Screening for Antagonistic Yeasts to Manage Alternaria spp. in Organic Farming

Round 1

Reviewer 1 Report

Comments and suggestions indicated on the manuscript

Comments for author File: Comments.pdf

Author Response

Thank you for all your valuable comments and suggestions. We found them very meaningful and helpful. They will help improve our experimental design and writing in the future. We have made the suggested changes to the manuscript. Responses to the comments (in red) are below.

Each section of the manuscript was improved according to suggestions made by the Reviewer in the text. All requested changes were done.

what surfaces??? - Examples were given (line 45).
Do all yeasts conform to these requirements? - Changed to “many yeast species” with additional citation (line 50, position 12).

rephrase this. Meaning not clear. – Rephrased (line 54).

analysed for what? Do you mean to say "used" for this study??? - Changed in the text (line 69).

Provide some details on the in planta experiment. What growth media was used? what potato cultivar? were the plants fertilised? If yes, provide details if the fertilizer? If no, what type of plant nutrients were used to ensure proper plant nutrition? Provide these details, please. how was the spraying done? by using hand held spray bottle? - Details are provided in the text. The plants were not fertilised in order to not introduce another factor to the experiment and the nutrients were from the potting mix (line 102).

 

how were the leaves selected to avoid biasness? - The leaves were selected randomly (line 110).

light microscope? what magnification? please provide details of how this was done? -  Details are provided in the text (line 130).

 

Rephrase. Sentence is poorly constructed. How ere the plants generated? Same as previously done under greenhouse conditions? - Rephrased, details added (line 135).

were the plants supplied with nutrients for proper plant growth and development? Please indicate - Plants were not additionally fertilized, they used nutrients from the potting mix (line 136).

provide details of how the leaves were collected? How many leaves were collected per plant? - Details are provided in the text (line 141).

what was used to shake at 6000 rotations per minute? - A mini centrifuge rotator (line 145).

what statistical package was used? Please specify - Statistica 12 (Line 152).

The graph is poorly drawn and does not really tell what happened during the in vitro bioassay. A table would have been better with statistical analysis to back the table. Moreover standard errors should accompany the values in the table - All graphs and tables were revised and improved. Standard errors were added to the tables.

This table says very little and does not give much information on what happened. Why were growth inhibition not quantitatively measured? There should have been a control plate where the percentage growth inhibition could have calculated  -Thank you for this valuable comment, this will be helpful in our future studies.  The inhibition was calculated on the original medium, however, after moving fungal discs to the fresh medium just the inhibition tendency was noted.

 Please relate this to your results – changes made in the text (line 234).

Not quantified. US 50 was positive for 2 out of 5 exoenzymes and Cobra positive for 3 out of 5 exoenzymes. which of these positive enzymes are likely to be involved in the inhibition process???? Hence care must be taken when inhibitions are attributed to the exoenzymes. - We agree that the inhibition mechanisms are complex, so further studies are needed and enzyme participation can not be clearly attributed, so proper changes were made in the text (line 296).

Did you determine this in the presence of the pathogen?? No pathogen was inoculated/this was done in the absence of the pathogen. Hence there shouldn't be much competition, if any at all between the inoculated yeast and other microorganisms. - Proper changes were made in the text (line 278).

Reviewer 2 Report

The manuscript titled “ Screening for antagonistic yeasts to manage Alternaria spp. in organic farming“. I find the idea interesting and in line with the aim of the journal. I have some concerns about the experimental setup to justify what the authors claim. Moreover, the rationale behind some of the data presented was not entirely clear. I also recommend to the authors improve their references by conducting a more extensive review of international literature. Particularly, the introduction statements are not supported by the references selected by the authors. The logic of some sentences is also questionable. Below is my point-to-point analysis of the manuscript.

Abstract

Ø  Abstract introductory statement is too long, it has to be improved with a more specific rationale of the study. The abstract should have crisp information about the aim materials method result and conclusion, which I don't find in the present form of abstract should contain an introduction aim hypothesis aim result and conclusion.

Ø  Conclusion is method is missing.

Ø  Result in the abstract should be more concise.

