Spice Defense: Resistance, Capsaicin, and Photosynthesis in Diverse Capsicum Genotypes Under Root-Knot Nematode Stress

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The work is very interesting, but before publishing, the authors should do another screening with the accessions selected in the first one in larger pots, with greater inoculum, for a longer period (three months) to be sure that the resistance was maintained after more nematode cycles.

Comments on the Quality of English Language

he work is very interesting, but before publishing, the authors should do another screening with the accessions selected in the first one in larger pots, with greater inoculum, for a longer period (three months) to be sure that the resistance was maintained after more nematode cycles.

Author Response

The work is very interesting, but before publishing, the authors should do another screening with the accessions selected in the first one in larger pots, with greater inoculum, for a longer period (three months) to be sure that the resistance was maintained after more nematode cycles.

We sincerely thank the reviewer for this valuable and insightful suggestion. We fully agree that evaluating resistance under conditions that allow for multiple nematode cycles—such as using larger pots, a higher inoculum density, and an extended period—would provide stronger confirmation of durable resistance. However, due to constraints relating to resources and experimental timelines, this extended validation could not be conducted within the scope of the current study. We will perform such an experiment in a future study.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

General comments

Overall, the manuscript contains useful data which will be helpful for Meloidogyne enterolobii management using resistant genotypes in Thailand and elsewhere. The resistant genotypes identified from present study will help to identify new sources of resistance and develop resistant pepper cultivars with desirable traits. However, the data was not presented in a clear and organized way to deliver the right massage. Hence, it needs major revision.

Shortcomings of the manuscript:

1. The title needs to reflect the main aspect of the study which is identifying resistant cultivars; Capsaicin is a small part of the study. (see line 11-14)
2. Generally, the manuscript lacks language clarity, flow of ideas and logical organization. Hence, it needs a lot of work on the language, flows of ideas and organization.
3. The authors need to use standard terminology in nematology studies. For example: they used “nematode-resilient” instead of “nematode resistant” line 18; “nematode infestation” instead of “nematode infection” (line 78); etc. The term nematode “infestation” is used to show the nematodes are in the soil and “infection” is used when the nematodes penetrate plants.
4. The authors did not clearly categorize the nematode resistance-susceptible categories. They did not show which method they followed (citation), did not mention if they modified that category and the reason for their modification. For example: what is the difference between “hypersusceptible” and “highly susceptible” (line 145-147); “highly resistant” and “very resistant” (line 236-237; 241-24; etc.), etc. The term like “hypersusceptible”, for example, is not common. If the authors have justification on changes in defining categories, they need to explain well and justify it or put the authority for that category (citation).
5. It is not clear how the authors measured tolerance using Reproductive factor (RF). Resistance and tolerance are two different concepts. Resistance refers to the trait that reduces nematode reproduction which is measured by rate of reproduction such as RF. Tolerance refers to reduction of the impact of nematode infection on hosts fitness and measured by the hosts trait such as yield parameters, root and shoot growth, or/etc. In the manuscript, the authors did not clarify this but used the RF factor which is not common for tolerance traits.
6. The authors used the term “root-knot nematode” too much instead of specifying they tested on Meloidogyne enterolobii. Once the general name “a root-knot nematode” mentioned and the work is specified the root-knot nematode is enterolobii, it should consistently be spelled out as “M. enterolobii” throughout the manuscript including topics of sections and subsections of Material and methods, Results, and Discussion.
7. Figure 2 is confusing. It looks like the susceptible genotype (Figure 2A) has better root biomass than the resistant one (Figure 2B). It is better to put new pictures that show a good contrast between susceptible and resistance genotypes.
8. Figure 3 is not clearly visible, especially the x-axis. It should be modified to make the fonts bigger and readable.
9. The Results and discussion sections are difficult to follow due to confusions in the use of terminologies, organization, the language and flow of ideas.
10. Follow the journals guide for reference formatting such as use of punctuation, italicizing scientific names, use of abbreviated journal name, etc.

