A Mixture of Piper Leaves Extracts and Rhizobacteria for Sustainable Plant Growth Promotion and Bio-Control of Blast Pathogen of Organic Bali Rice

Round 1

Reviewer 1 Report

The manuscript presents interesting, quite innovative research results.  Detailed comments are in teh MS text.

Comments for author File: Comments.pdf

Author Response

• Information of the corresponding author should be provided

Ans : E-mail id and Tel No of the corresponding author are provided. Line 17.

• Grammar mistake. Additional information about these metabolites should be added. More information and examples should be added.

Ans : Grammar correction, information on secondary metabolites, and more examples of PGPR is now provided. The whole introduction part is now revised.

• Do the authors know of any research publications on this topic? If so, please describe the previous research results of other authors.

Ans : Yes. The information on previous research publications on the topic and previous research results of the authors are now provided. Line 121-130.

• The aim of the study should be described more precisely.

Ans : The aim of the study is now mentioned. Line 130-132.

• Do the authors know what active substances are included in the extract?

Ans : Yes. It is mentioned now. Line 139-140.

• When was the experiment conducted?

Ans : This research was carried out during the rainy season (October-April) 2019 in the rice fields of the village of Senganan, Penebel, Tabanan Bali, Indonesia Line 163.

• The results presented in Table 1 should be accurately described.

Ans : The results presented in Table 1 are now accurately described. Line 217-230.

• The legend in Fig 1 needs a lot of work. It has to be shelf explanatory.

Ans : The legends are now properly presented. Line 235-244.

• The legend (Table 1) needs a lot of work. It has to be shelf explanatory. Please correct in all tables and figures.

Ans : The legend and Fig in table are now corrected. Line 122-131.

• All results should be described in more detail. It should be specified which variants of the experiment caused the most significant changes in the value of the measured parameter and the percentage differences.

Ans. All the results are described in more detail. Line 7-18 Pg 8

• References should be standardized according to editorial requirements.

Ans. All the references are now standardized and formatted according to editorial requirements. Many new and recent references are now added

Author Response File: Author Response.pdf

Reviewer 2 Report

I reviewed the manuscript entitled: “Synergistic Action of Leaf Extracts of Piper and Rhizobacteria for Plant Growth Promotion and Control of Blast Pathogen of Organic Bali Rice” and my recommendation is to reconsider after major revision. I believe that the manuscript in its present form does not provide sufficient background information to understand the context of this study, why this study is important, and how the study differs from other studies already published in literature. I also believe that it does not provide a thorough description of the experimental design and methods used to answer the research objectives, making difficult to understand the meaning and value of the provided results.

First, I would encourage the authors to review the structure of the introduction and information presented. I think the authors should provide more details about the challenges of rice production. What are the most common issues/diseases affecting rice production? How do the damages caused by the blast pathogen compare to the damages caused by the other most comment pathogens (globally, and/or in the authors’ region)? The authors could also provide more details about how the blast pathogen affect rice crops (effect of the pathogen on the plant, at which stage of the growing season does the farmer can detect the presence of the diseases, what is the best time frame to try to manage the disease, how does it spread in the field, etc). Then, I think the authors should talk about the different management strategies farmers can use to control the blast pathogen, and explain the advantages and limitations of these most common management strategies. Then, the authors can discuss a bit more the use of the two plants they are testing in this study. Why these two plants? Have they been proven to work? Are they commonly being used by growers to manage the blast pathogen? How are they usually being applied? What does the literature say? Where is the gap in literature? How does the authors’ study address such gap in literature?

I also don’t think that it is necessary to state the authors’ hypothesis in the introduction. I would reformulate lines 62 to 67 into a one sentence, objective statement.

Then, I would encourage the authors to review their material and methods section as described below.

Section 2.1: I would suggest that the authors clarify if one extract was created for each plant, or if both plants were mixed into one extract. It seems to the reader that the authors mixed both plants into one extract. What is the concentration of the crude extract(s) created?

Section 2.3: How did the authors know how to identify the active compounds with antifungal activity? Did they find this information in literature? If so, I think that information must be added to introduction. If not, the authors will need to explain this in more details

Section 2.4:  Was the experiment conducted in a rice field? If so, how did the authors choose the six plants per treatment? Was there a buffer distance between treatments? How far apart were the experimental units located? I think a diagram would help better understand the experimental design.

Sections: 2.1. to 2.3: Did the authors inoculate the rice plants with the blast pathogen? How did they do that? When? How did they make sure all plants were inoculated uniformly? Did the authors have control plants in the applications treatments, in addition to the control treatment? This is not clear in the manuscript, and I think it is very important to justify the value of the provided results.

