The Effect of Humic-Based Biostimulants on the Yield and Quality Parameters of Chili Peppers

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors 

 

Introduction 

Line 35: (1) why superindex??

I think authors must reinforce the relation between chili pepper and biostimulant spraying. Check literature related to this species and biostimulant. As support for objective of work. 

Line 76: please add IA of biostimulant not commercial name

I suggest a better redaction of objective, in fact it is confuse to read this section. 

M&M

Line 83-87: Chilli pepers species were cropped by authors who described these genotypes?

Line 90: Humix and Energon a registered?? TM? R?

Line 94: Ascophyllum in italic

Concentrations?, better description of commercial biostimulants

Line 112: What soil?, Taxonomy

Line 115: What are nutrient (N, P, K, S and Mg) source?

Line 121: I think a diagram could be more representative of clima (table 3).

Point 2.6: Better edition is needed 

Data analyses: First, add statistical method and post hoc test. Afterthat software. 

Results

Excessive use of dots i.e 8.14 t ha^-1. 

CAPS or DIH ug g-1 DW or FW?

Please, add information of SHU scale

From table 6: K, E and H are tretaments?. These codes didn´t properly described previously.  

Tables shows a poor edition, please it is a scientific document you need improve edition of table. Clumn are not coincident, use one, two or three decimals. Statistical differences were found, even with high standard error o deviation? 

What happened with 3rst harvest for Kristián?

Conclusions
Are very weak, Authors showed a lot of results, but they didnt consolidate these determination to explain what was real effect of assayed biostimulants

What happened with two biostimulants?, genotypes showed different performace after treatments?

Manuscript needs a further revision both in contents, result description and discussion, edition of tabler. Figures were edited in excel without quality to show in a scientific document. 

Author Response

Reiewer 1

Thank you for your feedback we tried to make all the corrections according to the requirements.

 Introduction 

Line 35: (1) why superindex??

Citation style was corrected

I think authors must reinforce the relation between chili pepper and biostimulant spraying. Check literature related to this species and biostimulant. As support for objective of work. 

Scientific sources related to the relationship between (chili) peppers and biostimulants have been included

Line 76: please add IA of biostimulant not commercial name

The active ingredients of the biostimulants used are described in detail in the framework of the methodology, however, we replaced the commercial names with the main active ingredients already in the introduction according to your suggestion

I suggest a better redaction of objective, in fact it is confuse to read this section. 

Objectives have been modified to make them more understandable

M&M

Line 83-87: Chilli pepers species were cropped by authors who described these genotypes?

The description of the species was sourced from a seed supplier, as it accurately reflected the characteristics of the peppers grown by the authors. Therefore, no modifications were made to the original description.

Line 90: Humix and Energon a registered?? TM? R?

The preparations used in the research were registered at the Central Institute for Supervising and Testing in Agriculture and are commercially available. All additional information about the preparations is available on the seller's website: Energen https://www.energen.info/cs/dokumenty-ke-stazeni/   and Humix  https://www.agrocultur.sk/certifikaty/ . Links to these registrations were added to the article (under the number 22, 23)

Line 94: Ascophyllum in italic

It was done

Concentrations?, better description of commercial biostimulants

The details that were provided by the company were added to the text, other information about more detailed composition of the commercial biostimulant used in this study is proprietary, and therefore cannot be disclosed. The primary objective of this research was to evaluate the effectiveness of a commercially available biostimulant on various pepper varieties, providing growers with data to support their selection of the most suitable product for their specific needs. It is important to note that the variability in the response of different pepper varieties to the same biostimulant is consistent with findings from other crop studies, where the same biostimulant has been shown to elicit differential effects across varieties within the same species. This highlights the importance of variety-specific assessments when selecting biostimulants for optimal agricultural outcomes

 

Line 112: What soil?, Taxonomy

It was included in the text.

Line 115: What are nutrient (N, P, K, S and Mg) source?

It was included in the text.

Line 121: I think a diagram could be more representative of clima (table 3).

We have not yet encountered such a request, since the second reviewer does not request to change table into diagram, please consider your request, or let the editor decide. Of course, if necessary, we will change the table to a graph

Point 2.6: Better edition is needed 

Data analyses: First, add statistical method and post hoc test. Afterthat software. 

It was done

Results

Excessive use of dots i.e 8.14 t ha^-1. 

