Biomass and Nutritional Status of Melon Hybrids Induced by Arbuscular Mycorrhizal Fungi Application Under Varying Irrigation Stress

Round 1

Reviewer 1 Report

There are serious flaws and a lack of scientific methods and representation. 

First, the title, content, and overall experiment lack consistency, giving the impression that the author does not fully understand the basic mechanisms of how AM fungi work or how to utilize them effectively. The manuscript contains numerous errors, some of which are highlighted below:

1. The title does not align with the scope of the work.
2. In the abstract, there is no introductory sentence, no problem statement, and no justification for conducting the experiment. Starting directly with materials and methods is unprofessional.
3. In the abstract, is "mycorrhizal fungi" referring to AMF? The abbreviation is incorrect, as "AM" is used inconsistently, while "mycorrhizal fungi" is a broader term.
4. In the abstract, the mention of "four treatments of AM fungi" is incorrect; there are only three treatments.
5. Why were 10 replicates used? Typically, 5 replicates are sufficient.
6. Spore names should be italicized.
7. The abstract mentions very few results—just one line. Is this sufficient? I don't think so.
8. Specific results should be mentioned, such as the significant changes in degree or percentage. Saying "just increased" is vague and inadequate.
9. The author chose these two AM fungi because they are commonly available. However, what is the molecular interaction of these spores with these plants? What mechanism justifies their selection? A solid, logical reason should be provided.
10. Why was Sorghum bicolor used for AMF spores instead of Trifolium?
11. Were 100 glomerospores checked and quantified?
12. Where is the statistical analysis?
13. Is Figure 2 sufficient to show colonization? How does it compare to the control (CK)?
14. Why are there no significant variances shown in Figure 3? How can the significant differences be evaluated?
15. Under waterlogged conditions, how do AM fungal spores survive when they require oxygen? How is this justified?

Author Response

Comments to the Reviewer 1:

 

Comments 1: Major comments. There are serious flaws and a lack of scientific methods and representation.

Response: The article has undergone an extensive revision to improve clarity and ensure a better understanding of the scientific methods and representations presented. We have carefully addressed the identified issues and enhanced the overall quality of the manuscript.

 

Comments 2: Detail comments. First, the title, content, and overall experiment lack consistency, giving the impression that the author does not fully understand the basic mechanisms of how AM fungi work or how to utilize them effectively. The manuscript contains numerous errors, some of which are highlighted below:

Response: The title has been rewritten, and the article has been significantly improved to address the concerns raised. The content has been revised for consistency, and we have carefully reviewed the experimental design and scientific basis to ensure accuracy and clarity. The manuscript is now submitted for a new round of verification.

 

Comments 3: The title does not align with the scope of the work.

Response: The title has been revised to better align with the scope of the work. The new title is: "Arbuscular mycorrhizal colonization, shoot biomass, and nutrient partitioning in melon plants under irrigation regimes with water stress."

 

Comments 4: In the abstract, there is no introductory sentence, no problem statement, and no justification for conducting the experiment. Starting directly with materials and methods is unprofessional.

Response: Thank you for your valuable feedback. We have addressed this concern by including the following introductory sentence in the abstract: "Water scarcity is a major challenge in northeastern Brazil, where efficient water management strategies are essential for sustainable agriculture." This provides context, highlights the problem, and justifies the need for the experiment.

 

Comments 5: In the abstract, is "mycorrhizal fungi" referring to AMF? The abbreviation is incorrect, as "AM" is used inconsistently, while "mycorrhizal fungi" is a broader term.

Response: We appreciate the observation, and the text has been corrected. In the abstract, the term "mycorrhizal fungi" now consistently refers to arbuscular mycorrhizal fungi (AMF), ensuring proper use of the abbreviation and avoiding ambiguity.

 

Comments 6: In the abstract, the mention of "four treatments of AM fungi" is incorrect; there are only three treatments.

Response: Thank you for your observation. We have taken it into account and made the necessary correction in the text. The study utilized four inoculation treatments (non-inoculated, and inoculated with E. etunicata, A. longula, and E. etunicata + A. longula), and this has been accurately reflected in the revised version. Why were 10 replicates used? Typically, 5 replicates are sufficient.

 

Comments 7: Spore names should be italicized. The abstract mentions very few results—just one line. Is this sufficient? I don't think so. Specific results should be mentioned, such as the significant changes in degree or percentage. Saying "just increased" is vague and inadequate.