Specific comments:

1. Lines 16-17: Why is using gall index which is used by many researchers less reliable in the present study?
2. Line 18: Better to use “nematode infection” instead of “nematode resilient”
3. Lines 21-23: I would suggest as “In contrast, capsaicin accumulation was influenced by genotype and nematode stress.”
4. Lines 35-37: Please support the sentence with citation.
5. Lines 37-39: Please support the sentence with citation.
6. Line 47: please change “Baht” amount into international unit (dollar amount)
7. Lines 52: add “the” as “Despite the growing awareness …”
8. Lines 55-58:  Please support the statements with citation.
9. Lines 58-65: Rewrite this part.
10. Lines 77-78: Put nematode “nematode infection” instead of “nematode infestation”
11. Lines 78-80: This section did not show the complete objectives of the present study. Please put the broader objective of your study by including screening for resistance, physiological properties and capsaicin accumulation.
12. Figure 1: Better to separate phrases in the legend with “;” instead of “,” and put “.” at the end.
13. Lines 110-123: Remove the details of this section. Just mention that you confirmed the identity. Like “ enterolobii was confirmed using ---------- (which region ITS, 28S) of the ribosomal DNA and cytochrome oxidase II genes. The identity was -----% and ----- % for --- ribosomal DNA and cytochrome oxidase II, respectively.
14. In subsection 2.2, 2.3,2.4 and 2.5, replace “root-knot nematode” with “ enterolobii”.
15. Line 135: replace “a random” with “a randomized”.
16. Line 144: Please put as” … number of eggs per gram of root” instead of “gram root weight”.
17. Line 17: Put “.” at the end.
18. Lines 153-154: It is not clear. Please rewrite it.
19. Line 170: Please put “.” before “Measurements…”

Note: Results and Discussion sections need major revision to follow the message.

Comments on the Quality of English Language

Overall, the manuscript contains useful data which will be helpful for Meloidogyne enterolobii management using resistant genotypes in Thailand and elsewhere. The resistant genotypes identified from present study will help to identify new sources of resistance and develop resistant pepper cultivars with desirable traits. However, the data was not presented in a clear and organized way to deliver the right massage. Hence, it needs major revision.

Author Response

Overall, the manuscript contains useful data which will be helpful for Meloidogyne enterolobii management using resistant genotypes in Thailand and elsewhere. The resistant genotypes identified from present study will help to identify new sources of resistance and develop resistant pepper cultivars with desirable traits. However, the data was not presented in a clear and organized way to deliver the right massage. Hence, it needs major revision.

Shortcomings of the manuscript:

1. Q: The title needs to reflect the main aspect of the study which is identifying resistant cultivars; Capsaicin is a small part of the study. (see line 11-14)

A: Thank you for your suggestion. We have changed the title to “Spice Defense: Resistance, Capsaicin, and Photosynthesis in Diverse Capsicum Genotypes Under Root-Knot Nematode Stress.”

1. Q: Generally, the manuscript lacks language clarity, flow of ideas and logical organization. Hence, it needs a lot of work on the language, flows of ideas and organization.

A: We appreciate your suggestion and have thoroughly revised the entire manuscript to enhance its clarity, coherence, and logical flow of ideas.

1. Q: The authors need to use standard terminology in nematology studies. For example: they used “nematode-resilient” instead of “nematode resistant” line 18; “nematode infestation” instead of “nematode infection” (line 78); etc. The term nematode “infestation” is used to show the nematodes are in the soil and “infection” is used when the nematodes penetrate plants.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: The authors did not clearly categorize the nematode resistance-susceptible categories. They did not show which method they followed (citation), did not mention if they modified that category and the reason for their modification. For example: what is the difference between “hypersusceptible” and “highly susceptible” (line 145-147); “highly resistant” and “very resistant” (line 236-237; 241-24; etc.), etc. The term like “hypersusceptible”, for example, is not common. If the authors have justification on changes in defining categories, they need to explain well and justify it or put the authority for that category (citation).

• Thank you for your suggestion. We have added an appropriate citation and changed "hypersusceptible" to "very susceptible."

1. Q: It is not clear how the authors measured tolerance using Reproductive factor (RF). Resistance and tolerance are two different concepts. Resistance refers to the trait that reduces nematode reproduction which is measured by rate of reproduction such as RF. Tolerance refers to reduction of the impact of nematode infection on hosts fitness and measured by the hosts trait such as yield parameters, root and shoot growth, or/etc. In the manuscript, the authors did not clarify this but used the RF factor which is not common for tolerance traits.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: The authors used the term “root-knot nematode” too much instead of specifying they tested on Meloidogyne enterolobii. Once the general name “a root-knot nematode” mentioned and the work is specified the root-knot nematode is enterolobii, it should consistently be spelled out as “M. enterolobii” throughout the manuscript including topics of sections and subsections of Material and methods, Results, and Discussion.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Figure 2 is confusing. It looks like the susceptible genotype (Figure 2A) has better root biomass than the resistant one (Figure 2B). It is better to put new pictures that show a good contrast between susceptible and resistance genotypes.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Figure 3 is not clearly visible, especially the x-axis. It should be modified to make the fonts bigger and readable.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: The Results and discussion sections are difficult to follow due to confusions in the use of terminologies, organization, the language and flow of ideas.

