the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 06 Nov 2025
Comparative Impact of Bio-Organic and Inorganic Fertilizer Application on Soil Health, Grain Quality and Yield Stability in Nutrient Deficient Regions
Abstract. Soil fertility limitations in arid regions restrict wheat productivity and grain nutritional quality, with zinc (Zn) deficiency being a major concern. Sustainable soil amendments combining organic and microbial inputs offer potential to address these constraints. This study aimed to evaluate the effectiveness of bio-organic fertilization in enhancing wheat growth, yield, grain Zn biofortification, and soil fertility under deficient arid field conditions. Two field trials were conducted in Bahawalpur and Bahawalnagar, Pakistan, using a randomized complete block design. Treatments included compost, ZnO (2 %), ZnSO4, zinc-solubilizing bacteria (ZSB), and their combinations. Wheat growth, yield, grain nutrient concentrations, and soil fertility indicators (organic matter, microbial biomass nitrogen (MBN), microbial biomass carbon (MBC), and nutrient availability) were measured. Microbial populations were determined through colony-forming units. Correlation and principal component analysis (PCA) were applied to explore associations among variables. The integrated application of compost + ZnO + ZSB significantly improved wheat height (19 %), biomass (20 %), yield attributes (10 %), and grain Zn concentration (39 %) compared with the control. Soil fertility parameters also increased (organic matter, 39 %; MBN, 32 %; MBC, 27 %). Correlation and PCA highlighted strong positive relationships among microbial populations, soil fertility, and crop performance. Bio-organic fertilization provides an eco-friendly and effective strategy to improve wheat yield, Zn biofortification, and soil fertility in arid agroecosystems.
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CC1: 'Comment on egusphere-2025-4852', Usman Jamshaid, 17 Nov 2025
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AC1: 'Reply on CC1', azhar Hussain, 18 Nov 2025
We sincerely thank the professor/scientist/researcher for this encouraging remark. We appreciate the positive evaluation and remain confident that the insights presented in this study will contribute meaningfully to ongoing efforts in sustainable soil management and climate-resilient agriculture.
Citation: https://doi.org/10.5194/egusphere-2025-4852-AC1
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AC1: 'Reply on CC1', azhar Hussain, 18 Nov 2025
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RC1: 'Comment on egusphere-2025-4852', Rubab Sarfraz, 21 Nov 2025
Please elaborate how PCA in this study predicts the relationship between crop performance and microbial population?
Why significance level upto 0.0001 has been described?
How LSD was calculated? Is it for overall treatment effect ? Why it is significant to calculate here?
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AC2: 'Reply on RC1', azhar Hussain, 26 Nov 2025
Response 1: We thank the reviewer for this suggestion. Principal Component Analysis (PCA) in our study is used as an exploratory multivariate tool to summarize covariation among many soil, microbial and plant variables and to visualize which variables move together (i.e., covary). PCA does not perform causal prediction in the strict sense, but it identifies the main axes (principal components) that explain the majority of variance in the dataset. In our results, PC-1 explained the large majority of variance (92.6% in Trial I, 91.5% in Trial II) and variables such as organic matter (OM), total organic carbon (TOC), nitrogen (N), phosphorus (P), microbial biomass carbon (MBC), microbial biomass nitrogen (MBN) and colony forming unit (CFU) had strong positive loadings on PC-1. Treatments with high positive PC-1 scores (e.g., Compost + 2% ZnO + ZSB) therefore cluster together with higher microbial indicators and better crop performance, demonstrating a strong co-variation between microbial population/biomass and crop performance. In short, PCA shows that microbial population and crop performance co-vary along the same major gradient (PC-1), supporting the interpretation that improved microbial activity and soil fertility are associated with improved crop growth and yield in our trials.
Response 2:
Thank you, The manuscript used conventional significance notation (asterisks) to indicate ranges of p-values; the table legend defined **** as p < 0.0001, while the global decision threshold for tests was α = 0.05. The appearance of **** (p < 0.0001) simply reflects that some comparisons produced very small p-values (highly significant differences) when evaluated by ANOVA / post-hoc tests in Statistix / OriginPro. We will clarify the legend and methods, so readers are not confused: we will keep α = 0.05 as the threshold for declaring significance but will report p-value ranges using the asterisk scheme.