Response: Thank you for your observations. We used 10 replicates to ensure greater statistical power and reliability, as this number was deemed necessary for the robustness of the experiment. The spore names have been corrected and italicized as suggested. Additionally, the abstract has been revised to include more specific results and data, addressing the concern that it was too brief and provided insufficient details.

 

Comments 8: The author chose these two AM fungi because they are commonly available. However, what is the molecular interaction of these spores with these plants? What mechanism justifies their selection? A solid, logical reason should be provided.

Response: The choice of AMF isolates Entrophospora etunicata (Univasf 09) and Acaulospora longula (URM-FMA 0700) used in this experiment was based on the availability of the inoculant produced. Both isolates have been shown to be effective in promoting the growth of some native Caatinga species (Honorato et al., 2020 - https://doi.org/10.1590/2179-8087.124017 and Silva et al., 2015 - https://doi.org/10.1080/01904167.2014.934472), and have been tested under saline and water stress conditions (Yano-Melo et al., 2003 - https://doi.org/10.1016/S0167-8809(02)00044-0; Silva et al., 2015 - https://doi.org/10.1016/j.apsoil.2015.05.004). The choice of inoculant does not require a molecular interaction, but rather focuses on the fungal isolates' ability to tolerate the conditions imposed on them, particularly water deficiency, which is the central theme of this research.

 

Comments 9: Why was Sorghum bicolor used for AMF spores instead of Trifolium?

Response: The multiplication of AMF spores can be done in association with several species of Poaceae that have a root system favorable to AMF spores multiplication. Under the conditions of the Brazilian semi-arid region, S. bicolor promotes the multiplication of AMF, including species native to the Caatinga (Medeiros et al., 2023 - https://doi.org/10.1111/1365-2664.14330), which is why we used sorghum for AMF spores multiplication.

 

Comments 10: Were 100 glomerospores checked and quantified?

Response: Yes, spores were extracted from AMF soil-inoculum, then separated and counted, in order to ensure that the inoculation was done with this aliquot of spores. For AMF inoculation a soil inoculum, containing spores, colonized root fragments, and AMF hyphae, was used. The text was changed to make it more clear

 

Comments 11: Where is the statistical analysis?

Response: Which analysis is this in reference to? If it refers to the quantity of AMF spores applied, it makes no sense, since the treatments must standardize the quantity.

 

Comments 12: Is Figure 2 sufficient to show colonization? How does it compare to the control (CK)?

Response: Figure 2 illustrates some of the internal structures formed by AMF, such as arbuscules, hyphae, and vesicles. However, the mycorrhizal colonization percentage data, which provides a clearer comparison, can be found in Figure 1 and Table 1. These results offer a more detailed assessment of colonization, including comparisons with the control (CK).

Comments 13: Why are there no significant variances shown in Figure 3? How can the significant differences be evaluated?

Response: The boxplot itself serves as a descriptive statistical analysis, providing a visual representation of the distribution of data. The purpose of the image was to show the spread and central tendency of the data across the different treatments. Significant differences can be evaluated through the statistical tests applied (such as ANOVA), and these results are provided in the text and tables. The boxplot highlights the general trends, while the statistical analysis in the results section offers the significance of the differences.

 

Comments 14: Under waterlogged conditions, how do AM fungal spores survive when they require oxygen? How is this justified?

Response: The writing in the manuscript has been corrected. Under waterlogged conditions, the AM fungal spores do not necessarily require constant exposure to oxygen. The irrigation level of 125% ETc was not sufficient to cause continuous waterlogging, which did not result in spore death but may have reduced the activity of some AMF species. The excess water (25% ETc) was drained from the pot but remained in trays underneath, undergoing evaporation and/or being utilized through capillary rise by the plants. As a result, soil aeration occurred after these processes, allowing the spores to survive in conditions where oxygen availability was not constant.

Author Response File: Author Response.pdf

Reviewer 2 Report

This is a very meaningful study with clear scientific questions, reasonable and feasible experimental design, and the author's unique interpretation of the results. Overall, it is a good article, and it is recommended to revise and publish it.

1. The term 'Biomass and nutrition' in the title is worth reconsidering, as the data tends to focus more on Ca rather than a large number of elements.

