Capsicum annuum L.: Phenological and Yield Performance of Native and Commercial Genotypes Under Open-Field and Low-Technology Greenhouse Hydroponic Systems

Response to Reviewer 1 Comments

1. Summary

Thank you for taking the time to review this manuscript and for your thorough and constructive comments. Your observation regarding the lack of true replication of the “production system” factor is highly relevant and has allowed us to substantially improve the statistical rigor and transparency of the manuscript. We fully agree with your main concern and have implemented major revisions in the statistical analysis, results presentation, discussion, and overall manuscript structure. All modifications have been highlighted in blue in the revised manuscript and are detailed below.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Not applicable

The experimental design limitation has now been explicitly acknowledged and addressed through revised statistical analysis (see Section 2.7 and Section 4.6).

Are the methods adequately described?

Yes

The statistical approach has been clarified and corrected (Section 2.7).

Are the results clearly presented?

Yes

Results were restructured to avoid invalid comparisons (Section 3).

Are the conclusions supported by the results?

Not applicable

Conclusions were revised to reflect the descriptive nature of system comparisons (Section 5).

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: The experimental design has a limitation that affects the validity of statistical inference regarding the “production system” factor, since only one greenhouse and one field were evaluated during a single cycle. The production system factor is not truly replicated. The reviewer suggests: (a) analyzing each production system separately to evaluate genotype effects, and (b) treating each “production system” as a fixed, independent effect.

Response 1: Thank you for pointing this out. We fully agree with this important methodological concern. Direct statistical comparison between open-field (OF) and greenhouse (GH) systems as if they were replicated treatments would indeed be invalid. Therefore, we have made substantial revisions throughout the manuscript to address this issue:

(1) Statistical analysis (Section 2.7)
We have completely removed the two-way ANOVA (genotype × system). Instead, two independent one-way ANOVAs were performed:

• OF system: randomized complete block design (RCBD), with genotype as fixed effect and block as random effect.
• GH system: completely randomized design (CRD), with genotype as fixed effect.

This approach allows statistically valid inference on genotype differences within each production system, which is the central question of the study.

(2) Results restructuring (Section 3)
The results have been reorganized to reflect the new analysis, eliminating any direct statistical comparisons between systems.

• Tables 2 and 3 now present results grouped by system within the same table. Tukey's letters indicate significant differences only among genotypes within each system.
• Figure 4 has been modified: the legend and caption clarify that letters at final time points represent significant differences among genotypes within that specific system. Comparisons of growth between systems are described qualitatively/descriptively.
• Text: All statements implying direct statistical comparisons between systems (e.g., “significantly higher yield in GH than OF”) have been replaced with descriptive language (e.g., “yield in OF was higher than that observed in GH for all genotypes under the conditions of this study”).

(3) Reformulation of the discussion and conclusions (Sections 4 and 5)

• The Discussion now emphasizes that differences between systems are interpreted in light of the contrasting microclimatic conditions (Section 3.1) and the genotype × environment (G×E) interaction revealed by canonical correlation analysis (CCA). No direct statistical claims are made comparing systems.
• The Conclusions have been nuanced to reflect the descriptive nature of the system comparison, while retaining the key finding that G×E interaction strongly influences genotype performance.

(4) Addition of a study limitations section (Section 4.6)

A new subsection, “4.6 Study limitations and contextualization of results”, has been added to explicitly acknowledge:

·         The lack of replication of the production system factor.

·         The single-season and single-site nature of the study.

·         That the GH system evaluated is a low-technology, passively ventilated structure, so results should not be extrapolated to high-technology hydroponic systems.

·         That the strength of the study lies in the detailed microclimatic characterization, the evaluation of genotype performance within each system, and the CCA-based exploration of G×E interactions.

(5) Language revision throughout the manuscript

The entire manuscript (Abstract, Introduction, Methods, Results, Discussion, Conclusions) has been carefully revised to eliminate any claims of direct statistical comparison between production systems. Descriptive and conditional language is now used consistently.

(6) Additional note on canonical correlation analysis (CCA)

As you correctly noted, the CCA remains valid because it explores correlations between environmental and agronomic variables within each genotype–system combination, without making direct statistical comparisons between systems. This analysis is preserved and continues to support the interpretation of G×E interactions.

We believe these revisions fully address your methodological concerns and significantly strengthen the transparency and robustness of the manuscript. We thank you again for your valuable time and expertise.

4. Response to Comments on the Quality of English Language

Point 1: The English language could be improved to enhance clarity of the research.

