Review Reports - Response of Sweet Pepper Varieties to Low-Input Conditions and Microbial Biostimulant Application

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The current manuscript addresses a highly relevant and timely topic in sustainable horticulture. The study is well designed, combining multiple stress conditions (reduced irrigation and fertilization) with microbial inoculation, and assessing a broad set of parameters, including yield, fruit quality traits, and rhizosphere enzymatic activities. The breadth of traits evaluated, along with the inclusion of both traditional (three) and commercial varieties (i.e., two experimental F1 hybrids, and two commercial F1 hybrids), adds value by highlighting genotype-dependent responses and opening perspectives for breeding programs focused on low-input agriculture.

One of the strengths of the manuscript is the clear evidence that genotype played a stronger role than treatments in determining yield and quality traits. For example, the experimental hybrid H2 consistently showed the highest average yield across treatments, while Cabañeros F1 excelled under optimal conditions but performed poorly under stress. Such results are important for identifying genotypes that maintain productivity with reduced inputs. Similarly, the sugar analyses revealed that traditional cultivars like Najerano and Piquillo displayed particularly favorable sugar profiles at the fully ripe stage, which is a valuable finding for both consumer quality and sustainable production.

The analysis of rhizosphere enzymatic activities provides an interesting dimension, linking soil microbial functioning with plant performance. The reported increase in alkaline phosphomonoesterase activity under reduced fertilization is particularly relevant, as it suggests a microbial adjustment to nutrient limitation. Likewise, the genotype-specific responses, such as the increase in catalase activity in BGV13004 under reduced fertilization, illustrate the complex interaction between host genotype and rhizosphere biology.

Despite these strengths, there are some areas where the manuscript could be improved for clarity and impact. First, while the ANOVA tables are detailed, the interpretation could be streamlined to emphasize the most relevant interactions. For instance, Table 2 shows that genotype × treatment interactions were significant for some enzymatic traits but not for yield; highlighting this contrast more explicitly in the discussion would improve readability. Second, while PGPR inoculation did not significantly mitigate yield losses, it did enhance certain sugar contents at the green ripe stage (e.g., increases in glucose and fructose in Isabel F1, Najerano, and Piquillo under PGPR treatments). This positive aspect could be more strongly emphasized, since it points to quality improvements even when yield stability is limited.

Another point for improvement concerns the discussion of practical implications. The conclusion rightly identifies H2, Najerano, Piquillo, and Isabel F1 as promising candidates for low-input systems. However, the discussion could further highlight the relevance of these results for Mediterranean greenhouse production, where water and nutrient management are pressing concerns. Integrating references to broader sustainability goals or current European agricultural policies could also strengthen the applied relevance.

I identified a few minor technical editing or terminology errors that need to be corrected. These can be found below:

Standardize spacing between numbers and units (e.g., use consistently “L h−1”, “g kg−1 fw”, “µmol g−1 soil h−1”).

Check consistency of superscripts and subscripts in formulas (e.g., “g−1 soil · h−1” appears with different spacing).

Avoid repeated definitions of abbreviations (e.g., PGPR explained more than once).

Minor typographic redundancies: e.g., “fw, relative to the control, with 12.4 g kg−1 fw” (double “fw”), should be simplified.

Adjust wording: replace “non-toxigenic and non-pathogenic microorganisms” with “non-toxigenic, non-pathogenic microorganisms”; replace “showed the most relevant increases” with “showed the most pronounced increases”; replace “no mitigation effect was described” with “no mitigation effect was observed”.

Ensure consistency between text and figure legends (e.g., order of genotypes in Figure 1 vs. their description in the text).

Revise expressions such as “commercial stage” → “commercial maturity stage” or “market maturity stage” for horticultural accuracy.

Clean duplicate links in references (e.g., “https://doi.org/https://doi.org/...”).

There would be another remark regarding terminology, since the terms varieties and cultivars appear several times in the manuscript, which can be considered synonymous in the context, but also hybrids, respectively commercial hybrid. The way in which the terms were used is appropriate, even if it is generally recommended to use a single term consistently (for example, either 'variety' or 'cultivar'). However, the alternation of these notions by the authors in the context of the manuscript is appropriate and very easy to understand for those who work in the field of genetics and plant breeding.

