Species Diversity in Colletotrichum Causing Anthracnose of Aromatic and Ornamental Lamiaceae in Italy

Round 1

Reviewer 1 Report

The manuscript is overall well written, but I have made some suggestions for improvement in the attached file. I would also like to add a couple of additional comments:

The authors use the term strain or isolate. I think it would be better to stick to one, and my preference would be isolate.

Seed isolation was conducted, but no results were presented, but then reference was made to that in the discussion. I believe seed isolation results should be added to the the Result section.

There is also a bit of confusion regarding disease data - what was collected during the survey and what was generated through artifical inoculations. However, this can easily be clarified and/or fixed.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 1

We are really thankful to you and your great work done to evaluate our MS.

We are also very happy that you appreciated various aspects of our article. We also appreciate your effort to provide many suggestions which are useful to improve the MS.

Following some responses to you comments:

The authors use the term strain or isolate. I think it would be better to stick to one, and my preference would be isolate.

Thanks for this comment, we agreed with you and modified to "isolate"

Seed isolation was conducted, but no results were presented, but then reference was made to that in the discussion. I believe seed isolation results should be added to the the Result section.

Thanks, this is now included into the "Results" section.

There is also a bit of confusion regarding disease data - what was collected during the survey and what was generated through artificial inoculations. However, this can easily be clarified and/or fixed.

The first section of "Results" has been edited to clarify these points. Thanks for helping to improve this part.  

In general, we passed through the whole manuscript and everything you suggested was fixed according with.

We really would like to thank you because all your suggests made more smooth and fix the text.

Best regards

Reviewer 2 Report

There are numerous changes that I would make throughout this paper. Full explanation of what you did and why needs to be ensured and your reasoning for doing the work needs to be much clearer. Throughout you need to be consistent with your formatting and also ensure that you are grammatically correct. In particular there are certain areas that need to be reworked and explained far more succinctly. For instance in your discussion this needs to be far more concise and succinct. For instance line 242-245, i.e. the opening sentence within the discussion needs to be changed and put further down. In my opinion you need to highlight that this is the first study of its kind and what the highlights were. For instance C. ocimi being demonstrated as an external contaminant of the seed for the first time in this study needs to be highlighted. You need to make your discussion more about bringing together your data and using the information from other studies to support it. In some cases you have sentences or paragraphs that are out of place and totally interrupt the flow of your discussion. The work you present is interesting but the article can definitely be shorter to present the most important findings. I would also ensure that you are more descriptive in your materials and methods in some cases to ensure that they are reproducible.  I have attached a Word document whereby I have tried to go through areas that need to be fixed up and what I think it detract from the overall study. I hope that this will help you make the necessary changes to improve the readability of the article.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer 2

We are really thankful for your effort and time spent to revise our paper and give a strong contribute to make it much better. We totally agree with what you have noticed.

We made much shorter the discussion, as to reach faster the main points of study.

We sent the MS for the English editing service for grammatical correction.

We highlighted the  importance of C. ocimi as contaminant of seeds.

More details are present in the Matherial and Methods

Following your comments with our responses:

Abstract

Line 15: add an “a” after in. Otherwise the abstract was fine and was very direct and to the point. What I think you should do is follow a similar style for your discussion as your abstract was very good. Your discussion however was not as well organised.

DONE

Introduction

The introduction overall is okay but needs to be far more succinct and focus on presenting information as to why you did this study. There are substantial formatting and grammatical errors throughout that need to be rectified.

L 36-37. At the end of this line remove “is” and “nowadays” and instead have the sentence as thefollowing.

“Although it has a primary tropical and subtropical distribution, it also affects temperate areas.”

The sentence directly after their starting with Colletotrichum spp. Is a direct quote from the article that you have cited. You must change that and my suggestion is to do the following:

Colletotrichum spp. Are included in the 10 most important plant pathogenic fungi in the world.

L49- L52. In the sentence on L49 at species then continue on with the rest of the sentence. However I would make sure that L 52-L 55 is moved to the paragraph above and that this entire put into context. At the moment it does not read smoothly and fit well with the overall writing.

L56-L64. Remove the word “the” from the beginning of this paragraph. I would also ensure thatyour grandma is succinct and makes sense. For instance that very last sentence in the paragraph needs to be improved.