Response 1: The manuscript has been thoroughly revised to improve English clarity, consistency, and overall readability. The text was carefully edited to enhance the academic tone and ensure precise and unambiguous wording throughout. Particular attention was given to improving sentence structure, terminology, and the correct interpretation of statistical results.

5. Additional clarifications

We would like to emphasize that the main objective of the study is to evaluate genotype performance within each production system and to explore genotype × environment (G×E) interactions. The revised analytical approach aligns fully with this objective and avoids unsupported statistical inference.

Response to Reviewer 2 Comments

1. Summary

Thank you for your valuable comments and suggestions, which have helped improve the clarity, accuracy, and overall quality of the manuscript. We have carefully addressed each of the points raised and implemented the corresponding revisions throughout the manuscript, as detailed below. All modifications have been highlighted in fuchsia in the revised version.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Are all the cited references relevant to the research?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

The Materials and Methods section has been revised to improve clarity and completeness. Additional details regarding the number of plants per genotype, experimental design, management practices, and key experimental dates have been incorporated (Sections 2.1, 2.3, 2.4, and 2.5).

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Provide a clearer description of the number of plants per genotype that were analyzed.

Response 1: Thank you for this valuable comment. We agree that this information should be more explicit. We have reviewed and unified the description of the number of plants used in each system and for each variable, ensuring it is clear throughout the Materials and Methods section and in the footnotes of the tables. The number of replicates and the total number of plants per genotype in each system are now specified. This information can now be found in:

·         Section 2.4.1 (Lines 143-148):
"The experiment was set up under a randomized complete block design (RCBD) with four repetitions (blocks). Each experimental unit consisted of one furrow containing 13 plants, from which the five central plants were considered as the sampling unit for measurements to avoid border effects. This resulted in a total of 20 plants evaluated per genotype in the open-field (OF) system (4 blocks × 5 plants)."

·         Section 2.4.2 (Lines 171-176):
"The experiment was conducted during the same agricultural cycle under a completely randomized design (CRD) with 12 repetitions per genotype. Each experimental unit consisted of a single bag containing two plants; however, one representative plant per unit was selected for measurements, resulting in a total of 12 plants evaluated per genotype in the greenhouse (GH) system."

·         Footnote to Tables 2, 3, and S4:
An explanatory note was added: "For the OF system, n=20 plants per genotype; for the GH system, n=12 plants per genotype. Values represent the mean ± standard deviation (SD)."

Comments 2: Remove the commercial names of the pesticides and specify the concentrations of the active ingredients.

Response 2: We accept the suggestion. We understand that for scientific reproducibility, it is more appropriate to report the active ingredients. We have modified the text to remove commercial names and specify the active ingredients and doses used. These changes were implemented in:

·         Section 2.4.1 (Lines 158-164):
" Preventive and corrective strategies were implemented. Whitefly (Bemisia tabaci) was controlled using imidacloprid (350 g·L⁻¹) applied at 0.75 L·ha⁻¹. Bacterial diseases (Xanthomonas spp.) were managed using copper sulfate pentahydrate (≈25% Cu) at 1 L·ha⁻¹. Soil-borne pathogens were controlled by alternating applications of Trichoderma harzianum (11.5 g·kg⁻¹, 1.15%) at 15 mL·plant⁻¹ with systemic fungicides, including propamocarb (530 g·L⁻¹) + fosetyl-Al (310 g·L⁻¹) applied at 6 L·ha⁻¹, azoxystrobin (46 g·L⁻¹) + chlorothalonil (460 g·L⁻¹) at 3 L·ha⁻¹, and carbendazim (500.76 g·L⁻¹) at 2.5 L·ha⁻¹."

·         Section 2.4.2 (Lines 186-181):
" Systemic insecticides included imidacloprid (350 g·L⁻¹) applied at 0.75 L·ha⁻¹ and a mixture of imidacloprid (210 g·L⁻¹) + bifenthrin (72 g·L⁻¹), applied in separate applications at 0.75 L·ha⁻¹ during the flowering stage to control whitefly (B. tabaci), aphids (Aphididae), and thrips (Thysanoptera). Additionally, elemental sulfur (800 g·kg⁻¹, 80% p/p) was applied at 2 g·L⁻¹ as a preventive measure against fungal pathogens."

Comments 3: Include key dates related to the experimental procedure (sowing, transplanting, harvest(s), and total number of harvests).