Finally, the manuscript makes a solid contribution to sustainable horticultural research. It convincingly demonstrates that genotype selection is crucial for pepper performance under low-input conditions and that microbial inoculants, while not universally effective, can enhance specific quality parameters. With a clearer emphasis on the positive findings (such as the stable yields of H2 and the improved sugar profiles under PGPR inoculation) and possible streamlining of methodological detail, this paper will be of significant interest to reaaders of Horticulturae.

Author Response

Dear Reviewer,

First of all, we would like to thank you your comments and suggestions and giving us the opportunity of improving our manuscript, horticulturae-3871531 “Response of Sweet Pepper Varieties to Low-Input Conditions and Microbial Biostimulant Application”, for publication in HORTICULTURAE.

We have considered all comments and carefully reviewed the manuscript. Moreover, we have provided a detailed, point-by-point, response to all comments in this cover letter. We believe that the revised manuscript has significantly improved and hope that can be now suitable for publication in Horticulturae. In this response letter, comments and suggestions from you appear in grey colour, while our answers appear in black bold letters. Changes made in this revised version appear now highlighted in yellow in the main manuscript to facilitate their identification.

Sincerely,

Adrián Rodríguez Burruezo (on behalf of the authors)

We sincerely thank the reviewer for their positive assessment of our manuscript. We greatly appreciate the recognition of the strengths of our study's design and results.

Despite these strengths, there are some areas where the manuscript could be improved for clarity and impact.

1.1. First, while the ANOVA tables are detailed, the interpretation could be streamlined to emphasize the most relevant interactions. For instance, Table 2 shows that genotype × treatment interactions were significant for some enzymatic traits but not for yield; highlighting this contrast more explicitly in the discussion would improve readability.

We thank the reviewer for this suggestion and agreed with his/her comment. The results and discussion sections have been revised to better emphasize the most relevant genotype × treatment interactions, as suggested. Below, we detail the new text added on this issue.

Page 11, lines 383-386: “The remarkable stability observed in most genotypes across treatments, in terms of yield and fruit weight, was in agreement with the non-significant G x T interaction detected by the ANOVA for the former, and with the slight but significant interaction found for the latter (Tables 3 and 4).”

Page 13, lines 456-460: “Overall, the significant and genotype-specific trends observed in some genotypes across treatments for ALP and CAT activities, supported the significant G x T interactions detected by the ANOVA. By contrast, genotypes barely changed their DH activity among treatments, in agreement with the non-significant G x T interaction in this trait (Tables 3 and 5).”

Page 17, lines 589-591: “On the whole, a significant effect of genotype was observed on the sugar profile, along with some specific genotype-treatment combinations, particularly at the green-ripe stage (Table 6).”

Page 18, lines 627-631: “Overall, the limited changes observed in yield and fruit weight across treatments in our collection were in agreement with the non-significant G x T interaction for yield, and the low significance of G x T interaction detected for fruit weight, indicating that the differences observed were mostly attributed to the main effects, i. e., genotype and treatment.”

Page 20, lines 701-709: “In general, rhizosphere enzymatic activities showed limited responses to treatments in most genotypes. Only a few genotype-specific patterns, particularly for ALP and CAT, were in agreement with the highly significant G x T interaction reported by the ANOVA. The stability of DH was consistent with its non-significant G x T interaction. This limited response could be attributed to environmental conditions, soil characteristics, or limited microbial interactions within the rhizosphere of our pepper cultivars [46,65]. Nevertheless, this consistency under stress conditions suggested a degree of resilience in maintaining soil functionality under low-input farming, which aligned with the stability observed in yield and fruit weight.”

Page 21, lines 758-760: “These differences in sugar accumulation were reflected in the variable responses to treatments depending on genotype and ripening stage, which corroborates the significant G x R interaction and, to a lesser extent, in some G x T interactions.”

1.2. Second, while PGPR inoculation did not significantly mitigate yield losses, it did enhance certain sugar contents at the green ripe stage (e.g. increases in glucose and fructose in Isabel F1, Najerano, and Piquillo under PGPR treatments). This positive aspect could be more strongly emphasized, since it points to quality improvements even when yield stability is limited.

