Review Reports - Improving Sustainable Vegetable Production with Biochar and Chitosan Xerogel Combination Under Water and Fertilizer Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

Suggestions for correction are in the attached file.

Kind regards,

Comments for author File: Comments.pdf

Author Response

Response

Detailed Response to Editor and Reviewer

Dear Reviewer,

Attached please find the manuscript entitled “Improving Sustainable Vegetable Production with Biochar and Chitosan Xerogel Combination under Water and Fertilizer Stress”, by I-Chun Pan , Chen-An Jiang , Wan-Yi Chiou * , Yi-Chun Chen *.

Enclosed please find the referenced manuscript (horticulturae-3913786). The comments from editor are gratefully acknowledged. The content has been modified followed by editor’s requirement. Our responses and the revised parts in the manuscript were described as followed:

Abstract

Dear authors, by including more quantitative data, deepening the theoretical foundation, and strengthening the discussion, the article could achieve a significant impact, contributing to the knowledge on bioinputs and sustainable practices in vegetable cultivation under environmental stress.

Dear authors, please clarify water and nutritional stress in more detail.

Response: We appreciate the reviewer’s insightful comment regarding clarification of water and nutritional stress. In this study, both water deficit and nutrient depletion were considered as key abiotic stress factors that limit crop productivity under global climate change. Global warming and the overexploitation of natural resources have exacerbated water scarcity, while excessive and unbalanced fertilizer use has resulted in soil acidification and nutrient leaching, leading to long-term fertility decline [1] [2]. Consequently, enhancing soil water retention and nutrient efficiency has become central to sustainable agriculture. (Page 2, Line 66-70)

Explain in more detail the data on productivity, nutrient content, and fruit quality and correlate this information with the combined effect of biochar and chitosan xerogel.

Response: We thank the reviewer for this valuable suggestion. To clarify, the analysis of productivity, nutrient content, and fruit quality was conducted to assess the synergistic effects of biochar and chitosan xerogel on plant growth and soil nutrient dynamics. The combined effect of biochar and chitosan reported from the previous studies that significant yield gains under biochar-chitosan polymer composite amendments due to substantial slow-release properties to overcome excessive nutrient loss[3]. The nutrient retention behavior observed in our study corroborates previous work, which demonstrated that biochar-chitosan composites improved nutrient-use efficiency by up to 29% after 30 day under limited fertilizer input [4]. Fruit yield and quality increased significantly with increasing concentrations of chitosan and biochar [5]. This combined effect of biochar and chitosan offers a sustainable approach to improving crop resilience under limited water and nutrient availability, echoing the conceptual framework of sustainable agro-technologies [6]. (Page 3, Line 108-117)

c) Dear Dr. I-Chun Pan and other co-authors, what is the relevance of the combination of bioinputs for sustainable agriculture?

Response: We thank the reviewer for this valuable suggestion.The combination of bioinputs, such as biochar, chitosan, beneficial water and fertilizers efficiently, plays a vital role in advancing sustainable agriculture by improving soil health, enhancing nutrient-use efficiency, and reducing dependence on synthetic agrochemicals. The integration of bioinputs represents a sustainable agricultural strategy that simultaneously addresses soil degradation, water scarcity, and nutrient inefficiency while valorizing biowastes. This holistic approach supports both productivity and environmental protection, fulfilling the goals of climate-resilient and resource-efficient agriculture. (Page 16, Line 493-503)

Introduction

Dear authors, what is the role of biochar and chitosan in stress tolerance in vegetables? Does the region where the experiment is being conducted present challenges regarding vegetable production due to water stress?

Response: We thank the reviewer for this valuable suggestion. The application of biochar and chitosan xerogel demonstrated distinct properties in enhancing stress tolerance of water spinach under water and fertilizer deficiency. Biochar improved soil aeration and structure but showed limited benefit under severe stress, as excessive biochar (≥8%) failed to enhance plant growth or soil moisture retention. Similar findings have been reported by previous study [7], indicating that biochar’s positive effect diminishes when water or nutrient availability is critically. In contrast, chitosan xerogel at concentrations of 0.4–0.8% effectively alleviated growth inhibition, restoring plant biomass and morphological traits to near-control levels. This improvement can be attributed to chitosan’s osmotic regulation, nutrient chelation, and biostimulant properties, which enhance plant resilience under abiotic stress [8]. The study was conducted in Taiwan’s subtropical region, where vegetable cultivation frequently faces intermittent drought and uneven rainfall, leading to water and nutrient stress. The combined use of biochar and chitosan therefore presents a sustainable soil amendment strategy, improving stress tolerance and stabilizing yield performance under the climatic variability characteristic of subtropical agriculture. (Page 16, Line 476-490)

Dear authors, how did you choose the plant species used in the experiment? Was it based on its economic and nutritional importance?

