al., 2016). A model has been proposed that exogenous PAs after being absorbed into plant cells are oxidized producing H₂O₂ which acts as a signaling molecule initiating signal transduction cascades leading to transcriptional up-regulation of genes involved in various modes of stress defense responses including ‘activation of genes in PA biosynthesis pathways’ (Tyagi et al., 2023).  

References:

Fu Y, Gu Q, Dong Q, Zhang Z, Lin C, Hu W, Pan R, Guan Y, Hu J. Spermidine Enhances Heat Tolerance of Rice Seeds by Modulating Endogenous Starch and Polyamine Metabolism. Molecules. 2019 Apr 9;24(7):1395. doi: 10.3390/molecules24071395. PMID: 30970602; PMCID: PMC6480098.

Raziq, A., Ud Din, A.M., Anwar, S., Wang, Y., Jahan, M.S., He, M., Ling, C.G., Sun, J., Shu, S., Guo, S. 2022. Exogenous spermidine modulates polyamine metabolism and improves stress responsive mechanisms to protect tomato seedlings against salt stress, Plant Physiology and Biochemistry,187,1-10  https://doi.org/10.1016/j.plaphy.2022.07.005.

Quinet M, Ndayiragije A, Lefèvre I, Lambillotte B, Dupont-Gillain CC, Lutts S. Putrescine differently influences the effect of salt stress on polyamine metabolism and ethylene synthesis in rice cultivars differing in salt resistance. J Exp Bot. 2010 Jun;61(10):2719-33. doi: 10.1093/jxb/erq118. Epub 2010 May 14. PMID: 20472577; PMCID: PMC2882274.

Puyang, X., An, M., Han, L., Zhang, X. 2015.Protective effect of spermidine on salt stress induced oxidative damage in two Kentucky bluegrass (Poa pratensis L.) cultivars. Ecotoxicology and Environmental Safety 117,96-106, https://doi.org/10.1016/j.ecoenv.2015.03.023.

Tyagi, A., Ali, S., Ramakrishna, G., Singh, A., Park, S., Mahmoudi, H., Bae, H. 2023. Revisiting the Role of Polyamines in Plant Growth and Abiotic Stress Resilience: Mechanisms, Crosstalk, and Future Perspectives. J Plant Growth Regul 42, 5074–5098 (2023). https://doi.org/10.1007/s00344-022-10847-3

8. Contrast PA accumulation patterns (Figure 11) with prior studies: Why did Put dominate over Spd/Spm in salt-stressed controls, contrary to typical rice PA metabolism under salinity?

Author response: Thank you very much for your remark. According to research on rice in the past (in the 1900s to early 2000s), data on changes in PAs induced by salt stress were performed in only one or two rice cultivars and the results varied a lot depending on genotypes, growth stages and stress levels. In 2014, Do et al. studied changes in endogenous PAs in response to salt stress at 50 and 100 mM NaCl. They compared changes in Put, Spd and Spm in 18 rice cultivars with a wide range of salt tolerance levels and found different patterns of changes. In response to salt stress, the tolerant cultivars displayed an increase in Put, Spd and Spm (most prominent increase in Spm) while the sensitive cultivars showed dramatic reductions in Put and slight reduction in Spd but a slight increase in Spm. The cultivar used in our study, KDML105, is salt sensitive. The pattern of change in PAs under salt stress in this study (Figure 11, comparing CK at 0 mM with CK at 100 mM) was in agreement with the result of Do et al. (2014) i.e. Put dramatically reduced after 7 and 14 days after salt stress (Figure 11A and B), Spd also reduced (Figure 11C and D) but Spm increased (Figure 11E and F). Also, similar to Do et al. (2014), for sensitive cultivars under control (non-stress) the amount of Put, and to a lesser extent Spd, dominated over Spm. This discussion was added on Page 20, Lines 481 – 495.

Reference:  Do PT, Drechsel O, Heyer AG, Hincha DK, Zuther E. Changes in free polyamine levels, expression of polyamine biosynthesis genes, and performance of rice cultivars under salt stress: a comparison with responses to drought. Front Plant Sci. 2014 May 8;5:182. doi: 10.3389/fpls.2014.00182. PMID: 24847340; PMCID: PMC4021140.