We appreciate the reviewer’s comment. The discussion has been revised to highlight the positive influence of PGPR inoculation on sugar content at the green-ripe stage. These results are now emphasized as evidence of quality improvements, even when yield effects are limited Page 21, lines 765-769: “Although PGPR did not improve yield or fruit weight in our collection, increases in specific sugars in some genotypes highlight improvements in fruit quality under low-input conditions. Moreover, the effect of PGPR was influenced by genotype and ripening stage, suggesting that these microorganisms can be strategically applied to enhance fruit quality in sustainable agriculture.”

Another point for improvement concerns the discussion of practical implications. The conclusion rightly identifies H2, Najerano, Piquillo, and Isabel F1 as promising candidates for low-input systems. However, the discussion could further highlight the relevance of these results for Mediterranean greenhouse production, where water and nutrient management are pressing concerns. Integrating references to broader sustainability goals or current European agricultural policies could also strengthen the applied relevance.

We thank the reviewer for this suggestion. The discussion has been expanded to emphasize the practical relevance of our findings for Mediterranean greenhouse systems. Furthermore, we have linked our results to broader sustainability objectives and current European agricultural policies.

Pages 17-18, lines 595-598: “However, the urgent need to transition to more sustainable crop management is promoting the optimization of agronomic inputs [30] and the identification of genotypes better adapted to low-input conditions, which includes local ecotypes [30]. This shift also reflects the priorities set by the Common Agriculture Policy (CAP 2023-2027) [31].”

Page 18, lines 639-643: “These results are particularly relevant in Mediterranean horticulture, where water scarcity and salinization are major challenges [53]. They also align with the European Green Deal and the Farm to Fork strategy, which set ambitious targets for reducing fertilizers, among other chemicals, by 2030 in EU Member States [54].”

I identified a few minor technical editing or terminology errors that need to be corrected. These can be found below:

Standardize spacing between numbers and units (e.g., use consistently “L h−1”, “g kg−1 fw”, “µmol g−1 soil h−1”).

All units have been standardized throughout the manuscript to ensure consistency.

Check consistency of superscripts and subscripts in formulas (e.g., “g−1 soil · h−1” appears with different spacing).

All superscripts and subscripts were checked and standardized throughout the manuscript.

Avoid repeated definitions of abbreviations (e.g., PGPR explained more than once).

Abbreviations are now defined only once, the first time they are mentioned in the text (beginning of the Introduction in the example mentioned, M&M in other cases, and only repeated in the abstract (if they are mentioned on it), ensuring clarity and avoiding unnecessary repetition.

Minor typographic redundancies: e.g., “fw, relative to the control, with 12.4 g kg−1 fw” (double “fw”), should be simplified.

We appreciate this observation. Minor typographic redundancies have been revised and simplified throughout the manuscript. The abbreviation fw is now indicated only in the Materials and Methods section and in Results and Discussion only in first mention in the text.

Adjust wording: replace “non-toxigenic and non-pathogenic microorganisms” with “non-toxigenic, non-pathogenic microorganisms”; replace “showed the most relevant increases” with “showed the most pronounced increases”; replace “no mitigation effect was described” with “no mitigation effect was observed”.

These changes have been done.

Ensure consistency between text and figure legends (e.g., order of genotypes in Figure 1 vs. their description in the text).

The genotypes have been ordered in both the Plant material table and figure (Table 1, Figure 1) according to their genetic background: starting with landraces (BGV13004, Najerano and Piquillo), followed by hybrids between landraces and breeding lines (H1 and H2), and finishing with commercial F1s (Isabel and Cabañeros). And they followed the same order in the tables of results (Tables 4, 5, 6 and 7). Please consider that then, in the main text, their names appear when necessary, to mention ranges of variation or to highlight specific remarkable performances, regardless the order followed in the tables. e.g. range of variation of yield in control conditions comprised between BGV13004 and H2 (the worst 1.69 kg plant^-1vs. the best 3.24 kg plant^-1); while other varieties exhibited the lowest and highest fruit weight Piquillo (27 g) and Cabañeros (197 g).

Revise expressions such as “commercial stage” → “commercial maturity stage” or “market maturity stage” for horticultural accuracy.