In terms of treatment selection, it seems that both P.caninum and P.betle were applied together – but in different concentration - to all treatments (except for the control).  Is that correct? If so, then the effect of each plant has not been tested in this experiment and the authors cannot make any conclusions on the effect of each plant or the synergetic effect of the two plants. They can only look at the effect of the two plants applied together. In this case, the title of this manuscript, abstract, objective, discussion, and conclusion sections should be reviewed. For instance, the statement provided line 29-31 in introduction is not supported by the results of this study: “However, the synergistic effect of the mixture of the two extracts exhibited greater effects than the effect of a single extract”. The following statement provided in conclusion is also not supported by the results of this study: “Although each botanical fungicide possesses potential antifungal activities, however, a combination of two extracts may increase their antifungal potential by many folds.”  

How were the 0.5% to 2% extract prepared?

Line 122: What do the authors mean by: “All the experiments were conducted in triplicates”. The authors mentioned line 89 that the experiment was replicated 6 times, not three. Are the authors referred to different locations or years? Furthermore, if the experiment was conducted as a randomized complete block design, I think the data should have been analyzed using a mixed-effect models (to account for the random effects) rather than an analysis of variance. Furthermore, what was the response variable for the models? If the authors analyzed the score values (or even the percentage of disease intensity), I think it is important for the authors to look at the residuals. I don’t think the residuals will be normally distributed and the authors will need to consider the use of generalized mixed-effect models to account for such non-normality.

Moreover, the authors do not mention collecting data at 8, 12, and 15 weeks in the material and method section. They only said that the rice was harvested after 4 months and 15 days. I would suggest the authors update the material and method section accordingly.

The abstract, results, discussion, and conclusions sections will also need to be updated accordingly to the changes suggested above.

 

Author Response

1) First, I would encourage the authors to review the structure of the introduction and information presented. I think the authors should provide more details about the challenges of rice production. What are the most common issues/diseases affecting rice production? How do the damages caused by the blast pathogen compare to the damages caused by the other most comment pathogens (globally, and/or in the authors’ region)?

Ans : The structure of the introduction and information presented is now revised. More details about the challenges of rice production, the most common issues affecting rice production, and the damages caused by the blast pathogen and its comparison to the damages caused by the other most comment pathogens have been mentioned now. Line 49-69.

2) The authors could also provide more details about how the blast pathogen affect rice crops (effect of the pathogen on the plant, at which stage of the growing season does the farmer can detect the presence of the diseases, what is the best time frame to try to manage the disease, how does it spread in the field, etc).

Ans : The details about the effect of the pathogen on the plant and the stage and environmental conditions that affect the plant and spread of disease in the field have been provided. Line 75-80.

3) Then, I think the authors should talk about the different management strategies farmers can use to control the blast pathogen, and explain the advantages and limitations of these most common management strategies. Then, the authors can discuss a bit more the use of the two plants they are testing in this study. Why these two plants? Have they been proven to work? Are they commonly being used by growers to manage the blast pathogen? How are they usually being applied? What does the literature say? Where is the gap in the literature? How does the authors’ study address such a gap in the literature?

Ans: Details on the different management practices for the control of blast pathogen etc and the gap in the literature on the control of blast disease is now provided. Line 82-94, 115-131.

4) I also don’t think that it is necessary to state the authors’ hypothesis in the introduction. I would reformulate lines 62 to 67 into a one-sentence, objective statement.

Ans:    This part is now revised as objectives of the study. Line 131-133.

Then, I would encourage the authors to review their material and methods section as described below.

5) Section 2.1: I would suggest that the authors clarify if one extract was created for each plant, or if both plants were mixed into one extract. It seems to the reader that the authors mixed both plants into one extract. What is the concentration of the crude extract(s) created?

Ans : 100 g of crude extract of each plant was mixed in the ratio of 1:1. Line 141-142.

6) Section 2.3: How did the authors know how to identify active compounds with antifungal activity? Did they find this information in literature? If so, I think that information must be added to the introduction. If not, the authors will need to explain this in more details

Ans : Leaves of piper plants are known to contain alkaloids, terpenoids, steroids, flavonoids, polyphenols, tannins, saponins [Surian et al 2019, Reference No. 19]. Line 142-144.

7) Section 2.4:Was the experiment conducted in a rice field? If so, how did the authors choose the six plants per treatment? Was there a buffer distance between treatments? How far apart were the experimental units located? I think a diagram would help better understand the experimental design.

Ans : Yes the experiments were conducted in the rice field. The experimental design is presented as the diagram. Line 169-172, 178-183.

8) Sections: 2.1. to 2.3:Did the authors inoculate the rice plants with the blast pathogen? How did they do that? When? How did they make sure all plants were inoculated uniformly? Did the authors have control plants in the application treatments, in addition to the control treatment? This is not clear in the manuscript, and I think it is very important to justify the value of the provided results.

Ans: Yes the rice plants were inoculated with a mixture of leaves extracts of piper plants, blast pathogen, and PGPR. Line 193-196, 198-203.