 

9. Provide full ANOVA results (F-values, p-values) and post-hoc tests for all figures. For example, Figure 7’s growth data requires clarity on whether SOF-CF equivalence is statistically validated (p>0.05).

Author response: The ANOVA tables are provided in the Supplementary.

10. Compare LOF performance against commercial biofertilizers (e.g., seaweed extracts, vermicompost teas) to benchmark efficacy. Currently, the CF-only comparison limits practical relevance.

Author response: Thank you very much for your constructive advice. We will consider involving other types of commercial biofertilizers in our future experiments for comparison with our LOFs.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Review of the manuscript "Polyamine-enriched liquid organic fertilizers (LOFs) promoted growth and alleviated salt stress impacts on rice" by Nuntavun Riddech et al., submitted to the journal "Sustainability".

Improving salt tolerance of rice still remains an urgent problem for the development of sustainable agriculture, especially in Asian countries, where rice is the main food crop. The authors propose to use liquid organic fertilizers produced from agricultural residues by fermentation of brassica vegetables, mushrooms and soybeans as a supplement or replacement for chemical fertilizers (foliar treatment) of rice plants in regions with saline soils. This direction is widely studied in the world, as evidenced by the large number of published articles on this topic. The authors studied the chemical characteristics and phytotoxicity of liquid organic fertilizers, and obtained new interesting scientific results, a positive effect on the nutritional value of rice, the results have a practical focus. An important result of the authors' work is the reduction of salt stress on rice plants when using liquid organic fertilizer, especially soybeans-based.

In general, the study was conducted competently and logically correctly. There are several wishes and comments:

The Latin name of rice plants should be included in the abstract and keywords.

L.120. The abbreviation "OM" is encountered for the first time - it should be deciphered as "organic matter".

L.194. The authors write "ten seeds ..." - why 10 seeds? This is very little for statistical analysis. Is this indicated in the methodology in the source [26]? How many replicates were there?

L.198. Why did the authors determine the germination of seeds of different crops at the same time - on the 7th day? It is customary to determine the germination of cabbage seeds on the 8th day, and the germination of tomato and rice seeds - on the 10th day.

L.224. In further research I advise the authors to use newer sources with precise formulas for calculating chlorophyll concentrations, rather than the 1949 source (Arnon). Modern formulas take into account the wavelength step of the spectrophotometer (0.5 nm or 1.0 nm). It is too bad that the authors did not indicate the brand and manufacturer of the spectrophotometer - please do so.

Figures.

All figure captions (except for Figures 7-11) should indicate what the letters above the columns mean. Also, the figure captions should include an explanation of all abbreviations in the figure.

Advice. Considering that under salt stress there is a decrease in the water content in shoots (Figure 7b), all calculations of physiological parameters should be performed on DW, not FW.

It is not clear from the manuscript whether liquid organic fertilizers can be stored for a long time (2-3 months) or should be used immediately after filtration. Is alkaline hydrolysis of fertilizers possible to preserve them for a long time?

The Discussion section is large and very detailed.

Can the authors explain the following fact? According to Figure 11bdf, the endogenous content of polyamines under salt stress increases in the leaves in the following order: Put, Spd, Spm. Does the accumulation of polyamines depend on the structure of their molecule - in this case, on the number of functionally active NH groups? It is known that the Put molecule contains 2 NH groups - it is contained in small quantities in leaves, no more than 2 μM, whereas the Spd molecule has 3 NH, Spm - 4 NH, respectively, their content reaches 4 μM.

L.522. The authors write "... (Spd + Spm): Put ratios were elevated under salt stress (Figure 11)." However, Figure 11 does not contain information on (Spd + Spm): Put ratios.

The authors made a separate Abbreviations section, which is very convenient.

The list of references is sufficient and includes titles mainly from the last 10 years.

Author Response

Comments and Suggestions for Authors

Review of the manuscript "Polyamine-enriched liquid organic fertilizers (LOFs) promoted growth and alleviated salt stress impacts on rice" by Nuntavun Riddech et al., submitted to the journal "Sustainability".