This term has been revised accordingly (Page 5, lines 168-169; page 6, line 245).

Clean duplicate links in references (e.g., “https://doi.org/https://doi.org/...”).

We appreciate this comment. Duplicate links in the reference list have been corrected in this revised version.

We thank the reviewer for this remark. We appreciate the confirmation that our usage in the manuscript is clear and appropriate.

We sincerely thank the reviewer for the positive evaluation of our work and for the constructive suggestions. In the revised manuscript, we have emphasized the positive findings more clearly to enhance the overall clarity and understanding of the study. Regarding, the methodological details, if the reviewer does not mind, we prefer keep them as they are currently. Because, by contrast, other reviewers are asking for more detail and could then disagree if we shorten the methodological info.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, you may see my review in attached file - Review Horticulturae IX 2025 Final. I accept your manuscript with minor revisions:

In Keywords change sweet pepper with Capsicum annuum
In Material and methods on 125 row there is technical mistake mean fruit weight is the correct.
In Results and discussion, I suggest in the Tables 4, 5 and 6 the letters of Duncan's test to be re-arranged according to ascending order - the highest values to be with a, A...
The sentences on rows 305-307; 384-386; 392-394; 396-398; 432-433; 589-590 explain your results but finish with cited literature. Please, explane and specify...

Comments for author File: Comments.pdf

Author Response

Sincerely,

Adrián Rodríguez Burruezo (on behalf of the authors)

REVIEWER 2

Dear authors, you may see my review in attached file - Review Horticulturae IX 2025 Final. I accept your manuscript with minor revisions:

Your manuscript makes a contribution to the scientific knowledge enrichment. The study is in line with the changing climatic conditions, the aspiration to protect the environment from pollution and sustainable agriculture. The topic is relevant to the scope of the journal. The title - Response of Sweet Pepper Varieties to Low Input Conditions and Microbial Biostimulant Application is explicit and effectively describes the content. The abstract is enough informative and self-reliant. The abstract and keywords correspond with the whole manuscript.

The introduction is adequate of the study. All materials and methodology are appropriately described. The used methods are suitable for analysis of the aimed traits. The experiments are well designed and statistical analysis is adequate. The results interpretation and discussion are acceptable in relation with existent literature. The figure and the tables sufficient to properly illustrate the research described. The interpretations and conclusions correspond with the presented results.

The quoted publications are adequate. The reference lists 80 literature sources as 73.75% of which are from the last 5 years; 12.50% - during the period 2014-2018; 7.50% from 2009 to 2013; 2.50% from 2004 to 2008 and 1.25% from 1994 to 2003. The list is properly formatted and more than 90% of the references are within the last 15 years.

I accept your manuscript for publication with minor revisions.

We sincerely thank the reviewer for the positive and constructive evaluation of our manuscript. We also appreciate the recognition of the significance of our study.

In Keywords I suggest to be included Capsicum annuum instead of sweet pepper.

The keyword has been updated as recommended (Page 1, line 31).

In Material and methods on 125 row change main fruit weight with mean fruit weight

We appreciate the reviewer pointing out this error. The word has been corrected accordingly (Page 5, lines 169-170).

In the Tables 4, 5 and 6 I suggest the letters of the Duncan’s test to be changed according to ascending order – the highest values to be with а, А.

We thank the reviewer for this recommendation. Perhaps, he/she is more used to the order proposed, but depending on the paper, one can find ascending orders or descending orders in the literature. Please also consider that due to the large number of changes needed and the risk of making mistakes during these changing process, we prefer to keep the current ordering, if he/she does not mind we prefer the current order. Nevertheless, we will certainly take this recommendation into account for future publications.

I don’t understand why the sentence in 305-307 rows finishes with cited literature – [30]. Please, point is it your result or discuss/explain the previous cited study… 5. The situation is similarly in the sentences in rows - 384-386; 392-394; 396-398; 432-433; 589-590.

