Round 1

Reviewer 1 Report

The manuscript (MS) entitled “Improvement of crop water productivity in greenhouse pepper” and submitted to the Agronomy MDPI by Susana Zapata-García, Abdelmalek Temnani, Pablo Berríos, Pedro J. Espinosa, Claudia Monllor, Alejandro Pérez-Pastor considers the efficiency of strategies for the use of seaweed and microbials biostimulants as well as regulated deficit irrigation application.

This is definitely a very high quality work that makes a significant contribution to the field of research.

The manuscript is well structured, the information is presented logically and consistently, the authors' positions are thoroughly argued, the experiments are described and discussed in great detail.

The manuscript meets the requirements of the journal Agronomy MDPI.

And I definitely recommend for this manuscript to be published in Agronomy.

Comments and suggestions through the first round of review:

1. It is very likely that the title of this manuscript has the potential for optimization in the context of a more complete correspondence and implementation of its content.

2. Perhaps "a bit unexplored" (lines 50 - 51) fact could be verified additionally.

3. Perhaps the phrase on page 72 could be optimized.

“Alternatively” - what is the alternative here and is it really an alternative?

4. How accurate and correct, according to the authors, is it to consider microorganisms (line 75) after substances as biostimulants in a semantic and functional context?

Substances and microorganisms are very different in nature.

What do the authors think about other definitions of biostimulants?

The authors work in organizations in Spain.

How are biostimulants regulated in Spain?

Is there national regulation in addition to EU regulation?

Is there a national biostimulant definition in national regulations?

This information would enrich this manuscript.

5. It could be recommended carefully checking the data in the appropriate tables and figures in the context of the significance of the obtained differences.

6. The term "biostimulation", of course, is used in a number of articles.

To what extent, in the opinion of the authors, is it correct and necessary to use it?

How does it differ conceptually from the term 'stimulation'?

7. It is advisable to present a more detailed coverage of related and close studies in the MS research area in the Introduction and Discussion sections.

8. Perhaps more justification for the novelty of the work would have been necessary.

Could be better.

Author Response

Reviewer 1

Comments and Suggestions for Authors

The manuscript (MS) entitled “Improvement of crop water productivity in greenhouse pepper” and submitted to the Agronomy MDPI by Susana Zapata-García, Abdelmalek Temnani, Pablo Berríos, Pedro J. Espinosa, Claudia Monllor, Alejandro Pérez-Pastor considers the efficiency of strategies for the use of seaweed and microbials biostimulants as well as regulated deficit irrigation application.

This is definitely a very high quality work that makes a significant contribution to the field of research.

The manuscript is well structured, the information is presented logically and consistently, the authors' positions are thoroughly argued, the experiments are described and discussed in great detail.

The manuscript meets the requirements of the journal Agronomy MDPI.

And I definitely recommend for this manuscript to be published in Agronomy.

Comments and suggestions through the first round of review:

Comments 1:

1. It is very likely that the title of this manuscript has the potential for optimization in the context of a more complete correspondence and implementation of its content

Responses:

Dear reviewer, thanks for review and suggestions. It is proposed to change the original title, ‘Improvement of crop water productivity in greenhouse pepper’ into ‘Optimizing the crop water productivity in greenhouse pepper’.

Comments 2:

2. Perhaps "a bit unexplored" (lines 50 - 51) fact could be verified additionally.

Responses:

According to reference [11] (Fereres et al. 2003), the sentence in line 50 has been changed into ‘relatively unexplored’, faithful to its author. Later in the MS (lines 69-71) the literature research about RDI in pepper are referenced.

Comments 3:

3. Perhaps the phrase on page 72 could be optimized.

Responses:

“Alternatively” - what is the alternative here and is it really an alternative?

Thanks for your comment. To improve the understanding, we have rephrased the sentence in line line 72: ‘Alternatively, to minimize the impact that water or other abiotic stresses may have on the crop, biostimulants have been proposed as a novel solution to tackle these issues [32,33],’

Into:

‘Biostimulants have been reported to effectively mitigate the abiotic stress [32,33], a problem that is often faced by crops that grow in semi-arid climates, where water resources are scarce and low quality. However, the quantification of these effects across all crop varieties remains to be accomplished, and they would be of great help for minimizing the drawbacks of RDI in crops growing in these areas.