A: We appreciate your suggestion and have thoroughly revised the entire manuscript to enhance its clarity, coherence, and logical flow of ideas.

1. Q: Follow the journals guide for reference formatting such as use of punctuation, italicizing scientific names, use of abbreviated journal name, etc.

A: Thank you for your suggestion. We have implemented the recommended changes.

Specific comments:

1. Q: Lines 16-17: Why is using gall index which is used by many researchers less reliable in the present study?

A: We thank the reviewer for this important question. The formation of galls does not always correlate with nematode reproduction, as the responses of different genotypes, enzymes, and hormones in plants can vary when infected with root-knot nematodes. The methods used to assess gall indices require expertise, which can sometimes lead to human error. However, we have already analyzed the correlations among multiple parameters in this study, as shown in Table 5.

1. Q: Line 18: Better to use “nematode infection” instead of “nematode resilient”

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Lines 21-23: I would suggest as “In contrast, capsaicin accumulation was influenced by genotype and nematode stress.”

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Lines 35-37: Please support the sentence with citation.

A: Thank you for your suggestion. We have added an appropriate citation.

5. Q: Lines 37-39: Please support the sentence with citation.

A: Thank you for your suggestion. We have added an appropriate citation.

1. Q: Line 47: please change “Baht” amount into international unit (dollar amount)

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Lines 52: add “the” as “Despite the growing awareness …”

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Lines 55-58:  Please support the statements with citation.

A: Thank you for your suggestion. We have added an appropriate citation.

1. Q: Lines 58-65: Rewrite this part.

A: Thank you for your valuable suggestion. We have implemented the recommended revisions.

1. Q: Lines 77-78: Put nematode “nematode infection” instead of “nematode infestation”

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Lines 78-80: This section did not show the complete objectives of the present study. Please put the broader objective of your study by including screening for resistance, physiological properties and capsaicin accumulation.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Figure 1: Better to separate phrases in the legend with “;” instead of “,” and put “.” at the end.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Lines 110-123: Remove the details of this section. Just mention that you confirmed the identity. Like “ enterolobii was confirmed using ---------- (which region ITS, 28S) of the ribosomal DNA and cytochrome oxidase II genes. The identity was -----% and ----- % for --- ribosomal DNA and cytochrome oxidase II, respectively.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: In subsection 2.2, 2.3,2.4 and 2.5, replace “root-knot nematode” with “ enterolobii”.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Line 135: replace “a random” with “a randomized”.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Line 144: Please put as” … number of eggs per gram of root” instead of “gram root weight”.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Line 17: Put “.” at the end.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Lines 153-154: It is not clear. Please rewrite it.

A: Thank you for your suggestion. We have implemented the recommended changes.

1. Q: Line 170: Please put “.” before “Measurements…”

A: Thank you for your suggestion. We have implemented the recommended changes.

20. Q: Note: Results and Discussion sections need major revision to follow the message.

A: We have revised the text to improve the Results and Discussion sections, based on the reviewer's suggestions.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript is a well-structured and informative study evaluating the physiological and biochemical responses of diverse Capsicum genotypes under Meloidogyne enterolobii infections. The integration of reproductive factor (Rf), capsaicinoid analysis, and photosynthetic data provides a valuable multi-angle approach for resistance screening. However, the manuscript would benefit from the following major and minor revisions.

Critical Points
Photosynthetic Performance Interpretation Needs Refinement
Some resistant and susceptible genotypes showed increased photosynthetic rates under nematode stress. However, the physiological relevance of this result (e.g., compensatory mechanisms vs. actual fitness advantage) is underexplored. A clearer discussion of what photosynthetic increase under stress signifies is necessary.

Insufficient Mechanistic Insight
The discussion mentions hormonal signaling and gene regulation, but remains speculative. Including more concrete references or preliminary transcriptomic data (if available) would strengthen this component.