Response 3:
The ANOVA F-test was used to evaluate the overall treatment effect (i.e., whether any treatment means differ). Where the ANOVA F-test was significant, we used the Least Significant Difference (LSD) procedure to perform pairwise comparisons between treatment means. LSD was calculated from the ANOVA residual mean square (MSE) and the number of replicates according to the standard formula:
LSDα=tα/2, df error × √2 × MSE/r
where tα/2, df error is the t-value at the chosen α (0.05) and the residual degrees of freedom, MSE is the mean square error from the ANOVA, and r is the number of replicates (here r = 3). The LSD values reported in Table 1 are the LSD at p ≤ 0.05 computed from ANOVA outputs in Statistix 8.1. LSD is appropriate here because the experiment used an RCBD with balanced replications and our objective was to identify which specific treatments differed (pairwise) after a significant overall ANOVA.
Citation: https://doi.org/10.5194/egusphere-2025-4852-AC2
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AC2: 'Reply on RC1', azhar Hussain, 26 Nov 2025
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CC2: 'Comment on egusphere-2025-4852', Atta Mohi Ud Din, 04 Dec 2025
A good study linking microbial activity with Zn biofortification and yield stability. Future work may explore scalability, cost–benefit analysis, and microbial persistence under variable arid climate condition.
Citation: https://doi.org/10.5194/egusphere-2025-4852-CC2 -
AC3: 'Reply on CC2', azhar Hussain, 05 Dec 2025
Thank you very much for your thoughtful and encouraging feedback on our work. We sincerely appreciate your recognition of the study's contribution in linking microbial activity with Zn biofortification and yield stability.
We fully agree that future research should address the broader applicability of these findings. It is worth mentioning that our current study already includes an economic analysis based on the data generated at our research sites, providing initial insights into the cost-effectiveness of the applied biofertilization approach. Building on this, our future work will further expand the assessment by evaluating scalability under diverse agroecological conditions and conducting a more comprehensive cost–benefit analysis across multiple regions.
Additionally, we acknowledge the importance of investigating microbial persistence and functional stability under variable arid and semi-arid climate conditions. These aspects are crucial for ensuring long-term adoption and real-world impact, and we plan to explore them in the next phase of our research.
Thank you once again for your valuable comments, which help strengthen the direction and applicability of our future work.
Citation: https://doi.org/10.5194/egusphere-2025-4852-AC3
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AC3: 'Reply on CC2', azhar Hussain, 05 Dec 2025
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CC3: 'Comment on egusphere-2025-4852', Sajad Hussain, 05 Dec 2025
Overall authors have addressed one of the major issues regarding soil fertility and provided suitable and affortable migitation strategy.
I would like to suggest some additions in the introduction section such as
Add 1–2 sentences to link the severity of Zn deficiency with why conventional fertilization is insufficient.
Briefly mention why Zn is crucial for wheat physiology (enzyme activation, antioxidant defense, reproductive growth) and add a sentence explaining why microbial inoculants + Zn enrichment work synergistically.
Citation: https://doi.org/10.5194/egusphere-2025-4852-CC3 -
AC4: 'Reply on CC3', azhar Hussain, 10 Dec 2025
Dear Professor/Researcher
Thank you very much for your constructive comments and for acknowledging the significance of our work in addressing soil fertility challenges through affordable and sustainable mitigation strategies.
We appreciate your valuable suggestions regarding the Introduction section. We agree that adding contextual information on the severity of zinc (Zn) deficiency and the limitations of conventional fertilization will improve the clarity and scientific depth of the manuscript. We will also incorporate brief statements highlighting the essential roles of Zn in wheat physiology—particularly its involvement in enzyme activation, antioxidant defense, and reproductive growth.