Response: We appreciate the reviewer’s insightful question regarding the selection of the plant species. Water spinach was selected as the experimental material due to its significant eco-nomic importance and nutritional value across Taiwan and other tropical regions, cou-pled with its short growth cycle and pronounced sensitivity to soil water and nutrient conditions. (Page 3, Line 142-145)

c) What is the study hypothesis? You can include the hypothesis of the experiment at the end of the introduction. The questions posed above will help improve the article's introduction. Feel free to accept or reject the revision suggestions.

Response: We thank the reviewer for the valuable suggestion. In accordance with the comment, the study hypothesis has now been clearly stated at the end of the Introduction to strengthen the logical framework of the manuscript and clarify the expected outcomes of the experiment. We hypothesize that the combined application of biochar and chitosan-based xerogel can synergistically enhance soil water retention, nutrient availability, and crop productivity under conditions of water and nutrient stress. (Page 3, Line 123-125)

Materials and methods

Dear authors, please provide more details on the experimental design, specifying the number of treatments, replicates, and blocks. What statistical software was used for the analysis?

Response: We thank the reviewer for this valuable suggestion. The experiment followed a completely randomized design (CRD) to investigate the combined effects of water volume, fertilizer concentration, and different ratios of biochar and chitosan xerogel on the growth and yield of water spinach. The design comprised eight treatment groups (T1–T8), each addressing specific environmental or material variables (Table 1). The experiment was designed to evaluate the individual and combined effects of water management, fertilizer (Hakaphos Blue), biochar, and chi-tosan xerogel on the growth and stress tolerance of water spinach under controlled condi-tions. Each treatment was replicated three times, with five plants per replicate, to ensure statistical reliability. Water volume and fertilizer concentration were set based on prelim-inary field recommendations for leafy vegetables in subtropical greenhouses. Biochar and chitosan xerogel levels were adjusted to represent low, medium, and high application ratios (0–12 % for biochar and 0–0.8 % for xerogel). T1 and T2 focused on baseline optimiza-tion of water and fertilizer input. T3 and T4 examined biochar dosage effects under fixed irrigation levels. T5 and T6 tested different xerogel concentrations under variable fertilizer levels. T7 and T8 combined biochar and xerogel treatments to assess potential synergistic effects. (Page 5-6, line 190-209)

The experiment was arranged in a CRD. Statistical analysis was performed using one-way ANOVA with SAS software (α = 0.05). Mean separations were conducted using the least significant difference (LSD) test, and different letters indicate significant differences among treatments. The corresponding statistical procedure has now been clarified in the revised manuscript. (Page 7, Line 255-259)

Provide the physical and chemical characteristics of the biochar and chitosan used (sources and dosages).

Response: We thank the reviewer for this valuable suggestion. Mushroom waste bags were collected in Taichung, Taiwan, and then dispersed, ground and dried in an oven. Pellets was prepared using a small flat-die pelletizing machine (Panshen Industrial Co., Ltd.), producing samples with a diameter of 0.8 cm and a length of ca. 3-5 cm. (Page 5, Line 170-173)

Wood vinegar is obtained by heat-treated the condensed liquid of Taiwan acacia (Acacia confusa) wood, which is then allowed to settle for 1 year and the supernatant liquid is collected and was kindly provided by Kunn Yih Wood Corporation Co., Ltd. in Yilan, Taiwan. Using shrimp shells and crab shells as raw materials, chitosan (Deacetylation ≥80 %, ash content ca. 2%) were purchased from Charm & Beauty Co., Ltd., Taipei, Taiwan. (Page 5, Line 178-182)

What are the geographic coordinates and climatic conditions of the experimental site?

