Prospection of Nematotoxic Aqueous Seeds Extracts Derived from the Preserved Arachis (Fabaceae) Germplasm Bank

Round 1

Reviewer 1 Report

All alternatives to synthetic nematicides are welcome.  The article is well written with minor mistakes. However there are certain things that need to clarified. The most part of my suggestions and comments are in the document.

1. The main thing is the use of ethanol at 70% as the positive control Nematodes are quite sensitive to ethanol even at low concentrations and usually in this kind of studies it is used a nematicide at the recommended concentrations as the positive control. With ethanol at 70% all nematodes are dead. So, whats the objective?

2. Why the choice of using blood and not another non target organism like, for example, C. elegans? In my opinion does not prove anything. 

3. The authors also should suggest more experiments in the soil because in the lab everything is controlled and in the soil the chemicals can react with other chemicals and act different. The references could be more extensive in the discussion. The authors almost don't compare their studies with others and there are so many with plant extracts and Meloidogyne and other nematodes. This part maybe should be better explored. This is only my opinion.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 1,

Thank you very much for taking the time to review this manuscript. Please find the detailed responses to your questions below. The eventual changes has been made in the re-submitted file.

1. The main thing is the use of ethanol at 70% as the positive control Nematodes are quite sensitive to ethanol even at low concentrations and usually in this kind of studies it is used a nematicide at the recommended concentrations as the positive control. With ethanol at 70% all nematodes are dead. So, whats the objective?

Answer:

The 70% ethanol is conventionally used in the in vitro nematotoxicity bioassays carried out in our laboratory as a substitute for synthetic nematicides such as Aldicarb, Crop Star and Rugby, which are highly toxic. This allows professionals to avoid exposure to these agrochemicals, which are harmful to human health. Finally, the intention of using 70% ethanol is to confirm the death of that portion of J₂ of Meloidogyne incognita, representing the biosensor (positive control).

2. Why the choice of using blood and not another non target organism like, for example, C. elegans? In my opinion does not prove anything.

Answer:

In vitro cytotoxic evaluation using a hemolysis assay demonstrated that the ACE has no hemolytic activity against bovine blood or isolated bovine red blood cells indicating that these molecules possibly are not risky to non-targeted mammalian organisms. This analysis is based on a study conducted by Rocha et al. (2017) (https://doi.org/10.1016/j.biori.2017.10.003). The study examined extract, fractions, and isolated metabolites from C. ensiformis seeds indicated their activity against M. incognita but did not present hemolytic activity. Similarly, studies using protein extract of Caryocar brasiliense seeds demonstrated no hemolytic activity, despite its insecticidal potential against pest insects in in vitro tests (Costa, Franco, Migliolo, & Dias, 2015 - doi:10.5539/jas.v7n6p197 - URL: http://dx.doi.org/10.5539/jas.v7n6p197).

3. The authors also should suggest more experiments in the soil because in the lab everything is controlled and in the soil the chemicals can react with other chemicals and act different. The references could be more extensive in the discussion. The authors almost don't compare their studies with others and there are so many with plant extracts and Meloidogyne and other nematodes. This part maybe should be better explored. This is only my opinion.

Answer:             

An in vivo greenhouse bioassay is being designed to evaluate the performance of the crude aqueous extract AM1 (Arachis stenosperma) in controlling the phytonematoid Meloidogyne incognita using soybean (Glycine max) and tobacco (Nicotina tabaccum) plants.

The suggestion was accepted and as suggested, the results generated in this work were compared in a more comprehensive and ostensible way with those available in the literature. In this sense, examples were added describing the effect of extracts from different parts of different species belonging to the Fabaceae family, as well as the nematotoxic effect of these extracts on the control of other phytonematoid species.

Author Response File: Author Response.pdf

Reviewer 2 Report

This article is about the Prospection of nematotoxic aqueous seeds extracts derived 2 from the preserved Arachis (Fabaceae) germplasm bank.