Furthermore, we acknowledge your important point about explaining the synergistic mechanism of microbial inoculants combined with Zn enrichment. We will integrate 1–2 sentences to clearly describe how microbial activity enhances Zn availability and uptake, thereby complementing Zn-enriched amendments.
All these suggestions will be carefully incorporated into the final revised version of the manuscript.
Thank you once again for your thoughtful feedback.
Citation: https://doi.org/10.5194/egusphere-2025-4852-AC4
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AC4: 'Reply on CC3', azhar Hussain, 10 Dec 2025
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RC2: 'Comment on egusphere-2025-4852', Anonymous Referee #2, 21 Dec 2025
Hussain et al. investigated the impacts of bio-organic and inorganic fertilizer application on soil health, grain quality, and yield stability in nutrient-deficient regions. The results showed that the integrated application of compost + ZnO + ZSB significantly improved wheat height, biomass, yield attributes, and grain Zn concentration. The study is well-designed, and the results are beneficial for soil improvement in arid and nutrient-deficient regions. I have several suggestions that may improve the manuscript:
- In the Introduction, knowledge gaps and the scientific importance of this study should be clearly presented.
- The results should be deeply discussed based on the published literature.
- The tables and figures should be carefully prepared; for example, in Figure 3, the bars are not clearly displayed.
- A key question is that a similar paper was published (Naeem, M., Iqbal, Z., Hussain, A., Jamil, M., Ahmad, H. T., Ismail, A. M., El-Mogy, M., El Ganainy, S., El-Beltagi, H., & Hadid, M. L. (2025). Upregulate Soil Health and Wheat Yield: Conversion of Organic Waste into Bio-activated Zn-Enriched Compost for Deficient Soils. Global NEST Journal, 27(7), 1-13.), which should be fully explained and discussed in the manuscript.
- Please carefully check the reference format.
- The manuscript still needs to be carefully checked to enhance its rigor, logic, and readability.
Citation: https://doi.org/10.5194/egusphere-2025-4852-RC2 -
AC5: 'Reply on RC2', azhar Hussain, 24 Dec 2025
We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments that have helped us improve its scientific rigor, clarity, and presentation. All suggestions have been carefully addressed, and the manuscript has been revised accordingly. Detailed responses are provided below.
Comment 1: In the Introduction, knowledge gaps and the scientific importance of this study should be clearly presented.
Response: Line 78 – 86; We appreciate this valuable suggestion. The Introduction has been revised to more clearly articulate the existing knowledge gaps and highlight the scientific novelty and importance of the present study. Specifically, we clarified that although Zn-enriched compost and microbial inoculants have been studied individually, field-scale validation of integrated bio-organic Zn fertilization under real, farmer-managed arid conditions remains limited. We also emphasized the lack of comparative assessments across multiple locations and the need to link soil biological indicators with yield stability and grain biofortification. These aspects establish the scientific importance of the study in advancing sustainable Zn management strategies for nutrient-deficient arid regions.
Comment 2: The results should be deeply discussed based on the published literature.
Response: Line 452 – 469; 499-511; We agree with the reviewer and have substantially strengthened the Discussion section. The revised Discussion now provides mechanistic explanations supported by recent and relevant literature, linking microbial activity, Zn solubilization, nutrient cycling, soil biochemical changes, and plant physiological responses. Redundancy with the Results section has been avoided, and emphasis has been placed on interpreting why integrated treatments performed better rather than restating numerical values.
Comment 3: The tables and figures should be carefully prepared; for example, in Figure 3, the bars are not clearly displayed.”
Response: Thank you for highlighting this issue. Figure 3 has been redrawn with improved graphical clarity. All figures and tables have been carefully rechecked to ensure readability, consistency, and proper labeling.
Comment 4: A key question is that a similar paper was published (Naeem et al., 2025), which should be fully explained and discussed in the manuscript.
Response: Line 87 - 99; We appreciate this important observation. A clear distinction between the previously published study (Naeem et al., 2025) and the present work has now been explicitly stated in the Introduction and reinforced in the Discussion. While the earlier study focused on development and validation of bio-activated Zn-enriched compost at a single research site, the present study advances this work by:
- Conducting multi-location field trials under farmer-managed conditions
- Integrating ZnO + ZSB + compost rather than compost alone
- Linking soil biological indicators (CFU, MBC, MBN) with yield stability and economic returns
- Demonstrating scalability and adaptability across variable arid environments
Comment 5: Please carefully check the reference format.”