Response: We thank the reviewer for the inquiry regarding the experimental site’s geographic coordinates and climatic conditions. The experiment was conducted in the greenhouse facilities of National Chung Hsing University (NCHU), located in Taichung City, at geographic coordinates 24°7'13"N 120°40'35"E, with an elevation of ca. 70 m above sea level. The greenhouse conditions during the cultivation period (March 2020 to June 2021) were maintained within 20-30 °C temperature range, with 60-80% relative humidity, and controlled irrigation to simulate moderate water availability. These environmental parameters ensured consistent growth conditions for Ipomoea aquatica, minimizing the influence of external climatic variability while allowing for evaluation of soil amendment effects under semi-controlled subtropical conditions. The details have been added to the Materials and Methods section for clarification. (Page 7, Line 216-224)

d) What analytical methods were used (e.g., Kjeldahl method, spectrophotometry, etc.)? And how did this help in evaluating the plants' response to growth and quality parameters?

Response: We thank the reviewer for the valuable suggestion. Plant growth and physiological parameters were assessed using standard analytical techniques. Total nitrogen (N) content was determined by the Kjeldahl method [9], which accurately quantifies organic and ammoniacal nitrogen, reflecting nitrogen assimilation efficiency. Phosphorus (P) and potassium (K) concentrations were measured after acid digestion using UV-Vis spectrophotometry and flame photometry, respectively, following the protocol of Chapman and Pratt (1961)[10]. Chlorophyll a, chlorophyll b, and total chlorophyll were quantified spectrophotometrically according to Arnon (1949) to assess photosynthetic efficiency[11]. Soluble sugars were analyzed using the anthrone–sulfuric acid method [12], while total phenolic content was determined by the Folin-Ciocalteu assay [13], which estimates the antioxidant capacity and oxidative stress tolerance of plant tissues. The experiment was arranged in a CRD. Statistical analysis was performed using one-way ANOVA with SAS software (α = 0.05). (Page 7, Line 234-243)

Discussion

Dear Dr. I-Chun Pan and other co-authors, please relate the results obtained regarding plant physiological mechanisms (water retention, nutrient uptake) to similar studies in the scientific literature. This will help discuss possible interactions between biochar and chitosan in mitigating abiotic stresses. It is also important to discuss the effects on water and nutrient use efficiency.

Response: Thank you for the insightful feedback regarding the discussion of plant physiological mechanisms in our manuscript. The discussion has been rewritten by more explicitly relating the observed physiological mechanisms (water retention, nutrient uptake, stress responses) to scientific literature. (Page 14-15, Line 408-429)

What are the implications of your results for commercial production?

Response: We thank the reviewer for the valuable suggestion. These results suggest that the incorporation of appropriate amounts of biochar and chitosan xerogel into the soil during commercial production may achievement of comparable yields under reduced fertilizer application and water usage, thereby potentially leading to cost savings. (Page 16, line 493-496)

Conclusion

Dear authors, please indicate the specific conditions under which the combination of biochar and xerogel was most effective. This will help to focus on implications and recommendations for different species or sustainable agricultural environments.

Response: We appreciate the reviewer’s insightful question. This suggests a viable pathway for reducing reliance on synthetic fertilizers, thereby sup-porting more sustainable and cost-effective agricultural production. 3) Enhanced Environmental Resilience: The significant improvements in growth and physiological performance were maintained under both water-deficient and fertilizer-deficient conditions, even in a high water-retention substrate. These results highlight the potential of this integrated amendment to enhance resource use efficiency and plant resilience in suboptimal environments. 4) Implications for Sustainable Agriculture: The findings support the integration of chitosan xerogel and biochar as a sustainable, low-input strategy to enhance crop productivity and maintain yield stability. This strategy is particularly relevant for mitigating the effects of climate change and can be tailored for semi-arid regions or con-trolled-environment cultivation systems where maintaining optimal soil water and nutrient balance is critical. (Page 16-17, Line 505-525)

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is a research project aimed at determining the effect of certain elements such as biochar and chitosan xerogel on soil and crop improvement, including fertilizers and water. In this case, Ipomoea aquatica is used as a research model. This is a topic of utmost importance today, given the scarcity of water and the transformation of soil by fertilizers. The manuscript is well developed, with interesting results that could contribute positively to urban vegetable gardens. However, apart from containing some typos, the experimental design is very difficult to follow and sometimes appears to have redundancies, making some figures, such as Figure 1, seem redundant. Therefore, I must ask the authors to be more specific about their design and for Table 1 to be explained in more detail. The treatment repetitions are incomprehensible; the experimental design is not the typical one with four variables (fertilizers, water, biochar, and chitosan), four repetitions, and eight groups, etc.