Abstract and introduction

The abstract and introduction are disorganised and don’t follow a clear line of thought. It is usual to start with the general view of the problem and then narrow it down until the specific topic of interest. For instance, you should start mentioning the Plant-parasitic nematodes in general, follow by the root-knot nematodes and finally the species M. incognita.

I think information on the importance of M. incognita, other species present and why M. incognita was chosen is missing.

Are the other species belonging to the tropical root-knot nematodes considered high risk organisms?

Materials and methods

Where did the isolate of Meloidogyne incognita come from? Is it laboratory/institution that provides pure cultures of root-knot nematodes?

How did you identify/confirm the species use was M. incognita?

How did you based the choice of the species of Arachis to be tested? Have some of them

If eggs were available why egg hatching test weren’t performed?

It is common to evaluate at 0, 24h, 48h, 72h, why just one time point was used?

It would have been useful to determine the chemical compounds present in the ACE’s evaluated.

Results

It wasn’t included the results of the statistical analysis, at least a paragraph on the results of the statistics applied should be added.

One of the most important parts of a manuscript are the results and as such they must be presented and discussed in a clear way. The results and discussion didn’t show appropriately the importance of this work or the relevance they may have for current management programmes.

Results should be further discussed. The conclusions are not a summary of the results, it should be rewritten.

Additional, comments are sent in the PDF version of the manuscript.

Comments for author File: Comments.pdf

 The manuscript could be improved as some paragraphs are confusing and long. The Figures legends are too long and should be written in a smaller font size. The manuscript lacks a well-structured discussion of the results.

Author Response

Dear Reviewer,

Thank you very much for taking the time to review this manuscript. Please find the detailed responses to your questions below. Questions are marked with a dot. The suggested modifications were made throughout the article and can be found in the re-submitted file.

• Does the introduction provide sufficient background and include all relevant references? Must be improved  

Answer:  The suggestion was accepted and the introduction was reformulated in a more fluid way and with more relevant references aimed at the proposed work.

• Are all the cited references relevant to the research? Can be improved

Answer:The suggestion was accepted and references not directly related to the research were removed from the text.

• Is the research design appropriate? Must be improved

Answer: The research design is appropriate since its aim is to select and characterize new extracts from Fabaceae plant species that effectively control the phytonematode Meloidogyne incognita. The research emphasizes the importance of valuing species in germplasm banks. The research followed the steps in a logical order. Selection of the botanical family to study was based on literature research and from professionals who maintain and work with the selected species. Extracts were obtained following established protocols, and a target (phytonematoid) was chosen for evaluation based on extensive literature research. The M. incognita is a serious problem for Brazilian and global agriculture, and the control method using plant extracts and phytochemicals is eco-friendly. Conducting a series of in vitro bioassays in a logical order to select the most effective candidates against the target, including nematotoxicity bioassay (viability and recovery), concentration curve, thermal stability, and cytotoxicity that are commonly used in this scientific approach.

• Are the methods adequately described? Can be improved

Answer: The methods used to carry out the bioassays were reviewed and followed the protocols already established and described in the literature.

• Are the results clearly presented? Must be improved

Answer: The presentation of the research results has been revised and rewritten in order to make the data presented clearer and more fluid.

• Are the conclusions supported by the results? Must be improved

Answer: The research findings have been updated, rewritten, and reorganized to reflect the results of the study accurately and clearly.

Abstract and introduction

• The abstract and introduction are disorganized and don’t follow a clear line of thought. It is usual to start with the general view of the problem and then narrow it down until the specific topic of interest. For instance, you should start mentioning the Plant-parasitic nematodes in general, follow by the root-knot nematodes and finally the species M. incognita. I think information on the importance of M. incognita, other species present and why M. incognita was chosen is missing.

Answer: All suggestions regarding the disorganization and lack of a line of thought in the summary and introduction of the work were reviewed and rewritten to make them clearer and more fluid.

• Are the other species belonging to the tropical root-knot nematodes considered high risk organisms?