Response: The reference list has been thoroughly revised to ensure consistency with the journal’s formatting guidelines. Author names, journal titles, volume/issue numbers, page ranges, and publication years have been standardized, and minor typographical errors have been corrected.
Comment 6: The manuscript still needs to be carefully checked to enhance its rigor, logic, and readability.
Response: We appreciate this general but important comment. The manuscript has undergone comprehensive language editing to improve clarity and rigor.
Citation: https://doi.org/10.5194/egusphere-2025-4852-AC5
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AC6: 'Comment on egusphere-2025-4852', azhar Hussain, 24 Dec 2025
December 24, 2025
Prof. Dr. Maria Jesus Gutierrez Gines
Editor,
Soil
Manuscript ID: EGUSPHERE-2025-4852
Subject: Manuscript resubmission — disclosure of related publication and revised manuscript (tracked changes attached)
Dear Prof. Maria,
On behalf of all authors, I am pleased to submit the revised version of our manuscript entitled: “Comparative Impact of Bio-Organic and Inorganic Fertilizer Application on Soil Health, Grain Quality and Yield Stability in Nutrient Deficient Regions”, Manuscript ID: EGUSPHERE-2025-4852.
We sincerely thank you and the reviewers for the constructive and insightful comments, which have greatly helped us to improve the quality, clarity, and scientific rigor of our manuscript.
In the revised version, we have carefully addressed all reviewers’ comments point by point. The revisions include improvements in the Results and Discussion sections, clarification of the experimental rationale, enhanced interpretation of findings, and language polishing throughout the manuscript. All changes made in response to the reviewers’ suggestions have been clearly highlighted in the revised manuscript, and a detailed response to reviewers' comments has been provided for your convenience.
Thank you very much for your time and consideration. We look forward to your favorable response.
Following is the point-by-point response to the reviewers' comments.
Reviewer 1:
We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments that have helped us improve its scientific rigor, clarity, and presentation. All suggestions have been carefully addressed, and the manuscript has been revised accordingly. Detailed responses are provided below.
Comment 1: In the Introduction, knowledge gaps and the scientific importance of this study should be clearly presented.
Response: Line 78 – 86; We appreciate this valuable suggestion. The Introduction has been revised to more clearly articulate the existing knowledge gaps and highlight the scientific novelty and importance of the present study. Specifically, we clarified that although Zn-enriched compost and microbial inoculants have been studied individually, field-scale validation of integrated bio-organic Zn fertilization under real, farmer-managed arid conditions remains limited. We also emphasized the lack of comparative assessments across multiple locations and the need to link soil biological indicators with yield stability and grain biofortification. These aspects establish the scientific importance of the study in advancing sustainable Zn management strategies for nutrient-deficient arid regions.
Comment 2: The results should be deeply discussed based on the published literature.
Response: Line 452 – 469; 499-511; We agree with the reviewer and have substantially strengthened the Discussion section. The revised Discussion now provides mechanistic explanations supported by recent and relevant literature, linking microbial activity, Zn solubilization, nutrient cycling, soil biochemical changes, and plant physiological responses. Redundancy with the Results section has been avoided, and emphasis has been placed on interpreting why integrated treatments performed better rather than restating numerical values.
Comment 3: The tables and figures should be carefully prepared; for example, in Figure 3, the bars are not clearly displayed.”
Response: Thank you for highlighting this issue. Figure 3 has been redrawn with improved graphical clarity. All figures and tables have been carefully rechecked to ensure readability, consistency, and proper labeling.
Comment 4: A key question is that a similar paper was published (Naeem et al., 2025), which should be fully explained and discussed in the manuscript.