 

Introduction

Ø  Have to be improved with a more specific rationale of the study.

Ø  Many sentences are wrongly written and do not flow as well.

Ø  I highlighted a few of them.

Ø  at line no 37 statement " Symptoms usually appear a few weeks after plant emergence on lower leaves as black or brown spots. The spots coalesce, which causes the leaves to die and disease to spread to other leaves“ needs a citation

Ø   at line no 39 statment " Cultural methods to control early blight in potatoes are limited. It includes long crop rotation, good weed management, removing alternative hosts, appropriate fertilizer application and irrigation. In organic farming, in which chemical pesticides are forbidden, other additional control methods, such as biological, need to be investigated. Needs citation

Ø  statement "  To be successful a microorganism, which is antagonistic against plant pathogens, needs to produce“needed to be rewritten.

 

Material and Method

Ø  Control is missing in figure 4.

Ø  Author has not explained on which basis he is justifying the result

Ø  Discussion and conclusion

Ø  Discussion is written well but it is not supported well by the previous study. So I suggest the author to read the discussion and provide more references in support of the present study.

Ø  conclusion is ok.

 

 

The overall manuscript is carelessly written there are many typo errors and language mistakes as well. I recommend carefully proofreading the manuscript before the submission of revision. 

Author Response

Thank you for all your valuable comments and suggestions. We found them very meaningful and helpful. They will help improve our experimental design and writing in the future. We have made the suggested changes to the manuscript. Responses to the comments (in red) are below.

Abstract

Ø  Abstract introductory statement is too long, it has to be improved with a more specific rationale of the study. The abstract should have crisp information about the aim materials method result and conclusion, which I don't find in the present form of abstract should contain an introduction aim hypothesis aim result and conclusion.

Ø  Conclusion is method is missing.

Ø  Result in the abstract should be more concise. - The abstract has been revised for clarity, length and its content (line 14).

 

Introduction

Ø  Have to be improved with a more specific rationale of the study. - The rationale of the study was improved (line 61).

Ø  Many sentences are wrongly written and do not flow as well. - The whole fragment has been reviewed and improved (lines 31-65).

Ø  I highlighted a few of them.

Ø  at line no 37 statement " Symptoms usually appear a few weeks after plant emergence on lower leaves as black or brown spots. The spots coalesce, which causes the leaves to die and disease to spread to other leaves“ needs a citation - The citation was added, position 4.

Ø   at line no 39 statment " Cultural methods to control early blight in potatoes are limited. It includes long crop rotation, good weed management, removing alternative hosts, appropriate fertilizer application and irrigation. In organic farming, in which chemical pesticides are forbidden, other additional control methods, such as biological, need to be investigated. Needs citation - The citations was added, positions 5-6.

Ø  statement "  To be successful a microorganism, which is antagonistic against plant pathogens, needs to produce“needed to be rewritten. - The fragment was rewritten (line 43). Positions 10-11 in literature.

 

Material and Method

Ø  Control is missing in figure 4. - The information in figure 4 was  completed

Ø  Author has not explained on which basis he is justifying the result

Materials and methods were improved.

Ø  Discussion and conclusion

Ø  Discussion is written well but it is not supported well by the previous study. So I suggest the author to read the discussion and provide more references in support of the present study. - Missing references have been added, the section was checked and corrected Positions 51-55, 63, 66 and 71).

Ø  conclusion is ok.

The overall manuscript is carelessly written there are many typo errors and language mistakes as well. I recommend carefully proofreading the manuscript before the submission of revision. - The manuscript was carefully checked and proofread. It has been checked by a native speaker Dr. Robert Lee from the University of Warmia and Mazury.

Reviewer 3 Report

Dear the authors,

In this article, the authors describe the research article on the topic “Screening for antagonistic yeasts to manage Alternaria spp. in organic farming”. I think the subject is interesting. However, the methods and results are unclear. The manuscript lacks the necessary details for readers to fully understand and difficult to follow the logic of the study. Major revision is required to improve the quality of the content and writing.