Improving salt tolerance of rice still remains an urgent problem for the development of sustainable agriculture, especially in Asian countries, where rice is the main food crop. The authors propose to use liquid organic fertilizers produced from agricultural residues by fermentation of brassica vegetables, mushrooms and soybeans as a supplement or replacement for chemical fertilizers (foliar treatment) of rice plants in regions with saline soils. This direction is widely studied in the world, as evidenced by the large number of published articles on this topic. The authors studied the chemical characteristics and phytotoxicity of liquid organic fertilizers, and obtained new interesting scientific results, a positive effect on the nutritional value of rice, the results have a practical focus. An important result of the authors' work is the reduction of salt stress on rice plants when using liquid organic fertilizer, especially soybeans-based.

In general, the study was conducted competently and logically correctly. There are several wishes and comments:

1. The Latin name of rice plants should be included in the abstract and keywords.

Author response: The Latin name of rice (Oryza sativa L.) has been added in the abstract (Line 20) and keywords (Line 28).

2. L.120. The abbreviation "OM" is encountered for the first time - it should be deciphered as "organic matter".

Author response: Thank you for your suggestion. ‘OM……..’ has been corrected to “Organic matter (OM)……..” (Line 124).

3. L.194. The authors write "ten seeds ..." - why 10 seeds? This is very little for statistical analysis. Is this indicated in the methodology in the source [26]? How many replicates were there?

Author response: The method followed the procedure in the source [26]. Three replicates were repeated. [Zucconi, F.; Forte, M.; Monaco, A.; De Bertoldi, M. Biological evaluation of compost maturity. Biocycle. 1981, 22, 27–29.]

4. L.198. Why did the authors determine the germination of seeds of different crops at the same time - on the 7th day? It is customary to determine the germination of cabbage seeds on the 8th day, and the germination of tomato and rice seeds - on the 10th day.

Author response: The determination of germination day followed procedure in the source [Zucconi et al., 1981]. Tomato, pakchoy and rice were selected because (1) they are salt sensitive crop (2) they are fastly germinated. Pakchoy seeds germinated after 1-2 days; tomato germinated after 2-3 days, and rice germinated after 2-4 days. On day 7, the difference in seedling growth of all three species treated with LOFs compared with the control were clearly evident (Figure 6). In addition, the experiment was conducted in petri dishes, 7 days was suitable to evaluate the seedling growth. Otherwise, the seedling roots would be too long and intertwined with each other making it difficult to safely separate individual seedlings for root length measurement.

5. L.224. In further research I advise the authors to use newer sources with precise formulas for calculating chlorophyll concentrations, rather than the 1949 source (Arnon). Modern formulas take into account the wavelength step of the spectrophotometer (0.5 nm or 1.0 nm). It is too bad that the authors did not indicate the brand and manufacturer of the spectrophotometer - please do so.

Author response: Your kind suggestions are highly appreciated. We will keep this in mind and use more modern techniques and calculations for determining chlorophyll concentrations. The brand and manufacturer of the spectrophotometer has been added in the revised version (Line 228).

Figures.

6. All figure captions (except for Figures 7-11) should indicate what the letters above the columns mean. Also, the figure captions should include an explanation of all abbreviations in the figure.

Author response: The letters above the column and all abbreviations are now explained in each figure caption.

7. Advice. Considering that under salt stress there is a decrease in the water content in shoots (Figure 7b), all calculations of physiological parameters should be performed on DW, not FW.

Author response: Your kind and constructive suggestions are highly appreciated. We will keep this in mind and perform calculations based on dry weight basis.  

8. It is not clear from the manuscript whether liquid organic fertilizers can be stored for a long time (2-3 months) or should be used immediately after filtration. Is alkaline hydrolysis of fertilizers possible to preserve them for a long time?

Author response: The liquid fertilizer can be stored within 6 months for best quality. Alkaline hydrolysis is often used to treat materials from sources like animal carcasses and sewage sludges when making liquid fertilizer to accelerate breakdown of complex molecules. In this study, with plant materials, we did not use alkaline hydrolysis.