Response: Line 87 - 99; We appreciate this important observation. A clear distinction between the previously published study (Naeem et al., 2025) and the present work has now been explicitly stated in the Introduction and reinforced in the Discussion. While the earlier study focused on development and validation of bio-activated Zn-enriched compost at a single research site, the present study advances this work by:
- Conducting multi-location field trials under farmer-managed conditions
- Integrating ZnO + ZSB + compost rather than compost alone
- Linking soil biological indicators (CFU, MBC, MBN) with yield stability and economic returns
- Demonstrating scalability and adaptability across variable arid environments
Comment 5: Please carefully check the reference format.”
Response: The reference list has been thoroughly revised to ensure consistency with the journal’s formatting guidelines. Author names, journal titles, volume/issue numbers, page ranges, and publication years have been standardized, and minor typographical errors have been corrected.
Comment 6: The manuscript still needs to be carefully checked to enhance its rigor, logic, and readability.
Response: We appreciate this general but important comment. The manuscript has undergone comprehensive language editing to improve clarity and rigor.
Reviewer 2:
Comment 1: Please elaborate on how PCA in this study predicts the relationship between crop performance and microbial population?
Response: We thank the reviewer for this suggestion. Principal Component Analysis (PCA) in our study is used as an exploratory multivariate tool to summarize covariation among many soil, microbial and plant variables and to visualize which variables move together (i.e., covary). PCA does not perform causal prediction in the strict sense, but it identifies the main axes (principal components) that explain the majority of variance in the dataset. In our results, PC-1 explained the large majority of variance (92.6% in Trial I, 91.5% in Trial II) and variables such as organic matter (OM), total organic carbon (TOC), nitrogen (N), phosphorus (P), microbial biomass carbon (MBC), microbial biomass nitrogen (MBN) and colony forming unit (CFU) had strong positive loadings on PC-1. Treatments with high positive PC-1 scores (e.g., Compost + 2% ZnO + ZSB) therefore cluster together with higher microbial indicators and better crop performance, demonstrating a strong co-variation between microbial population/biomass and crop performance. In short, PCA shows that microbial population and crop performance co-vary along the same major gradient (PC-1), supporting the interpretation that improved microbial activity and soil fertility are associated with improved crop growth and yield in our trials.
Comment 2: Why significance level up to 0.0001 described?
Response: Thank you, The manuscript used conventional significance notation (asterisks) to indicate ranges of p-values; the table legend defined **** as p < 0.0001, while the global decision threshold for tests was α = 0.05. The appearance of **** (p < 0.0001) simply reflects that some comparisons produced very small p-values (highly significant differences) when evaluated by ANOVA / post-hoc tests in Statistix / OriginPro. We will clarify the legend and methods, so readers are not confused: we will keep α = 0.05 as the threshold for declaring significance but will report p-value ranges using the asterisk scheme.
Comment 3: How was LSD calculated? Is it for the overall treatment effect? Why is it significant to calculate here?
Response: Good point, we will clarify. The ANOVA F-test was used to evaluate the overall treatment effect (i.e., whether any treatment means differ). Where the ANOVA F-test was significant, we used the Least Significant Difference (LSD) procedure to perform pairwise comparisons between treatment means. LSD was calculated from the ANOVA residual mean square (MSE) and the number of replicates according to the standard formula:
LSDα=tα/2, df error × √2 × MSE/r
where tα/2, df error is the t-value at the chosen α (0.05) and the residual degrees of freedom, MSE is the mean square error from the ANOVA, and r is the number of replicates (here r = 3). The LSD values reported in Table 1 are the LSD at p ≤ 0.05 computed from ANOVA outputs in Statistix 8.1. LSD is appropriate here because the experiment used an RCBD with balanced replications and our objective was to identify which specific treatments differed (pairwise) after a significant overall ANOVA.
With best regards
Dr. Azhar Hussain
Corresponding Author/ Associate Professor
Department of Soil Science
The Islamia University of Bahawalpur
Email: azharhaseen@gmail..com
Cell#: 0092 321 6814625
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This study makes a valuable scientific contribution by demonstrating how balanced fertilizer strategies can restore degraded soils and support sustainable agriculture.