We appreciate the reviewer’s remark in both cases. The Discussion section has been revised and reorganized to clearly distinguish our results from previously published studies. The sentences in the indicated lines have been revised to clarify that the results presented are our own findings, while the cited studies provide supporting evidence in the Discussion section.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript (MP) titled ' Response of Sweet Pepper Varieties to Low Input Conditions and Microbial Biostimulant Application', submitted by Marisa Jiménez-Pérez et al. investigates the response of sweet peppers to reduced fertilization, limited irrigation, and the regulatory role of plant growth-promoting rhizobacteria (PGPR), directly addressing the environmental issues caused by excessive use of water and chemical fertilizers in agricultural production. However, I have some concerns regarding this manuscript:

1, In Materials and Methods section, key information about the experimental site (Torreblanca Experimental Farm, IMIDA Institute, Murcia, Spain), such as soil type, pH value, initial organic matter content, and available NPK levels, is not mentioned. These factors significantly affect rhizosphere enzyme activity and crop nutrient absorption, potentially leading to biases in result interpretation. Moreover, the experiment was only conducted in greenhouses during the winter and summer seasons in 2021, without involving verification in different years or different climatic zones (such as arid and humid regions). The environmental adaptability of the results needs further confirmation.

2, In Materials and Methods section, only the commercial formulation Bactogreen® is mentioned to contain phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing bacteria, but specific concentrations, proportions, and viable cell counts of each strain are not provided. This hinders the reproducibility of experimental results, as other research teams cannot validate the method based on the information given.

3, The structure of the MP seems slightly unreasonable, with “Results and Discussion” section being excessively long. I suggest splitting it into “Results” section and “Discussion” section. Additionally, I recommend adding more literature and enriching the content of the discussion section.

4, Although relevant studies are cited for result comparison, the mechanism underlying the "limited effect of PGPR" is insufficiently discussed. For example, there is no integration of literature perspectives on PGPR colonization efficiency under low-fertilization and limited-irrigation conditions or its interaction with indigenous microorganisms to explain why PGPR fails to effectively improve yield. Additionally, the genetic mechanisms of "significant genotypic differences" (e.g., correlation with specific gene expression) are not explored in combination with existing molecular breeding studies, limiting the depth of discussion.

Author Response

Regarding English style review suggested by Reviewer 3, we can say that the manuscript has been reviewed by a UPV colleague, bilingual in English, who works at the Department of Foreign Languages bilingual in English. Nevertheless, if you still detect some English style issues, please let us know.

Sincerely,

Adrián Rodríguez Burruezo (on behalf of the authors)

REVIEWER 3

In Materials and Methods section, key information about the experimental site (Torreblanca Experimental Farm, IMIDA Institute, Murcia, Spain), such as soil type, pH value, initial organic matter content, and available NPK levels, is not mentioned. These factors significantly affect rhizosphere enzyme activity and crop nutrient absorption, potentially leading to biases in result interpretation. Moreover, the experiment was only conducted in greenhouses during the winter and summer seasons in 2021, without involving verification in different years or different climatic zones (such as arid and humid regions). The environmental adaptability of the results needs further confirmation.

We appreciate the reviewer’s comments and agreed with suggestions.

Now, in the revised version of the manuscript, key details regarding the experimental site (soil type, pH, initial organic matter content, and available NPK levels, among other parameters), have been added to the Materials and Methods section (see new Table 2 on Page 4).

Regarding the second issue, we understand your concern and that it has been made with a constructive aim. Nevertheless, please consider that the work was conducted to evaluate experimentally a comprehensive range of environment/soil conditions itself (i.e. control vs drought, low fertilization, PGPRs and combinations), covering also several genotypes to cover the genotype effect. And, moreover, under one the most usual and representative growing seasons in the Mediterranean area of Spain: Winter-late Spring in Campo de Cartagena/Murcia under greenhouse. Thus, the experiment was conceived to inspire and provide basic info to other breeders and horticulturist, for being tried further in their own growing conditions. In fact, it covers exhaustively per se 7 genotypes × 6 environments/treatments = 42 G×E combinations, encompassing 11 traits (yield, fruit weight, 3 enzymatic activities, 3 sugars unripe, 3 sugars fully ripe) and about 1500 total measurements.

