Rhamnolipid-Stabilized Essential Oils Nanoemulsions: Sustainable Biopesticides and Biostimulants with Potential for Crop Protection

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript on rhamnolipid-stabilized essential oil nanoemulsions as sustainable biopesticides and biostimulants for crop protection is well-structured and presents an innovative approach. It integrates multiple novel aspects, including the use of essential oils, a dual mechanism combining plant defense priming with environmentally friendly pest control strategies. This interdisciplinary approach further underscores the study’s originality and potential impact.

Some points to be addressed:

Authors should include rosemary essential oil (EO) in section 2.1 (Materials) or remove from section 2.3 (Design and production of RLs NEs).  

Pseudomonas fluorescens, Azospirillum argentinense, Trichoderma spp., and Arabidopsis must be italicized throughout the manuscript.

In line 145, the abbreviation Pst should be used instead of the full name, as it was already defined in line 124 as Pseudomonas syringae pv. tomato DC3000 (Pst).

The study attributes insecticidal effects to EO content, but further testing of the 3 individual vs. combined EO effects could clarify whether synergy is occurring and which EO(s) contribute most to the observed efficacy. This would provide valuable insights into optimizing EO selection for maximum efficacy in pest control.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

1. This investigation aims to develop rhamnolipid (RLs) stabilized nanoemulsions (NEs) encapsulating essential oils (EOs) as potential biopesticides and biostimulants for agroindustrial applications.
2. This study is original and novel. The authors have investigated the capacity of natural RLs to form stable NEs containing EOs. The RLs are interesting due to their dual functionality as emulsifying and bioprotective agents.

3. There is no other comparable study with other published material. This study provides the first evaluation of RLs to NEs containing EOs in this material plant (Arabidopsis thaliana), soil-beneficial microorganisms and an insect species (Myzus persicae).
4. The abstract should contain the following terms: Arabidopsis thaliana and Myzus persicae.
5. Authors should correct throughout the manuscript that scientific names are in italics.
6. The methods are detailed but can be improved. In 2.10 the authors must detail where, when or in what season the insect material was collected.
7. In Figure 3. A-1, the authors should clarify whether it is "n aphids/plate = 2" or "n aphids/plant = 2" and how the two adult aphids were transferred to the plant.
8. The conclusions are pertinent and consistent with the results.
9. References are appropriate, but they can be improved. For example, using italics in scientific names.
10. Tables and figures are appropriate.

Comments on the Quality of English Language

The English could be improved to more clearly express the research.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors examined the efficacy of rhamnolipids mixed with essential oils of some plant species on Pseudomonas syringae, indicating the potential of the combinations of these agents for measures to regulate the bacterial disease in plants. They also provide data that can be considered reducible effects of the substances on the population of the aphid, Myzus persicae, while no lethal effects were confirmed. All results were derived from experiments conducted in laboratory and, accordingly, the extension of their results seem to be rather confined. In this sense, the title should be changed, not indicating the tested agents as control measures but pointing out the effects of the agents on bacterial plant pathogens, such as “Bactericidal effects of rhamnolipid, stabilized with essential oils in nanoemulsion”. Revision of the manuscript is needed.

 

Comments

There are many typographical errors in the text and they must be corrected. All scientific names must be italicised but not in subheadings which are written in italics. Also, do not italicise “spp.” in Trichoderma spp. (L334)

Some descriptions in Results should be moved to Materials and Methods or Discussion, avoiding referring to citation.

Keywords

Any words used in the title should be avoided to list in Keywords. Instead, Pseudomonas syringae may be listed here. Also, it may be better to replace “insect” for “aphid” or “Myzus persicae”.

L85

Refer to the city and country of the company.

L85, 91, 97, 100, 109, 117, 123, 142, 148, 160 and 173

Refer to the time when the experiments in these subsections were performed.

L94

Specify the company and its location (city and country).

L159

What is the method? It must be explained in detail.

L163

Explain how the aphids were obtained.

L168

Use nymph for hemimetabolous insects.

L169

Did you use a “dissecting microscope” or “binocular” microscope?

L181

Explain how the solution was made.

L183

The URL can be deleted. It is shown at L115-116.

L183

Italicise “GP”.

L195-199

Move these sentences to Materials and Methods.

Show the data for L199-203.

L234-238

Move to Materials and Methods.

L239-246

Do not refer to citation in Results. Any sentences that need citation should be moved to Materials and Methods or Discussion.

L248-254

Move to Materials and Methods.

L277-288

Move to Discussion.

L290

Change “generalist” for “polyphagous”. Aphids are not omnivorous but phytophagous.

Figure 3

What is “####”?

L341-348

Move to Materials and Methods or Discussion.

L368-369

Refer to citation for this sentence.

L399

Write as “A. thaliana leaves” (‘s is not needed).

L440-443

Citation is needed for these sentences.

L446-447

Is “Dorey” the name of a researcher, or a genus name? If the case is former, it must not be italicised. But the name does not appear the reference [7].

 

more comments:

> Please consider providing some more specific comments addressing the following points:
> • What is the main question addressed by the research?
This is the most serious matter that this manuscript is considered difficult to accept. The authors state at L81-83, “Our findings confirm that RLs-stabilized EOs-NEs are effective, sustainable bioprotectants and biostimulants, supporting pesticide-free agroindustry”. The authors did not test the application of essential oils encapsulated with nanoemulsion on crop plants in field. Furthermore, the plant they tested was A. thaliana, not agricultural crop maintained in laboratory (L117 - 120). Thus, there is violation in two issues against their statement: the level of the experiment was never comparable to agricultural field and no agricultural crops were examined. Also, the efficacy of their agents was not examined on alive field-plants infected by any pathogens. The authors examined the application of their agents to crops but arranged no control plants without any agents in field. Therefore, they do not give any supporting data for “pesticide-free” in the manuscript.

> • Do you consider the topic original or relevant to the field? Does it address a specific gap in the field? Please also explain why this is/ is not the case.
As the authors write in the title “for crop production”, experiments to address the efficacy of the agents had to examine target crops in field cultivation. This is not the case for their experiments. In this regard, the authors, if trying to publish the study, have to change the title as “Suppressive effects of essential oils encapsulated with nanoemulsions on pathogens and aphids in laboratory”. However, it still remains the scientific value of the study to publish.

• What does it add to the subject area compared with other published material?
The absolute condition of the manuscript for publication is to provide evidence the efficacy of the agents on the pathogen comparing agent-treated “agricultural” crop with untreated check. Without such evidence, their study could not add scientifically meaningful knowledge to the study area, even though it would supply some additive unpublished information for future experiments.

• What specific improvements should the authors consider regarding the methodology?
Please see the previous review report and statements above.

• Are the conclusions consistent with the evidence and arguments presented and do they address the main question posed? Please also explain why this is/is not the case.
See above. The results in this manuscript do not reach the level of agricultural practice.

• Are the references appropriate?
Please see the first review in which I have pointed out any references to cite more.

• Any additional comments on the tables and figures.
If concentrated on laboratory experiments, they are sufficient. However, the acceptance of the manuscript in the journal is a different thing. 

Comments on the Quality of English Language

Althouth the current manuscript is written well in English, English should be edited by a native English speaker before the submission of a revised manuscript.

Author Response

Please see the attachment

Author Response File: Author Response.pdf