Diversity of Colletotrichum spp. on Aristolochia grandiflora: A Case Study in an Italian Botanical Garden

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors,

I consider that the manuscript presents very interesting results on the elucidation of the causative agent of extensive leaf spots found in Aristolochia grandiflora plants in a botanical garden in Italy; however, some remarks are made that could contribute to improve the final version.

 

Affiliation

The e-mail addresses and initials of all authors must be included. 

 

Abstract

Botanical garden is written in 3 different ways in the text. Their spelling should be unified.

 

Introduction

Line 32. It is recommended to include the classifier for Aristolochia grandiflora.

Line 33. It should be cm instead of centimeters.

Line 40. Why do you consider the environment of the Botanical Garden as controlled?

Line 41. The use of pathogens as a noun should be avoided. Extend to the rest of the document.

Line 50. Consider anthracnose-like symptoms instead of anthracnose symptoms

 

Materials and methods

Line 62. Consider the possibility of matching the subtitles of Materials and Methods with those of Results.

Line 66. It should be specified whether the leaves were symptomatic only.

Lines 72-73. I do not consider it necessary to give the composition of the PDA culture medium. I suggest that it should be handled as PDA culture medium, as for the other culture media in the rest of the document.

Line 80. It should be Figure 1. instead of Figure 1: ... I consider that there are not enough symptom differences between photos of A. grandiflora leaves to show three of them.

Lines 89-91. Gene names should be in italics.

Line 91. It should be forty nanograms instead of 40 ng because it is at the beginning of a sentence.

Line 94. It should be considered to include a table, which could be in supplementary documents, showing the sequence of the primers used with the size of the amplicons corresponding to each of the genes with their respective references.

Line 97. It should be CHS-354R primers, …

Lines 102-122. This paragraph should be written without anticipating results for the Colletrotrichum species found.

Line 107. This first BLASTn must be specified with the sequences of which gene it is performed, which according to the results is only with those of the ITS. Is this the case?

Line 142. It should be Drais et al. [19] and Brugneti et al. [6].

Line 168. It should be Moral et al. [20] …

Line 170. It should be 10 to the 5 instead of 105. The same comment in Line 180.

Line 181. It should be Garcia-Lopez et al. [21].

Lines 178-185. This paragraph should be written without anticipating results for the Colletrotrichum species found.

 

Results

Line 192. According to the result presented with the analysis of the ITS region, the three species of Colletrotrichum found can be defined. If so, I consider that this result could be shown, even if only in supplementary documents.

The classifier of each of the three Colletrotrichum species encountered during your description should be entered.

Line 228. Consider Conidiomata on Aristolochia leaf.  

Line 259. I consider that Table 1 should not be included in the body of the article but as supplementary material. 

Lines 266 and 271. Delete (ML).

Full scientific names should be given in figures and tables.

Line 279. It should be (Fig. S1) instead of (Figure S1).  

In figures 5 and 6 the substitution model used must be indicated.  

 

Discussion

Line 331. Care must be taken when writing as the species are not isolated.

Line 358. It should be Aristolochia by Zhi-jun et al. [28] …

Line 350. It should be while Tekade and Mohod [29] …

 

Conclusion

 

There is a final paragraph in the form of a conclusion with the subtitle named Discussion and conclusion. I proposed to split it in two separate subtitles. 

 

Author Response

Review 1

Dear Authors,

I consider that the manuscript presents very interesting results on the elucidation of the causative agent of extensive leaf spots found in Aristolochia grandiflora plants in a botanical garden in Italy; however, some remarks are made that could contribute to improve the final version. 

Dear Reviewer, thank you for the time dedicated to revise our manuscript, as well as for the helpful comments and suggestions provided. We sincerely appreciate your positive comments, and we carefully addressed all the issues raised.

Affiliation

Rev: The e-mail addresses and initials of all authors must be included. 

Answer: The e-mail addresses have been added.

 

Abstract

Rev: Botanical garden is written in 3 different ways in the text. Their spelling should be unified.

Answer: We corrected the names 

 

Introduction

Rev: Line 32. It is recommended to include the classifier for Aristolochia grandiflora.

Answer: The Aristolochia grandiflora classification has been added.

 

Rev: Line 33. It should be cm instead of centimeters.
Answer; We changed  the “ centimeters “ in “ cm”.