I have many doubts about experimental design

Other minor concerns

Line 27: says …spent mushroom… Should read: …spent a mushroom

Line 98: Global warming, not just global warming but human demand on…

Line 105: says The authors have demonstrated expertise… with NO references. Without support, this statement becomes, pardon the word, hearsay. Please remove this statement or support it with some references that demonstrate the authors' authority.

Line 111: says …aim is… it is better to say …aim of this study is…

Line 117: which variety is your cultivar?

Line 118: What volume is the pot: 4 liters? The entire volume of the pot was occupied.

How long did the experiment last?

Table 1: This table needs to be explained in more detail, as the treatments can be confusing. For example, in group T1, there are four different water treatments, but not for group T2. It's also important to explain the basis for deciding on the Hakaphos Blue, biochar, and xerogel treatments.

Figure 1. When was the irrigation? Figure 1F.

All experiments in pots and greenhouses must be transferred to the field. It is imperative that the authors at least speculate about what would happen in the field with the addition of biochar and chitosan and this spinach. Nothing is said about this in the discussion section.

Author Response

Response

Detailed Response to Editor and Reviewer

Dear Reviewer,

This is a research project aimed at determining the effect of certain elements such as biochar and chitosan xerogel on soil and crop improvement, including fertilizers and water. In this case, Ipomoea aquatica is used as a research model. This is a topic of utmost importance today, given the scarcity of water and the transformation of soil by fertilizers. The manuscript is well developed, with interesting results that could contribute positively to urban vegetable gardens. However, apart from containing some typos, the experimental design is very difficult to follow and sometimes appears to have redundancies, making some figures, such as Figure 1, seem redundant. Therefore, I must ask the authors to be more specific about their design and for Table 1 to be explained in more detail. The treatment repetitions are incomprehensible; the experimental design is not the typical one with four variables (fertilizers, water, biochar, and chitosan), four repetitions, and eight groups, etc.

Response: We sincerely appreciate the reviewer’s observation regarding the complexity of the experimental design and have clarified the structure, rationale, and replication scheme in the revised Materials and Methods section. Table 1 has been expanded with explanatory notes for better comprehension.

The experiment followed a completely randomized design (CRD) to investigate the combined effects of water volume, fertilizer concentration, and different ratios of biochar and chitosan xerogel on the growth and yield of Ipomoea aquatica (water spinach). The design comprised eight treatment groups (T1–T8), each addressing specific environmental or material variables (Table 1). The experiment was designed to evaluate the individual and combined effects of water management, fertilizer (Hakaphos Blue), biochar, and chitosan xerogel on the growth and stress tolerance of water spinach under controlled conditions. Each treatment was replicated three times, with five plants per replicate, to ensure statistical reliability. Water volume and fertilizer concentration were set based on preliminary field recommendations for leafy vegetables in subtropical greenhouses. Biochar and chitosan xerogel levels were adjusted to represent low, medium, and high application ratios (0–12 % for biochar and 0–0.8 % for xerogel). T1 and T2 focused on baseline optimization of water and fertilizer input. T3 and T4 examined biochar dosage effects under fixed irrigation levels. T5 and T6 tested different xerogel concentrations under variable fertilizer levels. T7 and T8 combined biochar and xerogel treatments to assess potential synergistic effects. Plant height, leaf number, chlorophyll content, and fresh weight were recorded when the control plants reached 30–40 cm. This design allowed the evaluation of both individual and interactive effects of water, nutrients, and bioinputs, enabling identification of the most effective combination (Notably T7-5 and T8-5, with 4 % biochar and 0.4 % xerogel) for improving soil water retention, nutrient efficiency, and plant growth performance. Revised Table Note: Table 1 summarizes the factorial combinations of water, fertilizer, biochar, and xerogel levels. Each treatment code (e.g., T3-3 or T7-5) represents a unique substrate mixture replicated three times. The ranges were chosen to simulate typical subtropical vegetable cultivation scenarios and to identify optimal bioinput ratios for sustainable production. (Page 5-6, Line 190-209)

I have many doubts about experimental design

Other minor concerns

Response: We sincerely appreciate the reviewer’s observation. Please refer the repose as follow:

Line 27: says …spent mushroom… Should read: …spent a mushroom.

Response: This word has been modified.

Line 98: Global warming, not just global warming but human demand on…

Response: The “human demand” has been added. (Page 3, Line 118)

Response: This sentence has been deleted.