Comments 4:

4. How accurate and correct, according to the authors, is it to consider microorganisms (line 75) after substances as biostimulants in a semantic and functional context?

Substances and microorganisms are very different in nature.

What do the authors think about other definitions of biostimulants?

The authors work in organizations in Spain. How are biostimulants regulated in Spain? Is there national regulation in addition to EU regulation? Is there a national biostimulant definition in national regulations?

This information would enrich this manuscript.

Responses:

Dear reviewer, indeed, they are very different, but the definition states in this manner.

For a deeper understanding of the point we have reach, we must reefer to previous classifications, as Calvo et al. (2014) that stablished 5 categories (i. microbial inoculants, ii. humic acids, iii. fulvic acids, iv. protein hydrolysates and amino acids, and v. seaweed extracts) or du Jardin (2015), who expand it into 7, differentiating the beneficial fungi and beneficial bacteria within the category of ‘microorganisms’ and including other categories for ‘substances’ (Humic and fulvic acids, protein hydrolysates and other N-containing compounds, seaweed extracts and botanicals, chitosan and other biopolymers, and inorganic compounds)

Until 2019 biostimulants were included into fertilizers regulation (Regulation (EU) 2009/1107), in a very broad way that led into different regulations of these products in each country.  For example, in Spain legislation, substances with biostimulant properties were included (RD 999/2017).

With the aim of harmonizing the definition (and possibly under additional pressure from the industry and scientific sectors), new EU legislation was introduced in 2019 (Regulation (EU) 2019/1009). However, considering the various classifications that have been made for these products, the definition of plant biostimulant has been simplified. It now takes into account the effect produced in the plant more than its nature, and that is the reason why ´a substance or microorganisms´ is mentioned, but the definition continues with the different effects that a biostimulant could have in the plant.

Dear reviewer, we believe it is not appropriate to add this information to the current manuscript as it could divert the reader’s focus from its primary objective. We consider this information to be useful, but it would be more suitable in a comprehensive dissertation or review on the topic. However, we remain receptive and open to any suggestions you might have for its incorporation into this manuscript. We appreciate your understanding and look forward to your feedback.

Comments 5:

5. It could be recommended carefully checking the data in the appropriate tables and figures in the context of the significance of the obtained differences.

Responses:

Dear review, there truly was an error, we apologize for the mistake. The sentence in Line 401 referenced a difference that do not exist in Figure 7A:

‘although they had been higher during the second harvest.’

It was corrected with the following sentence:

‘although it was higher when the remaining fruits of the last harvest were taken into account.’

Comments 6:

6. The term "biostimulation", of course, is used in a number of articles.

To what extent, in the opinion of the authors, is it correct and necessary to use it?

How does it differ conceptually from the term 'stimulation'?

Responses:

In our opinion, both words are synonymous in their meaning, but the concept of ´biostimulant´ has been extensively used to distinguish these substances (or microorganisms) from the fertilizer products, that act as ´stimulant´ of the plant growth.

Comments 7:

7. It is advisable to present a more detailed coverage of related and close studies in the MS research area in the Introduction and Discussion sections.

Responses:

The reference Lin KH et al., (2019) was included as [60] in Line 500:

‘Instead of subjecting the plant to drought conditions, the combination of RDI with biostimulation has been studied in some crops, such as maize, improving both the yield and water productivity [60], similarly to our trial 2, where…’

Comments 8:

8. Perhaps more justification for the novelty of the work would have been necessary.

Responses:

To emphasize the novelty of this study, the following paragraphs have been included in the MS, in introduction and conclusion.

Line 101:

RDI is widely known in woody crops as a technique to optimize the water use. Its implementation in horticulture has not been reached, as it has shown some disadvantages in production.