Rev: Line 40. Why do you consider the environment of the Botanical Garden as controlled?
Answer: The tropical greenhouse at the Botanical Garden has a control system that maintains constant temperature and humidity throughout the year.

Rev: Line 41. The use of pathogens as a noun should be avoided. Extend to the rest of the document.
Answer: We changed  the “pathogens” in “phytopathogens”.

Rev: Line 50. Consider anthracnose-like symptoms instead of anthracnose symptoms
Answer: We changed the “anthracnose symptoms“ to “anthracnose-like symptoms“.

Materials and methods

Line 62. Consider the possibility of matching the subtitles of Materials and Methods with those of Results.

Thank you for your suggestions. We believe that the current titles already matched

Rev: Line 66. It should be specified whether the leaves were symptomatic only.
Answer: We have specified the percentage of the disease’s incidence on the plant.

Lines 72-73. I do not consider it necessary to give the composition of the PDA culture medium. I suggest that it should be handled as PDA culture medium, as for the other culture media in the rest of the document.
Answer:  PDA composition has been removed.

Rev: Line 80. It should be Figure 1. instead of Figure 1: ... I consider that there are not enough symptom differences between photos of A. grandiflora leaves to show three of them.
Answer: We removed the additional photos.

Rev: Lines 89-91. Gene names should be in italics.
Answer: We changed  the characters of the genes in italics.

Rev: Line 91. It should be forty nanograms instead of 40 ng because it is at the beginning of a sentence.
Answer: We changed “ 40 ng “ to “Forty nanograms“

Rev: Line 94. It should be considered to include a table, which could be in supplementary documents, showing the sequence of the primers used with the size of the amplicons corresponding to each of the genes with their respective references.
Answer: A table with used primers and sequences has been added as supplementary material

Rev: Line 97. It should be CHS-354R primers, …
Answer: We added the correct caption.

Rev: Lines 102-122. This paragraph should be written without anticipating results for the Colletrotrichum species found.

Rev: Line 107. This first BLASTn must be specified with the sequences of which gene it is performed, which according to the results is only with those of the ITS. Is this the case?

The whole paragraph has been modified by introducing a more detailed ITS sequence analysis. Any mention to the species found has been removed.

Rev: Line 142. It should be Drais et al. [19] and Brugneti et al. [6]. Line 168. It should be Moral et al. [20] …
Answer: We corrected the citation.

Rev: Line 170. It should be 10 to the 5 instead of 105. The same comment in Line 180.
Answer: We corrected the citation.

Rev: Line 181. It should be Garcia-Lopez et al. [21].
Answer: We corrected the citation.

Rev: Lines 178-185. This paragraph should be written without anticipating results for the Colletrotrichum species found.
Answer: We removed the names of the species.

 

Results

Rev: Line 192. According to the result presented with the analysis of the ITS region, the three species of Colletotrichum found can be defined. If so, I consider that this result could be shown, even if only in supplementary documents. The classifier of each of the three Colletotrichum species encountered during your description should be entered.
Answer: We added two further phylogenetic trees of the ITS of all the obtained isolates in the supplementary matherials Figure S1 and S2.

Rev: Line 228. Consider Conidiomata on Aristolochia leaf. 
Answer: We corrected the caption.

Rev: Line 259. I consider that Table 1 should not be included in the body of the article but as supplementary material.
Answer: We have removed the table from the main manuscript and moved it to the supplementary materials.

Rev: Lines 266 and 271. Delete (ML). Full scientific names should be given in figures and tables.
Answer: We have deleted the (ML) and we corrected the names.

Line 279. It should be (Fig. S1) instead of (Figure S1).  

Rev: In figures 5 and 6 the substitution model used must be indicated.  

Answer: This information is indicated in the main text in Lines 115-120.

Discussion

Rev: Line 331. Care must be taken when writing as the species are not isolated., Line 358. It should be Aristolochia by Zhi-jun et al. [28] …, Line 350. It should be while Tekade and Mohod [29] …
Answer: We have corrected the citations and changed the word “isolates“ with “identified “.

Conclusion

Rev: There is a final paragraph in the form of a conclusion with the subtitle named Discussion and conclusion. I proposed to split it in two separate subtitles.
Answer: We have separated the two paragraphs.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors identified the causal agent of anthracnose of A. grandiflora in Italy and found three species of Colletotrichum. Overall, the manuscript is well-written with some minor revisions needed, and the experimental designs seem sound with reasonable conclusions.