CAPS or DIH ug g-1 DW or FW?

Analyzes related to the pungency of peppers were done in dry matter

Please, add information of SHU scale

It has been added

From table 6: K, E and H are treatments?. These codes didn´t properly described previously.  

Explanations of abbreviations were added to table 6, in other tables the abbreviations and explanations were included

 

 Tables shows a poor edition, please it is a scientific document you need improve edition of table. Clumn are not coincident, use one, two or three decimals. Statistical differences were found, even with high standard error o deviation? 

In several journals, we encountered the request to round data to Significant Figures, where Significant figures are all the digits in a measured value that carry meaning. These include all digits except zeros that are only used to place the decimal point.

When rounding measured values and uncertainties, the number of significant figures should follow the same rules for consistency.

 We rounded the numbers according to these rules (ISO: ISO/IEC Guide 98-3:2008).

If the rules of the magazine require it, we will round the numbers uniformly, to the same decimal place

What happened with 3rst harvest for Kristián?

The 'Kristián' peppers were harvested earlier in 2022, while the 'Habanero Orange' variety ripened more slowly, necessitating an additional late harvest. In 2023, due to the extremely cold weather conditions, ripening was delayed for both varieties. Consequently, three harvests were required for 'Kristián' as well, as the cold weather significantly slowed down the maturation process. This highlights the influence of climatic conditions on the ripening timeline, demonstrating that environmental factors such as temperature can significantly affect the number and timing of harvests in chili pepper production.

Conclusions
Are very weak, Authors showed a lot of results, but they didnt consolidate these determination to explain what was real effect of assayed biostimulants

What happened with two biostimulants?, genotypes showed different performace after treatments?

The conclusions were elaborated in accordance with the recommendations

Manuscript needs a further revision both in contents, result description and discussion, edition of tabler.

We tried to edit the text according to the comments of all reviewers

Figures were edited in excel without quality to show in a scientific document. 

Figures were originally created in Excel for data representation with the quality standards, as we have done in previous publications, including those published by reputable journals such as MDPI. In those instances, the quality of the figures was deemed acceptable and met the necessary publication standards. Please consider the reviewer's request in this case.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting study that examines the impact of applied biostimulants on the quality of chili peppers. However, there are major issues that the authors need to address before the paper can be considered for publication in the Special Issue ‘Effects of Biostimulants on the Growth and Development of Horticultural Crops’ in the journal Horticulturae.

The topic of biostimulants in Capsicum species is not novel. The authors should clearly highlight the novelty of this work compared to previous studies just before stating the objectives.

Another primary drawback of the study is its experimental design, which I will describe in detail later.

Other issues:

It would be beneficial for the paper to include pictures of the varieties.

The symbol for the product should be changed throughout the paper from (mg.kg⁻¹) to (mg·kg⁻¹).

 

L 28 and elsewhere, the term 'variant' is used. Do the authors mean 'experimental unit,' 'treatment,' ‘management’ ‘biostimulant application’ or 'lot'? It might be more appropriate to use a term that accurately reflects the intended meaning.

L89: the authors want to express maybe: “…were applied to the two selected varieties of chili peppers in this study”.

L105-106: the authors stated that: “The proliferation of soil bacteria and beneficial microorganisms is encouraged, leading to an overall enhancement in soil structure, particularly in denser clay soils.” Have the authors analyzed the texture of the soils used in the experiment?

Table 2 should include footnotes for the three variants

L123: The authors mention for the first time: 'In Nitra,...'. Shouldn't the site (municipality, region and country) where the experiment was conducted have been mentioned earlier, specifically in section 2.1, better than in L134-135?

Tables 3 and 4:

The information in L125-128 is confused, is the Climatic data from years 2022 and 2023 obtained from the meteorological station located in the Botanical Garden or provided by the Institute of Landscape Engineering of Slovak University of Agriculture in Nitra from years 1991-2020?

The titles of the tables 3 and 4 should be something like “Climatic conditions at the experimental site for years …”

Regarding the column labeled 'month': Wouldn't it be better to specify the months by name, from April to October?

By the way, is the information presented in Tables 3 and 4 sufficiently critical to warrant their inclusion?

Experimental design

The main drawback of the paper is the experimental design: It needs further clarification. Is a one-factor ANOVA used? What is the factor being analyzed? From what I observe in Tables 6 and subsequent tables, there appears to be a combination of variety and treatment. This should be appropriately explained in the Materials and Methods section.

