Identification of Differential-Expressed Genes in Banana-Biostimulant Interaction Using Suppression Subtractive Hybridization

Round 1

Reviewer 1 Report

Comments during the first round of peer review of the manuscript (MS) "Identification of Differential-Expressed Genes in Banana-Biostimulant Interaction using Suppression Subtractive Hybridization" (authors Tatiana Chavez-Navarrete, Luis Sanchez-Timm, Ricardo Pacheco-Coello, Niranjan Baisakh and Efré Sann tos-Ordóñez) submitted to the journal Agronomy MDPI:

1) This manuscript is related to the consideration of the molecular mechanisms of  biostimulants action.

This allows its authors to make a significant contribution to understanding the fundamentals of biostimulants action on plants.

The studies carried out are, of course, very important for the further development of the biostimulants science.

Therefore, this manuscript should be recommended for publication in the journal Agronomy MDPI.

2) At the same time, this manuscript should be significantly optimized and more deeply conceptualized.

A number of aspects need to be substantially clarified.

3) In particular, the authors' emphasis on "an organic alternative to control harmful pathogens", etc. (lines 16, 43-53, 368-371) raises questions about the correct categorization of the product in question.

As far as is known, biostimulants are characterized/distinguished by their effect on growth and resistance to abiotic stresses.

When the effect under conditions of biotic stress is discussed, it could be another category of products.

Although it is not excluded and an additional action in addition to the main one.

In any case, it is absolutely necessary to clearly define the functionality and category/categories of the product in question.

This is to some extent a difficult task, as there are many different definitions and classifications of biostimulants.

It is necessary to use for justification several approaches to the definitions of biostimulants, which are widely presented both in the scientific literature and in regulatory documents.

And based on their analysis and (this is closer to the end of the manuscript) the authors' own data, it is necessary to present their unique understanding of the essence of the problem.

4) More conceptual discussion is needed for "sustainable and environmentally friendly agriculture" essence (line 54).

5) It is necessary to more clearly present, formulate and organize the rationale, actualization, conceptualization, hypothesis, clear aims and objectives in this manuscript.

6) The authors of the manuscript work for organizations in Ecuador and the United States.

Therefore, it would be logical for the authors to provide definitions, both scientific and regulatory, in the manuscript, accepted in these countries and compare them with definitions, for example, in China and the European Union.

In addition, the authors use the terminology not only of biostimulants, but also of biofertilizers.

It is necessary to clearly distinguish between these concepts, including for product under consideration in this MS.

7) It is necessary to clarify the accuracy and reliability of the results and data in Table 1 and Figures 1-5

8) It is necessary to analyze and discuss the table and figures in the manuscript in more detail and to involve a wider range of literary sources in the discussion.

9) It is necessary to review the authors earlier related works for common aspects with this manuscript and discuss these in more detail.

10) The Conclusions section needs to be substantially rewritten and edited to be more conceptual and systemic.

The same applies for the Introduction and Discussion MS sections.

11) A more detailed description of the product under study should be provided.

Author Response

Dear Editor

We appreciate all the comments from the reviewers. All the comments were addressed and are indicated below:

Reviewer 1:

1) This manuscript is related to the consideration of the molecular mechanisms of biostimulants action. This allows its authors to make a significant contribution to understanding the fundamentals of biostimulants action on plants. The studies carried out are, of course, very important for the further development of the biostimulants science. Therefore, this manuscript should be recommended for publication in the journal Agronomy MDPI.

2) At the same time, this manuscript should be significantly optimized and more deeply conceptualized. A number of aspects need to be substantially clarified.

3) In particular, the authors' emphasis on "an organic alternative to control harmful pathogens", etc. (lines 16, 43-53, 368-371) raises questions about the correct categorization of the product in question. As far as is known, biostimulants are characterized/distinguished by their effect on growth and resistance to abiotic stresses. When the effect under conditions of biotic stress is discussed, it could be another category of products. Although it is not excluded and an additional action in addition to the main one. In any case, it is absolutely necessary to clearly define the functionality and category/categories of the product in question. This is to some extent a difficult task, as there are many different definitions and classifications of biostimulants. It is necessary to use for justification several approaches to the definitions of biostimulants, which are widely presented both in the scientific literature and in regulatory documents. And based on their analysis and (this is closer to the end of the manuscript) the authors' own data, it is necessary to present their unique understanding of the essence of the problem.

Response:

Line 16-18: Sentences were modified.

Line 43-73: A new paragraph was added.

Lines 99-104: New sentences were added and/or modified

Lines 119-129: A new paragraph was added.

 

4) More conceptual discussion is needed for "sustainable and environmentally friendly agriculture" essence (line 54).

Response:

Lines 88-93: New sentences were added

5) It is necessary to more clearly present, formulate and organize the rationale, actualization, conceptualization, hypothesis, clear aims and objectives in this manuscript.

Response:

Lines 130-138: A new paragraph was added.