Response 3: We thank the reviewer for this important observation. We agree that including this information improves the contextual understanding of the study. Accordingly, specific dates for sowing, transplanting, and harvest periods have been incorporated into the manuscript. In addition, new subsections were added to provide a clearer description of the experimental timeline. The following modifications were made:

·         Section 2.1 (Lines 110-111):
"The study was conducted in San Agustín Calvario, San Pedro Cholula, Puebla, Mexico, during the 2023 agricultural cycle (January–October)."

·         A new subsection 2.3. Seedling Production (Lines 132-140) was added to clarify the experimental timeline, including the sowing dates for both production systems.

·         Section 2.4.1 (Lines 143-144):
"The transplant was carried out on April 19th, 2023, 80 days after sowing (sowing date: January 29th, 2023)..."

·         Section 2.4.2 (Lines 171-172):
"Transplanting was conducted on May 19th, 2023 (89 days after sowing)."

·         A new subsection 2.5. Harvest Management (Lines 193-198) was added, detailing the harvest criteria, number of harvests, and the period during which they were conducted.

Comments 4: In Tables 1-3, the genotypes should be clearly separated from the Tukey HSD values.

Response 4: Thank you for this observation. We have reviewed and refined the table formatting to enhance visual clarity. Although the tables already presented a separation between genotype data and HSD values, the format has been improved by ensuring that the “HSD” row is clearly distinguished from the genotype data rows.

·         These changes were applied to Tables 1, 2, and 3: The table format was refined to ensure a clear visual separation between the headers, the genotype column, the data body, and the "HSD" row, which is already presented separately at the end of the table.

Comments 5: Carefully verify the relative values for fruit yield reduction reported.

Response 5: We thank the reviewer for pointing this out. The previously reported reduction range (32–54%) was inaccurate due to a miscalculation. We have rechecked the values based on the mean yield data from Table 3 and corrected them accordingly in the revised manuscript. The text has been revised to indicate that yield reduction in the greenhouse ranged from 34.1% (Diente de Perro) to 64.0% (Mixteco Largo), based on all evaluated genotypes. This correction can be found in:

·         Section 3.3 (Lines 340-341 - Original):
"Overall, the open-field system exceeded the greenhouse in yield for all genotypes, with reductions that varied between 32% (Serrano Tampico) and 54% (Diente de Perro)."

·         Section 3.3 (Lines 369-372 revised):

The original wording has been revised to avoid direct statistical comparisons between systems, and results are now described in a descriptive manner. The text has been corrected to reflect the accurate range of yield reduction (34.1%–64.0%).

4. Response to Comments on the Quality of English Language

Point 1: The English is correct and does not require improvement

Response 1: We thank the reviewer for the positive assessment of the English language quality in the manuscript.

5. Additional clarifications

The main revisions include improvements in the Materials and Methods section, such as the inclusion of detailed experimental dates, clarification of sample size per genotype, and replacement of commercial pesticide names with active ingredients. In addition, results were corrected and clarified where necessary, including the recalculation of yield reduction values and the addition of explanatory footnotes in tables.

Response to Reviewer 3 Comments

1. Summary

Thank you for your thoughtful and detailed review of our manuscript. Your insights are invaluable for strengthening the scientific rigor and clarity of our work. We have carefully considered all your comments and have prepared the following response and summary of the revisions made. We believe these changes will address your concerns and significantly improve the manuscript. All modifications have been highlighted in green in the revised manuscript.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

The Introduction has been revised by reducing general information on bioactive compounds and strengthening the discussion on agronomic adaptation, stress physiology, and genotype × environment interaction.

Are all the cited references relevant to the research?

Can be improved

The reference list was carefully revised to ensure relevance and focus. General references were reduced, and key literature related to stress physiology and genotype × environment interaction was incorporated to strengthen the scientific context of the study.

Is the research design appropriate?

Must be improved

The statistical approach was revised. The two-way ANOVA was removed and replaced with independent one-way ANOVAs per system. In addition, limitations of the experimental design are now explicitly acknowledged (Section 4.6).

Are the methods adequately described?

Must be improved

The Materials and Methods section was expanded to clarify experimental design, terminology (substrate-based hydroponic system), seedling production, and inclusion of ANOVA tables as supplementary material.

Are the results clearly presented?

Can be improved

The Results section was revised to improve clarity, consistency, and interpretation, including clarification of emergence data and avoidance of unsupported comparisons.

Are the conclusions supported by the results?