With the aim of improving the crop water productivity in pepper, in this study two different techniques have been combined, the implementation of deficit irrigation in pepper through controlling the soil water status, with the application of commercial biostimulation strategies, combining different products. Our hypothesis is that the biostimulation will promote plant the performance when facing the water stress created by the RDI.

Line 579:

This study represents a significant advancement in optimizing water usage in agriculture, combining deficit irrigation strategies and biostimulation. Despite the critical importance of this topic, there has been little research in this area so far, which further highlights the novelty and value of our findings. Our work not only opens new avenues for future research, but also has the potential to transform current agricultural practices, promoting more efficient use of water.

Comments on the Quality of English Language

Could be better.

To improve the quality of English language, the following changes have been done:

Line 350: ‘significantly increase’ into ‘significantly increased’

Lines 457-461: ‘The way in which deficit irrigation was applied was different, while in trial 1, it was promoting a more continuous deficit irrigation, maintaining the soil water potential (Ψ_m) homogeneously between –40 and –50 kPa (Figure 3B). On the other hand, the number of irrigations applied was also reduced in trial 2, alternating days of irrigation reduction respect to the Farmer, that resulted in Ψ_m values ranging from –37 kPa to field capacity values (Figure 3D).‘ into ‘The deficit irrigation was applied was differently in each trial, while in trial 1, a continuous deficit irrigation was applied, maintaining the soil water potential (Ψ_m) homogeneously between –40 and –50 kPa (Figure 3B). On the other hand, in trial 2, irrigation reduction days were alternated with Farmer irrigation, that resulted in Ψ_m values ranging from –37 kPa to field capacity values for this period (Figure 3D).’

Line 503: ‘the light water stress’ into ‘the mild water stress’

Line 548: ‘facilitating thereby the plant growth’ into ‘thereby facilitating the plant growth’.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors studied the effect of different strategies that combine the application of seaweed and microbials biostimulants with regulated deficit irrigation (RDI) strategies on the irrigation water productivity (WPI), fruit quality parameters and soil enzymatic activity in pepper plants (Capsicum annum sp.) under two commercial greenhouse conditions. The trials gained some merit and was recommended for publication in the journal, but some issues needed to be addressed.

The abstract described that the results obtained from the study were few and the content of the results needs to be increased.

Why was there no separation of RDI and biostimulation applications in the ii treatment in the trials?

The study did not have a quality index for peppers, only factors that produce yield. What did not measure quality?

Microbial stimulants were added to the study. How do you explain that using these agents can increase crop yields? Because you only have data on soil enzyme activity.Microbial stimulants were added to the study. How do you explain that using these agents can increase crop yields? Because you only have data on soil enzyme activity.

Measuring soil enzyme activity only once during the entire growth period of a crop was not strong evidence.

 Minor editing of English language required

Author Response

Reviewer 2

Comments and Suggestions for Authors

The authors studied the effect of different strategies that combine the application of seaweed and microbials biostimulants with regulated deficit irrigation (RDI) strategies on the irrigation water productivity (WPI), fruit quality parameters and soil enzymatic activity in pepper plants (Capsicum annum sp.) under two commercial greenhouse conditions. The trials gained some merit and was recommended for publication in the journal, but some issues needed to be addressed.

Comments 1:

1. The abstract described that the results obtained from the study were few and the content of the results needs to be increased.

Responses:

The root water absorption was not mentioned in the abstract, we have included it in line 24: This combined treatment also enhanced the root water absorption and improved the soil enzymatic activity in both greenhouses, suggesting that nutrients in the soil will become more available to plants.

Comments 2:

2. Why was there no separation of RDI and biostimulation applications in the ii treatment in the trials?

Responses:

Dear reviewer, the two trials presented were conducted in commercial greenhouses, which greatly complicated the establishment of more treatments in the agronomical design.

Comments 3:

3. The study did not have a quality index for peppers, only factors that produce yield. What did not measure quality?

Responses:

Dear reviewer, in order to get a quality indicator, the cooperatives' guidelines for pepper commercialization have been followed, classifying the harvested produce into first or second category products for their sale.