L69-71. This entire paragraph is one sentence. It needs to be broken into 2 to make it more readable. My suggestion issued at the sentence into two after Southern Italy.

L72-L78. This last paragraph really needs to address exactly why you did this work. You just need to mention and highlight the aims and my suggestion is you format that with specific dot points. For example:

i) species identification of Colletotrichum isolated from aromatic plants based on multi-locus phylogenetic analyses

ii) determination of the pathogenicity of the representative Colletotrichum isolates on the host species from which they were isolated and to confirm Koch’s postulates.

I think you should refrain from referring to the isolates in regard to their aggressiveness but rather use the term pathogenicity or virulence. This is much better than using the term aggressiveness as it has different connotations.

We fully agree with all your comments about the "Introduction". We followed your suggestions and made shorter the crucial part that you have mentioned (L72-78)

Materials and methods

Throughout the materials and methods consistency is the key. When you mention something like PDA at the first instance you should write this in full for instance it should be as follows. Potato dextrose agar (PDA). Also in regard to your use of sterile water (see Line 89) is it distilled wate,r reverse osmosis water, or tap water? Be more specific in exactly what you did so that your work is repeatable by others. Within this paragraph you must also refer to your disease incidence criteria here to enable the reader to ascertain the pathogenicity of the isolates from the field.

L 91. Change this to “were transferred onto PDA plates for monosporic cultures”. The rest of the sentence is not required and I would delete and for their collection.

L97: this is not the first mention of potato dextrose agar. See my previous comment. Where you refer to single-or tip you need to be explicitly clear what you are referring to here. I am unsure how you would get a single high fee for each of the cultures and I am assuming that you were taking high full lips. Please ensure this is abundantly clear. Throughout this section you are missing plurals and also your formatting needs to be correct.

Section 2.2

In regard to the ITS region, this sentence can be shortened dramatically. You have directed the reader to where they can get further information and this is sufficient information. Your reader can then refer to this paper if they require extra information.

Section 2.3.
You’ll methods for this section where clear and well described. Nothing needs to be changed here.

Thanks for these important comments that we have followed.

For the pathogenicity section, i.e. section 2.4, there are numerous issues that I have with this. For instance you have mention there a nine basil plants per pot is their only one plant for all of the other plant species that you tested? Were the plants all five months all when they were infected or were they cultivated when they were five months old? This needs to be clearer.

You really made stronger this part with your comments. A sentence has been added to clarify about number and age of plants used.

L147-L148. This sentence beginning with one strain for each needs to be clearer and reworded. For instance you need to make it clearer.

“One strain for each of the Colletotrichum species identified (based on molecular identificationparentheses were used for each of the part hosts (Table 2).”

Is this what you were trying to say as it was not clear in your write-up?

How many times was this pathogenicity trial undertaken as you say in the final sentence that data of the replications of repeated experiments were pooled and analysed together. This needs to be elucidate it for the reader to understand what you exactly did. Also in regard to the amount of inoculum per plant, you need to take into consideration that the basil will be much smaller, so far as I can understand how you cultivated all of the host plants, and therefore your inoculum per biomass unit will be very different between the different plant species. You need to ensure you make this very clear to the reader. Also within this particular section please ensure it is grammatically correct.

These points have been also clarified according with your comments. Thanks.

Results

As I have mentioned previously, you must refer to the disease incidence for your field survey, as you specifically refer to this in L161.

L169: this is not the first time you have mention relative humidity and as such can be shortened to RH here. L171: I am not sure what you mean in regard to shadow in the sentence. Are you referring to shade? Be more precise.

Figure 1 does now have Ocimum italicised. Also considering you did the molecular work can you please indicate on each of the figures from A to E what the Colletotrichum species was for each. This would be far more informative for the reader

These three points have been also clarified according with your comments. Thanks.

Table 1. I think that you could put this information into your results rather than have a separate table. However the decision of course is yours.

We have decided to keep the Table, but making it more complete based also on suggestions by other reviewer.

For table 2 (L178 –L188) it is extremely difficult to follow and my suggestion is to have two separate tables one for the molecular and one for the pathogenicity work. If you do leave it as one table I will ensure would have a more descriptive title that is currently given for instance I would not have virulence showed but virulence expressed after pathogenicity testing I would also indicate that they are Colletotrichum isolates that were included in this study. Be more precise in your titles so that they are stand-alone and give the reader a good understanding of the information you are presenting.