The following comments are for the authors to consider:

L18 and Table 1: What is Saccharomyces cerevisiae bayanus? Please correct it according to scientific writing principles. What should it be?

L19: Please correct to “Alternaria alternata and A. solani”

L62-63: The authors mentioned “using commercially available strains of yeast used in the food industry to prevent early blight in potatoes grown as organic crops.”. Is the early blight being main problem in food industry? I think it should be agriculture more than food industry.

L70-75: The authors mentioned about the species characteristics of each yeast strains such as a killer yeast, osmotolerant strain etc., but no references.

L95-97: The authors should explain the method in detail. How did you inoculate the fungal spore suspension to the plant?

L108-111: Although the author has cited the previous method. It needs to explain the method used in this study. The composition of the nutrients tested must be specified.

L113-114: Please revise this sentence

L199-131: This method was conducted under sterile condition? Are the authors confident that the yeast colonies growing on the medium are the target yeasts? It’s not contamination?

L129: Why did the authors incubate the PDA medium at 45ºC?

L138-151: How to calculate the percentage of the growth inhibition? Please provide the calculation formula in the method section.

L117-118: The strain codes are more important than strain numbers.

L185-192, L254-270 and Table 5: It is difficult to follow the logic of this study. In general, the main composition of fungal cell wall is chitin and glucan, follow by protein. Is the Alternaria spp. cell wall consisted of pectin, xylan, amylose? Why did the authors interested in detecting amylase, xylanase, pectinase? In my opinion, they are not important mechanisms for the destruction of pathogenic fungi.

In this section, the authors should indicate the name of tested medium. Did you measured the diameter of the observable transparent zones or blue halo surrounding yeast colonies? It would be better than providing results as positive or negative. Alternatively, you can add the figure showing the test results.

L195-199: In this study, the fungal mycelial were observed under microscopy and the authors mentioned that “The mycelia analyzed under the microscope were deformed and the loss of intracellular content was visible.”, but the author explained in text only. Thus, I strongly recommend to show the figure showing deformed hyphae.

L222: Replace “strain NCYC1006 (S. cerevisiae): with “S. cerevisiae NCYC1006”

L225-230: It is not necessary to writ strain before strain code. Pleas change to “

(i.e., Starmerella bacillaris FE08.05, Metschnikowia pulcherrima GP8, Hanseniaspora uvarum GM19, Hanseniaspora opuntiae GA22, Hanseniaspora opuntiae GM10, Hanseniaspora guilliermondii GA1, Hanseniaspora lachancei GM32, Hansenaspora pseudoguilliermondii GP14 and Candida awuaii GM3..”

L231: Meyerozyma guilliermondii SQUCC-33Y

L291-308: Please revise this section.

Fig.2-4: It's difficult to follow the results. I strongly suggest to specify the strain code instead of using the strain number 01-06 in the figure. Also, the author should specify what the x-axis or the y-axis is.

Table1 and Table 2: Suggest to combine Table 1 and Table 2 as a single Table (see Table 4), and change the star symbol in Table to the percentage of the growth inhibition.

 Fig. 4: Please add standard deviation (SD) in the Table.

Author Response

Thank you for all your valuable comments and suggestions. We found them very meaningful and helpful. They will help improve our experimental design and writing in the future. We have made the suggested changes to the manuscript. Responses to the comments (in red) are below.

 

The following comments are for the authors to consider:

L18 and Table 1: What is Saccharomyces cerevisiae bayanus? Please correct it according to scientific writing principles. What should it be? - It has been corrected.

L19: Please correct to “Alternaria alternata and A. solani” - It has been corrected (line 18).

L62-63: The authors mentioned “using commercially available strains of yeast used in the food industry to prevent early blight in potatoes grown as organic crops.”. Is the early blight being main problem in food industry? I think it should be agriculture more than food industry. - It has been clarified in the text (line 63).

L70-75: The authors mentioned about the species characteristics of each yeast strains such as a killer yeast, osmotolerant strain etc., but no references. - The references have been added (positions 32-36).

L95-97: The authors should explain the method in detail. How did you inoculate the fungal spore suspension to the plant? - The inoculation method has been explained in more detail (line 102).