Comments 4:

4. Microbial stimulants were added to the study. How do you explain that using these agents can increase crop yields? Because you only have data on soil enzyme activity.

Responses:

In in-vitro trials carried out during the development of this same product, have demonstrated that this specific bacterium successfully colonizes the root, creating a biofilm. In those trials a significant enhance of root development has been observed, here, the root development is inferred through the root water absorption (Table 3).

Find attached the link with the product specifications in different regions:

FMC Denmark. Product specifications, which include the mode of action.

https://ag.fmc.com/dk/sites/default/files/2023-09/Accudo_xt_lbl_dk.pdf

FMC Korea. Brochure that includes photographs of the biofilm formation.

https://www.fmckoreamall.com/goods/goods_view.php?goodsNo=1000000024

These studies have been referenced in the discussion, lines 548-550:

‘ and the specific bacterium that have been added in this trial has been reported to successfully colonize the root, creating a biofilm and promoting root development [70].’

Comments 5:

5. Measuring soil enzyme activity only once during the entire growth period of a crop was not strong evidence.

Responses:

Dear reviewer, as many processes in the soil, the soil enzymatic activities, as they are immersed in biogeochemical cycles, are influenced by multiple factors. A deeper deliberation about this topic can be found at Bell et al., 2014 (DOI: 10.1007/s10533-013-9935-0) and the special issue in which is contained. “Consequentially, many questions remain unanswered in regard to the environmental controls on enzyme production, turnover, and in situ activity that facilitate soil nutrient cycling processes, which limits our ability to incorporate enzymes into biogeochemical models.”.

We consider that by analysing those parameters in the most demanding period for these crops, as it is full harvest, was an approach to understanding the mechanism by which those biostimulants act in pepper. Correlations have been evidenced in the 2 trials between biostimulated treatment and enzyme activities, that are indeed a result of microbiological process in the soil, however, further research is still needed.

Comments on the Quality of English Language

 Minor editing of English language required

The text has been reviewed, modifying some expressions that were not clear.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments from the second round of review:

The authors' efforts and ideas to improve the manuscript after the first round of comments are very constructive and interesting.

This position certainly deserves respect.

Definitely this manuscript should be published.

At the same time, I would encourage authors to go through the manuscript again carefully, evaluate everything with fresh eyes, and make improvements in the context of the first-round review comment on a more complete, conceptual, and broader basis.

If all comments from the first round of review are answered fully and comprehensively (without any exceptions), this will strengthen and improve the manuscript - definitely.

All information discussed by the authors in their responses is appropriate and extremely important for this manuscript.

Its addition to this manuscript will not divert the reader's attention from its primary purpose.

On the contrary, this information and its more complete interpretation will increase readers' interest in the article if it is published.

In addition to the above, there are a few more important points.

The review by Calvo et al. (2014) builds on the concepts of another work, namely du Jardin (2012).

Therefore, it is important to mention exactly the work that is pioneering.

Also, the authors of the manuscript write that "... the concept of ´biostimulant´ has been extensively used to distinguish these substances (or microorganisms) from the fertilizer products, that act as ´stimulant´ of the plant growth."

But according to their definitions, fertilizers and stimulants work by completely different ways.

Authors should provide appropriate explanations of their concepts in more detail in this case.

Judging by a number of their responses to comments from the first round of peer review, the authors need to work more deeply on a number of aspects in the manuscript and ensure more responsible content there.

Could be better.

Author Response

Reviewer 1 – Round 2

Comments and Suggestions for Authors

Comments from the second round of review:

The authors' efforts and ideas to improve the manuscript after the first round of comments are very constructive and interesting.

This position certainly deserves respect.

Definitely this manuscript should be published.

At the same time, I would encourage authors to go through the manuscript again carefully, evaluate everything with fresh eyes, and make improvements in the context of the first-round review comment on a more complete, conceptual, and broader basis.

If all comments from the first round of review are answered fully and comprehensively (without any exceptions), this will strengthen and improve the manuscript - definitely.