Must be improved

The Conclusions were rewritten to avoid causal claims and instead provide a more precise and mechanistic interpretation supported by the data.

3. Point-by-point response to Comments and Suggestions for Authors

General Comments: The manuscript presents a comparative evaluation... The authors mention performing a two-way ANOVA however no ANOVA tables are presented... these measurements are not enough for publication in a highly ranked journal... yield reduction is attributed to impaired pollen viability, reduced photosynthesis, and photoassimilate limitation without physiological measurements.

Response 1: We thank the reviewer for this critical assessment. We acknowledge the lack of supporting data for our physiological claims and the absence of key statistical information. The manuscript was comprehensively revised to address these points. We have now: (1) added full ANOVA tables as supplementary material; (2) revised all causal physiological claims to correlational statements, explicitly noting the absence of direct physiological measurements; and (3) added a dedicated Study Limitations subsection to contextualize our findings.

Comments 1: The central conclusion is that greenhouse hydroponics underperformed due to extreme thermal stress (>48 °C). However, the greenhouse is described as a low-technology structure... This confounding factor limits the generalization of conclusions regarding hydroponic systems. The Discussion should explicitly state that the results apply to low-technology tunnel-type greenhouses with insufficient ventilation. The authors should avoid overgeneralizing about hydroponic systems in general. Furthermore, the experimental conditions differ substantially between the two systems... How is it scientifically justified to directly compare the two systems?

Response 1: We thank the reviewer for this crucial observation. We fully agree that our comparison is not between "hydroponics" and "soil" per se, but between a specific low-tech protected environment and a traditional open-field system.

We have made the following revisions, which we believe now fully address this concern:

1. Title changed to: " Capsicum annuum L.: Phenological and yield performance of native and commercial genotypes under open-field and low-technology greenhouse hydroponic systems" This explicitly flags the low-tech nature of the greenhouse.
2. Abstract revised to specify that the greenhouse was a low-technology, passively ventilated tunnel type, and that results should not be extrapolated to high-tech greenhouses.
3. New subsection 4.6 "Study Limitations and Contextualization of Results" added to the Discussion. In this subsection, we have gone beyond simply describing the limitations to explicitly state that the comparison is at the "system level" (i.e., comparing two integral agronomic packages, not isolated variables). We also explicitly caution against generalizing to high-tech greenhouses.
4. We have removed all instances where we claimed the greenhouse system "caused" the yield reduction, replacing them with statements like "was associated with" or "under the conditions recorded in the GH."

Comments 2: Please clearly state the objective (goal) of the study... Do not begin a sentence with an abbreviation... Since the environmental conditions were not identical, how can the results be directly compared?... The authors mention an initial hypothesis. However, it is not clearly stated.

Response 2: As requested, we have revised the Abstract as follows:

• Objective (Line 14): "... evaluated the phenological and productive responses of five genotypes (four native landraces and one commercial cultivar) under two systems representing locally relevant production conditions…" This reframes the comparison as an evaluation of G×E within specific local contexts, not a direct causal comparison of systems.
• Abbreviation (Line 19): The sentence now reads: " The GH system exhibited restrictive microclimatic conditions "
• Hypothesis (Lines 104-107, Introduction): We have added an explicit hypothesis statement: "It was hypothesized that contrasting production environments would differentially influence phenological and productive performance, revealing a significant genotype × environment interaction."
• Direct comparison justification: In the Abstract (Lines 23-26), we now state: " Environmental conditions were not standardized between systems; therefore, the results reflect the contrasting microclimates of locally relevant production systems and provide a context-specific assessment of genotype performance.”

Comments 3: The Introduction... could be more focused by reducing general information about bioactive compounds. Expanding instead on agronomic adaptation, stress physiology, and genotype × environment interaction theory.

Response 3: We have substantially revised the Introduction as follows:

• The paragraphs on nutraceutical properties and bioactive compounds have been substantially condensed and refocused, emphasizing key review contributions (Mandal et al., 2023; Baenas et al., 2019).
• The section on the physiological effects of high-temperature stress in Capsicum annuum has been expanded to explicitly include its impact on photosynthetic efficiency, pollen viability, and fruit set, supported by recent studies [15,16] (Rajametov et al., 2021; Ricardez-Miranda et al., 2021).
• The theoretical framework on genotype × environment (G×E) interaction has been expanded to clarify its role in explaining variability in agronomic performance, and its relevance for breeding programs and the conservation of native landraces has been explicitly incorporated.