2. How is the irrigation level determined? Why are there four levels? Do the authors have production data? I suggest calculating WUE (core indicator).

3. The author used a potted substrate with a 1:1 ratio of "pine substrate and sterilized soil at a ratio" or "Vermiculite and sterilized soil at a ratio of 1:1", which is not entirely soil. Therefore, the infection and colonization of AMF are influenced by the compactness of the substrate, which in turn affects the general adaptability of the experimental conclusions. I believe that AMF colonization in non sterilized soil does not necessarily increase linearly with the increase of soil moisture.

Suggest the author to discuss the limitations and representativeness of this experiment.

Other questions:

1. It is not recommended to divide the introduction and conclusion into too many paragraphs. Instead, paragraphs with similar meanings can be merged to increase readability.

2. Change line107 table5 to table1

3. Lines177-183 is recommended to be deleted as it is well known

4. Reference "33" in line 202 is an unnecessary citation

5. The irrigation water volume based on ETc lacks detailed time, detection equipment, and calculation formulas.

Author Response

Comments to the Reviewer 2:

 

Comments 1: Major comments. This is a very meaningful study with clear scientific questions, reasonable and feasible experimental design, and the author's unique interpretation of the results. Overall, it is a good article, and it is recommended to revise and publish

Response: Thank you for your positive feedback. The article has undergone several improvements to enhance clarity and ensure the presentation of the results is more comprehensible. We appreciate your recommendation for revision and publication.

 

Comments 2: The term 'Biomass and nutrition' in the title is worth reconsidering, as the data tends to focus more on Ca rather than a large number of elements.

Response: The title has been revised to better align with the scope of the work. The new title is: "Arbuscular mycorrhizal colonization, shoot biomass, and nutrient partitioning in melon plants under irrigation regimes with water stress."

 

Comments 3: How is the irrigation level determined? Why are there four levels?

Response: An extensive revision was added to the methodology to enhance the irrigation method. The irrigation levels were determined based on the evapotranspiration (ETc) rates, with four levels selected to evaluate the impact of varying water availability on plant growth. These levels (50%, 75%, 100%, and 125% ETc) were chosen to represent a range of water stress conditions, helping to better understand the effects of water management on melon plants.

 

Comments 4: The author used a potted substrate with a 1:1 ratio of "pine substrate and sterilized soil at a ratio" or "Vermiculite and sterilized soil at a ratio of 1:1", which is not entirely soil. Therefore, the infection and colonization of AMF are influenced by the compactness of the substrate, which in turn affects the general adaptability of the experimental conclusions. I believe that AMF colonization in non sterilized soil does not necessarily increase linearly with the increase of soil moisture.

Response: Only the substrate "pine substrate and sterilized soil at a ratio" was used for seedling formation, and the pots were filled with sterilized soil. The amount of substrate derived from seedling production was not sufficient to alter the nutrient availability in the soil. Regarding the concern about the compactness of the substrate, it is important to note that the soil was sterilized, and the focus was on the irrigation treatments and AMF colonization. The results indicate that AMF colonization does not necessarily follow a linear increase with soil moisture, as other factors such as the specific conditions of the substrate and the plant’s root system also play a role.

 

Comments 5: Suggest the author to discuss the limitations and representativeness of this experiment.

Response: Although it was not easy to address all the limitations, I have tried to clarify this point more clearly in the text. Additionally, the limitations and representativeness of the experiment will certainly be considered in future manuscripts.

 

Comments 6: It is not recommended to divide the introduction and conclusion into too many paragraphs. Instead, paragraphs with similar meanings can be merged to increase readability.

Response: I have made an effort to address your suggestions by merging paragraphs with similar meanings to improve readability. At the same time, I ensured that the paragraphs are not too long, maintaining a balance between clarity and readability.

 

Comments 7: Change line107 table5 to table1.

Response: Thank you for your observations. The correction has been made. Line 107 now references Table 1 instead of Table 5.

 

Comments 8: Lines177-183 is recommended to be deleted as it is well known Response: The recommended deletion of lines 177-183 has been made, as the information is well known.

 

Comments 9: Reference "33" in line 202 is an unnecessary citation.

Response: The unnecessary citation "33" in line 202 has been removed as per your suggestion.

 

Comments 10: The irrigation water volume based on ETc lacks detailed time, detection equipment, and calculation formulas.