L108-111: Although the author has cited the previous method. It needs to explain the method used in this study. The composition of the nutrients tested must be specified.- The method has been explained and the required details added in the text (line 119).

L113-114: Please revise this sentence - The sentence has been rewritten (line 128).

L199-131: This method was conducted under sterile condition? Are the authors confident that the yeast colonies growing on the medium are the target yeasts? It’s not contamination? - The plants were kept in a sterile environment and control plants were used to inspect for contamination with non-target yeasts. The explanation has been added to the text (line 137).

L129: Why did the authors incubate the PDA medium at 45ºC? - In order for the media to remain melted during incubation.

L138-151: How to calculate the percentage of the growth inhibition? Please provide the calculation formula in the method section. - The calculation has been added (line 94).

L117-118: The strain codes are more important than strain numbers. - It has been taken into consideration.

L185-192, L254-270 and Table 5: It is difficult to follow the logic of this study. In general, the main composition of fungal cell wall is chitin and glucan, follow by protein. Is the Alternaria spp. cell wall consisted of pectin, xylan, amylose? Why did the authors interested in detecting amylase, xylanase, pectinase? In my opinion, they are not important mechanisms for the destruction of pathogenic fungi. - The role of enzymes has been clarified in the text (line 278).

In this section, the authors should indicate the name of tested medium. Did you measured the diameter of the observable transparent zones or blue halo surrounding yeast colonies? It would be better than providing results as positive or negative. Alternatively, you can add the figure showing the test results. - Name of medium have been added. The diameters haven’t been measured but we’re take this valuable suggestion into consideration in the future (line 119).

L195-199: In this study, the fungal mycelial were observed under microscopy and the authors mentioned that “The mycelia analyzed under the microscope were deformed and the loss of intracellular content was visible.”, but the author explained in text only. Thus, I strongly recommend to show the figure showing deformed hyphae. - The figures aren’t available but we will implement this in our future studies.

L222: Replace “strain NCYC1006 (S. cerevisiae): with “S. cerevisiae NCYC1006” - Replaced (line 240).

L225-230: It is not necessary to writ strain before strain code. Pleas change to “ (i.e., Starmerella bacillaris FE08.05, Metschnikowia pulcherrima GP8, Hanseniaspora uvarum GM19, Hanseniaspora opuntiae GA22, Hanseniaspora opuntiae GM10, Hanseniaspora guilliermondii GA1, Hanseniaspora lachancei GM32, Hansenaspora pseudoguilliermondii GP14 and Candida awuaii GM3..” - Changed (lines 241-247).

L231: Meyerozyma guilliermondii SQUCC-33Y - Changed (line 248).

L291-308: Please revise this section. - The section has been revised (lines 309-320).

Fig.2-4: It's difficult to follow the results. I strongly suggest to specify the strain code instead of using the strain number 01-06 in the figure. Also, the author should specify what the x-axis or the y-axis is.

Table1 and Table 2: Suggest to combine Table 1 and Table 2 as a single Table (see Table 4), and change the star symbol in Table to the percentage of the growth inhibition. - All figures and tables have been revised.

 Fig. 4: Please add standard deviation (SD) in the Table. - SD has been added.

Round 2

Reviewer 1 Report

Please remove all the track and trace. Tremendous improvement on the manuscript after implementing the corrections, suggestions and changes. 

Author Response

Thank you very much for your overall beneficial suggestions and comments. We are grateful for your appreciation for our effort to improve the paper. All track and trace were removed.

Reviewer 3 Report

Thank you for responding to my comments.
The manuscript is well revised and structed.
However, I strongly recommend that the authors should repeat the test and show the results of the section 3.4 (Line511-515). I believe it will increase the quality of your article.

Author Response

Thank you very much for your overall beneficial suggestions and comments. We are grateful for your appreciation for our effort to improve the paper.  

The effect of yeast presence on Alternaria solani and alternata mycelium, which was observed during tests shown in section 3.4., was confirmed in additional tests which are planned to be published in another manuscript. An additional reference was added to the manuscript [41] which shows similar results to our observations.