Comments 4: Line 151 (line numbers refer to the original submission): If red tezontle sand was used as substrate, this is technically a soilless substrate system, but not a classical hydroponic system... Please clarify terminology. Lines 151-152: How many seeds were sown?... Lines 194-195 (line numbers refer to the original submission): The authors state that "variables were analyzed through a two-way analysis of variance (ANOVA)." However, no ANOVA tables are presented.

Response 4: We thank the reviewer for these technical points.

• Terminology (Section 2.4.2, line 167): We have changed the heading to "Substrate-based hydroponic system under low-technology greenhouse conditions (GH)" and added a clarifying sentence in the Methods: "This system corresponds to a substrate-based hydroponic scheme using an inert volcanic substrate (tezontle)." We have consistently used this precise terminology throughout.
• Seedling production (new Section 2.3, line 132): We have added the following details: "For each genotype, 200 seeds were sown in 200-cavity polystyrene trays... One seed per cavity was used; no thinning was required."
• ANOVA tables (Supplementary Material): We have created three new supplementary tables (Tables S1, S2, S3) containing the full ANOVA results, including sources of variation, degrees of freedom, mean squares, F-values, and p-values. These are now cited in the Results section. We have also corrected the description in the Methods to state that independent one-way ANOVAs were performed for each system, as the two systems were not directly compared statistically.

Comments 5: Lines 251-266 (line numbers refer to the original submission): Why are emergence data presented only for one system? Please clarify and present emergence data consistently.

Response 5: We have added the following clarifying sentence at the beginning of Section 3.2.1 (Results): "Emergence parameters were evaluated under controlled nursery conditions (identical for both production systems) prior to transplanting. Therefore, the data in Table 1 represent baseline seed quality and vigor for each genotype and are not specific to either the OF or GH system."

Comments 6: Line 488 (line numbers refer to the original submission): The statement "strong genotype-environment interaction" is very general... Please provide a more specific and mechanistic interpretation. Lines 490-491: How can the authors confidently attribute the yield reduction solely to greenhouse microclimate when the systems were not compared under standardized environmental conditions?

Response 6: We have rewritten the Conclusions section as follows:

·         We have removed the definitive causal attribution of yield reduction to microclimate alone. The Conclusions now state: "In the greenhouse hydroponic system (GH), the recorded microclimatic conditions were associated with a reduction in yield, indicating that, under limited ventilation, the environment may restrict the expression of productive potential."

·         We have added a specific, mechanistic interpretation of the G×E interaction: "The patterns identified through canonical correlation analysis confirm a strong genotype × environment interaction, revealing differential responses among genotypes. In this context, Serrano Tampico showed greater productive stability, whereas native genotypes such as Mixteco Largo and Cola de Ratón exhibited better performance under open-field conditions, suggesting specific adaptation to this environment."

·         We have also added a sentence explicitly acknowledging other potential contributing factors in the new Limitations subsection (4.6): " Comparisons between production systems were influenced not only by microclimatic conditions but also by differences in agronomic management, including planting density, substrate type, and irrigation regime, which are integral components of each system. Therefore, the observed differences cannot be attributed exclusively to microclimatic conditions and should be interpreted as the combined effect of these interacting factors."

Comments 7: Several minor grammatical inconsistencies are present... The term "allotment of resources" (line 275) would be better replaced with "allocation of resources."

Response 7: We have engaged a native English-speaking colleague to review the entire manuscript. All instances of "allotment" have been changed to "allocation". We have also broken up several overly long sentences for clarity and corrected minor grammatical errors throughout.

4. Response to Comments on the Quality of English Language

Point 1: The English could be improved.

Response 1:  The manuscript has been carefully revised to improve clarity, readability, and grammatical accuracy throughout.

5. Additional clarifications

We agree with the reviewer that our original manuscript overinterpreted the physiological mechanisms underlying yield reduction. We have now revised all relevant statements in the Discussion and Conclusions to reflect that our data are correlative, not mechanistic. Specifically:

• In the Discussion (Section 4.1), we now state: "From a physiological perspective, temperatures above 32 °C have been associated in the literature with alterations in pollen viability and fruit development... In this study, although direct physiological measurements were not performed, these mechanisms may partially explain the yield reduction observed in the GH."
• In the Conclusions, we removed definitive causal statements and replaced them with phrases such as "was associated with" or "suggesting."

We thank the reviewer again for the thorough and constructive feedback, which has significantly improved the manuscript. We believe the revised version is now much stronger, more rigorous, and appropriate for publication in Horticulturae.