Line 111: says …aim is… it is better to say …aim of this study is…

Response: The “study” has been added. (Page 3, Line 133)

Line 117: which variety is your cultivar?

Response: The test plant used in this study was Ipomoea aquatica Forsk. cv. Chungu Daye, a water spinach cultivar widely grown in Taiwan. (Page 5, Line 160-161)

Line 118: What volume is the pot: 4 liters? The entire volume of the pot was occupied.

Response: Yes. The pots used in this experiment measured 20 × 20 × 10 cm³. Each square pot has a volume of approximately 4 L, and the soil–substrate mixture completely filled the pot. (Page 5, Line 164-165)

How long did the experiment last?

Response: The experiment was conducted over a one-year cultivation period. The greenhouse conditions during the cultivation period (March 2020 to June 2021) were maintained within 20-30 °C temperature range, with 60-80% relative humidity, and controlled irrigation to simulate moderate water availability. (Page 7, Line 218-221)

Response: We appreciate the reviewer’s comment regarding the clarification of the treatment groups. The experiment followed a completely randomized design (CRD) to investigate the combined effects of water volume, fertilizer concentration, and different ratios of biochar and chitosan xerogel on the growth and yield of water spinach. The design comprised eight treatment groups (T1–T8), each addressing specific environmental or material variables (Table 1). The experiment was designed to evaluate the individual and combined effects of water management, fertilizer (Hakaphos Blue), biochar, and chitosan xerogel on the growth and stress tolerance of water spinach under controlled conditions. Each treatment was replicated three times, with five plants per replicate, to ensure statistical reliability. Water volume and fertilizer concentration were set based on preliminary field recommendations for leafy vegetables in subtropical greenhouses. Biochar and chitosan xerogel levels were adjusted to represent low, medium, and high application ratios (0–12 % for biochar and 0–0.8 % for xerogel). T1 and T2 focused on baseline optimization of water and fertilizer input. T3 and T4 examined biochar dosage effects under fixed irrigation levels. T5 and T6 tested different xerogel concentrations under variable fertilizer levels. T7 and T8 combined biochar and xerogel treatments to assess potential synergistic effects. Plant height, leaf number, chlorophyll content, and fresh weight were recorded when the control plants reached 30–40 cm. This design allowed the evaluation of both individual and interactive effects of water, nutrients, and bioinputs, enabling identification of the most effective combination (notably T7-5 and T8-5, with 4 % biochar and 0.4 % xerogel) for improving soil water retention, nutrient efficiency, and plant growth performance. (Page 5-6, Line 190-209)

Figure 1. When was the irrigation? Figure 1F.

Response: We appreciate the reviewer’s insightful comment. Future research will extend this study from controlled greenhouse conditions to open-field environments to validate the long-term agronomic and ecological performance of the biochar–chitosan system. In field applications, biochar (4%) is expected to enhance soil water retention, aeration, and nutrient holding capacity, while chitosan xerogel (0.4–0.8%) may act as a biodegradable moisture regulator that improves microbial activity and mitigates drought-induced stress. These synergistic effects are anticipated to stabilize vegetable yield and improve soil fertility under subtropical climatic variability. The next research phase will include multi-season field trials to evaluate productivity, soil health, and carbon sequestration potential. (Page 16-17, Line 504-512)

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Recommendations to authors

The manuscript presents an interesting study on the combined application of biochar derived from spent mushroom substrate and chitosan xerogel in enhancing crop performance. However, several sections require clarification and additional methodological and interpretative details to strengthen the paper’s scientific rigor and clarity. The following recommendations are provided to improve the manuscript.