Additionally, incorporating factors such as ‘Variety,’ ‘Year,’ and/or ‘Harvest’ into the ANOVA could enhance the robustness of the results, effectively converting the one-factor ANOVA into a multi-factor ANOVA. This approach might also help to streamline the information presented in the tables.

In fact, sometimes, as in Table 10, the authors present results of two years combined, and it should be stated it that corresponds to one-way ANOVA or

L136: Block method? Do the authors use a completely randomized design or another type of experiment design?

Table 5

The headings of the first column should have their first letters capitalized.

The dates should be formatted differently; for example, '2.2.' should be written as 02/02

The dates should be aligned using a consistent tabulation format

L145-150: The application of the products should be more clearly linked to Table 5. For example, does Energen Fruktus Plus  2 times applying (during vegetation by spraying on the leaf)correspond to spray on the leaf at the beginning of flowering and spray on the leaf at the beginning  of the first fruits formation?

L150: What criteria were used for harvesting? Was it based on color, size, or other factors?

L164: The authors might consider including subheadings in Section 2.6, 'Evaluation of Qualitative Parameters,' to improve clarity and organization.

L 167: It appears that there is a missing closing parenthesis after 'according to (16'. It should be corrected to 'according to (16).'

Table 6 and subsequent tables:

Footnotes for K, E, and H are missing and should be included

Horizontal lines should be removed to avoid confusion about where statistical differences are applied.

L 186-189: The number of decimal places in the text should match those presented in the table.

L187: Change “according to Table 6” to “(Table 6)”.

L191-195: Some references appear to be missing and should be included.

In Table 7 and so on, Does “±0.97a” indicate the standard deviation?

 

Figure 1:

There are three '0' values above the figure that may need to be deleted.

Is 'AA (mg/kg FM)' extraneous information?

Please standardize the format for units, either using 'mg·kg⁻¹' or 'mg/kg'

Only one 'Variant,' one 'Harvest,' and one 'Variety' should be represented on the X-axis, rather than three of each.

L419-429: The conclusions should be rewritten to improve clarity and understanding. For example, L420-423 could be revised to something like: "Since the two monitored hot pepper varieties showed an increase in economically significant yield and capsaicin levels, using the tested biostimulants could be a strategic approach to producing nutrient-rich vegetables with no adverse environmental effects."

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Reviewer 2

This is an interesting study that examines the impact of applied biostimulants on the quality of chili peppers. However, there are major issues that the authors need to address before the paper can be considered for publication in the Special Issue ‘Effects of Biostimulants on the Growth and Development of Horticultural Crops’ in the journal Horticulturae.

Thank you for your feedback we tried to make all the corrections according to the requirements.

The topic of biostimulants in Capsicum species is not novel. The authors should clearly highlight the novelty of this work compared to previous studies just before stating the objectives.

Regarding the novelty of our work on biostimulants in Capsicum species, we would like to emphasize that while the general topic of biostimulants in Capsicum is not entirely novel, our study specifically focuses on several unique aspects that distinguish it from previous research. First, our research evaluates the effects of two commercial humic-based biostimulants, Humix® and Energen, on both quantitative and qualitative traits of two distinct Capsicum species (Capsicum chinense 'Habanero Orange' and Capsicum annuum 'Kristián'), which have not been previously studied in this combination. The study also investigates the effect of these biostimulants over multiple growing seasons (2022 and 2023), which involved significant variation in climatic conditions, particularly the cold weather in 2023. This allows us to assess the interaction between biostimulants, pepper genotypes, and environmental factors, adding a new dimension to existing knowledge.

Moreover, our work offers a detailed analysis of both capsaicinoid accumulation (capsaicin and dihydrocapsaicin) and other important quality parameters, such as ascorbic acid and carotenoid content, under the influence of biostimulants, which are evaluated across different harvest terms. This granular examination of harvest timing and its significant effect on the bioactive compound profile presents a new understanding of how biostimulants can optimize chili pepper quality and yield.

We rewrote the abstract, where we inserted novelties of our study through sentence - Thus, our study provides insights into the dynamic responses of Capsicum species to biostimulants under variable climatic conditions, contributing new knowledge to both agricultural practices and the scientific understanding of biostimulant effects in Capsicum production.