Response: An extensive revision of the irrigation methodology has been conducted. The irrigation water volume based on ETc has now been detailed, including the time intervals, detection equipment, and calculation formulas used to ensure accuracy and consistency in the irrigation process.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

need revision carefully.

1.       The title still does not represent the current workflow. I suggest the title “Biomass and Nutritional Status of Melon Hybrids Induced by Arbuscular Mycorrhizal Fungi Application under varying irrigation stress”.

2.       Write nutrition (macronutrients) results in the abstract also Mycorrhizal colonization rate or %.

3.       Write fresh aerial biomass (FAB) and dry aerial biomass (DAB) results too in abstract.

4.       Line 283-286, write it under the subheading of ‘statistical analysis.’

5.       Use E. etunicata after using full form ‘Entrophospora etunicata’, same for other species.

6.       Discuss deeply the mechanism and interaction of nutrition (macronutrients) with Mycorrhizal colonization in discussion section.

7.       Use the full form of any nutrient with abbreviation as first. Later, use only Abbreavtion.  Such as line 456 used Calcium (Ca) then later 404; 424 used the same Calcium (Ca) which is scientific flaw, same for other nutrients.

8.       Please read and correct all manuscripts. There should be no scientific error before proceeding to the next step.

 

 

 

Author Response

Comments to the Reviewer 1 V.2:

 

Comments 1: The title still does not represent the current workflow. I suggest the title “Biomass and Nutritional Status of Melon Hybrids Induced by Arbuscular Mycorrhizal Fungi Application under varying irrigation stress”.

Response: Thank you for your valuable suggestion. The title has been updated as recommended to better reflect the current workflow. We appreciate your input in improving the manuscript.

 

Comments 2: Write nutrition (macronutrients) results in the abstract also Mycorrhizal colonization rate or %.

Response: The abstract has been revised to improve clarity, making the presentation of results, including the macronutrient levels and mycorrhizal colonization rates, more comprehensible.

 

Comments 3: Write fresh aerial biomass (FAB) and dry aerial biomass (DAB) results too in abstract.

Response: The abstract has been revised to improve clarity. The results for biomass have been included in the abstract.

 

Comments 4: Line 283-286, write it under the subheading of ‘statistical analysis.’

Response:

We are unsure whether you are suggesting creating a separate subsection for statistical analysis or expanding the writing. The authors understand that the methodology was not structured in subsections, and we have improved the writing of the statistical analyses. “The results were initially subjected to an analysis of variance (ANOVA) to deter-mine if there were any statistically significant differences among the treatments. For the qualitative factors that showed significant differences, a post-hoc mean comparison was performed using the Tukey test at a 5% probability level to identify which specific treatments differed from one another. For the quantitative factors, regression analysis was applied to determine the relationship between the variables and the optimal model that best fit the data. The most appropriate regression model was selected based on the significance of the variables and the goodness of fit. All statistical analyses were conducted using Excel spreadsheets for data organization and the "Assistat" 7.6 software for more complex statistical procedures, ensuring accurate and reliable results.”

 

Comments 5: Use E. etunicata after using full form ‘Entrophospora etunicata’, same for other species.

Response: The abbreviation "E. etunicata" has been standardized after using the full form "Entrophospora etunicata," and the same applies to the other species. We appreciate your thorough review.

 

Comments 6: Discuss deeply the mechanism and interaction of nutrition (macronutrients) with Mycorrhizal colonization in discussion section.

Response: Thank you for your suggestion. Two paragraphs have been added to the discussion section to provide a deeper understanding of the mechanism and interaction between nutrition (macronutrients) and mycorrhizal colonization. We appreciate your careful review.

 

Comments 7: Use the full form of any nutrient with abbreviation as first. Later, use only Abbreavtion.  Such as line 456 used Calcium (Ca) then later 404; 424 used the same Calcium (Ca) which is scientific flaw, same for other nutrients.

Response: It has been verified throughout the manuscript and corrected. The full form of the nutrient was used first, followed by the abbreviation, as suggested.

 

Comments 8: Please read and correct all manuscripts. There should be no scientific error before proceeding to the next step.

Response: Thank you for your attention. We have made some minor revisions to address the points raised, ensuring there are no scientific errors before proceeding to the next step.

Author Response File: Author Response.pdf