9. The Discussion section is large and very detailed.

Author response: We tried to shorten the discussion in the first part (characterization of LOFs and phytotoxicity test) as suggested by Reviewer #3. However, we added more details in the second part (effects of LOFs on rice growth and physiology) to elucidate a few important questions raised by Reviewers #1 and #3.

10. Can the authors explain the following fact? According to Figure 11bdf, the endogenous content of polyamines under salt stress increases in the leaves in the following order: Put, Spd, Spm. Does the accumulation of polyamines depend on the structure of their molecule - in this case, on the number of functionally active NH groups? It is known that the Put molecule contains 2 NH groups - it is contained in small quantities in leaves, no more than 2 μM, whereas the Spd molecule has 3 NH, Spm - 4 NH, respectively, their content reaches 4 μM.

Author response: According to literature in salt-sensitive rice cultivars under salt stress, the amount of endogenous Put would decrease dramatically, while Spd slightly decreased, and oppositely Spm would increase. Under salt stress Put is converted to Spd, and Spd to Spm.  And Spm is the most important type of PA that confers salt tolerance to rice through various molecular/physiological mechanisms. However, I cannot explain the relationship between the number of NH group and concentration of PA.   

11. L.522. The authors write "... (Spd + Spm): Put ratios were elevated under salt stress (Figure 11)." However, Figure 11 does not contain information on (Spd + Spm): Put ratios.

Author response: This sentence was deleted.

The authors made a separate Abbreviations section, which is very convenient.

The list of references is sufficient and includes titles mainly from the last 10 years.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The study has positive implications for the development of organic agriculture and the promotion of sustainable agricultural development. But the current form is not yet acceptable. If the author can supplement and revise in the following aspects, it will help with the acceptance of the manuscript.
1. The title does not match the content. The research content includes the production of LOFs through fermentation of plant materials and mushrooms rich in PA content. The physical and chemical properties, plant toxicity, plant hormones and nutrients contained in three types of LOFs, as well as their beneficial effects in alleviating salt stress in rice, were evaluated. Therefore, the title is proposed to be changed to: Physical and chemical properties evaluation of liquid organic fertilizers (LOFs) and their effects on promoting rice growth.
2. The abstract needs to be revised to highlight the effectiveness of LOF as a substitute for chemical fertilizers.
3. The experiment set up 5 treatments and repeated 3 times, but did not specify how many pots were planted in each treatment, how many plants were planted in each pot, and how many plants were measured in each treatment during sampling and measurement.
4. Has the effect on the growth of rice seedlings been measured, such as seedling height and tiller number, which are important traits reflecting the growth of rice seedlings.
5. In the results section, the description of the experimental results should be concise and consistent with the data. The ending of the first paragraph of "3.5 Effects of LOF on growth and physiology of rice under salt stress ": "MOF and SOF are more conducive to rice growth under salt stress than BOF" is inconsistent with result of Figure 7B. Although the DSW of BOF is slightly lower than that of MOF and SOF, there is no significant difference among the three LOFs.
6. The discussion is too long and the key points are not highlighted. There are too many descriptions of the physical and chemical properties, plant toxicity, plant hormones, and nutritional components of LOF, while there is relatively little description of its promotion of rice growth under salt stress. In addition, the following three points should be explained in the discussion.
(1) Rice is an important grain crop aimed at harvesting seeds. This study only measured seedling traits and did not have data on yield and yield factors. From the rice photo in Figure 6D, it seems that the growth of all treated seedlings is very abnormal. For rice crops that aim to harvest seeds, if they cannot bear seeds and grow well during the seedling stage, it is useless. Of course, the author's experiment focuses on demonstrating the effectiveness of LOF as a substitute for fertilizers, which is still beneficial. The author should explain this in the discussion.
(2) Compared with chemical fertilizers, organic fertilizers are a type of fertilizer that has a relatively slow effect on crop growth. Their role is more manifested in improving soil physicochemical properties and forming soil microbial communities. So, a year of experimentation is difficult to demonstrate the true function of organic fertilizers. The author should also clarify this point in the discussion.
(3) The reasons for the author's selection of raw materials, production methods, and the feasibility of mass production when producing LOF should also be explained in the discussion.