 

Another primary drawback of the study is its experimental design, which I will describe in detail later.

Other issues:

It would be beneficial for the paper to include pictures of the varieties.

The pictures have been added

The symbol for the product should be changed throughout the paper from (mg.kg⁻¹) to (mg·kg⁻¹).

The format for units, by using 'mg·kg⁻¹ was standardized in whole article

 L 28 and elsewhere, the term 'variant' is used. Do the authors mean 'experimental unit,' 'treatment,' ‘management’ ‘biostimulant application’ or 'lot'? It might be more appropriate to use a term that accurately reflects the intended meaning.

The term 'variant' was replaced by the more suitable term 'treatment' in the whole text

L89: the authors want to express maybe: “…were applied to the two selected varieties of chili peppers in this study”.

It was added

L105-106: the authors stated that: “The proliferation of soil bacteria and beneficial microorganisms is encouraged, leading to an overall enhancement in soil structure, particularly in denser clay soils.” Have the authors analysed the texture of the soils used in the experiment?

The description and reported effects of the tested preparations reflect the general characteristics provided by the official distributors of these biostimulants, and such effects should be attributed to biostimulants in general. Our study did not specifically investigate the influence of these preparations on soil texture. The soil used in the experiment is classified as medium heavy soil with a high content of the clay fraction, especially in the subsoil (at a depth of 30-60 cm), which has been added to text.

 

Table 2 should include footnotes for the three variants

It was added

L123: The authors mention for the first time: 'In Nitra,...'. Shouldn't the site (municipality, region and country) where the experiment was conducted have been mentioned earlier, specifically in section 2.1, better than in L134-135?

Thank you for the suggestion, we moved the sentence according to the instructions

 

Tables 3 and 4:

The information in L125-128 is confused, is the Climatic data from years 2022 and 2023 obtained from the meteorological station located in the Botanical Garden or provided by the Institute of Landscape Engineering of Slovak University of Agriculture in Nitra from years 1991-2020?

It was added and corrected in text - Climatic data from the years 2022 and 2023 were obtained from the meteorological station in the Botanical Garden, operated by the Institute of Landscape Engineering (ILE) of the Slovak University of Agriculture in Nitra. The climatic norm from the years 1991-2020, which is included in the tables, was also provided by the ILE, as they maintain all these data in their databases.

The titles of the tables 3 and 4 should be something like “Climatic conditions at the experimental site for years …”

It was edited

Regarding the column labeled 'month': Wouldn't it be better to specify the months by name, from April to October?

It was edited

By the way, is the information presented in Tables 3 and 4 sufficiently critical to warrant their inclusion?

Climatic conditions from 2022 and 2023 had a noticeable impact on the ripening time of the chili peppers. Tables help contextualize the environmental variables that may have influenced the plant responses to the biostimulants.

Experimental design

The main drawback of the paper is the experimental design: It needs further clarification. Is a one-factor ANOVA used? What is the factor being analysed? From what I observe in Tables 6 and subsequent tables, there appears to be a combination of variety and treatment. This should be appropriately explained in the Materials and Methods section.

Additionally, incorporating factors such as ‘Variety,’ ‘Year,’ and/or ‘Harvest’ into the ANOVA could enhance the robustness of the results, effectively converting the one-factor ANOVA into a multi-factor ANOVA. This approach might also help to streamline the information presented in the tables.

 

The experiment utilized a multifactorial design, considering both the variety (Capsicum chinense 'Habanero Orange' and Capsicum annuum 'Kristián') and the biostimulant treatment (Humix® and Energen) as factors. A multifactor analysis of variance (ANOVA) was applied to assess the effects of these factors independently and their interaction on both quantitative and qualitative parameters. The factors analysed include variety, treatment, and harvest term. This design allows for the examination of how different biostimulant treatments impact each variety, as well as how these effects vary across multiple harvests. The results are presented in Tables 6 and beyond, where the interaction between variety and treatment is reflected in the reported values. This approach ensures a comprehensive understanding of how each factor contributes to the observed outcomes.

 

In fact, sometimes, as in Table 10, the authors present results of two years combined, and it should be stated it that corresponds to one-way ANOVA or

Indeed, the results presented in Table 10 and similar sections reflect data where multiple years and harvests have been combined. In such cases, a one-way ANOVA was performed to analyse the overall effects of the treatments when summarized across the different variables. Our intention in doing so was to provide a holistic view of the data, capturing the combined influence of the treatments across the two years of experimentation.