Throughout the manuscript, you should abbreviate most of "Colletotrichum" as "C." and "Aristolochia" as "A.", except in table titles or figure legends.

L276-L299: In the Mycelium growth assay, df=376, df=45, etc. These degrees of freedom seem too high for these experimental designs. If you compare the same species on the three different types of media, df should be 2 = 1 x (3-1). If you compared three species on three different media, df should be 4 = (3-1) x (3-1). There must be a mistake in data entry before running statistical tests. The authors should correct these df values.

Comments on the Quality of English Language

Some minor edits may need to improve readability of the manuscript.

Author Response

Review 2

The authors identified the causal agent of anthracnose of A. grandiflora in Italy and found three species of Colletotrichum. Overall, the manuscript is well-written with some minor revisions needed, and the experimental designs seem sound with reasonable conclusions.

Dear Reviewer, thank you for the time dedicated to revise our manuscript, as well as for the helpful comments and suggestions provided. We sincerely appreciate your positive comments, and we carefully addressed all the issues raised.

Rev: Throughout the manuscript, you should abbreviate most of "Colletotrichum" as "C." and "Aristolochia" as "A.", except in table titles or figure legends.
Answer: we have corrected the names. 

L276-L299: In the Mycelium growth assay, df=376, df=45, etc. These degrees of freedom seem too high for these experimental designs. If you compare the same species on the three different types of media, df should be 2 = 1 x (3-1). If you compared three species on three different media, df should be 4 = (3-1) x (3-1). There must be a mistake in data entry before running statistical tests. The authors should correct these df values.
Answer: Thank you for this comment. We have revised the computation of the degrees of freedom and it is right, as you can also see from the raw dataset and the script published as supplementary material. The measurements of the radius were carried out on the 4 cardinal directions, so that for each plate we obtained 4 data points. Accordingly, for each species we have 3x4=12 measurements per substrate and 12x3=48 total measurements per species. Considering the overall dataset composed of the overall species, we have a total of 48x8=384 measurements. 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript describes isolation and identification of Colletrichum spp. infecting Aristolochia grandiflora in botanical garden in Viterbo.  Additionally, pathogenocity tests were also performed. The study is well described and only some imrovements should be done.

In the phylogenetic analysis I miss species belonging to C. boninense species complex, especially C. watphraense and C. doitungense that are closely related to C. boninense.  These should be included and the data re-analyzed.

Author Response

Review 3

The manuscript describes isolation and identification of Colletotrichum spp. infecting Aristolochia grandiflora in botanical garden in Viterbo.  Additionally, pathogenocity tests were also performed. The study is well described and only some imrovements should be done.

In the phylogenetic analysis I miss species belonging to C. boninense species complex, especially C. watphraense and C. doitungense that are closely related to C. boninense.  These should be included and the data re-analyzed. 

Dear Reviewer, thank you very much for the important suggestions. The two species have been included in the ITS phylogenetic tree (Figure S2). However, they were not included in the concatenated phylogenetic tree since they were missing the HIS3 and CHS genes. 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors, 

We thank you for your responses, which have undoubtedly improved the quality of the manuscript. 

However, there are still some small details to be improved to which you should pay attention.

Lines 95-103. Every time a gene is referred to, it must be in italics.

line 109. C. boninense and C. orchidearum It shoud be in italic.

Line 111. Colletotrichum shoud be in italic. 

lines 113-114. C. gloeosporioides shoud be in italic. 

line 140. It should be the standard PDA and ...

line 183. Instead of 105 should be 10 to the power 5.

Lines 191-194. Consider One isolate for each Colletotrichum species were ...

Line 195. Again, it should be 10 to the power 5.

Lines 203-211. it should be ... Colletotrichum spp. The BLASTn results of the twenty ITS sequences on the GenBank database indicated that the isolates belonged to two specific complexes.

Lines 207-211. Please note that scientific names must be in italics. 

 

Line 368. It should be Zhi-jun et al. [28] ... 

Author Response

Dear reviewer,

Thanks for your further patience in reviewing the Manuscript. We previously addressed your current suggestions but apparently there were some incongruences and software incompatibility.

Please check now the PDF for a more precise revision.

 

Author Response File: Author Response.pdf