Author Response

Comments and Suggestions for Authors

The study has positive implications for the development of organic agriculture and the promotion of sustainable agricultural development. But the current form is not yet acceptable. If the author can supplement and revise in the following aspects, it will help with the acceptance of the manuscript.

1. The title does not match the content. The research content includes the production of LOFs through fermentation of plant materials and mushrooms rich in PA content. The physical and chemical properties, plant toxicity, plant hormones and nutrients contained in three types of LOFs, as well as their beneficial effects in alleviating salt stress in rice, were evaluated. Therefore, the title is proposed to be changed to: Physical and chemical properties evaluation of liquid organic fertilizers (LOFs) and their effects on promoting rice growth.

Author response: The authors are grateful for the suggestion and agree with the new title.

2. The abstract needs to be revised to highlight the effectiveness of LOF as a substitute for chemical fertilizers.

Author response: The last sentence of Abstract has been modified to “The SOF was the most effective and enhanced growth and photosynthesis at a similar level as the chemical fertilizer and had good potential to be employed as an eco-friendly substitute for chemical fertilizer.”

3. The experiment set up 5 treatments and repeated 3 times, but did not specify how many pots were planted in each treatment, how many plants were planted in each pot, and how many plants were measured in each treatment during sampling and measurement.

Author response: The five pots were planted in each treatment. Five plants were planted in each pot and three plants were selected to measurement. This information is now added in the M & M section, Lines 214 – 216.

4. Has the effect on the growth of rice seedlings been measured, such as seedling height and tiller number, which are important traits reflecting the growth of rice seedlings.

Author response: Seedling height and tiller number were not recorded in this study, only the dry weight of shoots and roots. We are thankful for your comments and will include these parameters in our future experiments.

5. In the results section, the description of the experimental results should be concise and consistent with the data. The ending of the first paragraph of "3.5 Effects of LOF on growth and physiology of rice under salt stress ": "MOF and SOF are more conducive to rice growth under salt stress than BOF" is inconsistent with result of Figure 7B. Although the DSW of BOF is slightly lower than that of MOF and SOF, there is no significant difference among the three LOFs.

Author response: Thank you for your comment. The authors agree with your comments and has corrected this sentence to “Under 100 mM NaCl stress, all three LOFs signifiantly enhanced root growth compared with CK but only MOF and SOF enhanced shoot growth.” (Line 5 – 6 on Page 12)

6. The discussion is too long and the key points are not highlighted. There are too many descriptions of the physical and chemical properties, plant toxicity, plant hormones, and nutritional components of LOF, while there is relatively little description of its promotion of rice growth under salt stress. In addition, the following three points should be explained in the discussion.
   (1) Rice is an important grain crop aimed at harvesting seeds. This study only measured seedling traits and did not have data on yield and yield factors. From the rice photo in Figure 6D, it seems that the growth of all treated seedlings is very abnormal. For rice crops that aim to harvest seeds, if they cannot bear seeds and grow well during the seedling stage, it is useless. Of course, the author's experiment focuses on demonstrating the effectiveness of LOF as a substitute for fertilizers, which is still beneficial. The author should explain this in the discussion.

Author response: We are grateful for your comments. The photographs of rice in Figure 6D demonstrated the effects of three dilutions of LOFs on germination and rice seedlings as a ‘test for phytotoxicity’ of LOFs. The seeds were germinated and seedlings grown in petri dishes under low light intensity for 7 days, so the seedlings looked unhealthy. The need for further studies to test the effectiveness of LOFs at the reproductive stage to evaluate the effects on yield is now highlighted in the final paragraph of Discussion.

(2) Compared with chemical fertilizers, organic fertilizers are a type of fertilizer that has a relatively slow effect on crop growth. Their role is more manifested in improving soil physicochemical properties and forming soil microbial communities. So, a year of experimentation is difficult to demonstrate the true function of organic fertilizers. The author should also clarify this point in the discussion.

Author response: I am grateful for your insightful suggestion in relation to practical application of the work. The final paragraph of discussion was modified to involve the points you kindly raised here.

(3) The reasons for the author's selection of raw materials, production methods, and the feasibility of mass production when producing LOF should also be explained in the discussion.

