Pseudomonas spp. Producing Antimicrobial Compounds Regulate Fungal Communities Inhabiting Wheat Crown in Southern Chile

Round 1

Reviewer 1 Report

The manuscript entitled „Pseudomonas spp. producing antimicrobial compounds regulate fungal communities inhabiting wheat crown in Southern Chile” by Herman A. Doussoulin, Noberto L. Arismendi and Ernesto A. Moya-Elizondo describes the association between beneficial Pseudomonas sp. colonizing roots and fungal community structure residing in stems of wheat cultivated in southern Chile. The Author observed that 2,4 DAPG bacteria in the wheat rhizosphere have a beneficial effect on plant health.

 The manuscript is well written and organized appropriately. However, I have found some major lacks in the presentation and description of the results. I encourage the Authors to improve the manuscript according to the comments listed below. 

Major comments:

• In my opinion, more information about fungi isolated from studied wheat plants is worth showing. The Authors calculated the alpha diversity indices based on whole datasets, so the demonstration of whole obtained fungal community structure in studied groups (not only potentially pathogenic fungi) is needed.
• Please, add the GeneBank IDs of sequences obtained for every fungal isolate (supplementary table).
• I have found a lot of inaccuracies between data presented in tables and their description in the Results section (e.g. lines: 189, 192-193; 203-212). Please, check carefully the whole manuscript. 
•  There is a lack of some primer sequences used in the presented study. I recommend getting together whole used primer sequences and creating the additional table (in the main text or supplementary materials).  

Other comments:

Lines 19-20: From where the indicated fungi were isolated? From wheat roots or stems? It isn’t clear in the abstract and the main text.

Line 24: harmful microorganisms? 

Lines 154-156: I recommend transferring this sentence to Acknowledgments. 

Lines 329-331: unclear sentence

Author Response

Dear Editor and reviewer

We greatly appreciate the reviewers' comments and valuable suggestions on how to improve our manuscript "Pseudomonas spp. producing antimicrobial compounds regulate fungal communities inhabiting wheat crown in Southern Chile". We analyzed and evaluated the comments of the reviewers, corrected some inaccuracies suggested for them. In addition, we add new information based on the suggestions. For more information, see the authors' responses to each reviewer's particular comments.

Author Response File: Author Response.docx

Reviewer 2 Report

The manuscript presents a complete introduction and a sufficient explanation of the materials and methods. 

The discussion and the conclusions are extensively reported and the effort to give explanation to the not always congruent results is evident. Anyway, the authors should highlight, in the results and in the discussion, the pathogens detected in the different fields and they should relate them with the Pseudomonas spp. found in the tissues.

The reference in the text: the authors should check the way to report the references. The name of the authors that are cited have to be included in the text (if appropriate)?

Some punctual comments follow:

Line 72: add “.” after “spp”

Line 112: what do you mean for 20% PDA?

Line 113: Time of washing of the tissue is too short

Line 123: briefly, report the DNA extraction protocol

Line 126: MgCl2, use subscript character for “2”, if allowed by the journal

Line 129: “initial stage” should be “initial denaturation”

Line 131: “final step” should be “final elongation”

Line 134-135: it is preferable to explain better the PCR protocols used

Line 139: macrogen … laboratories?

Line 147: briefly explain the dilution end point assay

Table 2: The description of H/Ln is not clear

Line 207: did the authors explain in material and methods how they detect the phzCD gene?

Line 247: the authors should modify the phrase

Results: the authors should report, in the results, the biodiversity of fungi associated to wheat crown.

In general, the authors should write the results in a clearer drafting

Line 408: add “.” after “spp”

Author Response

Dear Editor and reviewer

We greatly appreciate the reviewers' comments and valuable suggestions on how to improve our manuscript "Pseudomonas spp. producing antimicrobial compounds regulate fungal communities inhabiting wheat crown in Southern Chile". We analyzed and evaluated the comments of the reviewers, corrected some inaccuracies suggested for them. In addition, we add new information based on the suggestions. For more information, see the authors' responses to each reviewer's particular comments.

Author Response File: Author Response.docx

Reviewer 3 Report

The paper is very interesting and gives contribution to the understanding of the relationships between microorganisms that are associated with wheat crown rot.

There is a large amount of data obtained; on the other hand, many data are mentioned but not clearly showed. I suppose, additional tables are necessary, for example – the number of obtained isolates.

Some expressions are unprecise, little corrections are necessary.

Abstract.

Line 13 – “associated with the control of wheat crop pathogens”. I do not agree – in this case, more precise could be “inhibit pathogen development” or similar.

Line 24 – expression “severity of microorganisms” is not precise – should be “severity of disease”.

Methods and materials

It is difficult to understand, how many stems are analysed altogether – only 25 stems during the whole period and across all fields? The same about the isolation of fungi – did the authors use 5 segments from one wheat culm?

How did authors combine the results from the identification by the morphology of colonies and by molecular methods?

Do “evenness” and “equity” mean the same?

What does “richness of species” mean?

Results.

Authors use the term “microorganisms’ severity” – it is incorrect. There is the severity of diseases but not of microorganisms.

Data about severity and incidence could be useful, because it is difficult to understand the data without understanding the situation on the whole.

It is difficult to understand Table 3. Correlation between severity and incidence versus yield was significant and positive for the total number of samples, but this correlation was not observed separately in each year. How could these results be explained? Could they be influenced by the spectrum of diseases’ causal agents in a particular year or on a particular field?

Data about the spectrum of determined fungi would be interesting, because it is difficult to understand the relationships without the knowledge about the full spectrum of isolates. Authors do not give any data about the dominant pathogens.

Lines 336–345 describe the results, there is no discussion.

What does it mean – low values and normal values (Lines 336-338)?

Author Response

Dear Editor and reviewer

We greatly appreciate the reviewers' comments and valuable suggestions on how to improve our manuscript "Pseudomonas spp. producing antimicrobial compounds regulate fungal communities inhabiting wheat crown in Southern Chile". We analyzed and evaluated the comments of the reviewers, corrected some inaccuracies suggested for them. In addition, we add new information based on the suggestions. For more information, see the authors' responses to each reviewer's particular comments.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors,

Thank you for improving the manuscript. I have only one suggestion. The number of obtained fungal isolates is very impressive (n= 1304), thus I understand that not all of them were identified using molecular tools. Especially, if Dr Ernesto Moya-Elizondo is experienced in the morphological evaluation of wheat associated fungi. However, there is a need to submit these 91 obtained ITS sequences to GenBank.

Author Response

We appreciatte the comments of the reviewers and we are going to to submit the obtained ITS sequences to GenBank, as soon will be possible.