Answer: Yes. However, according to a document released by the Ministry of Agriculture, Livestock and Supply - MAPA / Brazil, the species M. incognita is still considered to pose the greatest risk to crops of economic interest. The reason for this inference is related to some characteristics observed for this phytoparasite, discussed throughout the manuscript.

Materials and methods

• Where did the isolate of Meloidogyne incognita come from? Is it laboratory/institution that provides pure cultures of root-knot nematodes?

Answer: The isolate of M. incognita race 3 used in the research was kindly provided by the Nematology Laboratory, located at Embrapa Genetic Resources and Biotechnology. It is important to note that the mentioned species is kept on tomato and tobacco plants in a greenhouse belonging to the Laboratory for the Prospection of Bioactive Compounds (LPCB), located in the same research unit as the aforementioned.

• How did you identify/confirm the species use was M. incognita?

Answer: The M. incognita species is kept in a bank of microorganisms which strictly follows the protocols for maintaining the quality of the inoculum supplied for the research carried out at the Unit. The Nematology Lab routinely conducts enzymatic tests to authenticate the isolates offered for research purposes.

• How did you based the choice of the species of Arachis to be tested? Have some of them

Answer: The Arachis species were chosen with the help of researcher Dr. José Vals Montenegro, who is responsible for research into the conservation and genetics of these species at Embrapa Recursos Genéticos e Biotecnologia and has extensive knowledge of the subject. A more detailed description of the Arachis species has been added to the paper's methodology.

• If eggs were available why egg hatching test weren’t performed?

Answer: The focus of the work was to evaluate the action of the extracts against the infective phase of the gall nematode M. incognita using in vitro bioassays. In vitro bioassays on the hatching of second-stage juveniles should be carried out in the near future.

• It is common to evaluate at 0, 24h, 48h, 72h, why just one time point was used?

Answer: The in vitro nematotoxicity bioassay followed established protocols in the literature. In addition, 48 hours was established at the LPCB as the cut-off time for the nematotoxic action of the extracts evaluated. Once the most effective extracts have been chosen, an exposure time curve is drawn up.

• It would have been useful to determine the chemical compounds present in the ACE’s evaluated.

Answer: The aqueous crude extracts of Arachis seeds selected in the study are currently being fractionated by dialysis on membranes with a pore size of 3.5 KDa. The fractions, called internal dialysate and external dialysate, will then be evaluated by in vitro nematotoxicity bioassay and the active fractions will be purified by high-performance liquid chromatography on C18 reverse phase columns. The fractions obtained will be tested against J₂ juveniles of M. incognita and those showing nematotoxic activity will be analyzed by mass spectrometry to identify the active compounds.

Results

• It wasn’t included the results of the statistical analysis, at least a paragraph on the results of the statistics applied should be added.

Answer: The suggestion was accepted, and the results of the statistical analysis will be discussed for each of the conducted bioassays.

• One of the most important parts of a manuscript are the results and as such they must be presented and discussed in a clear way. The results and discussion didn’t show appropriately the importance of this work or the relevance they may have for current management programmes.

Answer: The suggestion was accepted, and the parts of the manuscript related to the results and discussion were re-evaluated and repaired to give more fluidity and concatenation to the information, highlighting the importance of the results generated for genetic resource valuation programs.

• Results should be further discussed. The conclusions are not a summary of the results, it should be rewritten.

Answer: As suggested, the results have been discussed in greater depth and the conclusion of the manuscript has been rewritten.

• The manuscript could be improved as some paragraphs are confusing and long. The Figures legends are too long and should be written in a smaller font size. The manuscript lacks a well-structured discussion of the results.

                                                                                                                      Answer: For the purpose of enhancing the clarity and flow of the article, we carefully revised the writing, with a focus on addressing lengthy paragraphs. Additionally, we condensed the figure captions by utilizing a smaller font. Furthermore, we reorganized the results discussion section, as mentioned earlier.

Author Response File: Author Response.pdf