We have decided to keep this table as well, however we provided the inputs that you suggested.

L198: eventually plants died. Do you mean after total defoliation? You need to be more specific here. Also you have a word in this sentence, into resting which is out of place.

3.2 Phylogenetic analyses.

This paragraph and the accompanying figure where fine and well explained. Nothing needs to be fixed.

3.3 pathogenicity. You have already indicated what disease severity stands for so you do not need to spell this in full at this stage. I am unsure what the X is representing in line 233.

We have fixed these three points that you have noticed. Thanks.

Discussion

the discussion make some good points but you nearly really need to change how you are presenting it. One of the most important things you need to do in regard to this discussion is high light the uniqueness of what you have done in the significance of this work for your area. You tend to spend a significant amount of time in regard to going over the results again. This is not necessary and is repetition of what you have already presented in some cases you have information that is not adding anything at all of value to your discussion. For instance, from line 296, this information can be

substantially condensed and I believe you can take out all this information right down to line 303. It does not add value to your work if you wish to use this then please paraphrase this more and put it into the relevant part where it makes more sense.

Your sentence beginning “the present study provides the first overview of Colletotrichum diversity associated with leaf anthracnose of several aromatic and ornamental plant host belonging to theLamiaceae family” is very important and should be put right at the beginning of your discussion this is what you’re research is about.

Your last sentence commencing with further studies are required, is very poor and re-needs to be redone. For instance what do you mean “which can interest”? I do not understand what you meanhere and this needs to be remedied.

We agree with the importance of your doubts on this section. According with this, we strongly modified the Discussion cutting redundant parts, and strongly improving the quality of the language, thanks also the editing service.

Once again, we would like to thank you for giving many precious comments.

Best regards.

Author Response File: Author Response.docx

Reviewer 3 Report

I whether “Agronomy” is the best journal for this paper? Is “Plants’ more appropriate?   There are a lot of small English expression, grammar and unusual word choices which I have highlighted.  M/s needs proper editing for English. 

This is a fairly ad-hoc study using isolates collected over a period of time and in no particular structured survey. Molecular analyses etc. is OK. Isolates were inoculated onto their original collection host but it is not clear whether multiple Coll species may infect a single host,   Cross pathogenicity studies would have strengthened this study. Include Coll species names in a table of hosts.  Was there any particular geographic distribution patterns of Coll species collected?? (although study was probably too small to be able to make conclusions of species distribution).

Author Response

Dear Reviewer 3

We really would like to thank you for your time and effort spent to read our article and your important comments.

Following our responses to your comments: 

I whether “Agronomy” is the best journal for this paper? Is “Plants’ more appropriate? 

I perfectly understand this comment which I totally respect. In this sense, we would like to specify that the article has been submitted for a SPECIAL ISSUE of the Journal Agronomy. The title of this SI is " Innovation in Detection and Management of Disease of Vegetables and Fruits". We consider this kind of identification ad a high level tool for detection, so we have considered this SI as suitable.

 There are a lot of small English expression, grammar and unusual word choices which I have highlighted.  M/s needs proper editing for English. 

We fully agree with this, so we have sent the MS out for the language editing and we have followed all the edits.

This is a fairly ad-hoc study using isolates collected over a period of time and in no particular structured survey. Molecular analyses etc. is OK. Isolates were inoculated onto their original collection host but it is not clear whether multiple Coll species may infect a single host,   Cross pathogenicity studies would have strengthened this study.

That's definitely a major point to investigate. We have stated now this point in the conclusion. Further studies will be conducted for sure in a second time, in detail when a we will be able to find a large (necessary) number of plants for this kind of trials. This is ongoing for subsequent studies.

Include Coll species names in a table of hosts.  

Thanks for this. We have included this now.

Was there any particular geographic distribution patterns of Coll species collected?? (although study was probably too small to be able to make conclusions of species distribution).

Yes, as you already said, this has not been possible in this first investigation. However, ongoing studies on more isolates of Colletotrichum collected from many ornamental plant species, will probably allow this kind of distribution study.

With our best regards.