9) In terms of treatment selection, it seems that both caninum and P.betle were applied together – but in different concentrations - to all treatments (except for the control).  Is that correct? If so, then the effect of each plant has not been tested in this experiment and the authors cannot make any conclusions on the effect of each plant or the synergetic effect of the two plants. They can only look at the effect of the two plants applied together. In this case, the title of this manuscript, abstract, objective, discussion, and conclusion sections should be reviewed. For instance, the statement provided line 29-31 in the introduction is not supported by the results of this study: “However, the synergistic effect of the mixture of the two extracts exhibited greater effects than the effect of a single extract”. The following statement provided in the conclusion is also not supported by the results of this study: “Although each botanical fungicide possesses potential antifungal activities, however, a combination of two extracts may increase their antifungal potential by many folds.”  

Ans : Yes the extract of both the plants was applied together. The effect of each plant was previously studied and the data is already published (please Refer Ref. No 19 Suriani et al 2019), hence the effect of the extract of the individual plant was not evaluated. The mixture of leaf extracts of different piper plants has already been tested with compost. A combination of a mixture of leaf extracts of Piper and PGPR has not been reported so far. Line 122-131.

10) How were 0.5% to 2% extract prepared?

Ans: A 100 gm of leaf extract of each plant was mixed equal ratio and dissolved in 1 L sterile distilled water and from this solution a concentration of 0.5%, 1%, 1.5%, 2% and added with 1% (w/v) Line 201-202.

11) Line 122: What do the authors mean by: “All the experiments were conducted in triplicates”. The authors mentioned line 89 that the experiment was replicated 6 times, not three. Are the authors referred to different locations or years? Furthermore, if the experiment was conducted as a randomized complete block design, I think the data should have been analyzed using mixed-effect models (to account for the random effects) rather than an analysis of variance. Furthermore, what was the response variable for the models? If the authors analyzed the score values (or even the percentage of disease intensity), I think it is important for the authors to look at the residuals. I don’t think the residuals will be normally distributed and the authors will need to consider the use of generalized mixed-effect models to account for such non-normality.

Ans: This section has been revised. Line 219-220.

12) Moreover, the authors do not mention collecting data at 8, 12, and 15 weeks in the material and method section. They only said that the rice was harvested after 4 months and 15 days. I would suggest the authors update the material and method section accordingly.

Ans :. Collecting data at 8, 12, and 15 weeks has been mentioned. Line 205.

13) The abstract, results, discussion, and conclusions sections will also need to be updated accordingly to the changes suggested above.

Ans :.The abstract, results, discussion, and conclusions sections have been revised according to the changes as suggested by the reviewer and revisions made.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The MS requires a significant language correction. There are many grammatical, stylistic and language errors in the MS, which at this stage prevent its publication in this form.

Keywords should be corrected. The words of the title should not be repeated. It is not corrected. This sugesstion was in first review.

At the end of Introduction Authors should added the aim of the study.

The legends in all Tables needs a lot of work. It has to be shelf explanatory. Please correct in all tables and figures.

Authors didn't read sugesstions in MS text in last review. 

The discussion should be re-written. Most of the information in the current version of the discussion should be moved to the introduction. Authors should focus on discussing their findings with those of other authors. This suggestion was also in last review.

References should be standardized according to editorial requirements.

 

Author Response

The MS requires a significant language correction. There are many grammatical, stylistic and language errors in the MS, which at this stage prevent its publication in this form.

Ans : Language and grammar corrections have been made

Keywords should be corrected. The words of the title should not be repeated. It is not corrected. This sugesstion was in first review.

Ans: Keywords are now revised

At the end of Introduction Authors should added the aim of the study.

Ans: Aim of the study is added at the end of introduction

The legends in all Tables needs a lot of work. It has to be shelf explanatory. Please correct in all tables and figures.

Ans : The legends in all Tables and Figures are now revised

Authors didn't read suggestions in MS text in last review. 

Ans : Remaining suggestions of last review are incorporated. 

The discussion should be re-written. Most of the information in the current version of the discussion should be moved to the introduction. Authors should focus on discussing their findings with those of other authors. This suggestion was also in last review.

Ans : Discussion section is now revised. Some information of the discussion is now moved to the introduction.

References should be standardized according to editorial requirements.

Ans : References are now standardized according to editorial requirements.

Author Response File: Author Response.pdf

Reviewer 2 Report

The authors have significantly improved the manuscript  I have really enjoyed reviewing the revised version of this manuscript. The introduction is now very clear and provides sufficient background to understand the context of this study, and why the work conducted by the authors is important and novel. The authors have also done a great job clarifying the material and methods section.  The results are very well presented and the discussion is thorough and informative. 

Author Response

Reviewer 2

Comments and Suggestions for Authors

The authors have significantly improved the manuscript I have really enjoyed reviewing the revised version of this manuscript. The introduction is now very clear and provides sufficient background to understand the context of this study, and why the work conducted by the authors is important and novel. The authors have also done a great job clarifying the material and methods section.  The results are very well presented and the discussion is thorough

Ans: Since there are no comments/suggestions of the Reviewer, the response of authors is NIL. Manuscript revised as per the comments of Reviewer 1 has been uploaded

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Thank You very much for Your work.