Line 27: Regarding “4% biochar derived from spent mushroom substrate and 0.4–0.8% chitosan xerogel”: I suggest specifying the optimal dose earlier. Since the conclusion (Line 30) states that the combined application used the 0.8% dose (4% biochar + 0.8% chitosan xerogel), mentioning the full range (0.4–0.8%) in the methods section (Line 27) can be slightly confusing. You could specify the optimal dose directly in the methods or clarify that the most effective dose was used in the combined treatment.
Line 31: Regarding “significantly increased fresh biomass”: It is preferable to quantify the improvement. Instead of simply stating “significantly increased,” indicate the magnitude of the effect — for example, by how much the combined application improved the main metric (e.g., fresh biomass) compared to the control or single treatments. Example: “…significantly increased fresh biomass by X% (or X-fold), leaf weight, and stem diameter…”.
Line 111: The only stated goal is: “The aim is to enhance the yield and quality of agricultural and horticultural crops effectively, while simultaneously addressing the challenges associated with agricultural waste management.” This is overly broad. The study’s specific aim (e.g., “to evaluate the synergistic effect of a novel biochar/chitosan xerogel combination on water spinach under combined water and fertilizer stress”) should be clearly defined, ideally beginning with: “Therefore, the aim of this study was to…”.
Line 113: The objectives are missing and should be explicitly added. For example, consider adding the following as objectives: 1) To determine the effects of the combined amendment on the biomass and yield of water spinach; 2) To assess the impact of the amendment on the plant’s nutrient uptake (N, P, K, etc.); 3) To compare the performance of the combined treatment with single treatments and optimal growth conditions; 4) To highlight the importance of the combined application of biochar derived from spent mushroom substrate and chitosan xerogel in enhancing crop performance.
Line 115: It would be helpful to include a paragraph describing the methodological framework or experimental strategy followed.
Line 146: Add appropriate references to the statement “Plant growth parameters (PGPs) were assessed using standardized methods.”
Line 150: Some methodological details are missing in this paragraph. For instance, specify which instruments or devices were used to measure leaf weight or plant height, and indicate whether measurements were performed by a single individual or a team.
Line 165: There is no mention of checking whether the data followed a normal distribution or whether the assumptions required for ANOVA were met. Please verify and report this before using ANOVA.
Line 364: The conclusions should be organized into a cohesive list that directly corresponds to the study’s objectives and highlights their broader implications.

Author Response

Response

Detailed Response to Editor and Reviewer

Dear Reviewer,

Line 27: Regarding “4% biochar derived from spent mushroom substrate and 0.4–0.8% chitosan xerogel”: I suggest specifying the optimal dose earlier. Since the conclusion (Line 30) states that the combined application used the 0.8% dose (4% biochar + 0.8% chitosan xerogel), mentioning the full range (0.4–0.8%) in the methods section (Line 27) can be slightly confusing. You could specify the optimal dose directly in the methods or clarify that the most effective dose was used in the combined treatment.

Response: We thank the reviewer for the valuable suggestion. Line 27: "4% biochar derived from spent mushroom substrate and 0.4%−0.8% chitosan xerogel" was deleted and replaced with the following text to specify the optimal dosage earlier: " Individually, adding biochar or chitosan xerogel in a peat-based substrate improved certain growth parameters but showed limited benefits under resource-deficient conditions. In contrast, the combined application of 4% biochar and 0.8% chitosan xerogel resulted in a significant restoration of growth under both resource-deficient conditions." (Page 1, Line 27-30) The misleading dosage description in the Results and Conclusion sections has also been deleted. The method of using the doses of biochar and chitosan xerogel is now explained in the Materials and Methods section: "Biochar and chitosan xerogel levels were adjusted to represent low, medium, and high application ratios (0−12% for biochar and 0−0.8% for xerogel)." (Page 5, Line 199-201)

Line 31: Regarding “significantly increased fresh biomass”: It is preferable to quantify the improvement. Instead of simply stating “significantly increased,” indicate the magnitude of the effect — for example, by how much the combined application improved the main metric (e.g., fresh biomass) compared to the control or single treatments. Example: “…significantly increased fresh biomass by X% (or X-fold), leaf weight, and stem diameter…”.

Response: We thank the reviewer for the valuable suggestion. We have rewritten the text, with an emphasis on the specific effects of the combined application of 4% biochar and 0.8% chitosan xerogel on water spinach under water- and fertilizer-deficient conditions as fellow: “The combined application of 4% biochar and 0.8% chitosan xerogel resulted in a significant restoration of growth under both resource-deficient conditions. Specifically, in water-deficient conditions, the treatment increased fresh weight by 1.2-fold, stem weight by 1.3-fold, leaf weight by 1.7-fold, plant height by 1.2-fold, and stem diameter by 1.3-fold. Similarly, under fertilizer-deficient conditions, the treatment increased fresh weight by 1.3-fold, stem weight by 2.0-fold, leaf weight by 1.4-fold, plant height by 1.3-fold, and stem diameter by 1.4-fold. Furthermore, this dual-amendment application restored both chlorophyll and β-carotene contents, showing 1.4-fold and 1.3fold increases under water deficiency, and 1.4-fold and 1.2-fold increases under fertilizer deficiency, respectively.” (Page 1, Line 27-38)

Line 111: The only stated goal is: “The aim is to enhance the yield and quality of agricultural and horticultural crops effectively, while simultaneously addressing the challenges associated with agricultural waste management.” This is overly broad. The study’s specific aim (e.g., “to evaluate the synergistic effect of a novel biochar/chitosan xerogel combination on water spinach under combined water and fertilizer stress”) should be clearly defined, ideally beginning with: “Therefore, the aim of this study was to…”.