Moreover, to limit the effect of other environmental factors and the year factor, the experiment was conducted under experimental greenhouse conditions, in the IMIDA experimental farm of Torre Blanca. With higher control of temperature and hydric state of the soil than, for instance, open field (in such case year verification would be a must, we fully agree). Also, temperature regime and light conditions measured in this 2021/22 season in these greenhouses were on the whole very similar to those of the 2020/21 and 2022/23 seasons, according to the records of local climate stations.

Thus, the manuscript consistently emphasizes the influence of different environmental conditions in the shape, soil type, and other conditions on the observed parameters. To further highlight this point, a brief note has been added at the end of the Discussion section (see now Page 21, lines 775-780).

“Identifying genotypes that combine stable yields and rhizosphere enzymatic activities with enhanced sugar contents under stress, alongside complementary strategies such as PGPR inoculation, represents a promising step toward sustainable pepper breeding adapted to the new Mediterranean conditions. Future multi-year and multi-location studies could further validate the broader applicability of these findings on other different pedoclimatic conditions.”

In Materials and Methods section, only the commercial formulation Bactogreen® is mentioned to contain phosphorus-solubilizing, potassium-solubilizing, and nitrogen-fixing bacteria, but specific concentrations, proportions, and viable cell counts of each strain are not provided. This hinders the reproducibility of experimental results, as other research teams cannot validate the method based on the information given.

We agree with the reviewer’s comment. As Bactogreen® is a proprietary product, detailed information on the exact proportions and viable cell counts of each strain is not publicly available. However, after consulting the manufacturer, we have achieved further info, now including the commercial concentrations of the microorganisms in the Materials and Methods section (see now Page 4, lines 158–162):

“PGPR was a commercial formulation based on a mixture of plant growth-promoting bacteria. Bactogreen® (Thader Biotechnology, S.L., Murcia, Spain) included phosphorus-solubilizing bacteria (Pseudomonas fluorescens, ≥1 x 10⁹ CFU mL⁻¹), potassium-solubilizing bacteria (Bacillus megaterium and Bacillus circulans, each ≥1 x 10⁹ CFU mL⁻¹), and nitrogen-fixing bacteria (Azospirillum brasilense, ≥1 x 10⁹ CFU mL⁻¹).”

The structure of the MP seems slightly unreasonable, with “Results and Discussion” section being excessively long. I suggest splitting it into “Results” section and “Discussion” section. Additionally, I recommend adding more literature and enriching the content of the discussion section.

We thank the reviewer for this suggestion. The manuscript has been revised to separate “Results” and “Discussion” sections. Additionally, the Discussion has been expanded with relevant literature, new comments suggested by the other reviewers and, therefore, providing a more comprehensive interpretation of the findings. As a result, the number of references cited has increased from 80 to 98 in this revised version of the manuscript.

Although relevant studies are cited for result comparison, the mechanism underlying the "limited effect of PGPR" is insufficiently discussed. For example, there is no integration of literature perspectives on PGPR colonization efficiency under low-fertilization and limited-irrigation conditions or its interaction with indigenous microorganisms to explain why PGPR fails to effectively improve yield. Additionally, the genetic mechanisms of "significant genotypic differences" (e.g., correlation with specific gene expression) are not explored in combination with existing molecular breeding studies, limiting the depth of discussion.

We appreciate the reviewer’s comment. The Discussion section has been further expanded to provide context on the limited effect of PGPR and its potential underlying causes:

Page 18, lines 621-627: “This lack of response may be explained by the restricted adaptability of most PGPR strains, whose efficacy is highly dependent on host genotype, cultivation practices and soil type [46]. It is also influenced by competition with endogenous microbial communities [47] and by strains’ ability to adopt survival strategies under water deficit, such as siderophore and microbial exopolysaccharide production [48], or under reduced fertilization, which may involve nitrogen fixation, linked to the presence of nifH genes [49].”

Although the exploration of the genetic mechanisms was not the focus of this study, we have briefly highlighted the benefits of integrating molecular breeding tools into studies such as ours:

Page 21, lines 769-774: “To improve the identification of PGPR strains with beneficial effects adapted to specific cultivars and environmental conditions, several authors recommend implementing molecular tools. These approaches include the analysis of specific plant stress-related genes responsive to PGPR, as well as broader studies of transcriptional responses of cultivars under stress combined with PGPR [49,98].”