Where individual harvests, years, and treatments are analysed separately (as seen in other tables), a multifactorial ANOVA was employed. This approach allowed us to examine the interactions between multiple factors—namely, variety, treatment, and harvest timing—thereby revealing more specific insights into how each of these factors influenced the outcome. By summarizing the data with a one-way ANOVA, we aimed to provide an overarching perspective that reflects the general trends observed across all available measurements while still accounting for detailed factorial analyses where appropriate. This dual approach ensures both depth and breadth in the interpretation of the results.

 

L136: Block method? Do the authors use a completely randomized design or another type of experiment design?

The experiment was designed using a randomized block design (RBD), not a completely randomized design. The block method was chosen to account for potential variability within the experimental field conditions, ensuring that any differences in environmental factors, such as soil composition or microclimate, would be minimized within each block. Each block contained all treatment combinations (variety and biostimulant), and the treatments were randomly assigned within each block. This design allows for better control of experimental variability and provides more reliable results by isolating the effects of the treatments from any potential block-to-block differences. We will update the manuscript to clarify the use of a randomized block design in the experimental setup.

 

Table 5

The headings of the first column should have their first letters capitalized.

It was edited

The dates should be formatted differently; for example, '2.2.' should be written as 02/02

The dates should be aligned using a consistent tabulation format

It was edited

L145-150: The application of the products should be more clearly linked to Table 5. For example, does Energen Fruktus Plus  2 times applying (during vegetation by spraying on the leaf)correspond to spray on the leaf at the beginning of flowering and spray on the leaf at the beginning  of the first fruits formation?

The links were added to text

L150: What criteria were used for harvesting? Was it based on color, size, or other factors?

Both varieties were harvested once the peppers had reached their typical size and shape. We avoided issues such as soft consistency, rotting, and over-ripening by conducting the harvest in three separate stages. This approach allowed us to selectively harvest ripe fruits while leaving unripe fruits on the plant for subsequent harvests. By carefully monitoring the ripening process, we were able to ensure the optimal quality of the fruits at each stage of collection.

L164: The authors might consider including subheadings in Section 2.6, 'Evaluation of Qualitative Parameters,' to improve clarity and organization.

It has been added to text

L 167: It appears that there is a missing closing parenthesis after 'according to (16'. It should be corrected to 'according to (16).'

Citation style was corrected in whole text, included L167

Table 6 and subsequent tables:

Footnotes for K, E, and H are missing and should be included

It was added

Horizontal lines should be removed to avoid confusion about where statistical differences are applied.

The tables have been edited

L 186-189: The number of decimal places in the text should match those presented in the table.

It has been edited

L187: Change “according to Table 6” to “(Table 6)”.

It has been edited

L191-195: Some references appear to be missing and should be included.

The references have been added

In Table 7 and so on, Does “±0.97a” indicate the standard deviation?

Yes, and it has been added to text

 

Figure 1:

There are three '0' values above the figure that may need to be deleted.

New figures have been added with the '0' values removed.

Is 'AA (mg/kg FM)' extraneous information?

It has been removed from graph

Please standardize the format for units, either using 'mg·kg⁻¹' or 'mg/kg'

It has been edited

Only one 'Variant,' one 'Harvest,' and one 'Variety' should be represented on the X-axis, rather than three of each.

Changing the X-axis as requested by the reviewer would result in the creation of six graphs instead of two, which we believe would unnecessarily complicate the presentation of the data. Additionally, the current graphs provide a clear and straightforward comparison, and the second reviewer did not raise any concerns. We kindly ask the reviewer to reconsider this request.

 

L419-429: The conclusions should be rewritten to improve clarity and understanding. For example, L420-423 could be revised to something like: "Since the two monitored hot pepper varieties showed an increase in economically significant yield and capsaicin levels, using the tested biostimulants could be a strategic approach to producing nutrient-rich vegetables with no adverse environmental effects."

The conclusions have been rewritten

Comments on the Quality of English Language

Minor editing of English language required.

The text has been edited

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have revised the manuscript and have addressed all the concerns raised in my initial review positively. The revisions have significantly improved the quality of the paper, and I am satisfied with the changes made. I believe the article can be accepted in its current form.