Response: We thank the reviewer for the valuable suggestion. Line 139, the research aim was be clearly defined as “The of this study was to evaluate the synergistic effect of combining biochar and chitosan xerogel on water spinach under water or fertilizer stress.” (Page 3, Line 133-134)

Line 113: The objectives are missing and should be explicitly added. For example, consider adding the following as objectives: 1) To determine the effects of the combined amendment on the biomass and yield of water spinach; 2) To assess the impact of the amendment on the plant’s nutrient uptake (N, P, K, etc.); 3) To compare the performance of the combined treatment with single treatments and optimal growth conditions; 4) To highlight the importance of the combined application of biochar derived from spent mushroom substrate and chitosan xerogel in enhancing crop performance.

Response: We thank the reviewer for the valuable suggestion. The objectives were modified as “To achieve this, the research sought to determine the positive impact of this dual amendment on fresh biomass and yield, while simultaneously assessing the corresponding improvements in nutrient uptake. By comparing the combined treatment's efficacy against single amendments and optimal growth conditions, the study aimed to confirm the synergistic benefit and highlight its practical importance as a sustainable strategy for enhancing crop performance while potentially reducing resource inputs. Collectively, this research sought to maximize plant vigor and nutrient efficiency in challenging agricultural settings.” (Page 3, Line 134-141)

Line 115: It would be helpful to include a paragraph describing the methodological framework or experimental strategy followed.

Response: We thank the reviewer for the valuable suggestion. The paragraph to describe the methodological framework were added. “Water spinach was selected as the experimental material due to its significant economic importance and nutritional value across Taiwan and other tropical regions, coupled with its short growth cycle and pronounced sensitivity to soil water and nutrient conditions. The experimental strategy was designed to systematically evaluate the effects of the amendments under defined stress levels. Initially, a quantitative number of plants were grown in a fixed volume of a peat-based substrate. Critical stress thresholds for water and fertilizer deficiency were established by treating the plants with varying irrigation volumes or nutrient concentrations to determine the point of impaired growth. Subsequently, the main experiment involved amending the substrate with biochar and chitosan xerogel, both individually and in combination, using defined dose ratios. Key plant growth parameters (PGPs), including fresh weight, stem weight, leaf weight, plant height, and stem diameter, were measured. Furthermore, plant physiological responses were assessed by quantifying leaf chlorophyll content and β-carotene content, while nutrient uptake was determined through elemental analysis of plant tissues. This framework allowed for a comprehensive understanding of how the combined amend-ments impact plant vigor, physiology, and nutrient acquisition under distinct water- or fertilizer-deficient states.” (Page 3-4, Line 145-157)

Line 146: Add appropriate references to the statement “Plant growth parameters (PGPs) were assessed using standardized methods.”

Response: We thank the reviewer for the valuable suggestion. The reference of the quantitative analysis of plant growth were cited [1]. (Page 7, Line 224)

Line 150: Some methodological details are missing in this paragraph. For instance, specify which instruments or devices were used to measure leaf weight or plant height, and indicate whether measurements were performed by a single individual or a team.

Response: We thank the reviewer for the valuable suggestion. The Stem diameters were measure by Vernier caliper. Fresh weight, stem weight, and leaf weight were determined using an electronic balance. All measurements were performed by a single person. (Page 7, Line 230-233)

Line 165: There is no mention of checking whether the data followed a normal distribution or whether the assumptions required for ANOVA were met. Please verify and report this before using ANOVA.

Response: We thank the reviewer for the valuable suggestion. The experiment was arranged in a CRD. Statistical analysis was performed using one-way ANOVA with SAS software (α = 0.05). Mean separa-tions were conducted using the least significant difference (LSD) test, and different letters indicate significant differences among treatments. The corresponding statistical procedure has now been clarified in the revised manuscript. (Page 7, Line 255-259)

Line 364: The conclusions should be organized into a cohesive list that directly corresponds to the study’s objectives and highlights their broader implications.