Figures Missing Replication or Statistical Error Bars
Some key graphs (e.g., gas exchange rates, capsaicinoid levels) do not clearly display biological replicates or error bars. These are critical for the interpretation of significance and reproducibility.

Minor Points
Line 28: Consider rephrasing “multifaceted nature” to something more specific, like “genotype-specific biochemical and physiological responses.”

Line 76: Avoid use of “hypersusceptible” unless clearly defined. Consider using "very susceptible" to maintain consistency with resistance grading scales.

Line 162: Specify the extraction solvent and mobile phase used in HPLC for better reproducibility.

Line 222: “tis” → “its”

Line 225: “efficiency to amplify” → “efficiency in amplifying”

Line 294: “root knot” → “root-knot”

Tables 1–4: Include summary statistics (e.g., mean ± SD) for gall index, egg/g root, and Rf per group (species).

Figure 3 captions: Use full terms instead of abbreviations (e.g., replace "Pn" with "net photosynthetic rate" in figure captions).

Author Response

This manuscript is a well-structured and informative study evaluating the physiological and biochemical responses of diverse Capsicum genotypes under Meloidogyne enterolobii infections. The integration of reproductive factor (Rf), capsaicinoid analysis, and photosynthetic data provides a valuable multi-angle approach for resistance screening. However, the manuscript would benefit from the following major and minor revisions.

1. Q: Critical Points
   Photosynthetic Performance Interpretation Needs Refinement
   Some resistant and susceptible genotypes showed increased photosynthetic rates under nematode stress. However, the physiological relevance of this result (e.g., compensatory mechanisms vs. actual fitness advantage) is underexplored. A clearer discussion of what photosynthetic increase under stress signifies is necessary.

A: We have revised the text to improve the Discussion section (lines 448-476), based on the reviewer's suggestions.

2. Q: Insufficient Mechanistic Insight
   The discussion mentions hormonal signaling and gene regulation, but remains speculative. Including more concrete references or preliminary transcriptomic data (if available) would strengthen this component.

A: We have revised the text to improve the Discussion section (lines 398-413), based on the reviewer's suggestions.

3. Q: Figures Missing Replication or Statistical Error Bars
   Some key graphs (e.g., gas exchange rates, capsaicinoid levels) do not clearly display biological replicates or error bars. These are critical for the interpretation of significance and reproducibility.
4. A: We have implemented your suggestion regarding Figure 3.

Minor Points

1. Q: Line 28: Consider rephrasing “multifaceted nature” to something more specific, like “genotype-specific biochemical and physiological responses.”

          A: We have implemented your suggestion.

2. Q: Line 76: Avoid use of “hypersusceptible” unless clearly defined. Consider using "very susceptible" to maintain consistency with resistance grading scales.

        A: We have implemented your suggestion.

3. Q: Line 162: Specify the extraction solvent and mobile phase used in HPLC for better reproducibility.

        A: We have implemented your suggestion (lines 150-163).

4. Q: Line 222: “tis” → “its”

         A: We sincerely apologize but, unfortunately, we were unable to find this error.

5. Q: Line 225: “efficiency to amplify” → “efficiency in amplifying”

         A: We sincerely apologize but, unfortunately, we were unable to find this error.

6. Q: Line 294: “root knot” → “root-knot”

       A: We have implemented your suggestion

7. Q: Tables 1–4: Include summary statistics (e.g., mean ± SD) for gall index, egg/g root, and Rf per group (species).

 A: We have implemented your suggestion, except for Table 1 (as large data cannot analyzed by ANOVA).

8. Q: Figure 3 captions: Use full terms instead of abbreviations (e.g., replace "Pn" with "net photosynthetic rate" in figure captions).

       A: We have implemented your suggestion.

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is ok now. There are some small  error in the text.

 

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript presents a well-structured and comprehensive study evaluating resistance, capsaicinoid accumulation, and photosynthetic responses in diverse Capsicum genotypes under M. enterolobii stress. The study features a robust experimental design and effectively integrates physiological, biochemical, and reproductive factor (Rf) data to support resistance screening. The analysis clearly captures genotype-specific responses and offers meaningful insights for future breeding strategies.
The authors have thoroughly addressed both the major and minor concerns raised in the previous review. The revisions have significantly improved the manuscript's clarity, scientific rigor, and overall quality.