All information discussed by the authors in their responses is appropriate and extremely important for this manuscript.

Its addition to this manuscript will not divert the reader's attention from its primary purpose.

On the contrary, this information and its more complete interpretation will increase readers' interest in the article if it is published.

In addition to the above, there are a few more important points.

The review by Calvo et al. (2014) builds on the concepts of another work, namely du Jardin (2012).

Therefore, it is important to mention exactly the work that is pioneering.

Also, the authors of the manuscript write that "... the concept of ´biostimulant´ has been extensively used to distinguish these substances (or microorganisms) from the fertilizer products, that act as ´stimulant´ of the plant growth."

But according to their definitions, fertilizers and stimulants work by completely different ways.

Authors should provide appropriate explanations of their concepts in more detail in this case.

Judging by a number of their responses to comments from the first round of peer review, the authors need to work more deeply on a number of aspects in the manuscript and ensure more responsible content there.

Dear reviewer, we consider that the report made for du Jardin (2012) to the European commission was pioneer, he presented and extensive review of different substances that categorized as biostimulant, and he arranged them in 8 different categories, whereas the novelty in Calvo et al., (2014) was the rearrangement of these substances, and the inclusion of microbial biostimulant in 5 different categories.

There is no wrong or right classification, we think that it is indeed a common process for an emerging concept.

This issue and the previous ones have been incorporated in the MS through the following changes:

Previous paragraph in line 72:

Biostimulants have been reported to effectively mitigate the abiotic stress [32,33], a problem that is often faced by crops that grow in semi-arid climates, where water re-sources are scarce and low quality. However, the quantification of these effects across all crop varieties remains to be accomplished, and they would be of great help for mini-mizing the drawbacks of RDI in crops growing in these areas. Defined by the European Biostimulant Industry Council [34], biostimulant are ‘A substance or microorganisms whose function when applied to crops or the rhizosphere is to stimulate natural pro-cesses to enhance nutrient uptake and efficiency, crop quality, and tolerance to abiotic stress, benefitting both plant yield and vigor’

Has been replaced with:

The term plant biostimulant has been incorporated into society not many years ago, going through different classifications and definitions in the scientific community (Calvo et al., 2014; du Jardin, 2015, 2012).  Until 2019, biostimulants were included in broad terms into the European regulation (Regulation (EU) 2009/1107) (European Parliament, 2009), which led into different regulations of these products for each country. With the aim of harmonizing the definition between countries, a new EU legislation for fertilising products was introduced in 2019 (Regulation (EU) 2019/1009) (European Parliament, 2019), in which plant biostimulants were defined as ‘Certain substances, mixtures and micro-organisms that not being an input of nutrients, stimulate plants’ natural nutrition processes’. This definition includes the terms substances and microorganisms indistinctively, due to it now takes into account the effect produced in the plant more than its nature, ‘Where such products aim solely at improving the plants’ nutrient use efficiency, tolerance to abiotic stress, quality traits or increasing the availability of confined nutrients in the soil or rhizosphere, they are by nature more similar to fertilising products than to most categories of plant protection products. They act in addition to fertilisers, with the aim of optimising the efficiency of those fertilisers and reducing the nutrient application rates.’, as industry claimed (EBIC – The European Biostimulants Industry Council, 2023).

Nowadays, both, the legislation and different scientific literature reports that biostimulants can effectively mitigate abiotic stress (du Jardin, 2015; Yakhin et al., 2017), a problem that need to be addressed, as it is often faced by crops that grow in semiarid climates, where water resources are scarce and low quality. However, the quantification of these effects across all crop varieties remains to be accomplished, and they would be of great help for minimizing the drawbacks of RDI in crops growing in these areas.

Comments on the Quality of English Language

Could be better.

The manuscript has been reviewed in its entirety, including reviewer 2 who found it adequate.

Reviewer 2 Report

Accept in present form.

Author Response

The reviewer has accepted the last requested changes, and has not found any issues with the quality of the english language.