 

6) The authors of the manuscript work for organizations in Ecuador and the United States

Therefore, it would be logical for the authors to provide definitions, both scientific and regulatory, in the manuscript, accepted in these countries and compare them with definitions, for example, in China and the European Union. In addition, the authors use the terminology not only of biostimulants, but also of biofertilizers. It is necessary to clearly distinguish between these concepts, including for product under consideration in this MS

 Response:Lines 377-395: A new paragraph was added. See response for comment # 3 7) It is necessary to clarify the accuracy and reliability of the results and data in Table 1 and Figures 1-5  Response:The bionformatic software used for the analysis and annotation of the sequences was blast2Go. The main idea was to see major pathways involved in the plant-biostimulant interaction. Several reports (as indicated in lines 43-73; 99-104; 130-138) indicate that the use of biostimulants in plants induce tolerance to (a)biotic stress and is related for the improvement of the nutritional status or growth. Therefore, genes and main pathways involved was detected by using the software Blast2GO, confirming the pathways related to growth and tolerance to (a)biotic stress. Furthermore, selected genes were tested for overexpression by using Rt-qPCR (Figure 5), confirming overexpression of genes related to specific pathways. 8) It is necessary to analyze and discuss the table and figures in the manuscript in more detail and to involve a wider range of literary sources in the discussion.

Response:

Lines 371-376: Paragraph was added in the discussion

 

9) It is necessary to review the authors earlier related works for common aspects with this manuscript and discuss these in more detail.

In lines 357-259 it was discussed with an early work from 2016 of a SSH on banana and the pathogen Pseudocercospora fijiensis.

Lines 371-376: A new paragraph was added.

 

10) The Conclusions section needs to be substantially rewritten and edited to be more conceptual and systemic. The same applies for the Introduction and Discussion MS sections.

Conclusions: Lines 500-508.

Introduction: Lines 43-73; lines 88-93; lines 99-104; lines 119-138.

Discussion: lines 371-396.

 

11) A more detailed description of the product under study should be provided.

Lines 409-421: New sentences were added.

 

Author Response File: Author Response.docx

Reviewer 2 Report

Comments to the Author

 

Biostimulants are emerging as an essential component in sustainable agricultural practices.This paper designs a foliar liquid biostimulant and evaluates its effects on plant at both physiology and molecular level. Understanding the influence of biostimulants on plant physiology and molecular pathways will gain a deeper knowledge of their molecular impacts may lead to an increase in their efficiency. The methods and analysis pipelines described in this manuscript are in general suitable for this kind of analysis.

Special comments:

(1) Most of the figures are not professional enough to publish and have a lot to improve. Please optimize picture layout and improve clarity. Other suggestions are as follows:

In figure 1, it is better to lowercase the alphabet representing statistically significant. Please adds Y-axis in the figure, as well as the statistical method in the figure legends.

In figure 2B and 4C, the word size is too small thus not clear enough to readers.

In figure 3 and figure 5, the name of the Y-axis is not accurate.

In figure 5, please revise “1s” into “1 week”

(2) The authors declare that they design the foliar liquid biostimulant (as described in the abstract), but the biostimulant used in this study seems to be a widely applied standard in banana plantations (as described in the introduction). Please fix it.

(3) In results part, no significant differences were observed for the changes on MSI and chlorophyll accumulation between control and the plants treated with the biostimulant. This is contrary to the conclusion as described in the abstract.

(4) SSH analysis identified 270 expressed sequence tags, but approximately 15% of coincidence was encountered with other plant organisms. Why?

(5) In general, it is lack of deep-level interpretation for the function of 270 up-regulated genes in the results part. Please reannotation of these expressed sequence tags by the genome sequence of banana.

(6) Please use an italic font for gene name in the whole manuscript.

(7) Line 22: Please revise “the of” into “application of”

(8) Line 78: It is italic font for the sentence “The Two-way ANOVA showed that the treatment was statistically significant (p = 0.0314)”. Please fix it. 

Author Response

Dear Editor

We appreciate all the comments from the reviewers. All the comments were addressed and are indicated below:

Reviewer 2:

(1) Most of the figures are not professional enough to publish and have a lot to improve. Please optimize picture layout and improve clarity. Other suggestions are as follows: In figure 1, it is better to lowercase the alphabet representing statistically significant. Please adds Y-axis in the figure, as well as the statistical method in the figure legends. In figure 2B and 4C, the word size is too small thus not clear enough to readers. In figure 3 and figure 5, the name of the Y-axis is not accurate. In figure 5, please revise “1s” into “1 week”

Response:

Figures were changed.

(2) The authors declare that they design the foliar liquid biostimulant (as described in the abstract), but the biostimulant used in this study seems to be a widely applied standard in banana plantations (as described in the introduction). Please fix it.

Lines 136-138: A sentence was modified.

(3) In results part, no significant differences were observed for the changes on MSI and chlorophyll accumulation between control and the plants treated with the biostimulant. This is contrary to the conclusion as described in the abstract.

Line 145 was added.

(4) SSH analysis identified 270 expressed sequence tags, but approximately 15% of coincidence was encountered with other plant organisms. Why?

Response:

Probable the annotation of the banana genome was not good enough in the database of Blast2GO. We think that those 15% were from banana origin and/or from microorganism (fungus).

 

(5) In general, it is lack of deep-level interpretation for the function of 270 up-regulated genes in the results part. Please reannotation of these expressed sequence tags by the genome sequence of banana.

Response: We use the software Blast2GO to make a complete analysis of the sequences from the database available in this software. Several sequences annotated in banana genomes correspond to growth and nutrition status of plants and/or related to tolerance/resistance to (a)biotic stress.

See lines 479-481.

 

(6) Please use an italic font for gene name in the whole manuscript.

Gene names were put in italic font.

 

(7) Line 22: Please revise “the of” into “application of”

Response:

Line 23 was modified.

(8) Line 78: It is italic font for the sentence “The Two-way ANOVA showed that the treatment was statistically significant (p = 0.0314)”. Please fix it.

Response:

Lines 145 was modified.

 

Author Response File: Author Response.docx