Author response: The reasons for using brassica vegetables, mushrooms and soybeans as raw materials were because they contain relatively high content of nutrients and PA as indicated in the reference [Atiya Ali, M.; Poortvliet, E.; Strömberg, R.; Yngve, A. Polyamines in foods: development of a food database. Food Nutr. Res. 2011, 55, 5572. http://doi: 10.3402/fnr.v55i0.5572]. The feasibility of mass production of LOFs is mentioned in the final paragraph of Discussion.

 

 

Reviewer 4 Report

Comments and Suggestions for Authors

In the front there is a manuscript with title Polyamine-enriched Liquid Organic Fertilizers (LOFs) Promoted Growth and Alleviated Salt Stress Impacts on Rice

Title: why authors use polyamine in the title?  They must delete it  

Abstract: Authors should use numbers of parentage decrease or increase between different treatments.

Keywords: well

Introduction: Is well but need some more information about  the beneficial uses of these extracts and their contents and  previous works about these extracts

Material: not clear authors should add all methods they work in the manuscript , I see some results not included in the material part how did you get from ? (not mentioned  in the Matrial part like Fig. 5 and 6 in the result).

Authors should explain more about extracts and their contents like how much minerals, phytohormones, vitamins, organic matter …..etc

Results: Well but there are some issue i can’t understand, Fig. 5 and 6 where did you get from.  

Discussion

Authors should divide the discussion to different parts like results

Conclusion:  well

General comments about the manuscript: This manuscript include good idea and good material, results and discussions but there is a big  mistake when we looking for new fertilizers or non traditional methods to replace with chemical  fertilizers we must use low costs materials  like  crop residues to make fermented  liquid fertilizers with low costs.  

 

 

 

 

Author Response

In the front there is a manuscript with title Polyamine-enriched Liquid Organic Fertilizers (LOFs) Promoted Growth and Alleviated Salt Stress Impacts on Rice

Title: why authors use polyamine in the title?  They must delete it

Author response: According to suggestion of Reviewer #3 - The title is changed to  ‘Physical and chemical properties evaluation of liquid organic fertilizers (LOFs) and their effects on promoting rice growth’

 

Abstract: Authors should use numbers of parentage decrease or increase between different treatments.

Author response: I tried to add some values into the Abstract but due to limited number of words and large number of treatments, I could only add a few chemical values of SOF which were highest among the three LOFs.  

Keywords: well

 

Introduction: Is well but need some more information about  the beneficial uses of these extracts and their contents and  previous works about these extracts

Author response: We added two examples of the beneficial effects of LOFs on plant growth promotion (Page 2, Lines 54 – 58).

Material: not clear authors should add all methods they work in the manuscript , I see some results not included in the material part how did you get from ? (not mentioned  in the Matrial part like Fig. 5 and 6 in the result).

Author response – The methods for Figure 5 and Figure 6 are described under section

2.6 Evaluation of phytotoxicity of LOF  (Page 5 – 6, Lines 191-204)

 

Authors should explain more about extracts and their contents like how much minerals, phytohormones, vitamins, organic matter …..etc

Author response: The characteristics of LOFs including pH and EC, minerals, phytohormones (IAA, GA, and PAs) (except vitamins which were not evaluated) were discussed in the first part of Discussion (4.1).

Results: Well but there are some issue i can’t understand, Fig. 5 and 6 where did you get from.  

Author response – The methods for Figure 5 and Figure 6 are described under section 2.6 Evaluation of phytotoxicity of LOF  (Page 5 – 6, Lines 191-204)

Discussion

Authors should divide the discussion to different parts like results

Author response – Your advice is highly appreciated. Discussion is now divided into three parts

Conclusion:  well

General comments about the manuscript: This manuscript include good idea and good material, results and discussions but there is a big  mistake when we looking for new fertilizers or non traditional methods to replace with chemical  fertilizers we must use low costs materials  like  crop residues to make fermented  liquid fertilizers with low costs.

Author response –  Thank you very much for your comment. For the experiments, fresh vegetables, mushrooms and soybeans were used so the quality of materials could be controlled. However, as the Reviewer suggested, when it comes to practical applications cheap materials like kitchen scraps, crop residues, and soybean mill should be utilized.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accept in present form.