Response: We thank the reviewer for the valuable suggestion. The conclusion was rewrite and highlights our research objective, result and implications. (Page 17, Line 514-534)

Evans, G.C. The quantitative analysis of plant growth; Univ of California Press: 1972; Volume 1.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

After reviewing this second revision of this manuscript, it can be seen that it has been significantly improved. Furthermore, the methodology has been revised and explained in greater detail. Therefore, I have no objection to this manuscript being published.

Author Response

Detailed Response to Reviewer

Dear Reviewer,

Enclosed please find the referenced manuscript (horticulturae-3913786). Our responses and the revised parts in the manuscript were described as followed:

Response: We sincerely appreciate the reviewer’s positive evaluation and recognition of our revision efforts.

Reviewer 3 Report

Comments and Suggestions for Authors

Recommendations to authors

Lines 31 – 37: This section has been improved but now it contains excessive detail, resulting in an abstract that exceeds the journal's 200-word limit.
Line 165 (Statistical Analysis): The statistical analysis remains insufficient. Specifically, there is no mention of checking data for normal distribution or confirming that the necessary assumptions for ANOVA were met. Please verify and report these diagnostic checks before utilizing the ANOVA test.

Author Response

Detailed Response to Reviewer

Dear Reviewer,

Attached please find the manuscript entitled “Improving Sustainable Vegetable Production with Biochar and Chitosan Xerogel Combination under Water and Fertilizer Stress”, by I-Chun Pan, Chen-An Jiang, Wan-Yi Chiou * , Yi-Chun Chen *.

Lines 31 – 37: This section has been improved but now it contains excessive detail, resulting in an abstract that exceeds the journal's 200-word limit.

Response: Thank you for your constructive feedback. We acknowledge that although the abstract has been improved in structure, it currently contains excessive details and exceeds the journal’s 200-word limit. The revised abstract has been be strictly adjusted to comply with the 200-word requirement and will be reflected in the updated manuscript.

Global warming has intensified water scarcity, while excessive fertilizer use has caused soil acidification and limited nutrient availability. This study investigated the effects of biochar and chitosan xerogel on water spinach (Ipomoea aquatica Forsk.) growth under water- and fertilizer-deficient conditions. Individually, either biochar or chitosan xerogel provided limited improvement. However, the combined application of 4% biochar and 0.8% chitosan xerogel significantly restored plant performance. Under water deficiency, fresh, stem, and leaf weights increased by 1.2-, 1.3-, and 1.7-fold, while plant height and stem diameter rose by 1.2- and 1.3-fold. Similar improvements were observed under fertilizer deficiency, with up to 1.3-fold, 2.0-fold, and 1.4-fold increases in fresh, stem and leaf weight. Chlorophyll and β-carotene contents were also enhanced under both stress conditions. Additionally, the dual amendment improved uptake of nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), and manganese (Mn), achieving growth comparable to optimal irrigation and fertilization. These findings demonstrate the synergistic potential of biochar and chitosan xerogel to enhance water and nutrient efficiency, supporting sustainable agriculture under resource limitations. (Page 1, Line 23-37)

Line 165 (Statistical Analysis): The statistical analysis remains insufficient. Specifically, there is no mention of checking data for normal distribution or confirming that the necessary assumptions for ANOVA were met. Please verify and report these diagnostic checks before utilizing the ANOVA test.

Response: We appreciate the reviewer’s concern. In this study, the data showed consistently low standard deviations across treatments, indicating limited variability. Under such conditions, ANOVA is considered robust even with slight deviations from normality (Blanca et al., 2017; Glass et al., 1972). Data distribution was visually inspected through histograms and residual plots, and no obvious skewness or variance inconsistency was observed; thus, the assumptions of ANOVA were reasonably satisfied. This clarification has now been added to the “Statistical Analysis” section, as follows:

All experimental data were subjected to one-way analysis of variance (ANOVA) using SAS software at a significance level of α = 0.05. Treatment means were compared using the least significant difference (LSD) test. Prior to ANOVA, data variability was examined, and the consistently low standard deviations across treatments indicated limited dispersion. Data normality and homogeneity of variance were visually assessed using histogram and residual plot inspections, which showed no obvious skewness or inconsistent variance patterns. Under these conditions, ANOVA is considered robust and appropriate for mean comparison in agricultural experiments. (Page 7, Line 248-256)